Introduction

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Enzyme induction enables some xenobiotics to accelerate their own biotransformation (auto-induction) or the biotransformation and elimination of other drugs. Drugs and new molecular entities are often screened for their ability to induce P450³ and other phase I and phase II enzymes with the aim of predicting or explaining drug-drug interactions in humans, and increases in liver weight and/or proliferation of the endoplasmic reticulum or peroxisomes, pharmacokinetic tolerance, and/or formation of liver and thyroid tumors in rodents. Enzyme induction is often evaluated by an ex vivo procedure whereby the xenobiotic is administered to rats and mice (or other laboratory animals) in vivo, followed by an evaluation of changes in the levels of liver microsomal P450 and other enzymes in vitro (Parkinson, 2001).

A number of P450 enzymes in human and/or rodent liver microsomes are inducible, including various members of the CYP1A, CYP2A, CYP2B, CYP2C, CYP2E, CYP3A, and CYP4A subfamilies (Parkinson, 2001). To date, eight P450 genes have been sequenced in the dog (compared with up to 60 sequenced genes in the rat) including CYP1A1, CYP1A2, CYP2B11, CYP2C21, CYP2C41, CYP2D15, CYP3A12, and CYP3A26 (http://www.icgeb.trieste.it/~P450srv/genesperspecies.html). However, for a species that is widely used by the pharmaceutical industry to assess the safety of drugs under development, relatively little information is available in the literature on the inducibility of P450 enzymes in the dog. Several studies have been performed in which a particular P450 cDNA has been cloned and the sequence compared with related enzymes in other species (Ohta et al., 1989; Graves et al., 1990; Ciaccio et al., 1991; Sakamoto et al., 1995; Fraser et al., 1997; Roussel et al., 1998; Lankford et al., 2000). However, much remains to be elucidated about the structure, function, and regulation of dog P450 enzymes. Two CYP1A enzymes, P450 D2 and P450 D3, have been purified from the liver of polychlorinated biphenyl-treated female beagle dogs (Ohta et al., 1989). Catalytic and structural properties of both proteins were shown to be similar to rat CYP1A2, although P450 D3 exhibits spectral properties similar to those of rat CYP1A1. It is not known whether either of these proteins is constitutively expressed in dog liver (Ohta et al., 1989).

Dog liver microsomes contain CYP2B11 (also called PBD-2), a constitutively expressed and phenobarbital-inducible enzyme with high metabolic activity toward 2,4,5,2',4',5'-hexachlorobiphenyl (Duignan et al., 1988). Like the rat CYP2B1, dog CYP2B11 (PDB-2) catalyzes both the 16- and 16-hydroxylation of testosterone. Another testosterone 16-hydroxylase has been purified from dog liver and identified as a member of the CYP2C subfamily (Uchida et al., 1990). Whereas rat CYP2B1 catalyzes the 16- and 16-hydroxylation of testosterone at roughly equal rates, dog CYP2B11 preferentially catalyzes the 16-hydroxylation of testosterone at 13 to 15 times the rate of testosterone 16-hydroxylation (Coulter et al., 1993; Ohmori et al., 1993). Another CYP2 protein, CYP2D15, has been cloned by Sakamoto et al. (1995). Recently, CYP2E1 cDNA has been cloned from beagle dogs followed by characterization and expression of the encoded protein (Lankford et al., 2000). Interestingly, the amino acid sequence of dog CYP2E1 exhibits 77% identity to the human ortholog, which is slightly higher than the identity to the rodent or rabbit sequence (75-76%). Characterization of the expressed CYP2E1 protein indicated that dog CYP2E1 has a lower affinity for chlorzoxazone than does human CYP2E1 (Lankford et al., 2000).

Dog liver is thought to express multiple forms of CYP3A, as has been shown in rat and human. PBD-1, a CYP3A enzyme, was purified from phenobarbital-treated dog liver but also appears to be expressed constitutively (Ciaccio and Halpert, 1989). Molecular and immunochemical analyses indicate the presence of at least one other CYP3A enzyme in dog liver (Ciaccio and Halpert, 1989; Ciaccio et al., 1991). In contrast to rats, there are no marked sex differences in CYP3A activity in dog liver microsomes. Like the corresponding rat enzyme, the dog CYP3A enzyme, CYP3A12 catalyzes the 6-hydroxylation of testosterone (Ciaccio and Halpert, 1989). In addition, CYP3A12 catalyzes the 16-hydroxylation of testosterone, which is also catalyzed in part by CYP2B11. Recently, a cDNA encoding a protein exhibiting 95.6% amino acid identity with CYP3A12 was isolated from phenobarbital-induced dogs (Fraser et al., 1997). This enzyme, called CYP3A26, is not as prominent as CYP3A12 in hydroxylating steroids.

A CYP4A protein has yet to be identified in dog liver (Adas et al., 1999). DUT-1, purified from liver microsomes of untreated male beagle dogs, catalyzes the 12-hydroxylation of lauric acid, but the N-terminal sequence of this protein is different from any other P450 characterized to date (Shiraga et al., 1994).

Due to the lack of information on the inducibility of P450 enzymes in the dog, we examined the induction profile of several P450 enzymes in cultured male dog hepatocytes (in vitro) and in microsomes prepared from the livers of male and female dogs that were treated with prototypical P450 inducers (in vivo). These inducers included -naphthoflavone, 3-methylcholanthrene, phenobarbital, isoniazid, rifampin, and clofibric acid. In each case, several marker substrate reactions and Western immunoblotting were used to assess P450 enzyme induction.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results and Discussion Conclusion References

Chemicals and Reagents. Insulin, Dulbecco's modified Eagle's medium, GlutaMAX-1 (dipeptide L-alanyl-L-glutamine 200 mM supplied in 0.85% NaCl), modified Chee's medium, minimal essential medium nonessential amino acids, and penicillin-streptomycin were purchased from Invitrogen (Carlsbad, CA). Matrigel and ITS⁺ (insulin, transferrin, selenium) were purchased from Collaborative Biomedical Products (Bedford, MA). Collagenase (type I) was purchased from Worthington Biochemicals (Freehold, NJ). Vitrogen 100 was purchased from Celtrix (Santa Clara, CA). Androstenedione, L-arginine, bovine serum albumin, clofibric acid, dexamethasone, DMSO, fetal bovine serum, EGTA, D-(+)-glucose, glucose 6-phosphate, glucose-6-phosphate dehydrogenase, L-glutamine, 11-hydroxytestosterone, lauric acid, NADP, -naphthoflavone, 4-nitrophenol, 4-nitrocatechol, Percoll, phenobarbital, testosterone, thymidine, and trypan blue were purchased from Sigma-Aldrich (St. Louis, MO). Bicinchoninic acid protein assay reagents were purchased as a kit from Pierce Chemical Co. (Rockford, IL). NuPage gels and related electrophoresis reagents were purchased from Novex (San Diego, CA). Polyvinylidene difluoride membranes were purchased from Bio-Rad (Hercules, CA). BCIP/NBT phosphatase substrate was purchased from Kirkegaard and Perry Laboratories (Gaithersburg, MD). 7-Ethoxyresorufin, 7-benzyloxyresorufin, and resorufin were purchased from Molecular Probes Inc. (Junction City, OR). [¹⁴C]Lauric acid (58 Ci/mol) was purchased from ICN Radiochemicals (Irvine, CA). 6-Hydroxytestosterone and 16-hydroxytestosterone were purchased from Steraloids, Inc. (Wilton, NH). Solvents were purchased either from Fisher Scientific (Pittsburgh, PA) or Aldrich Chemical Co. (Milwaukee, WI).

Hepatocyte Isolation and Culture. Three male beagle dogs (Covance Research Products, Inc., Cumberland, VA) were euthanized, and the livers were perfused by a modification of the previously described two-step collagenase digestion method (Seglen, 1976; Seglen et al., 1980; Quistorff et al., 1989; LeCluyse et al., 1996, Madan et al., 1999). Hepatocytes were maintained in culture for 3 days before treatment with P450 inducers. On day 4, the medium was aspirated and replaced with 3 ml of supplemented modified Chee's medium containing vehicle or the inducer. Cultures were treated daily for 3 consecutive days with either vehicle (0.1% DMSO or 0.1% saline), -naphthoflavone (33 µM), 3-methylcholanthrene (10 µM), phenobarbital (250 µM), isoniazid (100 µM), rifampin (50 µM), or clofibric acid (100 µM). (Prototypical inducers were dissolved in DMSO, except isoniazid, which was dissolved in saline.) At the end of the treatment period, the hepatocytes were harvested, and microsomes were prepared as described previously (Madan et al., 1999). The microsomal samples were stored at 80°C for later analysis of P450 activities. The protein concentration in the microsomal samples was determined with a BCA Protein Assay Kit, according to Technical Bulletin 23225X from Pierce Chemical Co. (Smith et al., 1985; Wiechelman et al., 1988).

Treatment of Dogs in Vivo and Preparation of Microsomes. Male and female beagle dogs (7-18 months old; Ridglan Farms, Mt. Horeb, WI) were treated by subcutaneous injection with corn oil vehicle (two males and two females), saline vehicle (two males and two females), 10 mg/kg/day -naphthoflavone (two males and one female), 10 mg/kg/day rifampin (two males and two females), 10 mg/kg/day clofibric acid (two males and one female), or phenobarbital (two males and two females). Animals treated with phenobarbital were treated for 14 consecutive days with dosing escalations from 10 mg/kg/day (days 0 and 1) to 20 mg/kg/day (days 2 through 5) to 30 mg/kg/day (days 6 through 13). Dogs treated with vehicle or enzyme inducers (other than phenobarbital) were treated for 4 consecutive days. After completion of the dosing regimen, the dogs were euthanized by intravenous injection with sodium pentobarbital (5 ml per dog) followed by excision of the livers, which were subsequently perfused with chilled saline. After perfusion, the livers were snap-frozen in liquid nitrogen and stored at 70°C. Microsomes were prepared as described previously (Pearce et al., 1996). This experimental design was reviewed and approved by the Institutional Animal Care and Use Committee of WIL Research Laboratories, Inc. (Ashland, OH).

Enzyme Assays. The O-dealkylation of 7-ethoxyresorufin and 7-benzyloxyresorufin, the 6-, 16-, and 16-hydroxylation of testosterone, the 4-hydroxylation of nitrophenol, and the 12-hydroxylation of lauric acid were determined by methods described previously (Burke and Mayer, 1974; Wood et al., 1983; Koop, 1986; Sonderfan et al., 1987; Romano et al., 1988; Sonderfan and Parkinson, 1988; Giera and Van Lier, 1991; Tierney et al., 1992; Burke et al., 1994; Pearce et al., 1996). The incubation conditions for each of the assays are given in Table 1.

Western Immunoblotting. Microsomal samples were analyzed by Western immunoblotting to determine levels of immunoreactive CYP1A, 2B, 3A, and 4A. Microsomes were subjected to SDS-polyacrylamide gel electrophoresis, based on the method originally described by Laemmli (1970). Briefly, microsomes were mixed in a 1:1 ratio with NuPage sample dilution buffer (pH = 8.5) containing 1.09 M glycerol, 141 mM Tris-base, 106 mM Tris-HCl, 73 mM SDS, 0.51 mM EDTA, 0.22 mM Serva Blue G250, and 0.175 mM phenol red and heated at 100°C for 2 to 5 min. The denatured proteins (up to 10 µg per lane, as specified in figure legends) were subjected to electrophoresis on precast 4 to 12% NuPage bis-Tris gels (pH 6.4 gels; constant voltage of 200 V; electrophoresis time ~55 min) (Novex). Proteins were transferred electrophoretically to polyvinylidene difluoride membranes and subjected to immunoblotting, based on the method by Towbin et al. (1979), with a Blot Module from Novex. Membranes were incubated in blocking buffer containing 10% (w/v) Carnation nonfat dry milk and 0.05% (v/v) Tween 20 in Tris-buffered saline (10 mM Tris-HCl and 150 mM NaCl, pH = 7.4) and then probed with polyclonal antibodies raised against purified rat liver microsomal CYP1A1, CYP2B1, CYP3A1 (Parkinson and Gemzik, 1991), or CYP4A (Affinity Bioreagents, Golden, CO) at final concentrations ranging from 0.25 µg/ml to 10 µg/ml. The secondary antibody was affinity-purified goat-anti-rabbit IgG (H + L) conjugated with alkaline phosphatase from Kirkegaard and Perry Laboratories, which was diluted in blocking buffer to a final concentration of 0.25 µg/ml. Membranes were washed three times with Tris-buffered saline, and the proteins were visualized by incubation with BCIP/NBT phosphatase substrate.

Statistical Analysis. For analysis of microsomes from the in vitro cultures, data are mean ± standard deviation of three preparations. An asterisk (*) indicates a statistically significant (p < 0.05) difference from control as determined by a one-way repeated measures analysis of variance test followed by a Dunnett's post hoc test. For ex vivo analysis of microsomes, data are duplicate measurements of a pool of microsomes from two dog livers (with the exception of the -naphthoflavone and clofibric acid females, in which case, only a single dog liver was available).

	Results and Discussion

Top Abstract Introduction Materials and Methods Results and Discussion Conclusion References

There are several reports on the cloning and sequencing of cDNAs encoding dog P450 enzymes, and on the characterization of the catalytic activity of dog P450 enzymes (Gee et al., 1984; Duignan et al., 1987; Ciaccio and Halpert, 1989; Ciaccio et al., 1991; Graves et al., 1990; Nicolas et al., 1995; Sakamoto et al., 1995; Ekins et al., 1996; Fraser et al., 1997; Nishibe et al., 1998; Roussel et al., 1998; Adas et al., 1999; Lankford et al., 2000; Hewitt et al., 2001). Several studies have shown that a select compound can cause induction of one or more P450 enzymes in dog (McKillop and Pickup, 1991; Robertson et al., 1995; Nishibe and Hirata, 1995; McKillop et al., 1998; Mae et al., 1998). However, little information is available on the profile of P450 enzymes induced by prototypical P450 enzyme inducers, namely, those that cause CYP1A, CYP2B, CYP2E, CYP3A, or CYP4A induction in other mammalian species (McKillop 1985; Nishibe and Hirata, 1993; Nishibe and Hirata, 1995; Nishibe et al., 1998). Along with enzyme induction, a large number of chemically diverse compounds have been shown to cause hepatic hyperplasia and proliferation of the endoplasmic reticulum and peroxisomes in rodents. Such compounds also cause formation liver tumors in rodents after chronic administration (Reddy and Rao, 1977; Reddy and Qureshi, 1979; Fitzgerald et al., 1981). On the other hand, nonrodent species, including dog, have been reported to be much less sensitive to peroxisome proliferation (Foxworthy et al., 1990).

Evaluation of Dog Hepatocytes. Viability of the final preparation of hepatocytes (after Percoll gradient centrifugation) was greater than 70% for each of the three preparations of dog hepatocytes. Dog hepatocytes attached to collagen-coated culture dishes. After 6 days in culture, representative culture dishes seeded with freshly isolated hepatocytes were photographed under light microscopy (Fig. 1). Hepatocytes exhibited morphology traits consistent with normal cells: the cells were cuboidal and contained granular cytoplasm with one or two centrally located nuclei (Fig. 1). Interestingly, the cellular morphology of the cultured dog hepatocytes closely resembled that of cultured human hepatocytes but not rat hepatocytes (LeCluyse et al., 1999, 2000). Unlike rat hepatocyte cultures, dog hepatocyte cultures tended to be completely confluent, covering nearly 100% of the culture dish. Even though the collagen substratum and Matrigel overlay caused cells to spread and flatten to a certain degree, the hepatocytes retained a high degree of three-dimensional architecture.

View larger version (181K): [in this window] [in a new window]   Fig. 1.   Photomicrograph of primary cultures of dog hepatocytes treated with 0.1% DMSO (vehicle control). Freshly isolated dog hepatocytes were isolated and cultured for 6 days as described under Materials and Methods. On day 6, the cultures were photographed under a phase contrast microscope. Magnification factor = 300×.

Induction of Cytochrome P450 Content in Dogs in Vivo. The effects of treating male and female dogs in vivo with -naphthoflavone, phenobarbital, rifampin, or clofibric acid on P450 content are shown in Table 1. Treatment with -naphthoflavone resulted in up to a 1.5-fold increase in P450 content, whereas treatment with phenobarbital or rifampin resulted in up to 2.0- and 3.1-fold increases, respectively. On the other hand, treatment with clofibric acid resulted in up to a 32% decrease in P450 content. The determination of P450 content requires relatively large amounts of microsomal protein (approximately 1 mg per assay); therefore, this assay was not performed with microsomal samples from cultured hepatocytes.

Induction of EROD (CYP1A1/2) Activity. Although EROD activity has not been shown to be specific for dog CYP1A as it has for the rat, there is indirect evidence suggesting that dog CYP1A catalyzes EROD. McKillop (1985) reported that EROD activity increases 3- to 5-fold in dogs treated with the CYP1A inducer -naphthoflavone but not with the CYP2B inducer phenobarbital. CYP1A1 and CYP1A2 mRNA are expressed at low levels in the liver of untreated dogs, but the levels increase after treatment with the polychlorinated biphenyl mixture, Kaneclor KC-500 (Uchida et al., 1990). Treatment of cultured dog hepatocytes with -naphthoflavone has been shown to increase EROD activity by 25-fold (Nishibe and Hirata, 1993).

View larger version (25K): [in this window] [in a new window]   Fig. 2.   The effect of treating dog hepatocytes or subcutaneous injection of dogs with prototypical P450 inducers on 7-ethoxyresorufin O-dealkylase (CYP1A) activity. Freshly isolated dog hepatocytes were isolated and cultured for up to 6 days, and beagle dogs were treated by subcutaneous injection as described under Materials and Methods. A, in vitro cultures treated with prototypical inducers. B, male and female beagle dogs were treated with prototypical inducers. C, microsomes were analyzed by Western immunoblotting. Gels were loaded with 10 µg per lane (except for Rat BNF samples, which were 0.05, 0.10, and 0.20 µg per lane for ex vivo samples and 0.1 µg per lane for in vitro samples). Samples of rat liver microsomes prepared from rats treated with one of four prototypical inducers were also analyzed as reference standards. Asterisk (*) indicates p < 0.05 as described under Materials and Methods. UT, untreated; BNF, -naphthoflavone; 3-MC, 3-methylcholanthrene; PB, phenobarbital; RIF, rifampin; INH, isoniazid; CLOF, clofibric acid; CO, corn oil.

Induction of BROD (CYP2B11) Activity. 7-Benzyloxyresorufin O-dealkylation has been shown to be a specific substrate for dog CYP2B11 (Klekotka and Halpert, 1995). Like the corresponding rat enzyme, dog CYP2B11 (PDB-2) catalyzes both the 16- and 16-hydroxylation of testosterone; however, dog CYP2B11 preferentially catalyzes the 16-hydroxylation of testosterone at 13 to 15 times the rate of testosterone 16-hydroxylation (Coulter et al., 1993; Ohmori et al., 1993).

View larger version (25K): [in this window] [in a new window]   Fig. 3.   The effect of treating dog hepatocytes or subcutaneous injection of dogs with prototypical P450 inducers on 7-benzyloxyresorufin O-dealkylase (CYP2B) activity. Freshly isolated dog hepatocytes were isolated and cultured for up to 6 days, and beagle dogs were treated by subcutaneous injection as described under Materials and Methods. A, in vitro cultures treated with prototypical inducers. B, male and female beagle dogs were treated with prototypical inducers. C, microsomes were analyzed by Western immunoblotting. Gels were loaded with 10 µg per lane (except for Rat PB samples, which were 0.05, 0.10, and 0.20 µg per lane for ex vivo samples and 0.1 µg per lane for in vitro samples). Samples of rat liver microsomes prepared from rats treated with one of four prototypical inducers were also analyzed as reference standards. Asterisk (*) indicates p < 0.05 as described under Materials and Methods. UT, untreated; BNF, -naphthoflavone; 3-MC, 3-methylcholanthrene; PB, phenobarbital; RIF, rifampin; INH, isoniazid; CLOF, clofibric acid; CO, corn oil.

Induction of 4-Nitrophenol Hydroxylase (CYP2E1) Activity. Lankford et al. (2000) have isolated and characterized a full-length CYP2E1 cDNA from a beagle liver cDNA library. The deduced amino acid sequence shares 77% identity to rat, rabbit, and human CYP2E1. In rodents, CYP2E1 catalyzes the hydroxylation of 4-nitrophenol (Koop, 1986). Treatment of cultured hepatocytes with the prototypical enzyme inducers had little or no effect on 4-nitrophenol hydroxylase activity (Fig. 4). Induction of CYP2E1 was also measured by chlorzoxazone 6-hydroxylase activity, a measure of human CYP2E1 activity. In agreement with the data shown in Fig. 4 for 4-nitrophenol hydroxylation, treatment with prototypical P450 inducers had little or no effect on chlorzoxazone 6-hydroxylase activity (data not shown). Western immunoblotting for detection of the CYP2E1 isozyme was not performed. Consistent with our results, Jayyosi et al. (1996) reported that treatment of dogs with isoniazid had only a slight effect on chlorzoxazone 6-hydroxylase activity and immunoreactive CYP2E1 levels.

View larger version (15K): [in this window] [in a new window]   Fig. 4.   The effect of treating dog hepatocytes with prototypical P450 inducers on 4-nitrophenol hydroxylase (CYP2E) activity. Freshly isolated dog hepatocytes were isolated and cultured for up to 6 days as described under Materials and Methods. Cultures were treated with prototypical P450 enzyme inducers and analyzed for CYP2E activity as described under Materials and Methods. BNF, -naphthoflavone; 3-MC, 3-methylcholanthrene; PB, phenobarbital; RIF, rifampin; INH, isoniazid; CLOF, clofibric acid.

Induction of Testosterone 6-Hydroxylase (CYP3A12) Activity. Treatment of cultured hepatocytes with phenobarbital or rifampin resulted in a 7.3- and 13-fold induction of testosterone 6-hydroxylase (CYP3A12) activity, respectively, whereas the other inducers examined had little or no effect (Fig. 5A). Western immunoblotting confirmed that treatment of cultured hepatocytes with phenobarbital and rifampin, but not -naphthoflavone or 3-methylcholanthrene, isoniazid, or clofibric acid, caused a marked increase in immunoreactive CYP3A12 (Fig. 5C). It should be noted that the induced levels of CYP3A12 on the Western immunoblot indicated a level of protein expression comparable with that observed in microsomes prepared from untreated male beagle dog, an observation that is consistent with the testosterone 6-hydroxylase results (Fig. 5, A and B). These data also suggest a marked difference in the degree to which CYP3A12 is expressed in vitro versus ex vivo.

View larger version (25K): [in this window] [in a new window]   Fig. 5.   The effect of treating dog hepatocytes or subcutaneous injection of dogs with prototypical P450 inducers on testosterone 6-hydroxylase (CYP3A) activity. Freshly isolated dog hepatocytes were isolated and cultured for up to 6 days, and beagle dogs were treated by subcutaneous injection as described under Materials and Methods. A, in vitro cultures treated with prototypical inducers. B, male and female beagle dogs were treated with prototypical inducers. C, microsomes were analyzed by Western immunoblotting. Gels were loaded with 10 µg per lane (except for Rat DEX samples, which were 0.05, 0.10, and 0.20 µg per lane for ex vivo samples and 0.1 µg per lane for in vitro samples). Samples of rat liver microsomes prepared from rats treated with one of four prototypical inducers were also analyzed as reference standards. Asterisk (*) indicates p < 0.05 as described under Materials and Methods. UT, untreated; BNF, -naphthoflavone; 3-MC, 3-methylcholanthrene; PB, phenobarbital; RIF, rifampin; INH, isoniazid; CLOF, clofibric acid; CO, corn oil; DEX, dexamethasone.

Induction of Lauric Acid 12-Hydroxylase (CYP4A) Activity. Treatment of freshly isolated hepatocytes with clofibric acid had little or no effect on lauric acid 12-hydroxyase activity (CYP4A); however, phenobarbital and rifampin appeared to increase this activity by 2-fold (Fig. 6A). Treatment of dogs in vivo with the prototypical inducers examined had little or no effect on lauric acid 12-hydroxylase activity (Fig. 6B). The lack of CYP4A induction by clofibric acid was confirmed by Western immunoblotting (Fig. 6C); however, the 2-fold increase in activity by phenobarbital and rifampin was not associated with a 2-fold increase in CYP4A levels. As in other cases, the rate of lauric acid 12-hydroxylation was substantially lower in vitro compared with that observed ex vivo.

View larger version (26K): [in this window] [in a new window]   Fig. 6.   The effect of treating dog hepatocytes or subcutaneous injection of dogs with prototypical P450 inducers on lauric acid 12-hydroxylase (CYP4A) activity. Freshly isolated dog hepatocytes were isolated and cultured for up to 6 days, and beagle dogs were treated by subcutaneous injection as described under Materials and Methods. A, in vitro cultures treated with prototypical inducers. B, male and female beagle dogs were treated with prototypical inducers. C, microsomes were analyzed by Western immunoblotting. Gels were loaded with 10 µg per lane (except for Rat CLOF samples, which were 0.05, 0.10, and 0.20 µg per lane for ex vivo samples and 0.1 µg per lane for in vitro samples). Samples of rat liver microsomes prepared from rats treated with one of four prototypical inducers were also analyzed as reference standards. Asterisk (*) indicates p < 0.05 as described under Materials and Methods. UT, untreated; BNF, -naphthoflavone; 3-MC, 3-methylcholanthrene; PB, phenobarbital; RIF, rifampin; INH, isoniazid; CLOF, clofibric acid; CO, corn oil.

	Conclusion

Top Abstract Introduction Materials and Methods Results and Discussion Conclusion References

In the present study, we attempted to evaluate the effects of prototypical P450 enzyme inducers on the in vivo and in vitro expression of P450 enzymes in dogs. -Naphthoflavone or 3-methlychloranthrene resulted in a large increase in microsomal 7-ethoxyresorufin O-dealkylase (CYP1A1/2) activity in vitro and in vivo. Phenobarbital caused up to a 13-fold increase in 7-benzyloxyresorufin O-dealkylase (CYP2B11) activity in vitro and in vivo. Isoniazid had little or no effect on 4-nitrophenol hydroxylase activity in vitro. Rifampin caused up to a 13-fold induction of testosterone 6-hydroxylase (CYP3A12) activity in vitro and in vivo. Treatment of dogs in vivo or dog hepatocytes in vitro with clofibric acid appeared to have no effect on CYP4A activity as determined by the 12-hydroxylation of lauric acid. In general, the absolute rates (picomoles per minute per milligram of microsomal protein) of P450 reactions catalyzed by microsomes from cultured hepatocytes (i.e., in vitro rates) were considerably lower than those catalyzed by microsomes from dog liver (i.e., ex vivo rates). These results suggest that beagle dogs have CYP1A, CYP2B, CYP2E, and CYP3A enzymes and that the induction profile resembles the profile observed in humans more than in rats.