Development of genotyping method for functionally relevant variants of cytochromes P450 in cynomolgus macaques

In cynomolgus macaques (Macaca fascicularis), widely used in drug metabolism studies, CYP2C9, CYP2C76, CYP2D6, CYP3A4, and CYP3A5, important drug‐metabolizing enzymes, are abundantly expressed in liver and metabolize cytochrome P450 substrates. CYP2C9 (c.334A>C), CYP2C76 (c.449TG>A), CYP2D6 (c.891A>G), CYP3A4 (IVS3 + 1G>del), and CYP3A5 (c.625A>T) substantially influence metabolic activity of enzymes, and thus are important variants in drug metabolism studies. In this study, a real‐time PCR method was developed for genotyping these variants. The validity of the methods was verified by genotyping two wild type, two heterozygous, and two homozygous DNAs and was used to genotype 41 cynomolgus macaques (from Cambodia, Indonesia, the Philippines, or Vietnam) for the five variants, along with another important variant CYP2C19 (c.308C>T). The CYP2C9 and CYP2C19 variants were found only in Cambodian and Vietnamese animals, while the CYP2C76 and CYP2D6 variants were found only in Indonesian and Philippine animals. The CYP3A4 and CYP3A5 variants were not found in any of the animals analyzed. Mauritian animals, genotyped using next‐generation sequencing data for comparison, possessed the CYP2C19 and CYP2D6 variants, but not the other variants. These results indicated differences in prevalence of these important variants among animal groups. Therefore, the genotyping tool developed is useful for drug metabolism studies using cynomolgus macaques.     

Isolation and characterization of arylacetamide deacetylase in cynomolgus macaques

Arylacetamide deacetylase (AADAC), a microsomal serine esterase, hydrolyzes drugs, such as flutamide, phenacetin and rifampicin. Because AADAC has not been fully investigated at molecular levels in cynomolgus macaques, the non-human primate species widely used in drug metabolism studies, cynomolgus AADAC cDNA was isolated and characterized. The deduced amino acid sequence, highly homologous (92%) to human AADAC, was more closely clustered with human AADAC than the dog, rat or mouse ortholog in a phylogenetic tree. AADAC was flanked by AADACL2 and SUCNR1 in the cynomolgus and human genomes. Moreover, relatively abundant expression of AADAC mRNA was found in liver and jejunum, the drug-metabolizing organs, in cynomolgus macaques, similar to humans. The results suggest molecular similarities of AADAC between cynomolgus macaques and humans.     

Identification and Analysis of CYP7A1, CYP17A1, CYP20A1, CYP27A1 and CYP51A1 in Cynomolgus Macaques

Cytochromes P450 (P450) are important for not only drug metabolism and toxicity, but also biosynthesis and metabolism of cholesterol and bile acids, and steroid synthesis. In cynomolgus macaques, widely used in biomedical research, we have characterized P450 cDNAs, which were isolated as expressed sequence tags of cynomolgus macaque liver. In this study, cynomolgus CYP7A1, CYP17A1, CYP20A1, CYP27A1 and CYP51A1 cDNAs were characterized by sequence analysis, phylogenetic analysis and tissue expression pattern. By sequence analysis, these five cynomolgus P450s had high sequence identities (94–99%) to the human orthologs in amino acids. By phylogenetic analysis, each cynomolgus P450 was more closely related to the human ortholog as compared with the dog or rat ortholog. By quantitative polymerase chain reaction, among the 10 tissue types, CYP7A1 and CYP17A1 mRNAs were preferentially expressed in liver and adrenal gland, respectively. Cynomolgus CYP27A1 and CYP51A1 mRNAs were most abundantly expressed in liver and testis, respectively. Cynomolgus CYP20A1 mRNA was expressed in all the tissues, including brain and liver. Tissue expression patterns of each cynomolgus P450 were generally similar to that of the human ortholog. These results suggest the molecular similarities of CYP7A1, CYP17A1, CYP20A1, CYP27A1 and CYP51A1 between cynomolgus macaques and humans.     

CYP1B1 is polymorphic in cynomolgus and rhesus macaques

Cytochrome P450 (CYP) 1B1 is involved in the metabolic activation of various procarcinogens, and some CYP1B1 genetic variants alter CYP1B1-dependent procarcinogen metabolism. Cynomolgus and rhesus macaques are frequently used in toxicity tests due to their evolutionary closeness to humans. In this study, we attempted to identify CYP1B1 genetic variants in 13 cynomolgus and 4 rhesus macaques. A total of 17 genetic variants were identified, including 8 non-synonymous genetic variants, indicating that, similar to humans, CYP1B1 is polymorphic in macaques. These CYP1B1 genetic variants could be the basis for understanding potential inter-animal differences in macaque CYP1B1-dependent metabolism of promutagens.     

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.     

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.     

High interindividual variability in plasma clopidogrel active metabolite concentrations in healthy cats is associated with sex and cytochrome P450 2C genetic polymorphism

Clopidogrel response variability has been identified in cats. In humans, evidence suggests that variable clopidogrel active metabolite (CAM) generation is the primary explanation for clopidogrel response variability with differences in body weight, sex, and variable metabolism of clopidogrel primarily due to polymorphisms of the gene encoding cytochrome P450 (CYP) 2C19 as some proposed mechanisms. The aim of this study was to evaluate whether variation in CAM concentrations exists in healthy cats and what the cause of such variation might be. Nineteen healthy cats were given 18.75 mg clopidogrel by mouth. Blood was collected 2 hr later. Plasma CAM concentrations were measured using high performance liquid chromatography and tandem mass spectrometry. Clopidogrel metabolism was estimated by calculating CAM metabolic ratio. DNA was collected, and feline CYP2C genotyping was performed. The cats demonstrated high interindividual variation of plasma CAM concentrations. Approximately 69% of this interindividual variation was primarily explained by differences in clopidogrel metabolism as measured by CAM metabolic ratio with some influence by sex but not by weight. A single nucleotide polymorphism was identified in the feline CYP2C gene that explained in part individual differences in CAM metabolic ratio and CAM plasma concentrations.     

Improvements of Vaginal Atrophy without Systemic Side Effects after Topical Application of Pueraria mirifica,a Phytoestrogen-rich Herb,in Postmenopausal Cynomolgus Macaques

The estrogenic efficacy of topical vaginal application of Pueraria mirifica extract (PM) on the restoration of vaginal atrophy, and the presence of any systemic side effects, were investigated in postmenopausal cynomolgus macaques. Twelve postmenopausal cynomolgus macaques, with complete cessation of menstruation for at least 5 years before start of this experiment, were divided into three groups. They received a topical vaginal application daily of 0.1 or 1% (w/w) PM cream or a conjugated equine estrogen (CEE) cream (a mixture of estrone, equilin, 17β-dihydroequilin, 17α-estradiol and 17α-dihydroequilin at 0.625 mg total estrogen/g cream) for 28 days. Estrogenic efficacy was assessed weekly by vaginal cytology assay and vaginal pH measurement, whilst the plasma luteinizing hormone (LH) and sex skin coloration levels were determined at the end of each treatment period to evaluate the systemic side effects. PM significantly increased the proportion of superficial cells in a dose-dependent manner, with a similar efficacy between 1% (w/w) PM and CEE. Together with increased vaginal maturation, PM decreased the vaginal pH to acidic levels, as observed in the CEE group. PM induced no detected systemic side effects, whilst CEE decreased the plasma LH level and increased the reddish color of the sex skin during the posttreatment period. Topical vaginal treatment with PM stimulated the maturation of the vaginal epithelium without causing systemic side effects in postmenopausal monkeys. The implication is that PM could be a safer alternative to treat vaginal atrophy in postmenopausal women.     

Few alterations in clinical pathology and histopathology observed in a CYP2C18&19 humanized mice model

Background

This study was performed to characterize a gene-addition transgenic mouse containing a BAC (bacterial artificial chromosome) clone spanning the human CYP2C18&19 genes (tg-CYP2C18&19).

Methods

Hemizygous tg-CYP2C18&19, 11 week old mice were compared with wild-type littermates to obtain information regarding clinical status, clinical pathology and anatomical pathology. After one week of clinical observations, blood samples were collected, organs weighed, and tissues collected for histopathology.

Results

In males, the tissue weights were lower in tg-CYP2C18&19 than in wild-type mice for brain (p ≤ 0.05), adrenal glands (p ≤ 0.05) and brown fat deposits (p ≤ 0.001) while the heart weight was higher (p ≤ 0.001). In female tg-CYP2C18&19, the tissue weights were lower for brain (p ≤ 0.001) and spleen (p ≤ 0.001) compared to wild-type females. Male tg-CYP2C18&19 had increased blood glucose levels (p ≤ 0.01) while females had decreased blood triglyceride levels (p ≤ 0.01).

Conclusion

Despite the observed alterations, tg-CYP2C18&19 did not show any macroscopic or microscopic pathology at the examined age. Hence, these hemizygous transgenic mice were considered to be viable and healthy animals.     

Evaluation of tizanidine as a marker of canine CYP1A2 activity

The dog CYP1A2 enzyme is likely an important contributor to the metabolism of veterinary drugs. Dog CYP1A2 is expressed in liver, plus it is inducible and polymorphic, creating the potential for intersubject differences in pharmacokinetics. Hence, the ability to probe dog CYP1A2 activity and inhibition is relevant toward veterinary drug development and drug–drug interaction assessment. Previous studies have relied on human probes with questionable specificity for CYP1A2, so it was hypothesized that recombinant CYP1A2 could be used to find a specific CYP1A2 substrate. Intrinsic clearance experiments demonstrated that tizanidine was a substrate of CYP1A2. Profiling of tizanidine metabolites generated by CYP1A2 identified the imidazole metabolite that was detectable in dog plasma. The imidazole metabolite was subsequently used to evaluate tizanidine as a CYP1A2 probe. Co‐administration of the CYP1A inhibitor enrofloxacin with tizanidine significantly decreased (30%; n = 3) the formation of the imidazole metabolite vs. control experiments. As enrofloxacin is a weak inhibitor, further studies are required to confirm the sensitivity of tizanidine as an in vivo probe. However, tizanidine may be a more selective CYP1A2 probe than phenacetin when conducting in vitro studies due to the presence of other phenacetin‐metabolizing enzymes in dog liver microsomes.     

Real-time PCR genotyping assay for feline erythrocyte pyruvate kinase deficiency and mutant allele frequency in purebred cats in Japan

Erythrocyte pyruvate kinase (PK) deficiency is an inherited glycolytic erythroenzymopathy caused by mutations of the PKLR gene. A causative mutation of the feline PKLR gene was originally identified in Abyssinian and Somali cats in the U.S.A. In the present study, a TaqMan probe-based real-time PCR genotyping assay was developed and evaluated for rapid genotyping and large-scale screening for this mutation. Furthermore, a genotyping survey was carried out in a population of four popular purebred cats in Japan to determine the current mutant allele frequency. The assay clearly displayed all genotypes of feline PK deficiency, indicating its suitability for large-scale survey as well as diagnosis. The survey demonstrated that the mutant allele frequency in Abyssinian and Somali cats was high enough to warrant measures to control and prevent the disease. The mutant allele frequency was relatively low in Bengal and American Shorthair cats; however, the testing should still be carried out to prevent the spread of the disease. In addition, PK deficiency should always be considered in the differential diagnosis of anemia in purebred cats in Japan as well as worldwide.     

RNAi-mediated knockdown of INHBB increases apoptosis and inhibits steroidogenesis in mouse granulosa cells

Inhibins are members of the TGFβ superfamily and act as suppressors of follicle stimulating hormone (FSH) secretion from pituitary glands via a negative feedback mechanism to regulate folliculogenesis. In this study, the INHBB gene was knocked down by three RNAi-Ready pSIREN-RetroQ-ZsGreen vector- mediated recombinant plasmids to explore the effects of INHBB silencing on granulosa cell (GC) cell cycle, apoptosis and steroid production in vitro. Quantitative real-time polymerase chain reaction, Western blot, flow cytometry and ELISA were performed to evaluate the role of INHBB in the mouse GC cell cycle, apoptosis and steroid production in vitro. The results showed that the relative mRNA and protein expression of INHBB in mouse GCs can be significantly reduced by RNAi with pshRNA-B1, pshRNA-B2 and pshRNA-B3 plasmids, with pshRNA-B3 having the best knockdown efficiency. Downregulation of the expression of INHBB significantly arrests cells in the G1 phase of the cell cycle and increases the apoptosis rate in GCs. This was further confirmed by downregulation of the protein expressions of Cyclin D1, Cyclin E and Bcl2, while the protein expression of Bax was upregulated. In addition, specific downregulation of INHBB markedly decreased the concentration of estradiol and progesterone, which was further validated by the decrease in the mRNA levels of CYP19A1and CYP11A1. These findings suggest that inhibin βB is important in the regulation of apoptosis and cell cycle progression in granulosa cells. Furthermore, the inhibin βB subunit has a role in the regulation of steroid hormone biosynthesis. Evidence is accumulating to support the concept that inhibin βB is physiologically essential for early folliculogenesis in the mouse.     

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.     

The Incidence of Lymphoplasmacytic Gastritis in the Fundus and Antrum of Cynomolgus Monkey ( Macaca fascicularis ) Stomachs

Lymphoplasmacytic gastritis is a concern for toxicological pathologists reading preclinical, non-human primate toxicity studies because subtle gastric changes which could be treatment-related may be masked and gastritis lesions may be confused with treatment-related effects and thus a gastric finding may be incorrectly assigned as a treatment-related lesion. This paper discusses the incidence of lymphoplasmacytic gastritis in cynomolgus monkeys at a contract research organization. The incidence of lymphoplasmacytic gastritis in the fundus and antrum of control cynomolgus monkeys on 18 non-gastric compound studies, was scored. The average fundus score ranged from 0.3 to 1.5 and the average antral score ranged from 0.9 to 3.5 in the cynomolgus monkey stomachs examined. The number of affected control animals in a study ranged from 0 to 5 control animals. No correlation between the route of vehicle administration and the severity or incidence of the lesions was noted. The percentage incidence of affected animals ranged from 0 to 100%. An increased incidence lymphoplasmatic gastritis from 2000 to 2004 was noted. The implications of lymphoplasmacytic gastritis in cynomolgus monkeys used for acute toxicity studies are discussed.     

Impact of age on hepatic cytochrome P450 of domestic male Camborough‐29 pigs

Swine is not only an important species in veterinary medicine research but also a popular animal model for human drug discovery. It is valuable to understand the impact of pig age on abundance and activity of porcine hepatic cytochrome P450 (CYP450). Liver microsomes were prepared from Camborough‐29 intact male pigs at the age of 1 day and 2 weeks and the castrated male pigs at the age of 5, 10, and 20 weeks. Hepatic CYP450 content in the liver microsomes was measured using a UV/visible spectroscopic method. The activities of CYP450s were evaluated by metabolism of phenacetin, coumarin, tolbutamide, bufuralol, chlorzoxazone, and midazolam. The porcine hepatic CYP450 content increased with age with a plateau between age 2 and 5 weeks. Activities of all CYP450 enzymes increased with age of pigs too. The bufuralol 1’‐hydroxylase showed the highest hepatic activities compared with other CYP enzymes at all ages of pigs. The average activities at the age of 20 weeks were about five times higher than those at the age of 5 weeks for most of the CYP enzymes. With compensation of the ratio of liver to body weights, the overall CYP450 metabolism capability of the pigs may be peaked around ages of 10 to 20 weeks. Those findings suggest that metabolism can be significantly different in growing phase of pigs and that the age may be an important factor in porcine medicine evaluation and pig model development.     

Identification of a novel miRNA from the ovine ovary by a combinatorial approach of bioinformatics and experiments

MicroRNAs (miRNAs) are a class of short endogenous, single-stranded, non-coding small RNA molecules, about 19–25 nucleotides in length that regulate gene expression at the translation level and influence many physiological process, such apoptosis, metabolism, signal transduction, and occurrence and development of diseases. In this study, we constructed a library from the ovine luteal phase ovary by using next-generation sequencing technology (Solexa high-throughput sequencing technique) and identified 267 novel miRNAs by bioinformatics. One of the novel miRNAs (ovis_aries_ovary-m0033_3p), which expressed in the sheep ovary and testis, was confirmed by real time PCR and northern blot. Ovis_aries_ovary-m0033_3p was 21 nucleotides in length and located on chromosome 12, and it had 100% similarity to hsa-miR-214-3p, mmu-miR-214-3p, dre-miR-214and ssc-miR-214. Meanwhile, the pre-miRNA was 82 nucleotides in length and had a standard hairpin stem-loop structure. From the consistency of the sequence and structure, we speculated that ovis_aries_ovary-m0033_3p had a function similar to hsa-miR-214-3p, which is involved in the fine regulation of cell survival, embryonic development, breeding activities and resistance to ovarian cancer, so we defined it as oar-miR-214-3p. These experimental results will enrich the miRNA database for ovis aries and provide the basis for researching the regulation mechanism of miRNA in relation to breeding activities of seasonal breeding animals.     

SNP genetic polymorphisms of MDR-1, CYP1A2 and CYPB11 genes in four canine breeds upon toxicological evaluation

The fields of pharmacogenetics and pharmacogenomics have become increasingly promising regarding the clinical application of genetic data to aid in prevention of adverse reactions. Specific screening tests can predict which animals express modified proteins or genetic sequences responsible for adverse effects associated with a drug. Among the genetic variations that have been investigated in dogs, the multidrug resistance gene (MDR) is the best studied. However, other genes such as CYP1A2 and CYP2B11 control the protein syntheses involved in the metabolism of many drugs. In the present study, the MDR-1, CYP1A2 and CYP2B11 genes were examined to identify SNP polymorphisms associated with these genes in the following four canine breeds: Uruguayan Cimarron, Border Collie, Labrador Retriever and German Shepherd. The results revealed that several SNPs of the CYP1A2 and CYP2B11 genes are potential targets for drug sensitivity investigations.     

CYP2C19基因多态性与肿瘤易感性关系的研究进展

细胞色素氧化酶P4502C19(CYP2C19)又称S-美芬妥英羟化酶,是一种重要的肝微粒体酶,该酶的羟化代谢在人群中呈二态分布,即S-美芬妥英4′-羟化强代谢者( EMs)和弱代谢者( PMs).CYP2C19ml和CYP2C19m2突变是PMs产生的主要原因.CYP2C19表型和基因型的遗传多态性决定了不同个体对致癌物代谢的差异性和肿瘤的化学致癌易感性.CYP2C19与消化道肿瘤(包括食管癌、胃癌、肝癌)、肺癌、急性白血病、膀胱癌等发病有关.通常认为CYP2C19的PM个体可能存在肿瘤易感性,但是膀胱癌的PM发生率显著低于正常对照组.论文综述了近年来CYP2C19基因多态性和肿瘤易感性的相关研究.     

Genotype frequency of ATP7A and ATP7B mutation-related copper-associated hepatitis in a Japanese guide dog Labrador retriever population

The incidence of copper-associated hepatitis in Labrador retriever in Japan has not been examined. This study examined the genotype frequencies of ATP7B:c.4358G>A, a mutation responsible for copper-associated hepatitis, and ATP7A:c.980C>T, a modifier of this disease, in Labrador retrievers of guide dog associations in Japan. Genetic material was collected by buccal swabs from 253 Labrador retrievers and genotyping was performed for the ATP7B and ATP7A mutations. The gene frequency was 0.107 for ATP7B:c.4358A. For ATP7A:c.980C, the gene frequencies were 0.703 in females and 0.368 in males. In this study, we established genotyping methods for the ATP7B:c.4358G>A and ATP7A:c.980C>T mutations. Based on the genotyping results, the risk of copper-associated hepatitis in the study population was 0.80% in males and 1.05% in females.     

Laser induced injury caused hyperglycemia-like effect in Drosophila larva: a possible insect model for posttraumatic diabetes

Diabetic patients need particular care in case of infection, digestive disorder or external injury, because external stress often exasperates the glucose metabolism, which is known as “sick day management”. In addition, severe trauma can be a cause of hyperglycemia with insulin resistance. In spite of critical component of the treatment, the precise mechanisms of how trauma develops posttraumatic diabetes remain unknown. Here, we ablated body wall muscles in Drosophila larvae by laser beam and found that the level of trehalose, the principal sugar circulating in the hemolymph or in the tissues of most insects, was increased. The model may provide a helpful tool to understand the relationship between trauma and sugar metabolism.