Introduction

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Paclitaxel, a member of the taxane family of anti-microtubule agents, is widely used in the treatment of several types of cancer, such as, ovarian, breast, and lung carcinomas. Cytochrome P450-mediated phase I metabolism represents a major route for inactivation and elimination of paclitaxel (Harris et al., 1994; Rahman et al., 1994). Members of the CYP3A subfamily are highly expressed in the liver and intestine and play a central role in the biotransformation of numerous endogenous substances and xenobiotics, including paclitaxel (Harris et al., 1994). In humans, CYP3A enzymes (CYP3A4, 3A5, and 3A7) collectively contribute to the metabolism of approximately two-thirds of all marketed drugs (Wrighton et al., 2000). A variety of structurally diverse compounds induce CYP3A genes, which provides a molecular basis for many clinically observed drug-drug interactions (Kocarek et al., 1995). Recent studies have shown that the pregnane X receptor (PXR¹ NR1I2), a member of the nuclear receptor family, mediates induction of CYP3A genes by a wide array of xenobiotics (Bertilsson et al., 1998; Blumberg et al., 1998; Lehmann et al., 1998). Many structurally dissimilar lipophilic compounds bind and activate PXR. In the presence of a ligand, PXR binds to xenobiotic-response elements present within the promoter region of CYP3A genes as a heterodimer with retinoid X receptor  to induce gene transcription. Recently, it was shown that paclitaxel is an effective inducer of CYP3A expression in primary cultures of rat and human hepatocytes (Kostrubsky et al., 1997; Kostrubsky et al., 1998; Nallani et al., 2001a). Furthermore, employing cell based reporter assays, Synold et. al. (2001) and our group showed that paclitaxel activates the human PXR (Nallani et al., 2001b; Synold et al., 2001). In the present study, we employed PXR-null mice to investigate whether targeted disruption of PXR influences the ability of paclitaxel to induce the hepatic CYP3A expression in mice. The results indicate that the induction of CYP3A by paclitaxel is mediated by PXR in vivo.

	Materials and Methods

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Cell Culture, Chemicals, and Reagents. Paclitaxel, pregnenolone 16-carbonitrile (PCN), testosterone, and 6-hydroxytestosterone were obtained from Sigma-Aldrich (St. Louis, MO). African green monkey kidney fibroblasts (CV-1 cells) were obtained from American Type Culture Collection (Manassas, VA). Cell culture media and supplements were purchased from CellGro (Herndon, VA). Polyclonal antibody for rat CYP3A was obtained from BD Gentest Corporation (Woburn, MA). The horseradish-peroxidase conjugated anti-goat secondary antibody was obtained from Oxford Biomedical Research Inc. (Oxford, MI). CYP3A11 oligonucleotide probes were custom made from Invitrogen (Carlsbad, CA). Enhanced chemiluminescence detection reagent was obtained from Amersham Biosciences Inc. (Piscataway, NJ).

Mouse PXR (mPXR) Activation Studies. Transient transfection of CV-1 cells was performed as described earlier (Jones et al., 2000). The luciferase reporter gene construct p3A4-362 (7836/7208ins) was used to examine activation of mPXR by paclitaxel and PCN, employing a concentration range of 1 nM to 10 µM. This construct contains the promoter proximal region of the CYP3A4 gene (bases 362 to + 53) linked to the distal xenobiotic-response element module region (Goodwin et al., 1999). Transfection mixes contained 8 ng luciferase reporter gene construct, 5 ng PXR expression vector (pSG5mPXR) (Lehmann et al., 1998), 8 ng p-actin-SPAP (an expression vector containing the secreted placental alkaline phosphatase cDNA under control of the -actin promoter), and 52 ng pBluescript (Stratagene, La Jolla, CA). Following overnight transfection, the cells were replenished with drug-containing medium and incubated for an additional 24 h. At the end of drug treatment, an aliquot of medium was withdrawn for SPAP assay and the cells lysed prior to luciferase determination. Luciferase activity was normalized to SPAP expression. Dose-response curves were generated and analyzed employing nonlinear data analysis (WinNonlin Standard, version 1.5; Pharsight Corporation, Mountain View, CA).

Drug Treatment of PXR Wild-type and PXR-null Mice. PXR-null (/) mice were generated and bred as previously described (Staudinger et al., 2001a). Adult male PXR wild-type (+/+) and PXR-null (/) mice weighing 25 to 35 g were employed in this study. The mice were provided with water and chow ad libitum during the experiment. Mice (n = 4) were randomly assigned to treatment groups receiving i.p. injection of paclitaxel (40 mg/kg) or PCN (100 mg/kg) in Cremophor solvent for 4 consecutive days. The Cremophor solvent, which comprised of Cremophor EL (5%) and ethanol (5%) in 5% dextrose for injection solution, is the clinically used paclitaxel formulation. Cremophor Solvent treated mice were maintained as controls. Twenty-four hours after the drug treatment, the mice were sacrificed employing carbon dioxide asphyxiation, and the livers were isolated and snap frozen in liquid nitrogen. Livers were processed for total RNA isolation and microsome preparation.

Hepatic Microsomal Metabolism of Testosterone. The CYP3A activity in liver microsomes isolated from control and treated mice was assessed employing testosterone 6-hydroxylation assay. The methods employed for the isolation of microsomal fraction and microsomal testosterone metabolism have been described previously (Genter et al., 2002). Based on the initial assessment of the linear range of testosterone 6-hydroxylation with respect to protein amount and incubation period, we employed 50 µg of microsomal protein and 15-min incubation period. Testosterone (1-100 µM) was incubated with liver microsomes in the presence of an NADPH-regenerating system. The reaction was initiated by the addition of glucose-6-phosphate dehydrogenase. At the end of the incubation period the reaction was terminated by the addition of the extraction solvent, methylene chloride (4 ml), which was immediately followed by the addition of the internal standard, 11-hydroxyprogesterone (1 µg/ml). The extracted organic layer was dried under nitrogen and reconstituted in 400 µl of methanol/water/acetonitrile mixture (39:60:1). An aliquot of the reconstituted sample (150 µl) was analyzed employing a previously described high performance liquid chromatography method (Nallani et al., 2001a). To confirm the role of CYP3A in testosterone 6-hydroxylation, microsomal testosterone metabolism was also examined in the presence of ketoconazole, a known inhibitor of CYP3A. For this purpose, testosterone (10 µM) metabolism was carried out as described in the presence of ketoconazole (10 µM).

Northern Blot Analysis. Total RNA from control and drug-treated mouse livers was isolated using TRIzol reagent (Invitrogen) and quantitated spectrophotometrically. Total RNA (10 µg) was then fractionated by electrophoresis in 1% agarose gels containing 2.2 M formaldehyde, followed by overnight transfer onto a nylon membrane (Millipore, Bedford, MA). Equal loading per lane was verified by ethidium bromide staining of 18S and 28S ribosomal RNA, which was visualized and photographed under UV illumination. For the detection of CYP3A11 mRNA, we used the previously described 30 base oligonucleotide probe (5'-TGTCCGATGTTCTTAGACACTGCCTTTCTG-3') (Sewer et al., 1998). The oligonucleotide probe was 5'-end labeled with [³²-P]ATP using T4 polynucleotide kinase. Hybridization was carried out in the presence of 100 µg/ml of ssDNA (Sigma-Aldrich) at 45°C. Northern membranes were washed at 45°C as described previously (Church and Gilbert, 1984). An autoradiogram was developed using Kodak X-OMAT X-ray film following 2- to 3-day exposure (Eastman Kodak, Rochester, NY).

Western Blot Analysis. Western blot analysis of the microsomal protein was performed exactly as described previously (Nallani et al., 2001a). Immunoreactive CYP3A was detected employing a polyclonal anti-rat CYP3A antibody (1:1500) for 1 h. This polyclonal antibody nonspecifically detects various CYP3A isoforms in mouse liver (Yamada et al., 2002). The antibody binding was visualized using a horseradish peroxidase-conjugated anti-goat secondary antibody for polyclonal CYP3A antibody (1:15000 dilution), followed by enhanced chemiluminescence detection (ECL; Amersham Biosciences Inc.). The immunoblots were quantitated employing NucleoVision image analyzer with Gel Expert photodensitometry software (NucleoTech, San Carlos, CA).

Data Analysis. Statistical analysis of 6-hydroxytestosterone formation and -fold increase in immunoreactive CYP3A protein and CYP3A11 mRNA among different treatment groups was performed employing one-way analysis of variance, followed by Tukey's test. A p < 0.05 was interpreted as the level of statistical significance. The kinetics of microsomal metabolism were analyzed employing WinNonlin Standard (version 1.5) (Pharsight Corporation). The velocity of 6-hydroxytestosterone formation was plotted as a function of substrate (testosterone) concentration. Data were fitted to Sigmoidal E_max equation V = V_max* [Sⁿ]/(K_mⁿ + [Sⁿ]) where V = reaction velocity (nmol/min/mg of microsomal protein), V_max = maximum velocity, S = substrate concentration (µM), and K_m = substrate concentration at half maximal velocity, n = hill coefficient or coefficient of sigmoidicity. Goodness of fit was assessed based on visual inspection, residual analysis, Akaike and Schwartz criteria, and percent coefficient of variation for V_max and K_m values.

	Results and Discussion

Top Abstract Introduction Materials and Methods Results and Discussion References

mPXR Activation. Activation of PXR and CYP3A induction by different xenobiotics is known to exhibit interspecies variability (Jones et al., 2000). Although it is recognized that paclitaxel activates human PXR, whether it activates PXRs from other species is not known. Therefore, we first examined the effect of paclitaxel on mPXR activity employing transient transfection of a reporter gene construct harboring PXR-9-cis retinoic acid responsive elements of CYP3A gene in CV-1 cells. The mPXR activation profiles of PCN, a known CYP3A inducer in rodents and an mPXR ligand, and paclitaxel at a concentration range of 1 nM to 10 µM were compared (Fig. 1). The concentration response for each compound was fit to a Sigmoidal E_max model employing WinNonlin Standard (version 1.5; Pharsight Corporation) and the maximal effect (E_max) and half-maximal effective concentration (EC₅₀) were determined from the model. The EC₅₀ values for PXR activation by paclitaxel and PCN were 5.6 and 0.27 µM, respectively. This indicates that paclitaxel is a strong activator of mPXR, albeit weaker than PCN.

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Dose-response profiles of mPXR activation by paclitaxel (open squares) and PCN (filled diamonds). CV-1 cells were transiently transfected with the pSG5-mPXR and the xenobiotic-response element module-CYP3A4-luciferase reporter plasmid and treated with paclitaxel and PCN (1 nM-10 µM) for 24 h. Cell extracts were subsequently assayed for luciferase activity and normalized to -actin-serum placental alkaline phosphatase activity to account for differences in the transfection efficiency (mean ± S.E.M., n = 3).

Paclitaxel-mediated CYP3A Induction in Vivo. Previous studies have shown that paclitaxel induces CYP3A in rat and human hepatocytes in vitro (Kostrubsky et al., 1998; Nallani et al., 2001a; Synold et al., 2001). However, to date the ability of paclitaxel to induce Cyp3a expression in vivo has not been characterized. The ability of paclitaxel to activate mouse PXR in cell-based assays suggested that the induction of Cyp3a may be mediated by this receptor. Therefore, we compared the induction of Cyp3a in wild-type mice and mice lacking functional PXR. In these experiments, PCN was employed as a positive control for PXR activation. Figure 2A shows the expression of CYP3A11 mRNA in wild-type and PXR-null mice determined using Northern blot analysis. As shown, PCN increased CYP3A11 mRNA levels by 5-fold (range 4- to 7-fold) in PXR wild-type mice. The observed PCN-mediated increase in CYP3A11 mRNA is in agreement with previously published reports (Xie et al., 2000a; Staudinger et al., 2001a,b). Paclitaxel caused a 2.5-fold (range 1.9- to 2.7-fold) induction of CYP3A11 mRNA in PXR wild-type mice. The induction of Cyp3a11 expression by paclitaxel and PCN was completely absent in PXR-null mice; the mRNA levels in paclitaxel- and PCN-treated PXR-null mice were not significantly different from controls (p > 0.05). Interestingly, we observed an approximately 2-fold increase in the basal CYP3A11 mRNA levels in PXR (/) mice compared to those in PXR wild type (+/+), which is consistent with previously reported observations (Staudinger et al., 2001a).

View larger version (40K): [in this window] [in a new window]   Fig. 2.   Expression of CYP3A11 mRNA and CYP3A immunoreactive protein levels in paclitaxel and PCN-treated mice compared with vehicle-treated controls. A, representative Northern blot depicting CYP3A11 mRNA levels in PXR wild-type and PXR-null mice treated with vehicle, paclitaxel, or PCN. B, representative Western blot depicting immunoreactive CYP3A protein levels in mouse liver microsomes prepared from PXR wild-type and PXR-null mice treated with vehicle, paclitaxel, or PCN.

View this table: [in this window] [in a new window]   TABLE 1 Kinetics of hepatic microsomal testosterone 6-hydroxylation Microsomal preparations from PXR wild-type and PXR-null mice (n = 4 for each treatment) treated with vehicle, paclitaxel, and PCN were employed for determining the kinetics of testosterone 6-hydroxylation. The velocity of 6-hydroxytestosterone formation in microsomal incubations was plotted against the substrate (testosterone) concentration. The profiles were analyzed by a nonlinear data analysis method employing the model V = Vmax*[Sn]/(Kmn + [Sn], to determine the Km [substrate (S) concentration when velocity (V) is one-half the maximal velocity (Vmax)] (n = hill coefficient or coefficient of sigmoidicity).