Transcript abundance of hepatic drug-metabolizing enzymes in two dog breeds compared with 14 species including humans.

Drug-metabolizing enzymes are important in drug development and therapy, but have not been fully identified and characterized in many species, lines, and breeds. Liver transcriptomic data were analyzed for phase I cytochromes P450, flavin-containing monooxygenases, and carboxylesterases and phase II UDP-glucuronosyltransferases, sulfotransferases, and glutathione S-transferases. Comparisons with a variety of species (humans, rhesus macaques, African green monkeys, baboons, common marmosets, cattle, sheep, pigs, cats, dogs, rabbits, tree shrews, rats, mice, and chickens) revealed both general similarities and differences in the transcript abundances of drug-metabolizing enzymes. Similarly, Beagle and Shiba dogs were examined by next-generation sequencing (RNA-seq). Consequently, no substantial differences in transcript abundance were noted in different breeds of pigs and dogs and in different lines of mice and rats. Therefore, the expression profiles of hepatic drug-metabolizing enzyme transcripts appear to be similar in Shiba and Beagle dogs and pig breeds and the rat and mouse lines analyzed, although some differences were found in other species.

A variety of cytochrome P450 enzymes and flavin-containing monooxygenases in dogs and pigs commonly used as preclinical animal models.

Drug oxygenation is mainly mediated by cytochromes P450 (P450s, CYPs) and flavin-containing monooxygenases (FMOs). Polymorphic variants of P450s and FMOs are known to influence drug metabolism.　Species differences exist in terms of drug metabolism and can be important when determining the contributions of individual enzymes. The success of research into drug-metabolizing enzymes and their impacts on drug discovery and development has been remarkable. Dogs and pigs are often used as preclinical animal models. This research update provides information on P450 and FMO enzymes in dogs and pigs and makes comparisons with their human enzymes. Newly identified dog CYP3A98, a testosterone 6ß- and estradiol 16α-hydroxylase, is abundantly expressed in small intestine and is likely the major CYP3A enzyme in small intestine, whereas dog CYP3A12 is the major CYP3A enzyme in liver. The roles of recently identified dog CYP2J2 and pig CYP2J33/34/35 were investigated. FMOs have been characterized in humans and several other species including dogs and pigs. P450 and FMO family members have been characterized also in cynomolgus macaques and common marmosets. P450s have industrial applications and have been the focus of attention of many pharmaceutical companies. The techniques used to investigate the roles of P450/FMO enzymes in drug oxidation and clinical treatments have not yet reached maturity and require further development. The findings summarized here provide a foundation for understanding individual pharmacokinetic and toxicological results in dogs and pigs as preclinical models and will help to further support understanding of the molecular mechanisms of human P450/FMO functionality.

Novel Tree Shrew Cytochrome P450 2Ds (CYP2D8a and CYP2D8b) Are Functional Drug-Metabolizing Enzymes that Metabolize Bufuralol and Dextromethorphan.

Tree shrews are a nonprimate species used in a range of biomedical studies. Recent genome analysis of tree shrews found that the sequence identities and the numbers of genes of cytochrome P450 (CYP or P450), an important family of drug-metabolizing enzymes, are similar to those of humans. However, tree shrew P450s have not yet been sufficiently identified and analyzed. In this study, novel CYP2D8a and CYP2D8b cDNAs were isolated from tree shrew liver and were characterized, along with human CYP2D6, dog CYP2D15, and pig CYP2D25. The amino acid sequences of these tree shrew CYP2Ds were 75%-78% identical to human CYP2D6, and phylogenetic analysis showed that they were more closely related to human CYP2D6 than rat CYP2Ds, similar to dog and pig CYP2Ds. For tree shrew CYP2D8b, two additional transcripts were isolated that contained different patterns of deletion. The gene and genome structures of CYP2Ds are generally similar in dogs, humans, pigs, and tree shrews. Tree shrew CYP2D8a mRNA was most abundantly expressed in liver, among the tissue types analyzed, similar to dog CYP2D15 and pig CYP2D25 mRNAs. Tree shrew CYP2D8b mRNA was also expressed in liver, but at a level 7.3-fold lower than CYP2D8a mRNA. Liver microsomes and recombinant protein of both tree shrew CYP2Ds metabolized bufuralol and dextromethorphan, selective substrates of human CYP2D6, but the activity level of CYP2D8a greatly exceeded that of CYP2D8b. These results suggest that tree shrew CYP2D8a and CYP2D8b are functional drug-metabolizing enzymes, of which CYP2D8a is the major CYP2D in liver. SIGNIFICANCE STATEMENT: Novel tree shrew CYP2D8a and CYP2D8b cDNAs were isolated from liver. Their amino acid sequences were 75%-78% identical to human CYP2D6. For CYP2D8b, two additional transcripts contained different patterns of deletion. Tree shrew CYP2D8a mRNA was abundantly expressed in liver, similar to dog CYP2D15 and pig CYP2D25 mRNAs. Recombinant tree shrew CYP2Ds catalyzed the oxidation of bufuralol and dextromethorphan. Tree shrew CYP2D8a and CYP2D8b are functional drug-metabolizing enzymes, of which CYP2D8a is the major CYP2D in liver.

Molecular and functional characterization of flavin-containing monooxygenases (FMO1-6) in tree shrews.

Flavin-containing monooxygenases (FMOs) are a family of important drug oxygenation enzymes that, in humans, consist of five functional enzymes (FMO1-5) and a pseudogene (FMO6P). The tree shrew is a non-rodent primate-like species that is used in various biomedical studies, but its usefulness in drug metabolism research has not yet been investigated. In this study, tree shrew FMO1-6 cDNAs were isolated and characterized by sequence analysis, tissue expression, and metabolic function. Compared with human FMOs, tree shrew FMOs showed sequence identities of 85-90 % and 81-89 %, respectively, for cDNA and amino acids. Phylogenetic analysis showed that each tree shrew and human FMO were closely clustered. The genomic and genetic structures of the FMO genes were conserved in tree shrews and humans. Among the five tissue types analyzed (lung, heart, kidney, small intestine, and liver), FMO3 and FMO1 mRNAs were most abundant in liver and kidney, respectively. Recombinant tree shrew FMO1-6 proteins expressed in bacterial membranes all mediated benzydamine and trimethylamine N-oxygenations and methyl p-tolyl sulfide S-oxygenation. The selective human FMO3 substrate trimethylamine was predominantly metabolized by tree shrew FMO3. Additionally, tree shrew FMO6 was active toward trimethylamine, as is cynomolgus macaque FMO6, in contrast with the absence of activity of the human FMO6P pseudogene product. Tree shrew FMO1-6, which are orthologous to human FMOs (FMO1-5 and FMO6P) were identified, and tree shrew FMO3 has functional and molecular features generally comparable to those of human FMO3 as the predominant FMO in liver.

Novel Cytochrome P450 2C119 Enzymes in Cynomolgus and Rhesus Macaques Metabolize Progesterone, Diclofenac, and Omeprazole.

Cynomolgus and rhesus macaques are used in drug metabolism studies due to their evolutionary and phylogenetic closeness to humans. Cytochromes P450 (P450s or CYPs), including the CYP2C family enzyme, are important endogenous and exogenous substrate-metabolizing enzymes and play major roles in drug metabolism. In cynomolgus and rhesus macaques, six CYP2Cs have been identified and characterized, namely, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2C76, and CYP2C93. In this study, CYP2C119, a new CYP2C, was identified and characterized in cynomolgus and rhesus macaques. Cynomolgus and rhesus CYP2C119 contained open reading frames of 489 amino acids with high sequence identities to human CYP2C8 and to cynomolgus and rhesus CYP2C8. Phylogenetic analysis showed that cynomolgus and rhesus CYP2C119 were closely related to cynomolgus and rhesus CYP2C8. In cynomolgus and rhesus genomes, CYP2C genes, including CYP2C119, form a cluster. Among the tissues analyzed, cynomolgus CYP2C119 mRNA was predominantly expressed in liver. Hepatic expressions of CYP2C119 mRNA in four cynomolgus and two rhesus macaques varied, with no expression in one rhesus macaque. Among the CYP2C mRNAs, CYP2C119 mRNA was expressed less abundantly than CYP2C8, CYP2C9, CYP2C19, and CYP2C76 mRNAs but more abundantly than CYP2C18 mRNA. Recombinant cynomolgus and rhesus CYP2C119 catalyzed progesterone 16α-, 17α-, and 21-hydroxylation and diclofenac and omeprazole oxidations, indicating that CYP2C119 is a functional enzyme. Therefore, the novel CYP2C119 gene, expressed in macaque liver, encodes a functional enzyme that metabolizes human CYP2C substrates and is likely responsible for drug clearances. SIGNIFICANCE STATEMENT: Cytochrome P450 2C119 was found in cynomolgus and rhesus macaques, in addition to the known P450 2C8, 2C9, 2C18, 2C19, 2C76, and 2C93. Cynomolgus and rhesus CYP2C119 contain open reading frames of 489 amino acids with high sequence identity to human CYP2C8. Cynomolgus CYP2C119 mRNA is predominantly expressed in the liver. Recombinant CYP2C119 catalyzed progesterone hydroxylation and diclofenac and omeprazole oxidations. Therefore, the novel CYP2C119 gene expressed in the macaque liver encodes a functional enzyme that metabolizes human CYP2C substrates.

Habu snakes (Protobothrops flavoviridis) show variation in thoracic aortic vasoreactivity between adjacent Japanese islands.

Habu snakes (Protobothrops flavoviridis) are pit vipers found in the geographically adjacent but ecologically divergent islands of Tokunoshima and Amami-Oshima in southwestern Japan. Abiotic factors can cause variation in animal populations between the two islands, and Habu snakes may show such intraspecific physiological variation. We therefore evaluated the vasoreactivity in aortas isolated from the Habu of both islands. Tokunoshima Habu showed significantly greater contractile responses to angiotensin (Ang) II, acetylcholine (ACh) and noradrenaline, and significantly higher affinities (pEC50) for Ang II and ACh, than Amami-Oshima Habu. ACh caused contractions in aortas from both populations, a finding previously unreported in snakes. Our findings indicate that vasoreactivity may differ between Tokunoshima and Amami-Oshima Habu.

Characterization of Vasoreactivity in a Semi-Arboreal Snake, the Tokara Habu (Protobothrops tokarensis).

Vasoreactivity is relatively well documented in terrestrial snakes but has previously been investigated in only one semi-arboreal snake species. Consequently, the extent to which vasoreactivity is common across snake taxa or varies by habitat is unclear. The Tokara habu (Protobothrops tokarensis) is a semi-arboreal snake endemic to only two small adjacent Japanese islands, and hence a useful species for further investigation of vasoreactivity. We evaluated responses to known vasoactive substances in thoracic aortas isolated from Tokara habu. Under resting tension, noradrenaline and angiotensin II induced concentration-dependent contraction, but acetylcholine, serotonin (5-hydroxytriptamine; 5-HT), and isoproterenol induced relaxation followed by contraction. Histamine and rattlesnake bradykinin had no effect. Experiments with receptor-specific antagonists suggest that M1 and M3 receptors are involved in the acetylcholine-induced response; 5-HT1, 5-HT2, and 5-HT7 receptors in the serotonin-induced response; and ß1 and ß2 adrenoceptors in isoproterenol-induced relaxation. This is the first report on such response patterns in snakes (including serotonin- and isoproterenol-induced relaxation). Nitric oxide may be involved in acetylcholine-induced relaxation but not in the responses to serotonin or isoproterenol. In contrast to the uniform vasoreactivity observed in terrestrial snakes, the vasoreactivity of semi-arboreal snakes may be governed by diverse regulatory mechanisms.

Identification of cytochrome P450 2C18 and 2C76 in tree shrews: P450 2C18 effectively oxidizes typical human P450 2C9/2C19 chiral substrates warfarin and omeprazole with less stereoselectivity.

Cytochromes P450 (P450s or CYPs), especially the CYP2C family, are important drug-metabolizing enzymes that play major roles in drug metabolism. Tree shrews, a non-rodent primate-like species, are used in various fields of biomedical research, notably hepatitis virus infection; however, its drug-metabolizing enzymes have not been fully investigated. In this study, tree shrew CYP2C18, CYP2C76a, CYP2C76b, and CYP2C76c cDNAs were identified and contained open reading frames of 489 or 490 amino acids with high sequence identities (70-78 %) to human CYP2Cs. Tree shrew CYP2C76a, CYP2C76b, and CYP2C76c showed higher sequence identities (79-80 %) to cynomolgus CYP2C76 and were not orthologous to any human CYP2C. Phylogenetic analysis revealed that tree shrew CYP2C18 and CYP2C76s were closely related to rat CYP2Cs and cynomolgus CYP2C76, respectively. Tree shrew CYP2C genes formed a gene cluster similar to human CYP2C genes. All four tree shrew CYP2C mRNAs showed predominant expressions in liver, among the tissue types examined; expression of CYP2C18 mRNA was also detected in small intestine. In liver, CYP2C18 mRNA was the most abundant among the tree shrew CYP2C mRNAs. In metabolic assays using human CYP2C substrates, all tree shrew CYP2Cs showed metabolic activities toward diclofenac, R,S-omeprazole, paclitaxel, and R,S-warfarin, with the activity of CYP2C18 exceeding that of the other CYP2Cs. Moreover, tree shrew CYP2C76 enzymes metabolized progesterone more efficiently than human, cynomolgus, or marmoset CYP2Cs. Therefore, these novel tree shrew CYP2Cs are expressed abundantly in liver, encode functional enzymes that metabolize human CYP2C substrates, and are likely responsible for drug clearances.

Tree shrew cytochrome P450 2E1 is a functional enzyme that metabolises chlorzoxazone and p-nitrophenol.

Cytochromes P450 (CYPs or P450s) are important enzymes for drug metabolism. Tree shrews are non-primate animal species used in various fields of biomedical research, including infection (especially hepatitis viruses), depression, and myopia. A recent tree shrew genome analysis indicated that the sequences and the numbers of P450 genes are similar to those of humans; however, P450s have not been adequately identified and analysed in this species.In this study, a novel CYP2E1 was isolated from tree shrew liver and was characterised in comparison with human, dog, and pig CYP2E1. Tree shrew CYP2E1 and human CYP2E1 showed high amino acid sequence identity (83%) and were closely related in a phylogenetic tree.Gene and genome structures of CYP2E1 were generally similar in humans, dogs, pigs, and tree shrews. Tissue expression patterns showed that tree shrew CYP2E1 mRNA was predominantly expressed in liver, just as for dog and pig CYP2E1 mRNAs. In tree shrews, recombinant CYP2E1 protein and liver microsomes metabolised chlorzoxazone and p-nitrophenol, probe substrates of human CYP2E1, just as they do in dogs and pigs.These results suggest that tree shrew CYP2E1 encodes a functional drug-metabolising enzyme that plays a role in the liver, similar to human CYP2E1.

Chronic Toxoplasma infection affects gene expression of drug-metabolizing enzymes in mouse liver.

Toxoplasma gondii is an intracellular protozoan parasite causing toxoplasmosis, an infectious disease affecting warm-blooded vertebrates worldwide. Many drug-metabolizing enzymes are located in the liver, a major organ of drug metabolism, and their function can be affected by pathogen infection.Using next-generation sequencing (RNA-seq) and quantitative polymerase chain reaction (qPCR), changes in the hepatic expressions of drug-metabolizing enzymes were analysed in mice chronically infected with T. gondii. The analysis found that, among drug-metabolizing enzymes, 22 genes were upregulated and 28 genes were downregulated (≥1.5-fold); of these 5 and 17 genes, respectively, were cytochromes P450 (Cyp or P450).Subsequent qPCR analysis showed that six P450 genes were upregulated significantly (≥1.5-fold, p < 0.05), namely, Cyp1b1, Cyp2c29, Cyp2c65, Cyp2d9, Cyp2d12, and Cyp3a59, whereas nine P450 genes were downregulated significantly (≥1.5-fold, p < 0.05), namely, Cyp2c38, Cyp2c39, Cyp2c44, Cyp2c69, Cyp2d40, Cyp2e1, Cyp3a11, Cyp3a41, and Cyp3a44.Moreover, metabolic assays in infected mouse liver using typical P450 substrates revealed that midazolam 1'-hydroxylation and testosterone 2-hydroxylation activities decreased significantly (≥1.5-fold, p < 0.05), whereas testosterone 16-hydroxylation activity increased significantly (≥1.5-fold, p < 0.05).Chronic Toxoplasma infection affects drug metabolism, at least partly, by altering the gene expressions of drug-metabolizing enzymes, including P450s.

Cytochrome P450 1A2 and 2C enzymes autoinduced by omeprazole in dog hepatocytes and human HepaRG and HepaSH cells are involved in omeprazole 5-hydroxylation and sulfoxidation.

The induction assay for the cytochromes P450 (P450s) is an important tool in drug discovery and development. The inductions of dog P450 1A2 and 3A12 by omeprazole and rifampicin were functionally characterised in dog hepatocytes and were compared with induction in human HepaRG and HepaSH cells.P450 1A2-dependent ethoxyresorufin O-deethylation was induced by R,S-omeprazole and P450 3 A-dependent midazolam 1'-hydroxylation was induced by rifampicin, and both reactions were significantly enhanced in cultured dog hepatocytes and human HepaRG and HepaSH cells.Recombinant dog P450 1A2 exhibited activities towards R- and S-omeprazole 5-hydroxylation with low Km values of 23-28 µM, whereas dog P450 2C21 and 3A12 efficiently mediated S-omeprazole 5-hydroxylation and sulfoxidation, respectively, with high Vmax values of 12-17 min-1.Although omeprazole 5-hydroxylation by human P450 2C19 (and sulfoxidation by P450 3A4) in human HepaSH cells were slightly (∼2-fold) induced by R,S-omeprazole, dog P450 1A2 was autoinduced by omeprazole in dog hepatocytes and showed enhanced R-omeprazole 5-hydroxylation activity (∼5-fold).These results indicate that omeprazole can be an autoinducer of P450 1A2 in hepatocytes, and this enzyme was found to be involved in omeprazole 5-hydroxylation and sulfoxidation in dog hepatocytes and human HepaRG and HepaSH cells.

Reduced Nitric Oxide Synthase Involvement in Aigamo Duck Basilar Arterial Relaxation.

The basilar arterial endothelium mediates blood vessel relaxation partly through the release of nitric oxide (NO). Apoptosis of cerebrovascular endothelial cells is linked to a high mortality rate in chickens infected with the highly pathogenic avian influenza virus, but interestingly, ducks exhibit a greater resistance to this virus. In this study, we examined the responsiveness of duck basilar arteries (BAs) to various vasoactive substances, including 5-hydroxytryptamine (5-HT), histamine (His), angiotensin (Ang) II, noradrenaline (NA), acetylcholine (ACh), and avian bradykinin ornithokinin (OK), aiming to characterize the receptor subtypes involved and the role of endothelial NO in vitro. Our findings suggest that arterial contraction is mediated with 5-HT1 and H1 receptors, while relaxation is induced with ß3-adrenergic and M3 receptors. Additionally, OK elicited a biphasic response in duck BAs, and Ang II had no effect. Endothelial NO appears to be crucial in relaxation mediated with M3 and OK receptors but not ß3-adrenergic receptors in the duck BA. The reduced endothelial NO involvement in the receptor-mediated relaxation response in duck BAs represents a clear difference from the corresponding response reported in chicken BAs. This physiological difference may explain the differences in lethality between ducks and chickens when vascular endothelial cells are infected with the virus.

Liver microsomal cytochrome P450 3A-dependent drug oxidation activities in individual dogs.

Drug oxidations are mediated mainly by cytochromes P450 (P450s or CYPs). CYP3As are an important P450 subfamily and include liver-specific CYP3A12 and intestine-specific CYP3A98 in dogs. Individual differences in drug oxidation activities were investigated, including correlations with immunoreactive CYP3A protein intensities and CYP3A mRNA expression levels in livers.Pooled and individual dog liver microsomes showed activities towards nifedipine, midazolam, alprazolam, and estradiol, but the levels of catalytic activities varied approximately twofold among the individual dogs. One dog harboured a CYP1A2 variant causing protein deletion but showed higher activities than the other dogs towards nifedipine oxidation, midazolam 1'-hydroxylation, alprazolam 4-hydroxylation, estradiol 16α-hydroxylation activities, and caffeine C8-hydroxylation; the latter is used as a reference reaction for CYP1A.In individual dog liver microsomes, the intensities of the immunochemical bands with anti-human CYP3A4 and anti-rat CYP3A2 antibodies along with CYP3A12 and CYP3A26 mRNA expression levels showed good correlations (p < 0.05) with nifedipine oxidation, midazolam 1'- and 4-hydroxylation, alprazolam 1'- and 4-hydroxylation, and estradiol 16α-hydroxylation activities.These results suggest that the oxidation activities of dog liver microsomes towards nifedipine and other typical CYP3A-catalyzed drugs exhibit approximately twofold individual differences and were predominantly mediated by liver-specific CYP3A12 in the dogs.

Involvement of beta3-adrenergic receptors in relaxation mediated by nitric oxide in chicken basilar artery.

The response of basilar arteries to noradrenaline varies among many animal species, but remains little studied in poultry. Accordingly, we aimed to characterize the adrenergic receptor (AR) subtypes that modulate vascular response in basilar arteries in the chicken, with isometric recording of arterial ring tension using an organ bath. We demonstrated the presence of both alpha and beta (α and ß) receptor subtypes through evaluating the response to noradrenaline, with and without a range of ß-AR and α-AR antagonists. The concentration-dependent relaxations then induced by a range of ß-AR agonists indicated a potency ranking of isoproterenol > noradrenaline > adrenaline > procaterol. We then investigated the effects of ß-AR antagonists that attenuate the effect of isoproterenol (propranolol for ß1,2,3-ARs, atenolol for ß1-ARs, butoxamine for ß2-ARs, and SR 59230A for ß3-ARs), with Schild regression analysis, ascertaining multiple ß-AR subtypes, with neither the ß1-AR nor the ß2-AR as the dominant subtype. SR 59230A was the only antagonist to yield a pA2 value (7.52) close to the reported equivalent for the relevant receptor subtype. Furthermore, treatment with SR 58611 (a ß3-AR agonist) induced relaxation, which was inhibited (P < 0.01) by L-NNA and SR 59230A. Additionally, treating basilar arterial strips (containing endothelium) with SR 58611 induced nitric oxide (NO) production, which was inhibited (P < 0.01) by L-NNA and SR 59230A. Based on this first characterization of AR subtypes in chicken basilar arteries (to our knowledge), we suggest that α- and ß-ARs are involved in contraction and relaxation, and that ß3-ARs, especially those on the endothelium, may play an important role in vasodilation via NO release.

2-Oxidation, 3-methyl hydroxylation, and 6-hydroxylation of skatole, a contributor to the odour of boar-tainted pork meat, mediated by porcine liver microsomal cytochromes P450 1A2, 2A19, 2E1, and 3A22.

The 2-oxidation, 3-methyl hydroxylation, and 6-hydroxylation of skatole (a contributor to boar taint) mediated by minipig liver microsomes and recombinant P450 enzymes expressed in bacterial membranes were investigated.At low substrate concentrations of 10 µM, the formation rates of indole-3-carbinol, 6-hydroxyskatole, and the sum of 3-methyloxindole, indole-3-carbinol, and 6-hydroxyskatole in male minipig liver microsomes were significantly lower than those in female minipig liver microsomes.Compensatory 3-methyloxindole and indole-3-carbinol formation in minipig liver microsomes, which lack 6-hydroxyskatole formation in males, was mediated partly by liver microsomal P450 1A2 and P450 1A2/2E1, respectively. These enzymes were suppressed by typical P450 inhibitors in female minipig liver microsomes.Among the 14 pig P450 forms evaluated, P450 2A19 was the dominant form mediating 3-methyloxindole, indole-3-carbinol, and 6-hydroxyskatole formation from skatole at substrate concentrations of 100 µM. Positive cooperativity was observed in 3-methyloxindole formation from skatole mediated by male minipig liver microsomes and by pig P450 3A22 with Hill coefficients of 1.2-1.5.These results suggest high skatole 2-oxidation, 3-methyl hydroxylation, and 6-hydroxylation activities of pig P450 2A19 and compensatory skatole oxidations mediated by pig P450 1A2, 2E1, or 3A22 in male minipig liver microsomes.

Novel Cytochrome P450 2C94 Functionally Metabolizes Diclofenac and Omeprazole in Dogs.

Cytochromes P450 (P450s or CYPs) are important drug-metabolizing enzymes. Because dogs are frequently used in drug metabolism studies, knowledge of dog CYP2C enzymes is essential because in humans these enzymes are abundant and play major roles in liver and intestine. The present study identified and characterized novel dog CYP2C94 along with previously identified dog CYP2C21 and CYP2C41. Dog CYP2C21, CYP2C41, and CYP2C94 cDNAs, respectively, contained open reading frames of 490, 489, and 496 amino acids and shared high-sequence identities (70%, 75%, and 58%) with human CYP2Cs. Dog CYP2C94 mRNA was preferentially expressed in liver, just as dog CYP2C21 and CYP2C41 mRNAs were. In dog liver, CYP2C21 mRNA was the most abundant, followed by CYP2C94 and CYP2C41 mRNAs. Moreover, the hepatic expressions of all three dog CYP2C mRNAs varied in four individual dogs, two of which did not express CYP2C41 mRNA. The three dog CYP2C genes had similar gene structures, and CYP2C94, although located on the same chromosome, was in a genomic region far from the gene cluster containing CYP2C21 and CYP2C41 Metabolic assays with recombinant proteins showed that dog CYP2C94, along with CYP2C21 and CYP2C41, efficiently catalyzed oxidations of diclofenac, warfarin, and/or omeprazole, indicating that dog CYP2C94 is a functional enzyme. Novel dog CYP2C94 is expressed abundantly in liver and encodes a functional enzyme that metabolizes human CYP2C substrates; it is, therefore, likely responsible for drug clearances in dogs. SIGNIFICANCE STATEMENT: Novel dog cytochrome P450 2C94 (CYP2C94) was identified and characterized along with dog CYP2C21 and CYP2C41. Dog CYP2C94, isolated from liver, had 58% sequence identity and a close phylogenetic relationship with its human homologs and was expressed in liver at the mRNA level. Dog CYP2C94 (and CYP2C21 and CYP2C41) catalyzed oxidations of diclofenac and omeprazole, human CYP2C9 and CYP2C19 substrates, respectively, but CYP2C41 also hydroxylated warfarin. CYP2C94 is therefore a functional drug-metabolizing enzyme likely responsible for drug clearances in dogs.

Newly identified cytochrome P450 3A genes of tree shrews and pigs are expressed and encode functional enzymes.

Novel cytochrome P450 3A5 (CYP3A5) cDNA in tree shrews (which are non-rodent primate-like species) and pig CYP3A227 cDNA were identified, along with known pig CYP3A22, CYP3A29, and CYP3A46 cDNAs. All five cDNAs contained open reading frames encoding a polypeptide of 503 amino acids that shared high sequence identity (72-78 %) with human CYP3A4 and were more closely related to human CYP3As than rat CYP3As by phylogenetic analysis. CYP3A5 was the only CYP3A in the tree shrew genome, but pig CYP3A genes formed a CYP3A gene cluster in the genomic region corresponding to that of human CYP3A genes. Tree shrew CYP3A5 mRNA was predominantly expressed in liver and small intestine, among the tissues analyzed, whereas pig CYP3A227 mRNA was most abundantly expressed in jejunum, followed by liver. Metabolic assays established that tree shrew CYP3A5 and pig CYP3A proteins heterologously expressed in Escherichia coli metabolized typical human CYP3A4 substrates nifedipine and midazolam. These results suggest that novel tree shrew CYP3A5 and pig CYP3A227 were functional enzymes able to metabolize human CYP3A4 substrates in liver and small intestine, similar to human CYP3A4, although pig CYP3A227 mRNA was minimally expressed in all tissues analyzed.

Newly Identified Tree Shrew Cytochrome P450 2A13 is Expressed in Liver and Lung and Encodes a Functional Drug-Metabolizing Enzyme Similar to Dog Cytochrome P450 2A13 and Pig Cytochrome P450 2A19.

The tree shrew, a non-rodent primate-like species, is used in various fields of biomedical research, including hepatitis virus infection, myopia, depression, and toxicology. Recent genome analysis found that the numbers of cytochrome P450 (P450 or CYP) genes are similar in tree shrews and humans and their sequence identities are high. Although the P450s are a family of important drug-metabolizing enzymes, they have not yet been fully investigated in tree shrews. In the current study, tree shrew CYP2A13 cDNA was isolated from liver, and its characteristics were compared with those of pig, dog, and human CYP2As. Tree shrew CYP2A13 amino acid sequences were highly identical (87-92%) to the human CYP2As and contained sequence motifs characteristic of P450s. Phylogenetic analysis revealed that tree shrew CYP2A13 was more closely related to human CYP2As than to rat CYP2As, similar to dog and pig CYP2As. Among the tissue types analyzed, tree shrew CYP2A13 mRNA was preferentially expressed in liver and lung, similar to dog CYP2A13 mRNA, whereas dog CYP2A25 and pig CYP2A19 mRNAs were predominantly expressed in liver. Tree shrew liver microsomes and tree shrew CYP2A13 proteins heterologously expressed in Escherichia coli catalyzed coumarin 7-hydroxylation and phenacetin O-deethylation, just as human, dog, and pig CYP2A proteins and liver microsomes do. These results demonstrate that tree shrew CYP2A13 is expressed in liver and lung and encodes a functional drug-metabolizing enzyme. SIGNIFICANCE STATEMENT: Novel tree shrew cytochrome P450 2A13 (CYP2A13) was identified and characterized in comparison with human, dog, and pig CYP2As. Tree shrew CYP2A13 isolated from liver had high sequence identities and close phylogenetic relationships to its human homologs and was abundantly expressed in liver and lung at the mRNA level. Tree shrew CYP2A13 metabolized coumarin and phenacetin, human selective CYP2A6 and CYP2A13 substrates, respectively, similar to dog and pig CYP2As, and is a functional drug-metabolizing enzyme likely responsible for drug clearances.

A Comprehensive Investigation of Dog Cytochrome P450 3A (CYP3A) Reveals a Functional Role of Newly Identified CYP3A98 in Small Intestine.

Dogs are frequently used in drug metabolism studies, and their important drug-metabolizing enzymes, including cytochromes P450 (P450), have been analyzed. In humans, CYP3A4 is an especially important P450 due to its abundance and major roles in liver and intestine. In the present study, dog CYP3A98 and CYP3A99 were identified and characterized, along with previously identified CYP3A12 and CYP3A26. The dog CYP3A cDNAs contained open reading frames of 503 amino acids and shared high sequence identity (78%-80%) with human CYP3As. Among the dog CYP3A mRNAs, CYP3A98 mRNA was expressed most abundantly in small intestine. In contrast, dog CYP3A12 and CYP3A26 mRNAs were expressed in liver, where CYP3A12 mRNA was the most abundant. The four CYP3A genes had similar gene structures and formed a gene cluster in the dog and human genomes. Metabolic assays of dog CYP3A proteins heterologously expressed in Escherichia coli indicated that the dog CYP3As tested were functional enzymes with respect to typical human CYP3A4 substrates. Dog CYP3A98 efficiently catalyzed oxidations of nifedipine, alprazolam, and midazolam, indicating major roles of CYP3A98 in the small intestine. Dog CYP3A12 and CYP3A26 metabolizing nifedipine and/or midazolam would play roles in these reactions in the liver. In contrast, dog CYP3A99 showed minimal mRNA expression and minimal metabolic activity, and its contribution to overall drug metabolism is, therefore, negligible. These results indicated that newly identified dog CYP3A98, a testosterone 6 ß - and estradiol 16 α -hydroxylase, was abundantly expressed in the small intestine and is likely the major CYP3A in the small intestine in combination with liver-specific CYP3A12. SIGNIFICANCE STATEMENT: Novel dog cytochromes P450 3A98 (CYP3A98) and CYP3A99 were identified and characterized to be functional and highly identical to human CYP3A4. Known CYP3A12 and new CYP3A98 efficiently catalyzed estradiol 16α-hydroxylation and midazolam 1'-hydroxylation. CYP3A98 mRNA was expressed in small intestine, whereas CYP3A12 mRNA was predominant in liver. Dog hepatic CYP3A12 and intestinal CYP3A98 are the enzymes likely responsible for the metabolic clearances of orally administered drugs, unlike human CYP3A4/5, which are in both the liver and intestine.

Novel cytochrome P450 1 (CYP1) genes in tree shrews are expressed and encode functional drug-metabolizing enzymes.

Tree shrews (Tupaia belangeri) are a non-rodent primate-like species sometimes used for biomedical research involving hepatitis virus infections and toxicology. Genome analysis has indicated similarities between tree shrews and humans in the numbers of cytochromes P450 (P450 or CYP), which constitute a family of important drug-metabolizing enzymes; however, P450s have not been fully investigated in tree shrews. In this study, we identified CYP1A1, CYP1A2, CYP1B1, and CYP1D1 cDNAs from tree shrew liver and compared their characteristics with dog, pig, and human CYP1As. The deduced amino acid sequences of tree shrew CYP1s were highly identical (82-87 %) to human CYP1s. In tree shrews, CYP1A1 and CYP1A2 mRNAs were preferentially expressed in liver, whereas CYP1D1 mRNA was preferentially expressed in kidney and lung. In contrast, CYP1B1 mRNA was expressed in various tissues, with the most abundant expression in spleen. Among the tree shrew CYP1 mRNAs, CYP1A2 mRNA was most abundant in liver, and CYP1B1 mRNA was most abundant in kidney, small intestine, and lung. All tree shrew CYP1 proteins heterologously expressed in Escherichia coli catalyzed caffeine and estradiol in a similar manner to tree shrew liver microsomes and human, dog, and pig CYP1 proteins. These results suggest that tree shrew CYP1A1, CYP1A2, CYP1B1, and CYP1D1 genes, different form human pseudogene CYP1D1P, are expressed in liver, small intestine, lung, and/or kidney and encode functional drug-metabolizing enzymes important in toxicology.