Abstract
Cytochrome P450 (CYP) 2E1 is a toxicologically important enzyme that inactivates a number of drugs and xenobiotics and also bioactivates many xenobiotic substrates to their hepatotoxic or carcinogenic forms. Although cDNAs for the human, rodent, and rabbit forms of CYP2E1 have been isolated and studied extensively, there is an absence of information about canine CYP2E1, despite the fact that the dog is routinely used in drug safety studies. In this study, we isolated and sequenced a full-length CYP2E1 cDNA from a beagle liver cDNA library. The deduced canine CYP2E1 amino acid sequence exhibited 75 to 76% identity with rat, mouse, and rabbit CYP2E1 sequences, and 77% identity with human CYP2E1. Two populations of clones, differing at a single nucleotide, were isolated from the unamplified library. The T1453C base change results in a Tyr485His amino acid substitution, which is well beyond the heme binding region but is possibly part of a β-sheet structure. An allele-specific polymerase chain reaction-based restriction enzyme test was developed for genotyping individual dogs from genomic DNA samples. One hundred mixed breed dogs were genotyped, and the frequencies of the Tyr485 and His485 alleles were found to be 0.85 and 0.15, respectively. The canine Tyr485 and His485 alleles and human CYP2E1 were expressed inEscherichia coli cells, and catalytic activities of the proteins were assessed using the substrate chlorzoxazone. Although the two canine enzymes had similar catalytic activity; significant kinetic differences were seen between canine and human CYP2E1s.
The cytochromes P450 (CYPs)2 are a superfamily of enzymes that play an important role in the oxidative metabolism of a wide variety of xenobiotics as well as endogenous compounds, such as steroids, arachidonic acid, and fatty acids. Members of this superfamily of enzymes are classified into families and subfamilies based on their amino acid sequence identities. With the development of molecular biological techniques over the past 20 years, the cDNAs of individual CYP members from numerous species, including humans, have been cloned and expressed in a variety of heterologous expression systems. Such studies have greatly increased our understanding concerning the role specific CYP enzymes play in the metabolism of specific substrates, the mechanisms involved in differences in drug metabolism between species, and the mechanisms underlying polymorphic differences in drug metabolism within a species.
In contrast to the large number of CYP cDNA clones that have been isolated, sequenced, and extensively studied from laboratory rodents, rabbits, and humans, the number of canine CYP cDNAs is limited. Because the dog is extensively used in pharmaceutical research as well as in drug safety assessment studies, information concerning specific canine CYP enzymes will be valuable in understanding their metabolic role in relation to that of other species. At present, the canine CYP enzymes involved in xenobiotic metabolism that have been cloned and sequenced include: 1) two members of the CYP1A subfamily (CYP1A1 and 1A2) (Uchida et al., 1990); 2) a member of the CYP2B subfamily (2B11) (Graves et al., 1990); 3) two members of the CYP2C subfamily (2C21 and 2C41) (Uchida et al., 1990; Blaisdell et al., 1998); 4) one member of the CYP2D subfamily (2D15) (Sakamoto et al., 1995); and 5) two members of the CYP3A subfamily (3A12 and 3A26) (Ciaccio et al., 1991; Fraser et al., 1997). Some of these canine cDNA clones have been heterologously expressed. One important CYP subfamily, CYP2E, has not been cloned from the dog, although it has been cloned and extensively studied from humans, laboratory rodents, and rabbits.
CYP2E is a constitutively expressed CYP that is induced by ethanol (Koop et al., 1982), by fasting (Hong et al., 1987), and during diabetes (Dong et al., 1988). These latter two conditions implicate CYP2E as having important roles in the intermediary metabolism of endogenous ketones and fatty acids. In most species studied, CYP2E is present as a single isoform, CYP2E1. In rabbits, however, a second highly similar isoform, CYP2E2, has also been identified (Khani et al., 1988). A number of therapeutic agents, including acetaminophen (Morgan et al., 1983), chlorzoxazone (Peter et al., 1990), theophylline (Zhang and Kaminsky, 1995), ethanol (Morgan et al., 1982), and the anesthetic agents enflurane (Thummel et al., 1993) and halothane (Gruenke et al., 1988), have been shown to be substrates for CYP2E. In addition to participating in the metabolism of therapeutic agents, CYP2E also plays a significant role in the metabolic activation of numerous precarcinogens such as benzene (Koop et al., 1989), nitrosamines (Yamazaki et al., 1992) and hepatotoxins such as acetaminophen (Hu et al., 1993), ethanol, and halogenated hydrocarbons (Olson et al., 1991), presumably through the production of free radicals.
The major objective of this study was to isolate, clone, and sequence members of the canine CYP2E subfamily from a canine liver cDNA library, and to compare the catalytic activities of heterologously expressed canine and human CYP2Es.
Materials and Methods
Chemicals and Reagents.
All restriction enzymes were obtained from New England Biolabs (Beverly, MA). δ-Aminolevulinic acid, isopropyl β-d-thiogalactopyranoside, NADPH, dilauroyll-α-phosphatidylcholine, ampicillin, cholate, phenylmethylsufonyl fluoride, Nonidet P40, and 4-methylpyrazole (4-MP) were obtained from Sigma (St. Louis, MO). Hydroxyapatite was purchased from Clarkson Chemical Company (Williamsport, PA). Recombinant human NADPH-CYP reductase and cytochromeb5 were obtained from Oxford Biomedical Research, Inc. (Oxford, MI). Chlorzoxazone was obtained from Aldrich Chemical Company (Milwaukee, WI), and 6-hydroxychlorzoxazone was purchased from Research Biochemicals International (Natick, MA). Other chemicals used were of the highest grade commercially available.
Tissue and Blood.
Frozen liver tissue from an untreated female beagle dog, generously supplied by M. Faletto and C. J. Serabjit-Singh at Glaxo-Wellcome (Research Triangle Park, NC), was used for the cDNA library. Canine whole blood samples from mixed breed dogs were obtained from the College of Veterinary Medicine at North Carolina State University, from a local animal hospital, and from a local animal shelter (Institutional Animal Care and Use Committee approval number 98-100-B).
RNA Isolation, cDNA Library, and Screening.
RNA was extracted from the female beagle liver by a guanidine isothiocyanate-phenol/chloroform method (Chomczynski and Sacchi, 1987). Poly (A+) RNA was isolated using an oligo(dT) cellulose column (Life Technologies, Gaithersburg, MD). A ZAP cDNA synthesis kit (Stratagene, La Jolla, CA) was used to construct an oligo(dT)-primed cDNA library from the mRNA. Approximately 2 × 105recombinant phage plaques from the unamplified library were screened on nitrocellulose filters by plaque hybridization. The probe was a CYP2E1 polymerase chain reaction (PCR) product made from a canine liver cDNA template using PCR primer pairs designed from two highly homologous regions in rat and human CYP2E1 cDNAs, in exons 2 and 6. The forward and reverse primers were 5′-CTTCGGGCCAGTGTTCAC-3′ and 5′-CCCATATCTCAGAGTTGTGC-3′, respectively. The PCR reaction mix contained 1× PCR buffer, 1.5 mM MgCl2, 0.2 mM dNTPs, 0.25 μM each primer, and 2 U Taq DNA polymerase (Qiagen, Valencia, CA) in a final volume of 20 μl. PCR, performed on a Perkin-Elmer 4800 thermal cycler (PE Applied Biosystems, Foster City, CA), consisted of an initial 5-min denaturation at 95°C followed by 32 cycles of: 95°C for 1 min., 45°C for 1 min., and 72°C for 1 min., with a final 5 min. extension at 72°C. The resulting 744 base pair (bp) product was isolated on a 1.5% ethidium bromide-stained agarose gel, gel-purified with a Micropure 0.22/Microcon 30 kit (Amicon, Beverly, MA), and labeled with 32P dCTP by random primer labeling (Life Technologies, Gaithersburg, MD). Hybridization was performed at 59°C, and filters were washed with 2× sodium chloride/sodium citrate buffer containing 0.1% SDS. The resulting clones were plaque-purified by three rounds of screening. pBluescript phagemids were excised from the Uni-ZAP XR vector using ExAssist helper phage and the SOLR strain of Escherichia coli.
Sequencing.
Phagemid DNA was isolated using a S.N.A.P. miniprep kit (Invitrogen, Carlsbad, CA). The cloned cDNA was sequenced in both directions by primer walking starting with modified T3 and T7 primers. Cycle sequencing using dye-labeled dideoxynucleotides was performed using an ABI PRISM cycle sequencing kit with AmpliTaqRestriciton Fragment Length Polymorphism DNA polymerase, FS (PE Applied Biosystems, Foster City, CA). Samples were autosequenced on an ABI model 310 Genetic Analyzer (PE Applied Biosystems). The Genetic Computer Group program (Madison, WI) was used for cDNA and protein sequence analyses.
Genotyping.
Genomic DNA for genotyping was extracted from 200 μl of whole blood with a Qiagen blood kit according to the manufacturer's directions.
Allele-Specific PCR Mismatch-Restriction Fragment Length Polymorphism Test.
Primers were designed to amplify a 118-bp product of theCYP2E1 gene to differentiate between two CYP2E1alleles found in this study. The forward and reverse primers were 5′-TAACCTGAAGTCTCTCGTCG-3′ (anneals at bases 1374–1393) and 5′-GGGTCTTCAGCCCGAGCGGGGAACGACACAGAGTTTTT-3′ (anneals at bases 1454–1491), respectively. The underlined nucleotide in the reverse primer represents a mismatch with the CYP2E1sequence. Genomic DNA (1–5 μl) was added to a PCR reaction mix containing 1× PCR buffer, 1.5 mM MgCl2, 0.2 mM dNTPs, 0.25 μM each primer, 5% dimethyl sulfoxide, and 2 UTaq DNA polymerase in a final volume of 20 μl. PCR, performed on a GeneAmp 2400 PCR System (PE Applied Biosystems), included an initial 5-min denaturation at 95°C, followed by 35 cycles of: 95°C for 20 s, 64°C for 10 s, and 72°C for 20 s, with a final 5-min extension at 72°C. The PCR product was digested overnight at 37°C with 8 U MseI in a 50-μl volume containing 1× NE buffer 2 and 100 μg/ml of BSA. After digestion, the reaction volume was reduced to 15 μl on a Speed Vac, loading buffer was added, and the entire sample was electrophoresed on a 4% agarose gel stained with ethidium bromide in 1× TBE buffer. Figure1 includes a schematic of the PCR-based restriction enzyme test for the two CYP2E1 alleles.
PCR Sequencing of Two CYP2E1 Alleles.
PCR was performed to amplify a 309-bp product of the CYP2E1gene. The forward and reverse primers were 5′-CTCGCATGGAGCTCTTCC-3′ (anneals at bases 1328–1345) and 5′-AAAGCAGCATCTCAGGACC-3′ (anneals at bases 1618–1636), respectively. Two microliters of genomic DNA was added to PCR reaction mix containing 1 × PCR buffer, 1.5 mM MgCl2, 0.2 mM dNTPs, 0.25 μM each primer, and 2 U Taq DNA polymerase in a final volume of 20 μl. PCR, performed on a GeneAmp 2400 PCR System, included an initial 5-min denaturation at 95°C, followed by 35 cycles of: 95°C for 20 s, 64°C for 10 s, and 72°C for 20 s, with a final 5-min extension at 72°C. Fifteen microliters of the reaction mix was electrophoresed on an ethidium bromide-stained 2% agarose gel in 1 × TBE buffer. The 309-bp band was cut from the gel and the DNA was extracted with a QIAEX II Gel Extraction kit (Qiagen). The PCR product was sequenced, in both directions, using dye-labeled dideoxynucleotides in an ABI PRISM cycle sequencing kit with AmpliTaq DNA polymerase, FS. The forward and reverse sequencing primers were 5′-TAACCTGAAGTCTCTCGTCG-3′ (anneals at bases 1374–1393) and 5′-TCCACCAGACTGTGCAGGG-3′ (anneals at bases 1570–1588), respectively. Samples were autosequenced on a ABI model 373 Genetic Analyzer (PE Applied Biosystems).
Construction of Expression Plasmids.
The N-termini of the canine CYP2E1 cDNAs were modified by replacing the first eight codons with the corresponding N-terminal codons of the bovine 17α-hydroxylase sequence. A PCR product was amplified using a forward primer designed with a XbaI restriction site 5 bases from the 5′-end followed by a NdeI restriction site, the modified N-terminal sequence, and an additional 19 bases of CYP2E1 sequence at the 3′-end. The forward primer for the bovine 17α-hydroxylase (B17α-H) modification is shown in Fig.2, the reverse primer was 5′-AGGTACAAGGTGAACACTGG-3′. The 229-bp PCR product, digested withXbaI and BglII, yielded a 83-bp fragment that was used to replace the corresponding N-terminal fragment of the CYP2E1s in Bluescript. Subsequently, the plasmid was digested withEco0109I (a restriction site 13-bp after the stop codon), blunt ended, and then digested with NdeI. The resulting 1499-bp insert was ligated into NdeI and blunt endedXbaI sites of the pCW vector.
A second expression construct with N-terminal modification similar to that reported by Gilliam et al. (1994) was made. The initial 21 codons of the canine CYP2E1 cDNA were deleted and the following two codons were replaced with an ATG and GCT (coding for Met and Ala). The first ten codons were then made A/T rich by conservative substitution. A PCR primer was designed with an NdeI restriction site eight bases from the 5′-end, followed by the modified CYP2E1 sequence and 19 bp of unmodified CYP2E1 sequence. The forward primer for this modification (Pho Del) is shown in Fig. 2, the reverse primer was 5′-TACCGTATCTCTCCTTCTGC-3′. The 762-bp PCR product, amplified from the canine CYP2E-Bluescript template, was digested with NdeI andBlpI, and the resultant 541-bp fragment was ligated into NdeI and BlpI sites of the above CYP2E1 constructs containing the bovine 17α-hydroxylase N-terminal modification. All PCR fragments were sequenced to ensure that no mutations had been introduced. The human CYP2E1 cDNA with this modification was provided by Dr. F. P. Guengerich (Vanderbilt University, Nashville, TN).
Protein Expression and Purification.
E. coli strain DH5α was transformed with the modified CYP2E1s in the pCW vector. Single colonies, selected on Luria-Bertani (LB)-ampicillin plates, were used to seed 10 ml of LB broth containing 100 ug/ml ampicillin and then cultured overnight at 37°C. The overnight cultures were diluted 100-fold with Terrific broth containing 0.2% bacto-peptone, 200 ug/ml ampicillin, and 0.5 mM δ-aminolevulinic acid, and were cultured at 37°C with vigorous shaking for 2 h. The cultures were brought to room temperature, 1 mM isopropyl β-d-thiogalactopyranoside was added, and the cultures were incubated at 30°C with shaking (150 rpm) for 48 h. CYP expression was monitored daily with a SLM Aminco DW-2000 split-beam spectrophotometer.
All steps of the CYP purification procedure, based on that ofRichardson et al., (1995), were performed at 4°C. Cells from the 1-liter cultures were pelleted (4000g for 15 min) and the pellets were suspended in suspension buffer (20 mM KPO4, pH 7.25, 100 mM KCl, 1 mM EDTA, and 1 mM dithiothreitol) containing 1 mM phenylmethylsufonyl fluoride and 50 μM 4-MP. The cells were sonicated (20-s bursts at 40-s intervals, 25 times) in an ice bath, and the resulting cell suspension was centrifuged at 100,000g for 1 h at 4°C. Cell membrane pellets were homogenized in resuspension buffer (10 mM KPO4, pH 7.4, 0.1 mM EDTA, 20% glycerol, and 1 mM dithiothreitol) containing 50 μM 4-MP, 0.3% Nonidet P40 was added, and the samples were gently rocked for 1 h to solubilize CYP. The supernatant fraction obtained by centrifugation at 100,000g for 1 h was applied to a hydroxylapatite column that had been prewashed with column wash buffer (10 mM KPO4, pH 7.4, 20% glycerol). The column was washed with 10 column volumes of resuspension buffer containing 50 μM 4-MP, and the CYP was then eluted with elution buffer (0.5 M KPO4, pH 7.4, 0.1 mM EDTA, 20% glycerol, and 0.1% cholate) containing 50 μM 4-MP. Fractions containing high concentrations of CYP were pooled and dialyzed against dialysis buffer (0.1 M KPO4, pH 7.4, 0.1 mM EDTA, and 20% glycerol) for 48 h.
Enzymatic Assays.
All samples were kept on ice except where otherwise noted. Recombinant CYP2E1s (50 pmol of canine CYP2E1 or 25 pmol of human CYP2E1) were preincubated with recombinant human NADPH-CYP reductase (reductase/CYP molar ratio, 4:1), rabbit cytochromeb5 (cytochromeb5/CYP molar ratio, 2:1), and dilauroylphosphatidylcholine (15 μg/ml, final concentration) for 3 min in a 37°C shaking water bath. After preincubation, 200 μl of reaction buffer (250 mM KPO4, pH 7.4, 3.75 mM MgCl2, and 0.25 mM EDTA) and chlorzoxazone (final concentration 0.0038–1 mM for human samples, 0.02–1 mM for canine samples) dissolved in fresh 60 mM KOH were added, and the total reaction volume was brought to 495 μl with water. After incubation in a 37°C shaking water bath for 4 min, reactions were initiated with the addition of NADPH (0.5 mM, final concentration). After a 5-min incubation, the reactions were quenched with the addition of 25 μl of 43% o-phosphoric acid.
The samples were extracted three times with 2 ml of 85% chloroform/15% 2-propanol, and the organic fractions were pooled and dried for HPLC analysis. The HPLC mobile phase consisted of 30% acetonitrile/70% 0.5% H3PO4 delivered at a flow rate of 1 ml/min. Separation of chlorzoxazone and 6OH-chlorzoxazone was achieved with a 250 × 4.6 mm Spherisorb 5 ODS(2) column (Phenomenex, Torrance, CA). Column eluate was monitored with a Perkin-Elmer LC135 ultraviolet detector (Perkin-Elmer Instruments, Norwalk, CT) set at 290 nm. The residues were dissolved in 75 μl of HPLC mobile phase, and the entire sample was injected into the HPLC system.
Results
cDNA Sequencing.
A number of CYP2E1 cDNA clones were isolated from a canine liver cDNA library, sized, and three were completely sequenced. The canine sequences were aligned with rat and human cDNA and gene sequences. Based on these alignments, the open reading frame was defined for the canine sequence. The assigned initiation ATG is in a strong context for translation initiation with an A residue at the −3 position and a G residue at the +4 position (Kozak, 1996). Two of the cloned inserts contained a complete 1485-bp open reading frame that codes for a polypeptide 494 amino acids in length. The two full-length clones differed from one another at position 1453, estimated to reside in exon 9 based on comparison with rat and human gene sequences, and at position 1719 in the poly-A tail. The T1453C change codes for a Tyr485His amino acid substitution. To determine whether this discrepancy in the coding region was a result of the cloning process, an additional 33 clones from the unamplified library were sequenced across this region, and two populations of clones, which differed at this position, were found. A third cDNA insert had an early stop codon due to a 1-bp deletion at position 1157 of the coding region. Fourteen clones were sequenced over this region but no other clones with this deletion were detected. The canine CYP2E1 cDNA and deduced protein sequence are shown in Fig. 3. Amino acid sequence identity and similarity comparisons between canine, human, rodent, and rabbit forms of CYP2E1 are summarized in Table1.
Allele Frequency Determination.
We designed an allele-specific PCR-based restriction enzyme test so that individual dogs could be readily genotyped for theTyr485His alleles from genomic DNA samples. The restriction enzyme maps of the two alleles differ at a single BccI site, which is present in theHis485 allele but not in theTyr485 allele. Because BccI is not commercially available, we created a MseI restriction site in the Tyr485 allele by designing a PCR primer with a 1-bp mismatch. A 118-bp product was amplified from exon 9 ofCYP2E1 using the mismatch reverse primer and was then digested with MseI, which digests the PCR product of theTyr485 allele into two fragments, 39 and 79 bp, but does not digest the PCR product of theHis485 allele. The predicted sizes of the fragments are shown in Fig. 1, A and B. Genomic DNA from dogs with each of the three possible genotypes was subjected to the PCR-restriction enzyme test; representative results are shown in Fig. 1C. The enzyme test results were confirmed in a number of individuals by sequencing a PCR product amplified from exon 9. One hundred mixed breed dogs were genotyped. The Tyr485 allele occurred at a frequency of 0.85 and the His485 allele at a frequency of 0.15. Thirteen purebred beagles were genotyped; the frequencies of the Tyr485 andHis485 alleles were determined to be 0.81 and 0.19, respectively.
Protein Expression and Characterization.
To determine whether the genetic polymorphism in CYP2E1 altered catalytic activity of the enzyme, we expressed N-terminally modified canine CYP2E1 Tyr485 andHis485 cDNAs in E. coli using the pCW vector. Replacement of the first eight codons of the CYP2E1 cDNA with those of modified bovine 17 α-hydroxylase resulted in very low expression. In an attempt to increase expression levels, we subsequently made N-terminal modifications as described by Gilliam et al. (1994). The first 63 bases encoding the N-terminal hydrophobic region were removed and the next two codons were altered to encode a methionine and an alanine. Additionally, silent mutations were made in the next 24 bases to enrich adenine and thymine content. Expression levels of up to 300 nmol P450 per liter of culture were obtained from canine cDNAs with this modification. A similarly modified human CYP2E1 cDNA, obtained from Dr. F. P. Guengerich (Vanderbilt University) was also expressed. The expressed and purified canine and human recombinant CYP2E1s were catalytically active in the presence of cytochrome b5 as demonstrated by their ability to 6-hydroxylate chlorzoxazone. In further experimentsKm and Vmaxvalues of the expressed and purified canine and human recombinant CYP2E1s were determined using the substrate chlorzoxazone. The kinetic parameters, determined by the direct linear plot method, of the canine Tyr485 and His485 variants and the human ortholog are compared in Table2.
Discussion
This is the first report of a canine CYP2E1 cDNA. The canine CYP2E1 amino acid sequence, deduced from the full-length cDNA, has a hydrophobic amino terminus as well as the conserved putative heme binding domain (Graham-Lorence and Peterson, 1996) (amino acid position 430–443) characteristic of the CYP proteins. The amino acid sequence of canine CYP2E1 has greater than 75% identity to human, rodent, and rabbit CYP2E1 sequences, and is one amino acid longer than the human, rodent, and rabbit orthologs. When the canine amino acid sequence is compared with sequences of the other CYP2 family members, identity is less than 54% and less than 34% when compared with the other CYP families. The canine CYP2E1 amino acid sequence has a higher identity with human CYP2E1 (77%) than with rodent or rabbit CYP2E1 (75–76%). However, the human CYP2E1 amino acid sequence has greater identity to rabbit and rodent CYP2E1 (78–79%) than to canine CYP2E1 (77%).
One hundred mixed breed dogs were genotyped and the results were analyzed. The Tyr485 allele occurred at a higher frequency (0.85) than the His485 allele (0.15). A test for Hardy-Weinberg equilibrium (HWE) shows a significant deviation from equilibrium (P = .031) with a higher proportion of heterozygotes than would be expected. This could be explained if some of the animals tested were first generation mixed breed and the parental breeds involved differed in allele frequency at the CYP2E1 locus. However, beagles, a breed used in drug testing studies, also varied in the incidence of these alleles.
The canine T1453C point mutation results in a Tyr485His amino acid substitution. Although this amino acid residue is beyond the putative heme-binding region, it is part of a β sheet structure in close proximity to substrate recognition site 6 as predicted by Gotoh (1992). Interestingly, the amino acid at position 485 of CYP2E1 is occupied by either a tyrosine or a phenylalanine in all species sequenced to date (human, rat, mouse, rabbit, hamster, macaque, pig,3 and cow,4) (Song et al., 1986; Khani et al., 1988; Freeman et al., 1992; Komori et al., 1992;Sakuma et al., 1994), which suggests that the presence of an aromatic residue at this position could have an important function.
Heterologous expression levels of the canine CYP2E1 alleles were highest when the cDNAs were modified by removing the first N-terminal hydrophobic segment (Nelson and Strobel, 1988) codons. Previous studies have shown that removal of this portion of the protein does not affect subcellular localization of CYP2E1 expressed in E. coli(Pernecky and Coon, 1996), nor does it affect catalytic activity of the heterologously expressed protein (Gilliam et al., 1994; Pernecky and Coon, 1996). The CYP2E1 ligand 4-MP was added to the buffers used for protein purification to stabilize the expressed enzyme. The expressed and purified canine and human recombinant CYP2E1s, in the presence of cytochrome b5, were shown to be catalytically active toward the prototypic substrate chlorzoxazone. No significant difference in Km orVmax was seen when the two allelic variants were compared by univariate ANOVAs. TheVmax value of the canine and human CYP2E1 were not significantly different from each other; however, theKm value of human CYP2E1 was significantly lower (P = .003) than that of canine CYP2E1. As a consequence, the intrinsic clearance (Vmax/Km ratio) of canine CYP2E1 is 4-fold lower than that of human CYP2E1, and the clearance of this substrate in vivo might be predicted to be lower in dogs than in humans. This study indicates that canine CYP2E1 has a lower affinity for chlorzoxazone than human CYP2E1.
Several polymorphisms of human CYP2E1 have also been identified, most of them occurring in noncoding regions of the gene. Single nucleotide polymorphisms in the 5′-flanking region include aPstI site (G-1259C) (Watanabe et al., 1990; Hayashi et al., 1991) and an RsaI site (C-1019 T) (Watanabe et al., 1990; Hayashi et al., 1991), as well as polymorphisms at positionsA-316G (Fairbrother et al., 1998),T-297A (Fairbrother et al., 1998), andG-35T (Hu et al., 1997). TheC-1019T variant, located within a putative binding site for the transcription factor hepatocyte nuclear factor 1, has been shown to exhibit higher in vitro transcriptional activity in a CAT construct compared with wild type (Hayashi et al., 1991). However, in vivo studies suggest no relationship between this polymorphism and chlorzoxazone 6-hydroxylase activity in a Caucasian population (Kim et al., 1995). The G-35T variant, located 9 bp upstream from the TATA box, showed increased transcriptional activity in vitro using luciferase constructs (Fairbrother et al., 1998).McCarver et al. (1998) identified a 100-bp insertion mutation in a region from −2270 to −1672 associated with increased in vivo chlorzoxazone metabolism only among individuals who were obese or had recently consumed alcohol. Polymorphisms in several introns have also been identified.
Four variant alleles have been identified in the coding region of humanCYP2E1, G1168A(Arg76His) in exon 2 (Hu et al., 1997),G4804A (Val179Ile) in exon 4 (Fairbrother et al., 1998), G10059A (Val389Ile) in exon 8 (Hu et al., 1997), andC10157T in exon 8 (silent) (Fairbrother et al., 1998). In vitro COS-1 cell expression studies indicate that theG1168A and the G10059Avariants encode proteins with normal catalytic activity. However, the G1168A variant protein had decreased stability compared with wild type CYP2E1 (Hu et al., 1997) and might therefore be expressed at different levels in vivo. Lymphoblastoid cell expression studies have shown that catalytic activity of the protein encoded by the G4804A variant is similar to that of wild type CYP2E1 (Fairbrother et al., 1998).
This study reports the full-length sequence of canine CYP2E1 cDNA. A variant allele of canine CYP2E1 has also been identified, and allele frequencies were determined in a population of dogs. In addition, a genetic test for the alleles has been developed so that individual dogs may be genotyped from blood samples before their use in drug safety assessment studies or studies in dogs of the metabolism of newly developed drugs. The cDNAs of the two canine variants along with the human CYP2E1 cDNA were heterologously expressed in a bacterial expression system, and the recombinant proteins were purified. The canine CYP2E1 variants had similar catalytic activity toward the prototype substrate chlorzoxazone but their affinities and intrinsic clearances for chlorzoxazone were lower than that of human CYP2E1, suggesting that the canine CYP2E1 is less efficient in metabolizing this substrate than human CYP2E1. These differences in canine and human CYP2E1 may affect predictions of clearance of CYP2E1 substrates in the canine model.
Acknowledgments
We are grateful to M. Faletto and C. J. Serabjit-Singh from Glaxo-Wellcome (Research Triangle Park, NC) for supplying the beagle liver tissue sample, to Dr. Betsy Sigmond (Apex, NC) for supplying mixed breed dog blood samples, and to Dr. F. P. Guengerich (Vanderbilt University, Nashville, TN) for the modified human CYP2E1 clone. We also thank Joyce Blaisdell for her valuable technical advice and Dr. Richard W. Morris, Analytical Sciences, Inc. (Research Triangle Park, NC) and Marlina D. Nasution (North Carolina State University, Raleigh, NC) for performing the statistical analyses.
Footnotes
-
Send reprint requests to: Susan M. Lankford, Department of Anatomy, Physiological Sciences and Radiology, North Carolina State University College of Veterinary Medicine, 4700 Hillsborough St., Raleigh, NC 27606. E-mail: susan-lankford{at}ncsu.edu
-
↵1 The nucleic acid sequences in this paper have been submitted to GenBank under accession numbers AFO29978 and AFO29979.
- Abbreviations used are::
- CYP
- cytochrome P450
- bp
- base pair(s)
- 4-MP
- 4-methylpyrazole
- PCR
- polymerase chain reaction
- Received February 3, 2000.
- Accepted May 10, 2000.
- The American Society for Pharmacology and Experimental Therapeutics