Short Communication Genetic Basis of Inter- and Intrastrain Differences in Diazepam p-Hydroxylation in Rats

Diazepam (7-chloro-1,3-dihydro-1-methyl-5-phenyl-2H-1,4-benzodiazepin-2-one) is widely used as a sedative, hypnotic, and antianxiety drug. At low diazepam concentrations, p-hydroxylation is the major metabolic pathway in rat liver microsomes. However, there are marked ( 300-fold) interand intrastrain differences in the activity among Sprague-Dawley, Brown Norway, Dark Agouti, and Wistar rats. In our previous study, we determined that a deficiency of CYP2D3 protein, not CYP2D2, was responsible for the interand intrastrain differences in diazepam p-hydroxylation (Drug Metab Dispos 33:1657–1660, 2005). Quantitative real-time polymerase chain reaction (PCR) did not provide enough evidence to explain the interand intrastrain differences in the expression of CYP2D3 protein. Nucleotide sequence analysis revealed the insertion of a thymine in exon 8 of the CYP2D3 gene in the poor diazepam metabolizers. This single nucleotide mutation caused a shift in the reading frame and introduced a premature termination signal. It is noteworthy that the heme binding region, which is essential to maintain proper heme binding and active cytochrome P450 enzymes, was consequently deleted by the premature termination signal. In contrast, no mutation was detected in the CYP2D3 gene of extensive metabolizers. Thus, the truncated CYP2D3 must be a nonfunctional enzyme in poor metabolizers. In addition, we developed a convenient and specific genotyping assay using PCRrestriction, fragment-length polymorphism to distinguish homozygotes from heterozygotes. The genotyping gave results fully consistent with those of the interand intrastrain differences in diazepam p-hydroxylation. A variety of strains of rats have been used as animal models in pharmacological and toxicological studies worldwide. To date, many strains of rats have been established and screened for various purposes, yet little is known about the differences in their drug metabolism characteristics that are ascribable to genetic variation. Currently, information on genetic variation in rat laboratory strains is accumulating as a set of microsatellite markers, simple sequence length polymorphism markers, and a variety of single nucleotide polymorphisms in coding regions (Thomas et al., 2003; Serikawa, 2004). Nevertheless, information on genetic variation in rat strains is still deficient compared with that on humans and mice. Cytochrome P450 (P450) comprises a superfamily of heme-containing monooxygenases that include enzymes responsible for drug metabolism. Although it occupies only approximately 2% of human liver P450, the CYP2D subfamily encodes for enzymes that are of clinical importance in the metabolism of numerous drugs (Shimada et al., 1994). The CYP2D subfamily has been extensively studied due to the involvement of CYP2D6 in the human debrisoquine/sparteine drug oxidation polymorphism (Eichelbaum and Gross, 1990). The genetic polymorphisms of CYP2D6 are among the most important factors in pharmacogenetics. In rats, six gene homologs to CYP2D6 have been isolated, namely, CYP2D1, CYP2D2, CYP2D3, CYP2D4, CYP2D5, and CYP2D18 (Nelson et al., 1996). In recent studies, we reported interand intrastrain differences in P450-dependent diazepam metabolism in rat liver microsomes (Saito et al., 2004a,b). Sprague-Dawley (SD) and Brown Norway (BN) rats had significantly higher levels of diazepam p-hydroxylation, which is the major metabolic pathway at concentrations of this drug close to the in vivo concentrations used for medication. In contrast, Dark Agouti (DA) rats completely lacked the activity. It is interesting to note that Wistar rats exhibited a phenotype polymorphism, having extensive metabolizers (EM) and poor metabolizers (PM). Thus, we classified EM Wistar rats as EM-W and PM Wistar rats as PM-W, respectively, in tests of diazepam p-hydroxylation. Comparing the expression levels of the CYP2D subfamily in liver microsomes by immunoblotting detected a band of proteins, the N-terminal amino acid sequences of which exactly corresponded to those of CYP2D3, in SD, BN, and EM-W rats. Moreover, using rat CYP2D isoforms expressed in yeast, the observation that diazepam p-hydroxylation is catalyzed by CYP2D3 was confirmed. Therefore, the polymorphic expression of CYP2D3 caused the interand intrastrain differences in diazepam p-hydroxylation among the four rat strains (Sakai et al., 2005). However, the question of what causes the polymorphic expression of CYP2D3 among the four rat strains remained to be solved. Clarification of the interand intrastrain differences in rats will be useful for predicting variability in human pharmacokinetics. Therefore, it is worthwhile to fully characterize animals used in pharmacoThis study was supported by grants-in-aid for Scientific Research from the Japanese Ministry of Education, Science, Sports, Culture and Technology [Grants 19671001, 19208028]. Article, publication date, and citation information can be found at http://dmd.aspetjournals.org. doi:10.1124/dmd.108.024273. ABBREVIATIONS: P450, cytochrome P450; SD, Sprague-Dawley; BN, Brown Norway; DA, Dark Agouti; EM, extensive metabolizers; PM, poor metabolizers; EM-W, EM from Wistar rats; PM-W, PM from Wistar rats; PCR, polymerase chain reaction; RFLP, restriction fragment length polymorphism; bp, base pair. 0090-9556/09/3702-268–271$20.00 DRUG METABOLISM AND DISPOSITION Vol. 37, No. 2 Copyright © 2009 by The American Society for Pharmacology and Experimental Therapeutics 24273/3424257 DMD 37:268–271, 2009 Printed in U.S.A. 268 at A PE T Jornals on A ril 1, 2017 dm d.aspurnals.org D ow nladed from kinetics studies from the point of view of the genetic expression of metabolic enzymes. In this study, we describe the relationship between a defect in the CYP2D3 gene (genotype) and the interand intrastrain differences in diazepam p-hydroxylation (phenotype) among four rat strains. Materials and Methods Materials and Animals. All reagents were of analytical grade. Adult male SD, DA, and Wistar rats (9 weeks old) were obtained from Nihon SLC Co. (Shizuoka, Japan). Adult male BN rats (9 weeks old) were obtained from Kyudo Co., Ltd. (Fukuoka, Japan). They were housed under standard laboratory conditions with free access to food and water, and they were used for experiments after 1 week of acclimatization. All experiments using animals were performed with the supervision and approval of the Animal Care and Use Committee of Hokkaido University. Isolation of Total RNA and Genomic DNA from Rat Liver. Total RNA was isolated from rat liver using Isogen (Nippon Gene, Toyama, Japan). Genomic DNA was isolated from rat liver using a DNeasy Tissue Kit (QIAGEN GmbH, Hilden, Germany). The concentration and purity of both DNA and RNA were determined by using a spectrophotometer. The integrity was examined by electrophoresis in a 1% agarose gel with ethidium bromide staining. Quantitative Real-Time Polymerase Chain Reaction Analysis. The cDNA samples were obtained using 1 g of total RNA as the template for reverse transcription with ReverTraAce and oligo(dT) 20 primer (Toyobo, Osaka, Japan). TaqMan MGB probes and primers for CYP2D3 and -glucuronidase, as an endogenous control, were purchased from ABI (Applied Biosystems, Foster City, CA). According to the manufacturer’s instructions, a primer for CYP2D3 was designed around the boundary between exons 4 and 5. TaqMan polymerase chain reaction (PCR) reaction was performed using Real-time PCR Master Mix (Toyobo). The first step was performed for 10 min at 95°C, followed by 40 cycles at 95°C for 15 s and 60°C for 1 min, as recommended by the manufacturer using an ABI Prism 7700 Sequence Detection system (Applied Biosystems). The real-time PCR products of CYP2D3 and -glucuronidase were cloned and sequenced to verify the analytical specificity. Sequencing of CYP2D3 cDNA. Sequencing analysis was performed by using the cDNA samples used in quantitative real-time PCR. The full length of CYP2D3 cDNA was amplified by using synthesized oligonucleotide primers of 5 -GGCCAGTGGTCTTTGGTAGC-3 (sense, nucleotide positions 15–34, NM_173093) and 5 -GGCAGCCACAGAACTGTTTTA-3 (antisense, nucleotide positions 1607–1627). The reaction condition was denaturation for 1 min at 94°C, annealing for 1 min at 57°C, and extension for 2 min at 72°C for 35 cycles. This PCR-amplified fragment was subsequently cloned into a pCR2.1TOPO vector (Invitrogen, Carlsbad, CA). More than 10 cDNA clones from each of the four strains were analyzed to identify a mutation and repeatedly cloned from other samples to exclude PCR errors. The nucleotide sequence was analyzed with a BigDye Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems) and an automated DNA sequencer (ABI Prism 310 Genetic Analyzer) (Applied Biosystems) following the manufacturer’s instructions. CYP2D3 Genotyping Using PCR-Restriction Fragment Length Polymorphism. The single PCR and restriction fragment length polymorphism (RFLP) methods were used to identify the wild-type and mutant CYP2D3 alleles. CYP2D3-specific PCR was accomplished with the primer pairs 5 -TCACATATCCCTGTCATA-3 and 5 -TCAGTCAGTCTGGGGCT-3 , yielding a single 384-base pair (bp) product. The reaction mixture contained approximately 100 ng of genomic DNA, 0.5 M of each primer, 0.2 M of each dNTP, 2 mM MgCl2, and 1 U of ExTaq polymerase (Takara Bio, Shiga, Japan) in a total volume of 50 l. Touchdown PCR was performed as follows: the first cycle of denaturation at 94°C for 60 s, annealing at 65°C for 30 s, elongation at 72°C for 30 s; the 2nd to 30th cycles of denaturation at 94°C for 30 s, annealing at 63 to 48.5°C for 30 s, elongation at 72°C for 30 s; the 31st cycle of denaturation at 94°C for 30 s, annealing at 45°C for 30 s, elongation at 72°C for 30 s; and the final elongation at 72°C for 5 min. In touchdown PCR, the annealing temperature began at 63°C and was reduced by 0.5°C every cycle. Six microliters of each of the PCR products was then digested by the restriction enzyme Xmn1 (NEB, Ipswitch, MA) without further purification. The digested PCR products were electrophoresed in a 2% agarose gel with ethidium bromide staining. The fragment patterns determined the presence of the CYP2D3 wild-type and mutant alleles, as indicated in Fig. 2. Results and Discussion Diazepam (7-chloro-1,3-dihydro-1-methyl-5-phenyl-2H-1,4-benzodiazepin-2-one) is one of the benzodiazepines that are widely used in the treatment of anxiety disorders, depression, and insomnia. Inaba et al. (1988) reported interindividual variability of diazepam metabFIG. 1. Comparison of nucleotide and amino acid (AA) sequences of wildand mutant-type CYP2D3. A, the wild-type CYP2D3 cDNA sequence corresponding to codons 421–25 is shown. Wild-type CYP2D3 cDNA was isolated from SD, BN, and EM-W rat liver. B, a single thymine insertion at nucleotide 1269 is marked by the arrow. Mutant-type CYP2D3 cDNA was isolated from DA, PM-W, and EM-W rat livers. C, the single thymine insertion is underlined. This mutation creates a premature termination at codon 434 (TGA) upstream of the heme binding region. The heme binding cysteine, which is perfectly conserved in P450, is enclosed in a box. The terminal signal is indicated by asterisks. 269 GENETIC POLYMORPHISM OF CYP2D3 IN RATS at A PE T Jornals on A ril 1, 2017 dm d.aspurnals.org D ow nladed from