Advertisement
Research ArticleArticle

The CYP2D6 Humanized Mouse: Effect of the HumanCYP2D6 Transgene and HNF4α on the Disposition of Debrisoquine in the Mouse

Javier Corchero, Camille P. Granvil, Taro E. Akiyama, Graham P. Hayhurst, Satish Pimprale, Lionel Feigenbaum, Jeffrey R. Idle and Frank J. Gonzalez
Molecular Pharmacology December 2001, 60 (6) 1260-1267; DOI: https://doi.org/10.1124/mol.60.6.1260

This article has a correction. Please see:

Abstract

CYP2D6 is a highly polymorphic human gene responsible for a large variability in the disposition of more than 100 drugs to which humans may be exposed. Animal models are inadequate for preclinical pharmacological evaluation of CYP2D6 substrates because of marked species differences in CYP2D isoforms. To overcome this issue, a transgenic mouse line expressing the human CYP2D6 gene was generated. The complete wild-type CYP2D6 gene, including its regulatory sequence, was microinjected into a fertilized FVB/N mouse egg, and the resultant offspring were genotyped by both polymerase chain reaction and Southern blotting. CYP2D6-specific protein expression was detected in the liver, intestine, and kidney from only the CYP2D6 humanized mice. Pharmacokinetic analysis revealed that debrisoquine (DEB) clearance was markedly higher (94.1 ± 22.3 l/h/kg), and its half-life significantly reduced (6.9 ± 1.6 h), in CYP2D6 humanized mice compared with wild-type animals (15.2 ± 0.9 l/h/kg and 16.5 ± 4.5 h, respectively). Mutations in hepatic nuclear factor 4α (HNF4α), a hepatic transcription factor known to regulate in vitro expression of the CYP2D6 gene, could affect the disposition of CYP2D6 drug substrates. To determine whether the HNF4α gene modulates in vivo pharmacokinetics of CYP2D6 substrates, a mouse line carrying both the CYP2D6gene and the HNF4α conditional mutation was generated and phenotyped using DEB. After deletion of HNF4α, DEB 4-hydroxylase activity in CYP2D6 humanized mice decreased more than 50%. The data presented in this study show that only CYP2D6 humanized mice but not wild-type mice display significant DEB 4-hydroxylase activity and that HNF4α regulates CYP2D6 activity in vivo. The CYP2D6 humanized mice represent an attractive model for future preclinical studies on the pharmacology, toxicology, and physiology of CYP2D6-mediated metabolism.

CYP2D6 is responsible for the DEB 4-hydroxylase polymorphism, which has important clinical consequences, including pronounced interindividual variation in the disposition of many important drugs such as β-adrenergic blocking agents, antiarrhythmics, antidepressants, and analgesics. Since it was first discovered, CYP2D6 has been the most studied human genetic polymorphism in drug metabolism with more than 75 identified alleles within most human populations and racial groups (http://www.imm.ki.se/CYPalleles/cyp2d6.htm). Approximately 7 to 10% of the Caucasian population inherit mutant CYP2D6alleles as an autosomal recessive trait (Mahgoub et al., 1977). This polymorphism stratifies the population depending on the copy number of wild-type alleles: poor (PM, zero), intermediate (one), extensive (EM, two), and ultra-rapid metabolizers (multiple copies) (Gonzalez, 1996;Wolf and Smith, 1999). The CYP2D gene locus has been isolated, sequenced, and mapped to human chromosome 22q13.1, and contains the only active CYP2D6 gene downstream of two inactive pseudogenes CYP2D7P and CYP2D8P(Gonzalez et al., 1988; Kimura et al., 1989).

Clinical studies are fundamental to the identification of human polymorphisms, to the establishment of pharmacokinetic profiles, and to drug-drug interaction effects. However, to determine how a drug is metabolized, what toxic effects it may produce, or how pathophysiological conditions affect drug metabolism, animal models or in vitro systems must be developed.

Because of marked differences between humans and experimental animals, the results from animal studies can be misleading and need to be interpreted very cautiously. For example, the CYP2D family in humans has a single active member, CYP2D6, whereas rats and mice have at least five genes (Gonzalez and Nebert, 1990; Nelson et al., 1996). DEB is hydroxylated to 4-hydroxydebrisoquine (4-OH-DEB) by humans and by Sprague-Dawley rats. However, Dark Agouti rats have been found to posses a low capacity to metabolize DEB (Al-Dabbagh et al., 1981). Similarly, no significant formation of 4-hydroxy DEB was detected by liver microsomes from three strains of mice and by purified cyp2d9–11 (Masubuchi et al., 1997).

The transcriptional factor hepatic nuclear factor 4α (HNF4α) is known to play a major role in liver organogenesis, maintenance, and gene transcription (Sladek, 1994). Several lines of evidence suggest that HNF4α controls constitutive levels of expression of theCYP2D6 gene. Indeed, it was shown that cotransfection of the minimal CYP2D6 promoter-CAT construct (−392 bp) with a mammalian HNF4 expression vector produced a 30-fold induction of CAT activity in COS-7 cells (Cairns et al., 1996). Moreover, using adenovirus-mediated antisense targeting that selectively diminishes HNF4α content in human hepatocytes,CYP2D6 gene expression, among other P450s, is down-regulated by the depletion of HNF4α (Jover et al., 2001).

In addition, the R154X mutation in the human HNF4α gene provokes maturity onset diabetes of the young, a genetically and clinically heterogeneous subtype of non-insulin-dependent diabetes mellitus. Persons carrying this mutation in HNF4α are predicted to have reduced levels of this transcription factor in the tissues where it is expressed (Yamagata et al., 1996; Lindner et al., 1997). Because this pathology occurs concomitantly with other diseases like obesity, hypertension, coronary insufficiency, and heart failure among others, multiple drug regimes are common within this population (Scheen and Lefebvre, 1995). Because CYP2D6 metabolizes a large number of clinically prescribed drugs, it would be relevant to determine whether the lack of HNF4α could modulate CYP2D6 metabolic activity in vivo. However, because of the embryonic lethality of a standard gene HNF4α knock-out approach, preclinical evaluation of this hypothesis using a intact animal model is not possible. Fortunately, the generation of a conditional liver-specific knock-out of the HNF4α gene mouse line has overcome this issue (Hayhurst et al., 2001).

To circumvent all of these methodological problems, a humanized mouse line expressing CYP2D6 would offer a unique approach to answering fundamental questions about the specific role of CYP2D6 in drug metabolism and drug interactions. Such experiments would be performed in the context of the entire animal and would overcome many limitations inherent in in vitro experiments. To this end, the complete wild-type allele of the human CYP2D6 gene, including its regulatory sequence, was microinjected into a fertilized FVB/N mouse egg to produce a humanized transgenic mouse line. In addition, to explore the in vivo regulation of CYP2D6 expression by HNF4α, a mouse line carrying both the CYP2D6 gene and the conditionalHNF4α mutation has been generated. Using DEB as a substrate, only the humanized CYP2D6 mice are able extensively to convert DEB to its 4-hydroxy metabolite and to display a pharmacokinetic profile similar to humans.

Materials and Methods

Animals.

Adult males from all the genotypes described in this work (25–30 g, 2–4 months) were maintained under conditions of controlled temperature (23 ± 1°C) and lighting (lights on 6:00 AM–6:00 PM), with food and water provided ad libitum. All animal experiments were conducted under National Institutes of Health guidelines for the use and care of laboratory animals, and approved by National Institutes of Health Animal Care and Use Committee.

Generation of the Humanized Mouse.

The CYP2D6gene (GenBank accession number M33388) previously isolated and sequenced (Kimura et al., 1989) was microinjected into a fertilized FVB/N mouse egg to produce a transgenic mouse line. Incorporation of the CYP2D6 DNA within the mouse genome was determined by both PCR and Southern blot analysis. The transgenic founder was mated to a nontransgenic FVB/N (wild-type), and animals from this cross were subsequently crossed to each other to produce homozygous mice. Mice homozygous for the transgene were confirmed by crossing them with wild-type mice and testing the progeny for transgene transmission. Heterozygous animals were generated by crossing homozygous and wild-type mice. Wild-type and homozygous littermates were bred and maintained by brother-sister mating.

Generation of Liver-Specific Deletion of HNF4αin the CYP2D6 Transgenic Line.

HNF4αconditional knock-out mice had been generated previously (Hayhurst et al., 2001). The breeding scheme to produce liver-specific deletion ofHNF4α in the CYP2D6 transgenic line was designed using two different mice genotypes: a) HNF4αfl/wt, AlbCre +/− mice generated by crossing HNF4α fl/fl homozygous to albumin-Cre heterozygous (Alb-Cre) kindly provided by Dr. Derek LeRoith (National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD) (Yakar et al., 1999); and b)HNF4α fl/fl, CYP2D6 +/− mice: generated by crossing HNF4α fl/fl homozygous mice to CYP2D6homozygous animals and back-crossing the resultant offspring to homozygous HNF4α fl/fl mice.

The breeding of mice carrying genotypes a and b generatesHNF4α knock-out mice carrying the CYP2D6transgene (HNF4α fl/fl, AlbCre+/−, CYP2D6+/−), and littermate control mice for AlbCre, and CYP2D6, as described under Results.

PCR Genotyping Procedures.

Genomic DNA was isolated from tails as described previously (Laird et al., 1991). ForCYP2D6 PCR analysis, approximately 50 ng of tail DNA was amplified in 25 μl of reaction mixture containing 2.5 mM MgCl2, 0.2 mM deoxynucleoside triphosphates (dNTPs), 1.25 U of AmpliTaq (PerkinElmer, Foster City, CA), and 20 pmol of CYP2D6 gene-specific primers CYP2D6F 5′-AGAAGGGGAAGCAGGTTTG-3′ and CYP2D6R 5′-CGGCACTCAGGACTA ACTCATC-3′, and microsomal epoxide hydrolase (mEH) gene-specific primers MEHF 5′-AAGTGAGTTTGCATGGCGCAGC-3′ and MEHR 5′-CCCTTTAGCCCCTTCCCTCTG-3′. Cycling conditions were 94°C for 5 min, and then 33 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 1 min, followed by a 5-min extension at 72°C. mEH primers served as a positive control for amplification, yielding a fragment of 341 bp in all samples (Miyata et al., 1999). An additional band of 241 bp was amplified exclusively in CYP2D6 humanized animals. For HNF4α fl/fl mice and AlbCre transgenic PCR procedures were described previously (Hayhurst et al., 2001).

Southern Blot Analysis and Determination of Transgene Copy Number.

Tail genomic DNA (15 μg/lane) was digested withBamHI and subjected to electrophoresis on a 0.5% agarose gel containing 0.5× Tris/borate/EDTA. The DNA was hydrolyzed in 0.2 M HCl and transferred on to a Gene Screen Plus nylon membrane (DuPont, Wilmington, DE) by capillary blotting in 0.4 M NaOH. Blots hybridized with random-primer 32P-labeled CYP2D6cDNA probe (Gonzalez et al., 1988) at 42°C overnight, washed twice in 2× SSC (1× SSC is 150 mM NaCl plus 15 mM sodium citrate) and 0.5% SDS at 65°C for 15 min, twice in 0.1 × SSC and 0.5% SDS for 5 min, and exposed to a PhosphorImager screen (Molecular Dynamics, Sunnyvale, CA) for 2 to 4 h.

Transgene copy number was determined by Southern blotting using the purified genomic clone DNA as a standard. Cloned DNA was diluted with mouse DNA to yield the equivalent of 1, 2, 5, and 10 copies of the gene per haploid genome (based on 3 × 109 base pairs per haploid genome). The DNA was digested with BamHI and subjected to Southern blot analysis with DNA isolated from heterozygous and homozygous CYP2D6 humanized mice. The signals were quantified by use of a PhosphorImager, and the copy number was determined from a standard curve of the standard.

Northern Blot Analysis.

Total RNA was isolated from the livers of all the HNF4 fl/fl, AlbCre, and CYP2D6humanized mice used in this study by the acidic guanidine isocyanate/phenol/chloroform extraction method using the Ultraspec RNA isolation system (Biotecx Laboratories, Houston, TX). Ten micrograms of total RNA per sample was subjected to electrophoresis on a 1% agarose gel containing 220 mM formaldehyde in 20 mM MOPS, 8 mM sodium acetate, and 1 mM EDTA buffer, pH 7.0, and transferred to Gene Screen Plus membranes by capillary blotting in 20× SSC. Blots were hybridized with a random-primer 32 P-labeled probe specific forHNF4α exon 4 and 5 probe (Hayhurst et al., 2001) at 42°C overnight, washed twice in 2× SSC and 0.5% SDS at 65°C for 15 min, twice in 0.1× SSC and 0.5% SDS for 5 min, and exposed to a PhosphorImager screen for 2 to 4 h.

Western Blot Analysis.

Tissues were homogenized in ice-cold buffer (1.15% KCl, 50 mM Tris-HCl, and 1 mM EDTA, pH 7.4) and microsomes were prepared by differential centrifugation as described previously (Sinal et al., 1999). Microsomal protein concentration was determined using a bicinchoninic acid protein kit (Pierce, Rockford, IL) using bovine serum albumin as a standard. SDS-polyacrylamide gel electrophoresis and Western blot analysis of microsomal proteins (40 μg) were performed with a 4% stack and 10% separating gel and transferred to nitrocellulose (Schleicher & Schuell, Keene, NH) using standard methods. The primary mouse monoclonal anti-CYP2D6 antibody (Gentest Corp., Woburn, MA) was diluted 3,000-fold in 0.5% nonfat dry milk, 1× phosphate-buffered saline. The primary monoclonal antibody against rat CYP2E1 (kindly provided by Dr. Harry V. Gelboin) was diluted 500-fold in 3% milk and 1× phosphate-buffered saline. Bound antibody was detected with a horseradish peroxidase-conjugated secondary antibody anti-mouse IgG, that was diluted 2,000- and 10,000-fold for CYP2D6 and CYP2E1 blotting, respectively. P450 proteins were visualized by use of the enhanced chemiluminescence reagent (Amersham Pharmacia Biotech, Piscataway, NJ).

Drug Administration and Blood and Urine Samples.

DEB hemisulfate (ICN, Irvine, CA) was dissolved in sterile water and administered orally by gavage at 2.5 mg/kg. For pharmacokinetic evaluations, blood samples were collected from suborbital veins 0, 0.5, 1, 2, 4, 6, 8, 12, and 24 h after DEB administration (2.5 mg/kg). Each time point was analyzed with three to four animals. Serum was separated by centrifugation at 1000g, 4°C, for 10 min. For the urine excretion analysis, three to four animals per group were dosed with DEB (2.5 mg/kg) and placed in metabolic chambers (Jencons, Leighton Buzzard, UK). Total urinary excretion from individual mice was collected for 24 h. Serum aliquots (0.2–0.5 ml) or urine volumes (0.8–1.5 ml) were stored at −80°C until analyzed. At the end of experiments, mice were euthanized by carbon dioxide.

Quantification of DEB and 4-OH-DEB by Liquid Chromatography/Tandem Mass Spectrometry.

Serum and urine concentrations of the DEB and 4-OH-DEB were determined using a previously described LC/MS/MS method with a minor modification using liquid-liquid extraction (Scott et al., 1999) and solid phase extraction (Pereira et al., 2000). Briefly, 100 μl of serum or urine (supplemented with 30 mg of sodium chloride) was spiked with 20 μl of internal standard (phenacetin, 30 μM in methanol), 500 μl of isopropanol, 50 μl of aqueous sodium hydroxide (400 mM), and 3 ml of methyl-t-butyl ether were added. The mixture was vortex-mixed for 1 min and the phases were separated by 10 min of centrifugation at 1000g, 4°C. The aqueous layer was frozen in dry ice and the organic phase was transferred to a fresh borosilicate tube and evaporated to dryness under a gentle stream of air at 30°C using a heating block (Pierce, Rockford, IL). The residue was reconstituted in 100 μl of acetonitrile-water (20:80, v/v) and transferred to polypropylene autosampler vials and 10 to 25 μl of sample was injected into the LC/MS/MS system.

The high-performance liquid chromatography system consisted on a PerkinElmer Series 200 quaternary pump, a vacuum degasser, and a series 200 autosampler with a 100-μl loop (PerkinElmer) both interfaced to a triple-quadrupole tandem mass spectrometer (API2000; PE SCIEX). DEB, 4-OH-DEB, and the internal standard were separated on a LUNA RX-Polar C18 column (2.0 × 50 mm, 3 μm; Phenomenex, Torrance, CA). The mobile phases consisted of the following: solvent A, 0.1% formic acid in water, and solvent B, 0.1% formic acid in acetonitrile. A linear gradient of solvent B from 5 to 95% over 4 min was applied on the column and was returned to the original condition and equilibrated for 1 min before the next injection. The samples were delivered with a flow rate of 0.20 ml/min and the run time was 5.0 min.

The mass spectrometer was operated in the turbo ion spray mode with positive ion detection. The turbo ion spray temperature was maintained at 300°C and a voltage of 4.8 kV was applied to the sprayer needle. Nitrogen was used as the turbo ion spray and nebulizing gas. The detection and quantification of compounds were performed using MS/MS in the multiple reaction monitoring mode. Multiple reaction monitoring data acquisition, monitoring the transitions m/z176/134 for DEB, 192/132 for 4-OH-DEB, and 180/110 for the internal standard, was employed. All raw data were processed with PerkinElmer SCIEX Analyst Software, version 1.2.

The method was linear for DEB and 4-OH-DEB concentrations ranging from 2.5 to 5000 nM. Calibration curves were constructed in duplicate and were completed using 1/χ2 weighting. Good linearity was achieved with correlation coefficients greater than 0.995 for both the analytes. The lower limit of quantitation was 2.5 nM for DEB and 4-OH-DEB in both serum and urine, where the coefficient of variation was less than 20%. The recoveries for DEB and 4-OH-DEB ranged between 80 and 102% in serum and urine. Intraday and interday coefficients of variation were less than 10% at a concentration of 30 nM for DEB and 4-OH-DEB in serum and urine.

Pharmacokinetic Analysis.

Pharmacokinetics parameters for DEB and its metabolite 4-OH-DEB were estimated from the serum concentration-time data by a noncompartmental approach with the software package WinNonlin Standard version 1.5 (Scientific Consulting Inc., Cary, NC). The peak concentration in serum (C max) and the time to reach serum concentration (T max) were obtained from the original data. The area under the serum concentration versus time curves from 0 to 24 h (AUC0–24 h) was determined with a combination of linear and logarithmic trapezoidal methods. The elimination rate constant (β) was determined by use of log-linear regression of the terminal portion of the concentration versus time curve using at least four data points. The elimination half-life (t 1/2) was calculated from 0.693/β. The apparent oral clearance was calculated from dose/AUC0–24 h. From urine concentrations, the percentage of the total dose excreted was determined.

Statistics.

Statistical analyses were performed with SigmaStat software (Jandel Corporation, San Rafael, CA) using one-way or two-way analysis of variance followed by the Student-Newman-Keul's test when comparing three or four groups, respectively. Differences were considered significant if the probability that they were due to chance was less than 5%.

Results

Generation of a CYP2D6 Humanized Mouse Line.

The CYP2D6 gene was isolated and sequenced in an earlier study (Kimura et al., 1989). The sequence indicated that it was a wild-type allele. The complete CYP2D6 gene (Fig.1A), including its promoter and regulatory elements, was microinjected into a fertilized FVB/N mouse egg to produce a transgenic mouse line. Successful incorporation of theCYP2D6 DNA into the germ line was assessed by Southern blot analysis. Genomic DNA from wild-type and CYP2D6 humanized mice was digested with BamHI, and probed with theCYP2D6 cDNA. Hybridization signal was found only in the lanes with transgenic DNA but not in the lanes with wild-type DNA. The size of the bands corresponds exactly with the predicted sizes calculated from the sequence of the CYP2D6 gene (Fig. 1B). Furthermore, the results of Southern blot analysis were corroborated by PCR. Genomic DNA from wild-type and CYP2D6 humanized mice was amplified with two different sets of specific primers:CYP2D6, and mEH (Miyata et al., 1999). Figure 1C shows the amplification of mEH PCR product (341 bp) in both wild-type and CYP2D6 humanized animals, which served as a positive control for amplification. CYP2D6 PCR product (241 bp) is only present in CYP2D6 humanized mice. The transgene was present at 5 ± 1 copies per haploid genome.

Figure 1
Figure 1

Generation and analysis of the CYP2D6humanized mouse. A, schematic diagram of the wild-typeCYP2D6 gene used for microinjection (GenBank accession number M33388). Restriction sites for EcoRI (E) andBamHI (B) are depicted. Black boxes representCYP2D6 exons. The bar represents 1 kb. B, Southern blot genotyping of wild-type and CYP2D6 humanized mice. Tail DNA (15 μg) was digested with BamHI and probed withCYP2D6 cDNA. Hybridization signals were present only inCYP2D6 humanized mice, and their sizes were as expected from the CYP2D6 sequence. C, PCR genotyping of wild-type and CYP2D6 humanized mice. Tail DNA was amplified withmEH (internal PCR control) and CYP2D6gene-specific primers. The PCR products (341 bp for mEH, 241 bp for CYP2D6) were separated on a 1.5% agarose gel. D, Western blot analysis of CYP2D6 protein expression in wild-type and CYP2D6 humanized mice. Liver (L), intestine (I), and kidney (K) microsomal proteins (40 μg) were separated by SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. A CYP2D6-specific monoclonal antibody was used to assess CYP2D6 protein expression. The antibody only reacted against CYP2D6 expressed protein but did not recognize any of the mouse CYP2D proteins. Human liver microsomes (HLM) were used as a control.

Expression of the transgenic protein was assessed by Western blotting. Microsomal protein preparations from wild-type and CYP2D6humanized animals were analyzed with a CYP2D6-specific monoclonal antibody, which did not cross react with the CYP2D proteins present in the wild-type mice. CYP2D6 was expressed in liver, intestine, and kidney microsomes only in humanized mice (Fig. 1D).

DEB Pharmacokinetics in CYP2D6 Humanized Mice.

Nonlinear pharmacokinetics in the elimination of CYP2D6 substrates has been reported (Brøsen and Gram, 1988; Brøsen, 1990). To determine a dose within the linear range of DEB elimination, a dose-effect study (up to 30 mg/kg) on DEB AUC was performed. We observed nonlinear pharmacokinetics of DEB elimination at doses of 10 mg/kg or higher. Therefore, the characterization of CYP2D6 humanized mouse pharmacokinetics was performed using a dose of 2.5 mg/kg, which is within the linear range of DEB elimination (data not shown). The average DEB serum concentration versus time curves in wild-type,CYP2D6 heterozygous and CYP2D6 homozygous humanized mice were determined (Fig. 2A). After a single oral dose of DEB (2.5 mg/kg), both CYP2D6heterozygous and homozygous mice had DEB serum levels significantly lower than in wild type. Consistently, 4-OH-DEB levels are highest inCYP2D6 homozygous, intermediate in CYP2D6heterozygous and extremely low in the wild-type (Fig. 2B).

Figure 2
Figure 2

Time course of serum concentrations of DEB (A) and 4-OH-DEB (B) from wild-type, CYP2D6 humanized heterozygous, and CYP2D6 humanized homozygous mice after one single oral administration of DEB (2.5 mg/kg). Venus blood was obtained 0, 0.5, 1, 2, 4, 6, 8, 12, and 24 h after DEB administration. Values represent the mean and the vertical lines (±S.E.M.) of DEB and 4-OH-DEB from three to four mice.

The pharmacokinetic data was calculated from the three groups (Table1). The AUC was 3- and 6-fold higher in wild-type mice than in heterozygous and homozygous CYP2D6humanized mice, respectively. This is illustrated by differences in the elimination half-life of DEB, which was 2.1 and 1.4 times shorter in the heterozygous and homozygous CYP2D6 humanized mice than in wild-type mice. Accordingly, CYP2D6 humanized mice showed a clearance 6- and 4-fold higher than wild-type mice. All the phenotypic differences were statistically significant.

View this table:
Table 1

Pharmacokinetic parameters for DEB and 4-OH-DEB after a single oral administration of DEB (2.5 mg/kg) in wild-type, CYP2D6humanized heterozygous, and CYP2D6 humanized homozygous mice

Urinary Metabolic Balance in the CYP2D6 Humanized Mice.

CYP2D6 integration in the mouse genome did not affect any of the physiological parameters concerning renal function (data not shown). Twenty four hours after a single oral dose of DEB,CYP2D6 humanized mice excreted significantly higher amounts of 4-OH-DEB (28.9 ± 12.5% of dose) and lower amounts of DEB (14.6 ± 6.4%) than the wild-type mice (6.2 ± 3.1% and 61.0 ± 9.0%, respectively). Total recoveries of DEB + 4-OH-DEB were 67.2 ± 10.7% and 43.5 ± 18.9% for the wild-type andCYP2D6 humanized mice, respectively (Fig.3). This latter finding perhaps indicates that the human CYP2D6 gene provokes metabolism of DEB to additional metabolites not detected in this study. The metabolic ratio (Mahgoub et al., 1977) for the wild-type mice fell from 9.8 to 0.5 after insertion of the human transgene.

Figure 3
Figure 3

Percentage of a single oral dose of DEB (2.5 mg/kg) excreted in urine over 24 h from wild-type, CYP2D6humanized heterozygous and CYP2D6 humanized homozygous mice. Columns represent the mean and S.E.M of DEB and 4-OH-DEB from three to four mice. ∗, values of 4-OH-DEB levels fromCYP2D6 humanized mice that are significantly different (p < 0.05) form wild-type mice. ∗∗, values of DEB levels from CYP2D6 humanized mice that are significantly different (p < 0.05) form wild-type mice.

Regulation of CYP2D6 Activity by Conditional Knock-Out of theHNF4α Allele.

To determine the mechanism of regulation of the CYP2D6 gene, the transgenic mouse was bred with a HNF4α conditional null mouse line. The HNF4α conditional null mouse allows the study of the role of this hepatic factor in an intact model, and circumvents the embryonic lethality of a standard HNF4α null mouse (Hayhurst et al., 2001). A mouse line carrying theCYP2D6 gene and the conditional HNF4α mutation was generated (see Materials and Methods). To this end,HNF4α fl/+; AlbCre +/− mice, previously produced, were crossed with HNF4α fl/fl; CYP2D6 +/− obtained in the present work. The F1 generation of this cross yieldedHNF4α fl/fl; AlbCre +/−; CYP2D6 +/− and littermate control mice, HNF4α fl/fl; AlbCre −/−;CYP2D6 +/−, HNF4α fl/fl; AlbCre +/−;CYP2D6 −/− and HNF4α fl/fl; AlbCre −/−;CYP2D6 −/−. Genotypes of all mice were assessed by PCR of tail DNA (data not shown).

The effect of Cre-mediated recombination at the HNF4αlocus was determined to be optimal at 45 days of age (Hayhurst et al., 2001). Therefore, all of these experiments were conducted using 45-day-old mice. Deletion of HNF4α mRNA in AlbCre +/− mice was confirmed by Northern blotting using a cDNA probe for exons 4 and 5 (Hayhurst et al., 2001).

Disruption of HNF4α expression resulted in a 50% decrease in CYP2D6 as analyzed by Western blotting. Because the antibody used is CYP2D6 specific, there was no signal detected in any of the wild-type mice. On the other hand, CYP2E1 protein levels were not affected byHNF4α deletion. The lack of effect of HNF4αon CYP2E1 protein levels is in agreement with a previous reports (Jover et al., 2001) and suggests that the hepatic lesions produced by the conditional mutation do not prevent the synthesis of nontarget proteins (Fig. 4B).

Figure 4
Figure 4

Regulation of CYP2D6 by conditional knock-out ofHNF4α. A, Northern blot analysis of mouse liverHNF4α expression. Hepatic mRNA (10 μg) fromHNF4α; AlbCre +/−; CYP2D6 +/− and littermate control mice, HNF4α fl/fl; AlbCre −/−;CYP2D6 +/−, HNF4α fl/fl; AlbCre +/−;CYP2D6 −/− and HNF4α fl/fl; AlbCre −/−; CYP2D6 −/− was separated on a 1% agarose gel, transferred to a nylon membrane, and hybridized with anHNF4α exon 4 and 5 cDNA probe. HNF4αwas completely deleted after Cre-mediated recombination. β-actin was used as a loading control. B, Western blot analysis of CYP2D6 protein expression in liver microsomal protein (40 μg) from the same animal groups of A. Deletion of HNF4α resulted in a more than 50% of CYP2D6 protein expression. The blots were re-probed with an antibody to CYP2E1 to verify loading and the presence of microsomal proteins in all the samples.

The phenotypic results of HNF4α mutation on CYP2D6 metabolic activity in intact animals were assessed by measuring the metabolic ratio after 24 h of an oral dose of DEB. As expected, intact CYP2D6 humanized animals showed the greatest metabolism of DEB (18.3 ± 3.7% dose as DEB, 7.2 ± 1.4% dose as 4-OH-DEB, MR 2.6 ± 0.2; see Table 3). However, in the absence of HNF4α, the metabolism of these animals decreased more than 50% (10.1 ± 0.3% DEB, 2.9 ± 0.5% 4-OH-DEB, MR 3.6 ± 0.6; P < 0.005). In control animals, basal DEB metabolic ratio levels were 5-fold lower than intact CYP2D6 humanized mice (51.6 ± 9.5% DEB, 2.9 ± 0.4% 4-OH-DEB, MR 18.3 ± 6.2); after HNF4α deletion, the metabolic ratio was also decreased (25.8 ± 1.9% DEB, 0.8 ± 0.1% 4-OH-DEB, MR 32.0 ± 3.0; P < 0.005) (Table 3).

View this table:
Table 3

DEB urine metabolic ratios from the animal groups in Figure 4A

Discussion

Although DEB has been extensively and successfully used as a clinical probe for the CYP2D6 polymorphism, the knowledge about its disposition in humans is limited. Most phenotyping studies have been carried out using urine excretion analysis; and during the last 20 years, only three studies of DEB pharmacokinetics have been reported (Silas et al., 1978; Sloan et al., 1983; Dalen et al., 1999). Human disposition of DEB depends on the number of functionalCYP2D6 alleles present in each subject. Using individuals that carried 0, 1, 2, 3 to 4, and 13 functional alleles, it was shown that there is a gene dose effect on DEB and 4-OH DEB pharmacokinetic profile. When the number of CYP2D6 active copies increases, 4-hydroxylase activity also increases, and the plasma levels of DEB decrease, so the drug is eliminated faster (Dalen et al., 1999).

In vitro, DEB 4-hydroxylase activity in wild-type mice is almost absent (Masubuchi et al., 1997). The data presented in this study provides an in vivo confirmation for this previous finding and show that, once theCYP2D6 gene is integrated, constitutively expressed, and transmitted into the germ line, DEB 4-hydroxylase activity is clearly present. After a single oral dose of DEB, the half-life, clearance, AUC values for wild-type, heterozygous and homozygous CYP2D6humanized mice reflect the gene dose effect found in the human population. All these data show that the humanized CYP2D6mouse model mimics the pharmacokinetic and metabolic properties of human CYP2D6-mediated metabolism.

Clinical studies on CYP2D6-mediated metabolism are based on phenotype and genotype tests. Although phenotyping analysis reflects the real metabolic status of the patient, and genotyping analysis offers pure genetically based prediction of individual metabolic profile, there are a number of nongenetic factors (age, sex, smoking and drinking habits, and pathological status) that can affect drug metabolism and interfere with an experimentally determined phenotype or genotype (Wolf and Smith, 1999). CYP2D6 humanized mice have a tremendous potential in phenotype-genotype correlations, because these animals share the same genetic background except for the presence or absence of the human CYP2D6 gene. Because the environmental conditions through the present work have been identical for all the animals used, the phenotypic differences determined in this study are caused by the presence/absence of the CYP2D6 gene.

An intriguing and apparently unaccountable observation is the major decline in the recovery of DEB plus 4-OH-DEB in the transgenic mice compared with the wild-type mice (Table2). The wild-type mice had a total recovery of 67.2 ± 10.7% dose, which fell to 40.9 ± 6.6% and 43.5 ± 18.9% in the heterozygous and homozygous humanized mice, respectively (unpairedt test, t = 2.90, df = 7,P = 0.02). Apparently, the fall in excretion of DEB due to CYP2D6 metabolism is not compensated by a concomitant rise in excreted 4-OH-DEB; some 25% of the dose is unaccounted for, a similar proportion of the dose as is excreted as 4-OH-DEB. The answer lies in alternative metabolic products produced by CYP2D6. Early studies (Allen et al., 1976; Idle et al., 1979) showed that DEB, in addition to 4-hydroxylation, was also metabolized to 5-, 6-, 7- and 8-hydroxy-DEB (phenolic metabolites) in humans, together with two ring-opened products, which are thought to arise from 1- and 3-hydroxylation of the drug and also are mediated by CYP2D6 (Eiermann et al., 1998). Therefore, DEB is metabolized at positions 1, 3, 4, 5, 6, 7, and 8 (i.e., at every C-H bond in the ring system). Quantitatively, the phenols represented 7.1 ± 5.8% dose eliminated in the 0- to 24-h human urine, with 7-hydroxylation predominating (Idle et al., 1979). In the female Wistar rat (Al-Dabbagh et al., 1981), 6-hydroxy-DEB (14.3 ± 1.5% dose) and an unidentified phenolic metabolite (11.3 ± 2.3% dose, calculated as a phenol; probably a dihydroxy metabolite) accounted for significant proportions of the 0- to 24-h excreted dose, compared with DEB (3 ± 0%) and 4-OH-DEB (23.7 ± 2.5%). The hydroxylations in positions 1 and 3 may contribute significantly to the excretion profile. Swedish investigators (Dalen et al., 1999) found that, as the number of functional CYP2D6 alleles increased in their human study population from 0 to 13, the total recovery of DEB and 4-OH-DEB decreased, a situation analogous to that reported here in the mouse. The early studies (Allen et al., 1976; Idle et al., 1979) had observed up to 15% dose excreted in 24 h as the ring-opened metabolites, and the Swedish study (Eiermann et al., 1998) suggests that the 1- and 3-hydroxylations may account for even more of the dose than this. However, until this point, it has not been possible to gain a true insight in vivo into relative quantitative importance of 4-hydroxylation and non-4-hydroxylation by CYP2D6. There were indications from older literature that the non-4-hydroxylation pathways may be major routes of DEB metabolism in man, rat, and dog (Allen et al., 1976; Idle et al., 1979; Al-Dabbagh et al., 1981). Using theCYP2D6 humanized mouse, it is possible to visualize that the non-4-hydroxylation of DEB by CYP2D6 may comprise about 50% of the total metabolism of this drug.

View this table:
Table 2

Urinary excretion (0–24 h) of percentage dose as DEB, 4-OH-DEB, and DEB + 4-OH-DEB, together with the metabolic ratio (MR; percentage dose as DEB/percentage dose as 4-OH-DEB) for wild-type (WT), heterozygous CYP2D6 humanized (HT), and homozygousCYP2D6 humanized mice (HM)

In addition to the pharmacokinetic study of CYP2D6 substrates, of which DEB is an archetype, the CYP2D6 humanized mouse offers us an opportunity to understand better the genetic foundation of the CYP2D6 polymorphism. The EM trait was recognized at the outset (Mahgoub et al., 1977) as the dominant phenotype, but few occasions have presented themselves for the quantification of the degree of dominance of the EM over the PM trait. The first determinations were made in family pedigrees that permitted identification of obligate heterozygotes (e.g., EM children of PM probands). The mean MR values for homozygous EMs (calculated), heterozygous EMs (observed), and PMs (observed) were 0.4, 1.9, and 33.9, respectively. These data permitted calculation of the degree of dominance of EM over PM to be approximately 30% (Evans et al., 1980). These findings were thought to explain the major phenotypic overlap between the homozygous and heterozygous EM genotypes and demonstrated just why molecular genetic methods would be required to determine the genotype reliably. However, the expression of the human transgene in the mouse background offers yet further insights. The MR values in the mouse for 0, 1, and 2 human CYP2D6genes are 15.2 ± 7.0 (n = 6), 0.7 ± 0.2 (n = 3), and 0.5 ± 0 (n = 3). Using log-transformed data according to the method of Evans et al. (1980), these results yield a degree of dominance of EM over PM, for the human gene in the mouse, of 78% (95% confidence interval, 60 to 100%) (Fig. 5). This is significantly greater than the human estimate of 30% and may reflect the fact that transcription and translation of the CYP2D6 transgene proceeds very efficiently. Perhaps the mouse hepatic background provides a more transcriptionally active environment than the endogenous human one.

Figure 5
Figure 5

Estimation of the mean degree of dominance of the EM [transgenic] over the PM [endogenous] phenotype in mice. Horizontal double-headed arrows represent 95% confidence intervals. The vertical dotted line shows the position of the mid-point between the two homozygous genotypes (+/+) and (−/−), the position of zero dominance. When the mean heterozygote (+/−) value falls to the left of this dotted line, then (+/+) is dominant over (−/−), the excursion to the left of this mid-point revealing the degree of dominance. For all calculations, only log-transformed data were used, because logMR is normally distributed, unlike its arithmetic values that are generally skewed to the right (Evans et al., 1980).

HNF4α is known to regulate the basal levels of expression of a several P450s. Promoter activity, mobility shift, and antisense targeting assays have shown that HNF4α regulates, among others, the expression of human CYP2D6 (Cairns et al., 1996; Jover et al., 2001) but not rat CYP2D5 (Lee et al., 1994) or humanCYP2E1 (Liu and Gonzalez, 1995). The results from the present study provide, for the first time, in vivo confirmation of these earlier findings.

The most intriguing finding with the HNF4a conditional knock-out experiments is not that in the wild-type andCYP2D6 humanized mice the metabolic ratio (MR) rises from 18.2 ± 6.2 to 32.0 ± 2.9 (t = 3.93, df = 5, P = 0.011) and from 2.6 ± 0.2 to 3.6 ± 0.6 (t = 3.31, df = 6, P = 0.016), respectively, but that the percentage dose recovered in urine as DEB plus 4-OH-DEB fell from 54.5 ± 9.1% to 26.6 ± 2.0% (t = 6.17, df = 5, P = 0.002) and from 25.5 ± 4.9% to 13.0 ± 0.6% (t = 5.06, df = 6, P = 0.002), respectively. This situation is somewhat similar to that described above for the decrease in urinary recovery associated with insertion of the CYP2D6transgene into the mouse. However, it is inconceivable that the conditional knock-out of HNF4a in the mouse liver, either in wild-type or humanized mice, could result in switching to alternative and undisclosed pathways of metabolism in the mouse. There is no evidence that DEB is metabolized by any P450s other than the CYP2D family. Conditional HNF4a knock-out mice harbor several biochemical abnormalities, together with hepatomegaly (Hayhurst et al., 2001). The single most dramatic biochemical change in these mice was an 18-fold elevation in serum bile acids from 16.7 ± 7 μM in wild-type mice to 283 ± 61 μM (t = 8.70, df = 6, P = 0.0001) in the HNF4α conditional null mice (conditionally hepatic HNF4a-null mice). Elevated bile acids, as is seen in biliary obstruction, for example, are known to impair renal function (Shen et al., 1990; Bomzon et al., 1997) and thus reduce urine output. The reduced urinary recovery of DEB and 4-OH-DEB, relative to controls, reported here in both the heterozygousCYP2D6 humanized mice with conditional hepaticHNF4a knock-out (fl/fl, CRE+/−, 2D6+/−) and wild-type mice with the same HNF4a knock-out (fl/fl, CRE+/−, 2D6−/−), is almost certainly secondary to the massively elevated serum bile acid concentration and concomitantly reduced renal function. In human populations with reduced urine volumes caused by dehydration, for example in Saudis and Egyptians (Islam et al., 1980), the 0- to 8-h urinary recovery of DEB plus 4-OH-DEB fell from 41% in British subjects to 15 to 16% in the Arab populations.

In conclusion, we report here the first humanized P450 model that predicts human drug metabolism and reflects the polymorphic status of the gene in the human population. Moreover, we show in vivo regulation of CYP2D6 by HNF4a. This preclinical model may be of considerable interest for the identification of in vivo drug interactions and for the preclinical evaluation of novel CYP2D6 substrates or inhibitors. The CYP2D6 humanized mouse will also permit investigation into the physiological significance of CYP2D6 and its polymorphism.

Acknowledgment

We thank John Buckley for technical assistance.

Footnotes

    • Received June 13, 2001.
    • Accepted August 10, 2001.

  • ↵1 Present address: Unité de Génétique de la Différenciation, Départment de Biologie Moléculaire, Institut Pasteur, 25/28 rue du Dr Roux 75724 Paris cedex 15, France.

  • ↵2 Present address: GENTEST Corporation, 6 Henshaw Street, Woburn, MA 01801.

  • ↵3 Present address: Zlatá 34, 36005 Karlovy Vary, Czech Republic.

  • This work was supported in part by a Cooperative Research and Development Agreement between the National Cancer Institute and Pfizer Inc. (Groton, CT). J.C. and C.P.G. contributed equally to this work.

Abbreviations

PM
poor metabolizer
EM
extensive metabolizer
DEB
debrisoquine
HNF4α
hepatic nuclear factor 4α
bp
base pair(s)
P450
cytochrome P450
fl
flanked by lox P
AlbCre
to albumin-Cre heterozygous
PCR
polymerase chain reaction
mEH
microsomal epoxide hydrolase
SSC
standard saline citrate
MOPS
4-morpholinepropanesulfonic acid
LC/MS/MS
liquid chromatography/tandem mass spectrometry
4-OH-DEB
4-hydroxydebrisoquine
AUC
area under the curve

References

  1. ↵
    1. Al-Dabbagh SG,
    2. Idle JR, and
    3. Smith RL
    (1981) Animal modelling of human polymorphic drug oxidation: the metabolism of debrisoquine and phenacetin in rat inbred strains. J Pharm Pharmacol 33:161–164.
    OpenUrlPubMed
  2. ↵
    1. Allen JG,
    2. Brown AN, and
    3. Marten TR
    (1976) Metabolism of debrisoquine sulphate in rat, dog and man. Xenobiotica 6:405–409.
    OpenUrlPubMed
  3. ↵
    1. Bomzon A,
    2. Holt S, and
    3. Moore K
    (1997) Bile acids, oxidative stress, and renal function in biliary obstruction. Semin Nephrol 17:549–562.
    OpenUrlPubMed
  4. ↵
    1. Brøsen K and
    2. Gram LF
    (1988) First-pass metabolism of imipramine and desipramine: impact of the sparteine oxidation phenotype. Clin Pharmacol Ther 43:400–406.
    OpenUrlPubMed
  5. ↵
    1. Brøsen K
    (1990) Recent developments in hepatic drug oxidation. Implications for clinical pharmacokinetics. Clin Pharmacokinet 18:220–239.
    OpenUrlPubMed
  6. ↵
    1. Cairns W,
    2. Smith CA,
    3. McLaren AW, and
    4. Wolf CR
    (1996) Characterization of the human cytochrome P4502D6 promoter. A potential role for antagonistic interactions between members of the nuclear receptor family. J Biol Chem 271:25269–25276.
    OpenUrlAbstract/FREE Full Text
  7. ↵
    1. Dalen P,
    2. Dahl ML,
    3. Eichelbaum M,
    4. Bertilsson L, and
    5. Wilkinson GR
    (1999) Disposition of debrisoquine in Caucasians with different CYP2D6-genotypes including those with multiple genes. Pharmacogenetics 9:697–706.
    OpenUrlPubMed
  8. ↵
    1. Eiermann B,
    2. Edlund PO,
    3. Tjernberg A,
    4. Dalén P,
    5. Dahl ML, and
    6. Bertilsson L
    (1998) 1- and 3-Hydroxylations, in addition to 4-hydroxylation, of debrisoquine are catalyzed by cytochrome P450 2D6 in humans. Drug Metab Dispos 26:1096–1101.
    OpenUrlAbstract/FREE Full Text
  9. ↵
    1. Evans DAP,
    2. Mahgoub A,
    3. Sloan TP,
    4. Idle JR, and
    5. Smith RL
    (1980) A family and population study of the genetic polymorphism of debrisoquine oxidation in a white British population. J Med Genet 17:102–105.
    OpenUrlAbstract/FREE Full Text
  10. ↵
    1. Ioannides C
    1. Gonzalez FJ
    (1996) The CYP2D subfamily. in Cytochromes P450 Metabolic and Toxicological Aspects, ed Ioannides C (CRC Press, Boca Raton, FL), pp 183–210.
  11. ↵
    1. Gonzalez FJ and
    2. Nebert DW
    (1990) Evolution of the P450 gene superfamily: animal-plant ‘warfare’, molecular drive and human genetic differences in drug oxidation. Trends Genet 6:182–186.
    OpenUrlCrossRefPubMed
  12. ↵
    1. Gonzalez FJ,
    2. Vilbois F,
    3. Hardwick JP,
    4. McBride OW,
    5. Nebert DW,
    6. Gelboin HV, and
    7. Meyer UA
    (1988) Human debrisoquine 4-hydroxylase (P450IID1): cDNA and deduced amino acid sequence and assignment of the CYP2D locus to chromosome 22. Genomics 2:174–179.
    OpenUrlCrossRefPubMed
  13. ↵
    1. Hayhurst GP,
    2. Lee YH,
    3. Lambert G,
    4. Ward JM, and
    5. Gonzalez FJ
    (2001) Hepatocyte nuclear factor 4alpha (nuclear receptor 2A1) is essential for maintenance of hepatic gene expression and lipid homeostasis. Mol Cell Biol 21:1393–1403.
    OpenUrlAbstract/FREE Full Text
  14. ↵
    1. Idle JR,
    2. Mahgoub A,
    3. Angelo MM,
    4. Dring LG,
    5. Lancaster R, and
    6. Smith RL
    (1979) The metabolism of [14C]-debrisoquine in man. Br J Clin Pharmacol 7:257–266.
    OpenUrlPubMed
  15. ↵
    1. Islam SI,
    2. Idle JR, and
    3. Smith RL
    (1980) The polymorphic 4-hydroxylation of debrisoquine in a Saudi arab population. Xenobiotica 10:819–825.
    OpenUrlPubMed
  16. ↵
    1. Jover R,
    2. Bort R,
    3. Gomez-Lechon MJ, and
    4. Castell JV
    (2001) Cytochrome P450 regulation by hepatocyte nuclear factor 4 in human hepatocytes: a study using adenovirus-mediated antisense targeting. Hepatology 33:668–675.
    OpenUrlCrossRefPubMed
  17. ↵
    1. Kimura S,
    2. Umeno M,
    3. Skoda RC,
    4. Meyer UA, and
    5. Gonzalez FJ
    (1989) The human debrisoquine 4-hydroxylase (CYP2D) locus: sequence and identification of the polymorphic CYP2D6 gene, a related gene, and a pseudogene. Am J Hum Genet 45:889–904.
    OpenUrlPubMed
  18. ↵
    1. Laird PW,
    2. Zijderveld A,
    3. Linders K,
    4. Rudnicki MA,
    5. Jaenisch R, and
    6. Berns A
    (1991) Simplified mammalian DNA isolation procedure. Nucleic Acids Res 19:4293.
    OpenUrlFREE Full Text
  19. ↵
    1. Lee YH,
    2. Yano M,
    3. Liu SY,
    4. Matsunaga E,
    5. Johnson PF, and
    6. Gonzalez FJ
    (1994) A novel cis-acting element controlling the rat CYP2D5 gene and requiring cooperativity between C/EBP beta and an Sp1 factor. Mol Cell Biol 14:1383–1394.
    OpenUrlAbstract/FREE Full Text
  20. ↵
    1. Lindner T,
    2. Gragnoli C,
    3. Furuta H,
    4. Cockburn BN,
    5. Petzold C,
    6. Rietzsch H,
    7. Weiss U,
    8. Schulze J, and
    9. Bell GI
    (1997) Hepatic function in a family with a nonsense mutation (R154X) in the hepatocyte nuclear factor-4alpha/MODY1 gene. J Clin Invest 100:1400–1405.
    OpenUrlCrossRefPubMed
  21. ↵
    1. Liu SY and
    2. Gonzalez FJ
    (1995) Role of the liver-enriched transcription factor HNF-1 alpha in expression of the CYP2E1 gene. DNA Cell Biol 14:285–293.
    OpenUrlCrossRefPubMed
  22. ↵
    1. Mahgoub A,
    2. Idle JR,
    3. Dring LG,
    4. Lancaster R, and
    5. Smith RL
    (1977) Polymorphic hydroxylation of debrisoquine in man. Lancet 2:584–586.
    OpenUrlCrossRefPubMed
  23. ↵
    1. Masubuchi Y,
    2. Iwasa T,
    3. Hosokawa S,
    4. Suzuki T,
    5. Horie T,
    6. Imaoka S,
    7. Funae Y, and
    8. Narimatsu S
    (1997) Selective deficiency of debrisoquine 4-hydroxylase activity in mouse liver microsomes. J Pharmacol Exp. Ther 282:1435–1441.
    OpenUrlAbstract/FREE Full Text
  24. ↵
    1. Miyata M,
    2. Kudo G,
    3. Lee YH,
    4. Yang TJ,
    5. Gelboin HV,
    6. Fernandez-Salguero P,
    7. Kimura S, and
    8. Gonzalez FJ
    (1999) Targeted disruption of the microsomal epoxide hydrolase gene. Microsomal epoxide hydrolase is required for the carcinogenic activity of 7,12-dimethylbenz[a]anthracene. J Biol Chem 274:23963–23968.
    OpenUrlAbstract/FREE Full Text
  25. ↵
    1. Nelson DR,
    2. Koymans L.,
    3. Kamataki T,
    4. Stegeman JJ,
    5. Feyereisen R,
    6. Waxman DJ,
    7. Waterman MR,
    8. Gotoh O,
    9. Coon MJ,
    10. Estabrook RW,
    11. et al.
    (1996) P450 superfamily: update on new sequences, gene mapping, accession numbers and nomenclature. Pharmacogenetics 6:1–42.
    OpenUrlCrossRefPubMed
  26. ↵
    1. Pereira VA,
    2. Auler JO,
    3. Carmona MJ,
    4. Mateus FH,
    5. Lanchote VL,
    6. Breimer DD, and
    7. Santos SR
    (2000) A micromethod for quantitation of debrisoquine and 4-hydroxydebrisoquine in urine by liquid chromatography. Braz J Med Biol Res 33:509–514.
    OpenUrlCrossRefPubMed
  27. ↵
    1. Scheen AJ and
    2. Lefebvre PJ
    (1995) Antihyperglycaemic agents. Drug interactions of clinical importance. Drug Safety 12:32–45.
    OpenUrlPubMed
  28. ↵
    1. Scott RJ,
    2. Palmer J,
    3. Lewis IA, and
    4. Pleasance S
    (1999) Determination of a “GW cocktail” of cytochrome P450 probe substrates and their metabolites in plasma and urine using automated solid phase extraction and fast gradient liquid chromatography tandem mass spectrometry. Rapid Commun Mass Spectrom 13:2305–2319.
    OpenUrlCrossRefPubMed
  29. ↵
    1. Shen YS,
    2. Chen CF,
    3. Liu HM, and
    4. Fang HS
    (1990) Effects of rat bile infusion on renal function in rats. Ren Physiol Biochem 13:213–222.
    OpenUrlPubMed
  30. ↵
    1. Silas JH,
    2. Lennard MS,
    3. Tucker GT,
    4. Smith AJ,
    5. Malcolm SL, and
    6. Marten TR
    (1978) The disposition of debrisoquine in hypertensive patients. Br J Clin Pharmacol 5:27–34.
    OpenUrlPubMed
  31. ↵
    1. Sinal CJ,
    2. Webb CD, and
    3. Bend JR
    (1999) Differential in vivo effects of alpha-naphthoflavone and beta-naphthoflavone on CYP1A1 and CYP2E1 in rat liver, lung, heart, and kidney. J Biochem Mol Toxicol 13:29–40.
    OpenUrlCrossRefPubMed
  32. ↵
    1. Tronche F and
    2. Yaniv M
    1. Sladek FM
    (1994) Hepatocyte nuclear factor 4. in Liver Gene Expression, eds Tronche F and Yaniv M (R. G. Landes Company, Austin, TX), pp 207–230.
  33. ↵
    1. Sloan TP,
    2. Lancaster R,
    3. Shah RR,
    4. Idle JR, and
    5. Smith RL
    (1983) Genetically determined oxidation capacity and the disposition of debrisoquine. Br J Clin Pharmacol 15:443–450.
    OpenUrlPubMed
  34. ↵
    1. Wolf CR and
    2. Smith G
    (1999) Chapter 18. Cytochrome P450 CYP2D6. IARC Sci Publ 128:209–229.
    OpenUrl
  35. ↵
    1. Yakar S,
    2. Liu JL,
    3. Stannard B,
    4. Butler A,
    5. Accili D,
    6. Sauer B, and
    7. LeRoith D
    (1999) Normal growth and development in the absence of hepatic insulin-like growth factor I. Proc Natl Acad Sci USA 96:7324–7329.
    OpenUrlAbstract/FREE Full Text
  36. ↵
    1. Yamagata K,
    2. Furuta H,
    3. Oda N,
    4. Kaisaki PJ,
    5. Menzel S,
    6. Cox NJ,
    7. Fajans SS,
    8. Signorini S,
    9. Stoffel M, and
    10. Bell GI
    (1996) Mutations in the hepatocyte nuclear factor-4alpha gene in maturity-onset diabetes of the young (MODY1). Nature (Lond) 384:458–460.
    OpenUrlCrossRefPubMed
Back to top

In this issue

Download PDF
Article Alerts
Email Article
Citation Tools

Research ArticleArticle

The CYP2D6 Humanized Mouse: Effect of the HumanCYP2D6 Transgene and HNF4α on the Disposition of Debrisoquine in the Mouse

Javier Corchero, Camille P. Granvil, Taro E. Akiyama, Graham P. Hayhurst, Satish Pimprale, Lionel Feigenbaum, Jeffrey R. Idle and Frank J. Gonzalez
Molecular Pharmacology December 1, 2001, 60 (6) 1260-1267; DOI: https://doi.org/10.1124/mol.60.6.1260
del.icio.us logo Digg logo Reddit logo Twitter logo Facebook logo Google logo Mendeley logo

Jump to section

Advertisement