Drug Metabolism and Disposition Fast Forward
First published on April 18, 2006; DOI: 10.1124/dmd.105.007542
0090-9556/06/3407-1190-1197$20.00
DMD 34:1190-1197, 2006
INDUCTION OF GENES FOR METABOLISM AND TRANSPORT BY TRANS-STILBENE OXIDE IN LIVERS OF SPRAGUE-DAWLEY AND WISTAR-KYOTO RATS
A. L. Slitt,
N. J. Cherrington,
C. D. Fisher,
M. Negishi, and
C. D. Klaassen
Department of Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical Center, Kansas City, Kansas (A.L.S., C.D.K.); Pharmacology and Toxicology, University of Arizona, Tucson, Arizona (N.J.C., C.D.F.); and The Pharmacogenetics Section, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina (M.N.)
(Received December 28, 2005;
accepted April 12, 2006)
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Abstract
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trans-Stilbene oxide (TSO) is a synthetic proestrogen that induces phase I and II drug-metabolizing enzymes in rat liver. The purpose of this study was to determine whether TSO also induces transporter expression in rat liver and whether gene induction in rat liver after TSO occurs in a constitutive androstane receptor (CAR)-dependent manner. Total RNA was isolated from male rat livers after treatment with TSO for up to 4 days (200 mg/kg, i.p., twice daily), and the mRNA levels for each gene were quantified. CYP2B1/2, CYP3A1, epoxide hydrolase, heme oxygenase-1, UGT1A6, UGT2B1, multiple drug resistance protein (Mdr) 1a and 1b, as well as multidrug resistance-associated protein (Mrp) 2, 3, and 4 mRNA were increased in livers after TSO treatment. To determine whether TSO activates gene expression in a CAR-dependent manner, male and female Wistar-Kyoto (WKY) rats were treated with TSO for 3 days. TSO induced CYP2B1/2, UGT2B1, and Mdr1b in males more than in females, suggesting that TSO could increase their expression via CAR. Conversely, TSO induced CYP3A1, epoxide hydrolase, UGT1A6, and Mrp3 similarly in both genders, indicating that induction of these genes occurs independently of CAR. TSO treatment also increased the activity of a CAR binding element luciferase reporter construct in HepG2 cells transfected with rat CAR and in mouse liver. Additionally, TSO increased antioxidant response element/electrophile response element luciferase reporter construct activity in HepG2 cells. In conclusion, in WKY rat liver, TSO increases CYP2B1/2, UGT2B1, and Mdr1b mRNA expression in a gender-dependent manner and CYP3A1, epoxide hydrolase, UGT1A6, and Mrp3 in a gender-independent manner.
trans-Stilbene oxide (TSO) is a synthetic estrogen that belongs to the class of compounds called stilbenes. TSO is commonly used to induce liver enzymes in rodents and is considered to be a phenobarbital (PB)-like chemical. In rats, exposure to TSO results in induction of several biotransformation enzymes in liver, such as CYP2B1, CYP3A1, epoxide hydrolase, heme oxygenase-1, NAD(P)H:quinone oxidoreductase 1 (NQO1), glutathione S-transferases, and UDP-glucuronosyltransferases (UGT) (Kuo et al., 1981
; Williams et al., 1984; Goon and Klaassen, 1992; Oguro et al., 1997; Schilter et al., 2000). Recently it was shown that TSO also increases mRNA and protein levels of the xenobiotic efflux transporters, multidrug resistance-associated protein (Mrp) 2 and 3 mRNA, in rat liver (Slitt et al., 2003). Thus, TSO induces mRNA expression of phase I and II drug-metabolizing enzymes, as well as xenobiotic transporters. Determining which transcription factor-mediated pathways are activated by TSO will lend insight into mechanisms by which these genes are regulated.
The mechanism by which PB induces CYP2B has been well described (for review, see Sueyoshi and Negishi, 2001; Swales and Negishi, 2004) and is dependent on the activation and nuclear translocation of the constitutive androstane receptor [CAR; nuclear receptor binding site 1 (NR1) I3], which is usually localized in the cytoplasm. PB activation of CAR is not well understood, but there is evidence suggesting that PB activates CAR via a signal transduction pathway (altering phosphorylation). After activation, CAR translocates to the nucleus, heterodimerizes with retinoid X receptor 
, and binds to the 51-base pair (bp) PB response element module (PBREM), and this enhances transcription of the CYP2B1 (rat) and Cyp2b10 (mouse) genes. Wistar-Kyoto (WKY) rats exhibit gender dimorphism in the induction of CYP2B1. Female WKY rats, which have low hepatic levels of CAR, induce CYP2B1 to a much lesser extent than male WKY rats, which have higher hepatic levels of CAR and exhibit a robust induction of CYP2B1 in liver (Yoshinari et al., 2001; Cherrington et al., 2003). Studies with WKY rats have revealed that Mrp3 induction in liver by PB-like compounds likely occurs through a CAR-independent mechanism (Xiong et al., 2002; Cherrington et al., 2003). Therefore, this CAR gender-divergent rat strain provides useful information about whether a compound is a potential CAR activator in rat liver in vivo, and whether a rat gene is likely regulated in a CAR-dependent manner.
For many years, it has been known that TSO increases the activity of liver biotransformation enzymes. However, the mechanism by which TSO induces this activity is not known. Because TSO induces some of the same genes involved in phase I and phase II metabolism that are known to be induced through CAR, it was hypothesized that TSO also might also induce genes in rat liver by a CAR-dependent mechanism. First, studies were performed to determine the expression of various phase I, II, and transporter genes at the mRNA level after TSO administration in livers from male Sprague-Dawley rats, which are commonly used to study liver enzyme induction by microsomal enzyme-inducing chemicals. The second goal was to determine whether TSO increases gene expression in a CAR-dependent manner in vivo by using male and female WKY rats that show a gender-dimorphic inducibility by PB and PB-like compounds. Finally, the third goal was to determine whether TSO activates a (NR1)5-tk-luciferase reporter construct, which contains five copies of the CAR NR1 binding site, in mouse liver (in vivo) and HepG2 cells (in vitro).
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Materials and Methods
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Materials. TSO was purchased from Aldrich Company (Milwaukee, WI). All the other chemicals were purchased from Sigma-Aldrich (St. Louis, MO).
Treatment of Animals. Male Sprague-Dawley rats weighing 200 to 250g were purchased from Charles River Laboratories (Wilmington, MA). Animals were housed in a temperature-, light-, and humidity-controlled environment in cages with hardwood chips. The rats were fed Harlan Teklad Rodent Diet W type (Harlan Laboratories, Madison, WI) ad libitum. Rats (n = 5/group) were treated with TSO in corn oil twice daily (200 mg/kg, 2 ml/kg, i.p.). Livers were removed at 3 h (one dose of TSO) and 12 h (two doses of TSO) after 4 days of twice-daily TSO administration, immediately frozen in liquid nitrogen, and stored at –70°C. Because in pilot studies the dosage of TSO used in the Sprague-Dawley rats was lethal to some WKY rats, the dosage of TSO administered to WKY rats was decreased. Male and female WKY rats (n = 4–5 per group, 200–250 g; Harlan, Indianapolis, IN) were treated with a single daily dose of TSO in corn oil (200 mg/kg, 5 ml/kg, i.p.) for 3 days. For both rat studies, the control rats received the same volume of corn oil vehicle as the treated animals. All the animal studies were conducted according to the National Institutes of Health guidelines.
RNA Extraction. Total RNA from liver tissue was extracted using RNA Bee Reagent (Tel-Test, Inc., Friendswood, TX) according to the manufacturer's protocol. RNA integrity was confirmed by formaldehyde-agarose gel electrophoresis.
Oligonucleotide Probe Sets for Branched DNA Signal Amplification Analysis. Rat CYP1A1, CYP2B1/2, CYP3A1, CYP4A2/3, NQO1, UGT1A6, UGT2B1, multiple drug resistance protein (Mdr) 1a, Mdr1b, Mrp2, Mrp3, and Mrp4 probes were used as described previously (Hartley and Klaassen, 2000; Brady et al., 2002; Cherrington et al., 2002; Li et al., 2002; Leazer and Klaassen, 2003; Shelby et al., 2003). Probe sets for rat epoxide hydrolase and heme oxygenase-1 are described in Table 1. These target sequences were analyzed by ProbeDesigner Software version 1.0 (Panomics, Fremont, CA). All the oligonucleotide probes were designed with a Tm of approximately 63°C, enabling hybridization conditions to be held constant (i.e., 53°C) during each hybridization step and for each oligonucleotide probe set. Probes developed in ProbeDesigner were submitted to the National Center for Biotechnology Information for nucleotide comparison by the basic linear alignment search tool (BLASTn) to ensure minimal cross-reactivity with other known rat sequences and expressed sequence tags.
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TABLE 1 Oligonucleotide probes generated for analysis: heme oxygenase-1 (HO-1) and microsomal epoxide hydrolase (EH) expression by bDNA signal amplification
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Branched DNA Assay. Oligonucleotide probes were diluted in lysis buffer supplied in the QuantiGene HV signal amplification kit (Panomics) All the reagents for analysis (i.e., lysis buffer, capture hybridization buffer, amplifier/label probe buffer, and substrate solution) were supplied in the QuantiGene HV signal amplification kit. Total RNA (1 µg/µl, 10 µl) was added to each well of a 96-well plate containing 50 µl of capture hybridization buffer and 50 µl of a diluted probe set. Total RNA was allowed to hybridize to each probe set overnight at 53°C. Subsequent hybridization steps were carried out according to the manufacturer's protocol, and luminescence was measured with a Quantiplex 320 branched DNA signal amplification (bDNA) luminometer inter-faced with Quantiplex data management software version 5.02 (Panomics) for analysis of luminescence from 96-well plates.
Transient Transfection Assays. Cultured HepG2 cells were transiently transfected with a rat CAR expression plasmid and a (NR1)5-tk-luciferase construct according to a previously published method with modifications (Kawamoto et al., 2000). Briefly, HepG2 cells were cultured in minimal essential medium supplemented with 10% fetal bovine serum according to the protocol provided by American Type Culture Collection (Manassas, VA). In collagen-coated 24-well plates, the (NR1)5-tk-luciferase plasmid (0.1 µg) was cotransfected with pRL-CMV (0.05 µg) (Promega Corp., Madison, WI) into HepG2 cells using Lipofectamine in combination with Plus agent (Invitrogen, Carlsbad, CA), with or without a rat CAR expression plasmid (0.2 µg). Approximately 16 h after transfection, cells were cultured in the presence of TSO (50 or 100 µM) with or without 5-androstan-3-ol (20 µM). After exposure to TSO for 24 h, the medium was removed, and cells were washed with phosphate-buffered saline (PBS). The PBS was aspirated from the cells, and 100 µl of passive lysis buffer was added to each well. To assess TSO activation of the antioxidant response element/electrophile response element (ARE/EpRE), HepG2 cells were transiently transfected with 0.1 µg of an ARE/EpRE luciferase reporter construct (Dieter et al., 2001) and 0.05 µg of pRL-CMV. Approximately 16 h after transfection, cells were cultured in the presence of TSO (100 µM) for 24 h. The medium was then removed, and cells were washed with PBS. The PBS was aspirated from the cells, and 100 µl of passive lysis buffer was added to each well. Luciferase activity was determined by the Dual-Glo Luciferase Assay (Promega Corp.).
In Vivo Luciferase Assay. Male C57BL/6 mice (20–25 g) were administered 1 µg of (NR1)5-tk-luciferase in the form of naked plasmid DNA in sterile saline by a rapid (5-s) tail vein injection in a volume equal to 10% of body weight (Schuetz et al., 2002). Twenty-four hours later, animals were anesthetized with a mixture of ketamine (72 mg/kg), acepromazine (6 mg/kg), and xylazine (6 mg/kg). Luciferin was injected into mice (70 µl of a 50-mg/ml stock solution, i.p.) 5 min before imaging. A VersArray 1300B camera from Roper Scientific (Tucson, AZ), thermoelectrically cooled to –100°C, was used to image the mice. A light-tight imaging chamber was used for all the images. Images were acquired in gray scale, and pseudocolor maps were created with the WinView 32 program (Tucson, AZ). Color maps were superimposed over the light image of the mouse using Adobe (Mountain View, CA) Photoshop 6.0. Light images were acquired with lights mounted inside the box, with an exposure time of about 20 ms, using a fast setting for the analog-digital converter. Bioluminescent images were acquired with interior lights turned off, an exposure of 10 min, and with a slow setting on the analog-digital converter. All the images were taken with an aperture of f1.2. This image was considered time 0. Each animal (n = 3) was administered a single dose of either TSO (200 mg/kg, i.p., 5 ml/kg) or corn oil (5 ml/kg), and images were taken 12 h after TSO treatment.
Statistics. Statistical differences between vehicle- and TSO-treated groups at each time point (3 h, 12 h, and 4 days) were determined by a Student's t test. Statistical differences between male and female WKY rats and vehicle- and TSO-treated groups were determined by analysis of log-transformed data using a two-way analysis of variance, followed by Duncan's multiple range, post hoc test. Asterisks (* represent a statistical difference (p < 0.05) between control and TSO-treated groups, and crosses (
) represent a statistical difference (p < 0.05) between male and female WKY rats treated with TSO.
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Results
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TSO Induction of Biotransformation and Transport Genes in Livers from Male Sprague-Dawley Rats. Previous studies have reported that genes for drug metabolism and transport are induced in rat liver after exposure to TSO (Kuo et al., 1981; Williams et al., 1984; Goon and Klaassen, 1992; Oguro et al., 1997; Schilter et al., 2000). In these studies, TSO was administered once or twice a day for several days for induction of liver microsomal enzymes (Gregus et al., 1990; Schilter et al., 2000). Therefore, to determine the time course for increased gene expression after TSO treatment, livers were removed 3 and 12 h after TSO administration, as well as after 4 days of TSO administration.
Figure 1 shows the mRNA levels of CYP1A1, CYP2B1/2, CYP3A1, CYP4A2/3, NQO1, epoxide hydrolase, and heme oxygenase-1 in male Sprague-Dawley livers at various times after the first dose of TSO. TSO administration did not induce CYP1A1 expression in liver. However, CYP2B1/2 was induced approximately 17-fold 12 h after TSO and 60-fold after 4 days of TSO administration. CYP3A1 was not induced at 3 or 12 h after TSO treatment but was induced approximately 8-fold after 4 days of TSO treatment. CYP4A2/3 mRNA levels in liver were not increased at any time after TSO treatment. Previous studies showed that TSO increases the expression and/or activity of NQO1, epoxide hydrolase, and heme oxygenase-1 in liver (Kuo et al., 1981; Williams et al., 1984; Oguro et al., 1997). Transcripts for all three genes were increased after TSO exposure. NQO1 mRNA levels were increased approximately 7-fold 12 h after TSO administration and 3-fold after 4 days of TSO treatment. Epoxide hydrolase mRNA levels doubled 12 h after TSO and tripled after 4 days of TSO treatment. NQO1 and epoxide hydrolase mRNA expression was similar to controls 3 h after TSO treatment. In contrast, heme oxygenase-1 mRNA levels increased 7-fold 3 h after TSO and markedly increased by approximately 35-fold 12 h after TSO treatment but returned to control levels after 4 days of TSO administration.
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FIG. 1. Effect of TSO administration on CYP1A1, CYP2B1/2, CYP3A1, CYP4A2/3, NQO1, epoxide hydrolase, and heme oxygenase-1 mRNA expression in livers of Sprague-Dawley rats. Tissue total RNA was isolated from adult male rats after 3 h, 12 h, or 4 days of TSO administration (200 mg/kg, i.p., twice daily) and analyzed by the bDNA signal amplification assay for CYP1A1, CYP2B1/2, CYP3A1, and CYP4A2/3, NQO1, epoxide hydrolase, and heme oxygenase-1 mRNA content. The data are presented as mean RLU ± S.E.M. (n = 4–5 animals). Asterisks (*) represent a statistical difference (p  5 0.05) between vehicle control (CON) and TSO groups.
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UGT are phase II drug-metabolizing enzymes that catalyze the conjugation of a glucuronic acid moiety to numerous endogenous and exogenous substrates. UGT are induced by TSO treatment (Seidegard and DePierre, 1982). Figure 2 shows UGT1A6 and UGT2B1 mRNA levels in male Sprague-Dawley liver after TSO administration. TSO doubled UGT1A6 and UGT2B1 mRNA levels in liver at 12 h. After 4 days of administration, TSO increased UGT1A6 and UGT2B1 mRNA expression in liver by approximately 20- and 8-fold, respectively.
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FIG. 2. Effect of TSO administration on UGT1A6 and UGT2B1 mRNA expression in livers of Sprague-Dawley rats. Tissue total RNA was isolated from adult male rats after 3 h, 12 h, or 4 days of TSO administration (200 mg/kg, i.p., twice daily) and analyzed by the bDNA signal amplification assay for UGT1A6 and UGT2B1 mRNA content. The data are presented as mean RLU ± S.E.M. (n = 4–5 animals). Asterisks (*) represent a statistical difference (p 0.05) between vehicle control (CON) and TSO groups.
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Mrps 2 through 4 are members of the multidrug resistance protein family of ATP binding cassette (or ABC) transporters. It has recently been reported that TSO increases Mrp2 and Mrp3 protein levels in liver (Slitt et al., 2003). Therefore, the effect of TSO on the mRNA expression of Mrp2 and Mrp3 along with other liver transporters was examined. Figure 3 illustrates the mRNA levels of several transporters in male Sprague-Dawley liver after TSO administration. In general, the expression of Mdr1a and Mdr1b in rat liver is low. Mdr1a mRNA expression approximately doubled 12 h and 4 days after TSO administration. Mdr1b expression increased approximately 6-fold at 12 h and 8-fold at 4 days after TSO administration. TSO did not affect Mdr2 expression at any time point (data not shown). Both Mrp2 and Mrp3 mRNA levels were unchanged at 3 or 12 h after TSO, but Mrp2 mRNA expression doubled after 4 days of TSO, whereas Mrp3 mRNA expression increased approximately 15-fold. Mrp4 mRNA levels tripled at 12 h after TSO administration and were doubled after 4 days of TSO treatment. TSO administration did not alter expression of organic anion transporting polypeptides 1, 2, and 4, Mrps 1, 5, and 6, or the bile salt export pump in liver of male Sprague-Dawley rats (data not shown).
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FIG. 3. Effect of TSO administration on Mdr1a, Mdr1b, and Mrps 2 through 4 mRNA expression in livers of Sprague-Dawley rats. Tissue total RNA was isolated from adult male rats after 3 h, 12 h, or 4 days of TSO administration (200 mg/kg, i.p., twice daily) and analyzed by the bDNA signal amplification assay for Mdr1a, Mdr1b, and Mrps 2 through 4 mRNA content. The data are presented as mean RLU ± S.E.M. (n = 4–5 animals). Asterisks (*) represent a statistical difference (p 0.05) between vehicle control (CON) and TSO groups.
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TSO Induction of Biotransformation and Transport Genes in Livers from Male and Female WKY Rats. WKY rats display a gender-dimorphic induction of CYP2B1 by PB, corresponding to CAR protein levels in liver: females have less CYP2B1 induction by PB and lower levels of CAR protein in liver compared with males (Yoshinari et al., 2001). Therefore, this rat strain was used to examine the role of CAR in the induction of genes in liver by TSO. Because most of the genes of interest were induced after 4 days of TSO administration, this time point was chosen to examine induction in the male and female WKY rats.
Induction of phase I and II drug-metabolizing enzymes in livers from male and female WKY rats after TSO administration is shown in Fig. 4. CYP2B1/2 inducibility in liver by TSO was higher in WKY males than in WKY females as expected (125-versus 12-fold). TSO approximately doubled CYP3A1 mRNA expression in livers from WKY males but did not induce CYP3A1 expression in livers from female WKY rats. Epoxide hydrolase mRNA expression was tripled in livers from male WKY rats after TSO administration; however, epoxide hydrolase was only doubled in female WKY rats. TSO induced NQO1 mRNA expression in livers from male WKY rats 7-fold. The induction was slightly less in females, being 3-fold higher in control female livers. Heme oxygenase-1 expression was not induced in WKY rat liver after 4 days of TSO administration (data not shown). UGT1A6 and UGT2B1 expression in male and female WKY rat livers was also examined after TSO administration. TSO induced UGT1A6 mRNA expression in livers from both genders approximately 9-fold. TSO increased UGT2B1 mRNA expression 5-fold in male WKY rats. In contrast, UGT2B1 induction in liver of female WKY rats by TSO was not statistically different from that detected in control liver.
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FIG. 4. Effect of TSO administration on CYP2B1/2, CYP3A1, NQO1, epoxide hydrolase, UGT1A6, and UGT2B1 mRNA expression in liver from WKY rats. Tissue total RNA was isolated from adult male and female (WKY) rats after 3 days of TSO administration (200 mg/kg, i.p.) and analyzed by the bDNA signal amplification assay for CYP2B1/2, CYP3A1, NQO1, and epoxide hydrolase mRNA content. The data are presented as mean RLU ± S.E.M. (n = 4–5 animals). Asterisks (*) represent a statistical difference (p 0.05) between vehicle control (CON) and TSO groups, and daggers () represent a significant difference between the TSO-treated male and female rats.
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In addition to phase I and II drug-metabolizing enzymes, some microsomal enzyme inducers increase transporter expression in rat liver. Therefore, the role of CAR in transporter induction by TSO was also investigated using male and female WKY rats. Figure 5 illustrates Mdr1a, Mdr1b, and Mrp2 through 4 induction in livers of male and female WKY rats. TSO markedly induced Mdr1b mRNA expression in livers from male WKY rats by 400-fold, and this induction was abated in female WKY rat livers. Mrp2 was not induced in WKY liver by TSO treatment in either gender. After TSO administration, Mrp3 mRNA was induced 10-fold in livers from both male and female WKY rats. The basal expression of Mrp4 mRNA was slightly higher in male WKY liver than in female WKY liver. TSO administration induced Mrp4 mRNA expression in livers from both male and female WKY rats, with induction being lower in livers from female rats.
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FIG. 5. Effect of TSO administration on Mdr1a, Mdr1b, and Mrps 2 through 4 mRNA expression in liver from WKY rats. Tissue total RNA was isolated from adult male and female (WKY) rats after 3 days of TSO administration (200 mg/kg, i.p.) and analyzed by the bDNA signal amplification assay for Mdr1a, Mdr1b, and Mrp 2s through 4 mRNA content. The data are presented as mean RLU ± S.E.M. (n = 4–5 animals). Asterisks (*) represent a statistical difference (p 0.05) between vehicle control (CON) and TSO groups.
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TSO Activation of CAR in Vitro and in Vivo. Because TSO administration markedly induced CYP2B1/2 mRNA levels in livers from male WKY rats, but not in females, the data suggest that TSO increases CYP2B1/2 mRNA levels in liver via activation of CAR and binding of CAR to the PBREM in the 5'-flanking region of the rat CYP2B1/2 gene. Therefore, in vitro activation assays were used to determine whether TSO activates rat CAR and the previously described NR1 that is part of the PBREM (Honkakoski et al., 1998). Figure 6 illustrates TSO activation of a luciferase construct containing five copies of the NR1 CAR binding element (NR1)5 with or without rat CAR in HepG2 cells. In wells transfected with the empty vector PCR3, there was no activation of the (NR1)5-tk-luciferase reporter construct. Cells were cultured in the presence of 20 µM5-androstan-3-ol to repress the constitutive activity of CAR (Kawamoto et al., 2000). In HepG2 cells treated with dimethyl sulfoxide (DMSO) vehicle, the activity of the (NR1)5-tk-luciferase reporter construct was significantly higher in cells transfected with rat CAR compared with cells transfected with the PCR3 plasmid. Importantly, in cells treated with 50 and 100 µM TSO, (NR1)5-tk-luciferase reporter activity was increased 2.2- and 2.7-fold, respectively, in cells cotransfected with rat CAR. Figure 7 illustrates in vivo activation of the (NR1)5-tk-luciferase reporter construct transfected into livers of mice administered vehicle or TSO. At 12 h, luciferase activity was increased in livers of TSO-treated as compared with vehicle-treated mice.
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FIG. 7. Activation of the (NR1)5-tk-luciferase reporter construct in mouse liver after vehicle or TSO administration. Male C57BL/6 mice were injected with 1 µg of naked plasmid DNA in a rapid (5-s) tail vein injection in sterile saline in a volume equal to 10% of body weight. Twenty-four hours later, animals were anesthetized with ketamine (72 mg/kg), acepromazine (6 mg/kg), and xylazine (6 mg/kg), and 5 min before imaging at times 0 and 12 h, mice were injected with luciferin i.p. At time 0, mice were injected with a single dose of corn oil vehicle (5 ml/kg, i.p.) or TSO (200 mg/kg, i.p.). Representative images depict (NR1)5-tk-luciferase activity at 0 and 12 h after vehicle or TSO administration.
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TSO Activation of the ARE/EpRE in HepG2 Cells. Induction of NQO1, epoxide hydrolase, and heme oxygenase-1 suggests that TSO may activate gene expression through a mechanism other than CAR. NQO1 induction after treatment with antioxidants and chemicals that cause oxidative stress is mediated through activation of the ARE/EpRE. Therefore, the ability of TSO to activate the ARE/EpRE was tested in vitro using HepG2 cells transiently transfected with an ARE/EpRE luciferase reporter construct. The data illustrated in Fig. 8 show that TSO treatment activated an ARE/EpRE luciferase reporter construct transiently transfected into HepG2 cells by approximately 3-fold.
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FIG. 8. Activation of the ARE/EpRE in HepG2 cells after treatment with TSO. HepG2 cells were transiently transfected with an ARE/EpRE luciferase reporter construct (0.1 µg) and pRL-CMV (0.05 µg) using Lipofectamine 2000. Sixteen hours after transfection, cells were treated with TSO (100 µm). Twenty-four hours later, luciferase activity was determined by the Dual-Glo Luciferase Assay. The data are presented as the mean -fold activation ± S.E.M. (n = 3 wells/treatment).
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Discussion
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For many years, it has been known that PB treatment induces CYP2B expression and activity in liver of mice and rats. It is now known PB increases CYP2B expression through activation of CAR (NR1I3), a member of the nuclear hormone receptor superfamily (Wei et al., 2000). Furthermore, experiments with CAR-null mice have shown that CAR expression is necessary for induction of Cyp2B10 in liver, as well as other hepatic biotransformation genes, such as Ugt1A1 (Maglich et al., 2002).
Like PB, TSO administration also induces CYP2B1/2 in rat liver. TSO is considered a "PB-like" compound because, like PB, it induces CYP2B1/2, CYP3A1, epoxide hydrolase, and NQO1 expression in liver (Pickett and Lu, 1981; Williams et al., 1984; Slawson et al., 1996; Schilter et al., 2000). However, it was not known how TSO induces gene expression in liver. In the present study, TSO administration increased CYP2B1/2 expression in liver from Sprague-Dawley rats at 12 h and 4 days. In addition, in male WKY rats, TSO administration caused a robust induction of CYP2B1/2 mRNA expression in liver. In contrast, CYP2B1/2 induction after TSO treatment was much lower in livers from female WKY rats, which express much lower levels of CAR protein in liver than male WKY rats. In WKY rats, inducibility of CYP2B by PB treatment correlates with CAR expression in liver (Yoshinari et al., 2001). Decreased induction of CYP2B1 expression in female WKY rats, as compared with male WKY rats, correlates with lower CAR protein expression levels and CAR binding to the PBREM in liver. These data suggest that TSO increases CYP2B1/2 in rat liver via the transcription factor CAR, similar to the previously published data that illustrate that PB also induces CYP2B1/2 in rat liver via CAR (Yoshinari et al., 2001).
TSO likely induces expression of some genes in rat liver via activation of CAR. First, the vitro and in vivo studies conducted show that TSO activates the NR1 CAR binding site in HepG2 cells by a CAR-dependent mechanism. Second, TSO induces the mRNA expression of some genes in liver from WKY males more than in liver from WKY females, suggesting a CAR-dependent induction. TSO induced CYP2B1/2 in WKY rat liver in a CAR-dependent manner. These data are consistent with previously published data by Yoshinari et al. (2001) and Cherrington et al. (2003), who reported CYP2B1/2 induction in rat liver by PB is mediated through CAR. NQO1 induction by TSO might also be mediated, in part, through CAR because there was less induction in liver from WKY females than WKY males. Third, TSO induction of Cyp2b10 in CAR-null mice is markedly abated (Slitt et al., 2006).
Gene expression data generated from male and female WKY rats must be interpreted with caution because differences aside from CAR expression levels exist between male and female rats. Thus, differences in hepatic gene induction between males and females could be completely independent of CAR. However, there is no in vivo model available to study the role of CAR in gene induction in rat that is equivalent to the CAR knockout mouse model. Hence, data in this manuscript that show gender-divergent induction of genes in male and female WKY rats suggest, but do not prove, that CAR mediates TSO induction of CYP2B1/2, UGT2B1, and Mdr1b. However, the combination of the WKY model with TSO provides insight into how genes such as UGT2B1 and Mdr1b are regulated.
The present study shows that TSO activates rat CAR, but future studies will be needed to address how TSO activates CAR. An interesting observation is that CAR is activated in vitro by 17ß-estradiol and estrone and repressed by progesterone and androgens (Kawamoto et al., 2000). Furthermore, compounds that are considered to be endocrine disrupters such as methoxychlor can activate CAR (Blizard et al., 2001). Stilbenes, as a class of compounds, are considered to have estrogenic properties, and it has been shown that rat liver microsomes can metabolize TSO to form estrogenic metabolites (Sugihara et al., 2000). Thus, TSO may increase gene expression in liver by exerting an estrogenic effect that activates CAR or releases androgen repression of CAR.
The present data also show that TSO increases mRNA levels of CYP3A1, NQO1, epoxide hydrolase, heme oxygenase-1, UGT1A6, UGT2B1/2, Mdr1a, Mdr1b, Mrp2, Mrp3, and Mrp4 in liver of male Sprague-Dawley rats. Interestingly, not all the genes induced in Sprague-Dawley liver were induced at the same time after TSO administration. CYP2B1/2, NQO1, epoxide hydrolase, heme oxygenase-1, UGT1A6, UGT2B1/2, Mdr1a, Mdr1b, and Mrp4 mRNA levels in livers of Sprague-Dawley rats were increased 12 h after initial TSO administration, whereas CYP3A1, Mrp2, and Mrp3 mRNA levels were not increased at 12 h but were increased after 4 days of TSO administration. This differential induction between genes in liver suggests that TSO may increase gene expression in liver through more than one mechanism. UGT1A6, Mrp2, and Mrp3 induction by TSO occurred in a CAR-independent manner. Because there is "cross-talk" between CAR and pregnane X receptor (PXR), it is possible that TSO could also activate PXR. Hepatic expression of PXR in male and female WKY rats has not been reported; therefore, it is not known whether PXR levels are similar or different in livers from male and female WKY rats. Thus, similar induction patterns in livers from male and female rats may be a result of PXR-mediated gene induction. TSO treatment did not increase the expression of mRNA expression of Oatp2 (Oatp1a4), a gene known to be regulated through PXR, in rat liver. The lack of Oatp2 induction after TSO administration suggests that TSO may not activate PXR. Because TSO administration did not increase CYP1A1 or CYP4A2/3 mRNA levels in liver, it is suspected that TSO does not activate the aryl hydrocarbon receptor or the peroxisome proliferator-activated receptor because CYP1A1 and CYP4A2/3 are target genes for these receptors, respectively.
Our data show that induction of CYP2B1/2 and NQO1 by TSO was lower in liver from female WKY rats, and induction of epoxide hydrolase, UGT1A6, Mrp2, and Mrp3 was similar in livers from male and female WKY rats, which suggests that there is another mechanism by which TSO increases gene expression in rat liver. Whereas TSO-mediated activation of PXR is possible, it is also possible that this occurs via activation of nuclear factor E2 p45-related factor 2 (Nrf2). The regulation of basal and inducible expression has been well defined for mouse and human NQO1, which is known to be regulated by Nrf2 binding to an ARE/EpRE (Gong et al., 2002). Nrf2 is a member of the family of basic leucine zipper transcription factors that regulate expression of globin genes during erythroid development and is now known to mediate induction of phase II enzymes in liver after anti-oxidant treatment (Ramos-Gomez et al., 2001). Moreover, Nrf2 regulates the basal and inducible expression of epoxide hydrolase and UGT1A6 and the inducible expression of heme oxygenase-1 in mouse liver (Ramos-Gomez et al., 2001; Gong et al., 2002). Because NQO1, epoxide hydrolase, and UGT1A6 are regulated/induced by Nrf2 activation in mouse liver, it is possible that TSO up-regulates NQO1, epoxide hydrolase, and UGT1A6 mRNA expression in rat liver via activation of Nrf2. It is known that TSO administration depletes glutathione levels in liver within 4 h after administration, and this is postulated to cause oxidative stress (Oguro et al., 1997; Sasaki et al., 2002). Because the ARE/EpRE is responsive to compounds such as diethyl maleate that deplete cellular glutathione, it is likely that TSO administration causes oxidative stress that also activates Nrf2 and the ARE/EpRE in addition to CAR. Furthermore, data in this study show that TSO activates the ARE/EpRE in HepG2 cells.
In summary, TSO treatment increases the mRNA levels for several genes that encode biotransformation enzymes, as well as drug transporters in Sprague-Dawley rat liver. Additionally, TSO regulates the expression of CYP2B1/2, UGT2B1, and Mdr1b mRNA expression in a gender-specific manner in WKY rats, suggesting that induction of these genes is CAR-dependent, whereas induction of CYP3A1, epoxide hydrolase, UGT1A6, and Mrp3 mRNA expression is gender-independent, suggesting a CAR-independent mechanism. TSO activates rat CAR and the NR1 CAR binding element in HepG2 cells and in the NR1 site in mouse liver. Together, these data indicate that TSO activates CAR both in vivo and in vitro.
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Acknowledgments
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We thank our colleagues in the Klaassen laboratory for assistance with tissue collection and manuscript preparation.
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Footnotes
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This work was supported by National Institutes of Health Grants ES-09716, ES-07079, and ES-11239 (A.L.S., N.J.C.).
This work was presented, in part, at the 2003 Annual Society of Toxicology Meeting in Salt Lake City, UT.
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
doi:10.1124/dmd.105.007542.
ABBREVIATIONS: TSO, trans-stilbene oxide; PB, phenobarbital; NQO1, NAD(P)H:quinone oxidoreductase 1; UGT, UDP-glucuronosyltransferase(s); Mrp, multidrug resistance-associated protein; CAR, constitutive androstane receptor; NR1, nuclear receptor binding site 1; bp, base pair(s); PBREM, phenobarbital response element module; WKY, Wistar-Kyoto; Mdr, multiple drug resistance protein; bDNA, branched DNA signal amplification; PBS, phosphate-buffered saline; ARE/EpRE, antioxidant response element/electrophile response element; DMSO, dimethyl sulfoxide; PXR, pregnane X receptor; Nrf2, nuclear factor E2-related factor 2; RLU, relative light units.
Address correspondence to: Curtis Klaassen, Department of Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical Center, 3901 Rainbow Boulevard, Kansas City, KS 66160-7417. E-mail: cklaasse{at}kumc.edu
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References
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Abstract
Materials and Methods
