in vivo responses of the human and murine pregnane x...

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In vivo responses of the human and murine pregnane X receptor to

dexamethasone in mice

Nico Scheer, Jillian Ross, Yury Kapelyukh, Anja Rode, and C. Roland Wolf

TaconicArtemis, Neurather Ring 1, Koeln 51063, Germany (NS, AR); CXR Biosciences

Limited, 2 James Lindsay Place, Dundee, DD1 5JJ, United Kingdom (JR, YK, CRW). Cancer

Research U.K., Molecular Pharmacology Unit, Biomedical Research Institute, Ninewells

Hospital and Medical School, University of Dundee, Dundee DD1 9SY, United Kingdom

(CRW).

DMD Fast Forward. Published on March 30, 2010 as doi:10.1124/dmd.109.031872

Copyright 2010 by the American Society for Pharmacology and Experimental Therapeutics.

This article has not been copyedited and formatted. The final version may differ from this version.DMD Fast Forward. Published on March 30, 2010 as DOI: 10.1124/dmd.109.031872

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Running title: Dexamethasone interactions with human PXR in mice

Address correspondence to:

Nico Scheer, PhD, TaconicArtemis, Neurather Ring 1, 51063 Koeln, Germany. Tel.: +49 221

9645343. Fax: +49 221 9645321. Email: [email protected]

Number of text pages: 28

Number of tables: 0

Number of figures: 6

Number of references: 39

Number of words in the abstract: 218

Number of words in the introduction: 638

Number of words in the discussion: 977

Nonstandard abbreviations used: ALT, alanine aminotransferase; BQ, 7-

benzyloxyquinoline; CAR, constitutive androstane receptor; CYP, cytochrome P450; DEX,

dexamethasone; ES cell, embryonic stem cell; GR, glucocorticoid receptor; hPXR, human

pregnane X receptor; huPXR, humanized pregnane X receptor mice; huPXRi, improved

humanized pregnane X receptor mice; PROD, pentoxyresorufin dealkylation; PXR, pregnane

X receptor; PXR KO, pregnane X receptor knock out mice; RIF, rifampicin


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Abstract

Dexamethasone (DEX) is a potent and widely used anti-inflammatory and

immunosuppressant glucocorticoid. It can bind and activate the pregnane X receptor (PXR),

which plays a critical role as xenobiotic sensor in mammals to induce the expression of many

enzymes including cytochrome P450s (CYPs) in the CYP3A family. This induction results in

its own metabolism. We have used a series of transgenic mouse lines, including a novel,

improved humanized PXR line to compare the induction profile of PXR-regulated drug

metabolising enzymes following dexamethasone administration as well as looking at hepatic

responses to rifampicin. The new humanized PXR model has uncovered further intriguing

differences between the human and mouse receptors in that rifampicin only induced Cyp2b10

in the new humanized model. Dexamethasone was found to be a much more potent inducer of

Cyp3a proteins in wild type mice than in mice humanized for PXR. In order to assess whether

PXR is involved in the detoxification of dexamethasone in the liver, we analysed the

consequences of high doses of the glucocorticoid on hepatotoxicity on different PXR genetic

backgrounds. We also studied these effects in an additional mouse model where functional

mouse Cyp3a genes have been deleted. These strains exhibited different sensitivities to

dexamethasone, indicating a protective role of the PXR as well as CYP3A proteins against the

hepatotoxicity of this compound.


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Introduction

Dexamethasone (DEX) is a synthetic glucocorticoid which is used to treat a wide variety of

medical conditions. It is a potent anti-inflammatory and immunosuppressant, which is

metabolised primarily by the liver and undergoes renal excretion (Minagawa et al., 1986).

Dexamethasone induces cytochrome P450 genes, such as Cyp3a11 and Cyp2b10 in mice and

CYP3A4 in humans (Watkins et al., 1985; Wrighton et al., 1985; Strom et al., 1996;

Yanagimoto et al., 1997). The mechanism of this induction is complex and is mediated by a

number of nuclear receptors. For example, the glucocorticoid receptor (GR) is essential for

the DEX-mediated induction of Cyp2b proteins in mouse liver, but at high doses the induction

of Cyp3a enzymes is mediated by other transcription factors (Schuetz et al., 2000). In

addition, it has been reported that DEX induction of CYP3A4 in human hepatocytes is

mediated by several transcription factors (Pascussi et al., 2001). At low (nmol) concentrations

CYP3A4 induction is mediated by a GR-dependent elevation of pregnane X receptor (PXR)

expression, while at high (supramicromolar) concentrations DEX directly binds to and

activates PXR. In addition, submicromolar concentrations of DEX also enhance the

expression of the constitutive androstane receptor (CAR) and the retinoid X receptor-alpha in

human hepatocytes (Pascussi et al., 2000a; Pascussi et al., 2000b).

It has been demonstrated that DEX is a more potent ligand for mouse PXR than the human

receptor (Meehan et al., 1988; Moore et al., 2000) and hepatic induction of Cyp3a, but not

Cyp2b, expression in mice is PXR-dependent (Schuetz et al., 2000; Scheer et al., 2008).

CYP3A plays a central role in DEX disposition in human liver as it catalyzes the formation of

the two major metabolites, 6α and 6ß-hydroxy-dexamethasone (Gentile et al., 1996). Indeed,

the relatively simple metabolic profile of DEX compared with many other substrates led to

the proposal that DEX would be a useful in vivo probe for CYP3A4 activity (Gentile et al.,

1996). Therefore, the ability of DEX to activate PXR in different species would be predicted


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to be a significant factor in determining both the metabolism and circulating levels of this

glucocorticoid.

DEX can influence the toxicity of other compounds by enzyme induction. For example, pre-

treatment of rats with DEX (5-20 mg/kg, p.o.) 24h before administration of the antitumor drug

yondelis (ET-743) markedly reduced the biochemical and histopathological manifestations of

yondelis-induced liver changes (Donald et al., 2003). This protective effect was speculated to

be a consequence of the decreased hepatic exposure to yondelis linked to induction of

metabolism by CYP enzymes.

On the other hand, pre-treatment of mice with high doses of DEX (75 mg/kg, ip, for 4 days)

increased acetaminophen-induced hepatotoxicity and at this dose DEX was also hepatotoxic

by itself (Madhu et al., 1992). At the therapeutic doses used in humans, DEX is not regarded

as a hepatotoxic. There are however some occasional cases in which DEX administration is

associated with severe hepatotoxicity. For example, DEX treatment has been associated with

methotrexate hepatotoxicity in children in a manner unrelated to changes in methotrexate

serum-levels (Wolff et al., 1998).

The in vivo factors which determine the metabolism and toxicity of DEX are complex and,

because of species differences in its interaction with nuclear receptors, such as CAR, PXR

and GR, studies in rodents might not accurately reflect the human situation. In this study we

have used mice either deleted or humanized at the Pxr gene locus as well as mice where seven

functional Cyp3a genes have been deleted to evaluate the roles of the human receptor and

Cyp3a proteins in DEX response. As part of this work we describe the generation of a new

and improved humanized PXR model. The data presented demonstrate that mouse and human

PXR exhibit marked differences in their in vivo interactions with DEX and the impact of these

differences on DEX-hepatotoxicity were analysed in this study.


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Materials and Methods

Animal husbandry. Mice were kept in Tecniplast Sealsafe microisolator cages. Food and

water were available ad libitum. Light cycles were on a 13:11 h light/dark cycle with the light

phasing starting at 06:00 h. Temperature and relative humidity were maintained between

21°C and 23°C and 45% and 65%.

Construction of the targeting vector used for the generation of an improved PXR

humanized mouse line (huPXRi). Intron 6 and 7 of the human PXR gene were amplified by

PCR from a bacterial artificial chromosome carrying human PXR and subcloned into the

targeting vector used for the generation of the PXR humanised (huPXR) mice previously

described (Scheer et al., 2008), to give rise to the targeting vector depicted in Figure 1.

Generation and molecular characterisation of huPXRi targeted embryonic stem (ES)

cells. Culture and targeted mutagenesis of ES cells were carried out as previously described

(Hogan et al., 1994). The targeting vector was linearised with NotI and electroporated into a

C57BL/6 mouse ES cell line. Of 132 hygromycin resistant and fluorescence negative ES cell

colonies screened by standard Southern blot analyses, 12 correctly targeted clones were

identified. Six of these were expanded and further analysis by Southern blot analyses with

different suitable restriction enzymes and 5’ and 3’ external probes and an internal

hygromycin probe, confirmed that these clones were correctly targeted at both homology arms

and didn’t carry additional random integrations (data not shown).

Generation and molecular characterisation of PXRi humanized mice. One of the PXRi

targeted ES cell clones described above was expanded, injected into BALBc-blastocysts and

transferred into foster mothers as previously described (Hogan et al., 1994). Litters from these

fosters were visually inspected and chimerism was determined by hair colour. Highly

chimeric animals were used for further breeding in a C57BL/6 genetic background. The


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hygromycin selection cassette was removed in vivo by crossing to an efficient FLP-deleter

(FLPe-deleter) strain carrying a transgene that expresses FLPe in the germline (Figure 1). The

FLPe-deleter has been generated in house on a C57BL/6 genetic background. The PXRi

humanized allele was detected by PCR with the primers 5’-GGACTTGCCCATCGAGGAC-

3’ and 5’-ACAGGATGGAGGGGCAGC-3’, giving rise to a 332 bp PCR fragment. PXR

knockout (PXR KO) and huPXR mice as described previously (Scheer et al., 2008) were used

in addition to the huPXRi mice described here. Homozygously humanized and knockout mice

for PXR were used for all studies and WT C57BL/6 animals for control experiments were

purchased from Harlan (UK).

Animal and Treatments. All animal procedures were carried out under a United Kingdom

Home Office license, and all animal studies were approved by the Ethical Review Committee,

University of Dundee. Male, sexually mature huPXR, huPXRi, PXR KO, Cyp3a-/-/Cyp3a13+/+

and WT (C57BL/6J) mice, were dosed daily by IP injection with either the vehicle, rifampicin

(3, 10, 20, 40 or 60 mg/kg/day) or dexamethasone (1, 3, 10, 30 or 60 mg/kg/day) for 4 days

and sacrificed 24 hrs post last dose.

Quantitative Reverse Transcriptase PCR (qRT-PCR). Human PXR RNA was analysed by

quantitative RT-PCR (TaqMan®). Total RNA was prepared from the liver using the Qiagen

RNA mini kit. cDNA was synthesized from 1µg total RNA using the Quantiscript Reverse

Transcriptase Kit (Qiagen). Primers for human PXR were from the Assay-On-Demand Kit

Hs00243666_ml (Applied Biosystems). qRT-PCR reactions were performed using TaqMan

Universal PCR Mastermix in an ABI PRISM 7000 Sequence Detection System (Applied

Biosystems). Data were analysed using comparative cycling time methology, in which

fluorescent output, measured as CT, was directly proportional to input cDNA concentrations.


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Input cDNA concentrations were normalized to murine ß-actin. Calculations were performed

by a comparative method (2-ΔΔCT).

Sequencing Analysis. RT-PCR was performed using RNA isolated from a huPXRi mouse

using oligos to amplify the potential full-length transcript. The oligos used were P6-F: 5’ GGA

ATT CAA CAT GGA GGT GAG ACC CAA AG 3’, hPXR-REV_3: 5’ TCA GCT ACC TGT GAT GCC

GAA CAA C 3’. One step RT-PCR reaction conducted using Superscript RT-PCR Kit

(Invitrogen). Sequence analysis was performed by Lark Technologies Ltd. (Takeley, UK).

Alignments were performed using T-COFFEE (http://tcoffee.vital-it.ch/cgi-

bin/Tcoffee/tcoffee_cgi/index.cgi) and Contig express and Align-X programs of Vector NTI 8

Software.

Hepatic Microsomal Preparation. Mouse livers used were freshly harvested, blotted and

weighed. The liver was scissor-minced in ice-cold KCl (1.15 % (w/v) then homogenized in

ice-cold SET buffer (0.25M sucrose, 5mM EDTA and 20mM Tris-HCL, pH7.4) to make a

10% (w/v) homogenate solution (9mL SET buffer/1g liver). Microsomes were prepared by

centrifugation firstly at 6441g (Heraeus 6445 rotor/Sorvall legend RT centrifuge) for 10mins

at 4°C, then the supernant was spun at 13864g (Sorvall F28/13 rotor) for 15mins at 4°C. The

resulting supernatant was spun at 82187g (Sorvall F28/13 rotor) for 90mins at 4°C and the

microsomal pellets were resuspended in ice-cold SET buffer and stored at -70°C.

Immunoblot Analysis. For Western Blot analysis, microsomal protein (5µg) from pooled

mouse samples (n=3) was separated by SDS-PAGE, electropheretically transferred to

nitrocellulose membranes and probed using an anti-rat CYP3A1 polyclonal Ab and anti-rat

CYP2B1/2 polyclonal Ab (BRI, University of Dundee) (Forrester et al., 1992), used at 1:2000

dilutions. The detection of mouse Cyp3a11 or Cyp2b10 by the corresponding rat antibodies


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was determined by Western blots using recombinant Cyp3a11 and Cyp2b10 proteins. The

secondary antibody was anti-rabbit HRP conjugate, used at a dilution of 1:2000 (GE

Healthcare). Detection of immunoreactive proteins was performed by an enhanced

chemiluminescence blot detection system (Amersham Biosciences). Chemiluminescence was

captured by Gel Doc XR (Bio-Rad Laboratories). Protein bands were automatically detected

and their intensity was quantified by Quantity One software (Version 4.60, Bio-Rad

Laboratories).

Cytochrome P450 Enzyme Activity Assays. Activity of Cyp2b10 in hepatic microsomes

was monitored using the preferred Cyp2b10 substrate pentoxyresorufin. Pentoxyresorufin

dealkyation (PROD) was monitored in a 1cm3 cuvette containing 66mM Tris-HCl buffer, pH

7.4, microsomes (20 to 50µg) and 1mM pentoxyresorufin. The reactions were initiated by the

addition of 50µM NADPH and the rate of fluorescence was measured. As an internal control,

each microsomal mixture was spiked with 10µM resorufin.

Dealkylation of 7-benzyloxyquinoline (BQ, 20mM), a Cyp3a11 preferred substrate was

measured in liver microsomes (20-50µg) suspended in 66mM Tris-HCl buffer, pH 7.4.

Assays were initiated by the addition of 50µM NADPH to the microsomal reaction and

followed using a Hitachi F-4500 fluorescence spectrophotometer with FL Solution 2.0

software. 7-Hydroxyquinoline (7-HQ, 1mM) was used as the internal standard.

Protein concentrations were measured using a modified version of the method of Lowry

(Lowry et al., 1951) with bovine serum albumin standards. Values were calculated and

expressed as nmol/min/mg protein or pmol/min/mg protein for the BQ and PROD assays,

respectively.

Plasma clinical chemistry. Blood was collected by cardiac puncture into lithium/heparin-

coated tubes for plasma. Following removal into suitable tubes for plasma preparation, venous


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samples were mixed on a roller for 10 minutes and cooled on ice. Red blood cells were

removed by centrifugation (2000 – 3000 rpm for 10 minutes at at 8-10°C). The supernatant

(plasma) was stored at -70°C prior to analysis. Plasma samples were analysed for alanine

aminotransferase (ALT) using the COBAS Integra 400+ (Roche).

Liver histopathology. Two samples of liver, one from the left lobe and one from the median

lobe, from different mice were taken and preserved in 4% neutral buffered formaldehyde. The

tissue samples were subsequently embedded in paraffin wax and sectioned at a nominal

thickness of about 5 µm. All sections were stained with haematoxylin and eosin. The

microscopic analysis and interpretation was carried out by an independent veterinary

pathologist.

Dexamethasone pharmacokinetics. Concentrations of dexamethasone in whole blood were

measured by tandem mass spectrometric detection (LC-MS/MS). Calibration standards were

prepared in whole blood: water (1:1 v/v, 95 µl) by adding an appropriate amount of

dexamethasone in methanol/H2O (1:1 v/v; 5 µl). 10 mM ammonium acetate in methanol (80

µl) was added to 20 µl of the test samples and the calibration standards. The mixture was

vortexed for 30 seconds and centrifuged at 13000 g for 6 minutes. The supernatant was

transferred to a 96-well plate and the concentrations of dexamethasone were measured by LC-

MS/MS from the calibration curve.

Statistics. Statistical significance was assessed to determine differences between mouse

groups using a two-tailed, paired, Student’s t test. The criterion for statistical significance was

P < 0.05.


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Results

Generation of the new humanized PXR mouse model. We recently described the

generation of a PXR humanized mouse (huPXR) expressing the human receptor under control

of the mouse Pxr promoter (Scheer et al., 2008). The DNA cassette used contained a fusion of

exons 2-4, intron 4, exon 5, intron 5, and a fusion of exons 6-9 of the human PXR gene. This

huPXR mouse expressed a functional human PXR protein and exhibited the predicted species

differences in its interaction with drugs such as rifampicin and dexamethasone. Furthermore,

we demonstrated that both the full length and the most abundant splice variants identified in

humans, human PXR (hPXR) SV2 (or hPXR.2) were also expressed (Dotzlaw et al., 1999;

Hustert et al., 2001; Fukuen et al., 2002). However, in this model we found that an as yet

unidentified splice variant was detected. Further investigation showed that a splice acceptor

site was created by the fusion of exon 7 and 8 which led to a variant where exon 5 was spliced

out of frame to exon 8, resulting in a premature stop codon in exon 8. As most of the ligand-

binding domain is altered, these transcripts either code for a non-functional protein or, though

unlikely, maybe even a dominant negative protein interfering with the normally spliced PXR

receptor. They also might be degraded by nonsense-mediated decay (see (Schell et al., 2002)

for review). As is it is difficult to measure how much of the truncated transcript is degraded,

we can’t estimate the total amount of transcript wasted by aberrant splicing. Although it is not

possible to quantify this effect, a fraction of the human PXR transcript will not code for a

functional protein potentially reducing the sensitivity of the model to enzyme inducers.

In order to avoid this possible problem, we have generated a new PXR-humanized mouse line

by altering the original targeting vector to include the two additional human introns 6 and 7.

The new PXR-humanized mouse line was generated by a knock-in strategy as shown in

Figure 1, such that a chimeric construct containing the cDNA representing exons 2-4 of

human PXR missing the start CTG, genomic sequences in-between exon 4 and 8 and again

cDNA of exon 8-9 followed by an SV40 polyA signal was introduced at the ATG of the


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mouse Pxr gene. This approach allowed the expression of the human receptor under control of

the mouse Pxr promoter and it deletes the endogenous Pxr gene. This model is referred to as

huPXRi.

Comparison of the huPXR and huPXRi sequences obtained by reverse transcription PCR

from hepatic mRNA showed that both lines expressed the full length and most abundant

human splice isoform hPXR SV2 in the liver. However, the previously observed variant

where splicing had occurred between exon 5 to exon 8 was not detected in the huPXRi line. In

addition, relative expression levels of hPXR transcripts in male and female livers were

quantified by reverse transcriptase PCR (Figure 2). Interestingly, human PXR mRNA

expression levels were increased by 5.6-fold in male huPXRi mice compared to the huPXR

males. In females the expression levels were slightly lower for both genotypes and in huPXRi

females relative expression of human PXR mRNA was increased by 4.6-fold relative to

huPXR mice of the same sex. The lower transcript levels in the huPXR mouse line might

reflect the degradation of a fraction of the transcripts in this mouse line by nonsense-mediated

decay. It was not possible to quantify the amount of full length PXR protein because no

suitable antibody is available.

Induction profiles of cytochrome P450 Cyp3a and Cyp2b proteins in rifampicin treated

WT, huPXR and huPXRi mice. In order to test if the new PXR model had an altered

sensitivity to PXR ligands, we treated WT, huPXR and huPXRi mice with rifampicin (RIF), a

compound showing higher affinity for the human versus the mouse receptor (Xie et al.,

2000a). In agreement with our previous findings (Scheer et al., 2008), a dose of 10 mg/kg

body weight resulted in a consistent Cyp3a11 induction in the huPXR mice, but only a

marginal induction in the WT (Figure 3A). However, in the huPXRi mice the induction at this

dose was more pronounced than in both WT and huPXR mice. In order to quantify these

differences we measured the band intensities of Cyp3a11 in animals treated with 10 mg/kg


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RIF relative to the untreated control of the corresponding genotype. The increase in band

intensity was 1,8-fold in the WT, 4,5-fold in the huPXR mouse line and 7,7-fold in huPXRi

animals. Furthermore, at a maximum dose of 60 mg/kg the Cyp3a11 band intensity in the

huPXRi mouse line was 1,9-fold higher compared to huPXR mice and 2,7-fold higher

compared to the WT. Therefore, the huPXRi mouse line has an increased sensitivity to RIF-

mediated Cyp3a11 induction compared to the huPXR mice both in the dose response and the

magnitude of induction. The dose dependency exemplifying the difference in sensitivity

between WT and huPXRi mice is shown in Figure 3B. The differences observed by Western

blotting were also reflected in the metabolism of the Cyp3a11 substrate 7-benzyloxyquinoline

(BQ); a significant increase in metabolism was observed at a dose of RIF of 10 mg/kg in

huPXRi compared to WT mice (Figure 3C). Namely, the increase of BQ metabolism at this

dose was 1,5-fold in the WT, 1,9-fold in the huPXR mice and 2,9-fold in huPXRi animals

compared to the untreated controls of the corresponding genotype. As reported previously, a

slight increase in the basal level of Cyp3a11 expression in both huPXR and huPXRi mice was

observed (Scheer et al., 2008).

Intriguingly, a marked induction of Cyp2b10 expression was observed in huPXRi mice at a

dose of 10 mg/kg RIF and a much higher induction at 60 mg/kg (Figure 3A). The induction of

this protein was substantiated by the marked increase in the Cyp2b10-specific

pentoxyresorufin dealkylation (PROD) in the huPXRi mouse line (Figure 3C). Consistent

with the expression observed in Cyp2b10 protein, no induction of PROD activity was

measured in WT and huPXR mice at 10 mg/kg RIF and only a marginal increase is observed

at 60 mg/kg. These data demonstrate the higher sensitivity of the new PXR model to

induction and consistent with the literature RIF has been reported to be an inducer of CYP2B6

expression in human hepatocytes (Goodwin et al., 2001; Pichard-Garcia et al., 2002).


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Comparison of cytochrome P450 induction by dexamethasone in wild type and

humanised mice. Characterisation of the interaction of dexamethasone with PXR is shown in

Figure 4. Cyp3a11 induction in WT mice was observed at much lower doses than in huPXRi

mice (Figure 4A). For example at a dose of 3 mg/kg a significant induction of Cyp3a11 was

observed in WT animals, but not in huPXRi mice. However, a similar level of induction was

found at a 10-fold higher dose of 30mg/kg. This was also reflected in the rate of metabolism

of the Cyp3a11 substrate BQ at the 30mg/kg dose (Figure 4B). We noted that relative to our

original huPXR mice, treatment with DEX resulted in a much more marked consistent

induction of Cyp3a11 in the new model. At a dose of 10 mg/kg Cyp3a11-induction was only

observed in huPXRi, but not in huPXR mice and at higher doses such as 30 mg/kg induction

in huPXRi mice was more profound (see also (Scheer et al., 2008)). Therefore, in addition to

an increased sensitivity and different pattern of induction in response to RIF, huPXRi

compared to huPXR mice were also more sensitive to the more potent mouse inducer, DEX.

The above effects were shown to be mediated by PXR as no induction of Cyp3a11 was seen

in the PXR null animals (Figure 4A). In agreement with previous findings Cyp2b10 induction

by DEX is comparable between WT, huPXRi and PXR KO mice and therefore is independent

of PXR (Figure 4A) (Schuetz et al., 2000; Scheer et al., 2008).

Differential expression of markers for hepatotoxicity in control and transgenic mice. In

preliminary studies we observed that dexamethasone was more hepatotoxic in huPXR and

PXR KO compared to WT mice, suggesting that PXR plays a role in the hepatotoxic effects

of DEX (unpublished). We have further investigated this observation in WT, PXR KO,

huPXR and huPXRi mice. In order to evaluate the role of the P450s we also included a new

mouse line where the murine Cyp3a57, Cyp3a16, Cyp3a41, Cyp3a44, Cyp3a11, Cyp3a25 and

Cyp3a59 genes have been deleted. These genes are in close proximity to each other on mouse

chromosome 5 and distant from Cyp3a13 which is the only remaining functional Cyp3a gene


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in this mouse line. In agreement with the literature we have found Cyp3a13 expression in

mouse liver to be marginal compared to the most abundant Cyp3a11 protein (Yanagimoto et

al., 1997; Anakk et al., 2003). Furthermore, it should be mentioned that a transcription

profiling study of Cyp3a-/-/Cyp3a13+/+ vs. WT mice did not show any significant

compensatory change of Cyp3a13 expression in the Cyp3a-/-/Cyp3a13+/+ mouse line (data not

shown). A more detailed analysis of this Cyp3a-/-/Cyp3a13+/+ mouse line will be described

elsewhere (manuscript in preparation). Dexamethasone administration (30mg/kg, daily i.p. for

4 days) significantly increased ALT levels, compared to the corresponding untreated controls,

in all mouse lines (Figure 5A). The sensitivity to DEX induced hepatotoxicity was in the

order WT ≤ huPXRi < huPXR < PXR KO < Cyp3a-/-/Cyp3a13+/+. At this dose the average

ALT levels were increased in the different mouse lines relative to the treated WT mice 1.2-

fold (huPXRi), 2.2-fold (huPXR), 4.9-fold (PXR KO) and 8.1–fold (Cyp3a-/-/Cyp3a13+/+),

respectively. However, due to the high variance between individuals from one group treated

with 30 mg/kg DEX, statistical significance in comparison to the treated WT animals was

only reached for the huPXR and Cyp3a-/-/Cyp3a13+/+ mice. At a lower (10 mg/kg) dose of

DEX, increases in ALT values were also observed with the same trend in sensitivity as

described above. The Cyp3a-/-/Cyp3a13+/+ mice were not tested at this dose. The same trend

as for the ALT values was also seen for aspartate aminotransferase (AST), though the

variance between individuals was even higher and, as a consequence, statistical significance

lower (data not shown).

The hepatotoxic effect of high doses of DEX (30mg/kg) was also apparent following

histological examination of liver sections in all strains. Macrovesicular hepatocellular

vaculation that was partly associated with a peripheral dislocation of the usually centrally

placed hepatocytic nuclei was observed in most treated mice of the different lines (Figure 5B)

and some individuals displayed necrosis of scattered single cells or subcapsular liver necrosis.

Single hepatocyte necrosis in the treated group was not observed in any of the analyzed WT


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and huPXRi mice, but in one huPXR mouse, two PXR KO and three Cyp3a-/-/Cyp3a13+/+

mice (n=4 for each group), which strengthens the observation of different susceptibilities to

DEX-induced hepatotoxicity as measured by ALT values.

Dexamethasone pharmacokinetics in WT, PXR KO and Cyp3a-/-/Cyp3a13+/+ mice. In the

above studies, DEX toxicity was observed after 4 days of administration suggesting that the

increased sensitivity of PXR KO and Cyp3a-/-/Cyp3a13+/+ mice could be explained by an

increased metabolism and detoxification in the WT, due to a DEX-mediated induction of

Cyp3a proteins in a PXR-dependent fashion. In order to test this possibility a pharmacokinetic

study was carried out in WT, PXR KO and Cyp3a-/-/Cyp3a13+/+ mice following 4 days of

DEX treatment. In all three mouse strains relatively slow DEX absorption after ip

administration was observed, probably due to the low water solubility of this compound.

Clear differences in DEX pharmacokinetics were observed in the different mouse lines, with

an increase in maximal DEX serum concentrations in PXR KO and Cyp3a-/-/Cyp3a13+/+ mice

(Figure 6A). Statistically significant differences in areas under the concentration versus time

curve (AUCs) between WT, PXR KO and Cyp3a-/-/Cyp3a13+/+ mice were also observed

(Figure 6B). It has been demonstrated that after intraperitoneal administration of an isoform-

specific CYP substrate the ratio of the expression level of the corresponding CYP in the liver

is inversely proportional to the ratio of the AUCs of the substrate (Lukas et al., 1971; Gibaldi

and Perrier, 1982). Thus, the AUC difference suggested that after induction with DEX,

enzymes responsible for DEX metabolism were expressed at lower level in the Cyp3a-/-

/Cyp3a13+/+ and PXR KO mice compared to the WT. These results are consistent with the

lack of induction of Cyp3a proteins in the DEX treated PXR KO mice (Figure 4). These data

also demonstrate that CYP3A enzymes play an important role in the metabolism of DEX. The

changes in pharmacokinetics are also consistent with the hypothesis that the higher


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susceptibility of the PXR KO and Cyp3a-/-/Cyp3a13+/+ lines to DEX-induced hepatotoxicity is

a result of an increased exposure to the parent compound.


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Discussion

As part of this report we have compared the properties of two mouse lines humanized for the

transcription factor PXR which only differ in that in the original model (Scheer et al., 2008) a

cryptic splice acceptor site was generated as part of the vector construction. Although the full-

length human PXR mRNA was expressed in both models and the expression of hPXR was

driven off identical promoter sequences, there were some significant differences between

these mouse lines in both the level of PXR expression and its capacity to regulate downstream

genes in response to ligands such as rifampicin and dexamethasone. This suggests that

alternative splicing of PXR can be an important factor in determining the level of functional

PXR receptor (Hustert et al., 2001; Fukuen et al., 2002). What was also particularly

interesting was that in the new humanized PXR model rifampicin was a good inducer of

Cyp2b10 and that this protein was not induced in either WT or the huPXR mouse line. This

indicates that the differential expression of PXR will not only affect the amplitude of

induction, but also which genes are induced. The induction of Cyp2b genes by rifampicin is

consistent with previous studies showing the induction of CYP2B6 in human hepatocytes by

this compound (Goodwin et al., 2001; Pichard-Garcia et al., 2002). Interestingly, though

dexamethasone elevates Cyp3a11-expression in a PXR-dependent manner, Cyp2b10

induction by this compound does not require PXR and might at least in part be the

consequence of an enhanced expression of CAR and the retinoid X receptor-alpha in response

to DEX (Pascussi et al., 2000a; Pascussi et al., 2000b). Taken together the data demonstrate

that regulation of Cyp2b10 (and possibly CYP2B6 in man) is complex and it involves several

transcription factors such as CAR (Honkakoski et al., 1998; Wei et al., 2000), GR (Schuetz et

al., 2000) and PXR (Xie et al., 2000b; Wagner et al., 2005). Furthermore, depending on the

compound the activation of a given receptor like PXR can either be required for Cyp2b10-

induction (RIF) or nonessential (DEX).


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The presented studies revealed a hepatotoxic effect of high doses of the glucocorticoid DEX

in mice. At 30 mg/kg a statistically significant increase in ALT levels could be observed in

WT, huPXR, huPXRi, PXR KO and Cyp3a-/-/Cyp3a13+/+ mice when compared with their

corresponding untreated controls. The liver damaging effect was also confirmed by the

induction of macrovesicular hepatocellular vaculation and different degrees of necrosis in

histological examination. This effect is in agreement with the previously observed

hepatotoxicity of high doses of DEX in mice (Madhu et al., 1992).

There is currently significant interest in the potential use of humanized and knockout animal

models as more reliable predictors of pathways of drug metabolism and chemical safety in

man (Cheung and Gonzalez, 2008; Lin, 2008; Scheer et al., 2008). This is based on the

increasing evidence that there are species differences in both nuclear receptors which regulate

the expression of drug metabolising enzymes as well as the enzymes themselves. There is also

evidence that these differences can lead to both increased sensitivity to some molecules or

reduced sensitivity to others such as certain non-genotoxic carcinogens (Zhang et al., 2002;

Cheung et al., 2004; Morimura et al., 2006; Gonzalez and Shah, 2008).

One important facet of chemical safety screening is that molecules, as a consequence of their

interaction with nuclear receptors like PXR and CAR can induce their own metabolism or that

of other molecules administered concomitantly. This can either result in reduced

pharmacological efficacy or, if the parent molecule is toxic, reduced toxicity, or, if the parent

molecule is metabolised to a reactive metabolite, increased toxicity. Therefore, species

differences in the capacity of compounds to activate rodent or human receptors can result in

marked differences in their pharmacological or toxicological effects.

Using a relatively small number of transgenic animals, significant insights into the pathways

regulating dexamethasone metabolism and toxicity could be obtained. The use of mice deleted

at the Cyp3a gene locus demonstrated the importance of the cytochrome P450 metabolic

pathway in DEX disposition and toxicity. The increased toxicity and reduced metabolic


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clearance of DEX in Cyp3a-/-/Cyp3a13+/+ mice provides evidence that metabolism of this

compound results in detoxification and that Cyp3a enzymes are involved. The fact that similar

results regarding toxicity and metabolism were obtained in PXR KO mice also strengthened

this evidence and demonstrated that enzyme induction via PXR is important. DEX therefore

induces its own metabolism leading to an increased rate of detoxification in mice.

This then raised the question whether human PXR interacts in a similar manner to murine

PXR? In this regard DEX was a weaker inducer of Cyp3a protein expression in huPXRi mice

than in wild type animals. With regard to hepatotoxicity, there was no difference between WT

and huPXRi mice at the 30 mg/kg doses of DEX which led to strongly increased ALT levels

in the Cyp3a-/-/Cyp3a13+/+ and PXR KO mice. This is probably, at least in part, because

induction of Cyp3a proteins at this dose was almost the same in WT and huPXRi mice.

The doses of DEX required to induce hepatotoxicity are, however, not representative of the

therapeutic doses used in humans. Even in shock therapy usually only 4 to 8 mg are injected

intravenously and if necessary this is repeated to a total maximum dose of 24 mg. Therefore,

the dose of up to 30 mg/kg body weight used in this study exceeds the maximum human dose

by two orders of magnitude. In addition, differences in the hepatic blood flow rates between

mice and humans might contribute to potential variations in hepatic sensitivity to this

glucocorticoid. Nevertheless, these studies exemplify the fact that species differences in the

interaction of compounds with PXR can alter the metabolism and potentially the toxicological

profile of drugs and chemicals, which could result in differences in the pathways of

metabolism and susceptibility to hepatotoxicity between animals and man.


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Acknowledgements

We wish to thank Sylvia Krüger, Anja Müller, Oliver Dahlmann (TaconicArtemis, Cologne)

and Sarah Waugh (CXR Biosciences, Dundee). This work was supported by ITI Life

Sciences, Scotland.


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Footnotes

This work was supported by ITI Life Sciences, Scotland.

Conflict of interest: N. Scheer and A. Rode receive income from TaconicArtemis and J. Ross,

Y. Kapelyukh and C.R. Wolf receive income from CXR Biosciences. The authors have

declared that no conflict of interest exists.


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Figure legends

Fig. 1 Strategy used to generate PXR humanised mice. A human PXR minigene, containing a

fusion of exons 2-4, genomic sequences between exons 4 and 8 and a fusion of exons 8 and 9

was knocked in onto the translational start ATG of the mouse Pxr wild type gene in order to

generate PXR targeted mice. Mouse exons are indicated in black vertical lines and with small

type. Human exons are white boxes with capitals and the targeting arms are shown as dark

and light grey lines. Targeted mice were crossed to a mouse strain expressing the FLPe

recombinase to delete the hygromycin selection cassette by FLP-mediated recombination at

the FRT-sites (white triangles) and to generate PXR humanised mice. For the sake of clarity

sequences of the targeting vector are not drawn to scale.

Fig. 2 Relative mRNA expression of hepatic human PXR in huPXR and huPXRi mice. RNA

was isolated from liver samples of male and female huPXR and huPXRi mice (n=3 per

genotype and sex) and relative human PXR mRNA levels were assesses as described in the

materials and methods. Values represent the mean expression level ± SEM relative to huPXR

males. The human PXR mRNA expression levels were normalized to murine β-actin. A

Student’s t-test (2-sided) was performed on the results; with *, ** and *** statistically

different from control at p<0.05, 0.01 and 0.001, respectively.

Fig. 3 Effect of rifampicin on hepatic Cyp3a11 and Cyp2b10 expression in WT, huPXR and

huPXRi mice. (A-B) Pooled liver microsomes (n=3) from WT, huPXR, huPXRi (A) and WT

and huPXRi mice (B), treated with either rifampicin or the vehicle, were analysed for both

Cyp3a11 and Cyp2b10 expression by immunoblotting. Microsomal protein (5µg) was

separated on 7.5% SDS-PAGE gel and subsequently immunoblotted with polyclonal Cyp3a11

and Cyp2b10 antibodies as described in the Materials and Methods. (C) BQ and PROD assays

in pooled WT, huPXR or huPXRi mouse liver microsomes (n=3 mice). Values are expressed


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as Mean ± SD; n=3 mice for all experiments. A Student’s t-test (2-sided) was performed on

the results; with *, ** and *** statistically different from control at p<0.05, 0.01 and 0.001,

respectively.

Fig. 4 Effect of dexamethasone on hepatic Cyp3a11 expression in WT, huPXRi and PXR KO

mice. (A) Pooled liver microsomes (n=3) from WT, huPXRi and PXR KO mice, treated with

either DEX or the vehicle, were analysed for Cyp2b10 and Cyp3a11 expression by

immunoblotting. Microsomal protein (5µg) was separated on 7.5% SDS-PAGE gel and

subsequently immunoblotted with polyclonal Cyp3a11 and Cyp2b10 antibodies as described

in the Materials and Methods. (B) Measurement of Cyp3a related activity in pooled WT,

huPXRi or PXR KO mouse liver microsomes. Values are expressed as Mean ± SD; n=3. A

Student’s t-test (2-sided) was performed; with *, ** and *** values statistically different from

control at p<0.05, 0.01 and 0.001, respectively.

Fig. 5 Hepatotoxic effects of dexamethasone in WT, humansized and knockout mice. (A)

Plasma ALT levels in control animals or mice treated with either 10 or 30 mg/kg DEX. Group

sizes are as follows: Untreated (WT, n=7; huPXRi, n=6; huPXR, n=4; PXR KO, n=9; Cyp3a-/-

/Cyp3a13+/+, n=4), 10 mg/kg DEX (WT, n=6; huPXRi, n=3; huPXR, n=3; PXR KO, n=3;

Cyp3a-/-/Cyp3a13+/+, not tested), 30 mg/kg DEX (WT, n=10; huPXRi, n=10; huPXR, n=10;

PXR KO, n=12; Cyp3a-/-/Cyp3a13+/+, n=4). Values are expressed as Mean ± SD. A Student’s

t-test (2-sided) was performed on the results; with *, ** and *** statistically different from

untreated control in each group at p<0.05, 0.01 and 0.001, respectively. In addition, for

equally treated mice the dotted lines highlight the statistical difference between the

corresponding genotypes. (B) Haematoxylin and eosin staining of representative liver sections

(5 µm) taken from WT, huPXRi, huPXR, PXR KO and Cyp3a-/-/Cyp3a13+/+ mice treated with

30 mg/kg DEX. For all images a 20x objective lens was used.


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Fig. 6 Dexamethasone pharmacokinetics in WT, PXR KO and Cyp3a-/-/Cyp3a13+/+ mice. (A)

Plasma concentration curve after 4 daily treatments with DEX at 30mg/kg I.P. (B) Total areas

under the concentration/time curve (AUC) calculated from the DEX pharmacokinetics.

Experimental details are given in the Materials and Methods section. Data are mean ± SD

(n=3 for Cyp3a-/-/Cyp3a13+/+; n=4 for other strains). AUC were compared using an unpaired t

test (two tailed P values), with *, ** and *** statistically different from the WT at p<0.05,

0.01 and 0.001, respectively.


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