Introduction

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The important role cytochrome P450 (P450¹) plays in the oxidative metabolism of xenobiotics as well as many endogenous substances such as steroids and fatty acids has long been recognized (Guengerich, 1990). Multiple P450 isozymes have been purified from various species. Of the most commonly used laboratory animals, rat, mouse, and rabbit P450s have been the most intensively studied. The development of molecular biology techniques has led to the identification of rare P450s that would be difficult to isolate using standard protein purification techniques and has allowed greater insights into genetic heterogeneity and polymorphisms of P450 expression.

Despite the use of the dog in safety evaluation and efficacy studies of new drugs, knowledge concerning the canine P450 system is limited. At present, only a small number of canine liver P450 isozymes have been identified and characterized. The most intensively studied of these has been CYP2B11. This P450 has been isolated from dog liver microsomes (Duignan et al., 1987), identified by cloning techniques (Graves et al., 1990), and expressed in cDNA expression systems (Kedzie et al., 1991, 1993; John et al., 1994). Other canine P450 cDNAs identified by cloning techniques include two members of the CYP1A subfamily (Fukuta et al., 1992; Uchida et al., 1990), a member of the CYP2D subfamily (CYP2D15) (Sakamoto et al., 1995), and a member of the CYP3A subfamily (Ciaccio et al., 1991).

Komori et al. (1989) purified a microsomal protein from male beagle dog liver (P-450-D1) and proposed that it belonged to the CYP2 subfamily based on similarities in N-terminal amino acid sequence and catalytic activities to rat CYP2C11. Subsequently, Uchida et al. (1990) isolated an 1875-base pair (bp) cDNA clone (DM1-1) from a cDNA library from the liver of a male beagle dog belonging to the CYP2 subfamily (CYP2C21). However, this clone was not full length. Therefore, it was not possible to determine whether it was identical to P-450-D1 isolated by Komori et al. (1989).

At present, four genes have been identified in the human CYP2C subfamily (Goldstein and de Morais, 1994) and five genes in the rat CYP2C subfamily (Soucek and Gut, 1992). Most of these genes are constitutively expressed, and some members are polymorphic in each of these species. In the rat, several members of the CYP2C subfamily are gender specific in their expression. The specific aim of this study was to determine whether additional members of the CYP2C subfamily exist in the dog using molecular cloning techniques.

	Materials and Methods

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Tissue and Blood. Frozen liver tissue samples from untreated male and female beagle dogs were generously provided by M. Faletto and C. J. Serabjit-Singh at Glaxo-Wellcome (Research Triangle Park, NC) and by D. D. Christ at the DuPont-Merck Pharmaceutical Company (Wilmington, DE). Male and female canine whole blood samples from mixed and beagle breeds were obtained from the College of Veterinary Medicine, North Carolina State University (Raleigh, NC).

Materials. Restriction endonucleases were purchased from New England Biolabs. ZAP-cDNA synthesis and ZAP-cDNA Gigapack Gold cloning kits were purchased from Stratagene, QIAamp blood and tissue kits and plasmid kits from Qiagen, and cDNA synthesis kits from GIBCO-BRL. Transfer membranes and [-³²P]dATP (6000 Ci/mmol) were from DuPont Chemical Co., and kanamycin, ampicillin, and tetracycline were obtained from Sigma.

RNA Isolation. RNA was extracted from livers from nine beagle dogs (five females and four males) by the method of Chomczynski and Sacchi (1987). Five µg of RNA was reversed-transcribed into cDNA, using a cDNA synthesis kit (GIBCO-BRL) and random hexamers or oligo(dT) (for library synthesis) as primers.

cDNA Library Construction and Screening. Three Uni-ZAP XR cDNA libraries were constructed using the ZAP cDNA synthesis kit from Stratagene. Three libraries were constructed from hepatic RNA from two male beagle dogs and one female beagle. Poly(A⁺) RNA was prepared using oligo(dT)-cellulose columns (GIBCO-BRL) and reverse-transcribed using oligo(dT) as a primer. The cDNA was sized by fractionation on a Sephacryl S-500 spin column, ligated to Uni-ZAP XR arms, and packaged into Escherichia coli XL1-Blue MRF' cells using a Gigapack Gold III packaging kit as described by the manufacturer (Stratagene).

Genomic DNA from Liver and Blood. Purified genomic DNA from either liver or blood was extracted using the QIAamp tissue and blood kits, according to the manufacturer's recommendations. Hepatic DNA was obtained from 9 beagles, and blood DNA was obtained from another 19 dogs of both sexes, 9 beagles and 10 of mixed breeds.

Genetic Test to Discriminate between CYP2C21 and CYP2C41 cDNAs. The strategy is outlined in fig. 1, A and B. Two specific primers were used to amplify exons 4 through 8 of both CYP2C21 and 2C41. The exon nomenclature is based on the human 2C genes (de Morais et al., 1993). The forward and reverse primers were 5'-CTGTGCTCCCTGCAATGTG-3' and 5'-CCATGAAGTAGTCACTCTTC-3', respectively. The underlined C in the forward primer represents a mismatch with the CYP2C41 sequence. The PCR reaction involved an initial denaturing step of 94°C for 1 min, followed by 38 cycles of denaturing at 94°C for 20 sec, annealing at 55°C for 10 sec, and extension at 72°C for 30 sec with a final extension of 5 min at 72°C. The 767-bp PCR product of this amplification was digested overnight at 37°C in separate reactions with NdeI, which digests the CYP2C21 fragment into two smaller fragments of 359 and 408 bp but does not digest CYP2C41, and BbsI, which digests the fragment from CYP2C41 into two smaller fragments of 654 and 113 bp but does not digest CYP2C21. The reactions were electrophoresed on a 3% agarose gel.

View larger version (31K): [in this window] [in a new window]   Fig. 1.   (A) Strategy used to genotype canine CYP2C21 and 2C41 liver cDNA utilizing PCR amplification of exon 4 through exon 8 followed by NdeI digestion for CYP2C21 and by BbsI for CYP2C41. (B) The predicted sizes of the digested liver cDNA fragments for the various genotypes are shown. (C) Ethidium bromide-stained agarose gel showing the PCR-restriction enzyme fragmentation patterns for NdeI and BbsI in three beagles from which the cDNA libraries were constructed. The size of the molecular markers (MW) is shown on the left, and the sizes of the PCR-restriction fragments are shown on the right.

Genetic Tests to Discriminate between CYP2C21 and CYP2C41 Genomic DNAs. Figure 2, A and B, outline the strategy used for this genetic test. Two specific primers were used to amplify a 124-bp product from exon 7 of both the CYP2C21 and 2C41 gene. The forward and reverse primers were 5'-TGAAAGTCCAGGAAGAGATT-3' and 5'-ACAAGGTCAATGTATCTCTG-3', respectively. Only the underlined nucleotides do not match the CYP2C21 sequence. The PCR reaction involved an initial denaturing step of 94°C for 2 min, followed by 39 cycles of denaturing at 94°C for 20 sec, annealing at 53°C for 10 sec, and extension at 72°C for 10 sec with a final extension of 5 min. The 124-bp PCR fragment from the amplification of exon 7 is digested overnight at 37°C in separate reactions with NlaIV, which digests the fragment from CYP2C21 gene into two smaller fragments of 45 and 79 bp but does not digest CYP2C41, and AciI, which digests the fragment from CYP2C41 into fragments of 87 and 37 bp but does not digest that from CYP2C21. The reactions were electrophoresed on a 4% agarose gel.

View larger version (35K): [in this window] [in a new window]   Fig. 2.   (A) Strategy used to genotype canine CYP2C21 and 2C41 genomic DNA utilizing PCR amplification of exon 7 followed by NlaIV digestion for CYP2C21 and by AciI for CYP2C41. (B) The predicted sizes of the digested DNA fragments for the various genotypes are shown. (C) Ethidium bromide-stained agarose gel showing PCR-restriction enzyme fragmentation patterns from the DNA from 25 dogs of both mixed breeds and beagles by NlaIV and AciI. The sizes of the PCR-restriction fragments are on the right. The DNA from dogs A-S was from blood, and the DNA from dogs 11, 13, 14, 51, 52, and 54 was from liver. Lanes marked 2C21 and 2C41 represent plasmids preps from the two CYP2C cDNA clones from the canine cDNA libraries.

	Results and Discussion

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Canine cDNA Libraries. Using the 275-bp canine CYP2C21 cDNA PCR product as a probe, 24 clones were isolated from three cDNA libraries. These could be divided into two members of the CYP2C subfamily. The first was similar to CYP2C21 (DM1-1) reported by Uchida et al. (1990). This CYP2C was present in all three cDNA libraries. The longest insert (clone 5b) was 1.9 kilobase pairs long and encoded a polypeptide of 487 amino acid residues.

View larger version (73K): [in this window] [in a new window]   View larger version (77K): [in this window] [in a new window]   Fig. 3.   (A) Nucleotide and deduced amino acid sequence of CYP2C21. The amino acid sequence is shown in the top line. The nucleotides underlined were not reported by Uchida et al. (1990). (B) Nucleotide and deduced amino acid sequence of CYP2C41. Nucleotides are numbered from the start codon. The amino acid sequence is shown in the top line. The stop codon is starred. The heme binding region is underlined, and the cysteine residue that acts as the fifth ligand to heme iron is boxed. An arrow marks a site where CYP2C41 lacks a single amino acid compared with the human CYP2Cs.(B)

Genetic Tests. CYP2C41 was found only in the cDNA library from the one female beagle liver, not in the libraries from the two male beagle livers, suggesting that CYP2C41 could be either sex-specific or polymorphic in its expression. An RT-PCR-based restriction enzyme test was developed to discriminate between CYP2C21 and CYP2C41 mRNAs, and a second PCR test was developed to discriminate between the two canine CYP2Cs at the genomic level. Exons 4 to 8 of the two CYP2C cDNAs are amplified using common primers. The PCR products are digested first with NdeI, which digests CYP2C21 to fragments of 359 and 408 but does not digest CYP2C41. The PCR products are also digested separately with BbsI, which digests CYP2C41 but not CYP2C21 (fig. 1, A and B). Figure 1C shows the results of this cDNA genetic test from the livers of the three beagles used to construct the cDNA libraries. CYP2C41 was found only in dog no. 11, the female dog liver used to construct the cDNA library, although all three dogs contained CYP2C21. cDNA prepared from livers of six additional beagles (four females and two males) were tested and found to contain CYP2C21 but not CYP2C41 (data not shown), suggesting that the difference in the expression of CYP2C41 was not sex-dependent but represents a polymorphism.