Introduction

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The cytochrome P450 (P450) superfamily of enzymes is responsible for the metabolism and inactivation of a chemically diverse group of xenobiotics and endobiotics. Inflammation has been observed clinically to reduce P450-related hepatic drug metabolism (Renton et al., 1980; Sonne et al., 1985). In human and animal experimental systems, we and others have demonstrated a significant decrease in hepatic P450 activity following induction of the acute phase response (APR) by the administration of bacterial endotoxin (LPS) (Shedlofsky et al., 1994, 1997; Sewer et al., 1996; Roe et al., 1998; Warren et al., 1999, 2001). Elevated levels of cytokines [e.g., tumor necrosis factor-alpha (TNF), interleukin-6 (IL-6), and interleukin-1 (IL-1)] have been implicated as the primary mediators of the APR and the accompanying decline in hepatic P450 expression (Heinrich et al., 1990; Schindler et al., 1990; van Deventer et al., 1990; Parmentier et al., 1993, 1997; Warren et al., 1999, 2001). Using "knockout" mice deficient in TNF or IL-6 responses, we have shown that endogenous TNF and IL-6 modulate constitutive as well as LPS-induced changes in hepatic P450 expression (Warren et al., 1999, 2001). The mechanism by which cytokines affect P450 expression is presently unknown.

As with many proteins, P450 expression can be induced by changes in animal physiology, administration of drugs, and exposure to environmental toxins (for review see Ioannides, 1996). The induction of hepatic P450 metabolism by xenobiotics is an important mechanism of protection in cases of harmful exposure. However, clinically, it can cause pronounced drug concentration changes when an inducer is added to, or removed from, a treatment regimen (Fuhr, 2000). Phenobarbital (PB) is a well studied inducer of a number of P450 subfamilies, the two most significant being the 50- to 100-fold induction of CYP2B and the 2- to 3-fold induction of CYP3A activity in rodents (for review see Waxman and Azaroff, 1992). Consistent with the negative influence of proinflammatory cytokines on constitutive P450 expression, these same cytokines have been shown in a number of cultured hepatocyte investigations to significantly blunt PB induction of CYP2B and CYP3A (Williams et al., 1991; Abdel-Razzak et al., 1995; Clark et al., 1995, 1996). In rat hepatocytes, TNF, IL-1, IL-6, and interferon have all been shown to inhibit PB induction of CYP2B and CYP3A (Williams et al., 1991; Abdel-Razzak et al., 1995; Clark et al., 1995, 1996; Pascussi et al., 2000). The recent discovery and characterization of the "orphan" nuclear receptor CAR, a critical transcriptional regulator of CYP2B gene expression, provides a mechanism by which cytokines may alter PB induction of CYP2B. CAR, or the constitutively activated receptor, is constitutively present at low levels in the hepatocyte nucleus (for review see Sueyoshi and Negishi, 2001). Upon exposure to PB, CAR levels are greatly increased in the nucleus, where they dimerize with the retinoid X receptor-alpha (RXR) and bind to one of two DR4 nuclear receptor binding motifs (denoted NR1 and NR2) located within the PB-responsive enhancer module (PBREM) of CYP2B genes (Honkakoski et al., 1998). Recently, Pascussi et al. (2000) demonstrated in human hepatocytes that IL-6 exposure decreased PB induction of CYP2B6 mRNA in parallel with a marked decrease in CAR mRNA expression and nuclear accumulation. Therefore, it appears that in human hepatocyte cultures, IL-6 modulates PB induction of CYP2B by decreasing nuclear concentrations of CAR in response to PB.

In addition to inducing P450 expression, PB has been shown to have a profound mitogenic effect on the liver, demonstrated by increased liver weight, expansion of the smooth endoplasmic reticulum, increased microsomal protein content, and tumor promotion (Nims et al., 1987; Waxman and Azaroff, 1992; Sueyoshi et al., 1995). The mechanism by which PB exerts these effects is presently unknown; however, an inflammatory (Laskin et al., 1988) oxidative stress mechanism involving nuclear factor kappa-B (NF-B) activation has been suggested (Li et al., 1996). The relationship between the hepatic inflammatory mitogenic effect of PB and induction of P450 expression has yet to be established. In light of this, "knockout" mice deficient in TNF signaling, a cytokine instrumental in the APR, were used to investigate the role that endogenous TNF plays in modulating the PB induction profile of CYP2B and CYP3A. Additionally, electro-mobility shift assay was used to investigate the relationship of nuclear NF-B and CAR binding to alterations in PB-induced CYP2B activity and protein in these mice.

	Materials and Methods

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Animals and Treatments. Unless otherwise specified, all chemicals were obtained from Sigma Chemical (St. Louis, MO). Adult male mice, 12- to 14-weeks old were used for all studies. TNF (p55^//p75^/) double receptor knockout mice (ko-TNF) were generated as previously described (Zheng et al., 1995; Bruce et al., 1996) and maintained on a C57BL/6 × 129 hybrid background wild-type mice (wt-TNF). Animals were allowed food and water ad libitum. PB-treated animals were administered by intraperitoneal injection, PB (75 mg per kg) dissolved in 0.9% normal saline between 8:00 to 9:00 AM on 4-consecutive days. Control animals received an equivalent volume of normal saline. Food was removed at midnight on day 4, and the animals were euthanatized under halothane anesthesia at 8:00 to 10:00 AM on day 5. The livers were excised and treated as described below. All animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee of the University of Kentucky.

Microsomal Preparation and Spectral Analysis. Livers were perfused with ice-cold normal saline prior to microsomal fraction preparation as previously described (Warren et al., 1999). In brief, tissues were placed in homogenization buffer (0.154 M KCl, 0.25 M potassium phosphate buffer, pH 7.4) with butylated hydroxy toluene added as an antioxidant just prior to homogenization. Tissues were homogenized using a Teflon grinder and centrifuged at 10,000g to pellet the membranes. The supernatant was then recentrifuged at 105,000g to separate the microsomal and cytosolic fractions. The resulting microsomal pellet was resuspended in 0.25 M sucrose in 0.02 M Tris buffer, pH 7.4, and stored at 80°C until analyzed. Spectral analysis of total P450 content was performed according to the method of Omura and Sato (1964). Total protein content was determined by the method of Lowry et al. (1951).

Enzyme Activity Assays. P450 activities were determined using the formation of monohydroxylated products from substrates associated with specific P450 isoforms. The hydroxylated products of testosterone were determined by the method of Sonderfan et al. (1987). 16-Hydroxytestosterone (16-OHT) formation is catalyzed by the CYP2B subfamily (Sonderfan et al., 1987). Formation of 6-OHT has been attributed to the CYP3A subfamily (Yanagimoto et al., 1992). The dealkylation of pentoxyresorufin (PROD) to resorufin was performed by the methods of Burke et al. (1985). In mice, cyp2b10 has been shown to be the major enzyme involved in PROD activity (Honkakoski, 1992).

Analysis of P450 Protein Content by Enzyme-Linked Immunosorbent Assay. Protein concentrations of CYP2B and CYP3A in liver microsomal samples were quantified by a noncompetitive enzyme-linked immunosorbent assay (ELISA) as previously described (Warren et al., 1999). In brief, 0.25 to 1 µg of microsomal protein was plated in triplicate onto 96-well flat-bottomed plates (Corning Inc., Corning, NY). Known concentrations of microsomal standards (Gentest, Woburn, MA) were plated in duplicate to generate a standard curve for quantification. Plates were blocked with 50% horse serum and then incubated with polyclonal goat anti-rat antibody for CYP2B1 and CYP3A2 (Gentest). The plates were washed and incubated with alkaline phosphatase-conjugated monoclonal anti-goat/sheep IgG antibody. Following extensive washing, p-nitrophenol phosphate substrate (Neogen Corp., Lexington, KY) was added, and the plates were analyzed using a Bio-Tek EL340 microplate reader (Bio-Tek Instruments Inc., Winooski, VT) set at 405 nm and 37°C. It should be noted that the specificity of these Gentest anti-rat P450 polyclonal antibodies has not been definitively established for use in evaluating mouse P450 proteins; however, the ELISA results are supported by Western blot (described below, Fig. 1) and correlative analysis (data not shown). Specific protein content will be referred to by the subfamily designation (e.g., CYP2B and CYP3A protein content).

View larger version (62K): [in this window] [in a new window]   Fig. 1.   Western blot analysis demonstrating the specific binding of Gentest polyclonal goat anti-rat CYP2B1 and CYP3A2 antibodies in two experimental sets of TNF (p55//p75/) double receptor ko-TNF and wt-TNF mice. Ten micrograms of microsomal protein per lane were separated on a 10% polyacrylamide gel and blotted onto nitrocellulose. PB, phenobarbital-treated; V, saline vehicle-treated.

Western Blot Analysis. Ten micrograms of microsomal protein and standards were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (10% polyacrylamide) and blotted onto nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA). The nitrocellulose membranes were blocked with 5% nonfat milk in phosphate-buffered saline (PBS) and probed with polyclonal goat anti-rat CYP2B1 or CYP3A2 antibodies (Gentest) in PBS/1% nonfat milk. Following extensive washing in PBS/0.1% Tween 20, the membranes were incubated with alkaline phosphatase-conjugated monoclonal anti-goat/sheep IgG antibody. The membranes were washed, soaked in CDP-Star/Nitro-Block II (Tropix, Bedford, MA), and exposed to X-ray film according to the manufacturer's directions.

Nuclear Protein Preparation. Nuclear protein extracts were prepared by a modified protocol derived from Sueyoshi et al. (1995). In brief, a 5 mm³ piece of mouse liver was homogenized in 5 volumes of ice-cold buffer A (10 mM HEPES, pH 7.6, 10 mM KCl, 0.25 M sucrose, 10% v/v glycerol, 0.15 mM spermine, 1 mM EDTA, 1 mM dithiothreitol, 1 µg/ml pepstatin, 1 µg/ml aprotinin, 0.2 mM phenylmethylsulfonyl fluoride, and 50 mM sodium fluoride) and centrifuged at 15,000g for 1 min. at 4°C. The pellet was resuspended in 5 volumes of buffer A with 1.6 M sucrose and centrifuged (15,000g, 10 min at 4°C) through a 500-µl cushion of the same buffer. The pellet was lysed with 200 µl of lysis buffer (10 mM HEPES, 100 mM NaCl, 10% glycerol, 3 mM MgCl, 0.1 mM EDTA, 1 mM dithiothreitol, 1 µg/ml pepstatin, 1 µg/ml aprotinin, 0.2 mM phenylmethylsulfonyl fluoride, and 50 mM sodium fluoride) and incubated on ice for 15 min with agitation. The nuclei were pelleted by centrifugation at 15,000g (2 min, 4°C), and the cytosolic supernatant was collected and snap frozen on dry ice. The nuclear proteins were extracted with 100 µl of lysis buffer containing 0.4 M NaCl and incubated on ice for 30 min with agitation. The extracted nuclei were pelleted by centrifugation at 15,000g (2 min., 4°C), and the supernatant nuclear protein extract was collected and snap frozen on dry ice. Total protein concentrations were determined using the micro-Bio-Rad Protein Assay (Bio-Rad Laboratories) according to the manufacturer's directions. All samples were stored at 80°C until analysis.

Electrophoretic-Mobility Shift Assay. Electrophoretic-mobility shift assay essentially as described by Honkakoski et al. (1998) was used to determine CAR binding to the NR1 sequence of the cyp2b10 PBREM. Ten micrograms of nuclear protein extracts from three individual animals within each treatment group were pooled for analysis. Five micrograms of these pooled extracts were incubated for 15 min at room temperature with 75,000 cpm of gamma-³²P labeled cyp2b10 NR1 oligonucleotide (5'-ACTGTACTTTCCTGACCTTG-3' (Honkakoski et al., 1998) in 20 µl of 10 mM HEPES, pH 7.6, 0.5 mM dithiothreitol, 15% v/v glycerol, 4 µg of poly(dI·dC), 0.05% Nonidet P-40 (NP-40), and 50 mM NaCl. Competition reactions were carried out with 10- to 100-fold excess of unlabeled oligonucleotide. An NF-B-specific oligonucleotide described below was used as a nonspecific competitor for NR1 binding. NF-B binding was performed essentially as described by Roe et al. (1998). Ten micrograms of pooled extracts were incubated for 15 min at room temperature with 75,000 cpm of gamma-³²P labeled NF-B oligonucleotide (5'-AGATGAGGGGACTTTCCCAGGC-3'; Promega, Madison, WI) in 20 µl of 10 mM HEPES, pH 7.9, 1.5 mM MgCl₂, 1.0 mM dithiothreitol, 1.0 mM EGTA, 10% v/v glycerol, and 2 µg of poly(dI · dC). A 100-fold excess of a single base pair mutant NF-B oligonucleotide (Santa Cruz Biotechnology Inc., Santa Cruz, CA) was used as a nonspecific competitor. In antibody supershift experiments, reactions were preincubated for 15 min at room temperature with 1 µg of specific antibody, rabbit anti-RXR, rabbit anti-p50, rabbit anti-p65, and preimmune rabbit serum (Santa Cruz Biotechnology). Due to the unavailability of a CAR-specific antibody, CAR supershift experiments were not carried out. Therefore, nuclear protein binding to the cyp2b10 NR1 oligonucleotide is referred to as apparent CAR binding. Proteins were resolved by electrophoresis through a nondenaturing 7% polyacrylamide gel (acrylamide/bisacrylamide ratio, 30:1) in 45.0 mM Tris, pH 8.0, containing 45.0 mM borate and 1.0 mM EDTA. Gels were dried and exposed to X-ray film. All results from pooled samples were verified using nuclear protein extracts from individual animals.

Statistical Analysis. Multiple comparisons were performed using SPSS software (SPSS Inc., Chicago, IL). All comparisons were made via a two factor analysis of variance with Fisher's LSD post hoc determination. Statistical significance was set at p < 0.05.

	Results

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Enhanced PB Induction of Hepatic CYP2B and CYP3A Activity and Protein in TNF (p55^//p75^/) Double Receptor Knockout Mice. No significant differences in constitutive CYP2B or CYP3A activity or protein content were demonstrated between ko-TNF and wt-TNF mice given saline (Fig. 2, A and B). PB treatment significantly induced CYP2B activity in both ko-TNF and wt-TNF mice as demonstrated by PROD and 16-OHT activities (p < 0.05, Fig. 2A). In parallel, CYP2B protein content was significantly (p < 0.05) induced by PB in both ko-TNF and wt-TNF mice (Fig. 2A.). A significant interaction effect (p < 0.05) of TNF double receptor knockout mice on PB induction of CYP2B activity and protein was identified, demonstrating an enhanced induction of CYP2B in ko-TNF mice compared with wt-TNF mice. Similarly, CYP3A activity (6-OHT) and protein content were induced to a much greater extent in the ko-TNF mice compared with wt-TNF mice (p < 0.05 for the interaction, Fig. 2B).

View larger version (19K): [in this window] [in a new window]   Fig. 2.   Enhanced PB induction of hepatic CYP2B (A) and CYP3A (B) activities and protein in TNF (p55//p75/) double receptor ko-TNF mice compared with wt-TNF mice. PROD, 7-pentoxyresorufin-O-dealkylase activity; 16-OHT, testosterone 16-hydroxylase activity. Asterisks denote significant difference from wt-TNF for a given treatment [PB or saline vehicle (V)]; letters indicate a significant induction by PB (a, both wt-TNF and ko-TNF; b, ko-TNF only); and significance for the interaction is given, p < 0.05, n = 9.

Blunted Induction of NF-B Binding following PB Administration in TNF (p55^//p75^/) Double Receptor Knockout Mice. A constitutive elevation in NF-B binding is demonstrated by EMSA in ko-TNF mice nuclear extracts compared with wt-TNF mice given saline (Fig. 3). PB treatment induced NF-B binding in wt-TNF mice but failed to induce NF-B binding in ko-TNF mice. The specificity of the binding reaction is demonstrated by the complete loss of binding in the presence of 100-fold excess unlabeled NF-B oligonucleotide. Competition with a 100-fold excess of a single base pair mutant NF-B oligonucleotide had no effect on the binding reaction. Supershift analysis using antibodies to each of the individual components of the NF-B heterodimer (e.g., p50 and p65) demonstrates the presence of these proteins in the binding complexes.

View larger version (93K): [in this window] [in a new window]   Fig. 3.   Blunted induction of hepatic NF-B binding following PB administration in TNF (p55//p75/) double receptor knockout mice. Nuclear extracts were prepared from three experimental sets of animals and pooled as described under Materials and Methods. Ten micrograms of pooled nuclear extracts were incubated with 32P-NF-B oligonucleotide and subjected to gel shift assay. As positive and negative controls for NF-B binding, 2.5 µg of nuclear extract prepared from rats treated with LPS and saline, respectively, were used. Ten micrograms of pooled ko-TNF, PB nuclear extract were used for competition and supershift analysis. Specific competitions were performed with unlabeled competitor NF-B oligonucleotide. A single base pair mutant NF-B oligonucleotide was used as a nonspecific competitor. PB, phenobarbital-treated; V, saline vehicle-treated; NF-B, specific NF-B complex; NS, nonspecific NF-B complex.

Enhanced Apparent CAR Binding to the NR1 Nuclear Receptor Binding Motif of the cyp2b10 PBREM following PB Administration in the Livers of TNF (p55^//p75^/) Double Receptor Knockout Mice. A constitutive elevation in apparent CAR binding is demonstrated by EMSA in ko-TNF mice nuclear extracts compared with wt-TNF mice given saline (Fig. 4). Apparent CAR binding was induced by PB treatment in both wt-TNF and ko-TNF mice. However, the PB-induced apparent CAR binding in ko-TNF mice nuclear extracts is substantially greater than in wt-TNF mice. The specificity of the binding reaction is demonstrated by the substantial decrease in binding when 40-fold excess of unlabeled NR1 oligonucleotide is included in the reaction (Fig. 4). Due to the unavailability of a CAR-specific antibody, the presence of CAR in the complexes could not be directly confirmed. However, the loss of binding in supershift analysis using anti-RXR antibodies does demonstrate the presence of the dimerization partner RXR of CAR in these complexes.

View larger version (94K): [in this window] [in a new window]   Fig. 4.   Enhanced CAR binding to the NR1 nuclear receptor binding motif of the cyp2b10 PBREM following PB administration in TNF (p55//p75/) double receptor knockout mice. Nuclear extracts were prepared from three experimental sets of animals and pooled as described under Materials and Methods. Five micrograms of pooled nuclear extracts were incubated with 32P-cyp2b10 NR1 oligonucleotide and subjected to gel shift assay. Specific competitions were performed with unlabeled competitor CAR oligonucleotide. An unrelated NF-B-specific oligonucleotide was used as a nonspecific competitor. PB, phenobarbital-treated; V, saline vehicle-treated.

	Discussion

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This investigation provides the first evidence that endogenous TNF signaling negatively modulates PB induction of hepatic CYP2B and CYP3A in the intact mouse liver. Furthermore, it is demonstrated that endogenous TNF may influence PB induction of CYP2B through inhibiting the nuclear accumulation of a nuclear protein shown to heterodimerize with RXR and bind to the NR1 motif of the cyp2b10 PBREM. This nuclear protein is believed to be CAR; however, its identity was not directly assessed due to the unavailability of a specific antibody.

The inhibition of PB-induced CYP2B expression by TNF is not unprecedented. In rat hepatocyte cultures, Kawamura et al. (1999) demonstrated a significant suppression of PB-induced CYP2B1/2B2 mRNA and protein when rat hepatocytes were concurrently exposed to TNF. The study described herein provides evidence that endogenous TNF signaling inhibits the PB induction response of CY2B in mice.

In a study by Li et al. (1996), PB was shown to stimulate the nuclear accumulation of NF-B, which is known to be activated by a host of inflammatory and noninflammatory stimuli including TNF (Baldwin, 1996). In a previous study, we have shown that hepatic NF-B binding increases in a time-dependent manner preceding decreases in CYP3A1/2 expression and activity following LPS administration in rats (Roe et al., 1998). Therefore, we examined the nuclear binding of NF-B in wt-TNF and ko-TNF mice following PB and saline administration to determine whether NF-B nuclear binding paralleled alterations in PB-induced CYP2B activity and protein. EMSA analysis demonstrated enhanced NF-B binding in nuclear extracts from wt-TNF, but not ko-TNF, mice following PB treatment (Fig. 3). These results suggest that PB stimulation of NF-B nuclear binding occurs through a mechanism involving TNF signaling. Considering that acute inflammation accompanied by increased NF-B binding in the liver is known to have a negative impact on hepatic P450 expression (Roe et al., 1998), the absence of enhanced NF-B binding in ko-TNF mice following PB treatment is consistent with enhanced CYP2B and CYP3A induction in these mice. However, a similar absence of enhanced NF-B binding following PB treatment has been demonstrated in our laboratory in interleukin-6 gene knockout mice without a parallel increase in CYP2B or CYP3A induction (unpublished data). Therefore, the mechanism underlying enhanced hepatic CYP2B and CYP3A induction in ko-TNF mice may involve a blunted NF-B response to PB treatment; however, other mechanisms may be involved.

In the rat, Iber et al. (2000) demonstrated NF-B binding to a putative NF-B-response element spanning the CYP2C11 transcription start site. Binding of NF-B to this site was shown to play a central role in the down-regulation of CYP2C11 expression following IL-1 treatment (Iber et al., 2000). In another study, Lee et al. (2000) identified an atypical NF-B binding site that overlaps with a recombination signal-sequence-binding protein-J site in the rat CYP2B1 and mouse cyp2b10 promoters. Interestingly, this atypical NF-B binding site suppresses transcription of a reporter construct only when recombination signal-sequence-binding protein-J is bound but not when NF-B is bound (Lee et al., 2000). These data suggest that if NF-B activation negatively modulates PB induction of hepatic CYP2B and CYP3A, it most likely involves an indirect mechanism whereby NF-B modulates the transcription of other genes, such as CAR. Very little is known regarding the transcriptional regulation of CAR, including any role for NF-B in its transcription. However, it is known that treatment of human hepatocytes with IL-6 markedly decreases the expression of CAR mRNA and the principal nuclear protein involved in CYP3A induction, the pregnane X receptor (PXR) (Pascussi et al., 2000). Therefore, there is evidence that inflammatory cytokine signaling through their respective nuclear proteins negatively influences CAR mRNA expression. Further studies investigating the role of TNF and NF-B activation on CAR and PXR mRNA expression will be required to evaluate this mechanism.

It has been shown that the transcriptional activation of CYP2B genes by PB is principally controlled by a 51-base pair regulatory sequence, referred to as the PBREM. The PBREM sequence is conserved in PB-inducible CYP2B genes in humans and rodents but is mutated in noninducible CYP2B genes, such as cyp2b9 in mice (Honkakoski et al., 1998). A number of investigators have sought to identify the principal nuclear protein, or proteins, that bind to this sequence and initiate transcription of CYP2B genes in response to PB. Until recently, the principal protein involved eluded discovery. In 1998, Honkakoski et al., in an elegant study using DNA affinity chromatography with a DR4 nuclear receptor binding motif (NR1) known to be located within the PBREM, enriched two orphan nuclear hormone receptors from PB-treated mouse hepatic nuclear extracts. They went on to show that in mice treated with PB, binding of these orphan receptors (CAR and RXR) to the NR1 element was rapidly increased by PB prior to the induction of cyp2b10 mRNA expression. Furthermore, cotransfection of CAR and RXR in HepG2 and HEK293 cells synergistically activated the PBREM. Since this study, several previously unexplained phenomena of PB induction have been attributed to changes in nuclear concentrations of CAR. Yoshinari et al. (2001) determined that differences in nuclear CAR accumulation explained the sexually dimorphic induction of the CYP2B1 gene in Wistar-Kyoto rats. In another study, Pascussi et al. (2000) demonstrated that the loss of CYP2B6 mRNA inducibility by PB in human hepatocytes exposed to IL-6 resulted from decreased expression of CAR mRNA and nuclear accumulation of CAR in response to PB. In the present study, enhanced apparent CAR binding to the NR1 element is demonstrated to occur in parallel with enhanced PB induction of CYP2B in ko-TNF mice (Fig. 4), suggesting that endogenous TNF signaling negatively regulates nuclear accumulation of CAR. Enhanced CYP3A activity and protein induction by PB in ko-TNF mice in the present study suggests that endogenous TNF signaling may also be a negative regulator of PXR (Pascussi et al., 2000). Whether endogenous TNF signaling retards nuclear CAR accumulation at the level of CAR gene expression or translocation from the cytosol to the nucleus requires further study.

In conclusion, the data presented herein suggest that endogenous TNF signaling modulates PB induction of hepatic CYP2B and CYP3A isoforms in vivo. Further, enhanced hepatic CYP2B induction by PB in the absence of an intact TNF response occurred in parallel with an elevation in apparent nuclear CAR binding to the NR1 element, providing evidence that TNF signaling may influence PB induction of CYP2B through inhibition of CAR nuclear accumulation. Additionally, TNF may influence PB induction of hepatic P450 enzymes through stimulation of NF-B activation.