Introduction

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The superfamily of cytochrome P-450 (P-450) proteins consists of enzymes involved in metabolism of an array of endogenous and xenobiotic compounds (Nelson et al., 1996). Processes mediated by P-450 enzymes include steroidogenesis and metabolism of cholesterol, vitamin D3, and fatty acids but also biotransformation of innumerable drugs, environmental chemicals, and pollutants. Four of the cytochrome P-450 families (CYP1, CYP2, CYP3, and CYP4) are of relevance in drug and xenobiotic metabolism. Expression of many members of xenobiotic-metabolizing P-450 isoforms is induced by their substrates, thus allowing adaptation of metabolism. The liver, constituting the major site of biotransformation of xenobiotics, exhibits abundant basal expression and induction of xenobiotic-metabolizing cytochrome P-450 isoforms. Phenobarbital (PB) is regarded as the prototype of structurally unrelated inducers that affect expression of a specific spectrum of genes (reviewed by Honkakoski and Negishi, 1998). Although members of several P-450 subfamilies are inducible by PB (CYP2A, CYP2B, CYP2C, CYP3A, CYP4B), phenobarbital-dependent induction of the rat CYP2B1 isoform is most pronounced [up to 100-fold or greater increase of CYP2B1 protein in rat liver microsomes (Waxman and Azaroff, 1992)]. Several laboratories recently demonstrated the pivotal role of a phenobarbital-responsive enhancer found in the distal region of the rat CYP2B2 promoter in conferring CYP2B2 gene activation by phenobarbital (Trottier et al., 1995; Park et al., 1996; Stoltz et al., 1998). The relevance of a homologous region in conveying PB-dependent CYP2B induction was later confirmed in the mouse Cyp2b10 promoter (Honkakoski and Negishi, 1997). The ability to confer induction has recently been localized to a 51-bp sequence in the mouse Cyp2B10 promoter, termed the phenobarbital-responsive enhancer module (PBREM; reviewed by Honkakoski and Negishi, 2000). Sequences highly homologous to the mouse PBREM are found in the human CYP2B6 and the rat CYP2B1/CYP2B2 promoters (Sueyoshi et al., 1999).

In addition to participating in elimination of xenobiotics, P-450 activity may also contribute to generation of potentially toxic products. Many procarcinogens are metabolically activated by P-450 isoforms to ultimate carcinogens. CYP2B isoforms in particular are involved in activation of aflatoxin B1 or cyclophosphamide to genotoxic metabolites (Chang et al., 1993). Furthermore, reactive oxygen species (ROS) are released during the catalytic reaction cycle of P-450 (Heinemeyer et al., 1980). It is assumed that activity of P-450 systems constitutes a major source of intracellular ROS in the liver (Bondy and Naderi, 1994; Puntarulo and Cederbaum, 1998), indicating that excessive P-450 activity resulting from P-450 induction may lead to cellular damage caused by generation of noxious metabolites or ROS. Accordingly, adaptive mechanisms, repressing P-450 induction during cellular oxidative stress and thus lowering P-450-dependent production of active intermediates, would be expected to minimize cellular damage. Indeed, several lines of evidence suggest that ROS might play a negative regulatory role in CYP2B induction by phenobarbital. Induction of CYP2B isoforms by phenobarbital, as well as basal and induced expression of other P-450 isoforms, have been shown to be decreased by inflammatory processes in the liver (reviewed by Morgan, 1997), during which also enhanced liberation of ROS occurs. Several proinflammatory cytokines, including interleukin 6 and tumor necrosis factor-, and also other cytokines (e.g., epidermal growth factor), that may lead to intracellular ROS production via physiological signaling (Bae et al., 1997; reviewed by Morel and Barouki, 1999), have also been identified as mediators of repression of phenobarbital-dependent CYP2B1 induction in rat hepatocytes (Aubrecht et al., 1995; Clark et al., 1995; Carlson and Billings, 1996).

To investigate whether ROS might affect CYP2B1 induction, we used as a model system primary rat hepatocyte cultures in which inducibility of CYP2B1 expression by phenobarbital was retained. Although treatment of hepatocytes with H₂O₂ and the catalase inhibitor 3-amino-1,2,4-triazole (AT) decreased CYP2B1 mRNA induction by PB, induction was markedly enhanced by the antioxidant N-acetylcysteine. In primary hepatocytes transfected with a CYP2B1 promoter-luciferase construct, promoter activity was significantly repressed by 3-amino-1,2,4-triazole and, conversely, enhanced by N-acetylcysteine. These data, therefore, demonstrate for the first time that CYP2B1 mRNA induction by phenobarbital is modulated in a redox-sensitive manner and that transcriptional activation participates in redox-dependent regulation of CYP2B1 mRNA induction.

	Experimental Procedures

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Materials. All chemicals were of reagent grade and purchased from commercial suppliers. Collagenase was obtained from Biochrom (Berlin, Germany) and fetal calf serum from PAA (Coelbe, Germany). Cell culture dishes were purchased from Nunc (Wiesbaden, Germany). T4 polynucleotide kinase was from Roche Molecular Biochemicals (Mannheim, Germany). Hybond N nylon membrane and [-³²P]ATP were obtained from Amersham Pharmacia Biotech (Freiburg, Germany). The transfection reagent Effectene was purchased from Qiagen (Hilden, Germany). Firefly and Renilla luciferase expression plasmids as well as the Dual Luciferase Reporter Assay kit were obtained from Promega (Mannheim, Germany).

Hepatocyte Culture and Induction Experiments. Primary hepatocytes were isolated from adult male Wistar rats (180-220 g) by collagenase perfusion (Seglen, 1976). Hepatocyte suspensions showed viabilities >90% as determined by trypan blue exclusion. Cells were plated onto culture dishes at a density of 8.6 × 10⁴ cells/cm² in MX-82 medium (Hoffmann et al., 1989) supplemented with 10% fetal calf serum. After an initial attachment period of 3 h at 37°C in a humidified atmosphere of 10% CO₂ and 90% air, the medium was replaced with serum-free MX-83 medium (Hoffmann et al., 1989) that lacked arginine, but contained 1 µM insulin and 20 µM hydrocortisone hemisuccinate. The cells were further cultured (37°C, humidified atmosphere of 10% CO₂/90% air) in the absence or presence of 0.75 or 1.5 mM PB, with or without oxidants (0.1-2 mM H₂O₂ or 0.5-5 mM 3-amino-1,2,4-triazole) or antioxidants (1-20 mM N-acetylcysteine or 100 µM tocopherol acetate), as indicated, for up to 3 days. Media changes were performed daily.

RNA Isolation and Northern Blot Analysis. Total cellular RNA was isolated by guanidinium thiocyanate-phenol-chloroform extraction (Chomczynski and Sacchi, 1987) and separated through formaldehyde agarose gels (20 µg/lane). The gels were stained with ethidium bromide to ensure equal loading of lanes, and RNA was subsequently blotted onto Hybond N nylon membranes by capillary transfer. RNA blots were hybridized to the rat CYP2B1 gene-specific oligonucleotide probe 5'-GGTTGGTAGCCGGTGTGA-3' (corresponding to bases 66-49 of exon 7 region, GeneBank L00318), which had been end-labeled by T4-polynucleotide kinase using -³²P-ATP (Omiecinski et al., 1985).

Hepatocyte Transfection and Luciferase Reporter Gene Assay. Firefly and Renilla luciferase expression plasmids (pGL3-Basic, pGL3-Control, pGL3-Promoter, and pRL-CMV) were purchased from Promega. A fragment representing approximately 2.7 kilobase pairs of the native rat CYP2B1 promoter sequence was amplified by PCR from genomic rat hepatocyte DNA using primers corresponding to sites of the promoter sequence published by Shaw et al., 1996 (bases 2648 to 2623 and 29 to 4, respectively). To facilitate cloning, the primers contained an additional 5' NheI recognition site and a three-base overhang: 5'-AAAGCTAGCAGGTTCCCAACCATTTGGCCTACGAA-3' (forward) and 5'-ATTGCTAGCTCCTGGTGTAACCACGGTAGACTTCA-3' (reverse).

	Results

Top Abstract Introduction Experimental Procedures Results Discussion References

In the present study, we addressed whether phenobarbital-dependent CYP2B1 induction might be subject to repression by reactive oxygen species. Experiments were performed with primary rat hepatocyte cultures in which induction of CYP2B1 by phenobarbital, a hepatocyte-specific function lost in hepatoma cell lines, was retained (Aubrecht et al., 1995). Maximal CYP2B1 mRNA induction was observed in hepatocyte cultures exposed to 0.75 or 1.5 mM PB for three days, which is consistent with previous reports (Aubrecht et al., 1995; Kietzmann et al., 1999). Therefore, primary hepatocytes were treated with PB and possible modulators of CYP2B1 mRNA induction for 3 days.

Repression by H₂O₂ of Phenobarbital-Dependent CYP2B1 mRNA Induction. To examine the possible role of oxidants in modulation of CYP2B1 mRNA induction by PB, hepatocyte cultures were treated with H₂O₂, a highly diffusible molecule that easily penetrates through cellular membrane structures. A H₂O₂ concentration of 1 mM was previously shown to up-regulate mRNA expression of antioxidant enzymes (catalase, Mn-superoxide dismutase; Röhrdanz and Kahl et al., 1998) as well as expression of the rat multidrug resistance mdr1b gene (Ziemann et al., 1999) in primary rat hepatocyte cultures. Thus, in the present study, initial concentrations of H₂O₂ from 0.1 to 2 mM were added to the medium once daily for 3 days. PB-dependent CYP2B1 mRNA induction was determined in Northern blot analyses. Induction, defined as the difference between PB-dependent CYP2B1 mRNA expression and basal CYP2B1 mRNA levels in the absence of PB, was set to 100% in cultures treated with PB in the absence of H₂O₂. Indeed, H₂O₂ elicited repression of PB-dependent CYP2B1 mRNA induction in a concentration-dependent manner (Fig. 1). This effect reached statistical significance at concentrations of 0.5 mM H₂O₂ and above, amounting to mRNA levels of about 37% at 0.5 mM H₂O₂, 30% at 1 mM H₂O₂, and 12% at 2 mM H₂O₂, respectively (Fig. 1, A and B). Expression of GAPDH mRNA in control hybridizations remained stable under H₂O₂ (Fig. 1B). On the other hand, we observed stimulation of mdr1b gene expression by H₂O₂ in the same culture system (shown for 0.5 mM H₂O₂ in Fig. 1C), with maximal stimulation at 0.5 to 1 mM H₂O₂ (Ziemann et al., 1999). Thus, H₂O₂ significantly repressed PB-dependent CYP2B1 mRNA induction at concentrations that did not result in a deleterious general repression of mRNA expression.

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Repression by H2O2 of PB-dependent CYP2B1 mRNA induction in primary rat hepatocyte cultures. Hepatocytes were cultured for 3 days in the presence or absence of PB with or without H2O2. H2O2 was added to the medium at initial concentrations of 0.1 to 2 mM. Total RNA (20 µg/lane) was subjected to RNA blot analysis. A, CYP2B1 mRNA expression was determined in Northern blots by hybridization to a 32P-labeled CYP2B1-specific oligonucleotide probe as described under Experimental Procedures. Hybridization signals were quantified using a Bio-Imaging Analyzer system. Induction was defined as the difference between expression under 1.5 mM PB and expression in the absence of PB. Induction with 1.5 mM PB in the absence of H2O2 was set to 100%. Data represent mean values ± S.E.M. of three or five (0 mM and 1 mM H2O2) independent experiments. Significant difference between PB and PB+H2O2 (**p < 0.01; *** p < 0.001; Student's t test). B, representative Northern blot. After hybridization to a CYP2B1-specific probe, the blot was rehybridized to a rat GAPDH-specific oligonucleotide probe. C, stimulation of mdr1b mRNA expression as an example of mRNA up-regulation by H2O2 (0.5 mM); representative Northern blot. c, control cells without H2O2.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Repression of PB-dependent CYP2B1 mRNA induction by AT in primary rat hepatocyte cultures. Hepatocytes were cultured for 3 days in the presence of 1.5 mM PB with or without 0.5 to 5 mM AT. CYP2B1 mRNA expression was determined by Northern blot analysis as outlined in Fig. 1. Induction of CYP2B1 mRNA was defined as the difference between CYP2B1 mRNA expression under PB and basal mRNA expression in the absence of PB. Induction without AT was set to 100%. Data represent mean values of two (0.5 mM and 5 mM AT) or of seven (1 mM AT) or three (2 mM AT) independent experiments ± S.E.M. Significant difference between cells treated with PB only and cells treated with PB+AT (**p < 0.01; ***p < 0.001; Student's t test).

Enhancement by N-Acetylcysteine of Phenobarbital-Dependent CYP2B1 mRNA Induction. Because H₂O₂ and 3-amino-1,2,4-triazole repressed PB-dependent CYP2B1 mRNA induction, it was hypothesized that an increase in hepatocyte antioxidant capacity might enhance CYP2B1 mRNA induction or counteract ROS-dependent inhibition of induction. Therefore, the effect of the antioxidant N-acetylcysteine (NAC), a precursor of glutathione, on PB-dependent CYP2B1 mRNA induction was examined. In hepatocytes concomitantly treated with PB and 1 to 20 mM NAC for 3 days, a dramatic concentration-dependent enhancement of CYP2B1 mRNA induction was observed that was maximal (approximately 1000%) between 10 and 20 mM NAC (Fig. 3, A and B). The incubation of hepatocytes with PB and a 100 µM concentration of the antioxidant tocopherol acetate (vitamin E), a membrane-bound radical scavenger, also led to a moderate increase (to approximately 200%) of PB-dependent CYP2B1 mRNA induction (Fig. 3C).

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Enhancement of PB-dependent CYP2B1 mRNA induction by N-acetylcysteine or tocopherol acetate in primary rat hepatocyte cultures. Hepatocytes were cultured for 3 days in the presence or absence of PB with or without further addition of NAC (1-20 mM) or tocopherol acetate (T, 100 µM) to the medium. A, enhancement of PB-dependent CYP2B1 mRNA induction by NAC (representative Northern blot analysis). B, CYP2B1 mRNA hybridization signals were quantified by Bio-Imaging Analyzer analysis of blot A. Induction was defined as the difference between CYP2B1 mRNA expression in the presence of PB and mRNA expression in the absence of PB. Induction without NAC was set to 100%. C, enhancement of PB-dependent CYP2B1 mRNA induction by tocopherol acetate (representative Northern blot analysis).

View larger version (29K): [in this window] [in a new window]   Fig. 4.   Aminotriazole-dependent abolishment of enhanced CYP2B1 induction under N-acetylcysteine in primary rat hepatocyte cultures. Cells were cultured in the presence or absence of 1.5 mM PB with or without 1 mM AT or 10 mM NAC for 3 days. CYP2B1 mRNA expression was determined by Northern blot analysis. A, hybridization signals were quantified by a Bio-Imaging Analyzer. Induction was defined as the difference between expression under 1.5 mM PB and expression in the absence of PB. Induction with 1.5 mM PB in the absence of AT or NAC was set to 100%. Data represent mean values ± S.E.M. of five (PB/AT and PB/NAC/AT) or 12 (PB and PB/NAC) independent experiments. Significant difference between PB and PB + modulator of induction (***p < 0.001). t, significant abolishment by AT of NAC-dependent enhancement of CYP2B1 induction (p < 0.05), Student's t-test. B, representative Northern blot. After hybridization to a CYP2B1-specific oligonucleotide probe, the blot was rehybridized to a rat GAPDH-specific probe.

Repression of PB-dependent CYP2B1 Promoter Activation by Aminotriazole and Enhancement of Promoter Activation by N-Acetylcysteine. To investigate whether the modulation of PB-dependent mRNA induction by ROS and N-acetylcysteine might be based on regulation of CYP2B1 transcription, a CYP2B1 promoter-luciferase reporter gene construct was generated, bearing the native CYP2B1 promoter sequence from +30 to 2648 (Fig. 5A). Primary rat hepatocyte cultures were transiently transfected with the original luciferase vector without the CYP2B1 promoter insert (pGL3-Basic) or with the CYP2B1 promoter-luciferase construct (pGL3C2B1) 24 h after seeding of hepatocytes and subsequently treated with 1.5 mM PB or with a combination of PB and AT and/or NAC for 48 h. Firefly luciferase activity was determined in hepatocyte lysates as a measure of promoter activation. Although AT or NAC alone did not influence promoter activation, PB significantly induced reporter gene expression (Fig. 5B). Mean promoter activation by PB alone was set to 100%. In accordance with modulation of PB-dependent CYP2B1 mRNA induction, 2 mM AT repressed PB-dependent promoter activation to approximately 25%, whereas 10 mM NAC enhanced promoter activation to about 900%. In combination with PB and NAC, AT counteracted enhancement by NAC of promoter activation (Fig. 5B). Thus, these results support the conclusion that modulation of CYP2B1 mRNA induction by AT and NAC occurs on the transcriptional level.

View larger version (21K): [in this window] [in a new window]   Fig. 5.   Modulation of PB-dependent CYP2B1 promoter activation by 3-amino-1,2,4-triazole and N-acetylcysteine. Primary rat hepatocytes cultured for 1 day were transiently cotransfected with the pRL-CMV Renilla luciferase vector and with the original pGL3-Basic vector (pGL3-Basic) or with the pGL3C2B1 construct, in which luciferase transcription was driven by the native CYP2B1 promoter sequence, respectively. Six hours after transfection, cells were incubated with 1.5 mM PB, 2 mM AT, or 10 mM NAC, as indicated, for an additional 48 h, and firefly luciferase activity was subsequently determined in cell lysates as described under Experimental Procedures. A, schematic representation of the CYP2B1 promoter fragment ligated into the pGL3-Basic luciferase vector, yielding the pGL3C2B1 construct. PBREM, phenobarbital-responsive enhancer module. The arrow indicates the transcription start site. B, relative firefly luciferase activity in lysates of transfected hepatocytes, standardized according to Renilla luciferase activity. Control, cells transfected with the pGL3C2B1 construct, but not treated with a modulator of CYP2B1 expression. Data represent mean values of five parallel culture wells ± S.D. of an experiment representative of three independent hepatocyte preparations. Significant difference from cells treated with PB (*p < 0.05;** p < 0.01, Student's t test). tt, significant abolishment by AT of the NAC-dependent enhancement of gene activation by PB, p < 0.01.

View larger version (23K): [in this window] [in a new window]   Fig. 6.   A 263-bp CYP2B1 promoter fragment confers inducibility by PB and redox-dependent modulation of promoter activation when linked to the heterologous SV40 promoter. Primary rat hepatocyte cultures were cotransfected with the Renilla luciferase expression vector pRL-CMV and with the pGL3CS1 construct in which the firefly luciferase was driven by the SV40 promoter and a 263-bp sequence of the distal CYP2B1 promoter, indicated under A. Transfected cells were incubated with 1.5 mM PB or modulators of CYP2B1 induction (2 mM AT, 10 mM NAC) for 48 h. A, schematic representation of the enhancer/promoter area of the firefly luciferase expression vector pGL3CS1. The CYP2B1 promoter fragment is indicated by a continuous line, while adjacent sequences of the pGL3-Promoter vector are dotted. PBREM, phenobarbital-responsive enhancer module. B, relative firefly luciferase activity, standardized according to Renilla luciferase activity in hepatocyte lysates. Control, cells transfected with pGL3CS1, but not treated with a modulator of CYP2B1 gene expression. Data represent mean values of six parallel culture wells ± S.D. of representative experiment. Significant difference from cells treated with PB (**p < 0.01; ***p < 0.001, Student's t test). tt, significant abolishment by AT of the NAC-dependent enhancement of gene activation by PB, p < 0.01.

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

Extreme exposure to ROS is deleterious to the cell, resulting either in necrosis or apoptosis. A transient or moderate increase in ROS, however, may allow reconstitution of the cellular redox status, either by direct detoxification processes or by long-term adaptive alterations in gene expression (Morel and Barouki, 1999). Thus, ROS-dependent repression of activation of genes encoding enzymes contributing to ROS production, as well as induction of proteins participating in ROS detoxification, may be regarded as part of a general defense response to maintain cellular redox homeostasis. In the present study, H₂O₂ and the catalase inhibitor 3-amino-1,2,4-triazole, which interferes with decomposition of intracellular H₂O₂, repressed PB-dependent induction of CYP2B1 mRNA.

Cytochrome P-450-dependent systems are regarded as a major source of intracellular production of free radicals in hepatocytes, even in the uninduced state (Bondy and Naderi, 1994; Puntarulo and Cederbaum, 1998). Under conditions of enhanced exposure to ROS (e.g., during inflammation), marked induction of P-450 enzymes, resulting in further P-450-dependent ROS production, would be expected to result in oxidant damage to the cell. Therefore, adaptive mechanisms by which ROS might prevent induction of several P-450 isoforms in the liver seem feasible. Indeed, the transcriptional suppression by H₂O₂ of basal expression of the CYP1A1 and CYP1A2 isoforms and of their -naphthoflavone-dependent induction has been described previously (Barker et al., 1994). The present study demonstrates ROS-dependent repression of induction by a different type of inducer (phenobarbital) of a different P-450 isoform (CYP2B1). At this point, the questions of whether PB-dependent induction of other xenobiotic-metabolizing genes apart from CYP2B1 might also be subject to regulation by ROS and whether overlapping mechanisms might be crucial in redox-dependent regulation of P-450 induction remain to be resolved.

The xenobiotic-responsive elements (XREs), to which heterodimers of the aryl hydrocarbon receptor and the aryl hydrocarbon receptor nuclear translocator protein bind, play a pivotal role in conferring induction of CYP1A enzymes by aromatic hydrocarbons (Hankinson, 1995). The ability of AT and NAC in the present study to modulate promoter activation in CYP2B1 promoter-reporter gene constructs transiently transfected into primary hepatocytes supports the conclusion that regulation of CYP2B1 induction by ROS occurs on the transcriptional level. However, in contrast to CYP1A gene regulation, XRE regions have not been shown to be of relevance in mediating PB-dependent induction (Waxman, 1999); rather, the major PB-responsive region in CYP2B promoters is a PB-responsive enhancer (Trottier et al., 1995; Park et al., 1996; Stoltz et al., 1998), which has been delimited to the PBREM in the mouse Cyp2b10 promoter (reviewed by Honkakoski and Negishi, 2000). To further specify the CYP2B1 promoter region conveying modulation of PB-dependent gene activation by AT and NAC, a promoter construct was employed in which a 263-bp fragment of the CYP2B1 promoter, encompassing the 51-bp PBREM, was placed in front of the heterologous SV40 promoter (Fig. 6A). Responsiveness not only to PB but also to AT and NAC was retained in hepatocytes transfected with the heterologous promoter construct, which suggests that either the PBREM itself or functional synergism between the PBREM and a promoter region in the vicinity of the PBREM is involved in conferring redox-sensitive regulation of PB-dependent CYP2B1 promoter activation.

The cellular redox status may affect transcription by different pathways. First, ROS might lead directly to oxidation of sensitive moieties of transcription factors, (e.g., of thiol groups), thus resulting in possible alterations in transcription factor DNA binding, in translocation to the nucleus, or in trans-activation (reviewed by Morel and Barouki, 1999). The PBREM region contains a nuclear factor 1 (NF1) binding motif, flanked by two nuclear receptor binding sites (reviewed by Honkakoski and Negishi, 2000). Interestingly, studies conducted with hepatoma cells suggest that oxidation of NF1 protein binding to a proximal NF1 site in the CYP1A1 promoter, which acts in synergy with XRE regions, constitutes the basis of suppression of CYP1A induction by ROS: the NF1/CTF trans-activating domain was repressed by oxidative stress, mediated by a critical cysteine (Morel et al., 1999). On the other hand, a different study indicates that the H₂O₂-dependent suppression of CYP1A1 induction is linked to the potential of the XREs to confer transcriptional activation by aromatic hydrocarbons (Xu and Pasco, 1998). Further examples of redox-sensitive regulation have been provided for members of the nuclear receptor superfamily (Morel and Barouki, 1999). Although oxidation of a cysteine residue in the nuclear localization signal domain NL1 of the glucocorticoid receptor interferes with nuclear translocation (Okamoto et al., 1999), thiol oxidation in the DNA binding domain of the estrogen receptor seems to constitute the major factor contributing to redox-dependent estrogen receptor regulation (Liang et al., 1998).

Alternatively, ROS may indirectly modulate the activity of transcription factors by leading to an alteration in their phosphorylation status. ROS (H₂O₂) have been shown to enhance protein phosphorylation by reducing protein tyrosine phosphatase and serine/threonine phosphatase activities (Whisler et al., 1995).

Hyperphosphorylation of as-yet-unspecified proteins is known to lead to repression of CYP2B1 mRNA induction by phenobarbital: inhibition of serin/threonine phosphatases (e.g., by okadaic acid) or inhibition of phosphotyrosine phosphatases (e.g., by orthovanadate) both result in inhibition of CYP2B induction (Sidhu and Omiecinski, 1997; Honkakoski and Negishi, 1998; Kawamura et al., 1999). Phosphorylation of members of the nuclear receptor family may interfere with nuclear translocation: phosphorylation of the nuclear orphan receptor NGFI-B, which is induced by nerve growth factor, results in redistribution of a NGFI-B-retinoid-X-receptor complex out of the nucleus, reducing transcriptional activation (Katagiri et al., 2000). Nuclear receptor dimers consisting of the constitutive androstane receptor and the retinoid-X-receptor are thought to play an essential role in PB-dependent transcriptional activation of CYP2B genes via the PBREM (reviewed by Honkakoski and Negishi, 2000). Nuclear translocation of the constitutive androstane receptor protein into the nucleus was shown to be inhibited by the protein phosphatase inhibitor okadaic acid (Kawamoto et al., 1999), indicating that either hyperphosphorylation of the nuclear receptor protein itself or hyperphosphorylation of a different protein essential for the translocation process interferes with nuclear translocation.

Thus, it may be hypothesized that ROS may repress PB-dependent transcriptional activation of CYP2B1 gene expression directly, by oxidation of redox-sensitive transcription factors, or indirectly, by leading to an alteration in transcription factor phosphorylation status and/or by interfering with translocation of a transcription factor into the nuclear compartment.

In summary, the present study demonstrates the regulation of PB-dependent CYP2B1 gene activation by the cellular redox status. Repression of CYP2B1 induction under oxidative stress may be interpreted as part of a general defense strategy that is activated under enhanced exposure to ROS or when the antioxidative status of the hepatocyte is compromised.