Allyl isothiocyanate (AITC) inhibits pregnane X receptor (PXR) and constitutive
androstane receptor (CAR) activation and protects against acetaminophen- and
amiodarone-induced cytotoxicity
Yun-Ping Lim1,2,* • Ching-Hao Cheng1 • Wei-Cheng Chen1 • Shih-Yu Chang3 •
Dong-Zong Hung1,2 • Jih-Jung Chen4 • Lei Wan5 • Wei-Chih Ma1 • Yu-Hsien Lin1 •
Cing-Yu Chen1 • Tsuyoshi Yokoi6 • Miki Nakajima6 • Chao-Jung Chen7,8,**
1
Department of Pharmacy, College of Pharmacy, China Medical University, Taichung,
Taiwan.
2
Department of Emergency, Toxicology Center, China Medical University Hospital,
Taichung, Taiwan.
3
4
Department of Public Health, Chung Shan Medical University, Taichung, Taiwan.
Graduate Institute of Pharmaceutical Technology, Tajen University, Pingtung,
Taiwan.
5
School of Chinese Medicine, China Medical University, Taichung, Taiwan.
6
Drug Metabolism and Toxicology, Faculty of Pharmaceutical Sciences, Kanazawa
University, Kakuma-machi, Kanazawa, Japan.
7
Graduate Institute of Integrated Medicine, China Medical University, Taichung,
Taiwan.
8
Proteomics Core Laboratory, Department of Medical Research, China Medical
University Hospital, Taichung, Taiwan.
* Corresponding author
Yun-Ping Lim, Ph.D.
Department of Pharmacy, College of Pharmacy, China Medical University, No. 91,
Hsueh-Shih Road, Taichung 40402, Taiwan, Republic of China.
Tel: +886-4-2205-3366 ext. 5802 / Fax: +886-4-2207-8083
E-mail: limyp@mail2000.com.tw, limyp@mail.cmu.edu.tw
** Co-corresponding author
Chao-Jung Chen, Ph.D.
Graduate Institute of Integrated Medicine, China Medical University, No. 91,
Hsueh-Shih Road, Taichung 40402, Taiwan, Republic of China.
Tel: +886-4-2205-3366 ext. 1542 / Fax: +886-4-2203-7690
E-mail: cjchen@mail.cmu.edu.tw, ironmanchen@yahoo.com.tw
Running title: Allyl isothiocyanate inhibits CYP3A4 and CYP2B6 through PXRand CAR-mediated pathway
Abbreviations
AITC
Allyl isothiocyanate
CAR
Constitutive androstane receptor
CITCO
6-(4-chlorophenyl)imidazo[2,1-b][1,3]thiazole-5-carbaldehydeO-(3,4-dich
lorobenzyl)-oxime
CYP450
Cytochrome P450
DMEs
Drug metabolizing enzymes
DMSO
Dimethylsulfoxide
DR
Direct repeat
ER
Everted repeat
GR
Glucocorticoid receptor
HNF4
Hepatocyte nuclear factor 4
PCN
Pregnenolone 16-carbonitrile
PGC-1
Peroxisome proliferator-activated receptor-gamma coactivator 1
PXR
Pregnane X receptor
RXR
Retinoic X receptor 
SRC-1
Steroid receptor coactivator-1
TCPOBOP
1,4-bis[2-(3,5-dichloropyridyloxy)]benzene
Abstract
Antagonizing the action of the pregnane X receptor (PXR) may have important
clinical implications for preventing inducer-drug interactions and improving
therapeutic efficacy. We identified a widely distributed isothiocyanate, allyl
isothiocyanate (AITC), which acts as an effective antagonist of the nuclear receptor
pregnane X receptor (PXR, NR1I2) and constitutive androstane receptor (CAR,
NR1I3). HepG2 cells were used to assay reporter function, mRNA levels, and protein
expression. Catalytic activities of the PXR and CAR target genes, CYP3A4 and
CYP2B6, respectively, were also assessed in differentiated HepaRG cells. Protective
effects of AITC on rifampin-induced cytotoxicity were observed, and transient
transfection assays showed that AITC was able to effectively attenuate the agonist
effects of rifampin and CITCO on human PXR and CAR activity, respectively.
AITC-mediated reduction in the transcriptional activity of PXR and CAR correlated
well with the suppression of CYP3A4 and CYP2B6 expression in HepG2 cells, which
reflected the reduced catalytic activities of both of these genes following AITC
treatment in differentiated HepaRG cells. Furthermore, AITC disrupts the
coregulations of PXR with several important coregulators. Furthermore, the
antagonist effect of AITC against PXR was found in HepaRG cells upon addition of
acetaminophen (APAP) and amiodarone, indicating that AITC protects cells from
drug-induced cytotoxicity. Taken together, our results show that AITC inhibits the
transactivation effects of PXR and CAR and reduces the expression and function of
CYP3A4 and CYP2B6. Additionally, AITC reversed the cytotoxic effects of APAP
and amiodarone induced by PXR ligand. Results from this study suggest that AITC
could be a powerful agent for reducing potentially dangerous interactions between
transcriptional inducers of CYP enzymes and therapeutic drugs.
Keywords:
Allyl isothiocyante • Pregnane X receptor • Cytochrome P450 3A4 • Constitutive
androstane Receptor • Cytochrome P450 2B6 • Inducer-Drug Interaction •
Drug-induced cytotoxicity
Introduction
Allyl isothiocyanate (AITC) is an aliphatic isothiocyanate produced by the
hydrolysis of a common glucosinolate (sinigrin) by myrosinase (EFSA 2010).
Sinigrin may be converted to AITC in vivo through the action of endogenous
myrosinase from intestinal microflora in animals and humans (Krul et al. 2002). The
majority of Brassica species, including cooked cabbage, cruciferous vegetables
(Brussels sprouts, cauliflower, and kale), turnip, watercress, horseradish, brown
mustard seeds (Brassica juncea), mustard oil, and wasabi contain sinigrin (EFSA
2010). AITC, which is the main naturally occurring constituent in volatile mustard oil,
is widely used as a food additive and flavoring substance (EFSA 2010). Moreover,
AITC has been reported to have several biological activities including anti-microbial
(Shin et al. 2004), fungicidal (Nielsen and Rios 2000), and anti-cancer activities due
to its multimode mechanisms of action (Xiao et al. 2003; Zhang 2010), including the
stimulation of cytoprotective proteins (Matsuda et al. 2007) and anti-inflammatory
activity (Wagner et al. 2012).
The cytochrome P450 family (CYP450) plays an important role in the
biotransformation of various endogenous components and xenobiotics (Guengerich
2008). Among these, CYP3A4 is involved in the metabolism of approximately half of
all pharmaceuticals used today, due to its large hydrophobic core and multiple
substrate binding sites (Guengerich 2008). Another member of this family, CYP2B6,
accounts for about 15% of CYP-mediated metabolism of all marketed drugs. Several
important clinically used drugs, including bupropion (anti-depressant), and
cyclophosphamide (anti-cancer), are CYP2B6 substrates (Walko et al. 2012).
Modulation of CYP3A4 and CYP2B6 activity due to co-administrated agents such as
enzyme inducers or inhibitors may change the victim drugs concentrations, making
the drug either ineffective or potentially toxic. Problems with drug-drug interactions
(DDIs) and the associated adverse events represent a substantial clinical concern that
needs to be addressed (Kliewer et al. 2002).
Major transcription factors that regulate the expression of CYP3A4 and CYP2B6
include the pregnane X receptor (PXR, NR1I2) and the constitutive androstane
receptor (CAR, NR1I3) (Goodwin et al. 1999; 2001). These transcriptional regulators
are activated by specific ligands and are subsequently translocated into the nucleus
where they interact with xenobiotic response elements (XREM) in promoter regions
of the CYP3A4 and CYP2B6 genes. PXR and CAR are known to interact with the
9-cis retinoic acid receptor  (RXR), forming a heterodimer that recognizes two
responsive elements in the CYP3A4 regulatory region (DR3 and ER6) leading to
chromatin remodeling and transcriptional activation (Bertilsson et al. 1998). The
PXR-mediated biotransformation and disposition of xenobiotics therefore occurs
through transcriptional control.
Compounds that transactivate PXR have highly diversified structures. These
include xenobiotics (rifampin, nifedipine, SR12813, etc.) (Lehmann et al. 1998),
environmental toxins (organochlorine and polybrominated compounds) (Jacobs et al.
2005), endogenous substances (Ex: bile acids) (Staudinger et al. 2001), and naturally
occurring compounds (Ex: hyperforin) (Watkins et al. 2003). PXR activation results
in up-regulation of CYP3A4, leading to the excessive metabolism of certain
medications, the enhanced toxicity of many xenobiotics, or drug-induced toxic events
(Zhang et al. 2002). PXR antagonists may therefore prove beneficial for preventing
unwanted side effects in patients who have chronically induced PXR activation, or to
fine-tune the efficacy of therapeutics that serve as PXR agonists. Like PXR, CAR is
highly expressed in the liver (Sueyoshi et al. 1999). CAR can heterodimerize with
RXR and regulate CYP2B6 induction by CITCO and phenobarbital (PB)-like
inducers via interactions with DR4 motifs (Kawamoto et al. 1999). It therefore acts as
a xenobiotic-sensing nuclear receptor. Exposure of xenobiotics that affect the activity
of these two nuclear receptors could therefore have a substantial influence on drug
metabolism and may enhance xenochemical toxicity.
Because the PXR-CYP3A4 and CAR-CYP2B6 pathway is extremely important
for controlling drug efficacy, development of an antagonist to these nuclear receptors
may reduce unwanted effects due to interactions between these transcriptional
activators and various drugs. Although many PXR agonists have been identified,
comparatively few antagonists of this receptor have been reported in the literature
(Wang et al. 2007; Zhou et al. 2007; Wang et al. 2008; Chen et al. 2010; Lim et al.
2012). In this study, we examined the effects of AITC on the transactivation activities
of PXR and CAR using reporter assays. We assessed mRNA and protein expression
of their target genes, CYP3A4 and CYP2B6 in HepG2 cells, and also their catalytic
activities in differentiated HepaRG cells. Furthermore, we characterized the activity
of AITC on the molecular transcriptional mechanism of the CYP3A4 promoter in the
context of several coregulators. We also examined the effect of AITC on PXR
ligand-induced cytotoxicity caused by acetaminophen (APAP) and amiodarone in
HepaRG cells.
Materials and Methods
Chemicals and cell culture
All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) and
dissolved in DMSO or water at concentrations appropriate for the specific studies in
which they were used. HepG2, LS174T, and COS-1 cells were purchased from the
Food Industry Research and Development Institute (FIRDI, Taiwan, R.O.C.) and
maintained in minimum essential medium (MEM)  medium (HepG2 and LS174T
cells), or Dulbecco’s Modified Essential Medium (DMEM) (COS-1 cells)
supplemented with 10% fetal bovine serum without antibiotics, in a 5% CO2
atmosphere at 37C. Cultures were used for experiments within the first 20 passages
after receipt. HepaRG cells were obtained from Gibco (Gibco Invitrogen, Germany).
Cells were detached by gentle trypsinization and seeded in the presence of William’s
E medium supplemented with 10% FetalCloneTM II serum (HycloneTM; Thermo
Fisher Scientific Inc., Waltham, MA), 5 g/mL insulin, and 5  10-5 M hydrocortisone
hemisuccinate. Cells were maintained in the abovementioned medium for 2 weeks.
Then, they were treated with 1% DMSO (in the same culture medium) for one week,
and then 2% DMSO for 2 more weeks. The purpose of treating HepaRG cells with
DMSO for several weeks was to induce differentiated hepatocyte-like properties of
this cell line.
Plasmid constructs
Plasmids pcDNA3-PXR, pcDNA3-rPXR, pcDNA3-HNF4, pCR3.1-SRC-1,
and pGL3B-CYP3A4 [(-444/+53)(-7836/-7208)], containing full-length human PXR,
rat PXR, human HNF4, human SRC-1, and a CYP3A4 promoter fragment,
respectively, have been described previously (Lim et al. 2012). Construction of
full-length human RXR (pGEM-3Z-RXR) and PGC-1 (pTARGET-PGC-1)
expression plasmids have been described previously (Itoh et al. 2006) and were kindly
provided by Dr. Tsuyoshi Yokoi (Drug Metabolism and Toxicology, Division of
Pharmaceutical Sciences, Graduate School of Medical Science, Kanazawa University,
Kakuma-machi, Kanazawa, Japan). One of the human constitutive androstane
receptor (CAR) variants, CAR3, contains a 5-amino acid insertion (APYLT) between
exon 7 and exon 8 within the ligand binding domain (LBD), and was found to be
highly expressed in human livers. CAR3 lacks high basal activation in immortalized
cell lines, but responds to the CAR agonist, CITCO (Chen et al. 2010). A full-length
human CAR3 construct (pCR3-hCAR) and the CYP2B6 reporter construct,
containing both PBREM and the distal XREM (pGL3-CYP2B6–2.2 kb), have been
constructed as previously described (Sueyoshi et al. 1999; Wang et al. 2003) and were
kindly provided by Dr. Hongbing Wang (Department of Pharmaceutical Sciences,
University of Maryland School of Pharmacy, Baltimore, MD). The mouse CAR
expressing
plasmid
(pCMX-mCAR)
and
the
cyp2b10
reporter
construct
(pGL3-cyp2b10) were generated as described previously (Xie et al. 2000). An internal
control plasmid, pRC-CMV--galactosidase, was purchased directly from Invitrogen
(Groningen, Netherlands).
Assessment of cell cytotoxicity
To verify that 80% of the cells were viable following xenobiotic exposure, cell
viability was evaluated using the modified acid-phosphatase (ACP) assay with
p-nitrophenyl phosphate (PNPP) disodium salt as a substrate. The cell culture medium
was aspirated, and the cells were washed with phosphate-buffered saline (PBS). After
washing, 100 L of ACP reagent (0.1 M sodium acetate [pH 5.5], 0.1% Triton X-100,
and 10 mM PNPP) was added. After a 1-h incubation at 37C, the reaction was
stopped by adding 10 L of 1 N NaOH, and the activity was determined
photometrically at 405 nm (Lo et al. 2012).
Transient transfection and reporter assays
HepG2 and LS174T cells were plated in 96-well plates (Nunc, Rochester, NY,
USA) at a density of 1.5  104 cells/well 18 h before transfection. Plasmid DNA was
introduced using the PolyJETTM transfection reagent (SignaGen Laboratories,
Rockville, MD, USA), according to the manufacturer’s instructions. For the reporter
assay, 0.15 g of reporter construct, 0.02 g of control -galactosidase plasmid, and
0.04 g of the PXR, CAR, rPXR, mCAR, RXR, SRC-1, HNF4, and PGC-1
expression plasmids were added per well. After 6 or 7 h, the cells were exposed to
ligands (rifampin, AITC, CITCO, PCN, SR12813, nifedipine, TCPOBOP, or a similar
volume of DMSO). After 20–24 h of incubation, the cells were lysed in situ with 80
L of cell culture lysis reagent (Promega). The cleared lysate (30 L) was used for the
-galactosidase assay. A 50-L aliquot of cleared lysate and 80 L of luciferase assay
reagent (Promega) were used for the reporter assay. Luminescent signals were
measured using a luminescence multi-mode microplate reader. Luciferase activity was
normalized to the corresponding -galactosidase activity. The data shown are the
means of at least triplicates  standard error (SE; error bars).
Quantitative reverse transcription-polymerase chain reaction (qRT-PCR)
Total RNA was isolated by using a Direct-zolTM RNA MiniPrep kit (ZYMO
Research, Irvine, CA, USA) according to the manufacturer’s instructions. We
confirmed that the ratio of the absorbance of the isolated nucleic acid at 260 and 280
nm (A260/A280) was 1.8. First-strand cDNA was synthesized using total RNA (1
g) with random primers and MultiScribeTM reverse transcriptase (Applied
Biosystems, Foster City, CA, USA) by incubation at 25°C for 10 min, 37°C for 120
min, and 85°C for 5 min. The cDNAs were then proceeded for quantitative
polymerase chain reaction (qPCR) using the TaqMan Gene Expression Assay kit
(Applied Biosystems). The TaqMan assay identification numbers are as follows:
CYP3A4, Hs00604506_m1; CYP2B6, Hs04183483_g1; PXR, Hs01114267_m1;
RXR, Hs01067640_m1; HNF4, Hs00230853_m1; CAR, Hs00901571_m1; SRC-1,
Hs00186661_m1;
GR,
Hs00353740_m1;
and
glyceraldehyde-3-phosphate
dehydrogenase (GAPDH), Hs02758991_g1. A 25-L PCR mix contained 12.5 L of
universal PCR master mix, 1.25 L of the gene-specific TaqMan assay mix (probe), 2
L of cDNA template, and 9.25 L of nuclease-free water. The reaction cycle was
50°C for 2 min, 95°C for 10 min, followed by 40 cycles of 15 s at 95°C and 1 min at
60°C, as recommended by the manufacturer. The PCR amplification and
quantification were carried out in a StepOnePlusTM system (Applied Biosystems).
Western blotting
The protein expression of CYP3A4 and CYP2B6 was measured by Western
blotting. HepG2 cells were seeded at a density of 2  106 cells in a 10-cm dish.
Various concentrations of AITC, either alone or in combination with 20 M rifampin,
or with 10 M CITCO were added to the HepG2 cell culture, which was incubated for
24 h. Following drug treatment, the medium was removed, and the cells were rinsed
twice with ice-cold PBS. After adding 200 L of ice-cold RIPA buffer with protease
inhibitors, the cells were scraped from the surface of the culture dish and incubated on
ice for 20 min, and then the cell lysates were centrifuged at 14,000 rpm for 30 min at
4°C. Total protein (50 μg) from the supernatant was resolved by electrophoresis on a
10% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and transferred onto
nitrocellulose (NC) paper. These blots were exposed to primary antibodies against
CYP3A4, CYP2B6, and -actin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA,
USA). Following detection using an enhanced chemiluminescence detection system
(Millipore, Billerica, MA, USA), the relative density of the immunoreactive bands
was quantitated using a densitometer (ImageQuant LAS 4000; GE Healthcare,
Waukesha, WI, USA).
Catalytic activity of CYP3A4 and CYP2B6 in differentiated HepaRG cells
Differentiated HepaRG cells were seeded into 10-cm dish, cultivated for 24 h,
and then incubated AITC alone or in combination with either 20 M rifampin or 10
M CITCO for 72 h. For the last 24 h of the 72-h incubation, CYP probe substrates (3
M midazolam for CYP3A4 and 200 M bupropion HCl for CYP2B6) were added.
The cells were lysed with hypotonic extraction buffer (10 mM HEPES-KOH, 1.5 mM
MgCl2, and 10 mM KCl), and total cell lysates were extracted by high-speed
centrifugation. The protein content in the lysates was measured using a BCA assay kit
(Pierce, Rockford, IL, USA) for normalization. The metabolites were extracted by
acetone (acetone:H2O, 5:1, v/v). The concentration of the metabolites was determined
using an established LC-MS/MS method (Zambon et al. 2010). A high performance
liquid chromatographic system (Ultimate 3000 LC; Dionex, Germany) coupled with a
hybrid
Q-TOF
mass
spectrometer
(maXis
impact;
Bruker)
was
used.
Chromatographic separation was achieved with an Atlantis T3 analytical column (C18,
5 µm, 2.1 × 150 mm; Waters, Millford, MA, USA). Mobile phase A consisted of 0.1%
(v/v) formic acid and 5% acetonitrile. Mobile phase B consisted of pure acetonitrile
and 0.1% (v/v) formic acid. From 0–10 min, a linear LC gradient from 1% (v/v) B to
95% B was used at a flow rate of 0.3 mL/min. It was then held at 95% B for 2.5 min
and returned to 1% B (v/v) to re-equilibrate the column for 2.5 min prior to the next
injection. The ESI source was operated in positive ion mode. Nitrogen was used as a
nebulizing (50 psi) and drying gas (8 L/min, 350C). Helium was used as the collision
gas. For MS/MS (in positive mode), multiple reaction transition (MRM) was used by
monitoring the parent ion and the corresponding daughter ion at 2 Hz with a mass
window of 5 Da. The parent ion/daughter ion/optimized collision energy was set as
follows:
bupropion
hydrochloride
(240.11/166.04/15eV);
-hydroxybupropion
(256.11/238.10/14eV); midazolam (326.08/291.11/42eV); and 1’-hydroxymidazolam
(342.08/324.07/33eV). MS/MS data were processed and extracted by DataAnalysis
4.1 (Bruker).
Statistical analysis
All error bars indicate the mean ± SE. The p value for each experimental
comparison was determined using ANOVA followed by Tukey’s test for multiple
comparisons. All p values were determined relative to the vehicle control or
ligand-treated group, as indicated in the figures. All statistical analyses were
performed using SPSS for Windows, version 20.0 (SPSS, Inc., Chicago, IL, USA). A
p value less than0.05 was considered statistically significant.
Results
Cell viability of HepG2 and LS174T cells following exposure to AITC
The present study was designed to report the functional but not cytotoxic effects
of anti-tumor agents. However, since AITC has been shown to inhibit the proliferation
of multiple malignant cell types (Zhang 2010), a cell viability test was performed to
rule out any cytotoxic effects of AITC itself. As shown in Fig. 1, HepG2 and LS174T
cells were exposed to a range of concentrations of AITC for 24 h, both alone and in
combination with the PXR transactivator rifampin, and cell viability was assessed
using the ACP assay. Rifampin alone did not result in substantial cytotoxicity in either
cell line. AITC with or without rifampin caused mild cytotoxicity, compared to cells
treated with DMSO alone. However, even after exposure to the highest concentration
of ATIC tested (40 M), cell viability was still approximately 80% in both cell lines.
The Effects of AITC on PXR transactivation using a CYP3A4 reporter gene
assay
To validate the effect of AITC on PXR-mediated transcriptional activation of the
CYP3A4 promoter, we transiently transfected human PXR expression plasmids into
HepG2 and LS174T cells. PXR transactivation reporter assays were then conducted to
assess inhibitory effects on the CYP3A4 promoter using AITC alone or in
combination with rifampin-induced PXR transactivation activity. Results of these
experiments are shown in Fig. 2. In HepG2 cells, AITC strongly and significantly
attenuated transactivation of the CYP3A4 promoter in a dose-dependent manner,
particularly when co-transfected with human PXR under rifampin treatment (Fig. 2A).
These experiments were repeated using a colon adenocarcinoma cell line (LS174T) to
exclude the possibility of a cell type-specific effect, yielding very similar results (Fig.
2B). Endogenous levels of PXR and CAR have been confirmed in HepG2 and
LS174T cells (Rigalli et al. 2011; Wang et al. 2011), which explains why rifampin
could activate the CYP3A4 reporter response in cells harboring the control vector
(pcDNA3). As shown in Fig. 2A and 2B, significant decreases in CYP3A4 reporter
response in HepG2 and LS174T cells transfected with either vector occurs in the
presence of rifampin, most notably at the higher doses of AITC (30 and 40 M). In
contrast, cells without rifampin induction failed to show a significant reduction of
CYP3A4 reporter activity in the presence of AITC. These results suggest that a
PXR-dependent mechanism is involved in the down-regulation of CYP3A4 by AITC.
In order to investigate whether the transactivation of PXR is rifampin-specific,
two other PXR ligands (SR12813 and nifedipine) were also examined in HepG2 cells
transfected with either plasmid. As shown in Fig. 2C, 10 M of SR12813 enhanced
CYP3A4 promoter activity up to 23-fold compared to controls. However, the presence
of AITC significantly attenuated these effects, with 20 and 40 M AITC reducing
CYP3A4 promoter activity to only 7.8- and 6.3-fold, respectively. The effects of
nifepidine were less pronounced, but followed the same pattern. Nifepidine at 10 M
increased CYP3A4 promoter activity up to 1.8-fold in HepG2 cells transfected with
PXR, whereas 20 and 40 M of AITC significantly decreased this transactivation to
0.5- and 0.4-fold, respectively. Pronounced species-specific differences in the
activation of PXR have been previously observed (Kliewer et al. 2002), so the
inhibition of rat PXR activation using AITC was also tested in HepG2 cells. As shown
in Fig. 2D, AITC also attenuated rat PXR-mediated CYP3A4 promoter activity
following induction of rat PXR using PCN, AITC (20 and 40 M) reducing activity
down to 24% and 14% of activity, respectively, observed in the group treated only
with PCN. These results suggest that AITC inhibits PXR activity across multiple
species.
The effects of AITC on CYP3A4 expression in HepG2 cells
We next determined whether AITC could inhibit the expression of
drug-metabolizing enzymes (DMEs) in HepG2 cells. PXR activation is known to
increase expression of CYP3A4 at both the mRNA and protein level (Bertilsson et al.
1998). Consistent with results obtained from PXR transactivation in the CYP3A4
reporter gene assay, AITC effectively inhibited rifampin-induced CYP3A4 mRNA
expression in HepG2 cells (Fig. 3A). We also determined the effect of AITC on
CYP3A4 protein expression in this cell line. As shown in Fig. 3B and 3C, rifampin
significantly induced CYP3A4 protein expression, and this effect was reduced in the
presence of AITC, with densitometry data showing a 40% reduction in CYP3A4
protein expression using 40 M AITC compared with the group treated with rifampin
only. HepG2 cells have endogenous PXR expression and AITC alone inhibited
CYP3A4 expression, suggesting that the observed inhibition was transcriptionally
dependent.
The effects of AITC on human and mouse CAR activation
CAR is a sister xenobiotic receptor of PXR which shares some of its ligands and
target genes including CYP3A4 (Goodwin et al. 1999). Expression of CYP3A4 is also
regulated by CAR using the same response elements. Previous studies showed that the
CAR gene produces a number of differentially spliced mRNAs, encoding structurally
diverse proteins (Chen et al. 2010). A human splice variant of CAR, termed CAR3,
functions as a ligand-dependent receptor that exhibits substantially lower constitutive
activity. Therefore, we used a human CAR3 expression vector (pCR3-hCAR) to
determine the effect of AITC on CAR activation using a luciferase reporter assay. As
shown in Fig. 4A, a human CAR3 specific ligand (CITCO) enhanced CYP3A4
promoter activity up to 5.3-fold as compared to cells transfected with pCR3-hCAR
with no CITCO induction. AITC (20 and 40 M) markedly attenuated
CITCO-induced, CAR-mediated CYP3A4 promoter activation in pCR3-hCAR
transfected cells by 40.5% and 45%, respectively.
CYP2B6 is the primary target gene of human CAR (Sueyoshi et al. 1999). To
examine the effect of AITC on CAR-mediated CYP2B6 promoter activity, reporter
assays were performed in HepG2 cells. A luciferase reporter plasmid harboring the
luciferase gene (pGL3-CYP2B6-2.2 kb) was used, which is driven by the
phenobarbital responsive element module (PBREM) to which CAR binds and
activates the transcription of downstream genes (Wang et al. 2003). As shown in Fig.
4B, treatment with CITCO induced greater CAR activation on the CYP2B6 promoter
compared to CYP3A4, relative to controls (5.3-fold on CYP3A4 and 12.5-fold on
CYP2B6). AITC effectively abolished this activation in a dose-dependent manner. We
then examined the effect of AITC on mouse CAR transactivation, with TCPOBOP
used as the specific ligand for mouse CAR. As shown in Fig. 4C, AITC was also
effective for inhibiting induced mCAR transactivation of the cyp2b10 promoter.
Taking together these findings, we suggest that AITC inhibits both CYP3A4 and
CYP2B6, which are predominantly modulated by PXR and CAR, respectively, and
provides further evidence that the inhibition of CAR activation is not highly
species-specific.
The effects of AITC on CYP2B6 mRNA and protein expression in HepG2 cells
To validate the effect of AITC on transcriptional control of CYP2B6 expression
at the mRNA and protein level, HepG2 cells were treated with CITCO and/or AITC
for 24 h. Levels of mRNA and protein from treated cells were then assessed for
further analysis. CITCO (10 M) significantly induced CYP2B6 mRNA expression
compared to no treatment (Fig. 5A), whereas this induction was abolished using 20
and 40 M AITC (decreased by 27% and 69%, respectively). We also determined the
effect of AITC on CYP2B6 protein expression in these cells. As shown in Fig. 5B and
5C, CITCO significantly induced CYP2B6 protein expression in this cell line, and this
effect was reduced in the presence of AITC (36% and 45.5% reductions at 20 and 40
M AITC, respectively). This data shows that AITC also inhibits CYP2B6 expression
through transcriptional control, in a manner similar to CYP3A4.
The effects of AITC on CYP3A4 and CYP2B6 catalytic activities in
differentiated HepaRG cells
The data suggests that AITC attenuates PXR and CAR transactivation, leading to
the decreased expression of CYP3A4 and CYP2B6 mRNA and protein. To evaluate
potential effects of AITC on CYP3A4 and CYP2B6 catalytic activity, we also
assessed the CYP3A4 and CYP2B6 probe substrates, midazolam and bupropion
hydrochloride metabolism, by measuring levels of 1’-hydroxymidazolam and
-hydroxybupropion, respectively, in cell lysates of differentiated HepaRG cells.
Rifampin and CITCO were included as human PXR and CAR-specific control ligands,
respectively. After 72 h of pretreatment with 20 M rifampin or 10 M CITCO, alone
or in combination with 40 M AITC, probe substrates were added and incubated for
an additional 24 h. As shown in Fig. 6A, 40 M of AITC reduced
1’-hydroxymidazolam levels especially in cells pretreated with 20 M rifampin.
CYP3A4 activity measured by 1’-hydroxymidazolam levels decreased by 58.1%
following AITC treatment compared to cells treated with rifampin only. Rifampin was
highly effective at inducing 1’-hydroxymidazolam formation compared to DMSO
controls in these differentiated HepaRG cells.
CYP2B6 mediates the metabolism of bupropion, so we also evaluated the effect
of AITC on CYP2B6 activity by measuring -hydroxybupropion levels in cell lysates
of differentiated HepaRG cells. Similar to observation made for CYP3A4,
-hydroxybupropion levels were significantly increased by 1.9-fold after pretreatment
with 10 M CITCO, and this induction was significantly attenuated by 40 M AITC
(Fig. 6B).
Inhibition of co-regulation of human RXR, SRC-1, HNF4, and PGC-1 with
PXR by AITC
Earlier studies showed that rifampin-mediated CYP3A4 induction in HepG2
cells was enhanced by the presence of RXR, SRC-1, HNF4, and PGC-1
(Takezawa et al. 2012). We therefore evaluated whether the effects of these four
CYP3A4 promoter co-regulators were altered in the presence of AITC. Full-length
human RXR (Fig. 7A), SRC-1 (Fig. 7B), HNF4 (Fig. 7C), and PGC-1 (Fig. 7D)
expression plasmids were co-transfected with full-length human PXR/vector control
and CYP3A4 reporter constructs into HepG2 cells. Transfected cells were then
exposed to AITC, alone or in combination with rifampin, followed by the
measurement of luciferase activity following 24 h of drug treatment. Co-transfection
of PXR with SRC-1 and PGC-1 resulted in significantly enhanced CYP3A4
promoter activity in the presence of rifampin (Fig. 7). AITC strongly disrupted this
promoter activity enhancement, particularly in rifampin-treated cells. These results
indicate that AITC disrupts the co-regulatory effects of PXR-coactivators, especially
SRC-1 and PGC-1, on CYP3A4 promoter activity. We also investigated whether
these effects could be caused by decreases in the mRNA expression of several
coregulators or nuclear receptors (Supplemental data, Table S1), indicating that
mRNA expression of PXR, RXR, HNF4, CAR, SRC-1, and GR were not
significantly changed following treatment of these factors.
Decreased cytotoxicity of acetaminophen (APAP) and amiodarone in the present
of CYP3A4 inducer and AITC
APAP and amiodarone are two well-characterized substrates of CYP3A4 that are
known to generate toxic metabolites resulting in cytotoxicity (Hosomi et al. 2010).
We treated differentiated HepaRG cells with 20 M rifampin alone or in combination
with 10 M AITC for 72 h, then 10 or 20 mM APAP, and 10 or 20 M amiodarone
was added to media for 20 h and cytotoxicity assays were then performed (Figure 8).
Concentration dependent decreases in cell viability were observed with the addition of
APAP and amiodarone in cells pretreated with rifampin (decreased by 20.6% and
39.3% at 10 and 20 mM APAP, 19.9% and 35.9% at 10 and 20 M amiodarone,
respectively). To investigate the potential of AITC for protecting cells from toxic
metabolites during rifampin induction, cell viability was also tested in the presence of
AITC. Results show increased cell viability with the addition of 10 M AITC
compared to rifampin alone, suggesting that AITC may protect cells from
rifampin-induced drug cytotoxic effects.
Discussion
This study showed that AITC is an antagonist of human PXR and CAR in vitro,
based on changes in mRNA and protein expression, in addition to reporter assays and
measurement of catalytic activity. This naturally occurring phytochemical can block
the effects of PXR and CAR agonists in well characterized HepG2 and HepaRG cells.
Evidence from this investigation indicates that AITC is an effective inhibitor of PXR
and CAR, subsequently reducing the expression and activity of their target genes,
CYP3A4 and CYP2B6. Although dramatic differences in the mechanisms of activation
have been reported in PXR and CAR orthologs between species, our results indicate
that AITC may also effectively antagonize rodent PXR and CAR activity. These data
suggest that AITC could act directly on PXR and CAR or affect the recruitment of
coregulatory proteins during promoter interactions. AITC is the second plant-derived
isothiocyanate shown to affect PXR/CAR activity. PXR has previously been shown to
mediate the effects of prescribed drugs or botanicals because of CYP3A4 induction
resulting in either the deactivation of therapeutic drugs or the accumulation of
potentially dangerous drug metabolites. These interactions have been associated with
clinically significant events, including those that were life threatening or lethal. We
also showed the cytotoxic effects of APAP and amiodarone following CYP3A4
induction with rifampin, an effect that was effectively attenuated by AITC.
Antagonizing the effect of PXR activation using this plant-derived isothiocyanate may
therefore reduce drug-associated hepatotoxicity.
AITC is an isothiocyanate known to possess anti-inflammatory, antimicrobial,
and anticancer activities (Zhang 2010). Most cruciferous vegetables, including
Brussels sprouts, cabbage, cauliflower, and kale contain AITC, which imparts a
pungent taste to these foods. The highest quantities of AITC have been found in
horseradish (Armoracia lapathifolia) (1500-9000 mg/kg), mustard, and wasabi
(Wasabi japonica) (Japanese horseradish) (9585 mg/kg, 34 μmol sinigrin/AITC per
gram of fresh wasabi) (Sultana et al. 2003; TNO 2009). It is also used as a food
preservative (Zhang 2010). Under normal circumstances, AITC is formed as sinigrin
(a kind of glucosinolate) and hydrolyzed to its final form by plant myrosinase. It is
also converted by myrosinase in vivo by intestinal microflora in humans and animals
(Krul et al. 2002). AITC is also thought to be rapidly metabolized via the mercapturic
acid pathway to an N-acetylcysteine conjugate, a form found solely in urine. Thus, it
has been suggested that AITC could be useful in the prevention of bladder cancer
(Zhang 2010). We did not rule out the possibility that the hepatoma cell lines used in
this study (HepG2 and HepaRG) metabolized AITC to its metabolites resulting in
PXR and CAR inactivation, so we also conducted the CYP3A4 reporter assay in
COS-1 cells (Supplemental data, Fig. S1). COS-1 cells are an established cell line
derived from monkey epithelial cells (CV-1) that harbor a defective SV40 mutant
(Hancock 1992). Results from this follow-up experiment showed that endogenous
expression of drug-metabolizing enzymes was extremely low or absent (Ahlin et al.
2009), and confirmed that AITC antagonized PXR activation as a result of the parent
compound rather than its metabolites.
The bioavailability of AITC is extremely high (>90%). Zhang (Zhang and
Talalay 1998; Zhang 2000) showed that intracellular concentration of AITC could
rapidly reach millimolar levels. Additionally, it has been reported that blood
concentration of AITC in mice and rats can reach levels as high as 0.04 and 0.5 mM,
respectively, from a single oral dose (Bollard et al. 1997). Concentrations of AITC
used in this study could therefore be reached at the cellular levels in humans. AITC
has also been shown to induce several Phase 2 enzymes through the Nrf2 signaling
pathway
(Munday
et
al.
2002).
We
tested
mRNA
expression
of
the
UDP-glucuronosyltransferase 1A1 (UGT1A1) gene following treatment with 20 M
rifampin alone and in combination with 10 M AITC for 24 h. Expression of
UGT1A1 mRNA was increased in the presence of rifampin, but similar to with the
addition of AITC (7.33-fold vs. 6.78-fold). Our cytotoxicity data in Fig. 8 suggests
that AITC rescued the cells from the cytotoxic effect of APAP and amiodarone,
indicates that this was not likely a result of protective effects from phase 2 enzymes.
PXR and CAR nuclear receptors recognize a broad spectrum of xenobiotics and
endogenous compounds. They control the expression of human CYP3A4/mouse
cyp3a11 and human CYP2B6/mouse cyp2b10 genes, which are responsible for 50%
and 15% of the overall CYP-mediated metabolism of prescribed drugs, respectively
(Guengerich 2008; Wang and Tompkins 2008). The activation/deactivation of these
two nuclear receptors may therefore affect drug metabolism. Inappropriate PXR
activation leads to undesirable pathophysiological outcomes, and can result in
clinically significant adverse drug interactions. Rifampin, an inducer of CYP3A4 and
one of the most potent human PXR activators, was shown to affect the therapeutic
efficacy of a number of substrate drugs (e.g. paclitaxel, cyclosporine), resulting in
poor efficacy and failure of bone marrow transplantation (Conney 2003). Failure of
oral contraceptives has also been reported in women who had taken rifampin
(Reimers and Jezek 1971), and loss of the analgesic effects of opioids have also been
reported in subjects treated with rifampin due to induction of PXR/CAR activation
(Bauer et al. 2006). Chronic administration of PXR activator can also lead to
osteomalacia by increasing clearance of 1,25-dihydroxyvitamin D3 (Pascussi et al.
2005). Another strong inducer of PXR derived from natural products, hyperforin,
significantly affects serum concentrations of a chemotherapeutic agent, irinotecan
(CPT-11), consequently reducing the anti-neoplastic effect and toxicity of this
CYP3A4 substrate (Hu et al. 2007).
The multitude of potential undesirable side effects in patients who must
chronically take PXR activating medications highlight the potential benefit of
developing PXR antagonists. Here, we show that AITC decreases constitutive
CYP3A4 and CYP2B6 mRNA and protein expression levels without changing
expression of upper control elements (PXR, CAR, RXR, HNF4, SRC-1, and GR).
Additionally, AITC attenuates rifampin-mediated CYP3A4 and CITCO-mediated
CYP2B6 catalytic activity in HepaRG cells. By disrupting the coregulation effects of
especially SRC-1, and PGC-1, AITC also significantly decreased rifampin-,
SR12813-, and nifedipine-mediated CYP3A4 promoter activity. These results suggest
that AITC may eliminate drug interactions caused by PXR or CAR ligands.
To detect and compare the basal expression levels of RXR, HNF4, SRC-1,
and PGC-1 in hepatocarcinoma-derived cell lines, we collected the whole cell lysate
of HepG2 and HepaRG cells and performed western blotting. The basal expression of
SRC-1 and PGC-1 could be detected in HepaRG cells but not in HepG2 cells. From
our unpublished data of basal expression of RXR, HNF4, SRC-1, and PGC-1 in
HepaRG cells (data not shown), the expression of RXR and HNF4 was higher than
that of SRC-1 and PGC-1. Due to the abundant endogenous expression of RXR
and HNF4 in the hepatocarcinoma cell line, the overexpression of these two proteins
may not enhance PXR activation of CYP3A4 promoter activity much more. In
contrast, along with the overexpression of SRC-1 and PGC-1 in our coregulation
promoter activity assays with PXR, synergistic effects of CYP3A4 promoter activity
were observed.
PXR stimulates transcription in response to ligand binding by interacting with
coactivators in a ligand-dependent manner. The complexity of the interacting sites of
PXR-coactivator complexes is not known. The disruption of these transcriptional
complexes might decrease target gene expression (Kliewer et al. 2002). Our data
indicated that AITC might be a receptor antagonist of human and rodent PXR/CAR
and that the recruitment of transcriptional complexes of PXR might be disrupted by
the presence of AITC, thereby abolishing PXR transactivation ability. Thus, the
expression of target genes was affected.
Dramatic differences in the activation of PXR and CAR orthologs among
different species have been reported (Moore et al. 2002). For example, rifampin and
CITCO are potent ligands for human, but not rodent PXR and CAR. Conversely, PCN
and TCPOBOP activate rodent PXR and CAR but not in human counterparts (Lake et
al. 1998). Our promoter activity data indicates that the activity of AITC is not highly
species-specific, and that it acts as an effective antagonist of PXR/CAR function in
both humans and rodents.
Development of PXR/CAR antagonists would also be useful for the study of
receptor molecular fundamental function. Relatively few PXR antagonists have been
identified to date. These include Trabectedin (ET-743) (Synold et al. 2001),
ketoconazole (and related azoles) (Wang et al. 2007), sulphoraphane (Zhou et al.
2007), A792611 (HIV protease inhibitor) (Healan-Greenberg et al. 2008), coumestrol
(Wang et al. 2008), metformin (Krausova et al. 2011), sesamin (a naturally occurring
lignan) (Lim et al. 2012), and fucoxanthin (Liu et al. 2012). Some of them are shown
to disrupt the activity of PXR coregulators in an agonist-dependent manner. In our
study, AITC inhibited both basal and inducible factors leading to PXR/CAR
activation.
In addition to the regulation of DMEs, the PXR/CAR receptors are also involved
in other biological processes, including bile acid and lipid metabolism, glucose and
energy homeostasis, and inflammation (Mahamuni et al. 2012; Gao and Xie 2012).
Targeting PXR/CAR activity could therefore be useful for the treatment of other
diseases such as steatosis, cholestasis, osteoporosis, and inflammatory bowel disease
(IBD) (Schulman 2010; Cheng et al. 2012; Gao and Xie 2012). Moreover, PXR
activation may also lead to cancer drug resistance, due to the induction of transporters
(e.g. P-glycoprotein, multidrug resistance protein). Modulating the activity of PXR
could therefore be useful for reversing PXR-mediated cancer drug resistance and
tumor growth for cancer therapy, in addition to its utility for reducing inducer-drug
interactions (Chen 2010; Chen et al. 2012). Thus, inhibition of PXR activity has been
considered a novel strategy to overcome chemotherapeutic drug resistance by
attenuating the expression of DMEs and transporters in tumors.
Another important issue is drug-induced hepatotoxicity. Although relatively rare,
it has caused serious adverse reactions following administration of a large number of
pharmaceutical drugs (Boelsterli and Lim 2007). One mechanism that has been
suggested drug-induced hepatotoxicity is associated with reactive metabolites
produced by DMEs (Guengerich and MacDonald 2007). For example, if reactive
metabolites covalently bind to intracellular proteins, cellular dysfunctions can result
in the loss of ionic gradients, a decline in ATP levels, actin disruption, cell swelling
and rupture (Yun et al. 1993). APAP is a widely used analgesic and antipyretic drug.
It is well tolerated at recommended therapeutic doses and metabolized by phase 2
conjugating enzymes (UGT and SULT) to yield nontoxic compounds that are
excreted via renal and biliary routes (James et al. 2003). CYP enzymes may also turn
APAP into a highly reactive metabolite, N-acetyl-p-benzoquinone-imine (NAPQI)
(Dahlin et al. 1984). NAPQI is eliminated by conjugating with glutathione by
glutathione S-transferase (GST), then further metabolized to a mercapturic acid and
followed by renal excretion (Beckett et al. 1985). When an APAP overdose is taken,
APAP undergoes P450-mediated formation of NAPQI and accumulation of this
intermediate will caused structural and metabolic cell disorders (Potter et al. 1974).
Previous studies have shown that PXR plays an important role in APAP metabolism.
Induction of cyp3a11 causes an increase of APAP reactive metabolites, thereby
causing liver injury in mice (Guo et al. 2004). Metabolic activation of CYP3A4 or
PXR therefore increases toxic effects, whereas CYP3A4 inhibitors or PXR
deactivators prevent APAP toxicity (Kostrubsky et al. 1997; Cheng et al. 2009).
Hepatotoxicity mediated by PXR ligand can be severe due to the induction of
CYP3A4, which produce saquinone metabolite of the parent drug (Cheng et al. 2009).
Development of PXR antagonists could therefore have substantial clinical potential
for reducing potentially dangerous adverse events resulting from overdoses of
otherwise benign and effective drugs.
Amiodarone is a widely used antiarrhythmic drug, but is associated with several
side effects, including liver toxicity (Mason 1987). Amiodarone is metabolized
primarily by CYP3A4 to mono-N-desethylamiodarone (MDEA) (Flanagan et al. 1982)
and di-N-desethylamiodarone (DDEA) (Ha et al. 2005). MDEA was reported to cause
cytotoxicity in HepG2 cells and rat hepatocytes (McCarthy et al. 2004), and
accumulation of toxic metabolites can result in cellular and organic toxicity. Our in
vitro results showed that rifampin enhanced the cytotoxicity of amiodarone, but this
effect was effectively reversed by AITC.
Low expression of CYP enzymes has been found in HepG2 cells due to
decreased transcription (Rodriguez-Antona et al. 2002), and transfection with
PXR/CAR or treatment with PXR/CAR agonists led to higher expression levels of
CYP3A4 and CYP2B6 (Moya et al. 2010). In the present study, induction of mRNA
and protein expression in HepG2 cells by rifampin and CITCO correlated well with
catalytic activities in differentiated HepaRG cells. HepaRG cells represent the best
surrogate for primary human hepatocytes, capable of expressing both phase 1 and 2
DMEs in addition to transporters (Guillouzo et al. 2007). As a consequence of lower
CYP expression in HepG2 cells, we could not detect midazolam and bupropion
metabolites in this cell line, which are the probe substrates of CYP3A4 and CYP2B6,
respectively. Thus, we used HepaRG cells to assess catalytic activity and cytotoxicity.
In conclusion, we have identified a common and a naturally occurring substance
(AITC) that acts as a potent inhibitor of human and rodent PXR and CAR. The study
provides evidence that AITC attenuates the induction of CYP3A4 and CYP2B6 by
inhibiting agonist-activated PXR and CAR. Additionally, AITC reduces the catalytic
activities of CYP3A4 and CYP2B6 in differentiated HepaRG cells, and disrupts the
coregulation of PXR with RXR/SRC-1/HNF4/PGC-1. These findings suggest a
complementary mechanism of AITC, which may be used as an adjuvant therapy to
decrease the frequency of adverse drug reactions. This agent could also reduce
excessive PXR-mediated drug clearance or cytotoxicity via the metabolic activities of
CYP3A4. Thus, we provide a new avenue of PXR antagonism that could prevent
harmful inducer-drug interactions and improve the therapeutic efficacy of currently
used medications.
Acknowledgements
This study was supported by National Science Council, Executive Yuan, Taiwan,
R.O.C. (NSC-101-2320-B-039-007-MY3), China Medical University Hospital,
Taichung, Taiwan (DMR-103-049), in part by Taiwan Department of Health Clinical
Trial and Research Center of Excellence (DOH102-TD-B-111-004), and “The Aim
for The Top University Plan” from Traditional Chinese Medicine Research Center of
China Medical University, Taichung, Taiwan. We thank Dr. Hongbing Wang
(Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy,
Baltimore, Maryland) for kindly providing human CAR3 and CYP2B6 reporter
constructs.
Conflict of interest
The authors declare that there are no conflicts of interest.
References
Ahlin G, Hilgendorf C, Karlsson J, Szigyarto CA, Uhlén M, Artursson P (2009)
Endogenous gene and protein expression of drug-transporting proteins in cell
lines routinely used in drug discovery programs. Drug Metab Dispos
37:2275-2283
Bauer B, Yang X, Hartz AM, Olson ER, Zhao R, Kalvass JC, Pollack GM, Miller DS
(2006) In vivo activation of human pregnane X receptor tightens the blood-brain
barrier to methadone through P-glycoprotein up-regulation. Mol Pharmacol
70:1212-1219
Beckett GJ, Chapman BJ, Dyson EH, Hayes JD (1985) Plasma glutathione
S-transferase measurements after paracetamol overdose: evidence for early
hepatocellular damage. Gut 26:26-31
Bertilsson G, Heidrich J, Svensson K, Asman M, Jendeberg L, Sydow-Backman M,
Ohlsson R, Postlind H, Blomquist P, Berkenstam A (1998) Identification of a
human nuclear receptor defines a new signaling pathway for CYP3A induction.
Proc Natl Acad Sci U S A 95:12208-12213
Boelsterli UA, Lim PL (2007) Mitochondrial abnormalities — a link to idiosyncratic
drug hepatotoxicity? Toxicol Appl Pharmacol 220:92-107
Bollard M, Stribbling S, Mitchell S, Caldwell J (1997) The disposition of allyl
isothiocyanate in the rat and mouse. Food Chem Toxicol 35:933-943
Chen T (2010) Overcoming drug resistance by regulating nuclear receptors. Adv Drug
Deliv Rev 62:1257-1264
Chen T, Tompkins LM, Li L, Li H, Kim G, Zheng Y, Wang H (2010) A single amino
acid controls the functional switch of human constitutive androstane receptor
(CAR) 1 to the xenobiotic-sensitive splicing variant CAR3. J Pharmacol Exp
Ther 332:106-115
Chen Y, Tang Y, Guo C, Wang J, Boral D, Nie D (2012) Nuclear receptors in the
multidrug resistance through the regulation of drug-metabolizing enzymes and
drug transporters. Biochem Pharmacol 83:1112-1126
Chen Y, Tang Y, Robbins GT, Nie D (2010) Camptothecin attenuates cytochrome
P450 3A4 induction by blocking the activation of human pregnane X receptor. J
Pharmacol Exp Ther 334:999-1008
Cheng J, Ma X, Krausz KW, Idle JR, Gonzalez FJ (2009) Rifampicin-activated
human
pregnane
X
receptor
and
CYP3A4
induction
enhance
acetaminophen-induced toxicity. Drug Metab Dispos 37:1611-1621
Cheng J, Shah YM, Gonzalez FJ (2012) Pregnane X receptor as a target for treatment
of inflammatory bowel disorders. Trends Pharmacol Sci 33:323-330
Conney AH (2003) Induction of drug-metabolizing enzymes: a path to the discovery
of multiple cytochromes P450. Annu Rev Pharmacol Toxicol 43:1-30
Dahlin DC, Miwa GT, Lu AY, Nelson SD (1984) N-acetyl-p-benzoquinone imine: a
cytochrome P-450-mediated oxidation product of acetaminophen. Proc Natl
Acad Sci U S A 81:1327-1331
EFSA (2010) Scientific Opinion on the safety of allyl isothiocyanate for the proposed
uses as a food additive. EFSA Journal 8:1943
Flanagan RJ, Storey GC, Holt DW, Farmer PB (1982) Identification and measurement
of desethylamiodarone in blood plasma specimens from amiodarone-treated
patients. J Pharm Pharmacol 34:638-643
Gao J, Xie W (2012) Targeting xenobiotic receptors PXR and CAR for metabolic
diseases. Trends Pharmacol Sci 33:552-558
Goodwin B, Hodgson E, Liddle C (1999) The orphan human pregnane X receptor
mediates the transcriptional activation of CYP3A4 by rifampicin through a distal
enhancer module. Mol Pharmacol 56:1329-1339
Goodwin B, Moore LB, Stoltz CM, McKee DD, Kliewer SA (2001) Regulation of the
human CYP2B6 gene by the nuclear pregnane X receptor. Mol Pharmacol
60:427-431
Guengerich FP (2008) Cytochrome P450 and chemical toxicology. Chem Res Toxicol
21:70-83
Guengerich FP, MacDonald JS (2007) Applying mechanisms of chemical toxicity to
predict drug safety. Chem Res Toxicol 20:344-369
Guillouzo A, Corlu A, Aninat C, Glaise D, Morel F, Guguen-Guillouzo C (2007) The
human hepatoma HepaRG cells: a highly differentiated model for studies of liver
metabolism and toxicity of xenobiotics. Chem Biol Interact 168:66-73
Guo GL, Moffit JS, Nicol CJ, Ward JM, Aleksunes LA, Slitt AL, Kliewer SA,
Manautou JE, Gonzalez FJ (2004) Enhanced acetaminophen toxicity by
activation of the pregnane X receptor. Toxicol Sci 82:374-380
Ha HR, Bigler L, Wendt B, Maggiorini M, Follath F (2005) Identification and
quantitation of novel metabolites of amiodarone in plasma of treated patients.
Eur J Pharm Sci 24:271-279
Hancock JF (1992) COS Cell Expression. Methods Mol Biol 8:153-158
Healan-Greenberg C, Waring JF, Kempf DJ, Blomme EA, Tirona RG, Kim RB (2008)
A human immunodeficiency virus protease inhibitor is a novel functional
inhibitor of human pregnane X receptor. Drug Metab Dispos 36:500-507
Hosomi H, Akai S, Minami K, Yoshikawa Y, Fukami T, Nakajima M, Yokoi T (2010)
An
in
vitro
drug-induced
hepatotoxicity
screening
system
using
CYP3A4-expressing and gamma-glutamylcysteine synthetase knockdown cells.
Toxicol in Vitro 24:1032-1038
Hu ZP, Yang XX, Chen X, Cao J, Chan E, Duan W, Huang M, Yu XQ, Wen JY, Zhou
SF (2007) A mechanistic study on altered pharmacokinetics of irinotecan by St.
John’s wort. Curr Drug Metab 8:157-171
Itoh M, Nakajima M, Higashi E, Yoshida R, Nagata K, Yamazoe Y, Yokoi T (2006)
Induction of human CYP2A6 is mediated by the pregnane X receptor with
peroxisome proliferator-activated receptor-gamma coactivator 1 alpha. J
Pharmacol Exp Ther 319:693-702
Jacobs MN, Nolan GT, Hood SR (2005) Lignans, bacteriocides and organochlorine
compounds activate the human pregnane X receptor (PXR). Toxicol Appl
Pharmacol 209:123-133
James LP, Mayeux PR, Hinson JA (2003) Acetaminophen-induced hepatotoxicity.
Drug Metab Dispos 31:1499-1506
Kawamoto T, Sueyoshi T, Zelko I, Moore R, Washburn K, Negishi M (1999)
Phenobarbital-responsive nuclear translocation of the receptor CAR in induction
of the CYP2B gene. Mol Cell Biol 19:6318-6322
Kliewer SA, Goodwin B, Willson TM (2002) The nuclear pregnane X receptor: a key
regulator of xenobiotic metabolism. Endocr Rev 23:687-702
Kostrubsky VE, Szakacs JG, Jeffery EH, Woods SG, Bement WJ, Wrighton SA,
Sinclair PR, Sinclair JF (1997) Protection of ethanol-mediated acetaminophen
hepatotoxicity by triacetyloleandomycin, a specific inhibitor of CYP3A. Ann
Clin Lab Sci 27:57-62
Krausova L, Stejskalova L, Wang H, Vrzal R, Dvorak Z, Mani S, Pavek P (2011)
Metformin suppresses pregnane X receptor (PXR)-regulated transactivation of
CYP3A4 gene. Biochem Pharmacol 82:1771-1180
Krul C, Humblot C, Philippe C, Vermeulen M, van Nuenen M, Havenaar R, Rabot S
(2002) Metabolism of sinigrin (2-propenyl glucosinolate) by the human colonic
microflora in a dynamic in vitro large-intestinal model. Carcinogenesis
23:1009-1016
Lake BG, Renwick AB, Cunninghame ME, Price RJ, Surry D, Evans DC (1998)
Comparison of the effects of some CYP3A and other enzyme inducers on
replicative DNA synthesis and cytochrome P450 isoforms in rat liver. Toxicology
131:9-20
Lehmann JM, McKee DD, Watson MA, Willson TM, Moore JT, Kliewer SA (1998)
The human orphan nuclear receptor PXR is activated by compounds that regulate
CYP3A4 gene expression and cause drug interactions. J Clin Invest
102:1016-1023
Lim YP, Ma CY, Liu CL, Lin YH, Hu ML, Chen JJ, Hung DZ, Hsieh WT, Huang JD
(2012) Sesamin: a naturally occurring lignan inhibits CYP3A4 by antagonizing
the pregnane X receptor activation. Evid Based Complement Alternat Med
2012:242810
Liu CL, Lim YP, Hu ML (2012) Fucoxanthin attenuates rifampin-induced cytochrome
P450 3A4 (CYP3A4) and multiple drug resistance 1 (MDR1) gene expression
through pregnane X receptor (PXR)-mediated pathways in human hepatoma
HepG2 and colon adenocarcinoma LS174T cells. Mar Drugs 10:242-257
Lo WS, Lim YP, Chen CC, Hsu CC, Souček P, Yun CH, Xie W, Ueng YF (2012) A
dual function of the furanocoumarin chalepensin in inhibiting Cyp2a and
inducing Cyp2b in mice: the protein stabilization and receptor-mediated
activation. Arch Toxicol 86:1927-1938
Mahamuni SP, Khose RD, Menaa F, Badole SL (2012) Therapeutic approaches to
drug targets in hyperlipidemia. BioMedicine 2:137-146
Mason JW (1987) Amiodarone. N Engl J Med 316:455-466
Matsuda H, Ochi M, Nagatomo A, Yoshikawa M (2007) Effects of allyl
isothiocyanate from horseradish on several experimental gastric lesions in rats.
Eur J Pharmacol 561:172-181
McCarthy TC, Pollak PT, Hanniman EA, Sinal CJ (2004) Disruption of hepatic lipid
homeostasis in mice after amiodarone treatment is associated with peroxisome
proliferator activated receptor alpha target gene activation. J Pharmacol Exp Ther
311:864-873
Moore LB, Maglich JM, McKee DD, Wisely B, Willson TM, Kliewer SA, Lambert
MH, Moore JT (2002) Pregnane X receptor (PXR), constitutive androstane
receptor (CAR), and benzoate X receptor (BXR) define three pharmacologically
distinct classes of nuclear receptors. Mol Endocrinol 16:977-986
Moya M, Gomez-Lechon MJ, Castell JV, Jover R (2010) Enhanced steatosis by
nuclear receptor ligands: a study in cultured human hepatocytes and hepatoma
cells with a characterized nuclear receptor expression profile. Chem Biol Interact
184:376-387
Munday R, Munday CM (2002) Selective induction of phase II enzymes in the
urinary bladder of rats by allyl isothiocyanate, a compound derived from
Brassica vegetables. Nutr Cancer 44:52-59
Nielsen PV, Rios R (2000) Inhibition of fungal growth on bread by volatile
components from spices and herbs, and the possible application in active
packaging, with special emphasis on mustard essential oil. Int J Food Microbiol
60:219-229
Pascussi JM, Robert A, Nguyen M, Walrant-Debray O, Garabedian M, Martin P,
Pineau T, Saric J, Navarro F, Maurel P, Vilarem MJ (2005) Possible involvement
of pregnane X receptor-enhanced CYP24 expression in drug-induced
osteomalacia. J Clin Invest 115:177-186
Potter WZ, Thorgeirsson SS, Jollow DJ, Mitchell JR (1974) Acetaminophen induced
hepatic necrosis. V. Correlation of hepatic necrosis, covalent binding and
glutathione depletion in hamsters. Pharmacology 12:129-143
Reimers D, Jezek A (1971) The simultaneous use of rifampicin and other
antitubercular agents with oral contraceptives. Prax Pneumol 25:255-262
Rigalli JP, Ruiz ML, Perdomo VG, Villanueva SS, Mottino AD, Catania VA (2011)
Pregnane X receptor mediates the induction of P-glycoprotein by spironolactone
in HepG2 cells. Toxicology 285:18-24
Rodriguez-Antona C, Donato MT, Boobis A, Edwards RJ, Watts PS, Castell JV,
Gomez-Lechon MJ (2002) Cytochrome P450 expression in human hepatocytes
and hepatoma cell lines: molecular mechanisms that determine lower expression
in cultured cells. Xenobiotica 32:505-520
Schulman IG (2010) Nuclear receptors as drug targets for metabolic disease. Adv
Drug Deliv Rev 62:1307-1315
Shin IS, Masuda H, Naohide K (2004) Bactericidal activity of wasabi (Wasabia
japonica) against Helicobacter pylori. Int J Food Microbiol 94:255-261
Staudinger JL, Goodwin B, Jones SA, Hawkins-Brown D, MacKenzie KI, LaTour A,
Liu Y, Klaassen CD, Brown KK, Reinhard J, Willson TM, Koller BH, Kliewer
SA (2001) The nuclear receptor PXR is a lithocholic acid sensor that protects
against liver toxicity. Proc Natl Acad Sci USA 98:3369-3374
Sueyoshi T, Kawamoto T, Zelko I, Honkakoski P, Negishi M (1999) The repressed
nuclear receptor CAR responds to phenobarbital in activating the human
CYP2B6 gene. J Biol Chem 274:6043-6046
Sultana T, Porter NG, Savage GP, McNeil DL (2003) Comparison of isothiocyanate
yield from wasabi rhizome tissues grown in soil or water. J Agric Food Chem
51:3586-3591
Synold TW, Dussault I, Forman BM (2001) The orphan nuclear receptor SXR
coordinately regulates drug metabolism and efflux. Nat Med 7:584-590
Takezawa T, Matsunaga T, Aikawa K, Nakamura K, Ohmori S (2012) Lower
expression of HNF4α and PGC1α might impair rifampicin-mediated CYP3A4
induction under conditions where PXR is overexpressed in human fetal liver
cells. Drug Metab Pharmacokinet 27:430-438
TNO (Netherlands Organisation for Applied Scientific Research) (2009) VCF Volatile
Compounds in Food : database / Nijssen, L.M.; Ingen-Visscher, C.A. van;
Donders, J.J.H. [eds]. – Version 12.1 –Zeist (The Netherlands) : TNO Quality of
Life, 1963-2009. http://www.vcf-online.nl/VcfHome.cfm
Wagner AE, Boesch-Saadatmandi C, Dose J, Schultheiss G, Rimbach G (2012)
Anti-inflammatory potential of allyl isothiocyanate  role of Nrf2, NF-κB and
microRNA-155. J Cell Mol Med 16:836-843
Walko CM, Combest AJ, Spasojevic I, Yu AY, Bhushan S, Hull JH, Hoskins J,
Armstrong D, Carey L, Collicio F, Dees EC (2012) The effect of a prepitant and
race on the pharmacokinetics of cyclophosphamide in breast cancer patients.
Cancer Chemother Pharmacol 69:1189-1196
Wang H, Faucette SR, Gilbert D, Jolley SL, Sueyoshi T, Negishi M, LeCluyse EL
(2003) Glucocorticoid receptor enhancement of pregnane X receptor-mediated
CYP2B6 regulation in primary human hepatocytes. Drug Metab Dispos
31:620-630
Wang H, Huang H, Li H, Teotico DG, Sinz M, Baker SD, Staudinger J, Kalpana G,
Redinbo MR, Mani S (2007) Activated pregnenolone X-receptor is a target for
ketoconazole and its analogs. Clin Cancer Res 13:2488-2495
Wang H, Li H, Moore LB, Johnson MD, Maglich JM, Goodwin B, Ittoop OR, Wisely
B, Creech K, Parks DJ, Collins JL, Willson TM, Kalpana GV, Venkatesh M,
XieW, Cho SY, Roboz J, Redinbo M, Moore JT, Mani S (2008) The
phytoestrogen coumestrol is a naturally occurring antagonist of the human
pregnane X receptor. Mol Endocrinol 22:838-857
Wang H, Tompkins LM (2008) CYP2B6: new insights into a historically overlooked
cytochrome P450 isozyme. Curr Drug Metab 9:598-610
Wang H, Venkatesh M, Li H, Goetz R, Mukherjee S, Biswas A, Zhu L, Kaubisch A,
Wang L, Pullman J, Whitney K, Kuro-O M, Roig AI, Shay JW, Mohammadi M,
Mani S (2011) Pregnane X receptor activation induces FGF19-dependent tumor
aggressiveness in humans and mice. J Clin Invest 121:3220-3232
Watkins RE, Maglich JM, Moore LB, Wisely GB, Noble SM, Davis-Searles PR,
Lambert MH, Kliewer SA, Redinbo MR (2003) A crystal structure of human
PXR in complex with the St. John’s wort compound hyperforin. Biochemistry
42:1430-1438
Xiao D, Srivastava SK, Lew KL, Zeng Y, Hershberger P, Johnson CS, Trump DL,
Singh SV (2003) Allyl isothiocyanate, a constituent of cruciferous vegetables,
inhibits proliferation of human prostate cancer cells by causing G2/M arrest and
inducing apoptosis. Carcinogenesis 24:891-897
Xie W, Barwick JL, Simon CM, Pierce AM, Safe S, Blumberg B, Guzelian PS, Evans
RM (2000) Reciprocal activation of xenobiotic response genes by nuclear
receptors SXR/PXR and CAR. Genes Dev 14:3014-3023
Yun CH, Okerholm RA, Guengerich FP (1993) Oxidation of the antihistaminic drug
terfenadine in human liver microsomes. Role of cytochrome P-450 3A(4) in
N-dealkylation and C-hydroxylation. Drug Metab Dispos 21:403-409
Zambon S, Fontana S, Kajbaf M (2010) Evaluation of cytochrome P450 inhibition
assays using human liver microsomes by a cassette analysis LC-MS/MS. Drug
Metab Lett 4:120-128
Zhang J, Huang W, Chua SS, Wei P, Moore DD (2002) Modulation of
acetaminophen-induced hepatotoxicity by the xenobiotic receptor CAR. Science
298:422-424
Zhang Y (2000) Role of glutathione in the accumulation of anticarcinogen
icisothiocyanates and their glutathione conjugates by murine hepatoma cells.
Carcinogenesis 21:1175-1182
Zhang Y (2010) Allyl isothiocyanate as a cancer chemopreventive phytochemical.
Mol Nutr Food Res 54:127-135
Zhang Y, Talalay P (1998) Mechanism of differential potencies of isothiocyanates as
inducers of anticarcinogenic Phase 2 enzymes. Cancer Res 58:4632-4639
Zhou C, Poulton EJ, Grun F, Bammler TK, Blumberg B, Thummel KE, Eaton DL
(2007) The dietary isothiocyanate sulforaphane is an antagonist of the human
steroid and xenobiotic nuclear receptor. Mol Pharmacol 71:220-229
Figure Legends
Figure 1. Viability of HepG2 and LS174T cells following exposure to AITC with or
without rifampin.
HepG2 and LS174T cells were exposed to AITC (10–40 µM), rifampin (20 µM), or
both rifampin (20 M) and AITC (10–40 µM) for 24 h. Cell viability was monitored
by cellular acid phosphatase activity using PNPP as a substrate. The data shown are
the mean  SE (error bars) from at least triplicates.
Figure 2. Transient transcription assays to determine the effects of AITC and PXR
ligand-mediated activation on a CYP3A4 reporter in HepG2 or LS174T cells. (A, C)
HepG2 and (B) LS174T cells were co-transfected with a vector control (pcDNA3) or
a PXR expression plasmid (pcDNA3-PXR), or (D) a rat PXR expression plasmid
(pcDNA3-rPXR),
a
CYP3A4
luciferase
reporter
plasmid,
and
a
pRC-CMV--galactosidase vector. The transfected cells were then exposed to AITC
with or without (A, B) rifampin, or (C) SR12813 or nifedipine for 24 h. Luciferase
activity was measured and normalized to -galactosidase activity. The data shown are
the mean  SE (error bars) of at least triplicates. #/*, p < 0.05; ##/**, p < 0.01; ###/***,
p < 0.001, vs. DMSO/rifampin/SR12813/nifedipine-treated cells as indicated.
Figure 3. Effect of AITC on CYP3A4 mRNA and protein expression.
(A) HepG2 cells were treated with AITC and rifampin, either individually or in
combination, for 24 h, total mRNA was collected, and the expression of CYP3A4 and
GAPDH (an internal control) were analyzed using real time-PCR. Values were
normalized to the expression of GAPDH, and CYP3A4 expression in DMSO-treated
cells was set at 1. The data shown are the mean  SE (error bars) of triplicates. *, p <
0.05 vs. 20 M rifampin-treated cells,
###
, p< 0.001 vs. DMSO-treated cells, as
indicated in the figure. (B) HepG2 cells were treated with AITC and rifampin, either
individually or in combination, for 24 h. Whole cell extracts were harvested, and the
expression of CYP3A4 and the internal control -actin were analyzed using western
blotting. (C) Quantitation of the CYP3A4 protein bands was normalized to -actin.
Basal expression of the CYP3A4 protein was set at 1. The data shown are the mean 
SE (error bars) of triplicates. #/*, p < 0.05; ##/**, p< 0.01, vs. DMSO/rifampin-treated
cells as indicated.
Figure 4. Transactivation of CYP3A4, CYP2B6, and cyp2b10 promoter activity by
human and mouse CAR in the presence of AITC with or without specific ligands.
HepG2 cells were co-transfected with a vector control and (A) a human CAR3
expression plasmid (pCR3-hCAR) and a CYP3A4 reporter plasmid; (B) pCR3-hCAR
and CYP2B6 reporter plasmids; and (C) a mouse CAR expression plasmid
(pCMX-mCAR), a cyp2b10 reporter plasmid, and a pRC-CMV--galactosidase vector.
The transfected cells were then exposed to AITC and/or to the human or mouse
CAR-specific ligands (CITCO or TCPOBOP, respectively) for 24 h. Then, luciferase
activity was measured. The data shown are the mean  SE (error bars) of at least
triplicates. *, p < 0.05; **, p< 0.01; ***, p < 0.001, vs. the ligand-treated cells.
Figure 5. Effect of AITC with or without CITCO on CYP2B6 mRNA and protein
expression.
(A) HepG2 cells were treated with AITC and CITCO, either individually or in
combination, for 24 h, total mRNA was collected, and the expression of CYP2B6 and
GAPDH (an internal control) were analyzed using real time-PCR. CYP2B6 expression
was normalized to GAPDH, and the CYP2B6 expression in DMSO-treated cells was
set at 1. The data shown are the mean  SE (error bars) of triplicates. #/*, p < 0.05;
##
/**, p< 0.01; ***, p< 0.001 vs. the DMSO/CITCO-treated groups as indicated in the
figure. (B) HepG2 cells were treated with AITC and CITCO, either individually or in
combination, for 24 h. Whole cell extracts were harvested, and the expression of
CYP2B6 and the internal control -actin were analyzed using Western blotting. (C)
CYP2B6 protein was normalized to -actin. Basal expression of CYP2B6 protein was
set at 1. The data shown are the mean  SE (error bars) of triplicates. #/*, p < 0.05;
##
/**, p< 0.01, vs. DMSO/CITCO-treated cells as indicated.
Figure 6. Reduction of CYP3A4 and CYP2B6 catalytic activity by AITC.
Differentiated HepaRG cells were pretreated with AITC alone or in combination with
(A) rifampin or (B) CITCO for 72 h. For the final 24 h of this incubation, the cells
were treated with (A) 3 M midazolam and (B) 200 M bupropion HCl. Total cell
lysate was isolated, and the metabolites were extracted with acetone. The metabolites
of midazolam and bupropion HCl, 1‘-hydroxymidazolam and -hydroxybupropion,
respectively, were determined by LC-MS/MS as described in the “Materials and
Methods.” The data shown are the mean  SE (error bars) of triplicates. *, p < 0.05;
***, p< 0.001, vs. rifampin/CITCO-treated cells as indicated.
Figure 7. Co-regulation of RXR, SRC-1, HNF4, and PGC-1 with PXR is
disrupted by AITC in CYP3A4 reporter assays.
HepG2 cells were co-transfected with a vector control (pcDNA3) or a PXR
expression plasmid (pcDNA3-PXR) along with (A) a RXR expression plasmid
(pGEM-3Z-RXR); (B) a SRC-1 expression plasmid (pCR3.1-SRC-1); (C) a HNF4
expression plasmid (pcDNA3-HNF4); or (D) a PGC-1 expression plasmid
(pTARGET-PGC-1) in combination with the CYP3A4 luciferase reporter plasmid
and the pRC-CMV--galactosidase vector. Then, the transfected cells were exposed to
AITC and/or rifampin for 24 h. Luciferase activity was measured and normalized to
-galactosidase activity. The data shown are the mean  SE (error bars) of at least
triplicates. *, p< 0.05; ***, p < 0.001, vs. DMSO/rifampin-treated cells as indicated.
Figure 8. Decreased cytotoxicity of rifampin-induced APAP and amiodarone in the
presence of AITC in HepaRG cells.
Differentiated HepaRG cells were pretreated with rifampin, either alone or in
combination with AITC, for 72 h, and for the last 24 h of this treatment, the cells were
also treated with APAP or amiodarone. Cell viability was monitored by assessing
cellular acid phosphatase activity using PNPP as a substrate. The data shown are the
mean  SE (error bars) of at least triplicates. **, p < 0.01; ***, p< 0.001, vs.
DMSO-treated cells.
Supplemental data
Table S1. Effect of AITC,either alone or in combination with rifampin, on PXR,
RXR, HNF4, CAR, SRC-1, and GR mRNA expression.
HepG2 cells were treated with AITC and rifampin, either individually or in
combination, for 24 h, total mRNA was collected, and the expression of the indicated
genes as well as an internal control (GAPDH) were analyzed using real time-PCR.
Experimental values were normalized relative to the expression of GAPDH, and
expression in DMSO-treated cells was set at 1. The data shown are the mean  SE
(error bars) of triplicates.
Figure S1. Viability of COS-1 cells following exposure to AITC alone and in
combination with rifampin, and CYP3A4 reporter transient transcription assays in
COS-1 cells.
(A) COS-1 cells were exposed to AITC alone (20 and 40 µM), or in combination with
rifampin (20 µM), for 24 h. Cell viability was monitored as cellular acid phosphatase
activity using PNPP as a substrate. The data shown are the mean  SE (error bars) of
at least triplicates. (B) COS-1 cells were co-transfected with a vector control
(pcDNA3) or a PXR expression plasmid (pcDNA3-PXR), a CYP3A4 luciferase
reporter plasmid, and a pRC-CMV--galactosidase vector. Then, the transfected cells
were exposed to AITC and/or rifampin for 24 h. Luciferase activity was measured and
normalized to -galactosidase activity. The data shown are the mean  SE (error bars)
of at least triplicates. **, p< 0.01; ***, p < 0.001, vs. rifampin-treated cells.