Masahiro Hiratsuka

Functional characterization of 32 CYP2C9 allelic variants

Genetic variations in cytochrome P450 2C9 (CYP2C9) contribute to interindividual variability in the metabolism of clinically used drugs such as warfarin and tolbutamide. We functionally characterized 32 types of allelic variant CYP2C9 proteins. Recombinant CYP2C9 proteins generated using a heterologous expression system are useful for comparing functional changes in CYP2C9 variant proteins expressed from low-frequency alleles. Wild-type CYP2C9 and its 31 variants were found to be transiently expressed in COS-7 cells, and the enzymatic activity of the CYP2C9 variants was characterized using S-warfarin as a representative substrate. Among the 32 types of CYP2C9 allelic variants tested, CYP2C9.18, CYP2C9.21, CYP2C9.24, CYP2C9.26, CYP2C9.33 and CYP2C9.35 exhibited no enzyme activity, and 12 types showed significantly decreased enzyme activity. In vitro analysis of CYP2C9 variant proteins should be useful for predicting CYP2C9 phenotypes and for application to personalized drug therapy.

Functional Characterization of CYP2B6 Allelic Variants in Demethylation of Antimalarial Artemether

Artemether (AM) is one of the most effective antimalarial drugs. The elimination half-life of AM is very short, and it shows large interindividual variability in pharmacokinetic parameters. The aim of this study was to identify cytochrome P450 (P450) isozymes responsible for the demethylation of AM and to evaluate functional differences between 26 CYP2B6 allelic variants in vitro. Of 14 recombinant P450s examined in this study, CYP2B6 and CYP3A4 were primarily responsible for production of the desmethyl metabolite dihydroartemisinin. The intrinsic clearance (Vmax/Km) of CYP2B6 was 6-fold higher than that of CYP3A4. AM demethylation activity was correlated with CYP2B6 protein levels (P = 0.004); however, it was not correlated with CYP3A4 protein levels (P = 0.27) in human liver microsomes. Wild-type CYP2B6.1 and 25 CYP2B6 allelic variants (CYP2B6.2-CYP2B6.21 and CYP2B6.23-CYP2B6.27) were heterologously expressed in COS-7 cells. In vitro analysis revealed no enzymatic activity in 5 variants (CYP2B6.8, CYP2B6.12, CYP2B6.18, CYP2B6.21, and CYP2B6.24), lower activity in 7 variants (CYP2B6.10, CYP2B6.11, CYP2B6.14, CYP2B6.15, CYP2B6.16, CYP2B6.20, and CYP2B6.27), and higher activity in 4 variants (CYP2B6.2, CYP2B6.4, CYP2B6.6, and CYP2B6.19), compared with that of wild-type CYP2B6.1. In kinetic analysis, 3 variants (CYP2B6.2, CYP2B6.4, and CYP2B6.6) exhibited significantly higher Vmax, and 3 variants (CYP2B6.14, CYP2B6.20 and CYP2B6.27) exhibited significantly lower Vmax compared with that of CYP2B6.1. This functional analysis of CYP2B6 variants could provide useful information for individualization of antimalarial drug therapy.

Glucagon-like peptide-1 production in the GLUTag cell line is impaired by free fatty acids via endoplasmic reticulum stress

OBJECTS Glucagon-like peptide-1 (GLP-1) is secreted from intestinal L cells, enhances glucose-stimulated insulin secretion, and protects pancreas beta cells. However, few studies have examined hypernutrition stress in L cells and its effects on their function. Here, we demonstrated that a high-fat diet reduced glucose-stimulated secretion of GLP-1 and induced expression of an endoplasmic reticulum (ER) stress markers in the intestine of a diet-induced obesity mouse model. METHODS To clarify whether ER stress in L cells caused the attenuation of GLP-1 secretion, we treated the mouse intestinal L cell line, GLUTag cells with palmitate or oleate. RESULTS Palmitate, but not oleate caused ER stress and decreased the protein levels of prohormone convertase 1/3 (PC1/3), an essential enzyme in GLP-1 production. The same phenomena were observed in GLUTag cells treated with in ER stress inducer, thapsigargin. Moreover, oleate improved palmitate-induced ER stress, reduced protein and activity levels of PC1/3, and attenuated GLP-1 secretion from GLUTag cells. CONCLUSIONS/INTERPRETATION These results suggest that the intake of abundant saturated fatty acids induces ER stress in the intestine and decreases GLP-1 production.

Evaluation of Influence of Single Nucleotide Polymorphisms in Cytochrome P450 2B6 on Substrate Recognition Using Computational Docking and Molecular Dynamics Simulation

In this study, we investigated the influence of single nucleotide polymorphisms on the conformation of mutated cytochrome P450 (CYP) 2B6 proteins using molecular dynamics (MD) simulation. Some of these mutations influence drug metabolism activities, leading to individual variations in drug efficacy and pharmacokinetics. Using computational docking, we predicted the structure of the complex between the antimalarial agent artemether and CYP2B6 whose conformations were obtained by MD simulation. The simulation demonstrated that the entire structure of the protein changes even when a single residue is mutated. Moreover, the structural flexibility is affected by the mutations and it may influence the enzyme activity. The results suggest that some of the inactive mutants cannot recognize artemether due to structural changes caused by the mutation.

Induction of Thymic Stromal Lymphopoietin Production by Xylene and Exacerbation of Picryl Chloride-Induced Allergic Inflammation in Mice

Background: Some chemical compounds in the environment worsen allergic inflammation. In this study, we examined whether organic solvents induce the production of thymic stromal lymphopoietin (TSLP) which elicits Th2-type immune responses. Methods: Organic solvents were painted on the earlobes of BALB/c mice. The expression of TSLP in the ear was determined by ELISA. Results: Xylene and toluene, but not chloroform or ethyl acetate, induced the expression of mRNA for TSLP in the earlobe tissue. Among the aromatic compounds, xylene, especially m-xylene, and trimethylbenzene caused apparent TSLP production. The level of TSLP in the xylene-treated earlobes reached a maximum at 24 h, and TSLP was expressed in epithelial tissues. Production of TSLP was unaffected in mast cell-deficient W/Wv mice but apparently diminished in TNF-α knockout mice and IL-4 receptor knockout mice. Repeated painting of xylene for 7 days induced an increase in the weight of cervical lymph nodes and expression of OX40 ligand, both of which were inhibited in TSLP receptor knockout mice. Xylene promoted the picryl chloride-induced thickening of the ear and IL-4 production, which were reversed in TSLP receptor knockout mice. Conclusion: Xylene induced TSLP production, resulting in an exacerbation of allergic inflammation. Thus, xylene might be a good tool for examining the roles of TSLP in eliciting allergy in experimental animals.

Regulation of dipeptidyl peptidase 4 production in adipocytes by glucose

Objective Type 1 and 2 diabetes are characterized by elevated blood glucose levels and increased dipeptidyl peptidase 4 (DPP4) activity levels in the serum. However, previous studies reported a negative correlation between glucose concentrations and DPP4 levels. The purpose of this study was to elucidate the connection between glucose and DPP4 in adipocytes under physiological and diabetic conditions, because DPP4 is an adipokine. Methods Blood glucose and serum DPP4 levels were measured, and adipocytes were collected from mice under normal, high-fat diet fed, and diabetic conditions. The adipocytes obtained were incubated for 24 hours in medium containing 5.5 or 25 mM glucose, and 3T3-L1 preadipocytes were differentiated under 5.5 or 25 mM glucose. Adipocytes from mice and 3T3-L1 were stimulated by tumor necrosis factor-α (TNF-α) for 24 hours. The levels of released and intracellular DPP4 were determined by enzyme-linked immunosorbent assay. Results Mice fed high-fat diet had lower serum DPP4 levels in the first and second week than controls. However, this difference gradually disappeared over 6 weeks. The differentiation of 3T3-L1 adipocytes under 25 mM glucose produced lower DPP4 levels than those differentiated under 5.5 mM; this was also observed in isolated adipocytes from mice. However, these effects of glucose were lost in adipocytes from diabetic mice, and an increase in total DPP4 levels was observed. The stimulation of adipocytes with TNF-α increased the release of DPP4 irrespective of glucose concentration. Conclusion The production of DPP4 in adipocytes was negatively regulated by 25 mM glucose under physiological conditions, but not in diabetic mice. Our results suggest that the observed increase in serum DPP4 levels may be attributed to increased production of DPP4 in adipocytes and an enhancement in TNF-α-induced release.

Induction of Thymic Stromal Lymphopoietin Production by Nonanoic Acid and Exacerbation of Allergic Inflammation in Mice

BACKGROUND Thymic stromal lymphopoietin (TSLP) plays critical roles in the induction and exacerbation of allergic diseases. We tested various chemicals in the environment and found that xylene and 1,2,4-trimethylbenzene induced the production of TSLP in vivo. These findings prompted us to search for additional chemicals that induce TSLP production. In this study, we examined whether fatty acids could induce the production of TSLP in vivo and exacerbate allergic inflammation. METHODS Various fatty acids and related compounds were painted on the ear lobes of mice and the amount of TSLP in the homogenate of ear lobe tissue was determined. The effects of nonanoic acid on allergic inflammation were also examined. RESULTS Octanoic acid, nonanoic acid, and decanoic acid markedly induced TSLP production, while a medium-chain aldehyde and alcohol showed only weak activity. Nonanoic acid induced the production of TSLP with a maximum at 24 h. TSLP production was even observed in nonanoic acid-treated C3H/HeJ mice that lacked functional toll-like receptor 4. The aryl hydrocarbon receptor agonist β-naphthoflavone did not induce TSLP production. Nonanoic acid promoted sensitization to ovalbumin, resulting in an enhancement in the cutaneous anaphylactic response. In addition, painting of nonanoic acid after the sensitization augmented picryl chloride-induced thickening of the ear, which was reversed in TSLP receptor-deficient mice. CONCLUSIONS Nonanoic acid and certain fatty acids induced TSLP production, resulting in the exacerbation of allergic inflammation. We propose that TSLP-inducing chemical compounds such as nonanoic acid be recognized as chemical allergo-accelerators.

Functional characterization of 21 CYP2C19 allelic variants for clopidogrel 2-oxidation

Genetic variations in cytochrome P450 2C19 (CYP2C19) contribute to interindividual variability in the metabolism of therapeutic agents such as clopidogrel. Polymorphisms in CYP2C19 are associated with large interindividual variations in the therapeutic efficacy of clopidogrel. This study evaluated the in vitro oxidation of clopidogrel by 21 CYP2C19 variants harboring amino acid substitutions. These CYP2C19 variants were heterologously expressed in COS-7 cells, and the kinetic parameters of clopidogrel 2-oxidation were estimated. Among the 21 CYP2C19 variants, 12 (that is, CYP2C19.5A, CYP2C19.5B, CYP2C19.6, CYP2C19.8, CYP2C19.9, CYP2C19.10, CYP2C19.14, CYP2C19.16, CYP2C19.19, CYP2C19.22, CYP2C19.24 and CYP2C19.25) showed no or markedly low activity compared with the wild-type protein CYP2C19.1B. This comprehensive in vitro assessment provided insights into the specific metabolic activities of CYP2C19 proteins encoded by variant alleles, and this may to be valuable when interpreting the results of in vivo studies.

Identification of a cell line producing high levels of TSLP: Advantages for screening of anti-allergic drugs

Thymic stromal lymphopoietin (TSLP) plays critical roles in the induction and exacerbation of allergic diseases. These findings suggest that an inhibitor of TSLP production may be a novel drug for allergic diseases. However, conducting high-throughput screening of such compounds is difficult because there is currently no appropriate in vitro system. In the present study, we demonstrated that the mouse keratinocyte cell line KCMH-1 produced higher amounts of TSLP than the mouse keratinocyte cell line PAM-212, human keratinocyte cell line HaCaT, or bronchial cell line BEAS-2B. A reporter gene assay revealed that transcriptional activity of the TSLP gene was also markedly higher in KCMH-1 than in PAM212 cells. Both dexamethasone and the retinoid X receptor agonist HX600 inhibited the production of TSLP in KCMH-1 cells, which indicated that its production could be pharmacologically regulated. Moreover, the biological activity of TSLP released from KCMH-1 cells in the medium was endorsed by the induction of OX40L expression in bone marrow-derived dendritic cells. These results indicate that KCMH-1 can be utilized in high-throughput screening of inhibitors of TSLP production and also as a source of native TSLP.

Enhancement of nickel elution by lipopolysaccharide-induced inflammation

BACKGROUND Implantations of metallic biomedical devices into bodies are increasing. The elution of Ni ions from these devices can lead to metal allergies. However, the molecular mechanisms of the elution have not been fully examined. Furthermore, it is not clear whether infection and inflammation affect the corrosion of metals. OBJECTIVE We examined whether the elution of Ni from metal wires and plates was enhanced by inflammation in vivo and in vitro. METHODS A Ni or SUS316L wire was implanted subcutaneously in the dorsum of mice. Lipopolysaccharide (LPS) was injected at the site immediately following the implantation. After 8, 24, and 72 h, the tissue around the wire was excised. RAW 264 cells were seeded on a Ni plate and incubated for 24h in medium containing LPS. The amount of Ni in the tissue or conditioned medium was determined fluorometrically. RESULTS The release of Ni ions from the wire was significantly increased from 8 to 72 h, and further increased by LPS. LPS also enhanced the release of Ni ions by the cells, but only when they were attached to the Ni plate. Chloroquine, bafilomycin A(1) and amiloride markedly inhibited the effects of LPS. CONCLUSION The activation of inflammatory cells on metals enhanced the elution of Ni probably via the release of protons at the interface of the cells and material.