Enzyme and plasma protein induction by multiple oral administrations of phenobarbital at a therapeutic dosage regimen in dogs

In dogs effects of phenobarbital (PB) on hepatic cytochrome P450 (CYP) activities and on concentrations of plasma alpha 1-acid glycoprotein (AGP) were examined. Total body clearance (Cl(B)) of antipyrine and plasma AGP concentrations were monitored during oral PB treatment at a therapeutic dose for 35 days. Cl(B) of antipyrine, which reflects hepatic CYP activities, gradually increased and was maintained at about threefold concentrations compared with that before treatment, suggesting that PB induced CYP activities at a large extent even in a therapeutic dose, necessary for an antiepileptic effect. Plasma AGP concentrations also increased significantly (about fourfold). Dogs were killed at the 35th day of the PB treatment, and hepatic CYP content and enzyme kinetics of several CYPs were determined using liver microsomes. CYP content was about twofold higher than that from untreated dogs. The V(max) values for CYP1A-like activity (ethoxyresorufin O-deethylation), 2B-like activity (ethoxycoumarin O-deethylation), 2C-like activity (tolbutamide hydroxylation) and 3A-like activity (midazolam 4-hydroxylation) were higher (2-4-fold) than that in untreated dogs. In summary, a therapeutic dose of PB for antiepileptic therapy significantly induced hepatic CYPs and plasma AGP in dogs. Therefore, during antiepileptic therapy with PB, special attention must be paid to the pharmacokinetics of drugs simultaneously administered.     

In vitro characterization of the inhibitory effects of ketoconazole on metabolic activities of cytochrome P-450 in canine hepatic microsomes

OBJECTIVE: To evaluate the inhibitory potency of ketoconazole (KTZ) on the metabolic activities of isozymes of cytochrome P-450 (CYP) in dogs. ANIMALS: 4 healthy 1-year-old male Beagles. PROCEDURE: Hepatic microsomes were harvested from 4 dogs after euthanasia. To investigate the effects of KTZ on CYP metabolic activities, 7-ethoxyresorufin, tolbutamide, bufuralol, and midazolam hydrochloride were used as specific substrates for CYP1A1/2, CYP2C21, CYP2D15, and CYP3A12, respectively. The concentrations of metabolites formed by CYP were measured by high-performance liquid chromatography, except for the resorufin concentrations that were measured by a fluorometric method. The reaction velocity-substrate concentration data were analyzed to obtain kinetic variables, including maximum reaction velocity, Michaelis-Menten constant, and inhibitory constant (Ki). RESULTS: KTZ competitively inhibited 7-ethoxyresorufin O-deethylation and midazolam 4-hydroxylation; it noncompetitively inhibited tolbutamide methylhydroxylation. Bufuralol 1'-hydroxylation was inhibited slightly by KTZ. The mean Ki values of KTZ were 10.6+/-6.0, 170+/-2.5, and 0.180+/-0.131 microM for 7-ethoxyresorufin O-deethylation, tolbutamide methylhydroxylation, and midazolam 4-hydroxylation, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: In dogs, KTZ at a therapeutic dose may change the pharmacokinetics of CYP3A12 substrates as a result of inhibition of their biotransformation. Furthermore, no influence of KTZ on the pharmacokinetics of CYP1A1/2, CYP2C21, and CYP2D15 substrates are likely. In clinical practice, adverse drug effects may develop when KTZ is administered concomitantly with a drug that is primarily metabolized by CYP3A12.     

Characterization of equine cytochrome P450: role of CYP3A in the metabolism of diazepam

Research on drug metabolism and pharmacokinetics in large animal species including the horse is scarce because of the challenges in conducting in vivo studies. The metabolic reactions catalyzed by cytochrome P450s (CYPs) are central to drug pharmacokinetics. This study elucidated the characteristics of equine CYPs using diazepam (DZP) as a model compound as this drug is widely used as an anesthetic and sedative in horses, and is principally metabolized by CYPs. Diazepam metabolic activities were measured in vitro using horse and rat liver microsomes to clarify the species differences in enzyme kinetic parameters of each metabolite (temazepam [TMZ], nordiazepam [NDZ], p‐hydroxydiazepam [p‐OH‐DZP], and oxazepam [OXZ]). In both species microsomes, TMZ was the major metabolite, but the formation rate of p‐OH‐DZP was significantly less in the horse. Inhibition assays with a CYP‐specific inhibitors and antibody suggested that CYP3A was the main enzyme responsible for DZP metabolism in horse. Four recombinant equine CYP3A isoforms expressed in Cos‐7 cells showed that CYP3A96, CYP3A94, and CYP3A89 were important for TMZ formation, whereas CYP3A97 exhibited more limited activity. Phylogenetic analysis suggested diversification of CYP3As in each mammalian order. Further study is needed to elucidate functional characteristics of each equine CYP3A isoform for effective use of diazepam in horses.     

Characterization of cytochrome P450-mediated drug metabolism in cats

In this study we examined activities of cytochrome P450 (CYP)1A, 2C, 2D and 3A using hepatic microsomes from five male and five female cats. CYP1A, 2C, 2D and 3A activities were referred by ethoxyresorufin O-deethylation (EROD), tolbutamide hydroxylation (TBH), bufuralol 1'-hydroxylation (BLH) and midazolam 1'- and 4-hydroxylation respectively. The anti-rat CYP1A2 and CYP3A2 serum significantly inhibited EROD and midazolam 1'- and 4-hydroxylation, suggesting that EROD and midazolam 1'- and 4-hydroxylation were catalysed by CYP1A and 3A in cats respectively. Quinidine inhibited BLH in cats microsomes at quite low concentrations, suggesting that BLH was catalysed by CYP2D in cats. Tolbutamide hydroxylation activities were negligible in hepatic microsomes from both male and female cats, suggesting CYP2C activities of cats are extremely low. This suggests that CYP2C substrates should be carefully administered to cats. Although there is no sexual difference in CYP1A activities, there are differences in CYP2D and 3A activities of cats. CYP2D activities were higher (3-fold), but CYP3A activities were lower (one-fifth) in female cats. These results might suggest that CYP2D and 3A substrates should be prescribed for male and female cats using different dosage regimen.     

Comparison of in vitro activities of biotransformation enzymes in pig, cattle, goat and sheep

In vitro activities of cytochromes P450 (7-alkyl/aryloxyresorufin dealkyl(aryl)ases, testosterone hydroxylase/oxidase, 6-chlorzoxazone hydroxylase, 7-methoxy-4-trifluoromethyl-coumarin demethylase, and lauric acid hydroxylases), reductases of carbonyl group (toward metyrapone, daunorubicin, glyceraldehyde, and 4-pyridine-carboxaldehyde) and conjugation enzymes (p-nitrophenol-UDP-glucuronosyl transferase, 1-chloro-2,4-dinitrobenzene glutathione-S-tranferase) in young adults, males, non-castrated (N=6) farm animals were studied and compared. Presence of proteins cross-reacting with anti-human CYP3A4, CYP2C9, and CYP2E1 IgG was detected in all farm species. Bovine microsomes differed from other microsomes of farm species in very high 7-ethoxyresorufin-O-deethylase activity (CYP1A1/2). Significantly higher 7-methoxy-4-trifluoromethyl-coumarin demethylase (2-3 times) and 12-lauric acid hydroxylases (4-10 times) activities (probably corresponding to CYP2C and CYP4A, respectively) were found in ovine microsomes. The highest 6beta-testosterone hydroxylase activity, which is usually considered to be a CYP3A activity marker, was found in pig. Reductases of all farm animals display considerable ability to reduce carbonyl group of xenobiotics. Significant differences in level and activity of many biotransformation enzymes tested suggest that extrapolation of pharmacokinetic data obtained in one species to another (even related) could be misleading.     

Effect of oral administration of clinically relevant doses of dexamethasone on regulation of cytochrome P450 subfamilies in hepatic microsomes from dogs and rats

OBJECTIVE: To evaluate the effect of oral administration of dexamethasone (DEX) at clinically relevant doses on metabolic activities of cytochrome P450 (CYP) isoenzymes in dogs and rats. ANIMALS: 15 healthy 1-year-old male Beagles and 20 healthy 10-week-old male Wistar rats. PROCEDURE: Hepatic microsomes were harvested from dogs treated orally with DEX at 2.5 and 7.5 mg for 5 days and from rats treated orally with DEX at 0.75, 6, and 48 mg/kg for 5 days. 7-ethoxyresorufin, tolbutamide, bufuralol, and midazolam were used as CYP1A, CYP2C, CYP2D, and CYP3A substrates, respectively. Concentrations of metabolites formed by CYPs were measured by use of high-performance liquid chromatography, except for the resorufin concentrations measured by use of a fluorometric method. Reaction velocity-substrate concentration data were analyzed to obtain maximum reaction velocity (Vmax) and Michaelis-Menten constant (Km). RESULTS: Values of Vmax for midazolam 4-hydroxylation were significantly decreased by treatment with DEX at 2.5 and 7.5 mg in dogs, although values of Km were not affected. Values of Vmax for bufuralol 1'-hydroxylation were also decreased by treatment with DEX. In rats, values of Vmax for midazolam 4- hydroxylation were significantly decreased by treatment with DEX at 0.75 and 6 mg/kg but significantly increased at 48 mg/kg. Other reactions were not affected by treatment with DEX. CONCLUSIONS AND CLINICAL RELEVANCE: Our results indicate that DEX downregulates the CYP3A subfamily when administered at clinically relevant doses to dogs. The effect of downregulation of CYP3A in dogs treated with DEX should be considered to avoid adverse effects from coadministration of drugs.     

Comparative metabolism of mycophenolic acid by glucuronic acid and glucose conjugation in human,dog, and cat liver microsomes

Use of the immunosuppressant mycophenolic acid (MPA) in cats is limited because MPA elimination depends on glucuronidation, which is deficient in cats. We evaluated formation of major (phenol glucuronide) and minor (acyl glucuronide, phenol glucoside, and acyl glucoside) MPA metabolites using liver microsomes from 16 cats, 26 dogs, and 48 humans. All MPA metabolites were formed by human liver microsomes, while dog and cat liver microsomes formed both MPA glucuronides, but only one MPA glucoside (phenol glucoside). Intrinsic clearance (CLint) of MPA for phenol glucuronidation by cat liver microsomes was only 15–17% that of dog and human liver microsomes. However, CLint for acyl glucuronide and phenol glucoside formation in cat liver microsomes was similar to or greater than that for dog and human liver microsomes. While total MPA conjugation CLint was generally similar for cat liver microsomes compared with dog and human liver microsomes, relative contributions of each pathway varied between species with phenol glucuronidation predominating in dog and human liver microsomes and phenol glucosidation predominating in cat liver microsomes. MPA conjugation variation between cat liver microsomes was threefold for total conjugation and for phenol glucosidation, sixfold for phenol glucuronidation, and 11‐fold for acyl glucuronidation. Our results indicate that total MPA conjugation is quantitatively similar between liver microsomes from cats, dogs, and humans despite large differences in the conjugation pathways that are utilized by these species.     

Inhibitory effect of several fluoroquinolones on hepatic microsomal cytochrome P-450 1A activities in dogs

We examined inhibitory effects of ofloxacin (OFX), orbifloxacin (OBFX), ciprofloxacin (CFX), enrofloxacin (EFX) and norfloxacin (NFX) on cytochrome P-450 1A (CYP1A) activities using hepatic microsomes from four beagle dogs. Ethoxyresorufin O-de-ethylation was referred as CYP1A activities. All the fluoroquinolones inhibited the reaction in a noncompetitive manner. The determined inhibitory constants were the followings; 10.1 +/- 3.8 mM for OFX, 6.43 +/- 2.01 mM for OBFX, 0.726 +/- 0.134 mM for CFX, 4.06 +/- 1.19 mM for EFX and 4.75 +/- 1.63 mM for NFX respectively. As these values are >100-fold of plasma concentrations after a clinical single dose of the fluoroquinolones, it is suggested that the inhibitory effect on CYP1A activities is not so high to elicit drug-drug interaction with CYP1A substrates, when these fluoroquinolones are co-administered. Mechanism based inhibition was also examined in this study. Of the five fluoroquinolones examined, OFX, OBFX and CFX had this inhibition manner. As this inhibition is irreversible, inhibitory effects of the three fluoroquinolones may accumulate, when they are repeatedly administered. Therefore, OFX, OBFX and CFX may result in substantial drug-drug interaction with a CYP1A substrate even in clinical states. As EFX is metabolized to CFX in the body, it may also have the same possibility.     

Isolation of two cytochrome P450 forms, CYP2A19 and CYP1A, from pig liver microsomes

Cytochromes P450 comprise a superfamily of proteins that play a crucial role in the biotransformation of numerous chemicals. Purified CYPs can be used e.g. in studies on structure or determining the drug metabolism pathways. In this work, purification of the porcine CYP1A and CYP2A19 to electrophoretic homogeneity from the pig hepatic microsomes using octylamino Sepharose and hydroxylapatite column chromatography is reported. The proteins have been clearly recognized by commercial antibodies against rat and human CYP1A2 (porcine CYP1A) and human CYP2A6 (CYP2A19) respectively, using Western blot. Activities of both enzymes were determined by specific substrates, 7-ethoxyresorufin, 7-methoxyresorufin (CYP1A) and coumarin (CYP2A19). The isolated enzymes show kinetic parameters similar to human counterparts. Taken together, pig cytochromes can be used for the testing of veterinary drug metabolism, useful for the determination of drug residues in meat of pigs. The results obtained show that the pigs may be a suitable model for biotransformation of xenobiotics in humans.     

Reaction phenotyping of vinblastine metabolism in dogs

Vinblastine is a vinca alkaloid used either as a single agent or in combination therapy for the treatment of canine mast cell tumours and lymphomas. The objective of this study was to determine which isoform of cytochrome P450 enzyme is responsible for the majority of vinblastine metabolism in dogs. A panel of eight recombinant canine cytochrome P450 enzymes (CYP1A1, CYP1A2, CYP3A12, CYP3A26, CYP2B11, CYP2C41, CYP2C21 and CYP2D15) were incubated in vitro with vinblastine. Findings were confirmed by the use of canine polyclonal antibodies of cytochrome P450 enzymes (CYP1A1, CYP3A12, CYP2B11 and CYP2C21) that were pre‐incubated with individual and pooled hepatic microsomes that were purified from canine liver. Substrate depletion was observed in the presence of recombinant CYP3A12, whereas depletion did not substantially occur when microsomes were pre‐incubated with polyclonal antibodies against CYP3A12. These findings confirmed that CYP3A12 is the major cytochrome P450 isoform responsible for the metabolism of vinblastine in dogs.     

Lack of inhibitory effects of several fluoroquinolones on cytochrome P-450 3A activities at clinical dosage in dogs

Inhibitory effects of several fluoroquinolones (FQs) on liver CYP3A activities were examined by in vitro and in vivo tests in dogs. Midazolam (MDZ) hydroxylation rate was used to determine the CYP3A activities in liver microsomes. Enrofloxacin (EFX), ofloxacin (OFX) orbifloxacin (OBFX) and ciprofloxacin (CFX) were tested. None of the FQs changed Vmax, Km or intrinsic clearance (Vmax/Km) of MDZ. For in vivo test, we examined the effects of oral administration of EFX and OFX on the pharmacokinetics of quinidine (QN), a CYP3A substrate. EFX or OFX (10 mg/kg) was administered once a day for 3 days. QN (2 mg/kg) was intravenously injected at 2 h after the final dose of FQs administration. The same dose of QN was intravenously injected 3 weeks before the start of FQs administration for control. Neither EFX nor OFX changed the pharmacokinetic parameters of QN. These in vitro and in vivo consisted results suggest that these FQs lack the inhibitory effects on CYP3A activities in dogs. Hence, given these results, the risk of drug-drug interaction is unlikely to occur between FQs and CYP3A substrates in clinical situation in dogs.     

Cynomolgus macaque CYP4 isoforms are functional, metabolizing arachidonic acid

Cytochrome P450 (CYP) is important for metabolism of not only xenobiotics such as drugs, but also endogenous compounds including arachidonic acids. CYP4A11, CYP4F3v2, CYP4F11, and CYP4F45 have been identified in cynomolgus macaque, an animal species widely used for investigation of drug metabolism due to its evolutionary closeness to human. However, their metabolic functions have not been investigated. In this study, proteins were heterologously expressed in Escherichia coli and characterized by metabolic assays using arachidonic acids as substrates that are metabolized by CYP4 isoforms in human. The results showed that all four CYPs metabolized arachidonic acids. Therefore, cynomolgus macaque CYP4A11, CYP4F3v2, CYP4F11, and CYP4F45 are functional enzymes.     

Midazolam oxidation in cattle liver microsomes: The role of cytochrome P450 3A

In humans, the cytochrome P450 3A (CYP3A) subfamily is involved in midazolam (MDZ) biotransformation into 1′- and 4-hydroxy metabolites, and the former serves as a probe for CYP3A catalytic activity. In veterinary species is still crucial to identify enzyme- and species-specific CYP substrates; thus, the aim of this study was to characterize MDZ oxidation in cattle liver. A HPLC-UV method was used to measure 1′- and 4-hydroxy MDZ (1′- and 4-OHMDZ, respectively) formation in cattle liver microsomes and assess the role of CYP3A by an immunoinhibition study. Moreover, MDZ hydroxylation was evaluated in 300 cattle liver samples and results were correlated with testosterone hydroxylation. Formation of both metabolites conformed to a single-enzyme Michaelis–Menten kinetics. Values of V_max and K_m were 0.67 nmol/min/mg protein and 6.16 μM for 4-OHMDZ, and 0.06 nmol/min/mg protein and 10.08 μM for 1′-OHMDZ. An anti-rat CYP3A1 polyclonal antibody inhibited up to 50% and 94% 1′- and 4-OHMDZ formation, respectively. MDZ oxidation in liver microsomes was poorly correlated with testosterone hydroxylation. In conclusion, cattle metabolized MDZ to 1′-OHMDZ and 4-OHMDZ. The immunoinhibition results indicated a major contribution of CYP3As to 4-OHMDZ formation and the involvement of other CYPs in 1′-OHMDZ production, paving the way for further investigations.     

Cytochrome P450 and its role in veterinary drug interactions.

Cytochrome P450 (CYP) enzymes are common sites of drug interactions in human beings. Drugs may act as inhibitors or inducers of CYPs, leading to altered clearance of a second drug. Clinically relevant drug interactions involving various CYP isoforms in people, including CYP1A2, CYP2C9, CYP2D6, and CYP3A4, have been well documented. Analogous interactions are beginning to be characterized in dogs, for which canine CYPs share many of the same substrate ranges as in human beings.     

Comparative expression of liver cytochrome P450-dependent monooxygenases in the horse and in other agricultural and laboratory species

The apoprotein expression and the catalytic activities of cytochrome P450s involved in the biotransformation of xenobiotics were investigated in horse liver microsomes and compared with those of food producing (cattle, pigs, broiler chicks, and rabbits) and laboratory species (rats). Western blot analysis revealed the presence of proteins immunorelated to rat CYP 1A, CYP 2B, CYP 2E, and CYP 3A subfamilies in hepatic microsomes from horses and from any other examined species. With the exception of the N-demethylation of N-nitrosodimethylamine in broiler chicks, all the recorded interspecies differences were quantitative in nature. Equine preparations proved the most active in the biotransformation of the CYP 1A substrates ethoxy- and methoxyresorufin and the least active in the metabolism of aminopyrine and ethoxycoumarin. On a comparative basis, large differences were observed in the rate of the in vitro metabolism of model substrates between "minor" (rabbits, horses) and "major" food producing species. Taken in due consideration the limitations of the in vitro approach, results from this study reinforce the conclusion that studies on drug efficacy and residue depletion should be performed in each target species.     

High performance liquid chromatography determination of cytochrome P450 1A and 2C activities in bovine liver microsomes

This study reports fluorescence high performance liquid chromatography (HPLC) and UV-Vis HPLC methods for the determination of 7-ethoxyresorufin O-deethylase (EROD) and tolbutamide methylhydroxylase (TMH) activities, respectively, using bovine liver microsomes. The detection limits were 0.022 and 5.5 pmol on the column, respectively; intra-day and inter-day precisions (expressed as relative standard deviation) were <10%. Both methods showed enough sensitivity to allow for an accurate determination of enzyme kinetic parameters according to Michaelis-Menten plots and the results were: K(m)=0.23+/-0.051 microM, V(max)=0.488+/-0.035 nmol/min/mg protein for EROD activity, and K(m)=1010+/-155.7 microM, V(max)=0.089+/-0.006 nmol/min/mg protein for TMH activity. An Eadie-Hofstee plot analysis showed that in bovine liver microsomes, EROD and TMH activities followed a monophasic kinetic pattern. alpha-Naphthoflavone, a cytochrome P450 1A1/2 (CYP1A1/2) inhibitor, and sulfaphenazole, a cytochrome P450 2C9 (CYP2C9) inhibitor, decreased EROD and TMH activities, respectively. The sensitivity of the methods allowed the use of microsomes with low enzyme activity, such as those from veal calf liver. Thus, EROD and TMH activities may be adopted as markers for the evaluation of CYP1A and CYP2C9-like activities in liver microsomes from veal and beef cattle.     

Inhibition of cytochrome P450 2A participating in coumarin 7-hydroxylation in pig liver microsomes

Five commonly used human cytochrome P450 (CYP) inhibitors were examined for their effects on coumarin 7-hydroxylase (CYP2A) activity in pig liver microsomes. The K(m) and V(max) values for coumarin 7-hydroxylation in pig liver microsomes were estimated to be 1 μm and 0.26 nmol·mg/min, respectively. The following human CYP inhibitors caused little or no inhibition of CYP2A as defined by a K(i) > 200 μm: quinidine (CYP2D6), troleandomycin (CYP3A4), and sulfaphenazole (CYP2C9). The other two human CYP inhibitors were classified as strong inhibitors of CYP2A: 8-methoxypsoralen (CYP2A6) and α-naphthoflavone (CYP1A1/2). In the absence of a preincubation period, 8-MOP inhibited the 7-hydroxylation of coumarin with a K(i) value of 1.1 μm, which decreased to 0.1 μm when 8-MOP was preincubated with pig liver microsomes for 3 min. α-Naphthoflavone inhibited the 7-hydroxylation of coumarin with a K(i) value of 32 μm, which did not increase ability to inhibitor CYP2A when α-naphthoflavone was preincubated with pig liver microsomes for 3 min. These results of this study suggest that 8-MOP is a potent, mechanism-based inhibitor of pig CYP2A activity in pig liver microsomes.     

CYP2D-related Metabolism in Animals of the Canoidea Superfamily – Species Differences

CYP2D-related drug metabolism in liver microsomes from animals of the Canoidea super family, i.e. mink (Mustela vison), bears (Ursus arctos), foxes (Vulpes vulpes) and dogs, were investigated. Propranolol, bunitrolol and imipramine, which are typically substrates of CYP2D subfamilies, were used in the experiment. All the animals of the Canoidea superfamily that were tested lacked the ability to catalyse 7-hydroxylation of propranolol, which is one of the major metabolic pathways in rats. Stereoselectivity of propranolol metabolism was towards (S)-propranolol in all the reactions of the animals tested with the exception of mink, which showed a selective tendency towards (R)-propranolol in N-dealkylation. As far as metabolic patterns of (R)- and (S)-propranolol are concerned, bears, foxes and dogs are alike, but minks are somewhat different. Liver microsomes from mink showed, among the animals of the Canoidea superfamily, the lowest propranolol hydroxylase activity at 4- and 5-positions and imipramine 2-hydroxylation and {N-}demethylation activities. We could not detect bunitrolol 4-hydroxylation in mink liver microsomes at the low substrate concentration used. We conclude that mink have the lowest activity of CYP2D-related xenobiotic metabolism among the Canoidea superfamily.     

In vitro cytochrome P450-mediated hepatic activities for five substrates in specific pathogen free chickens

Total hepatic microsomal cytochrome P450 (CYP) content as well as in vitro CYP mediated activities for five substrates [bufuralol 1-hydroxylation, ethoxyresorufin O-deethylation, S-mephenytoin 4-hydroxylation, testosterone 6beta-hydroxylation, and tolbutamide hydroxylation] were measured in specific pathogen free male Japanese leghorn chickens and male beagle dogs. The Vmax, Km and intrinsic clearance (Vmax/Km) for these substrates were calculated and compared between animal species in order to evaluate the drug catalytic activity in chicken liver. The total CYP content in chicken (0.296 +/- 0.04 nmol/mg microsomal protein) was close to levels reported for other species including humans, cats, pigs and some nonmammalian vertebrates (e.g. snakes, frogs and trout fish), but was lower than levels measured in dogs (1.11 +/- 0.22) or recorded in guinea-pigs, hamsters, monkeys, mice, rabbits, rats, horse and ruminants. Bufuralol 1-hydroxylation, ethoxyresorufin O-deethylation, S-mephenytoin 4-hydroxylation, and testosterone 6beta-hydroxylation were lower in chickens than in dogs based on intrinsic clearance. On the other hand, tolbutamide hydroxylation was markedly higher in chickens than in dogs.