Involvement of pregnane X receptor in the regulation of CYP2B6 gene expression by oltipraz in human hepatocytes

Toxicology in Vitro 24 (2010) 452–459

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Involvement of pregnane X receptor in the regulation of CYP2B6 gene expression by oltipraz in human hepatocytes Amélie Piton a,b,1, Claudine Rauch a,b,1, Sophie Langouet a,b, André Guillouzo a, Fabrice Morel a,b,* a b

INSERM U620, Université de Rennes 1, F-35043, France IFR 140, Rennes, F-35043, France

a r t i c l e

i n f o

Article history: Received 1 April 2009 Accepted 28 September 2009 Available online 12 October 2009 Keywords: Oltipraz CYP2B6 Human hepatocytes Pregnane X receptor Nrf2 Testosterone hydroxylation

a b s t r a c t Oltipraz, a synthetic derivative of the cruciferous vegetable product 1,2-dithiole-3-thione, is considered as a potent chemoprotectant. Previously, we have demonstrated that CYP2B6 expression is induced in cultured human hepatocytes by a 24 h treatment with oltipraz. The aim of this study was to further determine mechanisms involved in the regulation of CYP2B6 by this compound. An increase of CYP2B6 mRNA is observed after a 4 h exposure and maximum induction is reached after 24 h. The rapid induction of CYP2B6 mRNA in oltipraz-treated cells suggests a transcriptional activation of corresponding gene. To test this hypothesis, we performed transient transfections with constructs containing the CYP2B6 gene 50 flanking region upstream of the luciferase gene in order to measure the transcriptional activity of CYP2B6 gene in human hepatoma HepG2 cells, in absence or presence of oltipraz. The results demonstrate that transcriptional activation of CYP2B6 gene is mediated mainly by the pregnane X receptor (PXR) and the Phenobarbital Responsive Element Module (PBREM). The nuclear factor-erythroid 2-related factor 2 (Nrf2) and an antioxidant responsive element (ARE), located upstream the PBREM, might also have a role in this activation but their involvement remains unclear. Despite increasing CYP2B6 apoprotein levels in human hepatocytes, oltipraz has little effect, if any, on testosterone 16b-hydroxylation which is catalyzed by CYP2B6. This can be explained by a dose-dependent inhibition of CYP2B6 activity in presence of oltipraz as demonstrated with human hepatocyte microsomes. Altogether, this study provides the first demonstration of PXR involvement in oltipraz transcriptional activation of CYP2B6 gene and of the inhibitory effect of oltipraz on CYP2B6 activity. Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction Oltipraz is a synthetic derivative of 1,2-dithiole-3-thione, a constituent of cruciferous vegetables, and has been developed as a chemopreventive agent for many malignancies, including liver and colorectal cancers, on the basis of its in vivo protective activity against chemically induced tumors, in a variety of animal models. This protection has been associated with an enhanced capacity to detoxify reactive carcinogens and with increased DNA repair (Boone et al., 1990; Kensler et al., 1987; Liu et al., 1988; O’Dwyer et al., 1997; Rao et al., 1993). In humans, phase I trials have proved the tolerability and safety of oltipraz and have led to a phase II chemoprevention placebo-controlled trial conducted in Qidong, an Abbreviations: ARE, antioxidant responsive element; CAR, constitutive androstane receptor; CYP, cytochrome P450; Nrf2, nuclear factor-erythroid 2-related factor 2; PBREM, Phenobarbital Responsive Enhancer Module; PXR, pregnane X receptor; RXR, retinoid acid X receptor. * Corresponding author. Address: INSERM U522, Hôpital Pontchaillou, 35033 Rennes, France. Tel.: +33 299 45 74 01; fax: +33 299 54 74 01 37. E-mail address: [email protected] (F. Morel). 1 These two authors contributed equally to this work. 0887-2333/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2009.09.025

area with one of the highest incidences of hepatocellular carcinoma in the People’s Republic of China (Glintborg et al., 2006; Jacobson et al., 1997; Kensler et al., 1998; Zhang et al., 1997). The chemopreventive action of oltipraz is mainly due to its ability to induce phase II detoxication and antioxidant enzymes (Clapper, 1998; Kensler et al., 1987). However, oltipraz has also been shown to have effects on phase I enzymes by inhibiting both human CYP1A2, CYP1A1, CYP2B1, CYP2E1 and CYP3A4 and rat CYP1A, CYP2B and CYP3A2 (Langouet et al., 1995, 1997, 2000; Lee et al., 2007). Moreover, oltipraz is able to induce expression of several rat CYPs both in vivo and in vitro (Buetler et al., 1996; Langouet et al., 1997; Le Ferrec et al., 2001; Maheo et al., 1998) and human CYP1A1 in Caco-2 cells (Le Ferrec et al., 2002). The pathways currently known to be involved in gene regulation by the chemopreventive agent oltipraz are: the nuclear factor-erythroid 2-related factor 2 (Nrf2), which binds the antioxidant responsive element (ARE) and induces phase II enzymes expression (Iida et al., 2004; Manandhar et al., 2007; Pietsch et al., 2003); the nuclear factor kappa B, which leads to an induction of NADPH-quinone reductase expression (Nho and O’Dwyer, 2004; Yao and O’Dwyer, 1995) and the Ah receptor,

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which binds the Xenobiotic Responsive Element, and is involved in hCYP1A1 up-regulation (Auyeung et al., 2003; Le Ferrec et al., 2002). Moreover, the transcription factor CCAAT/Enhancer-Binding Protein beta has been identified in the regulation of rGSTA2, hCYP1A1 and rat Transforming Growth Factor beta1 expression by oltipraz (Auyeung et al., 2003; Bae and Kim, 2005; Kang et al., 2002; Ko et al., 2006). Recently, we have demonstrated an increase of CYP2B6 expression in human hepatocytes treated with oltipraz (Piton et al., 2005). CYP2B6 metabolizes a number of clinically important drugs such as cyclophosphamide, ifosfamide, phenobarbital, rifampicin and bupropion (Chang et al., 1993; Faucette et al., 2004). Extensive variations in CYP2B6 activity are clinically important as they affect drug metabolism and alter responses to certain drugs (Coller et al., 2002; Hesse et al., 2000, 2004). Recent studies have demonstrated that CYP2B6 is also important in the metabolism of pesticides including chlorpyrifos, carbaryl, alachlor, metolachlor, acetochlor, and butachlor, as well as for the insect repellent N,N-diethyl-mtoluamide in human (Coleman et al., 2000; Tang et al., 2001; Usmani et al., 2002). The CYP2B family is described as the phenobarbital-induced cytochrome family. Induction of CYP2B6 is mediated through transcriptional activation of the corresponding gene by the nuclear receptors pregnane X receptor (PXR) and constitutive androstane receptor (CAR), which are capable of binding to the same response elements in the promoter regions of CYP2B6 gene (Goodwin et al., 2001; Sueyoshi et al., 1999; Wang et al., 2003). In human CYP2B6 promoter, a PBREM (Phenobarbital Responsive Enhancer Module) has been described 1.6 kb upstream of the initiation transcription site. CAR and PXR which dimerize with retinoic acid X receptor alpha (RXRa) bind to the PBREM sequence and activate CYP2B6 transcription (Goodwin et al., 2001; Sueyoshi et al., 1999). The aim of this study was to determine mechanisms involved in the regulation of CYP2B6 by oltipraz. Our results clearly demonstrate that oltipraz exerts both an inhibitory effect on CYP2B6 enzymatic activity and a PXR-dependent transcriptional activation of the CYP2B6 gene. 2. Materials and methods 2.1. Chemicals Culture media, glutamine and penicillin–streptomycin were purchased from Gibco (Gibco BRL, Life Technologies, Rockland, USA), fetal calf serum (FCS) from PAN Biotechnology (Stanford, USA), bovine insulin from Sigma (Saint-Louis, USA), bovine serum albumin (BSA) from Eurobio (Les Ulis, France) and hydrocortisone hemisuccinate from Roche (Basel, Switzerland). Oltipraz was kindly supplied by Dr. F. Ballet (Aventis, Evry, France). Testosterone, 2a-, 6b- and 16a-testosterone, as well as NADPH were purchased from Sigma (Saint-Louis, USA). Nrf-2 (sc-722) and PXR antibodies were purchased from Santa Cruz Biotechnologies (Tebu, France) and Active Motif (Rixensart, Belgium), respectively. AntiCYP2B6 antibody was a gift from Pr. F. Guengerich (Nashville, USA). 2.2. Plasmids Fragments of the CYP2B6 promoter were cloned into pGL3 plasmid containing the firefly luciferase reporter gene. The fragments (1995, -1663) were amplified by PCR using a high fidelity polymerase (TaqHifi, Invitrogen) from human genomic DNA (Human Genomic DNA, Promega) with the following primers: forward primers containing a KpnI restriction site (italics): CYP2B6-1995: 50 -actggtacct tggacaatgtagccccaac-30 ; CYP2B6-1663: 50 -actggtacctggatccagcaaagggttt-30 ; reverse primer containing a MluI restriction site: 50 -

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actacgcgtcctccaactgggccttttat-30 . Amplified products were subcloned in pGL3 plasmid upstream of the firefly luciferase gene using KpnI and MluI enzymes, and sequenced entirely. Plasmid expressing dominant-negative Nrf2 (Nrf2M) was a gift from Dr. Alam (Ochsner Clinic Foundation, New Orleans, USA). 2.3. Human hepatocyte isolation and culture Human liver samples were obtained in France from patients undergoing liver resections for secondary hepatomas. Hepatocytes were isolated by a two-step collagenase perfusion procedure, and the experimental procedures used were done in compliance with French laws and approved by the National Ethics Committee. Cell viability was 70–85%, as estimated by the trypan blue exclusion test while cell yields varied with the size of the fragment. Liver parenchymal cells were seeded at a density of 107 viable cells/ 75 cm2 dish in a nutrient medium consisting of Williams E medium supplemented with 0.2% bovine serum albumin, 0.01% bovine insulin, 2 mM of L-glutamine, 100U/ml of penicillin, 10 lg/ml of streptomycin and 10% FCS. The medium, supplemented with 0.1 lM hydrocortisone hemisuccinate but lacking serum, was renewed daily. Oltipraz, dissolved in dimethylsulfoxide (DMSO), was added 36 h after cell seeding and at each medium renewal (every 24 h) to give a final concentration of 50 lM (in 0.1% DMSO). Control cultures received the same concentration of solvent. 2.4. RNA isolation, reverse-transcription and real-time PCR Total RNA was extracted from 106 human hepatocytes with the SV total RNA isolation system (Promega, Madison, WI, USA) which directly includes a DNase treatment step. RNAs were reversed-transcribed into cDNA using High-Capacity cDNA Archive kit (Applied Biosystems). Real-time quantitative PCR was performed by the fluorescent dye SYBR Green methodology using the SYBRÒ Green PCR Master Mix (Applied Biosystem) and the ABI Prism 7000 (Applied Biosystem). Primer pairs were chosen using the Primers3 software (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi): CYP2B6-forward: 50 -ttcctactgcttccgtctatcaaa-30 ; CYP2B6-reverse 50 -gtgcagaatcccacagctca-30 ; CYP3A4-foward 50 -cttcatccaatggactgcataaat-30 ; CYP3A4-reverse 50 -tcccaagtataacactctacacagacaa-30 ; CYP2C9-foward 50 -ggacagagacgacaagcaca-30 ; CYP2C9-reverse 50 aatggacatgaacaaccctca-30 . The amplification curves were read with the ABI Prism 7000 SDS Software using the comparative Cycle Threshold method. The relative quantification of the steady-state of target mRNA levels was calculated after normalization of the total amount of cDNA tested by an active reference, 18S. Furthermore, a dissociation curve was performed after the PCR reaction to verify the specificity of the amplification. 2.5. Preparation of hepatocyte microsomes Human hepatocytes were homogenized in 50 mM Tris–HCl buffer (pH 7.4) containing 0.25 M sucrose, 1 mM EDTA, 25 lM phenylmethylsulfonyl fluoride and 1 mM dithiothreitol. Nuclei and mitochondria were eliminated by centrifugation at 3000g for 10 min and at 8000g for 20 min. The supernatant containing microsomes and cytosol was subjected to centrifugation at 30 000g for 1 h. Cytosols were discarded and microsomal pellets were dissolved in 0.1 M phosphate buffer (pH 7.4) containing 10% glycerol and stored at 80 °C until use. 2.6. Western Blot analysis For Western Blot analysis, 10 lg of microsomal proteins extracted from human hepatocytes were subjected to electrophoresis on a polyacrylamide slab gel and electroblotted overnight onto

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Hybond enhanced chemiluminescence (ECL) nitrocellulose membranes (Amersham). Equal transfer of proteins was confirmed by staining the nitrocellulose with Ponceau Red (0.2% w/v H2O diluted 99:1 in 10% trifluoroacetic acid). Filters were blocked in 5% low fat milk in Tris-buffered saline (TBS) (25 mM Tris, pH 7.5, 0.9% NaCl) and then incubated for 2 h with the first antibody diluted at 1/ 1000 in 5% low fat milk in TBS containing 1% Tween 20. The filters were washed with TBS containing 1% Tween 20 and incubated with peroxidase-conjugated secondary antibody in 5% low fat milk in TBS containing 1% Tween 20 for 1hour. Peroxidase activity was detected using an ECL Western Blotting detection procedure (Amersham). 2.7. Testosterone hydroxylation assays Human primary hepatocytes, treated or not by oltipraz for 24 h or 72 h, were washed by PBS and then incubated with 0.2 mM testosterone in 300 ll of phenol red-free MEM medium. After 2 h, each cell medium was recovered for high performance liquid chromatography (hplc) analysis and the cells were kept for protein measurement. Microsomal extracts (50 lg) from human hepatocytes were incubated with 100 lM testosterone, 1 mM NADPH and different concentrations of oltipraz (0–20 lM) in 500 ll of phosphate buffer (0.1 M, pH 7.6) for 30 min. Twenty pmol of CYP2B6 Supersomes (Human CYP2B6 + P450 reductase BD SupersomesTM Enzyme, BD Biosciences) were incubated for 1 h in the same conditions as microsomes. The reaction was stopped with 100 ll of 1 M Na2CO3/2 M NaCl, and the reaction products were then extracted as previously described (Langouet et al., 1995). Testosterone metabolites were separated by hplc on a 5 lm particle Nucleosil C18 column (150  4.6 mm) (CIL Cluzeau, France) with a flow rate of 1 ml/min. The aqueous phase was water, containing 0.1% glacial acetic acid, and the organic phase was ethanol/acetonitrile (50/50). The reaction was followed spectrophotometrically at 254 mm and results were analysed using Agilent 1100 software.

Fig. 1. CYP2B6 mRNA and protein induction by oltipraz in primary cultures of human hepatocytes. A. Hepatocytes obtained from two different liver donors were treated with 50 lM oltipraz for 1, 2, 4, 8, 12, 24, 48 or 72 h or the vehicle only. Total RNA was extracted and CYP2B6 mRNA was quantified by real-time PCR as described in Section 2. mRNA levels are expressed as fold increase over the corresponding control (untreated cells) given the arbitrary value of 1. The expression level of CYP2B6 transcripts in human hepatocyte populations obtained from two different livers was normalized to that of 18S RNA, which was measured using the same cDNA. B. Human hepatocytes were untreated (UT) or treated with 50 lM oltipraz for 2, 4, 8, 12, 24, 48 or 72 h and CYP2B6 apoprotein levels were determined by Western Blot analysis. Calnexin (CANX) was used as a loading control. Western Blot data represent one of three independent experiments with similar results.

2.8. HepG2 cell culture and transfection assay

2.10. Bioinformatic analyses

HepG2 cells were cultured in a medium containing 10% FCS, 1% non essential aminoacids, penicillin and streptomycin (50U/ml). HepG2 were transfected in 24-well multiwell plates with lipofectamine 2000 (Invitrogen). For each well, we used 3 lg of pGL3 constructs containing different sizes of the CYP2B6 promoter upstream Firefly luciferase gene, 0.3 lg of pRL-tk (Renilla luciferase under the control of thymidine kinase promoter) with or without 0.7 lg of expression vector encoding PXR, CAR or Nrf2M. ‘‘Dual Luciferase-Reporter Assay System” (Promega), which allows measurement of Firefly and Renilla luciferase activities, was used as described in the manufacturer’s protocol.

The identification of potential regulatory elements in CYP2B6 promoter was conducted using the internet software Matinspector (Genomatix, http://www.genomatix.de/) with the following parameters: 100% core sequence similarity and 85% matrix similarity. The CYP2B6 promoter sequence was extracted from the contig nucleotide sequence NT_01139.

2.9. Electrophoretic mobility shift assays Human hepatocytes were cultured as described above and nuclear extracts were isolated with the ‘‘TransFactor Extraction kit” (Clontech) following the manufacturer recommendations. Binding of the nuclear extracts to [gamma-32P] ATP (GE Healthcare) end-labeled double-stranded oligonucleotides was performed with the ‘‘Gel Shift Assay System” (Promega). Top strand sequences for the binding sites were as follows: ARE-like sequence from -1797 to 1773 (50 -gtgtcagatgacacagcacagcaag-30 ), the mutated ARE (50 -tagtgtcagaggacacagcacagcaag-30 ), the nuclear receptor binding site PBREM sequence spanning from -1765 to -1706 (50 -cccttggttcaggaaagtccatgctgccacctcttcagggtcaggaaagtacagtttcca-30 ) and a mutated PXR binding site. Supershift experiments were performed with 500 ng of anti-Nrf2 or anti-PXR antibodies.

2.11. Statistical analysis The results are presented as mean ± SD from at least three independent experiments. The non parametric Mann–Whitney test was applied to compare CYP2B6 or luciferase activities between two groups, i.e. cells treated with oltipraz vs. control cells. P values <0.05 were interpreted as significant. 3. Results 3.1. Effect of oltipraz on CYP2B6 mRNA and protein expression in human hepatocytes We have demonstrated that CYP2B6 mRNA and protein levels are increased after 24 and 48 h of oltipraz treatment in primary human hepatocytes (Piton et al., 2005). In order to extend this observation, CYP2B6 mRNA expression levels were analysed by realtime PCR after different treatment times with oltipraz (1, 2, 4, 8, 12, 24, 48 and 72 h) in human hepatocyte populations obtained from two different livers (Fig. 1A). Although a difference in the

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Fig. 2. Effects of oltipraz on CYP2B6 and CYP3A4 activities in human hepatocytes. Human hepatocyte cultures from five different livers (HL7–HL11) were treated with vehicle (0.2% DMSO, v/v) or oltipraz for 24 h or 72 h, then incubated with testosterone as described in Section 2. A. CYP2B6 activity measured by 16bhydroxylation of testosterone. B. CYP3A4 activity measured by 6b-hydroxylation of testosterone. Activity values are expressed in pmol of products per minute per mg of proteins and correspond to the mean ± SD of measurements obtained from three different wells. Statistical analysis (Mann–Whitney test) was performed by comparison of oltipraz-treated and control hepatocytes (*, p < 0.05; **, p < 0.01).

level of CYP2B6 mRNA increase was observed, the kinetics of induction were very similar in both cases: an increase of CYP2B6 mRNA steady-state levels was observed 4 h after oltipraz addition and reached the maximal value after 24 h of treatment. The CYP2B6 protein was also induced with a maximum increase after 48 h of treatment (Fig. 1B). 3.2. Effect of oltipraz on CYP2B6 enzymatic activity in human hepatocytes In order to determine whether CYP2B6 mRNA and protein induction by OPZ lead to an increase of CYP2B6 activity, we measured this activity in cultures of human hepatocytes isolated from five different human livers. CYP2B6 activity can be measured by different ways such as S-mephenytoin, N-demethylation, bupropion hydroxylation or testosterone 16b-hydroxylation (Hanna et al., 2000; Imaoka et al., 1996). Testosterone was used as a substrate since it allows measuring CYP2B6 activity (16b-hydroxylation of testosterone) and CYP3A4 activity (6b-hydroxylation of testosterone) at the same time. Testosterone hydroxylation was assayed for 2 h in untreated and 24 h or 72 h oltipraz-treated hepatocytes. As shown in Fig. 2A, oltipraz treatment had no significant effect on CYP2B6 activity after either 24 or 72 h of treatment. In the same conditions, CYP3A4 activity (6b-hydroxytestosterone production) was significantly inhibited in the presence of oltipraz (Fig. 2B). Moreover, we also observed an inhibitory effect on 2a-hydroxylation of testosterone activity (data not shown). This activity is mainly dependent on CYP2C11 in rat while the human CYP involved has not been identified yet. Since we observed an increase in CYP2B6 mRNA and protein levels and no effect on the 16bhydroxylation of testosterone after oltipraz treatment, we hypothesized that oltipraz might have a direct inhibitory effect on this en-

Fig. 3. Effects of oltipraz on 16b-hydroxylation of testosterone in human hepatocyte microsomes or CYP2B6 supersomes. A. Hepatocyte microsomes were extracted as described in Section 2. Fifty lg of microsomal proteins were incubated with different concentrations of oltipraz (2.5, 5, 10 and 20 lM) or vehicle (0.2% DMSO) before measurement of 16b-hydroxylation of testosterone. Activities are expressed in pmol of products formed per minute per mg of microsomal proteins and correspond to the mean ± SD of three measurements. Statistical analysis (Mann– Whitney test) was performed by comparison of oltipraz-treated and control microsomes (*, p < 0.05; **, p < 0.01). B. Twenty pmol of CYP2B6 Supersomes were incubated with DMSO (0.2%) or oltipraz (OPZ 20 lM) and 16b-hydroxylation testosterone was measured. Results are expressed as the percentage of activity in CYP2B6 supersomes incubated with oltipraz versus CYP2B6 supersomes incubated with DMSO and correspond to the mean ± SD of three measurements. Statistical analysis (Mann–Whitney test) was performed by comparison of oltipraz-treated and control CYP2B6 supersomes (**, p < 0.01).

zyme activity. Such an inhibitory effect of oltipraz has already been observed for other human CYPs, such as CYP1A2, 1B1, 1A1 and 3A4 (Langouet et al., 1995, 2000). 3.3. Inhibitory effect of oltipraz on 16b-hydroxylation of testosterone To test the hypothesis of an inhibitory effect of oltipraz on CYP2B6 enzyme activity, we measured 16b-hydroxylation of testosterone in human hepatocyte microsomes incubated with 0.2% DMSO (vehicle) or with different concentrations of oltipraz (Fig. 3A). Interestingly, a dose-dependent inhibition of testosterone 16b-hydroxylation was observed. A 50% inhibition was attained with 20 lM oltipraz. Since a recent study has suggested that CYP3A4 could have a very slight residual testosterone 16b-hydroxylation activity (Hanna et al., 2000), we verified our results using recombinant supersomes which only expressed CYP2B6 and the NADPH cytochrome P450 reductase. These supersomes were incubated in the same conditions as microsomes, and we confirmed the direct inhibitory

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Table 1 Effects of oltipraz treatment (24 or 72 h) on CYP3A4 and CYP2C9 mRNA levels in hepatocytes obtained from five different human livers. Values are expressed as ratio of mRNA levels (oltipraz-treated/control). *Data from Piton et al. (2005), nd: not determined. HL1

CYP3A4 CYP2C9 CYP2B6*

HL3

HL4

HL5

HL6

Mean ± SD

24 h

72 h

24 h

72 h

24 h

72 h

24 h

72 h

24 h

72 h

24 h

72 h

0.55 1.3 6.9

0.57 0.73 6

3.05 1.0 5.2

2.2 0.60 4.5

1.5 0.62 12.7

1.0 0.53 10.6

2.1 0.76 6.8

1.2 0.68 3.7

2.2 0.56 nd

2.9 0.50 nd

1.9 + 0.9 0.86 + 0.3 7.9 + 3.2

1.6 + 1.0 0.61 + 0.1 6.2 + 3

effect of oltipraz on 16b-hydroxylation activity (32% inhibition) (Fig. 3B). The 16b-hydroxylation activity of CYP2B6 obtained here, i.e. 0.22 nmol/min/nmol CYP2B6, was consistent with those described in other studies (Hanna et al., 2000; Imaoka et al., 1996). 3.4. Effects of oltipraz treatment on human CYP3A4 and CYP2C9 mRNA levels Like the two other human CYPs, CYP3A4 and CYP2C9, CYP2B6 is known to be regulated by xenobiotics via orphan nuclear receptor pathways, particularly PXR. We therefore decided to study the effects of an oltipraz treatment on expression of these two CYPs by real-time PCR. This experiment was performed with hepatocytes obtained from five different human livers used in a previous study (Piton et al., 2005). Oltipraz increased CYP3A4 mRNA levels in three hepatocyte populations and fold-inductions were lower than

those observed for CYP2B6 (Table 1). A slight inhibitory effect was observed for one liver (HL1). No induction of CYP2C9 by oltipraz was observed. 3.5. Identification of CYP2B6 promoter elements responding to oltipraz treatment The rapid increase of CYP2B6 mRNA levels in oltipraz-treated cells suggests the involvement of a transcriptional mechanism. To test this hypothesis, we performed transient transfections with pGL3 constructs containing the CYP2B6 gene 50 -flanking region upstream of the luciferase gene in order to measure the transcriptional activity of CYP2B6 in human hepatoma HepG2 cells in the absence or presence of oltipraz. Neither pGL3-basic nor pGL3-promoter luciferase activities were affected after oltipraz treatment (data not shown). In HepG2 cells transfected with the 2B6-1995

Fig. 4. Effects of oltipraz on CYP2B6 promoter activity in transiently transfected HepG2 cells. Constructs consisting of the firefly luciferase gene controlled by different sizes of the CYP2B6 gene 50 -flanking region were transfected into HepG2 cells. Twenty-four hours later, the cells were treated or not with 50 lM oltipraz for 16 h. A. The different sizes of CYP2B6 promoter sequence used in transfection experiments are shown with ARE (grey boxes) and PBREM (black boxes) sites. White boxes represent mutated ARE (2B61995-AREm) and PBREM (2B6-1995-PBREMm) sequences. B. Comparison of the luciferase activities between control and oltipraz-treated HepG2 cells transfected with 2B6luciferase construct or co-transfected with 2B6-luciferase construct and either PXR expression vector or Nrf2M expression vector. Luciferase activity has been normalized to that of the co-transfected plasmid expressing Renilla luciferase. The values represent the mean ± SD of five independent experiments performed in triplicate. Statistical analysis (Mann–Whitney test) was performed by comparing oltipraz-treated and control cells (*, p < 0.01).

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construct (which contains 1995 bp of the CYP2B6 gene 50 -flanking region upstream of the luciferase gene), oltipraz had no effect on the luciferase activity compared to untreated cells (Fig. 4). However, since the nuclear factors PXR and CAR are not expressed in the human hepatoma HepG2 cell line, we co-transfected the 2B61995 construct with PXR and CAR expression vectors. Co-transfection of the 2B6-1995 construct with a PXR expression vector resulted in a 3-fold increase of the luciferase activity (p < 0.01) when cells were treated by oltipraz, showing that PXR plays a role in CYP2B6 induction by oltipraz. By contrast, co-transfection of 2B6-1995 with a CAR expression vector resulted in an increase of basal luciferase activity but no differences between untreated and oltipraz-treated cells were observed (data not shown). Interestingly, there was no significant variation of luciferase activity between untreated and oltipraz-treated cells when transfection was performed with a construct containing only 1663 bp from the 50 flanking region of the CYP2B6 gene. Analysis of the CYP2B6 50 flanking region sequence between -1995 and -1663 with MatInspector (Genomatix) demonstrated the presence of an ARE-like sequence from -1797 to -1773 and a Phenobarbital Responsive Enhancer Module (PBREM). The latter, located between -1765 and -1706 bp, has been previously described by Sueyoshi et al. (Sueyoshi et al., 1999) who showed that CAR and PXR are involved in the transcriptional regulation of CYP2B genes through the PBREM. Consistent with the implication of PXR, mutation of the PBREM suppressed oltipraz induction of CYP2B6 promoter activity (Fig. 4). To investigate the involvement of Nrf2/ARE pathway in oltipraz-dependent induction of CYP2B6, we co-transfected 2B61995 with a dominant-negative Nrf2 (Nrf2M) expression vector, which has been obtained by deleting the activation domain (amino acid residues 1–392) and only contains the heterodimerization domains (Alam et al., 1999). Interestingly, there was no significant variation of luciferase activity between untreated and oltipraztreated cells co-transfected with 2B6-1995, PXR and Nrf2M expression vectors. When HepG2 cells were co-transfected with 2B61995-AREm (containing a mutated ARE) and PXR, a slight but not significant increase of luciferase activity was observed in the presence of oltipraz when compared to control cells, suggesting that the Nrf2/ARE pathway might also play a role in CYP2B6 regulation by oltipraz. The ability of the putative ARE sequence to bind Nrf2

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was assessed by an electrophoretic mobility shift assay (EMSA) using nuclear extracts from cultured human hepatocytes treated or not with oltipraz. For these studies, two double-stranded DNA probes spanning the ARE-like sequence from -1797 to -1773 and a mutated ARE were prepared. Fig. 5 shows that the ARE-like sequence bound a complex whereas no band was observed with mutated ARE. A 50-fold molar excess of unlabelled ARE-like was able to efficiently compete for the bound nuclear protein component(s). Furthermore, an increase of the binding was observed after oltipraz treatment. The same approach was used to investigate the binding capacity of a double-stranded DNA probe spanning the PBREM sequence from -1765 to -1706. This sequence also bound a complex which disappeared in the presence of a 50-fold molar excess of unlabelled PBREM. As previously observed with the ARE-like sequence, treatment with oltipraz slightly increased the binding capacity of PBREM. Furthermore, the use of antibodies directed against Nrf2 or PXR induced a decreased intensity of the shifted bands and the appearance of supershifted bands.

4. Discussion The pleiotropic effects of oltipraz, a chemopreventive agent, are now well recognized, and previous studies have demonstrated that this compound can induce both phase I and phase II enzymes in either the liver or extrahepatic tissues. Recently, we have demonstrated an increase of CYP2B6 expression in human hepatocytes treated for 24 and 72 h with oltipraz (Piton et al., 2005). The results presented here extend this observation and show that oltipraz is also an inhibitor of CYP2B6 activity. We demonstrate that CYP2B6 mRNA expression is increased as early as 4 h after oltipraz addition suggesting a transcriptional activation of the corresponding gene. This augmentation of CYP2B6 mRNA levels is followed by an induction of the corresponding protein. Surprisingly, when CYP2B6 activity was measured by the 16b-hydroxylation of testosterone, no significant variations were observed. This led us to hypothesize an inhibition of CYP2B6 activity by oltipraz. Indeed, previous works reported that oltipraz influences CYP activities and is a potent inhibitor of both CYP1A and 2B in rat liver in vivo and in vitro (Langouet et al., 1997) and of CYP1A2 and 3A4 in primary human

Fig. 5. In vitro binding of nuclear proteins to the CYP2B6-ARE and -PBREM promoter fragments. Nuclear extracts, prepared from human hepatocytes cultured in absence or presence of oltipraz for 0.5, 2 and 4 h, were incubated with CYP2B6-ARE oligonucleotide (A) or with an oligonucleotide corresponding to the CYP2B6-PBREM sequence (B). Incubation steps were performed without or with a 50-fold excess of cold competitor or with 0.5 lg of anti-Nrf2 or anti-PXR antibodies. Incubation of nuclear extracts with anti-Nrf2 or anti-PXR antibody induced a decreased intensity of the shifted bands and the appearance of supershifted bands. Binding specificities were also assessed by using mutated ARE or PBREM sequences.

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hepatocyte cultures (Langouet et al., 1995). Interestingly, by using testosterone as a CYP2B6 substrate we also demonstrated a decrease of 16b-hydroxylation with either hepatic microsomes or CYP2B6 supersomes, confirming the inhibitory effect of oltipraz. In the experimental conditions we used, the dual effect of oltipraz on both expression (induction) and activity (inhibition) of CYP2B6 led to an absence of effect of this compound on CYP2B6 global activity in human hepatocytes in culture. This finding suggests the likelihood of significant drug interactions between oltipraz and drugs whose metabolism is mediated by CYP2B6. Previous reports indicate that oltipraz is able to increase transcriptional activity of phase II enzymes and CYP1A1 genes through the binding of transcriptional factors to the ARE and XRE sequences, respectively. The present study clearly demonstrates the involvement of PXR/PBREM pathways in oltipraz induction of CYP2B6. The PBREM, located between -1765 and -1706 bp in the CYP2B6 gene’s 50 -flanking region, contains a core of two direct repeat nuclear receptor binding sites, NR-1 and NR-2. This sequence mediates phenobarbital induction and is transactivated by CAR or PXR which bind as CAR/RXR or PXR/RXR heterodimers. When constructs containing the 50 -flanking region of CYP2B6 upstream the luciferase gene where transfected in HepG2 cells, oltipraz treatment had no effect on luciferase activity, since HepG2 cells expressed very low levels of PXR. The presence of PXR in oltipraztreated cells is necessary to observe an increase of luciferase activity. Furthermore, oltipraz had no effect on luciferase activity in HepG2 cells transfected with a vector containing a mutated PBREM (2B6-1995-PBREMm). Finally, the ability of PBREM sequence to bind nuclear extracts from untreated and oltipraz-treated cells was examined by EMSA. In close agreement with the cell-based reporter gene assays described above, we observed a slight increase of binding complex in the presence of nuclear extracts isolated from oltipraz-treated cells when compared to untreated cells. In order to further confirm that PXR is present in the binding complex observed by EMSA, we used an antibody directed against PXR. In these conditions, we observed a decrease of the shifted band intensity and the appearance of supershifted band which confirmed that the complex formed with PBREM sequence involves PXR. Altogether, these observations indicate that both PXR and PBREM are involved in the regulation of CYP2B6 gene by oltipraz. Interestingly, we also found the presence of a functional ARE-like sequence located between -1797 to -1773 bp in the 50 -flanking region of the CYP2B6 gene, very close to the PBREM. Co-transfection of 2B61995 with Nrf2M led to a strong diminution of oltipraz effects. Regarding the role of ARE/Nrf2 pathway in CYP2B6 induction by oltipraz, our results suggest that it might be involved in the regulation of CYP2B6 expression since an increase of an ARE-protein binding complex, determined by EMSA, was observed with nuclear extracts isolated from hepatocytes incubated with oltipraz when compared with untreated cells. However, oltipraz still had a slight but not significant effect on luciferase activity in HepG2 cells transfected with 2B6-1995-AREm, a construct containing a mutated ARE sequence. The close proximity of ARE and PBREM sequences in the 50 flanking region of the CYP2B6 gene as well as the involvement of PXR and potentially of Nrf2 in CYP2B6 induction might suggest a physical interaction between these two transcription factors. Interestingly, a previous study related to the regulation of rat glutathione transferase A2 gene by glucocorticoids mentioned a putative interaction between PXR and factors binding to the ARE to elicit the pregnane inducible response of glutathione transferase A2 gene (Falkner et al., 2001). Such a putative interaction between PXR and Nrf2 will have to be further investigated. Interestingly, a recent study has also demonstrated the involvement of CAR in induction of mouse cyp2b10 and human CYP2B6 by oltipraz (Merrell et al., 2008). In this study, the authors used a CYP2B6 pro-

moter-reporter construct for in vivo transcription assay in mouse liver and transient transfection of HepG2 cells, and suggested a potential role of PBREM in CYP2B6 induction by oltipraz. In our study, when constructs containing the 50 -flanking region of CYP2B6 gene upstream the luciferase gene and a CAR expression vector were co-transfected in HepG2 cells, oltipraz treatment had no effect on luciferase activity (data not shown). This lack of response to oltipraz in the presence of CAR might be related to HepG2 cells. Indeed, it has been shown that an overexpression of CAR in HepG2 cells, by using a CAR expression vector, leads to its accumulation in nucleus and then to its constitutive activation, even in absence of inducer (Kawamoto et al., 1999; Sueyoshi et al., 1999). In conclusion, we demonstrate for the first time the involvement of the human orphan receptor PXR in the induction of CYP2B6 expression in human hepatocytes treated with oltipraz. Therefore, our study adds one more transcriptional activation pathway to the list of already reported nuclear factors activated by oltipraz (i.e. Nrf2, CAR, AhR, NFkb and CEBP). Coordinate regulation of expression of several genes by multiple nuclear factors certainly explains the pleiotropic effects of this compound. We also identified a functional ARE in CYP2B6 promoter that might also be involved in this induction, via the binding of Nrf2. Finally, we have demonstrated an inhibitory effect of oltipraz on CYP2B6 activity. The balance between induction of CYP2B6 expression and inhibition of its activity raises new questions about oltipraz concerning its interaction with the metabolism of CYP2B6-dependent drugs and/or xenobiotics in the context of chemopreventive assays in human. Acknowledgements We thank the Biological Resource Centre (BRC) of Rennes for the supply of isolated human hepatocytes and Dr. Jean-Marc Pascussi for providing us plasmid expressing PXR. We also thank Drs. Dan Spiegelman and Vincent Hyenne for critical reading of the manuscript. This work was supported in part by the Institut National de la Sante et de la Recherche Medicale, the Association pour la Recherche sur le Cancer (ARC) and the Ligue 35 Contre le Cancer. Amelie Piton was the recipient of a fellowship from the Association pour la Recherche sur le Cancer. References Alam, J., Stewart, D., Touchard, C., Boinapally, S., Choi, A.M., Cook, J.L., 1999. Nrf2, a Cap’n’Collar transcription factor, regulates induction of the heme oxygenase-1 gene. The Journal of Biological Chemistry 274, 26071–26078. Auyeung, D.J., Kessler, F.K., Ritter, J.K., 2003. Mechanism of rat UDPglucuronosyltransferase 1A6 induction by oltipraz: evidence for a contribution of the Aryl hydrocarbon receptor pathway. Molecular Pharmacology 63, 119–127. Bae, E.J., Kim, S.G., 2005. Enhanced CCAAT/enhancer-binding protein beta-liverenriched inhibitory protein production by Oltipraz, which accompanies CUG repeat-binding protein-1 (CUGBP1) RNA-binding protein activation, leads to inhibition of preadipocyte differentiation. Molecular Pharmacology 68, 660– 669. Boone, C.W., Kelloff, G.J., Malone, W.E., 1990. Identification of candidate cancer chemopreventive agents and their evaluation in animal models and human clinical trials: a review. Cancer Research 50, 2–9. Buetler, T.M., Bammler, T.K., Hayes, J.D., Eaton, D.L., 1996. Oltipraz-mediated changes in aflatoxin B(1) biotransformation in rat liver: implications for human chemointervention. Cancer Research 56, 2306–2313. Chang, T.K., Weber, G.F., Crespi, C.L., Waxman, D.J., 1993. Differential activation of cyclophosphamide and ifosphamide by cytochromes P-450 2B and 3A in human liver microsomes. Cancer Research 53, 5629–5637. Clapper, M.L., 1998. Chemopreventive activity of oltipraz. Pharmacology & Therapeutics 78, 17–27. Coleman, S., Linderman, R., Hodgson, E., Rose, R.L., 2000. Comparative metabolism of chloroacetamide herbicides and selected metabolites in human and rat liver microsomes. Environmental Health Perspectives 108, 1151–1157. Coller, J.K., Krebsfaenger, N., Klein, K., Endrizzi, K., Wolbold, R., Lang, T., Nussler, A., Neuhaus, P., Zanger, U.M., Eichelbaum, M., Murdter, T.E., 2002. The influence of CYP2B6, CYP2C9 and CYP2D6 genotypes on the formation of the potent

A. Piton et al. / Toxicology in Vitro 24 (2010) 452–459 antioestrogen Z-4-hydroxy-tamoxifen in human liver. British Journal of Clinical Pharmacology 54, 157–167. Falkner, K.C., Pinaire, J.A., Xiao, G.H., Geoghegan, T.E., Prough, R.A., 2001. Regulation of the rat glutathione S-transferase A2 gene by glucocorticoids: involvement of both the glucocorticoid and pregnane X receptors. Molecular Pharmacology 60, 611–619. Faucette, S.R., Wang, H., Hamilton, G.A., Jolley, S.L., Gilbert, D., Lindley, C., Yan, B., Negishi, M., LeCluyse, E.L., 2004. Regulation of CYP2B6 in primary human hepatocytes by prototypical inducers. Drug Metabolism and Disposition 32, 348–358. Glintborg, B., Weimann, A., Kensler, T.W., Poulsen, H.E., 2006. Oltipraz chemoprevention trial in Qidong, People’s Republic of China: unaltered oxidative biomarkers. Free Radical Biology & Medicine 41, 1010–1014. Goodwin, B., Moore, L.B., Stoltz, C.M., McKee, D.D., Kliewer, S.A., 2001. Regulation of the human CYP2B6 gene by the nuclear pregnane X receptor. Molecular Pharmacology 60, 427–431. Hanna, I.H., Reed, J.R., Guengerich, F.P., Hollenberg, P.F., 2000. Expression of human cytochrome P450 2B6 in Escherichia coli: characterization of catalytic activity and expression levels in human liver. Archives of Biochemistry and Biophysics 376, 206–216. Hesse, L.M., Venkatakrishnan, K., Court, M.H., von Moltke, L.L., Duan, S.X., Shader, R.I., Greenblatt, D.J., 2000. CYP2B6 mediates the in vitro hydroxylation of bupropion: potential drug interactions with other antidepressants. Drug Metabolism and Disposition 28, 1176–1183. Hesse, L.M., He, P., Krishnaswamy, S., Hao, Q., Hogan, K., von Moltke, L.L., Greenblatt, D.J., Court, M.H., 2004. Pharmacogenetic determinants of interindividual variability in bupropion hydroxylation by cytochrome P450 2B6 in human liver microsomes. Pharmacogenetics 14, 225–238. Iida, K., Itoh, K., Kumagai, Y., Oyasu, R., Hattori, K., Kawai, K., Shimazui, T., Akaza, H., Yamamoto, M., 2004. Nrf2 is essential for the chemopreventive efficacy of oltipraz against urinary bladder carcinogenesis. Cancer Research 64, 6424– 6431. Imaoka, S., Yamada, T., Hiroi, T., Hayashi, K., Sakaki, T., Yabusaki, Y., Funae, Y., 1996. Multiple forms of human P450 expressed in Saccharomyces cerevisiae. Systematic characterization and comparison with those of the rat. Biochemical Pharmacology 51, 1041–1050. Jacobson, L.P., Zhang, B.C., Zhu, Y.R., Wang, J.B., Wu, Y., Zhang, Q.N., Yu, L.Y., Qian, G.S., Kuang, S.Y., Li, Y.F., Fang, X., Zarba, A., Chen, B., Enger, C., Davidson, N.E., Gorman, M.B., Gordon, G.B., Prochaska, H.J., Egner, P.A., Groopman, J.D., Munoz, A., Helzlsouer, K.J., Kensler, T.W., 1997. Oltipraz chemoprevention trial in Qidong, People’s Republic of China: study design and clinical outcomes. Cancer Epidemiology Biomarkers & Prevention 6, 257–265. Kang, K.W., Kim, Y.G., Cho, M.K., Bae, S.K., Kim, C.W., Lee, M.G., Kim, S.G., 2002. Oltipraz regenerates cirrhotic liver through CCAAT/enhancer binding proteinmediated stellate cell inactivation. FASEB Journal 16, 1988–1990. 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. Molecular and Cellular Biology 19, 6318–6322. Kensler, T.W., Egner, P.A., Dolan, P.M., Groopman, J.D., Roebuck, B.D., 1987. Mechanism of protection against aflatoxin tumorigenicity in rats fed 5-(2pyrazinyl)-4-methyl-1,2-dithiol-3-thione (oltipraz) and related 1,2-dithiol-3thiones and 1,2-dithiol-3-ones. Cancer Research 47, 4271–4277. Kensler, T.W., He, X., Otieno, M., Egner, P.A., Jacobson, L.P., Chen, B., Wang, J.S., Zhu, Y.R., Zhang, B.C., Wang, J.B., Wu, Y., Zhang, Q.N., Qian, G.S., Kuang, S.Y., Fang, X., Li, Y.F., Yu, L.Y., Prochaska, H.J., Davidson, N.E., Gordon, G.B., Gorman, M.B., Zarba, A., Enger, C., Munoz, A., Helzlsouer, K.J., et al., 1998. Oltipraz chemoprevention trial in Qidong, People’s Republic of China: modulation of serum aflatoxin albumin adduct biomarkers. Cancer Epidemiology Biomarkers & Prevention 7, 127–134. Ko, M.S., Lee, S.J., Kim, J.W., Lim, J.W., Kim, S.G., 2006. Differential effects of the oxidized metabolites of oltipraz on the activation of CCAAT/enhancer binding protein-beta and NF-E2-related factor-2 for GSTA2 gene induction. Drug Metabolism and Disposition 34, 1353–1360. Langouet, S., Coles, B., Morel, F., Becquemont, L., Beaune, P., Guengerich, F.P., Ketterer, B., Guillouzo, A., 1995. Inhibition of CYP1A2 and CYP3A4 by oltipraz results in reduction of aflatoxin B1 metabolism in human hepatocytes in primary culture. Cancer Research 55, 5574–5579. Langouet, S., Maheo, K., Berthou, F., Morel, F., Lagadic-Gossman, D., Glaise, D., Coles, B., Ketterer, B., Guillouzo, A., 1997. Effects of administration of the chemoprotective agent oltipraz on CYP1A and CYP2B in rat liver and rat hepatocytes in culture. Carcinogenesis 18, 1343–1349.

459

Langouet, S., Furge, L.L., Kerriguy, N., Nakamura, K., Guillouzo, A., Guengerich, F.P., 2000. Inhibition of human cytochrome P450 enzymes by 1,2-dithiole-3-thione, oltipraz and its derivatives, and sulforaphane. Chemical Research in Toxicology 13, 245–252. Le Ferrec, E., Ilyin, G., Maheo, K., Bardiau, C., Courtois, A., Guillouzo, A., Morel, F., 2001. Differential effects of oltipraz on CYP1A and CYP2B in rat lung. Carcinogenesis 22, 49–55. Le Ferrec, E., Lagadic-Gossmann, D., Rauch, C., Bardiau, C., Maheo, K., Massiere, F., Le Vee, M., Guillouzo, A., Morel, F., 2002. Transcriptional induction of CYP1A1 by oltipraz in human Caco-2 cells is aryl hydrocarbon receptor- and calciumdependent. The Journal of Biological Chemistry 277, 24780–24787. Lee, D.Y., Kim, J.W., Lee, M.G., 2007. Pharmacokinetic interaction between oltipraz and omeprazole in rats: competitive inhibition of metabolism of oltipraz by omeprazole via CYP1A1 and 3A2, and of omeprazole by oltipraz via CYP1A1/2, 2D1/2, and 3A1/2. European Journal of Pharmaceutical Sciences 32, 328–339. Liu, Y.L., Roebuck, B.D., Yager, J.D., Groopman, J.D., Kensler, T.W., 1988. Protection by 5-(2-pyrazinyl)-4-methyl-1,2-dithiol-3-thione (oltipraz) against the hepatotoxicity of aflatoxin B1 in the rat. Toxicology and Applied Pharmacology 93, 442–451. Maheo, K., Morel, F., Antras-Ferry, J., Langouet, S., Desmots, F., Corcos, L., Guillouzo, A., 1998. Endotoxin suppresses the oltipraz-mediated induction of major hepatic glutathione transferases and cytochromes P450 in the rat. Hepatology 28, 1655–1662. Manandhar, S., Cho, J.M., Kim, J.A., Kensler, T.W., Kwak, M.K., 2007. Induction of Nrf2-regulated genes by 3H-1,2-dithiole-3-thione through the ERK signaling pathway in murine keratinocytes. European Journal of Pharmacology 577, 17– 27. Merrell, M.D., Jackson, J.P., Augustine, L.M., Fisher, C.D., Slitt, A.L., Maher, J.M., Huang, W., Moore, D.D., Zhang, Y., Klaassen, C.D., Cherrington, N.J., 2008. The nrf2 activator oltipraz also activates the constitutive androstane receptor. Drug Metabolism and Disposition 36, 1716–1721. Nho, C.W., O’Dwyer, P.J., 2004. NF-kappaB activation by the chemopreventive dithiolethione oltipraz is exerted through stimulation of MEKK3 signaling. The Journal of Biological Chemistry 279, 26019–26027. O’Dwyer, P.J., Johnson, S.W., Khater, C., Krueger, A., Matsumoto, Y., Hamilton, T.C., Yao, K.S., 1997. The chemopreventive agent oltipraz stimulates repair of damaged DNA. Cancer Research 57, 1050–1053. Pietsch, E.C., Chan, J.Y., Torti, F.M., Torti, S.V., 2003. Nrf2 mediates the induction of ferritin H in response to xenobiotics and cancer chemopreventive dithiolethiones. The Journal of Biological Chemistry 278, 2361–2369. Piton, A., Le Ferrec, E., Langouet, S., Rauch, C., Petit, E., Le Goff, F., Guillouzo, A., Morel, F., 2005. Oltipraz regulates different categories of genes relevant to chemoprevention in human hepatocytes. Carcinogenesis 26, 343–351. Rao, C.V., Rivenson, A., Katiwalla, M., Kelloff, G.J., Reddy, B.S., 1993. Chemopreventive effect of oltipraz during different stages of experimental colon carcinogenesis induced by azoxymethane in male F344 rats. Cancer Research 53, 2502–2506. 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. The Journal of Biological Chemistry 274, 6043–6046. Tang, J., Cao, Y., Rose, R.L., Brimfield, A.A., Dai, D., Goldstein, J.A., Hodgson, E., 2001. Metabolism of chlorpyrifos by human cytochrome P450 isoforms and human, mouse, and rat liver microsomes. Drug Metabolism and Disposition 29, 1201– 1204. Usmani, K.A., Rose, R.L., Goldstein, J.A., Taylor, W.G., Brimfield, A.A., Hodgson, E., 2002. In vitro human metabolism and interactions of repellent N,N-diethyl-mtoluamide. Drug Metabolism and Disposition 30, 289–294. Wang, H., Faucette, S., Sueyoshi, T., Moore, R., Ferguson, S., Negishi, M., LeCluyse, E.L., 2003. A novel distal enhancer module regulated by pregnane X receptor/ constitutive androstane receptor is essential for the maximal induction of CYP2B6 gene expression. The Journal of Biological Chemistry 278, 14146– 14152. Yao, K.S., O’Dwyer, P.J., 1995. Involvement of NF-kappa B in the induction of NAD(P)H:quinone oxidoreductase (DT-diaphorase) by hypoxia, oltipraz and mitomycin C. Biochemical Pharmacology 49, 275–282. Zhang, B.C., Zhu, Y.R., Wang, J.B., Wu, Y., Zhang, Q.N., Qian, G.S., Kuang, S.Y., Li, Y.F., Fang, X., Yu, L.Y., De Flora, S., Jacobson, L.P., Zarba, A., Egner, P.A., He, X., Wang, J.S., Chen, B., Enger, C.L., Davidson, N.E., Gordon, G.B., Gorman, M.B., Prochaska, H.J., Groopman, J.D., Munoz, A., Kensler, T.W., 1997. Oltipraz chemoprevention trial in Qidong, Jiangsu Province, People’s Republic of China. Journal of Cellular Biochemistry 28–29, 166–173.