7-Benzyloxyresorufin-O-dealkylase activity as a marker for measuring cytochrome P450 CYP3A induction in mouse liver

Analytical Biochemistry 398 (2010) 104–111

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7-Benzyloxyresorufin-O-dealkylase activity as a marker for measuring cytochrome P450 CYP3A induction in mouse liver Christoph E. Hagemeyer a, Carolin Bürck b,c, Ricarda Schwab b,c, Rolf Knoth b, Ralf P. Meyer b,* a

Baker IDI Heart and Diabetes Institute, Melbourne, Vic. 8008, Australia Abteilung Neuropathologie, Neurozentrum, Universitätsklinik Freiburg, D-79106 Freiburg, Germany c Department of Mechanical and Process Engineering, Hochschule Furtwangen University, D-78054 Villingen–Schwenningen, Germany b

a r t i c l e

i n f o

Article history: Received 11 August 2009 Received in revised form 2 November 2009 Accepted 4 November 2009 Available online 10 November 2009 Keywords: Phenytoin CYP3A11 Mouse 7-Benzyloxyresorufin In vitro testing Prediction

a b s t r a c t The cytochrome P450 subfamily CYP3A belongs to the most important detoxification enzymes. Because the main CYP3A isoforms are not polymorphic and therefore detract themselves from genetic screening as a potent prediction marker for drug metabolism or induction effects, effective in vitro testing of a putative drug–CYP3A interaction is indicated. We used mouse liver microsomes treated with the model drug phenytoin to set up an effective and reliable in vitro test system. A metabolic assay analyzing 7-alkoxyresorufin-O-dealkylation showed specific CYP3A-dependent 7-benzyloxyresorufin oxidation (BROD). This was confirmed by testing other alkoxyresorufins (7-ethoxy-, 7-methoxy-, and 7-pentoxyresorufin) in mice and correlation of the data with testosterone 6b-hydroxylation and a plethora of isoform-specific chemical inhibitors (orphenadrine, chloramphenicol, nifedipine, ketoconazole, and sulfaphenazole). Isoform-specific expression and induction of CYP3A11 in mouse liver was tested by RNase protection assay, reverse transcription polymerase chain reaction (RT-PCR), and immunoblot. With the BROD assay, we could clearly dissect CYP3A11 from other P450s induced by phenytoin-like CYP2C29, CYP2B9, CYP1A1, and CYP4A. We conclude that the BROD assay is a specific tool to assign CYP3A induction by drugs or other chemicals, at least in a mouse model system. Ó 2009 Elsevier Inc. All rights reserved.

Cytochrome P450 (P450, CYP)1 is the collective term for a large gene superfamily of heme-containing proteins that play an important role in the oxidative metabolism of numerous endogenous and foreign compounds. Four of the P450 families, namely the families 1–4, are involved in drug metabolism and are preferentially expressed in the liver but also in extrahepatic tissues such as the lung, the kidney, and the brain [1,2]. Among these P450s, the subfamily CYP3A is the most important one. This is true for both the amount (25% of all hepatic human P450s) and the importance in drug oxi* Corresponding author. Fax: +49 761 6964482. E-mail address: [email protected] (R.P. Meyer). 1 Abbreviations used: CYP, P450, cytochrome P450; BROD, O-dealkylation of 7benzyloxyresorufin; DPH, 5,50 -diphenylhydantoin (phenytoin); BBT, N-benzyl-1aminobenzotriazole; IgG, immunoglobulin G; FPIA, polarization immunoassay; PBS, phosphate-buffered saline; EDTA, ethylenediaminetetraacetic acid; PMSF, phenylmethylsulfonyl fluoride; HPLC, high-performance liquid chromatography; PDA, photodiode array; UV–vis, ultraviolet–visible; SDS, sodium dodecyl sulfate; ECL, enhanced chemiluminescence; NCBI, National Center for Biotechnology Information; cDNA, complementary DNA; PCR, polymerase chain reaction; DIG, digoxigenin; cRNA, complementary RNA; RPA, RNase A protection assay; PAGE, polyacrylamide gel electrophoresis; ANOVA, analysis of variance; EROD, O-dealkylation of 7-ethoxyresorufin; MROD, O-dealkylation of 7-methoxyresorufin; PROD, O-dealkylation of 7pentoxyresorufin; Dex, dexamethasone; CF, clofibrate; b-NF, b-naphthoflavone; 3MC, 3-methylcholantrene; mRNA, messenger RNA. 0003-2697/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2009.11.004

dations. CYP3A isoforms are responsible for approximately 50% of the metabolism of clinically used drugs [3]. CYP3A isoforms are highly inducible by a broad variety of drugs and other chemicals in many species (e.g., mice, humans) [4]. Apart from in the liver, drug-inducible CYP3A4 (human) or CYP3A11 (mouse) isoforms are reported to influence endocrine function by drug-mediated alterations in androgen level and signaling in the brain [5]. Because of these manifold actions of CYP3A isoforms, it is proven that efficient testing and prediction of CYP3A metabolic properties are prerequisites for the use of drugs or special nutrition in, for example, clinical therapy or self-medication. However, genetic screening usually provides no sufficient information on CYP3A function. This is because the major human isoform CYP3A4 is not regarded as a polymorphic enzyme, unlike other prominent P450s such as CYP2C9/19 and CYP2D6. Furthermore, there is very large interindividual variability in expression of CYP3A isoforms, with hepatic microsomal apoprotein content varying by 40- to 50-fold [3]. These findings show that CYP3A needs to be tested by the use of adequate in vitro prediction models. These must be as cost-efficient as possible and capable of high-throughput analysis [6]. In the current study, we introduced the O-dealkylation of 7benzyloxyresorufin (BROD) as a specific marker to assign induction of CYP3A11 in a mouse liver microsomal preparation. Because a

Measuring cytochrome P450 CYP3A induction / C.E. Hagemeyer et al. / Anal. Biochem. 398 (2010) 104–111

long history is available on phenytoin (DPH, 5,50 -diphenylhydantoin) induction potency and pharmacology [7–9], with a broad set of data, we used the antiepileptic drug phenytoin as a model to investigate P450 induction profiles. This enabled us to unequivocally check BROD assay specificity in mouse liver microsomes. We concluded from our data that the BROD assay is an adequate and specific tool to assign CYP3A induction by drugs or other chemicals, at least in a mouse model system.

Materials and methods Chemicals Testosterone, all of the resorufins (7-benzyloxy-, 7-ethoxy-, 7methoxy-, and 7-pentoxyresorufin as well as resorufin itself), phenytoin sodium salt, and the inhibitors N-benzyl-1-aminobenzotriazole (BBT), orphenadrine, chloramphenicol, nifedipine, ketoconazole, and sulfaphenazole were obtained from Sigma–Aldrich (Deisenhofen, Germany). NADH and NADPH were obtained from Serva Electrophoresis (Heidelberg, Germany). The antibodies were obtained from the following sources: polyclonal rabbit antimouse CYP1A1 (RPN 256) and polyclonal rabbit anti-rat CY3A (RPN 258) from Amersham Pharmacia Biotech (Freiburg, Germany); polyclonal rabbit anti-rat CYP3A1 (CR3310) for individual detection of mouse CYP3A11/13 from Biotrend (Cologne, Germany); monoclonal mouse anti-rat CYP2B1/2 (mab 204/44), cross-reactive to murine CYP2B9/10, from Urs A. Meyer (Basel, Switzerland); polyclonal rabbit anti-mouse CYP4A (PA3-033) from Affinity Bioreagents (Golden, CO, USA); polyclonal rabbit antimouse CYP2C29 (K-6, purified immunoglobulin G [IgG] fraction), from own manufacturing [9]; and monoclonal mouse anti-GAPDH (MAB374) from Chemicon (Temecula, CA, USA). The avian myeloblastosis virus reverse transcriptase was obtained from Promega (Mannheim, Germany), and the Thermus aquaticus DNA polymerase (Taq) was obtained from Invitek (Berlin, Germany). All other reagents were purchased from commercial sources at the highest purity available.

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All homogenization and centrifugation procedures were carried out on ice or at 4 °C. Livers were homogenized in homogenization buffer (100 mM phosphate-buffered saline [PBS, pH 7.4] containing 20% volume per volume glycerol, 1 mM ethylenediaminetetraacetic acid [EDTA], and 0.2 mM phenylmethylsulfonyl fluoride [PMSF]) by Potter treatment and sonication (4 bursts for 5 s each) in a cell disrupter (Branson, Dunbury, CT, USA). Microsomes were obtained by differential centrifugation of the homogenate at 750g and 25,000g prior to a final centrifugation at 150,000g [14,15]. The differential centrifugation steps led to highly enriched (purity >95%) subcellular fractions that were characterized by determination of the activity of the marker enzymes succinate dehydrogenase for mitochondria (EC 1.3.99.1), NADPH–cytochrome c reductase for microsomes (EC 1.6.2.4), and lactate dehydrogenase (EC 1.1.1.27) for cytosol as well as electron microscopy (data not shown). Protein content was measured according to the Bradford method with bovine serum albumin as standard.

7-Alkyloxyresorufin-O-dealkylase assay Alkyloxyresorufin-O-dealkylase activities were measured by Burke and coworkers’ method [16]. Microsomal protein (10 lg) was incubated in 50 mM Hepes buffer (pH 7.5) containing 0.1 mM EDTA, 15 mM MgCl2, and 6 lM of the respective 7-alkyloxyresorufin as substrate. Reaction was started by adding NADPH (0.5 mM) and was allowed to proceed for 5 min at 37 °C. Fluorescence of the product and identically handled resorufin standards was measured with a PerkinElmer LS50B luminescence photometer (PerkinElmer, Überlingen, Germany). An identical set of probes, boiled for 15 min at 95 °C, was used as blanks. For inhibition studies, different chemicals were added 15 min prior to application of substrate and NADPH. Control incubations were carried out either by using NADH instead of NADPH as cofactor or by adding the general P450 inhibitor BBT.

Metabolism of testosterone in mouse liver microsomes Animals, tissue preparation, and subfractioning Adult male C57Bl/6 mice (25–30 g, 3–5 months of age) were fed for 2 weeks with a solid standard diet (Eggersmann, Rinteln, Germany) supplemented with 500 ppm phenytoin, which permits a daily drug consumption of 20 mg/kg. This treatment resulted in a phenytoin level of 15 ± 3 lg/ml serum, which is within the therapeutic range of epileptic patients [10] and adequate for seizure protection in mice [11,12]. According to previous studies, the phenytoin level in serum was measured at the end of the treatment period by fluorescence polarization immunoassay (FPIA), which enables the control of sufficient intake of phenytoin [9]. Control mice were housed identically but did not get phenytoin supplementation. Both groups received tap water ad libitum. The animals were kept under a 12-h light/dark cycle at a temperature of 22 °C. According to previous studies, phenytoin caused a gain in body weight from 23.2 ± 0.21 g (untreated) to 28.0 ± 0.45 g (phenytoin treated) (P 6 0.001) [13]. The mice were anesthetized as described previously [7]. After transcardial perfusion with Ringer’s solution, the livers were removed immediately, frozen in liquid nitrogen, and stored at 70 °C. The experiments were vetted by the institutional animal ethics committee and were carried out in accordance with the Declaration of Helsinki and with the European Community Council Directive of 1986 (86/609/EEC). All efforts were made to minimize the number of animals used and their suffering.

The testosterone assay was performed as described recently [17]. Microsomal protein (10 lg) was mixed with testosterone (100 nmol in 10 ll of methanol), NADPH–P450 reductase (150 pmol), isocitrate (10 lmol), and isocitrate dehydrogenase (1 U) as an NADPH-regenerating system to give a final volume of 1 ml of incubation buffer containing 50 mM Tris–HCl (pH 7.4), 150 mM KCl, 10 mM MgCl2, and 1 mM EDTA (saturated with oxygen). After preincubation at 37 °C for 2 min, the reaction was started by adding NADPH (1 mM) for 5, 10, and 15 min. Under the experimental conditions, the dependence of reaction velocity from either substrate or protein concentration was proved to be linear and the NADPH level remained constant during the incubation time [17]. For inhibition experiments of testosterone metabolism, microsomes were preincubated with P450 inhibitors at room temperature for 15 min before adding testosterone and NADPH. The incubation was stopped by the addition of 9 ml of ethyl acetate, and 1 nmol of progesterone in 10 ll of methanol was added as internal standard. The metabolites were extracted by vigorous shaking for 20 min. The mixture was centrifuged at 300g for 5 min, and the organic phase was evaporated to dryness by vacuum centrifugation (Jouan, Unterhaching, Germany). To obtain extracts free of particles, the dried extracts were redissolved in 20 ll of methanol, sonicated for 1 min, and centrifuged at 14,000g for 2 min. A volume of 10 ll was used for high-performance liquid chromatography (HPLC) analysis.

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HPLC–PDA analysis

Reverse transcription PCR, cloning, and sequencing of PCR products

Testosterone and its metabolites were separated as described recently [17]. Steroids were separated on a Nova-Pak C18 column heated to 30 °C (150  3.9 mm, 4 lm particle size, used with a precolumn C18, Waters, Eschborn, Germany) by use of the Waters 2690 separations module (Alliance) and detected with the Waters 996 photodiode array (PDA) detector, which acquires the whole absorption spectrum of each steroid metabolite. Products were eluted with water/methanol/acetonitrile/tetrahydrofurane under the following conditions: 0–9 min isocratic at 64:30:4:2, 9–16 min linear gradient to 24:73:1:2, 16–16.5 min linear gradient to 100% methanol, followed by an isocratic flow for 1.5 min at 100% methanol. The flow rate was adjusted to 0.8 ml/min, and the elution was monitored at 254 nm. Testosterone and its metabolites were quantified using Waters Millennium 2010 software. The hydroxylation products of testosterone metabolism were identified by comigration and comparison of their specific ultraviolet–visible (UV–vis) absorption spectra with authentic standards.

Total RNA from livers of untreated and phenytoin-treated mice was extracted using the peqGOLD RNA Pure system (PEQLAB, Erlangen, Germany) according to the manufacturer’s protocol. Before cDNA synthesis, the samples were digested with DNase I to eliminate any residual genomic DNA contamination (Qiagen, Hilden, Germany). Then 1 lg of total RNA was reverse transcribed and the obtained cDNA was diluted 1:10 for further analysis. The amplification mixture contained cDNA, 0.4 lM of each specific primer, 0.2 mM dNTPs, 1.5 mM MgCl2, 1 U of Taq polymerase, and 2.5 ll of 10  Taq buffer (Invitek) in a 25-ll setup. Initial denaturation (3 min at 95 °C) was followed by a 30-cycle polymerase chain reaction (PCR) with each 1 min annealing at 56 °C, 1 min elongation at 72 °C, and 45 s denaturation at 95 °C. PCR products were separated by electrophoresis on a 1.5% (w/v) agarose gel containing 1 lg/ml ethidium bromide. The bands of the amplified fragments were recovered and purified using the Sephaglas BandPrep Kit (GE Healthcare, Munich, Germany) according to the manufacturer’s protocol. Then DNA fragments were ligated into pCRII– TOPO plasmid according to the supplier’s instructions (TOPO TA Cloning Kit, Invitrogen, Karlsruhe, Germany). The ligated DNAs were used to transform host Escherichia coli strain XL1–Blue for propagation of recombinant plasmids. Sequencing of the cloned PCR products was performed by GeneScan (Freiburg, Germany).

Gel electrophoresis and immunoblots To get detailed information on specific expression of mouse P450s due to phenytoin treatment, the content of CYP1A, CYP2B, CYP2C, CYP3A, and CYP4A isoforms was measured by immunoblot investigation in untreated and phenytoin-treated conditions. Denatured microsomal protein (20 lg) was resolved on 10% sodium dodecyl sulfate (SDS)–polyacrylamide gels. Proteins were transferred to polyvinylidene difluoride membranes (Immobilon P, Millipore, Schwalbach, Germany) in buffer (125 mM Tris and 960 mM glycine). Incubations were performed with CYP1A1, CYP2C29, CYP3A, CYP3A1, and CYP4A polyclonal antibodies (dilutions of 1:50 for CYP1A1, 1:2000 for CYP2C29, 1:50 for CYP3A, 1:2000 for CYP3A1, and 1:1500 for CYP4A), followed by exposure to horseradish peroxidase-conjugated IgG goat anti-rabbit (Jackson Immunoresearch, Newmarket, Suffolk, UK) at a dilution of 1:10,000. Incubation with the monoclonal CYP2B1/2 and GAPDH antibodies (dilutions of 1:4000 and 1:5000, respectively) was followed by exposure to horseradish peroxidase-conjugated goat anti-mouse (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at a dilution of 1:4000. The specificity of the antibodies for the antigens was proven previously [17,18]. The immunopositive bands were visualized with enhanced chemiluminescence (ECL, Amersham Biosciences, Freiburg, Germany). The intensity of the immunostained bands was evaluated by scanning densitometry using Tina 2.10 software (Raytest, Straubenhardt, Germany).

Murine Cyp3a consensus oligonucleotides We have designed a consensus primer pair that specifically recognizes the known murine P450s of the Cyp3a subfamily sequences available in the nucleotide database of the National Center for Biotechnology Information (NCBI). The following primer pair was selected: 50 -TGCCTACAGCATGGATGTG (forward primer) and 50 -TGAGAGCAAACCTCATGCC (reverse primer). The primers were blasted against the mouse genome database (NBLAST) and demonstrated, at most, one mismatch per CYP3A isoform. Therefore, the designed primer pair allows specific amplification of a theoretical 819-bp complementary DNA (cDNA) of each CYP3A isoform.

cRNA probe preparation The SP6/T7 polymerase digoxigenin (DIG) labeling and transcription kit (Roche Diagnostics, Mannheim, Germany) was used to synthesize DIG-labeled sense and antisense complementary RNA (cRNA) probes of Cyp3a11/13 following the supplier’s instructions. The sense cRNA probe was generated by SP6 RNA polymerase from pCRII–TOPO (20 lg) linearized with XhoI (20 U). The antisense probe was generated by T7 RNA polymerase from pCRII–TOPO (20 lg) linearized with BamHI (20 U). After transcription, residual plasmids were digested at 37 °C for 15 min with 20 U of RNase-free DNase I. The DIG-labeled cRNA probes were precipitated with 1/10 volumes of LiCl (4 M) and 2.5 volumes of cold ethanol (20 °C) at 70 °C for 30 min. After centrifugation at 14,000g for 10 min and washing with ice-cold 70% (v/v) ethanol, the probes were dried and dissolved in diethyl-pyrocarbonate-treated water. Efficiency of labeling and the probe concentration were checked by dot blot antibody assay as suggested by the supplier. RNase A protection assay Cyp3a11 and Cyp3a13 pCRII–TOPO plasmids were cut with HincII (Cyp3a11) and BbsI (Cyp3a13) restriction enzymes (New England Biolabs, Beverly, MA, USA) to generate 265- and 322-bp probes, respectively. A radiolabeled antisense RNA probe was transcribed after linearization of these vectors and transcription with T7 DNA-dependent RNA polymerase in the presence of a-32P-labeled UTP. Labeled transcript was mixed with total RNA, dried, and then dissolved in hybridization buffer (40 mM Pipes [pH 6.4], 1 mM EDTA, 400 mM NaCl, and 80% formamide) to a concentration of 100 dpm/ll. It was then heated for 10 min at 90 °C and hybridized overnight at 56 °C to 8 lg of total RNA. For RNase A protection assay (RPA), 300 ll of RNase A buffer (10 mM Tris–HCl [pH 7.5], 5 mM EDTA, and 300 mM NaCl) containing 1.0 pg of RNase A was added. The digestion was performed at 32 °C for 45 min. The ribonuclease digestion was terminated by the addition of 20 ll of proteinase K. Then the reaction mixture was incubated for 30 min at 37 °C, phenol chloroform was extracted, and ethanol was precipitated. The samples were subjected to denaturing SDS–polyacrylamide gel electrophoresis (PAGE). The polyacrylamide gels were

Measuring cytochrome P450 CYP3A induction / C.E. Hagemeyer et al. / Anal. Biochem. 398 (2010) 104–111

dried and used to expose Kodak X-OMAT X-ray films (Eastman Kodak, Rochester, NY, USA) for 6–72 h to generate autoradiograms. Autoradiograms were analyzed by densitometry using a computer-based video-imaging system (Tina 2.10 software). Statistical analysis Each experiment was confirmed and measured at least in triplicate. Data are presented as means ± standard deviations of a representative experiment. For statistical analysis, we used an unpaired two-group t test (two-tailed t test) or, for group-wide comparisons, an analysis of variance (ANOVA) one-way test with a post hoc correction (Bonferroni). Data processing was performed using MS Excel 2003 and SPSS 11.0.1 software (SAS Institute, Cary, NC, USA). The accepted significance level was at least P 6 0.05.

Results Influence of phenytoin on P450 expression We initially checked the influence of the model drug phenytoin on the expression of those liver P450 subfamilies that are generally considered to be sensitive to this type of antiepileptic drug treatment, namely CYP1A, CYP2B, CYP2C, CYP3A, and CYP4A. We used antibodies known to be cross-reactive within the specified subfamily. Immunoblot investigation of liver microsomes demonstrated a strong phenytoin-dependent induction of CYP2C29 (13-fold), CYP3A (5-fold), and CYP4A (4-fold). CYP2B9 induction was revealed to be less, whereas the expression CYP2B10 and CYP1A1 proteins did not show any measurable alteration on phenytoin treatment (Fig. 1).

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Dealkylation of alkoxyresorufin derivatives in phenytoin-induced mouse liver microsomes To establish a rapid, sensitive, and efficient enzymatic assay to assign CYP3A induction after xenobiotic treatment, we analyzed the O-dealkylation of alkoxyresorufin derivatives in mouse microsomes. In untreated controls, O-dealkylation of 7-ethoxyresorufin (EROD) and BROD activities was approximately three times higher than O-dealkylation of 7-methoxyresorufin (MROD) and O-dealkylation of 7-pentoxyresorufin (PROD) activities (Table 1). After treatment with phenytoin, we found a very strong induction of BROD activity (50-fold) and a moderate induction of PROD activity (24-fold) (one-way ANOVA: FSt = 2885.641). A similar highly significant induction rate of BROD was detected only after dexamethasone (Dex) treatment (39-fold, one-way ANOVA: FSt = 1304.583) (Table 1), whereas all other treatments and assays led to much weaker induction levels of the resorufin O-dealkylation. The treatment-dependent F values of the one-way ANOVA were much lower, ranging from 0.00 (clofibrate [CF]) to 34.00 (b-naphthoflavone [b-NF]) to 103.33 (3-methylcholantrene [3-MC]). These results assign that BROD might be a valuable marker for CYP3A induction in liver because phenytoin and Dex share the potency of CYP3A induction in mice [4]. Testosterone 6b-hydroxylase activity Next, we checked testosterone 6b-hydroxylase activity in mouse liver microsomes because this hydroxylation is commonly accepted as a marker for CYP3A activity. However, it is known from studies in rats and other species that P450 isoforms of the families CYP1A1, CYP2C, and CYP4 are also able to hydroxylate testosterone in the 6b position, making this assay less valuable to assign exclusive CYP3A induction [19]. This was strikingly confirmed by the

Fig. 1. Expression of CYP3A, CYP2B9/10, CYP2C29, CYP1A1, and CYP4A in phenytoin-treated mice: Immunoblots of microsomes from untreated and phenytoin-treated mouse liver. (A) Densitometric analysis of P450 expression. Data are presented as alterations of P450 expression after treatment referring to untreated controls (means ± standard deviations, n = 3). Statistical significance is demonstrated by asterisks: ***P 6 0.001; n.s., alteration not significant. DPH, phenytoin; utr, untreated. (B) Representative immunoblots with the respective antibodies (see Materials and methods for details). Here 10 lg of microsomal protein was loaded per lane. Immunosignals were visualized using ECL. Lanes showing CYP2B9/10: *CYP2B10; **CYP2B9.

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Table 1 Resorufin-7-dealkylase activities in mouse liver microsomes (pmol/min mg protein). Treatment

BROD

EROD

Activity Untreated DPH b-NF 3-MC Dex CF

14.00 ± 3.00 700.00 ± 75.00 60.00 ± 14.00 70.00 ± 4.00 543.00 ± 65.00 17.00 ± 2.00

Induction (x-fold)

Activity

50 4 5 39 

13.00 ± 2.00 28.00 ± 4.00 87.00 ± 6.00 147.00 ± 18.00 20.00 ± 2.00 14.00 ± 3.00

MROD Induction (x-fold)

Activity

2 7 11 2 

4.00 ± 1.00 27.00 ± 3.00 30.00 ± 5.00 63.00 ± 8.00 34.00 ± 4.00 6.00 ± 2.00

PROD Induction (x-fold)

Activity

Induction(x-fold)

7 8 16 9 

5.00 ± 1.00 118.00 ± 10.00 20.00 ± 2.00 61.00 ± 6.00 64.00 ± 9.00 7.00 ± 1.00

24 4 12 13 1

Note. Data are presented as means ± standard deviations (n = 5). P values 60.05 (two-tailed t test, treated vs. untreated for each assay group in single comparison) except where insignificant (). Groupwise ANOVA: Resorufins: BROD (FSt = 278.331, fl = 5, P 6 0.001); EROD (FSt = 224.127, fl = 5, P 6 0.001); MROD (FSt = 110.278, fl = 5, P 6 0.001); PROD (FSt = 256.922, fl = 5, P 6 0.001). Treatments: DPH (FSt = 2885.641, fl = 3, P 6 0.001); b-NF (FSt = 34.000, fl = 3, P 6 0.001); 3-MC (FSt = 103.330, fl = 3, P 6 0.001); Dex (FSt = 1304.583, fl = 3, P 6 0.001); CF (no influence).

data obtained in the current study. Nearly all of the drugs used for induction testing led to enhancement of testosterone 6b-hydroxylation in liver microsomes (Table 2), ranging from a 17-fold induction (3-MC) to a 77-fold induction (b-NF). Nevertheless, one advantage of this assay is its usefulness in inhibition studies, where selective inhibitors can assign the role of a selected P450 isoform [17,20]. Specificity of BROD and testosterone 6b-hydroxylase activities To analyze the specificity of BROD for assigning CYP3A induction, we tested mouse liver microsomes supplemented with a carefully defined set of chemical inhibitors known to inhibit selective P450 isoforms: chloramphenicol as inhibitor of CYB2B and CYP2C activity [21]; orphenadrine for CYP2B, CYP2D, and CYP1A isoforms [22,23]; ketoconazole for CYP3A and CYP2C; nifedipine for CYP3A and CYP1A [24,25]; and sulfaphenazole as inhibitor of CYP2C isoforms [24,26]. Ketoconazole and nifedipine showed by far the highest potency in inhibiting both BROD and testosterone 6b-hydroxylase activities (Table 3). Sulfaphenazole exerted a less pronounced moderate effect on BROD (33% inhibition) compared with ketoconazole or nifedipine, whereas it failed, as expected, to inhibit testosterone 6b-hydroxylation. Chloramphenicol and orphenadrine had only a weak inhibitory effect on both BROD and testosterone 6b-hydroxylase activity (Table 3). The complete inhibition of BROD by the addition of NADH instead of NADPH as cofactor, or by the general P450 inhibitor BBT [27], clearly pointed to the characteristics of P450-catalyzed reactions (Table 3). These data further confirmed BROD as a valuable marker for CYP3A induction in mouse liver.

database). This procedure enabled us to amplify specific 819-bp PCR products of CYP3A isoforms in mouse liver. As expected from the activity and immunoblot screening data (Fig. 1 and Tables 1 and 2), the amplification of the Cyp3a PCR product was clearly increased in phenytoin-treated animals compared with the untreated animals (Fig. 2A). These PCR products could be unequivocally identified by subsequent cloning and cycle sequencing. Close investigation of a representative amount of clones revealed identity of our Cyp3a PCR products with the published cDNA sequences of Cyp3a11 (2 clones) and Cyp3a13 (3 clones). To gain quantitative information about Cyp3a11 and Cyp3a13 messenger RNA (mRNA) expression in mouse liver, we performed an RPA. By using this methodology, we overcame the problems of reverse transcription and cDNA amplification by directly measuring the mRNA content of our target genes in liver. We designed an RPA multiprobe set with Cyp3a11, Cyp3a13, and ribosomal protein L32 as a loading control. With this probe set, we found a marginal increase of Cyp3a13 mRNA after phenytoin treatment. In contrast, Cyp3a11 mRNA appeared to be highly induced (Fig. 2B). Protein expression of CYP3A11/13 in liver microsomes We next analyzed the specific protein expression of CYP3A11 and CYP3A13 in mouse liver. We performed immunoblots using a rat CYP3A1 antibody that specifically detects both of these isoforms in murine tissue, as reported in several recent studies [17,28]. Thus, CYP3A13 was constitutively expressed in untreated liver homogenates and microsomes, but it was not inducible by phenytoin. On the other hand, CYP3A11 was only barely detectable in untreated homogenates and microsomes, but it revealed major inducibility after phenytoin treatment (Fig. 3). Our immunoblot

Identification of CYP3A11 as the main CYP3A affected by phenytoin in mouse liver Expression of Cyp3a11/13 mRNA Initially, we selected a consensus primer pair of all known murine liver Cyp3a cDNA sequences (published in the NCBI nucleotide Table 2 Testosterone 6b-hydroxylase activity in mouse liver microsomes (pmol/min mg protein). Treatment

Testosterone 6b-hydroxylase activity Activity

Induction (x-fold)

Untreated DPH b-NF 3-MC Dex CF

1.63 ± 0.17 59.49 ± 6.26 124.90 ± 13.15 28.08 ± 2.96 81.96 ± 8.63 65.16 ± 6.86

36 77 17 50 40

Note. Data are presented as means ± standard deviations (n = 3). P values = 0.001 (two-tailed t test, treated vs. untreated for each assay group in single comparison). Groupwise ANOVA: FSt = 98.488, fl = 5, P 6 0.001.

Table 3 Inhibition of BROD and testosterone 6b-hydroxylase activities in phenytoin-induced mouse liver microsomes. Chemical inhibitor

BROD Activity

None NADH BBT Chloramphenicol Orphenadrine Ketoconazole Nifedipine Sulfaphenazole

100.00 ± 9.48 0.27 ± 0.10 0.27 ± 0.10 85.30 ± 13.20 81.46 ± 7.93 26.24 ± 14.66 32.97 ± 8.75 66.90 ± 10.88

Testosterone 6bhydroxylase Inhibition (%) 100 100  19 74 67 33

Activity 100.00 ± 0.24 – – 82.00 ± 3.46 85.00 ± 3.22 6.00 ± 0.80 65.00 ± 4.13 100.00 ± 2.13

Inhibition (%)

18 15 94 35 

Note. Data are presented as percentages from complete system ± standard deviations (BROD: n = 5; testosterone 6b-hydroxylase: n = 3). P values 6 0.05 (two-tailed t test, treated vs. untreated for each assay group in single comparison) except where insignificant (). Groupwise ANOVA: BROD (FSt = 185.030, fl = 7, P 6 0.001); testosterone 6b-hydroxylation (FSt = 336.940, fl = 5, P 6 0.001). –, not measured.

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CYP3A11 expression in mouse liver after phenytoin treatment becomes evident.

Discussion

Fig. 2. Expression of Cyp3a mRNA in mouse liver. (A) Analysis of 1 lg of cDNA prepared from mouse liver total RNA of untreated and phenytoin-treated animals. The resulting amplification products were separated on ethidium bromide agarose gel (1.5%, w/v). NTC, nontemplate control; utr, untreated; DPH, phenytoin; *500 bp. (B) Representative RPA of 10 lg of total RNA isolated from liver of untreated and phenytoin-treated animals. Antisense cRNA of Cyp3a11, Cyp3a13, and L32 (loading control) were used as probes (see lane with undigested probe set). The resulting hybrids were protected from RNase digestion and visualized by autoradiography after acryl amide gel electrophoresis. Both gels (A and B) represent one of three independent experiments with similar results.

Fig. 3. Expression of CYP3A11 and CYP3A13 proteins: Representative immunoblots of homogenates and microsomes of mouse liver from untreated and phenytointreated animals. Electrophoresis and immunoblotting of 20 lg of each protein are shown. Immunosignals were visualized using ECL. Immunoblots represent one of three independent experiments with similar results. utr, untreated; DPH, phenytoin.

data allowed us to conclude that CYP3A11 is the CYP3A isoform mainly affected by phenytoin treatment in mouse liver.

Correlation of BROD activity and CYP3A11 expression data We summarized the results of this study in a cross-matrix table and added published information concerning P450 induction and inhibition by the drugs used here (Table 4). By summarizing the data in this way, the strong correlation between BROD and

In this article, we have described BROD as a valuable marker assigning CYP3A11 induction in mouse liver. This was conducted from the analysis of P450 mRNA and protein expression, correlated with the enzymatic activity data using diverse 7-alkoxyresorufins and testosterone in phenytoin-treated mice (Table 4). The CYP3A isoforms belong to the most important P450 subfamilies in both amount and importance for drug metabolism [3]. By far, most of the drugs used in clinical practice interact with CYP3A isoforms either as substrate or as inducer, in particular with CYP3A4 in humans or CYP3A11 in mice [4,17,29]. As mentioned above, effective in vitro testing of a putative drug–CYP3A interaction is shown because the main CYP3A isoforms are not polymorphic and, therefore, detract themselves from genetic screening as a potent prediction marker for drug metabolism or induction effects [6]. However, a major problem of P450 in vitro testing with chemical substances is the low isoform specificity of many substances. This complicates the search for effective and valuable test chemicals. An example of this is the 6b-hydroxylation of testosterone that is generally used to assign CYP3A-mediated metabolism of this androgen [30,31]. However, the current study revealed that, in addition to the induction of CYP3A, 6b-hydroxylation of testosterone is enhanced in liver microsomes treated with the CYP1A1 inducers b-NF and 3-MC and with the CYP4A inducer CF (Tables 2 and 4). This considerably limits this assay as a prediction marker of specific induction profiles evoked by a substance in consideration. Other examples for very rapid and sensitive detection methods are the demethylation of aminopyrine and the O-dealkylation of different alkoxyresorufins [32–34]. In particular, alkoxyresorufin dealkylation is considered as a valuable and cost-effective test system because of its fluorimetric quantification that significantly enhances sensitivity and specificity [35–37]. This assay system has been widely used to analyze the induction of P450 in hepatic microsomes predominantly in the rat system. In accordance with this previous work in rats, we detected enhanced EROD activity in b-NF- and 3-MC-treated mouse liver microsomes and increased methoxyresorufin oxidation after 3-MC treatment, both assigning CYP1A induction [16,38]. In our hands, we found by far the strongest increase of dealkylation activity using benzyloxyresorufin as substrate in phenytoinand Dex-treated mice (Table 1). Phenytoin is known to induce mainly CYP2C29 and CYP3A11 in mice; fewer effects are described for CYP2B9 and CYP4A [9,17,39]. Dex is reported to induce CYP3A11 in mice and CYP3A4 and CYP2D6 in humans [4,40,41]. From our data, we conclude that, in the mouse liver, BROD is an effective tool to filter out CYP3A11 induction profiles within a multitude of induction effects caused by substances like the antiepileptic drug phenytoin. This is an interesting finding because in rat liver BROD activity usually assigns CYP1A and CYP2B induction [34]. This conclusion is driven mostly by the correlation of our complete data set explored from protein expression, enzymatic assays, and inhibition studies in mouse liver microsomes, as demonstrated in the cross-matrix evaluation (Table 4). Although each of these investigations per se can leave room for interpretation due to unsharp isoform specificity, the combined results clearly show highly significant overlap in assigning CYP3A11 induction in mouse liver. By using the BROD assay for CYP3A11, we can precisely dissect from CYP2C29 the other main P450 induced by substances like phenytoin and phenobarbital in mouse liver (Fig. 1) [9]. CYP2C29

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Measuring cytochrome P450 CYP3A induction / C.E. Hagemeyer et al. / Anal. Biochem. 398 (2010) 104–111

Table 4 Cross-matrix table: Correlation of BROD activity and CYP3A11 expression data.

Note. +++++, very strong; ++++, strong; +++, moderate; ++, weak; +, very weak (barely observable). Data in grayscale demonstrate strong correlation between BROD and CYP3A11 data. Data below the dashed line are extracted from the literature (refer to Supplementary Table 1 in supplementary material for detailed information). References for induction or inhibition potency: CYP3A11 and Dex [41]; induction of CYP1A1/2 and CYP2D6: [4]; induction of CYP4A and inhibition of CYPs by chloramphenicol [21,43]; ketoconazole and nifedipine inhibition [17]; inhibition of CYPs by orphenadrine [22,23]; sulfaphenazole as CYP inhibitor [24,26]. Additional references are made in the text.

appears as the major P450 induced after phenytoin or phenobarbital treatment, which could be nicely shown by regioselective oxidative hydrolysis of the coumarin derivative scoparone, a novel mechanism specific for murine CYP2C isoforms [9,42]. The relative importance of CYP3A isoforms in endogenous and exogenous metabolism, in cross-talk between drugs and steroids affecting androgen signaling cascades and patient well-being, and in the large interindividual variability in expression of CYP3A isoforms [3,5] gives high demand on reliable in vitro test systems. We found the BROD assay to be an effective and valuable tool for in vitro testing of CYP3A. The results of the current study indicate a novel method for identifying CYP3A induction in mice. Our observations are important when assessing the function of P450s in drug metabolism and may be useful in developing reliable test systems for this major P450 subfamily. Acknowledgments This work was supported by grants from the Deutsche Forschungsgemeinschaft (DFG Me 1544/4 and Vo 272/7). We thank Margarethe Ditter (Department of Neuropathology, University of Freiburg) for her extraordinary technical assistance and thank Benedikt Volk for his continuous discussion, advice, and support. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.ab.2009.11.004. References [1] D.R. Nelson, P450 nomenclature and overview, http://drnelson.utmem.edu/ CytochromeP450.html (accessed 10 August 2009). [2] R.P. Meyer, M. Gehlhaus, R. Knoth, B. Volk, Expression and function of cytochrome P450 in brain drug metabolism, Curr. Drug Metab. 8 (2007) 297– 306. [3] M. Ingelman-Sundberg, Human drug metabolising cytochrome P450 enzymes: properties and polymorphisms, Naunyn Schmiedebergs Arch. Pharmacol. 369 (2004) 89–104. [4] D.A. Flockhart, P450–drug interaction table, http://medicine.iupui.edu/ clinpharm/ddis (accesssed 10 August 2009). [5] N. Killer, M. Hock, M. Gehlhaus, P. Capetian, R. Knoth, G. Pantazis, B. Volk, R.P. Meyer, Modulation of androgen and estrogen receptor expression by antiepileptic drugs and steroids in hippocampus of patients with temporal lobe epilepsy, Epilepsia 50 (2009) 1875–1890.

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