QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: dmd.105.006528v1 34/1/27    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Kourylko, O. Articles by du Souich, P. Search for Related Content PubMed PubMed Citation Articles by Kourylko, O. Articles by du Souich, P.

MODULATION OF CYP1A2 AND CYP3A6 CATALYTIC ACTIVITIES BY SERUM FROM RABBITS WITH A TURPENTINE-INDUCED INFLAMMATORY REACTION AND INTERLEUKIN 6

Département de Pharmacologie, Faculté de Médecine, Université de Montréal, Montréal, Québec, Canada

(Received July 12, 2005; accepted September 29, 2005)

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
Inflammatory reactions reduce the activity of cytochrome P450 isoforms. The aim of the study was to determine the mechanisms underlying the decrease in CYP1A2 and CYP3A6 catalytic activities produced by serum from rabbits with a turpentine-induced inflammatory reaction (S_TIIR) and interleukin 6 (IL-6). S_TIIR and IL-6 were incubated with cultured primary hepatocytes from control rabbits (H_CONT), and from rabbits with a turpentine-induced inflammatory reaction (H_TIIR) in the absence or presence of pyrrolidine dithiocarbamate (PDTC), an antioxidant and inhibitor of nuclear factor B transcription; 2'-amino-3'-methoxyflavone (PD98059), an inhibitor of extracellular signal-related kinase (Erk1/2); 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole (SB203580), an inhibitor of p38MAPK; N-nitro-L-arginine methyl ester, an inhibitor of nitric-oxide synthase 2 (NOS2); the combination of PDTC, PD98059, and SB203580; and genistein, an inhibitor of Janus-associated protein tyrosine kinase (JAK). After 4 and 24 h of incubation of H_CONT with S_TIIR and IL-6, CYP1A2 activity was reduced without changes in expression; the reduction in activity was partially prevented by the inhibition of JAK, Erk1/2, and NOS2. In H_CONT, S_TIIR and IL-6 did not affect CYP3A6 activity; however, PDTC reduced CYP3A6 activity by 40 and 80% after 4 and 24 h of incubation. In H_TIIR, S_TIIR and IL-6 reduced both CYP1A2 and CYP3A6 activities; this decrease is partially prevented by inhibitors of protein tyrosine kinases, Erk1/2, and NOS2. In H_TIIR, SB203580 increased CYP3A6 activity in a dose-dependent manner without changes in protein expression. These results show that the signal transduction pathways mediating the decrease in CYP1A2 and 3A6 activity, produced by S_TIIR and IL-6, involve JAK, Erk1/2, and NOS2.

Already in 1978, the decrease in theophylline clearance was attributed to the down-regulation of P450 isoforms (Renton, 1978). Since then, many reports have confirmed that an inflammatory reaction triggers the release of proinflammatory mediators, e.g., IL-6, IL-1ß, and interferon-, among others, which will cause a transcriptional down-regulation of P450 genes and post-transcriptional reductions in enzyme expression (Riddick et al., 2004). Actually, it is known that, in vivo and in vitro, the down-regulation of P450 isoforms is preceded by a decrease in activity that is mediated primarily by IL-6, IL-1ß and interferon- (El-Kadi et al., 1997; Bleau et al., 2000; Barakat et al., 2001). This post-translational reduction in P450 activity is still not well understood, but has been associated with reversible inhibitory effects of reactive oxygen species (ROS) and nitric oxide (NO·) (Takemura et al., 1999; El-Kadi et al., 2000), and implicates the activation of extracellular signal-related kinases (Erk1/2) and protein kinase C (Levitchi et al., 2004).

It is actually emerging that the effect of an inflammatory reaction on P450 activity is enzyme-selective. Several reasons underlie such specificity: 1) the serum mediators released by inflammation, e.g., cytokines, down-regulate the expression of P450 isoforms differentially (Morgan et al., 2001; Renton, 2001); 2) the factors regulating the expression of P450 isoforms show a relative specificity (Handschin and Meyer, 2003); and 3) the effect of known inhibitors of P450 activity, such as NO·, is enzyme-specific (Vuppugalla and Mehvar, 2004).

The aim of the present study was to understand the differential effect of serum from rabbits with a turpentine-induced inflammatory reaction (S_TIIR) and IL-6 on the catalytic activity of CYP1A2 and CYP3A6, and the signal transduction pathways modulating such activity, in hepatocytes in primary culture from control rabbits (H_CONT) and hepatocytes from rabbits with a turpentine-induced inflammatory reaction (H_TIIR) after 4 and 24 h of incubation.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Production of the Aseptic Inflammatory Reaction. Male New Zealand rabbits (2.2–2.5 kg) from the Ferme Charles Rivers (St. Constant, QC, Canada) were housed in separate cages and fed water and chow ad libitum for at least 7 days before use. A local inflammatory reaction was induced by means of the subcutaneous injection of 2.5 ml of turpentine at two sites of the back of the rabbits. All the experiments were conducted according to the Canadian Council on Animal Care guidelines for use of laboratory animals.

Primary Rabbit Hepatocyte Culture. Forty-eight hours after the injection of turpentine, a blood sample (20 ml) was withdrawn in a sterile BD Vacutainer brand SST (BD Biosciences, Mississauga, ON, Canada) from the central artery of the ear, and the hepatocytes were isolated according to a two-step liver perfusion method, with minor modifications (El-Kadi et al., 1997). Rabbits were anesthetized with 30 mg/kg sodium pentobarbital, and after a middle laparotomy, the portal and inferior cava veins were cannulated. The liver was perfused in situ via the portal vein with a washing solution: 115 mM NaCl, 5 mM KCl, 1 mM KH₂PO₄, 25 mM HEPES, 0.5 mM EGTA, 5.5 mM glucose, and 56.8 mg/ml heparin, followed by a perfusion of a solution of 0.013% collagenase, CaCl₂ (1 mM), and trypsin inhibitor (0.25 mM). Harvested cells were centrifuged on a 40% Percoll gradient to isolate viable hepatocytes. Viability was always greater than 90% as assessed by trypan blue exclusion. Thereafter, hepatocytes (2 x 10⁶ in 2 ml of Dulbecco's modified Eagle's medium supplemented with 10% calf serum) were plated in 12- and 24-well plastic culture plates (Falcon; BD Biosciences Discovery Labware, Bedford, MA) coated with type I rat-tail collagen.

Blood samples were allowed to clot at room temperature for 3 h, centrifuged at 2500 rpm for 5 min, and the serum was decanted and stored frozen at –80°C in 1-ml aliquots until use. When samples are handled as described, serum mediators conserve their activity for up to 12 months (El-Kadi et al., 1997).

Signal Transduction Pathways Modulating CYP1A2 and CYP3A6 Activities. To assess the mechanisms underlying the decrease in hepatocyte CYP1A2 and 3A6 activities caused by S_TIIR and IL-6, inhibitors of protein tyrosine kinases, mitogen-activated protein kinases (MAPKs), NF-B transcription, and nitric-oxide synthase (NOS) were used: 1) genistein (4',5,7-trihydroxyisoflavone; 90 µM), a nonspecific inhibitor of Janus-associated protein tyrosine kinase (JAK); 2) PD98059 (10 µM), an inhibitor of extracellular signal-related kinase 1/2 (Erk1/2 or p42/44MAPK); 3) SB203580 (25 µM), a specific inhibitor of p38MAPK; 4) pyrrolidine dithiocarbamate (PDTC; 10 µM), an antioxidant that inhibits NF-B transcription by scavenging ROS and/or by inhibiting ubiquitin ligase and so impeding phosphorylated IB activation; and 5) N-nitro-L-arginine methyl ester (L-NAME; 1 mM), an inhibitor of NOS and NO· production. Genistein, PD98059, and SB203580 were dissolved in dimethyl sulfoxide, and pyrrolidine dithiocarbamanate and L-NAME in 0.9% NaCl, and 5 µl of the solutions were added to the hepatocytes in primary culture. The concentrations of dimethyl sulfoxide in the cell culture were always <1%.

Harvested hepatocytes were plated in 12-well plastic culture plates; the medium was changed 2 h after plating and the inhibitors were added. After a 30-min preincubation of the inhibitors with the hepatocytes, 200-µl aliquots of rabbit serum were added to H_CONT and H_TIIR, and further incubated for 4 and 24 h. Since IL-6 is the serum mediator responsible for the decrease in cytochrome P450 activity in rabbits with a turpentine-induced inflammatory reaction (Bleau et al., 2000), recombinant IL-6 (20 ng) was incubated with H_CONT and H_TIIR for 4 and 24 h, and CYP1A2 and 3A6 activities were determined. The effect of sodium nitroprusside (SNP; 1 mM) on CYP1A2 and 3A6 activities was used as a positive control.

In preliminary experiments, we confirmed by conventional phase-contrast light microscopy that incubation of H_CONT and H_TIIR with serum, IL-6, and the various inhibitors for 4 and 24 h does not modify the monolayer of hepatocytes and the polygonally shaped cells, suggesting that viability of H_CONT and H_TIIR is not affected by the experimental conditions.

CYP1A2 Activity. CYP1A2 activity was determined by measuring the concentration of resorufin formed by the O-demethylation of methoxyresorufin (Van Vleet et al., 2002). After 4 and 24 h of incubation, growth medium was removed, the hepatocytes were washed twice with 300 µl of Krebs' solution, and 3.3 µM methoxyresorufin O-demethylase in 300 µl of Krebs' solution were then added into each well. After a 10-min incubation period at 37°C, 100 µl of supernatant were transferred to a 96-well fluorescent plate containing 67 µl of a perchloric acid/glycine solution and 33 µl of 5.4% K₂CO₃. Production of resorufin was measured fluorimetrically at excitation and emission wavelengths of 530 and 584 nm, respectively, with a fluorescent plate reader (Victor2 1420 Multilabel Counter; PerkinElmer Wallac, Gaithersburg, MD).

CYP3A6 Activity. The activity of CYP3A6 was assessed by measuring the ability of the hepatocytes to convert 3,4-difluorobenzyloxy-5,5-dimethyl-4-(4-methylsulfonyl phenyl)-(5H)-furan-2-one (DFB), a CYP3A substrate, to 3-hydroxy-4-(4-methylsulfonyl phenyl)-(5H)-furan-2-one (DFH), its fluorescent metabolite (Levitchi et al., 2004). After 4 and 24 h of incubation, the growth medium was removed, and hepatocytes were washed twice with 300 µl of Krebs' solution. Then, 300 µl of DFB (60 µM)/Krebs mixture were added to each well for a 20-min incubation at 37°C. Thereafter, 100 µl of supernatant were transferred to a 96-well fluorescent plate containing 100 µl of Tris buffer/acetonitrile solution (0.05 M/40%). DFH was measured at excitation and emission wavelengths of 360 and 440 nm, respectively, with a fluorescent plate reader (Victor2 1420 Multilabel Counter; PerkinElmer Wallac).

Measurement of NO·. Nitric oxide was determined by measuring the nitrite and nitrate in the culture media using a colorimetric method based on the Griess reaction (Nims et al., 1996). To reduce the nitrate, the samples were incubated at 37°C in the presence of 0.1 U/ml nitrate reductase, 50 µM NADPH, and 5 µl of FAD. To avoid any interference with the determination of nitrite, NADPH was oxidized by incubating the samples with 10 U/ml lactate dehydrogenase and 10 mM sodium pyruvate for 5 min at 37°C. Because premixed Griess reagent results in an incomplete azo dye formation at exposure to light and pH >1, the following steps were observed: the samples were cooled at 4°C, and 1 mM sulfanilamide, 0.1 M HCl, and 1 mM naphthylethylene-diamine were added. NO· was measured at 540 nm with a fluorescent plate reader (Victor2 1420 Multilabel Counter, PerkinElmer Wallac).

Expression of CYP1A2 and 3A6 Proteins. Protein content in cells was measured by the method of Lowry et al. (1951). After 4 and 24 h of incubation, the expression of CYP1A1/2 and CYP3A6 proteins in the hepatocytes was assessed by Western blot analysis as described elsewhere (Bleau et al., 2000; Levitchi et al., 2004). Proteins (50 µg) were separated by SDS-polyacrylamide gel electrophoresis (PAGE) (7.5% polyacrylamide). Thereafter, proteins were transferred by electrophoresis to a nitrocellulose membrane using the Mini Trans-Blot Electrophoretic Transfer System (Bio-Rad, Hercules, CA). CYP3A6 protein was detected with a monoclonal anti-rat CYP3A1 with cross-reactivity to rabbit CYP3A6, and a horseradish peroxidase-conjugated secondary antibody. Chemiluminescence was visualized by autoradiography. CYP1A2 proteins were detected with polyclonal anti-rabbit CYP1A1 and visualized with an alkaline phosphatase-conjugated secondary goat antibody using blue tetrazolium as the substrate. As reference protein, in each gel, 50 µg of proteins extracted from the same set of H_CONT with a constant amount of CYP1A2, and CYP3A6 were used. The assays were linear in the range of protein amounts assessed under the actual experimental conditions, and the results are presented as a ratio of the P450 isoform to the reference protein. Band intensities were measured with the software Un-Scan-It-Gel (Silk Scientific Inc., Orem, UT). Data are presented as arbitrary values of densitometry for each sample over that of the reference protein.

Immunoprecipitation and Nitrotyrosine-Containing Proteins. Protein content in cultured hepatocytes was determined by the method of Lowry et al. (1951) and diluted to obtain a 10 mg/ml concentration in a final volume of 150 µl, and immunoprecipitation was performed according to manufacturer's instructions. Samples were centrifuged at 12000 rpm at 4°C for 10 min, the supernatant was transferred into an Eppendorf tube, and 10 µl (1:15 dilution) of the primary antibody was added to each sample, e.g., a monoclonal anti-rat CYP3A1 with cross-reactivity to rabbit CYP3A6 or a polyclonal anti-rabbit CYP1A1. Thereafter, the tubes were maintained at 4°C for 24 h with constant agitation. In the meantime, 80 µl of Protein A-Sepharose were washed twice with 80 µl of lysis buffer (20 mM KH₂PO₄ and 80 mM K₂HPO₄ at pH 7.4), centrifuged at 4000 rpm for 1 min, and the supernatant was discarded and the pellet diluted (1:1 dilution) with lysis buffer. After a 24-h incubation of samples with a monoclonal anti-rat CYP3A1 with cross-reactivity to the rabbit CYP3A6 or a polyclonal anti-rabbit CYP1A1, lysis buffer containing Protein A-Sepharose was added to the samples to obtain 20 µl of Protein A-Sepharose per 10 µl of antibody. The samples containing the Protein A-Sepharose were maintained for 24 h at 4°C with constant agitation, centrifuged at 4000 rpm for 1 min, and finally washed with the lysis buffer (1:2 dilution) five times. Thereafter, the supernatant was aspirated and 50 µl of ß-mercaptoethanol/Laemmli buffer (5:45) were added to each sample; after mixing, the samples were heated for 3 min at 100°C and centrifuged at 4000 rpm for 1 min. Proteins were separated by SDS-PAGE (7.5% of polyacrylamide) and transferred to a nitrocellulose membrane using the Mini Trans-Blot Electrophoretic Transfer System (Bio-Rad). Tyrosine nitration of CYP1A1/2 and 3A6 proteins was determined by adding the rabbit anti-nitrotyrosine antibody (1:1000 dilution) and a goat anti-rabbit horseradish peroxidase (1:4000 dilution). Chemiluminescence was visualized by autoradiography.

To identify protein tyrosine nitration of P450 isoforms, the same nitrocellulose membranes were washed four times (10 min each time) with Tris-buffered saline-Tween 20 (10 mM Tris-HCl, 150 mM NaCl, 0.1% Tween 20 at pH 8.0) and incubated overnight at 4°C with a monoclonal anti-rat CYP3A1 with cross-reactivity to the rabbit CYP3A6 or a polyclonal anti-rabbit CYP1A1, and P450 proteins were detected with a horseradish peroxidase-conjugated secondary antibody. Chemiluminescence was visualized by autoradiography.

Immunoblot Analysis of Erk1/Erk2. H_TIIR were incubated with rabbit serum in the absence and presence of the inhibitors. Protein extracts were prepared by homogenization of hepatocytes in lysis buffer. Equal amounts of protein were resolved by SDS-PAGE and transferred to nitrocellulose membranes, which were blocked in Tris-buffered saline, 0.1% Tween 20, 5% nonfat dried milk, and probed with antibody SM6 (1:1000 dilution), which recognizes Erk1 and Erk2 isoforms, for 2 h at room temperature (Meloche, 1995) or anti-phospho-p38 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) overnight at 4°C. To control for protein loading, the blot was stripped and reprobed with anti-ß-actin (1:10,000 dilution). We also used an Erk2 N-terminal-specific polyclonal antibody (AB3055; Chemicon International, Temecula, CA) to confirm the absence of expression of N-terminal fragments of Erk2. The anti-Erk1/2 phosphothreonine-183, phosphotyrosine-185 (pTpY Erk1/2) E10 monoclonal antibody (Cell Signaling Technology Inc., Beverly, MA) was diluted 1:1000 according to the manufacturer's instructions.

Materials. Percoll gradient, Williams' medium E, calf serum, type I rat-tail collagen, trypsin inhibitor, NaCl, KCl, KH₂PO₄, EGTA, glucose, genistein, PDTC, SNP, and L-NAME were purchased from Sigma-Aldrich (St. Louis, MO); insulin, nitrate reductase, lactate dehydrogenase, and sodium pyruvate were obtained from Roche Diagnostics (Mannheim, Germany). Collagenase A was acquired from Worthington Biochemicals (Freehold, NJ). The 12- and 24-well plastic culture plates were obtained from BD Biosciences Discovery Labware; turpentine was obtained from Recochem (Montréal, QC, Canada). Phloretin, PD98059, SB203580, and human recombinant IL-6 were purchased from EMD Biosciences (San Diego, CA). Polyclonal anti-rabbit CYP1A1 and monoclonal anti-rat CYP3A1 were obtained from Oxford Biochemical Research (Oxford, MI). DFB and DFH were gracefully provided by Merck Frosst Canada (Kirkland, QC, Canada).

Statistical Analysis. All results are reported as mean ± S.E. Comparison of results from the various experimental groups and their corresponding controls was carried out using a one-way analysis of variance followed by the Student-Newman-Keuls test for all pair-wise comparisons of the mean responses among the different treatment groups. Since baseline values of CYP1A2 and 3A6 activity and the effect of the sera on P450 activity varied according to the intensity of the turpentine-induced inflammatory reaction, the effect of S_TIIR is presented as percentage change by reference to the normalized effect of serum from control rabbits (S_CONT), which is corrected by baseline activity. The differences were considered significant when p < 0.05.

View larger version (39K): [in this window] [in a new window]   FIG. 1. Catalytic activity of CYP1A2 (black columns) and CYP3A6 (dashed columns) measured in HCONT, and incubated with SCONT and STIIR for 4 (upper panel) and 24 h (lower panel). HCONT incubated with STIIR were preincubated for 30 min with PDTC, PD98059, or SB203580, alone or combined with PDTC and PD98059 (3X), L-NAME, and genistein (GEN). The effect of STIIR is presented as percentage of change by reference to the normalized effect of SCONT, which was corrected by baseline activity. All results are reported as mean ± S.E. of five to six experiments. * and &, CYP1A2 and/or CYP3A6 activity is significantly different (p < 0.05) from SCONT and STIIR, respectively.

	Results

Top Abstract Materials and Methods Results Discussion References

Incubation of IL-6 with H_CONT for 4 and 24 h reduced CYP1A2 activity by 15 and 28%, respectively (p < 0.05, n = 3; Table 1). The decrease of CYP1A2 activity was partially prevented by PDTC, PD98059, L-NAME, and genistein (data not shown). When H_CONT were incubated for 4 and 24 h with S_TIIR or IL-6 and SB203580 (Fig. 1), alone or combined with PDTC and PD98059 (3X in Fig. 1), CYP1A2 activity was reduced by 30 to 40% (p < 0.05).

Incubation of S_CONT and S_TIIR with H_CONT for 24 h did not affect the expression of CYP1A1/2 (Fig. 2A, n = 3). Preincubation of S_TIIR or IL-6 with SB203580, alone or in combination with PDTC and PD98059, did not modify the expression of CYP1A1/2 in H_CONT (data not shown).

View larger version (55K): [in this window] [in a new window]   FIG. 2. Effect of SCONT and STIIR on CYP1A1/2 and CYP3A6 expression as determined by SDS-PAGE. SCONT and STIIR were incubated for 24 h with HCONT and from HTIIR. A, representative blots and mean (±S.E.) densitometry ratios (n = 3) depicting the amount of CYP1A1/2 and CYP3A6, 1) in HCONT and in HTIIR, and 2) in HCONT and in HTIIR after incubation with SCONT and STIIR. B, representative blots and mean (±S.E.) densitometry ratios (n = 3) depicting the effect of STIIR on CYP3A6 expression in HCONT in the presence of PDTC, PD98059, or SB203580, alone or combined with PDTC and PD98059 (3X), L-NAME, and genistein. C, representative blots and mean (±S.E.) densitometry ratios (n = 3) depicting the effect of STIIR on CYP1A1/2 and CYP3A6 expression in HTIIR in the presence of PDTC, PD98059, or SB203580 combined with PDTC and PD98059 (3X), and genistein.

View larger version (31K): [in this window] [in a new window]   FIG. 3. CYP1A2 (black columns) and CYP3A6 (dashed columns) activities measured in HTIIR, and incubated with SCONT and STIIR for 4 (upper panel) and 24 h (lower panel). HTIIR incubated with STIIR were preincubated for 30 min with PDTC, PD98059, or SB203580 (SB), alone or combined with PDTC and PD98059 (3X), L-NAME, and genistein (GEN). The effect of STIIR is presented as percentage of change by reference to the normalized effect of SCONT, which was corrected by baseline activity. All results are reported as mean ± S.E. of 8 to 10 experiments. * and &, CYP1A2 and/or CYP3A6 activity is significantly different (p < 0.05) from SCONT and STIIR, respectively.

View larger version (20K): [in this window] [in a new window]   FIG. 4. Dose-dependent effect of SB203580 on CYP1A2 and CYP3A6 activities. Several doses of SB203580 were incubated for 24 h with hepatocytes in primary culture harvested from rabbits with a turpentine-induced inflammatory reaction in the presence of serum from rabbits with a turpentine-induced inflammatory reaction. Emax is the predicted maximal inhibition elicited by SB203580 and EC50 is the concentration decreasing by 50% Emax.

CYP3A6 Activity in H_CONT. Incubation of H_CONT with S_CONT, S_TIIR, and IL-6 for 4 and 24 h did not affect CYP3A6 activity. On the other hand, when H_CONT and S_TIIR or IL-6 were incubated in the presence of PDTC for 4 and 24 h, CYP3A6 activity decreased by around 50% (p < 0.05, n = 5; Fig. 1). Interestingly, when H_CONT was incubated for 24 h with S_TIIR or IL-6 combined with SB203580, CYP3A6 activity increased by 46% (p < 0.05, n = 5; Fig. 1) and 30% (p < 0.05, n = 4; data not shown), respectively. Incubation of SNP with H_CONT for 4 and 24 h reduced CYP3A6 activity by around 80% (p < 0.05, n = 5; Table 1).

The expression of CYP3A6 in H_CONT was not affected by the 24-h incubation with S_CONT, S_TIIR (Fig. 2A), and IL-6 (data not shown) alone or combined with PDTC, PD98059, SB203580, L-LNAME, the combination of PDTC, PD98059 and SB203580, and genistein (Fig. 2B).

CYP3A6 Activity in H_TIIR. CYP3A6 activity was not altered by the incubation of H_TIIR with S_CONT, but S_TIIR diminished its activity by 18% (p < 0.05, n = 9; Fig. 3) after 4 h of incubation; this reduction was prevented by PDTC, and partially prevented by L-NAME and genistein. When the incubation of H_TIIR with S_TIIR was prolonged to 24 h, CYP3A6 activity was reduced by 14% (p < 0.05, n = 8; Fig. 3); this decrease was prevented by PDTC, PD98059, L-NAME, and genistein. SNP reduced CYP3A6 activity by 53% and 68%, following 4 and 24 h of incubation, respectively (p < 0.05; Table 1).

CYP3A6 activity increased by 200% and 500% (p < 0.05) when H_TIIR were incubated with S_TIIR and SB203580, alone or combined with PDTC and PD98059, for 4 h and 24 h, respectively (Fig. 3). The increment in CYP3A6 activity elicited by SB203580 was directly associated with the dose (r² = 0.97, p < 0.013), and at the greatest dose of SB203580 tested (100 µM), CYP3A6 activity was enhanced more than 10-fold (Fig. 4).

After 4 and 24 h of incubation, IL-6 produced a modest decrease in CYP3A6 activity in H_TIIR (p < 0.05, n = 6; Fig. 5), an effect prevented by PDTC, PD98059, L-NAME, and genistein. In the presence of SB203580, IL-6 enhanced the activity of CYP3A6 by 400 and 800% following 4 and 24 h of incubation, respectively (p < 0.05, n = 6; Fig. 5).

View larger version (24K): [in this window] [in a new window]   FIG. 5. Activity of CYP3A6 measured in HTIIR and incubated with SCONT and IL-6 for 4 (upper panel) and 24 h (lower panel). HTIIR incubated with IL-6 were preincubated for 30 min with PDTC, PD98059, or SB203580 (SB), alone or combined with PDTC and PD98059 (3X), L-NAME, and genistein (GEN). The effect of IL-6 is presented as percentage of change by reference to the normalized effect of SCONT, which was corrected by baseline activity. All results are reported as mean ± S.E. of six experiments. * and &, CYP3A6 activity is significantly different (p < 0.05) from SCONT and IL-6, respectively.

Production of NO·. Incubation of S_TIIR and IL-6 with H_CONT for 4 and 24 h increased the concentration of NO· in the supernatant (p < 0.05, n = 6). This effect was partially prevented by PDTC, PD98059, L-NAME, and genistein (Table 2). Preincubation of H_CONT with SB203580 potentiated the increase in NO· produced by S_TIIR and IL-6 (p < 0.05, n = 6). Although, when SB203580 was combined with PDTC and PD98059, S_TIIR and IL-6 did not enhance NO· concentrations, in fact, they were lower than those measured in H_CONT incubated with S_TIIR and IL-6 (p < 0.05, n = 6). Incubation of H_CONT with SNP alone for 4 and 24 h increased NO· by almost 200% (p < 0.05, n = 6; Table 2).

In the supernatant of H_TIIR, baseline concentrations of NO· were about 2-fold those measured in H_CONT (p < 0.05). Incubation of S_TIIR with H_TIIR for 4 and 24 h increased the concentration of NO· in the supernatant by 37 and 47% (p < 0.05, n = 9). This effect was partially prevented by PDTC, PD98059, L-NAME, and genistein (Table 2). The presence of SB203580 alone or combined with PDTC and PD98059 did not modify the increase in NO· concentrations elicited by S_TIIR. The incubation of H_TIIR with IL-6 did not increase NO· concentration. Incubation of H_TIIR with SNP alone for 4 and 24 h increased NO· by around 100% (p < 0.05, n = 5).

Protein Tyrosine Nitration and Activation of Erk1/2. Nitrotyrosine formation was barely detectable in H_CONT and H_TIIR after 24 h of incubation with saline (Fig. 6A). In H_CONT, S_CONT and S_TIIR activated protein tyrosine nitration, and none of the inhibitors was able to prevent the tyrosine nitration induced by S_TIIR. However, in H_TIIR, L-NAME and genistein partially prevented the protein nitrotyrosine formation induced by S_TIIR (Fig. 6B). Immunoprecipitation of CYP1A2 and CYP3A6 confirmed that both S_CONT and S_TIIR cause the nitration of tyrosine residues of CYP3A6 and, to a minor extent, of CYP1A1/2 (Fig. 6, A and B).

View larger version (49K): [in this window] [in a new window]   FIG. 6. Protein tyrosine nitration of CYP1A1/2 and CYP3A6 by serum from rabbits. A, effect of SCONT and STIIR on nitrotyrosine formation in HCONT and from HTIIR after 24 h of incubation (upper blots); and effect of SCONT and STIIR on tyrosine nitration of CYP1A1/2 and CYP3A6 assessed by immunoprecipitation (IP) (lower blots). B, effect of SCONT and STIIR in the absence and presence of PDTC, PD98059 (PD), L-NAME (L-NA), or SB203580 (SB), alone or combined with PDTC and PD (3X), genistein (Gen), and SNP on protein tyrosine nitration in HTIIR (upper blot), and on CYP3A6 tyrosine nitration as determined by immunoprecipitation (middle blot) and Western blot of CYP3A6 (lower blot).

View larger version (42K): [in this window] [in a new window]   FIG. 7. Effect of SCONT and STIIR and of IL-6 on pTpY Erk1/2 in HTIIR. Representative blots and mean (±S.E.) densitometry ratios (n = 3) of the effect of STIIR (A) and IL-6 (B), which were incubated with HTIIR for 24 h in the absence and presence of PDTC, PD98059 (PD), SB203580 (SB), L-NAME (L-NA), the combination of PDTC, PD98059, and SB203580 (3X), and genistein (Gen).

By comparison with H_TIIR alone, the presence of IL-6 produced a nonsignificant (40%) increase in pTpY Erk1/2 (Fig. 7B, lanes 1 and 3); this effect was prevented by PD98059, L-NAME, and genistein (n = 3; Fig. 7B). Incubation of IL-6 for 24 h with H_TIIR in the presence of SB203580 increased pTpY Erk1/2 (p < 0.05, n = 3; Fig. 7B, lane 6).

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
This study demonstrates that the regulation of CYP1A2 activity differs from that of CYP3A6 in H_CONT and in H_TIIR in a variety of aspects. First, in H_CONT, CYP1A2 activity is reduced by S_TIIR and IL-6, after 4 and 24 h of incubation, without changes in protein expression. The reduction in CYP1A2 activity is partially prevented by PDTC, PD98059, L-NAME, and genistein. In contrast, in H_CONT, S_TIIR and IL-6 do not affect CYP3A6 activity; however, PDTC reduces and SB203580 enhances its activity. In H_TIIR, after 4 and 24 h of incubation, S_TIIR and IL-6 reduce the activity of CYP1A2 without changing its protein expression. However, after 24 h of incubation with H_TIIR, S_TIIR reduced CYP3A6 activity and tended to decrease its expression. The reduction of CYP1A2 and 3A6 activities by S_TIIR and IL-6 is partially prevented by inhibitors of protein tyrosine kinases, Erk1/2 and NOS2, and by PDTC. Diverging from the results obtained with H_CONT and from CYP1A2, in H_TIIR, PDTC does not reduce CYP3A6 activity, although SB203580 enhances CYP3A6 activity 3- to 7-fold.

The decrease in CYP1A2 activity produced by S_TIIR is likely due to IL-6 (Bleau et al., 2000). IL-6 regulates the acute phase response to injury by binding to membrane receptor containing the signal transducing receptor chain glycoprotein 130. Signal transduction involves the activation of JAK family members leading to 1) the activation of transcription factors of the signal transducers and activators of transcription family, 2) the Ras-Raf-MAP3K pathway, with downstream activation of Erk1/2, 3) the stress-activated members p38MAPK and c-Jun N-terminal kinase, and 4) the phosphatidylinositol 3-phosphate/Akt pathway (Heinrich et al., 2003). Incubation of S_TIIR with H_TIIR for 4 and 24 h reduces CYP1A2 activity in the absence of CYP1A1/2 expression changes, by means of a post-translational mechanism. Both S_TIIR and IL-6 increase Erk1/2 phosphorylation, and the reduction of CYP1A2 activity is partially prevented by genistein, PD98059, PDTC, and L-NAME. These results are in agreement with the signal transduction pathways activated by IL-6 (Heinrich et al., 2003).

Following an inflammatory aggression of an aseptic (turpentine) or a septic (LPS) nature, the in vivo and in vitro decrease in P450 activity has been associated with the production of NO· (Takemura et al., 1999; El-Kadi et al., 2000; Barakat et al., 2001; Ferrari et al., 2001). In the current study, incubation of H_CONT and H_TIIR with S_TIIR and IL-6 increased NO· concentrations. Several mechanisms may contribute to the increase of NO· by S_TIIR and IL-6. First, by activating the transcription factors signal transducer and activator of transcription and activator protein-1, IL-6 increases NOS expression and NO· production in less than 4 h (Kleinert et al., 2004). Second, the increase in ROS triggered by the turpentine-induced inflammatory reaction (Proulx and du Souich, 1995) can induce the formation of NO· by means of the cooperative effects of phosphatidylinositol 3-phosphate/Akt- and Erk1/2-dependent activation of NOS (Cai et al., 2003). Finally, ROS, by activating Erk1/2, can increase the formation of NO· in a matter of minutes due to an enhanced transport of L-arginine into the cell (Flores et al., 2003).

In agreement with the mechanisms listed above, preincubation of hepatocytes with PDTC, PD98059, the combination of PDTC, PD98059, and SB203580, and genistein, before the addition of S_TIIR and IL-6, partially prevented the increase in NO· and the reduction in CYP1A2 and 3A6 activity. The involvement of NO· in the S_TIIR- and IL-6-induced reduction of CYP1A2 and 3A6 activity is further supported by the ability of sodium nitroprusside to reduce the activity of these isoforms. However, NO· cannot account for the complete reduction of CYP1A2 and 3A6 activity because the experimental conditions that prevented the increase in NO·, for instance PDTC, PD98059, and genistein in the presence of IL-6, do not restore completely CYP1A2 and 3A6 activity. Moreover, L-NAME does not prevent completely the reduction of CYP1A2 and 3A6 activity. The current results agree with those of Nicholson et al. (2004), who concluded that, besides NO·, there are other mechanisms contributing to the lipopolysaccharide-induced decrease in CYP1A1/2 activity, a conclusion reached because the reduction in activity was only partially prevented by the inhibition of NOS. There is evidence that ROS are also involved in the reduction of P450 activity, since in vitro hydrogen peroxide (H₂O₂) reduces CYP1A2 activity in a dose-dependent manner, and the antioxidants N-acetylcysteine and dimethylthiourea prevent, in a dose-dependent manner, but incompletely, the decrease of CYP1A2 activity produced by S_TIIR (El-Kadi et al., 2000). Moreover, in vivo, the antioxidant U74389G prevents the decrease in CYP1A2 activity produced by the turpentine-induced inflammatory reaction (Galal and Souich, 1999).

In H_CONT, but not in H_TIIR, SB203580 potentiated the increase in NO· production induced by S_TIIR and IL-6. This observation may be explained, taking into account that inhibition of p38MAPK by SB203580 activates Erk1/2, which increases nuclear translocation of NF-B and activator protein-1 (Birkenkamp et al., 2000), known inducers of NOS (Kleinert et al., 2004). Supporting this hypothesis is the fact that when S_TIIR and IL-6 were incubated with SB203580 combined with PD98059, which inhibits Erk1/2, and PDTC, an antioxidant that blocks NF-B translocation, the increase in NO· was prevented.

The mechanism underlying the decrease in CYP1A2 and CYP3A6 activities by NO· remains unclear. The decrease in activity may be due to binding of NO· to the heme prosthetic group (Minamiyama et al., 1997). In addition, NO· may interact with sulfhydryl groups of cysteine amino acid residues in P450 enzymes, forming reversible S-nitrosothiols (Minamiyama et al., 1997). Irreversible nitration of tyrosine residues positioned at the active site of the enzyme may be another mechanism contributing to the decrease in CYP1A2 and 3A6 activities (Roberts et al., 1998). Tyrosine nitration of CYP1A2 and CYP3A6 was observed after 4 and 24 h of incubation with both S_CONT and S_TIIR, suggesting that tyrosine nitration is not responsible for the decrease in CYP1A2 and 3A6 activities elicited by S_TIIR and IL-6. As a consequence, we postulate that the inhibition of CYP1A2 and 3A6 by S_TIIR and IL-6 is associated with the binding of NO· to heme; a similar mechanism was proposed to explain P450 inhibition by LPS (Takemura et al., 1999) and sodium nitroprusside (Vuppugalla and Mehvar, 2004).

In H_CONT, PDTC potentiated the decrease of CYP3A6 activity elicited by S_TIIR and IL-6, despite a decrease in NO·. In contrast, the S_TIIR- and IL-6-induced decrease of CYP1A2 activity was not affected by PDTC. The decrease in CYP3A6 activity, in the absence of CYP3A6 expression changes, produced by PDTC was apparent after 4 h of incubation, suggesting that the PDTC effect is associated with its antioxidant properties. This hypothesis is indirectly supported by the fact that antioxidants, such as resveratrol, polyphenols, isoflavans, quercetin, and ginsenoids are potent inhibitors of CYP3A4 activity (Muto et al., 2001; Kent et al., 2002; Piver et al., 2003). Further studies are required to elucidate how antioxidants may affect CYP3A6 activity but not that of CYP1A2.

Incubation of H_TIIR with S_TIIR for 24 h did not affect CYP1A1/2 expression but reduced the expression of CYP3A6 by around 30%, and even if the decrease in expression was not statistically significant, it may have contributed to the decrease in CYP3A6 activity. The decrease in CYP3A6 expression was reverted by the preincubation of H_TIIR with PDTC and PD98059. It has been shown that activation of NF-B by LPS, IL-1ß, and tumor necrosis factor- leads to the suppression of gene expression and down-regulation of CYP1A1 and CYP2C11 (Iber et al., 2000; Ke et al., 2001). Unpublished results from this laboratory show that S_TIIR or IL-6 do not activate NF-B nuclear translocation, results that are in agreement with the observation that the down-regulation of CYP2C11 produced by IL-6 is not mediated by NF-B (Iber et al., 2000). Altogether, the down-regulation of CYP3A6 appears NF-B independent but mediated by Erk1/2 and ROS.

The incubation of H_CONT and H_TIIR with SB203580, alone or combined with PDTC and PD98059, potentiated the effect of S_TIIR and IL-6 on the decrease in CYP1A2 activity without changes in its expression. The reduction of CYP1A2 activity by SB203580 is dose-dependent with an IC₅₀ of 21 µM and a predicted maximal inhibition of 73%. Two mechanisms may contribute to the decrease in CYP1A2 activity. SB203580 is a pyridinylimidazole analog and, as such, it inhibits the activity of recombinant CYP1A2 (Adams et al., 1998). On the other hand, in H_CONT, SB203580 increased NO· concentration by around 25%, and that may have contributed to the decrease in CYP1A2 activity.

When H_TIIR were incubated with S_TIIR or IL-6 and SB203580, CYP3A6 activity was 3- and 7-fold greater than control values after 4 and 24 h of incubation, respectively, without changes in protein expression, e.g., compared with H_TIIR incubated with S_TIIR. Moreover, SB203580 increased, in a dose-dependent manner, CYP3A6 activity 10-fold. The mechanism underlying the increase in CYP3A6 activity by SB203580 remains unknown. Increases in CYP3A6 activity by SB203580 of such amplitude point to a mechanism related to the rate-limiting steps of the P450 catalytic cycle; e.g., step 2, electron transfer by NADPH via NADPH-cytochrome P450 reductase, and step 4, electron transfer by cytochrome b₅ (Guengerich, 1999; Backes and Kelley, 2003). SB203580 could elicit a heterotropic cooperativity with DFB used to measure CYP3A6 activity (Guengerich, 1999). Three facts argue against a cooperativity phenomenon; i.e., the magnitude of the increase in activity, e.g., 1000%, the increase of activity of CYP3A6 was greater after 24 h than after 4 h of incubation, and the increase in CYP3A6 activity was much greater in primed hepatocytes (H_TIIR) than in control hepatocytes (H_CONT). Further studies are required to elucidate how p38MAPK inhibition by SB203580 modulates the activity of CYP3A6, because of the interest of p38MAPK inhibitors as modulators of the inflammatory reaction in humans (Parasrampuria et al., 2003).

In conclusion, the present study demonstrates that in the turpentine-induced inflammatory reaction, the reduction of CYP1A1/2 and CYP3A6 activity by S_TIIR and IL-6 is partially prevented by inhibitors of protein tyrosine kinases, Erk1/2 and NOS2, and PDTC. In addition, this study emphasizes that the effect of S_TIIR and IL-6 is model- and isoform-dependent, e.g., the antioxidant PDTC is a potent inhibitor of CYP3A6 in H_CONT but not of CYP1A2, and PDTC has no effect on any isoform in H_TIIR; moreover, inhibition of p38MAPK by SB203580 triggers a significant increase in CYP4A6 activity without changing its expression in H_TIIR, a phenomenon not observed in H_CONT or with CYP1A2.

	Acknowledgments

 
We thank Lucie Héroux for excellent technical assistance. We are grateful to Dr. N. Chauret from Merck Frosst Canada for providing 3,4-difluorobenzyloxy-5,5 dimethyl-4-(4-methylsulfonyl phenyl)-(5H)-furan-2-one (DFB).

	Footnotes

 
This study was supported by the Canadian Institutes of Health Research (MOP-43925).

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

doi:10.1124/dmd.105.006528.

ABBREVIATIONS: P450, cytochrome P450; LPS, lipopolysaccharide; IL, interleukin; ROS, reactive oxygen species; NO·, nitric oxide; MAPK, mitogen-activated protein kinase; NOS, nitric-oxide synthase; NF-B, nuclear factor B; PD98059, 2'-amino-3'-methoxyflavone; SB203580, 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole; PDTC, pyrrolidine dithiocarbamate; SNP, sodium nitroprusside; PAGE, polyacrylamide gel electrophoresis; pTpY Erk1/2, phosphorylated Erk1/2; DFB, 3,4-difluorobenzyloxy-5,5 dimethyl-4-(4-methylsulfonyl phenyl)-(5H)-furan-2-one; DFH, 3-hydroxy-4-(4-methylsulfonyl phenyl)-(5H)-furan-2-one; Erk1/2, extracellular signal-related kinase 1/2; H_CONT, hepatocytes from a control rabbit; H_TIIR, hepatocytes from rabbits with a turpentine-induced inflammatory reaction; JAK, Janus-associated protein tyrosine kinase; L-NAME, N-nitro-L-arginine methyl ester; S_CONT, serum from control rabbits; S_TIIR, serum from rabbits with a turpentine-induced inflammatory reaction; U74389G, 21-[4-(2,6-di-1-pyrrolidinyl-4-pyrimidinyl)-1-(piperazinyl]-pregna-1,4,9(11)-triene-3,20-dione (2)-2-butenedioate.

Address correspondence to: Patrick du Souich, Département de pharmacologie, Local R-412, Faculté de médecine, Université de Montréal, C.P. 6128, Succ. Centre-ville, Montréal, Québec, Canada, H3C 3J7. E-mail: patrick.du.souich{at}umontreal.ca

	References

Top Abstract Materials and Methods Results Discussion References

 


Adams JL, Boehm JC, Kassis S, Gorycki PD, Webb EF, Hall R, Sorenson M, Lee JC, Ayrton A, Griswold DE, et al. (1998) Pyrimidinylimidazole inhibitors of CSBP/p38 kinase demonstrating decreased inhibition of hepatic cytochrome P450 enzymes. Bioorg Med Chem Lett 8: 3111–3116.[CrossRef][Medline]

Backes WL and Kelley RW (2003) Organization of multiple cytochrome P450s with NADPHcytochrome P450 reductase in membranes. Pharmacol Ther 98: 221–233.[CrossRef][Medline]

Barakat MM, El-Kadi AO, and du Souich P (2001) L-NAME prevents in vivo the inactivation but not the down-regulation of hepatic cytochrome P450 caused by an acute inflammatory reaction. Life Sci 69: 1559–1571.[CrossRef][Medline]

Birkenkamp KU, Tuyt LM, Lummen C, Wierenga AT, Kruijer W, and Vellenga E (2000) The p38 MAP kinase inhibitor SB203580 enhances nuclear factor-kappa B transcriptional activity by a non-specific effect upon the ERK pathway. Br J Pharmacol 131: 99–107.[CrossRef][Medline]

Bleau AM, Levitchi MC, Maurice H, and du Souich P (2000) Cytochrome P450 inactivation by serum from humans with a viral infection and serum from rabbits with a turpentine-induced inflammation: the role of cytokines. Br J Pharmacol 130: 1777–1784.[CrossRef][Medline]

Cai H, Li Z, Davis ME, Kanner W, Harrison DG, and Dudley SC Jr (2003) Akt-dependent phosphorylation of serine 1179 and mitogen-activated protein kinase kinase/extracellular signal-regulated kinase 1/2 cooperatively mediate activation of the endothelial nitric-oxide synthase by hydrogen peroxide. Mol Pharmacol 63: 325–331.[Abstract/Free Full Text]

Chang KC, Bell TD, Lauer BA, and Chai H (1978) Altered theophylline pharmacokinetics during acute respiratory viral illness. Lancet 1: 1132–1133.[Medline]

Cheng PY and Morgan ET (2001) Hepatic cytochrome P450 regulation in disease states. Curr Drug Metab 2: 165–183.[CrossRef][Medline]

El-Kadi AO, Bleau AM, Dumont I, Maurice H, and du Souich P (2000) Role of reactive oxygen intermediates in the decrease of hepatic cytochrome P450 activity by serum of humans and rabbits with an acute inflammatory reaction. Drug Metab Dispos 28: 1112–1120.[Abstract/Free Full Text]

El-Kadi AO, Maurice H, Ong H, and du Souich P (1997) Down-regulation of the hepatic cytochrome P450 by an acute inflammatory reaction: implication of mediators in human and animal serum and in the liver. Br J Pharmacol 121: 1164–1170.[Medline]

Ferrari L, Peng N, Halpert JR, and Morgan ET (2001) Role of nitric oxide in down-regulation of CYP2B1 protein, but not RNA, in primary cultures of rat hepatocytes. Mol Pharmacol 60: 209–216.[Abstract/Free Full Text]

Flores C, Rojas S, Aguayo C, Parodi J, Mann G, Pearson JD, Casanello P, and Sobrevia L (2003) Rapid stimulation of L-arginine transport by D-glucose involves p42/44(mapk) and nitric oxide in human umbilical vein endothelium. Circ Res 92: 64–72.[Abstract/Free Full Text]

Galal A and Souich P (1999) 21-aminosteroids prevent the down-regulation of hepatic cytochrome P450 induced by hypoxia and inflammation in conscious rabbits. Br J Pharmacol 128: 374–379.[CrossRef][Medline]

Guengerich FP (1999) Cytochrome P-450 3A4: regulation and role in drug metabolism. Annu Rev Pharmacol Toxicol 39: 1–17.[CrossRef][Medline]

Haas CE, Kaufman DC, Jones CE, Burstein AH, and Reiss W (2003) Cytochrome P450 3A4 activity after surgical stress. Crit Care Med 31: 1338–1346.[CrossRef][Medline]

Handschin C and Meyer UA (2003) Induction of drug metabolism: the role of nuclear receptors. Pharmacol Rev 55: 649–673.[Abstract/Free Full Text]

Heinrich PC, Behrmann I, Haan S, Hermanns HM, Muller-Newen G, and Schaper F (2003) Principles of interleukin (IL)-6-type cytokine signalling and its regulation. Biochem J 374: 1–20.[CrossRef][Medline]

Iber H, Chen Q, Cheng PY, and Morgan ET (2000) Suppression of CYP2C11 gene transcription by interleukin-1 mediated by NF-kappaB binding at the transcription start site. Arch Biochem Biophys 377: 187–194.[CrossRef][Medline]

Ke S, Rabson AB, Germino JF, Gallo MA, and Tian Y (2001) Mechanism of suppression of cytochrome P-450 1A1 expression by tumor necrosis factor-alpha and lipopolysaccharide. J Biol Chem 276: 39638–39644.[Abstract/Free Full Text]

Kent UM, Aviram M, Rosenblat M, and Hollenberg PF (2002) The licorice root derived isoflavan glabridin inhibits the activities of human cytochrome P450S 3A4, 2B6 and 2C9. Drug Metab Dispos 30: 709–715.[Abstract/Free Full Text]

Kleinert H, Pautz A, Linker K, and Schwarz PM (2004) Regulation of the expression of inducible nitric oxide synthase. Eur J Pharmacol 500: 255–266.[CrossRef][Medline]

Levitchi M, Fradette C, Bleau AM, Michaud D, Kourylko O, Arcand M, and du Souich P (2004) Signal transduction pathways implicated in the decrease in CYP1A1, 1A2 and 3A6 activity produced by serum from rabbits and humans with an inflammatory reaction. Biochem Pharmacol 68: 573–582.[CrossRef][Medline]

Lowry OH, Rosebrough NJ, Farr AL, and Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193: 265–275.[Free Full Text]

Meloche S (1995) Cell cycle reentry of mammalian fibroblasts is accompanied by the sustained activation of p44mapk and p42mapk isoforms in the G1 phase and their inactivation at the G1/S transition. J Cell Physiol 163: 577–588.[CrossRef][Medline]

Minamiyama Y, Takemura S, Imaoka S, Funae Y, Tanimoto Y, and Inoue M (1997) Irreversible inhibition of cytochrome P450 by nitric oxide. J Pharmacol Exp Ther 283: 1479–1485.[Abstract/Free Full Text]

Morgan ET, Ullrich V, Daiber A, Schmidt P, Takaya N, Shoun H, McGiff JC, Oyekan A, Hanke CJ, Campbell WB, et al. (2001) Cytochromes P450 and flavin monooxygenases—targets and sources of nitric oxide. Drug Metab Dispos 29: 1366–1376.[Abstract/Free Full Text]

Muto S, Fujita K, Yamazaki Y, and Kamataki T (2001) Inhibition by green tea catechins of metabolic activation of procarcinogens by human cytochrome P450. Mutat Res 479: 197–206.[Medline]

Nicholson TE, Dibb S, and Renton KW (2004) Nitric oxide mediates an LPS-induced depression of cytochrome P450 (CYP1A) activity in astrocytes. Brain Res 1029: 148–154.[CrossRef][Medline]

Nims RW, Cook JC, Krishna MC, Christodoulou D, Poore CM, Miles AM, Grisham MB, and Wink DA (1996) Colorimetric assays for nitric oxide and nitrogen oxide species formed from nitric oxide stock solutions and donor compounds. Methods Enzymol 268: 93–105.[CrossRef][Medline]

Parasrampuria DA, de Boer P, Desai-Krieger D, Chow AT, and Jones CR (2003) Single-dose pharmacokinetics and pharmacodynamics of RWJ 67657, a specific p38 mitogen-activated protein kinase inhibitor: a first-in-human study. J Clin Pharmacol 43: 406–413.[Abstract/Free Full Text]

Piver B, Berthou F, Dreano Y, and Lucas D (2003) Differential inhibition of human cytochrome P450 enzymes by epsilon-viniferin, the dimer of resveratrol: comparison with resveratrol and polyphenols from alcoholized beverages. Life Sci 73: 1199–1213.[CrossRef][Medline]

Proulx M and du Souich P (1995) Inflammation-induced decrease in hepatic cytochrome P450 in conscious rabbits is accompanied by an increase in hepatic oxidative stress. Res Commun Mol Pathol Pharmacol 87: 221–236.[Medline]

Renton K (1978) Altered theophylline kinetics. Lancet ii: 160–161.

Renton KW (2001) Alteration of drug biotransformation and elimination during infection and inflammation. Pharmacol Ther 92: 147–163.[CrossRef][Medline]

Riddick DS, Lee C, Bhathena A, Timsit YE, Cheng PY, Morgan ET, Prough RA, Ripp SL, Miller KK, Jahan A, et al. (2004) Transcriptional suppression of cytochrome P450 genes by endogenous and exogenous chemicals. Drug Metab Dispos 32: 367–375.[Abstract/Free Full Text]

Roberts ES, Lin H, Crowley JR, Vuletich JL, Osawa Y, and Hollenberg PF (1998) Peroxynitrite-mediated nitration of tyrosine and inactivation of the catalytic activity of cytochrome P450 2B1. Chem Res Toxicol 11: 1067–1074.[CrossRef][Medline]

Sonne J, Dossing M, Loft S, and Andreasen PB (1985) Antipyrine clearance in pneumonia. Clin Pharmacol Ther 37: 701–704.[Medline]

Takemura S, Minamiyama Y, Imaoka S, Funae Y, Hirohashi K, Inoue M, and Kinoshita H (1999) Hepatic cytochrome P450 is directly inactivated by nitric oxide, not by inflammatory cytokines, in the early phase of endotoxemia. J Hepatol 30: 1035–1044.[CrossRef][Medline]

Van Vleet TR, Mace K, and Coulombe RA Jr (2002) Comparative aflatoxin B(1) activation and cytotoxicity in human bronchial cells expressing cytochromes P450 1A2 and 3A4. Cancer Res 62: 105–112.[Abstract/Free Full Text]

Vuppugalla R and Mehvar R (2004) Hepatic disposition and effects of nitric oxide donors: rapid and concentration-dependent reduction in the cytochrome P450-mediated drug metabolism in isolated perfused rat livers. J Pharmacol Exp Ther 310: 718–727.[Abstract/Free Full Text]

This Article Abstract Full Text (PDF) All Versions of this Article: dmd.105.006528v1 34/1/27    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Kourylko, O. Articles by du Souich, P. Search for Related Content PubMed PubMed Citation Articles by Kourylko, O. Articles by du Souich, P.