Short Communication Expression and Characterization of Dog Cytochrome P450 2A13 and 2A25 in Baculovirus-Infected Insect Cells

Dog CYP2A13 and CYP2A25 were coexpressed with dog NADPHcytochrome P450 reductase (OR) in baculovirus-infected Sf9 insect cells. CYP2A13 effectively catalyzed 7-ethoxycoumarin (7EC) deethylation and coumarin hydroxylation with apparent Km values of 4.8 and 2.1 M, respectively, similar to those observed using dog liver microsomes (7.5 and 0.75 M, respectively). CYP2A25 exhibited much lower affinity toward 7EC, with an apparent Km value of 150 M, which indicates that CYP2A13 plays a more significant role in the metabolism of these CYP2A substrates. Similar to the dog CYP1A2 enzyme, CYP2A13 efficiently catalyzed phenacetin deethylation with a Km value of 3.9 M, which suggests that phenacetin is not a selective probe for dog CYP1A2 activity. Both dog CYP2A13 and CYP2A25 exhibited little or no catalytic activity toward other common cytochrome P450 probe substrates, including bupropion, amodiaquine, diclofenac, S-mephenytoin, bufuralol, dextromethorphan, midazolam, and testosterone. These results provided additional information about the selectivity of these commonly used probe substrates. Dog is a commonly used species for the preclinical assessment of metabolism, pharmacokinetic, and toxicological testing of new drugs. At least 12 dog cytochrome P450 (P450) genes have been identified to date (Martignoni et al., 2006). Knowledge of dog P450 enzymes and their differences from P450 enzymes in human and other species is of great importance in drug development. Shou et al. (2003) have expressed and characterized seven dog P450 enzymes (CYP1A1, 2B11, 2C21, 2C41, 2D15, 3A12, and 3A26) in baculovirus-infected Sf21 insect cells. More recently, the same seven dog P450s have also been expressed in Escherichia coli with coexpression of dog NADPHcytochrome P450 reductase (OR) (Locuson et al., 2009). Dog CYP2A enzymes have not been reported in any cDNA-directed expression system. Drug-metabolizing capability and specificity of dog CYP2A enzymes have not been characterized as extensively as the human and rodent isoforms. Human CYP2A6 is mainly expressed in human liver and contributes 4% of total hepatic P450 (Martignoni et al., 2006), whereas human CYP2A13 is primarily expressed in the respiratory tract (Ling et al., 2007). Both dog CYP2A13 and CYP2A25 are present predominantly in liver tissues (Martignoni et al., 2006). Human CYP2A6 and CYP2A13 differ at 32 amino acid positions and exhibit different metabolic specificity. Carcinogenic epoxide metabolites of aflatoxin B1 were only produced by human CYP2A13 but not CYP2A6 (He et al., 2006). Human CYP2A6 exhibited little activity toward phenacetin, whereas human CYP2A13 metabolized phenacetin more efficiently than human CYP1A2 (Fukami et al., 2007). CYP2A isoforms in rats and mice have been reported to catalyze 7 and 15 testosterone hydroxylation, in addition to coumarin 7-hydroxylation (Martignoni et al., 2006). In contrast, dog liver microsomes catalyze coumarin 7-hydroxylation, but not 7 and 15 -testosterone hydroxylation, which is similar to that observed in human liver microsomes (Pearce et al., 1992). However, the enzyme(s) responsible for coumarin metabolism have not been clearly demonstrated in dog. In the present study, dog CYP2A13 and CYP2A25 were coexpressed with dog OR by using the baculovirus-insect cell system. The activities of these enzymes toward various P450 probe compounds were evaluated more broadly in this study. Materials and Methods Materials. Chemicals were obtained from Sigma-Aldrich (St. Louis, MO), Toronto Research Chemicals (Toronto, Canada), and Steraloids (Newport, RI). Polymerase chain reaction (PCR)-Ready First-Strand cDNA from female beagle dog normal liver was obtained from BioChain Institute, Inc. (Hayward, CA). Bac-to-Bac baculovirus expression system and Sf9 (Spodoptera frugiperda) insect cells, protein mini-gels, and XCell SureLock Mini-Cell with XCell II Blot Module Kit were obtained from Invitrogen (Carlsbad, CA). A BaculoELISA titer kit was purchased from Clontech (Mountain View, CA). CYP2A goat polyclonal antibody raised against a peptide mapping at the C terminus of CYP2A of human origin and donkey anti-goat IgG-horseradish peroxidase were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Isolation of cDNAs and Construction of Recombinant Baculoviruses. cDNAs for dog CYP2A13, CYP2A25, and OR were PCR-amplified from dog liver single-strand cDNA with PfuUltra High-Fidelity DNA polymerase (Agilent Technologies, La Jolla, CA) following the manufacturer’s protocols. Forward and reverse primers, respectively, were as follows: 5 -ATGTTGGCCTCAGGGCTTCTTCTG-3 and 5 -TCAGCGGGGCTGGAAGCTCATGGT-3 (CYP2A13); 5 -ATGGTGGCCTCAGGGATCCTTCTG-3 and 5 -TCAGCGGGGCTGGAAGCTCATGGT-3 (CYP2A25); and 5 -ATGGAGGACTCCAGTATGGATGCCA-3 and 5 -CTAGCTCCACACGTCCAGGGAGTAGCG-3 (OR). The PCR product was ligated This work was presented in part as follows: Zhou D, Linnenbach AJ, Luzietti R, Booth-Genthe C, and Grimm SW (2009) Characterization of heterologously expressed dog CYP1A2, CYP2A13, CYP2A25, CYP2B11, CYP2C21, CYP2C41, CYP2D15, CYP3A12 and CYP3A26 enzymes. 16th North American Regional ISSX Meeting; 2009 Oct 18–22; Baltimore, MD. International Society for the Study of Xenobiotics, Washington, DC. Article, publication date, and citation information can be found at http://dmd.aspetjournals.org. doi:10.1124/dmd.110.033068. ABBREVIATIONS: P450, cytochrome P450; OR, NADPH-cytochrome P450 reductase; PCR, polymerase chain reaction; 7EC, 7-ethoxycoumarin. 0090-9556/10/3807-1015–1018$20.00 DRUG METABOLISM AND DISPOSITION Vol. 38, No. 7 Copyright © 2010 by The American Society for Pharmacology and Experimental Therapeutics 33068/3598099 DMD 38:1015–1018, 2010 Printed in U.S.A. 1015 at A PE T Jornals on O cber 9, 2017 dm d.aspurnals.org D ow nladed from into pCR-Blunt and subcloned into restriction enzyme-digested and dephosphorylated pFastBac1. Clones and subclones were propagated in TOP10 E. coli cells. The cDNA sequences were determined by using BigDye Terminator version 3.1 Cycle Sequencing Kits (ABI, Foster City, CA). The cDNA sequences of CYP2A13 and CYP2A25 cloned in the present study were identical to GenBank reference sequences NM_001037345 and NM_001048027.1, respectively. Dog OR was cloned and deposited in GenBank (GU827421). In this study, two synonymous nucleotide differences (T256C and C1694T) were identified in the cloned dog OR compared with the GenBank reference sequence for dog OR (XM_844050.1). Baculovirus recombinants were obtained by using the Invitrogen Bac-toBac baculovirus expression system according to manufacturer’s protocols. In brief, E. coli DH10Bac competent cells that harbored bacmid vector DNA were transformed with pFastBacI plasmid recombinant DNAs via heat shock. Recombinant bacmid DNA was isolated and used to transform Sf9 insect cells via Cellfectin II-mediated gene transfer to generate recombinant baculovirus. Recombinant baculovirus-containing culture supernatants were harvested, amplified, and titered by using BaculoELISA kits. High-titer passage 3 stock was used for protein expression. Expression of Dog P450s with OR in Sf9 Insect Cells. Sf9 insect cell liquid cultures were grown at 27°C in Sf-900 II SFM medium (Invitrogen) to a cell density of 1 to 1.5 10 cells/ml. Cells were coinfected with virus encoding P450 and OR at a multiplicity of infection of 0.02 and 0.002, respectively. At 24-h postinfection, a hemin-albumin complex was added to achieve 1 g/ml hemin (Shou et al., 2003). Cells were harvested by centrifugation after 72-h infection. Microsomes were prepared by homogenization and 2-speed centrifugation (10,000 and 105,000g) and were reconstituted in phosphate-buffered saline (pH 7.4). Microsomal protein concentrations were measured by using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA). P450 content of dog recombinant CYP2A13 and CYP2A25 was measured by CO-difference spectrum (Omura and Sato, 1964). OR activities were measured by cytochrome c reduction assay (Yasukochi and Masters, 1976). Microsomes were stored at 70°C until use. Western Blots. The expression of recombinant dog CYP2A13 and CYP2A25 in microsomes isolated from baculovirus-infected insect cells was determined by electrophoresis under denaturing/reducing conditions and immunoblot analysis, according to the manufacturer’s specifications. In brief, protein (0.2 pmol of P450) was treated with NuPAGE (Invitrogen) sample reducing agent, separated on a 4 to 12% NuPAGE Bis-Tris mini-gel in NuPAGE MOPS SDS running buffer, and then electrotransferred to polyvinylidene difluoride membrane. Both upper gel running and transfer buffers contained NuPAGE antioxidant. The blot was blocked with phosphate-buffered saline containing 0.05% Tween 20 and nonfat dry milk and then sequentially incubated with goat polyclonal anti-CYP2A (1:200; Santa Cruz Biotechnology, Inc.) and horseradish peroxidase-conjugated donkey anti-goat IgG (1:10,000; Santa Cruz Biotechnology, Inc.). CYP2A protein was visualized by using luminol chemiluminescent reagent and Amersham Hyperfilm ECL (GE Healthcare, Chalfont St. Giles, UK). Pooled dog liver microsomes (10 g) and human recombinant CYP2A6 (0.2 pmol) were included as references. Microsomes (10 g) from uninfected Sf9 insect cells were used as a negative control. Enzyme Activity and Kinetics. Phenacetin, 7-ethoxycoumarin (7EC), coumarin, bupropion, amodiaquine, diclofenac, S-mephenytoin, bufuralol, dextromethorphan, midazolam, and testosterone were incubated at 50 M in the presence of 25 pmol/ml dog recombinant CYP2A13 or CYP2A25 for 30 min at 37°C in a final volume of 0.2 ml in duplicate. Nicotine was incubated under the same conditions as other compounds, and 1 mg/ml dog liver cytosol was added as an excess aldehyde oxidase source (Messina et al., 1997). The reaction mixtures contained 100 mM potassium phosphate (pH 7.4) and 5 mM MgCl2. Reactions were initiated with 1 mM NADPH and stopped with 0.2 ml of acetonitrile containing 200 nM -hydroxytriazolam (internal standard). Supernatants were analyzed by using Waters UPLC (Milford, MA) and Applied Biosystems/MDS Sciex API-4000 Q-Trap mass spectrometer (Foster City, CA) (Wang and Zhang, 2007; Zhou et al., 2009). To further evaluate dog recombinant CYP2A13 and CYP2A25 kinetics, reaction rates were measured under linear conditions using a number of substrates. Phenacetin (0.3 100 M), 7EC (0.01 100 M), and coumarin (0.3 100 M) were incubated with CYP2A13 (10 pmol/ml) for 10 min. 7EC (1 500 M) was incubated in the presence of CYP2A25 (25 pmol/ml) for 30 min. Phenacetin, 7EC, and coumarin were also incubated with 0.2 mg/ml dog liver microsomes for 15 min. Incubations with 7EC were performed in triplicates, whereas other substrates were performed in duplicates. Sample preparation and analysis were the same as described above. Kinetic Analysis. Enzyme kinetics for each metabolite was obtained by fitting the Michaelis-Menten equation [v Vmax [S]/(Km [S])] to experimental data using nonlinear regression (Prism 4; GraphPad Software Inc., San Diego, CA). Kinetic constants were reported as the mean S.E. of the parameter estimated. Results and Discussion The cDNA-directed expression of individual P450 enzymes has provided valuable tools for the assessment of enzyme selectivity toward certain compounds (Bjornsson et al., 2003). Baculovirus has been successfully used to express full-length human P450 cDNAs (Gonzalez and Korzekwa, 1995; Crespi and Penman, 1997). In the current study, dog CYP2A13 and CYP2A25 were cloned and coexpressed with dog OR in baculovirus-Sf9 insect cells, and enzyme activities toward commonly used human P450 probe compounds were evaluated. To our knowledge, this is the first report of the expression and substrate selectivity for dog CYP2A13 and CYP2A25. Protein Expression. Dog CYP2A13 and CYP2A25 were coexpressed with dog OR in Sf9 insect cells. Dog CYP2A13 and CYP2A25 exhibited P450 content of 40 and 48 pmol/mg microsomal protein and OR activity of 46 and 30 nmol/min/mg protein, respectively. Expression was also confirmed by immunoblot analysis using antiCYP2A antibody (Fig. 1). Human CYP2A6 and dog liver microsomes were used as control test systems. Microsomes prepared from uninfected Sf9 cells exhibited no cross-reactivity to the anti-CYP2A antibody. Activity Characterization and Kinetic Analysis. Initially, a set of known human P450 probe substrates were incubated with dog recombinant CYP2A13 and CYP2A25 to evaluate substrate selectivity. CYP2A13 catalyzed 7EC deethylation, phenacetin deethylation, coumarin hydroxylation, and nicotine oxidation. Under the same conditions, CYP2A25 catalyzed 7EC deethylation (2.1 pmol/min/pmol) and showed relatively low activities toward phenacetin, coumarin, and nicotine metabolism with 0.01, 0.025, and 0.17 pmol/min/pmol, respectively (Fig. 2A). CYP2A13 exhibited high affinity (Km 4.8 M) and high capacity (Vmax 47 pmol/min/pmol), whereas CYP2A25 demonstrated low affinity (Km 150 M) and low capacity (Vmax 8.4 pmol/min/pmol) toward 7EC deethylation (Table 1). The 7EC deethylation catalyzed by CYP2A13 was found to be 170-fold more efficient than that by CYP2A25. Dog liver FIG. 1. Western blot analysis of CYP2A13 and CYP2A25 expression in microsomes prepared from baculovirus-infected Sf9 insect cells. Dog liver microsomes (DLM) and human CYP2A6 were used as positive controls. Microsomes from uninfected insect cells were used as a negative control. Lanes were loaded with 0.2 pmol of CYP2A13, CYP2A25, or CYP2A6 or 10 g of DLM or microsomes from uninfected insect cells. 1016 ZHOU ET AL. at A PE T Jornals on O cber 9, 2017 dm d.aspurnals.org D ow nladed from microsomes showed an apparent Km of 7.5 M, similar to that calculated for CYP2A13. Coumarin 7-hydroxylation by dog liver microsomes and dog recombinant CYP2A13 both exhibited oneenzyme Michaelis-Menten kinetics, with apparent Km values of 0.75 and 2.1 M, respectively. These results suggest dog CYP2A13 plays a more significant role in the metabolism of these CYP2A substrates in dog liver. CYP2A13 exhibited high affinity (Km 3.9 M) and low capacity (Vmax 3.6 pmol/min/pmol) for phenacetin deethylation (Table 1). In our previous report (Zhou et al., 2009), dog CYP1A2 exhibited relative low affinity (Km 12.3 M) and high capacity (Vmax 13.9 pmol/min/pmol). The intrinsic clearance (Vmax/Km) of phenacetin deethylation from this study was comparable for dog CYP2A13 (0.92 l/min/pmol) and dog CYP1A2 (1.1 l/min/pmol). Similar results were reported for human CYP2A13 enzyme, which catalyzed phenacetin more efficiently than human CYP1A2 (Fukami et al., 2007). Human CYP2A13 is mainly expressed in the respiratory tract (Ling et al., 2007), whereas dog CYP2A13 is predominantly present in liver tissue (Martignoni et al., 2006). For this reason, phenacetin should not be used as a selective probe drug for dog CYP1A2 enzyme activity in vitro or in vivo. There was no measurable dextrorphan or 1 -hydroxybufuralol metabolite formation in CYP2A13 or CYP2A25 incubations (Fig. 2A). Both bufuralol and dextromethorphan are well known human CYP2D6 substrates (Bjornsson et al., 2003) and were reported as dog CYP2D15-selective substrates (Shou et al., 2003). The present study provides additional information to confirm their selectivity. Both CYP2A13 and CYP2A25 catalyzed metabolism of bupropion, amodiaquine, diclofenac, and S-mephenytoin at a low rate ( 0.13 pmol/ min/pmol) at 50 M substrate concentration. Diclofenac has been reported to be metabolized by dog CYP2B11 and CYP2C21 efficiently (Shou et al., 2003). CYP2A13 and CYP2A25 would be minor contributors for the metabolism of diclofenac in dog liver microsomes. CYP2A13 metabolized midazolam to 1 -hydroxylation and 4-hydroxylation metabolite at rates of 0.11 and 0.28 pmol/min/pmol, respectively (Fig. 2A), whereas CYP2A25 exhibited much lower catalytic capability toward midazolam. Both enzymes would be minor contributors for the metabolism of midazolam in dog liver microsomes compared with other dog P450s, such as CYP3A12 (Locuson et al., 2009). Dog CYP2A13 and CYP2A25 exhibited low catalytic activities in hydroxylating testosterone at multiple sites (Fig. 2B). No measurable 7 -hydroxylation was observed in either CYP2A13 or CYP2A25 incubations, which is consistent with previous observations (Pearce et al., 1992). The overall contribution of CYP2A13 and CYP2A25 to the metabolism of testosterone is likely to be insignificant in dog liver microsomes. Dog CYP2A13, dog CYP2A25, human CYP2A6, and human CYP2A13 retain more than 85% amino acid sequence identity. Dog CYP2A13 and CYP2A25 differ only at 34 of 494 amino acids and FIG. 2. Metabolite formation of phenacetin, 7-ethoxycoumarin, coumarin, nicotine, bupropion, amodiaquine, diclofenac, S-mephenytoin, bufuralol, dextromethorphan, and midazolam (A) or testosterone (B) at 50 M substrate concentration in the presence of 25 pmol/ml dog recombinant CYP2A13 or CYP2A25. Data are presented as average of duplicate incubations, and the variation between duplicate incubations was within 10% of each other in all cases.