In Vivo and in Vitro Induction of Cytochrome P450 Enzymes in Beagle Dogs

Abstract

ABSTRACT:The aim of this study was to determine the in vitro and in vivo effects of several prototypical inducers, namely ␤-naphthoflavone, 3-methylcholanthrene, phenobarbital, isoniazid, rifampin, and clofibric acid, on the expression of cytochrome P450 (P450) enzymes in beagle dogs. For the in vitro induction study, primary cultures of dog hepatocytes were treated with enzyme inducers for 3 days, after which microsomes were prepared and analyzed for P450 activities. For the in vivo induction study, male and female beagle dogs were treated with enzyme inducers for 4 days (with the exception of phenobarbital, which was given for 14 days), after which the livers were removed and microsomal P450 activities were determined ex vivo. Treatment of male beagle dog hepatocyte cultures (n ‫؍‬ 3) with ␤-naphthoflavone or 3-methlychloranthrene resulted in up to a 75-fold increase in microsomal 7-ethoxyresorufin O-dealkylase (CYP1A1/2) activity, whereas in vivo treatment of male and female beagle dogs with ␤-naphthoflavone followed by ex vivo analysis resulted in up to a 24-fold increase. Phenobarbital caused a 13-fold increase in 7-benzyloxyresorufin O-dealkylase (CYP2B11) activity in vitro and up to a 9.9-fold increase in vivo. Isoniazid had little or no effect on 4-nitrophenol hydroxylase activity in vitro. Rifampin caused a 13-fold induction of testosterone 6␤-hydroxylase (CYP3A12) activity in vitro and up to a 4.5-fold increase 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.

Polymorphic Expression of Cyp1a2 Leading to Interindividual Variability in Metabolism of a Novel Benzodiazepine Receptor Partial Inverse Agonist in Dogs

“…Remarkable differences were observed in the expression level of CYP1A-related protein between EM and PM dogs. In addition, the CYP1A-related protein detected by immunoblotting was possibly CYP1A2, because CYP1A2, but not CYP1A1, has been reported to be constitutively expressed in untreated dogs (Uchida et al, 1990;Graham et al, 2002). These results indicate that the decrease in CYP1A2 activity in PM dogs is caused by a decrease in the expression level of CYP1A2 protein and not by a decrease in its intrinsic activity.…”

“…Activities of EROD and POD, both of which reflect CYP1A2 activity (Chauret et al, 1997;Graham et al, 2002), and activities of ECOD and APND, both of which reflect CYP2B and/or CYP3A activity (Nishibe and Hirata, 1995;Mae et al, 1998;Nakata et al, 2000), were determined. A reaction mixture consisting of 50 mM phosphate buffer (pH 7.4), 0.8 mM NADPH, EM or PM dog liver microsomes, and a substrate [ethoxyresorufin (0.01 mM), phenacetin (0.1 mM), ethoxycoumarin (0.1 mM), and aminopyrine (5 mM)] was incubated at 37°C for 15 min.…”

Characterization of Substrate Specificity of Dog CYP1A2 Using CYP1A2-Deficient and Wild-Type Dog Liver Microsomes

“…Moreover, as the major metabolite of dog urine after administration of phenacetin is acetaminophen, an O-deethylated metabolite (Podder et al, 1988), dog CYP1A2 is the major P450 isoform involved in phenacetin metabolism in dogs. EROD activity is used as an in vitro CYP1A1/2 marker activity not only in humans but also in dogs (Graham et al, 2002). Although heterologously expressed dog CYP1A1 and CYP1A2 catalyzed EROD activity, the contribution of CYP1A2 in EROD activity in dog liver microsomes has been unknown (Gibson et al, 2005).…”

Induction of Cyp1a in the Beagle Dog by an Inhibitor of Kinase Insert Domain-Containing Receptor: Differential Effects in Vitro and in Vivo on Mrna and Functional Activity

“…The use of primary hepatocytes has shown great utility in the ability to model the induction of P450 enzymes in vitro (Silva et al, 1998;Silva and NicollGriffith, 2002). Both CYP1A1 and CYP1A2 mRNAs were detectable in the livers of untreated dogs, albeit at low levels, suggesting some degree of constitutive expression, and both enzymes appear to be highly inducible by polychlorinated biphenyls and BNF (Uchida et al, 1990;Graham et al, 2002). However, when varying concentrations of compound I were incubated in the presence of dog primary hepatocytes for 48 h, no functional or transcriptional induction of CYP1A1/2 was observed.…”

“…It has recently been shown that beagle dogs possess inducible forms of CYP1A, CYP2B, CYP2E, and CYP3A and that their profiles of induction resemble those in humans (Graham et al, 2002). The use of both fresh and cryopreserved primary dog hepatocytes has proven to be a valuable in vitro model in evaluating the ability of a drug candidate to induce the expression and activity of both phase I and II drug-metabolizing enzymes (Nishibe and Hirata, 1993;Harauchi and Hirata, 1994;Nishibe and Hirata, 1995;Hengstler et al, 2000;Lu and Li, 2001;Graham et al, 2002).…”