Differential effect of liver cirrhosis on the PXR-mediated...

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Differential effect of liver cirrhosis on the PXR-mediated induction of

cytochromes P450 3A1 and 3A2 in the rat.

Sara De Martin, Daniela Gabbia, Giovanna Albertin, Maria Martina Sfriso, Claudia Mescoli,

Laura Albertoni, Giovanna Paliuri, Sergio Bova, Pietro Palatini.

Affiliations

Department of Pharmaceutical and Pharmacological Sciences (S.D.M., D.G., G.P., S.B., P.P),

Department of Molecular Medicine, Section of Anatomy (G.A., M.M.S.),

Department of Medicine, Surgical Pathology & Cytopathology Unit (C.M., L.A.),

University of Padova, Padova, Italy.

This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on July 16, 2014 as DOI: 10.1124/dmd.114.058511

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Running Title: Effect of cirrhosis on CYP3A1 and CYP3A2 induction in rats

Corresponding author:

Prof. Pietro Palatini, Department of Pharmaceutical and Pharmacological Sciences, University

of Padova, Largo Meneghetti 2, 35131 Padova, Italy;

Telephone: +39 049 827 5077; Fax: +39 049 827 5093. E-mail: [email protected]

Number of text pages: 32

Number of tables: 3

Number of figures: 7

Number of references: 54

Number of words in the Abstract: 242

Number of words in the Introduction: 736

Number of words in the Discussion: 1489

Abbreviations: DEX, dexamethasone; PXR, Pregnane X Receptor; CYP, Cytochrome P450;

AhR, Aryl Hydrocarbon Receptor; GR, glucocorticoid receptor; CCl4, carbon tetrachloride;

DMSO, Dimethylsulfoxide; SDS-PAGE, sodium dodecylsulfate polyacrylamide gel

electrophoresis; TST, testosterone; ALT, alanine aminotransferase; AST, aspartate

aminotransferase.


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Abstract

Conflicting results have been obtained by clinical studies investigating the effect of liver

cirrhosis on enzyme induction. Since ethical concerns do not consent methodologically

rigorous studies in humans, we addressed this question by examining the effect of the

prototypical inducer dexamethasone (DEX) on the Pregnane X receptor (PXR)-mediated

induction of cytochromes P450 (CYPs) 3A1 and 3A2 in a validated animal model of liver

cirrhosis obtained by exposure of rats to carbon tetrachloride. For this purpose, we assessed

mRNA levels, protein expressions and enzymatic activities of both CYP3A enzymes, as well

as mRNA and protein expressions of PXR in rat populations rigorously stratified according to

the severity of liver insufficiency. Constitutive mRNA and protein expressions of CYP3A1

and CYP3A2, and their basal enzyme activities were not affected by liver dysfunction. DEX

treatment markedly increased steady-state mRNA level, protein content and enzymatic

activity of CYP3A1 in healthy and cirrhotic rats, irrespective of the degree of liver

dysfunction. On the contrary, the inducing effect of DEX on gene and protein expressions and

enzyme activity of CYP3A2 was preserved in moderate liver insufficiency, whereas was

greatly curtailed when liver insufficiency became severe. mRNA and protein expressions of

PXR were neither reduced by liver dysfunction nor increased by DEX treatment. These

results indicate that even the inducibility of CYP isoforms under the transcriptional control of

the same nuclear receptor may be differentially affected by cirrhosis and may partly explain

why conflicting results were obtained by human studies.


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Introduction

Metabolic interactions, i.e. inhibition and induction of drug-metabolizing enzymes, especially

those of the cytochrome P450 (CYP) superfamily, are the main cause of serious drug-drug

interactions encountered in clinical practice (Lin and Lu, 1998). Although inhibition-mediated

drug interactions are generally considered more dangerous than those due to induction

(Bachmann and Lewis, 2005), serious or even fatal consequences of CYP induction have been

reported, such as transplant rejection and exacerbation of liver failure caused by

acetaminophen overdose (Park et al., 1996; Fuhr, 2000).

In principle, drug interactions can be managed by dose adjustment. However, the wide

interindividual variability in the magnitude of metabolic drug interactions makes this task

difficult. Identification of the factors responsible for this variability is therefore crucial for an

appropriate dose adjustment in clinical practice. The influence of liver functional status on the

magnitude of metabolic interactions has long been investigated. However, whereas it has been

clearly established that liver dysfunction markedly decreases the magnitude of inhibitory drug

interactions (Orlando et al., 2004; Orlando et al., 2006; Orlando et al., 2009; Palatini et al.,

2010), the effect of liver insufficiency on the inducibility of drug-metabolizing enzymes is

still a controversial matter. It has been pointed out that two main factors are probably

responsible for the conflicting results obtained by studies on human beings (Hoyumpa and

Schenker, 1982; Palatini et al., 2008): 1) the variable nature and degree of hepatic dysfunction

in the clinical studies thus far performed; 2) the possible differential susceptibility to liver

insufficiency of the diverse types of inducing effects examined, which are mediated by

different mechanisms.

Early animal studies (Marshall and McLean, 1969; Farrell and Zaluzny, 1984; Wu et al.,

1991) found no cirrhosis-associated modification of the degree of induction of CYP-mediated


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metabolic reactions. However, they examined rats with essentially mild liver cirrhosis, which

generally causes minor alterations in the regulation of drug metabolism (Palatini et al., 2008).

Thus, they left unanswered the question of whether inducibility may be compromised in

severe liver insufficiency.

Since ethical constraints do not consent methodologically rigorous studies in human beings,

we recently tried to clarify this question by investigating the Aryl hydrocarbon Receptor

(AhR)-mediated induction of CYP1A1 and CYP1A2 in rats with experimentally induced

cirrhosis, rigorously stratified according to the severity of liver dysfunction (Floreani et al.,

2013). That study showed that the inducibility of CYP1A enzymes is strictly related to the

degree of liver impairment, as it is essentially preserved in the compensated state of cirrhosis,

whereas it becomes severely curtailed when decompensation occurs. However, these results

cannot a priori be extended to the induction of other CYP isoforms, since different nuclear

receptors are involved in the transcription of their CYP genes.

The aim of the present study was to assess the influence of liver dysfunction on the Pregnane

X Receptor (PXR)-mediated induction of CYP3A enzymes, the CYP subfamily most

abundantly expressed in the liver (Shimada et al., 1994), by dexamethasone (DEX), which is

the most effective inducer of rat CYP3A enzymes (Ghosal et al., 1996). For this purpose, we

evaluated the effect of DEX on control and cirrhotic rats obtained by exposure to carbon

tetrachloride (CCl4), which produces animals with compensated or decompesated cirrhosis

depending on the length of treatment (Floreani et al., 2013).

Although 4 CYP3A enzymes are expressed in rat liver (Mahnke et al., 1997), CYP3A2 and

CYP3A1 are regarded as the most metabolically relevant isoforms in male rats; the former,

which is male specific, is the isoform expressed at the highest constitutive level, the latter is

the isoform most susceptible to induction (Ghosal et al., 1996; Jan et al., 2006). Therefore, we


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studied the inducing effect of DEX on these two CYP3A enzymes by means of the three

techniques previously used for the assessment of the AhR-mediated CYP1A induction: 1)

determination of CYP3A1 and CYP3A2 mRNA steady-state levels by quantitative real-time

reverse transcription-polymerase chain reaction (Real-Time PCR); b) measurement of the

protein expressions of CYP3A enzymes by means of Western Blot analysis; c) determination

of the kinetic parameters of CYP3A1 and CYP3A2 enzyme reactions by means of kinetic

analysis of testosterone (TST) 6β-hydroxylase activity. Since in vitro experiments (Pascussi et

al., 2000; Huss and Kasper, 2000) have shown that PXR-mediated induction of CYP3A

enzymes by DEX is partly due to a positive regulation of PXR expression by the

glucocorticoid receptor (GR), we also assessed the effects of cirrhosis and DEX on the

mRNA and protein expressions of these two nuclear receptors.

Materials and Methods

Reagents.

NADPH was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Dimethylsulfoxide (DMSO), 40% acrylamide solution, sodium dodecyl sulfate (SDS), and

Tween 20 were purchased from Sigma-Aldrich Italy (Milan, Italy). Phenobarbital was

obtained from Bracco S.p.A. (Milan, Italy). Acetonitrile (HPLC grade) was from Carlo Erba

Reagenti (Milan, Italy). Ultrapure water was obtained by means of a Pure-Lab Option Q

apparatus (Elga Lab Water, High Wycombe, UK).

Microsomes prepared from baculovirus-infected insect cells expressing rat CYP3A1 or

CYP3A2 (SupersomesTM) were purchased from BD Gentest (Woburn, MA, USA). The

expression levels of CYP3A1 and CYP3A2 were provided by the manufacturer’s data sheets.


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Rabbit polyclonal anti-CYP3A1 antibody, sheep polyclonal anti-CYP3A2 antibody, mouse

polyclonal anti-PXR and anti-GR antibodies and rabbit anti-mouse IgG, used as secondary

antibody, were obtained from Abcam (Cambridge, UK); goat anti-rabbit and rabbit anti-sheep

IgG, also used as secondary antibodies, were obtained from Millipore (Billerica, MA, USA).

Mouse monoclonal anti-β-actin antibody was purchased from Santa Cruz Biotechnology, Inc.

(Santa Cruz, CA, USA).

Animals and treatments.

The procedures involving animals and their care were in conformity with institutional

guidelines that comply with national and international laws and policies (European Economic

Community Council Directive 86/609,OJ L 358, 1, Dec.12, 1987; NIH Guide for the Care and

Use of Laboratory Animals, NIH Publication no. 85-23, 1985). The study design was

approved by the Ethics Committee of the University of Padova for the care and use of

laboratory animals (Prot. no. 18758 – March 26, 2010).

Study design

The investigation was performed on male Wistar rats according to the experimental protocol

shown in Fig. 1, which was previously described in detail (Floreani et al., 2013). Briefly,

cirrhosis was induced by exposing animals to CCl4 in an inhalation chamber twice a week,

following a method previously described (Jimenez et al., 1992). Exposure time increased

from 30 sec to 4 min during the first 5 weeks, and was then 5 min from the 6th week to the end

of the treatment. Phenobarbital (0.3 g/l) was added to the drinking water of rats, since it

greatly increases the formation rate of the reactive metabolites of CCl4 responsible for the

development of cirrhosis, thereby drastically reducing the cirrhosis induction time (Jimenez et


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al., 1992; Rosa et al., 2010). After a 2-week washout period, which was shown to be

sufficient to allow full withdrawal of the inducing effect of phenobarbital on hepatic CYPs

(Marshall and McLean, 1969; Farrell and Zaluzny, 1984), healthy, non-ascitic and ascitic rats

were divided into two subgroups: induced and control rats. The former received 100 mg/kg

DEX dissolved in 3 ml of corn oil once daily for 3 consecutive days by the intraperitoneal

route; the latter received corn oil according to the same administration protocol. Treatment

with CCl4 was continued until 8 rats per group were obtained.

At the end of treatment, rats were weighed and sacrificed under sevoflurane anaesthesia (2.5%

per litre of oxygen) 24 hours after the last dose of DEX or corn oil. Blood was collected by

cardiac puncture for biochemical tests of liver function [alanine aminotransferase (ALT),

aspartate aminotransferase (AST), serum albumin and total bilirubin concentrations]. After

exsanguination, livers were rapidly removed and weighed, and a piece was excised for

histological examination. The liver was then rinsed in ice-cold 0.9% NaCl and a small piece

(less than 100 mg of hepatic tissue) was placed in an Eppendorf tube for Real-Time PCR

analysis. This sample and the remaining liver were quickly frozen and stored at -80°C until

used.

Histological evaluation.

Immediately after excision, a piece (3-4 g) of each liver was fixed in 4% neutral buffered

formalin for histological examination, as previously described (Floreani et al., 2013). Images

were obtained by means of a Leica SCN400 slide scanner. In order to evaluate the degree of

the CCl4-induced liver damage, the Ishak scoring system was adopted, which scores livers

from 0 to 6 according to the severity of cirrhotic alterations (Ishak et al., 1995).


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Preparation of liver microsomal fractions and total protein extracts.

Microsomal fractions were prepared exactly as previously described (Floreani et al., 2013).

Western Blot analysis of PXR expression in hepatic tissue was performed on total liver

protein extracts. For this purpose, frozen hepatic tissue (about 0.2 g) was allowed to thaw in a

modified ice-cold RIPA buffer consisting of 25 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM

EDTA, 1% TritonX-100, 1% sodium deoxycholate, 0.1% SDS, and a Complete Protease

Inhibitor Cocktail (Roche, Milan, Italy). Hepatic tissue was then homogenized in 10 volumes

(w/v) of the same buffer in ice for two 5-sec intervals with an IKA T25 Ultra-Turrax disperser

and processed as previously described (Floreani et al., 2012). Protein concentrations of both

microsomal fractions and total protein extracts were determined by means of a commercial

available kit (Novagen BCA Protein Assay kit) using a standard calibration curve obtained

with known amounts of bovine serum albumin.

Determination of mRNA levels by Real-Time PCR.

In order to determine the mRNA expression of CYP3A1, CYP3A2, PXR and GR, hepatic

tissue was homogenized and total RNA was extracted and purified by means of the SV Total

RNA Isolation System (Promega Corporation, Madison, WI). During RNA extraction, a

DNAse treatment was carried out to remove genomic DNA contamination. Total RNA was

then reverse transcribed to cDNA. Negative controls, used to verify the absence of

contaminating DNA, were obtained by processing samples of RNA in the absence of Reverse

Transcriptase. Real-time PCR was carried out in an I-Cycler iQ system (BioRad Laboratories,

Milan, Italy), as described in detail previously (Albertin et al., 2005), using the primers

reported in Table 1. The PCR program included a denaturation step at 95 °C for 3 min, 40

cycles of two amplification steps (95 °C for 15 s and annealing extension at 60 °C for 30 s)


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and a melting curve (60–90 °C with a heating rate of 0.5 °C/10 s). During the exponential

phase, the fluorescence signal threshold was calculated, and the fractional cycle number at

which each sample crossed the threshold (cycle threshold, Ct) was determined. Ct values were

used to calculate the relative mRNA expression, according to the mathematical quantification

model proposed by Pfaffl (Pfaffl, 2001). All samples were amplified in duplicate and

hydroxymethylbilane synthase (HMBS) and β-actin expressions were tested as housekeeping

genes. The results reported in this paper are calculated in reference to β-actin. The specificity

of the amplification procedure was tested at the end of each run by melting curve analysis,

using the I-Cycler software 3.0.

Western Blot analysis.

20 µg per lane of hepatic microsomal proteins, as well as 0.1 pmol of rat recombinant

CYP3A1 and CYP3A2 enzymes as reference standards, were subjected to sodium

dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 10% polyacrylamide gels

according to Laemmli (Laemmli, 1970). Anti-CYP3A1 or anti-CYP3A2 primary antibody

was alternatively used to detect CYP3A1 or CYP3A2 protein in the hepatic microsomal

fractions.

Signal intensity of immunoreactive bands was analyzed by the Quantity One software (Bio-

Rad Laboratories S.r.l.) and was expressed as intensity * mm2 (INT*mm2). According to a

previously described procedure for quantification of microsomal CYPs (see e.g., Yoshinari et

al., 2004; Paine et al., 2006), the signal intensity of CYP3A1 or CYP3A2 band was

normalized to a known amount of recombinant CYP3A1 or CYP3A2 microsomal protein

loaded into the gel.


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For the evaluation of PXR and GR expressions in liver samples, total liver protein extracts (60

µg of proteins per lane) were used. The signal intensity (INT*mm2) of the immunoreactive

band was normalized to that of the β-actin band.

Determination of testosterone 6β-hydroxylase activity of liver microsomes.

The kinetics of TST 6β-hydroxylase activity, a marker reaction for CYP3A enzymes

(Kobayashi et al., 2002; Walsky and Obach, 2004), was studied under rigorously controlled

initial-rate conditions, by determining the rate of 6β-hydroxy-testosterone (6β-OH-TST)

formation at increasing concentrations of TST. The incubation mixture (total volume: 0.2 ml)

contained 0.1 M KH2PO4 (pH 7.4), 0.5 mM NADPH, increasing concentrations of TST (from

5 to 800 µM, n = 11), and microsomal proteins or recombinant enzymes. For each

microsomal preparation preliminary experiments were performed, at the lowest and highest

substrate concentrations used for the kinetic assays, in order to assess linearity with respect to

incubation time and microsomal protein concentration. Incubation time was 3, 5 or 10 min,

and protein concentration varied according to the activity of the microsomal preparation,

ranging from 10 µg/ml (microsomes from DEX-induced animals) to 125 µg/ml (microsomes

from non-induced rats). Incubation conditions for recombinant enzymes were as described

above, except that 2.5 pmol/ml CYP3A1 and 5 pmol/ml CYP3A2 were used. After a 3-min

thermal equilibration at 37°C, the enzyme reaction was started by the addition of NADPH

and, after shaking in a water bath at 37°C in aerobic conditions, it was stopped by adding 0.1

ml ice-cold acetonitrile and cooling the sample in ice. The mixture was then centrifuged for

10 min at 20000 x g (4°C) to remove denatured proteins. An aliquot (100 ml) of the

supernatant was analyzed for 6β-OH-TST quantification by means of a previously described

HPLC method coupled to UV-VIS detection (Li and Letcher, 2002). Quantitative


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determination of 6β-OH-TST was carried out by standard calibration curves obtained with

authentic 6β-OH-TST at concentrations ranging from 1 to 50 nmol/ml (n = 8), processed in

exactly the same way as the samples obtained from kinetic experiments. The calibration

curves were linear in this concentration range (r2 ≥ 0.99), the lowest value of the range

representing the limit of quantification of the assay. Both inter- and intra-assay CVs for 6β-

OH-TST determination (n = 5) were lower than 5% at 1 nmol/ml and lower than 3% at 50

nmol/ml. TST 6β-hydroxylase activity was expressed as pmol of 6β-OH-TST produced per

mg of protein per min.

The inhibitory effect of the selective CYP3A inhibitor clotrimazole (Turan et al., 2001) on

TST 6β-hydroxylase activity was evaluated by using 6 pooled microsomal preparations

obtained from all rats belonging to each of the 6 study groups (control and DEX-treated

healthy, non-ascitic and ascitic rats). The substrate concentration used for these inhibition

experiments (50 µM) was chosen on the basis of the Km value obtained from the kinetic

analyses (see Results). Protein concentrations were 125 µg/ml and 10 µg/ml for controls and

DEX-treated rats, respectively. For assessment of inhibition of recombinant enzymes, 2.5

pmol/ml CYP3A1 and 5 pmol/ml CYP3A2 were used. Clotrimazole, dissolved in 2 µl of

acetonitrile, was added to the incubation mixture, and an equal volume of solvent was added

to control samples. Final inhibitor concentration ranged from 1 to 50 nM.

Kinetic and statistical analyses.

Initial velocity data for 6β-OH-TST formation catalyzed by recombinant CYP3A1 and

CYP3A2, and liver microsomes were evaluated by best-fitting procedures, by means of the

GraphPad Prism software, version 5 (GraphPad Software Inc., San Diego, CA, USA). The F

test was used to discriminate between different kinetic models [one- or two-enzyme


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Michaelis-Menten model, one-enzyme Michaelis-Menten kinetics with substrate inhibition or

autoactivation (sigmoidal) kinetics]. Kinetic parameters were estimated by non-linear

regression analysis of untransformed initial velocity data (GraphPad Prism software) using

the appropriate equation. The following kinetic parameters were determined: Vmax, maximum

velocity of the reaction; Km, substrate concentration yielding 50% of Vmax, and CLint, intrinsic

metabolic clearance, calculated as the ratio of Vmax to Km. IC50 values (inhibitor

concentrations causing 50% inhibition of control enzyme activities) were determined by the

use of non-linear regression of relative reaction velocities at increasing inhibitor

concentrations (Eagling et al., 1998).

Statistical analyses were also performed by means of the GraphPad Prism software. Unless

otherwise indicated, the data are presented as mean values ± S.D.. Comparison of the

experimental data obtained from healthy, non-ascitic and ascitic cirrhotic groups was made by

one-way analysis of variance (ANOVA). In case of significant differences (α = 0.05), the

analysis of variance was followed by the Newman-Keuls post-hoc test. Comparison of the

results obtained from control and induced animals within each group was performed by

means of Student’s t-test. P < 0.05 was considered statistically significant.

Results

Serum chemistry and histology of rat livers.

The results of biochemical tests of liver function and histological examination of healthy and

cirrhotic rats are shown in Table 2. Apart from serum albumin, there were only minor,

statistically non-significant differences between healthy and non-ascitic cirrhotic rats. All

indexes of liver function were significantly altered in rats with ascites, indicating advanced

hepatocellular insufficiency. The significantly higher AST and ALT values in DEX-treated


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rats of all 3 groups can be ascribed to the inducing effect of DEX on these hepatic enzymes

(Ennulat et al., 2010). Histological examination revealed normal liver architecture in healthy

animals (Fig. 2A), whereas non-ascitic (Fig. 2B) and ascitic (Fig 2C) cirrhotic rats showed

progressive alterations of liver structure, with increasing presence of fibrous septa, which

were never present in healthy animals. Quite similar cirrhosis-associated alterations were

observed in control and DEX-treated rats of each group.

mRNA and protein expressions of CYP3A1 and CYP3A2.

Fig. 3 shows the values of CYP3A1 mRNA and protein expressions relative to those of

control healthy rats. The level of CYP3A1 mRNA did not decrease significantly in cirrhotic

with respect to healthy animals (Fig. 3A). DEX treatment caused a significant increase in

CYP3A1 mRNA expression in all 3 groups of rats, although to a somewhat lesser extent in

ascitic animals. A representative Western Blot (Fig. 3B) shows that the specificity of the anti-

rat CYP3A1 antibody was not absolute, since it cross-reacted, although to a very limited

extent, with CYP3A2. Quantification of the immunodetectable CYP3A1 protein (Fig. 3C)

gave results in substantial agreement with those of Real-Time PCR, since it revealed that

protein expression was similar in the three groups of control rats, and increased significantly

in all groups of DEX-treated animals.

In contrast to CYP3A1, the constitutive mRNA expression of CYP3A2 (Fig. 4A) tended to

increase with the decline of liver function, although to a non-significant extent. At further

variance with CYP3A1, significant DEX induction of CYP3A2 mRNA was only observed in

healthy and non-ascitic rats, whereas it was absent in ascitic animals. The results of Western

Blot analysis (Fig. 4B and C) were in accordance with those of Real-Time PCR experiments,

regarding both the effects of cirrhosis and those of DEX treatment, since there was a slight,


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non-significant cirrhosis-associated increase in CYP3A2 protein expression in control

animals, and a significant inducing effect of DEX in healthy and non-ascitic animals, but not

in ascitic ones.

mRNA and protein expressions of PXR and GR.

Since DEX has been reported to induce CYP3A enzymes through both direct activation of

PXR and GR-mediated increase in PXR expression (Pascussi et al., 2000; Huss and Kasper,

2000), we measured the mRNA levels and the protein contents of both PXR and GR. Fig. 5A

shows that PXR gene expression was affected neither by cirrhosis nor by DEX treatment. The

results of Western Blot analysis (Fig. 5B-C) were in agreement with those of Real-Time PCR,

since no significant difference could be observed between healthy and cirrhotic rats or control

and induced ones. Similarly, neither cirrhosis nor DEX treatment caused any statistically

significant modification of mRNA or protein expression of GR (Fig. 6).

Kinetic analysis of testosterone 6β-hydroxylase activity.

Although TST 6β-hydroxylation is currently used as a marker reaction of CYP3A enzymes in

both humans and rats (Kobayashi et al., 2002; Walsky and Obach, 2004), its specificity in the

rat has been questioned (Chovan et al., 2007). Thus, in order to assess the specificity of TST

6β-hydroxylation, we tested the effect of clotrimazole, a potent and selective inhibitor of rat

CYP3A enzymes (Turan et al., 2001), on microsomal preparations of all 6 groups of rats. Fig.

7 shows that clotrimazole-induced inhibition of TST 6β-hydroxylation was always complete

at 50 nM, a concentration which does not appreciably affect the activity of other major CYP

isoforms (Turan et al., 2001). IC50 values were very similar for all microsomal preparations

(6-7 nM and 2-3 nM for microsomes obtained from control and induced rats, respectively),


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and similar to those observed with recombinant CYP3A1 (4.6 ± 0.5 nM) and CYP3A2 (2.0 ±

0.2 nM).

In preliminary experiments, we also measured midazolam 4- and 1’-hydroxylations,

alternative marker reactions for rat CYP3A enzymes (Kobayashi et al., 2002). Both these

reactions exhibited Michaelis-Menten kinetics with substrate inhibition, that could not be

fitted to a two-enzyme kinetic model. In the concentration range used in this study (5-800

µM), the TST 6β-hydroxylase activity of rat liver microsomes was best fitted to a two-enzyme

Michaelis-Menten equation, which is consistent with the presence of two main CYP3A

isoforms: CYP3A1 and CYP3A2. In order to distinguish between the activities of these two

enzymes, we determined the kinetic parameters of the reaction using rat recombinant

CYP3A1 and CYP3A2 enzymes. The results (shown in Table 3) indicate that, whereas the

Vmax values of the two enzymes were similar, the mean Km value was 13 times lower for

CYP3A2. Best fitting of the kinetic data obtained with microsomal preparations yielded two

Km values differing by an order of magnitude and similar to those observed with recombinant

enzymes, thereby consenting us to ascribe to CYP3A1 and CYP3A2 the microsomal activities

with greater and lower Km, respectively (Table 3). The results of the best-fitting procedure

were confirmed by the Eadie-Hofstee graphical analysis, the plotting technique most sensitive

to deviations from linearity, which gave markedly curved plots, consistent with the presence

of two enzymes with considerably different Km (Segel, 1975), for all rats examined in this

study (results not shown). Table 3 also shows that liver function impairment, rather than

decreasing the Vmax of CYP3A1 and CYP3A2 activities in control rats, caused a non-

significant increment, particularly in non-ascitic cirrhotic animals, as previously observed

with non-specific CYP probes in the same rat model of cirrhosis (Wu et al., 1991). In full

agreement with mRNA and protein expression data, DEX treatment significantly increased


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the Vmax of the CYP3A1-catalyzed reaction in all three groups of rats, although to a somewhat

lower extent in ascitic animals, whereas it increased significantly the Vmax of CYP3A2 only in

healthy and non-ascitic cirrhotic rats. The Km values of both CYP3A1 and CYP3A2 activities

were similar in all groups of rats. As a consequence, the modifications of CLint (the ratio of

Vmax to Km) paralleled those of Vmax. The absolute values of CLint of the recombinant enzymes

were very similar to those previously obtained by Carr et al. (2006) (3.3 and 0.1 µl/min/pmol

CYP for rCYP3A2 and rCYP3A1, respectively), thus confirming the greater catalytic

efficiency of CYP3A2.

Discussion

Induction in human beings has been assessed by measuring modifications of the

pharmacokinetic parameters (half-life or systemic clearance) of a metabolic probe or of the

enzyme activities of liver tissues obtained at biopsy. Although such determinations provide a

reliable measure of the extent of induction, they give no information regarding the mechanism

by which liver cirrhosis may impair enzyme induction. We have recently shown that the use

of an animal model of liver cirrhosis produced by exposure to CCl4 makes it possible to study

the mechanisms by which cirrhosis may affect enzyme induction by means of the

simultaneous determination of the mRNA level, protein expression and enzymatic activity of

the induced enzyme(s) in the liver obtained from sacrificed animals (Floreani et al., 2013).

Previous studies using Long-Evans (Cooper et al., 1993) or Sprague-Dawley (Jan et al., 2006)

male rats found no detectable mRNA level or negligible protein expression of CYP3A1,

respectively. The results of our Real-Time PCR determinations and Western Blot analyses

show that both CYP3A1 and CYP3A2 are constitutively expressed in male Wistar rats and

their mRNA and protein levels are not significantly modified by liver cirrhosis. The latter


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finding contrasts with the previous observation regarding CYP1A2 expression in the same

animal model of cirrhosis, since the basal mRNA and protein levels of CYP1A2 were found

to decrease significantly (up to 80%) in proportion to the severity of liver dysfunction

(Floreani et al., 2013). That result was in agreement with the observations of human studies,

which have consistently found a profound fall in CYP1A2 expression in liver cirrhosis

(reviewed in Hasler, 1999). On the contrary, studies of the effect of cirrhosis on the protein

expression of human CYP3A enzymes have generated conflicting results, since no significant

modifications of protein levels have been observed by Guengerich and Turvy (1991) or Lown

et al. (1992), whereas George et al. (1995) observed a significant decrease in hepatocellular,

but not cholestatic cirrhosis. The observation that the metabolic clearance of CYP3A

substrates is generally reduced in decompensated cirrhosis (Verbeeck, 2008) is not

incompatible with a preserved CYP3A expression, since a large body of evidence has

accumulated that other factors, such as reduced number of functional hepatocytes and

impaired drug uptake by the liver, may be more important determinants of the cirrhosis-

associated decrease in drug biotransformation (Morgan and McLean, 1995; Orlando et al.,

2006).

DEX treatment caused highly significant increases in both gene and protein expressions of

CYP3A1 in healthy as well as cirrhotic rats, irrespective of the degree of liver dysfunction. In

contrast, CYP3A2 gene and protein expressions were significantly induced in healthy and

non-ascitic cirrhotic rats, but not in those with ascitic cirrhosis, indicating that the induction of

this CYP isoform is greatly curtailed when liver dysfunction becomes severe. A comparison

of the results of Real-Time PCR and Western Blot determinations reveals a further difference

between these two CYP3A isoforms in their response to DEX treatment, since a quantitatively

similar degree of induction is observed for CYP3A2 mRNA level and protein expression,


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whereas the increase in CYP3A1 mRNA level exceeds by one order of magnitude that in

protein expression. Differences in the degree of mRNA and protein expression have been

observed with various proteins including CYP enzymes, and have been attributed to

translational repression by miRNAs (Singh et al., 2011). Thus, our observations suggest that

the translations of CYP3A1 and CYP3A2 mRNAs are subject to different miRNA-mediated

controls.

Since the expression of CYP3A enzymes is under the transcriptional control of PXR, and

PXR upregulation, mediated by DEX activation by GR, has been observed in vitro with both

human (Pascussi et al., 2000) and rat hepatic cell lines (Huss and Kasper, 2000), we assessed

the effects of liver dysfunction and DEX on the mRNA and protein expressions of both PXR

and GR. However, we could not confirm in vivo the results previously obtained with cultured

hepatocytes, since we observed no significant modification of the mRNA or protein

expression of these nuclear receptors. Thus, unlike AhR, whose transcription has been found

to be reduced in vivo in the presence of liver cirrhosis (Floreani et al., 2013), and upregulated

following the administration of various AhR ligands (Floreani et al., 2012; Franc et al., 2001),

PXR mRNA and protein levels are neither appreciably reduced by liver cirrhosis nor

increased by DEX treatment. The former finding is consistent with the observation that the

constitutive expression of both CYP3A enzymes is unaffected by liver dysfunction, and is

also consistent with the observation that CYP3A1 induction is not compromised in liver

cirrhosis, but leaves unanswered the question of why the induction of CYP3A2, which is

transcriptionally activated by the same nuclear receptor, is compromised in decompensated

cirrhosis. Although no certain explanation can be provided on the basis of the present

knowledge of CYP3A2 transcription machinery, it may be hypothesized that, in addition to

mRNA translation, the gene transcription of CYP3A2 is also under the control of a different


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regulatory mechanism involving either coactivators or a RNA polymerase more sensitive than

those of CYP3A1 to the modifications associated with severe liver cirrhosis. The observation

that the mRNA level of CYP3A1 is increased to a far greater extent in response to DEX

treatment than that of CYP3A2 is consistent with a different regulatory control of their

transcription. Evidence that the mRNA expressions of CYP3A1 and CYP3A2 are not

coordinately regulated in response to DEX treatment has also been obtained by (Choudhuri et

al., 1995).

Although Real-Time PCR and Western Blot analyses provide information relevant to the

mechanism (altered transcription or translation) underlying changes in CYP expression,

determination of CYP activity provides the unique quantitative measure of changes in CYP-

mediated drug metabolism (Murray et al., 2001), and is the methodological approach

recommended by US FDA (Hewitt et al., 2007). In previous kinetic analyses of TST 6β-

hydroxylation by rat liver microsomes, velocity data were fitted to an one-enzyme hyperbolic

kinetic model (Jimenez et al., 1992; Murray and Butler, 1996; Jiko et al., 2005; Velenosi et

al., 2012) or to a sigmoidal kinetics with low degree of autoactivation [Hill coefficients of

1.2-1.4 (Carr et al., 2006)]. This did not consent a determination of the separate contributions

of the two main CYP3A isoforms (CYP3A1 and CYP3A2) to the measured enzymatic

activity. Based on velocity curves obtained with 11 substrate concentrations in rigorously

controlled initial rate conditions, we could show that the kinetic data were best described by a

two-enzyme Michaelis-Menten model, and that the Km values obtained with liver microsomes

were in good agreement with those of recombinant CYP3A1 and CYP3A2. This has enabled

us to determine for the first time the changes in activity of each of these two CYP isoforms.

The results of these determinations confirm the indications of Real-Time PCR and Western

Blot analyses that both CYP3A1 and CYP3A2 are constitutively expressed in Wistar rats. The


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observed changes in Vmax are also in full agreement with those in mRNA and protein

expressions since, a) the constitutive activities of both CYP3A1 and CYP3A2 are not

significantly modified by liver dysfunction; b) the inducibility of CYP3A1 activity is

preserved in both non-ascitic and ascitic cirrhotic rats, whereas that of CYP3A2 is preserved

in rats with moderate liver dysfunction, but significantly decreased when liver dysfunction

becomes severe.

In conclusion, the results of our Real-Time PCR, Western Blot and kinetic analyses all concur

in showing that the induction of CYP3A enzymes is differentially affected by liver function

impairment since, unlike CYP3A1, CYP3A2 is not significantly induced in severe liver

dysfunction. The induction of the latter enzyme is compromised at a pretranslational level,

since proportional reductions in induced mRNA level, protein expression and enzyme activity

are observed in ascitic rats. The effect of liver dysfunction on both the constitutive expression

and the PXR-mediated induction of CYP3A enzymes differs from that on the basal expression

and the AhR-mediated induction of CYP1A enzymes, observed in the same animal model of

liver cirrhosis (Floreani et al., 2013). That study showed that the constitutive expression of

CYP1A2 (the sole CYP1A isoform constitutively expressed in the liver) is decreased in

cirrhosis and the induction of both CYP1A enzymes is well preserved in compensated, but

markedly reduced in decompensated cirrhosis. These effects on both constitutive and induced

enzyme expressions could be ascribed to a decreased transcription of CYP1A genes as a

consequence of a reduced AhR expression. Unlike AhR, PXR expression is not reduced in

liver dysfunction, and the precise mechanism responsible for the selective impairment of

CYP3A2 transcription remains to be determined. Whatever the explanation may be for this

selective effect, the present results make it clear that no general conclusion can be drawn from

the study of any particular CYP isoform, since even the induction of enzymes under the


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transcriptional control of the same nuclear receptor may be differentially affected by liver

function impairment. These findings may also partly explain why conflicting results have

been obtained by human studies assessing the effect of liver dysfunction on the induction of

drug metabolism.


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Acknowledgments

We thank Mr. Mauro Berto for skilful technical assistance.

Authorship Contributions

Participated in research design: Palatini

Conducted experiments: Gabbia, Sfriso, Mescoli, Albertoni, Paliuri

Performed data analysis: De Martin, Albertin, Mescoli, Bova, Palatini

Wrote or contributed to the writing of the manuscript: De Martin, Palatini


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Footnotes

This work was supported by a grant [# CPDA093519] from the University of Padova, Padova,

Italy.

Presented as a poster at the Italian Society of Pharmacology (Società Italiana di Farmacologia,

SIF) meeting, 2013. Abstract published in the SIF 2013 meeting homepage

(http://congresso.sifweb.org/abs/191.pdf)

Address for reprint requests: Prof. Pietro Palatini, Department of Pharmaceutical and

Pharmacological Sciences, University of Padova, Largo Meneghetti 2, 35131 Padova, Italy;

(email: [email protected]).


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Figure legends

Figure 1. Study design. PB: Phenobarbital; DEX: dexamethasone.

Fig. 2. Histological analysis. Representative photomicrographs (Van Gieson stain, 200 x

magnification) of liver sections taken from an healthy (Ishak score = 0) (A), a non-ascitic

(Ishak score = 4) (B), and an ascitic cirrhotic rat (Ishak score = 6) (C).

Figure 3. CYP3A1 gene and protein expressions in control and DEX-treated rats. (A)

Experimental data showing CYP3A1 mRNA levels are reported as fold variation compared

with healthy control rats. (B) Representative Western blot showing the separation of

microsomal proteins obtained from control and DEX-treated rats. As reference standards,

0.125 pmol of rat recombinant CYP3A1 or CYP3A2 protein were loaded in lanes 1 and 2,

respectively. Lanes 3 to 5: healthy, non-ascitic and ascitic control rats; lanes 6 to 8: healthy,

non-ascitic and ascitic DEX-treated rats. (C) Amount of CYP3A1 protein in livers from

control and DEX-treated rats normalized to the signal intensity of the CYP3A1 band and

shown as fold variation compared with healthy control rats. All results are means ± S.E. of

data obtained from 8 rats per group. ***p<0.001 vs. control rats of the same group; §p<0.05 vs.

DEX-treated healthy rats.

Figure 4. CYP3A2 gene and protein expressions in control and DEX-treated rats. (A)

Experimental data showing CYP3A2 mRNA levels are reported as fold variation compared

with healthy control rats. (B) Representative Western blots showing the separation of

microsomal proteins obtained from control and DEX-treated rats. As reference standards,

0.125 pmol of rat recombinant CYP3A1 or CYP3A2 protein were loaded in lanes 1 and 2,

respectively. Lanes 3 to 5: healthy, non-ascitic and ascitic control rats; lanes 6 to 8: healthy,

non-ascitic and ascitic DEX-treated rats. (C) Amount of CYP3A2 protein in livers from

control and DEX-treated rats normalized to the signal intensity of the CYP3A2 band and


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shown as fold variation compared with healthy control rats. All results are means ± S.E. of

data obtained from 8 rats per group. **p<0.01, ***p<0.001 vs. control rats of the same group;

§p<0.05 vs. DEX-treated healthy rats; #p<0.05 vs. DEX-treated non-ascitic cirrhotic rats.

Figure 5. PXR mRNA and protein expressions in control and DEX-treated rats. (A)

Experimental data showing PXR mRNA levels are reported as fold variation compared with

healthy control rats. (B) Representative Western blots showing the separation of total liver

protein extracts obtained from control and DEX-treated rats. Lanes 1 to 3: healthy, non-ascitic

and ascitic control rats; lanes 4 to 6: healthy, non-ascitic and ascitic DEX-treated rats. (C)

Amount of PXR protein in livers from control and DEX-treated rats normalized to the signal

intensity of the corresponding β-actin band (43 kDa) and shown as fold variation compared

with healthy control rats. All results are means ± S.E. of data obtained from 8 rats per group.

Figure 6. GR mRNA and protein expressions in control and DEX-treated rats. (A)

Experimental data showing GR mRNA levels are reported as fold variation compared with

healthy control rats. (B) Representative Western blots showing the separation of total liver

protein extracts obtained from control and DEX-treated rats. Lanes 1 to 3: healthy, non-ascitic

and ascitic control rats; lanes 4 to 6: healthy, non-ascitic and ascitic DEX-treated rats. (C)

Amount of GR protein in livers from control and DEX-treated rats normalized to the signal

intensity of the corresponding β-actin band (43 kDa) and shown as fold variation compared

with healthy control rats. All results are means ± S.E. of data obtained from 8 rats per group.

Figure 7. Inhibitory effect of clotrimazole on TST-6β-hydroxylase activities of pooled

microsomal preparations obtained from control (left column) and DEX-treated (right

column) rats. Results are the means ± S.E. of 3 separate determinations performed in

duplicate. S.E. is not shown where the size of data points is larger than the S.E. bar.


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Table 1. Primer sequences used in this study, NCBI reference sequences and amplicon sizes

(base pairs).

Gene Forward primer Reverse primer RefSeq Size (bp)

CYP3A1 5’- cca-tca-cgg-aca-cag-aaa-tg - 3’ 5’ – ctt-tcc-cca-taa-tcc-cca-ct - 3’ NM013105 102

CYP3A2 5’ – agt-ggg-gat-tat-ggg-gaa-ag - 3’ 5’ – caa-tga-tgg-gga-aca-tct-cc - 3’ NM153312 119

PXR 5’ – caa-atc-tgc-cgt-gta-tgt-gg - 3’ 5’ – gtt-tca-tgg-ccc-ttc-tga-aa - 3’ NM052980 97

GR 5’- ggt-gat-tga-acc-cga-ggt-gt - 3’ 5’ – tgg-tat-cgc-ctt-tgc-cca-tt - 3’ NM012576 136

HMBS 5’ – ccc-ttg-tga-tgc-tgt-tgt-ttt - 3’ 5’ – tgt-tga-ggt-ttc-ccc-caa-ta - 3’ NM013168 161

β-ACTIN 5’ – gcc-acc-agt-tcg-cca-tgg-a - 3’ 5’ – ttc-tga-ccc-ata-ccc-acc-at - 3’ NM031144 163


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Table 2. Biochemical tests of liver function and histological examination.

Healthy rats

Non-ascitic rats

Ascitic rats

Liver test

Control DEX-treated

Control DEX-treated

Control DEX-treated

Albumin (g/l)

40 ± 3 38 ± 6

33 ± 3§§§ 30 ± 4§§

31 ± 2§§§ 28 ± 5§§

Total bilirubin (mmol/l)

0.91 ± 0.62 1.06 ± 0.12

1.06 ± 0.64 1.40 ± 0.95§§

1.57 ± 1.28 2.40 ± 1.01§§§,#

AST (U/l)

116 ± 28 184 ± 52***

127 ± 33 197 ± 58**

168 ± 22§§§,# 240 ± 40***

ALT (U/l)

41 ± 9 185 ± 46***

58 ± 11 160 ± 36***

85 ± 24§§§,## 221 ± 3***

Ishak Score (0-6)

0 (0-0) 0 (0-0)

4 (3-5) 4 (3-5)

6 (6-6) 6 (6-6)

Results are means ± S.D. of data obtained from 8 rats per group. *p<0.05, **p<0.01, ***p<0.001

vs. control rats of the same group; §§p<0.01, §§§p<0.001 vs. healthy rats; #p<0.05, ##p<0.01, vs.

non-ascitic cirrhotic rats.


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Table 3. Kinetic parameters for TST-6β-hydroxylase activities of recombinant CYPs and

microsomal preparations obtained from control and DEX-treated rats.

CYP3A1 CYP3A2

Vmax

a

Km (µM)

CLintb

Vmax

a

Km (µM)

CLintb

Recombinant CYP3A1

5.3 ± 1.0 34 ± 1 0.15 ± 0.04 - - -

Recombinant CYP3A2

- - - 7.4 ± 0.3 2.6 ± 0.1 2.8 ± 0.1

Healthy Control 984 ± 636 54 ± 25 19 ± 12 374 ± 175 2.9 ± 1.9 128 ± 64

Healthy DEX-treated 5275 ± 1091*** 45 ± 9 119 ± 32*** 1561 ± 903*** 2.7 ±1.4 643 ± 274***

Non-ascitic Control 1224 ± 325 48 ± 13 27 ± 11 508 ± 267 4.2 ± 1.6 119 ± 35

Non-ascitic DEX-treated

4302 ± 1215*** 47 ± 17 99 ± 35* 2453 ± 1302** 3.8 ± 2.5 645 ± 333**

Ascitic Control 1081 ± 298 55 ± 15 22 ± 11 468 ± 224 4.6 ± 0.6 106 ± 43

Ascitic DEX-treated 3330 ± 423***,§ 48 ± 9 72 ± 19*** 990 ± 598#,§ 2.8 ± 2.4 350 ± 211**

Results for recombinant enzymes are the means ± S.D. of three separate determinations performed in duplicate;

those for liver microsomes are the means ± S.D. of data obtained from 8 rats per group.

a Vmax is expressed as pmol/min/mg protein for liver microsomes and pmol/min/pmol CYP for recombinant

enzymes.

b CLint is expressed as µl/min/mg protein for liver microsomes and µl/min/pmol CYP for recombinant enzymes.

*p<0.05, **p<0.01, ***p<0.001 vs. control rats of the same group; §p<0.05 vs. healthy rats; #p<0.05 vs. non ascitic

cirrhotic rats.


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