0090-9556/05/3307-956962$20.

00
DRUG METABOLISM AND DISPOSITION
Copyright 2005 by The American Society for Pharmacology and Experimental Therapeutics
DMD 33:956962, 2005

Vol. 33, No. 7

3798/3038069
Printed in U.S.A.

INDUCTION OF THE MULTIDRUG RESISTANCE-ASSOCIATED PROTEIN FAMILY OF

TRANSPORTERS BY CHEMICAL ACTIVATORS OF RECEPTOR-MEDIATED PATHWAYS IN
MOUSE LIVER
Jonathan M. Maher, Xingguo Cheng, Angela L. Slitt, Matthew Z. Dieter, and Curtis D. Klaassen
Department of Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical Center, Kansas City, Kansas
Received January 24, 2005; accepted April 6, 2005

ABSTRACT:
[2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), polychlorinated biphenyl 126 (PCB126), and -naphthoflavone] induced Mrp2, -3, -5,
and -6 mRNA expression. The CAR activator 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) induced Mrp2, -3, -4, -6, and -7
mRNA expression. Mrp3 was also induced by two other CAR activators phenobarbital and diallyl sulfide, two PXR ligands, pregnenalone-16-carbonitrile and spironolactone, and the PPAR ligands clofibrate, ciprofibrate, and diethylhexylphthalate. The Nrf2
activators (butylated hydroxyanisole, oltipraz, and ethoxyquin) induced Mrp26. In conclusion, a variety of mechanisms are suggested for Mrp3 induction, including AhR, CAR, PXR, PPAR, and
Nrf2, whereas on a whole, a predominant role for AhR and Nrf2 in
hepatic induction of the Mrp family was observed. Thus, these
specific transcription factors are implicated in regulation of both
drug metabolism and efflux transport.

Multidrug resistance-associated proteins (Mrps) are ATP-dependent transporters known to transport a diverse set of substrates. Mrps
have an affinity for conjugated compounds, including glutathione-,
glucuronide- and sulfate-conjugated metabolites. Mrps as a group are
known to efflux these conjugated substrates from cells, including the
transport of chemicals such as bilirubin, 2,4-dinitrophenol, 17-estradiol, taurocholate, and protease inhibitors, such as ritonavir, with high
affinity (Jedlitschky et al., 1997; Hirohashi et al., 1999; Keppler,
1999; Huisman et al., 2002). Four Mrp transporters (Mrp2, -3, -4, and
-6) are expressed to an appreciable extent in liver (Maher et al., 2005).
In liver, Mrp2 is the only Mrp localized to the canalicular membrane
and participates in excretion of chemicals into bile. Alternatively,
Mrp3 and Mrp4 are localized to the basolateral membrane and efflux
chemicals from hepatocytes into blood. Mrp6 is thought to be local-

ized to the basolateral membrane as well, but a high-affinity substrate

for this transporter has not been identified (Madon et al., 2000). The
Mrps play an important role in the hepatic elimination of metabolites,
and modulation of Mrp expression in liver can alter drug disposition
(Xiong et al., 2002a; Slitt et al., 2003).
Several transcription factors have an important role in modulating
the basal and inducible expression of genes involved in phase I drug
metabolism. One of the best understood mechanisms of gene induction in liver is the marked induction of Cyp1A1 by dioxin-like
compounds, which is mediated through the aryl hydrocarbon receptor
(AhR) (Jaiswal et al., 1985). The AhR is known to have a physiological role, although the endogenous ligand has been elusive (Hahn,
2002). Studies in AhR-null mice have revealed that AhR is important
in immune function and in hepatic development, and loss of AhR
leads to nonresponsiveness to dioxins (Fernandez-Salguero et al.,
1995; Schmidt et al., 1996). Furthermore, loss of the AhR protects
against benzo-a-pyrene-induced tumors (Shimizu et al., 2000).
The pregnane X receptor (PXR) and the constitutive androstane
receptor (CAR) are believed to be xenosensors that can cooperate to
protect the liver from hepatotoxicity by up-regulating the expression

This work was supported by the National Institutes of Health Grants ES-09716
and ES-07079.
Article, publication date, and citation information can be found at
http://dmd.aspetjournals.org.
doi:10.1124/dmd.105.003798.

ABBREVIATIONS: Mrp, multidrug resistance-associated protein; AhR, aryl hydrocarbon receptor; PXR, pregnane X receptor; CAR, constitutive
androstane receptor; PPAR, peroxisome proliferator-activated receptor ; Nrf2, nuclear factor-E2-related factor 2; BHA, butylated hydroxyanisole; Nqo1, NAD(P)H:quinone oxidoreductase 1; PCB126, polychlorinated biphenyl 126; bDNA, branched DNA; BNF, -napthoflavone; TCDD,
2,3,7,8-tetrachlorodibenzo-p-dioxin; PB, phenobarbital; TCPOBOP, 1,4-bis[2,5-dichloropyridyloxy)]benzene; DAS, diallylsulfide; PCN, pregnenalone-16-carbonitrile; SPR, spironolactone; DEX, dexamethasone; CLOF, clofibrate; CPFP, ciprofibrate; DEHP, di(2-ethylhexyl)phthalate; EXQ,
ethoxyquin; PMEA, 9-(2-phosphonylmethoxyethyl)adenine; BQ-123, cyclo(L-Leu-D-Trp-D-Asp-L-Pro-D-Val); RLU, relative light unit.
956

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The multidrug resistance-associated proteins (Mrp) are ATP-dependent transporters that export a variety of conjugated and unconjugated compounds out of cells. There are nine identified Mrp
transporters in humans, with murine orthologs for all except Mrp8.
Because nuclear receptors mediate induction of phase I enzymes,
Mrp transporter expression might be similarly regulated by these
receptors to coordinate metabolism and export of chemicals from
liver. To test the hypothesis that Mrp expression may be coordinately regulated with phase I enzyme expression in liver, 15 different compounds were given representing known transcriptionally
mediated pathways: aryl hydrocarbon receptor (AhR), pregnane X
receptor (PXR), constitutive androstane receptor (CAR), peroxisome proliferator-activated receptor (PPAR), and nuclear factor-E2-related factor 2 (Nrf2). Each of these compounds induced
expression of their respective target enzyme in liver, demonstrating that the chemical regimens were effective. The AhR ligands

957

INDUCTION OF Mrps IN MOUSE LIVER

Materials and Methods

Chemicals. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) was a gift from
Dr. Karl Rozman (University of Kansas Medical Center, Kansas City, KS).
Oltipraz was a gift from Dr. Steven Safe (Texas A&M University, College
Station, TX). Polychlorinated biphenyl 126 (PCB126) was obtained from
AccuStandard (New Haven, CT). All other chemicals, unless otherwise indicated, were purchased from Sigma-Aldrich Co. (St. Louis, MO).
Animals. Male C57BL/6 mice were purchased from Charles River Laboratories (Wilmington, MA) and were exposed to microsomal enzyme inducers
at approximately 8 weeks of age (n 5/treatment). The chemicals, dosing
regimen, vehicle, and corresponding control for the treatment are included in
Table 1. After dosing each day for 4 days with the microsomal enzyme
inducers, livers were collected on the 5th day (96 h after the first dose). Tissues
were snap-frozen in liquid nitrogen and stored at 80C.

TABLE 1
List of chemicals, dosing regimen, known receptor activated,
and the target gene of activation
Compound

Dosing Regimen

Receptor

Marker Gene

TCDD
PCB126
BNF

40 g/kg CO i.p.
300 g/kg CO p.o.
200 mg/kg CO i.p.

AhR

Cyp1A1

PB
TCPOBOP
DAS

100 mg/kg SAL i.p.

3 mg/kg CO i.p.
200 mg/kg CO i.p.

CAR

Cyp2B10

PCN
SPR
DEX

200 mg/kg CO i.p.

200 mg/kg CO i.p.
75 mg/kg CO i.p.

PXR

Cyp3A11

CLOF
CPFP
DEHP

500 mg/kg SAL i.p.

40 mg/kg SAL i.p.
1000 mg/kg CO p.o.

PPAR

Cyp4A14

BHA
OTZ
EXQ

350 mg/kg CO i.p.

150 mg/kg CO p.o.
250 mg/kg CO p.o.

Nrf2

Nqo1

CO, corn oil; SAL, saline.

RNA Extraction. Total RNA was isolated using the RNA Bee reagent
(Tel-test, Friendswood, TX) according to the manufacturers instructions.
RNA concentrations were determined spectrophotometrically, and the quality
of the RNA was determined by gel electrophoresis.
Development of Specific Oligonucleotide Probe Sets for bDNA Analysis. The Mrp gene sequences were accessed from GenBank. The target sequences were analyzed by ProbeDesigner Software Version 1.0. (Genospectra,
Fremont, CA). The oligonucleotide probes were specific for only one mRNA
transcript. All oligonucleotide probes were designed with a melting temperature of approximately 63C, enabling optimal hybridization conditions. Each
probe set was submitted to the National Center for Biotechnology Information
(NCBI) for nucleotide comparison by the basic local alignment search tool
(BLASTn) to ensure minimal cross-reactivity with other mouse sequences and
expressed sequence tags (Maher et al., 2005).
Branched DNA Assay. The specific Mrp oligonucleotide probes were
diluted in Tris-EDTA buffer according to instructions provided by the QuantiGene bDNA Signal Amplification Kit (Genospectra). Total RNA (1 g/l;
10 l) was added to each well of a 96-well plate containing 50 l of capture
hybridization buffer and 50 l of each diluted probe set. Total RNA was
allowed to hybridize overnight at 53C in a hybridization oven. Subsequent
hybridization steps were carried out according to the manufacturers protocol,
and luminescence was measured with a Quantiplex 320 bDNA luminometer
interfaced with Quantiplex Data Management Software Version 5.02 for
analysis of luminescence from 96-well plates.
Statistical Analysis. Data were analyzed by one-way analysis of variance,
followed by Duncans multiple range post hoc test. Bars represent standard
error of the mean. Asterisks (*) represent statistical differences ( p 0.05) in
mRNA levels between the control and treated groups.

Results
Validation of Chemical Treatment Regimen by Phase I Inducers. The microsomal enzyme inducers utilized in this experiment can
be divided into five distinct mechanisms of activation, those being
AhR, CAR, PXR, PPAR, and Nrf2 activators. The chemicals, dosages, and marker gene demonstrating activation of the respective
receptor have been summarized in Table 1. The results of the corresponding treatments on Cyp1a1, 2b10, 3a11, 4a14, and Nqo1 in
mouse liver are illustrated in Fig. 1. Cyp1A1 mRNA expression was
increased by treatment with the Ah ligands TCDD (1230-fold), BNF
(1230-fold), and PCB126 (1170-fold). Cyp2B10 was induced by CAR
activators PB (15-fold), TCPOBOP (150-fold), and DAS (22-fold).
Cyp3A11 was induced by PXR ligands PCN (5-fold), SPR (5-fold),
and DEX (5-fold). The mRNA levels of Cyp4A14 were induced by
PPAR ligands CLOF (22-fold), CPFP (93-fold), and DEHP (87-

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of Cyp3a and Cyp2b, which are important drug metabolizing enzymes

(Baes et al., 1994; Honkakoski et al., 1998; Lehmann et al., 1998;
Staudinger et al., 2001). PXR is activated by a diverse number of
compounds, including rifampicin, phenobarbital, and mifepristone
(RU486) in humans (Kliewer et al., 1998). The dually named constitutively active receptor/constitutive androstane receptor or (CAR) is
unique because, as the names suggest, it does not need to have bound
ligand to be active, and its activity is repressed by androstenol (Wei
et al., 2000).
The peroxisome proliferator-activated receptor (PPAR) is
known to mediate transcription of numerous genes, including those
encoding peroxisomal and mitochondrial -oxidation enzymes, the
CYP4A family, and fatty acid-binding proteins (Keller et al., 1993). A
class of compounds that include the fibrate class of triglyceride- and
cholesterol-lowering drugs has been shown to bind to PPAR and
alter transcription and metabolism of other xenobiotics (Lee et al.,
1995). PPAR-null mouse livers did not demonstrate the peroxisomal
proliferation or the neoplasms that are typically present in rodents
after long-term treatment with fibrate drugs (Peters et al., 1997).
Nuclear factor-E2-related factor 2 (Nrf2) has been identified as a
transcription factor that is activated by microsomal enzyme inducers
such as butylated hydroxyanisole, oltipraz, and ethoxyquin. Nrf2
heterodimerizes with a member of the small musculoaponeurotic
fibrosarcoma oncogene family of proteins to bind cognate electrophile
response elements in DNA (Motohashi et al., 1998, 2002). Nrf2 is
responsible for both constitutive and inducible expression of electrophile response element-regulated genes. For example, the induction of
glutathione S-transferase by butylated hydroxyanisole (BHA) was
abolished in Nrf2-null mice (Chanas et al., 2002). Furthermore, oltipraz-induced induction of phase I and II enzymes, such as NAD(P)H:
quinone oxidoreductase 1 (Nqo1) and glutathione S-transferase was
completely abolished, and the chemopreventive effect of oltipraz was
completely lost in Nrf2-null mice (Ramos-Gomez et al., 2001). Thus,
Nrf2 plays a central role in regulating constitutive and inducible
expression of some phase I and II enzymes, and the chemoprotective
effects of chemicals such as oltipraz are directly due to transcriptional
mediation of the antioxidant battery of genes regulated via Nrf2.
Whereas the mechanisms up-regulating certain phase I drugmetabolizing enzymes have been examined in detail, the regulation
of efflux transporters is poorly understood, particularly in mice. To
determine potential mechanisms for transcriptional regulation of
Mrps, 15 compounds that are known activators of nuclear and other
transcription factor-mediated pathways were administered to mice.
Thus, the aim of this study is to quantitatively determine the expression of mouse Mrps after treatment with structurally distinct types of
chemicals to determine whether potential coordinate induction of
phase I and II metabolism and efflux transporters exists in livers of
mice.

958

MAHER ET AL.

fold). Nqo1 was induced by Nrf2 activators BHA (3-fold), EXQ

(5-fold), and OPZ (2-fold).
Hepatic Expression of Mrp1, -2, and -3 mRNA in Mice Treated
with Microsomal Enzyme Inducers. Hepatic mRNA expression
levels of Mrp1 are low in mouse liver and were not significantly
induced by any of the treatments (Fig. 2). However, Mrp1 expression
in liver was repressed by CPFP (80% decrease), DEHP (80% decrease), and BHA (71% decrease) administration.
Mrp2 mRNA expression in mouse liver was significantly increased
by a variety of compounds, including the three AhR ligands TCDD
(114%), PCB126 (130%), and BNF (134%); the CAR ligand
TCPOBOP (294%); and all three Nrf2 activators BHA (150%), OPZ
(119%), and EXQ (129%) (Fig. 2). In previous studies, TCPOBOP
also induced Mrp2 in mice in vivo, correlating well with the present
data (Guo et al., 2003).
All classes of nuclear receptor activators induced the expression of
Mrp3. This includes AhR ligands TCDD (229%), PCB126 (315%),
BNF (257%); the CAR activators PB (98%), TCPOBOP (512%), and
DAS (82%); the PXR ligands PCN (91%) and SPR (98%); the
PPAR ligands CLOF (193%), CPFB (200%), and DEHP (146%); as
well as the Nrf2 activators BHA (224%), OPZ (233%), and EXQ
(367%) (Fig. 2). These studies correlate well with previous work
showing up-regulation of mouse Mrp3 by TCPOBOP and PCN
(Staudinger et al., 2003).
Hepatic Expression of Mrp4, -5, and -6 mRNA in Mice Treated
with Microsomal Enzyme Inducers. Only a few compounds induced
Mrp4 (Fig. 3). The CAR activator TCPOBOP induced Mrp4 in liver
338%, and all three Nrf2 activators BHA (133%), OPZ (382%), and
EXQ (594%) induced expression of Mrp4. These data correlate with
up-regulation of Mrp4 by TCPOBOP by others (Assem et al., 2004).
Mrp5 expression is also low in liver, but was inducible. The three

AhR ligands TCDD (641%), PCB126 (515%), and BNF (611%); two
of three CAR ligands TCPOBOP (450%) and DAS (184%); and all
three Nrf2 activators BHA (413%), OPZ (361%), and EXQ (349%)
induced expression of Mrp5 (Fig. 3).
Mrp6, which is highly expressed in liver, was also induced by a
similar subset of compounds. The three AhR ligands TCDD (226%),
PCB126 (290%), and BNF (248%); two of three CAR ligands
TCPOBOP (186%), and PB (105%); and all three of the Nrf2 activators OPZ (239%), EXQ (180%), and BHA (171%) induced Mrp6
expression (Fig. 3).
Hepatic Expression of Mrp7 and -9 mRNA in Mice Treated
with Microsomal Enzyme Inducers. Mrp7 expression is low in liver,
yet was induced by several chemical activators, including all three
AhR ligands TCDD (170%), PCB126 (183%), and BNF (201%); all
three CAR activators PB (224%), TCPOBOP (230%), and DAS
(265%); and one of three Nrf2 activators, BHA (188%) (Fig. 4). Mrp9
expression is extremely low in liver, but was repressed by EXQ
(99.9%), CPFP (99.9%), DEHP (99.8%) (Fig. 4).
Discussion
The purpose of this study was to determine whether Mrps, like
phase I and II enzymes, are induced in mouse liver following administration of microsomal enzyme inducers. Five different types of
microsomal enzyme inducers with known mechanisms of induction
were used to identify potential transcriptional mechanisms that regulate the expression of various Mrps in mouse liver. In this study, all of
the compounds used induced their cognate phase I enzyme, indicating
that the dosing regimen used was efficacious.
Data from this study demonstrate that Mrp expression is inducible
in mouse liver by microsomal enzyme inducers and suggest that

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FIG. 1. mRNA expression of Cyp1A1, Cyp2B10, Cyp3A1/23, Cyp4A14, and NAD(P)H quinone oxidoreductase (Nqo) 1 in C57BL/6 mouse liver after administration of
prototypical phase I and phase II drug-metabolizing enzyme inducers. The abbreviations used are listed in the Abbreviation footnote. Total RNA from five chemically
treated male livers was analyzed by the bDNA assay. Data are presented as mean relative light units (RLUs) S.E.M. , statistically significant difference between treated
and control mice (p 0.05).

INDUCTION OF Mrps IN MOUSE LIVER

959

nuclear receptors regulate Mrp expression. However, the magnitude

of induction of Mrps in mouse liver is not as marked as the upregulation of some cytochromes P450 by administration of microsomal enzyme inducers. Also, some transporters such as Mrp2 and Mrp3
are more highly expressed in mouse liver, and therefore, a two-fold
increase in expression may translate into higher effective levels of
efflux transport. Mrp1, -5, -7, and -9 are not expressed to an appreciable extent at the mRNA level in mouse liver; nevertheless, Mrp4,
whose mRNA is also only minimally expressed, has been detected at
the protein level in liver, with localization to the basolateral membrane (Rius et al., 2003; Assem et al., 2004). It is important to note
that the expression of many P450s, such as Cyp1a1 and Cyp2b10, is
similarly quite low in uninduced liver, but is up-regulated hundredsfold after chemical treatment. Similar induction of these poorly expressed Mrps in liver is not marked and may not correlate to functional changes in hepatic transport. Therefore, the discussion will
be limited to the transporters in liver with known expression and
function.
Mrp2 is a canalicular Mrp transporter known to transport chemicals
into bile (Jansen et al., 1993). Mrp2 has been shown to transport a vast
array of organic anions, including monobilirubin glucuronide and
2,4-dinitrophenyl S-glutathione (Jedlitschky et al., 1997; Keppler,

1999). In the present study, AhR ligands, TCPOBOP, and Nrf2

activators increased Mrp2 expression in mouse liver, suggesting that
AhR, CAR, and Nrf2 may be important for modulating Mrp2 expression by chemicals.
Whereas few studies have examined the regulation of mouse Mrp2,
more is known regarding the regulation of rat and human Mrp2.
Induction of rat Mrp2 has been observed with numerous chemicals,
such as PCN, SPR, and DEX (PXR ligands), PB (CAR ligand), and
OTZ (Nrf2 activator) (Courtois et al., 1999; Johnson and Klaassen,
2002). Similar induction of Mrp2 with indole-3-carbinol and -naphthoflavone, both AhR ligands, has also been observed in rat liver
(Cherrington et al., 2002). Treatment with the chemical carcinogen
2-acetylaminofluorene, the antineoplastic drug cisplatin, and the protein-synthesis inhibitor cycloheximide increased expression of Mrp2
in rat liver (Kauffmann et al., 1997), although the mechanism(s) are
not understood. Human Mrp2 is similarly up-regulated by the PXR
activators rifampicin and tamoxifen, which differ from known rodent
ligands for PXR (Jones et al., 2000). Similarly, Mrp2 is induced by PB
(Schrenk et al., 2001) and by tert-butyl hydroquinone in HepG2 cells
(Kauffmann et al., 2002), which suggests that CAR and Nrf2, respectively, may regulate expression of the human Mrp2 gene. Taken

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FIG. 2. Expression of Mrp1 (above), Mrp2 (middle), and Mrp3 (bottom) mRNA in liver after treatment with prototypical drug-metabolizing enzyme inducers. The
abbreviations used are the same as in Fig. 1. Total RNA from liver of chemically treated male C57BL/6 mice (n 5/treatment) was analyzed by the bDNA assay. Data
are presented as mean RLUs S.E.M. , statistically significant difference between treated and control mice (p 0.05).

960

MAHER ET AL.

together, these experiments suggest that the gene for Mrp2 may be
similarly up-regulated by PXR agonists in human and rat, but mouse
Mrp2 may not be as sensitive to PXR ligands.
Mrp3 is a basolateral efflux transporter that is up-regulated during
cholestatic liver conditions and may serve to protect the liver from
bile acid toxicity during cholestasis by transporting bile acids from
liver into blood (Soroka et al., 2001). Similarly, Mrp3 can transport a
host of conjugated xenobiotics out of the liver. Data from the present
study demonstrate that Mrp3 mRNA expression is induced by AhR,
CAR, PXR, PPAR, and Nrf2 activators. Previous studies have
shown up-regulation of Mrp3 in rodent by PCN, which has been
shown to be dependent on PXR, as well as up-regulation of Mrp3 by
TCPOBOP and PB (Maglich et al., 2002; Xiong et al., 2002b; Cherrington et al., 2003; Staudinger et al., 2003). The regulation of Mrp3
by TCPOBOP has been described as CAR-dependent (Maglich et al.,
2002), whereas induction by PB is CAR-independent (Xiong et al.,
2002b; Cherrington et al., 2003). This could be due to differences in
activation of CAR, whether by liganded activation as for TCPOBOP,
or potentially by cell signaling, leading to activation of CAR (Swales
and Negishi, 2004). Induction of mouse Mrp3 by AhR, PPAR, and
Nrf2 activators has not been previously described; therefore, these

may represent new, novel mechanisms of regulation of Mrp3 in mouse

liver.
In rats, mice, and humans, Mrp3 has been shown to be regulated by
PB, DAS, and polychlorinated biphenyl 99 (PCB99), compounds that
induce Cyp2B1/2 and are known or hypothesized CAR activators
(Cherrington et al., 2002). Similar to Mrp2, Mrp3 is highly upregulated by OTZ, suggesting that Nrf2 might be an important transcription factor that regulates Mrp3 (Cherrington et al., 2002). In
humans, induction by -naphthoflavone and rifampicin suggests that
Mrp3 might be regulated via AhR or PXR, respectively (Schrenk et
al., 2001; Hitzl et al., 2003). Overall, Mrp3 seems to be regulated
similarly in rats, mice, and humans, with potential transcriptional
regulation by AhR, PXR, CAR, PPAR, and Nrf2.
Mrp4 is localized to the basolateral membrane and can transport
chemicals from hepatocytes into blood (Denk et al., 2004). Much like
Mrp3, Mrp4 can transport bile acids, as well as many antiviral drugs
such as PMEA (Reid et al., 2003; Zelcer et al., 2003). Data from the
present study demonstrate that TCPOBOP and Nrf2 activators induce
Mrp4 in mouse liver, indicating potential roles for CAR and Nrf2 in
the regulation of mouse Mrp4. In rats, Mrp4 is induced in liver by the
Nrf2 activators EXQ and OTZ (Chen and Klaassen, 2004). Little data

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FIG. 3. Expression of Mrp4 (above), Mrp5 (middle), and Mrp6 (bottom) mRNA in liver after treatment with prototypical drug-metabolizing enzyme inducers. The
abbreviations used are the same as in Fig. 1. Total RNA from liver of chemically treated male C57BL/6 mice (n 5/treatment) was analyzed by the bDNA assay. Data
are presented as mean RLUs S.E.M. , statistically significant difference between treated and control mice (p 0.05).

961

INDUCTION OF Mrps IN MOUSE LIVER

exists on induction of Mrp4 in humans; however, studies in CAR-null

mice have definitively shown that induction of Mrp4 by TCPOBOP
and PB is via CAR (Assem et al., 2004). Taken together, the most
likely means of induction of Mrp4 is by transcriptional activation by
CAR and Nrf2.
Mrp6 is thought to be localized on the basolateral membrane in
hepatocytes, but may have canalicular localization as well (Madon et
al., 2000). The only substrate that has been found to be transported
with relatively high affinity by Mrp6 is the endothelin antagonist
BQ-123; however, overexpression of Mrp6 does lead to weak resistance to chemotherapeutic drugs (Belinsky et al., 2002). Few chemicals have been observed to modulate expression of Mrp6 in rats or
humans, and a battery of inducers similar to those used in this study
showed no changes in Mrp6 expression in rat (data not shown).
However, in this study, AhR, CAR, and Nrf2 activators induced
expression of Mrp6 in mouse liver.
One of the patterns of Mrp expression of note is that AhR and Nrf2
activators often induce the same transporter (i.e., Mrp2, -3, -5, and -6).
Several genes known to be regulated by Nrf2 are also regulated in a
similar manner compared with these Mrps. Rat UDP-glucuronosyltransferase 1A6 is induced by oltipraz, a classical Nrf2 activator, and
oltipraz induction of UDP-glucuronosyltransferase 1A6 is dependent
on the binding of AhR to the xenobiotic response element (Auyeung
et al., 2003). Furthermore, one of the known target genes of Nrf2
activation, Nqo1, can be induced by the classical AhR ligand TCDD,
and that induction was Nrf2-dependent (Ma et al., 2004). Although the
mechanism of this cross-activation is not well defined, Mrps may
share a similar pattern of inducibility to the phase I and II enzymes
known to be regulated by these two receptors. Thus, it is unclear
whether the induction of Mrps by some of the microsomal enzyme
inducers is mediated through direct mechanisms (transcription factor

TABLE 2
The number of receptor-activating chemicals in a class that alter mRNA
expression of Mrps after treatment

Mrp1
Mrp2
Mrp3
Mrp4
Mrp5
Mrp6
Mrp7
Mrp9

AhR

PXR

CAR

1(3/3)
1(3/3)

2(2/3)
1(2/3)

1(1/3)
1(3/3)
1(1/3)
1(2/3)
1(2/3)
1(3/3)

1(3/3)
1(3/3)
1(3/3)
2(1/3)

PPAR

NRF2

2(2/3)

2(1/3)
1(2/3)
1(3/3)
1(3/3)
1(3/3)
1(3/3)
1(1/3)
2(1/3)

1(3/3)

2(2/3)

binding to its cognate response element) or indirect mechanisms that

involve some sort of cross talk (activation of multiple receptors by
a chemical and/or transcriptional up-regulation of another gene or
transcription factor that acts on the gene of interest).
In conclusion, these results demonstrate Mrp2, -3, -5, -6, and -7 are
induced by AhR ligands (Table 2). The potent and efficacious CAR
activator TCPOBOP induces mRNA expression of Mrp27, although
only Mrp3 was induced by all three CAR activators administered
(Table 2). PXR ligands PCN and SPR induced mRNA expression of
Mrp3 in mouse liver. Mrp3 was induced by all three PPAR agonists
administered, whereas the Nrf2 activators induced expression of
Mrp2 6. In conclusion, the data demonstrate that a variety of microsomal enzyme inducers increase expression of Mrps in mouse liver
and suggest that coordinate regulation mediated by AhR, CAR, PXR,
PPAR, and Nrf2 may exist for genes involved in drug metabolism
and disposition.
Acknowledgments. We thank Mindy Shelby, David Buckley, and
Drs. Susan Buist, Tyra Leazer, Jim Brady, Hong Lu, and Chuan Chen
for technical expertise in tissue collection. A special thanks to Dr. Jay

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FIG. 4. Expression of Mrp7 (above), and Mrp9 (bottom) mRNA in liver after treatment with prototypical drug-metabolizing enzyme inducers. The abbreviations used are
the same as in Fig. 1. Total RNA from liver of chemically treated male C57BL/6 mice (n 5/treatment) was analyzed by the bDNA assay. Data are presented as mean
RLUs S.E.M. , statistically significant difference between treated and control mice (p 0.05).

962

MAHER ET AL.

Petrick for help with data analysis and to the personnel in Dr.
Klaassens lab for critical review of this manuscript.
References

Address correspondence to: Dr. Curtis Klaassen, Department of Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical Center, 3901
Rainbow Blvd., Kansas City, KS 66160-7417. E-mail: cklaasse@kumc.edu

Downloaded from dmd.aspetjournals.org at ASPET Journals on April 6, 2015

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