Ferlini Agne G, Somogyi AA, Sykes B, Knych H, Franklin S.In humans, omeprazole is metabolised by cytochrome P450 (CYP450) CYP2C19 and CYP3A4 with differences in CYP2C19 genotypes leading to variable response to therapy. Despite a wide use of omeprazole in horses with evidence of variable therapeutic efficiency, information regarding enzymatic metabolism is not currently available. This study aims to describe the in vitro kinetics of omeprazole metabolism and determine which enzyme(s) are responsible for omeprazole metabolism in horses. Omeprazole (0-800 uM) was incubated with liver microsomes and a panel of equine recombinant CYP450s (eq-rCYP). Meta...
Leiberich M, Marais HJ, Naidoo V.The plight of the white rhinoceros () and the increasing need of treatment options for injured poaching victims led to the necessity to expand the knowledge on applicable drugs in this endangered species. With very little information available on drug pharmacokinetics in rhino, veterinarians have to rely on information generated from other species. The horse being a closely related species, has served as the model for dose extrapolations. However, from recent research on enrofloxacin and carprofen, the white rhino showed considerable differences in the pharmacokinetic properties of these drugs...
Shibany KA, Tu00f6temeyer S, Pratt SL, Paine SW.1. Scaling factor values for the in vitro-in vivo extrapolation of hepatic metabolic clearance for xenobiotics have not yet been determined in horses. Scaling factors were determined by comparing the total protein and or cytochrome (CYP) P450 content in microsomes and cryopreserved hepatocytes against the content in the liver. 2. Microsomal protein per gram of liver (MPPGL) and hepatocellularity number per gram of liver (HPGL) using CYP P450 content method ranged 41-73 mg/gram of liver (mean= 57 mg/gram of liver, n = 39) and 146-320 × 10 cells/g of liver (mean = 227× 10 c...
Nakayama SM, Ikenaka Y, Hayami A, Mizukawa H, Darwish WS, Watanabe KP, Kawai YK, Ishizuka M.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 enzy...
Stefanski A, Mevissen M, Mu00f6ller AM, Kuehni-Boghenbor K, Schmitz A.In this study, we established cell culture conditions for primary equine hepatocytes allowing cytochrome P450 enzyme (CYP) induction experiments. Hepatocytes were isolated after a modified method of Bakala et al. (2003) and cultivated on collagen I coated plates. Three different media were compared for their influence on morphology, viability and CYP activity of the hepatocytes. CYP activity was evaluated with the fluorescent substrate 7-benzyloxy-4-trifluoromethylcoumarin. Induction experiments were carried out with rifampicin, dexamethasone or phenobarbital. Concentration-response curves for...
Hayami A, Darwish WS, Ikenaka Y, Nakayama SM, Ishizuka M.There is little information about drug metabolism and pharmacokinetics in horses. Therefore, it is necessary to characterize the profiles of drug metabolites for the safe use of drugs. In this study, we focused on cytochrome P450 enzymes (CYPs), which represent an important enzyme group to determine pharmacological effects of drugs. We chose diazepam as the drug of choice for this study. The aim of this study was to elucidate the metabolic pathway of diazepam in horses in comparison with rats, and to clarify CYP subfamilies responsible for diazepam metabolism in horses. Our results showed tema...
Arai KY, Tanaka Y, Taniyama H, Tsunoda N, Nambo Y, Nagamine N, Watanabe G, Taya K.In this study, the expression patterns of inhibins, activins, insulin-like growth factor-I (IGF-I) and steroidogenic enzymes in equine placentae recovered during the latter two-thirds of gestation were examined. Concentrations of inhibin A and inhibin pro-alphaC in endometrial and fetal placental tissue homogenates were very low during the period examined, whereas these tissues contained high concentrations of activin A. In both maternal endometrial and fetal placental tissues, activin A levels decreased as pregnancy progressed. Expression of inhibin alpha-subunit was not observed in the place...
Seralini GE, Tomilin A, Auvray P, Nativelle-Serpentini C, Sourdaine P, Moslemi S.We characterized testicular equine aromatase and its expression. A 2707 bp cDNA was isolated, it encoded a polypeptide of 503 residues with a deduced molecular mass of 57.8 kDa. The sequence features were those of a cytochrome P450 aromatase, with a 78% polypeptide identity with the human counterpart. The gene has a minimal length of 74 kb comprising at least 9 exons and expresses a 2.8 kb mRNA in the testis. Transient cDNA transfections in E293 cells and in vitro translations in a reticulocyte lysate system allowed aromatase protein and activity detections. The activity increased with androst...
Gokbulut C, Nolan AM, McKellar QA.Oxibendazole (OBZ) was administered to eight horses at an oral dose of 10 mg kg(-1) bodyweight each. Parent OBZ could only be detected in plasma at the 0.5 and 1.0 hours post administration sampling times and the mean maximum plasma concentration was 0.008 microg ml(-1). Parent OBZ was detected in faeces between 12 and 72 hours after administration and the highest dry faecal concentration was detected at 24 hours. An unidentified metabolite was detected in plasma between 0.5 and 72 hours. The unidentified metabolite in the plasma of treated horses corresponded to the second eluted metabolite i...
Nebbia C, Ceppa L, Dacasto M, Nachtmann C, Carletti M.The oxidative metabolism of monensin, an ionophore antibiotic extensively used in veterinary practice as a coccidiostat and a growth promoter, was studied in hepatic microsomal preparations from horses, pigs, broiler chicks, cattle and rats. As assayed by the measurement of the amount of the released formaldehyde, the rate of monensin O-demethylation was nearly of the same order of magnitude in all species, but total monensin metabolism, which was estimated by measuring the rate of substrate disappearance by a high-performance liquid chromatography (HPLC) method, was highest in cattle, interme...
Hunter RP, Short CR, McClure JR, Koch CE, Keowen ML, VanSteenhouse JL, Dees AA.Cimetidine (CIM) is an H2-receptor antagonist that has been used in racehorses in an attempt to reduce the occurrence of stress-related gastric ulceration. It has also been shown to produce several useful effects other than its gastric acid suppression properties. Further, it is a well documented antagonist of cytochrome P-450 (CYP) mediated oxygenation reactions. Nitric oxide (NO), a recently discovered mediator or modifier of numerous physiological functions, is generated by several forms of nitric oxide synthase (NOS), one of which is inducible (iNOS). Inducible NOS, expressed in neutrophil...
Court MH, Von Moltke LL, Shader RI, Greenblatt DJ.The 6-hydroxylation of chlorzoxazone (CLZ) is currently being used in both in vivo and in vitro studies to quantify cytochrome P450 2E1 (CYP2E1) activity in humans. Comparatively little is known with regard to the biotransformation of this drug in other species. The NADPH-dependent biotransformation of CLZ was therefore studied using hepatic microsomes derived from humans and ten other mammalian species. In all species, 6-hydroxychlorzoxazone (6OH-CLZ) was the only metabolic product that could be identified by HPLC with ultraviolet detection. Enzyme kinetic analysis was used to characterize th...
Eisenhauer KM, McCue PM, Nayden DK, Osawa Y, Roser JF.Stallion testes secrete large amounts of estrogens, but the cellular location of the enzyme that converts androgens to estrogens, cytochrome P450 aromatase, has not been determined. The goal of the present study was to immunocytochemically localize stallion testicular aromatase using a polyclonal antibody generated against human placental cytochrome P450 aromatase. Testes were obtained from 12 stallions from 2 to 23 years of age, during both the breeding and non-breeding seasons. Immunoreactivity was confined to the Leydig cells in all testes examined. No immunostaining was observed in the Ser...
Turk MA, Thomas DE.To study the role of cytochrome P-450-dependent mixed function oxidase reactions in equine 3-methylindole (3MI) toxicosis, ponies were given 20 mg of phenobarbital/kg of body weight at 72, 60, 48, 36, and 24 hours before 100 mg of oral 3MI/kg to induce cytochrome P-450 or no treatment (controls). Maximal 3MI plasma concentration was decreased and clearance was faster in phenobarbital-treated ponies. Plasma 3MI was still detectable 12 and 36 hours after dosing in phenobarbital-treated and control ponies, respectively. Phenobarbital treatment induced a distribution phase with transition from a 1...
Court MH, Von Moltke LL, Shader RI, Greenblatt DJ.The 6-hydroxylation of chlorzoxazone (CLZ) is currently being used in both in vivo and in vitro studies to quantify cytochrome P450 2E1 (CYP2E1) activity in humans. Comparatively little is known with regard to the biotransformation of this drug in other species. The NADPH-dependent biotransformation of CLZ was therefore studied using hepatic microsomes derived from humans and ten other mammalian species. In all species, 6-hydroxychlorzoxazone (6OH-CLZ) was the only metabolic product that could be identified by HPLC with ultraviolet detection. Enzyme kinetic analysis was used to characterize th...
Nebbia C, Ceppa L, Dacasto M, Nachtmann C, Carletti M.The oxidative metabolism of monensin, an ionophore antibiotic extensively used in veterinary practice as a coccidiostat and a growth promoter, was studied in hepatic microsomal preparations from horses, pigs, broiler chicks, cattle and rats. As assayed by the measurement of the amount of the released formaldehyde, the rate of monensin O-demethylation was nearly of the same order of magnitude in all species, but total monensin metabolism, which was estimated by measuring the rate of substrate disappearance by a high-performance liquid chromatography (HPLC) method, was highest in cattle, interme...
Arai KY, Tanaka Y, Taniyama H, Tsunoda N, Nambo Y, Nagamine N, Watanabe G, Taya K.In this study, the expression patterns of inhibins, activins, insulin-like growth factor-I (IGF-I) and steroidogenic enzymes in equine placentae recovered during the latter two-thirds of gestation were examined. Concentrations of inhibin A and inhibin pro-alphaC in endometrial and fetal placental tissue homogenates were very low during the period examined, whereas these tissues contained high concentrations of activin A. In both maternal endometrial and fetal placental tissues, activin A levels decreased as pregnancy progressed. Expression of inhibin alpha-subunit was not observed in the place...
Eisenhauer KM, McCue PM, Nayden DK, Osawa Y, Roser JF.Stallion testes secrete large amounts of estrogens, but the cellular location of the enzyme that converts androgens to estrogens, cytochrome P450 aromatase, has not been determined. The goal of the present study was to immunocytochemically localize stallion testicular aromatase using a polyclonal antibody generated against human placental cytochrome P450 aromatase. Testes were obtained from 12 stallions from 2 to 23 years of age, during both the breeding and non-breeding seasons. Immunoreactivity was confined to the Leydig cells in all testes examined. No immunostaining was observed in the Ser...
Stefanski A, Mevissen M, Mu00f6ller AM, Kuehni-Boghenbor K, Schmitz A.In this study, we established cell culture conditions for primary equine hepatocytes allowing cytochrome P450 enzyme (CYP) induction experiments. Hepatocytes were isolated after a modified method of Bakala et al. (2003) and cultivated on collagen I coated plates. Three different media were compared for their influence on morphology, viability and CYP activity of the hepatocytes. CYP activity was evaluated with the fluorescent substrate 7-benzyloxy-4-trifluoromethylcoumarin. Induction experiments were carried out with rifampicin, dexamethasone or phenobarbital. Concentration-response curves for...
Hunter RP, Short CR, McClure JR, Koch CE, Keowen ML, VanSteenhouse JL, Dees AA.Cimetidine (CIM) is an H2-receptor antagonist that has been used in racehorses in an attempt to reduce the occurrence of stress-related gastric ulceration. It has also been shown to produce several useful effects other than its gastric acid suppression properties. Further, it is a well documented antagonist of cytochrome P-450 (CYP) mediated oxygenation reactions. Nitric oxide (NO), a recently discovered mediator or modifier of numerous physiological functions, is generated by several forms of nitric oxide synthase (NOS), one of which is inducible (iNOS). Inducible NOS, expressed in neutrophil...
Seralini GE, Tomilin A, Auvray P, Nativelle-Serpentini C, Sourdaine P, Moslemi S.We characterized testicular equine aromatase and its expression. A 2707 bp cDNA was isolated, it encoded a polypeptide of 503 residues with a deduced molecular mass of 57.8 kDa. The sequence features were those of a cytochrome P450 aromatase, with a 78% polypeptide identity with the human counterpart. The gene has a minimal length of 74 kb comprising at least 9 exons and expresses a 2.8 kb mRNA in the testis. Transient cDNA transfections in E293 cells and in vitro translations in a reticulocyte lysate system allowed aromatase protein and activity detections. The activity increased with androst...
Gokbulut C, Nolan AM, McKellar QA.Oxibendazole (OBZ) was administered to eight horses at an oral dose of 10 mg kg(-1) bodyweight each. Parent OBZ could only be detected in plasma at the 0.5 and 1.0 hours post administration sampling times and the mean maximum plasma concentration was 0.008 microg ml(-1). Parent OBZ was detected in faeces between 12 and 72 hours after administration and the highest dry faecal concentration was detected at 24 hours. An unidentified metabolite was detected in plasma between 0.5 and 72 hours. The unidentified metabolite in the plasma of treated horses corresponded to the second eluted metabolite i...
Shibany KA, Tu00f6temeyer S, Pratt SL, Paine SW.1. Scaling factor values for the in vitro-in vivo extrapolation of hepatic metabolic clearance for xenobiotics have not yet been determined in horses. Scaling factors were determined by comparing the total protein and or cytochrome (CYP) P450 content in microsomes and cryopreserved hepatocytes against the content in the liver. 2. Microsomal protein per gram of liver (MPPGL) and hepatocellularity number per gram of liver (HPGL) using CYP P450 content method ranged 41-73 mg/gram of liver (mean= 57 mg/gram of liver, n = 39) and 146-320 × 10 cells/g of liver (mean = 227× 10 c...
Leiberich M, Marais HJ, Naidoo V.The plight of the white rhinoceros () and the increasing need of treatment options for injured poaching victims led to the necessity to expand the knowledge on applicable drugs in this endangered species. With very little information available on drug pharmacokinetics in rhino, veterinarians have to rely on information generated from other species. The horse being a closely related species, has served as the model for dose extrapolations. However, from recent research on enrofloxacin and carprofen, the white rhino showed considerable differences in the pharmacokinetic properties of these drugs...
Nakayama SM, Ikenaka Y, Hayami A, Mizukawa H, Darwish WS, Watanabe KP, Kawai YK, Ishizuka M.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 enzy...
Ferlini Agne G, Somogyi AA, Sykes B, Knych H, Franklin S.In humans, omeprazole is metabolised by cytochrome P450 (CYP450) CYP2C19 and CYP3A4 with differences in CYP2C19 genotypes leading to variable response to therapy. Despite a wide use of omeprazole in horses with evidence of variable therapeutic efficiency, information regarding enzymatic metabolism is not currently available. This study aims to describe the in vitro kinetics of omeprazole metabolism and determine which enzyme(s) are responsible for omeprazole metabolism in horses. Omeprazole (0-800 uM) was incubated with liver microsomes and a panel of equine recombinant CYP450s (eq-rCYP). Meta...
Turk MA, Thomas DE.To study the role of cytochrome P-450-dependent mixed function oxidase reactions in equine 3-methylindole (3MI) toxicosis, ponies were given 20 mg of phenobarbital/kg of body weight at 72, 60, 48, 36, and 24 hours before 100 mg of oral 3MI/kg to induce cytochrome P-450 or no treatment (controls). Maximal 3MI plasma concentration was decreased and clearance was faster in phenobarbital-treated ponies. Plasma 3MI was still detectable 12 and 36 hours after dosing in phenobarbital-treated and control ponies, respectively. Phenobarbital treatment induced a distribution phase with transition from a 1...
Hayami A, Darwish WS, Ikenaka Y, Nakayama SM, Ishizuka M.There is little information about drug metabolism and pharmacokinetics in horses. Therefore, it is necessary to characterize the profiles of drug metabolites for the safe use of drugs. In this study, we focused on cytochrome P450 enzymes (CYPs), which represent an important enzyme group to determine pharmacological effects of drugs. We chose diazepam as the drug of choice for this study. The aim of this study was to elucidate the metabolic pathway of diazepam in horses in comparison with rats, and to clarify CYP subfamilies responsible for diazepam metabolism in horses. Our results showed tema...
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