RESUMEN
CYP2C19 is a member of the human microsomal cytochrome P450 (CYP). Significant variation in CYP2C19 levels and activity can be attributed to polymorphisms in this gene. Wildtype CYP2C19 and 13 mutants (CYP2C19.1B, CYP2C19.5A, CYP2C19.5B, CYP2C19.6, CYP2C19.8, CYP2C19.9, CYP2C19.10, CYP2C19.11, CYP2C19.13, CYP2C19.16, CYP2C19.19, CYP2C19.23, CYP2C19.30, and CYP2C19.33) were coexpressed with NADPH-cytochrome P450 reductase in Escherichia coli. Hydroxylase activity toward testosterone and progesterone was also examined. Ten CYP2C19 variants showed Soret peaks (450 nm) typical of P450 in the reduced CO-difference spectra. CYP2C19.11 and CYP2C19.23 showed higher testosterone 11α, 16α-/17- and progesterone 6ß-,21-,16α-/17α-hydroxylase activities than CYP2C19.1B. CYP2C19.6, CYP2C19.16, CYP2C19.19, and CYP2C19.30 showed lower activity than CYP2C19.1B. CYP2C19.9, CYP2C19.10. CYP2C19.13, and CYP2C19.33 showed different hydroxylation activities than CYP2C19.1B. These results indicated that CYP2C19 variants have very different substrate specificities for testosterone and progesterone.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas , Progesterona , Humanos , Progesterona/metabolismo , Testosterona/metabolismo , Citocromo P-450 CYP2C19/genética , Citocromo P-450 CYP2C19/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Hidrocarburo de Aril Hidroxilasas/metabolismo , HidroxilaciónRESUMEN
CYP2C9 is a human microsomal cytochrome P450c (CYP). Much variation in CYP2C9 levels and activity can be attributed to polymorphisms of this gene. Wild-type CYP2C9 and ten mutants were co-expressed with NADPH-cytochrome P450 reductase in Escherichia coli. The hydroxylase activities toward steroids were examined. CYP2C9.2, CYP2C9.3, CYP2C9.4, CYP2C9.16, CYP2C9.28, CYP2C9.48 and CYP2C9.52 had higher testosterone 6ß-hydroxylation than CYP2C9.1. CYP2C9.4 showed higher progesterone 6ß-hydroxylation activity than CYP2C9.1. CYP2C9.28 and CYP2C9.48 showed higher progesterone 11α-hydroxylation activity than CYP2C9.1. CYP2C9.48 showed higher progesterone 16α-hydroxylation activity than CYP2C9.1. CYP2C9.2, CYP2C9.3, CYP2C9.16 and CYP2C9.30 had higher estrone 16α-hydroxylation activity than CYP2C9.1. CYP2C9.3 had higher estrone 11α-hydroxylation activity than CYP2C9.1. CYP2C9.39 and CYP2C9.57 showed similar activities to CYP2C9.1. These results indicate that the substrate specificity of CYP2C9.39 and CYP2C9.57 was not changed, but CYP2C9.2, CYP2C9.3, CYP2C9.4, CYP2C9.16, CYP2C9.28, CYP2C9.30, CYP2C9.48 and CYP2C9.52 showed different hydroxylation activities toward steroids compared with CYP2C9.1.
Asunto(s)
Citocromo P-450 CYP2C9/metabolismo , Esteroides/metabolismo , Citocromo P-450 CYP2C9/genética , Escherichia coli/genética , Hidroxilación , Polimorfismo de Nucleótido Simple , Proteínas Recombinantes/metabolismo , Esteroide Hidroxilasas/metabolismoRESUMEN
Chlorpyrifos (CPS) is a broad-spectrum organophosphate insecticide that is neurotoxic in humans. Chlorpyrifos oxon (CPO) is a toxic metabolite of CPS that is produced by CYP2B6. In this study, we examined the variability of CPS metabolism resulting from single-nucleotide polymorphisms in CYP2B6. Wild-type CYP2B6 (CYP2B6.1) and two variants each with a single amino acid substitution: CYP2B6.5 (R487C) and CYP2B6.8 (K139E) were co-expressed together with human NADPH-dependent cytochrome P450 reductase in Escherichia coli (E. coli). Both of the CYP2B6 variants were successfully expressed in E. coli. The conversion of CPS to CPO by the CYP2B6 variants was analyzed with high-performance liquid chromatography. Km and Vmax of the reaction by CYP2B6.1 were 18.50±2.94µM and 17.07±1.15mol/min/mol P450, respectively. The CYP2B6 variants produced CPO with the following kinetic parameters: Km for CYP2B6.5 and CYP2B6.8 were 20.44±6.43 and 44.69±9.97µM, respectively; and Vmax were 1.10±0.10 and 1.77±0.26mol/min/mol P450, respectively. These results indicate that the amino acid substitutions in the CYP2B6 variants suppressed the metabolic activation of CPS. CYP2B6 variants have altered capacity to bioactivate CPF and may affect individual susceptibility of CPF.
Asunto(s)
Cloropirifos/análogos & derivados , Citocromo P-450 CYP2B6/genética , Insecticidas/farmacocinética , Isoenzimas/genética , Polimorfismo de Nucleótido Simple , Activación Metabólica , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Western Blotting , Cloropirifos/farmacocinética , Cromatografía Líquida de Alta Presión , Citocromo P-450 CYP2B6/química , Citocromo P-450 CYP2B6/metabolismo , Escherichia coli/genética , Humanos , Isoenzimas/química , Isoenzimas/metabolismo , Cinética , Homología de Secuencia de AminoácidoRESUMEN
Aspergillus oryzae is a fungus widely used in traditional Japanese fermentation industries. Cytochrome P450 (CYP) proteins are ubiquitously distributed in nature and display a broad range of enzymatic activities. A novel CYP52 (CYP52G3) gene was found in A. oryzae. In this study, we report the functional characterization of CYP52G3. The recombinant protein was expressed heterologously in Escherichia coli, and its membrane fraction isolated. CYP52G3 showed activities for 7-ethoxycoumarin and α-naphtoflavone. Furthermore, CYP52G3 hydroxylated flavanone at the 4' and 6 position and metabolized some hydroxyl-flavanones and steroids. Bioconversion experiments indicated that CYP52G3 could convert flavanone and testosterone in a synthetic medium. The conversion rates of flavanone and testosterone at 24 H were 50% and 70%, respectively. These results support that CYP52G3 could prove a useful enzyme for the efficient production of new compounds from flavonoids and steroids.
Asunto(s)
Aspergillus oryzae/enzimología , Sistema Enzimático del Citocromo P-450/química , Flavanonas/química , Proteínas Fúngicas/química , Testosterona/químicaRESUMEN
CYP2C9 is a human microsomal cytochrome P450c (CYP). Much of the variation in CYP2C9 levels and activity can be attributed to polymorphisms of this gene. Wild-type CYP2C9 and mutants were coexpressed with NADPH-cytochrome P450 reductase in Escherichia coli. The hydroxylase activities toward 7-ethoxycoumarin, flavanone and steroids were examined. Six CYP2C9 variants showed Soret peaks (450 nm) typical of P450 in reduced CO-difference spectra. CYP2C9.38 had the highest 7-ethoxycoumarin de-ethylase activity. All the CYP2C9 variants showed lower flavanone 6-hydroxylation activities than CYP2C9.1 (the wild-type). CYP2C9.38 showed higher activities in testosterone 6ß-hydroxylation, progesterone 6ß-/16α-hydroxylation, estrone 11α-hydroxylation and estradiol 6α-hydroxylation than CYP2C9.1. CYP2C9.40 showed higher testosterone 17-oxidase activity than CYP2C9.1; CYP2C9.8 showed higher estrone 16α-hydroxylase activity and CYP2C9.12 showed higher estrone 11α-hydroxylase activity. CYP2C9.9 and CYP2C9.10 showed similar activities to CYP2C9.1. These results indicate that the substrate specificity of CYP2C9.9 and CYP2C9.10 was not changed, but CYP2C9.8, CYP2C9.12 and CYP2C9.40 showed different substrate specificity toward steroids compared with CYP2C9.1; and especially CYP2C9.38 displayed diverse substrate specificities towards 7-ethoxycoumarin and steroids.
Asunto(s)
Cumarinas/metabolismo , Citocromo P-450 CYP2C9/genética , Citocromo P-450 CYP2C9/metabolismo , Flavanonas/metabolismo , Esteroides/metabolismo , 7-Alcoxicumarina O-Dealquilasa/metabolismo , Escherichia coli/genética , Humanos , Hidroxilación , Polimorfismo de Nucleótido SimpleRESUMEN
CYP2A6 is a major hepatic member of the cytochrome P450 family in humans. Much variation in CYP2A6 levels and activity can be attributed to genetic polymorphisms of this gene. CYP2A6*25 comprises an amino acid substitution, F118L. To clarify the effect of the leucine substitution at position 118 in CYP2A6.25, this variant, wild type CYP2A6 and three additional variants consisting of artificial mutations at the substrate binding site (position 481) suggested by earlier reports using random mutagenesis studies [CYP2A6.1, CYP2A6.25, CYP2A6.1(F118A), CYP2A6.1(A481G) and CYP2A6.25(A481G)], were co-expressed with NADPH-cytochrome P450 reductase in E. coli. The hydroxylase activity of these variants toward 7-ethoxycoumarin, coumarin, flavone, α-naphthoflavone, flavanone and hydroxyflavanone were examined. All the mutants had lower activities for coumarin 7-hydroxylation than the wild type. All the mutants showed higher activities for flavone and α-naphthoflavone compared with CYP2A6.1. CYP2A6.1 had the highest flavanone 2'-hydroxylase activity, whereas CYP2A6.25 had the highest 6- and 4'-hydroxylase activities. CYP2A6.1(F118A), CYP2A6.1(A481G) and CYP2A6.25(A481G) had higher flavanone 3'-hydroxylase activities than CYP2A6.1 and CYP2A6.25. Furthermore, 4'-hydroxyflavanone was metabolized by CYP2A6.25. These results indicate that the CYP2A6.25 mutation confers new substrate specificity towards flavonoids.
Asunto(s)
Citocromo P-450 CYP2A6/genética , Citocromo P-450 CYP2A6/metabolismo , Flavonoides/metabolismo , Mutación Puntual , Polimorfismo de Nucleótido Simple , Sustitución de Aminoácidos , Sitios de Unión , Cromatografía Líquida de Alta Presión , Escherichia coli/genética , Hidroxilación , Especificidad por Sustrato , TransfecciónRESUMEN
Tobacco cytochrome P450 (CYP) 71AH11 metabolized the herbicide chlorotoluron, and its mRNA level was increased in tobacco culture cells by the treatment of 2,4-D. In order to clarify molecular mechanisms of induced gene expression of CYP71AH11 by herbicide treatment, a 1574-bp 5'-flanking region of CYP71AH11 was cloned, ligated to the reporter ß-glucuronidase (GUS) gene, and then transformed into tobacco plants. The GUS activity in the transgenic tobacco plants was highly induced by bromoxynil treatment, followed by 2,4-D. Chlorotoluron was slightly increased the GUS activity. The bromoxynil-increased GUS activity was partially repressed by the antioxidants, suggesting that reactive oxygen species may be involved in activation of the 5'-flanking region of CYP71AH11 by bromoxynil treatment. Deletion and mutation assays showed that the region CD (-1281 to -770bp from the start codon of CYP71AH11) was important, but not sufficient, for response to bromoxynil. Electrophoretic mobility shift assays and southwestern blotting revealed that the sequences AAAAAG, and GAACAAAC and GAAAATTC in the CD region were important for interaction to the nuclear proteins of <30 and ≈75 kDa, respectively. Particularly, interaction between AAAAAG and <30 kDa proteins was increased by bromoxynil treatment. These results gave a cue for understanding the bromoxynil-induced gene expression of CYP71AH11, which may contribute to herbicide tolerance and selectivity in crop plants.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Regulación Enzimológica de la Expresión Génica , Herbicidas/metabolismo , Nicotiana/enzimología , Compuestos de Fenilurea/metabolismo , Proteínas de Plantas/metabolismo , Secuencia de Bases , Sistema Enzimático del Citocromo P-450/genética , Regulación de la Expresión Génica de las Plantas , Datos de Secuencia Molecular , Proteínas de Plantas/genética , Regiones Promotoras Genéticas , Nicotiana/genética , Nicotiana/metabolismoRESUMEN
We report an efficient and reproducible method to generate a microarray of model biological membranes on a solid substrate by applying the inkjet printing technology. Although inkjet printing is currently widely used for industrial fabrication processes, including biological materials, printing lipid membranes remains technically challenging due to the hydrophobic nature of droplets and instability of the lipid bilayer structure against dehydration. In the present study, we printed lipids onto a glass substrate covered with a micropatterned membrane of a polymeric phospholipid bilayer. Polymeric bilayers were formed by the lithographic photopolymerization of a diacetylene-containing phospholipid, 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine (DiynePC). After removal of nonpolymerized DiynePC with a detergent solution, natural lipid membranes were incorporated into the polymer-free regions (corrals) by using an electric-field-based inkjet printing device that can eject subfemtoliter volume droplets. To avoid rapid dehydration and destabilization, we preprinted an aqueous solution containing agarose and trehalose onto the corrals and subsequently printed lipid suspensions ("two-step-printing method"). After rinsing, stable lipid bilayer membranes were formed in the corrals. The bilayers were continuous and fluid as confirmed by fluorescence recovery after photobleaching. We could introduce multiple bilayer patches having different lipid compositions into the neighboring corrals. The present results demonstrate that the combination of a patterned polymeric bilayer and inkjet printing technology enables efficient, reliable, and scalable generation of the model membrane microarrays having varied compositions.
Asunto(s)
Bioimpresión , Membrana Dobles de Lípidos/química , Fosfolípidos/química , Polímeros/química , Membrana Dobles de Lípidos/síntesis química , Microscopía Fluorescente , Polimerizacion , Polímeros/síntesis químicaRESUMEN
We describe a stable and functional model biological membrane based on a polymerized lipid bilayer with a chemically modified surface. A polymerized lipid bilayer was formed from a mixture of two diacetylene-containing phospholipids, 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine (DiynePC) and 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphoethanolamine (DiynePE). DiynePC formed a stable bilayer structure, whereas the ethanolamine headgroup of DiynePE enabled functional molecules to be grafted onto the membrane surface. Copolymerization of DiynePC and DiynePE resulted in a robust bilayer. Functionalization of the polymeric bilayer provided a route to a robust and biomimetic surface that can be linked with biomolecules, cells, and three-dimensional (3D) microstructures. Biotin and peptides were grafted onto the polymeric bilayer for attaching streptavidin and cultured mammalian cells by molecular recognition, respectively. Nonspecific adsorption of proteins and cells on polymeric bilayers was minimum. DiynePE was also used to attach a microstructure made of an elastomer (polydimethylsiloxan: PDMS) onto the membrane, forming a confined aqueous solution between the two surfaces. The microcompartment enabled us to assay the activity of a membrane-bound enzyme (cyochrome P450). Natural (fluid) lipid bilayers were incorporated together with membrane-bound proteins by lithographically polymerizing DiynePC/DiynePE bilayers. The hybrid membrane of functionalized polymeric bilayers and fluid bilayers offers a novel platform for a wide range of biomedical applications including biosensor, bioassay, cell culture, and cell-based assay.
Asunto(s)
Materiales Biomiméticos/química , Fibroblastos/citología , Membrana Dobles de Lípidos/química , Polímeros/química , Proteínas/química , Estreptavidina/química , Adsorción , Animales , Materiales Biomiméticos/síntesis química , Adhesión Celular , Membrana Dobles de Lípidos/síntesis química , Ratones , Modelos Moleculares , Estructura Molecular , Células 3T3 NIH , Polimerizacion , Polímeros/síntesis química , Propiedades de SuperficieRESUMEN
CYP 2A6 is a human enzyme that metabolizes many xenobiotics including coumarin, indole, nicotine and carcinogenic nitrosamines. The gene for CYP2A6 is polymorphic. There are few data available to clarify the relationship between P450 genetic variants and the metabolism of materials in food. The CYP 2A6 wild-type protein and 13 mutants (CYP2A6.1, CYP2A6.2, CYP2A6.5, CYP2A6.6, CYP2A6.7, CYP2A6.8, CYP2A6.11, CYP2A6.15, CYP2A6.16, CYP2A6.17, CYP2A6.18, CYP2A6.21, CYP2A6.23 and CYP2A6.25) were co-expressed with NADPH-cytochrome P450 reductase in E. coli. The hydroxylase activities toward 7-ethoxycoumarin, coumarin, safrole, flavanone and hydroxyflavanone were examined. Ten types of CYP2A6 variants except for CYP2A6.2, CYP2A6.5 and CYP2A6.6 showed Soret peaks (450 nm) typical of P450 in the reduced CO-difference spectra and had 7-ethoxycoumarin O-deethylase activities. CYP2A6.15 and CYP2A6.18 showed higher activities for safrole 1'-hydroxylation than CYP2A6.1. CYP2A6.25 and CYP2A6.7 had lower safrole 1'-hydroxylase activities. CYP2A6.7 had lower flavanone 6- and 2'-hydroxylase activities, whereas CYP2A6.25 had higher 6-hydroxylase activity and lower 2'-hydroxylase activity. Hydroxyflavanone was metabolized by CYP2A6.25, but was not metabolized by wild-type CYP2A6.1. These results indicate that CYP2A6.25 possessed new substrate specificity toward flavonoids.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas/metabolismo , Cumarinas/metabolismo , Flavanonas/metabolismo , Safrol/metabolismo , Hidrocarburo de Aril Hidroxilasas/genética , Citocromo P-450 CYP2A6 , Escherichia coli/genética , Variación Genética , Humanos , Hidroxilación , NADPH-Ferrihemoproteína Reductasa/genética , NADPH-Ferrihemoproteína Reductasa/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Cytochrome P450 (P450) species play an important role in the metabolism of xenobiotics, and assaying the activities of P450 is important for evaluating the toxicity of chemicals in drugs and food. However, the lag time caused by the introduction and mixing of sample solutions can become sources of error as the throughput is heightened by increasing the sample number and decreasing the sample volume. To amend this technological obstacle, we developed a methodology to photoregulate the activity of P450 by using photoprotected (caged) compounds. We synthesized caged molecules of nicotinamide adenine dinucleotide phosphate (NADP(+)) and glucose 6-phosphate (G6P), which are involved in the generation of NADPH (cofactor of P450). The use of caged-G6P completely blocked the P450 catalysis before the UV illumination, whereas caged-NADP(+) resulted in a little background reaction. Upon UV illumination, more than 90% of the enzymatic activity could be restored. The use of caged-G6P enabled assays in isolated microchambers (width, 50 µm; height, 50 µm) by encapsulating necessary ingredients in advance and initiating the reaction by UV illumination. The initiation of enzymatic reaction could be observed in a single microchamber. Minimizing uncertainties caused by the introduction and mixing of solutions led to significantly reduced errors of obtained kinetic constants.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Luz , Secuencia de Bases , Cartilla de ADN , Escherichia coli/enzimología , Glucosa-6-Fosfato/metabolismo , Humanos , Cinética , NADP/metabolismoRESUMEN
A microarray chip containing human P450 isoforms was constructed for the parallel assay of their metabolic activities. The chip had microwells that contained vertically integrated P450 and oxygen sensing layers. The oxygen sensing film was made of an organically modified silica film (ORMOSIL) doped with tris(4,7-diphenyl-1,10-phenanthroline) ruthenium dichloride (Ru(dpp)(3)Cl(2)). Human P450s (23 types) expressed in E. coli and purified as membrane fractions were immobilized in agarose matrixes on the oxygen sensing layer. The activities of P450s were determined by evaluating the fluorescence intensity enhancement of the oxygen sensor due to the oxygen consumption by the metabolic reaction. By normalizing the responses with the amounts of oxygen sensor and P450 enzymes in microwells, we could obtain fluorescence enhancement patterns that were characteristic to the combination of P450 isoforms and substrate material. The patterns obtained from two psoralen derivatives resembled each other, whereas a structurally different substrate (capsaicin) resulted in a distinct pattern. These results suggest the potential of the microarray to analyze the activities of diverse P450 isoforms in a high-throughput fashion. Furthermore, mechanism-based inactivation (MBI) of P450 could be detected by successively incubating a chip with different substrate solutions and measuring the residual activities.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Oxígeno/metabolismo , Análisis por Matrices de Proteínas/métodos , Cicloparafinas/química , Humanos , Compuestos Organometálicos/química , Fenantrolinas/química , Polímeros/química , Dióxido de Silicio/químicaRESUMEN
To overcome bladder cancer, one of the most common cancer and deadly cancers in the world, early diagnosis and treatment interventions are crucial. The development of efficient diagnostic methods is required. Previously, we developed a cytochrome P450 (P450 or CYP) inhibition assay that detected alterations in the quality and quantity of P450 relevant substances in the serum, caused by inflammation and exposure to endogenous or exogenous substances. Since bladder cancer is known to alter the expression levels of P450s in patients, we tested whether the P450 inhibition assay could distinguish between the sera of patients with bladder cancer and healthy individuals. When assays were performed using sera recovered from mice with bladder cancer and control mice, significant differences were observed in the inhibition rates of CYP2A13, CYP2C18 and CYP2E1. Moreover, the results of the assay using human clinical samples revealed that the P450 inhibition assay can detect bladder cancer with an area under the receiver operating characteristic (ROC) curve of 0.867-0.950. These findings demonstrated that the P450 inhibition assay can aid the future development of liquid biopsy-based diagnostic methods for bladder cancer.
Asunto(s)
Neoplasias de la Vejiga Urinaria , Humanos , Ratones , Animales , Neoplasias de la Vejiga Urinaria/diagnóstico , Sistema Enzimático del Citocromo P-450/metabolismoRESUMEN
Drug-induced liver injury (DILI) causes abortive drug development in both clinical and non-clinical phases. Therefore, it is important to develop an evaluation system that can be used to accurately and easily assess DILI during drug development and at an early stage. The diagnosis of diseases using P450 inhibition assays, with focus on changes in cytochrome P450 (CYP, P450) during disease onset, has been previously validated in mouse models of ulcerative colitis and type 2 diabetes. In this study, we tested the effectivity of the P450 inhibition assay in a DILI mouse model treated with acetaminophen (APAP). We found that TNF-α expression was upregulated in the APAP-treated group, and CYP2E1 gene expression was significantly decreased at 8 h after treatment. In the P450 inhibition assay, in which sera were used, significant changes in CYP2E1 and CYP3A5 levels were observed 8 and 24 h after APAP administration, respectively. Receiver operating characteristic curve analysis showed significant inhibition rate changes; the area under the curve values of CYP2E1 and CYP3A5 were above 0.8, at 0.832 and 0.849, respectively. In conclusion, we suggest that P450 inhibition assay could be used for the diagnosis of liver diseases, such as acute DILI.
Asunto(s)
Enfermedad Hepática Inducida por Sustancias y Drogas , Diabetes Mellitus Tipo 2 , Acetaminofén/farmacología , Animales , Enfermedad Hepática Inducida por Sustancias y Drogas/diagnóstico , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Citocromo P-450 CYP2E1/metabolismo , Citocromo P-450 CYP3A/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Modelos Animales de Enfermedad , Hígado , RatonesRESUMEN
Parkinson's disease (PD) is the second most common neurodegenerative disease, and diagnostic methods and biomarkers for patients without subjective motor symptoms have not yet been established. Previously, we developed a cytochrome P450 inhibition assay that detects alterations in metabolite levels associated with P450s caused by inflammation and exposure to endogenous or exogenous substances. However, it is unknown whether the P450 inhibition assay can be applied in PD diagnosis. Here, we determined whether the P450 inhibition assay can discriminate sera between patients with PD and healthy individuals. The results of the assay revealed that the P450 inhibition assay can discriminate PD with an area under the receiver operating characteristic curve (AUC) value of 0.814-0.914 in rats and an AUC value of 0.910 in humans. These findings demonstrate that the P450 inhibition assay can aid in the future development of liquid biopsy-based diagnostic methods for PD.
Asunto(s)
Enfermedades Neurodegenerativas , Enfermedad de Parkinson , Animales , Biomarcadores , Sistema Enzimático del Citocromo P-450 , Humanos , Enfermedad de Parkinson/diagnóstico , Curva ROC , RatasRESUMEN
An assaying method of cytochrome P450 (P450 or CYP) monooxygenase activities for toxicological evaluation of drugs and environmental pollutants was developed by immobilizing P450 on an oxygen sensoring layer. Membrane fractions from E. coli expressing human P450 were entrapped in agarose or silica-based gels and immobilized on 96-well microarrays having an oxygen sensing film at the bottom. The oxygen sensing film was made of an organically modified silica film (ORMOSIL) doped with Tris(4,7-diphenyl-1,10-phenanthroline) ruthenium dichloride (Ru(dpp)(3)Cl(2)). P450 activity toward the substrates was monitored through the fluorescence intensity enhancement due to the oxygen consumption by the metabolic reactions. For the metabolism of chlortoluron, a selective herbicide used to control grass weeds, CYP1A1 immobilized in agarose gel showed a higher activity and stability compared with those in silica gels and free suspensions. The luminescence changing rate evaluated by the dynamic transient method (DTM) could be correlated with the substrate concentration. We also compared the metabolic responses of human P450s (CYP1A1,CYP2C8, CYP2E1, CYP3A4) toward various substances. The use of immobilized P450 on an oxygen sensing layer provides a versatile assaying platform owing to the following features. First, the oxygen sensor can detect metabolic reactions of any P450 species, in contrast with assays using fluorogenic substrates. Second, vertical integration of the oxygen sensor and immobilized P450 enhanced the sensitivity because of the effective depletion of oxygen in the vicinity of the oxygen sensing layer. Third, immobilization enables repeated use of P450 by replacing the substrate solutions using a flow cell. Furthermore, the activity of immobilized P450 was retained at least for 3 weeks at 4 °C, suggesting its long-term stability, which is an additional attractive feature.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Membranas Artificiales , Oxígeno/análisis , Humanos , Oxígeno/metabolismo , Sefarosa/química , Dióxido de Silicio/químicaRESUMEN
Docosahexaenoic acid (DHA) has been shown to have neuroprotective effects in Parkinson's disease, but the underlying mechanism has not been fully elucidated. DHA is metabolized to DHA epoxides (EDPs) and hydroxides by cytochrome P450s (P450s), and EDPs are further hydroxylated to the corresponding diols, dihydroxydocosapentaenoic acids (DHDPs) by soluble epoxide hydrolase (sEH). In the present study, we investigated the roles of these DHA metabolites in the beneficial effects of DHA supplementation on a rotenone-induced rat model of Parkinson's disease. Metabolite analysis by LC-MS revealed that CYP2A1, 2C11, 2C13, 2C23, and 2E1 contributed to the formation of EDPs, and these P450s and sEH were expressed in the rat brain. We found that DHA supplementation in rats improved the motor dysfunction induced by rotenone. In addition, DHA reversed the decrease in tyrosine hydroxylase and the increase in lipid peroxidation generated by rotenone in the striatum. DHA supplementation also induced mRNA expression of antioxidant genes, such as sod1 and catalase, and Nrf2 protein expression in the striatum. However, these effects of DHA supplementation were eliminated by cosupplementation with the sEH inhibitor TPPU. Supplementation with DHA increased the amount of 19,20-DHDP in the rat brain, while the amount of EDPs was not significantly increased. In addition, TPPU suppressed the increase in DHDPs and increased EDPs in the brain. In PC12 cells, 19,20-DHDP increased the mRNA levels of sod1 and catalase along with Nrf2 induction. This study suggests that DHA metabolites-DHDPs generated by P450s and sEH-have an important role in improving rotenone-induced Parkinson's disease.
Asunto(s)
Ácidos Docosahexaenoicos/administración & dosificación , Ácidos Grasos Insaturados/metabolismo , Fármacos Neuroprotectores/administración & dosificación , Enfermedad de Parkinson Secundaria/tratamiento farmacológico , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/patología , Catalasa/metabolismo , Sistema Enzimático del Citocromo P-450/metabolismo , Modelos Animales de Enfermedad , Ácidos Docosahexaenoicos/metabolismo , Epóxido Hidrolasas/antagonistas & inhibidores , Epóxido Hidrolasas/metabolismo , Humanos , Masculino , Factor 2 Relacionado con NF-E2/metabolismo , Fármacos Neuroprotectores/metabolismo , Oxidación-Reducción/efectos de los fármacos , Enfermedad de Parkinson Secundaria/inducido químicamente , Enfermedad de Parkinson Secundaria/patología , Ratas , Rotenona/toxicidad , Superóxido Dismutasa-1/metabolismoRESUMEN
A number of studies have demonstrated that cytochrome P450 (P450) converts furanocoumarin derivatives into reactive molecules, which form covalent bonds to biomolecules. 5-Methoxypsoralen (5-MOP) is a natural furanocoumarin from apiaceous plants. In this study, we examined the effect on 5-MOP metabolism of single nucleotide polymorphisms (SNPs) in CYP2A13. We used Escherichia coli-generated recombinant enzymes of wild-type CYP2A13*1 and five variants, CYP2A13*4 (R101Q), CYP2A13*5 (F453Y), CYP2A13*6 (R494C), CYP2A13*8 (D158E), and CYP2A13*9 (V323L). In high-performance liquid chromatography analyses of 5-MOP metabolic products, CYP2A13*1 converted 5-MOP into 5-MOP dihydrodiol; K(m) and V(max) values of the reaction were 1.44 ± 0.17 µM and 4.23 ± 0.36 nmol/(min · nmol P450), respectively. The generation of a dihydrodiol from 5-MOP implies that conversion by CYP2A13 causes toxicity due to the formation of covalent bonds with DNA or proteins. Most of the CYP2A13 variants could metabolize 5-MOP; K(m) values for CYP2A13*5, *6, *8, and *9 were 1.63 ± 0.12, 1.36 ± 0.10, 0.85 ± 0.09, and 0.58 ± 0.06 µM, respectively, and V(max) values were 3.20 ± 0.13, 4.69 ± 0.13, 2.34 ± 0.07, and 1.84 ± 0.09 nmol/(min · nmol P450), respectively. However, the processing of 5-MOP by CYP2A13*4 was not detectable. Based on this data, we hypothesize that SNPs within the CYP2A13 gene affect metabolism of 5-MOP in humans.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas/genética , Metoxaleno/análogos & derivados , Polimorfismo de Nucleótido Simple , 5-Metoxipsoraleno , Hidrocarburo de Aril Hidroxilasas/fisiología , Escherichia coli/genética , Humanos , Metoxaleno/metabolismo , Proteínas Recombinantes/metabolismoRESUMEN
Human cytochrome P450 (or CYP) inhibition rates were investigated in sera from high fat diet (HFD)-induced type 2 diabetes (T2D), T2D recovered, and asymptomatic mice models to verify whether P450 inhibition assays could be used for the detection of disease, evaluation of therapeutic effect, and early diagnosis of T2D. In T2D mice, the blood glucose levels markedly increased; while blood glucose levels of recovered mice exceeded 200 mg dL-1, these eventually returned to the levels seen in control mice. In asymptomatic mice fed with short term HFD (stHFD), no changes in blood glucose levels were observed. The inhibition rates of CYP1A2, CYP2A13, and CYP2C18 in T2D mice significantly increased. Whereas in recovered mice, these changes returned to the same levels noted in the control mice. Changes in the inhibition rates of CYP2A13 and CYP2C18 in stHFD mice were similar to those in T2D mice. A receiver operating characteristic (ROC) curve analysis showed high area under the ROC curve (AUC) values (0.879-1.000) of CYP2A13 and CYP2C18 in T2D and stHFD mice, indicating their high diagnostic accuracy. Collectively, this study validates the P450 inhibition assay as a method for the therapeutic evaluation and early diagnosis of T2D mouse models.
Asunto(s)
Hidrocarburo de Aril Hidroxilasas/antagonistas & inhibidores , Inhibidores Enzimáticos del Citocromo P-450/sangre , Diabetes Mellitus Experimental/diagnóstico , Diabetes Mellitus Tipo 2/diagnóstico , Animales , Hidrocarburo de Aril Hidroxilasas/metabolismo , Enfermedades Asintomáticas , Biomarcadores/sangre , Citocromo P-450 CYP1A2/metabolismo , Inhibidores del Citocromo P-450 CYP1A2/sangre , Diabetes Mellitus Experimental/sangre , Diabetes Mellitus Experimental/etiología , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/etiología , Dieta Alta en Grasa , Diagnóstico Precoz , Femenino , Humanos , Ratones Endogámicos C57BL , Valor Predictivo de las Pruebas , Proteínas Recombinantes/metabolismoRESUMEN
Aflatoxin B1 (AFB1) is a mycotoxin produced by Aspergillus flavus (A. flavus). AFB1 is reported to have high thermal stability and is not decomposed by heat treatment during food processing. Therefore, in this study, knowing that AFB1 is metabolized by cytochrome P450 (CYP), our aim was to develop a method to detoxify A. flavus-contaminated maize, under normal temperature and pressure, using Escherichia coli expressing human CYP3A4. First, the metabolic activity of AFB1 by recombinant human CYP3A4 was evaluated. As a result, we confirmed that recombinant human CYP3A4 metabolizes 98% of AFB1. Next, we found that aflatoxin Q1, a metabolite of AFB1 was no longer mutagenic. Furthermore, we revealed that about 50% of the AFB1 metabolic activity can be maintained for 3 months when E. coli expressing human CYP3A4 is freeze-dried in the presence of trehalose. Finally, we found that 80% of AFB1 in A. flavus-contaminated maize was metabolized by E. coli expressing human CYP3A4 in the presence of surfactant triton X-405 at a final concentration of 10% (v/v). From these results, we conclude that AFB1 in A. flavus-contaminated maize can be detoxified under normal temperature and pressure by using E. coli expressing human CYP3A4.