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THE SIGNAL TRANSDUCTION PATHWAYS INVOLVED IN HEPATIC CYTOCHROME P450 REGULATION IN THE RAT DURING A LIPOPOLYSACCHARIDE-INDUCED MODEL OF CENTRAL NERVOUS SYSTEM INFLAMMATION

Department of Pharmacology (D.A., K.B.G., E.G.D.B.C., K.W.R.) and Department of Pediatrics, Isaak Walton Killam Health Center (K.B.G.), Dalhousie University, Halifax, Nova Scotia, Canada

(Received March 1, 2005; accepted July 6, 2005)

	Abstract

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It is well known that inflammatory and infectious conditions of the central nervous system (CNS) differentially regulate hepatic drug metabolism through changes in cytochrome P450 (P450); however, the pathways leading to this regulation remain unknown. We provide evidence delineating a signal transduction pathway for hepatic P450 gene expression down-regulation in an established rat model of CNS inflammation using lipopolysaccharide (LPS) injected (i.c.v.) directly into the lateral cerebral ventricle. Brain cytokine levels were elevated, and the expression of tumor necrosis factor and inhibitor of B alpha (IB) were increased in the liver following the i.c.v. administration of LPS, indicating the presence of an inflammatory response in the brain and liver. The expression of CYP2D1/5, CYP2B1/2, and CYP1A1 was down-regulated following CNS inflammation. The binding of several transcription factors [nuclear factor of the enhancer in B cells (NF-B), activator protein-1, cAMP response element binding protein, CCAAT-enhancer binding protein (C/EBP)] to responsive elements on P450 promoter regions was examined using electromobility shift assays. Binding of both NF-B and C/EBP to the promoter regions of CYP2D5 and CYP2B1, respectively, was increased, indicating that they play an important role in the regulation of these two isoforms during inflammatory responses. Evidence is also provided suggesting that the rapid transfer of LPS from the CNS into the periphery likely accounts for the down-regulation of P450s in the liver.

The effects of host defense and immune stimulation on P450 isoforms have been well documented (Renton, 2001; Morgan et al., 2002). Both viral and bacterial inflammatory conditions can lead to differential regulation of hepatic P450 isoforms (Renton and Nicholson, 2000; Nicholson and Renton, 2001). Cytokines are known to play a dominant role in this regulation; IFN, IL-1ß, IL-6, and TNF have been shown to down-regulate hepatic CYP1A1, CYP1A2, CYP2B1/2, and CYP3A1/2 in rat models of systemic inflammation when given alone or in combination (Barker et al., 1992, 1994; Pan et al., 2000; Morgan, 2001; Renton, 2001; Morgan et al., 2002; Nicholson and Renton, 2002). The injection of immunostimulants directly into the CNS produces a highly regulated inflammatory response characterized by the production of cytokines, immune cell infiltration, and tissue damage (Nicholson and Renton, 1999, 2002; Renton, 2001; Gavrilyuk et al., 2002; Rivest, 2003). We have previously demonstrated that the catalytic activity of both hepatic and CNS P450 isoforms are down-regulated in rat models of CNS inflammation/infection (Renton and Nicholson, 2000; Nicholson and Renton, 2001; Garcia Del Busto Cano and Renton, 2003). A reduction in brain P450 metabolism may exacerbate susceptibility to neurotoxic agents such as 1-methyl-4-phenylpyridinium (Goralski and Renton, 2004). A loss or reduction in hepatic P450 levels has direct consequences for reduced metabolism of therapeutic agents (Chang and Kam, 1999; Renton, 2001). The pathways directly causing the down-regulation of P450 isoforms following conditions of CNS inflammation are not completely understood; however, multiple mechanisms are implicated.

Our objective was to determine the signal transduction mechanisms that contribute to hepatic P450 regulation in a rat model of lipopolysaccharide (LPS)-induced CNS inflammation. To obtain this objective, we examined the regulation of hepatic P450 gene expression following the i.c.v. administration of LPS, a well known and utilized model of CNS inflammation/infection (Shimamoto et al., 1999; Renton and Nicholson, 2000; Nadeau and Rivest, 2002). Our major findings are that several hepatic transcription factors play a vital role in P450 gene regulation during conditions of CNS inflammation and that LPS is rapidly transferred from the CNS into the periphery, where it likely contributes to the effects observed in this rodent model of CNS inflammation.

	Materials and Methods

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Reagents. All laboratory reagents were purchased from Sigma-Aldrich (St. Louis, MO) with the exceptions noted in the text. Gel-purified Escherichia coli LPS of serotype 0127:B8 (Sigma-Aldrich) was utilized in the experiments as outlined below.

Animals and Treatments. Male Sprague-Dawley rats (125–150 g) were obtained from Charles River Canada (Montreal, QC, Canada) and were housed on corncob bedding for a period of 5 days on a 12-h light/dark cycle. All animal procedures were performed according to the Dalhousie University Committee on Laboratory Animals following the guidelines established by the Canadian Council on Animal Care. Rats were allowed ad libitum access to food and water before and after the experimental procedure. On the day of the experiment, rats were anesthetized using enflurane and maintained on a 4% level of the anesthetic during the surgery. Injections (i.c.v.) into the lateral ventricle were performed using a Kopf stereotaxic instrument (David Kopf Instruments, Tujunga, CA). The coordinates utilized relative to bregma were 1.7 mm lateral and 4.7 mm below the skull surface. A dose of 25 µg of LPS was dissolved in pyrogen-free saline and injected in a volume of 5 µl. In a separate set of experiments, rats were injected i.v. through the tail vein with 25 µg of LPS dissolved in 100 µl of saline and tissue samples obtained at either 3, 6, or 24 h after injection. Some experiments required intraperitoneal injection of rats with 25 µg of LPS dissolved in 100 µl of saline. All experiments utilized four to six male rats per treatment. For the cytokine experiments, animals received a cytokine cocktail containing 100 ng of TNF, 50 ng of IL-1ß, 45 ng of IL-1, 50 ng of IL-6, and 50 ng of IFN (all cytokines obtained from Cedarlane Laboratories Ltd., Hornby, ON, Canada) made in 0.1% sterile filtered bovine serum albumin. For the cytokine inhibitor experiments, rats received 25 µg of LPS i.c.v. in combination with one of the following treatments: the TNF-soluble antibody etanercept (Enbrel, Immunex; Wyeth-Ayerst, Montreal, QC, Canada), administered either i.p. (25 mg kg^-1) or i.c.v. (40 µg), or the IL-1 inhibitor YVAD (Alexis Biochemicals, San Diego, CA) administered i.c.v. (0.63 µg). Doses for the cytokine cocktail and the cytokine inhibitor experiments were selected based on results from dose-response curves (data not shown).

Tissue Isolation, Microsomal Fraction Preparation, and Microsomal Metabolism Assays. At 2, 4, 6, and 9 h after the i.c.v. injection of either saline or LPS, rats were anesthetized and decapitated, and liver (100 mg) for RNA isolation was obtained, serum was collected, and whole brain homogenates for cytokine measurement were obtained. For the i.v. experiments, total liver RNA was isolated at 3 and 6 h after treatment. Liver microsomal fractions were obtained at 24 h following either the i.v. or i.c.v. injection of LPS as described previously (Renton and Nicholson, 2000) and were suspended in a glycerolphosphate buffer (50 mM KH₂PO₄ buffer, pH 7.4, containing 20% glycerol and 0.4% KCl). Liver microsomal fractions were stored at -80°C until usage. Total protein concentrations were determined according to a modified Lowry protocol (Lowry et al., 1951). The activity of CYP1A1/2 and CYP2B1/2 were determined using the 7-ethoxyresorufin O-dealkylase (EROD) assay and the pentoxyresorufin O-dealkylase (PROD) assay, respectively, as described by Burke et al. (1985). Total P450 values were determined according to the method of Omura and Sato (1964).

RNA Extraction and Northern Blot Analysis. Total liver RNA was extracted using the TRIzol method according to the manufacturer's instructions and quality was determined using 260/280 nm ratios. Total RNA (10 µg) was electrophoresed on a 1.1% formaldehyde gel and transferred onto an Immobolin-NY+ membrane (Millipore Corporation, Billerica, MA) overnight and fixed to the membrane by UV cross-linking and heating for 1 h at 65°C. Blots were prehybridized for 1 h in 10 ml of Sigma Perfecthyb Plus (Sigma-Aldrich), after which the [³²P]dCTP (PerkinElmer Life and Analytical Sciences, Woodbridge, ON, Canada)-labeled probes (RmT Random Primer Labeling kit; Stratagene, La Jolla, CA) were added to a specific activity of 1 x 10⁷ cpm. Blots were exposed to a storage phosphor screen (Amersham Biosciences Inc., Piscataway, NJ) for 16 to 24 h and scanned using a PhosphorImager (Amersham Biosciences Inc.). Bands were quantified using ImageQuant 5.2 software (Amersham Biosciences Inc.). CYP2D1/5 (Chow et al., 1999), TNF (Cearley et al., 2003), and I B (Gavrilyuk et al., 2002) probes were constructed from forward and reverse primers (CYP2D1/5 forward, 5'-ATCGCTGGACTTCTCGCTAC-3', CYP2D1/5 reverse, 5'-GTCTTCTGACCTTGGAAGAC-3'; TNF forward, 5'-TACTGAACTTCGGGGTGATTGGTCC-3', TNF reverse, 5'-CAGCCTTGTCCCTTGAAGAGAACC-3'; I B forward 5'-CATGAAGAGAAGACACTGACCATGGAA-3', IB reverse, 5'-TGGATAGAGGCTAAGTGTAGACACG-3') using a TOPO TA Cloning kit (Invitrogen Canada, Inc., Burlington, ON, Canada) according to the manufacturer's instructions. CYP2B1/2, MAPKK, and GAPDH probes were a generous gift from Dr. C. J. Sinal (Dalhousie University, Halifax, NS, Canada).

Real-Time Quantitative PCR. A total of 5 µg of liver RNA was reverse transcribed in a 25-µl reaction containing 62.5 nM random primers, 20 units of RNaseOUT (Invitrogen Canada, Inc.), 1x StrataScript RT-buffer, and 12.5 units of StrataScript Reverse Transcriptase (both Stratagene) according to the instructions provided with the reverse transcriptase enzyme. Real-time quantitative PCR was performed using an MX3000P instrument (Stratagene) in a total volume of 20 µl. Reactions contained 10 µl of 2x Brilliant SYBR Green QPCR mix (Stratagene), 62.5 ng of both forward and reverse primers, and 25 nM reference dye. Cycle parameters consisted of an initial 10-min denaturation step at 95°C followed by either 35 cycles for GAPDH or 45 cycles for CYP1A1 as follows: 30-s denaturation at 95°C, 18-s annealing at 60°C, and 30-s extension at 72°C. Dissociation curves were also performed to verify the amplicon being amplified. Primers specific for CYP1A1 and GAPDH were specifically designed using the published sequence for rat CYP1A1 (accession number X00469 [GenBank] ) and rat GAPDH (accession number X02231 [GenBank] ) as follows: CYP1A1 forward, 5'-GGAGCTGGGTTTGACACAAT-3', CYP1A1 reverse, 5'-GATAGGGCAGCTGAGGTCTG-3' (amplicon size 157 bp); GAPDH forward, 5'-AGACAGCCGCATCTTCTTGT-3', GAPDH reverse 5'-CTTGCCGTGGGTAGAGTCAT-3' (amplicon size 207 bp). Data were analyzed using the 2^-C_T method (Livak and Schmittgen, 2001), where the cycle threshold (C_T) values for CYP1A1 and GAPDH were 32 and 19, respectively.

Cytokine Measurements. Whole brain was obtained following the i.c.v. injection of LPS at 2, 4, 6, and 9 h and homogenized in 2 ml of phosphate-buffered saline, pH 7.4. The homogenates were spun at 13,000 rpm for 10 min at 4°C and the supernatant was stored at -80°C until used for cytokine measurements. Protein levels in the brain homogenates were determined using a modified Lowry method (Lowry et al., 1951). Levels of TNF and IL-1ß in the brain were measured using a sandwich enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, MN), and results are reported as picograms of cytokine per milligram of protein present in the brain homogenate. The limit of detection for both TNF and IL-1ß cytokine enzyme-linked immunosorbent assays was 5 pg ml^-1.

Nitrite Measurement. The total amount of NO in plasma was indirectly determined by measuring total nitrites and nitrates (end products of NO oxidation) using a NO assay kit (Cayman Chemical, Ann Arbor, MI) according to the manufacturer's instructions. The kit involved converting nitrates into nitrites and then measuring the converted products using Griess reagent. Nitrite accumulation was determined in a 96-well plate format with the absorbances read at 540 nm. The limit of detection for this assay was 2.5 µM.

Determination of Endotoxin Levels. LPS concentrations were determined in serum at 15 min, 30 min, 2 h, 4 h, 6 h, 15 h, and 24 h after the administration of LPS by either the i.c.v. or i.p. route. Rats were injected with 25 µg of LPS either i.c.v. (in 5 µl of pyrogen-free saline) or i.p. (in 100 µl of pyrogen-free saline). Following decapitation, trunk blood was collected and allowed to clot for 1 h at room temperature and 1 h on ice, and serum was obtained following a 10-min spin at 3000 rpm, and stored at –80°C until usage. All groups were compared with noninjected rats. LPS levels were determined per manufacturer's instructions from a commercially available kinetic assay kit based on ChromoLAL as a substrate (Associates of Cape Cod, MA, USA). The assay was linear from 0.0395 pg ml^-1 to 100 ng ml^-1 of LPS. The levels of endotoxin in the saline used to resuspend LPS were below the limit of detection of the assay, indicating that samples preparation was sufficiently aseptic for endotoxin detection.

Liver Nuclear Fraction Isolation. Liver nuclear fractions were isolated from rats 1 and 3 h after the i.c.v. injection of LPS according to a previously described method (Gorski et al., 1986). Briefly, animals were decapitated and livers were homogenized in 20 ml of homogenization buffer (100 mM HEPES, pH 7.4, containing 25 mM KCl, 0.15 mM spermine, 0.5 mM spermidine, 1 mM EDTA, 2 M sucrose, 10% glycerol, 5 µg ml^-1 pepstatin A, and 5 µg ml^-1 leupeptin) and centrifuged at 17,000 rpm for a period of 20 min at 4°C. The nuclear pellet was resuspended in 10 ml of nuclear lysis buffer (100 mM HEPES, pH 7.4, containing 100 mM KCl, 3 mM MgCl₂, 0.1 mM EDTA, 1 mM DTT, 0.1 mM phenylmethylsulfonyl fluoride, 10% glycerol, 5 µg ml^-1 pepstatin A, and 5 µg ml^-1 leupeptin) and homogenized using a Dounce homogenizer. The extraction was initialized by the addition of (NH₄)₂SO₄ to the nuclear lysate in drop-wise fashion to a final concentration of 0.4 M. The viscous lysates were incubated for a period of 30 min on ice with constant shaking, after which they were ultracentrifuged at 35,000 rpm for a period of 60 min at 4°C. Solid (NH₄)₂SO₄ was added to the supernatants at a concentration of 0.3 g ml^-1. The solutions were inverted several times and incubated on ice for a period of 20 min until all the (NH₄)₂SO₄ had dissolved. The solutions were then further centrifuged at 35,000 rpm for a period of 25 min at 4°C, and the pellets were resuspended in suspension buffer (25 mM HEPES, pH 7.6, containing 40 mM KCl, 0.1 mM EDTA, 1 mM DTT, 10% glycerol, 5 µg ml^-1 pepstatin A, and 5 µg ml^-1 leupeptin) and stored at -80°C until usage. Total protein concentrations were determined according to a modified Lowry protocol (Lowry et al., 1951).

Electromobility Shift Assays. Reactions were carried out in a total volume of 20 µl and contained 5 µg of protein, 50,000 cpm of ³²P-labeled probes, binding buffer (50 mM Tris-HCl, pH 7.9, containing 5 mM MgCl₂, 2.5 mM EDTA, 2.5 mM DTT, 250 mM NaCl, and 20% glycerol), and 2 µg of poly(dI/dC). Reactions were preincubated with the poly(dI/dC) for a period of 15 min, after which the radiolabeled probe was added to initiate the reaction. In cases of specific competitions, a 20x excess amount of nonradioactive self-oligonucleotide was utilized and was included in the reaction mixture. Reactions were incubated at room temperature for a period of 30 min and run on a 5% TBE-acrylamide gel at a voltage of 170 V. In the case of nonspecific competitions, a 20x excess amount of nonradioactive non-self oligonucleotide was utilized in the reaction mixture. For supershift reactions, the reaction mixture was incubated with either a p65 antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or a CCAAT-enhancer binding protein (C/EBP) antibody (generous gift from Dr. M. W. Nachtigal, Dalhousie University, Halifax, NS, Canada) for a 30-min period following the addition of the radioactive specific probe. The gels were then dried using a Bio-Rad gel dryer [Bio-Rad Laboratories (Canada, Ltd.), Mississauga, ON, Canada] operating under vacuum and set at 80°C for 105 min. Gels were then exposed to a phosphor-storage screen for a period of 16 to 24 h and scanned using a PhosphorImager (Amersham Biosciences Inc.). Bands were quantified using ImageQuant 5.2 software (Amersham Biosciences Inc.). The probes (described in Table 1) were obtained either commercially from Santa Cruz Biotechnology, Inc. or as single-stranded oligonucleotides from Sigma-Genosys (The Woodlands, TX) and annealed according to a standard protocol. Briefly, 300 pmol of each oligonucleotide were incubated in annealing buffer (100 mM Tris, pH 7.9, and 50 mM MgCl₂) for 10 min at 95°C and allowed to gradually cool down to 25°C.

Statistical Analysis. All data are reported as the mean ± the standard error of the mean. Each time point constituted a separate experiment in which LPS treatment was compared with saline and was carried out on a different day, and, therefore, an unpaired t test was utilized to compare saline versus LPS groups, where p < 0.05 determined statistical significance. Data are presented in one graph for all time points for convenience in illustrating the data.

	Results

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The Intracerebroventricular Injection of LPS Initiates an Inflammatory Response in the Brain and Liver. Levels of TNF and IL-1ß proteins were increased in the brains of animals at 2, 4, and 6 h after the i.c.v. administration of LPS as compared with saline control rats (Fig. 1, A and B). These data indicate that a CNS inflammatory response occurs in response to the i.c.v. administration of LPS.

View larger version (17K): [in this window] [in a new window]   FIG. 1. The up-regulation of TNF and IL-1ß levels in the brain following the administration of LPS into the lateral cerebral. Rats were injected i.c.v. with either 25 µg of LPS or saline (sal), and brain homogenates were obtained 1 to 6 h later. Levels of TNF (A) and IL-1ß (B) are shown as picograms of cytokine per milligram of protein present in the brain homogenates. The levels of these two cytokines in naive rats that have not received any i.c.v. treatment are shown in each panel. Each bar represents the mean ± S.E.M. from four rats. *, cytokine level is higher compared with the respective saline treatment using an unpaired t test (p < 0.05).

View larger version (32K): [in this window] [in a new window]   FIG. 2. The induction of an inflammatory response in the liver in response to the administration of LPS into the lateral cerebral ventricle. Rats were injected i.c.v. with either 25 µg of LPS or saline (sal), and liver RNA was isolated at various time points following treatment. The ratio of the intensity of each band to its respective GAPDH was obtained by densitometry, and the results are plotted as percentage control of each respective saline group for IB (A) and MAPKK (B). The average absolute value for IB expression (with respect to GAPDH) was 0.29 for the 2-h saline samples, 0.67 for the 4-h saline samples, 0.64 for the 6-h saline samples, and 0.50 for the 9-h saline samples. The average absolute value for MAPKK expression (with respect to GAPDH) was 0.66 for the 2-h saline samples, 0.55 for the 4-h saline samples, 0.79 for the 6-h saline samples, and 0.71 for the 9-h saline samples. Each bar represents the mean ± S.E.M. mRNA expression from four rats. Representative blots for TNF (C) relative to GAPDH are shown for the 2-, 4-, 6-, and 9-h time points following the induction of CNS inflammation. *, mRNA expression is higher compared with corresponding saline-treated rats using an unpaired t test (p < 0.05).

View larger version (11K): [in this window] [in a new window]   FIG. 3. An increase in nitrite levels in plasma of rats occurs in response to the administration of LPS into the lateral cerebral ventricle. Rats were injected with 25 µg of LPS i.c.v., and 2 to 15 h later, rats were sacrificed and plasma was obtained. Nitrite levels in the plasma were detected using a commercially available kit that converts total nitrates into nitrites (both end products of nitric oxide). Each bar represents the mean ± S.E.M. total nitrite level from four rats. *, nitrite level is higher compared with that of corresponding saline (sal)-treated animals using an unpaired t test (p < 0.05).

View larger version (14K): [in this window] [in a new window]   FIG. 4. Rapid changes in the expression of CYP2D1/5, CYP2B1/2, and CYP1A1 occur following the administration of LPS into the lateral cerebral ventricle to induce CNS inflammation. Animals were administered either 25 µg of LPS or 5 µl of saline (sal), and liver RNA was isolated. Specific probes for CYP2D1/5 and CYP2B1/2 were utilized for Northern blot analyses (A and B). Specific primers for CYP1A1 were utilized for the quantitative PCR in C. LPS-treated animals were compared with the respective group of saline-treated animals at each time point, with each bar showing the mean results from four rats. For CYP2D1/5 (A) and CYP2B1/2 (B), the ratio of the intensity of each band to its respective GAPDH was obtained, and the results are plotted as percentage control of the respective saline groups. The 2-CT method was used to obtain the fold decrease of CYP1A1 (C) at each time point compared with its respective saline-treated group. The average absolute value for CYP2D1/5 expression (with respect to GAPDH) was 1.53 for the 2-h saline samples, 0.78 for the 4-h saline samples, 2.23 for the 6-h saline samples, and 1.83 for the 9-h saline samples. The average absolute value for CYP2B1/2 expression (with respect to GAPDH) was 0.48 for the 2-h saline samples, 0.23 for the 4-h saline samples, 0.57 for the 6-h saline samples, and 0.36 for the 9-h saline samples.*, P450 expression is lower compared with respective saline-treated animals using an unpaired t test (p < 0.05).

View larger version (74K): [in this window] [in a new window]   FIG. 5. The binding of specific transcription factors in the liver is increased in response to the administration of LPS into the lateral cerebral ventricle. Rats were injected with either 25 µg of LPS or 5 µl of saline (sal) i.c.v., and liver nuclear fractions were isolated 3 h later and were used in electromobility shift assays using commercially obtained oligonucleotides (Table 1). Representative blots for NF-B (A), CREB (B), and AP-1 (C) are shown, where the solid arrow indicates specific binding. Specific competitions (spec comp) and nonspecific competitions (non-spec comp) indicate competitions performed with excess nonradioactive specific oligonucleotide and nonradioactive nonspecific oligonucleotide, respectively. Each blot shows the results from four saline and four LPS-treated rats.

The binding of transcription factors to the promoter regions of specific P450 isoforms is illustrated in Figs. 6 and 7. An up-regulation in the binding of an NF-B response element (identified using MacVector; Accelrys, San Diego, CA) on the CYP2D5 promoter occurred in liver nuclear fractions isolated 3 h after the treatment of rats with LPS i.c.v. (Fig. 6A). The binding of NF-B to this response element on CYP2D5 was confirmed using specific and nonspecific competitions and was supershifted using a p65 antibody. NF-B and C/EBP response elements have been identified on the CYP2B1 promoter (Park and Kemper, 1996; Shaw et al., 1996). An increased binding of NF-B and C/EBP response elements occurred in hepatic nuclear fractions 3 h after the administration of LPS i.c.v. (Figs. 6B and 7A). The binding of both proteins was confirmed using specific and nonspecific competitions and was supershifted using a p65 and a C/EBP antibody. No binding was observed to the CREB response element on the promoter region of CYP1A1 (identified using MacVector) (data not shown). Finally, an up-regulation in the binding of an NF-B response element (identified using MacVector) on the CYP1A1 promoter was observed in liver nuclear fractions isolated 3 h after the treatment of rats with LPS i.c.v. (Fig. 7B). The binding was confirmed using specific and nonspecific competitions.

View larger version (78K): [in this window] [in a new window]   FIG. 6. NF-B plays a vital role in the regulation of P450 isoforms. Rats were injected with either 25 µg of LPS or 5 µl of saline (sal) i.c.v., and liver nuclear fractions were isolated 3 h later and were used in electromobility shift assays using commercially obtained oligonucleotides (Table 1). EMSA binding reactions were performed using single-stranded oligonucleotides (Table 1) that were annealed according to a standard annealing procedure as outlined under Materials and Methods. An up-regulation in NF-B binding to an NF-B-responsive region on the promoter of CYP2D5 (A) and CYP2B1 (B) is shown, where the solid arrow indicates specific binding, and the dashed arrow indicates nonspecific binding. Specific competitions (spec comp) and nonspecific competitions (non-spec comp) indicate competitions performed with excess nonradioactive specific oligonucleotide and nonradioactive nonspecific oligonucleotide, respectively. The binding of NF-B was confirmed using supershift assays, where C and D show a supershift (indicated by the diamond-head arrow) in binding to the CYP2D5 NF-B region and the CYP2B1 NF-B region, respectively, observed following incubation with a p65 antibody. Each blot shows the results from four saline- and four LPS-treated rats.

View larger version (71K): [in this window] [in a new window]   FIG. 7. The roles of C/EBP and CREB in the regulation of CYP2B1 and CYP1A1. Rats were injected with either 25 µg of LPS or 5 µl of saline (sal) i.c.v., and liver nuclear fractions were isolated 3 h later. EMSA binding reactions were performed using single-stranded oligonucleotides (Table 1) that were annealed according to a standard annealing procedure. The binding of C/EBP to a C/EBP responsive region on the promoter of CYP2B1 (A) and NF-B to an NF-B response element on the promoter region of CYP1A1 (B) is shown, where the solid line indicates specific binding. Specific competitions (spec comp) and nonspecific competitions (non-spec comp) indicate competitions performed with excess nonradioactive specific oligonucleotide and nonradioactive nonspecific oligonucleotide, respectively, as described under Materials and Methods. The binding of C/EBP to the CYP2B1 C/EBP region was confirmed using a supershift assay as shown in C, where the diamond-head arrow indicates the shift following incubation with a C/EBP antibody. Each blot shows the results from four saline- and four LPS-treated rats.

View larger version (19K): [in this window] [in a new window]   FIG. 8. The noninvolvement of several pathways in mediating the effects of LPS i.c.v. on hepatic P450 isoforms. Rats were administered a cytokine cocktail consisting of TNF, IL-1ß, IL-1, IL-6, and IFN, and total cytochrome P450 levels were measured 24 h later (A). In a separate set of experiments, rats were injected with 25 µg of LPS i.c.v., in addition to etanercept (Enbrel), given either as a dose of 40 µg i.c.v. (B) or 2.5 mg/kg i.p. (C) or the IL-1 inhibitor YVAD administered as a dose of 0.63 µg i.c.v. (D), and total P450 levels were measured 24 h later as described under Materials and Methods. * is statistically different compared with respective saline-treated animals (p < 0.05; two-way analysis of variance performed for panels B, C, and D; t test performed for panel A).

LPS Levels Are Detected in the Serum of Animals Given 25 µg of LPS by i.c.v. or i.p. Injection. Since the distribution of LPS following its administration i.c.v. to rats has not been previously characterized, we determined whether LPS transfer to the periphery might contribute to the observed changes in hepatic P450s. We measured the amounts of LPS (in pg/ml) in the serum of animals administered 25 µg of LPS either intracerebroventricularly or intraperitoneally. Following the i.c.v. administration of LPS, the serum endotoxin levels were 100-fold greater than the amounts detected in the serum following the i.p. administration of the same dose of LPS (Fig. 9). We have previously shown that the administration of 25 µg of LPS i.p. does not affect liver P450 activity (Renton and Nicholson, 2000). Minimal LPS was detected in control rats that did not receive LPS.

View larger version (21K): [in this window] [in a new window]   FIG. 9. The levels of endotoxin detected in the serum of animals injected with 25 µg of LPS i.p. or i.c.v. Animals were injected with 25 µg of LPS either i.p. or i.c.v., and serum was obtained at 15 min (15m), 30 min (30m), 2 h (2h), 4 h (4h), 6 h (6h), 15 h (15h), and 24 h (24h) after injection. Endotoxin levels were measured according to a commercially available kinetic assay kit. The graph shown represents the results from three animals. In the 30-min i.p. and the 2-h i.p. groups, the presence of endotoxin was detected in only two of the three animals. Noninjected rats served as control (Ctrl) animals for the experiment.

	Discussion

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Inflammatory mediators such as cytokines, prostaglandins, and reactive oxygen species are known to be released from activated microglia, the resident macrophages of the brain, during conditions of CNS inflammation (Rivest, 2003). Our current results indicate that an inflammatory response occurs in the brain and in the peripheral tissues following the administration of LPS i.c.v., manifested by increases in the levels of proinflammatory cytokines in rat brain and blood (Renton and Nicholson, 2000) and increases in the mRNA expression of the acute phase response genes TNF, IB, and MAPKK in rat liver, as outlined in Fig. 10. Changes in P450 gene regulation are well documented during inflammation responses occurring in vitro (Ke et al., 2001; Kelicen and Tindberg, 2004). It is likely that the regulation of hepatic P450s observed in vivo (Renton and Nicholson, 2000; Nicholson and Renton, 2001) also occurs as a result of changes in gene expression. We tested this idea by examining the regulation of hepatic CYP2D1/5, CYP2B1/2, and CYP1A1 using a well utilized model of CNS inflammation induced by the i.c.v. injection of LPS. These particular isoforms were selected since they are known to play a significant role in the metabolism of many clinically relevant drugs (Anzenbacher and Anzenbacherova, 2001). Our results indicate a significant reduction in the mRNA expression of CYP2D1/5, CYP2B1/2, and CYP1A1 in the liver following the i.c.v. administration of LPS, which is in agreement with the loss in enzymatic activity and/or protein levels previously reported for these isoforms (Renton and Nicholson, 2000).

View larger version (21K): [in this window] [in a new window]   FIG. 10. A proposed mechanism by which LPS regulates hepatic cytochrome P450 following its administration into the lateral cerebral ventricle. (1) The i.c.v. injection of LPS into the lateral cerebral ventricles of rats is thought to activate Toll-like receptor 4 (TLR4). (2) Activation of TLR4, normally present on microglia and astrocytes in the brain, would lead to the up-regulation of cytokine and transcription factors in the brain. (3) The rapid "leakage" of LPS from the CNS into the periphery plays a role in down-regulation of hepatic P450s. Although cytokines are elevated in the CNS during this model of LPS-induced CNS inflammation, it appears from our results that centrally produced cytokines are not important for the regulation of hepatic P450s. (4) The presence of LPS in the periphery induces a peripheral inflammatory response characterized by the production of cytokines (from immune cells such as macrophages) and the induction of an acute phase response in the liver. (5) Cytokines from peripheral sources, in addition to LPS present in the serum, act on the hepatocyte to differentially regulate P450s. (6) Some of the intrahepatic mechanisms by which P450 regulation occurs involve up-regulation of various transcription factors such as NF-B, AP-1, and C/EBP. These transcription factors can then bind to the promoter regions on specific P450 isoforms, leading to changes in the transcription of these P450s.

Previous studies have suggested that a signaling pathway must exist between the brain and liver to account for the loss in hepatic P450s during conditions of LPS-induced CNS inflammation (Terrazzino et al., 1997; Shimamoto et al., 1998, 1999; Renton and Nicholson, 2000). TNF, IL-1ß, and IL-6 have all been shown to regulate several P450 isoforms at the level of the enzyme and mRNA in both peripheral and CNS models of inflammation (Barker et al., 1992; Ke et al., 2001; Morgan, 2001; Nicholson and Renton, 2002). We observed no effects on total cytochrome P450 levels following the i.c.v. administration of a cytokine cocktail (TNF, IL-1, IL-1ß, IFN, and IL-6). In addition, the central and peripheral blockade of the TNF pathway (using the TNF-soluble antibody etanercept or Enbrel) and the IL-1 signal transduction pathway (using the IL-1 inhibitor YVAD) could not prevent the down-regulation in total cytochrome P450 levels induced by the i.c.v. administration of LPS. Similar results were observed when we examined the effects of the cytokine cocktail and cytokine inhibitors on CYP1A EROD-catalyzed activity (data not shown). Shimamoto et al. (1998, 1999) demonstrated that adrenalectomy did not block the loss in hepatic P450s in response to the i.c.v. administration of LPS. In support of these results, we observed that hypophysectomized rats maintained the response to the i.c.v. administration of LPS (data not shown), indicating the lack of involvement of the hypothalamic-pituitary axis in the down-regulation of hepatic P450s during CNS inflammation. All these results support the idea that hepatic P450 regulation during the i.c.v. administration of LPS is occurring at an intrahepatic level with various acute phase proteins such as NF-B playing a dominant role in this regulation.

The injection of LPS i.c.v. is a commonly used model to produce CNS inflammation; however, it has not been determined whether endotoxin leakage into the periphery plays a role in the effects on hepatic P450s observed by us and others (Terrazzino et al., 1997; Shimamoto et al., 1998, 1999; Renton and Nicholson, 2000). To test whether endotoxin leakage from the CNS might account for the effects observed on hepatic P450s in LPS-treated rats, we measured endotoxin levels in serum obtained from animals administered LPS directly into the lateral cerebral ventricle. We were able to detect significant amounts of LPS in the serum of rats as early as 15 min and for up to 2 h after the i.c.v. administration of LPS. An energy-mediated transport mechanism or bulk reabsorption of the cerebrospinal fluid likely accounts for the extremely rapid transfer of LPS from the CNS into the periphery. When animals were treated with the same dose of LPS (25 µg) given by the i.p. route, only small amounts of LPS were detected in the serum following LPS administration. Based on these results, the bioavailability of LPS from its i.p. administration is relatively small compared with the bioavailability which occurs from i.c.v. administration. This difference in bioavailability could explain previous results by our laboratory and Shimamoto and coworkers, where the administration of LPS by the i.p. route did not cause the down-regulation in hepatic P450s that is observed by administering the same dose directly into the lateral cerebral ventricle (Shimamoto et al., 1998, 1999; Renton and Nicholson, 2000). It is likely that the rapid transfer of LPS from the CNS into the periphery in significant amounts is what accounts for the observed effects of i.c.v. LPS on hepatic P450s (as shown in Fig. 10). In support of this idea, we have observed that the administration of 25 µg of LPS by the intravenous route also causes a significant down-regulation in CYP2B1/2, CYP2D1/5, and CYP1A1 expression and a significant up-regulation in the expression of TNF and IB at 3 to 6 h.

In summary, we suggest a signal transduction mechanism to explain the differential regulation of cytochrome P450 intrahepatically following a well utilized LPS-induced model of CNS inflammation and provide insight into some of the molecular mechanisms by which rapid regulation of cytochrome P450 occurs in this model. Our results indicate that P450 regulation following the i.c.v. administration of LPS occurs at an intrahepatic level, with proteins such as NF-B and C/EBP playing a dominant role in this regulation. We also show that the regulation of hepatic P450s during LPS-mediated CNS inflammation results by a novel mechanism through which rapid transfer of LPS from the CNS into the periphery occurs following its i.c.v. administration.

	Acknowledgments

 
We thank Sandra Dibb for technical assistance. We would also like to thank Dr. C. J. Sinal for helpful discussions and help with the electromobility shift assays.

	Footnotes

 
This work was supported by a grant from the Canadian Institute for Health Research (CIHR). D.A. was funded by an Eliza Ritchie doctoral research award. K.B.G. was funded by fellowships from CIHR, IWK Health Centre, and the Nova Scotia Health Research Foundation.

Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.

doi:10.1124/dmd.105.004564.

ABBREVIATIONS: P450, cytochrome P450; LPS, lipopolysaccharide; CNS, central nervous system; NO, nitric oxide; TNF, tumor necrosis factor ; IB, inhibitor of B alpha; IL-1ß, interleukin-1ß; IL-6, interleukin-6; IFN; interferon ; NF-B, nuclear factor of the enhancer in B cells; AP-1, activator protein-1; CREB, cAMP response element binding protein; C/EBP, CAAT enhancer binding protein; EROD, 7-ethoxyresorufin O-dealkylase; PROD, pentoxyresorufin O-dealkylase; ICE-1, IL-1 converting enzyme; PCR, polymerase chain reaction; DTT, dithiothreitol; MAPKK, mitogen-activated protein kinase kinase; EMSA, electromobility shift assay.

Address correspondence to: Kenneth W. Renton, Dept. of Pharmacology, Sir Charles Tupper Medical Bldg., Dalhousie University, Halifax, N.S., B3H 4H7, Canada. E-mail: Ken.Renton{at}dal.ca

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