WorldWideScience

Sample records for citocromo p450 aromatasa

  1. Interacciones farmacológicas mediadas por el citocromo P-450 en un grupo de pacientes VIH positivos

    OpenAIRE

    Juárez Giménez, Juan Carlos

    2010-01-01

    Las combinaciones de fármacos antirretovirales (ARV) pueden provocar interacciones farmacológicas (IF) mediadas por el complejo enzimático del citocromo P450. Algunos ARV actúan como sustratos, inductores e inhibidores de este complejo. Así, se ha establecido que hasta un 96 % de los pacientes tratados con la terapia antirretroviral de gran actividad, presentan como mínimo una situación clínica no deseada o un efecto adverso generado por una interacción farmacológica (IF). El objetivo del pre...

  2. Doenças cardiovasculares: metabolitos do citocromo P450 da via do ácido araquidónico no controlo do tónus vascular

    OpenAIRE

    Fernandes, José Miguel da Mota

    2013-01-01

    As doenças cardiovasculares (DCV) continuam a ser a principal causa de morte e são responsáveis por quase 30% de todas as causas de mortalidade nos países desenvolvidos. Os citocromos P450 (CYP) epoxigenases metabolizam o ácido araquidónico em eicosanóides biologicamente ativos, os EET. Muitos membros de diversas famílias de enzimas CYP foram identificados no coração, endotélio e do músculo liso dos vasos sanguíneos. Os efeitos cardiovasculares dos EET incluem a vasodilatação, tendo estes ...

  3. Respuesta terapéutica de pacientes con malaria por plasmodium falciparum a los antimaláricos y fenotipo y genotipo del citocromo p450

    Directory of Open Access Journals (Sweden)

    Fanny Cuesta González

    2004-02-01

    Full Text Available

    En la evaluación de la eficacia a los antimaláricos, la falta
    de concordancia entre el fenotipo observado In vivo en el paciente (respuesta exitosa, falla e In vitro en el parásito (sensible, resistente, sugiere que factores como el metabolismo del medicamento en el hospedero pueden cumplir un papel
    determinante. El metabolismo de varios antimaláricos es
    realizado por el complejo citocromo P-450 (CYP450 en el
    hígado (1 y su actividad es vulnerable a la inhibición y a la
    inducción por: el estado nutricional, el genotipo enzimático y la
    administración concomitante de otros medicamentos (2. La
    amodiaquina (AQ es metabolizada al compuesto activo Ndesetilamodiaquina por el CYP2C8 y la mefloquina (MQ es
    metabolizada a carboximefloquina (su forma de excreción, por
    el CYP3A4; la inhibición del CYP450 puede convertir un metabolismo rápido en lento llevando a concentraciones
    inefectivas del medicamento, mientras que la inducción puede
    acelerar la conversión a su metabolito y favorecer la excreción
    rápida del mismo.

     

     

  4. Evaluación de la actividad de la enzima citocromo P-450 1A2 en una población colombiana

    Directory of Open Access Journals (Sweden)

    Fanny Cuesta González

    2000-02-01

    Full Text Available

    Introducción: La enzima citocromo P450 1A2 (CYP1A2, presente en tejidos hepáticos humanos, participa en el metabolismo de fármacos tales como Clozapina, Imipramina, Teofilina y Cafeína. Estudios realizados en diferentes poblaciones han mostrado una distribución bimodal y a veces trimodal en la actividad de esta enzima. Este comportamiento ha sido relacionado con variaciones tanto del gen codificante como de las regiones reguladoras, aunque hasta el momento los resultados no son concluyentes. Además del metabolismo de fármacos, la CYP1A2 interviene en la activación de sustancias procarcinogénicas tales como N-nitrosaminas, aminas heterocíclicas y aromáticas; es conocida la correlación entre una actividad incrementada de esta enzima (metabolizadores rápidos con una mayor exposición al riesgo de cáncer.

    Debido a la baja incidencia de efectos secundarios y la facilidad en la medición de los metabolitos en orina, la Cafeína es utilizada como fármaco prueba para la determinación de la actividad enzimática CYP1A2, calculando la relación entre las concentraciones molares de sus metabolitos (Actividad CYP1A2 = AFMU+MU+MX/17DMU.

    En el presente estudio se evaluará la actividad de la enzima CYP1A2 en una muestra de individuos sanos, con el fin de describir su comportamiento y compararlo con el encontrado en otras poblaciones, las cuales tienen una influencia genética y ambiental diferente a la de la población colombiana.

    Metodología: Una vez recolectadas las muestras de orina, se separarán de ellas los

  5. Identificación funcional de citocromos involucrados en la biotransformación in vitro de AFB1 por medio de sustratos modelo e inhibidores específicos en cuatro especies de aves: gallinas, pavos, patos y codornices / Functional identification of cytochrome P450 enzymes involved in AFB1 in vitro biotransformation using prototype substrates and specific human CYP450 inhibitors in four poultry species: chicken, turkey, duck and quail

    OpenAIRE

    Murcia Gutiérrez, Hansen Wilber

    2010-01-01

    Las aflatoxina B1 (AFB1) es un metabolito secundario producido por algunas cepas de Aspergillus flavus y Aspergillus parasiticus que crecen sobre diferentes tipos de granos utilizados en alimentación de animales de granja. Al momento de ser ingeridas estas toxinas son metabolizadas por un tipo de enzimas llamadas citocromo P450 (CYP450), una familia de enzimas relacionadas con la biotransformación de productos endógenos y xenobióticos. El producto de biotransformación es una molécula altamen...

  6. Evaluation of cytochrome P-450 concentration in Saccharomyces cerevisiae strains

    Directory of Open Access Journals (Sweden)

    Míriam Cristina Sakuragui Matuo

    2010-09-01

    Full Text Available Saccharomyces cerevisiae has been widely used in mutagenicity tests due to the presence of a cytochrome P-450 system, capable of metabolizing promutagens to active mutagens. There are a large number of S. cerevisiae strains with varying abilities to produce cytochrome P-450. However, strain selection and ideal cultivation conditions are not well defined. We compared cytochrome P-450 levels in four different S. cerevisiae strains and evaluated the cultivation conditions necessary to obtain the highest levels. The amount of cytochrome P-450 produced by each strain varied, as did the incubation time needed to reach the maximum level. The highest cytochrome P-450 concentrations were found in media containing fermentable sugars. The NCYC 240 strain produced the highest level of cytochrome P-450 when grown in the presence of 20 % (w/v glucose. The addition of ethanol to the media also increased cytochrome P-450 synthesis in this strain. These results indicate cultivation conditions must be specific and well-established for the strain selected in order to assure high cytochrome P-450 levels and reliable mutagenicity results.Linhagens de Saccharomyces cerevisiae tem sido amplamente empregadas em testes de mutagenicidade devido à presença de um sistema citocromo P-450 capaz de metabolizar substâncias pró-mutagênicas à sua forma ativa. Devido à grande variedade de linhagens de S. cerevisiae com diferentes capacidades de produção de citocromo P-450, torna-se necessária a seleção de cepas, bem como a definição das condições ideais de cultivo. Neste trabalho, foram comparados os níveis de citocromo P-450 em quatro diferentes linhagens de S. cerevisiae e avaliadas as condições de cultivo necessárias para obtenção de altas concentrações deste sistema enzimático. O maior nível enzimático foi encontrado na linhagem NCYC 240 em presença de 20 % de glicose (p/v. A adição de etanol ao meio de cultura também produziu um aumento na s

  7. Cytochrome P450 database.

    Science.gov (United States)

    Lisitsa, A V; Gusev, S A; Karuzina, I I; Archakov, A I; Koymans, L

    2001-01-01

    This paper describes a specialized database dedicated exclusively to the cytochrome P450 superfamily. The system provides the impression of superfamily's nomenclature and describes structure and function of different P450 enzymes. Information on P450-catalyzed reactions, substrate preferences, peculiarities of induction and inhibition is available through the database management system. Also the source genes and appropriate translated proteins can be retrieved together with corresponding literature references. Developed programming solution provides the flexible interface for browsing, searching, grouping and reporting the information. Local version of database manager and required data files are distributed on a compact disk. Besides, there is a network version of the software available on Internet. The network version implies the original mechanism, which is useful for the permanent online extension of the data scope. PMID:11769119

  8. Moonlighting Cytochrome P450 Monooxygenases

    OpenAIRE

    Zhao, Bin; Waterman, Michael R.

    2011-01-01

    Recently, cytochrome P450 170A1 (CYP170A1) has been found to be a bifunctional protein, which catalyzes both monooxygenase activity and terpene synthase activity by two distinct active sites in the well established P450 protein structure. Therefore, CYP170A1 is identified clearly as a moonlighting protein. The known activities of a small number of the 13,000 members of the P450 superfamily fall into two general classes; promiscuous enzymes that are not considered as moonlighting and forms tha...

  9. Immobilized Cytochrome P450 for Monitoring of P450-P450 Interactions and Metabolism.

    Science.gov (United States)

    Bostick, Chris D; Hickey, Katherine M; Wollenberg, Lance A; Flora, Darcy R; Tracy, Timothy S; Gannett, Peter M

    2016-05-01

    Cytochrome P450 (P450) protein-protein interactions have been shown to alter their catalytic activity. Furthermore, these interactions are isoform specific and can elicit activation, inhibition, or no effect on enzymatic activity. Studies show that these effects are also dependent on the protein partner cytochrome P450 reductase (CPR) and the order of protein addition to purified reconstituted enzyme systems. In this study, we use controlled immobilization of P450s to a gold surface to gain a better understanding of P450-P450 interactions between three key drug-metabolizing isoforms (CYP2C9, CYP3A4, and CYP2D6). Molecular modeling was used to assess the favorability of homomeric/heteromeric P450 complex formation. P450 complex formation in vitro was analyzed in real time utilizing surface plasmon resonance. Finally, the effects of P450 complex formation were investigated utilizing our immobilized platform and reconstituted enzyme systems. Molecular modeling shows favorable binding of CYP2C9-CPR, CYP2C9-CYP2D6, CYP2C9-CYP2C9, and CYP2C9-CYP3A4, in rank order.KDvalues obtained via surface plasmon resonance show strong binding, in the nanomolar range, for the above pairs, with CYP2C9-CYP2D6 yielding the lowestKD, followed by CYP2C9-CYP2C9, CYP2C9-CPR, and CYP2C9-CYP3A4. Metabolic incubations show that immobilized CYP2C9 metabolism was activated by homomeric complex formation. CYP2C9 metabolism was not affected by the presence of CYP3A4 with saturating CPR concentrations. CYP2C9 metabolism was activated by CYP2D6 at saturating CPR concentrations in solution but was inhibited when CYP2C9 was immobilized. The order of addition of proteins (CYP2C9, CYP2D6, CYP3A4, and CPR) influenced the magnitude of inhibition for CYP3A4 and CYP2D6. These results indicate isoform-specific P450 interactions and effects on P450-mediated metabolism. PMID:26961240

  10. Cytochrome P450 humanised mice

    Directory of Open Access Journals (Sweden)

    Gonzalez Frank J

    2004-05-01

    Full Text Available Abstract Humans are exposed to countless foreign compounds, typically referred to as xenobiotics. These can include clinically used drugs, environmental pollutants, food additives, pesticides, herbicides and even natural plant compounds. Xenobiotics are metabolised primarily in the liver, but also in the gut and other organs, to derivatives that are more easily eliminated from the body. In some cases, however, a compound is converted to an electrophile that can cause cell toxicity and transformation leading to cancer. Among the most important xenobiotic-metabolising enzymes are the cytochromes P450 (P450s. These enzymes represent a superfamily of multiple forms that exhibit marked species differences in their expression and catalytic activities. To predict how humans will metabolise xenobiotics, including drugs, human liver extracts and recombinant P450s have been used. New humanised mouse models are being developed which will be of great value in the study of drug metabolism, pharmacokinetics and pharmacodynamics in vivo, and in carrying out human risk assessment of xenobiotics. Humanised mice expressing CYP2D6 and CYP3A4, two major drug-metabolising P450s, have revealed the feasibility of this approach.

  11. Polymorphism of human cytochrome P-450.

    Science.gov (United States)

    Guengerich, F P; Umbenhauer, D R; Churchill, P F; Beaune, P H; Böcker, R; Knodell, R G; Martin, M V; Lloyd, R S

    1987-03-01

    The cytochrome P-450 forms involved in debrisoquine 4-hydroxylation (P-450DB), phenacetin O-deethylation (P-450PA), S-mephenytoin 4-hydroxylation (P-450MP), and nifedipine 1,4-oxidation (P-450NF) have been purified to electrophoretic homogeneity from human liver microsomes. All of these reactions show in vivo polymorphism in humans. Evidence for the roles of the purified proteins in these processes comes from in vitro reconstitution and immunoinhibition studies. The rat orthologs of these enzymes are as follows--P-450DB: P-450UT-H; P-450PA: P-450ISF-G; P-450MP: P-450UT-I; P-450NF: P-450PCN-E. Only in the case of P-450UT-H is the primary rat ortholog the same cytochrome P-450 which catalyses the catalytic reaction under consideration. Reconstitution and immunochemical studies establish that the following reactions are catalysed by the individual P-450s--P-450DB: debrisoquine 4-hydroxylation, sparteine delta 5-oxidation, bufuralol 1'-hydroxylation, encainide O-demethylation, and propanolol 4-hydroxylation; P-450PA: phenacetin O-deethylation; P-450MP: S-mephenytoin 4-hydroxylation and tolbutamide methyl hydroxylation; P-450NF: oxidation of nifedipine and 16 other substituted dihydropyridines, estradiol 2- and 4-hydroxylation, aldrin epoxidation, benzphetamine N-demethylation and 6 beta-hydroxylation of testosterone, androstenedione and cortisol. A cDNA clone has been isolated that corresponds to rat P-450UT-H, as shown by a number of criteria. Studies with this probe establish that the sex and strain variation in debrisoquine 4-hydroxylase and related activities is related to differences in the levels of a 2.0 kb length mRNA present.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:3577206

  12. Cytochrome P450 enzyme systems in fungi

    NARCIS (Netherlands)

    Brink, H.M. van den; Gorcom, R.F.M. van; Hondel, C.A.M.J.J. van den; Punt, P.J.

    1998-01-01

    The involvement of cytochrome P450 enzymes in many complex fungal bioconversion processes has been characterized in recent years. Accordingly, there is now considerable scientific interest in fungal cytochrome P450 enzyme systems. In contrast to S. cerevisiae, where surprisingly few P450 genes have

  13. Light-driven cytochrome P450 hydroxylations

    DEFF Research Database (Denmark)

    Jensen, Kenneth; Jensen, Poul Erik; Møller, Birger Lindberg

    2011-01-01

    Plants are light-driven "green" factories able to synthesize more than 200,000 different bioactive natural products, many of which are high-value products used as drugs (e.g., artemisinin, taxol, and thapsigargin). In the formation of natural products, cytochrome P450 (P450) monooxygenases play a...... key role in catalyzing regio- and stereospecific hydroxylations that are often difficult to achieve using the approaches of chemical synthesis. P450-catalyzed monooxygenations are dependent on electron donation typically from NADPH catalyzed by NADPH-cytochrome P450 oxidoreductase (CPR). The...

  14. Characterisation of Novel Cytochrome P450-fusion enzymes

    OpenAIRE

    Luciakova, Dominika

    2015-01-01

    Characterisation of Novel Cytochrome P450-fusion enzymesSeptember 2014This study focuses on the characterisation of three novel cytochrome P450-partner (P450-fusion) enzymes of unknown structure and function. Despite several well-established P450 functions, new structures of P450s are published frequently, with the P450-redox partner fusion systems being among the most discussed, due to their enhanced activity and biotechnological potential. Other, more intriguing, P450-fusions involve partne...

  15. Cytochrome P450-mediated metabolic engineering

    DEFF Research Database (Denmark)

    Renault, Hugues; Bassard, Jean-Étienne André; Hamberger, Björn Robert;

    2014-01-01

    Cytochromes P450 catalyze a broad range of regiospecific, stereospecific and irreversible steps in the biosynthetic routes of plant natural metabolites with important applications in pharmaceutical, cosmetic, fragrance and flavour, or polymer industries. They are consequently essential drivers for...

  16. Drug interactions due to cytochrome P450

    OpenAIRE

    Ogu, Chris C.; Maxa, Jan L.

    2000-01-01

    Cytochrome P450 is a family of isozymes responsible for the biotransformation of several drugs. Drug metabolism via the cytochrome P450 system has emerged as an important determinant in the occurrence of several drug interactions that can result in drug toxicities, reduced pharmacological effect, and adverse drug reactions. Recognizing whether the drugs involved act as enzyme substrates, inducers, or inhibitors can prevent clinically significant interactions from occurring. Avoiding coadminis...

  17. Biotransformation of the sesquiterpene (+)-valencene by cytochrome P450cam and P450BM-3.

    Science.gov (United States)

    Sowden, Rebecca J; Yasmin, Samina; Rees, Nicholas H; Bell, Stephen G; Wong, Luet-Lok

    2005-01-01

    The sesquiterpenoids are a large class of naturally occurring compounds with biological functions and desirable properties. Oxidation of the sesquiterpene (+)-valencene by wild type and mutants of P450cam from Pseudomonas putida, and of P450BM-3 from Bacillus megaterium, have been investigated as a potential route to (+)-nootkatone, a fine fragrance. Wild type P450cam did not oxidise (+)-valencene but the mutants showed activities up to 9.8 nmol (nmol P450)(-1) min(-1), with (+)-trans-nootkatol and (+)-nootkatone constituting >85% of the products. Wild type P450BM-3 and mutants had higher activities (up to 43 min(-1)) than P450cam but were much less selective. Of the many products, cis- and trans-(+)-nootkatol, (+)-nootkatone, cis-(+)-valencene-1,10-epoxide, trans-(+)-nootkaton-9-ol, and (+)-nootkatone-13S,14-epoxide were isolated from whole-cell reactions and characterised. The selectivity patterns suggest that (+)-valencene has one binding orientation in P450cam but multiple orientations in P450BM-3. PMID:15602599

  18. The P450 gene superfamily: recommended nomenclature.

    Science.gov (United States)

    Nebert, D W; Adesnik, M; Coon, M J; Estabrook, R W; Gonzalez, F J; Guengerich, F P; Gunsalus, I C; Johnson, E F; Kemper, B; Levin, W

    1987-02-01

    A nomenclature for the P450 gene superfamily is proposed based on evolution. Recommendations include Roman numerals for distinct gene families, capital letters for subfamilies, and Arabic numerals for individual genes. An updating of this list, which presently includes 65 entries, will be required every 1-2 years. Assignment of orthologous genes is presently uncertain in some cases--between widely diverged species and especially in the P450II family due to the large number of genes. As more is known, it might become necessary to change some gene assignments that are based on our present knowledge. PMID:3829886

  19. Intronic polymorphisms of cytochromes P450

    Directory of Open Access Journals (Sweden)

    Ingelman-Sundberg Magnus

    2010-08-01

    Full Text Available Abstract The cytochrome P450 enzymes active in drug metabolism are highly polymorphic. Most allelic variants have been described for enzymes encoded by the cytochrome P450 family 2 (CYP2 gene family, which has 252 different alleles. The intronic polymorphisms in the cytochrome P450 genes account for only a small number of the important variant alleles; however, the most important ones are CYP2D6*4 and CYP2D6*41, which cause abolished and reduced CYP2D6 activity, respectively, and CYP3A5*3 and CYP3A5*5, common in Caucasian populations, which cause almost null activity. Their discoveries have been based on phenotypic alterations within individuals in a population, and their identification has, in several cases, been difficult and taken a long time. In light of the next-generation sequencing projects, it is anticipated that further alleles with intronic mutations will be identified that can explain the hitherto unidentified genetic basis of inter-individual differences in cytochrome P450-mediated drug and steroid metabolism.

  20. Prediction of cytochrome P450 mediated metabolism

    DEFF Research Database (Denmark)

    Olsen, Lars; Oostenbrink, Chris; Jørgensen, Flemming Steen

    2015-01-01

    Cytochrome P450 enzymes (CYPs) form one of the most important enzyme families involved in the metabolism of xenobiotics. CYPs comprise many isoforms, which catalyze a wide variety of reactions, and potentially, a large number of different metabolites can be formed. However, it is often hard to...

  1. Papel central de los citocromos P450 en el catabolismo del colesterol y su regulación en micobacterias

    OpenAIRE

    García Fernández, Esther

    2013-01-01

    El colesterol es un esteroide ampliamente distribuido en la Naturaleza, que tiene una gran importancia fisiológica porque se encuentra formando parte de las membranas de las células animales y además es precursor inmediato de vitaminas, hormonas esteroideas y ácidos biliares. La degradación del colesterol llevada a cabo por bacterias aeróbicas se divide en varias etapas que consisten en la oxidación del colesterol a 4-colesten-3-ona, la oxidación de la cadena lateral y la degradación del sist...

  2. Monoclonal antibodies to drosophila cytochrome P-450's

    International Nuclear Information System (INIS)

    Hybridomas producing monoclonal antibodies were prepared by the fusion of SP2/0 myeloma cells and spleen cells from a female BALB/c mouse immunized by cytochrome P-450-A and P-450-B purified from Drosophila Hikone-R (BG) microsomes. P-450-A and P-450-B are electrophoretically distinct subsets of Drosophila P-450. P-450-A is ubiquitous among strains tested, while P-450-B is present in only a few strains displaying unique enzyme activities and increased insecticide resistance. The Oregon-R strain contains only cytochromes P-450-A and is susceptible to insecticides. The authors Hikone-R (BG) strain expresses both cytochromes P-450-A and P-450-B and is insecticide resistant. Antibody producing hybridomas were detected in a solid-phase radioimmunoassay (RIA) by binding to Hikone-R (BG) or Oregon-R microsomes. Four independent hybridomas were identified as producing monoclonal antibodies that recognized proteins in the P-450 complex by immunoblot experiments. Three monoclonal antibodies recognized P-450-A proteins, while one monoclonal antibody bound predominantly P-450-B. This monoclonal antibody also recognized southern armyworm (Spodoptera eridania, Cramer) microsomal proteins

  3. Monoclonal antibodies to drosophila cytochrome P-450's

    Energy Technology Data Exchange (ETDEWEB)

    Sundseth, S.S.; Kennel, S.J.; Waters, L.C.

    1987-05-01

    Hybridomas producing monoclonal antibodies were prepared by the fusion of SP2/0 myeloma cells and spleen cells from a female BALB/c mouse immunized by cytochrome P-450-A and P-450-B purified from Drosophila Hikone-R (BG) microsomes. P-450-A and P-450-B are electrophoretically distinct subsets of Drosophila P-450. P-450-A is ubiquitous among strains tested, while P-450-B is present in only a few strains displaying unique enzyme activities and increased insecticide resistance. The Oregon-R strain contains only cytochromes P-450-A and is susceptible to insecticides. The authors Hikone-R (BG) strain expresses both cytochromes P-450-A and P-450-B and is insecticide resistant. Antibody producing hybridomas were detected in a solid-phase radioimmunoassay (RIA) by binding to Hikone-R (BG) or Oregon-R microsomes. Four independent hybridomas were identified as producing monoclonal antibodies that recognized proteins in the P-450 complex by immunoblot experiments. Three monoclonal antibodies recognized P-450-A proteins, while one monoclonal antibody bound predominantly P-450-B. This monoclonal antibody also recognized southern armyworm (Spodoptera eridania, Cramer) microsomal proteins.

  4. Expresión de la Aromatasa P450 en los prolactinomas y su relación con la proliferación celular y apoptosis

    OpenAIRE

    Larrínaga Mendizábal, José Manuel

    2009-01-01

    [ES] La hipófisis o glándula pituitaria es una glándula de secreción interna, de pequeño tamaño, situada en la cara inferior del cerebro, por detrás del quiasma óptico y debajo del hipotálamo, al que se encuentra unida a través del tallo infundibular. Está alojada en la silla turca del esfenoides y en ella se diferencian varias partes; según la clasificación, aceptada hace más de 60 años, que hicieran Rioch se diferencian en ella, un lóbulo anterior o adenohipófisis, subdividida en tres pa...

  5. Recent Structural Insights into Cytochrome P450 Function.

    Science.gov (United States)

    Guengerich, F Peter; Waterman, Michael R; Egli, Martin

    2016-08-01

    Cytochrome P450 (P450) enzymes are important in the metabolism of drugs, steroids, fat-soluble vitamins, carcinogens, pesticides, and many other types of chemicals. Their catalytic activities are important issues in areas such as drug-drug interactions and endocrine function. During the past 30 years, structures of P450s have been very helpful in understanding function, particularly the mammalian P450 structures available in the past 15 years. We review recent activity in this area, focusing on the past 2 years (2014-2015). Structural work with microbial P450s includes studies related to the biosynthesis of natural products and the use of parasitic and fungal P450 structures as targets for drug discovery. Studies on mammalian P450s include the utilization of information about 'drug-metabolizing' P450s to improve drug development and also to understand the molecular bases of endocrine dysfunction. PMID:27267697

  6. Synthetic Biology with Cytochromes P450 Using Photosynthetic Chassis

    DEFF Research Database (Denmark)

    Gnanasekaran, Thiyagarajan

    these commercially important high value bioactive compounds are plant derived, and in plants, some of the key enzymes that catalyze the production of these compounds are cytochromes P450 (P450s). This thesis focuses on three subprojects in which we expressed plant metabolic pathways involving P450...

  7. Whole genome co-expression analysis of soybean cytochrome P450 genes identifies nodulation-specific P450 monooxygenases

    Science.gov (United States)

    Cytochrome P450 monooxygenases (P450s) catalyze oxidation of various substrates using oxygen and NAD(P)H. Plant P450s are involved in the biosynthesis of primary and secondary metabolites performing diverse biological functions. The recent availability of soybean genome sequence allows us to ident...

  8. Model complexes of key intermediates in fungal cytochrome P450 nitric oxide reductase (P450nor).

    Science.gov (United States)

    McQuarters, Ashley B; Wirgau, Nathaniel E; Lehnert, Nicolai

    2014-04-01

    Denitrifying bacteria and fungi efficiently detoxify the toxic metabolite nitric oxide (NO) through reduction to nitrous oxide (N2O) using nitric oxide reductase (NOR) enzymes. In fungi, for example Fusarium oxysporum, NO is reduced by a Cytochrome P450 NOR (P450nor). This enzyme contains a heme b center coordinated to a proximal cysteinate ligand in the active site. In the proposed mechanism of P450nor, the ferric heme binds NO first to form a ferric heme-nitrosyl complex, which is subsequently reduced by NAD(P)H to generate a ferrous HNO species as the next key intermediate. Recently, key progress has been made in our understanding of the electronic structures and fundamental reactivity of these important intermediates, using suitable model complexes. In this review, model complexes of ferric heme-nitrosyls with varied axial anionic ligands (such as N-donors, O-donors, and S-donors) are discussed first. Then, the generation and reactivity of ferrous heme-HNO complexes is summarized and related back to the mechanism of P450nor. PMID:24658055

  9. Cytochrome P450-Mediated Phytoremediation using Transgenic Plants: A Need for Engineered Cytochrome P450 Enzymes

    OpenAIRE

    Kumar, Santosh; Jin, Mengyao; Weemhoff, James L.

    2012-01-01

    There is an increasing demand for versatile and ubiquitous Cytochrome P450 (CYP) biocatalysts for biotechnology, medicine, and bioremediation. In the last decade there has been an increase in realization of the power of CYP biocatalysts for detoxification of soil and water contaminants using transgenic plants. However, the major limitations of mammalian CYP enzymes are that they require CYP reductase (CPR) for their activity, and they show relatively low activity, stability, and expression. O...

  10. Cytochrome P450-based cancer gene therapy: current status.

    Science.gov (United States)

    Kan, On; Kingsman, Susan; Naylor, Stuart

    2002-12-01

    Results from a number of preclinical studies have demonstrated that a P450-based gene-directed enzyme prodrug therapy (GDEPT) strategy for the treatment of cancer is both safe and efficacious. This strategy has now moved forward into the clinic. At least two different approaches using different delivery methods (retroviral vector MetXia [Oxford BioMedica] and encapsulated P450 expressing cells), different cytochrome P450 isoforms (human CYP2B6 versus rat CYP2B1) and different prodrugs (cyclophosphamide [CPA] versus ifosfamide [IFA]) have concluded Phase I/II clinical trial with encouraging results. In the future, P450-based GDEPT can potentially be further enhanced by improved vectors for P450 gene delivery and disease-targeted promoters for focused gene expression at the target site. In addition, there is scope for developing synthetic P450s and their respective prodrugs to improve both enzyme kinetics and the profile of the active moiety. PMID:12517265

  11. Epidermal CYP2 family cytochromes P450

    International Nuclear Information System (INIS)

    Skin is the largest and most accessible drug-metabolizing organ. In mammals, it is the competent barrier that protects against exposure to harmful stimuli in the environment and in the systemic circulation. Skin expresses many cytochromes P450 that have critical roles in exogenous and endogenous substrate metabolism. Here, we review evidence for epidermal expression of genes from the large CYP2 gene family, many of which are expressed preferentially in extrahepatic tissues or specifically in epithelia at the environmental interface. At least 13 CYP2 genes (CYP2A6, 2A7, 2B6, 2C9, 2C18, 2C19, 2D6, 2E1, 2J2, 2R1, 2S1, 2U1, and 2W1) are expressed in skin from at least some human individuals, and the majority of these genes are expressed in epidermis or cultured keratinocytes. Where epidermal expression has been localized in situ by hybridization or immunocytochemistry, CYP2 transcripts and proteins are most often expressed in differentiated keratinocytes comprising the outer (suprabasal) cell layers of the epidermis and skin appendages. The tissue-specific transcriptional regulation of CYP2 genes in the epidermis, and in other epithelia that interface with the environment, suggests important roles for at least some CYP2 gene products in the production and disposition of molecules affecting competency of the epidermal barrier

  12. Cytochrome P450: taming a wild type enzyme

    OpenAIRE

    Jung, Sang Taek; Lauchli, Ryan; Arnold, Frances H.

    2011-01-01

    Protein engineering of cytochrome P450 monooxygenases (P450s) has been very successful in generating valuable non-natural activities and properties, allowing these powerful catalysts to be used for the synthesis of drug metabolites and in biosynthetic pathways for the production of precursors of artemisinin and paclitaxel. Collected experience indicates that the P450s are highly 'evolvable'--they are particularly robust to mutation in their active sites and readily accept new substrates and e...

  13. Sterol 14α-Demethylase Cytochrome P450 (CYP51), a P450 in all Biological Kingdoms

    OpenAIRE

    Lepesheva, Galina I.; Waterman, Michael R.

    2006-01-01

    The CYP51 family is an intriguing subject for fundamental P450 structure/function studies and is also an important clinical drug target. This review updates information on the variety of the CYP51 family members, including their physiological roles, natural substrates and substrate preferences, and catalytic properties in vitro. We present experimental support for the notion that specific conserved regions in the P450 sequences represent a CYP51 signature. Two possible roles of CYP51 in P450 ...

  14. The SMARTCyp cytochrome P450 metabolism prediction server

    DEFF Research Database (Denmark)

    Rydberg, Patrik; Gloriam, David Erik Immanuel; Olsen, Lars

    2010-01-01

    The SMARTCyp server is the first web application for site of metabolism prediction of cytochrome P450-mediated drug metabolism.......The SMARTCyp server is the first web application for site of metabolism prediction of cytochrome P450-mediated drug metabolism....

  15. Predicting drug metabolism by cytochrome P450 2C9

    DEFF Research Database (Denmark)

    Rydberg, Patrik; Olsen, Lars

    2012-01-01

    By the use of knowledge gained through modeling of drug metabolism mediated by the cytochrome P450 2D6 and 3A4 isoforms, we constructed a 2D-based model for site-of-metabolism prediction for the cytochrome P450 2C9 isoform. The similarities and differences between the models for the 2C9 and 2D6...

  16. Kinetic Deuterium Isotope Effects in Cytochrome P450 Oxidation Reactions

    Science.gov (United States)

    Guengerich, F. Peter

    2016-01-01

    Cytochrome P450 (P450) enzymes account for ~ 75% of the metabolism of drugs. Most of the reactions catalyzed by P450s are mixed-function oxidations, and a C-H bond is (usually) broken. The rate-limiting nature of this step can be analyzed using the kinetic isotope effect (KIE) approach. The most relevant type of KIE is one termed intermolecular non-competitive, indicative of rate-limiting C-H bond breaking. A KIE vs. kcat for several P450s showed a correlation coefficient (r2) of 0.62. Deuterium substitution has been considered as a potential means of slowing drug metabolism or redirecting sites of metabolism in some cases, and several general points can be made regarding the potential for application of deuterium in drug design/development based on what is known about P450 KIEs. PMID:24285515

  17. Isolation of the alkane inducible cytochrome P450 (P450alk) gene from the yeast Candida tropicalis

    Science.gov (United States)

    The gene for the alkane-inducible cytochrome P450, P450alk, has been isolated from the yeast Candida tropicalis by immunoscreening a λgt11 library. Isolation of the gene has been identified on the basis of its inducibility and partial DNA sequence. Transcripts of this gene were i...

  18. Cytochrome P450 and Non-Cytochrome P450 Oxidative Metabolism: Contributions to the Pharmacokinetics, Safety, and Efficacy of Xenobiotics.

    Science.gov (United States)

    Foti, Robert S; Dalvie, Deepak K

    2016-08-01

    The drug-metabolizing enzymes that contribute to the metabolism or bioactivation of a drug play a crucial role in defining the absorption, distribution, metabolism, and excretion properties of that drug. Although the overall effect of the cytochrome P450 (P450) family of drug-metabolizing enzymes in this capacity cannot be understated, advancements in the field of non-P450-mediated metabolism have garnered increasing attention in recent years. This is perhaps a direct result of our ability to systematically avoid P450 liabilities by introducing chemical moieties that are not susceptible to P450 metabolism but, as a result, may introduce key pharmacophores for other drug-metabolizing enzymes. Furthermore, the effects of both P450 and non-P450 metabolism at a drug's site of therapeutic action have also been subject to increased scrutiny. To this end, this Special Section on Emerging Novel Enzyme Pathways in Drug Metabolism will highlight a number of advancements that have recently been reported. The included articles support the important role of non-P450 enzymes in the clearance pathways of U.S. Food and Drug Administration-approved drugs over the past 10 years. Specific examples will detail recent reports of aldehyde oxidase, flavin-containing monooxygenase, and other non-P450 pathways that contribute to the metabolic, pharmacokinetic, or pharmacodynamic properties of xenobiotic compounds. Collectively, this series of articles provides additional support for the role of non-P450-mediated metabolic pathways that contribute to the absorption, distribution, metabolism, and excretion properties of current xenobiotics. PMID:27298339

  19. Cytochrome p450 inhibitory properties of common efflux transporter inhibitors.

    Science.gov (United States)

    Englund, Gunilla; Lundquist, Patrik; Skogastierna, Cristine; Johansson, Jenny; Hoogstraate, Janet; Afzelius, Lovisa; Andersson, Tommy B; Projean, Denis

    2014-03-01

    Drug transporter inhibitors are important tools to elucidate the contribution of transporters to drug disposition both in vitro and in vivo. These inhibitors are often unselective and affect several transporters as well as drug metabolizing enzymes, which can make experimental results difficult to interpret with confidence. We therefore tested 14 commonly used P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and multidrug-resistance associated protein (MRP) inhibitors as inhibitors of cytochrome P450 (P450) enzyme activities using recombinant enzymes. A subset of P-gp and/or CYP3A inhibitors were selected (cyclosporin A, elacridar, ketoconazole, quinidine, reserpine, and tacrolimus) for a comparison of P450 inhibition in human microsomes and hepatocytes. Most P-gp inhibitors showed CYP3A4 inhibition, with potencies often in a similar range as their P-gp inhibition, as well as less potent CYP2C19 inhibition. Other P450 enzymes were not strongly inhibited except a few cases of CYP2D6 inhibition. MRP and BCRP inhibitors showed limited P450 inhibition. Some inhibitors showed less P450 inhibition in human hepatocytes than human liver microsomes, for example, elacridar, probably due to differences in binding, permeability limitations, or active, P-gp mediated efflux of the inhibitor from the hepatocytes. Quinidine was a potent P450 inhibitor in hepatocytes but only showed weak inhibition in microsomes. Quinidine shows an extensive cellular uptake, which may potentiate intracellular P450 inhibition. Elacridar, described as a potent and selective P-gp inhibitor, displayed modest P450 inhibition in this study and is thus a useful model inhibitor to define the role of P-gp in drug disposition without interference with other processes. PMID:24396142

  20. Fast prediction of cytochrome P450 mediated drug metabolism

    DEFF Research Database (Denmark)

    Rydberg, Patrik Åke Anders; Poongavanam, Vasanthanathan; Oostenbrink, Chris;

    2009-01-01

    Cytochrome P450 mediated metabolism of drugs is one of the major determinants of their kinetic profile, and prediction of this metabolism is therefore highly relevant during the drug discovery and development process. A new rule-based method, based on results from density functional theory...... calculations, for predicting activation energies for aliphatic and aromatic oxidations by cytochromes P450 is developed and compared with several other methods. Although the applicability of the method is currently limited to a subset of P450 reactions, these reactions describe more than 90 % of the...

  1. Regulation of rabbit lung cytochrome P-450 prostaglandin omega-hydroxylase (P-450/sub PG-omega/) during pregnancy

    International Nuclear Information System (INIS)

    The mechanism of induction during pregnancy of a rabbit lung prostaglandin omega-hydroxylase cytochrome P-450 has been investigated. This activity has been demonstrated to be induced over 100-fold in 28-day pregnant rabbits, as compared to nonpregnant rabbits. The induction is reflected by an increase in the amount of P-450/sub PG-omega/ protein as measured by Western blotting. P-450/sub PG-omega/ microsomal protein increases throughout gestation concomitant with an increase in PGE1 omega-hydroxylase activity. Elucidation of the level of induction involved extraction of RNA from rabbit lungs obtained at various days of gestation followed by in vitro translation of the RNA in the presence of 35S-methionine. Immunoprecipitation of newly synthesized P-450 and analysis of the immunoisolates by SDS-PAGE, autoradiography and densitometry of the P-450/sub PG-omega/ band revealed that the P-450/sub PG-omega/ mRNA levels followed the gestational time-dependent increase observed for both PGE1 omega-hydroxylase activity and P-450/sub PG-omega/ protein, i.e., a gradual increase peaking at 28-days, dropping precipitously to near control levels following parturition. These data suggest that control of P-450/sub PG-omega expression occurs at the transcriptional level. Western blots of human lung bronchioloalveolar-carcinoma cell lines NCL-H322 and NCL-H358 utilizing a guinea pig IgG to P-450/sub PG-omega/ detect a cross-reactive species

  2. Artificial Self-Sufficient P450 in Reversed Micelles

    Directory of Open Access Journals (Sweden)

    Teruyuki Nagamune

    2010-04-01

    Full Text Available Cytochrome P450s are heme-containing monooxygenases that require electron transfer proteins for their catalytic activities. They prefer hydrophobic compounds as substrates and it is, therefore, desirable to perform their reactions in non-aqueous media. Reversed micelles can stably encapsulate proteins in nano-scaled water pools in organic solvents. However, in the reversed micellar system, when multiple proteins are involved in a reaction they can be separated into different micelles and it is then difficult to transfer electrons between proteins. We show here that an artificial self-sufficient cytochrome P450, which is an enzymatically crosslinked fusion protein composed of P450 and electron transfer proteins, showed micelle-size dependent catalytic activity in a reversed micellar system. Furthermore, the presence of thermostable alcohol dehydrogenase promoted the P450-catalyzed reaction due to cofactor regeneration.

  3. In Vitro Cytochrome P450-Mediated Metabolism of Exemestane

    OpenAIRE

    Kamdem, Landry K.; Flockhart, David A.; Desta, Zeruesenay

    2011-01-01

    Exemestane is a potent and irreversible steroidal aromatase inhibitor drug used for the treatment of estrogen receptor-positive breast cancer. Our aim was to identify and assess the contribution of the specific cytochromes P450 (P450s) responsible for exemestane primary in vitro metabolism. With the use of high-performance liquid chromatography and liquid chromatography-tandem mass spectrometry analytical techniques, 17-hydroexemestane (MI) formation and 6-hydroxymethylexemestane (MII) format...

  4. The Cytochrome P450 Engineering Database: integration of biochemical properties

    OpenAIRE

    Lisitsa Andrey; Wagner Florian; Sirim Demet; Pleiss Jürgen

    2009-01-01

    Abstract Background Cytochrome P450 monooxygenases (CYPs) form a vast and diverse enzyme class of particular interest in drug development and a high biotechnological potential. Although very diverse in sequence, they share a common structural fold. For the comprehensive and systematic comparison of protein sequences and structures the Cytochrome P450 Engineering Database (CYPED) was established. It was built up based on an extensible data model that enables its functions readily enhanced. Des...

  5. Interindividual Variability in Cytochrome P450-Mediated Drug Metabolism.

    Science.gov (United States)

    Tracy, Timothy S; Chaudhry, Amarjit S; Prasad, Bhagwat; Thummel, Kenneth E; Schuetz, Erin G; Zhong, Xiao-Bo; Tien, Yun-Chen; Jeong, Hyunyoung; Pan, Xian; Shireman, Laura M; Tay-Sontheimer, Jessica; Lin, Yvonne S

    2016-03-01

    The cytochrome P450 (P450) enzymes are the predominant enzyme system involved in human drug metabolism. Alterations in the expression and/or activity of these enzymes result in changes in pharmacokinetics (and consequently the pharmacodynamics) of drugs that are metabolized by this set of enzymes. Apart from changes in activity as a result of drug-drug interactions (by P450 induction or inhibition), the P450 enzymes can exhibit substantial interindividual variation in basal expression and/or activity, leading to differences in the rates of drug elimination and response. This interindividual variation can result from a myriad of factors, including genetic variation in the promoter or coding regions, variation in transcriptional regulators, alterations in microRNA that affect P450 expression, and ontogenic changes due to exposure to xenobiotics during the developmental and early postnatal periods. Other than administering a probe drug or cocktail of drugs to obtain the phenotype or conducting a genetic analysis to determine genotype, methods to determine interindividual variation are limited. Phenotyping via a probe drug requires exposure to a xenobiotic, and genotyping is not always well correlated with phenotype, making both methodologies less than ideal. This article describes recent work evaluating the effect of some of these factors on interindividual variation in human P450-mediated metabolism and the potential utility of endogenous probe compounds to assess rates of drug metabolism among individuals. PMID:26681736

  6. Cytochrome P450 detection in liver of the catfish Ancistrus multispinis (Osteichthyes, Loricariidae

    Directory of Open Access Journals (Sweden)

    Claudio Klemz

    2010-04-01

    Full Text Available Sensitive biological responses to environmental contaminants are useful as early warning signals to predict the damages by long-term exposure. Protocols standardization to quantify biochemical parameters in different fish species is required to validate its use as biomarkers. Comparative studies from different fish species and its interpretation are a challenge for the validation of its use as general biomarkers, representative of environmental impact. In this study, the protocol for liver cytochrome P450 (CYP analysis from the native Brazilian fish Ancistrus multispinis was established. The microsome contamination by hemoglobin during the analysis of CYP in liver was detected, leading to misinterpretation of the results. The spectrophotometric method for CYP analysis was adapted in order to diminish the hemoglobin interference. Additionally, the western blotting method for CYP1A analysis was tested with success for this fish species.Respostas biológicas sensíveis aos contaminantes ambientais são úteis para prever efeitos prejudiciais devido a exposições crônicas. Padronização de protocolos para quantificar parâmetros bioquímicos em diferentes espécies de peixes é necessária para validar o uso como biomarcador. Estudos comparativos de diferentes espécies de peixe e sua interpretação são um avanço para a validação do uso de biomarcadores gerais, representativos do impacto ambiental. Neste estudo o protocolo para a análise do citocromo P450 (CYP do peixe nativo brasileiro Ancistrus multispinis foi estabelecido. Cyp é um biomarcador de exposição principalmente de hidrocarbonetos policíclicos aromáticos (HAP, bifenilas policloradas (PCB e dioxinas. A contaminação do microssomo pela hemoglobina durante as análises do CYP no fígado foi detectada, levando a uma interpretação errônea dos resultados. O método espectrofotométrico para análise do CYP foi adaptado para diminuir a interferência da hemoglobina. Al

  7. Whole genome co-expression analysis of soybean cytochrome P450 genes identifies nodulation-specific P450 monooxygenases

    Directory of Open Access Journals (Sweden)

    Pandey Sona

    2010-11-01

    Full Text Available Abstract Background Cytochrome P450 monooxygenases (P450s catalyze oxidation of various substrates using oxygen and NAD(PH. Plant P450s are involved in the biosynthesis of primary and secondary metabolites performing diverse biological functions. The recent availability of the soybean genome sequence allows us to identify and analyze soybean putative P450s at a genome scale. Co-expression analysis using an available soybean microarray and Illumina sequencing data provides clues for functional annotation of these enzymes. This approach is based on the assumption that genes that have similar expression patterns across a set of conditions may have a functional relationship. Results We have identified a total number of 332 full-length P450 genes and 378 pseudogenes from the soybean genome. From the full-length sequences, 195 genes belong to A-type, which could be further divided into 20 families. The remaining 137 genes belong to non-A type P450s and are classified into 28 families. A total of 178 probe sets were found to correspond to P450 genes on the Affymetrix soybean array. Out of these probe sets, 108 represented single genes. Using the 28 publicly available microarray libraries that contain organ-specific information, some tissue-specific P450s were identified. Similarly, stress responsive soybean P450s were retrieved from 99 microarray soybean libraries. We also utilized Illumina transcriptome sequencing technology to analyze the expressions of all 332 soybean P450 genes. This dataset contains total RNAs isolated from nodules, roots, root tips, leaves, flowers, green pods, apical meristem, mock-inoculated and Bradyrhizobium japonicum-infected root hair cells. The tissue-specific expression patterns of these P450 genes were analyzed and the expression of a representative set of genes were confirmed by qRT-PCR. We performed the co-expression analysis on many of the 108 P450 genes on the Affymetrix arrays. First we confirmed that CYP93C5 (an

  8. Cytochrome P450-2D6 Screening Among Elderly Using Antidepressants (CYSCE)

    Science.gov (United States)

    2015-12-09

    Depression; Depressive Disorder; Poor Metabolizer Due to Cytochrome P450 CYP2D6 Variant; Intermediate Metabolizer Due to Cytochrome P450 CYP2D6 Variant; Ultrarapid Metabolizer Due to Cytochrome P450 CYP2D6 Variant

  9. Repellents inhibit P450 enzymes in Stegomyia (Aedes aegypti.

    Directory of Open Access Journals (Sweden)

    Gloria Isabel Jaramillo Ramirez

    Full Text Available The primary defence against mosquitoes and other disease vectors is often the application of a repellent. Despite their common use, the mechanism(s underlying the activity of repellents is not fully understood, with even the mode of action of DEET having been reported to be via different mechanisms; e.g. interference with olfactory receptor neurones or actively detected by olfactory receptor neurones on the antennae or maxillary palps. In this study, we discuss a novel mechanism for repellence, one of P450 inhibition. Thirteen essential oil extracts from Colombian plants were assayed for potency as P450 inhibitors, using a kinetic fluorometric assay, and for repellency using a modified World Health Organisation Pesticide Evaluations Scheme (WHOPES arm-in cage assay with Stegomyia (Aedes aegypti mosquitoes. Bootstrap analysis on the inhibition analysis revealed a significant correlation between P450-inhibition and repellent activity of the oils.

  10. Guidelines for development and implementation of biocatalytic P450 processes

    DEFF Research Database (Denmark)

    Lundemo, Marie Therese; Woodley, John

    2015-01-01

    application, including use in synthetic processes. The biological characteristics of P450s (e.g., cofactor dependence) motivate the use of whole-cell systems for synthetic processes, and those processes implemented in industry are so far dominated by growing cells and native host systems. However, for an...... economically feasible process, the expression of P450 systems in a heterologous host with sufficient biocatalyst yield (g/g cdw) for non-growing systems or space-time yield (g/L/h) for growing systems remains a major challenge. This review summarizes the opportunities to improve P450 whole-cell processes and...... strategies in order to apply and implement them in industrial processes, both from a biological and process perspective. Indeed, a combined approach of host selection and cell engineering, integrated with process engineering, is suggested as the most effective route to implementation....

  11. Marmoset cytochrome P450 2J2 mainly expressed in small intestines and livers effectively metabolizes human P450 2J2 probe substrates, astemizole and terfenadine.

    Science.gov (United States)

    Uehara, Shotaro; Uno, Yasuhiro; Inoue, Takashi; Okamoto, Eriko; Sasaki, Erika; Yamazaki, Hiroshi

    2016-11-01

    1. Common marmoset (Callithrix jacchus), a New World Monkey, has potential to be a useful animal model in preclinical studies. However, drug metabolizing properties have not been fully understood due to insufficient information on cytochrome P450 (P450), major drug metabolizing enzymes. 2. Marmoset P450 2J2 cDNA was isolated from marmoset livers. The deduced amino acid sequence showed a high-sequence identity (91%) with cynomolgus monkey and human P450 2J2 enzymes. A phylogenetic tree revealed that marmoset P450 2J2 was evolutionarily closer to cynomolgus monkey and human P450 2J2 enzymes, than P450 2J forms in pigs, rabbits, rats or mice. 3. Marmoset P450 2J2 mRNA was abundantly expressed in the small intestine and liver, and to a lesser extent in the brain, lung and kidney. Immunoblot analysis also showed expression of marmoset P450 2J2 protein in the small intestine and liver. 4. Enzyme assays using marmoset P450 2J2 protein heterologously expressed in Escherichia coli indicated that marmoset P450 2J2 effectively catalyzed astemizole O-demethylation and terfenadine t-butyl hydroxylation, similar to human and cynomolgus monkey P450 2J2 enzymes. 5. These results suggest the functional characteristics of P450 2J2 enzymes are similar among marmosets, cynomolgus monkeys and humans. PMID:26899760

  12. Correlation of Cytochrome P450 Oxidoreductase Expression with the Expression of 10 Isoforms of Cytochrome P450 in Human Liver.

    Science.gov (United States)

    Zhang, Hai-Feng; Li, Zhi-Hui; Liu, Jia-Yu; Liu, Ting-Ting; Wang, Ping; Fang, Yan; Zhou, Jun; Cui, Ming-Zhu; Gao, Na; Tian, Xin; Gao, Jie; Wen, Qiang; Jia, Lin-Jing; Qiao, Hai-Ling

    2016-08-01

    Human cytochrome P450 oxidoreductase (POR) provides electrons for all microsomal cytochromes P450 (P450s) and plays an indispensable role in drug metabolism catalyzed by this family of enzymes. We evaluated 100 human liver samples and found that POR protein content varied 12.8-fold, from 12.59 to 160.97 pmol/mg, with a median value of 67.99 pmol/mg; POR mRNA expression varied by 26.4-fold. POR activity was less variable with a median value of 56.05 nmol/min per milligram. Cigarette smoking and alcohol consumption clearly influenced POR activity. Liver samples with a 2286822 TT genotype had significantly higher POR mRNA expression than samples with CT genotype. Homozygous carriers of POR2286822C>T, 2286823G>A, and 3823884A>C had significantly lower POR protein levels compared with the corresponding heterozygous carriers. Liver samples from individuals homozygous at 286823G>A, 1135612A>G, and 10954732G>A generally had lower POR activity levels than those from heterozygous or wild-type samples, whereas the common variant POR*28 significantly increased POR activity. There was a strong association between POR and the expression of P450 isoforms at the mRNA and protein level, whereas the relationship at the activity level, as well as the effect of POR protein content on P450 activity, was less pronounced. POR transcription was strongly correlated with both hepatocyte nuclear factor 4 alpha and pregnane X receptor mRNA levels. In conclusion, we have elucidated some potentially important correlations between POR single-nucleotide polymorphisms and POR expression in the Chinese population and have developed a database that correlates POR expression with the expression and activity of 10 P450s important in drug metabolism. PMID:27271371

  13. Correlation of Cytochrome P450 Oxidoreductase Expression with the Expression of 10 Isoforms of Cytochrome P450 in Human Liver

    Science.gov (United States)

    Zhang, Hai-Feng; Li, Zhi-Hui; Liu, Jia-Yu; Liu, Ting-Ting; Wang, Ping; Fang, Yan; Zhou, Jun; Cui, Ming-Zhu; Gao, Na; Tian, Xin; Gao, Jie; Wen, Qiang; Jia, Lin-Jing

    2016-01-01

    Human cytochrome P450 oxidoreductase (POR) provides electrons for all microsomal cytochromes P450 (P450s) and plays an indispensable role in drug metabolism catalyzed by this family of enzymes. We evaluated 100 human liver samples and found that POR protein content varied 12.8-fold, from 12.59 to 160.97 pmol/mg, with a median value of 67.99 pmol/mg; POR mRNA expression varied by 26.4-fold. POR activity was less variable with a median value of 56.05 nmol/min per milligram. Cigarette smoking and alcohol consumption clearly influenced POR activity. Liver samples with a 2286822 TT genotype had significantly higher POR mRNA expression than samples with CT genotype. Homozygous carriers of POR2286822C>T, 2286823G>A, and 3823884A>C had significantly lower POR protein levels compared with the corresponding heterozygous carriers. Liver samples from individuals homozygous at 286823G>A, 1135612A>G, and 10954732G>A generally had lower POR activity levels than those from heterozygous or wild-type samples, whereas the common variant POR*28 significantly increased POR activity. There was a strong association between POR and the expression of P450 isoforms at the mRNA and protein level, whereas the relationship at the activity level, as well as the effect of POR protein content on P450 activity, was less pronounced. POR transcription was strongly correlated with both hepatocyte nuclear factor 4 alpha and pregnane X receptor mRNA levels. In conclusion, we have elucidated some potentially important correlations between POR single-nucleotide polymorphisms and POR expression in the Chinese population and have developed a database that correlates POR expression with the expression and activity of 10 P450s important in drug metabolism. PMID:27271371

  14. Application of nanodisc technology for direct electrochemical investigation of plant cytochrome P450s and their NADPH P450 oxidoreductase.

    Science.gov (United States)

    Bavishi, Krutika; Laursen, Tomas; Martinez, Karen L; Møller, Birger Lindberg; Della Pia, Eduardo Antonio

    2016-01-01

    Direct electrochemistry of cytochrome P450 containing systems has primarily focused on investigating enzymes from microbes and animals for bio-sensing applications. Plant P450s receive electrons from NADPH P450 oxidoreductase (POR) to orchestrate the bio-synthesis of a plethora of commercially valuable compounds. In this report, full length CYP79A1, CYP71E1 and POR of the dhurrin pathway in Sorghum bicolor were reconstituted individually in nanoscale lipid patches, "nanodiscs", and directly immobilized on unmodified gold electrodes. Cyclic voltammograms of CYP79A1 and CYP71E1 revealed reversible redox peaks with average midpoint potentials of 80 ± 5 mV and 72 ± 5 mV vs. Ag/AgCl, respectively. POR yielded two pairs of redox peaks with midpoint potentials of 90 ± 5 mV and -300 ± 10 mV, respectively. The average heterogeneous electron transfer rate constant was calculated to be ~1.5 s(-1). POR was electro-catalytically active while the P450s generated hydrogen peroxide (H2O2). These nanodisc-based investigations lay the prospects and guidelines for construction of a simplified platform to perform mediator-free, direct electrochemistry of non-engineered cytochromes P450 under native-like conditions. It is also a prelude for driving plant P450 systems electronically for simplified and cost-effective screening of potential substrates/inhibitors and fabrication of nano-bioreactors for synthesis of high value natural products. PMID:27386958

  15. Rational redesign of the biodegradative enzyme cytochrome P450 cam:

    International Nuclear Information System (INIS)

    Cytochromes P450, a superfamily of monooxygenase enzymes present in all kingdoms of living organisms, are very versatile with respect to substrate range and catalytic functionality. Many recalcitrant halogenated hydrocarbons, on DOE sites and throughout the nation, result in serious environmental impact. Cytochromes P450 have been shown to be catalytically capable of, at least partial, dehalogenation of some such compounds. Clearly, however, their active site stereochemistry and related functional components are not well suited for this role because the rates of dehalogenation are generally rather modest. The evolution of modified active site and access channel structures may proceed very slowly if multiple genetic changes are simultaneously required for enzyme adaptation. Since each mutational event is by itself a rare event, a basic premise of our research is that designing multiple changes into an enzyme may be more timely than waiting for them to occur biologically either via natural selection or under laboratory-controlled conditions. Starting with available high-resolution x-ray crystal structures, molecular modeling and molecular dynamics simulations have been used to probe the basic structure/function principles and conformational fluctuations of the biodegradative enzyme, cytochrome P450cam (camphor hydroxylase from Pseudomonas putida) and active site mutants, to provide the fundamental understanding necessary for rational engineering of the enzyme for modified substrate specificity. In the present paper, we review our progress to data, in the area of molecular dynamics simulations and active site redesign of P450cam. 36 refs., 2 figs

  16. Bioprocess engineering for the application of P450s

    DEFF Research Database (Denmark)

    Lundemo, Marie Therese; Ringle, M.; Notonier, S.;

    The highly specific hydroxylation performed by P450 monooxygenases is a very powerful tool for synthetic chemists, not only at laboratory scale, but potentially also at industrial scale. However, despite this potential, only in a few cases has this class of enzymes been implemented at an industrial...

  17. Cytochrome P450 polymorphism and postoperative cognitive dysfunction

    DEFF Research Database (Denmark)

    Steinmetz, J; Jespersgaard, Cathrine; Dalhoff, Kim Peder;

    2012-01-01

    cytochrome P450 encoding genes. METHODS:We included patients who underwent non-cardiac surgery under total intravenous anesthesia with propofol. POCD was identified using a neuropsychological test-battery administered preoperatively, one week, and three months after surgery. Genotyping of CYP2C19*2, *3, CYP2...

  18. Trends in predicted chemoselectivity of cytochrome P450 oxidation

    DEFF Research Database (Denmark)

    Rydberg, Patrik; Lonsdale, Richard; Harvey, Jeremy N;

    2014-01-01

    Prediction of epoxide formation in drug metabolism is a difficult but important task, as epoxide formation is linked to drug toxicity. A comparison of the energy barriers for cytochrome P450 mediated epoxidation of alkenes to the barriers for the hydroxylation of an aliphatic carbon atom next to a...

  19. Cytochrome P450 aromatase expression in human seminoma

    Directory of Open Access Journals (Sweden)

    Montanaro Daniela

    2005-12-01

    Full Text Available Abstract Background The enzyme cytochrome P450 aromatase, catalysing the conversion of androgens into estrogens, has been detected in normal human testicular cells suggesting a physiological role of local estrogen biosynthesis on spermatogenesis control. Estrogens, regulating cell growth and apoptosis, can also be involved in tumorigenesis process, but the possible link between estrogens and testicular neoplastic process is, up to now, scarcely known. This study examined aromatase expression in human seminoma, which is the most common germ cell tumour of the testis. Methods The tumour-bearing testes were obtained from 20 patients with classic seminoma undergoing to therapeutic orchidectomy. Paraffin embedded tissues were processed for immunohistochemistry using a mouse monoclonal antibody generated against human placental cytochrome P450 arom, as primary antibody, and a biotinylated goat-anti-mouse IgG, as secondary antibody. Furthermore, Western blot analysis of seminoma extracts was carried out. Results Intense P450 arom immunoreactivity was observed in the seminoma cells and Western blot analysis confirmed the immunodetection. A strong immunostaining was also detected in cells of intratubular germ cell neoplasia (IGCN, adjacent to seminoma. Conclusion The present study demonstrated, for the first time in human, aromatase expression in neoplastic cells of seminoma suggesting a relation between local estrogen biosynthesis and germ cell tumorigenesis. The P450 arom immunolocalization in the cells of IGCN, representing the common precursor of most germ cell tumors, seems to support these findings.

  20. Effects of bromocriptine on hepatic cytochrome P-450 monooxygenase system.

    Science.gov (United States)

    Moochhala, S M; Lee, E J; Hu, G T; Koh, O S; Becket, G

    1989-02-01

    We have evaluated the in vitro effects of bromocriptine (Br), on the hepatic cytochrome P-450 monooxygenase system of rats pretreated with saline phenobarbitone (PB) and beta-naphthoflavone (BNF). Br inhibited ethoxyresorufin O-dealkylase (EROD) activity in liver microsomes of rats pretreated with saline and PB but not in BNF pretreated animals. Maximum inhibition of EROD activity by Br in the microsomes of saline and PB pretreated rats were 50%-60% of the control. In contrast, a dual effect was observed on aminopyrine N-demethylase activity (APD) by Br in microsomes of saline, PB and BNF pretreated rats. At a low concentration (25 microM), Br inhibited the activity of APD to a similar extent in all pretreatment groups; however, with higher concentrations of Br (50 microM to 300 microM), enhancement of APD activity was observed. Br (300 microM) increased the APD activity to 2-3 times the control level in microsomes of rats pretreated with saline, PB or BNF. Spectral studies revealed a Type II binding of Br to cytochrome P-450 from microsomes of saline and PB pretreated rats. A reverse type I binding was observed for BNF induced microsomes. In addition, Br also enhanced NADPH cytochrome c (P-450) reductase activity to a similar extent in all pretreatment groups. These results suggest that the inhibition of EROD activity may be due to direct binding by Br to certain isozymes of cytochrome P-450 and that the enhancing effect of Br on APD activity may be in part due to the activation of the NADPH cytochrome c reductase component of the cytochrome P-450 monooxygenase system. PMID:2499727

  1. Differentially regulated NADPH: cytochrome p450 oxidoreductases in parsely

    International Nuclear Information System (INIS)

    Two NADPH:cytochrome P450 oxidoreductases (CPRs) from parsley (Petroselinum crispum) were cloned, and the complete proteins were expressed and functionally identified in yeast. The two enzymes, designated CPR1 and CPR2, are 80% identical in amino acid sequence with one another and about 75% identical with CPRs from several other plant species. The mRNA accumulation patterns for CPR1 and CPR2 in fungal elicitor-treated or UV-irradiated cultured parsley cells and in developing or infected parsley plants were compared with those for cinnamate 4-hydroxylase (C4H), one of the most abundant CPR-dependent P450 enzymes in plants. All treatments strongly induced the mRNAs for C4H and CPR1 but not for CPR2, suggesting distinct metabolic roles of CPR1 and CPR2 and a functional relationship between CPR1 and C4H

  2. Contributions of Human Cytochrome P450 Enzymes to Glyburide Metabolism*

    OpenAIRE

    Zhou, Lin; Naraharisetti, Suresh B.; Liu, Li; Wang, Honggang; Lin, Yvonne S.; Isoherranen, Nina; Unadkat, Jashvant D.; Hebert, Mary F.; Mao, Qingcheng

    2010-01-01

    Glyburide (GLB) is a widely used oral sulfonylurea for the treatment of gestational diabetes. Therapeutic use of GLB is often complicated by a substantial inter-individual variability in the pharmacokinetics and pharmacodynamics of the drug in human populations, which might be caused by inter-individual variations in factors such as GLB metabolism. Therefore, there has been a continued interest in identifying human cytochrome P450 (CYP) isoforms that play a major role in the metabolism of GLB...

  3. Methodological approaches to cytochrome P450 profiling in embryos

    OpenAIRE

    Goldstone, Jared V.; Stegeman, John J.

    2012-01-01

    Cytochrome P450 enzymes (CYPs) are heme thiolate proteins essential for vertebrate development, and also play important roles in toxicology as well as normal metabolic function. CYP enzymes catalyze the oxidative biotransformation of many endogenous and exogenous chemicals, including steroids and eicosanoids, and drugs and other xenobiotic toxicants. Many CYPs have known or potential roles in development, and many chemicals that cause developmental abnormalities are substrates for CYPs. The r...

  4. Ipriflavone as an inhibitor of human cytochrome P450 enzymes

    OpenAIRE

    Monostory, Katalin; Vereczkey, László; Lévai, Ferenc; SZATMÁRI, ISTVÁN

    1998-01-01

    Reduction of theophylline metabolism and elimination were observed in a theophylline-treated patient during ipriflavone administration. After withdrawal of ipriflavone, the serum theophylline level decreased to an extent similar to that found before administration of ipriflavone. The effects of ipriflavone and its major metabolites 7-hydroxy-isoflavone and 7-(1-carboxy-ethoxy)-isoflavone on cytochrome P450 activities were studied in vitro in human liver microsomes from three donors.Ipriflavon...

  5. NADPH Cytochrome P-450 Oxidoreductase and Susceptibility to Ketoconazole

    OpenAIRE

    Venkateswarlu, K; Kelly, Diane E.; Manning, Nigel J.; Kelly, Steven L.

    1998-01-01

    The phenotype of a strain of Saccharomyces cerevisiae containing a disruption of the gene encoding NADPH cytochrome P-450 oxidoreductase (CPR) was quantified biochemically and microbiologically, as were those of various transformants of this strain after expression of native CPR, cytochrome P-45051 (CYP51), and a fusion protein of CYP51-CPR (FUS). Only a 4-fold decrease in ergosterol biosynthesis was observed for the cpr strain, but ketoconazole sensitivity increased 200-fold, indicating hype...

  6. Cytochrome P450 aromatase expression in human seminoma

    OpenAIRE

    Montanaro Daniela; Aquila Saveria; Romeo Francesco; Rago Vittoria; Andò Sebastiano; Carpino Amalia

    2005-01-01

    Abstract Background The enzyme cytochrome P450 aromatase, catalysing the conversion of androgens into estrogens, has been detected in normal human testicular cells suggesting a physiological role of local estrogen biosynthesis on spermatogenesis control. Estrogens, regulating cell growth and apoptosis, can also be involved in tumorigenesis process, but the possible link between estrogens and testicular neoplastic process is, up to now, scarcely known. This study examined aromatase expression ...

  7. Valence tautomerism in synthetic models of cytochrome P450.

    Science.gov (United States)

    Das, Pradip Kumar; Samanta, Subhra; McQuarters, Ashley B; Lehnert, Nicolai; Dey, Abhishek

    2016-06-14

    CytP450s have a cysteine-bound heme cofactor that, in its as-isolated resting (oxidized) form, can be conclusively described as a ferric thiolate species. Unlike the native enzyme, most synthetic thiolate-bound ferric porphyrins are unstable in air unless the axial thiolate ligand is sterically protected. Spectroscopic investigations on a series of synthetic mimics of cytP450 indicate that a thiolate-bound ferric porphyrin coexists in organic solutions at room temperature (RT) with a thiyl-radical bound ferrous porphyrin, i.e., its valence tautomer. The ferric thiolate state is favored by greater enthalpy and is air stable. The ferrous thiyl state is favored by entropy, populates at RT, and degrades in air. These ground states can be reversibly interchanged at RT by the addition or removal of water to the apolar medium. It is concluded that hydrogen bonding and local electrostatics protect the resting oxidized cytP450 active site from degradation in air by stabilizing the ferric thiolate ground state in contrast to its synthetic analogs. PMID:27302948

  8. The Cytochrome P450 Engineering Database: integration of biochemical properties

    Directory of Open Access Journals (Sweden)

    Lisitsa Andrey

    2009-11-01

    Full Text Available Abstract Background Cytochrome P450 monooxygenases (CYPs form a vast and diverse enzyme class of particular interest in drug development and a high biotechnological potential. Although very diverse in sequence, they share a common structural fold. For the comprehensive and systematic comparison of protein sequences and structures the Cytochrome P450 Engineering Database (CYPED was established. It was built up based on an extensible data model that enables its functions readily enhanced. Description The new version of the CYPED contains information on sequences and structures of 8613 and 47 proteins, respectively, which strictly follow Nelson's classification rules for homologous families and superfamilies. To gain biochemical information on substrates and inhibitors, the CYPED was linked to the Cytochrome P450 Knowledgebase (CPK. To overcome differences in the data model and inconsistencies in the content of CYPED and CPK, a metric was established based on sequence similarity to link protein sequences as primary keys. In addition, the annotation of structurally and functionally relevant residues was extended by a reliable prediction of conserved secondary structure elements and by information on the effect of single nucleotide polymorphisms. Conclusion The online accessible version of the CYPED at http://www.cyped.uni-stuttgart.de provides a valuable tool for the analysis of sequences, structures and their relationships to biochemical properties.

  9. Applications of microbial cytochrome P450 enzymes in biotechnology and synthetic biology.

    Science.gov (United States)

    Girvan, Hazel M; Munro, Andrew W

    2016-04-01

    Cytochrome P450 enzymes (P450s) are a superfamily of monooxygenase enzymes with enormous potential for synthetic biology applications. Across Nature, their substrate range is vast and exceeds that of other enzymes. The range of different chemical transformations performed by P450s is also substantial, and continues to expand through interrogation of the properties of novel P450s and by protein engineering studies. The ability of P450s to introduce oxygen atoms at specific positions on complex molecules makes these enzymes particularly valuable for applications in synthetic biology. This review focuses on the enzymatic properties and reaction mechanisms of P450 enzymes, and on recent studies that highlight their broad applications in the production of oxychemicals. For selected soluble bacterial P450s (notably the high-activity P450-cytochrome P450 reductase enzyme P450 BM3), variants with a multitude of diverse substrate selectivities have been generated both rationally and by random mutagenesis/directed evolution approaches. This highlights the robustness and malleability of the P450 fold, and the capacity of these biocatalysts to oxidise a wide range of chemical scaffolds. This article reviews recent research on the application of wild-type and engineered P450s in the production of important chemicals, including pharmaceuticals and drug metabolites, steroids and antibiotics. In addition, the properties of unusual members of the P450 superfamily that do not follow the canonical P450 catalytic pathway are described. PMID:27015292

  10. Coupled motions direct electrons along human microsomal P450 Chains.

    Directory of Open Access Journals (Sweden)

    Christopher R Pudney

    2011-12-01

    Full Text Available Protein domain motion is often implicated in biological electron transfer, but the general significance of motion is not clear. Motion has been implicated in the transfer of electrons from human cytochrome P450 reductase (CPR to all microsomal cytochrome P450s (CYPs. Our hypothesis is that tight coupling of motion with enzyme chemistry can signal "ready and waiting" states for electron transfer from CPR to downstream CYPs and support vectorial electron transfer across complex redox chains. We developed a novel approach to study the time-dependence of dynamical change during catalysis that reports on the changing conformational states of CPR. FRET was linked to stopped-flow studies of electron transfer in CPR that contains donor-acceptor fluorophores on the enzyme surface. Open and closed states of CPR were correlated with key steps in the catalytic cycle which demonstrated how redox chemistry and NADPH binding drive successive opening and closing of the enzyme. Specifically, we provide evidence that reduction of the flavin moieties in CPR induces CPR opening, whereas ligand binding induces CPR closing. A dynamic reaction cycle was created in which CPR optimizes internal electron transfer between flavin cofactors by adopting closed states and signals "ready and waiting" conformations to partner CYP enzymes by adopting more open states. This complex, temporal control of enzyme motion is used to catalyze directional electron transfer from NADPH→FAD→FMN→heme, thereby facilitating all microsomal P450-catalysed reactions. Motions critical to the broader biological functions of CPR are tightly coupled to enzyme chemistry in the human NADPH-CPR-CYP redox chain. That redox chemistry alone is sufficient to drive functionally necessary, large-scale conformational change is remarkable. Rather than relying on stochastic conformational sampling, our study highlights a need for tight coupling of motion to enzyme chemistry to give vectorial electron

  11. Cytochrome P450 polymorphism and postoperative cognitive dysfunction

    DEFF Research Database (Denmark)

    Steinmetz, J; Jespersgaard, Cathrine; Dalhoff, Kim Peder;

    2012-01-01

    cytochrome P450 encoding genes. METHODS:We included patients who underwent non-cardiac surgery under total intravenous anesthesia with propofol. POCD was identified using a neuropsychological test-battery administered preoperatively, one week, and three months after surgery. Genotyping of CYP2C19*2, *3, CYP2......D6*3, *4, *5 and *6 was performed using pyrosequencing, and patients were characterized according to their phenotype as ultra, extensive, intermediate, or poor metabolizers. RESULTS: In total, 337 patients with a median age of 67 years were included. 30 (9.4%) out of the 319 patients who underwent...

  12. The pharmacogenetics of cytochrome P450 enzymes in personalized medicine

    Directory of Open Access Journals (Sweden)

    Majid Moridani

    2007-06-01

    Full Text Available Personalized medicine is partially enabled by in vitro diagnostics including pharmacogenomic, proteomic and other functional testing such as therapeutic drug management and toxicological testing. This paper will introduce the conceptual aspects of developing personalized treatment using pharmacogenetics information. The initial discussion will give an overview of the application of pharmacogenetics in personalized medicine, followed by specific examples involving cytochrome P450s drug metabolizing enzymes (CYP enzymes. The paper also discusses the influence of racial and ethnic characteristics of a population on the variation in drug effectiveness and toxicity. The need for implementation of pharmacogenetics in medical education is highlighted.

  13. Oxidation of indolic bases by cytochrome P450 and ferrous picolinate

    OpenAIRE

    Jankowski, K; Delaforge, M.; Jaouen, M.; Virelizier, H

    1997-01-01

    Oxidations performed on four indolic bases, β-carboline 1, ibogaine 2, reserpine 3 and ajmaline 4, by hepatic cytochrome P450 (Cyt. P450) and ferrous picolinate (Fe(PA)2), lead to alicyclic hydroxylation as major reaction products.

  14. Induction of human cytochrome P450 enzymes : Predictive in vitro models and rifampicin induction in vivo

    OpenAIRE

    Kanebratt, Kajsa P

    2008-01-01

    The cytochrome P450 (P450) enzymes comprise the most important enzyme system with regard to phase I metabolism of drugs. Induction of P450s can result in decreased plasma concentrations of the drug itself or a coadminstered drug, followed by lack of effect. In the present study different in vitro models have been investigated for their ability to predict P450 induction in humans. It was found that human liver slices respond to prototypical inducers, although the model is not...

  15. Nanoscale electron transport measurements of immobilized cytochrome P450 proteins

    International Nuclear Information System (INIS)

    Gold nanopillars, functionalized with an organic self-assembled monolayer, can be used to measure the electrical conductance properties of immobilized proteins without aggregation. Measurements of the conductance of nanopillars with cytochrome P450 2C9 (CYP2C9) proteins using conducting probe atomic force microscopy demonstrate that a correlation exists between the energy barrier height between hopping sites and CYP2C9 metabolic activity. Measurements performed as a function of tip force indicate that, when subjected to a large force, the protein is more stable in the presence of a substrate. This agrees with the hypothesis that substrate entry into the active site helps to stabilize the enzyme. The relative distance between hopping sites also increases with increasing force, possibly because protein functional groups responsible for electron transport (ETp) depend on the structure of the protein. The inhibitor sulfaphenazole, in addition to the previously studied aniline, increased the barrier height for electron transfer and thereby makes CYP2C9 reduction more difficult and inhibits metabolism. This suggests that P450 Type II binders may decrease the ease of ETp processes in the enzyme, in addition to occupying the active site. (paper)

  16. Spaceflight Effects on Cytochrome P450 Content in Mouse Liver.

    Directory of Open Access Journals (Sweden)

    Natalia Moskaleva

    Full Text Available Hard conditions of long-term manned spaceflight can affect functions of many biological systems including a system of drug metabolism. The cytochrome P450 (CYP superfamily plays a key role in the drug metabolism. In this study we examined the hepatic content of some P450 isoforms in mice exposed to 30 days of space flight and microgravity. The CYP content was established by the mass-spectrometric method of selected reaction monitoring (SRM. Significant changes in the CYP2C29, CYP2E1 and CYP1A2 contents were detected in mice of the flight group compared to the ground control group. Within seven days after landing and corresponding recovery period changes in the content of CYP2C29 and CYP1A2 returned to the control level, while the CYP2E1 level remained elevated. The induction of enzyme observed in the mice in the conditions of the spaceflight could lead to an accelerated biotransformation and change in efficiency of pharmacological agents, metabolizing by corresponding CYP isoforms. Such possibility of an individual pharmacological response to medication during long-term spaceflights and early period of postflight adaptation should be taken into account in space medicine.

  17. Ab initio dynamics of the cytochrome P450 hydroxylation reaction

    Energy Technology Data Exchange (ETDEWEB)

    Elenewski, Justin E.; Hackett, John C, E-mail: jchackett@vcu.edu [Department of Physiology and Biophysics and The Massey Cancer Center, School of Medicine, Virginia Commonwealth University, 401 College Street, Richmond, Virginia 23219-1540 (United States)

    2015-02-14

    The iron(IV)-oxo porphyrin π-cation radical known as Compound I is the primary oxidant within the cytochromes P450, allowing these enzymes to affect the substrate hydroxylation. In the course of this reaction, a hydrogen atom is abstracted from the substrate to generate hydroxyiron(IV) porphyrin and a substrate-centered radical. The hydroxy radical then rebounds from the iron to the substrate, yielding the hydroxylated product. While Compound I has succumbed to theoretical and spectroscopic characterization, the associated hydroxyiron species is elusive as a consequence of its very short lifetime, for which there are no quantitative estimates. To ascertain the physical mechanism underlying substrate hydroxylation and probe this timescale, ab initio molecular dynamics simulations and free energy calculations are performed for a model of Compound I catalysis. Semiclassical estimates based on these calculations reveal the hydrogen atom abstraction step to be extremely fast, kinetically comparable to enzymes such as carbonic anhydrase. Using an ensemble of ab initio simulations, the resultant hydroxyiron species is found to have a similarly short lifetime, ranging between 300 fs and 3600 fs, putatively depending on the enzyme active site architecture. The addition of tunneling corrections to these rates suggests a strong contribution from nuclear quantum effects, which should accelerate every step of substrate hydroxylation by an order of magnitude. These observations have strong implications for the detection of individual hydroxylation intermediates during P450 catalysis.

  18. Cytochrome P450 Monooxygenase Immobilization as a Model of Herbicide Metabolism in vitro

    Institute of Scientific and Technical Information of China (English)

    Xiang Wensheng(向文胜); Wang Xiangjing; Ju Xiulian; Ren Tianrui

    2004-01-01

    To investigate herbicide metabolism in vitro by cytochrome P450 with stable enzymatic activity, cytochrome P450 is immobilized in silk fibroin. The enzymatic activity of immobilized cytochrome P450 is maintained above 80% after repeated batch experiments for 10 times. Moreover, the enzymatic activity of immobilized cytochrome P450 is kept as relatively high as 73.8% after storage for two months at 4℃. In addition, immobilization can improve the temperature and pH stability of cytochrome P450. Immobilized cytochrome P450 has the similar affinity Km values for herbicide chlorsulfuron and triasulfuron as the free cytochrome P450. In the case of chlorosulfuron, affinity Km value is 53μmol/L for free cytochrome P450, and 63μmol/L for immobilized cytochrome P450, respectively. In the case of triasulfuron affinity, Km value is 36μmol/L for free cytochrome P450, and 44μmol/L for immobilized cytochrome P450, respectivily. Immobilized cytochrome P450 will be convenient, rapid, stable and continuous for herbicide metabolism in micro-bioreactor in vitro.

  19. High-throughput fluorescence assay of cytochrome P450 3A4

    OpenAIRE

    Cheng, Qian; Guengerich, F. Peter

    2013-01-01

    Microtiter plate-based fluorescence assays allow rapid measurement of the catalytic activities of cytochrome P450 oxygenases (P450s). We describe a high-throughput fluorescence assay of P450 3A4, one of the key enzymes involved in xenobiotic metabolism. The assay involves the oxidative debenzylation of 7-hydroxy-4-trifluoromethyl coumarin, producing an increase in fluorescence.

  20. Conformational changes of the NADPH-dependent cytochrome P450 reductase in the course of electron transfer to cytochromes P450

    DEFF Research Database (Denmark)

    Laursen, Tomas; Jensen, Kenneth; Møller, Birger Lindberg

    2011-01-01

    The NADPH-dependent cytochrome P450 reductase (CPR) is a key electron donor to eucaryotic cytochromes P450 (CYPs). CPR shuttles electrons from NADPH through the FAD and FMN-coenzymes into the iron of the prosthetic heme-group of the CYP. In the course of these electron transfer reactions, CPR...

  1. Personalized Cancer Therapy Considering Cytochrome P450 Variability.

    Science.gov (United States)

    Preissner, Saskia; Simmaco, Maurizio; Gentile, Giovanna; Preissner, Robert

    2015-01-01

    The individual variability of pharmacokinetics is underestimated and few systematic studies exist in this field. In most cases, this leads to unwanted side effects or toxicity. In polychemotherapy, prodrugs (like ifosfamide), which have to be activated by cytochrome P450 enzymes (CYPs), play an important role. If patients are poor metabolizers for these drugs, the therapy will be ineffective. Furthermore, CYPs and transporters can be (over)expressed in target tissues, which is also not examined and considered in clinical routine. Here, we present a body map showing relevant enzymes in some organs and tissues. Finally, a typical case of a Caucasian chemotherapy patient with breast cancer is presented and discussed regarding a personalized cancer therapy considering the single nucleotide polymorphisms found via genotyping. PMID:26233905

  2. Novel Bioactivation Pathway of Benzbromarone Mediated by Cytochrome P450.

    Science.gov (United States)

    Kitagawara, Yumina; Ohe, Tomoyuki; Tachibana, Kumiko; Takahashi, Kyoko; Nakamura, Shigeo; Mashino, Tadahiko

    2015-09-01

    Benzbromarone (BBR) is a hepatotoxic drug, but the detailed mechanism of its toxicity remains unknown. We identified 2,6-dibromohydroquinone (DBH) and mono-debrominated catechol (2-ethyl-3-(3-bromo-4,5-dihydroxybenzoyl)benzofuran; CAT) as novel metabolites of BBR in rat and human liver microsomal systems by comparison with chemically synthesized authentic compounds, and we also elucidated that DBH is formed by cytochrome P450 2C9 and that CAT is formed mainly by CYP1A1, 2D6, 2E1, and 3A4. Furthermore, CAT, DBH, and the oxidized form of DBH are highly cytotoxic in HepG2 compared with BBR. Taken together, our data demonstrate that DBH, a novel reactive metabolite, may be relevant to BBR-induced hepatotoxicity. PMID:26106235

  3. Bioprocess Engineering for the Application of P450s

    DEFF Research Database (Denmark)

    Lundemo, Marie Therese

    Biocatalytic processes are advancing because of their high selectivity and mild operating conditions, in contrast to many chemical catalyzed processes. This is a clear advantage and frequently results in improved environmental performance. Biocatalytic processes have been implemented replacing...... traditional chemical catalysts as well as enabling new synthesis. Regardless of the process routes, the economic feasibility is crucial for successful industrial implementation. This has also been demonstrated by implemented biocatalytic processes, showing a clear cost advantage compared to the chemical...... alternative. One family of enzymes described to have a lot of potential for industrial biocatalysis is cytochrome P450 monooxygenases. The main motivation for this statement is their ability to hydroxylate nonactivated hydrocarbons in a specific manner, using molecular oxygen as oxidant. Containing more than...

  4. Active site dynamics of toluene hydroxylation by cytochrome P-450

    International Nuclear Information System (INIS)

    Rat liver cytochrome P-450 hydroxylates toluene to benzyl alcohol plus o-, m-, and p-cresol. Deuterated toluenes were incubated under saturating conditions with liver microsomes from phenobarbital-pretreated rats, and product yields and ratios were measured. Stepwise deuteration of the methyl leads to stepwise decreases in the alcohol/cresol ratio without changing the cresol isomer ratios. Extensive deuterium retention in the benzyl alcohols from PhCH2D and PhCHD2 suggests there is a large intrinsic isotope effect for benzylic hydroxylation. After replacement of the third benzylic H by D, the drop in the alcohol/cresol ratio was particularly acute, suggsting that metabolic switching from D to H within the methyl group was easier than switching from the methyl to the ring. Comparison of the alcohol/cresol ratio for PhCH3 vs PhCD3 indicated a net isotope effect of 6.9 for benzylic hydroxylation. From product yield data for PhCH3 and PhCD3, DV for benzyl alcohol formation is only 1.92, whereas DV for total product formation is 0.67 (i.e., inverse). From competitive incubations of PhCH3/PhCD3 mixtures D(V/K) isotope effects on benzyl alcohol formation and total product formation (3.6 and 1.23, respectively) are greatly reduced, implying strong commitment to catalysis. In contrast, D(V/K) for the alcohol/cresol ratio is 6.3, indicating that the majority of the intrinsic isotope effect is expressed through metabolic switching. Overall, these data are consistent with reversible formation of a complex between toluene and the active oxygen form of cytochrome P-450, which rearranges internally and reacts to form products faster than it dissociates back to release substrate

  5. Mode of Antifungal Drugs Interaction with Cytochrome P- 450

    Directory of Open Access Journals (Sweden)

    M- Mahmodian

    1991-07-01

    Full Text Available Computer was used to identify the interactions of substrates and antifungal drugs with the enzyme, Cytochrome P-450; and then Molplot.bas computer program was applied to get three dimensional figures of 5-hydroxy camphor.oxidation products of camphor analogues, and antifungal drugs.Cartesian characteristics of atoms building molecules, are taken from Buildz. for program, which can calculate X,Y,Z coordinates of atoms by Zmatrix data. The other program which can calculate X,Y,Z coordinates, using fractional characteristics, is the Coord, for program that, gives our cartesian characteristics of the atoms of molecule, then by using these data, we obtain three dimensional figures and distance between active atoms in compounds under consideration. Results show that distance between two oxygen atoms in 5-exo-hydroxy- camphor and the other compounds obtained from oxidation of camphor analogues, with the distance of two oxygen atoms in antifungal compounds under discussion are equal. Therefore, we can conclude that, the antifungal molecule also interacts with enzyme's active site, by its own sites, in a similar manner to the 5-hydroxy camphor molecule, which is:"n1. Nitrogen atom (N of Imidazole and Triazole ring in antifungal molecule with Iron atom in heam molecule belonging to Cytochrome P-450 enzyme, are coordinated."n2. The other atoms such as : 0,S or N in structure of the antifungal drug are coordinated with hydrogen atom of hydroxyl group belong ing to Tyr-96 in the structure of enzyme, forming hydrogen bonding.

  6. Genome-Wide Annotation and Comparative Analysis of Cytochrome P450 Monooxygenases in Basidiomycete Biotrophic Plant Pathogens

    OpenAIRE

    Qhanya, Lehlohonolo Benedict; Matowane, Godfrey; Chen, Wanping; Sun, Yuxin; Letsimo, Elizabeth Mpholoseng; Parvez, Mohammad; Yu, Jae-Hyuk; Mashele, Samson Sitheni; Syed, Khajamohiddin

    2015-01-01

    Fungi are an exceptional source of diverse and novel cytochrome P450 monooxygenases (P450s), heme-thiolate proteins, with catalytic versatility. Agaricomycotina saprophytes have yielded most of the available information on basidiomycete P450s. This resulted in observing similar P450 family types in basidiomycetes with few differences in P450 families among Agaricomycotina saprophytes. The present study demonstrated the presence of unique P450 family patterns in basidiomycete biotrophic plant ...

  7. Crystal structure of cytochrome P450 MoxA from Nonomuraea recticatena (CYP105)

    International Nuclear Information System (INIS)

    Cytochrome P450 MoxA (P450moxA) from a rare actinomycete Nonomuraea recticatena belongs to the CYP105 family and exhibits remarkably broad substrate specificity. Here, we demonstrate that P450moxA acts on several luciferin derivatives, which were originally identified as substrates of the human microsomal P450s. We also describe the crystal structure of P450moxA in substrate-free form. Structural comparison with various bacterial and human microsomal P450s reveals that the P450moxA structure is most closely related to that of the fungal nitric oxide reductase P450nor (CYP55A1). Final refined model of P450moxA comprises almost all the residues, including the 'BC-loop' and 'FG-loop' regions pivotal for substrate recognition, and the current structure thus defines a well-ordered substrate-binding pocket. Clear electron density map reveals that the MES molecule is bound to the substrate-binding site, and the sixth coordination position of the heme iron is not occupied by a water molecule, probably due to the presence of MES molecule in the vicinity of the heme. The unexpected binding of the MES molecule might reflect the ability of P450moxA to accommodate a broad range of structurally diverse compounds

  8. Hepatic cytochrome P450 activity, abundance, and expression throughout human development

    Energy Technology Data Exchange (ETDEWEB)

    Sadler, Natalie C.; Nandhikonda, Premchendar; Webb-Robertson, Bobbie-Jo M.; Ansong, Charles; Anderson, Lindsey N.; Smith, Jordan N.; Corley, Richard A.; Wright, Aaron T.

    2016-07-01

    Cytochrome P450s are Phase I metabolic enzymes that play critical roles in the biotransformation of endogenous compounds and xenobiotics. The expression and activity of P450 enzymes can vary considerably throughout human development, especially when comparing fetal development to neonates, children, and adults. In an effort to develop a more comprehensive understanding of the ontogeny of P450 expression and activity we employed a multi-omic characterization of P450 transcript expression, protein abundance, and functional activity. To quantify the functional activity of individual P450s we employ activity-based protein profiling, which uses modified mechanism-based inhibitors of P450s as chemical probes, in tandem with proteomic analyses to quantify activity. Our results reveal life-stage-dependent variability in P450 expression, abundance, and activity throughout human development and frequent discordant relationships between expression and activity. The results were used to distribute P450s into three general classes based upon developmental stage of expression and activity. We have significantly expanded the knowledge of P450 ontogeny, particularly at the level of individual P450 activity. We anticipate that our ontogeny results will be useful for enabling predictive therapeutic dosing, and for avoiding potentially adverse and harmful reactions during maturation from both therapeutic drugs and environmental xenobiotics.

  9. Two mutant alleles of the human cytochrome P-450dbl gene (P450C2D1) associated with genetically deficient metabolism of debrisoquine and other drugs

    International Nuclear Information System (INIS)

    The debrisoquine polymorphism is a clinically important genetic defect of drug metabolism affecting 5-10% of individuals in Caucasian populations. It is inherited as an autosomal recessive trait. A full-length cDNA for human cytochrome P-450db1, the deficient enzyme (also designated P450IID1 for P450 family II subfamily D isozyme 1), has recently been cloned. Leukocyte DNA from extensive metabolizers (EMs) or poor metabolizers (PMs) of debrisoquine was examined by Southern analysis. Two polymorphic restriction fragments were associated with the PM phenotype when DNAs from 24 unrelated PM and 29 unrelated EM individuals were probed with P-450db1 cDNA after digestion with Xba I restriction endonuclease and Southern blotting. Seventy-five percent of PMs had either the 44-kb or the 11.5-kb fragment or both. Segregation of these restriction fragment length polymorphisms in the families of six PM probands demonstrated that each of the two fragments is allelic with the 29-kb fragment present in all EM individuals and suggests that they identify two independent mutated alleles of the P-450db1 gene (designated P450C2D1). The Xba I 44-kb fragment and 11.5-kb fragment were in linkage disequilibrium with restriction fragment length polymorphisms generated by four and five additional restriction endonucleases, respectively, which can be used to identify the same mutant alleles for the P-450db1 gene

  10. Immunochemical characterization of multiple forms of cytochrome P-450 in rabbit nasal microsomes and evidence for tissue-specific expression of P-450s NMa and NMb.

    Science.gov (United States)

    Ding, X X; Coon, M J

    1990-04-01

    Two unique forms of cytochrome P-450 (P-450), designated NMa and NMb, were recently isolated in this laboratory from nasal microsomes of rabbits. In the present study, polyclonal antibodies to the purified nasal cytochromes were prepared. Immunochemical analysis with specific rabbit anti-NMa and sheep anti-NMb antibodies indicated that P-450 isozymes identical to or having a high structural homology with NMa are present in both olfactory and respiratory mucosa, as well as in liver, but NMb was detected only in the olfactory mucosa. Neither form was detected in other tissues examined, including brain, esophageal mucosa, heart, intestinal mucosa, kidney, and lung. The specific occurrence of NMb in the olfactory mucosa was further substantiated by the detection and specific inhibition by anti-NMb of the formation of unique NMb-dependent metabolites of testosterone in olfactory microsomes but not in microsomes from liver or respiratory mucosa. Similar experiments with antibodies to previously purified rabbit hepatic P-450 isozymes indicated that not all of the hepatic cytochromes are expressed in the nasal tissues. Thus, P-450 isozymes structurally homologous to hepatic forms 2, 3a, and 4, but not 3b and 6, were found in the olfactory mucosa. On the other hand, only form 2 was detected in the respiratory mucosa. Immunoquantitation experiments revealed that NMa and NMb are the major P-450 forms in olfactory microsomes, whereas NMa and P-450 form 2 (or its homolog) constitute the major portion of the respiratory nasal microsomal P-450. The level of NMa in the liver is relatively low, accounting for less than 3% of total microsomal P-450 in this tissue. In addition, evidence is provided that NMa is the major catalyst in the dealkylation of two nasal carcinogens, hexamethylphosphoramide and phenacetin, in both olfactory and respiratory nasal microsomes. PMID:2109181

  11. Effects of methotrexate on rat P-450 cytochrome mono-oxygenases; Action du methotrexate sur les monooxygenases a cytochromes P-450 chez le rat

    Energy Technology Data Exchange (ETDEWEB)

    Guitton, J.; Guilluy, R.; Brazier, J.L. [Faculte de Pharmacie, 69 - Lyon (France); Souillet, G. [Hopital Debrousse, 69 - Lyon (France); Riviere, J.L. [INRA, 69 - Marcy l`Etoile (France); Gerard, F. [Institut Pasteur, 69 - Lyon (France)

    1994-12-31

    Methotrexate, an anti-cancerous agent, acts as an anti-metabolite of the nucleic acids which synthesis is then inhibited. Using aminopyrine breath test after methotrexate processing, the effects of the molecule on activities of the hepatocyte P-450 cytochrome mono-oxygenases, are studied. Breath micro-tests with carbon 13-labelled aminopyrine have been carried out to observe the metabolism evolution. Micro-test results have been compared to microsomal enzymatic activities for various substrates, and also to P-450 cytochrome ratio. Results show that methotrexate induces a reduction in the P-450 cytochrome ratio, and thus reduce the hepatic biotransformation process. 1 fig., 30 refs.

  12. Regulation of the cytochrome P450 2A genes

    International Nuclear Information System (INIS)

    Cytochrome P450 monooxygenases of the CYP2A subfamily play important roles in xenobiotic disposition in the liver and in metabolic activation in extrahepatic tissues. Many of the CYP2A transcripts and enzymes are inducible by xenobiotic compounds, and the expression of at least some of the CYP2A genes is influenced by physiological status, such as circadian rhythm, and pathological conditions, such as inflammation, microbial infection, and tumorigenesis. Variability in the expression of the CYP2A genes, which differs by species, animal strain, gender, and organ, may alter the risks of chemical toxicity for numerous compounds that are CYP2A substrates. The mechanistic bases of these variabilities are generally not well understood. However, recent studies have yielded interesting findings in several areas, such as the role of nuclear factor 1 in the tissue-selective expression of CYP2A genes in the olfactory mucosa (OM); the roles of constitutive androstane receptor, pregnane X receptor (PXR), and possibly, peroxisome proliferator-activated receptors in transcriptional regulation of the Cyp2a5 gene; and the involvement of heterogeneous nuclear ribonucleoprotein A1 in pyrazole-induced stabilization of CYP2A5 mRNA. The aims of this minireview are to summarize current knowledge of the regulation of the CYP2A genes in rodents and humans, and to stimulate further mechanistic studies that will ultimately improve our ability to determine, and to understand, these variabilities in humans

  13. Cytochrome P450 genetic polymorphisms of Mexican indigenous populations.

    Science.gov (United States)

    Sosa-Macías, Martha; Llerena, Adrián

    2013-01-01

    This review focuses on the genetic polymorphisms of the cytochrome P450 (CYP) genes in Mexican indigenous populations, who are a part of the wide ethnic diversity of this country. These native groups have a particular historical trajectory that is different from the Mexican Mestizos. This variability may be reflected in the frequency distribution of polymorphisms in the CYP genes that encode enzymes involved in the metabolism of drugs and other xenobiotics. Therefore, these polymorphisms may affect drug efficacy and safety in indigenous populations in Mexico. The present study aimed to analyze the prevalence of CYP polymorphisms in indigenous Mexicans and to compare the results with studies in Mexican Mestizos. Because the extrapolation of pharmacogenetic data from Mestizos is not applicable to the majority of indigenous groups, pharmacogenetic studies directed at indigenous populations need to be developed. The Amerindians analyzed in this study showed a low phenotypic (CYP2D6) and genotypic (CYP2D6, CYP2C9) diversity, unlike Mexican Mestizos. The frequency of polymorphisms in the CYP1A1, CYP2C19, CYP2E1, and CYP3A4 genes was more similar among the Amerindians and Mexican Mestizos, with the exception of the CYP1A2 gene, whose *1F variant frequency in Mexican Amerindians was the highest described to date. PMID:24145057

  14. CARACTERIZACIÓN DE VARIANTES ALÉLICAS DE CITOCROMO CYP2D6 EN LA POBLACIÓN DE LA REGIÓN CENTROCCIDENTAL DE VENEZUELA

    Directory of Open Access Journals (Sweden)

    GRIMÁN PEDRO

    2009-04-01

    Full Text Available

    RESUMEN

    El gen CYP2D6 codifica para una monooxigenasa perteneciente al citocromo P450, la cual está involucrada en la biotransformación de un gran número de drogas comúnmente prescritas, como antidepresivos, antineoplásicos y antihipertensivos. Algunos efectos adversos, así como falla terapéutica pueden ser relacionados con la actividad anormal de CYP2D6 producto de polimorfismos en el gen de dicha enzima. Con el fin de predecir la frecuencia de algunos fenotipos metabolizadores pobres de CYP2D6 en la población de la región centroccidental de Venezuela se determinaron las frecuencias alélicas y genotípicas de las variantes alélicas CYP2D6*3, *4 y *6. Se extrajo ADN genómico a partir de sangre periférica de 100 individuos voluntarios aparentemente sanos, y se procedió a la genotipificación por PCR tetra-primer alelo-específica y análisis por electroforesis en geles de agarosa. Se compararon las frecuencias obtenidas con poblaciones de otros países. El alelo más frecuente fue CYP2D6*4 con 16,5%, mostrando una diferencia significativa con la reportada con poblaciones asiáticas. Este trabajo constituye un estudio preliminar en la caracterización de un grupo más amplio de alelos de CYP2D6 con el fin de asistir al desarrollo de una farmacoterapia individualizada en nuestro país.

    Palabras clave: Citocromo P450, CYP2D6, Farmacogenética.


    ABSTRACT

    The CYP2D6 gene encodes for a monooxygenase belonging to the cytochrome P450, which is involved in the biotransformation of a large number of commonly prescribed drugs such as antidepressants, antihypertensive and antineoplastic. Some side effects, as well as therapeutic failure may be related to abnormal activity of CYP2D6 product of polymorphisms in the CYP2D6 gene. In order to predict the frequency of some poor metabolisers phenotypes of CYP2D6 in the population of the

  15. New Trends in Cytochrome P450 Research at the Half-Century Mark*

    OpenAIRE

    Guengerich, F. Peter

    2013-01-01

    Cytochrome P450 enzymes have major roles in the metabolism of steroids, drugs, carcinogens, eicosanoids, and numerous other chemicals. The P450s are collectively considered the most diverse catalysts known in biochemistry, although they operate from a basic structural fold and catalytic mechanism. The four minireviews in this thematic series deal with the unusual aspects of catalytic reactions and electron transfer pathway organization, the structural diversity of P450s, and the expanding rol...

  16. A Stereoselective Hydroxylation Step of Alkaloid Biosynthesis by a Unique Cytochrome P450 in Catharanthus roseus*

    OpenAIRE

    Giddings, Lesley-Ann; Liscombe, David K.; Hamilton, John P; Childs, Kevin L.; DellaPenna, Dean; Buell, C. Robin; O'Connor, Sarah E.

    2011-01-01

    Plant cytochrome P450s are involved in the production of over a hundred thousand metabolites such as alkaloids, terpenoids, and phenylpropanoids. Although cytochrome P450 genes constitute one of the largest superfamilies in plants, many of the catalytic functions of the enzymes they encode remain unknown. Here, we report the identification and functional characterization of a cytochrome P450 gene in a new subfamily of CYP71, CYP71BJ1, involved in alkaloid biosynthesis. Co-expression analysis ...

  17. N-acetyl-p-benzoquinone imine: a cytochrome P-450-mediated oxidation product of acetaminophen.

    OpenAIRE

    Dahlin, D C; Miwa, G T; Lu, A Y; Nelson, S. D.

    1984-01-01

    N-acetyl-p-benzoquinone imine (NAPQI) has been proposed as the toxic metabolite of acetaminophen for the past 10 years, although it has never been detected as an enzymatic oxidation product of acetaminophen. We report (i) direct detection of NAPQI formed as an oxidation product of acetaminophen by cytochrome P-450 and cumene hydroperoxide and (ii) indirect evidence that is compelling for NAPQI formation from acetaminophen by cytochrome P-450, NADPH, and NADPH-cytochrome P-450 reductase. Evide...

  18. Hot-spot residues in the cytochrome P450cam-putidaredoxin binding interface.

    Science.gov (United States)

    Hiruma, Yoshitaka; Gupta, Ankur; Kloosterman, Alexander; Olijve, Caroline; Olmez, Betül; Hass, Mathias A S; Ubbink, Marcellus

    2014-01-01

    Cytochrome P450cam (P450cam) is a heme-containing monooxygenase that catalyzes the hydroxylation of D-camphor to produce 5-exo-hydroxycamphor. The catalytic cycle of P450cam requires two electrons, both of which are donated by putidaredoxin (Pdx), a ferredoxin containing a [2 Fe-2 S] cluster. Atomic-resolution structures of the Pdx-P450cam complex have recently been solved by X-ray crystallography and paramagnetic NMR spectroscopy. The binding interface showed the potential electron transfer pathways and interactions between Pdx Asp38 and P450cam Arg112, as well as hydrophobic contacts between the Pdx Trp106 and P450cam residues. Several polar residues not previously recognized as relevant for binding were found in the interface. In this study, site-directed mutagenesis, kinetic measurements, and NMR studies were employed to probe the energetic importance and role of the polar residues in the Pdx-P450cam interaction. A double mutant cycle (DMC) analysis of kinetic data shows that favorable interactions exist between Pdx Tyr33 and P450cam Asp125, as well as between Pdx Ser42 and P450cam His352. The results show that alanine substitutions of these residues and several others do not influence the rates of electron transfer. It is concluded that these polar interactions contribute to partner recognition rather than to electronic coupling of the redox centers. PMID:24302683

  19. Absence of hepatic cytochrome P450bufI causes genetically deficient debrisoquine oxidation in man

    International Nuclear Information System (INIS)

    The common genetic deficiency of drug oxidation known as debrisoquine/sparteine-type polymorphism was investigated with bufuralol as prototype substrate. In human liver microsomes the 1'-hydroxylation of bufuralol is catalyzed by two functionally distinct P-450 isozymes, the high-affinity/highly stereoselective P450bufI and the low-affinity/nonstereoselective P450bufII. The authors demonstrate that P450bufI is unique in hydroxylating bufuralol in a cumene hydroperoxide (CuOOH) mediated reaction whereas P450bufII is active only in the classical NADPH- and O2-supported monooxygenation. In microsomes of liver biopsies of in vivo phenotyped poor metabolizers of debrisoquine or sparteine, the CuOOH-mediated activity was drastically reduced. Rabbit antibodies against a rat P-450 isozyme with high bufuralol 1'-hydroxylase activity (P450db1) precipitated exclusively P450bufI-type activity from solubilized microsomes. Western blotting of microsomes with these antibodies revealed a close correlation between the immunoreactive protein and CuOOH-mediated (+)-bufuralol 1'-hydroxylation. No immunoreactive protein was detected in liver microsomes of in vivo phenotyped poor metabolizers. These data provide evidence for a specific deficiency of P450bufI and are consistent with the complete or almost complete absence of this protein in the liver of poor metabolizers

  20. The role of renal proximal tubule P450 enzymes in chloroform-induced nephrotoxicity: Utility of renal specific P450 reductase knockout mouse models

    International Nuclear Information System (INIS)

    The kidney is a primary target for numerous toxic compounds. Cytochrome P450 enzymes (P450) are responsible for the metabolic activation of various chemical compounds, and in the kidney are predominantly expressed in proximal tubules. The aim of this study was to test the hypothesis that renal proximal tubular P450s are critical for nephrotoxicity caused by chemicals such as chloroform. We developed two new mouse models, one having proximal tubule-specific deletion of the cytochrome P450 reductase (Cpr) gene (the enzyme required for all microsomal P450 activities), designated proximal tubule-Cpr-null (PTCN), and the other having proximal tubule-specific rescue of CPR activity with the global suppression of CPR activity in all extra-proximal tubular tissues, designated extra-proximal tubule-Cpr-low (XPT-CL). The PTCN, XPT-CL, Cpr-low (CL), and wild-type (WT) mice were treated with a single oral dose of chloroform at 200 mg/kg. Blood, liver and kidney samples were obtained at 24 h after the treatment. Renal toxicity was assessed by measuring BUN and creatinine levels, and by pathological examination. The blood and tissue levels of chloroform were determined. The severity of toxicity was less in PTCN and CL mice, compared with that of WT and XPT-CL mice. There were no significant differences in chloroform levels in the blood, liver, or kidney, between PTCN and WT mice, or between XPT-CL and CL mice. These findings indicate that local P450-dependent activities play an important role in the nephrotoxicity induced by chloroform. Our results also demonstrate the usefulness of these novel mouse models for studies of chemical-induced kidney toxicity. - Highlights: • New mouse models were developed with varying P450 activities in the proximal tubule. • These mouse models were treated with chloroform, a nephrotoxicant. • Studies showed the importance of local P450s in chloroform-induced nephrotoxicity

  1. The role of renal proximal tubule P450 enzymes in chloroform-induced nephrotoxicity: Utility of renal specific P450 reductase knockout mouse models

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Senyan [Kidney Institute and Division of Nephrology, Changzheng Hospital, Shanghai 200003 (China); Wadsworth Center, New York State Department of Health, and School of Public Health, State University of New York, Albany, NY 12201 (United States); Yao, Yunyi; Lu, Shijun; Aldous, Kenneth; Ding, Xinxin [Wadsworth Center, New York State Department of Health, and School of Public Health, State University of New York, Albany, NY 12201 (United States); Mei, Changlin, E-mail: chlmei1954@126.com [Kidney Institute and Division of Nephrology, Changzheng Hospital, Shanghai 200003 (China); Gu, Jun, E-mail: jungu@wadsworth.org [Wadsworth Center, New York State Department of Health, and School of Public Health, State University of New York, Albany, NY 12201 (United States)

    2013-10-01

    The kidney is a primary target for numerous toxic compounds. Cytochrome P450 enzymes (P450) are responsible for the metabolic activation of various chemical compounds, and in the kidney are predominantly expressed in proximal tubules. The aim of this study was to test the hypothesis that renal proximal tubular P450s are critical for nephrotoxicity caused by chemicals such as chloroform. We developed two new mouse models, one having proximal tubule-specific deletion of the cytochrome P450 reductase (Cpr) gene (the enzyme required for all microsomal P450 activities), designated proximal tubule-Cpr-null (PTCN), and the other having proximal tubule-specific rescue of CPR activity with the global suppression of CPR activity in all extra-proximal tubular tissues, designated extra-proximal tubule-Cpr-low (XPT-CL). The PTCN, XPT-CL, Cpr-low (CL), and wild-type (WT) mice were treated with a single oral dose of chloroform at 200 mg/kg. Blood, liver and kidney samples were obtained at 24 h after the treatment. Renal toxicity was assessed by measuring BUN and creatinine levels, and by pathological examination. The blood and tissue levels of chloroform were determined. The severity of toxicity was less in PTCN and CL mice, compared with that of WT and XPT-CL mice. There were no significant differences in chloroform levels in the blood, liver, or kidney, between PTCN and WT mice, or between XPT-CL and CL mice. These findings indicate that local P450-dependent activities play an important role in the nephrotoxicity induced by chloroform. Our results also demonstrate the usefulness of these novel mouse models for studies of chemical-induced kidney toxicity. - Highlights: • New mouse models were developed with varying P450 activities in the proximal tubule. • These mouse models were treated with chloroform, a nephrotoxicant. • Studies showed the importance of local P450s in chloroform-induced nephrotoxicity.

  2. Mechanistic Scrutiny Identifies a Kinetic Role for Cytochrome b5 Regulation of Human Cytochrome P450c17 (CYP17A1, P450 17A1.

    Directory of Open Access Journals (Sweden)

    Alexandr N Simonov

    Full Text Available Cytochrome P450c17 (P450 17A1, CYP17A1 is a critical enzyme in the synthesis of androgens and is now a target enzyme for the treatment of prostate cancer. Cytochrome P450c17 can exhibit either one or two physiological enzymatic activities differentially regulated by cytochrome b5. How this is achieved remains unknown. Here, comprehensive in silico, in vivo and in vitro analyses were undertaken. Fluorescence Resonance Energy Transfer analysis showed close interactions within living cells between cytochrome P450c17 and cytochrome b5. In silico modeling identified the sites of interaction and confirmed that E48 and E49 residues in cytochrome b5 are essential for activity. Quartz crystal microbalance studies identified specific protein-protein interactions in a lipid membrane. Voltammetric analysis revealed that the wild type cytochrome b5, but not a mutated, E48G/E49G cyt b5, altered the kinetics of electron transfer between the electrode and the P450c17. We conclude that cytochrome b5 can influence the electronic conductivity of cytochrome P450c17 via allosteric, protein-protein interactions.

  3. Cytochrome P450-dependent metabolism of caffeine in Drosophila melanogaster.

    Directory of Open Access Journals (Sweden)

    Alexandra Coelho

    Full Text Available Caffeine (1, 3, 7-trimethylxanthine, an alkaloid produced by plants, has antioxidant and insecticide properties that can affect metabolism and cognition. In vertebrates, the metabolites derived from caffeine have been identified, and their functions have been characterized. However, the metabolites of caffeine in insects remain unknown. Thus, using radiolabelled caffeine, we have identified some of the primary caffeine metabolites produced in the body of Drosophila melanogaster males, including theobromine, paraxanthine and theophylline. In contrast to mammals, theobromine was the predominant metabolite (paraxanthine in humans; theophylline in monkeys; 1, 3, 7-trimethyluric acid in rodents. A transcriptomic screen of Drosophila flies exposed to caffeine revealed the coordinated variation of a large set of genes that encode xenobiotic-metabolizing proteins, including several cytochromes P450s (CYPs that were highly overexpressed. Flies treated with metyrapone--an inhibitor of CYP enzymes--showed dramatically decreased caffeine metabolism, indicating that CYPs are involved in this process. Using interference RNA genetic silencing, we measured the metabolic and transcriptomic effect of three candidate CYPs. Silencing of CYP6d5 completely abolished theobromine synthesis, whereas CYP6a8 and CYP12d1 silencing induced different consequences on metabolism and gene expression. Therefore, we characterized several metabolic products and some enzymes potentially involved in the degradation of caffeine. In conclusion, this pioneer approach to caffeine metabolism in insects opens novel perspectives for the investigation of the physiological effects of caffeine metabolites. It also indicates that caffeine could be used as a biomarker to evaluate CYP phenotypes in Drosophila and other insects.

  4. Characterization of human cytochrome P450 induction by pesticides.

    Science.gov (United States)

    Abass, Khaled; Lämsä, Virpi; Reponen, Petri; Küblbeck, Jenni; Honkakoski, Paavo; Mattila, Sampo; Pelkonen, Olavi; Hakkola, Jukka

    2012-03-29

    Pesticides are a large group of structurally diverse toxic chemicals. The toxicity may be modified by cytochrome P450 (CYP) enzyme activity. In the current study, we have investigated effects and mechanisms of 24 structurally varying pesticides on human CYP expression. Many pesticides were found to efficiently activate human pregnane X receptor (PXR) and/or constitutive androstane receptor (CAR). Out of the 24 compounds tested, 14 increased PXR- and 15 CAR-mediated luciferase activities at least 2-fold. While PXR was predominantly activated by pyrethroids, CAR was, in addition to pyrethroids, well activated by organophosphates and several carbamates. Induction of CYP mRNAs and catalytic activities was studied in the metabolically competent, human derived HepaRG cell line. CYP3A4 mRNA was induced most powerfully by pyrethroids; 50 μM cypermethrin increased CYP3A4 mRNA 35-fold. CYP2B6 was induced fairly equally by organophosphate, carbamate and pyrethroid compounds. Induction of CYP3A4 and CYP2B6 by these compound classes paralleled their effects on PXR and CAR. The urea herbicide diuron and the triazine herbicide atrazine induced CYP2B6 mRNA more than 10-fold, but did not activate CAR indicating that some pesticides may induce CYP2B6 via CAR-independent mechanisms. CYP catalyzed activities were induced much less than the corresponding mRNAs. At least in some cases, this is probably due to significant inhibition of CYP enzymes by the studied pesticides. Compared with human CAR activation and CYP2B6 expression, pesticides had much less effect on mouse CAR and CYP2B10 mRNA. Altogether, pesticides were found to be powerful human CYP inducers acting through both PXR and CAR. PMID:22310298

  5. Defective Cytochrome P450-Catalysed Drug Metabolism in Niemann-Pick Type C Disease

    Science.gov (United States)

    Wassif, Christopher A.; Gray, James; Burkert, Kathryn R.; Smith, David A.; Morris, Lauren; Cologna, Stephanie M.; Peer, Cody J.; Sissung, Tristan M.; Uscatu, Constantin-Daniel; Figg, William D.; Pavan, William J.; Vite, Charles H.; Porter, Forbes D.; Platt, Frances M.

    2016-01-01

    Niemann-Pick type C (NPC) disease is a neurodegenerative lysosomal storage disease caused by mutations in either the NPC1 or NPC2 gene. NPC is characterised by storage of multiple lipids in the late endosomal/lysosomal compartment, resulting in cellular and organ system dysfunction. The underlying molecular mechanisms that lead to the range of clinical presentations in NPC are not fully understood. While evaluating potential small molecule therapies in Npc1-/- mice, we observed a consistent pattern of toxicity associated with drugs metabolised by the cytochrome P450 system, suggesting a potential drug metabolism defect in NPC1 disease. Investigation of the P450 system in the context of NPC1 dysfunction revealed significant changes in the gene expression of many P450 associated genes across the full lifespan of Npc1-/- mice, decreased activity of cytochrome P450 reductase, and a global decrease of multiple cytochrome P450 catalysed dealkylation reactions. In vivo drug metabolism studies using a prototypic P450 metabolised drug, midazolam, confirmed dysfunction in drug clearance in the Npc1-/- mouse. Expression of the Phase II enzyme uridinediphosphate-glucuronosyltransferase (UGT) was also significantly reduced in Npc1-/- mice. Interestingly, reduced activity within the P450 system was also observed in heterozygous Npc1+/- mice. The reduced activity of P450 enzymes may be the result of bile acid deficiency/imbalance in Npc1-/- mice, as bile acid treatment significantly rescued P450 enzyme activity in Npc1-/- mice and has the potential to be an adjunctive therapy for NPC disease patients. The dysfunction in the cytochrome P450 system were recapitulated in the NPC1 feline model. Additionally, we present the first evidence that there are alterations in the P450 system in NPC1 patients. PMID:27019000

  6. Defective Cytochrome P450-Catalysed Drug Metabolism in Niemann-Pick Type C Disease.

    Science.gov (United States)

    Nicoli, Elena-Raluca; Al Eisa, Nada; Cluzeau, Celine V M; Wassif, Christopher A; Gray, James; Burkert, Kathryn R; Smith, David A; Morris, Lauren; Cologna, Stephanie M; Peer, Cody J; Sissung, Tristan M; Uscatu, Constantin-Daniel; Figg, William D; Pavan, William J; Vite, Charles H; Porter, Forbes D; Platt, Frances M

    2016-01-01

    Niemann-Pick type C (NPC) disease is a neurodegenerative lysosomal storage disease caused by mutations in either the NPC1 or NPC2 gene. NPC is characterised by storage of multiple lipids in the late endosomal/lysosomal compartment, resulting in cellular and organ system dysfunction. The underlying molecular mechanisms that lead to the range of clinical presentations in NPC are not fully understood. While evaluating potential small molecule therapies in Npc1-/- mice, we observed a consistent pattern of toxicity associated with drugs metabolised by the cytochrome P450 system, suggesting a potential drug metabolism defect in NPC1 disease. Investigation of the P450 system in the context of NPC1 dysfunction revealed significant changes in the gene expression of many P450 associated genes across the full lifespan of Npc1-/- mice, decreased activity of cytochrome P450 reductase, and a global decrease of multiple cytochrome P450 catalysed dealkylation reactions. In vivo drug metabolism studies using a prototypic P450 metabolised drug, midazolam, confirmed dysfunction in drug clearance in the Npc1-/- mouse. Expression of the Phase II enzyme uridinediphosphate-glucuronosyltransferase (UGT) was also significantly reduced in Npc1-/- mice. Interestingly, reduced activity within the P450 system was also observed in heterozygous Npc1+/- mice. The reduced activity of P450 enzymes may be the result of bile acid deficiency/imbalance in Npc1-/- mice, as bile acid treatment significantly rescued P450 enzyme activity in Npc1-/- mice and has the potential to be an adjunctive therapy for NPC disease patients. The dysfunction in the cytochrome P450 system were recapitulated in the NPC1 feline model. Additionally, we present the first evidence that there are alterations in the P450 system in NPC1 patients. PMID:27019000

  7. Defective Cytochrome P450-Catalysed Drug Metabolism in Niemann-Pick Type C Disease.

    Directory of Open Access Journals (Sweden)

    Elena-Raluca Nicoli

    Full Text Available Niemann-Pick type C (NPC disease is a neurodegenerative lysosomal storage disease caused by mutations in either the NPC1 or NPC2 gene. NPC is characterised by storage of multiple lipids in the late endosomal/lysosomal compartment, resulting in cellular and organ system dysfunction. The underlying molecular mechanisms that lead to the range of clinical presentations in NPC are not fully understood. While evaluating potential small molecule therapies in Npc1-/- mice, we observed a consistent pattern of toxicity associated with drugs metabolised by the cytochrome P450 system, suggesting a potential drug metabolism defect in NPC1 disease. Investigation of the P450 system in the context of NPC1 dysfunction revealed significant changes in the gene expression of many P450 associated genes across the full lifespan of Npc1-/- mice, decreased activity of cytochrome P450 reductase, and a global decrease of multiple cytochrome P450 catalysed dealkylation reactions. In vivo drug metabolism studies using a prototypic P450 metabolised drug, midazolam, confirmed dysfunction in drug clearance in the Npc1-/- mouse. Expression of the Phase II enzyme uridinediphosphate-glucuronosyltransferase (UGT was also significantly reduced in Npc1-/- mice. Interestingly, reduced activity within the P450 system was also observed in heterozygous Npc1+/- mice. The reduced activity of P450 enzymes may be the result of bile acid deficiency/imbalance in Npc1-/- mice, as bile acid treatment significantly rescued P450 enzyme activity in Npc1-/- mice and has the potential to be an adjunctive therapy for NPC disease patients. The dysfunction in the cytochrome P450 system were recapitulated in the NPC1 feline model. Additionally, we present the first evidence that there are alterations in the P450 system in NPC1 patients.

  8. Effects of electro-acupuncture on ovarian P450arom, P450c17α and mRNA expression induced by letrozole in PCOS rats.

    Directory of Open Access Journals (Sweden)

    Jie Sun

    Full Text Available Hyperandrogenism is a core factor in the series of reproductive and endocrine metabolic disorders involved in polycystic ovary syndrome (PCOS. Abnormalities in enzymatic activity and the expression of ovarian granular cell layer P450arom and theca cell P450c17α can lead to an atypical environment of local ovarian hormones, including excessive androgen levels. Rat models prepared with letrozole exhibit similar endocrine and histological changes to those that occur in human PCOS. We used such a model to study the role of electro-acupuncture (EA in regulating ovarian P450arom and P450c17α enzymatic activity and mRNA expression in PCOS rats. Female Sprague Dawley (SD rats aged 42 days were randomly divided into 3 groups (control, PCOS, and PCOS EA consisting of 10 rats each. The PCOS and PCOS EA groups were administered a gavage of 1.0 mg/kg(-1 of letrozole solution once daily for 21 consecutive days. Beginning in the ninth week, the PCOS EA group was administered low-frequency EA treatment daily for 14 consecutive days. After the treatment, we obtained the following results. The estrous cycles were restored in 8 of the 10 rats in the PCOS EA group, and their ovarian morphologies and ultrastructures normalized. The peripheral blood measurements (with ELISA showed significantly decreased androgens (i.e., androstenedione and testosterone with significantly increased estrogens (i.e., estrone, estradiol and increased P450arom with decreased P450C17α. Immunohistochemistry and Western blotting methods showed enhanced expression of ovarian granular cell layer P450arom as well as decreased expression of theca cell layer P450C17α. Fluorescence quantitative PCR methods showed enhanced expression of ovarian granular cell layer P450arom mRNA as well as decreased expression of theca cell layer P450C17α mRNA. These results may help explain the effects of electro-acupuncture in changing the local ovarian hyperandrogenic environment and improving reproductive

  9. Redução do citocromo C e resistência ao metotrexato em cancro da mama

    OpenAIRE

    Barros, Susana Raquel Costa

    2012-01-01

    O metotrexato é um agente quimioterápico usado para tratar uma variedade de cancros. No entanto, a ocorrência de resistência limita a sua eficácia. A relação entre o estado redox do citocromo c, apoptose e o desenvolvimento de resistência ao metotrexato foi avaliada em células humanas de cancro da mama. O citocromo c no seu estado reduzido é menos capaz de induzir a cascata apoptótica. Aqui nós mostramos que células incubadas com agentes redutores de citocromo c, tais como t...

  10. Fusion of Ferredoxin and Cytochrome P450 Enables Direct Light-Driven Biosynthesis.

    Science.gov (United States)

    Mellor, Silas Busck; Nielsen, Agnieszka Zygadlo; Burow, Meike; Motawia, Mohammed Saddik; Jakubauskas, Dainius; Møller, Birger Lindberg; Jensen, Poul Erik

    2016-07-15

    Cytochrome P450s (P450s) are key enzymes in the synthesis of bioactive natural products in plants. Efforts to harness these enzymes for in vitro and whole-cell production of natural products have been hampered by difficulties in expressing them heterologously in their active form, and their requirement for NADPH as a source of reducing power. We recently demonstrated targeting and insertion of plant P450s into the photosynthetic membrane and photosynthesis-driven, NADPH-independent P450 catalytic activity mediated by the electron carrier protein ferredoxin. Here, we report the fusion of ferredoxin with P450 CYP79A1 from the model plant Sorghum bicolor, which catalyzes the initial step in the pathway leading to biosynthesis of the cyanogenic glucoside dhurrin. Fusion with ferredoxin allows CYP79A1 to obtain electrons for catalysis by interacting directly with photosystem I. Furthermore, electrons captured by the fused ferredoxin moiety are directed more effectively toward P450 catalytic activity, making the fusion better able to compete with endogenous electron sinks coupled to metabolic pathways. The P450-ferredoxin fusion enzyme obtains reducing power solely from its fused ferredoxin and outperforms unfused CYP79A1 in vivo. This demonstrates greatly enhanced electron transfer from photosystem I to CYP79A1 as a consequence of the fusion. The fusion strategy reported here therefore forms the basis for enhanced partitioning of photosynthetic reducing power toward P450-dependent biosynthesis of important natural products. PMID:27119279

  11. Evolution of NADPH-cytochrome P450 oxidoreductases (POR) in Apiales - POR 1 is missing

    DEFF Research Database (Denmark)

    Andersen, Trine Bundgaard; Hansen, Niels Bjørn; Laursen, Tomas;

    2016-01-01

    The NADPH-dependent cytochrome P450 oxidoreductase (POR) is the obligate electron donor to eukaryotic microsomal cytochromes P450 enzymes. The number of PORs within plant species is limited to one to four isoforms, with the most common being two PORs per plant. These enzymes provide electrons to ...

  12. Hepatotoxicity of oral and intravenous voriconazole in relation to cytochrome P450 polymorphisms

    OpenAIRE

    Levin, M.-D.; Hollander, Jan; van der Holt, Bronno; Rijnders, Bart; Vliet, Martin; Sonneveld, Pieter; Van Schaik, Ron,

    2007-01-01

    textabstractObjectives: Voriconazole, like all other antifungals of the azole group, is potentially hepatotoxic. A large interpatient variability of liver enzyme elevations during oral or intravenous (iv) voriconazole administration is observed. This interpatient variability may be explained by differences in voriconazole metabolism because of cytochrome P450 polymorphisms. We examined the relationship between cytochrome P450 polymorphisms and hepatotoxicity in immunocompromised patients pred...

  13. Immunoreactivities of androgen receptor, estrogen receptors, p450arom, p450c17 proteins in wild ground squirrels ovaries during the nonbreeding and breeding seasons

    Directory of Open Access Journals (Sweden)

    Li Xiaonan

    2012-09-01

    Full Text Available Abstract The aim of this study was to elucidate the regulatory role of androgen in the follicular development of wild female ground squirrels. Immunohistochemical staining of FSHR, LHR, P450c17, P450arom, androgen receptor (AR, estrogen receptors (ERa and ERb were executed in ovaries of female ground squirrels from both breeding and nonbreeding seasons. In addition, total ovarian proteins were extracted from the ovaries of squirrels from breeding and nonbreeding seasons, and Western blot analysis were performed in order to probe for FSHR, LHR, P450c17, P450arom, AR, ERa and ERb. The results of immunohistochemical staining and Western blotting of P450c17 showed that there was no significant difference between the breeding and nonbreeding seasons. It was found that granulosa cells expressed P450arom during the breeding season. In contrast, there was no positive staining of P450arom in the nonbreeding season. There was no significant difference in immunoreactivity of AR between the breeding and nonbreeding seasons. However, the immunoreactivities of ERa and ERb were both significantly reduced in the nonbreeding season compared to the breeding season. The positive stains of FSHR and LHR were found in the granulosa cells and theca cells of the ovaries of the breeding and nonbreeding seasons. In addition, the Western blotting results of FSHR and LHR showed a significant reduction in the nonbreeding season compared with the breeding season. These findings suggested that androgen might be predominantly converted into estrogen in order to regulate the follicular development via binding of estrogen receptors during the breeding season, whereas androgen might predominantly directly bind androgen receptor to regulate the follicular development during the nonbreeding season in the ovaries of wild female ground squirrels.

  14. Cytochrome P450BM-3 reduces aldehydes to alcohols through a direct hydride transfer

    Energy Technology Data Exchange (ETDEWEB)

    Kaspera, Ruediger; Sahele, Tariku; Lakatos, Kyle [Department of Medicinal Chemistry, University of Washington, Box 357610, Seattle, WA 98195-7610 (United States); Totah, Rheem A., E-mail: rtotah@u.washington.edu [Department of Medicinal Chemistry, University of Washington, Box 357610, Seattle, WA 98195-7610 (United States)

    2012-02-17

    Highlights: Black-Right-Pointing-Pointer Cytochrome P450BM-3 reduced aldehydes to alcohols efficiently (k{sub cat} {approx} 25 min{sup -1}). Black-Right-Pointing-Pointer Reduction is a direct hydride transfer from R-NADP{sup 2}H to the carbonyl moiety. Black-Right-Pointing-Pointer P450 domain variants enhance reduction through potential allosteric/redox interactions. Black-Right-Pointing-Pointer Novel reaction will have implications for metabolism of xenobiotics. -- Abstract: Cytochrome P450BM-3 catalyzed the reduction of lipophilic aldehydes to alcohols efficiently. A k{sub cat} of {approx}25 min{sup -1} was obtained for the reduction of methoxy benzaldehyde with wild type P450BM-3 protein which was higher than in the isolated reductase domain (BMR) alone and increased in specific P450-domain variants. The reduction was caused by a direct hydride transfer from preferentially R-NADP{sup 2}H to the carbonyl moiety of the substrate. Weak substrate-P450-binding of the aldehyde, turnover with the reductase domain alone, a deuterium incorporation in the product from NADP{sup 2}H but not D{sub 2}O, and no inhibition by imidazole suggests the reductase domain of P450BM-3 as the potential catalytic site. However, increased aldehyde reduction by P450 domain variants (P450BM-3 F87A T268A) may involve allosteric or redox mechanistic interactions between heme and reductase domains. This is a novel reduction of aldehydes by P450BM-3 involving a direct hydride transfer and could have implications for the metabolism of endogenous substrates or xenobiotics.

  15. Effector Roles of Putidaredoxin on Cytochrome P450cam Conformational States.

    Science.gov (United States)

    Liou, Shu-Hao; Mahomed, Mavish; Lee, Young-Tae; Goodin, David B

    2016-08-17

    In this study, the effector role of Pdx (putidaredoxin) on cytochrome P450cam conformation is refined by attaching two different spin labels, MTSL or BSL (bifunctional spin-label) onto the F or G helices and using DEER (double electron-electron resonance) to measure the distance between labels. Recent EPR and crystallographic studies have observed that oxidized Pdx induces substrate-bound P450cam to change from the closed to the open state. However, this change was not observed by DEER in the reduced Pdx complex with carbon-monoxide-bound P450cam (Fe(2+)CO). In addition, recent NMR studies have failed to observe a change in P450cam conformation upon binding Pdx. Hence, resolving these issues is important for a full understanding the effector role of Pdx. Here we show that oxidized Pdx induces camphor-bound P450cam to shift from the closed to the open conformation when labeled on either the F or G helices with MTSL. BSL at these sites can either narrow the distance distribution widths dramatically or alter the extent of the conformational change. In addition, we report DEER spectra on a mixed oxidation state containing oxidized Pdx and ferrous CO-bound P450cam, showing that P450cam remains closed. This indicates that CO binding to the heme prevents P450cam from opening, overriding the influence exerted by Pdx binding. Finally, we report the open form P450cam crystal structure with substrate bound, which suggests that crystal packing effects may prevent conformational conversion. Using multiple labeling approaches, DEER provides a unique perspective to resolve how the conformation of P450cam depends on Pdx and ligand states. PMID:27452076

  16. Engineering Macaca fascicularis cytochrome P450 2C20 to reduce animal testing for new drugs.

    Science.gov (United States)

    Rua, Francesco; Sadeghi, Sheila J; Castrignanò, Silvia; Di Nardo, Giovanna; Gilardi, Gianfranco

    2012-12-01

    In order to develop in vitro methods as an alternative to P450 animal testing in the drug discovery process, two main requisites are necessary: 1) gathering of data on animal homologues of the human P450 enzymes, currently very limited, and 2) bypassing the requirement for both the P450 reductase and the expensive cofactor NADPH. In this work, P450 2C20 from Macaca fascicularis, homologue of the human P450 2C8 has been taken as a model system to develop such an alternative in vitro method by two different approaches. In the first approach called "molecular Lego", a soluble self-sufficient chimera was generated by fusing the P450 2C20 domain with the reductase domain of cytochrome P450 BM3 from Bacillus megaterium (P450 2C20/BMR). In the second approach, the need for the redox partner and also NADPH were both obviated by the direct immobilization of the P450 2C20 on glassy carbon and gold electrodes. Both systems were then compared to those obtained from the reconstituted P450 2C20 monooxygenase in presence of the human P450 reductase and NADPH using paclitaxel and amodiaquine, two typical drug substrates of the human P450 2C8. The K(M) values calculated for the 2C20 and 2C20/BMR in solution and for 2C20 immobilized on electrodes modified with gold nanoparticles were 1.9 ± 0.2, 5.9 ± 2.3, 3.0 ± 0.5 μM for paclitaxel and 1.2 ± 0.2, 1.6±0.2 and 1.4 ± 0.2 μM for amodiaquine, respectively. The data obtained not only show that the engineering of M. fascicularis did not affect its catalytic properties but also are consistent with K(M) values measured for the microsomal human P450 2C8 and therefore show the feasibility of developing alternative in vitro animal tests. PMID:22819650

  17. [Induction and measurement of cytochrome P450 in white rot fungi].

    Science.gov (United States)

    Ning, Da-liang; Wang, Hui; Li, Dong

    2009-08-15

    The induction and measurement of cytochrome P450 in white rot fungus Phanerochaete chrysosporium were studied in this work. The spectrophotometric results demonstrated that n-hexane was able to induce the fungal P450 to high level, which facilitated isolation and measurement of microsomal P450. The highest concentration of microsomal P450 could reach 140-160 pmol/mg after 6-h-induction by addition of 2 microL/mL hexane each hour, and the concentration of hexane and incubation time had significant effect on the induction of P450s. After effective induction, the method for isolation and measurement of microsomal P450 with CO difference spectrum was studied and the optimized method was obtained as followed. High-speed disperser and glass homogenizer were used to disrupt cells, which obtained higher amount of microsomal P450 than those from cells disrupted by glass homogenizer, ultrasonicator and bead-beater respectively. To record CO difference spectrum,the sample was bubbled with CO for 40 s at a rate of 3 mL/min (300 microL sample), and the reference cuvette was bubbled with N2 to the same extent. Then, the reducer sodium dithionite was added to a concentration 0.4 mol/L. PMID:19799321

  18. Effects of 2-acetylaminofluorene, dietary fats and antioxidants on nuclear envelope cytochrome P-450

    Energy Technology Data Exchange (ETDEWEB)

    Carubelli, R.; Graham, S.A.; Griffin, M.J.; McCay, P.B.

    1986-05-01

    The authors reported a marked loss of cytochrome P-450 in hepatic nuclear envelope (NE) but not in microsomes of male Sprague-Dawley rats fed a semipurified diet containing 0.05% w/w 2-acetylaminofluorene (AAF) for 3 weeks. This may reflect loss of NE capacity to detoxify AAF metabolites generated by microsomal P-450. They are now investigating if dietary effects such as progressive decrease in the incidence of AAF-induced tumors in rats fed high polyunsaturated fat diet (HPUF) vs. high saturated fat diet (HSF) vs. low fat diet (LF), and the anticarcinogenic activity of butylated hydroxytoluene (BHT; 0.3% w/w) correlate with preservation of NE P-450. Rats fed AAF HSF (25.6% w/w corn oil) showed marked loss of NE P-450 after 3 weeks; BHT protected against this loss. Rats fed AAF in HSF (25.6% w/w; 18 parts beef tallow + 2 parts corn oil), on the other hand, experienced a marked drop in NE P-450 after 9 weeks; BHT protected against this loss. Comparison of NE P-450 levels in control rats fed HPUF or HSF for 3 weeks with those of rats fed a semipurified diet with 10% fat or Purina chow (ca. 5% fat), support the prediction of an inverse correlation between the levels of dietary fat and the NE P-450 content. Studies on AAF and BHT effects using LF (2% w/w corn oil) are in progress.

  19. Pyrethroid Activity-Based Probes for Profiling Cytochrome P450 Activities Associated with Insecticide Interactions

    Energy Technology Data Exchange (ETDEWEB)

    Ismail, Hanafy M.; O' Neill, Paul M.; Hong, David; Finn, Robert; Henderson, Colin; Wright, Aaron T.; Cravatt, Benjamin; Hemingway, Janet; Paine, Mark J.

    2014-01-18

    Pyrethroid insecticides are used to control a diverse spectrum of diseases spread by arthropods. We have developed a suite of pyrethroid mimetic activity based probes (PyABPs) to selectively label and identify P450s associated with pyrethroid metabolism. The probes were screened against pyrethroid metabolizing and non-metabolizing mosquito P450s, as well as rodent microsomes to measure labeling specificity, plus CPR and b5 knockout mouse livers to validate P450 activation and establish the role for b5 in probe activation. Using a deltamethrin mimetic PyABP we were able to profile active enzymes in rat liver microsomes and identify pyrethroid metabolizing enzymes in the target tissue. The most reactive enzyme was a P450, CYP2C11, which is known to metabolize deltamethrin. Furthermore, several other pyrethroid metabolizers were identified (CYPs 2C6, 3A4, 2C13 and 2D1) along with related detoxification enzymes, notably UDP-g’s 2B1 - 5, suggesting a network of associated pyrethroid metabolizing enzymes, or ‘pyrethrome’. Considering the central role that P450s play in metabolizing insecticides, we anticipate that PyABPs will aid the identification and profiling of P450s associated with insecticide pharmacology in a wide range of species, improving understanding of P450-insecticide interactions and aiding the development of new tools for disease control.

  20. Type II ligands as chemical auxiliaries to favor enzymatic transformations by P450 2E1.

    Science.gov (United States)

    Ménard, Amélie; Fabra, Camilo; Huang, Yue; Auclair, Karine

    2012-11-26

    The remarkable ability of P450 enzymes to oxidize inactivated C-H bonds and the high substrate promiscuity of many P450 isoforms have inspired us and others to investigate their use as biocatalysts. Our lab has pioneered a chemical-auxiliary approach to control the promiscuity of P450 3A4 and provide product predictability. The recent realization that type II ligands are sometimes also P450 substrates has prompted the design of a new generation of chemical auxiliaries with type II binding properties. This approach takes advantage of the high affinity of type II ligands for the active site of these enzymes. Although type II ligands typically block P450 activity, we report here that type II ligation can be harnessed to achieve just the opposite, that is, to favor biocatalysis and afford predictable oxidation of small hydrocarbon substrates with P450 2E1. Moreover, the observed predictability was rationalized by molecular docking. We hope that this approach might find future use with other P450 isoforms and yield complimentary products. PMID:23129539

  1. A Cytochrome P450-Independent Mechanism of Acetaminophen-Induced Injury in Cultured Mouse Hepatocytes.

    Science.gov (United States)

    Miyakawa, Kazuhisa; Albee, Ryan; Letzig, Lynda G; Lehner, Andreas F; Scott, Michael A; Buchweitz, John P; James, Laura P; Ganey, Patricia E; Roth, Robert A

    2015-08-01

    Mouse hepatic parenchymal cells (HPCs) have become the most frequently used in vitro model to study mechanisms of acetaminophen (APAP)-induced hepatotoxicity. It is universally accepted that APAP hepatocellular injury requires bioactivation by cytochromes P450 (P450s), but this remains unproven in primary mouse HPCs in vitro, especially over the wide range of concentrations that have been employed in published reports. The aim of this work was to test the hypothesis that APAP-induced hepatocellular death in vitro depends solely on P450s. We evaluated APAP cytotoxicity and APAP-protein adducts (a biomarker of metabolic bioactivation by P450) using primary mouse HPCs in the presence and absence of a broad-spectrum inhibitor of P450s, 1-aminobenzotriazole (1-ABT). 1-ABT abolished formation of APAP-protein adducts at all concentrations of APAP (0-14 mM), but eliminated cytotoxicity only at small concentrations (≦5 mM), indicating the presence of a P450-independent mechanism at larger APAP concentrations. P450-independent cell death was delayed in onset relative to toxicity observed at smaller concentrations. p-Aminophenol was detected in primary mouse HPCs exposed to large concentrations of APAP, and a deacetylase inhibitor [bis (4-nitrophenyl) phosphate (BNPP)] significantly reduced cytotoxicity. In conclusion, APAP hepatocellular injury in vitro occurs by at least two mechanisms, a P450-dependent mechanism that operates at concentrations of APAP ≦ 5 mM and a P450-independent mechanism that predominates at larger concentrations and is slower in onset. p-Aminophenol most likely contributes to the latter mechanism. These findings should be considered in interpreting results from APAP cytotoxicity studies in vitro and in selecting APAP concentrations for use in such studies. PMID:26065700

  2. An extensive (co-expression analysis tool for the cytochrome P450 superfamily in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Provart Nicholas J

    2008-04-01

    Full Text Available Abstract Background Sequencing of the first plant genomes has revealed that cytochromes P450 have evolved to become the largest family of enzymes in secondary metabolism. The proportion of P450 enzymes with characterized biochemical function(s is however very small. If P450 diversification mirrors evolution of chemical diversity, this points to an unexpectedly poor understanding of plant metabolism. We assumed that extensive analysis of gene expression might guide towards the function of P450 enzymes, and highlight overlooked aspects of plant metabolism. Results We have created a comprehensive database, 'CYPedia', describing P450 gene expression in four data sets: organs and tissues, stress response, hormone response, and mutants of Arabidopsis thaliana, based on public Affymetrix ATH1 microarray expression data. P450 expression was then combined with the expression of 4,130 re-annotated genes, predicted to act in plant metabolism, for co-expression analyses. Based on the annotation of co-expressed genes from diverse pathway annotation databases, co-expressed pathways were identified. Predictions were validated for most P450s with known functions. As examples, co-expression results for P450s related to plastidial functions/photosynthesis, and to phenylpropanoid, triterpenoid and jasmonate metabolism are highlighted here. Conclusion The large scale hypothesis generation tools presented here provide leads to new pathways, unexpected functions, and regulatory networks for many P450s in plant metabolism. These can now be exploited by the community to validate the proposed functions experimentally using reverse genetics, biochemistry, and metabolic profiling.

  3. Effects of Chronic Renal Failure on Brain Cytochrome P450 in Rats.

    Science.gov (United States)

    Naud, Judith; Harding, Jessica; Lamarche, Caroline; Beauchemin, Stephanie; Leblond, Francois A; Pichette, Vincent

    2016-08-01

    Chronic renal failure (CRF) impedes renal excretion of drugs and their metabolism by reducing the expression of liver cytochrome P450 (P450). Uremic serum contains factors, such as parathyroid hormone (PTH), that decrease liver P450s. The P450s are also involved in the metabolism of xenobiotics in the brain. This study investigates: 1) the effects of CRF on rat brain P450, 2) the role of PTH in the downregulation of brain P450s in CRF rats, and 3) the effects of PTH on P450s in astrocytes. Protein and mRNA expression of P450s were assessed in the brain of CRF and control (CTL) rats, as well as from CTL or CRF rats that underwent parathyroidectomy (PTX) 1 week before nephrectomy. CYP3A activity was measured using 3-[(3, 4-difluorobenzyl) oxy]-5, 5-dimethyl-4-[4-methylsulfonyl) phenyl] furan-2(5H)-1 metabolism in brain microsomal preparation. CYP3A protein expression was assessed in primary cultured astrocytes incubated with serum obtained from CRF or CTL rats or with PTH. Significant downregulations (≥40%) of CYP1A, CYP2C11, and CYP3A proteins were observed in microsomes from CRF rat brains. CYP3A activity reduction was also observed. CYP3A expression and activity were unaffected in PTX-pretreated CRF rats. Serum of PTX-treated CRF rats had no impact on CYP3A levels in astrocytes compared with that of untreated CRF rats. Finally, PTH addition to normal calf serum induced a reduction in CYP3A protein similar to CRF serum, suggesting that CRF-induced hyperparathyroidism is associated with a significant decrease in P450 drug-metabolizing enzymes in the brain, which may have implications in drug response. PMID:27271372

  4. A cytochrome P450 terpenoid hydroxylase linked to the suppression of insect juvenile hormone synthesis

    OpenAIRE

    Sutherland, T. D.; Unnithan, G C; Andersen, J F; Evans, P H; Murataliev, M. B.; Szabo, L. Z.; Mash, E. A.; Bowers, W. S.; Feyereisen, R

    1998-01-01

    A cDNA encoding a cytochrome P450 enzyme was isolated from a cDNA library of the corpora allata (CA) from reproductively active Diploptera punctata cockroaches. This P450 from the endocrine glands that produce the insect juvenile hormone (JH) is most closely related to P450 proteins of family 4 and was named CYP4C7. The CYP4C7 gene is expressed selectively in the CA; its message could not be detected in the fat body, corpora cardiaca, or brain, but trace levels of expression were found in the...

  5. Effects of methotrexate on rat P-450 cytochrome mono-oxygenases

    International Nuclear Information System (INIS)

    Methotrexate, an anti-cancerous agent, acts as an anti-metabolite of the nucleic acids which synthesis is then inhibited. Using aminopyrine breath test after methotrexate processing, the effects of the molecule on activities of the hepatocyte P-450 cytochrome mono-oxygenases, are studied. Breath micro-tests with carbon 13-labelled aminopyrine have been carried out to observe the metabolism evolution. Micro-test results have been compared to microsomal enzymatic activities for various substrates, and also to P-450 cytochrome ratio. Results show that methotrexate induces a reduction in the P-450 cytochrome ratio, and thus reduce the hepatic biotransformation process. 1 fig., 30 refs

  6. Structural and Kinetic Studies of Novel Cytochrome P450 Small-Alkane Hydroxylases

    Energy Technology Data Exchange (ETDEWEB)

    Arnold, Frances H.

    2012-02-27

    The goals of this project are to investigate (1) the kinetics and stabilities of engineered cytochrome P450 (P450) small alkane hydroxylases and their evolutionary intermediates, (2) the structural basis for catalytic proficiency on small alkanes of these engineered P450s, and (3) the changes in redox control resulting from protein engineering. To reach these goals, we have established new methods for determining the kinetics and stabilities of multicomponent P450s such as CYP153A6. Using these, we were able to determine that CYP153A6 is proficient for hydroxylation of alkanes as small as ethane, an activity that has never been observed previously in any natural P450. To elucidate the structures of the engineered P450s, we obtained x-ray diffraction data for two variants in the P450PMO (propane monooxygenase) lineage and a preliminary structure for the most evolved variant. This structure shows changes in the substrate binding regions of the enzyme and a reduction in active site volume that are consistent with the observed changes in substrate specificity from fatty acids in the native enzyme to small alkanes in P450PMO. We also constructed semi-rational designed libraries mutating only residues in the enzyme active site that in one round of mutagenesis and screening produced variants that achieved nearly half of the activity of the most evolved enzymes of the P450PMO lineage. Finally, we found that changes in redox properties of the laboratory-evolved P450 alkane hydroxylases did not reflect the improvement in their electron transfer efficiency. The heme redox potential remained constant throughout evolution, while activity increased and coupling efficiency improved from 10% to 90%. The lack of correlation between heme redox potential and enzyme activity and coupling efficiency led us to search for other enzyme properties that could be better predictors for activity towards small alkanes, specifically methane. We investigated the oxidation potential of the radical

  7. A comparative study of P450 gene expression in field and laboratory Musca domestica L. strains

    DEFF Research Database (Denmark)

    Højland, Dorte Heidi; Vagn Jensen, Karl-Martin; Kristensen, Michael

    2014-01-01

    BACKGROUND The housefly is a global pest that has developed resistance to most insecticides applied for its control. Resistance has been associated with cytochrome P450 monooxygenases (P450s). The authors compare the expression of six genes possibly associated with insecticide resistance in three...... unselected strains: a multiresistant strain (791a), a neonicotinoid-resistant strain (766b) and a new field strain (845b). RESULTS CYP4G2 was highly expressed throughout the range of strains and proved to be the one of the most interesting expression profiles of all P450s analysed. CYP6G4 was expressed up to...

  8. Identificación de regiones isofuncionales en el citocromo c6 y en la plastocianina

    OpenAIRE

    Molina Heredia, Fernando Publio

    2001-01-01

    Una de las líneas de investigación mas intereses de la bioquímica contemporánea es el estudio de la relación entre la estructura y la función de las macromoléculas biológicas, o reconocimiento molecular. Como sistema objeto de este estudio, se ha escogido el citocromo c6(Cit) y la plastocianina (Pc), dos proteínas con estructuras diferentes pero con idéntica función, cual es transportar electrones entre los complejos de membrana citocromo b6-f y fotosistema I(PSI). La conexión entre ambos ...

  9. Regiospecificity of placental metabolism by cytochromes P450 and glutathione S-transferase.

    Science.gov (United States)

    McRobie, D J; Glover, D D; Tracy, T S

    1996-01-01

    The placenta possesses the ability to metabolize numerous xenobiotics and endogenous steroids. However, it is unknown whether regional differences in these enzymatic reactions exist in the human placenta. To this end, we undertook a study of four regions of the placenta, the chorionic plate, maternal surface, placental margin and whole tissue, to assess the activities of cytochrome P450 1A1 and 19A1 (aromatase) and glutathione S-stransferase in these fractions. No differences in either P450 1A1 or glutathione S-transferase activities were noted among any of the placental fractions. However, with respect to P450 19A1 activity, the placental margin differed significantly from all other fractions (p < 0.05). This study demonstrates that whole tissue samples of the human placenta are adequate for placental cytochrome P450 and glutathione S-transferase metabolism studies. PMID:8938464

  10. Genotype-Phenotype Analysis in Congenital Adrenal Hyperplasia due to P450 Oxidoreductase Deficiency

    NARCIS (Netherlands)

    Krone, Nils; Reisch, Nicole; Idkowiak, Jan; Dhir, Vivek; Ivison, Hannah E.; Hughes, Beverly A.; Rose, Ian T.; O'Neil, Donna M.; Vijzelaar, Raymon; Smith, Matthew J.; MacDonald, Fiona; Cole, Trevor R.; Adolphs, Nicolai; Barton, John S.; Blair, Edward M.; Braddock, Stephen R.; Collins, Felicity; Cragun, Deborah L.; Dattani, Mehul T.; Day, Ruth; Dougan, Shelley; Feist, Miriam; Gottschalk, Michael E.; Gregory, John W.; Haim, Michaela; Harrison, Rachel; Olney, Ann Haskins; Hauffa, Berthold P.; Hindmarsh, Peter C.; Hopkin, Robert J.; Jira, Petr E.; Kempers, Marlies; Kerstens, Michiel N.; Khalifa, Mohamed M.; Koehler, Birgit; Maiter, Dominique; Nielsen, Shelly; O'Riordan, Stephen M.; Roth, Christian L.; Shane, Kate P.; Silink, Martin; Stikkelbroeck, Nike M. M. L.; Sweeney, Elizabeth; Szarras-Czapnik, Maria; Waterson, John R.; Williamson, Lori; Hartmann, Michaela F.; Taylor, Norman F.; Wudy, Stefan A.; Malunowicz, Ewa M.; Shackleton, Cedric H. L.; Arlt, Wiebke; Smith, M.J.

    2012-01-01

    Context: P450 oxidoreductase deficiency (PORD) is a unique congenital adrenal hyperplasia variant that manifests with glucocorticoid deficiency, disordered sex development (DSD), and skeletal malformations. No comprehensive data on genotype-phenotype correlations in Caucasian patients are available.

  11. Clinical, genetic, and enzymatic characterization of P450 oxidoreductase deficiency in four patients.

    LENUS (Irish Health Repository)

    Sahakitrungruang, Taninee

    2009-12-01

    P450 oxidoreductase (POR) deficiency causes disordered steroidogenesis; severe mutations cause genital ambiguity in both sexes plus the Antley-Bixler skeletal malformation syndrome, whereas mild mutations can cause adult infertility.

  12. Genome-Wide Annotation and Comparative Analysis of Cytochrome P450 Monooxygenases in Basidiomycete Biotrophic Plant Pathogens.

    Science.gov (United States)

    Qhanya, Lehlohonolo Benedict; Matowane, Godfrey; Chen, Wanping; Sun, Yuxin; Letsimo, Elizabeth Mpholoseng; Parvez, Mohammad; Yu, Jae-Hyuk; Mashele, Samson Sitheni; Syed, Khajamohiddin

    2015-01-01

    Fungi are an exceptional source of diverse and novel cytochrome P450 monooxygenases (P450s), heme-thiolate proteins, with catalytic versatility. Agaricomycotina saprophytes have yielded most of the available information on basidiomycete P450s. This resulted in observing similar P450 family types in basidiomycetes with few differences in P450 families among Agaricomycotina saprophytes. The present study demonstrated the presence of unique P450 family patterns in basidiomycete biotrophic plant pathogens that could possibly have originated from the adaptation of these species to different ecological niches (host influence). Systematic analysis of P450s in basidiomycete biotrophic plant pathogens belonging to three different orders, Agaricomycotina (Armillaria mellea), Pucciniomycotina (Melampsora laricis-populina, M. lini, Mixia osmundae and Puccinia graminis) and Ustilaginomycotina (Ustilago maydis, Sporisorium reilianum and Tilletiaria anomala), revealed the presence of numerous putative P450s ranging from 267 (A. mellea) to 14 (M. osmundae). Analysis of P450 families revealed the presence of 41 new P450 families and 27 new P450 subfamilies in these biotrophic plant pathogens. Order-level comparison of P450 families between biotrophic plant pathogens revealed the presence of unique P450 family patterns in these organisms, possibly reflecting the characteristics of their order. Further comparison of P450 families with basidiomycete non-pathogens confirmed that biotrophic plant pathogens harbour the unique P450 families in their genomes. The CYP63, CYP5037, CYP5136, CYP5137 and CYP5341 P450 families were expanded in A. mellea when compared to other Agaricomycotina saprophytes and the CYP5221 and CYP5233 P450 families in P. graminis and M. laricis-populina. The present study revealed that expansion of these P450 families is due to paralogous evolution of member P450s. The presence of unique P450 families in these organisms serves as evidence of how a host

  13. Genome-Wide Annotation and Comparative Analysis of Cytochrome P450 Monooxygenases in Basidiomycete Biotrophic Plant Pathogens.

    Directory of Open Access Journals (Sweden)

    Lehlohonolo Benedict Qhanya

    Full Text Available Fungi are an exceptional source of diverse and novel cytochrome P450 monooxygenases (P450s, heme-thiolate proteins, with catalytic versatility. Agaricomycotina saprophytes have yielded most of the available information on basidiomycete P450s. This resulted in observing similar P450 family types in basidiomycetes with few differences in P450 families among Agaricomycotina saprophytes. The present study demonstrated the presence of unique P450 family patterns in basidiomycete biotrophic plant pathogens that could possibly have originated from the adaptation of these species to different ecological niches (host influence. Systematic analysis of P450s in basidiomycete biotrophic plant pathogens belonging to three different orders, Agaricomycotina (Armillaria mellea, Pucciniomycotina (Melampsora laricis-populina, M. lini, Mixia osmundae and Puccinia graminis and Ustilaginomycotina (Ustilago maydis, Sporisorium reilianum and Tilletiaria anomala, revealed the presence of numerous putative P450s ranging from 267 (A. mellea to 14 (M. osmundae. Analysis of P450 families revealed the presence of 41 new P450 families and 27 new P450 subfamilies in these biotrophic plant pathogens. Order-level comparison of P450 families between biotrophic plant pathogens revealed the presence of unique P450 family patterns in these organisms, possibly reflecting the characteristics of their order. Further comparison of P450 families with basidiomycete non-pathogens confirmed that biotrophic plant pathogens harbour the unique P450 families in their genomes. The CYP63, CYP5037, CYP5136, CYP5137 and CYP5341 P450 families were expanded in A. mellea when compared to other Agaricomycotina saprophytes and the CYP5221 and CYP5233 P450 families in P. graminis and M. laricis-populina. The present study revealed that expansion of these P450 families is due to paralogous evolution of member P450s. The presence of unique P450 families in these organisms serves as evidence of how a host

  14. Systematic identification and evolutionary analysis of catalytically versatile cytochrome p450 monooxygenase families enriched in model basidiomycete fungi.

    Directory of Open Access Journals (Sweden)

    Khajamohiddin Syed

    Full Text Available Genome sequencing of basidiomycetes, a group of fungi capable of degrading/mineralizing plant material, revealed the presence of numerous cytochrome P450 monooxygenases (P450s in their genomes, with some exceptions. Considering the large repertoire of P450s found in fungi, it is difficult to identify P450s that play an important role in fungal metabolism and the adaptation of fungi to diverse ecological niches. In this study, we followed Sir Charles Darwin's theory of natural selection to identify such P450s in model basidiomycete fungi showing a preference for different types of plant components degradation. Any P450 family comprising a large number of member P450s compared to other P450 families indicates its natural selection over other P450 families by its important role in fungal physiology. Genome-wide comparative P450 analysis in the basidiomycete species, Phanerochaete chrysosporium, Phanerochaete carnosa, Agaricus bisporus, Postia placenta, Ganoderma sp. and Serpula lacrymans, revealed enrichment of 11 P450 families (out of 68 P450 families, CYP63, CYP512, CYP5035, CYP5037, CYP5136, CYP5141, CYP5144, CYP5146, CYP5150, CYP5348 and CYP5359. Phylogenetic analysis of the P450 family showed species-specific alignment of P450s across the P450 families with the exception of P450s of Phanerochaete chrysosporium and Phanerochaete carnosa, suggesting paralogous evolution of P450s in model basidiomycetes. P450 gene-structure analysis revealed high conservation in the size of exons and the location of introns. P450s with the same gene structure were found tandemly arranged in the genomes of selected fungi. This clearly suggests that extensive gene duplications, particularly tandem gene duplications, led to the enrichment of selective P450 families in basidiomycetes. Functional analysis and gene expression profiling data suggest that members of the P450 families are catalytically versatile and possibly involved in fungal colonization of plant

  15. Genome-Wide Annotation and Comparative Analysis of Cytochrome P450 Monooxygenases in Basidiomycete Biotrophic Plant Pathogens

    Science.gov (United States)

    Sun, Yuxin; Letsimo, Elizabeth Mpholoseng; Parvez, Mohammad; Yu, Jae-Hyuk; Mashele, Samson Sitheni; Syed, Khajamohiddin

    2015-01-01

    Fungi are an exceptional source of diverse and novel cytochrome P450 monooxygenases (P450s), heme-thiolate proteins, with catalytic versatility. Agaricomycotina saprophytes have yielded most of the available information on basidiomycete P450s. This resulted in observing similar P450 family types in basidiomycetes with few differences in P450 families among Agaricomycotina saprophytes. The present study demonstrated the presence of unique P450 family patterns in basidiomycete biotrophic plant pathogens that could possibly have originated from the adaptation of these species to different ecological niches (host influence). Systematic analysis of P450s in basidiomycete biotrophic plant pathogens belonging to three different orders, Agaricomycotina (Armillaria mellea), Pucciniomycotina (Melampsora laricis-populina, M. lini, Mixia osmundae and Puccinia graminis) and Ustilaginomycotina (Ustilago maydis, Sporisorium reilianum and Tilletiaria anomala), revealed the presence of numerous putative P450s ranging from 267 (A. mellea) to 14 (M. osmundae). Analysis of P450 families revealed the presence of 41 new P450 families and 27 new P450 subfamilies in these biotrophic plant pathogens. Order-level comparison of P450 families between biotrophic plant pathogens revealed the presence of unique P450 family patterns in these organisms, possibly reflecting the characteristics of their order. Further comparison of P450 families with basidiomycete non-pathogens confirmed that biotrophic plant pathogens harbour the unique P450 families in their genomes. The CYP63, CYP5037, CYP5136, CYP5137 and CYP5341 P450 families were expanded in A. mellea when compared to other Agaricomycotina saprophytes and the CYP5221 and CYP5233 P450 families in P. graminis and M. laricis-populina. The present study revealed that expansion of these P450 families is due to paralogous evolution of member P450s. The presence of unique P450 families in these organisms serves as evidence of how a host

  16. Role of cytochrome P450 genotype in the steps toward personalized drug therapy

    Directory of Open Access Journals (Sweden)

    Cavallari LH

    2011-11-01

    Full Text Available Larisa H Cavallari1,2, Hyunyoung Jeong1,2, Adam Bress11Department of Pharmacy Practice, 2Department of Biopharmaceutical Sciences, College of Pharmacy, University of Illinois at Chicago, Chicago, IL, USAAbstract: Genetic polymorphism for cytochrome 450 (P450 enzymes leads to interindividual variability in the plasma concentrations of many drugs. In some cases, P450 genotype results in decreased enzyme activity and an increased risk for adverse drug effects. For example, individuals with the CYP2D6 loss-of-function genotype are at increased risk for ventricular arrhythmia if treated with usual does of thioridazine. In other cases, P450 genotype may influence the dose of a drug required to achieve a desired effect. This is the case with warfarin, with lower doses often necessary in carriers of a variant CYP2C9*2 or *3 allele to avoid supratherapeutic anticoagulation. When a prodrug, such as clopidogrel or codeine, must undergo hepatic biotransformation to its active form, a loss-of-function P450 genotype leads to reduced concentrations of the active drug and decreased drug efficacy. In contrast, patients with multiple CYP2D6 gene copies are at risk for opioid-related toxicity if treated with usual doses of codeine-containing analgesics. At least 25 drugs contain information in their US Food and Drug Administration-approved labeling regarding P450 genotype. The CYP2C9, CYP2C19, and CYP2D6 genes are the P450 genes most often cited. To date, integration of P450 genetic information into clinical decision making is limited. However, some institutions are beginning to embrace routine P450 genotyping to assist in the treatment of their patients. Genotyping for P450 variants may carry less risk for discrimination compared with genotyping for disease-associated variants. As such, P450 genotyping is likely to lead the way in the clinical implementation of pharmacogenomics. This review discusses variability in the CYP2C9, CYP2C19, and CYP2D6 genes and the

  17. Cytochrome P450 System Proteins Reside in Different Regions of the Endoplasmic Reticulum

    OpenAIRE

    Ji Won PARK; Reed, James R.; Brignac-Huber, Lauren M.; Backes, Wayne L.

    2014-01-01

    Cytochrome P450 function is dependent on the ability of these enzymes to successfully interact with their redox partners, NADPH-cytochrome P450 reductase (CPR) and cytochrome b5, in the endoplasmic reticulum (ER). Because the ER is heterogeneous in lipid composition, membrane microdomains with different characteristics are formed. Ordered microdomains are more tightly packed, and enriched in saturated fatty acids, sphingomyelin and cholesterol, whereas disordered regions contain higher levels...

  18. A cytochrome P450 phenotyping cocktail causing unexpected adverse reactions in female volunteers

    DEFF Research Database (Denmark)

    Pedersen, Rasmus Steen; Damkier, Per; Hougaard Christensen, Mette Marie;

    2013-01-01

    A four-drug cytochrome P450 (CYP) phenotyping cocktail was developed to rapidly and safely determine CYP2D6, CYP2C19, CYP2C9 and CYP1A2 enzyme activity and phenotype.......A four-drug cytochrome P450 (CYP) phenotyping cocktail was developed to rapidly and safely determine CYP2D6, CYP2C19, CYP2C9 and CYP1A2 enzyme activity and phenotype....

  19. Human steroidogenic cytochromes P450 : biotransformation of drugs and biotechnological application

    OpenAIRE

    Schiffer, Lina Maria Klara Renate

    2015-01-01

    In humans, monooxygenases from the cytochrome P450 superfamily contribute to the biosynthesis of steroid hormones by catalyzing the side-chain cleavage of cholesterol and subsequent regio- and stereoselective hydroxylations, which determine the bioactivity of the hormones. In this work, the additional contribution of these steroidogenic cytochromes P450s to the metabolism of xenobiotics was established and their employment as highly selective and efficient biocatalysts in recombinant bacteria...

  20. Use of Human Plasma Samples to Identify Circulating Drug Metabolites that Inhibit Cytochrome P450 Enzymes.

    Science.gov (United States)

    Eng, Heather; Obach, R Scott

    2016-08-01

    Drug interactions elicited through inhibition of cytochrome P450 (P450) enzymes are important in pharmacotherapy. Recently, greater attention has been focused on not only parent drugs inhibiting P450 enzymes but also on possible inhibition of these enzymes by circulating metabolites. In this report, an ex vivo method whereby the potential for circulating metabolites to be inhibitors of P450 enzymes is described. To test this method, seven drugs and their known plasma metabolites were added to control human plasma at concentrations previously reported to occur in humans after administration of the parent drug. A volume of plasma for each drug based on the known inhibitory potency and time-averaged concentration of the parent drug was extracted and fractionated by high-pressure liquid chromatography-mass spectrometry, and the fractions were tested for inhibition of six human P450 enzyme activities (CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4). Observation of inhibition in fractions that correspond to the retention times of metabolites indicates that the metabolite has the potential to contribute to P450 inhibition in vivo. Using this approach, norfluoxetine, hydroxyitraconazole, desmethyldiltiazem, desacetyldiltiazem, desethylamiodarone, hydroxybupropion, erythro-dihydrobupropion, and threo-dihydrobupropion were identified as circulating metabolites that inhibit P450 activities at a similar or greater extent as the parent drug. A decision tree is presented outlining how this method can be used to determine when a deeper investigation of the P450 inhibition properties of a drug metabolite is warranted. PMID:27271369

  1. The interplay between tubulins and P450 cytochromes during Plasmodium berghei invasion of Anopheles gambiae midgut.

    Directory of Open Access Journals (Sweden)

    Rute C Félix

    Full Text Available BACKGROUND: Plasmodium infection increases the oxidative stress inside the mosquito, leading to a significant alteration on transcription of Anopheles gambiae detoxification genes. Among these detoxification genes several P450 cytochromes and tubulins were differently expressed, suggesting their involvement in the mosquito's response to parasite invasion. P450 cytochromes are usually involved in the metabolism and detoxification of several compounds, but are also regulated by several pathogens, including malaria parasite. Tubulins are extremely important as components of the cytoskeleton, which rearrangement functions as a response to malaria parasite invasion. METHODOLOGY/PRINCIPAL FINDINGS: Gene silencing methods were used to uncover the effects of cytochrome P450 reductase, tubulinA and tubulinB silencing on the A. gambiae response to Plasmodium berghei invasion. The role of tubulins in counter infection processes was also investigated by inhibiting their effect. Colchicine, vinblastine and paclitaxel, three different tubulin inhibitors were injected into A. gambiae mosquitoes. Twenty-four hours post injection these mosquitoes were infected with P. berghei through a blood meal from infected CD1 mice. Cytochrome P450 gene expression was measured using RT-qPCR to detect differences in cytochrome expression between silenced, inhibited and control mosquitoes. Results showed that cytochrome P450 reductase silencing, as well as tubulin (A and B silencing and inhibition affected the efficiency of Plasmodium infection. Silencing and inhibition also affected the expression levels of cytochromes P450. CONCLUSIONS: Our results suggest the existence of a relationship between tubulins and P450 cytochromes during A. gambiae immune response to P. berghei invasion. One of the P450 cytochromes in this study, CYP6Z2, stands out as the potential link in this association. Further work is needed to fully understand the role of tubulin genes in the response to

  2. Biosynthesis of Hexahydroxyperylenequinone Melanin via Oxidative Aryl Coupling by Cytochrome P-450 in Streptomyces griseus

    OpenAIRE

    Funa, Nobutaka; Funabashi, Masanori; OHNISHI, Yasuo; Horinouchi, Sueharu

    2005-01-01

    Dihydroxyphenylalanine (DOPA) melanins formed from tyrosine by tyrosinases are found in microorganisms, plants, and animals. Most species in the soil-dwelling, gram-positive bacterial genus Streptomyces produce DOPA melanins and melanogenesis is one of the characteristics used for taxonomy. Here we report a novel melanin biosynthetic pathway involving a type III polyketide synthase (PKS), RppA, and a cytochrome P-450 enzyme, P-450mel, in Streptomyces griseus. In vitro reconstitution of the P-...

  3. Regulation of P450 oxidoreductase by gonadotropins in rat ovary and its effect on estrogen production

    OpenAIRE

    Uesaka Miki; Kangawa Kenji; Kokame Koichi; Mizutani Tetsuya; Yazawa Takashi; Inaoka Yoshihiko; Umezawa Akihiro; Miyamoto Kaoru

    2008-01-01

    Abstract Background P450 oxidoreductase (POR) catalyzes electron transfer to microsomal P450 enzymes. Its deficiency causes Antley-Bixler syndrome (ABS), and about half the patients with ABS have ambiguous genitalia and/or impaired steroidogenesis. POR mRNA expression is up-regulated when mesenchymal stem cells (MSCs) differentiate into steroidogenic cells, suggesting that the regulation of POR gene expression is important for steroidogenesis. In this context we examined the regulation of POR...

  4. Cytochrom P450-Monooxygenasen : Modellierung, Datenbankanalyse und experimentelle Charakterisierung neuer Enzymvarianten

    OpenAIRE

    Seifert, Alexander

    2008-01-01

    Die vorliegende Arbeit befasst sich mit Cytochrom P450-Monooxygenasen. Vertreter dieser Enzymsuperfamilie sind aufgrund ihrer Beteiligung am Medikamentenstoffwechsel des Menschen, sowie ihrer Fähigkeit eine Vielzahl chemischer Verbindungen stereo- und regioselektiv zu oxidieren Gegenstand intensiver Forschung. Die genaue Vorhersage des von P450-Monooxygenasen verursachten Medikamentenstoffwechsels ist von großer Bedeutung für die Entwicklung neuer Wirksubstanzen. Weiterhin sind einige Vertret...

  5. In Vitro Metabolism of Montelukast by Cytochrome P450s and UDP-Glucuronosyltransferases.

    Science.gov (United States)

    Cardoso, Josiane de Oliveira; Oliveira, Regina Vincenzi; Lu, Jessica Bo Li; Desta, Zeruesenay

    2015-12-01

    Montelukast has been recommended as a selective in vitro and in vivo probe of cytochrome P450 (P450) CYP2C8 activity, but its selectivity toward this enzyme remains unclear. We performed detailed characterization of montelukast metabolism in vitro using human liver microsomes (HLMs), expressed P450s, and uridine 5'-diphospho-glucuronosyltransferases (UGTs). Kinetic and inhibition experiments performed at therapeutically relevant concentrations reveal that CYP2C8 and CYP2C9 are the principal enzymes responsible for montelukast 36-hydroxylation to 1,2-diol. CYP3A4 was the main catalyst of montelukast sulfoxidation and stereoselective 21-hydroxylation, and multiple P450s participated in montelukast 25-hydroxylation. We confirmed direct glucuronidation of montelukast to an acyl-glucuronide. We also identified a novel peak that appears consistent with an ether-glucuronide. Kinetic analysis in HLMs and experiments in expressed UGTs indicate that both metabolites were exclusively formed by UGT1A3. Comparison of in vitro intrinsic clearance in HLMs suggest that direct glucuronidation may play a greater role in the overall metabolism of montelukast than does P450-mediated oxidation, but the in vivo contribution of UGT1A3 needs further testing. In conclusion, our in vitro findings provide new insight toward montelukast metabolism. The utility of montelukast as a probe of CYP2C8 activity may be compromised owing to involvement of multiple P450s and UGT1A3 in its metabolism. PMID:26374173

  6. Genome-wide identification and expression analyses of cytochrome P450 genes in mulberry (Morus notabilis)

    Institute of Scientific and Technical Information of China (English)

    Bi Ma; Yiwei Luo; Ling Jia; Xiwu Qi; Qiwei Zeng; Zhonghuai Xiang; Ningjia He

    2014-01-01

    Cytochrome P450s play critical roles in the biosyn-thesis of physiological y important compounds in plants. These compounds often act as defense toxins to prevent herbivory. In the present study, a total of 174 P450 genes of mulberry (Morus notabilis C.K.Schn) were identified based on bioinfor-matics analyses. These mulberry P450 genes were divided into nine clans and 47 families and were found to be expressed in a tissue-preferential manner. These genes were compared to the P450 genes in Arabidopsis thaliana. Families CYP80, CYP92, CYP728, CYP733, CYP736, and CYP749 were found to exist in mulberry, and they may play important roles in the biosynthesis of mulberry secondary metabolites. Analyses of the functional and metabolic pathways of these genes indicated that mulberry P450 genes may participate in the metabolism of lipids, other secondary metabolites, xenobiotics, amino acids, cofactors, vitamins, terpenoids, and polyketides. These results provide a foundation for understanding of the structures and biological functions of mulberry P450 genes.

  7. Use of heterologously-expressed cytochrome P450 and glutathione transferase enzymes in toxicity assays.

    Science.gov (United States)

    Guengerich, F Peter; Wheeler, James B; Chun, Young-Jin; Kim, Donghak; Shimada, Tsutomu; Aryal, Pramod; Oda, Yoshimitsu; Gillam, Elizabeth M J

    2002-12-27

    Our groups have had a long-term interest in utilizing bacterial systems in the characterization of bioactivation and detoxication reactions catalyzed by cytochrome P450 (P450) and glutathione transferase (GST) enzymes. Bacterial systems remain the first choice for initial screens with new chemicals and have advantages, including high-throughput capability. Most human P450s of interest in toxicology have been readily expressed in Escherichia coli with only minor sequence modification. These enzymes can be readily purified and used in assays of activation of chemicals. Bicistronic systems have been developed in order to provide the auxiliary NADPH-P450 reductase. Alternative systems involve these enzymes expressed together within bacteria. In one approach, a lac selection system is used with E. coli and has been applied to the characterization of inhibitors of P450s 1A2 and 1B1, as well as in basic studies involving random mutagenesis. Another approach utilizes induction of the SOS (umu) response in Salmonella typhimurium, and systems have now been developed with human P450s 1A1, 1A2, 1B1, 2C9, 2D6, 2E1, and 3A4, which have been used to report responses from heterocyclic amines. S. typhimurium his reporter systems have also been used with GSTs, first to demonstrate the role of rat GST 5-5 in the activation of dihalomethanes. These systems have been used to compare these GSTs with regard to activation of dihaloalkanes and potential toxicity. PMID:12505322

  8. Metabolic engineering of light-driven cytochrome P450 dependent pathways into Synechocystis sp. PCC 6803.

    Science.gov (United States)

    Wlodarczyk, Artur; Gnanasekaran, Thiyagarajan; Nielsen, Agnieszka Zygadlo; Zulu, Nodumo Nokolunga; Mellor, Silas Busck; Luckner, Manja; Thøfner, Jens Frederik Bang; Olsen, Carl Erik; Mottawie, Mohammed Saddik; Burow, Meike; Pribil, Mathias; Feussner, Ivo; Møller, Birger Lindberg; Jensen, Poul Erik

    2016-01-01

    Solar energy provides the energy input for the biosynthesis of primary and secondary metabolites in plants and other photosynthetic organisms. Some secondary metabolites are high value compounds, and typically their biosynthesis requires the involvement of cytochromes P450s. In this proof of concept work, we demonstrate that the cyanobacterium Synechocystis sp. PCC 6803 is an eminent heterologous host for expression of metabolically engineered cytochrome P450-dependent pathways exemplified by the dhurrin pathway from Sorghum bicolor comprising two membrane bound cytochromes P450s (CYP79A1 and CYP71E1) and a soluble glycosyltransferase (UGT85B1). We show that it is possible to express multiple genes incorporated into a bacterial-like operon by using a self-replicating expression vector in cyanobacteria. We demonstrate that eukaryotic P450s that typically reside in the endoplasmic reticulum membranes can be inserted in the prokaryotic membranes without affecting thylakoid membrane integrity. Photosystem I and ferredoxin replaces the native P450 oxidoreductase enzyme as an efficient electron donor for the P450s both in vitro and in vivo. The engineered strains produced up to 66mg/L of p-hydroxyphenylacetaldoxime and 5mg/L of dhurrin in lab-scale cultures after 3 days of cultivation and 3mg/L of dhurrin in V-shaped photobioreactors under greenhouse conditions after 9 days cultivation. All the metabolites were found to be excreted to the growth media facilitating product isolation. PMID:26548317

  9. Cytochrome P450 peroxidase/peroxygenase mediated xenobiotic metabolic activation and cytotoxicity in isolated hepatocytes.

    Science.gov (United States)

    Anari, M R; Khan, S; Liu, Z C; O'Brien, P J

    1995-12-01

    Cytochrome P450 (P450) can utilize organic hydroperoxides and peracids to support hydroxylation and dealkylation of various P450 substrates. However, the biological significance of this P450 peroxygenase/peroxidase activity in the bioactivation of xenobiotics in intact cells has not been demonstrated. We have shown that tert-butyl hydroperoxide (tBHP) markedly enhances 3-20-fold the cytotoxicity of various aromatic hydrocarbons and their phenolic metabolites. The tBHP-enhanced hepatocyte cytotoxicity of 4-nitroanisole (4-NA) and 4-hydroxyanisole (4-HA) was also accompanied by an increase in the hepatocyte O-demethylation of 4-NA and 4-HA up to 7.5- and 21-fold, respectively. Hepatocyte GSH conjugation by 4-HA was also markedly increased by tBHP. An LC/MS analysis of the GSH conjugates identified hydroquinone-GSH and 4-methoxy-catechol:GSH conjugates as the predominant adducts. Pretreatment of hepatocytes with P450 inhibitors, e.g., phenylimidazole, prevented tBHP-enhanced 4-HA metabolism, GSH depletion, and cytotoxicity. In conclusion, hydroperoxides can therefore be used by intact cells to support the bioactivation of xenobiotics through the P450 peroxidase/peroxygenase system. PMID:8605292

  10. Process development for oxidations of hydrophobic compounds applying cytochrome P450 monooxygenases in-vitro.

    Science.gov (United States)

    Brummund, Jan; Müller, Monika; Schmitges, Thomas; Kaluzna, Iwona; Mink, Daniel; Hilterhaus, Lutz; Liese, Andreas

    2016-09-10

    Cytochrome P450 monooxygenases are a unique family of enzymes that are able to catalyze regio- and stereospecific oxidations for a broad substrate range. However, due to limited enzyme activities and stabilities, hydrophobicity of substrates, as well as the necessity of a continuous electron and oxygen supply the implementation of P450s for industrial processes remains challenging. Aim of this study was to point out key aspects for the development of an efficient synthesis concept for cytochrome P450 catalyzed oxidations. In order to regenerate the natural cofactor NADPH, a glucose dehydrogenase was applied. The low water soluble terpene α-ionone was used as substrate for the model reaction system. The studies reveal that an addition of surfactants in combination with low volumetric amounts of co-solvent can significantly increase substrate availability and reaction rates. Furthermore, these additives facilitated a reliable sampling procedure during the process. Another key factor for the process design was the oxygen supply. Based on various investigations, a bubble-aerated stirred tank reactor in batch mode represents a promising reactor concept for P450 oxidations. Main restriction of the investigated reaction system was the low process stability of the P450 monooxygenase, characterized by maximum total turnover numbers of ∼4100molα-ionone/molP450. PMID:27396939

  11. Human cytochrome P450 enzymes of importance for the bioactivation of methyleugenol to the proximate carcinogen 1′-hydroxymethyleugenol

    NARCIS (Netherlands)

    Jeurissen, S.M.F.; Bogaards, J.J.P.; Boersma, M.G.; Horst, J.P.F. ter; Awad, H.M.; Fiamegos, Y.C.; Beek, T.A. van; Alink, G.M.; Sudhölter, E.J.R.; Cnubben, N.H.P.; Rietjens, I.M.C.M.

    2006-01-01

    In vitro studies were performed to elucidate the human cytochrome P450 enzymes involved in the bioactivation of methyleugenol to its proximate carcinogen 1′-hydroxymethyleugenol. Incubations with Supersomes, expressing individual P450 enzymes to a high level, revealed that P450 1A2, 2A6, 2C9, 2C19,

  12. HPLC Determination of Caffeine and Paraxanthine in Urine: An Assay for Cytochrome P450 1A2 Activity

    Science.gov (United States)

    Furge, Laura Lowe; Fletke, Kyle J.

    2007-01-01

    Cytochrome P450 enzymes are a family of heme-containing proteins located throughout the body with roles in metabolism of endogenous and exogenous compounds. Among exogenous compounds, clinically relevant pharmaceutical agents are nearly all metabolized by P450 enzymes. However, the activity of the different cytochrome P450 enzymes varies among…

  13. In vivo roles of Bm3R1 repressor in the barbiturate-mediated induction of the cytochrome P450 genes (P450(BM-3) and P450(BM-)1) of Bacillus megaterium.

    Science.gov (United States)

    Liang, Q; Chen, L; Fulco, A J

    1998-04-10

    We previously showed [Q. Liang, A.J. Fulco, J. Biol. Chem., 270 (1995) 18606-18614) that the binding of Bm3R1 repressor to Barbie box elements and operator sites in the 5'-flanking regions of the P450BM-3 and P450BM-1 (CYP102 and CYP106) genes in Bacillus megaterium was a critical factor in their regulation at the level of transcription. We now describe experiments that delineate specific roles for Bm3R1 in the barbiturate-mediated induction of these genes. We directly demonstrate the interaction of Bm3R1 with Barbie box and operator sequences and show that high in vivo levels of Bm3R1 prevent putative positive factors from binding to Barbie box elements, strongly inhibit the expression of the P450 genes, prolong the lag phase of growth in Bacillus megaterium cultures and increase the sensitivity of the cells to the growth-inhibitory effects of barbiturates. Finally, our data suggest that there may be two forms of Bm3R1, either of which can interact with OIII, the bicistronic operator sequence. PMID:9565684

  14. Molecular cloning and xenobiotic induction of seven novel cytochrome P450 monooxygenases in Aedes albopictus.

    Science.gov (United States)

    Chan, Hiang Hao; Wajidi, Mustafa Fadzil Farid; Zairi, Jaal

    2014-01-01

    Cytochrome P450 monooxygenase (P450) is a superfamily of enzymes that is important in metabolism of endogenous and exogenous compounds. In insects, these enzymes confer resistance to insecticides through its metabolic activities. Members of P450 from family 6 in insects are known to play a role in such function. In this study, we have isolated seven novel family 6 P450 from Aedes albopictus (Skuse) (Diptera: Culicidae), a vector of dengue and chikungunya fever. Induction profile of these seven genes was studied using several insecticides and xenobiotics. It was found that deltamethrin and permethrin did not induce expression of any genes. Another insecticide, temephos, inhibited expression of CYP6P15 for fivefold and twofold for CYP6N29, CYP6Y7, and CYP6Z18. In addition, copper II sulfate induced expression of CYP6M17 and CYP6N28 for up to sixfold. Benzothiazole (BZT), a tire leachate induced the expression of CYP6M17 by fourfold, CYP6N28 by sevenfold, but inhibited the expression of CYP6P15 for threefold and CYP6Y7 for twofold. Meanwhile, piperonyl butoxide (PBO) induced the expression CYP6N28 (twofold), while it inhibited the expression of CYP6P15 (fivefold) and CYP6Y7 (twofold). Remarkably, all seven genes were induced two- to eightfold by acetone in larval stage, but not adult stage. Expression of CYP6N28 was twofold higher, while expression of CYP6P15 was 15-fold lower in adult than larva. The other five P450s were not differentially expressed between the larvae and adult. This finding showed that acetone can be a good inducer of P450 in Ae. albopictus. On the other hand, temephos can act as good suppressor of P450, which may affect its own bioefficacy because it needs to be bioactivated by P450. To the best of our knowledge, this is the first report on acetone-inducible P450 in insects. Further study is needed to characterize the mechanisms involved in acetone induction in P450. PMID:25399430

  15. Cytochrome p450 mRNA expression in the rodent brain: species-, sex-, and region-dependent differences.

    Science.gov (United States)

    Stamou, Marianna; Wu, Xianai; Kania-Korwel, Izabela; Lehmler, Hans-Joachim; Lein, Pamela J

    2014-02-01

    Cytochrome P450 (P450) enzymes play a critical role in the activation and detoxication of many neurotoxic chemicals. Although research has largely focused on P450-mediated metabolism in the liver, emerging evidence suggests that brain P450s influence neurotoxicity by modulating local metabolite levels. As a first step toward better understanding the relative role of brain P450s in determining neurotoxic outcome, we characterized mRNA expression of specific P450 isoforms in the rodent brain. Adult mice (male and female) and rats (male) were treated with vehicle, phenobarbital, or dexamethasone. Transcripts for CYP2B, CYP3A, CYP1A2, and the orphan CYP4X1 and CYP2S1 were quantified in the liver, hippocampus, cortex, and cerebellum by quantitative (real-time) polymerase chain reaction. These P450s were all detected in the liver with the exception of CYP4X1, which was detected in rat but not mouse liver. P450 expression profiles in the brain varied regionally. With the exception of the hippocampus, there were no sex differences in regional brain P450 expression profiles in mice; however, there were marked species differences. In the liver, phenobarbital induced CYP2B expression in both species. Dexamethasone induced hepatic CYP2B and CYP3A in mice but not rats. In contrast, brain P450s did not respond to these classic hepatic P450 inducers. Our findings demonstrate that P450 mRNA expression in the brain varies by region, regional brain P450 profiles vary between species, and their induction varies from that of hepatic P450s. These novel data will be useful for designing mechanistic studies to examine the relative role of P450-mediated brain metabolism in neurotoxicity. PMID:24255117

  16. Human Cytochrome P450c17: Single Step Purification and Phosphorylation of Serine 258 by Protein Kinase A

    OpenAIRE

    Wang, Yue-Hao; Tee, Meng Kian; Miller, Walter L.

    2010-01-01

    Cytochrome P450c17 (P450c17) is the single microsomal enzyme that catalyzes steroid 17α-hydroxylase and 17,20 lyase activities. The ratio of lyase to hydroxylase activity of human P450c17 determines whether steroidogenesis leads to the synthesis of cortisol or sex steroids. This ratio is regulated posttranslationally by factors that influence the efficiency of electron transfer from P450 oxidoreductase to P450c17. One factor favoring more efficient electron transfer and 17,20 lyase activity i...

  17. Fusion to Hydrophobin HFBI Improves the Catalytic Performance of a Cytochrome P450 System

    Science.gov (United States)

    Schulz, Sebastian; Schumacher, Dominik; Raszkowski, Daniel; Girhard, Marco; Urlacher, Vlada B.

    2016-01-01

    Cytochrome P450 monooxygenases (P450) are heme-containing enzymes that oxidize a broad range of substrates in the presence of molecular oxygen and NAD(P)H. For their activity, most P450s rely on one or two redox proteins responsible for the transfer of electrons from the cofactor NAD(P)H to the heme. One of the challenges when using P450s in vitro, especially when non-physiological redox proteins are applied, is the inefficient transfer of electrons between the individual proteins resulting in non-productive consumption of NAD(P)H – referred to as uncoupling. Herein, we describe the improvement of the coupling efficiency between a P450 and its redox partner – diflavin reductase – by fusing both enzymes individually to the hydrophobin HFBI – a small self-assembling protein of the fungus Trichoderma reesei. The separated monooxygenase (BMO) and reductase (BMR) domains of P450 BM3 from Bacillus megaterium were chosen as a P450-reductase model system and individually fused to HFBI. The fusion proteins could be expressed in soluble form in Escherichia coli. When HFBI-fused BMO and BMR were mixed in vitro, substantially higher coupling efficiencies were measured as compared with the respective non-fused enzymes. Consequently, myristic acid conversion increased up to 20-fold (after 6 h) and 5-fold (after 24 h). Size exclusion chromatography demonstrated that in vitro the hydrophobin-fused enzymes build multimeric protein assemblies. Thus, the higher activity is hypothesized to be due to HFBI-mediated self-assembly arranging BMO and BMR in close spatial proximity in aqueous solution. PMID:27458582

  18. Novel Cytochrome P450 Reaction Phenotyping for Low-Clearance Compounds Using the Hepatocyte Relay Method.

    Science.gov (United States)

    Yang, Xin; Atkinson, Karen; Di, Li

    2016-03-01

    A novel cytochrome P450 (P450) reaction phenotyping method for low-clearance compounds has been developed for eight P450 enzymes (CYP1A2, 2B6, 2D6, 2C8, 2C9, 2C19, 3A, and 3A4) and pan-cytochrome using the hepatocyte relay approach. Selective mechanism-based inhibitors were used to inactivate the individual P450 enzymes during preincubation, and inactivators were removed from the incubation before adding substrates to minimize reversible inhibition and maximize inhibitor specificity. The inhibitors were quite selective for specific P450 isoforms using the following inhibitor concentrations and preincubation times: furafylline (1 µM, 15 minutes) for CYP1A2, phencyclidine (20 µM, 15 minutes) for 2B6, paroxetine (1.8 µM, 15 minutes) for CYP2D6, gemfibrozil glucuronide (100 µM, 30 minutes) for 2C8, tienilic acid (15 µM, 30 minutes) for 2C9, esomeprazole (8 µM, 15 minutes) for 2C19, troleandomycin (25 µM, 15 minutes) for 3A4/5, CYP3cide (2 µM, 15 minutes) for 3A4, and 1-aminobenzotriazole (1 mM, 30 minutes) supplemented with tienilic acid (15 µM, 30 minutes) for pan-cytochrome. The inhibitors were successfully applied to the hepatocyte relay method in a 48-well format for P450 reaction phenotyping of low-clearance compounds. This novel method provides a new approach for determining the fraction metabolized of low-turnover compounds that are otherwise challenging with the traditional methods, such as chemical inhibitors with human liver microsomes and hepatocytes or human recombinant P450 enzymes. PMID:26700955

  19. A New Marmoset P450 4F12 Enzyme Expressed in Small Intestines and Livers Efficiently Metabolizes Antihistaminic Drug Ebastine.

    Science.gov (United States)

    Uehara, Shotaro; Uno, Yasuhiro; Yuki, Yukako; Inoue, Takashi; Sasaki, Erika; Yamazaki, Hiroshi

    2016-06-01

    Common marmosets (Callithrix jacchus) are attracting attention as animal models in preclinical studies for drug development. However, cytochrome P450s (P450s), major drug-metabolizing enzymes, have not been fully identified and characterized in marmosets. In this study, based on the four novel P450 4F genes found on the marmoset genome, we successfully isolated P450 4F2, 4F3B, 4F11, and 4F12 cDNAs in marmoset livers. Deduced amino acid sequences of the four marmoset P450 4F forms exhibited high sequence identities (87%-93%) to the human and cynomolgus monkey P450 4F homologs. Marmoset P450 4F3B and 4F11 mRNAs were predominantly expressed in livers, whereas marmoset P450 4F2 and 4F12 mRNAs were highly expressed in small intestines and livers. Four marmoset P450 4F proteins heterologously expressed in Escherichia coli catalyzed the ω-hydroxylation of leukotriene B4 In addition, marmoset P450 4F12 effectively catalyzed the hydroxylation of antiallergy drug ebastine, a human P450 2J/4F probe substrate. Ebastine hydroxylation activities by small intestine and liver microsomes from marmosets and cynomolgus monkeys showed greatly higher values than those of humans. Ebastine hydroxylation activities by marmoset and cynomolgus monkey small intestine microsomes were inhibited (approximately 60%) by anti-P450 4F antibodies, unlike human small intestine microsomes, suggesting that contribution of P450 4F enzymes for ebastine hydroxylation in the small intestine might be different between marmosets/cynomolgus monkeys and humans. These results indicated that marmoset P450 4F2, 4F3B, 4F11, and 4F12 were expressed in livers and/or small intestines and were functional in the metabolism of endogenous and exogenous compounds, similar to those of cynomolgus monkeys and humans. PMID:27044800

  20. Polar bear hepatic cytochrome P450: Immunochemical quantitation, EROD/PROD activity and organochlorines

    Energy Technology Data Exchange (ETDEWEB)

    Letcher, R.J.; Norstrom, R.J. [Carleton Univ., Ottawa, Ontario (Canada). Centre for Analytical and Environmental Chemistry]|[Environment Canada, Ottawa, Ontario (Canada). Canadian Wildlife Service

    1994-12-31

    Polar bears (Ursus maritimus) are an ubiquitous mammal atop the arctic marine food chain and bioaccumulate lipophilic environmental contaminants. Antibodies prepared against purified rat liver cytochrome P450-1 Al, -1 A2, -2Bl and -3Al enzymes have been found to cross-react with structurally-related orthologues present in the hepatic microsomes of wild polar bears, immunochemically determined levels of P450-1 A and -2B proteins in polar bear liver relative to liver of untreated rats suggested enzyme induction, probably as a result of exposure to xenobiotic contaminants. Optical density quantitation of the most immunochemically responsive isozymes P450-I Al, -IA2 and -2Bi to polygonal rabbit anti-rat P450-IA/IA2 sera and -2BI antibodies in hepatic microsomes of 13 adult male polar bars from the Resolute Bay area of the Canadian Arctic is presented. Correlations with EROD and PROD catalytic activities and levels of organochlorines, such as polychlorinated biphenyls (PCBs), 1,1-dichloro-2,2-bis(4-chlorophenyl)ethene (p,p-DDE) and their methyl sulfone (MeSO2-) metabolites are made to determine if compound-specific enzyme induction linkages exist. Inter-species immunochemical quantitation of isozymic P450 cytochromes can serve as an indicator of exposure to biologically active contaminant.

  1. Cytokine-mediated bone resorption is cytochrome P-450 dependent. Student Research Award 1998.

    Science.gov (United States)

    Young, N; Chole, R

    1999-12-01

    Localized bone loss leads to much of the morbidity of chronic otitis media. Although the cellular events of bone remodeling have been well established, their regulation remains poorly understood. Various cytokines, including tumor necrosis factor-alpha, interleukin-1beta, and interferon-gamma, used alone and in combination, are powerful inducers of bone resorption. One of the modulators of cytokine-induced bone resorption is nitric oxide (NO), a product of the action of NO synthase (NOS) on L -arginine to form NO. Cytochrome P-450, an enzyme that is similar to NOS both structurally and functionally, may also have a role in NO production in various cellular systems. The goal of this study was to elucidate a possible role of cytochrome P-450 in bone. In this study cytokine-induced bone resorption was blocked with cimetidine and clotrimazole, which are selective inhibitors of the cytochrome P-450 IIIA family and 7-ethoxyresorufin, a nonspecific cytochrome P-450 inhibitor. A concomitant reduction of NO was also observed. This effect may be explained by cytochrome P-450 being a preferred alternative pathway or providing an essential cofactor to NOS in bone. PMID:10580224

  2. Purification of human placental aromatase cytochrome P-450 with monoclonal antibody and its characterization

    International Nuclear Information System (INIS)

    A simple and efficient method is described for the purification of microsomal aromatase cytochrome P-450 from human placenta. The enzyme was solubilized with Emulgen 913 and sodium cholate and subjected to chromatography on a column of Sepharose 4B couples with a specific monoclonal antibody, followed by hydroxyapatite column chromatography. The specific cytochrome P-450 content of purified aromatase was 13.1 (12-14.8) nmol/mg of protein. Aromatase assays were carried out with reconstituted systems of bovine liver P-450 reductase and dilauroyl-L-α-phosphatidylcholine with [1β-3H,4-14C]androstenedione as substrate. The total recovery of purified aromatase activity was 32.2%, and P-450 recovery was 17.6%. The very high Km value for 16α-hydroxytestosterone aromatization gives a reasonable indication that estriol is not the directly aromatized product in the fetoplacental unit of human pregnancy. The aromatase P-450 was subjected to SDS-polyacrylamide gel electrophoresis in increasing quantities. Silver stain detection techniques indicated a single band having a molecular mass of 55 kDa with greater than 97% purity. The stability analysis showed a half-life of over 4 years on storage at -80C

  3. A Stereoselective Hydroxylation Step of Alkaloid Biosynthesis by a Unique Cytochrome P450 in Catharanthus roseus*

    Science.gov (United States)

    Giddings, Lesley-Ann; Liscombe, David K.; Hamilton, John P.; Childs, Kevin L.; DellaPenna, Dean; Buell, C. Robin; O'Connor, Sarah E.

    2011-01-01

    Plant cytochrome P450s are involved in the production of over a hundred thousand metabolites such as alkaloids, terpenoids, and phenylpropanoids. Although cytochrome P450 genes constitute one of the largest superfamilies in plants, many of the catalytic functions of the enzymes they encode remain unknown. Here, we report the identification and functional characterization of a cytochrome P450 gene in a new subfamily of CYP71, CYP71BJ1, involved in alkaloid biosynthesis. Co-expression analysis of putative cytochrome P450 genes in the Catharanthus roseus transcriptome identified candidate genes with expression profiles similar to known terpene indole alkaloid biosynthetic genes. Screening of these candidate genes by functional expression in Saccharomyces cerevisiae yielded a unique P450-dependent enzyme that stereoselectively hydroxylates the alkaloids tabersonine and lochnericine at the 19-position of the aspidosperma-type alkaloid scaffold. Tabersonine, which can be converted to either vindoline or 19-O-acetylhörhammericine, represents a branch point in alkaloid biosynthesis. The discovery of CYP71BJ1, which forms part of the pathway leading to 19-O-acetylhörhammericine, will help illuminate how this branch point is controlled in C. roseus. PMID:21454651

  4. Involvement of Cytochrome P450 in Glucosinolate Biosynthesis in White Mustard (A Biochemical Anomaly).

    Science.gov (United States)

    Bennett, R. N.; Kiddle, G.; Wallsgrove, R. M.

    1997-08-01

    One of the first steps in glucosinolate biosynthesis is the conversion of amino acids to their aldoximes. The biochemistry of this process is controversial, and several very different enzyme systems have been described. The major glucosinolate in white mustard (Sinapis alba) is sinalbin, which is derived from tyrosine via its aldoxime, and this conversion is catalyzed by a cytochrome P450 (Cyt P450) monooxygenase. Phenylethyl- and alkenylglucosinolates are also present in white mustard leaves, as are the enzymes catalyzing the relevant aldoxime formation from homophenylalanine and methionine homologs, respectively. These enzymes are similar to those found in Brassica sp. and are distinct from the tyrosine-dependent enzyme in that they contain no heme and are unaffected by Cyt P450 inhibitors. They are instead inhibited by the flavoprotein inhibitor diphenylene iodonium and by Cu2+. In both white mustard and oilseed rape (Brassica napus) methyl jasmonate specifically stimulates indolylglucosinolate biosynthesis and yet has no effect on sinalbin accumulation in either cotyledons or leaves of white mustard. White mustard appears to be unique among crucifers in having a Cyt P450 aldoxime-forming enzyme for biosynthesis of one glucosinolate, although it also contains all of the non-Cyt P450 enzyme systems found in other members of the family. Sinalbin biosynthesis in white mustard is therefore an inappropriate model system for the synthesis of other glucosinolates in crucifers, including canola and oilseed rape. PMID:12223771

  5. Development of gold-immobilized P450 platform for exploring the effect of oligomer formation on P450-mediated metabolism for in vitro to in vivo drug metabolism predictions

    Science.gov (United States)

    Kabulski, Jarod L.

    The cytochrome P450 (P450) enzyme family is responsible for the biotransformation of a wide range of endogenous and xenobiotic compounds, as well as being the major metabolic enzyme in first pass drug metabolism. In vivo drug metabolism for P450 enzymes is predicted using in vitro data obtained from a reconstituted expressed P450 system, but these systems have not always been proven to accurately represent in vivo enzyme kinetics, due to interactions caused by oligomer formation. These in vitro systems use soluble P450 enzymes prone to oligomer formation and studies have shown that increased states of protein aggregation directly affect the P450 enzyme kinetics. We have developed an immobilized enzyme system that isolates the enzyme and can be used to elucidate the effect of P450 aggregation on metabolism kinetics. The long term goal of my research is to develop a tool that will help improve the assessment of pharmaceuticals by better predicting in vivo kinetics in an in vitro system. The central hypothesis of this research is that P450-mediated kinetics measured in vitro is dependent on oligomer formation and that the accurate prediction of in vivo P450-mediated kinetics requires elucidation of the effect of oligomer formation. The rationale is that the development of a P450 bound to a Au platform can be used to control the aggregation of enzymes and bonding to Au may also permit replacement of the natural redox partners with an electrode capable of supplying a constant flow of electrons. This dissertation explains the details of the enzyme attachment, monitoring substrate binding, and metabolism using physiological and electrochemical methods, determination of enzyme kinetics, and the development of an immobilized-P450 enzyme bioreactor. This work provides alternative approaches to studying P450-mediated kinetics, a platform for controlling enzyme aggregation, electrochemically-driven P450 metabolism, and for investigating the effect of protein

  6. Chromogenic nitrophenolate-based substrates for light-driven hybrid P450 BM3 enzyme assay.

    Science.gov (United States)

    Lam, Quan; Cortez, Alejandro; Nguyen, Thanh Truc; Kato, Mallory; Cheruzel, Lionel

    2016-05-01

    The incorporation of a p-nitrophenoxy moiety in substrates has enabled the development of colorimetric assays to rapidly screen for O-demethylation activity of P450 enzymes. For the light-driven hybrid P450 BM3 enzymes, where a Ru(II) photosensitizer powers the enzyme upon visible light irradiation, we have investigated a family of p-nitrophenoxy derivatives as useful chromogenic substrates compatible with the light-driven approach. The validation of this assay and its adaptability to a 96-well plate format will enable the screening of the next generation of hybrid P450 BM3 enzymes towards C-H bond functionalization of non-natural substrates. PMID:26712653

  7. Structure, dynamics, and function of the monooxygenase P450 BM-3: insights from computer simulations studies

    International Nuclear Information System (INIS)

    The monooxygenase P450 BM-3 is a NADPH-dependent fatty acid hydroxylase enzyme isolated from soil bacterium Bacillus megaterium. As a pivotal member of cytochrome P450 superfamily, it has been intensely studied for the comprehension of structure–dynamics–function relationships in this class of enzymes. In addition, due to its peculiar properties, it is also a promising enzyme for biochemical and biomedical applications. However, despite the efforts, the full understanding of the enzyme structure and dynamics is not yet achieved. Computational studies, particularly molecular dynamics (MD) simulations, have importantly contributed to this endeavor by providing new insights at an atomic level regarding the correlations between structure, dynamics, and function of the protein. This topical review summarizes computational studies based on MD simulations of the cytochrome P450 BM-3 and gives an outlook on future directions. (topical review)

  8. Adrenodoxin supports reactions catalyzed by microsomal steroidogenic cytochrome P450s

    International Nuclear Information System (INIS)

    The interaction of adrenodoxin (Adx) and NADPH cytochrome P450 reductase (CPR) with human microsomal steroidogenic cytochrome P450s was studied. It is found that Adx, mitochondrial electron transfer protein, is able to support reactions catalyzed by human microsomal P450s: full length CYP17, truncated CYP17, and truncated CYP21. CPR, but not Adx, supports activity of truncated CYP19. Truncated and the full length CYP17s show distinct preference for electron donor proteins. Truncated CYP17 has higher activity with Adx compared to CPR. The alteration in preference to electron donor does not change product profile for truncated enzymes. The electrostatic contacts play a major role in the interaction of truncated CYP17 with either CPR or Adx. Similarly electrostatic contacts are predominant in the interaction of full length CYP17 with Adx. We speculate that Adx might serve as an alternative electron donor for CYP17 at the conditions of CPR deficiency in human

  9. Purification and immunochemical detections of ?-naphthoflavone- and phenobarbital-induced avian cytochrome P450 enzymes

    Science.gov (United States)

    Brown, R.L.; Levi, P.E.; Hodgson, E.; Melancon, M.J.

    1996-01-01

    Livers from mallards (Anas platyrhynchos) were treated with either -naphthoflavone (50 mg/kg) or phenobarbital (70 mg/kg). Purification of induced hepatic cytochrome P450 was accomplished using both DEAE and hydroxyapatite columns, as well as sodium dodecyl sulfate polyacrylamide gel electrophoresis separation. Polyclonal antibodies to these proteins were then produced in young male New Zealand White rabbits. ?-naphthoflavone (?NF)- and phenobarbital(PB)-treated red-winged blackbird, screech owl, European starling and lesser scaup liver microsomes were analyzed in western blots for species cross-reactivity. Although all four of these avian species exhibited cross-reactivity with antibodies to ?NF-induced mallard P450, all but the lesser scaup revealed a protein of higher molecular weight than that of the ?NF-induced mallard. In addition, only the lesser scaup exhibited cross-reactivity with the anti-PB-induced mallard P450 antibodies.

  10. Structure, dynamics, and function of the monooxygenase P450 BM-3: insights from computer simulations studies

    Science.gov (United States)

    Roccatano, Danilo

    2015-07-01

    The monooxygenase P450 BM-3 is a NADPH-dependent fatty acid hydroxylase enzyme isolated from soil bacterium Bacillus megaterium. As a pivotal member of cytochrome P450 superfamily, it has been intensely studied for the comprehension of structure-dynamics-function relationships in this class of enzymes. In addition, due to its peculiar properties, it is also a promising enzyme for biochemical and biomedical applications. However, despite the efforts, the full understanding of the enzyme structure and dynamics is not yet achieved. Computational studies, particularly molecular dynamics (MD) simulations, have importantly contributed to this endeavor by providing new insights at an atomic level regarding the correlations between structure, dynamics, and function of the protein. This topical review summarizes computational studies based on MD simulations of the cytochrome P450 BM-3 and gives an outlook on future directions.

  11. In Silico Prediction of Cytochrome P450-Mediated Biotransformations of Xenobiotics: A Case Study of Epoxidation.

    Science.gov (United States)

    Zhang, Jing; Ji, Li; Liu, Weiping

    2015-08-17

    Predicting the biotransformation of xenobiotics is important in toxicology; however, as more compounds are synthesized than can be investigated experimentally, powerful computational methods are urgently needed to prescreen potentially useful candidates. Cytochrome P450 enzymes (P450s) are the major enzymes involved in xenobiotic metabolism, and many substances are bioactivated by P450s to form active compounds. An example is the conversion of olefinic substrates to epoxides, which are intermediates in the metabolic activation of many known or suspected carcinogens. We have calculated the activation energies for epoxidation by the active species of P450 enzymes (an iron-oxo porphyrin cation radical oxidant, compound I) for a diverse set of 36 olefinic substrates with state-of-the-art density functional theory (DFT) methods. Activation energies can be estimated by the computationally less demanding method of calculating the ionization potentials of the substrates, which provides a useful and simple predictive model based on the reaction mechanism; however, the preclassification of these diverse substrates into weakly polar and strongly polar groups is a prerequisite for the construction of specific predictive models with good predictability for P450 epoxidation. This approach has been supported by both internal and external validations. Furthermore, the relation between the activation energies for the regioselective epoxidation and hydroxylation reactions of P450s and experimental data has been investigated. The results show that the computational method used in this work, single-point energy calculations with the B3LYP functional including zero-point energy and solvation and dispersion corrections based on B3LYP-optimized geometries, performs well in reproducing the experimental trends of the epoxidation and hydroxylation reactions. PMID:26200167

  12. Relationships among Ergot Alkaloids, Cytochrome P450 Activity, and Beef Steer Growth

    Science.gov (United States)

    Rosenkrans, Charles; Ezell, Nicholas

    2015-03-01

    Determining a grazing animal’s susceptibility to ergot alkaloids has been a research topic for decades. Our objective was to determine if the Promega™ P450-Glo assay could be used to indirectly detect ergot alkaloids or their metabolites in urine of steers. The first experiment validated the effects of ergot alkaloids [0, 20, and 40 μM of ergotamine (ET), dihydroergotamine (DHET), and ergonovine (EN)] on human CYP3A4 using the P450-Glo assay (Promega™ V9800). With this assay, luminescence is directly proportional to CYP450 activity. Relative inhibition of in vitro cytochrome P450 activity was affected (P experiment 2, urine was collected from Angus-sired crossbred steers (n = 39; 216 ± 2.6 d of age; 203 ± 1.7 kg) after grazing tall fescue pastures for 105 d. Non-diluted urine was added to the Promega™ P450-Glo assay, and observed inhibition (3.7 % ± 2.7 of control). Urine content of total ergot alkaloids (331.1 ng/mg of creatinine ± 325.7) was determined using enzyme linked immunosorbent assay. Urine inhibition of CYP450 activity and total alkaloids were correlated (r = -0.31; P animals that are susceptible to ergot alkaloids. Although, additional research is needed, we demonstrate that the Promega™ P450-Glo assay is sensitive to ergot alkaloids and urine from steers grazing tall fescue. With some refinement the P450-Glo assay has potential as a tool for screening cattle for their exposure to fescue toxins.

  13. Enhancement of DMNQ-induced hepatocyte toxicity by cytochrome P450 inhibition

    International Nuclear Information System (INIS)

    Two mechanisms have been proposed to explain quinone cytotoxicity: oxidative stress via the redox cycle and the arylation of intracellular nucleophiles. As the redox cycle is catalyzed by NADPH cytochrome P450 reductase, cytochrome P450 systems are expected to be related to the cytotoxicity induced by redox-cycling quinones. Thus, we investigated the relationship between cytochrome P450 systems and quinone toxicity for rat primary hepatocytes using an arylator, 1,4-benzoquinone (BQ), and a redox cycler, 2,3-dimethoxy-1,4-naphthoquinone (DMNQ). The hepatocyte toxicity of both BQ and DMNQ increased in a time- and dose-dependent manner. Pretreatment with cytochrome P450 inhibitors, such as SKF-525A (SKF), ketoconazole and 2-methy-1,2-di-3-pyridyl-1-propanone, enhanced the hepatocyte toxicity induced by DMNQ but did not affect BQ-induced hepatocyte toxicity. The production of superoxide anion and the levels of glutathione disulfide and thiobarbituric-acid-reactive substances were increased by treatment with DMNQ, and SKF pretreatment further enhanced their increases. In addition, NADPH oxidation in microsomes was increased by treatment with DMNQ and further augmented by pretreatment with SKF, and a NADPH cytochrome P450 reductase inhibitor, diphenyleneiodonium chloride completely suppressed NADPH oxidations increased by treatment with either DMNQ- or DMNQ + SKF. Pretreatment with antioxidants, such as α-tocopherol, reduced glutathione, N-acetyl cysteine or an iron ion chelator deferoxamine, totally suppressed DMNQ- and DMNQ + SKF-induced hepatocyte toxicity. These results indicate that the hepatocyte toxicity of redox-cycling quinones is enhanced under cytochrome P450 inhibition, and that this enhancement is caused by the potentiation of oxidative stress

  14. Metabolism and binding of cyclophosphamide and its metabolite acrolein to rat hepatic microsomal cytochrome P-450

    International Nuclear Information System (INIS)

    The hepatic cytochrome P-450-mediated metabolism and metabolic activation of [chloroethyl-3H]cyclophosphamide [( chloroethyl-3H]CP) and [4-14C]cyclophosphamide [( 4-14C]CP) were investigated in vitro in the reconstituted system containing cytochrome P-450 isolated from phenobarbital-treated rats. In addition, hepatic microsomal binding and the hepatic microsome-mediated metabolism of [14C]acrolein, a metabolite of [4-14C]CP, were also investigated. The metabolism of [chloroethyl-3H]CP and [4-14C]CP to polar metabolites was found to depend on the presence of NADPH and showed concentration dependence with respect to cytochrome P-450 and NADPH:cytochrome P-450 reductase. Km and Vmax values were essentially similar. The patterns of inhibition by microsomal mixed-function oxidase inhibitors, anti-cytochrome P-450 antibody, and heat denaturation of the cytochrome P-450 were essentially similar, with subtle differences between [4-14C]CP and [chloroethyl-3H]CP metabolism. The in vitro metabolic activation of CP in the reconstituted system demonstrated predominant binding of [chloroethyl-3H]CP to nucleic acids and almost exclusive binding of [4-14C]CP to proteins. Gel electrophoresis-fluorography of the proteins in the reconstituted system treated with [4-14C]CP demonstrated localization of the 14C label in the cytochrome P-450 region. To examine this association further, hepatic microsomes were modified with [14C]acrolein in the presence and the absence of NADPH. The results confirmed covalent association between [14C]acrolein and cytochrome P-450 in the microsomes and also demonstrated further metabolism of [14C]acrolein, apparently to an epoxide, which is capable of binding covalently to proteins. The results of these investigations not only confirm the significance of primary metabolism but also emphasize the potential role of the secondary metabolism of cyclophosphamide in some of its toxic manifestations

  15. DrugMetZ DB: an anthology of human drug metabolizing Chytochrome P450 enzymes

    OpenAIRE

    Antony, Tresa Remya Thomas; Nagarajan, Shanthi

    2006-01-01

    Understandings the basics of Cytochrome P450 (P450 or CYP) will help to discern drug metabolism. CYP, a super-family of heme-thiolate proteins, are found in almost all living organisms and is involved in the biotransformation of a diverse range of xenobiotics, therapeutic drugs and toxins. Here, we describe DrugMetZ DB, a database for CYP metabolizing drugs. The DB is implemented in MySQL, PHP and HTML. Availability www.bicpu.edu.in/DrugMetZDB/

  16. Theoretical study of the cytochrome P450 mediated metabolism of phosphorodithioate pesticides

    DEFF Research Database (Denmark)

    Rydberg, Patrik

    2012-01-01

    The toxicity of phosphorodithioate pesticides is due to the formation of the active oxane product through desulfurization by cytochrome P450 enzymes, both in humans and insects. During this desulfurization, inhibition of cytochrome P450 and a loss of heme has been observed. Here, we study the...... mechanism of desulfurization and inhibition with density functional theory, using the B3LYP functional with and without dispersion correction. The results show that a reaction mechanism initiated by sulfur oxidation is most likely, with a reaction barrier of 47 kJ/mol. The sulfur oxidation is followed by a...

  17. Detection of substrate-dependent conformational changes in the P450 fold by nuclear magnetic resonance

    OpenAIRE

    Allison M. Colthart; Drew R. Tietz; Yuhua Ni; Friedman, Jessica L.; Marina Dang; Pochapsky, Thomas C.

    2016-01-01

    Cytochrome P450 monooxygenases typically catalyze the insertion of one atom of oxygen from O2 into unactivated carbon-hydrogen and carbon-carbon bonds, with concomitant reduction of the other oxygen atom to H2O by NAD(P)H. Comparison of the average structures of the camphor hydroxylase cytochrome P450cam (CYP101) obtained from residual dipolar coupling (RDC)-restrained molecular dynamics (MD) in the presence and absence of substrate camphor shows structural displacements resulting from the es...

  18. Flavoproteins in directed evolution : iterative CASTing to evolve YqjM and P450BM3

    OpenAIRE

    Kille, Sabrina

    2010-01-01

    In der vorliegenden Arbeit wurden zwei Enzyme, eine Enoatreduktase (YqjM) und eine Monooxigenase (P450BM3), mittels "Gerichteter Evolution" auf die Akzeptanz neuer Substrate angepasst. Mittel iterativem CASTing (combinatorial active site saturation test) konnten in beiden Fällen hoch regio- und stereoselektive Mutanten erhalten werden. Zusätzlich wurde Kontrolle über die Enantioselektivität (YqjM) und Regioselektivität (P450BM3) erreicht. Ein weiterer Aspekt der Arbeit war die Unt...

  19. Cytochrome P-450 dependent ethanol oxidation. Kinetic isotope effects and absence of stereoselectivity

    International Nuclear Information System (INIS)

    Deuterium isotope effects [/sup D/(V/K)] and stereoselectivity of ethanol oxidation in cytochrome P-450 containing systems and in the xanthine-xanthine oxidase system were compared with those of yeast alcohol dehydrogenase. The isotope effects were determined by using both a noncompetitive method, including incubation of unlabeled of [1,1-2H2] ethanol at various concentrations, and a competitive method, where 1:1 mixtures of [1-13C]- and [2H6] ethanol or [2,2,2-2H3]- and [1,1-2H2] ethanol were incubated and the acetaldehyde formed was analyzed by gas chromatography/mass spectrometry. The /sup D/(V/K) isotope effects of the cytochrome P-450 dependent ethanol oxidation were about 4 with liver microsomes from imidazole-, phenobarbital- or acetone-treated rabbits or with microsomes from acetone- or ethanol-treated rats. Similar isotope effects were reached with reconstituted membranes containing the rabbit ethanol-inducible cytochrome P-450 (LMeb), whereas control rat microsomes and membranes containing rabbit phenobarbital-inducible P-450 LM2 oxidized the alcohol with /sup D/(V/K) of about 2.8 and 1.8, respectively. Addition of Fe/sup III/EDTA either to microsomes from phenobarbital-treated rabbits or to membranes containing P-450 LMeb significantly lowered the isotope effect. Incubations of all cytochrome P-450 containing systems of the xanthine-xanthine oxidase systems with (1R)- and (1S)-[1-2H] ethanol, revealed, taking the isotope effects into account, that 44-66% of the ethanol oxidized had lost the 1-pro-R hydrogen. The data indicate that cytochrome P-450 dependent ethanol oxidation is not stereospecific and that cleavage of the C1-H bond appears to be a rate-determining step in the catalysis by the ethanol-inducible form of P-450. The contribution of hydroxyl radicals in ethanol oxidation by the various enzymic systems is discussed

  20. Exploring the substrate range of selected myxobacterial cytochromes P450 from Sorangium cellulosum So ce56

    OpenAIRE

    Litzenburger, Martin

    2016-01-01

    Cytochromes P450 are highly versatile biocatalysts due to their ability to introduce molecular oxygen into a non-activated C-H bond. In this work, nine P450 enzymes from Sorangium cellulosum So ce56 were investigated concerning their substrate range and catalytic biodiversity. Hence, terpenes, terpenoids, carotenoid-derived aroma compounds, aromatic compounds and drugs were tested as potential substrates for CYP109C1, CYP109C2, CYP109D1, CYP260A1, CYP260B1, CYP264A1, CYP264B1, CYP267A1 and CY...

  1. Cytochrome P450 Reductase: A Harbinger of Diffusible Reduced Oxygen Species

    OpenAIRE

    Manoj, Kelath Murali; Gade, Sudeep Kumar; Mathew, Lazar

    2010-01-01

    The bi-enzymatic system of cytochrome P450 (CYP, a hemoprotein) and cytochrome P450 reductase (CPR, a diflavoenzyme) mediate the redox metabolism of diverse indigenous and xenobiotic molecules in various cellular and organ systems, using oxygen and NADPH. Curiously, when a 1∶1 ratio is seen to be optimal for metabolism, the ubiquitous CYP:CPR distribution ratio is 10 to 100∶1 or higher. Further, the NADPH equivalents consumed in these in vitro or in situ assemblies usually far exceeded the am...

  2. Mechanisms of π-bond oxidation by cytochrome p-450: acetylenes as probes

    International Nuclear Information System (INIS)

    Phenylacetylene and biphenylacetylene are oxidized by microsomal and purified P-450 to the corresponding arylacetic acids. During this transformation, the acetylenic hydrogen undergoes a 1,2 shift which causes a kinetic isotope effect of 1.8 on the overall enzymatic rate. The same products and kinetic isotope effects are observed when the arylacetylenes are oxidized by m-chloroperbenzoic acid. Suicide inactivation of P-450 by the arylacetylenes, which occurs simultaneously with metabolite formation, is insensitive to isotopic substitution so the partition ratio changes from 26 for phenylacetylene of 14 for [1-2H] phenylacetylene

  3. Sequence analysis of ripening-related cytochrome P-450 cDNAs from avocado fruit.

    OpenAIRE

    Bozak, K R; Yu, H.; Sirevåg, R; Christoffersen, R E

    1990-01-01

    The ripening of avocado fruit is associated with the expression of a number of mRNAs concomitant with overt changes in texture and flavor. Two overlapping cDNAs for a mRNA that accumulates during ripening were identified. Sequence analysis of these two cDNAs revealed a polypeptide of 471 amino acids with characteristics of a typical P-450: an N-terminal hydrophobic membrane anchor, a conserved heme-binding domain in the C-terminal region, and patches of similarity to various P-450 family memb...

  4. Role of hepatic cytochromes P450 in bioactivation of the anticancer drug ellipticine: Studies with the hepatic NADPH:Cytochrome P450 reductase null mouse

    International Nuclear Information System (INIS)

    Ellipticine is an antineoplastic agent, which forms covalent DNA adducts mediated by cytochromes P450 (CYP) and peroxidases. We evaluated the role of hepatic versus extra-hepatic metabolism of ellipticine, using the HRN (Hepatic Cytochrome P450 Reductase Null) mouse model, in which cytochrome P450 oxidoreductase (POR) is deleted in hepatocytes, resulting in the loss of essentially all hepatic CYP function. HRN and wild-type (WT) mice were treated i.p. with 1 and 10 mg/kg body weight of ellipticine. Multiple ellipticine-DNA adducts detected by 32P-postlabelling were observed in organs from both mouse strains. Highest total DNA binding levels were found in liver, followed by lung, kidney, urinary bladder, colon and spleen. Ellipticine-DNA adduct levels in the liver of HRN mice were up to 65% lower relative to WT mice, confirming the importance of CYP enzymes for the activation of ellipticine in livers, recently shown in vitro with human and rat hepatic microsomes. When hepatic microsomes of both mouse strains were incubated with ellipticine, ellipticine-DNA adduct levels with WT microsomes were up to 2.9-fold higher than with those from HRN mice. The ratios of ellipticine-DNA adducts in extra-hepatic organs between HRN and WT mice of up to 4.7 suggest that these organs can activate ellipticine and that more ellipticine is available in the circulation. These results and the DNA adduct patterns found in vitro and in vivo demonstrate that both CYP1A or 3A and peroxidases participate in activation of ellipticine to reactive species forming DNA adducts in the mouse model used in this study

  5. Inhibitory potency of quinolone antibacterial agents against cytochrome P450IA2 activity in vivo and in vitro.

    OpenAIRE

    Fuhr, U.; Anders, E M; Mahr, G; Sörgel, F; Staib, A H

    1992-01-01

    Inhibition of cytochrome P450IA2 activity is an important adverse effect of quinolone antibacterial agents. It results in a prolonged half-life for some drugs that are coadministered with quinolones, such as theophylline. The objective of the study described here was to define the parameters for quantifying the inhibitory potencies of quinolones against cytochrome P450IA2 in vivo and in vitro and to investigate the relationship between the results of both approaches. Cytochrome P450IA2 activi...

  6. A simplified process design for P450 driven hydroxylation based on surface displayed enzymes.

    Science.gov (United States)

    Ströhle, Frank W; Kranen, Eva; Schrader, Jens; Maas, Ruth; Holtmann, Dirk

    2016-06-01

    New production routes for fine and bulk chemicals are important to establish further sustainable processes in industry. Besides the identification of new biocatalysts and new production routes the optimization of existing processes in regard to an improved utilization of the catalysts are needed. In this paper we describe the successful expression of P450BM3 on the surface of E. coli cells with the Autodisplay system. The successful hydroxylation of palmitic acid by using surface-displayed P450BM3 was shown. Besides optimization of surface protein expression, several cofactor regeneration systems were compared and evaluated. Afterwards, the development of a suitable process for the biocatalytic hydroxylation of fatty acids based on the re-use of the catalysts after a simple centrifugation was investigated. It was shown that the catalyst can be used for several times without any loss in activity. By using surface-displayed P450s in combination with an enzymatic cofactor regeneration system a total turnover number of up to 54,700 could be reached, to the knowledge of the authors the highest value reported for a P450 monooxygenase to date. Further optimizations of the described reaction system can have an enormous impact on the process design for more sustainable bioprocesses. Biotechnol. Bioeng. 2016;113: 1225-1233. © 2015 Wiley Periodicals, Inc. PMID:26574191

  7. Overcoming heterologous protein interdependency to optimize P450-mediated Taxol precursor synthesis in Escherichia coli.

    Science.gov (United States)

    Biggs, Bradley Walters; Lim, Chin Giaw; Sagliani, Kristen; Shankar, Smriti; Stephanopoulos, Gregory; De Mey, Marjan; Ajikumar, Parayil Kumaran

    2016-03-22

    Recent advances in metabolic engineering have demonstrated the potential to exploit biological chemistry for the synthesis of complex molecules. Much of the progress to date has leveraged increasingly precise genetic tools to control the transcription and translation of enzymes for superior biosynthetic pathway performance. However, applying these approaches and principles to the synthesis of more complex natural products will require a new set of tools for enabling various classes of metabolic chemistries (i.e., cyclization, oxygenation, glycosylation, and halogenation) in vivo. Of these diverse chemistries, oxygenation is one of the most challenging and pivotal for the synthesis of complex natural products. Here, using Taxol as a model system, we use nature's favored oxygenase, the cytochrome P450, to perform high-level oxygenation chemistry inEscherichia coli An unexpected coupling of P450 expression and the expression of upstream pathway enzymes was discovered and identified as a key obstacle for functional oxidative chemistry. By optimizing P450 expression, reductase partner interactions, and N-terminal modifications, we achieved the highest reported titer of oxygenated taxanes (∼570 ± 45 mg/L) inE. coli.Altogether, this study establishesE. colias a tractable host for P450 chemistry, highlights the potential magnitude of protein interdependency in the context of synthetic biology and metabolic engineering, and points to a promising future for the microbial synthesis of complex chemical entities. PMID:26951651

  8. Cloning and expression of an atrazine inducible cytochrome P450 from Chironomus tentans (Diptera: Chironomidae)

    Science.gov (United States)

    Previous studies performed in our lab have measured the effect of atrazine exposure on cytochrome P450-dependent monooxygenase activity and have found increased activity in midge larvae (Chironomus tentans) as a result of atrazine exposure (1-10 ppm). Here we report the cloning and expression of a ...

  9. Dynamics of water molecules in the active-site cavity of human cytochromes P450

    DEFF Research Database (Denmark)

    Rydberg, Patrik; Rod, Thomas Holm; Olsen, Lars;

    2007-01-01

    We have studied the dynamics of water molecules in six crystal structures of four human cytochromes P450, 2A6, 2C8, 2C9, and 3A4, with molecular dynamics simulations. In the crystal structures, only a few water molecules are seen and the reported sizes of the active-site cavity vary a lot. In the...

  10. Photosystem I from plants as a bacterial cytochrome P450 surrogate electron donor

    DEFF Research Database (Denmark)

    Jensen, Kenneth; Johnston, Jonathan B.; Montellano, Paul R. Ortiz de; Møller, Birger Lindberg

    2012-01-01

    The ability of cytochrome P450 enzymes to catalyze highly regio- and stereospecific hydroxylations makes them attractive alternatives to approaches based on chemical synthesis but they require expensive cofactors, e.g. NAD(P)H, which limits their commercial potential. Ferredoxin (Fdx) is a multif...

  11. PROPICONAZOLE-INDUCED CYTOCHROME P450 GENE EXPRESSION AND ENZYMATIC ACTIVITIES IN RAT AND MOUSE LIVER

    Science.gov (United States)

    Conazoles are N-substituted azole antifungal agents used as both pesticides and drugs. Some of these compounds are hepatocarcinogenic in mice and some can induce thyroid tumors in rats. Many of these compounds are able to induce and/or inhibit mammalian hepatic cytochrome P450s t...

  12. Een familie met acties, reacties en interacties : Deel 2. Het cytochroom P450-enzymsysteem

    NARCIS (Netherlands)

    Touw, D.J.; Breimer, D.D.

    1997-01-01

    Large interindividual variability exists in the capacity to oxidise drugs in man. An important contributing factor is the occurrence of a genetically determined polymorphism of some enzymes of the microsomal cytochrome P450 (CYP) enzyme system. In addition, numerous environmental factors influence o

  13. Het cytochroom P450-enzymsysteem : Een familie met acties, reacties en interacties. Deel 1

    NARCIS (Netherlands)

    Touw, D.J.; Breimer, D.D.

    1997-01-01

    Large interindividual variability exists in the capacity to oxidise drugs in man. An important contributing factor is the occurrence of a genetically determined polymorphism of some enzymes of the microsomal cytochrome P450 (CYP) enzyme system. In addition, numerous environmental factors influence o

  14. Human cytochrome P450 enzyme specificity for the bioactivation of estragole and related alkenylbenzenes

    NARCIS (Netherlands)

    Jeurissen, S.M.F.; Punt, A.; Boersma, M.G.; Bogaards, J.J.P.; Fiamegos, Y.C.; Schilter, B.; Bladeren, P.J. van; Cnubben, N.H.P.; Rietjens, I.M.C.M.

    2007-01-01

    Human cytochrome P450 enzymes involved in the bioactivation of estragole to its proximate carcinogen 1′-hydroxyestragole were identified and compared to the enzymes of importance for 1′-hydroxylation of the related alkenylbenzenes methyleugenol and safrole. Incubations with Supersomes revealed that

  15. Cytochrome P450 levels are altered in patients with esophageal squamous-cell carcinoma

    DEFF Research Database (Denmark)

    Bergheim, I.; Wolfgarten, E.; Bollschweiler, E.;

    2007-01-01

    AIM: To investigate the role of cytochrome P450 (CYP) in the carcinogenesis of squamous-cell carcinoma (SCC) in human esophagus by determining expression patterns and protein levels of representative CYPs in esophageal tissue of patients with SCC and controls. METHODS: mRNA expression of CYP2E1, ...

  16. Molecular evolution of the insect Halloween family of cytochrome P450s

    DEFF Research Database (Denmark)

    Rewitz, Kim; O'Connor, Michael B.; Gilbert, Lawrence I.

    2007-01-01

    mosquito CYP307A subfamily genes. Evolutionary links between the insect Halloween genes and vertebrate steroidogenic P450s suggest that they originated from common ancestors, perhaps destined for steroidogenesis, before the deuterostome-arthropod split. Conservation of putative substrate recognition sites...

  17. Screening and identification of novel cytochrome P450s in ticks

    Science.gov (United States)

    Cytochrome P450s are the major phase I drug metabolizing enzymes found in most species, including those belonging to the phylum Arthropoda. Much of the work within the area of xenobiotic metabolism in this phylum has centered on mosquito species such as Anopheles gambiae due to their role as vectors...

  18. Prediction of activation energies for hydrogen abstraction by cytochrome p450

    DEFF Research Database (Denmark)

    Olsen, Lars; Rydberg, Patrik; Rod, Thomas Holm; Ryde, Ulf

    2006-01-01

    We have estimated the activation energy for hydrogen abstraction by compound I in cytochrome P450 for a diverse set of 24 small organic substrates using state-of-the-art density functional theory (B3LYP). We then show that these results can be reproduced by computationally less demanding methods...

  19. Prediction of activation energies for aromatic oxidation by cytochrome P450

    DEFF Research Database (Denmark)

    Rydberg, Patrik; Ryde, Ulf; Olsen, Lars

    2008-01-01

    We have estimated the activation energy for aromatic oxidation by compound I in cytochrome P450 for a diverse set of 17 substrates using state-of-the-art density functional theory (B3LYP) with large basis sets. The activation energies vary from 60 to 87 kJ/mol. We then test if these results can be...

  20. Structure and function of NADPH-cytochrome P450 reductase and nitric oxide synthase reductase domain

    International Nuclear Information System (INIS)

    NADPH-cytochrome P450 reductase (CPR) and the nitric oxide synthase (NOS) reductase domains are members of the FAD-FMN family of proteins. The FAD accepts two reducing equivalents from NADPH (dehydrogenase flavin) and FMN acts as a one-electron carrier (flavodoxin-type flavin) for the transfer from NADPH to the heme protein, in which the FMNH ·/FMNH2 couple donates electrons to cytochrome P450 at constant oxidation-reduction potential. Although the interflavin electron transfer between FAD and FMN is not strictly regulated in CPR, electron transfer is activated in neuronal NOS reductase domain upon binding calmodulin (CaM), in which the CaM-bound activated form can function by a similar mechanism to that of CPR. The oxygenated form and spin state of substrate-bound cytochrome P450 in perfused rat liver are also discussed in terms of stepwise one-electron transfer from CPR. This review provides a historical perspective of the microsomal mixed-function oxidases including CPR and P450. In addition, a new model for the redox-linked conformational changes during the catalytic cycle for both CPR and NOS reductase domain is also discussed

  1. Expression of cytochrome P450 genes in CD34(+) hematopoietic stem and progenitor cells

    Czech Academy of Sciences Publication Activity Database

    Souček, P.; Anzenbacher, P.; Skoumalová, I.; Dvořák, Michal

    2005-01-01

    Roč. 23, č. 9 (2005), s. 1417-1422. ISSN 1066-5099 Institutional research plan: CEZ:AV0Z50520514 Keywords : CD34+ stem/progenitor cells * cytochrome P450 isoforms Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 6.094, year: 2005

  2. Freeze-quenched iron-oxo intermediates in cytochromes P450

    International Nuclear Information System (INIS)

    Since the discovery of cytochromes P450 and their assignment to heme proteins a reactive iron-oxo intermediate as the hydroxylating species has been discussed. It is believed that the electronic structure of this intermediate corresponds to an iron(IV)-porphyrin-π-cation radical system (Compound I). To trap this intermediate the reaction of P450 with oxidants (shunt pathway) has been used. The common approaches are stopped-flow experiments with UV-visible spectroscopic detection or rapid-mixing/freeze-quench studies with EPR and Moessbauer spectroscopic characterization of the trapped intermediate. Surprisingly, the two approaches seem to give conflicting results. While the stopped-flow data indicate the formation of a porphyrin-π-cation radical, no such species is seen by EPR spectroscopy, although the Moessbauer data indicate iron(IV) for P450cam (CYP101) and P450BMP (CYP102). Instead, radicals on tyrosine and tryptophan residues are observed. These findings are reviewed and discussed with respect to intramolecular electron transfer from aromatic amino acids to a presumably transiently formed porphyrin-π-cation radical

  3. Moessbauer and EPR Study of Reaction Intermediates of Cytochrome P450

    International Nuclear Information System (INIS)

    We present a complementary Moessbauer and EPR study on reaction intermediates of substrate-free and substrate-bound cytochrome P450cam from Pseudomonas putida prepared by the freeze-quench method from 57Fe-labeled P450cam using peroxy acetic acid as oxidizing agent. When reacting the substrate-free P450cam for 8 ms reaction time the reaction mixture consists of ∼85% of ferric low-spin iron (Fe(III)) with g-factors and hyperfine parameters of the starting material; the remaining ∼15% are identified as ferryl iron (Fe(IV); SFe=1) by its Moessbauer signature. Parallel to the ferryl iron a tyrosine radical (Srad=1/2) is formed. The two paramagnetic species are not exchange-coupled; however, they are close enough to significantly influence the (EPR) relaxation behavior of the radical spin. In the case of substrate-bound P450cam only trace amounts of the tyrosine radical are formed within 8 ms (<3%); within the accuracy of Moessbauer spectroscopy (5%) iron(IV) can not be detected. The results point to Tyr-96, which is hydrogen-bonded to the substrate camphor, as the candidate for the observed tyrosine radical.

  4. The effects of selected flavonoids on cytochromes P450 in rat liver and small intestine

    Czech Academy of Sciences Publication Activity Database

    Křížková, J.; Burdová, K.; Stiborová, M.; Křen, Vladimír; Hodek, P.

    2009-01-01

    Roč. 2, č. 3 (2009), s. 201-204. ISSN 1337-6853 R&D Projects: GA ČR GD305/09/H008 Institutional research plan: CEZ:AV0Z50200510 Keywords : flavonoids * cytochrome p450 * small intestine Subject RIV: EE - Microbiology, Virology

  5. Fusion of ferredoxin and cytochrome P450 enables direct light-driven biosynthesis

    DEFF Research Database (Denmark)

    Mellor, Silas Busck; Nielsen, Agnieszka Janina Zygadlo; Burow, Meike;

    2016-01-01

    ferredoxin with P450 CYP79A1 from the model plant Sorghum bicolor, which catalyzes the initial step in the pathway leading to biosynthesis of the cyanogenic glucoside dhurrin. Fusion with ferredoxin allows CYP79A1 to obtain electrons for catalysis by interacting directly with photosystem I. Furthermore...

  6. Consequences of Vitamin D and xenobiotic metabolism by cytochrome P450 in HIV infection

    NARCIS (Netherlands)

    Bout-van den Beukel, C. van den

    2009-01-01

    Antiretroviral drugs (ARVs) and medicinal herbs as well as vitamin D are metabolized by the cytochrome P450 enzyme system in the liver. This thesis focuses on the interaction between these compounds. We also explored the hypothesis that HIV-patients might develop insufficiënt vitamin D levels as a r

  7. Ecologically appropriate xenobiotics induce cytochrome P450s in Apis mellifera.

    Directory of Open Access Journals (Sweden)

    Reed M Johnson

    Full Text Available BACKGROUND: Honey bees are exposed to phytochemicals through the nectar, pollen and propolis consumed to sustain the colony. They may also encounter mycotoxins produced by Aspergillus fungi infesting pollen in beebread. Moreover, bees are exposed to agricultural pesticides, particularly in-hive acaricides used against the parasite Varroa destructor. They cope with these and other xenobiotics primarily through enzymatic detoxificative processes, but the regulation of detoxificative enzymes in honey bees remains largely unexplored. METHODOLOGY/PRINCIPAL FINDINGS: We used several approaches to ascertain effects of dietary toxins on bee susceptibility to synthetic and natural xenobiotics, including the acaricide tau-fluvalinate, the agricultural pesticide imidacloprid, and the naturally occurring mycotoxin aflatoxin. We administered potential inducers of cytochrome P450 enzymes, the principal biochemical system for Phase 1 detoxification in insects, to investigate how detoxification is regulated. The drug phenobarbital induces P450s in many insects, yet feeding bees with phenobarbital had no effect on the toxicity of tau-fluvalinate, a pesticide known to be detoxified by bee P450s. Similarly, no P450 induction, as measured by tau-fluvalinate tolerance, occurred in bees fed xanthotoxin, salicylic acid, or indole-3-carbinol, all of which induce P450s in other insects. Only quercetin, a common pollen and honey constituent, reduced tau-fluvalinate toxicity. In microarray comparisons no change in detoxificative gene expression was detected in phenobarbital-treated bees. However, northern blot analyses of guts of bees fed extracts of honey, pollen and propolis showed elevated expression of three CYP6AS P450 genes. Diet did not influence tau-fluvalinate or imidacloprid toxicity in bioassays; however, aflatoxin toxicity was higher in bees consuming sucrose or high-fructose corn syrup than in bees consuming honey. CONCLUSIONS/SIGNIFICANCE: These results

  8. Oxidase uncoupling in heme monooxygenases: Human cytochrome P450 CYP3A4 in Nanodiscs

    Energy Technology Data Exchange (ETDEWEB)

    Grinkova, Yelena V.; Denisov, Ilia G.; McLean, Mark A. [Departments of Biochemistry and Chemistry, University of Illinois, 505 South Goodwin Avenue (United States); Sligar, Stephen G., E-mail: s-sligar@illinois.edu [Departments of Biochemistry and Chemistry, University of Illinois, 505 South Goodwin Avenue (United States)

    2013-01-25

    Highlights: ► Substantial reducing equivalents are lost in human P450 CYP3A4 via an oxidase channel. ► Substrate binding has a pronounced effect on uncoupling in cytochrome P450. ► Anionic phospholipids improve the overall coupling in CYP3A4 Nanodiscs. -- Abstract: The normal reaction mechanism of cytochrome P450 operates by utilizing two reducing equivalents to reduce atmospheric dioxygen, producing one molecule of water and an oxygenated product in an overall stoichiometry of 2 electrons:1 dioxygen:1 product. However, three alternate unproductive pathways exist where the intermediate iron–oxygen states in the catalytic cycle can yield reduced oxygen products without substrate metabolism. The first involves release of superoxide from the oxygenated intermediate while the second occurs after input of the second reducing equivalent. Superoxide rapidly dismutates and hence both processes produce hydrogen peroxide that can be cytotoxic to the organism. In both cases, the formation of hydrogen peroxide involves the same overall stoichiometry as oxygenases catalysis. The key step in the catalytic cycle of cytochrome P450 involves scission of the oxygen–oxygen bond of atmospheric dioxygen to produce a higher valent iron-oxo state termed “Compound I”. This intermediate initiates a radical reaction in the oxygenase pathway but also can uptake two additional reducing equivalents from reduced pyridine nucleotide (NADPH) and the flavoprotein reductase to produce a second molecule of water. This non-productive decay of Compound I thus yields an overall oxygen to NADPH ratio of 1:2 and does not produce hydrocarbon oxidation. This water uncoupling reaction provides one of a limited means to study the reactivity of the critical Compound I intermediate in P450 catalysis. We measured simultaneously the rates of NADPH and oxygen consumption as a function of substrate concentration during the steady-state hydroxylation of testosterone catalyzed by human P450 CYP3A4

  9. Oxidase uncoupling in heme monooxygenases: Human cytochrome P450 CYP3A4 in Nanodiscs

    International Nuclear Information System (INIS)

    Highlights: ► Substantial reducing equivalents are lost in human P450 CYP3A4 via an oxidase channel. ► Substrate binding has a pronounced effect on uncoupling in cytochrome P450. ► Anionic phospholipids improve the overall coupling in CYP3A4 Nanodiscs. -- Abstract: The normal reaction mechanism of cytochrome P450 operates by utilizing two reducing equivalents to reduce atmospheric dioxygen, producing one molecule of water and an oxygenated product in an overall stoichiometry of 2 electrons:1 dioxygen:1 product. However, three alternate unproductive pathways exist where the intermediate iron–oxygen states in the catalytic cycle can yield reduced oxygen products without substrate metabolism. The first involves release of superoxide from the oxygenated intermediate while the second occurs after input of the second reducing equivalent. Superoxide rapidly dismutates and hence both processes produce hydrogen peroxide that can be cytotoxic to the organism. In both cases, the formation of hydrogen peroxide involves the same overall stoichiometry as oxygenases catalysis. The key step in the catalytic cycle of cytochrome P450 involves scission of the oxygen–oxygen bond of atmospheric dioxygen to produce a higher valent iron-oxo state termed “Compound I”. This intermediate initiates a radical reaction in the oxygenase pathway but also can uptake two additional reducing equivalents from reduced pyridine nucleotide (NADPH) and the flavoprotein reductase to produce a second molecule of water. This non-productive decay of Compound I thus yields an overall oxygen to NADPH ratio of 1:2 and does not produce hydrocarbon oxidation. This water uncoupling reaction provides one of a limited means to study the reactivity of the critical Compound I intermediate in P450 catalysis. We measured simultaneously the rates of NADPH and oxygen consumption as a function of substrate concentration during the steady-state hydroxylation of testosterone catalyzed by human P450 CYP3A4

  10. An open conformation of mammalian cytochrome P450 2B4 at 1.6-Å resolution

    OpenAIRE

    Scott, Emily E.; He, You Ai; Wester, Michael R.; White, Mark A.; Chin, Christopher C.; Halpert, James R.; Johnson, Eric F.; Stout, C. David

    2003-01-01

    The xenobiotic metabolizing cytochromes P450 (P450s) are among the most versatile biological catalysts known, but knowledge of the structural basis for their broad substrate specificity has been limited. P450 2B4 has been frequently used as an experimental model for biochemical and biophysical studies of these membrane proteins. A 1.6-Å crystal structure of P450 2B4 reveals a large open cleft that extends from the protein surface directly to the heme iron between the α-helical and β-sheet dom...

  11. Five of 12 forms of vaccinia virus-expressed human hepatic cytochrome P450 metabolically activate aflatoxin B1

    International Nuclear Information System (INIS)

    Twelve forms of human hepatic cytochrome P450 were expressed in hepatoma cells by means of recombinant vaccinia viruses. The expressed P450s were analyzed for their abilities to activate the potent hepatocarcinogen aflatoxin B1 to metabolites having mutagenic or DNA-binding properties. Five forms, P450s IA2, IIA3, IIB7, IIIA3, and IIIA4, activated aflatoxin B1 to mutagenic metabolites as assessed by the production of His revertants of Salmonella typhimurium in the Ames test. The same P450s catalyzed conversion of aflatoxin B1 to DNA-bound derivatives as judged by an in situ assay in which the radiolabeled carcinogen was incubated with cells expressing the individual P450 forms. Seven other human P450s, IIC8, IIC9, IID6, IIE1, IIF1, and IIIA5, and IVB1, did not significantly activate aflatoxin B1 as measured by both the Ames test and the DNA-binding assay. Moreover, polyclonal anti-rat liver P450 antibodies that crossreact with individual human P450s IA2, IIA3, IIIA3, and IIIA4 each inhibited aflatoxin B1 activation catalyzed by human liver S-9 extracts. Inhibition ranged from as low as 10% with antibody against IIA3 to as high as 65% with antibody against IIIA3 and IIIA4. These results establish that metabolic activation of aflatoxin B1 in human liver involves the contribution of multiple forms of P450

  12. CYTOCHROME P450 REGULATION: THE INTERPLAY BETWEEN ITS HEME AND APOPROTEIN MOIETIES IN SYNTHESIS, ASSEMBLY, REPAIR AND DISPOSAL123

    OpenAIRE

    Correia, Maria Almira; Sinclair, Peter R.; De Matteis, Francesco

    2010-01-01

    Heme is vital to our aerobic universe. Heme cellular content is finely tuned through an exquisite control of synthesis and degradation. Heme deficiency is deleterious to cells, whereas excess heme is toxic. Most of the cellular heme serves as the prosthetic moiety of functionally diverse hemoproteins, including cytochromes P450 (P450s). In the liver, P450s are its major consumers with >50% of hepatic heme committed to their synthesis. Prosthetic heme is the sine qua non of P450 catalytic biot...

  13. Challenges and pitfalls of P450-dependent (+)-valencene bioconversion by Saccharomyces cerevisiae.

    Science.gov (United States)

    Gavira, Carole; Höfer, René; Lesot, Agnès; Lambert, Fanny; Zucca, Joseph; Werck-Reichhart, Danièle

    2013-07-01

    Natural nootkatone is a high value ingredient for the flavor and fragrance industry because of its grapefruit flavor/odor, low sensorial threshold and low availability. Valencene conversion into nootkatol and nootkatone is known to be catalyzed by cytochrome P450 enzymes from both prokaryotic and eukaryotic organisms, but so far development of a viable bioconversion process using either native microorganisms or recombinant enzymes was not successful. Using an in silico gene-mining approach, we selected 4 potential candidate P450 enzymes from higher plants and identified two of them that selectively converted (+)-valencene into β-nootkatol with high efficiency when tested using recombinant yeast microsomes in vitro. Recombinant yeast expressing CYP71D51v2 from tobacco and a P450 reductase from arabidopsis was used for optimization of a bioconversion process. Bioconversion assays led to production of β-nootkatol and nootkatone, but with low yields that decreased upon increase of the substrate concentration. The reasons for this low bioconversion efficiency were further investigated and several factors potentially hampering industry-compatible valencene bioconversion were identified. One is the toxicity of the products for yeast at concentrations exceeding 100 mg L⁻¹. The second is the accumulation of β-nootkatol in yeast endomembranes. The third is the inhibition of the CYP71D51v2 hydroxylation reaction by the products. Furthermore, we observed that the formation of nootkatone from β-nootkatol is not P450-dependent but catalyzed by a yeast component. Based on these data, we propose new strategies for implementation of a viable P450-based bioconversion process. PMID:23518241

  14. Alcoolismo e violência doméstica : investigação de variantes genéticas em genes da família do citocromo P450

    OpenAIRE

    Marques, Andreia Torres da Silva

    2010-01-01

    O alcoolismo é um grave problema de saúde pública global, com implicações a nível social e económico. O consumo excessivo de álcool está associado à violência doméstica, que à semelhança do alcoolismo representa um problema social e de saúde pública igualmente preocupante na actualidade. A dependência alcóolica é uma doença complexa e multifactorial, que resulta de interacções gene-gene e gene-ambiente, com uma hereditariedade estimada entre 40- 60%. A identificação de facto...

  15. Relationships among Ergot Alkaloids, Cytochrome P450 Activity, and Beef Steer Growth

    Directory of Open Access Journals (Sweden)

    Charles F. Rosenkrans

    2015-03-01

    Full Text Available Determining a grazing animal’s susceptibility to ergot alkaloids has been a research topic for decades. Our objective was to determine if the Promega™ P450-Glo assay could be used to indirectly detect ergot alkaloids or their metabolites in urine of steers. The first experiment validated the effects of ergot alkaloids [0, 20, and 40 μM of ergotamine (ET, dihydroergotamine (DHET, and ergonovine (EN] on human CYP3A4 using the P450-Glo assay (Promega™ V9800. With this assay, luminescence is directly proportional to CYP450 activity. Relative inhibition of in vitro cytochrome P450 activity was affected (P < 0.001 by an interaction between alkaloids and concentration. That interaction resulted in no concentration effect of EN, but within ET and DHET 20 and 40 µM concentrations inhibited CYP450 activity when compared with controls. In experiment 2, urine was collected from Angus-sired crossbred steers (n = 39; 216 ± 2.6 d of age; 203 ± 1.7 kg after grazing tall fescue pastures for 105 d. Non-diluted urine was added to the Promega™ P450-Glo assay, and observed inhibition (3.7 % ± 2.7 of control. Urine content of total ergot alkaloids (331.1 ng/mg of creatinine ± 325.7 was determined using enzyme linked immunosorbent assay. Urine inhibition of CYP450 activity and total alkaloids were correlated (r = -0.31; P < 0.05. Steers were genotyped at CYP450 single nucleotide polymorphism, C994G. Steer genotype affected (P < 0.03 inhibition of CYP450 activity by urine; heterozygous steers had the least amount of CYP450 inhibition suggesting that genotyping cattle may be a method of identifying animals that are susceptible to ergot alkaloids. Although, additional research is needed, we demonstrate that the Promega™ P450-Glo assay is sensitive to ergot alkaloids and urine from steers grazing tall fescue. With some refinement the P450-Glo assay has potential as a tool for screening cattle for their exposure to fescue toxins.

  16. Application of Osmotic Pumps for Sustained Release of 1-Aminobenzotriazole and Inhibition of Cytochrome P450 Enzymes in Mice: Model Comparison with the Hepatic P450 Reductase Null Mouse.

    Science.gov (United States)

    Stringer, Rowan A; Ferreira, Suzie; Rose, Jonathan; Ronseaux, Sebastien

    2016-08-01

    The effectiveness of controlled release 1-aminobenzotriazole (ABT) administration to inhibit cytochrome P450 (P450) enzymes has been evaluated in mice. To maximize the duration of P450 inhibition in vivo, ABT was administered via an osmotic pump. The degree of P450 inhibition was compared with that achieved with a single bolus dose of ABT. Two-hour prior subcutaneous treatment of mice with ABT (50 mg/kg) inhibited antipyrine clearance by 88%. A less pronounced inhibitory effect (29% reduction in clearance) was observed when ABT was administered 24-hours before antipyrine administration, indicating partial restoration of P450 activity during this longer pretreatment time. The duration of ABT in mice was very short (mean residence time = 1.7 hours) after subcutaneous bolus administration. When the inhibitor was delivered by an osmotic pump, maximum blood concentrations of the inhibitor were observed 24 hours after device implantation and were maintained at steady state for 6 days. Inhibition of P450 activity, as measured by antipyrine clearance, was confirmed at 24 hours and 120 hours after pump implantation, highlighting the utility of this method as a longer-term model for P450 inhibition in mice. The magnitude of P450 inhibition in ABT-treated mice was compared with that in hepatic P450 reductase null mice and both models were comparable. In vivo ABT administration by an osmotic pump offers an effective approach for longer-term P450 inhibition in mice and avoids the necessity for multiple dosing of the inhibitor. PMID:27271368

  17. Cytochrome P450-mediated metabolism of tumour promoters modifies the inhibition of intercellular communication: a modified assay for tumour promotion

    DEFF Research Database (Denmark)

    Vang, Ole; Wallin, H.; Doehmer, J.; Autrup, Herman

    1993-01-01

    carcinogens, ochratoxin A, aflatoxin B1 and 4-nitrobiphenyl, did not inhibit metabolic cooperation in either V79 cells expressing or cells not expressing cytochrome P450. We conclude that cytochrome P450-associated metabolism plays an important role in the inhibition of gap junctional intercellular...

  18. A chicory cytochrome P450 mono-oxygenase CYP71AV8 for the oxidation of (+)-valencene

    NARCIS (Netherlands)

    Cankar, K.; Houwelingen, van A.M.M.L.; Bosch, H.J.; Sonke, Th.; Bouwmeester, H.J.; Beekwilder, M.J.

    2011-01-01

    Chicory (Cichorium intybus L.), which is known to have a variety of terpene-hydroxylating activities, was screened for a P450 mono-oxygenase to convert (+)-valencene to (+)-nootkatone. A novel P450 cDNA was identified in a chicory root EST library. Co-expression of the enzyme with a valencene syntha

  19. Biosynthesis of a steroid metabolite by an engineered Rhodococcus erythropolis strain expressing a mutant cytochrome P450 BM3 enzyme

    NARCIS (Netherlands)

    Venkataraman, Harini; Te Poele, Evelien M; Rosłoniec, Kamila Z; Vermeulen, Nico; Commandeur, Jan N M; van der Geize, Robert; Dijkhuizen, Lubbert

    2014-01-01

    In the present study, the use of Rhodococcus erythropolis mutant strain RG9 expressing the cytochrome P450 BM3 mutant M02 enzyme has been evaluated for whole-cell biotransformation of a 17-ketosteroid, norandrostenedione, as a model substrate. Purified P450 BM3 mutant M02 enzyme hydroxylated the ste

  20. Significantly shorter Fe-S bond in cytochrome P450-I is consistent with greater reactivity relative to chloroperoxidase

    Science.gov (United States)

    Krest, Courtney M.; Silakov, Alexey; Rittle, Jonathan; Yosca, Timothy H.; Onderko, Elizabeth L.; Calixto, Julio C.; Green, Michael T.

    2015-09-01

    Cytochrome P450 (P450) and chloroperoxidase (CPO) are thiolate-ligated haem proteins that catalyse the activation of carbon hydrogen bonds. The principal intermediate in these reactions is a ferryl radical species called compound I. P450 compound I (P450-I) is significantly more reactive than CPO-I, which only cleaves activated C-H bonds. To provide insight into the differing reactivities of these intermediates, we examined CPO-I and P450-I using variable-temperature Mössbauer and X-ray absorption spectroscopies. These measurements indicate that the Fe-S bond is significantly shorter in P450-I than in CPO-I. This difference in Fe-S bond lengths can be understood in terms of variations in the hydrogen-bonding patterns within the ‘cys-pocket’ (a portion of the proximal helix that encircles the thiolate ligand). Weaker hydrogen bonding in P450-I results in a shorter Fe-S bond, which enables greater electron donation from the axial thiolate ligand. This observation may in part explain P450's greater propensity for C-H bond activation.

  1. Aberrant splicing and missense mutations cause steroid 21-hydroxylase [P-450(C21)] deficiency in humans: Possible gene conversion products

    International Nuclear Information System (INIS)

    Four steroid 21-hydroxylase B [P-450(C21)B] genes (designated P.7, P.10-1, P.10-2, and P.3) from three P-450(C21)-deficient patients were isolated to analyze their structures and functions. Several base changes were observed in the sequences of the four P-450(C21)B genes as compared to that of the functional B gene. Many of these base changes were identical to those of the P-450(C21)A pseudogene. The three DNAs (P.10-1, P.10.2, and P.3) produced no P-450(C21) activity in a functional assay for P-450(C21) by the COS cell expression system, while the P.7 DNA expressed the activity. The P.10-1 and P.10-2 DNAs were shown to have a point mutation in the second intron, causing aberrant splicing. The P.3 DNA carried three clustered missense mutations in the sixty exon, which impaired P-450(C21) activity. All these critical mutations could be seen in the corresponding site of the P-450(C21)A pseudogene. These data strongly suggest the involvement of gene conversion in this genetic disease

  2. Decreased expression of cytochrome P450 protein in non-malignant colonic tissue of patients with colonic adenoma

    DEFF Research Database (Denmark)

    Bergheim, I.; Bode, C.; Parlesak, Alexandr

    2005-01-01

    BACKGROUND: Cytochrome P450 (CYP) enzymes in epithelial cells lining the alimentary tract play an important role in both the elimination and activation of (pro-)carcinogens. To estimate the role of cytochrome P450 in carcinogenesis of the colon, expression patterns and protein levels of four repr...

  3. Optical probe for the cytochrome P-450 cholesterol side chain cleavage enzyme

    Science.gov (United States)

    Marrone, Babetta L.; Simpson, Daniel J.; Unkefer, Clifford J.; Whaley, Thomas W.

    1992-01-01

    An optical probe enables the study of enzyme activity by absorbance spectroscopy or by sensitive fluorescence methods. In particular, the probe provides the ability to monitor the activity of cytochrome P-450.sub.scc enzyme, the rate limiting enzyme for steroid biosynthesis. Located on the inner mitochondrial membrane, P-450.sub.scc catalyzes the conversion of cholesterol to pregnenolone and isocapraldehyde by sequential oxidations of the cholesterol side chain. The fluorogenic probe includes a cholesterol-like steroid linked to a chromophore through a linking group. The chromophore is selected to have little optical response when linked to the steroid substrate and an enhanced optical response when cleaved from the substrate and linking group. Thus, a fluorescent anion that can be optically detected is generated by the side-chain cleavage reaction during steroidogenesis.

  4. Nitrogen inversion barriers affect the N-oxidation of tertiary alkylamines by cytochromes P450

    DEFF Research Database (Denmark)

    Rydberg, Patrik; Jørgensen, Martin S.; Jacobsen, T.A.;

    2013-01-01

    Calculations: Cytochrome P450 enzymes facilitate a number of chemically different reactions. For example, amines can be either N-dealkylated or N-oxidized, but it is complex to rationalize which of these competing reactions occurs. It is shown that the barrier for inversion of the alkylamine nitr...... nitrogen atom seems to be of vital importance for the amount of N-oxidized product formed relative to dealkylation and hydroxylation products. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.......Calculations: Cytochrome P450 enzymes facilitate a number of chemically different reactions. For example, amines can be either N-dealkylated or N-oxidized, but it is complex to rationalize which of these competing reactions occurs. It is shown that the barrier for inversion of the alkylamine...

  5. Alterations of cytochrome P450-dependent monooxygenase activities in Eriocheir japonicus in response to water pollution.

    OpenAIRE

    Ishizuka, M; Hoshi, H.; Minamoto, N; Masuda, M; Kazusaka, A; Fujita, S.

    1996-01-01

    Eriocheir japonicus, fresh-water crabs inhabiting rivers and estuaries in Japan, were investigated for cytochrome P450 (CYP)-dependent drug-metabolizing enzyme activities to see if these activities reflect the river pollution gradient. From the laboratory dose-response experiments, we found that the polycyclic aromatic hydrocarbon (PAH) 3-methylcholanthrene induced total CYP contents, ethoxycoumarin O-deethylase activity, and bunitrolol 4-hydroxylase activity in crab hepatopancreas. In the fi...

  6. P-Link: A method for generating multicomponent cytochrome P450 fusions with variable linker length

    DEFF Research Database (Denmark)

    Belsare, Ketaki D.; Ruff, Anna Joelle; Martinez, Ronny;

    2014-01-01

    Fusion protein construction is a widely employed biochemical technique, especially when it comes to multi-component enzymes such as cytochrome P450s. Here we describe a novel method for generating fusion proteins with variable linker lengths, protein fusion with variable linker insertion (P...... but also requires only a single cloning and transformation step in order to generate multiple linker variants (1 to 16 amino acids long), making the approach technically simple and robust....

  7. Arachidonic Acid-metabolizing Cytochrome P450 Enzymes Are Targets of ω-3 Fatty Acids*

    OpenAIRE

    Arnold, Cosima; Markovic, Marija; Blossey, Katrin; Wallukat, Gerd; Fischer, Robert; Dechend, Ralf; Konkel, Anne; von Schacky, Clemens; Luft, Friedrich C.; Muller, Dominik N.; Rothe, Michael; Schunck, Wolf-Hagen

    2010-01-01

    Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) protect against cardiovascular disease by largely unknown mechanisms. We tested the hypothesis that EPA and DHA may compete with arachidonic acid (AA) for the conversion by cytochrome P450 (CYP) enzymes, resulting in the formation of alternative, physiologically active, metabolites. Renal and hepatic microsomes, as well as various CYP isoforms, displayed equal or elevated activities when metabolizing EPA or DHA instead of AA. CYP2C/2J...

  8. Human bone marrow niche chemoprotection mediated by cytochrome p450 enzymes

    OpenAIRE

    Alonso, Salvador; Su, Meng; Jones, Jace W.; Ganguly, Sudipto; Kane, Maureen A.; Jones, Richard J; Ghiaur, Gabriel

    2015-01-01

    Substantial evidence now demonstrates that interactions between the tumor microenvironment and malignant cells are a critical component of clinical drug resistance. However, the mechanisms responsible for microenvironment-mediated chemoprotection remain unclear. We showed that bone marrow (BM) stromal cytochrome P450 (CYP)26 enzymes protect normal hematopoietic stem cells (HSCs) from the pro-differentiation effects of retinoic acid. Here, we investigated if stromal expression of CYPs is a gen...

  9. Contribution of human cytochrome P-450 isoforms to the metabolism of the simplest phenothiazine neuroleptic promazine

    OpenAIRE

    Wójcikowski, Jacek; Pichard-Garcia, Lydiane; Maurel, Patrick; Daniel, Władysława A

    2003-01-01

    The aim of the present study was to identify human cytochrome P-450 isoforms (CYPs) involved in 5-sulphoxidation and N-demethylation of the simplest phenothiazine neuroleptic promazine in human liver.The experiments were performed in the following in vitro models: (A) a study of promazine metabolism in liver microsomes—(a) correlations between the rate of promazine metabolism and the level and activity of CYPs; (b) the effect of specific inhibitors on the rate of promazine metabolism (inhibit...

  10. Interpulse interval in circulating growth hormone patterns regulates sexually dimorphic expression of hepatic cytochrome P450.

    OpenAIRE

    Waxman, D. J.; Pampori, N A; Ram, P A; Agrawal, A. K.; Shapiro, B. H.

    1991-01-01

    Plasma growth hormone (GH) profiles are sexually differentiated in many species and regulate the sex-dependence of peripubescent growth rates and liver function, including steroid hydroxylase cytochrome P450 expression, by mechanisms that are poorly understood. By use of an external pump to deliver to hypophysectomized rats pulses of rat GH of varying frequency and amplitude, a critical element for liver discrimination between male and female GH patterns was identified. Liver expression of th...

  11. Low dose trichloroethylene alters cytochrome P450 - 2C subfamily expression in the developing chick heart

    OpenAIRE

    Makwana, Om; Ahles, Lauren; Lencinas, Alejandro; Selmin, Ornella I.; Runyan, Raymond B.

    2013-01-01

    Trichloroethylene (TCE) is an organic solvent and common environmental contaminant. TCE exposure is associated with heart defects in humans and animal models. Primary metabolism of TCE in adult rodent models is by specific hepatic cytochrome P450 enzymes (Lash et al., 2000). As association of TCE exposure with cardiac defects is in exposed embryos prior to normal liver development, we investigated metabolism of TCE in the early embryo. Developing chick embryos were dosed in ovo with environme...

  12. Reaction of Mycobacterium tuberculosis Cytochrome P450 Enzymes with Nitric Oxide†

    OpenAIRE

    Ouellet, Hugues; Lang, Jérôme; Couture, Manon; Ortiz de Montellano, Paul R.

    2009-01-01

    During the initial growth infection stage of Mycobacterium tuberculosis (Mtb), •NO produced by host macrophages inhibits heme-containing terminal cytochrome oxidases, inactivates iron/sulfur proteins and promotes entry into latency. Here we evaluate the potential of •NO as an inhibitor of Mtb cytochrome P450 enzymes, as represented by CYP130, CYP51 and the two previously uncharacterized enzymes CYP125 and CYP142. Using UV-visible absorption, resonance Raman, and stopped-flow spectroscopy, we ...

  13. Formation of Azole-Resistant Candida albicans by Mutation of Sterol 14-Demethylase P450

    OpenAIRE

    Asai, Kentaro; Tsuchimori, Noboru; Okonogi, Kenji; Perfect, John R.; Gotoh, Osamu; Yoshida, Yuzo

    1999-01-01

    The sterol 14-demethylase P450 (CYP51) of a fluconazole-resistant isolate of Candida albicans, DUMC136, showed reduced susceptibility to this azole but with little change in its catalytic activity. Twelve nucleotide substitutions, resulting in four amino acid changes, were identified in the DUMC136 CYP51 gene in comparison with a reported CYP51 sequence from a wild-type, fluconazole-susceptible C. albicans strain. Seven of these substitutions, including all of those causing amino acid changes...

  14. CYP51: A Major Drug Target in the Cytochrome P450 Superfamily

    OpenAIRE

    Lepesheva, Galina I.; Hargrove, Tatyana Y.; Kleshchenko, Yuliya; Nes, W. David; Villalta, Fernando; Waterman, Michael R.

    2008-01-01

    The cytochrome P540 (CYP) superfamily currently includes about 9,000 proteins forming more than 800 families. The enzymes catalyze monooxygenation of a vast array of compounds and play essentially two roles. They provide biodefense (detoxification of xenobiotics, antibiotic production) and participate in biosynthesis of important endogenous molecules, particularly steroids. Based on these two roles, sterol 14|*alpha*|-demethylases (CYP51) belong to the second group of P450s. The CYP51 family,...

  15. Cytochrome P450 1A1 expression in cetacean skin biopsies from the Indian Ocean

    OpenAIRE

    Jauniaux, Thierry; Farnir, Frédéric; Fontaine, Michael; Kiszka, Jeremy; Sarlet, Michaël; Coignoul, Freddy

    2011-01-01

    The study describes cytochrome P450 1A1 (CYPA1) expression in the skin of different cetacean species (Megaptera novaeangliae, n = 15; Stenella attenuata, n = 7 and Stenella longirostris, n = 24) from the Mozambique Channel island of Mayotte. Immunohistochemical examination was performed with a monoclonal antibody against scup cytochrome CYPA1. The sex was determined using a molecular approach consisting in the genotyping sex-specific genes. CYPA1 was detected at the junction between epidermis...

  16. Prediction and analysis of the modular structure of cytochrome P450 monooxygenases

    OpenAIRE

    Wagner Florian; Widmann Michael; Sirim Demet; Pleiss Jürgen

    2010-01-01

    Abstract Background Cytochrome P450 monooxygenases (CYPs) form a vast and diverse family of highly variable sequences. They catalyze a wide variety of oxidative reactions and are therefore of great relevance in drug development and biotechnological applications. Despite their differences in sequence and substrate specificity, the structures of CYPs are highly similar. Although being in research focus for years, factors mediating selectivity and activity remain vague. Description This systemat...

  17. A novel role of Drosophila cytochrome P450-4e3 in permethrin insecticide tolerance

    DEFF Research Database (Denmark)

    Terhzaz, Selim; Cabrero, Pablo; Brinzer, Robert A; Halberg, Kenneth A; Dow, Julian A T; Davies, Shireen-A

    2015-01-01

    The exposure of insects to xenobiotics, such as insecticides, triggers a complex defence response necessary for survival. This response includes the induction of genes that encode key Cytochrome P450 monooxygenase detoxification enzymes. Drosophila melanogaster Malpighian (renal) tubules are crit...... under ER stress. This work increases our understanding of the molecular mechanisms of insecticide detoxification and provides further evidence of the oxidative stress responses induced by permethrin metabolism....

  18. Predictive Models for Cytochrome P450 Isozymes Based on Quantitative High Throughput Screening Data

    OpenAIRE

    Sun, Hongmao; Veith, Henrike; Xia, Menghang; Austin, Christopher P.; Huang, Ruili

    2011-01-01

    The human cytochrome P450 (CYP450) isozymes are the most important enzymes in the body to metabolize many endogenous and exogenous substances including environmental toxins and therapeutic drugs. Any unnecessary interactions between a small molecule and CYP450 isozymes may raise a potential to disarm the integrity of the protection. Accurately predicting the potential interactions between a small molecule and CYP450 isozymes is highly desirable for assessing the metabolic stability and toxici...

  19. Optimization of a cytochrome P450 oxidation system for enhancing protopanaxadiol production in Saccharomyces cerevisiae.

    Science.gov (United States)

    Zhao, Fanglong; Bai, Peng; Liu, Ting; Li, Dashuai; Zhang, Xiangmei; Lu, Wenyu; Yuan, Yingjin

    2016-08-01

    Ginsenosides, the major bioactive components of Panax ginseng, are regarded as promising high-value pharmaceutical compounds. In ginseng, ginsenosides are produced from their precursor protopanaxadiol. Recently, an artificial biosynthetic pathway of protopanaxadiol was built in Saccharomyces cerevisiae by introducing a P. ginseng dammarenediol-II synthase, a P. ginseng cytochrome P450-type protopanaxadiol synthase (PPDS), and a Arabidopsis thaliana NADPH-cytochrome P450 reductase (ATR1). In this engineered yeast strain, however, the low metabolic flux through PPDS resulted in a low productivity of protopanaxadiol. Moreover, health of the yeast cells was significantly affected by reactive oxygen species released by the pool coupling between PPDS and ATR1. To overcome the obstacles in protopanaxadiol production, PPDS was modified through transmembrane domain truncation and self-sufficient PPDS-ATR1 fusion construction in this study. The fusion enzymes conferred approximately 4.5-fold increase in catalytic activity, and 71.1% increase in protopanaxadiol production compared with PPDS and ATR1 co-expression. Our in vivo experiment indicated that the engineered yeast carrying fusion protein effectively converted 96.8% of dammarenediol-II into protopanaxadiol. Protopanaxadiol production in a 5 L bioreactor in fed-batch fermentation reached 1436.6 mg/L. Our study not only improved protopanaxadiol production in yeast, but also provided a generic method to improve activities of plant cytochrome P450 monooxygenases. This method is promising to be applied to other P450 systems in yeast. Biotechnol. Bioeng. 2016;113: 1787-1795. © 2016 Wiley Periodicals, Inc. PMID:26757342

  20. P450-catalyzed asymmetric cyclopropanation of electron-deficient olefins under aerobic conditions.

    Science.gov (United States)

    Renata, Hans; Wang, Z Jane; Kitto, Rebekah Z; Arnold, Frances H

    2014-10-01

    A variant of P450 from Bacillus megaterium five mutations away from wild type is a highly active catalyst for cyclopropanation of a variety of acrylamide and acrylate olefins with ethyl diazoacetate (EDA). The very high rate of reaction enabled by histidine ligation allowed the reaction to be conducted under aerobic conditions. The promiscuity of this catalyst for a variety of substrates containing amides has enabled synthesis of a small library of precursors to levomilnacipran derivatives. PMID:25221671

  1. The scent of royalty: a p450 gene signals reproductive status in a social insect.

    Science.gov (United States)

    Hoffmann, Katharina; Gowin, Johannes; Hartfelder, Klaus; Korb, Judith

    2014-10-01

    Cooperation requires communication; this applies to animals and humans alike. The main communication means differ between taxa and social insects (ants, termites, and some bees and wasps) lack the cognitive abilities of most social vertebrates. Central to the regulation of the reproductive harmony in insect societies is the production of a royalty scent which signals the fertility status of the reproducing queen to the nonreproducing workers. Here, we revealed a central genetic component underlying this hallmark of insect societies in the termite Cryptotermes secundus. Communication between queens and workers relied upon the expression of a gene, Neofem4, which belongs to the cytochrome P450 genes. We inhibited Neofem4 in queens by RNA interference. This resulted in the loss of the royalty scent in queens and the workers behaved as though the queen were absent. The queen's behavior was not generally affected by silencing Neofem4. This suggests that the lack of the royalty scent lead to workers not recognizing her anymore as queen. P450 genes are known to be involved in the production of chemical signals in cockroaches and their expression has been linked to a major fertility regulator, juvenile hormone. This makes P450 genes, both a suitable and available evolutionary substrate in the face of natural selection for production of a queen substance. Our data suggest that in an organism without elaborate cognitive abilities communication has been achieved by the exploitation of a central gene that links the fertility network with the chemical communication pathway. As termites and social Hymenoptera seem to share the same class of compounds in signaling fertility, this role of P450 genes might be more widespread across social insects. PMID:25053804

  2. Molecular Analysis and Heterologous Expression of an Inducible Cytochrome P-450 Protein from Periwinkle (Catharanthus roseus L.) 1

    Science.gov (United States)

    Vetter, Hans-Peter; Mangold, Ursula; Schröder, Gudrun; Marner, Franz-Josef; Werck-Reichhart, Danielle; Schröder, Joachim

    1992-01-01

    We screened cDNA libraries from periwinkle (Catharanthus roseus) cell cultures induced for indole alkaloid synthesis and selected clones for induced cytochrome P-450 (P-450) proteins by differential hybridization, size of the hybridizing mRNA, and presence of amino acid motifs conserved in many P-450 families. Four cDNAs satisfying these criteria were analyzed in detail. They were grouped in two classes (pCros1, pCros2) that represented two closely related genes of a new P-450 family designated CYP72. Antiserum against a cDNA fusion protein overexpressed in Escherichia coli recognized in C. roseus a protein band of 56 kD. Quantification of western blots showed that it represented 1.5 ± 0.5 and 6 ± 1 μg/mg of protein in the membranes from noninduced and induced cells, respectively, and analysis of the total P-450 content suggested that the cDNA-encoded protein was one of the dominant P-450 proteins. The pathway to indole alkaloids contains two known P-450 enzymes, geraniol-10-hydroxylase (GE10H) and nerol-10-hydroxylase (NE10H). The induction kinetics of the cloned P-450 protein and of GE10H activity were similar, but those of NE10H were different. Western blots with membranes from other plants suggested that P-450 CYP72 is specific for C. roseus and other plants with GE10H activity. A tentative assignment of CYP72 as GE10H is discussed. The cDNA was recloned for expression in Saccharomyces cerevisiae, and the presence of the protein was demonstrated by western blots. Assays for GE10H failed to detect enzyme activity, and the same negative result was obtained for NE10H and other P-450 enzymes that are present in C. roseus. Images Figure 5 Figure 7 PMID:16653087

  3. Secondary deuterium isotope effects on olefin epoxidation by cytochrome P-450

    International Nuclear Information System (INIS)

    Secondary deuterium isotope effects have been determined for the epoxidation of p-phenylstyrene (1a) and p-methylstyrene (1b) by cytochrome P-450 of rat liver microsomes. With both substrates there was an inverse isotope effect of 7 per cent/deuterium (i.e. ksub(H)/ksub(D)=0.93) at Cα of the olefin, but no isotope effect was observed at Cβ. The epoxidation of (1a) by m-chloroperbenzoic acid has previously been shown to be accompanied by an inverse secondary isotope effect of 9 per cent/deuterium at Cβ, with no detectable isotope effect at C α. Thus in both the enzymatic (P-450) and non-enzymatic (peracid) epoxidation of styrene derivatives, the oxygen atom is transferred to the vinyl group in an asymmetric non-concerted fashion. However, the fact that the isotope effects for these two systems are reversed, together with previous comparisons of substituent effects on the two reactions, suggests that there is little mechanistic similarity between cytochrome P-450 enzymes and organic peracids as chemical models for these enzymes. (author)

  4. Novel cytochrome P450, cyp6a17, is required for temperature preference behavior in Drosophila.

    Directory of Open Access Journals (Sweden)

    Jongkyun Kang

    Full Text Available Perception of temperature is an important brain function for organisms to survive. Evidence suggests that temperature preference behavior (TPB in Drosophila melanogaster, one of poikilothermal animals, is regulated by cAMP-dependent protein kinase (PKA signaling in mushroom bodies of the brain. However, downstream targets for the PKA signaling in this behavior have not been identified. From a genome-wide search for the genes regulated by PKA activity in the mushroom bodies, we identified the cyp6a17 Cytochrome P450 gene as a new target for PKA. Our detailed analysis of mutants by genetic, molecular and behavioral assays shows that cyp6a17 is essential for temperature preference behavior. cyp6a17 expression is enriched in the mushroom bodies of the adult brain. Tissue-specific knockdown and rescue experiments demonstrate that cyp6a17 is required in the mushroom bodies for normal temperature preference behavior. This is the first study, to our knowledge, to show PKA-dependent expression of a cytochrome P450 gene in the mushroom bodies and its role as a key factor for temperature preference behavior. Taken together, this study reveals a new PKA-Cytochrome P450 pathway that regulates the temperature preference behavior.

  5. Active-Site Hydration and Water Diffusion in Cytochrome P450cam: A Highly Dynamic Process

    Energy Technology Data Exchange (ETDEWEB)

    Miao, Yinglong [ORNL; Baudry, Jerome Y [ORNL

    2011-01-01

    Long-timescale molecular dynamics simulations (300 ns) are performed on both the apo- (i.e., camphor-free) and camphor-bound cytochrome P450cam (CYP101). Water diffusion into and out of the protein active site is observed without biased sampling methods. During the course of the molecular dynamics simulation, an average of 6.4 water molecules is observed in the camphor-binding site of the apo form, compared to zero water molecules in the binding site of the substrate-bound form, in agreement with the number of water molecules observed in crystal structures of the same species. However, as many as 12 water molecules can be present at a given time in the camphor-binding region of the active site in the case of apo-P450cam, revealing a highly dynamic process for hydration of the protein active site, with water molecules exchanging rapidly with the bulk solvent. Water molecules are also found to exchange locations frequently inside the active site, preferentially clustering in regions surrounding the water molecules observed in the crystal structure. Potential-of-mean-force calculations identify thermodynamically favored trans-protein pathways for the diffusion of water molecules between the protein active site and the bulk solvent. Binding of camphor in the active site modifies the free-energy landscape of P450cam channels toward favoring the diffusion of water molecules out of the protein active site.

  6. The P450 superfamily: update on new sequences, gene mapping, and recommended nomenclature.

    Science.gov (United States)

    Nebert, D W; Nelson, D R; Coon, M J; Estabrook, R W; Feyereisen, R; Fujii-Kuriyama, Y; Gonzalez, F J; Guengerich, F P; Gunsalus, I C; Johnson, E F

    1991-01-01

    We provide here a list of 154 P450 genes and seven putative pseudogenes that have been characterized as of October 20, 1990. These genes have been described in a total of 23 eukaryotes (including nine mammalian and one plant species) and six prokaryotes. Of 27 gene families so far described, 10 exist in all mammals. These 10 families comprise 18 subfamilies, of which 16 and 14 have been mapped in the human and mouse genomes, respectively; to date, each subfamily appears to represent a cluster of tightly linked genes. We propose here a modest revision of the initially proposed (Nebert et al., DNA 6, 1-11, 1987) and updated (Nebert et al., DNA 8, 1-13, 1989) nomenclature system based on evolution of the superfamily. For the gene we recommend that the italicized root symbol CYP for human (Cyp for mouse), representing cytochrome P450, be followed by an Arabic number denoting the family, a letter designating the subfamily (when two or more exist), and an Arabic numeral representing the individual gene within the subfamily. A hyphen should precede the final number in mouse genes. We suggest that the human nomenclature system be used for other species. This system is consistent with our earlier proposed nomenclature for P450 of all eukaryotes and prokaryotes, except that we are discouraging the future use of cumbersome Roman numerals. PMID:1991046

  7. 细胞色素P450酶系介导的除草剂抗性研究进展%Research Progress on Herbicide Resistance of Cytochrome P450-mediated

    Institute of Scientific and Technical Information of China (English)

    刘小民; 梁双波; 王贵启; 樊翠芹; 赵青松

    2012-01-01

    细胞色素P450酶系在生物界中广泛分布,并且是大多数除草剂代谢第一阶段的重要酶系,对除草剂抗性的形成发挥着重要作用。结合国内外细胞色素P450酶系在除草剂代谢中的研究进展,对细胞色素P450酶系参与的杂草抗性形成及其在抗除草剂转基因作物培育中的应用等方面的研究进行了综述,旨为该领域的相关研究提供参考。%Cytochrome P450 are one of the most important enzymes participated in the phaseⅠmetabolism of herbicides which have been found in all organisms from bacteria to humans.The Cytochrome P450 play an important role in the formation of herbicide resistance.Based on the progresses of cytochrome P450-mediated in the herbicide metabolism at home and abroad,research on the formation of weed resistance and the application of transgenic herbicide-resistant crops which cytochrome P450 mediated are summed up.

  8. 油棕P450基因在果皮发育过程中的动态表达%Dynamic expression of one cytochrome P450-like gene in mesocarp of oil palm nut

    Institute of Scientific and Technical Information of China (English)

    梁远学; 袁怡君; 鲍玉佳; 李东栋

    2012-01-01

    The dynamic expression of one cytochrom P450-like gene in mesocarp of oil palm (Elaeis guineensis) nut at five different development stages were analyzed. The content of fatty acid in mesocarp tissues was investigated with chloroform-methanol method. The results showed that mRNA level of the P450-like gene was the highest at the fourth period with 201. 07 times of the first stage. The ratio of fatty acid accumulation reached the maximum during the 3rd-4th stage (15. 79%). The change of fatty acid accumulation during five stages fitted well with P450 gene expression. According to previous studies,the expression of P450 in the development of oil palm nuts may affect the oxidation, epoxidation or alkyla-tion of fatty acid in oil palm. This study will provide a basis for further research about the regulation of P450 gene during the maturation of oil palm,and open up a new field of modifying oil palm fatty acid metabolism pathway by genetic improvement.%对油棕果实5个不同发育时期果皮中脂肪酸含量和类细胞色素P450(cytochrome P450,P450)基因的表达情况进行了分析.结果表明:在所选择的5个不同发育时期中第4个时期P450的表达量最高,为第1个时期表达量的201.07倍.脂肪酸总含量分析表明第3、4个时期之间增加速率最高(15.79%).在第3个时期脂肪酸的合成较少,脂肪酸含量变化趋势与同样组织中的P450基因表达趋势类似.由于细胞色素P450在植物脂肪酸代谢中起重要作用,油棕果实发育中细胞色素P450的表达极有可能对其脂肪酸的氧化、环氧化、烃基化等代谢产生影响,进而对脂肪酸的组成、产量及一些保护性化合物等的合成产生影响.

  9. Reduction of aromatic and heterocyclic aromatic N-hydroxylamines by human cytochrome P450 2S1.

    Science.gov (United States)

    Wang, Kai; Guengerich, F Peter

    2013-06-17

    Many aromatic amines and heterocyclic aromatic amines (HAAs) are known carcinogens for animals, and there is also strong evidence of some in human cancer. The activation of these compounds, including some arylamine drugs, involves N-hydroxylation, usually by cytochrome P450 enzymes (P450) in Family 1 (1A2, 1A1, and 1B1). We previously demonstrated that the bioactivation product of the anticancer agent 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203), an N-hydroxylamine, can be reduced by P450 2S1 to its amine precursor under anaerobic conditions and, to a lesser extent, under aerobic conditions [Wang, K., and Guengerich, F. P. (2012) Chem. Res. Toxicol. 25, 1740-1751]. In the study presented here, we tested the hypothesis that P450 2S1 is involved in the reductive biotransformation of known carcinogenic aromatic amines and HAAs. The N-hydroxylamines of 4-aminobiphenyl (4-ABP), 2-naphthylamine (2-NA), and 2-aminofluorene (2-AF) were synthesized and found to be reduced by P450 2S1 under both anaerobic and aerobic conditions. The formation of amines due to P450 2S1 reduction also occurred under aerobic conditions but was less apparent because the competitive disproportionation reactions (of the N-hydroxylamines) also yielded amines. Further, some nitroso and nitro derivatives of the arylamines could also be reduced by P450 2S1. None of the amines tested were oxidized by P450 2S1. These results suggest that P450 2S1 may be involved in the reductive detoxication of several of the activated products of carcinogenic aromatic amines and HAAs. PMID:23682735

  10. Single molecule activity measurements of cytochrome P450 oxidoreductase reveal the existence of two discrete functional states

    DEFF Research Database (Denmark)

    Laursen, Tomas; Singha, Aparajita; Rantzau, Nicolai;

    2014-01-01

    Electron transfer between membrane spanning oxi-doreductase enzymes crucially controls vital meta-bolic processes. Here we studied for the first time with single molecule resolution the function of P450 oxidoreductase (POR), the canonical membrane spanning activator of all microsomal cytochrome P......450 enzymes. Measurements and statistical analy-sis of individual catalytic turnover cycles shows POR to sample at least two major functional states. This phenotype may underlie regulatory interactions with different cytochromes P450 but to date remained masked in bulk kinetics. To ensure that we...

  11. Human cytochrome P-450PA (P-450IA2), the phenacetin O-deethylase, is primarily responsible for the hepatic 3-demethylation of caffeine and N-oxidation of carcinogenic arylamines.

    OpenAIRE

    Butler, M.A. (Mary A.); Iwasaki, M; Guengerich, F P; Kadlubar, F F

    1989-01-01

    Aromatic amines are well known as occupational carcinogens and are found in cooked foods, tobacco smoke, synthetic fuels, and agricultural chemicals. For the primary arylamines, metabolic N-oxidation by hepatic cytochromes P-450 is generally regarded as an initial activation step leading to carcinogenesis. The metabolic activation of 4-aminobiphenyl, 2-naphthylamine, and several heterocyclic amines has been shown recently to be catalyzed by rat cytochrome P-450ISF-G and by its human ortholog,...

  12. Cytochrome b(5) and epoxide hydrolase contribute to benzo[a]pyrene-DNA adduct formation catalyzed by cytochrome P450 1A1 under low NADPH:P450 oxidoreductase conditions

    Czech Academy of Sciences Publication Activity Database

    Stiborová, M.; Moserová, M.; Černá, V.; Indra, R.; Dračínský, Martin; Šulc, M.; Henderson, C. J.; Wolf, C. R.; Schmeiser, H. H.; Phillips, D. H.; Frei, E.; Arlt, V. M.

    2014-01-01

    Roč. 318, Apr 6 (2014), s. 1-12. ISSN 0300-483X Grant ostatní: GA ČR(CZ) GAP301/10/0356 Institutional support: RVO:61388963 Keywords : benzo[a]pyrene * cytochrome P450 1A1 * NADPH:cytochrome P450 oxidoreductase * epoxide hydrolase * cytochrome b(5) Subject RIV: CC - Organic Chemistry Impact factor: 3.621, year: 2014

  13. Intestinal cytochromes P450 regulating the intestinal microbiota and its probiotic profile

    Directory of Open Access Journals (Sweden)

    Eugenia Elefterios Venizelos Bezirtzoglou

    2012-09-01

    Full Text Available Cytochromes P450 (CYPs enzymes metabolize a large variety of xenobiotic substances. In this vein, a plethora of studies were conducted to investigate their role, as cytochromes are located in both liver and intestinal tissues. The P450 profile of the human intestine has not been fully characterized. Human intestine serves primarily as an absorptive organ for nutrients, although it has also the ability to metabolize drugs. CYPs are responsible for the majority of phase I drug metabolism reactions. CYP3A represents the major intestinal CYP (80% followed by CYP2C9. CYP1A is expressed at high level in the duodenum, together with less abundant levels of CYP2C8-10 and CYP2D6. Cytochromes present a genetic polymorphism intra- or interindividual and intra- or interethnic. Changes in the pharmacokinetic profile of the drug are associated with increased toxicity due to reduced metabolism, altered efficacy of the drug, increased production of toxic metabolites, and adverse drug interaction. The high metabolic capacity of the intestinal flora is due to its enormous pool of enzymes, which catalyzes reactions in phase I and phase II drug metabolism. Compromised intestinal barrier conditions, when rupture of the intestinal integrity occurs, could increase passive paracellular absorption. It is clear that high microbial intestinal charge following intestinal disturbances, ageing, environment, or food-associated ailments leads to the microbial metabolism of a drug before absorption. The effect of certain bacteria having a benefic action on the intestinal ecosystem has been largely discussed during the past few years by many authors. The aim of the probiotic approach is to repair the deficiencies in the gut flora and establish a protective effect. There is a tentative multifactorial association of the CYP (P450 cytochrome role in the different diseases states, environmental toxic effects or chemical exposures and nutritional status.

  14. Regulation of gap junction function and Connexin 43 expression by cytochrome P450 oxidoreductase (CYPOR)

    International Nuclear Information System (INIS)

    Highlights: → Humans with severe forms of cytochrome P450 oxidoreductase (CYPOR) mutations show bone defects as observed in Antley-Bixler Syndrome. → First report showing knockdown of CYPOR in osteoblasts decreased Connexin 43 (Cx43) protein levels. Cx43 is known to play an important role in bone modeling. → Knockdown of CYPOR decreased Gap Junctional Intercellular Communication and hemichannel activity. → Knockdown of CYPOR decreased Cx43 in mouse primary calvarial osteoblasts. → Decreased Cx43 expression was observed at the transcriptional level. -- Abstract: Cytochrome P450 oxidoreductase (CYPOR) is a microsomal electron-transferring enzyme containing both FAD and FMN as co-factors, which provides the reducing equivalents to various redox partners, such as cytochromes P450 (CYPs), heme oxygenase (HO), cytochrome b5 and squalene monooxygenase. Human patients with severe forms of CYPOR mutation show bone defects such as cranio- and humeroradial synostoses and long bone fractures, known as Antley-Bixler-like Syndrome (ABS). To elucidate the role of CYPOR in bone, we knocked-down CYPOR in multiple osteoblast cell lines using RNAi technology. In this study, knock-down of CYPOR decreased the expression of Connexin 43 (Cx43), known to play a critical role in bone formation, modeling, and remodeling. Knock-down of CYPOR also decreased Gap Junction Intercellular Communication (GJIC) and hemichannel activity. Promoter luciferase assays revealed that the decrease in expression of Cx43 in CYPOR knock-down cells was due to transcriptional repression. Primary osteoblasts isolated from bone specific Por knock-down mice calvariae confirmed the findings in the cell lines. Taken together, our study provides novel insights into the regulation of gap junction function by CYPOR and suggests that Cx43 may play an important role(s) in CYPOR-mediated bone defects seen in patients.

  15. Size-dependent effects of nanoparticles on the activity of cytochrome P450 isoenzymes

    International Nuclear Information System (INIS)

    Nanoparticles are known to be able to interfere with cellular metabolism and to cause cytotoxicity and moreover may interfere with specific cellular functions. Serious effects on the latter include changes in liver cell function. The cytochrome P450 system is expressed in many cells but is especially important in hepatocytes and hormone-producing cells. The interaction of polystyrene nanoparticles with the most important drug-metabolizing cytochrome P450 isoenzymes, CYP3A4, CYP2D6, CYP2C9 and CYP2A1 expressed individually in insect cells (BACULOSOMES) was studied by the cleavage of substrates coupled to a fluorescent dye. The data obtained for individual isoenzymes were compared to metabolism in microsomes isolated from normal liver and from the hepatoma cell line H4-II-E-C3. Small (20-60 nm) carboxyl polystyrene particles but not larger (200 nm) ones reached high intracellular concentrations in the vicinity of the endoplasmic reticulum. These small particles inhibited the enzymatic activity of CYP450 isoenzymes in BACULOSOMES and substrate cleavage in normal liver microsomes. They moreover increased the effect of known inhibitors of the cytochrome P450 system (cimetidine, phenobarbital and paclitaxel). Substrate cleavage by the hepatoma cell line H4-II-E-C3 in contrast was undetectable, making this cell line unsuitable for this type of study. Our results thus demonstrate that nanoparticles can inhibit the metabolism of xenobiotics by the CYP450 system in model systems in vitro. Such inhibition could also potentially occur in vivo and possibly cause adverse effects in persons receiving medication.

  16. Mass Spectrometric Analysis of Oxylipins : Application to Cytochrome P450-Dependent Metabolism

    OpenAIRE

    Nilsson, Tomas

    2009-01-01

    Cytochrome P450 (CYP) family 4 constitutes monoxygenases responsible for hydroxylation of fatty acids and other lipids. For example, CYP4F3 metabolizes leukotrienes and CYP4F8 prostaglandin H. Importantly, six of the twelve CYP4 enzymes are orphans, i.e., with an unknown biological function. The catalytic activity of the enzyme CYP4F8 is known in seminal vesicles, but not in skin or psoriatic lesions, where CYP4F8 is highly expressed. The orphan CYP4F22 is also expressed in skin, and mutation...

  17. The binding of UDP-glucosyltransferase to the cytochrome P450s in dhurrin biosynthesis

    DEFF Research Database (Denmark)

    Baden, Camilla Knudsen; Laursen, Tomas; Kannangara, Rubini Maya;

    2015-01-01

    Cyanogenic glucosides are amino-acid derived plant chemical defense compounds against generalist herbivores. The subtropical crop plant Sorghum bicolor synthesizes the L-tyrosine derived cyanogenic glucoside dhurrin through the activity of two multifunctional cytochrome P450s (CYPs), CYP79A1 and...... ER localized by an N-terminal anchor, whereas the UGT85B1 is thought to be cytosolic with tight association to the ER. Fluorescence resonance energy transfer (FRET) studies have shown that the soluble UGT is associated close to the ER localized CYPs, which enables efficient glycosylation and...

  18. The contribution of atom accessibility to site of metabolism models for cytochromes P450

    DEFF Research Database (Denmark)

    Rydberg, Patrik; Rostkowski, M.; Gloriam, D.E.; Olsen, L.

    2013-01-01

    Three different types of atom accessibility descriptors are investigated in relation to site of metabolism predictions. To enable the integration of local accessibility we have constructed 2DSASA, a method for the calculation of the atomic solvent accessible surface area that is independent of 3D...... coordinates. The method was implemented in the SMARTCyp site of metabolism prediction models and improved the results by up to 4 percentage points for nine cytochrome P450 isoforms. The final models are made available at http://www.farma.ku.dk/smartcyp....

  19. Cyclosporin A-induced free radical generation is not mediated by cytochrome P-450

    OpenAIRE

    Krauskopf, Alexandra; Buetler, Timo M; Nguyen, Nathalie S D; Macé, Katherine; Ruegg, Urs T.

    2002-01-01

    Reactive oxygen species (ROS) have been proposed to play a role in the side effects of the immunosuppressive drug cyclosporin A (CsA).The aim of this study was to investigate whether cytochrome P-450 (CYP) dependent metabolism of CsA could be responsible for ROS generation since it has been suggested that CsA may influence the CYP system to produce ROS.We show that CsA (1 – 10 μM) generated antioxidant-inhibitable ROS in rat aortic smooth muscle cells (RASMC) using the fluorescent probe 2,7-d...

  20. Cytochrome P450-generated metabolites derived from ω-3 fatty acids attenuate neovascularization

    OpenAIRE

    Yanai, Ryoji; Mulki, Lama; Hasegawa, Eiichi; Takeuchi, Kimio; Sweigard, Harry; Suzuki, Jun; Gaissert, Philipp; Vavvas, Demetrios G.; Sonoda, Koh-Hei; Rothe, Michael; Schunck, Wolf-Hagen; Miller, Joan W.; Connor, Kip M.

    2014-01-01

    The ω-3 long-chain polyunsaturated fatty acids are a class of dietary lipids that are highly enriched in the central nervous system and the retina. We demonstrate that dietary enrichment with ω-3s suppresses choroidal neovascularization in a mouse model of age-related macular degeneration (AMD), a leading cause of blindness. The ω-3s have anti-inflammatory properties and compete with ω-6s for downstream lipid metabolite synthesis at the cytochrome P450 (CYP) level. Specifically, 17,18- epoxye...

  1. Cytochrome P450s in the Regulation of Cellular Retinoic Acid Metabolism

    OpenAIRE

    Ross, A. Catharine; Zolfaghari, Reza

    2011-01-01

    The active metabolite of vitamin A, retinoic acid (RA), is a powerful regulator of gene transcription. RA is also a therapeutic drug. The oxidative metabolism of RA by certain members of the cytochrome P450 (CYP) superfamily helps to maintain tissue RA concentrations within appropriate bounds. The CYP26 family—CYP26A1, CYP26B1, and CYP26C1—is distinguished by being both regulated by and active toward all-trans-RA (at-RA) while being expressed in different tissue-specific patterns. The CYP26A1...

  2. Regulation of Porcine Hepatic Cytochrome P450 by Chicory Root - Implication of Boar Taint

    OpenAIRE

    Rasmussen, Martin Krøyer

    2012-01-01

    Dietary components have been shown to affect the hepatic detoxification system. Hepatic clearance of xenobiotics is performed in two steps, Phase I and II. Phase I is usually carried out by enzymes belonging to the cytochrome P450 (CYP) family, while Phase II is carried out by a more diverse group. Phase I and II enzymes are extensively studied for their importance in drug clearance and in pigs for their impact on the occurrence of boar taint. It is generally believed that sufficient Phase I ...

  3. Differentially regulated NADPH:cytochrome P450 oxidoreductases in parsley

    OpenAIRE

    Koopmann, Edda; Hahlbrock, Klaus

    1997-01-01

    Two NADPH:cytochrome P450 oxidoreductases (CPRs) from parsley (Petroselinum crispum) were cloned, and the complete proteins were expressed and functionally identified in yeast. The two enzymes, designated CPR1 and CPR2, are 80% identical in amino acid sequence with one another and about 75% identical with CPRs from several other plant species. The mRNA accumulation patterns for CPR1 and CPR2 in fungal elicitor-treated or UV-irradiated cultured parsley cells and in developing or infected parsl...

  4. Identification of the human cytochromes P450 catalysing the rate-limiting pathways of gliclazide elimination

    OpenAIRE

    Elliot, David J.; Suharjono,; Lewis, Benjamin C; Gillam, Elizabeth M. J.; Birkett, Donald J; Gross, Annette S.; Miners, John O

    2007-01-01

    What is already known about this subjectGliclazide is a widely used oral hypoglycaemic agent.The major metabolites of gliclazide formed in vivo have been identified.However, the cytochrome P450 enzymes catalysing the rate-limiting pathways of gliclazide elimination are unknown.What this study addsCYP2C9 is the major enzyme involved in the various hydroxylation pathways of gliclazide, although a contribution of CYP2C19 to tolymethylhydroxylation, the major metabolic route, cannot be discounted...

  5. Multiple Oxidative Modifications in the Ophiobolin Biosynthesis: P450 Oxidations Found in Genome Mining.

    Science.gov (United States)

    Narita, Koji; Chiba, Ryota; Minami, Atsushi; Kodama, Motoichiro; Fujii, Isao; Gomi, Katsuya; Oikawa, Hideaki

    2016-05-01

    Heterologous expression of four candidate genes found in ophiobolin gene clusters from three fungal strains was employed to elucidate the late-stage biosynthetic pathway of phytotoxin ophiobolin. Expression of oblBAc (cytochrome P450) from the cryptic gene cluster gave unexpected products, and that of oblBBm/oblBEv from the gene cluster of ophiobolin producers, with oblDBm as the transporter, yielded intermediate ophiobolin C through an unusual four-step oxidation process. The observation made in this study may provide a useful guideline for the elucidation of genuine biosynthetic pathways of natural products. PMID:27116000

  6. Probing cytochrome P450-mediated activation with a truncated azinomycin analogue

    OpenAIRE

    Vinader, Victoria; Sadiq, Maria; Sutherland, Mark; Huang, Menying; Loadman, Paul; Elsalem, Lina; Shnyder, Steven; Cui, Hongjuan; Afarinkia, Kamyar; Searcey, Mark; Patterson, Laurence; Pors, Klaus

    2015-01-01

    A deactivated alkene precursor (IC50 = 81 μM) to the azinomycin epoxide natural product can be bioactivated by several cytochromes P450 (CYP) to generate antiproliferative metabolites with increased potency (IC50 = 1-30 μM) in CHOwt cells. CYP1A1 and 3A4 were shown to generate exclusively the unnatural and the natural-configured azinomycin epoxide diastereoisomer respectively, while CYP1B1 produced both epoxides in a 3:1 mixture. The antiproliferative activity is linked to DNA damage as demon...

  7. Identification of two Nereis virens [Annelida: Polychaeta] cytochrome P450 enzymes and induction by xenobiotics

    DEFF Research Database (Denmark)

    Rewitz, Kim; Kjellerup, C; Jørgensen, A; Petersen, C; Andersen, Ole

    2004-01-01

    Cytochrome P450 (CYP) enzyme catalysed metabolism of xenobiotics such as polycyclic aromatic hydrocarbons (PAHs) are known to occur in polychaetes. Yet specific polychaete CYP enzymes have so far not been identified. Here, we report two partial CYP cDNA sequences, both of 453 bp, characterised fr...... contaminants such as PAHs. The present study demonstrates that these N. virens CYP genes are transcriptionally inducible, and suggests that N. virens CYP4 enzymes may be involved in the metabolism of both exogenous and endogenous compounds....

  8. Opposing Regulation of Cytochrome P450 Expression by CAR and PXR in Hypothyroid Mice

    OpenAIRE

    Park, Young Joo; Lee, Eun Kyung; Lee, Yoon Kwang; Park, Do Joon; Jang, Hak Chul; Moore, David D.

    2012-01-01

    Clinical hypothyroidism affects various metabolic processes including drug metabolism. CYP2B and CYP3A are important cytochrome P450 drug metabolizing enzymes that are regulated by the xenobiotic receptors constitutive androstane receptor (CAR, NR1I3) and pregnane X receptor (PXR, NR1I2). We evaluated the regulation of the hepatic expression of CYPs by CAR and PXR in the hypothyroid state induced by a low-iodine diet containing 0.15% propylthiouracil. Expression of Cyp3a11 was suppressed in h...

  9. Catalytic Characterization and Cytokine Mediated Regulation of Cytochrome P450 4Fs in Rat Hepatocytes

    OpenAIRE

    Kalsotra, Auinash; Anakk, Sayeepriyadarshini; Brommer, Chad L; Kikuta, Yasushi; Morgan, Edward T.; Strobel, Henry W.

    2007-01-01

    Cytochrome P450 (CYP) 4F mediated leukotriene B4 (LTB4) metabolism modulates inflammation during injury and infection. Here we show that in addition to LTB4, the recombinant rat CYP4Fs catalyze omega-hydroxylations of lipoxin A4, and hydroxyeicosatetraeonic acids. CYP4F gene regulation studies in primary hepatocytes reveal that pro-inflammatory cytokines interleukin (IL) -1β, IL-6 and tumor necrosis factor (TNF) -α produce a general inductive response whereas IL-10, an anti-inflammatory cytok...

  10. Inhibition selectivity of grapefruit juice components on human cytochromes P450.

    Science.gov (United States)

    Tassaneeyakul, W; Guo, L Q; Fukuda, K; Ohta, T; Yamazoe, Y

    2000-06-15

    Five compounds including furanocoumarin monomers (bergamottin, 6', 7'-dihydroxybergamottin (DHB)), furanocoumarin dimers (4-¿¿6-hydroxy-71-¿(1-hydroxy-1-methyl)ethyl-4-methyl-6-(7-oxo-7H- furo¿3,2-g1benzopyran-4-yl)-4-hexenyl]oxy]-3,7-dimethyl- 2-octenyl]oxy]-7H-furo[3,2-g]¿1benzopyran-7-one (GF-I-1) and 4-¿¿6-hydroxy-7¿¿4-methyl-1-(1-methylethenyl)-6-(7-oxo-7H-furo¿3, 2-g1benzopyran-4-yl)-4-hexenylŏxy-3, 7-dimethyl-2-octenylŏxy-7H-furo¿3,2-g1benzopyran-7-one (GF-I-4)), and a sesquiterpene nootkatone have been isolated from grapefruit juice and screened for their inhibitory effects toward human cytochrome P450 (P450) forms using selective substrate probes. Addition of ethyl acetate extract of grapefruit juice into an incubation mixture resulted in decreased activities of CYP3A4, CYP1A2, CYP2C9, and CYP2D6. All four furanocoumarins clearly inhibited CYP3A4-catalyzed nifedipine oxidation in concentration- and time-dependent manners, suggesting that these compounds are mechanism-based inhibitors of CYP3A4. Of the furanocoumarins investigated, furanocoumarin dimers, GF-I-1 and GF-I-4, were the most potent inhibitors of CYP3A4. Inhibitor concentration required for half-maximal rate of inactivation (K(I)) values for bergamottin, DHB, GF-I-1, and GF-I-4 were calculated, respectively, as 40.00, 5. 56, 0.31, and 0.13 microM, whereas similar values were observed on their inactivation rate constant at infinite concentration of inhibitor (k(inact), 0.05-0.08 min(-1)). Apparent selectivity toward CYP3A4 does occur with the furanocoumarin dimers. In contrast, bergamottin showed rather stronger inhibitory effect on CYP1A2, CYP2C9, CYP2C19, and CYP2D6 than on CYP3A4. DHB inhibited CYP3A4 and CYP1A2 activities at nearly equivalent potencies. Among P450 forms investigated, CYP2E1 was the least sensitive to the inhibitory effect of furanocoumarin components. A sesquiterpene nootkatone has no significant effect on P450 activities investigated except for CYP2A6 and CYP2C19

  11. Environmentally persistent free radicals inhibit cytochrome P450 activity in rat liver microsomes

    International Nuclear Information System (INIS)

    Combustion processes generate particulate matter that affects human health. When incineration fuels include components that are highly enriched in aromatic hydrocarbons (especially halogenated varieties) and redox-active metals, ultrafine particulate matter containing air-stable, environmentally persistent free radicals (EPFRs) is generated. The exposure to fine EPFRs (less than 2.5 μm in diameter) has been shown to negatively influence pulmonary and cardiovascular functions in living organisms. The goal of this study was to determine if these EPFRs have a direct effect on cytochrome P450 function. This was accomplished by direct addition of the EPFRs to rat liver microsomal preparations and measurement of several P450 activities using form-selective substrates. The EPFRs used in this study were formed by heating vapors from an organic compound (either monochlorophenol (MCP230) or 1,2-dichlorobenzene (DCB230)) and 5% copper oxide supported on silica (approximately 0.2 μm in diameter) to 230 °C under vacuum. Both types of EPFRs (but not silica, physisorbed silica, or silica impregnated with copper oxide) dramatically inhibited the activities of CYP1A, CYP2B, CYP2E1, CYP2D2 and CYP3A when incubated at concentrations less than 0.1 mg/ml with microsomes and NADPH. Interestingly, at the same concentrations, the EPFRs did not inhibit HO-1 activity or the reduction of cytochrome c by NADPH-cytochrome P450 reductase. CYP2D2-selective metabolism by rat liver microsomes was examined in more detail. The inhibition of CYP2D2-selective metabolism by both DCB230- and MCP230-EPFRs appeared to be largely noncompetitive and was attenuated in the presence of catalase suggesting that reactive oxygen species may be involved in the mechanism of inhibition. - Highlights: • Combustion of organic pollutants generates long-lived particulate radicals (EPFRs). • EPFRs inhibit metabolism by all cytochromes P450 tested in rat liver microsomes. • EPFR-mediated inhibition is related to

  12. Environmentally persistent free radicals inhibit cytochrome P450 activity in rat liver microsomes

    Energy Technology Data Exchange (ETDEWEB)

    Reed, James R., E-mail: rreed@lsuhsc.edu [Department of Pharmacology and Experimental Therapeutics, Louisiana State University Health Science Center, 533 Bolivar St., New Orleans, LA 70112 (United States); The Stanley S. Scott Cancer Center, Louisiana State University Health Science Center, 533 Bolivar St., New Orleans, LA 70112 (United States); Cawley, George F.; Ardoin, Taylor G. [Department of Pharmacology and Experimental Therapeutics, Louisiana State University Health Science Center, 533 Bolivar St., New Orleans, LA 70112 (United States); The Stanley S. Scott Cancer Center, Louisiana State University Health Science Center, 533 Bolivar St., New Orleans, LA 70112 (United States); Dellinger, Barry; Lomnicki, Slawomir M.; Hasan, Farhana; Kiruri, Lucy W. [Department of Chemistry, Louisiana State University, Baton Rouge, LA 70803 (United States); Backes, Wayne L. [Department of Pharmacology and Experimental Therapeutics, Louisiana State University Health Science Center, 533 Bolivar St., New Orleans, LA 70112 (United States); The Stanley S. Scott Cancer Center, Louisiana State University Health Science Center, 533 Bolivar St., New Orleans, LA 70112 (United States)

    2014-06-01

    Combustion processes generate particulate matter that affects human health. When incineration fuels include components that are highly enriched in aromatic hydrocarbons (especially halogenated varieties) and redox-active metals, ultrafine particulate matter containing air-stable, environmentally persistent free radicals (EPFRs) is generated. The exposure to fine EPFRs (less than 2.5 μm in diameter) has been shown to negatively influence pulmonary and cardiovascular functions in living organisms. The goal of this study was to determine if these EPFRs have a direct effect on cytochrome P450 function. This was accomplished by direct addition of the EPFRs to rat liver microsomal preparations and measurement of several P450 activities using form-selective substrates. The EPFRs used in this study were formed by heating vapors from an organic compound (either monochlorophenol (MCP230) or 1,2-dichlorobenzene (DCB230)) and 5% copper oxide supported on silica (approximately 0.2 μm in diameter) to 230 °C under vacuum. Both types of EPFRs (but not silica, physisorbed silica, or silica impregnated with copper oxide) dramatically inhibited the activities of CYP1A, CYP2B, CYP2E1, CYP2D2 and CYP3A when incubated at concentrations less than 0.1 mg/ml with microsomes and NADPH. Interestingly, at the same concentrations, the EPFRs did not inhibit HO-1 activity or the reduction of cytochrome c by NADPH-cytochrome P450 reductase. CYP2D2-selective metabolism by rat liver microsomes was examined in more detail. The inhibition of CYP2D2-selective metabolism by both DCB230- and MCP230-EPFRs appeared to be largely noncompetitive and was attenuated in the presence of catalase suggesting that reactive oxygen species may be involved in the mechanism of inhibition. - Highlights: • Combustion of organic pollutants generates long-lived particulate radicals (EPFRs). • EPFRs inhibit metabolism by all cytochromes P450 tested in rat liver microsomes. • EPFR-mediated inhibition is related to

  13. Moessbauer- and EPR-Snapshots of an Enzymatic Reaction: The Cytochrome P450 Reaction Cycle

    International Nuclear Information System (INIS)

    In this communication we present a complimentary Moessbauer- and EPR-study of the time dependance of the reaction of substrate free P450cam with peracetic acid within a time region ranging from 8 ms up to 5 min. An Fe(IV) species as well as a tyrosyl radical residing on the amino acid residue Tyr96 have been identified as reaction intermediates. These species possibly are formed by the reduction of compound I by means of transferring an electron from Tyr 96 to the heme moiety.

  14. Drug Interactions of Thalidomide with Midazolam and Cyclosporine A: Heterotropic Cooperativity of Human Cytochrome P450 3A5

    OpenAIRE

    Okada, Yusuke; Murayama, Norie; Yanagida, Chihiro; Shimizu, Makiko; Guengerich, F. Peter; Yamazaki, Hiroshi

    2008-01-01

    There is growing clinical interest of thalidomide because of its immunomodulatory and antiangiogenic properties, despite its teratogenicity. However, little information about thalidomide has been reported regarding its precise effects on drug-metabolizing enzymes. We investigated the effects of thalidomide on cytochrome P450 (P450) enzymes in human liver microsomes to clarify the potential for possible drug interactions. Thalidomide inhibited S-mephenytoin 4′-hydroxyla...

  15. Spectroscopic and Kinetic Studies of Nor1, a Cytochrome P450 Nitric Oxide Reductase From the Fungal Pathogen Histoplasma capsulatum†

    OpenAIRE

    Chao, Lily Y.; Rine, Jasper; Marletta, Michael A

    2008-01-01

    The fungal respiratory pathogen Histoplasma capsulatum evades the innate immune response and colonizes macrophages during infection. Although macrophage production of the antimicrobial effector nitric oxide (NO) restricts H. capsulatum growth, the pathogen is able to establish a persistent infection. H. capsulatum contains a P450 nitric oxide reductase homologue (NOR1) that may be important for detoxifying NO during infection. To characterize the activity of this putative P450 enzyme, a 404 a...

  16. Production de terpènes fonctionnalisés par les cytochromes P450 de plantes recombinants

    OpenAIRE

    Gavira, Carole

    2013-01-01

    Our aim was to identify cytochromes P450 catalyzing hydroxylation of mono-and sesquiterpenes to produce functionalized "natural" compounds with interesting organoleptic properties for the flavor and fragrance industry. We identified 7 P450-substrate pairs showing . 45 % in vitro conversion and/or forming an expected product. The amounts of products resulting from yeast bioconversion were however too low for implementation of an industrial process. Factors limiting the nootkatone production fr...

  17. Mouse Knockout of the Cholesterogenic Cytochrome P450 Lanosterol 14α-Demethylase (Cyp51) Resembles Antley-Bixler Syndrome*

    OpenAIRE

    Keber, Rok; Motaln, Helena; Wagner, Kay D.; Debeljak, Nataša; Rassoulzadegan, Minoo; Ačimovič, Jure; Rozman, Damjana; Horvat, Simon

    2011-01-01

    Antley-Bixler syndrome (ABS) represents a group of heterogeneous disorders characterized by skeletal, cardiac, and urogenital abnormalities that have frequently been associated with mutations in fibroblast growth factor receptor 2 or cytochrome P450 reductase genes. In some ABS patients, reduced activity of the cholesterogenic cytochrome P450 CYP51A1, an ortholog of the mouse CYP51, and accumulation of lanosterol and 24,25-dihydrolanosterol has been reported, but the role of CYP51A1 in the AB...

  18. Molecular Docking Based Virtual Design of Polysubstituted Triazoles as Cytochrome P-450 14-Alpha-Sterol Demethylase (Cyp51) Inhibitor

    OpenAIRE

    Bibek Pati*,1; Subhasis Banerjee2

    2012-01-01

    Computational ligand docking methodology, AutoDock 4.0, based on Lamarckian genetic algorithm was employed for virtual screening of a compound library with 13 entries including reference compound as fluconazole with the enzyme Cytochrome P-450-14-Alpha-Sterol Demethylase (Cyp51), a potential antifungal drug target. Considering free energy of binding as a criteria of evaluation, a total of 12 compounds were predicted to be potential inhibitors of Cytochrome P-450-14-Alpha-Sterol Demethylase (C...

  19. Chronic ethanol exposure downregulates hepatic expression of pregnane X receptor and P450 3A11 in female ICR mice

    International Nuclear Information System (INIS)

    Pregnane X receptor (PXR) is a nuclear receptor that regulates cytochrome P450 3A (CYP3A) gene transcription in a ligand-dependent manner. Ethanol has been reported to be either an inducer or an inhibitor of CYP3A expression. In this study, we investigated the effects of chronic ethanol exposure on PXR and P450 3A11 gene expression in mouse liver. Female ICR mice were administered by gavage with different doses (1000, 2000 and 4000 mg/kg) of ethanol for up to 5 weeks. Hepatic PXR and P450 3A11 mRNA levels were measured using RT-PCR. Erythromycin N-demethylase (ERND) activity was used as an indicator of CYP3A protein expression. Results showed that chronic ethanol exposure markedly decreased hepatic PXR and P450 3A11 mRNA levels. Consistent with downregulation of P450 3A11 mRNA, chronic ethanol exposure significantly decreased ERND activity in a dose-dependent manner. Additional experiment showed that chronic ethanol exposure significantly increased plasma endotoxin level and hepatic CD14 and TLR-4 mRNA expression, all of which were blocked by elimination of Gram-negative bacteria and endotoxin with antibiotics. Correspondingly, pretreatment with antibiotics reversed the downregulation of PXR and P450 3A11 mRNA expression and ERND activity in mouse liver. Furthermore, the downregulation of hepatic PXR and P450 3A11 mRNA expression was significantly attenuated in mice pretreated with GdCl3, a selective Kupffer cell toxicant. GdCl3 pretreatment also significantly attenuated chronically ethanol-induced decrease in ERND activity. These results indicated that activation of Kupffer cells by gut-derived endotoxin contributes to downregulation of hepatic PXR and P450 3A11 expression during chronic alcohol intoxication

  20. Transcriptome Analysis of an Insecticide Resistant Housefly Strain: Insights about SNPs and Regulatory Elements in Cytochrome P450 Genes

    Science.gov (United States)

    Asp, Torben; Kristensen, Michael

    2016-01-01

    Background Insecticide resistance in the housefly, Musca domestica, has been investigated for more than 60 years. It will enter a new era after the recent publication of the housefly genome and the development of multiple next generation sequencing technologies. The genetic background of the xenobiotic response can now be investigated in greater detail. Here, we investigate the 454-pyrosequencing transcriptome of the spinosad-resistant 791spin strain in relation to the housefly genome with focus on P450 genes. Results The de novo assembly of clean reads gave 35,834 contigs consisting of 21,780 sequences of the spinosad resistant strain. The 3,648 sequences were annotated with an enzyme code EC number and were mapped to 124 KEGG pathways with metabolic processes as most highly represented pathway. One hundred and twenty contigs were annotated as P450s covering 44 different P450 genes of housefly. Eight differentially expressed P450s genes were identified and investigated for SNPs, CpG islands and common regulatory motifs in promoter and coding regions. Functional annotation clustering of metabolic related genes and motif analysis of P450s revealed their association with epigenetic, transcription and gene expression related functions. The sequence variation analysis resulted in 12 SNPs and eight of them found in cyp6d1. There is variation in location, size and frequency of CpG islands and specific motifs were also identified in these P450s. Moreover, identified motifs were associated to GO terms and transcription factors using bioinformatic tools. Conclusion Transcriptome data of a spinosad resistant strain provide together with genome data fundamental support for future research to understand evolution of resistance in houseflies. Here, we report for the first time the SNPs, CpG islands and common regulatory motifs in differentially expressed P450s. Taken together our findings will serve as a stepping stone to advance understanding of the mechanism and role of P450s

  1. Molecular Docking Based Virtual Design of Polysubstituted Triazoles as Cytochrome P-450 14-Alpha-Sterol Demethylase (Cyp51 Inhibitor

    Directory of Open Access Journals (Sweden)

    Bibek Pati*,1

    2012-01-01

    Full Text Available Computational ligand docking methodology, AutoDock 4.0, based on Lamarckian genetic algorithm was employed for virtual screening of a compound library with 13 entries including reference compound as fluconazole with the enzyme Cytochrome P-450-14-Alpha-Sterol Demethylase (Cyp51, a potential antifungal drug target. Considering free energy of binding as a criteria of evaluation, a total of 12 compounds were predicted to be potential inhibitors of Cytochrome P-450-14-Alpha-Sterol Demethylase (Cyp51 and 10 compounds displayed greater binding affinities than fluconazole as Cytochrome P-450-14-Alpha-Sterol Demethylase (Cyp51 Inhibitor. Compound 1a & 1b were the most potent in inhibiting the Cytochrome P-450-14-Alpha-Sterol Demethylase (Cyp51, in silico. Putative interactions between Cytochrome P-450-14-Alpha-Sterol Demethylase (Cyp51 and inhibitors were identified by inspection of docking-predicted poses. This understanding of protein–ligand interaction and value of binding energy imparts impetus to the rapid development of novel Cytochrome P-450-14-Alpha-Sterol Demethylase (Cyp51.

  2. Reconstitution of the Cytb5 -CytP450 Complex in Nanodiscs for Structural Studies using NMR Spectroscopy.

    Science.gov (United States)

    Zhang, Meng; Huang, Rui; Ackermann, Rose; Im, Sang-Choul; Waskell, Lucy; Schwendeman, Anna; Ramamoorthy, Ayyalusamy

    2016-03-24

    Cytochrome P450s (P450s) are a superfamily of enzymes responsible for the catalysis of a wide range of substrates. Dynamic interactions between full-length membrane-bound P450 and its redox partner cytochrome b5 (cytb5 ) have been found to be important for the enzymatic activity of P450. However, the stability of the circa 70 kDa membrane-bound complex in model membranes renders high-resolution structural NMR studies particularly difficult. To overcome these challenges, reconstitution of the P450-cytb5 complex in peptide-based nanodiscs, containing no detergents, has been demonstrated, which are characterized by size exclusion chromatography and NMR spectroscopy. In addition, NMR experiments are used to identify the binding interface of the P450-cytb5 complex in the nanodisc. This is the first successful demonstration of a protein-protein complex in a nanodisc using NMR structural studies and should be useful to obtain valuable structural information on membrane-bound protein complexes. PMID:26924779

  3. Molecular Characterization and Functional Analysis of Three Pathogenesis-Related Cytochrome P450 Genes from Bursaphelenchus xylophilus (Tylenchida: Aphelenchoidoidea

    Directory of Open Access Journals (Sweden)

    Xiao-Lu Xu

    2015-03-01

    Full Text Available Bursaphelenchus xylophilus, the causal agent of pine wilt disease, causes huge economic losses in pine forests. The high expression of cytochrome P450 genes in B. xylophilus during infection in P. thunbergii indicated that these genes had a certain relationship with the pathogenic process of B. xylophilus. Thus, we attempted to identify the molecular characterization and functions of cytochrome P450 genes in B. xylophilus. In this study, full-length cDNA of three cytochrome P450 genes, BxCYP33C9, BxCYP33C4 and BxCYP33D3 were first cloned from B. xylophilus using 3' and 5' RACE PCR amplification. Sequence analysis showed that all of them contained a highly-conserved cytochrome P450 domain. The characteristics of the three putative proteins were analyzed with bioinformatic methods. RNA interference (RNAi was used to assess the functions of BxCYP33C9, BxCYP33C4 and BxCYP33D3. The results revealed that these cytochrome P450 genes were likely to be associated with the vitality, dispersal ability, reproduction, pathogenicity and pesticide metabolism of B. xylophilus. This discovery confirmed the molecular characterization and functions of three cytochrome P450 genes from B. xylophilus and provided fundamental information in elucidating the molecular interaction mechanism between B. xylophilus and its host plant.

  4. Interaction between chlorpyrifos and hepatic microsomal cytochrome P450 enzyme system in fish%毒死蜱与鱼肝微粒体P450的相互作用

    Institute of Scientific and Technical Information of China (English)

    边文杰; 徐燕; 李少南; 朱国念

    2011-01-01

    为了评估毒死蜱对水生生态系统的风险,比较研究了毒死蜱对斑马鱼、麦穗鱼及太阳鱼的急性毒性,3种鱼肝微粒体P450酶系对毒死蜱的脱硫转化作用及毒死蜱对肝微粒体P450酶系的影响.结果表明,毒死蜱对斑马鱼、麦穗鱼及太阳鱼的96 h-LC50分别为1.94,0.0273,0.0681 mg·L-1.3种鱼肝微粒体P450酶系对毒死蜱代谢的Vmax/Km值分别为:1.67×104,5.00×104,2.00× 104,即代谢能力大小为麦穗鱼>太阳鱼>斑马鱼,与急性毒性试验结果相吻合,从而P450酶系的脱硫转化活性可以一定程度上解释毒死蜱对3种鱼的毒性差异.试验浓度毒死蜱对3种鱼的P450酶系的影响不显著.%Acute toxicity of chlorpyrifos to Danio rerio, Pseudorasboraparva, and Lepomis gibbosus, oxidative activation of chlorpyrifos catalyzed by hepatic microsomal cytochrome P450 system, and influence of chlorpyrifos to the P450 system were studied to evaluate the risk of chlorpyrifos on ecology system. 96 h LC50 of chlorpyrifos to the Dani-o rerio, the Pseudorasbora parva, and the Lepomis gibbosus was measured to be 1. 94, 0. 0273, 0. 0681 mg·L-1, respectively. The Vmax/Km of chlorpyrifos oxidation catalyzed by P450 system was measured to be 1. 67 × 104 , 5. 00 × 104, 2. 00 × 104 , respectively, indicating that the oxidative metabolism capacity of the P450 system was the highest in Pseudorasbora parva and the lowest in Danio rerio, which matched the results of acute toxicity tests. The influence of chlorpyrifos to the P450 system was not obvious.

  5. Regio- and stereoselectivity of P450-catalysed hydroxylation of steroids controlled by laboratory evolution

    Science.gov (United States)

    Kille, Sabrina; Zilly, Felipe E.; Acevedo, Juan P.; Reetz, Manfred T.

    2011-09-01

    A current challenge in synthetic organic chemistry is the development of methods that allow the regio- and stereoselective oxidative C-H activation of natural or synthetic compounds with formation of the corresponding alcohols. Cytochrome P450 enzymes enable C-H activation at non-activated positions, but the simultaneous control of both regio- and stereoselectivity is problematic. Here, we demonstrate that directed evolution using iterative saturation mutagenesis provides a means to solve synthetic problems of this kind. Using P450 BM3(F87A) as the starting enzyme and testosterone as the substrate, which results in a 1:1 mixture of the 2β- and 15β-alcohols, mutants were obtained that are 96-97% selective for either of the two regioisomers, each with complete diastereoselectivity. The mutants can be used for selective oxidative hydroxylation of other steroids without performing additional mutagenesis experiments. Molecular dynamics simulations and docking experiments shed light on the origin of regio- and stereoselectivity.

  6. Regulation of P450 4A expression by peroxisome proliferator activated receptors

    International Nuclear Information System (INIS)

    The induction of P450 4A enzymes by peroxisome proliferators (PPs) and fatty acids is mediated by the peroxisome proliferator activated receptor α (PPARα) that binds to response elements in target genes as a heterodimer with the retinoid X receptor (RXR). The consensus sequence recognized by PPAR/RXR heterodimers, contains an imperfect direct repeat of two nuclear receptor binding motifs separated by a single nucleotide. This repeat is preceded by a conserved A/T rich sequence that is required for function. In mice, chronic exposure to PPs results in PPARα mediated liver hypertrophy, hyperplasia and carcinogenesis accompanied by a proliferation of peroxisomes. In contrast, humans exhibit a reduced sensitivity to PP pathogenesis. This could reflect >10-fold lower PPARα levels relative to mice as well as differences in targeted genes. In order to identify PPAR responsive human genes, the human hepatoma cell line, HepG2, was engineered to express increased levels of PPARα. Several genes encoding rate-limiting enzymes and branch points in ketone body formation are regulated by PPARα in these cells. In contrast, significant induction by PP is not evident for peroxisomal fatty acid oxidation that is associated with peroxisome proliferation in mice. Human P450 4A11 is not expressed in dividing cultures of cells with enhanced PPARα levels, but it is expressed in confluent cultures expressing elevated amounts of PPARα

  7. Prediction and analysis of the modular structure of cytochrome P450 monooxygenases

    Directory of Open Access Journals (Sweden)

    Wagner Florian

    2010-10-01

    Full Text Available Abstract Background Cytochrome P450 monooxygenases (CYPs form a vast and diverse family of highly variable sequences. They catalyze a wide variety of oxidative reactions and are therefore of great relevance in drug development and biotechnological applications. Despite their differences in sequence and substrate specificity, the structures of CYPs are highly similar. Although being in research focus for years, factors mediating selectivity and activity remain vague. Description This systematic comparison of CYPs based on the Cytochrome P450 Engineering Database (CYPED involved sequence and structure analysis of more than 8000 sequences. 31 structures have been applied to generate a reliable structure-based HMM profile in order to predict structurally conserved regions. Therefore, it was possible to automatically transfer these modules on CYP sequences without any secondary structure information, to analyze substrate interacting residues and to compare interaction sites with redox partners. Conclusions Functionally relevant structural sites of CYPs were predicted. Regions involved in substrate binding were analyzed in all sequences among the CYPED. For all CYPs that require a reductase, two reductase interaction sites were identified and classified according to their length. The newly gained insights promise an improvement of engineered enzyme properties for potential biotechnological application. The annotated sequences are accessible on the current version of the CYPED. The prediction tool can be applied to any CYP sequence via the web interface at http://www.cyped.uni-stuttgart.de/cgi-bin/strpred/dosecpred.pl.

  8. Detection of substrate-dependent conformational changes in the P450 fold by nuclear magnetic resonance.

    Science.gov (United States)

    Colthart, Allison M; Tietz, Drew R; Ni, Yuhua; Friedman, Jessica L; Dang, Marina; Pochapsky, Thomas C

    2016-01-01

    Cytochrome P450 monooxygenases typically catalyze the insertion of one atom of oxygen from O2 into unactivated carbon-hydrogen and carbon-carbon bonds, with concomitant reduction of the other oxygen atom to H2O by NAD(P)H. Comparison of the average structures of the camphor hydroxylase cytochrome P450(cam) (CYP101) obtained from residual dipolar coupling (RDC)-restrained molecular dynamics (MD) in the presence and absence of substrate camphor shows structural displacements resulting from the essential collapse of the active site upon substrate removal. This collapse has conformational consequences that extend across the protein structure, none of which were observed in analogous crystallographic structures. Mutations were made to test the involvement of the observed conformational changes in substrate binding and recognition. All of the mutations performed based upon the NMR-detected perturbations, even those remote from the active site, resulted in modified substrate selectivity, enzyme efficiency and/or haem iron spin state. The results demonstrate that solution NMR can provide insights into enzyme structure-function relationships that are difficult to obtain by other methods. PMID:26911901

  9. Cytochrome P450 2C9 gene polymorphism in phenytoin induced gingival enlargement: A case report

    Directory of Open Access Journals (Sweden)

    S. P. K. Kennedy Babu

    2013-01-01

    Full Text Available Gingival enlargement comprises any clinical condition in which an increase in the size of the gingiva is observed. Among the drugs that induce gingival enlargement, the antiepileptic agent phenytoin has been widely related to this condition. The Cytochrome P450(CYP superfamily is the most commonly involved enzymes in metabolism of drugs. Common coding region CYP variants that affects drug elimination and response has been studied in great detail. Pharmacogenetic influences on drug metabolism have been widely reviewed and gene polymorphism of cytochrome P450 2C9 appeared to be responsible for much of the interindividual variability on drug elimination. Genetic variation in the CYP2C9 gene can affect metabolism, leading to altered phenotypes. Individuals with poor metaboliser alleles of CYP2C9 gene were shown to have a reduced metabolism of phenytoin compared with wild-type alleles. Thus identification of patients genotype prior to anti-epileptic drug administration could potentially prevent higher serum drug concentrations leading to adverse side effects such as gingival enlargement. This case report addresses the influence of CYP2C9 genetic polymorphism on Phenytoin drug metabolism thereby causing gingival enlargement.

  10. Pi-pi Stacking Mediated Cooperative Mechanism for Human Cytochrome P450 3A4

    Directory of Open Access Journals (Sweden)

    Botao Fa

    2015-04-01

    Full Text Available Human Cytochrome P450 3A4 (CYP3A4 is an important member of the cytochrome P450 superfamily with responsibility for metabolizing ~50% of clinical drugs. Experimental evidence showed that CYP3A4 can adopt multiple substrates in its active site to form a cooperative binding model, accelerating substrate metabolism efficiency. In the current study, we constructed both normal and cooperative binding models of human CYP3A4 with antifungal drug ketoconazoles (KLN. Molecular dynamics simulation and free energy calculation were then carried out to study the cooperative binding mechanism. Our simulation showed that the second KLN in the cooperative binding model had a positive impact on the first one binding in the active site by two significant pi-pi stacking interactions. The first one was formed by Phe215, functioning to position the first KLN in a favorable orientation in the active site for further metabolism reactions. The second one was contributed by Phe304. This pi-pi stacking was enhanced in the cooperative binding model by the parallel conformation between the aromatic rings in Phe304 and the dioxolan moiety of the first KLN. These findings can provide an atomic insight into the cooperative binding in CYP3A4, revealing a novel pi-pi stacking mechanism for drug-drug interactions.

  11. Active sites of two orthologous cytochromes P450 2E1: Differences revealed by spectroscopic methods

    International Nuclear Information System (INIS)

    Cytochromes P450 2E1 of human and minipig origin were examined by absorption spectroscopy under high hydrostatic pressure and by resonance Raman spectroscopy. Human enzyme tends to denature to the P420 form more easily than the minipig form; moreover, the apparent compressibility of the heme active site (as judged from a redshift of the absorption maximum with pressure) is greater than that of the minipig counterpart. Relative compactness of the minipig enzyme is also seen in the Raman spectra, where the presence of planar heme conformation was inferred from band positions characteristic of the low-spin heme with high degree of symmetry. In this respect, the CYP2E1 seems to be another example of P450 conformational heterogeneity as shown, e.g., by Davydov et al. for CYP3A4 [Biochem. Biophys. Res. Commun. 312 (2003) 121-130]. The results indicate that the flexibility of the CYP active site is likely one of its basic structural characteristics

  12. CYP63A2, a catalytically versatile fungal P450 monooxygenase capable of oxidizing higher-molecular-weight polycyclic aromatic hydrocarbons, alkylphenols, and alkanes

    Science.gov (United States)

    Cytochrome P450 monooxygenases (P450s) are known to oxidize hydrocarbons albeit with limited substrate specificity across classes of these compounds. Here we report a P450 monooxygenase (CYP63A2) from the model ligninolytic white rot fungus Phanerochaete chrysosporium that was fo...

  13. The role of Barbie box sequences as cis-acting elements involved in the barbiturate-mediated induction of cytochromes P450BM-1 and P450BM-3 in Bacillus megaterium.

    Science.gov (United States)

    Liang, Q; He, J S; Fulco, A J

    1995-03-01

    In a previous publication (He, J.-S., and Fulco, A. J. (1991) J. Biol. Chem. 266, 7864-7869), we reported that a 15-17-base pair DNA sequence (designated a Barbie box element) in the 5'-regulatory regions of cytochrome P450BM-1 and P450BM-3 genes from Bacillus megaterium was recognized by a barbiturate-regulated protein. It is now recognized that essentially all eukaryotic and prokaryotic genes whose 5'-flanking regions are known and that encode barbiturate-inducible proteins contain the Barbie box element. A 4-base pair sequence (AAAG) is found in the same relative position in all Barbie box elements. In B. megaterium, mutation of the Barbie box located in the P450BM-1 gene leads to the constitutive synthesis of cytochrome P450BM-1 and a 10-fold increase of expression of Bm1P1, a small gene located upstream of the P450BM-1 gene, that encodes a putative regulatory protein. Mutation of the P450BM-3 Barbie box significantly increased the expression of both P450BM-3 and Bm3P1 (another small gene located upstream of the P450BM-3 gene that encodes a second putative regulatory protein) in response to pentobarbital induction but left the basal levels unaffected. In gel mobility shift assays, Bm3R1, a repressor of the P450BM-3 gene, was found to specifically interact with the Barbie box sequences of the B. megaterium P450 genes. Mutated Barbie boxes showed a decreased binding affinity for Bm3R1 compared to their wild type (unmutated) counterparts. Barbie box sequences were also shown to specifically interact with putative positive regulatory factors of B. megaterium cells. These putative positive factors were induced by pentobarbital and were also present at high levels during late stationary phase of B. megaterium cell cultures grown in the absence of barbiturates. The mutated Barbie box sequences had greater binding affinity for these positive factors than did unmutated Barbie box sequences. DNase I footprinting analysis of the 5'-flanking region of the P450BM-1 gene

  14. NMR Studies of Ligand Binding to P450eryF Provides Insight into the Mechanism of Cooperativity

    Energy Technology Data Exchange (ETDEWEB)

    Arthur G.,Roberts; M. Dolores,Díaz; Jed N.,Lampe; Laura M.,Shireman; Jeffrey S.,Grinstead; Michael J.,Dabrowski; Josh T.,Pearson; Michael K.,Bowman; William M.,Atkins; A. Patricia,Campbell

    2006-02-01

    Cytochrome P450's (P450's) catalyze the oxidative metabolism of most drugs and toxins. Although extensive studies have proven that some P450's demonstrate both homotropic and heterotropic cooperativity toward a number of substrates, the mechanistic and molecular details of P450 allostery are still not well-established. Here, we use UV/vis and heteronuclear nuclear magnetic resonance (NMR) spectroscopic techniques to study the mechanism and thermodynamics of the binding of two 9-aminophenanthrene (9-AP) and testosterone (TST) molecules to the erythromycin-metabolizing bacterial P450eryF. UV/vis absorbance spectra of P450eryF demonstrated that binding occurs with apparent negative homotropic cooperativity for TST and positive homotropic cooperativity for 9-AP with Hill-equation-derived dissociation constants of KS = 4 and 200 μM, respectively. The broadening and shifting observed in the 2D-{1H,15N}-HSQC-monitored titrations of 15N-Phe-labeled P450eryF with 9-AP and TST indicated binding on intermediate and fast chemical exhange time scales, respectively, which was consistent with the Hill-equation-derived KS values for these two ligands. Regardless of the type of spectral perturbation observed (broadening for 9-AP and shifting for TST), the 15N-Phe NMR resonances most affected were the same in each titration, suggesting that the two ligands ''contact'' the same phenylalanines within the active site of P450eryF. This finding is in agreement with X-ray crystal structures of bound P450eryF showing different ligands occupying similar active-site niches. Complex spectral behavior was additionally observed for a small collection of resonances in the TST titration, interpreted as multiple binding modes for the low-affinity TST molecule or multiple TST-bound P450eryF conformational substates. A structural and energetic model is

  15. NMR Studies of Ligand Binding to P450eryF Provides Insight into the Mechanism of Cooperativity

    Energy Technology Data Exchange (ETDEWEB)

    Roberts, Arthur G.; Diaz, Maria D.; Lampe, Jed N.; Shireman, Laura; Grinstead, Jeffrey S.; Dabrowski, Michael J.; Pearson, Josh T.; Bowman, Michael K.; Atkins, William M.; Campbell, Ann P.

    2006-02-14

    Cytochrome P450’s (P450’s) catalyze the oxidative metabolism of most drugs and toxins. Although extensive studies have proven that some P450’s demonstrate both homotropic and heterotropic cooperativity toward a number of substrates, the mechanistic and molecular details of P450 allostery are still not well-established. Here, we use UV/vis and heteronuclear nuclear magnetic resonance (NMR) spectroscopic techniques to study the mechanism and thermodynamics of the binding of two 9-aminophenanthrene (9-AP) and testosterone (TST) molecules to the erythromycin-metabolizing bacterial P450eryF. UV/vis absorbance spectra of P450eryF demonstrated that binding occurs with apparent negative homotropic cooperativity for TST and positive homotropic cooperativity for 9-AP with Hill-equation-derived dissociation constants of KS ) 4 and 200 íM, respectively. The broadening and shifting observed in the 2D-{1H,15N}-HSQC-monitored titrations of 15N-Phe-labeled P450eryF with 9-AP and TST indicated binding on intermediate and fast chemical exhange time scales, respectively, which was consistent with the Hillequation- derived KS values for these two ligands. Regardless of the type of spectral perturbation observed (broadening for 9-AP and shifting for TST), the 15N-Phe NMR resonances most affected were the same in each titration, suggesting that the two ligands “contact” the same phenylalanines within the active site of P450eryF. This finding is in agreement with X-ray crystal structures of bound P450eryF showing different ligands occupying similar active-site niches. Complex spectral behavior was additionally observed for a small collection of resonances in the TST titration, interpreted as multiple binding modes for the lowaffinity TST molecule or multiple TST-bound P450eryF conformational substates. A structural and energetic model is presented that combines the energetics and structural aspects of 9-AP and TST binding derived from these observations.

  16. 丽蝇蛹集金小蜂P450基因家族的发现和分子进化分析%Genome-level Identification and Evolution Analysis of P450 Gene Family in Nasonia vitripennis

    Institute of Scientific and Technical Information of China (English)

    刘金定; 李飞

    2013-01-01

    Nasonia vitripennisis is an important class of natural enemies of pests, which has been widely used in biological control. Though N. vitripennisis is not the direct target of insecticides, it is still likely to get in touch with insecticides in wide environment. P450 gene family is not only an important factor conferring insecticide resistance but also participates in synthesis and degradation of hormones. Therefore, investigation of P450 genes in N. vitripennisis is great helpful for fully using in biological control and protects it from insecticides. We identified 91 P450 genes from the genome of N. vitripennis. Gene family analysis demonstrated that there were 15 P450 super gene families and 27 subfamilies in N. vitripennis. We also found that five P450 gene groups meet positive selection and two gene groups have gene conversion. There is a positive relation between selection and gene conversion. The results indicated that CYP9P had the highest selection pressure. We also identified five motifs in N. vitripennis P450 gene family using MEME program. The occurrence frequencies of these five motifs were>90%. The“LAE[NH]P”was a new motif identified in N. vitripennis P450 gene family.%丽蝇蛹集金小蜂是一类重要的天敌昆虫,大量地应用于生物防治中。虽然寄生蜂不是化学农药的直接作用靶标,但其在自然环境中可能接触到化学农药。P450基因家族是昆虫产生抗药性的重要原因之一,同时也参与了昆虫激素的合成与降解。因此,研究 P450基因家族对充分利用丽蝇蛹集金小蜂,以及保护其免受农药毒杀具有重要的意义。本文利用生物学信息方法从丽蝇蛹集金小蜂基因组筛选到91个P450基因。家族分析表明,丽蝇蛹集金小蜂具有15个P450基因超家族、27个亚家族。对P450基因的正选择和基因转换开展了进一步分析,研究发现5个基因族存在正选择位点,2个基因族存在基因转换,正选择和

  17. Steroid biotransformations in biphasic systems with Yarrowia lipolytica expressing human liver cytochrome P450 genes

    Directory of Open Access Journals (Sweden)

    Braun Andreas

    2012-08-01

    Full Text Available Abstract Background Yarrowia lipolytica efficiently metabolizes and assimilates hydrophobic compounds such as n-alkanes and fatty acids. Efficient substrate uptake is enabled by naturally secreted emulsifiers and a modified cell surface hydrophobicity and protrusions formed by this yeast. We were examining the potential of recombinant Y. lipolytica as a biocatalyst for the oxidation of hardly soluble hydrophobic steroids. Furthermore, two-liquid biphasic culture systems were evaluated to increase substrate availability. While cells, together with water soluble nutrients, are maintained in the aqueous phase, substrates and most of the products are contained in a second water-immiscible organic solvent phase. Results For the first time we have co-expressed the human cytochromes P450 2D6 and 3A4 genes in Y. lipolytica together with human cytochrome P450 reductase (hCPR or Y. lipolytica cytochrome P450 reductase (YlCPR. These whole-cell biocatalysts were used for the conversion of poorly soluble steroids in biphasic systems. Employing a biphasic system with the organic solvent and Y. lipolytica carbon source ethyl oleate for the whole-cell bioconversion of progesterone, the initial specific hydroxylation rate in a 1.5 L stirred tank bioreactor was further increased 2-fold. Furthermore, the product formation was significantly prolonged as compared to the aqueous system. Co-expression of the human CPR gene led to a 4-10-fold higher specific activity, compared to the co-overexpression of the native Y. lipolytica CPR gene. Multicopy transformants showed a 50-70-fold increase of activity as compared to single copy strains. Conclusions Alkane-assimilating yeast Y. lipolytica, coupled with the described expression strategies, demonstrated its high potential for biotransformations of hydrophobic substrates in two-liquid biphasic systems. Especially organic solvents which can be efficiently taken up and/or metabolized by the cell might enable more

  18. Effects of quinolones on liver microsome cytochrome P450 in rats

    Directory of Open Access Journals (Sweden)

    Yi ZHANG

    2012-11-01

    Full Text Available Objective  To study and compare the effects of fluoroquinolones (levofloxacin, gatifloxacin, moxifloxacin and pazufloxacin on the enzyme system of liver microsome cytochrome P450 in rat. Methods  Thirty male Wistar rats were equally assigned into five groups: control group, levofloxacin (LV group, gatifloxacin (GT group, moxifloxacin (MX group and pazufloxacin (PZ group. Each drug was consecutively administered by tail vein injection for 7 days in a dosage of 120 mg/(kg•d. Liver microsomes were prepared by differential centrifugation, the concentration of protein in the liver microsome was measured by Lowry method, the content and activity of cy tochrome P450 were detected by spectrophotometric determination, and the results were analyzed by one-way ANOVA. Results  Compared with control group, the weight of liver in MX group and GT group was significantly reduced (P 0.05. Assay of aminopyrine-N-demethylase activity showed that the difference in enzyme activity was statistically significant between the control group and groups LV, GT and MX (P < 0.01. Erythromycin-N-demethylase activity measurement revealed that the enzyme activity was lowered in GT group and slightly elevated in MX group, and the difference was statistically significant compared with that of control group (P < 0.01. Measurement of activity of rat liver microsomal CYP450 enzyme system subfamily showed that the BROD activity increased in LV, MX and PZ groups (P < 0.01, and slightly decreased in GT group as compared with control group (P < 0.05. The PROD activity increased in GT group, but decreased in PZ group (P < 0.01. The EROD activity increased in all the four groups (P < 0.01. Conclusions  The four fluoroquinolones have some effects on the enzyme system of liver microsome cytochrome P450 in rats, but the effects may be different (enhancement or attenuation of the enzymatic activity depending on the enzymes, and the extent of the decrease of effect is in the

  19. Key Residues Controlling Phenacetin Metabolism By Human Cytochrome P450 2A Enzymes

    Energy Technology Data Exchange (ETDEWEB)

    DeVore, N.M.; Smith, B.D.; Urban, M.J.; Scott, E.E.

    2009-05-14

    Although the human lung cytochrome P450 2A13 (CYP2A13) and its liver counterpart cytochrome P450 2A6 (CYP2A6) are 94% identical in amino acid sequence, they metabolize a number of substrates with substantially different efficiencies. To determine differences in binding for a diverse set of cytochrome P450 2A ligands, we have measured the spectral binding affinities (K{sub D}) for nicotine, phenethyl isothiocyanate (PEITC), coumarin, 2{prime}-methoxyacetophenone (MAP), and 8-methoxypsoralen. The differences in the K{sub D} values for CYP2A6 versus CYP2A13 ranged from 74-fold for 2{prime}-methoxyacetophenone to 1.1-fold for coumarin, with CYP2A13 demonstrating the higher affinity. To identify active site amino acids responsible for the differences in binding of MAP, PEITC, and coumarin, 10 CYP2A13 mutant proteins were generated in which individual amino acids from the CYP2A6 active site were substituted into CYP2A13 at the corresponding position. Titrations revealed that substitutions at positions 208, 300, and 301 individually had the largest effects on ligand binding. The collective relevance of these amino acids to differential ligand selectivity was verified by evaluating binding to CYP2A6 mutant enzymes that incorporate several of the CYP2A13 amino acids at these positions. Inclusion of four CYP2A13 amino acids resulted in a CYP2A6 mutant protein (I208S/I300F/G301A/S369G) with binding affinities for MAP and PEITC much more similar to those observed for CYP2A13 than to those for CYP2A6 without altering coumarin binding. The structure-based quantitative structure-activity relationship analysis using COMBINE successfully modeled the observed mutant-ligand trends and emphasized steric roles for active site residues including four substituted amino acids and an adjacent conserved Leu{sup 370}.

  20. Sexually dimorphic regulation and induction of P450s by the constitutive androstane receptor (CAR)

    International Nuclear Information System (INIS)

    The constitutive androstane receptor (CAR) is a xenosensing nuclear receptor and regulator of cytochrome P450s (CYPs). However, the role of CAR as a basal regulator of CYP expression nor its role in sexually dimorphic responses have been thoroughly studied. We investigated basal regulation and sexually dimorphic regulation and induction by the potent CAR activator TCPOBOP and the moderate CAR activator Nonylphenol (NP). NP is an environmental estrogen and one of the most commonly found environmental toxicants in Europe and the United States. Previous studies have demonstrated that NP induces several CYPs in a sexually dimorphic manner, however the role of CAR in regulating NP-mediated sexually dimorphic P450 expression and induction has not been elucidated. Therefore, wild-type and CAR-null male and female mice were treated with honey as a carrier, NP, or TCPOBOP and CYP expression monitored by QPCR and Western blotting. CAR basally regulates the expression of Cyp2c29, Cyp2b13, and potentially Cyp2b10 as demonstrated by QPCR. Furthermore, we observed a shift in the testosterone 6α/15α-hydroxylase ratio in untreated CAR-null female mice to the male pattern, which indicates an alteration in androgen status and suggests a role for androgens as CAR inverse agonists. Xenobiotic-treatments with NP and TCPOBOP induced Cyp2b10, Cyp2c29, and Cyp3a11 in a CAR-mediated fashion; however NP only induced these CYPs in females and TCPOBOP induced these CYPs in both males and females. Interestingly, Cyp2a4, was only induced in wild-type male mice by TCPOBOP suggesting Cyp2a4 induction is not sensitive to CAR-mediated induction in females. Overall, TCPOBOP and NP show similar CYP induction profiles in females, but widely different profiles in males potentially related to lower sensitivity of males to either indirect or moderate CAR activators such as NP. In summary, CAR regulates the basal and chemically inducible expression of several sexually dimorphic xenobiotic metabolizing P

  1. P450XXI (steroid 21-hydroxylase) gene deletions are not found in family studies of congenital adrenal hyperplasia

    International Nuclear Information System (INIS)

    Congenital adrenal hyperplasia (CAH) is a common genetic disorder due to defective 21-hydroxylation of steroid hormones. The human P450XXIA2 gene encodes cytochrome P450c21 [steroid 21-monooxygenase (steroid 21-hydroxylase)], which mediates 21-hydroxylation. The P450XXIA2 gene may be distinguished from the duplicated P450XXIA1 pseudogene by cleavage with the restriction endonuclease Taq I, with the XXIA2 gene characterized by a 3.7-kilobase (kb) fragment and the XXIA1 pseudogene characterized by a 3.2-kb fragment. Restriction endonuclease mapping by several laboratories has suggested that deletion of the P450XXIA2 gene occurs in about 25% of patients with CAH, as their genomic DNA lacks detectable 3.7-kb Taq I fragments. The authors have cloned human P450c21 cDNA and used it to study genomic DNA prepared from 51 persons in 10 families, each of which includes 2 or more persons with CAH. After Taq I digestion, apparent deletions are seen in 7 of the 20 alleles of the probands; using EcoRI, apparent deletions are seen in 9 of the 20 alleles. However, the apparently deleted alleles seen with Taq I do not coincide with those seen with EcoRI. Furthermore, studies with Bgl II, EcoRI, Kpn I, and Xba I yield normal patterns with at least two enzymes in all cases. Since all probands yielded normal patterns with at least two of the five enzymes used, they conclude that the P450XXIA2 gene deletions widely reported in CAH patients probably represent gene conversions, unequal crossovers,or polymorphisms rather than simple gene deletions

  2. Multiple Cytochrome P450 genes: their constitutive overexpression and permethrin induction in insecticide resistant mosquitoes, Culex quinquefasciatus.

    Directory of Open Access Journals (Sweden)

    Nannan Liu

    Full Text Available Four cytochrome P450 cDNAs, CYP6AA7, CYP9J40, CYP9J34, and CYP9M10, were isolated from mosquitoes, Culex quinquefasciatus. The P450 gene expression and induction by permethrin were compared for three different mosquito populations bearing different resistance phenotypes, ranging from susceptible (S-Lab, through intermediate (HAmCq(G0, the field parental population to highly resistant (HAmCq(G8, the 8(th generation of permethrin selected offspring of HAmCq(G0. A strong correlation was found for P450 gene expression with the levels of resistance and following permethrin selection at the larval stage of mosquitoes, with the highest expression levels identified in HAmCq(G8, suggesting the importance of CYP6AA7, CYP9J40, CYP9J34, and CYP9M10 in the permethrin resistance of larva mosquitoes. Only CYP6AA7 showed a significant overexpression in HAmCq(G8 adult mosquitoes. Other P450 genes had similar expression levels among the mosquito populations tested, suggesting different P450 genes may be involved in the response to insecticide pressure in different developmental stages. The expression of CYP6AA7, CYP9J34, and CYP9M10 was further induced by permethrin in resistant mosquitoes. Taken together, these results indicate that multiple P450 genes are up-regulated in insecticide resistant mosquitoes through both constitutive overexpression and induction mechanisms, thus increasing the overall expression levels of P450 genes.

  3. Metabolism of agrochemicals and related environmental chemicals based on cytochrome P450s in mammals and plants.

    Science.gov (United States)

    Ohkawa, Hideo; Inui, Hideyuki

    2015-06-01

    A yeast gene expression system originally established for mammalian cytochrome P450 monooxygenase cDNAs was applied to functional analysis of a number of mammalian and plant P450 species, including 11 human P450 species (CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1 and CYP3A4). The human P450 species CYP1A1, CYP1A2, CYP2B6, CYP2C18 and CYP2C19 were identified as P450 species metabolising various agrochemicals and environmental chemicals. CYP2C9 and CYP2E1 specifically metabolised sulfonylurea herbicides and halogenated hydrocarbons respectively. Plant P450 species metabolising phenylurea and sulfonylurea herbicides were also identified mainly as the CYP71 family, although CYP76B1, CYP81B1 and CYP81B2 metabolised phenylurea herbicides. The transgenic plants expressing these mammalian and plant P450 species were applied to herbicide tolerance as well as phytoremediation of agrochemical and environmental chemical residues. The combined use of CYP1A1, CYP2B6 and CYP2C19 belonging to two families and three subfamilies covered a wide variety of herbicide tolerance and phytoremediation of these residues. The use of 2,4-D-and bromoxynil-induced CYP71AH11 in tobacco seemed to enhance herbicide tolerance and selectivity. PMID:25077812

  4. Influence of N,N-dimethylaniline on the association of phenobarbital-induced cytochrome P-450 and NADPH-cytochrome c(P-450) reductase in a reconstituted rabbit liver microsomal enzyme system.

    Science.gov (United States)

    Hlavica, P; Golly, I; Wolf, J

    1987-09-01

    N,N-Dimethylaniline when added to reaction mixtures provokes deviation from Michaelis-Menten law of the interaction kinetics of NADPH-cytochrome c(P-450) reductase (NADPH:ferrihaemoprotein oxidoreductase, EC 1.6.2.4) with highly purified phenobarbital-induced rabbit liver microsomal cytochrome P-450 (P-450LM2). This phenomenon is not associated with the low-to-high spin transition in the iron-coordination sphere of the haemoprotein, as elicited by the arylamine. Substrate-triggered departure from linearity of the kinetics is abolished by inclusion into the assay media of p-chloromercuribenzoate, hinting at a vital role in the process of thiols. Similarly, the parabolic progress curve (nH = 1.7) is transformed to a straight line (nH = 1.01) when the N-terminal reductase-binding domain in the P-450LM2 molecule is selectively blocked through covalent attachment of fluorescein isothiocyanate (FITC); such a modification does not alter the affinity of the haemoprotein for the amine substrate. Steady-state fluorescence polarization measurements reveal that N,N-dimethylaniline perturbs the motional properties of the fluorophore-bearing reductase-binding region, suggesting the induction of a conformational change. Summarizing these results, the data possibly indicate N,N-dimethylaniline-induced cooperativity in the association of reductase with P-450LM2. PMID:3113486

  5. Virtual Screening and Prediction of Site of Metabolism for Cytochrome P450 1A2 Ligands

    DEFF Research Database (Denmark)

    Vasanthanathan, P.; Hritz, Jozef; Taboureau, Olivier; Olsen, Lars; Jørgensen, F.S.; Vermeulen, N.P.E.; Oostenbrink, C.

    2009-01-01

    questions have been addressed: 1. Binding orientations and conformations were successfully predicted for various substrates. 2. A virtual screen was performed with satisfying enrichment rates. 3. A classification of individual compounds into active and inactive was performed. It was found that while docking...... can be used successfully to address the first two questions, it seems to be more difficult to perform the classification. Different scoring functions were included, and the well-characterized water molecule in the active site was included in various ways. Results are compared to experimental data and...... earlier classification data using machine learning methods. The possibilities and limitations of using structure-based drug design tools for cytochrome P450 1A2 come to light and are discussed....

  6. Cytochrome P450 genes in coronary artery diseases: Codon usage analysis reveals genomic GC adaptation.

    Science.gov (United States)

    Malakar, Arup Kumar; Halder, Binata; Paul, Prosenjit; Chakraborty, Supriyo

    2016-09-15

    Establishing codon usage biases are imperative for understanding the etiology of coronary artery diseases (CAD) as well as the genetic factors associated with these diseases. The aim of this study was to evaluate the contribution of 18 responsible cytochrome P450 (CYP) genes for the risk of CAD. Effective number of codon (Nc) showed a negative correlation with both GC3 and synonymous codon usage order (SCUO) suggesting an antagonistic relationship between codon usage and Nc of genes. The dinucleotide analysis revealed that CG and TA dinucleotides have the lowest odds ratio in these genes. Principal component analysis showed that GC composition has a profound effect in separating the genes along the first major axis. Our findings revealed that mutational pressure and natural selection could possibly be the major factors responsible for codon bias in these genes. The study not only offers an insight into the mechanisms of genomic GC adaptation, but also illustrates the complexity of CYP genes in CAD. PMID:27275533

  7. Inhibition of cytochrome P450 by furanocoumarins in grapefruit juice and herbal medicines

    Institute of Scientific and Technical Information of China (English)

    Lian-qing GUO; Yasushi YAMAZOE

    2004-01-01

    Furanocoumarins (psoralens) exist in various plants and some of them are used to cure skin diseases. These chemicals draw attentions recently because of their abilities to arouse drug interaction through inhibition of cytochrome P450. Grapefruit juice is a well-known example for food-drug interaction. But in vitro and in vivo studies have shown that the causative components are mainly furanocoumarin derivatives with geranyloxy side chains. In vitro experiments confirmed that furanocoumarins from grapefruit juice are both competitive and mechanismbased inhibitors of CYP3A4. Although the inhibition appeared to be stronger in the dimers than that in the monomers,all contribute comprehensively to the grapefruit juice-drug interaction. Further experiments with other furanocoumarins and related citrus fruits or umbelliferous herbal medicines indicate that drug interaction might also occur with stuffs other than grapefruit juice, especially with traditional medicine.

  8. The Halloween genes code for cytochrome P450 enzymes mediating synthesis of the insect molting hormone

    DEFF Research Database (Denmark)

    Rewitz, Kim; Rybczynski, Robert; Warren, James T.; Gilbert, Lawrence I.

    2006-01-01

    The developmental events occurring during molting and metamorphosis of insects are controlled by precisely timed changes in levels of ecdysteroids, the molting hormones. The final four sequential hydroxylations of steroid precursors into the active ecdysteroid of insects, 20-hydroxyecdysone (20E......), are mediated by four cytochrome P450 enzymes, encoded by genes in the Halloween family. Orthologs of the Drosophila Halloween genes phantom (phm: CYP306A1), disembodied (dib: CYP302A1), shadow (sad: CYP315A1) and shade (shd: CYP314A1) were obtained from the endocrinological model insect, the tobacco...... hornworm Manduca sexta. Expression of these genes was studied and compared with changes in ecdysteroid titer that controls transition from the larval to pupal stage. Phm, dib and sad (mediating the final hydroxylations in the biosynthesis of ecdysone; E) were selectively expressed in the prothoracic gland...

  9. In vitro metabolism of genistein and tangeretin by human and murine cytochrome p450s

    DEFF Research Database (Denmark)

    Breinholt, Vibeke; Rasmussen, Salka; Brøsen, Kim;

    2003-01-01

    Recombinant cytochrome P450 (CYP) 1A2, 3A4, 2C9 or 2D6 enzymes obtained from Escherichia coli and human liver microsomes samples were used to investigate the ability of human CYP enzymes to metabolize the two dietary flavonoids, genistein and tangeretin. Analysis of the metabolic profile from...... incubations with genistein and human liver microsomes revealed the production of five different metabolites, of which three were obtained in sufficient amounts to allow a more detailed elucidation of the structure. One of these metabolites was identified as orobol, the 3'-hydroxylated metabolite of genistein....... The remaining two metabolites were also hydroxylated metabolites as evidenced by LC/MS. Orobol was the only metabolite formed after incubation with CYP1A2. The two major product peaks after incubation of tangeretin with human microsomes were identical with 4'-hydroxy-5,6,7,8-tetramethoxyflavone and 5...

  10. Comparable constitutive expression and activity of cytochrome P450 between the lobes of the porcine liver.

    Science.gov (United States)

    Rasmussen, Martin Krøyer; Zamaratskaia, Galia; Ekstrand, Bo

    2014-10-01

    Due to limited availability of human liver tissue for the study of cytochrome P450 (CYP450), porcine liver tissue has been suggested as an alternative source to prepare microsomes and hepatocytes. The porcine liver is made by four different lobes. The present study investigated the expression and activity of specific CYP450 isoforms in the four lobes, with the purpose to examine if one lobe of the porcine liver resembles the human more than others. Samples from the four major lobes were taken from female pigs and mRNA expression and activity of CYP1A, 2A, 2C, 2D, 2E and 3A determined. The results showed no differences in specific mRNA expression and activity of any of the investigated CYP450 isoforms. In conclusion, the study shows that all parts of the porcine liver are equally useful as model tissue. PMID:24952075

  11. The Influence of Cytochrome P450 Pharmacogenetics on Disposition of Common Antidepressant and Antipsychotic Medications

    Science.gov (United States)

    van der Weide, Jan; Hinrichs, John WJ

    2006-01-01

    Since the identification of all the major drug-metabolising cytochrome P450 (CYP) enzymes and their major gene variants, pharmacogenetics has had a major impact on psychotherapeutic drug therapy. CYP enzymes are responsible for the metabolism of most clinically used drugs. Individual variability in CYP activity is an important reason for drug therapy failure. Variability in CYP activity may be caused by various factors, including endogenous factors such as age, gender and morbidity as well as exogenous factors such as co-medication, food components and smoking habit. However, polymorphisms, present in most CYP genes, are responsible for a substantial part of this variability. Although CYP genotyping has been shown to predict the majority of aberrant phenotypes, it is currently rarely performed in clinical practice. PMID:16886044

  12. Effects of genetic polymorphism of cytochrome P450 enzymes on the pharmacokinetics of benzodiazepines.

    Science.gov (United States)

    Fukasawa, T; Suzuki, A; Otani, K

    2007-08-01

    Pharmacogenetic studies have shown that several cytochrome P450 (CYP) enzymes exhibit genetic polymorphisms. Several benzodiazepines (BZPs) are metabolized predominantly or partly by polymorphic CYP2C19 and CYP3A4/5. The pharmacokinetics of diazepam, etizolam, quazepam and desmethylclobazam have been shown to be affected by CYP2C19 polymorphism. The CYP3A5 polymorphism has been reported to affect the pharmacokinetics of alprazolam, but its effect on midazolam kinetics has been inconclusive. For etizolam and desmethylclobazam, some data suggest that CYP2C19 deficiency leads to side-effects or toxicity. For the remaining BZPs the clinical significance of the observed pharmacokinetic changes remains unclear. Further studies on the effects of genetic polymorphisms of CYP enzymes on the pharmacokinetics and pharmacodynamics of BZPs are necessary to guide treatment individualization and optimization. PMID:17635335

  13. Cytochrome P450 1A1 expression in cetacean skin biopsies from the Indian Ocean.

    Science.gov (United States)

    Jauniaux, Thierry; Farnir, Frédéric; Fontaine, Michaël; Kiszka, Jeremy; Sarlet, Michael; Coignoul, Freddy

    2011-06-01

    The study describes cytochrome P450 1A1 (CYPA1) expression in the skin of different cetacean species (Megaptera novaeangliae, n=15; Stenella attenuata, n=7 and Stenella longirostris, n=24) from the Mozambique Channel island of Mayotte. Immunohistochemical examination was performed with a monoclonal antibody against scup cytochrome CYPA1. The sex was determined using a molecular approach consisting in the genotyping sex-specific genes. CYPA1 was detected at the junction between epidermis and blubber on dolphins only, mostly in the endothelial cells. Similar observation was obtained in the dermis of one M. novaeangliae. Immunohistochemical slides were scored to evaluate the expression of the CYPA1 and a higher expression was observed in S. longirostris, suggesting a higher exposure to pollutants for this species. The difference of expression between sexes was not significant. PMID:21565363

  14. Genetically Modified Caco-2 Cells With Improved Cytochrome P450 Metabolic Capacity.

    Science.gov (United States)

    Küblbeck, Jenni; Hakkarainen, Jenni J; Petsalo, Aleksanteri; Vellonen, Kati-Sisko; Tolonen, Ari; Reponen, Petri; Forsberg, Markus M; Honkakoski, Paavo

    2016-02-01

    The human intestinal Caco-2 cell line has been extensively used as a model of small intestinal absorption but it lacks expression and function of cytochrome P450 enzymes, particularly CYP3A4 and CYP2C9, which are normally expressed in the intestinal epithelium. In order to increase the expression and activity of CYP isozymes in these cells, we created 2 novel Caco-2 sublines expressing chimeric constitutive androstane or pregnane X receptors and characterized these cells for their metabolic and absorption properties. In spite of elevated mRNA expression of transporters and differentiation markers, the permeation properties of the modified cell lines did not significantly differ from those of the wild-type cells. In contrast, the metabolic activity was increased beyond the currently used models. Specifically, CYP3A4 activity was increased up to 20-fold as compared to vitamin D treated wild-type Caco-2 cells. PMID:26869438

  15. Structural basis for human NADPH-cytochrome P450 oxidoreductase deficiency

    Energy Technology Data Exchange (ETDEWEB)

    Xia, Chuanwu; Panda, Satya P.; Marohnic, Christopher C.; Martásek, Pavel; Masters, Bettie Sue; Kim, Jung-Ja P. (MCW); (Charles U); (UTSMC)

    2012-03-15

    NADPH-cytochrome P450 oxidoreductase (CYPOR) is essential for electron donation to microsomal cytochrome P450-mediated monooxygenation in such diverse physiological processes as drug metabolism (approximately 85-90% of therapeutic drugs), steroid biosynthesis, and bioactive metabolite production (vitamin D and retinoic acid metabolites). Expressed by a single gene, CYPOR's role with these multiple redox partners renders it a model for understanding protein-protein interactions at the structural level. Polymorphisms in human CYPOR have been shown to lead to defects in bone development and steroidogenesis, resulting in sexual dimorphisms, the severity of which differs significantly depending on the degree of CYPOR impairment. The atomic structure of human CYPOR is presented, with structures of two naturally occurring missense mutations, V492E and R457H. The overall structures of these CYPOR variants are similar to wild type. However, in both variants, local disruption of H bonding and salt bridging, involving the FAD pyrophosphate moiety, leads to weaker FAD binding, unstable protein, and loss of catalytic activity, which can be rescued by cofactor addition. The modes of polypeptide unfolding in these two variants differ significantly, as revealed by limited trypsin digestion: V492E is less stable but unfolds locally and gradually, whereas R457H is more stable but unfolds globally. FAD addition to either variant prevents trypsin digestion, supporting the role of the cofactor in conferring stability to CYPOR structure. Thus, CYPOR dysfunction in patients harboring these particular mutations may possibly be prevented by riboflavin therapy in utero, if predicted prenatally, or rescued postnatally in less severe cases.

  16. Evidence for concerted kinetic oxidation of progesterone by purified rat hepatic cytochrome P-450g

    International Nuclear Information System (INIS)

    Purified cytochrome P-450g, a male-specific rat hepatic isozyme, was observed to metabolize progesterone to two primary metabolites (6β-hydroxyprogesterone and 16α-hydroxyprogesterone), two secondary metabolites (6β,16α-dihydroxyprogesterone and 6-ketoprogesterone), and one tertiary metabolite (6-keto-16α-hydroxyprogesterone). The K/sub m,app/ for the formation of these products from progesterone was determined to be approximately 0.5 μM, while the K/sub m,app/ for metabolism of 6β- and 16α-hydroxyprogesterone was found to be 5-10 μM. The ratio of primary to secondary metabolites did not change significantly at progesterone concentrations from 6 to 150 μM, and a lag in formation of secondary metabolites was not observed in 1-min incubations. Concerted oxidation of progesterone to secondary products without the intermediate products leaving the active site was suggested by these results and confirmed by isotopic dilution experiments in which little or no dilution of metabolically formed 6β,16α-dihydroxyprogesterone and 6-keto-16α-hydroxyprogesterone was observed in incubations containing a mixture of radiolabeled progesterone and unlabeled 6β-hydroxyprogesterone or 16α-hydroxyprogesterone. Incubation of 6β-hydroxyprogesterone with a reconstituted system in an atmosphere of 18I2 resulted in > 90% incorporation of 18O in the 16α-position of 6β,16α-dihydroxyprogesterone but no incorporation of 18O into 6-ketoprogesterone, even though the reaction was dependent upon enzyme and O2, and not inhibited by mannitol, catalase, or superoxide dismutase. Factors which characterize the metabolism of progesterone by cytochrome P-450g in terms of active-site constraints and the catalytic competence of the enzyme in microsomes were also explored

  17. Cytochrome P450-mediated hepatic metabolism of new fluorescent substrates in cats and dogs.

    Science.gov (United States)

    van Beusekom, C D; Schipper, L; Fink-Gremmels, J

    2010-12-01

    This study aimed to investigate the biotransformation of cat liver microsomes in comparison to dogs and humans using a high throughput method with fluorescent substrates and classical inhibitors specific for certain isozymes of the human cytochrome P450 (CYP) enzyme family. The metabolic activities associated with CYP1A, CYP2B, CYP2C, CYP2D, CYP2E and CYP3A were measured. Cat liver microsomes metabolized all substrates selected for the assessment of cytochrome P450 activity. The activities associated with CYP3A and CYP2B were higher than the activities of the other measured CYPs. Substrate selectivity could be demonstrated by inhibition studies with α-naphthoflavone (CYP1A), tranylcypromine/quercetine (CYP2C), quinidine (CYP2D), diethyldithiocarbamic acid (CYP2E) and ketoconazole (CYP3A) respectively. Other prototypical inhibitors used for characterization of human CYP activities such as furafylline (CYP1A), tranylcypromine (CYP2B) and sulfaphenazole (CYP2C) did not show significant effects in cat and dog liver microsomes. Moreover, IC50-values of cat CYPs differed from dog and human CYPs underlining the interspecies differences. Gender differences were observed in the oxidation of 7-ethoxy-4-trifluoromethylcoumarin (CYP2B) and 3-[2-(N, N-diethyl-N-methylamino)ethyl]-7-methoxy-4-methylcoumarin (CYP2D), which were significantly higher in male cats than in females. Conversely, oxidation of the substrates dibenzylfluorescein (CYP2C) and 7-methoxy-4-trifluoromethylcoumarin (CYP2E) showed significant higher activities in females than in male cats. Overall CYP-activities in cat liver microsomes were lower than in those from dogs or humans, except for CYP2B. The presented difference between feline and canine CYP-activities are useful to establish dose corrections for feline patients of intensively metabolized drugs licensed for dogs or humans. PMID:21062303

  18. Biosynthesis of Sandalwood Oil: Santalum album CYP76F cytochromes P450 produce santalols and bergamotol.

    Directory of Open Access Journals (Sweden)

    Maria L Diaz-Chavez

    Full Text Available Sandalwood oil is one of the world's most highly prized essential oils, appearing in many high-end perfumes and fragrances. Extracted from the mature heartwood of several Santalum species, sandalwood oil is comprised mainly of sesquiterpene olefins and alcohols. Four sesquiterpenols, α-, β-, and epi-β-santalol and α-exo-bergamotol, make up approximately 90% of the oil of Santalum album. These compounds are the hydroxylated analogues of α-, β-, and epi-β-santalene and α-exo-bergamotene. By mining a transcriptome database of S. album for candidate cytochrome P450 genes, we cloned and characterized cDNAs encoding a small family of ten cytochrome P450-dependent monooxygenases annotated as SaCYP76F37v1, SaCYP76F37v2, SaCYP76F38v1, SaCYP76F38v2, SaCYP76F39v1, SaCYP76F39v2, SaCYP76F40, SaCYP76F41, SaCYP76F42, and SaCYP76F43. Nine of these genes were functionally characterized using in vitro assays and yeast in vivo assays to encode santalene/bergamotene oxidases and bergamotene oxidases. These results provide a foundation for production of sandalwood oil for the fragrance industry by means of metabolic engineering, as demonstrated with proof-of-concept formation of santalols and bergamotol in engineered yeast cells, simultaneously addressing conservation challenges by reducing pressure on supply of sandalwood from native forests.

  19. Association of cytochrome P450 genetic polymorphisms with neoadjuvant chemotherapy efficacy in breast cancer patients

    Directory of Open Access Journals (Sweden)

    Seredina Tatyana A

    2012-06-01

    Full Text Available Abstract Background The enzymes of the cytochrome P450 family (CYPs play an important role in the metabolism of a great variety of anticancer agents; therefore, polymorphisms in genes encoding for metabolizing enzymes and drugs transporters can affect drug efficacy and toxicity. Methods The genetic polymorphisms of cytochrome P450 were studied in 395 patients with breast cancer by RLFP analysis. Results Here, we studied the association of functionally significant variant alleles of CYP3A4, CYP3A5, CYP2B6, CYP2C8, CYP2C9 and CYP2C19 with the clinical response to neoadjuvant chemotherapy in breast cancer patients. A significant correlation was observed between the CYP2C9*2 polymorphism and chemotherapy resistance (OR = 4.64; CI 95% = 1.01 – 20.91, as well as between CYP2C9*2 heterozygotes and chemotherapy resistance in women with nodal forms of breast cancer and a cancer hereditary load (OR = 15.50; CI 95% = 1.08 – 826.12 when the potential combined effects were examined. No significant association between chemotherapy resistance and the other examined genotypes and the potential combined clinical and tumour-related parameters were discovered. Conclusion In conclusion, CYP2C9*2 was associated with neoadjuvant chemotherapy resistance (OR = 4.64; CI 95% = 1.01 – 20.91 in the population of interest.

  20. Structure and function of cytochrome P450S in insect adaptation to natural and synthetic toxins: insights gained from molecular modeling.

    Science.gov (United States)

    Schuler, Mary A; Berenbaum, May R

    2013-09-01

    Over evolutionary time, insect herbivores have adapted to the presence of natural toxins and more recently to synthetic insecticides in or on the plants they consume. Biochemical analyses and molecular modeling of the cytochrome P450 monooxygenases (P450s) that metabolize these compounds have provided insight into the many variations affecting their catalytic activity. Phylogenetically distinct P450s may metabolize similar substrates, and phylogenetically similar P450s may metabolize different substrates; as well, some P450s process broad arrays of both phytochemicals and synthetic insecticides, while closely related P450s are restricted to a narrow range of phytochemicals. Mapped on the predicted three-dimensional structures of insect P450s developed from available mammalian P450 crystal structures, differences in multiple regions of the insect proteins reveal the evolutionary processes occurring as P450 genes have duplicated and diverged. Analyses of site-directed mutants in select lepidopteran and dipteran P450s demonstrate that slight changes in the catalytic site, the putative product release channel, and the proximal surface (interacting with electron transfer partners such as cytochrome P450 reductase and cytochrome b5) yield pronounced activity differences. Additionally, changes in the catalytic site and in the linker region preceding the proline-hinge influence P450 folding. With predicted structures available for many mammalian P450s involved in metabolism of xenobiotics, it is possible to record allelic variation relative to catalytically important regions in the overall P450 structure and to predict functionally critical differences. Together with information on the relative levels of allelic variant transcripts, comprehensive characterization of the mechanisms that modulate metabolism of natural and synthetic xenobiotics in insects can yield insights into plant-insect coevolution and into novel approaches for chemical pest management. PMID:24036972

  1. Effects of dietary quercetin on performance and cytochrome P450 expression of the cotton bollworm, Helicoverpa armigera.

    Science.gov (United States)

    Liu, D; Yuan, Y; Li, M; Qiu, X

    2015-12-01

    Quercetin is ubiquitous in terrestrial plants. The cotton bollworm Helicoverpa armigera as a highly polyphagous insect has caused severe crop losses. Until now, interactions between this pest and quercetin are poorly understood at the biochemical and molecular levels. In this study, we investigated the in vivo effects of quercetin on performance of cotton bollworm and on cytochrome P450 (P450) expression. Deleterious effects of quercetin on the performance of the cotton bollworm, including growth, survival, pupation and adult emergence were observed after oral administration of 3 and 10 mg g(-1) quercetin to larvae since the third instar, whereas no significant toxic effect was found at 0.1 mg g(-1) quercetin treatment. Piperonyl butoxide treatment enhanced the toxicity of quercetin. In vitro metabolism studies showed that quercetin was rapidly transformed by gut enzymes of fifth instar larvae of the cotton bollworm. qRT-PCR results revealed that the effect of quercetin on P450 expression was tissue- and dose-specific. Quercetin regulated P450 expression in a mild manner, and it could serve as P450 inducer (CYP337B1, CYP6B6) or repressor (CYP337B1, CYP6B7, CYP6B27, CYP9A14, CYP6AE11, and CYP4M7). These findings are important for advancing our understanding of the biochemical and molecular response of insects to plant toxins and have implications for a smart pest control. PMID:26440448

  2. Role of lipid peroxidation in cytochrome P-450 degradation in hepatocytes and lymphocytes and stabilizing effects of antioxidants

    Energy Technology Data Exchange (ETDEWEB)

    Kagan, V.; Novikov, K.; Bogdanova, E.; Prilipko, L.

    1986-01-01

    Incubation of primary culture of hepatocytes was accompanied by spontaneous degradation of cytochrome P-450 (P-450) and by a decrease of its monooxygenase activities. Accumulation of lipid peroxidation (LPO) products also occurred. Addition of LPO inducers (Fe/sup 2 +/-ADP, NADPH) to the medium led to a drastic acceleration of both processes. Synthetic free radical scavengers (butylated hydroxytoluene, 3-hydroxybenzene, alpha-naphthol) prevented LPO activation and protected P-450 against degradation. Phenolic derivatives of 3,4-benzpyrene, formed as a result of its hydroxylation, also had an antioxidant and a protective effect on P-450. Similarly, incubation of human peripheral blood lymphocytes stimulated by PHA resulted in a rapid inhibition of arylhydrocarbon hydroxylase (AHH). In lymphocytes stimulated by PHA and then induced by 3-methylcholanthrene, the sharply increased AHH activity remained unchanged for a long period and no LPO products were accumulated. Exogenous LPO inducers did not either stimulate LPO and were ineffective on the AHH activity. This was due to the presence of 3-methyl cholanthrene phenolic derivatives formed during lymphocytes induction. It is concluded that free radical scavengers could be used as P-450 stabilizers in cell cultures.

  3. Kinetic analysis of the three-step steroid aromatase reaction of human cytochrome P450 19A1.

    Science.gov (United States)

    Sohl, Christal D; Guengerich, F Peter

    2010-06-01

    Cytochrome P450 19A1 (P450 19A1), the aromatase, catalyzes the conversion of androgens to estrogens through a sequential three-step reaction, generating 19-hydroxy and 19-aldehyde intermediates en route to the product estrogen. A procedure for the heterologous expression and purification of P450 19A1 in Escherichia coli was developed (k(cat) of 0.06 s(-1) for the conversion of androstenedione to estrone). Binding of the substrate and intermediates show low micromolar dissociation constants and are at least two-step processes. Rates of reduction of the iron were fast in the presence of substrate, either intermediate, or product. P450 19A1 is a distributive rather than a processive enzyme, with the sequential reaction allowing free dissociation of the intermediates as revealed by pulse-chase experiments. Conversion of androstenedione to estrone (under single turnover conditions) generated a progress curve showing changes in the concentrations of the substrate, intermediates, and product. A minimal kinetic model containing the individual rate constants for the steps in P450 19A1 catalysis was developed to globally fit the time course of the overall reaction, the dissociation constants, the two-step ligand binding, the distributive character, the iron-reduction rates, and the steady-state conversion of the 19-hydroxy androstenedione and 19-aldehyde androstenedione intermediates to estrone. PMID:20385561

  4. Direct retroviral delivery of human cytochrome P450 2B6 for gene-directed enzyme prodrug therapy of cancer.

    Science.gov (United States)

    Kan, O; Griffiths, L; Baban, D; Iqball, S; Uden, M; Spearman, H; Slingsby, J; Price, T; Esapa, M; Kingsman, S; Kingsman, A; Slade, A; Naylor, S

    2001-07-01

    Human cytochrome P450 2B6 (CYP2B6) metabolizes the prodrug cyclophosphamide (CPA) to produce phosphoramide mustard that cross-links DNA leading to cell death. We have constructed a novel retroviral vector encoding CYP2B6 (designated "MetXia-P450") and used it to transduce the human tumor cell lines HT29 and T47D. MetXia-P450 transduction sensitised these cells to the cytotoxic effects of the prodrug CPA. Results from in vitro experiments demonstrated adverse effects on the clonogenic survival of cyclophosphamide-treated cells transduced with MetXia-P450. Cytotoxic activity accompanied by bystander effect was particularly evident in 3-D multicellular spheroid models suggesting that this in vitro system may be a more appropriate model for assessing the efficacy of gene directed-enzyme prodrug therapy (GDEPT). We have applied this approach in a clinically relevant gene therapy protocol on established subcutaneous tumor xenografts. These studies show for the first time the efficacy of a P450-based GDEPT strategy mediated by a direct retroviral gene transfer in vivo. PMID:11498768

  5. FROM GENE TO PROTEIN – CLONNING, EXPRESSION AND PUFICATION OF A P450 CYTOCHROM FROM CAMPYLOBACTER JEJUNI

    Directory of Open Access Journals (Sweden)

    N. CORCIONIVOSCHI

    2009-05-01

    Full Text Available Recently, the complete genome sequence of Campylobacter jejuni NCTC 11168 was published revealingthe presence of only one open reading frame (Cj1411c encoding for a cytochrome P450, in contrast to 20found in M. tuberculosis. The gene Cj1411c encodes for a soluble 52.6 kDa protein with a predictedisoelectric point of 9.3. The P450 gene is part of reading frame which hosts genes involved in thesynthesis of cell surface components (capsula. Campylobacter capsule are important in adherence,invasion and colonisation of host cells and for maintenance of cell surface charge and serum resistance.These capsule are thought to cause autoimmunity leading to Guillan-Barre and Miller-Fischersyndromes. The structure of the lipoolygosaccharides and capsule polysaccharide was published last yearrevealing that the strain possessed a type II/III capsule locus found in other microorganisms suchNisseria meningitidis. This project focuses on the cloning and characterisation of the only P450 enzymeof the human pathogen Campylobacter jejuni NCTC 11168. We aim to understand the metabolic role ofthis P450 cytochrome in order to elucidate its possible use as a new target for drug design. To achievethis aim we have cloned, expressed and purify the product of P450 coding gene.

  6. FROM GENE TO PROTEIN – CLONNING, EXPRESSION AND PUFICATION OF A P450 CYTOCHROM FROM Campylobacter jejuni

    Directory of Open Access Journals (Sweden)

    N. CORCIONIVOSCHI

    2013-07-01

    Full Text Available Recently, the complete genome sequence of Campylobacter jejuni NCTC 11168 was published revealing the presence of only one open reading frame (Cj1411c encoding for a cytochrome P450, in contrast to 20 found in M. tuberculosis. The gene Cj1411c encodes for a soluble 52.6 kDa protein with a predicted isoelectric point of 9.3. The P450 gene is part of reading frame which hosts genes involved in the synthesis of cell surface components (capsula. Campylobacter capsule are important in adherence, invasion and colonisation of host cells and for maintenance of cell surface charge and serum resistance. These capsule are thought to cause autoimmunity leading to Guillan-Barre and Miller-Fischer syndromes. The structure of the lipoolygosaccharides and capsule polysaccharide was published last year revealing that the strain possessed a type II/III capsule locus found in other microorganisms such Nisseria meningitidis. This project focuses on the cloning and characterisation of the only P450 enzyme of the human pathogen Campylobacter jejuni NCTC 11168. We aim to understand the metabolic role of this P450 cytochrome in order to elucidate its possible use as a new target for drug design. To achieve this aim we have cloned, expressed and purify the product of P450 coding gene.

  7. Over-expression of a cytochrome P450 is associated with resistance to pyriproxyfen in the greenhouse whitefly Trialeurodes vaporariorum.

    Directory of Open Access Journals (Sweden)

    Nikos Karatolos

    Full Text Available BACKGROUND: The juvenile hormone mimic, pyriproxyfen is a suppressor of insect embryogenesis and development, and is effective at controlling pests such as the greenhouse whitefly Trialeurodes vaporariorum (Westwood which are resistant to other chemical classes of insecticides. Although there are reports of insects evolving resistance to pyriproxyfen, the underlying resistance mechanism(s are poorly understood. RESULTS: Bioassays against eggs of a German (TV8 population of T. vaporariorum revealed a moderate level (21-fold of resistance to pyriproxyfen. This is the first time that pyriproxyfen resistance has been confirmed in this species. Sequential selection of TV8 rapidly generated a strain (TV8pyrsel displaying a much higher resistance ratio (>4000-fold. The enzyme inhibitor piperonyl butoxide (PBO suppressed this increased resistance, indicating that it was primarily mediated via metabolic detoxification. Microarray analysis identified a number of significantly over-expressed genes in TV8pyrsel as candidates for a role in resistance including cytochrome-P450 dependent monooxygenases (P450s. Quantitative PCR highlighted a single P450 gene (CYP4G61 that was highly over-expressed (81.7-fold in TV8pyrsel. CONCLUSION: Over-expression of a single cytochrome P450 gene (CYP4G61 has emerged as a strong candidate for causing the enhanced resistance phenotype. Further work is needed to confirm the role of the encoded P450 enzyme CYP4G61 in detoxifying pyriproxyfen.

  8. 丙泊酚对大鼠肝微粒体细胞色素酶P450的影响%Effect of propofol on liver microsomal cytochrome P450 in rats

    Institute of Scientific and Technical Information of China (English)

    李振洲; 陈学新; 陈雅儒; 闫瑞; 孟尽海; 马汉祥; 邓丽琴

    2011-01-01

    目的:研究丙泊酚对大鼠肝微粒体细胞色素酶P450含量的影响.方法:健康雄性SD大鼠18只,体重180 ~ 220 g,随机分为苯巴比妥钠组、丙泊酚组、生理盐水组,每组6只,分别给予苯巴比妥钠75 mg/kg,丙泊酚3.789 mg/kg及等量生理盐水,持续3 d.测定肝微粒体蛋白和P450的含量以及氨基比林-N脱甲基酶的活性.结果:与生理盐水组比较,苯巴比妥钠组、丙泊酚组肝微粒体蛋白和P450的含量升高;苯巴比妥钠组氨基比林-N脱甲基酶活性明显增高.结论:丙泊酚对大鼠肝微粒体蛋白、细胞色素P450具有诱导作用,对氨基比林-N脱甲基酶的活性无影响.%Objective To explore the effect of propofol on liver microsomal cytochrome P450 in rats.Methods Eighteen male SD rats were randomly assigned to receive phenobarbital of 75 mg/kg (phenobarbital group, n= 6), propofol of 3.789 mg/kg (propofol group, n= 6), or normal saline (control group, n= 6) for three days. Levels of liver microsomal proteins and P450 were detected and activity of aminopyrine N-demethylase was detemined by spectrophotometry. Results As compared with the control group, the levels of microsomal proteins and cytochrome P450 were increased in phenobarbital group and propofol group; the activity of aminopyrine N-demethylase was significantly elevated in phenobarbital group. Conclusions Propofol can induce liver microsomal cytochrome P450 in rats but has no effect on the activity of aminiopyrine N-demethylase.

  9. [Activity of 5-aminolevulinate synthase in rat liver during degradation of cytochrome P-450 caused by administration of cadmium chloride].

    Science.gov (United States)

    Kaliman, P A; Inshina, N N

    2003-01-01

    The 5-aminolevulinate synthase, tryptophan-2,3-dioxygenase activities and cytochrome P-450 content in the rat liver was studied in different terms after CdCl2 administration and after administration of metal salt against a background of 2-hours action of alpha-tocopherol. The lowering of activity of 5-aminolevulinate synthase in 2 h with the consequent increase of the enzyme activity in 6 h and 24 h was detected. The holoenzyme activity and heme saturation of tryptophan-2,3-dioxygenase increased 6 h after CdCl2 administration. The holoenzyme activity and the total activity of tryptophan-2,3-dioxygenase rised in 24 h. The level of cytochrome P-450 lowered. Preliminary administration of alpha-tocopherol prevented changes of studied parameters 24 h after CdCl2 administration. The relationship between decrease of cytochrome P-450 level and 5-aminolevulinate synthase activation are discussed. PMID:14577179

  10. A chicory cytochrome P450 mono-oxygenase CYP71AV8 for the oxidation of (+)-valencene.

    Science.gov (United States)

    Cankar, Katarina; van Houwelingen, Adèle; Bosch, Dirk; Sonke, Theo; Bouwmeester, Harro; Beekwilder, Jules

    2011-01-01

    Chicory (Cichorium intybus L.), which is known to have a variety of terpene-hydroxylating activities, was screened for a P450 mono-oxygenase to convert (+)-valencene to (+)-nootkatone. A novel P450 cDNA was identified in a chicory root EST library. Co-expression of the enzyme with a valencene synthase in yeast, led to formation of trans-nootkatol, cis-nootkatol and (+)-nootkatone. The novel enzyme was also found to catalyse a three step conversion of germacrene A to germacra-1(10),4,11(13)-trien-12-oic acid, indicating its involvement in chicory sesquiterpene lactone biosynthesis. Likewise, amorpha-4,11-diene was converted to artemisinic acid. Surprisingly, the chicory P450 has a different regio-specificity on (+)-valencene compared to germacrene A and amorpha-4,11-diene. PMID:21115006

  11. A Novel Semi-biosynthetic Route for Artemisinin Production Using Engineered Substrate-Promiscuous P450BM3

    Energy Technology Data Exchange (ETDEWEB)

    Dietrich, Jeffrey; Yoshikuni, Yasuo; Fisher, Karl; Woolard, Frank; Ockey, Denise; McPhee, Derek; Renninger, Neil; Chang, Michelle; Baker, David; Keasling, Jay

    2009-11-30

    Production of fine heterologus pathways in microbial hosts is frequently hindered by insufficient knowledge of the native metabolic pathway and its cognate enzymes; often the pathway is unresolved and enzymes lack detailed characterization. An alternative paradigm to using native pathways is de novo pathway design using well-characterized, substrate-promiscuous enzymes. We demonstrate this concept using P450BM3 from Bacillus megaterium. Using a computer model, we illustrate how key P450BM3 activ site mutations enable binding of non-native substrate amorphadiene, incorporating these mutations into P450BM3 enabled the selective oxidation of amorphadiene arteminsinic-11s,12-epoxide, at titers of 250 mg L"1 in E. coli. We also demonstrate high-yeilding, selective transformations to dihydroartemisinic acid, the immediate precursor to the high value anti-malarial drug artemisinin.

  12. 尿1-羟基芘与外周血细胞色素P450酶关系初步研究%Relationship between urinary 1-hydroxrpyrene level and cytochrome P450 enzyme family in peripheral blood

    Institute of Scientific and Technical Information of China (English)

    王芳; 陈靖晗; 侍洪斌; 吴丽娜; 吕严

    2012-01-01

    Objective To investigate the relationship between the urinary 1 -hydroxypyrene level and cytochrome P450 enzyme family in peripheral blood among non-occupational polycyclic aromatic hydrocarbons exposure individuals in Tianjin. Methods Fifty people undergone physical examinations to the hospital were selected. Urinary levels of 1 -hydroxypyrene were measured by alkaline hydrolysis combined with high performance liquid chromatography, and cytochrome P450 enzyme family were measured with ELISA method. Results The direct correlation was observed between the urinary level of 1-hydroxypyrene and cytochrome P450 enzyme family in peripheral blood in the study subjects(r=0.431, P=0.001) after adjusting sex, age and alcohol drinking.The activity of cytochrome P450 enzyme could be induced by exposure to electromagnetic radiation, inhibited by intake of garlic. Conclusion Urinary levels of 1 -hydroxypyrene of non -occupational polycyclic aromatic hydrocarbons exposure individuals positively correlated with the acivity of cytochrome P450 enzyme in peripheral blood.%目的 研究非职业多环芳烃暴露者尿中1-羟基芘水平与外周血细胞色素P450酶含量间的关系.方法 于2011年12月选取50名到天津市某医院健康管理中心体检的人员,使用碱水解-高效液相色谱法测定尿1-羟基芘浓度;以双抗体夹心法测定外周血细胞色素P450酶活力;以问卷调查形式收集个人的一般情况、生活、职业史等信息.结果 调整了年龄、性别和饮酒状况后,尿1-羟基芘浓度与外周血细胞色素P450酶活力存在正相关(r=0.431,P=0.001),另外电磁辐射诱导外周血细胞色素P450酶活力,食用大蒜抑制其活力.结论 非职业多环芳烃暴露者尿1-羟基芘浓度与外周血细胞色素P450酶活力存在正相关,为外周血细胞色素P450酶系作为PAHs暴露早期生物标志物的可行性提供依据.

  13. Identification of human cytochrome P450 enzymes involved in the hepatic and intestinal biotransformation of 20(S)-protopanaxadiol.

    Science.gov (United States)

    Chiu, Nga Ting Colette; Tomlinson Guns, Emma S; Adomat, Hans; Jia, William; Deb, Subrata

    2014-03-01

    20(S)-Protopanaxadiol (aPPD), a ginseng sapogenin, has been shown to be a promising anti-cancer compound and anti-depressant agent. Although the bacterial biotransformation of ginsenosides has been studied thoroughly, few have reported on the cytochrome P450 (P450) mediated metabolism of aPPD. Taken orally, aPPD must first undergo absorption and metabolism in the intestine before further metabolism in the liver. The present study investigated the comparative biotransformation profile of aPPD in human intestinal microsomes (HIM) and human liver microsomes (HLM) and characterized the human P450 enzymes involved in aPPD metabolism. Three major monooxygenated metabolites and five minor dioxygenated metabolites were identified as the predominant products in aPPD incubations with HIM and HLM using liquid chromatography-mass spectrometry. Reaction phenotyping studies were performed with a panel of specific P450 chemical inhibitors, antibody inhibition and human recombinant P450 enzymes. Ketoconazole, a CYP3A inhibitor, blocked the formation of oxygenated metabolites of aPPD in both HIM and HLM in a concentration dependent manner. Among the human recombinant P450 enzymes assayed, CYP3A4 exhibited the highest activity towards aPPD oxidative metabolite formation, followed by CYP3A5. In summary, the results have shown that aPPD is extensively metabolized by HIM and the metabolite profile following in vitro incubations is similar in HIM and HLM. CYP3A4 and CYP3A5 isoforms are the predominant enzymes responsible for oxygenation of aPPD in HIM and HLM. The characterization of aPPD as a CYP3A substrate may facilitate better prediction of drug-herb interactions when aPPD is taken concomitantly with other therapeutic agents. PMID:24151189

  14. CHARACTERIZATION AND EXPRESSION PROFILES OF FIVE POSSIBLE CYTOCHROME P450 GENES FROM Liposcelis entomophila (ENDERLEIN) (PSOCOPTERA: LIPOSCELIDIDAE).

    Science.gov (United States)

    Li, Ting; Liu, Yan; Wei, Dan-Dan; Shang, Feng; Smagghe, Guy; Dou, Wei; Wang, Jin-Jun; Smagghe, Guy

    2016-08-01

    In this study, the cDNAs of five cytochromes P450 genes (named CYP345P1, CYP358B1, CYP4FD2, CYP4CD2, and CYP6JN1) contained open reading frames from 1,500 to 1,554 nucleotides that encoded 499 to 517 amino acids were cloned from the psocid Liposcelis entomophila. They are characterized by predicted molecular weights from 57.67 to 59.64 kDa and theoretical isoelectric points of 5.57-9.07. Quantitative real-time PCR analysis showed these five genes were expressed at all tested developmental stages and higher expressions were observed in adults. CYP358B1 was expressed at higher levels in egg and adult compared to the larval stages. mRNA abundances of five genes were detected in both sexes and were relatively more abundant in adult females than in adult males. Synergism bioassay showed that the synergic ratio was 2.20 and 2.45 when insects were treated with the mixture of deltamethrin or malathion with the synergist piperonyl butoxide (PBO). Because PBO induces cytochrome P450s in some insects, this suggested to us that cytochromes P450 might participate in detoxification of these insecticides. The transcripts of the five cytochromes P450 genes in adult psocids could be induced to the highest level at 12 h after the exposure to malathion. After exposure to deltamethrin, CYP358B1 reached maximum expression at 24 h. The maximum expression of the other four genes occurred at 36 h. Treatments with the carbamate propoxur did not influence transcription of the cytochromes P450 gene. The induction profiles suggested that these five cytochrome P450 genes may be associated with deltamethrin and malathion metabolism in psocids. PMID:27087161

  15. The effect of lycopene on the total cytochrome P450, CYP1A2 and CYP2E1

    Directory of Open Access Journals (Sweden)

    Melva Louisa

    2009-12-01

    Full Text Available Aim: Some carotenoids such as canthaxantin, astaxanthin and beta apo-8’-carotenal were reported to have modulatoryeffect on the cytochrome P450. The present study was conducted to investigate the effects of lycopene, a nonprovitamin A carotenoid, on microsomal cytochrome P450, CYP1A2 and CYP2E1.Methods: Total cytochrome P450 levels, CYP1A2 and CYP2E1-catalyzed reactions (acetanilide 4-hydroxylation and p-nitrophenol hydroxylation were studied in the liver microsomes of male Sprague Dawley rats. Microsomes were prepared using differential centrifugation combined with calcium aggregation method. Lycopene was orally administered in the dosages of 0, 25, 50 or 100 mg/kgBW/day for 14 days in a repeated fashion. Data were analyzed using ANOVA test.Results: Total cytochrome P450 level and acetanilide 4-hydroxylase activity were unaffected by any of the treatments. The CYP2E1 probe enzyme (p-nitrophenol hydroxylase was significantly reduced by repeated administration of 100mg/ kgBW/day lycopene (7.88 + 2.04 vs 12.26 + 2.77 n mol/min/mg prot.Conclusion: The present results suggest that lycopene does not affect the total cytochrome P450 or CYP1A2 activity but it inhibits the activity of CYP2E1 (p-nitrophenol hydroxylase in the rat. (Med J Indones 2009; 18: 233-8Keywords: lycopene, cytochrome P450, CYP1A2, CYP2E1

  16. Deletion of P399E401 in NADPH cytochrome P450 oxidoreductase results in partial mixed oxidase deficiency

    International Nuclear Information System (INIS)

    Highlights: → Mutations in human POR cause congenital adrenal hyperplasia. → We are reporting a novel 3 amino acid deletion mutation in POR P399E401del. → POR mutation P399E401del decreased P450 activities by 60-85%. → Impairment of steroid metabolism may be caused by multiple hits. → Severity of aromatase inhibition is related to degree of in utero virilization. -- Abstract: P450 oxidoreductase (POR) is the electron donor for all microsomal P450s including steroidogenic enzymes CYP17A1, CYP19A1 and CYP21A2. We found a novel POR mutation P399E401del in two unrelated Turkish patients with 46,XX disorder of sexual development. Recombinant POR proteins were produced in yeast and tested for their ability to support steroid metabolizing P450 activities. In comparison to wild-type POR, the P399E401del protein was found to decrease catalytic efficiency of 21-hydroxylation of progesterone by 68%, 17α-hydroxylation of progesterone by 76%, 17,20-lyase action on 17OH-pregnenolone by 69%, aromatization of androstenedione by 85% and cytochrome c reduction activity by 80%. Protein structure analysis of the three amino acid deletion P399E401 revealed reduced stability and flexibility of the mutant. In conclusion, P399E401del is a novel mutation in POR that provides valuable genotype-phenotype and structure-function correlation for mutations in a different region of POR compared to previous studies. Characterization of P399E401del provides further insight into specificity of different P450s for interaction with POR as well as nature of metabolic disruptions caused by more pronounced effect on specific P450s like CYP17A1 and aromatase.

  17. Induction of rabbit lung cytochrome P450 prostaglandin in omega-hyroxylase during pregnancy: evidence for regulation at the genetic level

    International Nuclear Information System (INIS)

    The induction of a cytochrome P450 prostaglandin omega-hydroxylase (P450/sub PG omega/) isolated from pregnant rabbit lung has been shown by Western blots to be concomitant with an increase in the amount of P450 protein. Peaks in enzyme activity and P450/sub PG omega/ protein occur between the 20th and 28th days of gestation with increases of more than 100-fold compared to nonpregnant rabbits. To elucidate the mechanisms controlling induction, total cellular RNA was extracted from rabbit lungs at various days of gestation, translated in vitro using 35S-met, and the newly synthesized P450/sub PG omega/ immunoprecipitated from the lysate. Utilizing an immunopurified goat IgG to P450/sub PG omega/, immunopellets of in vitro translation reactions charged with RNA from lungs at 6,11,19,22,25, or 28-days gestation were isolated. A single band corresponding to P450/sub PG omega/ was seen in autoradiographs of SDS-PAGE gels containing these immunopellets, but no band was visible in lanes containing immunopellets from reactions charged with RNA from nonpregnant or 1-day post-partum animals. The gestational time-dependent increase in in vitro-translated P450/sub PG omega/ suggests that control of its induction during pregnancy is at the transcriptional level. A monoclonal antibody to the P450/sub PG omega/ has been produced for the isolation of the P450/sub PG omega/ mRNA for cDNA production

  18. Sobreexpresión, purificación y reconstitución del citocromo b559

    OpenAIRE

    Luján Serrano, María Ángeles

    2009-01-01

    El citocromo b559 (cytb559) forma parte del centro de reacción del fotosistema II. Está compuesto por dos subunidades: la subunidad ¿, codificada por el gen psbE, y la subunidad ß, codificada por el gen psbF. Ambos genes se encuentran en el genoma cloroplástico. Entre las dos subunidades se encuentra ensamblado un grupo hemo de tipo b, coordinado por dos histidinas, una de cada subunidad. Desempeña un papel estructural dentro del centro de reacción pero a pesar de los numerosos estudios reali...

  19. Significance of cytochrome P450 system responses and levels of bile fluorescent aromatic compounds in marine wildlife following oil spills

    International Nuclear Information System (INIS)

    The relationships among cytochrome P450 induction in marine wildlife species, levels of fluorescent aromatic compounds (FAC) in their bile, the chemical composition of the inducing compounds, the significance of the exposure pathway, and any resulting injury, as a consequence of exposure to crude oil following a spill, are reviewed. Fish collected after oil spills often show increases in cytochrome P450 system activity, cytochrome P4501A (CYP1A) and bile fluorescent aromatic compounds (FAC), that are correlated with exposure to polycyclic aromatic hydrocarbons (PAH) in the oil. There is also some evidence for increases in bile FAC and induction of cytochrome P450 in marine birds and mammals after oil spills. However, when observed, increases in these exposure indicators are transitory and generally decrease to background levels within one year after the exposure. Laboratory studies have shown induction of cytochrome P450 systems occurs after exposure of fish to crude oil in water, sediment or food. Most of the PAH found in crude oil (dominantly 2- and 3-ring PAH) are not strong inducers of cytochrome P450. Exposure to the 4-ring chrysenes or the photooxidized products of the PAH may account for the cytochrome P450 responses in fish collected from oil-spill sites. The contribution of non-spill background PAH, particularly combustion-derived (pyrogenic) PAH, to bile FAC and cytochrome P450 system responses can be confounding and needs to be considered when evaluating oil spill effects. The ubiquity of pyrogenic PAH makes it important to fully characterize all sources of PAH, including PAH from natural resources, e.g. retene, in oil spill studies. In addition, such parameters as species, sex, age, ambient temperature and season need to be taken into account. While increases in fish bile FAC and cytochrome P450 system responses, can together, be sensitive general indicators of PAH exposure after an oil spill, there is little unequivocal evidence to suggest a linkage to

  20. Functional characterization of NADPH-cytochrome P450 reductase from Bactrocera dorsalis: Possible involvement in susceptibility to malathion

    OpenAIRE

    Yong Huang; Xue-Ping Lu; Luo-Luo Wang; Dong Wei; Zi-Jiao Feng; Qi Zhang; Lin-Fan Xiao; Wei Dou; Jin-Jun Wang

    2015-01-01

    NADPH cytochrome P450 reductase (CPR) is essential for cytochrome P450 catalysis, which is important in the detoxification and activation of xenobiotics. In this study, two transcripts of Bactrocera dorsalis CPR (BdCPR) were cloned, and the deduced amino-acid sequence had an N-terminus membrane anchor for BdCPR-X1 and three conserved binding domains (FMN, FAD, and NADP), as well as an FAD binding motif and catalytic residues for both BdCPR-X1 and BdCPR-X2. BdCPR-X1 was detected to have the hi...

  1. A chicory cytochrome P450 mono-oxygenase CYP71AV8 for the oxidation of (+)-valencene

    OpenAIRE

    Cankar, K.; Houwelingen, van, K.; Bosch, H.J.; Sonke, Th.; Bouwmeester, H.J.; Beekwilder, M.J.

    2011-01-01

    Chicory (Cichorium intybus L.), which is known to have a variety of terpene-hydroxylating activities, was screened for a P450 mono-oxygenase to convert (+)-valencene to (+)-nootkatone. A novel P450 cDNA was identified in a chicory root EST library. Co-expression of the enzyme with a valencene synthase in yeast, led to formation of trans-nootkatol, cis-nootkatol and (+)-nootkatone. The novel enzyme was also found to catalyse a three step conversion of germacrene A to germacra-1(10),4,11(13)-tr...

  2. Genome analysis of cytochrome P450s and their expression profiles in insecticide resistant mosquitoes, Culex quinquefasciatus.

    Directory of Open Access Journals (Sweden)

    Ting Yang

    Full Text Available Here we report a study of the 204 P450 genes in the whole genome sequence of larvae and adult Culex quinquefasciatus mosquitoes. The expression profiles of the P450 genes were compared for susceptible (S-Lab and resistant mosquito populations, two different field populations of mosquitoes (HAmCq and MAmCq, and field parental mosquitoes (HAmCq(G0 and MAmCq(G0 and their permethrin selected offspring (HAmCq(G8 and MAmCq(G6. While the majority of the P450 genes were expressed at a similar level between the field parental strains and their permethrin selected offspring, an up- or down-regulation feature in the P450 gene expression was observed following permethrin selection. Compared to their parental strains and the susceptible S-Lab strain, HAmCq(G8 and MAmCq(G6 were found to up-regulate 11 and 6% of total P450 genes in larvae and 7 and 4% in adults, respectively, while 5 and 11% were down-regulated in larvae and 4 and 2% in adults. Although the majority of these up- and down-regulated P450 genes appeared to be developmentally controlled, a few were either up- or down-regulated in both the larvae and adult stages. Interestingly, a different gene set was found to be up- or down-regulated in the HAmCq(G8 and MAmCq(G6 mosquito populations in response to insecticide selection. Several genes were identified as being up- or down-regulated in either the larvae or adults for both HAmCq(G8 and MAmCq(G6; of these, CYP6AA7 and CYP4C52v1 were up-regulated and CYP6BY3 was down-regulated across the life stages and populations of mosquitoes, suggesting a link with the permethrin selection in these mosquitoes. Taken together, the findings from this study indicate that not only are multiple P450 genes involved in insecticide resistance but up- or down-regulation of P450 genes may also be co-responsible for detoxification of insecticides, insecticide selection, and the homeostatic response of mosquitoes to changes in cellular environment.

  3. Electrochemiluminescent Arrays for Cytochrome P450-Activated Genotoxicity Screening. DNA Damage from Benzo[a]pyrene Metabolites

    OpenAIRE

    Hvastkovs, Eli G.; So, Minjeong; Krishnan, Sadagopan; Bajrami, Besnik; Tarun, Maricar; Jansson, Ingela; Schenkman, John B.; Rusling, James F.

    2007-01-01

    Arrays suitable for genotoxicity screening are reported that generate metabolites from cytochrome P450 enzymes (CYPs) in thin-film spots. Array spots containing DNA, various human cyt P450s, and electrochemiluminescence (ECL) generating metallopolymer [Ru(bpy)2PVP10]2+ were exposed to H2O2 to activate the enzymes. ECL from all spots was visualized simultaneously using a CCD camera. Using benzo[a]pyrene as a test substrate, enzyme activity for producing DNA damage in the arrays was found in th...

  4. Production of a highly active, soluble form of the cytochrome P450 reductase (CPR A) from Candida tropicalis

    Science.gov (United States)

    Donnelly, Mark

    2006-08-01

    The present invention provides soluble cytochrome p450 reductase (CPR) proteins from Candida sp. having an altered N-terminal region which results in reduced hydrophobicity of the N-terminal region. Also provided are host cells comprising the subject soluble CPR proteins. In addition, the present invention provides nucleotide and corresponding amino acid sequences for soluble CPR proteins and vectors comprising the nucleotide sequences. Methods for producing a soluble CPR, for increasing production of a dicarboxylic acid, and for detecting a cytochrome P450 are also provided.

  5. Reduction of Aromatic and Heterocyclic Aromatic N-Hydroxylamines by Human Cytochrome P450 2S1

    OpenAIRE

    Kai WANG; Guengerich, F. Peter

    2013-01-01

    Many aromatic amines and heterocyclic aromatic amines (HAAs) are known carcinogens for animals and there is also strong evidence for some in human cancer. The activation of these compounds, including some arylamine drugs, involves N-hydroxylation, usually by cytochrome P450 enzymes (P450) in Family 1 (1A2, 1A1, and 1B1). We previously demonstrated that the bioactivation product of the anti-cancer agent 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203), an N-hydroxylamine, can be reduc...

  6. FROM GENE TO PROTEIN – CLONNING, EXPRESSION AND PUFICATION OF A P450 CYTOCHROM FROM Campylobacter jejuni

    OpenAIRE

    N. CORCIONIVOSCHI

    2013-01-01

    Recently, the complete genome sequence of Campylobacter jejuni NCTC 11168 was published revealing the presence of only one open reading frame (Cj1411c) encoding for a cytochrome P450, in contrast to 20 found in M. tuberculosis. The gene Cj1411c encodes for a soluble 52.6 kDa protein with a predicted isoelectric point of 9.3. The P450 gene is part of reading frame which hosts genes involved in the synthesis of cell surface components (capsula). Campylobacter capsule are important in adherence,...

  7. Regulation of P450 oxidoreductase by gonadotropins in rat ovary and its effect on estrogen production

    Directory of Open Access Journals (Sweden)

    Uesaka Miki

    2008-12-01

    Full Text Available Abstract Background P450 oxidoreductase (POR catalyzes electron transfer to microsomal P450 enzymes. Its deficiency causes Antley-Bixler syndrome (ABS, and about half the patients with ABS have ambiguous genitalia and/or impaired steroidogenesis. POR mRNA expression is up-regulated when mesenchymal stem cells (MSCs differentiate into steroidogenic cells, suggesting that the regulation of POR gene expression is important for steroidogenesis. In this context we examined the regulation of POR expression in ovarian granulosa cells by gonadotropins, and its possible role in steroidogenesis. Methods Changes in gene expression in MSCs during differentiation into steroidogenic cells were examined by DNA microarray analysis. Changes in mRNA and protein expression of POR in the rat ovary or in granulosa cells induced by gonadotropin treatment were examined by reverse transcription-polymerase chain reaction and western blotting. Effects of transient expression of wild-type or mutant (R457H or V492E POR proteins on the production of estrone in COS-7 cells were examined in vitro. Effects of POR knockdown were also examined in estrogen producing cell-line, KGN cells. Results POR mRNA was induced in MSCs following transduction with the SF-1 retrovirus, and was further increased by cAMP treatment. Expression of POR mRNA, as well as Cyp19 mRNA, in the rat ovary were induced by equine chorionic gonadotropin and human chorionic gonadotropin. POR mRNA and protein were also induced by follicle stimulating hormone in primary cultured rat granulosa cells, and the induction pattern was similar to that for aromatase. Transient expression of POR in COS-7 cells, which expressed a constant amount of aromatase protein, greatly increased the rate of conversion of androstenedione to estrone, in a dose-dependent manner. The expression of mutant POR proteins (R457H or V492E, such as those found in ABS patients, had much less effect on aromatase activity than expression of wild

  8. Inhibitory effect of mitragynine on human cytochrome P450 enzyme activities

    Directory of Open Access Journals (Sweden)

    N A Hanapi

    2013-01-01

    Full Text Available Context: To date, many findings reveal that most of the modern drugs have the ability to interact with herbal drugs. Aims: This study was conducted to determine the inhibitory effects of mitragynine on cytochrome P450 2C9, 2D6 and 3A4 activities. Methods and Material: The in vitro study was conducted using a high-throughput luminescence assay. Statistical Analysis: Statistical analysis was conducted using one-way ANOVA and Dunnett′s test with P < 0.05 vs. control. The IC 50 values were calculated using the GraphPad Prism® 5 (Version 5.01, GraphPad Software, Inc., USA. Results: Assessment using recombinant enzymes showed that mitragynine gave the strongest inhibitory effect on CYP2D6 with an IC 50 value of 0.45±0.33 mM, followed by CYP2C9 and CYP3A4 with IC 50 values of 9.70±4.80 and 41.32±6.74 mM respectively. Positive inhibitors appropriate for CYP2C9, CYP2D6, and CYP3A4 which are sulfaphenazole, quinidine and ketoconazole were used respectively. V max values of CYP2C9, CYP2D6 and CYP3A4 were 0.0005, 0.01155 and 0.0137 mM luciferin formed/pmol/min respectively. K m values of CYP2C9, CYP2D6, and CYP3A4 were 32.65, 56.01, and 103.30 mM respectively. Mitragynine noncompetitively inhibits CYP2C9 and CYP2D6 activities with the K i values of 61.48 and 12.86 mM respectively. On the other hand, mitragynine inhibits CYP3A4 competitively with a K i value of 379.18 mM. Conclusions: The findings of this study reveal that mitragynine might inhibit cytochrome P450 enzyme activities, specifically CYP2D6. Therefore, administration of mitragynine together with herbal or modern drugs which follow the same metabolic pathway may contribute to herb-drug interactions.

  9. Mass spectrometry-based proteomic analysis of human liver cytochrome(s) P450

    International Nuclear Information System (INIS)

    The major objective of personalized medicine is to select optimized drug therapies and to a large degree such mission is determined by the expression profiles of cytochrome(s) P450 (CYP). Accordingly, a proteomic case study in personalized medicine is provided by the superfamily of cytochromes P450. Our knowledge about CYP isozyme expression on a protein level is very limited and based exclusively on DNA/mRNA derived data. Such information is not sufficient because transcription and translation events do not lead to correlated levels of expressed proteins. Here we report expression profiles of CYPs in human liver obtained by mass spectrometry (MS)-based proteomic approach. We analyzed 32 samples of human liver microsomes (HLM) of different sexes, ages and ethnicity along with samples of recombinant human CYPs. We have experimentally confirmed that each CYP isozyme can be effectively differentiated by their unique isozyme-specific tryptic peptide(s). Trypsin digestion patterns for almost 30 human CYP isozymes were established. Those findings should assist in selecting tryptic peptides suitable for MS-based quantitation. The data obtained demonstrate remarkable differences in CYP expression profiles. CYP2E1, CYP2C8 and CYP4A11 were the only isozymes found in all HLM samples. Female and pediatric HLM samples revealed much more diverse spectrum of expressed CYPs isozymes compared to male HLM. We have confirmed expression of a number of “rare” CYP (CYP2J2, CYP4B1, CYP4V2, CYP4F3, CYP4F11, CYP8B1, CYP19A1, CYP24A1 and CYP27A1) and obtained first direct experimental data showing expression of such CYPs as CYP2F1, CYP2S1, CYP2W1, CYP4A22, CYP4X1, and CYP26A1 on a protein level. - Highlights: ► First detailed proteomic analysis of CYP isozymes expression in human liver ► Trypsin digestion patterns for almost 30 human CYP isozymes established ► The data obtained demonstrate remarkable differences in CYP expression profiles. ► Female HLM samples revealed more

  10. Identification and developmental expression of the full complement of Cytochrome P450 genes in Zebrafish

    Directory of Open Access Journals (Sweden)

    Parente Thiago

    2010-11-01

    Full Text Available Abstract Background Increasing use of zebrafish in drug discovery and mechanistic toxicology demands knowledge of cytochrome P450 (CYP gene regulation and function. CYP enzymes catalyze oxidative transformation leading to activation or inactivation of many endogenous and exogenous chemicals, with consequences for normal physiology and disease processes. Many CYPs potentially have roles in developmental specification, and many chemicals that cause developmental abnormalities are substrates for CYPs. Here we identify and annotate the full suite of CYP genes in zebrafish, compare these to the human CYP gene complement, and determine the expression of CYP genes during normal development. Results Zebrafish have a total of 94 CYP genes, distributed among 18 gene families found also in mammals. There are 32 genes in CYP families 5 to 51, most of which are direct orthologs of human CYPs that are involved in endogenous functions including synthesis or inactivation of regulatory molecules. The high degree of sequence similarity suggests conservation of enzyme activities for these CYPs, confirmed in reports for some steroidogenic enzymes (e.g. CYP19, aromatase; CYP11A, P450scc; CYP17, steroid 17a-hydroxylase, and the CYP26 retinoic acid hydroxylases. Complexity is much greater in gene families 1, 2, and 3, which include CYPs prominent in metabolism of drugs and pollutants, as well as of endogenous substrates. There are orthologous relationships for some CYP1 s and some CYP3 s between zebrafish and human. In contrast, zebrafish have 47 CYP2 genes, compared to 16 in human, with only two (CYP2R1 and CYP2U1 recognized as orthologous based on sequence. Analysis of shared synteny identified CYP2 gene clusters evolutionarily related to mammalian CYP2 s, as well as unique clusters. Conclusions Transcript profiling by microarray and quantitative PCR revealed that the majority of zebrafish CYP genes are expressed in embryos, with waves of expression of different

  11. Mass spectrometry-based proteomic analysis of human liver cytochrome(s) P450

    Energy Technology Data Exchange (ETDEWEB)

    Shrivas, Kamlesh; Mindaye, Samuel T.; Getie-Kebtie, Melkamu; Alterman, Michail A., E-mail: Michail.Alterman@fda.hhs.gov

    2013-02-15

    The major objective of personalized medicine is to select optimized drug therapies and to a large degree such mission is determined by the expression profiles of cytochrome(s) P450 (CYP). Accordingly, a proteomic case study in personalized medicine is provided by the superfamily of cytochromes P450. Our knowledge about CYP isozyme expression on a protein level is very limited and based exclusively on DNA/mRNA derived data. Such information is not sufficient because transcription and translation events do not lead to correlated levels of expressed proteins. Here we report expression profiles of CYPs in human liver obtained by mass spectrometry (MS)-based proteomic approach. We analyzed 32 samples of human liver microsomes (HLM) of different sexes, ages and ethnicity along with samples of recombinant human CYPs. We have experimentally confirmed that each CYP isozyme can be effectively differentiated by their unique isozyme-specific tryptic peptide(s). Trypsin digestion patterns for almost 30 human CYP isozymes were established. Those findings should assist in selecting tryptic peptides suitable for MS-based quantitation. The data obtained demonstrate remarkable differences in CYP expression profiles. CYP2E1, CYP2C8 and CYP4A11 were the only isozymes found in all HLM samples. Female and pediatric HLM samples revealed much more diverse spectrum of expressed CYPs isozymes compared to male HLM. We have confirmed expression of a number of “rare” CYP (CYP2J2, CYP4B1, CYP4V2, CYP4F3, CYP4F11, CYP8B1, CYP19A1, CYP24A1 and CYP27A1) and obtained first direct experimental data showing expression of such CYPs as CYP2F1, CYP2S1, CYP2W1, CYP4A22, CYP4X1, and CYP26A1 on a protein level. - Highlights: ► First detailed proteomic analysis of CYP isozymes expression in human liver ► Trypsin digestion patterns for almost 30 human CYP isozymes established ► The data obtained demonstrate remarkable differences in CYP expression profiles. ► Female HLM samples revealed more

  12. Progress on research of the alternative splicing of human cytochrome P450 pre-mRNA%人细胞色素P450前mRNA的可变剪接研究进展

    Institute of Scientific and Technical Information of China (English)

    诸葛坚; 余应年

    2005-01-01

    Human genes typically contain multiple introns, and in many cases the exons can be joined more than one way to generate multiple rnRNAs, encoding distinct protein isoforms. This process is called alternative splicing. The article summarized the human cytochrome P450 pre-mRNA alternative splicing and their regulatory mechanism and impacts on biological functions.

  13. 食品动物细胞色素P450的研究进展%The Research Progress of Cytochrome P450 in Food-producing Animals

    Institute of Scientific and Technical Information of China (English)

    刘兆颖; 伍勇

    2011-01-01

    细胞色素P450酶系是参与药物及各种内源性和外源性化合物在体内的主要代谢酶,目前研究猪、牛、羊、鸡和鱼等食品动物的细胞色素P450代谢酶越来越受到重视.研究食品动物细胞色素P450,对明确其代谢转化机制、指导兽医临床合理用药、新兽药设计与剂型改进、预防兽药相互作用、种属外推用药、食品动物兽药残留消除分析及人类食品安全评价,都具有十分重要的意义.%Cytochrome P450s are the major metabolic enzymes involved in the metabolism of drugs, endogenous and exogenous compounds.Currently, the studies of cytochrome P450 in food-producing animals ( such as pig, cattle, sheep, chicken and fish )are drawing more attention.These works are of great significance in giving scientific guidance for the identification of metabolic mechanisms, clinical use of veterinary drugs, design of new drugs, and improvement of drug preparation, prevention of drug-drug interaction, extrapolation of different species, analysis of residues elimination and food safety assessment.

  14. Cytochrome P450 promiscuity leads to a bifurcating biosynthetic pathway for tanshinones.

    Science.gov (United States)

    Guo, Juan; Ma, Xiaohui; Cai, Yuan; Ma, Ying; Zhan, Zhilai; Zhou, Yongjin J; Liu, Wujun; Guan, Mengxin; Yang, Jian; Cui, Guanghong; Kang, Liping; Yang, Lei; Shen, Ye; Tang, Jinfu; Lin, Huixin; Ma, Xiaojing; Jin, Baolong; Liu, Zhenming; Peters, Reuben J; Zhao, Zongbao K; Huang, Luqi

    2016-04-01

    Cytochromes P450 (CYPs) play a key role in generating the structural diversity of terpenoids, the largest group of plant natural products. However, functional characterization of CYPs has been challenging because of the expansive families found in plant genomes, diverse reactivity and inaccessibility of their substrates and products. Here we present the characterization of two CYPs, CYP76AH3 and CYP76AK1, which act sequentially to form a bifurcating pathway for the biosynthesis of tanshinones, the oxygenated diterpenoids from the Chinese medicinal plant Danshen (Salvia miltiorrhiza). These CYPs had similar transcription profiles to that of the known gene responsible for tanshinone production in elicited Danshen hairy roots. Biochemical and RNA interference studies demonstrated that both CYPs are promiscuous. CYP76AH3 oxidizes ferruginol at two different carbon centers, and CYP76AK1 hydroxylates C-20 of two of the resulting intermediates. Together, these convert ferruginol into 11,20-dihydroxy ferruginol and 11,20-dihydroxy sugiol en route to tanshinones. Moreover, we demonstrated the utility of these CYPs by engineering yeast for heterologous production of six oxygenated diterpenoids, which in turn enabled structural characterization of three novel compounds produced by CYP-mediated oxidation. Our results highlight the incorporation of multiple CYPs into diterpenoid metabolic engineering, and a continuing trend of CYP promiscuity generating complex networks in terpenoid biosynthesis. PMID:26682704

  15. Human bone marrow niche chemoprotection mediated by cytochrome P450 enzymes.

    Science.gov (United States)

    Alonso, Salvador; Su, Meng; Jones, Jace W; Ganguly, Sudipto; Kane, Maureen A; Jones, Richard J; Ghiaur, Gabriel

    2015-06-20

    Substantial evidence now demonstrates that interactions between the tumor microenvironment and malignant cells are a critical component of clinical drug resistance. However, the mechanisms responsible for microenvironment-mediated chemoprotection remain unclear. We showed that bone marrow (BM) stromal cytochrome P450 (CYP)26 enzymes protect normal hematopoietic stem cells (HSCs) from the pro-differentiation effects of retinoic acid. Here, we investigated if stromal expression of CYPs is a general mechanism of chemoprotection. We found that similar to human hepatocytes, human BM-derived stromal cells expressed a variety of drug-metabolizing enzymes. CYP3A4, the liver's major drug-metabolizing enzyme, was at least partially responsible for BM stroma's ability to protect multiple myeloma (MM) and leukemia cells from bortezomib and etoposide, respectively, both in vitro and in vivo. Moreover, clarithromycin overcame stromal-mediated MM resistance to dexamethasone, suggesting that CYP3A4 inhibition plays a role in its ability to augment the activity of lenalidomide and dexamethasone as part of the BiRd regimen. We uncovered a novel mechanism of microenvironment-mediated drug resistance, whereby the BM niche creates a sanctuary site from drugs. Targeting these sanctuaries holds promise for eliminating minimal residual tumor and improving cancer outcomes. PMID:25915157

  16. Cytochrome P450 inhibition potential of new psychoactive substances of the tryptamine class.

    Science.gov (United States)

    Dinger, Julia; Woods, Campbell; Brandt, Simon D; Meyer, Markus R; Maurer, Hans H

    2016-01-22

    New psychoactive substances (NPS) are not tested for their cytochrome P450 (CYP) inhibition potential before consumption. Therefore, this potential was explored for tryptamine-derived NPS (TDNPS) including alpha-methyl tryptamines (AMTs), dimethyl tryptamines (DMTs), diallyl tryptamines (DALTs), and diisopropyl tryptamines (DiPTs) using test substrates preferred by the Food and Drug Administration in a cocktail assay. All tested TDNPS with the exception of DMT inhibited CYP2D6 activity with IC50 values below 100μM. DALTs inhibited CYP2D6 activity similar to paroxetine and quinidine and CYP1A2 activity comparable to fluvoxamine. 5-Methoxy-N,N-diallyltryptamine reduced in vivo the caffeine metabolism in rats consistent with in vitro results. Five of the AMTs also inhibited CYP1A2 activity comparable to amiodarone. AMT and 6-F-AMT inhibited CYP2A6 activity in the range of the test inhibitor tranylcypromine. CYP2B6 activity was inhibited by 19 tryptamines, but weakly compared to efavirenz. CYP2C8 activity was inhibited by five of the tested TDNPS and three showed values comparable to trimethoprim and gemfibrozil. Six tryptamines inhibited CYP2C9 and seven CYP2C19 activities comparable to fluconazole and chloramphenicol, respectively. Nineteen compounds showed inhibition of CYP2E1 and 18 of CYP3A activity, respectively. These results showed that the CYP inhibition by TDNPS might be clinically relevant, but clinical studies are needed to explore this further. PMID:26599973

  17. The human genome project and novel aspects of cytochrome P450 research

    International Nuclear Information System (INIS)

    Currently, 57 active cytochrome P450 (CYP) genes and 58 pseudogenes are known to be present in the human genome. Among the genes discovered by initiatives in the human genome project are CYP2R1, CYP2W1, CYP2S1, CYP2U1 and CYP3A43, the latter apparently encoding a pseudoenzyme. The function, polymorphism and regulation of these genes are still to be discovered to a great extent. The polymorphism of drug metabolizing CYPs is extensive and influences the outcome of drug therapy causing lack of response or adverse drug reactions. The basis for the differences in the global distribution of the polymorphic variants is inactivating gene mutations and subsequent genetic drift. However, polymorphic alleles carrying multiple active gene copies also exist and are suggested in case of CYP2D6 to be caused by positive selection due to development of alkaloid resistance in North East Africa about 10,000-5000 BC. The knowledge about the CYP genes and their polymorphisms is of fundamental importance for effective drug therapy and for drug development as well as for understanding metabolic activation of carcinogens and other xenobiotics. Here, a short review of the current knowledge is given

  18. Effects of gestational and overt diabetes on placental cytochromes P450 and glutathione S-transferase.

    Science.gov (United States)

    Glover; McRobie; Tracy

    1998-07-01

    Objective: Animal and in vivo human studies have observed that diabetes alters the expression of hepatic metabolizing cytochrome P450 (CYP) and glutathione S-transferase (GST) enzymes. The placenta has the ability to metabolize a number of xenobiotic and endogenous compounds by processes similar to those seen in the liver. Our objective was to compare placental xenobiotic metabolizing activity in diabetics to matched non-diabetic controls to determine if the presence of diabetes alters placental xenobiotic metabolizing activity.Methods: The catalytic activities of 7-ethoxyresorufin-O-deethylation [EROD] (CYP1A1), chlorzoxazone 6-hydroxylation (CYP2E1), dextromethorphan N-demethylation (CYP3A4), dextromethorphan O-demethylation (CYP2D6), and 1-chloro-2,4-dinitrobenzene (CDNB) conjugation with glutathione (GST) from placentas of diet controlled (class A1) and insulin-dependent (class A2) gestational diabetics and overt diabetics were compared to matched controls.Results: No differences in EROD activity were observed among overt or gestational diabetics and their respectively matched controls. CYP2E1, 2D6, and 3A4 enzyme activity were not detected in human placentas. In contrast, GST activity was significantly reduced by 30% (P <.05) in overt diabetics as compared to their matched controls and gestational diabetics.Conclusion: Pregnant women with overt diabetes have reduced GST activity in the placenta, which could potentially result in exposure of the fetus to harmful reactive electrophilic metabolites. PMID:10838356

  19. The Effect of Vinpocetine on Human Cytochrome P450 Isoenzymes by Using a Cocktail Method

    Directory of Open Access Journals (Sweden)

    Lingti Kong

    2016-01-01

    Full Text Available Vinpocetine is a derivative of the alkaloid vincamine, which had been prescribed for chronic cerebral vascular ischemia and acute ischemic stroke or used as a dietary supplement for its several different mechanisms of biological activities. However, information on the cytochrome P450 (CYP enzyme-mediated drug metabolism has not been previously studied. The present study was performed to investigate the effects of vinpocetine on CYPs activity, and cocktail method was used, respectively. To evaluate the effects of vinpocetine on the activity of human CYP3A4, CYP2C9, CYP2C19, CYP2D6, and CYP2E1, human liver microsomes were utilized to incubate with the mixed CYPs probe substrates and the target components. The results indicate that vinpocetine exhibited weak inhibitory effect on the CYP2C9, where the IC50 value is 68.96 μM, whereas the IC50 values for CYP3A4, CYP2C19, CYP2D6, and CYP2E1 were all over range of 100 μM, which showed that vinpocetine had no apparent inhibitory effects on these CYPs. In conclusion, the results indicated that drugs metabolized by CYP2C9 coadministrated with vinpocetine may require attention or dose adjustment.

  20. Subgrouping of patients with oral lichen planus according to cytochrome P450 enzyme phenotype and genotype

    DEFF Research Database (Denmark)

    Kragelund, Camilla; Jensen, Siri Beier; Hansen, Claus;

    2014-01-01

    Objective. This study aimed to determine if the activity of the environmentally influenced cytochrome P450 enzyme CYP1A2, alone or in combination with CYP2D6*4 genotype, discriminates subgroups of oral lichen planus (OLP) according to lifestyle factors and clinical manifestations. Study Design. A...... total of 111 patients with OLP were categorized according to normal, low, or high CYP1A2 activity and CYP2D6*4 genotype. Lifestyle parameters influencing the CYP1A2 activity and symptoms and manifestations of OLP were recorded. Results. Of the 111 patients, 21% had low, 65% normal, and 14% high CYP1A2...... activity. The high-CYP1A2-activity group was more exposed to CYP1A2 inducers than the low-CYP1A2-activity group. In the normal-CYP1A2-activity group, more patients had a CYP2D6*4 genotype (58%) (P = .02), and they presented more symptoms (P = .003) and gingival lesions (P = .03). More patients in the low...

  1. Elucidation of cladofulvin biosynthesis reveals a cytochrome P450 monooxygenase required for anthraquinone dimerization.

    Science.gov (United States)

    Griffiths, Scott; Mesarich, Carl H; Saccomanno, Benedetta; Vaisberg, Abraham; De Wit, Pierre J G M; Cox, Russell; Collemare, Jérôme

    2016-06-21

    Anthraquinones are a large family of secondary metabolites (SMs) that are extensively studied for their diverse biological activities. These activities are determined by functional group decorations and the formation of dimers from anthraquinone monomers. Despite their numerous medicinal qualities, very few anthraquinone biosynthetic pathways have been elucidated so far, including the enzymatic dimerization steps. In this study, we report the elucidation of the biosynthesis of cladofulvin, an asymmetrical homodimer of nataloe-emodin produced by the fungus Cladosporium fulvum A gene cluster of 10 genes controls cladofulvin biosynthesis, which begins with the production of atrochrysone carboxylic acid by the polyketide synthase ClaG and the β-lactamase ClaF. This compound is decarboxylated by ClaH to yield emodin, which is then converted to chrysophanol hydroquinone by the reductase ClaC and the dehydratase ClaB. We show that the predicted cytochrome P450 ClaM catalyzes the dimerization of nataloe-emodin to cladofulvin. Remarkably, such dimerization dramatically increases nataloe-emodin cytotoxicity against mammalian cell lines. These findings shed light on the enzymatic mechanisms involved in anthraquinone dimerization. Future characterization of the ClaM enzyme should facilitate engineering the biosynthesis of novel, potent, dimeric anthraquinones and structurally related compound families. PMID:27274078

  2. Insights on Cytochrome P450 Enzymes and Inhibitors Obtained Through QSAR Studies

    Directory of Open Access Journals (Sweden)

    Maryam Foroozesh

    2012-08-01

    Full Text Available The cytochrome P450 (CYP superfamily of heme enzymes play an important role in the metabolism of a large number of endogenous and exogenous compounds, including most of the drugs currently on the market. Inhibitors of CYP enzymes have important roles in the treatment of several disease conditions such as numerous cancers and fungal infections in addition to their critical role in drug-drug interactions. Structure activity relationships (SAR, and three-dimensional quantitative structure activity relationships (3D-QSAR represent important tools in understanding the interactions of the inhibitors with the active sites of the CYP enzymes. A comprehensive account of the QSAR studies on the major human CYPs 1A1, 1A2, 1B1, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4 and a few other CYPs are detailed in this review which will provide us with an insight into the individual/common characteristics of the active sites of these enzymes and the enzyme-inhibitor interactions.

  3. Identification of the cytochrome P450 monooxygenase that bridges the clavine and ergoline alkaloid pathways.

    Science.gov (United States)

    Haarmann, Thomas; Ortel, Ingo; Tudzynski, Paul; Keller, Ullrich

    2006-04-01

    Clavines and D-lysergic acid-derived alkaloid amides and alkaloid peptides are two different families of compounds that have the indole-derived tetracyclic metergoline ring system in common. Previous work has shown that D-lysergic acid is biosynthetically derived from clavine alkaloids. Recent cloning and analysis of the ergot alkaloid biosynthesis gene cluster from the D-lysergic acid peptide (ergopeptines)-producing Claviceps purpurea, has shown that it most probably contains all genes necessary for D-lysergic acid synthesis as well as those that encode the assembly of D-lysergic acid peptides, such as ergotamine. To address the role of the oxygenase genes of alkaloid-gene clusters, the only cytochrome P450 monooxygenase gene of this cluster was inactivated by disruption. The resultant mutant accumulated agroclavine, elymoclavine, and chanoclavine in substantial amounts but not ergopeptines. Feeding the mutant with D-lysergic acid restored ergopeptine synthesis; this suggests a block in the conversion of elymoclavine to D-lysergic acid. The gene was designated cloA (for encoding a clavine oxidase, CLOA). Retransformation of the mutant with the intact cloA gene also restored ergopeptine synthesis. These data show that CLOA catalyses the conversion of clavines to D-lysergic acid, it acts as a critical enzyme in the ergot alkaloid gene cluster, and bridges the biosynthesis of the two different families of alkaloids. PMID:16538694

  4. Effect of biliary obstruction and internal biliary drainage on hepatic cytochrome P450 isozymes in rats

    Institute of Scientific and Technical Information of China (English)

    Shintaro Fukushima; Hiroyasu Okuno; Nobuyuki Shibatani; Yoshitsugu Nakahashi; Toshihito Seki; Kazuichi Okazaki

    2008-01-01

    AIM: To investigate the total cytochrome P450 (CYP)content, microsomal mixed-function oxidase (MFO)activity, and expression of mRNAs for various CYP isozymes in a simple rat model of reversible obstructive jaundice.METHODS: Obstructive jaundice was created in male rats by causing bile duct obstruction with polyester tape.In another group of rats, bile duct obstruction was followed by internal biliary drainage after releasing the tape.The expression of various CYP isozyme mRNAs was semi-quantitatively assessed by competitive RTPCR.RESULTS: The total CYP content and microsomal MFO activity showed a significant decrease after biliary obstruction, but returned to respective control levels after biliary drainage.A marked reduction in the expression of CYPIA2, 2B1/2, 2Cll, 2E1, 3A1, and 3A2 mRNA was detected during biliary obstruction,while expression increased significantly toward the control level after biliary drainage.Although expression of CYP4A1 mRNA showed no reduction during biliary obstruction, it still increased significantly after biliary drainage.CONCLUSION: These results suggest that not only obstructive jaundice, but also the subsequent internal biliary drainage may affect regulatory medications of the synthesis of individual CYP isozymes differently.

  5. Effects of Pristane on Cytochrome P450 Isozyme Expression in Rat Tissues

    Directory of Open Access Journals (Sweden)

    Marvin A. Cuchens

    2005-04-01

    Full Text Available Chemical carcinogenesis studies are powerful tools to obtain information on potential mechanisms of chemical factors for malignancies. In this study Western blot analyses, using monoclonal antibodies specific for three different cytochrome P450 (CYP isozymes (CYP1A1, CYP1A2 and CYP2B, were employed to examine the effect(s of 3-methylcholanthrene and/or pristane (2,6,10,14-tetramethylpentadecane on the basal and inducible levels of expression of CYP proteins within Copenhagen rat tissues. Pristane exposure led to tissue specific differences in the CYP isozymes expressed and elicited increased CYP protein expression over 3-methylcholanthrene induced levels in microsomes isolated from liver, Peyer's Patches, and thymus. Within the context of the chemical carcinogenesis model employed in this study, these observations correlated with the induction of B-cell malignancies by low doses of 3-methylcholanthrene and of thymic lymphomas by a high 3-methylcholanthrene dose. The data suggest that pristane treatment affects CYP isozyme expression. This pristane-mediated effect clearly could be a contributing factor in the chemical carcinogenesis of the previously observed lymphoid malignancies, and a possible basis for the tumor enhancing effects of pristane.

  6. Orthogonal Assays Clarify the Oxidative Biochemistry of Taxol P450 CYP725A4.

    Science.gov (United States)

    Biggs, Bradley Walters; Rouck, John Edward; Kambalyal, Amogh; Arnold, William; Lim, Chin Giaw; De Mey, Marjan; O'Neil-Johnson, Mark; Starks, Courtney M; Das, Aditi; Ajikumar, Parayil Kumaran

    2016-05-20

    Natural product metabolic engineering potentially offers sustainable and affordable access to numerous valuable molecules. However, challenges in characterizing and assembling complex biosynthetic pathways have prevented more rapid progress in this field. The anticancer agent Taxol represents an excellent case study. Assembly of a biosynthetic pathway for Taxol has long been stalled at its first functionalization, putatively an oxygenation performed by the cytochrome P450 CYP725A4, due to confounding characterizations. Here, through combined in vivo (Escherichia coli), in vitro (lipid nanodisc), and metabolite stability assays, we verify the presence and likely cause of this enzyme's inherent promiscuity. Thereby, we remove the possibility that promiscuity simply existed as an artifact of previous metabolic engineering approaches. Further, spontaneous rearrangement and the stabilizing effect of a hydrophobic overlay suggest a potential role for nonenzymatic chemistry in Taxol's biosynthesis. Taken together, this work confirms taxadiene-5α-ol as a primary enzymatic product of CYP725A4 and provides direction for future Taxol metabolic and protein engineering efforts. PMID:26930136

  7. Cytochrome P450 metabolism of the post-lanosterol intermediates explains enigmas of cholesterol synthesis

    Science.gov (United States)

    Ačimovič, Jure; Goyal, Sandeep; Košir, Rok; Goličnik, Marko; Perše, Martina; Belič, Ales; Urlep, Žiga; Guengerich, F. Peter; Rozman, Damjana

    2016-06-01

    Cholesterol synthesis is among the oldest metabolic pathways, consisting of the Bloch and Kandutch-Russell branches. Following lanosterol, sterols of both branches are proposed to be dedicated to cholesterol. We challenge this dogma by mathematical modeling and with experimental evidence. It was not possible to explain the sterol profile of testis in cAMP responsive element modulator tau (Crem τ) knockout mice with mathematical models based on textbook pathways of cholesterol synthesis. Our model differs in the inclusion of virtual sterol metabolizing enzymes branching from the pathway. We tested the hypothesis that enzymes from the cytochrome P450 (CYP) superfamily can participate in the catalysis of non-classical reactions. We show that CYP enzymes can metabolize multiple sterols in vitro, establishing novel branching points of cholesterol synthesis. In conclusion, sterols of cholesterol synthesis can be oxidized further to metabolites not dedicated to production of cholesterol. Additionally, CYP7A1, CYP11A1, CYP27A1, and CYP46A1 are parts of a broader cholesterol synthesis network.

  8. Two Cytochrome P450 Monooxygenases Catalyze Early Hydroxylation Steps in the Potato Steroid Glycoalkaloid Biosynthetic Pathway.

    Science.gov (United States)

    Umemoto, Naoyuki; Nakayasu, Masaru; Ohyama, Kiyoshi; Yotsu-Yamashita, Mari; Mizutani, Masaharu; Seki, Hikaru; Saito, Kazuki; Muranaka, Toshiya

    2016-08-01

    α-Solanine and α-chaconine, steroidal glycoalkaloids (SGAs) found in potato (Solanum tuberosum), are among the best-known secondary metabolites in food crops. At low concentrations in potato tubers, SGAs are distasteful; however, at high concentrations, SGAs are harmful to humans and animals. Here, we show that POTATO GLYCOALKALOID BIOSYNTHESIS1 (PGA1) and PGA2, two genes that encode cytochrome P450 monooxygenases (CYP72A208 and CYP72A188), are involved in the SGA biosynthetic pathway, respectively. The knockdown plants of either PGA1 or PGA2 contained very little SGA, yet vegetative growth and tuber production were not affected. Analyzing metabolites that accumulated in the plants and produced by in vitro enzyme assays revealed that PGA1 and PGA2 catalyzed the 26- and 22-hydroxylation steps, respectively, in the SGA biosynthetic pathway. The PGA-knockdown plants had two unique phenotypic characteristics: The plants were sterile and tubers of these knockdown plants did not sprout during storage. Functional analyses of PGA1 and PGA2 have provided clues for controlling both potato glycoalkaloid biosynthesis and tuber sprouting, two traits that can significantly impact potato breeding and the industry. PMID:27307258

  9. Contribution of human hepatic cytochrome p450 isoforms to the metabolism of psychotropic drugs.

    Science.gov (United States)

    Niwa, Toshiro; Shiraga, Toshifumi; Ishii, Ikuko; Kagayama, Akira; Takagi, Akira

    2005-09-01

    The metabolic activities of six psychotropic drugs, diazepam, clotiazepam, tofisopam, etizolam, tandospirone, and imipramine, were determined for 14 isoforms of recombinant human hepatic cytochrome P450s (CYPs) and human liver microsomes by measuring the disappearance rate of parent compounds. In vitro kinetic studies revealed that Vmax/Km values in human liver microsomes were the highest for tofisopam, followed by tandospirone>clotiazepam>imipramine, diazepam, and etizolam. Among the recombinant CYPs, CYP3A4 exhibited the highest metabolic activities of all compounds except for clotiazepam and imipramine. The metabolism of clotiazepam was catalyzed by CYP2B6, CYP3A4, CYP2C18, and CYP2C19, and imipramine was metabolized by CYP2D6 most efficiently. In addition, the metabolic activities of diazepam, clotiazepam, and etizolam in human liver microsomes were inhibited by 2.5 microM ketoconazole, a CYP3A4 inhibitor, by 97.5%, 65.1%, and 83.5%, respectively, and the imipramine metabolism was not detected after the addition of 1 or 10 microM quinidine, a CYP2D6 inhibitor. These results suggest that the psychotropic drugs investigated are metabolized predominantly by CYP3A4, except that CYP2D6 catalyzes the metabolism of imipramine. In addition, this approach based on the disappearance rate appears to be useful for the identification of the responsible CYP isoform(s) of older drugs, for which metabolic profiles have not been reported. PMID:16141545

  10. Rice cytochrome P450 MAX1 homologs catalyze distinct steps in strigolactone biosynthesis

    KAUST Repository

    Zhang, Yanxia

    2014-10-26

    Strigolactones (SLs) are a class of phytohormones and rhizosphere signaling compounds with high structural diversity. Three enzymes, carotenoid isomerase DWARF27 and carotenoid cleavage dioxygenases CCD7 and CCD8, were previously shown to convert all-trans-β-carotene to carlactone (CL), the SL precursor. However, how CL is metabolized to SLs has remained elusive. Here, by reconstituting the SL biosynthetic pathway in Nicotiana benthamiana, we show that a rice homolog of Arabidopsis More Axillary Growth 1 (MAX1), encodes a cytochrome P450 CYP711 subfamily member that acts as a CL oxidase to stereoselectively convert CL into ent-2\\'-epi-5-deoxystrigol (B-C lactone ring formation), the presumed precursor of rice SLs. A protein encoded by a second rice MAX1 homolog then catalyzes the conversion of ent-2\\'-epi-5-deoxystrigol to orobanchol. We therefore report that two members of CYP711 enzymes can catalyze two distinct steps in SL biosynthesis, identifying the first enzymes involved in B-C ring closure and a subsequent structural diversification step of SLs.

  11. Cytochrome P450 CYP1A1: wider roles in cancer progression and prevention

    International Nuclear Information System (INIS)

    CYP1A1 is one of the main cytochrome P450 enzymes, examined extensively for its capacity to activate compounds with carcinogenic properties. Continuous exposure to inhalation chemicals and environmental carcinogens is thought to increase the level of CYP1A1 expression in extrahepatic tissues, through the aryl hydrocarbon receptor (AhR). Although the latter has long been recognized as a ligand-induced transcription factor, which is responsible for the xenobiotic activating pathway of several phase I and phase II metabolizing enzymes, recent evidence suggests that the AhR is involved in various cell signaling pathways critical to cell cycle regulation and normal homeostasis. Disregulation of these pathways is implicated in tumor progression. In addition, it is becoming increasingly evident that CYP1A1 plays an important role in the detoxication of environmental carcinogens, as well as in the metabolic activation of dietary compounds with cancer preventative activity. Ultimately the contribution of CYP1A1 to cancer progression or prevention may depend on the balance of procarcinogen activation/detoxication and dietary natural product extrahepatic metabolism

  12. Multiple P450 substrates in a single run: rapid and comprehensive in vitro interaction assay.

    Science.gov (United States)

    Turpeinen, Miia; Uusitalo, Jouko; Jouko, Uusitalo; Jalonen, Jorma; Jorma, Jalonen; Pelkonen, Olavi; Olavi, Pelkeonen

    2005-01-01

    The dramatically increased number of new chemical entities (NCE) used in drug discovery has raised a demand for efficient and rapid drug metabolism screening techniques. The aim of this study was to develop a global in vitro metabolic interaction screening test utilising the N-in-1 approach. A cocktail consisting of 10 CYP-selective probes with known kinetic, metabolic and interaction properties in vivo was incubated in a pool of human liver microsomes, and metabolites of melatonin (CYP1A2), coumarin (CYP2A6), bupropion (CYP2B6), amodiaquine (CYP2C8), tolbutamide (CYP2C9), omeprazole (CYP2C19 and CYP3A4), dextromethorphan (CYP2D6), chlorzoxazone (CYP2E1), midazolam (CYP3A4) and testosterone (CYP3A4) were analysed simultaneously using LC/TOF-MS. Performance of the method was assessed with cDNA expressed P450s and diagnostic CYP-specific inhibitors. The results were in good accordance with literature and our previous studies. The cocktail developed is suitable for fast and reliable in vitro screening of the interaction potential and characteristics of NCEs. PMID:15626586

  13. Lack of evidence for metabolism of p-phenylenediamine by human hepatic cytochrome P450 enzymes

    International Nuclear Information System (INIS)

    p-Phenylenediamine (PPD) is a widely used ingredient in permanent hair dyes; however, little has been published on its metabolism, especially with respect to hepatic cytochrome P450 (CYP)-mediated oxidation. This is regarded as a key step in the activation of carcinogenic arylamines that ultimately leads to the development of bladder cancer. Most epidemiology studies show no significant association between personal use of hair dyes and bladder cancer, but one recent study reported an increased risk of bladder cancer in women who were frequent users of permanent hair dyes. The aim of the present study was to use intact human hepatocytes, human liver microsomes, and heterologously expressed human CYPs to determine whether PPD is metabolised by hepatic CYPs to form an N-hydroxylamine. p-Phenylenediamine was N-acetylated by human hepatocytes to form N-acetylated metabolites, but there was no evidence for the formation of mono-oxygenated metabolites or for enzyme-mediated covalent binding of 14C-PPD to microsomal protein. In contrast, 2-aminofluorene underwent CYP-mediated metabolism to ≥4 different hydroxylated metabolites. The lack of evidence for hepatic CYP-mediated metabolism of PPD is inconsistent with the hypothesis that this compound plays a causal role in the development of bladder cancer via a mode of action involving hepatic metabolism to an N-hydroxyarylamine

  14. Jacobsen Catalyst as a Cytochrome P450 Biomimetic Model for the Metabolism of Monensin A

    Directory of Open Access Journals (Sweden)

    Bruno Alves Rocha

    2014-01-01

    Full Text Available Monensin A is a commercially important natural product isolated from Streptomyces cinnamonensins that is primarily employed to treat coccidiosis. Monensin A selectively complexes and transports sodium cations across lipid membranes and displays a variety of biological properties. In this study, we evaluated the Jacobsen catalyst as a cytochrome P450 biomimetic model to investigate the oxidation of monensin A. Mass spectrometry analysis of the products from these model systems revealed the formation of two products: 3-O-demethyl monensin A and 12-hydroxy monensin A, which are the same ones found in in vivo models. Monensin A and products obtained in biomimetic model were tested in a mitochondrial toxicity model assessment and an antimicrobial bioassay against Staphylococcus aureus, S. aureus methicillin-resistant, Staphylococcus epidermidis, Pseudomonas aeruginosa, and Escherichia coli. Our results demonstrated the toxicological effects of monensin A in isolated rat liver mitochondria but not its products, showing that the metabolism of monensin A is a detoxification metabolism. In addition, the antimicrobial bioassay showed that monensin A and its products possessed activity against Gram-positive microorganisms but not for Gram-negative microorganisms. The results revealed the potential of application of this biomimetic chemical model in the synthesis of drug metabolites, providing metabolites for biological tests and other purposes.

  15. Molecular identity and gene expression of aldosterone synthase cytochrome P450

    International Nuclear Information System (INIS)

    11β-Hydroxylase (CYP11B1) of bovine adrenal cortex produced corticosterone as well as aldosterone from 11-deoxycorticosterone in the presence of the mitochondrial P450 electron transport system. CYP11B1s of pig, sheep, and bullfrog, when expressed in COS-7 cells, also performed corticosterone and aldosterone production. Since these CYP11B1s are present in the zonae fasciculata and reticularis as well as in the zona glomerulosa, the zonal differentiation of steroid production may occur by the action of still-unidentified factor(s) on the enzyme-catalyzed successive oxygenations at C11- and C18-positions of steroid. In contrast, two cDNAs, one encoding 11β-hydroxylase and the other encoding aldosterone synthase (CYP11B2), were isolated from rat, mouse, hamster, guinea pig, and human adrenals. The expression of CYP11B1 gene was regulated by cyclic AMP (cAMP)-dependent signaling, whereas that of CYP11B2 gene by calcium ion-signaling as well as cAMP-signaling. Salt-inducible protein kinase, a cAMP-induced novel protein kinase, was one of the regulators of CYP11B2 gene expression

  16. Mixing apples and oranges: Analysis of heterotropic cooperativity in cytochrome P450 3A4.

    Science.gov (United States)

    Frank, Daniel J; Denisov, Ilia G; Sligar, Stephen G

    2009-08-15

    Heterotropic cooperative phenomena have been documented in studies with cytochrome P450 3A4, with few attempts to quantify this behavior other than to show the apparent stimulatory effect of certain CYP3A4 substrates on the enzyme's catalytic activity for others. Here CYP3A4 solubilized in Nanodiscs is studied for its ability to interact with two substrates, alpha-naphthoflavone and testosterone, which produce transitions in the heme spin state with apparent spectral affinities (corrected for membrane partitioning) of 7 and 38 microM, respectively. Simultaneous addition of both substrates at fixed molar ratios allows for the separation of specific heterotropic cooperative interactions from the simple additive affinities for the given substrate ratios. The absence of any changes in the normalized spectral dissociation constant due to changes in substrate ratio reveals that the observed stimulatory effect is largely due to differences in the relative substrate affinities and the presence of additional substrate in the system, rather than any specific positive heterotropic interactions between the two substrates. PMID:19560436

  17. The Effects of Acute Hydrogen Sulfide Poisoning on Cytochrome P450 Isoforms Activity in Rats

    Directory of Open Access Journals (Sweden)

    Xianqin Wang

    2014-01-01

    Full Text Available Hydrogen sulfide (H2S is the second leading cause of toxin related death (after carbon monoxide in the workplace. H2S is absorbed by the upper respiratory tract mucosa, and it causes histotoxic hypoxemia and respiratory depression. Cocktail method was used to evaluate the influences of acute H2S poisoning on the activities of cytochrome P450 isoforms CYP2B6, CYP2D6, CYP3A4, CYP1A2, CYP2C19, and CYP2C9, which were reflected by the changes of pharmacokinetic parameters of six specific probe drugs, bupropion, metoprolol, midazolam, phenacetin, omeprazole, and tolbutamide, respectively. The experimental rats were randomly divided into two groups, control group and acute H2S poisoning group (inhaling 300 ppm for 2 h. The mixture of six probes was given to rats by oral administration and the blood samples were obtained at a series of time points through the caudal vein. The concentrations of probe drugs in rat plasma were measured by LC-MS. The results for acute H2S poisoning and control groups were as follows: there was a statistically significant difference in the AUC and Cmax for bupropion, metoprolol, phenacetin, and tolbutamide, while there was no statistical pharmacokinetic difference for midazolam and omeprazole. Acute H2S poisoning could inhibit the activity of CYP2B6, CYP2D6, CYP1A2, and CYP2C9 in rats.

  18. Single-Walled Carbon Nanotubes Inhibit the Cytochrome P450 Enzyme, CYP3A4

    Science.gov (United States)

    El-Sayed, Ramy; Bhattacharya, Kunal; Gu, Zonglin; Yang, Zaixing; Weber, Jeffrey K.; Li, Hu; Leifer, Klaus; Zhao, Yichen; Toprak, Muhammet S.; Zhou, Ruhong; Fadeel, Bengt

    2016-02-01

    We report a detailed computational and experimental study of the interaction of single-walled carbon nanotubes (SWCNTs) with the drug-metabolizing cytochrome P450 enzyme, CYP3A4. Dose-dependent inhibition of CYP3A4-mediated conversion of the model compound, testosterone, to its major metabolite, 6β-hydroxy testosterone was noted. Evidence for a direct interaction between SWCNTs and CYP3A4 was also provided. The inhibition of enzyme activity was alleviated when SWCNTs were pre-coated with bovine serum albumin. Furthermore, covalent functionalization of SWCNTs with polyethylene glycol (PEG) chains mitigated the inhibition of CYP3A4 enzymatic activity. Molecular dynamics simulations suggested that inhibition of the catalytic activity of CYP3A4 is mainly due to blocking of the exit channel for substrates/products through a complex binding mechanism. This work suggests that SWCNTs could interfere with metabolism of drugs and other xenobiotics and provides a molecular mechanism for this toxicity. Our study also suggests means to reduce this toxicity, eg., by surface modification.

  19. Theoretical and isotope effect studies on cytochrome P-450 mediated aromatic oxidation

    International Nuclear Information System (INIS)

    Both theoretical and experimental approaches were used to probe the mechanism of cytochrome P-450 mediated aromatic oxidation. The theoretical studies used semiempirical molecular orbital calculations (MNDO) to model possible addition-rearrangement pathways and to clarify the role of epoxide intermediates during phenol formation. Both biradical and protonated cationic pathways leading to epoxides, ketones, and phenols were characterized for a series of substituted benzenes and naphthalene. The heats of formation and other thermodynamic properties of the geometry optimized products, intermediate, and transition states provided free energies of activation and allowed the calculation of kinetic isotope effects. The results of this study indicated that biradical pathways show little or no substituent effects and the order or reaction rates is epoxide > ketone > phenol. The cationic pathways show marked substituent effects and the order of reaction rates is reversed with phenol > ketone > epoxide. The experimental studies involved the measurement of isotope effects associated with the metabolism of a series of deuterated chlorobenzenes. Positive isotope effects were observed for the meta hydroxylation of 3,5-2H2-,2,4,6-2H3-, and 2H5-chlorobenzenes, whereas inverse isotope effects were measured for the ortho and para hydroxylation of 2,4,6-2H3-chlorobenzene

  20. The Effect of Vinpocetine on Human Cytochrome P450 Isoenzymes by Using a Cocktail Method.

    Science.gov (United States)

    Kong, Lingti; Song, Chunli; Ye, Linhu; Guo, Daohua; Yu, Meiling; Xing, Rong

    2016-01-01

    Vinpocetine is a derivative of the alkaloid vincamine, which had been prescribed for chronic cerebral vascular ischemia and acute ischemic stroke or used as a dietary supplement for its several different mechanisms of biological activities. However, information on the cytochrome P450 (CYP) enzyme-mediated drug metabolism has not been previously studied. The present study was performed to investigate the effects of vinpocetine on CYPs activity, and cocktail method was used, respectively. To evaluate the effects of vinpocetine on the activity of human CYP3A4, CYP2C9, CYP2C19, CYP2D6, and CYP2E1, human liver microsomes were utilized to incubate with the mixed CYPs probe substrates and the target components. The results indicate that vinpocetine exhibited weak inhibitory effect on the CYP2C9, where the IC50 value is 68.96 μM, whereas the IC50 values for CYP3A4, CYP2C19, CYP2D6, and CYP2E1 were all over range of 100 μM, which showed that vinpocetine had no apparent inhibitory effects on these CYPs. In conclusion, the results indicated that drugs metabolized by CYP2C9 coadministrated with vinpocetine may require attention or dose adjustment. PMID:27006677

  1. Sex difference in the principal cytochrome P-450 for tributyltin metabolism in rats

    International Nuclear Information System (INIS)

    Tributyltin is metabolized by cytochrome P-450 (CYP) system enzymes, and its metabolic fate may contribute to the toxicity of the chemical. In the present study, it is examined whether sex differences in the metabolism of tributyltin exist in rats. In addition, the in vivo and in vitro metabolism of tributyltin was investigated using rat hepatic CYP systems to confirm the principal CYP involved. A significant sex difference in metabolism occurred both in vivo and in vitro, suggesting that one of the CYPs responsible for tributyltin metabolism in rats is male specific or predominant at least. Eight cDNA-expressed rat CYPs, including typical phenobarbital (PB)-inducible forms and members of the CYP2C subfamily, were tested to determine their capability for tributyltin metabolism. Among the enzymes studied, a statistically significant dealkylation of tributyltin was mediated by CYP2C6 and 2C11. Furthermore, the sex difference in metabolism disappeared in vitro after anti-rat CYP2C11 antibody pretreatment because CYP2C11 is a major male-specific form in rats. These results indicate that CYP2C6 is the principal CYP for tributyltin metabolism in female rats, whereas CYP2C11 as well as 2C6 is involved in tributyltin metabolism in male rats, and it is suggested that CYP2C11 is responsible for the significant sex difference in the metabolism of tributyltin observed in rats

  2. Induction of cytochrome P-450 1A2 by oxidized tryptophan in Hepa lclc7 cells.

    Science.gov (United States)

    Sindhu, R K; Mitsuhashi, M; Kikkawa, Y

    2000-03-01

    Recent studies from this laboratory have demonstrated that L-tryptophan, after oxidation either by UV-irradiation or ozone, induces aryl hydrocarbon receptor (AhR) activation and binding of the liganded AhR complex to its specific DNA recognition site, thereby initiating transcription of the cytochrome P-450 1a1 (Cyp1a1) gene with concomitant increase of CYP1A1 protein and 7-ethoxyresorufin O-deethylase activity in wild-type mouse hepatoma cells, Hepa lclc7 (Hepa-1), in culture. Temporary inhibition of protein synthesis by cycloheximide resulted in superinduction of oxidized tryptophan-inducible CYP1A1 mRNA, protein, and 7-ethoxyresorufin O-deethylase activity in Hepa-1 cells. In the present communication, the results obtained by immunoblot analyses with monoclonal CYP1A1/1A2 antibody (NIH 1-7-1) demonstrate that both UV- or ozone-oxidized tryptophan also induce CYP1A2 protein in Hepa-1 cells. CYP1A2 mRNA, detected by reverse transcription-polymerase chain reaction, was markedly induced in the UV- or ozone-oxidized tryptophan-treated cells. Temporary inhibition of protein synthesis by cycloheximide further induced oxidized tryptophan-inducible CYP1A2 mRNA as well as the protein in Hepa-1 cells. This is the first report demonstrating the induction of CYP1A2 mRNA and protein in Hepa-1 cells. PMID:10688617

  3. A Fungal Cytochrome P-450nor Confers Denitrifying Ability to Tobacco By-2 Cells

    Directory of Open Access Journals (Sweden)

    Babiker M.A. Abdel-Banat

    2008-01-01

    Full Text Available Reactive nitrogen gases progressively contribute to the global warming. Development of gas-gas denitrifying plants that can efficiently reduce reactive nitrogen gases to dinitrogen (N2 could help to mitigate the effect of these gases. Taking the advances in gene manipulation technology, tobacco BY-2 cells were transformed with the fungus Cylindrocarpon tonkinense cytochrome P-450nor2 (Cnor2 gene. The product of this gene acts as nitric oxide reductase (nor. Transgenic BY-2 cell clones cultured in 15N-labelled nitrate (15NO3- actively evolved 15N2O gas up to 35-folds compared to the wild-type cells. In 15N-labelled ammonium (15NH4+, the transgenic and wild-type cells produced comparable amounts of 15N2O. This indicates that ammonium is not a direct substrate for nor and the small amount of N2O observed may be due to the nitrification of ammonium to nitrite. Addition of tungstate (a nitrate reductase inhibitor and cyanide to the transgenic cell cultures strongly inhibited 15N2O production. Activity of nor enzyme was also confirmed by in vitro activity assay. These observations together suggest that Cnor2 is actively expressed and enhanced the reduction of nitrate to N2O in plant cells. This finding indicates that plant cells are capable to tackle the denitrification pathway.

  4. Geneva cocktail for cytochrome p450 and P-glycoprotein activity assessment using dried blood spots.

    Science.gov (United States)

    Bosilkovska, M; Samer, C F; Déglon, J; Rebsamen, M; Staub, C; Dayer, P; Walder, B; Desmeules, J A; Daali, Y

    2014-09-01

    The suitability of the capillary dried blood spot (DBS) sampling method was assessed for simultaneous phenotyping of cytochrome P450 (CYP) enzymes and P-glycoprotein (P-gp) using a cocktail approach. Ten volunteers received an oral cocktail capsule containing low doses of the probes bupropion (CYP2B6), flurbiprofen (CYP2C9), omeprazole (CYP2C19), dextromethorphan (CYP2D6), midazolam (CYP3A), and fexofenadine (P-gp) with coffee/Coke (CYP1A2) on four occasions. They received the cocktail alone (session 1), and with the CYP inhibitors fluvoxamine and voriconazole (session 2) and quinidine (session 3). In session 4, subjects received the cocktail after a 7-day pretreatment with the inducer rifampicin. The concentrations of probes/metabolites were determined in DBS and plasma using a single liquid chromatography-tandem mass spectrometry method. The pharmacokinetic profiles of the drugs were comparable in DBS and plasma. Important modulation of CYP and P-gp activities was observed in the presence of inhibitors and the inducer. Minimally invasive one- and three-point (at 2, 3, and 6 h) DBS-sampling methods were found to reliably reflect CYP and P-gp activities at each session. PMID:24722393

  5. Induction of cytochrome P450 enzymes in primary equine hepatocyte culture.

    Science.gov (United States)

    Stefanski, A; Mevissen, M; Möller, A-M; Kuehni-Boghenbor, K; Schmitz, A

    2013-10-01

    In this study, we established cell culture conditions for primary equine hepatocytes allowing cytochrome P450 enzyme (CYP) induction experiments. Hepatocytes were isolated after a modified method of Bakala et al. (2003) and cultivated on collagen I coated plates. Three different media were compared for their influence on morphology, viability and CYP activity of the hepatocytes. CYP activity was evaluated with the fluorescent substrate 7-benzyloxy-4-trifluoromethylcoumarin. Induction experiments were carried out with rifampicin, dexamethasone or phenobarbital. Concentration-response curves for induction with rifampicin were created. Williams' medium E showed the best results on morphology and viability of the hepatocytes and was therefore used for the following induction experiments. Cells cultured in Dulbecco's Modified Eagle Medium were not inducible. Incubation with rifampicin increased the CYP activity in two different hepatocyte preparations in a dose dependent manner (EC₅₀=1.20 μM and 6.06 μM; Emax=4.1- and 3.4-fold induction). No increase in CYP activity was detected after incubation with dexamethasone or phenobarbital. The hepatocyte culture conditions established in this study proved to be valuable for investigation of the induction of equine CYPs. In further studies, other equine drugs can be evaluated for CYP induction with this in vitro system. PMID:23916975

  6. Assessment of cytochrome p450 enzyme inhibition and inactivation in drug discovery and development.

    Science.gov (United States)

    Nettleton, David O; Einolf, Heidi J

    2011-01-01

    Evaluation of the potential of a drug candidate to inhibit or inactivate cytochrome P450 (CYP) enzymes remains an important part of pharmaceutical drug Discovery and Development programs. CYP enzymes are considered to be one of the most important enzyme families involved in the metabolic clearance of the vast majority of prescribed drugs. Clinical drug-drug interactions (DDI) involving inhibition or time-dependent inactivation of these enzymes can result in dangerous side effects resulting from reduced clearance/increased exposure of the drug being affected (the 'victim' drug). In this regard, pharmaceutical companies have become quite vigilant in mitigating CYP inhibition/inactivation liabilities of drug candidates early in Discovery including continued risk assessment throughout Development. In this review, common strategies and decision making processes for the assessment of DDI risk in the different stages of pharmaceutical development are discussed. In addition, in vitro study designs, analysis, and interpretation of CYP inhibition and inactivation data are described in stage appropriate context. The in vitro tools and knowledge available now enable the Discovery Chemist to place the potential CYP DDI liability of a drug candidate into perspective and to aid in the optimization of chemical drug design to further mitigate this risk. PMID:21320066

  7. Regulation of Porcine Hepatic Cytochrome P450 — Implication for Boar Taint

    Directory of Open Access Journals (Sweden)

    Martin Krøyer Rasmussen

    2014-09-01

    Full Text Available Cytochrome P450 (CYP450 is the major family of enzymes involved in the metabolism of several xenobiotic and endogenous compounds. Among substrates for CYP450 is the tryptophan metabolite skatole (3-methylindole, one of the major contributors to the off-odour associated with boar-tainted meat. The accumulation of skatole in pigs is highly dependent on the hepatic clearance by CYP450s. In recent years, the porcine CYP450 has attracted attention both in relation to meat quality and as a potential model for human CYP450. The molecular regulation of CYP450 mRNA expression is controlled by several nuclear receptors and transcription factors that are targets for numerous endogenously and exogenously produced agonists and antagonists. Moreover, CYP450 expression and activity are affected by factors such as age, gender and feeding. The regulation of porcine CYP450 has been suggested to have more similarities with human CYP450 than other animal models, including rodents. This article reviews the available data on porcine hepatic CYP450s and its implications for boar taint.

  8. Pomegranate juice effects on cytochrome P450S expression: in vivo studies.

    Science.gov (United States)

    Faria, Ana; Monteiro, Rosário; Azevedo, Isabel; Calhau, Conceição

    2007-12-01

    Beneficial health effects have recently been claimed for pomegranate juice. In vitro and in vivo studies have demonstrated its anti-atherosclerotic capacity, chemoprevention and chemotherapy of prostate cancer, and antiproliferative, apoptotic, and antioxidant activity, among others. On the other hand, there is a complex interplay between tumor initiation, promotion, and progression and xenobiotic biotranformation. This led us to investigate the effect of pomegranate juice consumption on cytochrome P450 (CYP) activity and expression. For this purpose, male mice consumed this fruit juice for 4 weeks, and pentobarbital-induced sleeping time and total hepatic CYP content, activity, and expression were evaluated. Moreover, the activity of CYP isoform 2E1 and expression of the main CYP isoforms, namely, CYP1A1/2, CYP2E1, and CYP3A, were also assessed. It was found that pomegranate juice consumption decreased total hepatic CYP content as well as the expression of CYP1A2 and CYP3A. Prevention of procarcinogen activation through CYP activity/expression inhibition may be involved in pomegranate juice's effect on tumor initiation, promotion, and progression. PMID:18158835

  9. Potent inhibition by star fruit of human cytochrome P450 3A (CYP3A) activity.

    Science.gov (United States)

    Hidaka, Muneaki; Fujita, Ken-ichi; Ogikubo, Tetsuya; Yamasaki, Keishi; Iwakiri, Tomomi; Okumura, Manabu; Kodama, Hirofumi; Arimori, Kazuhiko

    2004-06-01

    There has been very limited information on the capacities of tropical fruits to inhibit human cytochrome P450 3A (CYP3A) activity. Thus, the inhibitory effects of tropical fruits on midazolam 1'-hydroxylase activity of CYP3A in human liver microsomes were evaluated. Eight tropical fruits such as common papaw, dragon fruit, kiwi fruit, mango, passion fruit, pomegranate, rambutan, and star fruit were tested. We also examined the inhibition of CYP3A activity by grapefruit (white) and Valencia orange as controls. The juice of star fruit showed the most potent inhibition of CYP3A. The addition of a star fruit juice (5.0%, v/v) resulted in the almost complete inhibition of midazolam 1'-hydroxylase activity (residual activity of 0.1%). In the case of grape-fruit, the residual activity was 14.7%. The inhibition depended on the amount of fruit juice added to the incubation mixture (0.2-6.0%, v/v). The elongation of the preincubation period of a juice from star fruit (1.25 or 2.5%, v/v) with the microsomal fraction did not alter the CYP3A inhibition, suggesting that the star fruit did not contain a mechanism-based inhibitor. Thus, we discovered filtered extracts of star fruit juice to be inhibitors of human CYP3A activity in vitro. PMID:15155547

  10. Identification and Characterization of CYP9A40 from the Tobacco Cutworm Moth (Spodoptera litura), a Cytochrome P450 Gene Induced by Plant Allelochemicals and Insecticides

    OpenAIRE

    Rui-Long Wang; Christian Staehelin; Qing-Qing Xia; Yi-Juan Su; Ren-Sen Zeng

    2015-01-01

    Cytochrome P450 monooxygenases (P450s) of insects play crucial roles in the metabolism of endogenous and dietary compounds. Tobacco cutworm moth (Spodoptera litura), an important agricultural pest, causes severe yield losses in many crops. In this study, we identified CYP9A40, a novel P450 gene of S. litura, and investigated its expression profile and potential role in detoxification of plant allelochemicals and insecticides. The cDNA contains an open reading frame encoding 529 amino acid re...

  11. Inhibition of NADPH cytochrome P450 reductase by the model sulfur mustard vesicant 2-chloroethyl ethyl sulfide is associated with increased production of reactive oxygen species

    OpenAIRE

    Gray, Joshua P.; Mishin, Vladimir; Heck, Diane E.; Laskin, Debra L.; Laskin, Jeffrey D.

    2010-01-01

    Inhalation of vesicants including sulfur mustard can cause significant damage to the upper airways. This is the result of vesicant-induced modifications of proteins important in maintaining the integrity of the lung. Cytochrome P450’s are the major enzymes in the lung mediating detoxification of sulfur mustard and its metabolites. NADPH cytochrome P450 reductase is a flavin-containing electron donor for cytochrome P450. The present studies demonstrate that the sulfur mustard analog, 2-chloroe...

  12. Cj1411c GENE OF CAMPYLOBACTER JEJUNI 11168 ENCODES FOR A CYTOCHROME P450 INVOLVED IN BACTERIAL CAPSULE SUGAR METABOLISM

    Directory of Open Access Journals (Sweden)

    CORCIONIVOSCHI N.

    2007-01-01

    Full Text Available After isolation in 1970s, Campylobacter jejuni become the most commonlyrecognized cause of bacterial gastroenteritis in man. In animals is frequently foundin bovines on ovines. Publishing of the genome sequence of Campylobacter jejuni11168 (Parkhill, 2000 revealed the presence of only one cytochrome P450 in anoperon involved in sugar and cell surface biosynthesis. The gene name is Cj1411c, is1359 bp long and encodes 453 aa. The sequence is strictly conserved inCampylobacter jejuni RM221. Similarities with two cytochrome P450s, one formSilicobacter sp. and one form Poloromonas sp., were identified. These two enzymesare known to be involved in ascorbate and aldarate metabolism. The recombinantconstruct allowed the expression of active P450 enzyme with a 450 nm peak whenbinds CO. The protein was purified in proportion of ~ 70 %. By deleting the P450gene from the Campylobacter jejuni 11168 genome clear changes in cellmorphology were identified cells becoming wider and shorter. The capsular sugarprofile of the NCI strain reveals the presence of arabinose which was not found inthe wild type strain. The arabinose was identified by both High Performance LiquidChromatography (HPLC and Nuclear Magnetic Resonance (NMR.

  13. Involvement of cytochrome P-450 enzyme activity in the selectivity and safening action of pyrazosulfuron-ethyl.

    Science.gov (United States)

    Yun, M S; Shim, I S; Usui, K

    2001-03-01

    To investigate the selectivity and safening action of the sulfonylurea herbicide pyrazosulfuron-ethyl (PSE), pyrazosulfuron-ethyl O-demethylase (PSEOD) activity involving oxidative metabolism by cytochrome P-450 was studied in rice (Oryza sativa L cv Nipponbare) and Cyperus serotinus Rottb. Cytochrome P-450-dependent activity was demonstrated by the use of the inducers 1,8-naphthalic anhydride and ethanol, the herbicides PSE, bensulfuron-methyl, dimepiperate and dymron, or the inhibitor piperonyl butoxide (PBO). Growth inhibition in C serotinus seedlings was more severe than that in rice seedlings. O-Dealkylation activities of PSE were induced differently in rice and in C serotinus, with distinctly higher activity in rice seedlings. The induced PSEOD activities were slightly inhibited by PBO in rice seedlings, whereas they were strongly inhibited in C serotinus seedlings. Dimepiperate and dymron were effective safeners of rice against PSE treatment. Treatments with herbicide alone resulted in less induction of PSEOD activity compared with combined treatments of the herbicide and safener. PSEOD activity in rice seedlings induced with herbicide alone was strongly inhibited by PBO, whereas it was weakly inhibited in rice seedlings induced with combinations of PSE and two safeners. These results suggest that O-demethylation by cytochrome P-450 enzymes may be involved in the metabolism of PSE and may contribute to its selectivity and safening action. Furthermore, these results suggest the existence of a multiple form of cytochrome P-450 in plants. PMID:11455659

  14. Whole genome identification, phylogeny and evolution of the cytochrome P450 family 2 (CYP2) sub-families in birds

    DEFF Research Database (Denmark)

    Almeida, Daniela; Maldonado, Emanuel; Khan, Imran; Silva, Liliana; Gilbert, M Thomas P; Zhang, Guojie; Jarvis, Erich D; O'Brien, Stephen J; Johnson, Warren E; Antunes, Agostinho

    2016-01-01

    The cytochrome P450 (CYP) superfamily defends organisms from endogenous and noxious environmental compounds, and thus is crucial for survival. However, beyond mammals the molecular evolution of CYP2 subfamilies is poorly understood. Here, we characterized the CYP2 family across 48 novel avian who...

  15. Antipsychotic-induced extrapyramidal syndromes and cytochrome P-450 2D6 genotype : a case-control study

    NARCIS (Netherlands)

    Schillevoort, [No Value; de Boer, A; van der Weide, J; Steijns, LSW; Roos, RAC; Jansen, PAF; Leufkens, HGM

    2002-01-01

    To study the association between polymorphism of the cytochrome P-450 2D6 gene (CYP2D6) and the risk of antipsychotic-induced extrapyramidal syndromes, as measured by the use of anti parkinsonian medication. Data for this case-control study were obtained from a psychiatric hospital where newly admit

  16. Evolution of substrate recognition sites (SRSs) in cytochromes P450 from Apiaceae exemplified by the CYP71AJ subfamily

    DEFF Research Database (Denmark)

    Dueholm, Bjørn; Krieger, Celia; Drew, Damian; Olry, Alexandre; Kamo, Tsunashi; Taboureau, Olivier; Weitzel, Corinna; Bourgaud, Frederic; Hehn, Alain; Simonsen, Henrik Toft

    2015-01-01

    Background: Large proliferations of cytochrome P450 encoding genes resulting from gene duplications can be termed as 'blooms', providing genetic material for the genesis and evolution of biosynthetic pathways. Furanocoumarins are allelochemicals produced by many of the species in Apiaceaous plant...

  17. Purification, crystallization and preliminary X-ray analysis of cytochrome P450 219A1 from Novosphingobium aromaticivorans DSM 12444

    International Nuclear Information System (INIS)

    A 2.4 Å resolution data set was collected from a crystal of CYP219A1. This is the first protein of a new P450 family from the oligotrophic organism N. aromaticivorans which can oxidize steroids and bind sesquiterpenoids. Cytochrome P450 enzymes catalyze a variety of reactions and are widely distributed in living organisms. In recent studies, the first members of five new families of cytochrome P450 enzymes have been identified, including cytochrome P450 219A1 (CYP219A1) from Novosphingobium aromaticivorans DSM 12444. It has also been reported that isolongifolen-9-one (C15H22O), a sesquiterpenoid ketone derivative, is a potential substrate for CYP219A1, inducing a ≥95% shift of the haem spin state to high spin upon binding. The CYP219A1 protein has been crystallized and single crystals have been studied by X-ray crystallography. Diffraction data were collected to 2.4 Å resolution. The crystals belonged to space group P6, with unit-cell parameters a = 93.1, b = 93.1, c = 98.0 Å. Preliminary X-ray diffraction data analysis revealed that the asymmetric unit contained one protein molecule

  18. Induction and characterization of a cytochrome P-450-dependent camphor hydroxylase in tissue cultures of common sage (Salvia officinalis)

    Energy Technology Data Exchange (ETDEWEB)

    Funk, C.; Croteau, R. (Washington State Univ., Pullman (United States))

    1993-04-01

    (+)-Camphor, a major monoterpene of the essential oil of common sage (Salvia officinalis), is catabolized in senescent tissue, and the pathway for the breakdown of this bicyclic ketone has been previously elucidated in sage cell-suspension cultures. In the initial step of catabolism, camphor is oxidized to 6-exo-hydroxycamphor, and the corresponding NADPH- and O[sub 2]-dependent hydroxylase activity was demonstrated in microsomal preparations of sage cells. Several well-established inhibitors of cytochrome P-450-dependent reactions, including cytochrome c, clotrimazole, and CO, inhibited the hydroxylation of camphor, and CO-dependent inhibition was partially reversed by blue light. Upon treatment of sage suspension cultures with 30 mM MnCl[sub 2], camphor-6-hydroxylase activity was induced up to 7-fold. A polypeptide with estimated molecular mass of 58 kD from sage microsomal membranes exhibited antigenic cross-reactivity in western blot experiments with two heterologous polyclonal antibodies raised against cytochrome P-450 camphor-5-exo-hydroxylase from Pseudomonas putida and cytochrome P-450 limonene-6S-hydroxylase from spearmint (Mentha spicata). Dot blotting indicated that the concentration of this polypeptide increased with camphor hydroxylase activity in microsomes of Mn[sup 2+]-induced sage cells. These results suggest that camphor-6-exo-hydroxylase from sage is a microsomal cytochrome P-450 monooxygenase that may share common properties and epitopes with bacterial and other plant monoterpene hydroxylases. 44 refs., 6 figs., 2 tabs.

  19. Genetic Basis for Varied Levels of Injury to Sweet Corn Hybrids from Three Cytochrome P-450 Metabolized Herbicides

    Science.gov (United States)

    Some sweet corn (Zea mays L.) hybrids and inbreds can be severely injured or killed following post-emergence applications of certain P450-metabolized-herbicides. Consequently, existing hybrids are regularly evaluated for reaction to new herbicides, and new hybrids are evaluated for reactions to exi...

  20. Effects of interferon-tau and steroids on cytochrome P450 activity in bovine endometrial epithelial cells

    Science.gov (United States)

    The objective of the current study was to examine cyclooxygenase (COX), cytochrome P450 1A (CYP1A) and 2C (CYP2C) activity in bovine endometrial cell cultures following exposure to oxytocin (OT), interferon-t (IFN), estradiol (E2) and/or progesterone (P4). Bovine endometrial epithelial cells were tr...

  1. Improved cyclopropanation activity of histidine-ligated cytochrome P450 enables the enantioselective formal synthesis of levomilnacipran.

    Science.gov (United States)

    Wang, Z Jane; Renata, Hans; Peck, Nicole E; Farwell, Christopher C; Coelho, Pedro S; Arnold, Frances H

    2014-06-23

    Engineering enzymes capable of modes of activation unprecedented in nature will increase the range of industrially important molecules that can be synthesized through biocatalysis. However, low activity for a new function is often a limitation in adopting enzymes for preparative-scale synthesis, reaction with demanding substrates, or when a natural substrate is also present. By mutating the proximal ligand and other key active-site residues of the cytochrome P450 enzyme from Bacillus megaterium (P450-BM3), a highly active His-ligated variant of P450-BM3 that can be employed for the enantioselective synthesis of the levomilnacipran core was engineered. This enzyme, BM3-Hstar, catalyzes the cyclopropanation of N,N-diethyl-2-phenylacrylamide with an estimated initial rate of over 1000 turnovers per minute and can be used under aerobic conditions. Cyclopropanation activity is highly dependent on the electronic properties of the P450 proximal ligand, which can be used to tune this non-natural enzyme activity. PMID:24802161

  2. Evolution of substrate recognition sites (SRSs) in cytochromes P450 from Apiaceae exemplified by the CYP71AJ subfamily

    DEFF Research Database (Denmark)

    Dueholm, Bjørn; Krieger, Celia; Drew, Damian;

    2015-01-01

    significant modification of the accession to the iron atom, which might explain the difference of the substrate specificity between the cytochromes P450 restricted to furanocoumarins as substrates and the orphan CYP71AJ. Conclusion: Two subclades functionally assigned to the biosynthesis of furanocoumarins...

  3. The novel antifungal agent PLD-118 is neither metabolized by liver microsomes nor inhibits cytochrome P450 in vitro

    NARCIS (Netherlands)

    Parnham, M.J.; Bogaards, J.J.P.; Schrander, F.; Schut, M.W.; Orešković, K.; Mildner, B.

    2005-01-01

    PLD-118 is a novel, oral antifungal drug, under development for the treatment of Candida infections. Possible metabolism of PLD-118 by rat, dog and human S9 liver homogenates and inhibition of human cytochrome P450 (CYP) enzymes were investigated. PLD-118 (10 and 100 μm) incubated for 0-60 min with

  4. Effect of butylated hydroxytoluene, curcumin, propyl gallate and thiabendazole on cytochrome P450 forms in cultured human hepatocytes

    NARCIS (Netherlands)

    Price, R.J.; Scott, M.P.; Giddings, A.M.; Walters, D.G.; Stierum, R.H.; Meredith, C.; Lake, B.G.

    2008-01-01

    1. The objective of this study was to investigate the effects of four food chemicals, namely butylated hydroxytoluene (BHT), curcumin (CC), propyl gallate (PG) and thiabendazole (TB), on cytochrome P450 (CYP) forms in cultured human hepatocytes. 2. Treatment of human hepatocytes for 72 h with 2-200

  5. Aromatase enzyme (P450arom) mRNA expression in mouse gonads and the effect of intra-peritoneal amino acid injection

    OpenAIRE

    Hüseyin Baki Çiftci

    2011-01-01

    The aim of this study was show the presence of (P450arom) mRNA expressions in mouse gonads and to measure the effect of intra-peritoneal serine/threonine or glycine injections on the expression of aromatase enzyme P450arommRNA. Three different (1.4, 2.4 and 4.4kb) P450arom mRNA species were indentified in mouse testis and the ovary. Relative to saline injected group (S), all the species of P450arom enzyme mRNA decreased in serine/threonine (A) or glycine (G) injected female mice, while all o...

  6. Evidence against the Bm1P1 protein as a positive transcription factor for barbiturate-mediated induction of cytochrome P450BM-1 in bacillus megaterium.

    Science.gov (United States)

    Shaw, G C; Sung, C C; Liu, C H; Lin, C H

    1998-04-01

    The Bm1P1 protein was previously proposed to act as a positive transcription factor involved in barbiturate-mediated induction of cytochrome P450BM-1 in Bacillus megaterium. We now report that the bm1P1 gene encodes a protein of 217 amino acids, rather than the 98 amino acids as reported previously. In vitro gel shift assays indicate that the Bm1P1 protein did not interact with probes comprising the regulatory regions of the P450BM-1 gene. Moreover, disruption of the bm1P1 gene did not markedly affect barbiturate induction of P450BM-1 expression. A multicopy plasmid harboring only the P450BM-1 promoter region could increase expression of the chromosome-encoded P450BM-1. The level of expression is comparable with that shown by a multicopy plasmid harboring the P450BM-1 promoter region along with the bm1P1 gene. These results strongly suggest that the Bm1P1 protein is unlikely to act as a positive regulator for barbiturate induction of P450BM-1 expression. Finally, deletion of the Barbie box did not markedly diminish the effect of pentobarbital on expression of a reporter gene transcriptionally fused to the P450BM-1 promoter. This suggests that the Barbie box is unlikely to be a key element in barbiturate-mediated induction of P450BM-1. PMID:9525898

  7. The cytochrome P450 complement of the myxobacterium Sorangium cellulosum So ce56 and characterization of two members, CYP109D1 and CYP260A1

    OpenAIRE

    Khatri, Yogan

    2009-01-01

    This is a very first systematic study of cytochrome P450s from Sorangium cellulosum So ce56. In chapter I, the open reading frames (ORFs) encoding twenty-one cytochrome P450s with nine novel families were identified, and a phylogenetic tree and a physical map were constructed. All P450 ORFs were cloned, expressed in Escherichia coli in a soluble form, purified and characterized. The heterologous redox partners for nine of the P450s were identified. A compound library (~17,000 ligands) was scr...

  8. Metabolism of Vitamin D2 to 17,20,24-Trihydroxyvitamin D2 by Cytochrome P450scc (CYP11A1)

    OpenAIRE

    Nguyen, Minh N.; Slominski, Andrzej; Li, Wei; Ng, Yun Rui; Tuckey, Robert C.

    2008-01-01

    As well as catalyzing the conversion of cholesterol to pregnenolone for steroid synthesis, cytochrome P450scc (P450scc) can also metabolize vitamins D2 (D2) and D3 (D3). Two products of D2 metabolism by P450scc, 20-hydroxyvitamin D2 and 17,20-dihydroxyvitamin D2, have been identified and shown to exert biological activity on cultured keratinocytes. The aim of this study was to fully characterize the metabolism of D2 by P450scc, including identifying additional products...

  9. Substrate binding to cytochrome P450-2J2 in Nanodiscs detected by nanoplasmonic Lycurgus cup arrays.

    Science.gov (United States)

    Plucinski, Lisa; Ranjan Gartia, Manas; Arnold, William R; Ameen, Abid; Chang, Te-Wei; Hsiao, Austin; Logan Liu, Gang; Das, Aditi

    2016-01-15

    Cytochrome P450s are the primary enzymes involved in phase I drug metabolism. They are an important target for early drug discovery research. However, high-throughput drug screening of P450s is limited by poor protein stability and lack of consistent measurement of binding events. Here we present the detection of substrate binding to cytochrome P450-2J2 (CYP2J2), the predominant P450 in the human heart, using a combination of Nanodisc technology and a nanohole plasmonic sensor called nanoplasmonic Lycurgus cup array (nanoLCA). The Nanodisc, a nanoscale membrane bilayer disc, is used to stabilize the protein on the metallic plasmonic surface. Absorption spectroscopy of seven different substrates binding to CYP2J2 in solution showed that they are all type I, resulting in shifting of the protein bands to lower wavelengths (blue shift). Detection on the nanoLCA sensor also showed spectral blue shifts of CYP2J2 following substrate binding. Finite Difference Time Domain (FDTD) electromagnetic simulation suggested that the blue shift on the nanoLCA is because of the hybridization of plasmon polariton Bloch wave and the electronic resonance of the heme group of CYP2J2. We found the plasmonic properties of the nanoLCA sensor to be highly reproducible, which allowed comparisons among the different substrates at different concentrations. Further, due to the unique spectral properties of the nanoLCA sensor, including the transmission of a single color, we were able to perform colorimetric detection of the binding events. These results indicate that a resonance plasmonic sensing mechanism can be used to distinguish between different substrates of the same binding type at different concentrations binding to P450s and that the nanoLCA sensor has the potential to provide consistent high-throughput measurements of this system. PMID:26334592

  10. Mechanism of chloroform-induced renal toxicity: Non-involvement of hepatic cytochrome P450-dependent metabolism

    International Nuclear Information System (INIS)

    Chloroform causes hepatic and renal toxicity in a number of species. In vitro studies have indicated that chloroform can be metabolized by P450 enzymes in the kidney to nephrotoxic intermediate, although direct in vivo evidence for the role of renal P450 in the nephrotoxicity has not been reported. This study was to determine whether chloroform renal toxicity persists in a mouse model with a liver-specific deletion of the P450 reductase (Cpr) gene (liver-Cpr-null). Chloroform-induced renal toxicity and chloroform tissue levels were compared between the liver-Cpr-null and wild-type mice at 24 h following differing doses of chloroform. At a chloroform dose of 150 mg/kg, the levels of blood urea nitrogen (BUN) were five times higher in the exposed group than in the vehicle-treated one for the liver-Cpr-null mice, but they were only slightly higher in the exposed group than in the vehicle-treated group for the wild-type mice. Severe lesions were found in the kidney of the liver-Cpr-null mice, while only mild lesions were found in the wild-type mice. At a chloroform dose of 300 mg/kg, severe kidney lesions were observed in both strains, yet the BUN levels were still higher in the liver-Cpr-null than in the wild-type mice. Higher chloroform levels were found in the tissues of the liver-Cpr-null mice. These findings indicated that loss of hepatic P450-dependent chloroform metabolism does not protect against chloroform-induced renal toxicity, suggesting that renal P450 enzymes play an essential role in chloroform renal toxicity

  11. Does cytochrome P450 liver isoenzyme induction increase the risk of liver toxicity after paracetamol overdose?

    Directory of Open Access Journals (Sweden)

    Kalsi SS

    2011-10-01

    Full Text Available Sarbjeet S Kalsi1,2, David M Wood2–4, W Stephen Waring5, Paul I Dargan2–4 1Emergency Department, 2Clinical Toxicology, Guy's and St Thomas' NHS Foundation Trust, London; 3King's Health Partners, 4King's College London, London; 5York Teaching Hospital NHS Foundation Trust, York, UK Abstract: Paracetamol (acetaminophen, N-acetyl-p-aminophenol, 4-hydroxyacetanilide is the most common cause of acute liver failure in developed countries. There are a number of factors which potentially impact on the risk of an individual developing hepatotoxicity following an acute paracetamol overdose. These include the dose of paracetamol ingested, time to presentation, decreased liver glutathione, and induction of cytochrome P450 (CYP isoenzymes responsible for the metabolism of paracetamol to its toxic metabolite N-acetyl-p-benzoquinoneimine (NAPQI. In this paper, we review the currently published literature to determine whether induction of relevant CYP isoenzymes is a risk factor for hepatotoxicity in patients with acute paracetamol overdose. Animal and human in vitro studies have shown that the CYP isoenzyme responsible for the majority of human biotransformation of paracetamol to NAPQI is CYP2E1 at both therapeutic and toxic doses of paracetamol. Current UK treatment guidelines suggest that patients who use a number of drugs therapeutically should be treated as “high-risk” after paracetamol overdose. However, based on our review of the available literature, it appears that the only drugs for which there is evidence of the potential for an increased risk of hepatotoxicity associated with paracetamol overdose are phenobarbital, primidone, isoniazid, and perhaps St John's wort. There is no evidence that other drugs often quoted as increasing risk, such as carbamazepine, phenytoin, primidone, rifampicin, rifabutin, efavirenz, or nevirapine, should be considered risk factors for hepatotoxicity in patients presenting with acute paracetamol overdose. Keywords

  12. Cytochrome P450-dependent toxic effects of primaquine on human erythrocytes

    International Nuclear Information System (INIS)

    Primaquine, an 8-aminoquinoline, is the drug of choice for radical cure of relapsing malaria. Use of primaquine is limited due to its hemotoxicity, particularly in populations with glucose-6-phosphate dehydrogenase deficiency [G6PD(-)]. Biotransformation appears to be central to the anti-infective and hematological toxicities of primaquine, but the mechanisms are still not well understood. Metabolic studies with primaquine have been hampered due to the reactive nature of potential hemotoxic metabolites. An in vitro metabolism-linked hemotoxicity assay has been developed. Co-incubation of the drug with normal or G6PD(-) erythrocytes, microsomes or recombinant cytochrome P450 (CYP) isoforms has allowed in situ generation of potential hemotoxic metabolite(s), which interact with the erythrocytes to generate hemotoxicity. Methemoglobin formation, real-time generation of reactive oxygen intermediates (ROIs) and depletion of reactive thiols were monitored as multiple biochemical end points for hemotoxicity. Primaquine alone did not produce any hemotoxicity, while a robust increase was observed in methemoglobin formation and generation of ROIs by primaquine in the presence of human or mouse liver microsomes. Multiple CYP isoforms (CYP2E1, CYP2B6, CYP1A2, CYP2D6 and CYP3A4) variably contributed to the hemotoxicity of primaquine. This was further confirmed by significant inhibition of primaquine hemotoxicity by the selective CYP inhibitors, namely thiotepa (CYP2B6), fluoxetine (CYP2D6) and troleandomycin (CYP3A4). Primaquine caused similar methemoglobin formation in G6PD(-) and normal human erythrocytes. However, G6PD(-) erythrocytes suffered higher oxidative stress and depletion of thiols than normal erythrocytes due to primaquine toxicity. The results provide significant insights regarding CYP isoforms contributing to hemotoxicity and may be useful in controlling toxicity of primaquine to increase its therapeutic utility.

  13. Cytochrome P450 1A2 Metabolizes 17β-Estradiol to Suppress Hepatocellular Carcinoma

    Science.gov (United States)

    Ren, Jianwai; Chen, George G.; Liu, Yi; Su, Xianwei; Hu, Baoguang; Leung, Billy C. S.; Wang, Y.; Ho, Rocky L. K.; Yang, Shengli; Lu, Gang; Lee, C. G.; Lai, Paul B. S.

    2016-01-01

    Hepatocellular carcinoma (HCC) occurs more frequently in men than in women. It is commonly agreed that estrogen plays important roles in suppressing HCC development, however, the underlying mechanism remains largely unknown. Since estrogen is mainly metabolized in liver and its metabolites affect cell proliferation, we sought to investigate if the liver-specific cytochrome P450 1A2 (CYP1A2) mediated the inhibitory effect of estrogen on HCC. In this study, the expression of estrogen-metabolizing enzyme CYP1A2 was determined in HCC tissues and cell lines. Cell proliferation and apoptosis were assessed in cells with or without CYP1A2 overexpression. The levels of 17β-estradiol (E2) and its metabolite 2-methoxyestradiol (2-ME) were determined. A xenograft tumor model in mice was established to confirm the findings. It was found that CYP1A2 expression was greatly repressed in HCC. E2 suppressed HCC cell proliferation and xenograft tumor development by inducing apoptosis. The inhibitory effect was significantly enhanced in cells with CYP1A2 overexpression, which effectively conversed E2 to the cytotoxic 2-ME. E2 in combination with sorafenib showed an additive effect on HCC. The anti-HCC effect of E2 was not associated with estrogen receptors ERα and ERβ as well as tumor suppressor P53 but enhanced by the approved anti-HCC drug sorafenib. In addition, HDAC inhibitors greatly induced CYP1A2 promoter activities in cancer cells, especially liver cancer cells, but not in non-tumorigenic cells. Collectively, CYP1A2 metabolizes E2 to generate the potent anti-tumor agent 2-ME in HCC. The reduction of CYP1A2 significantly disrupts this metabolic pathway, contributing the progression and growth of HCC and the gender disparity of this malignancy. PMID:27093553

  14. Albendazole metabolism in patients with neurocysticercosis: antipyrine as a multifunctional marker drug of cytochrome P450

    Directory of Open Access Journals (Sweden)

    M.P. Marques

    2002-02-01

    Full Text Available The present study investigates the isoform(s of cytochrome P450 (CYP involved in the metabolism of albendazole sulfoxide (ASOX to albendazole sulfone (ASON in patients with neurocysticercosis using antipyrine as a multifunctional marker drug. The study was conducted on 11 patients with neurocysticercosis treated with a multiple dose regimen of albendazole for 8 days (5 mg/kg every 8 h. On the 5th day of albendazole treatment, 500 mg antipyrine was administered po. Blood and urine samples were collected up to 72 h after antipyrine administration. Plasma concentrations of (+-ASOX, (--ASOX and ASON were determined by HPLC using a chiral phase column and detection by fluorescence. The apparent clearance (CL/f of ASON and of the (+ and (--ASOX enantiomers were calculated and compared to total antipyrine clearance (CL T and the clearance for the production of the three major antipyrine metabolites (CLm. A correlation (P<=0.05 was obtained only between the CL T of antipyrine and the CL/f of ASON (r = 0.67. The existence of a correlation suggests the involvement of CYP isoforms common to the metabolism of antipyrine and of ASOX to ASON. Since the CL T of antipyrine is a general measure of CYP enzymes but with a slight to moderate weight toward CYP1A2, we suggest the involvement of this enzyme in ASOX to ASON metabolism in man. The study supports the establishment of a specific marker drug of CYP1A2 in the study of the in vivo metabolism of ASOX to ASON.

  15. A cytochrome P450 regulates a domestication trait in cultivated tomato

    Science.gov (United States)

    Chakrabarti, Manohar; Zhang, Na; Sauvage, Christopher; Muños, Stéphane; Blanca, Jose; Cañizares, Joaquin; Diez, Maria Jose; Schneider, Rhiannon; Mazourek, Michael; McClead, Jammi; Causse, Mathilde; van der Knaap, Esther

    2013-01-01

    Domestication of crop plants had effects on human lifestyle and agriculture. However, little is known about the underlying molecular mechanisms accompanying the changes in fruit appearance as a consequence of selection by early farmers. We report the fine mapping and cloning of a tomato (Solanum lycopersicum) fruit mass gene encoding the ortholog of KLUH, SlKLUH, a P450 enzyme of the CYP78A subfamily. The increase in fruit mass is predominantly the result of enlarged pericarp and septum tissues caused by increased cell number in the large fruited lines. SlKLUH also modulates plant architecture by regulating number and length of the side shoots, and ripening time, and these effects are particularly strong in plants that transgenically down-regulate SlKLUH expression carrying fruits of a dramatically reduced mass. Association mapping followed by segregation analyses revealed that a single nucleotide polymorphism in the promoter of the gene is highly associated with fruit mass. This single polymorphism may potentially underlie a regulatory mutation resulting in increased SlKLUH expression concomitant with increased fruit mass. Our findings suggest that the allele giving rise to large fruit arose in the early domesticates of tomato and becoming progressively more abundant upon further selections. We also detected association of fruit weight with CaKLUH in chile pepper (Capsicum annuum) suggesting that selection of the orthologous gene may have occurred independently in a separate domestication event. Altogether, our findings shed light on the molecular basis of fruit mass, a key domestication trait in tomato and other fruit and vegetable crops. PMID:24082112

  16. Inhibition of cytochrome P450s enhances (+)-usnic acid cytotoxicity in primary cultured rat hepatocytes.

    Science.gov (United States)

    Shi, Qiang; Greenhaw, James; Salminen, William F

    2014-08-01

    (+)-Usnic acid (UA) is consumed as a dietary supplement to promote weight loss; however, dietary supplements containing UA have been associated with clinical cases of severe liver injury. UA has been shown to be hepatotoxic in rats and is extensively metabolized by hepatic cytochrome P450s (CYPs); therefore, we examined if UA metabolism results in the formation of cytotoxic metabolites or if metabolism is a detoxification process in primary rat hepatocytes. When CYP activity was suppressed by the non-isoenzyme-selective inhibitor SKF-525A (20 μM), or the CYP1A inhibitor alpha-naphthoflavone (10 μM), or the CYP3A inhibitor ketoconazole (25 μM), the cytotoxicity of UA at 3~6 μM after 3~20 h of exposure was significantly increased as measured by lactate dehydrogenase (LDH) leakage. At 2 h after UA exposure, an earlier time point prior to LDH release, these CYP inhibitors potentiated UA-induced inhibition of cellular respiration as determined by the Clark type oxygen electrode. Cellular adenosine triphosphate (ATP) depletion by UA was also exacerbated by these CYP inhibitors. The CYP2B/2C inhibitor, ticlopidine at 20 μM, showed no effects in parallel experiments. These data demonstrate that UA is bio-transformed to less toxic metabolites in rat primary hepatocytes, probably mainly by CYP1A and 3A, but not 2B/2C. Published 2013. This article is a U.S. Government work and is in the public domain in the USA. PMID:23686521

  17. In vivo effect of Schisandrin B on cytochrome P450 enzyme activity.

    Science.gov (United States)

    Li, Wei-Liang; Xin, Hua-Wen; Yu, Ai-Rong; Wu, Xiao-Chun

    2013-06-15

    To investigate the possible drug interaction, this study is designed to evaluate the ability of Schisandrin B (Sch B) to modulate cytochrome P450 3A activity (CYP3A) in vivo and to alter the pharmacokinetic profiles of CYP3A substrate (midazolam) in treated rats. Rats were repeated administered with physiological saline (negative control group), ketoconazole (75 mg/kg, positive control group) or varied doses of Sch B (experimental groups) for three consecutive days. Subsequently, changes in hepatic microsomal CYP3A activity and the pharmacokinetic profiles of midazolam and 1'-hydroxy midazolam in plasma were studied to evaluate CYP3A activity. The results indicated that Sch B significantly dose-dependently inhibited rat hepatic microsomal CYP3A activity with Ki value of 16.64 mg/kg and showed the characteristic of a noncompetitive inhibitor. Oral administration of Sch B for 3 days in rats produced significant effect on the pharmacokinetics of oral midazolam. Sch B resulted in a significant, dose-dependent increase in midazolam AUC0-∞ except at the dose of 2 mg/kg, while AUC0-∞ increased by 26.1% (8 mg/kg) and 60.6% (16 mg/kg), respectively. In the pharmacokinetic profiles of 1'-hydroxy midazolam, the significant, dose-dependent decrease in AUC0-∞ was observed except at the dose of 2 mg/kg, while AUC0-∞ reduced by 44.5% (8 mg/kg) and 49.2% (16 mg/kg), respectively. These results suggested that 3-day treatment of Sch B could increase concentration and oral bioavailability of drug metabolized by CYP3A. When the drug, consisting of Sch B, is used in the clinic for more than 3 days, the possible drug-drug interactions should be taken into consideration. PMID:23523258

  18. Effects of Eupatilin and Jaceosidin on Cytochrome P450 Enzyme Activities in Human Liver Microsomes

    Directory of Open Access Journals (Sweden)

    Ji Hyun Jeong

    2010-09-01

    Full Text Available Eupatilin and jaceosidin are bioactive flavones found in the medicinal herbs of the genus Artemisia. These bioactive flavones exhibit various antioxidant, antiinflammatory, antiallergic, and antitumor activities. The inhibitory potentials of eupatilin and jaceosidin on the activities of seven major human cytochrome P450 enzymes in human liver microsomes were investigated using a cocktail probe assay. Eupatilin and jaceosidin potently inhibited CYP1A2-catalyzed phenacetin O-deethylation with 50% inhibitory concentration (IC50 values of 9.4 mM and 5.3 mM, respectively, and CYP2C9-catalyzed diclofenac 4-hydroxylation with IC50 values of 4.1 mM and 10.2 mM, respectively. Eupatilin and jaceosidin were also found to moderately inhibit CYP2C19-catalyzed [S]-mephenytoin 4¢-hydroxylation, CYP2D6-catalyzed bufuralol 1¢-hydroxylation, and CYP2C8-catalyzed amodiaquine N-deethylation. Kinetic analysis of human liver microsomes showed that eupatilin is a competitive inhibitor of CYP1A2 with a Ki value of 2.3 mM and a mixed-type inhibitor of CYP2C9 with a Ki value of 1.6 mM. Jaceosidin was shown to be a competitive inhibitor of CYP1A2 with a Ki value of 3.8 mM and a mixed-type inhibitor of CYP2C9 with Ki value of 6.4 mM in human liver microsomes. These in vitro results suggest that eupatilin and jaceosidin should be further examined for potential pharmacokinetic drug interactions in vivo due to inhibition of CYP1A2 and CYP2C9.

  19. The curious case of benzbromarone: insight into super-inhibition of cytochrome P450.

    Directory of Open Access Journals (Sweden)

    Abhinav Parashar

    Full Text Available Cytochrome P450 (CYP family of redox enzymes metabolize drugs and xenobiotics in liver microsomes. Isozyme CYP2C9 is reported to be inhibited by benzbromarone (BzBr and this phenomenon was hitherto explained by classical active-site binding. Theoretically, it was impossible to envisage the experimentally derived sub-nM Ki for an inhibitor, when supra-nM enzyme and 10X KM substrate concentrations were employed. We set out to find a more plausible explanation for this highly intriguing "super-inhibition" phenomenon. In silico docking of various BzBr analogs with known crystal structure of CYP2C9 did not provide any evidence in support of active-site based inhibition hypothesis. Experiments tested the effects of BzBr and nine analogs on CYPs in reconstituted systems of lab-purified proteins, complex baculosomes & crude microsomal preparations. In certain setups, BzBr and its analogs could even enhance reactions, which cannot be explained by an active site hypothesis. Generally, it was seen that Ki became smaller by orders of magnitude, upon increasing the dilution order of BzBr analogs. Also, it was seen that BzBr could also inhibit other CYP isozymes like CYP3A4, CYP2D6 and CYP2E1. Further, amphipathic derivatives of vitamins C & E (scavengers of diffusible reactive oxygen species or DROS effectively inhibited CYP2C9 reactions in different reaction setups. Therefore, the inhibition of CYP activity by BzBr analogs (which are also surface-active redox agents is attributed to catalytic scavenging of DROS at phospholipid interface. The current work expands the scope of interpretations of inhibitions in redox enzymes and ushers in a new cellular biochemistry paradigm that small amounts of DROS may be obligatorily required in routine redox metabolism for constructive catalytic roles.

  20. Role of rat liver cytochrome P450 3A and 2D in metabolism of imrecoxib

    Institute of Scientific and Technical Information of China (English)

    Hai-yan XU; Zhi-yong XIE; Peng ZHANG; Jin SUN; Feng-ming CHU; Zong-ru GUO; Da-fang ZHONG

    2006-01-01

    Aim: To investigate the in vitro metabolism of imrecoxib in rat liver microsomes and to identify the cytochrome P450 (CYP) forms involved in its metabolism. Methods: Liver microsomes of Wistar rats were prepared using an ultracentrifuge. The in vitro metabolism of imrecoxib was studied by incubation with rat liver microsomes. To characterize the CYP forms involved in the 4'-methyl hydroxylation of imrecoxib, the effects of typical CYP inducers (such as dexamethasone, isoniazid and (3-naphthoflavone) and of CYP inhibitors (such as ketoconazole, quinine, a-naphthoflavone, methylpyrazole, and cimetidine) on the formation rate of 4'-hydroxymethyl imrecoxib were investigated. Results: Imrecoxib wasmetabolized to 3 metabolites by rat liver microsomes: 4'-hydroxymethyl imrecoxib (M4), 4'-hydroxymethyl-5-hydoxyl imrecoxib (M3), and 4'-hydroxymethyl-5-carbonyl imrecoxib (M5). Over the imrecoxib concentration range studied (5-600 umol/L), the rate of 4'-methyl hydroxylation conformed to monophasic Michaelis-Menten kinetics. Dexamethasone significantly induced the formation of M4. Ketoconazole markedly lowered the metabolic rate of imrecoxib in a concentration-dependent manner. Moreover, a significant inhibitory effect of quinine on the formation of M4 was observed in microsomes obtained from control rats, isoniazid-induced rats, and (3-naphthoflavone-induced rats. In contrast, α-naphthoflavone, cimetidine, and methylpyrazole had no inhibitory effects on this metabolic pathway. Conclusion: Imrecoxib is metabolized via 4'-methyl hydroxylation in rat liver microsomes. The reaction is mainly catalyzed by CYP 3A. CYP 2D also played a role in control rats, in isoniazid-induced rats and in β-naphthoflavone-induced rats.

  1. Modulation of cytochrome P450 metabolism and transport across intestinal epithelial barrier by ginger biophenolics.

    Directory of Open Access Journals (Sweden)

    Rao Mukkavilli

    Full Text Available Natural and complementary therapies in conjunction with mainstream cancer care are steadily gaining popularity. Ginger extract (GE confers significant health-promoting benefits owing to complex additive and/or synergistic interactions between its bioactive constituents. Recently, we showed that preservation of natural "milieu" confers superior anticancer activity on GE over its constituent phytochemicals, 6-gingerol (6G, 8-gingerol (8 G, 10-gingerol (10 G and 6-shogaol (6S, through enterohepatic recirculation. Here we further evaluate and compare the effects of GE and its major bioactive constituents on cytochrome P450 (CYP enzyme activity in human liver microsomes by monitoring metabolites of CYP-specific substrates using LC/MS/MS detection methods. Our data demonstrate that individual gingerols are potent inhibitors of CYP isozymes, whereas GE exhibits a much higher half-maximal inhibition value, indicating no possible herb-drug interactions. However, GE's inhibition of CYP1A2 and CYP2C8 reflects additive interactions among the constituents. In addition, studies performed to evaluate transporter-mediated intestinal efflux using Caco-2 cells revealed that GE and its phenolics are not substrates of P-glycoprotein (Pgp. Intriguingly, however, 10 G and 6S were not detected in the receiver compartment, indicating possible biotransformation across the Caco-2 monolayer. These data strengthen the notion that an interplay of complex interactions among ginger phytochemicals when fed as whole extract dictates its bioactivity highlighting the importance of consuming whole foods over single agents. Our study substantiates the need for an in-depth analysis of hepatic biotransformation events and distribution profiles of GE and its active phenolics for the design of safe regimens.

  2. Role of Cytochrome P450 2C8 in Drug Metabolism and Interactions.

    Science.gov (United States)

    Backman, Janne T; Filppula, Anne M; Niemi, Mikko; Neuvonen, Pertti J

    2016-01-01

    During the last 10-15 years, cytochrome P450 (CYP) 2C8 has emerged as an important drug-metabolizing enzyme. CYP2C8 is highly expressed in human liver and is known to metabolize more than 100 drugs. CYP2C8 substrate drugs include amodiaquine, cerivastatin, dasabuvir, enzalutamide, imatinib, loperamide, montelukast, paclitaxel, pioglitazone, repaglinide, and rosiglitazone, and the number is increasing. Similarly, many drugs have been identified as CYP2C8 inhibitors or inducers. In vivo, already a small dose of gemfibrozil, i.e., 10% of its therapeutic dose, is a strong, irreversible inhibitor of CYP2C8. Interestingly, recent findings indicate that the acyl-β-glucuronides of gemfibrozil and clopidogrel cause metabolism-dependent inactivation of CYP2C8, leading to a strong potential for drug interactions. Also several other glucuronide metabolites interact with CYP2C8 as substrates or inhibitors, suggesting that an interplay between CYP2C8 and glucuronides is common. Lack of fully selective and safe probe substrates, inhibitors, and inducers challenges execution and interpretation of drug-drug interaction studies in humans. Apart from drug-drug interactions, some CYP2C8 genetic variants are associated with altered CYP2C8 activity and exhibit significant interethnic frequency differences. Herein, we review the current knowledge on substrates, inhibitors, inducers, and pharmacogenetics of CYP2C8, as well as its role in clinically relevant drug interactions. In addition, implications for selection of CYP2C8 marker and perpetrator drugs to investigate CYP2C8-mediated drug metabolism and interactions in preclinical and clinical studies are discussed. PMID:26721703

  3. Cytochrome P450 2C epoxygenases mediate photochemical stress-induced death of photoreceptors.

    Science.gov (United States)

    Chang, Qing; Berdyshev, Evgeny; Cao, Dingcai; Bogaard, Joseph D; White, Jerry J; Chen, Siquan; Shah, Ravi; Mu, Wenbo; Grantner, Rita; Bettis, Sam; Grassi, Michael A

    2014-03-21

    Degenerative loss of photoreceptors occurs in inherited and age-related retinal degenerative diseases. A chemical screen facilitates development of new testing routes for neuroprotection and mechanistic investigation. Herein, we conducted a mouse-derived photoreceptor (661W cell)-based high throughput screen of the Food and Drug Administration-approved Prestwick drug library to identify putative cytoprotective compounds against light-induced, synthetic visual chromophore-precipitated cell death. Different classes of hit compounds were identified, some of which target known genes or pathways pathologically associated with retinitis pigmentosa. Sulfaphenazole (SFZ), a selective inhibitor of human cytochrome P450 (CYP) 2C9 isozyme, was identified as a novel and leading cytoprotective compound. Expression of CYP2C proteins was induced by light. Gene-targeted knockdown of CYP2C55, the homologous gene of CYP2C9, demonstrated viability rescue to light-induced cell death, whereas stable expression of functional CYP2C9-GFP fusion protein further exacerbated light-induced cell death. Mechanistically, SFZ inhibited light-induced necrosis and mitochondrial stress-initiated apoptosis. Light elicited calcium influx, which was mitigated by SFZ. Light provoked the release of arachidonic acid from membrane phospholipids and production of non-epoxyeicosatrienoic acid metabolites. Administration of SFZ further stimulated the production of non-epoxyeicosatrienoic acid metabolites, suggesting a metabolic shift of arachidonic acid under inhibition of the CYP2C pathway. Together, our findings indicate that CYP2C genes play a direct causative role in photochemical stress-induced death of photoreceptors and suggest that the CYP monooxygenase system is a risk factor for retinal photodamage, especially in individuals with Stargardt disease and age-related macular degeneration that deposit condensation products of retinoids. PMID:24519941

  4. Effects of soy containing diet and isoflavones on cytochrome P450 enzyme expression and activity.

    Science.gov (United States)

    Ronis, Martin J J

    2016-08-01

    Cytochromes P450 (CYPs) play an important role in metabolism and clearance of most clinically utilized drugs and other xenobiotics. They are important in metabolism of endogenous compounds including fatty acids, sterols, steroids and lipid-soluble vitamins. Dietary factors such as phytochemicals are capable of affecting CYP expression and activity, which may be important in diet-drug interactions and in the development of fatty liver disease, cardiovascular disease and cancer. One important diet-CYP interaction is with diets containing plant proteins, particularly soy protein. Soy diets are traditionally consumed in Asian countries and are linked to lower incidence of several cancers and of cardiovascular disease in Asian populations. Soy is also an important protein source in vegetarian and vegan diets and the sole protein source in soy infant formulas. Recent studies suggest that consumption of soy can inhibit induction of CY1 enzymes by polycyclic aromatic hydrocarbons (PAHs) which may contribute to cancer prevention. In addition, there are data to suggest that soy components promiscuously activate several nuclear receptors including PXR, PPAR and LXR resulting in increased expression of CYP3As, CYP4As and CYPs involved in metabolism of cholesterol to bile acids. Such soy-CYP interactions may alter drug pharmacokinetics and therapeutic efficacy and are associated with improved lipid homeostasis and reduced risk of cardiovascular disease. The current review summarizes results from in vitro; in vivo and clinical studies of soy-CYP interactions and examines the evidence linking the effects of soy diets on CYP expression to isoflavone phytoestrogens, particularly, genistein and daidzein that are associated with soy protein. PMID:27440109

  5. Responsiveness of cerebral and hepatic cytochrome P450s in rat offspring prenatally exposed to lindane

    International Nuclear Information System (INIS)

    ABSTRACT: Prenatal exposure to low doses of lindane has been shown to affect the ontogeny of xenobiotic metabolizing cytochrome P450s (CYPs), involved in the metabolism and neurobehavioral toxicity of lindane. Attempts were made in the present study to investigate the responsiveness of CYPs in offspring prenatally exposed to lindane (0.25 mg/kg b. wt.; 1/350th of LD50; p. o. to mother) when challenged with 3-methylcholanthrene (MC) or phenobarbital (PB), inducers of CYP1A and 2B families or a sub-convulsant dose of lindane (30 mg/kg b. wt., p. o.) later in life. Prenatal exposure to lindane was found to produce an increase in the mRNA and protein expression of CYP1A1, 1A2, 2B1, 2B2 isoforms in brain and liver of the offspring at postnatal day 50. The increased expression of the CYPs in the offspring suggests the sensitivity of the CYPs during postnatal development, possibly, to low levels of lindane, which may partition into mother's milk. A higher increase in expression of CYP1A and 2B isoenzymes and their catalytic activity was observed in animals pretreated prenatally with lindane and challenged with MC (30 mg/kg, i. p. x 5 days) or PB (80 mg/kg, i. p. x 5 days) when young at age (approx. 7 weeks) compared to animals exposed to MC or PB alone. Further, challenge of the control and prenatally exposed offspring with a single sub-convulsant dose of lindane resulted in an earlier onset and increased incidence of convulsions in the offspring prenatally exposed to lindane have demonstrated sensitivity of the CYPs in the prenatally exposed offspring. Our data assume significance as the subtle changes in the expression profiles of hepatic and cerebral CYPs in rat offspring during postnatal development could modify the adult response to a later exposure to xenobiotics

  6. 23. Establishment of two transgenic cells stable expression of human cytochrome P450 2C

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    AIM: To clone the human cytochrome P450 2C9 (CYP2C9) and CYP2C18 cDNA and establish two transgenic CHL cell line stable expressing human CYP2C9 and CYP2C18. METHODS:Extracting total RNA from human liver tissue, the human CYP2C9 and CYP2C18 cDNA was amplified with reverse transcription polymerase chain reaction (RT-PCR), and cloned into cloning vector pGEM-T. The cDNA segment was identified by DNA sequencing and subcloned into a mammalian expression vector pREP9. Two transgenic cell line were established by transfecting the recombinant vectors of pREP9-CYP2C9 and pREP9-CYP2C18 to Chinese hamster lung cell CHL. The enzyme activity of CYP2C9 and CYP2C18 catalyze tolbutamide to 4-hydroxy tolbutamide in S9 protein of the cells were determinated by HPLC. RESULTS: The sequence of the two cDNA segments cloned, which were 1540 bp and 1671 bp in length, were identical to those reported by Romkes et al(GenBank accession number: M61855, M61856, J05326) in coding amino acids. The S9 fraction of the established cell lines can metabolize tolbutamide to 4-hydroxy tolbutamide, the tolbutamide-4-hydroxylase activity was found to be 0.465±0.109 and 0.509±0.052 nmol*min-1*(mg S9 protein)-1 (n=3), but was not detectable in parental CHL cell. CONCLUSION: The cDNA of CYP2C9 and CYP2C18 were successfolly cloned and cell lines of CHL-CYP2C9 and CHL-CYP2C18 which efficiently expressed the protein of CYP2C9 and CYP2C18 were established.

  7. Cytochrome P450 2E1 polymorphism and nasopharyngeal carcinoma development in Thailand: a correlative study

    International Nuclear Information System (INIS)

    Nasopharyngeal carcinoma (NPC) is a rare tumor in most parts of the world but occurs at relatively high frequency among people of Chinese descent. The cytochrome P450 2E1 enzyme (CYP2E1) is responsible for the metabolic activation of nitrosamines, and has been shown to be a susceptibility gene for NPC development in Taiwan [RR = 2.6; 95%CI = 1.2-5.7]. Since there has been only one report of this link, it was decided to investigate the susceptibility of CYP2E1 to NPC development in other populations. Therefore, the correlation between the RsaI polymorphism of this gene and NPC was studied in-patients including Thai and Chinese in Thailand. The present study comprised 217 cases diagnosed with NPC and 297 healthy controls. Similar to the result found in Taiwanese, a homozygous uncut genotype demonstrated a higher relative risk both when all cases were analyzed [RR = 2.19; 95%CI = 0.62-8.68] or individual racial groups, Thai [RR = 1.51; 95%CI = 0.08-90.06] or Chinese [RR = 1.99; 95%CI = 0.39-10.87]. The ethnicity-adjusted odds ratio is 2.39 with 95%CI, 0.72-7.89. Though our finding was not statistically significant due to the moderate sample size of the study, similarity to the study in Taiwan with only a slight loss in precision was demonstrated. The higher RR found for the same genotype in distinct populations confirmed that CYP2E1 is one of several NPC susceptibility genes and that the RsaI minus variant is one mutation that affects phenotype

  8. A Highly Selective Ratiometric Two-Photon Fluorescent Probe for Human Cytochrome P450 1A.

    Science.gov (United States)

    Dai, Zi-Ru; Ge, Guang-Bo; Feng, Lei; Ning, Jing; Hu, Liang-Hai; Jin, Qiang; Wang, Dan-Dan; Lv, Xia; Dou, Tong-Yi; Cui, Jing-Nan; Yang, Ling

    2015-11-18

    Cytochrome P450 1A (CYP1A), one of the most important phase I drug-metabolizing enzymes in humans, plays a crucial role in the metabolic activation of procarcinogenic compounds to their ultimate carcinogens. Herein, we reported the development of a ratiometric two-photon fluorescent probe NCMN that allowed for selective and sensitive detection of CYP1A for the first time. The probe was designed on the basis of substrate preference of CYP1A and its high capacity for O-dealkylation, while 1,8-naphthalimide was selected as fluorophore because of its two-photon absorption properties. To achieve a highly selective probe for CYP1A, a series of 1,8-naphthalimide derivatives were synthesized and used to explore the potential structure-selectivity relationship, by using a panel of human CYP isoforms for selectivity screening. After screening and optimization, NCMN displayed the best combination of selectivity, sensitivity and ratiometric fluorescence response following CYP1A-catalyzed O-demetylation. Furthermore, the probe can be used to real-time monitor the enzyme activity of CYP1A in complex biological systems, and it has the potential for rapid screening of CYP1A modulators using tissue preparation as enzyme sources. NCMN has also been successfully used for two-photon imaging of intracellular CYP1A in living cells and tissues, and showed high ratiometric imaging resolution and deep-tissue imaging depth. In summary, a two-photon excited ratiometric fluorescent probe NCMN has been developed and well-characterized for sensitive and selective detection of CYP1A, which holds great promise for bioimaging of endogenous CYP1A in living cells and for further investigation on CYP1A associated biological functions in complex biological systems. PMID:26488456

  9. Mammalian Cytochrome P450-Dependent Metabolism of Polychlorinated Dibenzo-p-dioxins and Coplanar Polychlorinated Biphenyls

    Directory of Open Access Journals (Sweden)

    Hideyuki Inui

    2014-08-01

    Full Text Available Polychlorinated dibenzo-p-dioxins (PCDDs and coplanar polychlorinated biphenyls (PCBs contribute to dioxin toxicity in humans and wildlife after bioaccumulation through the food chain from the environment. The authors examined human and rat cytochrome P450 (CYP-dependent metabolism of PCDDs and PCBs. A number of human CYP isoforms belonging to the CYP1 and CYP2 families showed remarkable activities toward low-chlorinated PCDDs. In particular, human CYP1A1, CYP1A2, and CYP1B1 showed high activities toward monoCDDs, diCDDs, and triCDDs but no detectable activity toward 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-tetraCDD. Large amino acids located at putative substrate-recognition sites and the F-G loop in rat CYP1A1 contributed to the successful metabolism of 2,3,7,8-tetraCDD. Rat, but not human, CYP1A1 metabolized 3,3',4,4',5-pentachlorobiphenyl (CB126 to two hydroxylated metabolites. These metabolites are probably less toxic than is CB126, due to their higher solubility. Homology models of human and rat CYP1A1s and CB126 docking studies indicated that two amino acid differences in the CB126-binding cavity were important for CB126 metabolism. In this review, the importance of CYPs in the metabolism of dioxins and PCBs in mammals and the species-based differences between humans and rats are described. In addition, the authors reveal the molecular mechanism behind the binding modes of dioxins and PCBs in the heme pocket of CYPs.

  10. Cytochromes P450 are Expressed in Proliferating Cells in Barrett's Metaplasia

    Directory of Open Access Journals (Sweden)

    Steven J. Hughes

    1999-06-01

    Full Text Available The expression of cytochromes P450 (CYP in Barrett's esophagus and esophageal squamous mucosa was investigated. Esophagectomy specimens from 23 patients were examined for CYP expression of CYP1A2, CYP3A4, CYP2C9/10, and CYP2E1 by immunohistochemical analysis, and the expression of CYP1A1, CYP3A4, CYP1B1, CYP2E1, and CYP2C9/10 in these tissues was further confirmed by reverse transcription polymerase chain reaction. Immunohistochemical analysis of esophageal squamous mucosa (n = 12 showed expression of CYP1A2, CYP3A4, CYP2E1, and CYP2C9/10 proteins, but it was noted that cells within the basal proliferative zone did not express CYPs. Immunohistochemical analysis of Barrett's esophagus (n = 13 showed expression of CYP1A2, CYP3A4, CYP2E1, and CYP2C9/10 that was prominent in the basal glandular regions, which are areas containing a high percentage of actively proliferating cells. Immunohistochemical staining for both proliferating cell nuclear antigen and the CYPs further supported the colocalization of CYP expression to areas of active cell proliferation in Barrett's esophagus, whereas in the esophageal squamous epithelium, CYP expression is limited to cells that are not proliferating. RT-PCR with amplification product sequence analysis confirmed CYP1A1, CYP3A4, CYP1B1, CYP2E1, and CYP2C9/10 mRNA expression in Barrett's esophagus. These data suggest that the potential ability of cells in Barrett's esophagus to both activate carcinogens and proliferate may be important risk factors affecting carcinogenesis in this metaplastic tissue.

  11. Conformational Mobility in Cytochrome P450 3A4 Explored by Pressure-Perturbation EPR Spectroscopy.

    Science.gov (United States)

    Davydov, Dmitri R; Yang, Zhongyu; Davydova, Nadezhda; Halpert, James R; Hubbell, Wayne L

    2016-04-12

    We used high hydrostatic pressure as a tool for exploring the conformational landscape of human cytochrome P450 3A4 (CYP3A4) by electron paramagnetic resonance and fluorescence spectroscopy. Site-directed incorporation of a luminescence resonance energy transfer donor-acceptor pair allowed us to identify a pressure-dependent equilibrium between two states of the enzyme, where an increase in pressure increased the spatial separation between the two distantly located fluorophores. This transition is characterized by volume change (ΔV°) and P1/2 values of -36.8 ± 5.0 mL/mol and 1.45 ± 0.33 kbar, respectively, which corresponds to a Keq° of 0.13 ± 0.06, so that only 15% of the enzyme adopts the pressure-promoted conformation at ambient pressure. This pressure-promoted displacement of the equilibrium is eliminated by the addition of testosterone, an allosteric activator. Using site-directed spin labeling, we demonstrated that the pressure- and testosterone-sensitive transition is also revealed by pressure-induced changes in the electron paramagnetic resonance spectra of a nitroxide side chain placed at position 85 or 409 of the enzyme. Furthermore, we observed a pressure-induced displacement of the emission maxima of a solvatochromic fluorophore (7-diethylamino-3-((((2-maleimidyl)ethyl)amino)carbonyl) coumarin) placed at the same positions, which suggests a relocation to a more polar environment. Taken together, the results reveal an effector-dependent conformational equilibrium between open and closed states of CYP3A4 that involves a pronounced change at the interface between the region of α-helices A/A' and the meander loop of the enzyme, where residues 85 and 409 are located. Our study demonstrates the high potential of pressure-perturbation strategies for studying protein conformational landscapes. PMID:27074675

  12. Cytochrome P450 levels are altered in patients with esophageal squamous-cell carcinoma

    Institute of Scientific and Technical Information of China (English)

    I Bergheim; E Wolfgarten; E Bollschweiler; AH H(o)lscher; C Bode; A Parlesak

    2007-01-01

    AIM:To investigate the role of cytochrome P450(CYP)in the carcinogenesis of squamous-cell carcinoma(SCC)in human esophagus by determining expression patterns and protein levels of representative CYPs in esophageal tissue of patients with SCC and controls.METHODS:mRNA expression of CYP2E1,CYP2C,CYP3A4,and CYP3A5 was determined using RT-PCR in both normal and malignant esophageal tissues of patients with untreated esophageal SCC(n = 21)and in controls(n = 10).Protein levels of CYP2E1,CYP2C8,CYP3A4,and CYP3A5 were measured by Western blot.RESULTS:Within the group of SCC patients,mRNA expression of CYP 3A4 and CYP2C was significantly lower in malignant tissue(-39% and -74%,respectively,P < 0.05)than in normal tissue.Similar results were found in CYP3A4 protein levels.Between groups,CYP3A4,CYP3A5,and CYP2C8 protein concentration was significantly higher in non-malignant tissue of SCC patients(4.8-,2.9-,and 1.9-fold elevation,P < 0.05)than in controls.In contrast,CYP2E1 protein levels were significantly higher in controls than in SCC patients (+46%,P < 0.05).CONCLUSION:Significant differences exist in protein levels of certain CYPs in non-malignant esophageal tissue (e.g.CYP2C8,CYP3A4,CYP3A5,and CYP2E1)between SCC patients and healthy subjects and may contribute to the development of SCC in the esophagus.

  13. Food-drug interactions via human cytochrome P450 3A (CYP3A).

    Science.gov (United States)

    Fujita, Ken-ichi

    2004-01-01

    Food-drug interactions have been reported to occur in various systems in the body. The causes of these interactions are mainly divided into pharmacodynamic and pharmacokinetic processes. Among these processes, drug metabolism plays a crucial role in drug interactions. Metabolic food-drug interactions occur when a certain food alters the activity of a drug-metabolizing enzyme, leading to a modulation of the pharmacokinetics of drugs metabolized by the enzyme. A variety of interactions have been documented so far. Foods consisting of complex chemical mixtures, such as fruits, alcoholic beverages, teas, and herbs, possess the ability to inhibit or induce the activity of drug-metabolizing enzymes. According to results obtained thus far, cytochrome P450 3A4 (CYP3A4) appears to be a key enzyme in food-drug interactions. For example, interactions of grapefruit juice with felodipine and cyclosporine, red wine with cyclosporine, and St John's wort with various medicines including cyclosporine, have been demonstrated. The results indicate the requirement of dosage adjustment to maintain drug concentrations within their therapeutic windows. The CYP3A4-related interaction by food components may be related to the high level of expression of CYP3A4 in the small intestine, as well as its broad substrate specificity, as CYP3A4 is responsible for the metabolism of more than 50% of clinical pharmaceuticals. This review article summarizes the findings obtained to date concerning food-drug interactions and their clinical implications. It seems likely that more information regarding such interactions will accumulate in the future, and awareness is necessary for achieving optimal drug therapy. PMID:15663291

  14. Inhibition of Human Cytochrome P450 Enzymes by Bacopa monnieri Standardized Extract and Constituents

    Directory of Open Access Journals (Sweden)

    Seetha Ramasamy

    2014-02-01

    Full Text Available Bacopa monnieri and the constituents of this plant, especially bacosides, possess various neuropharmacological properties. Like drugs, some herbal extracts and the constituents of their extracts alter cytochrome P450 (CYP enzymes, causing potential herb-drug interactions. The effects of Bacopa monnieri standardized extract and the bacosides from the extract on five major CYP isoforms in vitro were analyzed using a luminescent CYP recombinant human enzyme assay. B. monnieri extract exhibited non-competitive inhibition of CYP2C19 (IC50/Ki = 23.67/9.5 µg/mL, CYP2C9 (36.49/12.5 µg/mL, CYP1A2 (52.20/25.1 µg/mL; competitive inhibition of CYP3A4 (83.95/14.5 µg/mL and weak inhibition of CYP2D6 (IC50 = 2061.50 µg/mL. However, the bacosides showed negligible inhibition of the same isoforms. B. monnieri, which is orally administered, has a higher concentration in the gut than the liver; therefore, this herb could exhibit stronger inhibition of intestinal CYPs than hepatic CYPs. At an estimated gut concentration of 600 µg/mL (based on a daily dosage of 300 mg/day, B. monnieri reduced the catalytic activities of CYP3A4, CYP2C9 and CYP2C19 to less than 10% compared to the total activity (without inhibitor = 100%. These findings suggest that B. monnieri extract could contribute to herb-drug interactions when orally co-administered with drugs metabolized by CYP1A2, CYP3A4, CYP2C9 and CYP2C19.

  15. Estudio de análogos de la sulfonanilida que actúan como supresores de la expresión de la aromatasa Study of sulfonanilide analogues acting as suppressants of the aromatase expression

    Directory of Open Access Journals (Sweden)

    Ricardo Vivas-Reyes

    2009-12-01

    Full Text Available Se realizó un estudio tridimensional cuantitativo de relación-estructura con nuevos análogos de la sulfonanilida que actúan como supresores de la expresión y actividad de la aromatasa en células de cáncer de mama, independientemente de la inhibición de la ciclooxigenasa-2 (COX-2. La superposición molecular de los ligandos en la plantilla fue llevada a cabo por el método Database Alignment. El mejor modelo estuvo constituido por la combinación de los campos estérico e hidrofóbico, los cuales arrojaron los siguientes parámetros: q² = 0,613 con siete componentes, R² no validado igual a 0,976, valor de F igual a 81,695; 0,347 y 0,047 para los errores estándar de predicción y estimación, respectivamente. Las contribuciones estérica e hidrofóbica fueron de 50,5 y 49,5% respectivamente. Los datos generados por el presente estudio podrían impulsar el diseño de nuevos y más potentes reguladores selectivos de la expresión de la aromatasa.A three-dimensional quantitative structure-activity relationship study was performed with new Sulfonanilide analogue molecules acting as aromatase expression and activity suppressors in breast cancer cells independent of COX-2 inhibition. The molecular supression of the ligands in the grid was carried out by the DATA-BASE ALIGNMENT method. The best model formed by combining the esteric fields and hydrophobic fields yielded the following parameters: q² = 0.613 with seven components, not validated R² equal to 0.976, with an F value of 81.695, 0.347 and 0.047 for the standard errors of prediction and estimate, respectively. The contribution of esteric and hydrophobic fields was 50.5 and 49.5% respectively. data generated from this study may be used to design new and more potent selective aromatase expression regulators.

  16. Kinetics of cytochrome P450 2E1-catalyzed oxidation of ethanol to acetic acid via acetaldehyde.

    Science.gov (United States)

    Bell-Parikh, L C; Guengerich, F P

    1999-08-20

    The P450 2E1-catalyzed oxidation of ethanol to acetaldehyde is characterized by a kinetic deuterium isotope effect that increases K(m) with no effect on k(cat), and rate-limiting product release has been proposed to account for the lack of an isotope effect on k(cat) (Bell, L. C., and Guengerich, F. P. (1997) J. Biol. Chem. 272, 29643-29651). Acetaldehyde is also a substrate for P450 2E1 oxidation to acetic acid, and k(cat)/K(m) for this reaction is at least 1 order of magnitude greater than that for ethanol oxidation to acetaldehyde. Acetic acid accounts for 90% of the products generated from ethanol in a 10-min reaction, and the contribution of this second oxidation has been overlooked in many previous studies. The noncompetitive intermolecular kinetic hydrogen isotope effects on acetaldehyde oxidation to acetic acid ((H)(k(cat)/K(m))/(D)(k(cat)/K(m)) = 4.5, and (D)k(cat) = 1.5) are comparable with the isotope effects typically observed for ethanol oxidation to acetaldehyde, and k(cat) is similar for both reactions, suggesting a possible common catalytic mechanism. Rapid quench kinetic experiments indicate that acetic acid is formed rapidly from added acetaldehyde (approximately 450 min(-1)) with burst kinetics. Pulse-chase experiments reveal that, at a subsaturating concentration of ethanol, approximately 90% of the acetaldehyde intermediate is directly converted to acetic acid without dissociation from the enzyme active site. Competition experiments suggest that P450 2E1 binds acetic acid and acetaldehyde with relatively high K(d) values, which preclude simple tight binding as an explanation for rate-limiting product release. The existence of a rate-determining step between product formation and release is postulated. Also proposed is a conformational change in P450 2E1 occurring during the course of oxidation and the discrimination of P450 2E1 between acetaldehyde and its hydrated form, the gem-diol. This multistep P450 reaction is characterized by kinetic

  17. Hop (Humulus lupulus L.) Extract and 6-Prenylnaringenin Induce P450 1A1 Catalyzed Estrogen 2-Hydroxylation.

    Science.gov (United States)

    Wang, Shuai; Dunlap, Tareisha L; Howell, Caitlin E; Mbachu, Obinna C; Rue, Emily A; Phansalkar, Rasika; Chen, Shao-Nong; Pauli, Guido F; Dietz, Birgit M; Bolton, Judy L

    2016-07-18

    Humulus lupulus L. (hops) is a popular botanical dietary supplement used by women as a sleep aid and for postmenopausal symptom relief. In addition to its efficacy for menopausal symptoms, hops can also modulate the chemical estrogen carcinogenesis pathway and potentially protect women from breast cancer. In the present study, an enriched hop extract and the key bioactive compounds [6-prenylnarigenin (6-PN), 8-prenylnarigenin (8-PN), isoxanthohumol (IX), and xanthohumol (XH)] were tested for their effects on estrogen metabolism in breast cells (MCF-10A and MCF-7). The methoxyestrones (2-/4-MeOE1) were analyzed as biomarkers for the nontoxic P450 1A1 catalyzed 2-hydroxylation and the genotoxic P450 1B1 catalyzed 4-hydroxylation pathways, respectively. The results indicated that the hop extract and 6-PN preferentially induced the 2-hydroxylation pathway in both cell lines. 8-PN only showed slight up-regulation of metabolism in MCF-7 cells, whereas IX and XH did not have significant effects in either cell line. To further explore the influence of hops and its bioactive marker compounds on P450 1A1/1B1, mRNA expression and ethoxyresorufin O-dealkylase (EROD) activity were measured. The results correlated with the metabolism data and showed that hop extract and 6-PN preferentially enhanced P450 1A1 mRNA expression and increased P450 1A1/1B1 activity. The aryl hydrocarbon receptor (AhR) activation by the isolated compounds was tested using xenobiotic response element (XRE) luciferase construct transfected cells. 6-PN was found to be an AhR agonist that significantly induced XRE activation and inhibited 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induced XRE activity. 6-PN mediated induction of EROD activity was also inhibited by the AhR antagonist CH223191. These data show that the hop extract and 6-PN preferentially enhance the nontoxic estrogen 2-hydroxylation pathway through AhR mediated up-regulation of P450 1A1, which further emphasizes the importance of

  18. Cyp15F1: A novel cytochrome P450 gene linked to juvenile hormone-dependent caste differention in the termite R. flavipes

    Science.gov (United States)

    Termites are eusocial insects that perform social interactions that facilitate chemical signaling. Previous research identified two cytochrome P450s that have homology to other insect p450s responsible for the production of juvenile hormone. Juvenile hormone is an important morphogenic hormone tha...

  19. Process limitations of a whole-cell P450 catalyzed reaction using a CYP153A-CPR fusion construct expressed in Escherichia coli

    DEFF Research Database (Denmark)

    Lundemo, M. T.; Notonier, S.; Striedner, G.;

    2016-01-01

    Cytochrome P450s are interesting biocatalysts due to their ability to hydroxylate non-activated hydrocarbons in a selective manner. However, to date only a few P450-catalyzed processes have been implemented in industry due to the difficulty of developing economically feasible processes. In this s...

  20. Modes of Heme-Binding and Substrate Access for Cytochrome P450 CYP74A Revealed by Crystal Structures of Allene Oxide Synthase

    Science.gov (United States)

    Cytochrome P450s exist ubiquitously in all organisms and are involved in many biological processes. Allene oxide synthase (AOS) is a P450 enzyme that plays a key role in the biosynthesis of oxylipin jasmonates which are involved in signal and defense reactions in higher plants. The crystal structure...

  1. Role of cytochrome P450 2D6 genetic polymorphism in carvedilol hydroxylation in vitro

    Directory of Open Access Journals (Sweden)

    Wang Z

    2016-06-01

    Full Text Available Zhe Wang,1,* Li Wang,2,3,* Ren-ai Xu,4 Yun-yun Zhan,2 Cheng-ke Huang,1 Da-peng Dai,5 Jian-ping Cai,5 Guo-xin Hu2 1Department of Pharmacy, The Second Affiliated Hospital & Yuying Children’s Hospital of Wenzhou Medical University, 2Department of Pharmacology, School of Pharmacy, Wenzhou Medical University, Wenzhou, 3Department of Pharmacy, Women’s Hospital, School of Medicine, Zhejiang University, Hangzhou, 4Department of Pharmacy, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, 5The Key Laboratory of Geriatrics, Beijing Hospital & Beijing Institute of Geriatrics, Ministry of Health, Beijing, People’s Republic of China *These authors contributed equally to this work Abstract: Cytochrome P450 2D6 (CYP2D6 is a highly polymorphic enzyme that catalyzes the metabolism of a great number of therapeutic drugs. Up to now, >100 allelic variants of CYP2D6 have been reported. Recently, we identified 22 novel variants in the Chinese population in these variants. The purpose of this study was to examine the enzymatic activity of the variants toward the CYP2D6 substrate carvedilol in vitro. The CYP2D6 proteins, including CYP2D6.1 (wild type, CYP2D6.2, CYP2D6.10, and 22 other novel CYP2D6 variants, were expressed from insect microsomes and incubated with carvedilol ranging from 1.0 µM to 50 µM at 37°C for 30 minutes. After termination, the carvedilol metabolites were extracted and detected using ultra-performance liquid chromatography tandem mass-spectrometry. Among the 24 CYP2D6 variants, CYP2D6.92 and CYP2D6.96 were catalytically inactive and the remaining 22 variants exhibited significantly decreased intrinsic clearance values (ranging from ~25% to 95% compared with CYP2D6.1. The present data in vitro suggest that the newly found variants significantly reduced catalytic activities compared with CYP2D6.1. Given that CYP2D6 protein activities could affect carvedilol plasma levels, these findings are greatly relevant to

  2. Upregulation of a tonoplast-localized cytochrome P450 during petal senescence in Petunia inflata

    Directory of Open Access Journals (Sweden)

    Ishida Hiroyuki

    2006-04-01

    Full Text Available Abstract Background Gene expression in Petunia inflata petals undergoes major changes following compatible pollination. Severe flower wilting occurs reproducibly within 36 hours, providing an excellent model for investigation of petal senescence and programmed cell death. Expression of a number of genes and various enzyme activities involved in the degradation and remobilization of macromolecules have been found to be upregulated during the early stages of petal senescence. Results By performing differential display of cDNAs during Petunia inflata petal senescence, a highly upregulated gene encoding a cytochrome P450 was identified. Analysis of the complete cDNA sequence revealed that the predicted protein is a member of the CYP74C family (CYP74C9 and is highly similar to a tomato CYP74C allene oxide synthase (AOS that is known to be active on 9-hydroperoxides. Cloning of the petunia genomic DNA revealed an intronless gene with a promoter region that carries signals found in stress-responsive genes and potential binding sites for Myb transcription factors. Transcripts were present at detectable levels in root and stem, but were 40 times more abundant in flowers 36 hours after pollination. Ethylene and jasmonate treatment resulted in transitory increases in expression in detached flowers. A protein fusion of the CYP74C coding region to a C-terminal GFP was found to be located in the tonoplast. Conclusion Though oxylipins, particularly jasmonates, are known to be involved in stress responses, the role of other products of CYP74 enzymes is less well understood. The identification of a CYP74C family member as a highly upregulated gene during petal senescence suggests that additional products of fatty acid metabolism may play important roles during programmed cell death. In contrast to the chloroplast localization of AOS proteins in the CYP74A subfamily, GFP fusion data indicates that the petunia CYP74C9 enzyme is in the tonoplast. This result

  3. Inducible bilirubin oxidase: A novel function for the mouse cytochrome P450 2A5

    International Nuclear Information System (INIS)

    We have previously shown that bilirubin (BR), a breakdown product of haem, is a strong inhibitor and a high affinity substrate of the mouse cytochrome P450 2A5 (CYP2A5). The antioxidant BR, which is cytotoxic at high concentrations, is potentially useful in cellular protection against oxygen radicals if its intracellular levels can be strictly controlled. The mechanisms that regulate cellular BR levels are still obscure. In this paper we provide preliminary evidence for a novel function of CYP2A5 as hepatic “BR oxidase”. A high-performance liquid chromatography/electrospray ionisation mass spectrometry screening showed that recombinant yeast microsomes expressing the CYP2A5 oxidise BR to biliverdin, as the main metabolite, and to three other smaller products with m/z values of 301, 315 and 333. The metabolic profile is significantly different from that of chemical oxidation of BR. In chemical oxidation the smaller products were the main metabolites. This suggests that the enzymatic reaction is selective, towards biliverdin production. Bilirubin treatment of primary hepatocytes increased the CYP2A5 protein and activity levels with no effect on the corresponding mRNA. Co-treatment with cycloheximide (CHX), a protein synthesis inhibitor, resulted in increased half-life of the CYP2A5 compared to cells treated only with CHX. Collectively, the observations suggest that the CYP2A5 is potentially an inducible “BR oxidase” where BR may accelerate its own metabolism through stabilization of the CYP2A5 protein. It is possible that this metabolic pathway is potentially part of the machinery controlling intracellular BR levels in transient oxidative stress situations, in which high amounts of BR are produced. -- Highlights: ► CYP2A5 metabolizes bilirubin to biliverdin and dipyrroles. ► Bilirubin increased the hepatic CYP2A5 protein and activity levels. ► Bilirubin does not change the hepatic CYP2A5 mRNA levels. ► Co-treatment with a protein synthesis inhibitor

  4. Opposing regulation of cytochrome P450 expression by CAR and PXR in hypothyroid mice

    Energy Technology Data Exchange (ETDEWEB)

    Park, Young Joo [Department of Internal Medicine, Seoul National University College of Medicine (Korea, Republic of); Seoul National University Bundang Hospital, Seoul (Korea, Republic of); Lee, Eun Kyung [Department of Internal Medicine, Seoul National University College of Medicine (Korea, Republic of); Lee, Yoon Kwang [Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030 (United States); Park, Do Joon; Jang, Hak Chul [Department of Internal Medicine, Seoul National University College of Medicine (Korea, Republic of); Moore, David D., E-mail: moore@bcm.edu [Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX 77030 (United States)

    2012-09-01

    Clinical hypothyroidism affects various metabolic processes including drug metabolism. CYP2B and CYP3A are important cytochrome P450 drug metabolizing enzymes that are regulated by the xenobiotic receptors constitutive androstane receptor (CAR, NR1I3) and pregnane X receptor (PXR, NR1I2). We evaluated the regulation of the hepatic expression of CYPs by CAR and PXR in the hypothyroid state induced by a low-iodine diet containing 0.15% propylthiouracil. Expression of Cyp3a11 was suppressed in hypothyroid C57BL/6 wild type (WT) mice and a further decrement was observed in hypothyroid CAR{sup −/−} mice, but not in hypothyroid PXR{sup −/−} mice. In contrast, expression of Cyp2b10 was induced in both WT and PXR{sup −/−} hypothyroid mice, and this induction was abolished in CAR{sup −/−} mice and in and CAR{sup −/−} PXR{sup −/−} double knockouts. CAR mRNA expression was increased by hypothyroidism, while PXR expression remained unchanged. Carbamazepine (CBZ) is a commonly used antiepileptic that is metabolized by CYP3A isoforms. After CBZ treatment of normal chow fed mice, serum CBZ levels were highest in CAR{sup −/−} mice and lowest in WT and PXR{sup −/−} mice. Hypothyroid WT or PXR{sup −/−} mice survived chronic CBZ treatment, but all hypothyroid CAR{sup −/−} and CAR{sup −/−} PXR{sup −/−} mice died, with CAR{sup −/−}PXR{sup −/−} mice surviving longer than CAR{sup −/−} mice (12.3 ± 3.3 days vs. 6.3 ± 2.1 days, p = 0.04). All these findings suggest that hypothyroid status affects xenobiotic metabolism, with opposing responses of CAR and PXR and their CYP targets that can cancel each other out, decreasing serious metabolic derangement in response to a xenobiotic challenge. -- Highlights: ► Hypothyroid status activates CAR in mice and induces Cyp2b10 expression. ► Hypothyroid status suppresses PXR activity in mice and represses Cyp3a11 expression. ► These responses balance each other out in normal mice.

  5. Cytochrome P450 2D6 variants in a Caucasian population: Allele frequencies and phenotypic consequences

    Energy Technology Data Exchange (ETDEWEB)

    Sachse, C.; Brockmoeller, J.; Bauer, S.; Roots, I. [Humboldt Univ., Berlin (Germany)

    1997-02-01

    Cytochrome P450 2D6 (CYP2D6) metabolizes many important drugs. CYP2D6 activity ranges from complete deficiency to ultrafast metabolism, depending on at least 16 different known alleles. Their frequencies were determined in 589 unrelated German volunteers and correlated with enzyme activity measured by phenotyping with dextromethorphan or debrisoquine. For genotyping, nested PCR-RFLP tests from a PCR amplificate of the entire CYP2D6 gene were developed. The frequency of the CYP2D6*1 allele coding for extensive metabolizer (EM) phenotype was .364. The alleles coding for slightly (CYP2D6*2) or moderately (*9 and *10) reduced activity (intermediate metabolizer phenotype [IM]) showed frequencies of .324, .018, and .015, respectively. By use of novel PCR tests for discrimination, CYP2D6 gene duplication alleles were found with frequencies of.005 (*1 x 2), .013 (* 2 x 2), and .001 (*4 x 2). Frequencies of alleles with complete deficiency (poor metabolizer phenotype [PM]) were .207 (*4), .020 (*3 and *5), .009 (*6), and .001 (*7, *15, and *16). The defective CYP2D6 alleles *8, *11, *12, *13, and *14 were not found. All 41 PMs (7.0%) in this sample were explained by five mutations detected by four PCR-RFLP tests, which may suffice, together with the gene duplication test, for clinical prediction of CYP2D6 capacity. Three novel variants of known CYP2D6 alleles were discovered: *1C (T{sub 1957}C), *2B (additional C{sub 2558}T), and *4E (additional C{sub 2938}T). Analysis of variance showed significant differences in enzymatic activity measured by the dextromethorphan metabolic ratio (MR) between carriers of EN/PM (mean MR = .006) and IM/PM (mean MR = .014) alleles and between carriers of one (mean MR = .009) and two (mean MR = .003) functional alleles. The results of this study provide a solid basis for prediction of CYP2D6 capacity, as required in drug research and routine drug treatment. 35 refs., 4 figs., 5 tabs.

  6. Cytochrome P450 2A5 and bilirubin: Mechanisms of gene regulation and cytoprotection

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Sangsoo Daniel; Antenos, Monica [Department of Biomedical Sciences, University of Guelph, Guelph, Ontario, N1G 2W1 (Canada); Squires, E. James [Department of Animal and Poultry Sciences, University of Guelph, Guelph, Ontario, N1G 2W1 (Canada); Kirby, Gordon M., E-mail: gkirby@uoguelph.ca [Department of Biomedical Sciences, University of Guelph, Guelph, Ontario, N1G 2W1 (Canada)

    2013-07-15

    Bilirubin (BR) has recently been identified as the first endogenous substrate for cytochrome P450 2A5 (CYP2A5) and it has been suggested that CYP2A5 plays a major role in BR clearance as an alternative mechanism to BR conjugation by uridine-diphosphate glucuronyltransferase 1A1. This study investigated the mechanisms of Cyp2a5 gene regulation by BR and the cytoprotective role of CYP2A5 in BR hepatotoxicity. BR induced CYP2A5 expression at the mRNA and protein levels in a dose-dependent manner in primary mouse hepatocytes. BR treatment also caused nuclear translocation of Nuclear factor-E2 p45-related factor 2 (Nrf2) in hepatocytes. In reporter assays, BR treatment of primary hepatocytes transfected with a Cyp2a5 promoter-luciferase reporter construct resulted in a 2-fold induction of Cyp2a5 reporter activity. Furthermore, cotransfection of the hepatocytes with a Nrf2 expression vector without BR treatment resulted in an increase in Cyp2a5 reporter activity of approximately 2-fold and BR treatment of Nrf2 cotransfectants further increased reporter activity by 4-fold. In addition, site-directed mutation of the ARE in the reporter construct completely abolished both the BR- and Nrf2-mediated increases in reporter activity. The cytoprotective role of CYP2A5 against BR-mediated apoptosis was also examined in Hepa 1–6 cells that lack endogenous CYP2A5. Transient overexpression of CYP2A5 partially blocked BR-induced caspase-3 cleavage in Hepa 1–6 cells. Furthermore, in vitro degradation of BR was increased by microsomes from Hepa 1–6 cells overexpressing CYP2A5 compared to control cells transfected with an empty vector. Collectively, these results suggest that Nrf2-mediated CYP2A5 transactivation in response to BR may provide an additional mechanism for adaptive cytoprotection against BR hepatotoxicity. - Highlights: • The mechanism of Cyp2a5 gene regulation by BR was investigated. • The cytoprotective role of CYP2A5 in BR hepatotoxicity was determined. • BR

  7. Cytochrome P450 2E1 genetic polymorphism and gastric cancer in Changle, Fujian Province

    Institute of Scientific and Technical Information of China (English)

    Lin Cai; Shun-Zhang Yu; Zuo-Feng Zhang

    2001-01-01

    AIM: Genetic polymorphism in enzymes of carcinogen metabolism has been found to have the influence on the susceptibility to cancer. Cytochrome P450 2E1 ( CYP2 E1) is considered to play an important role in the metabolic activation of procarcinogens such as N-nitroscoamines and Iow molecular weight organic compounds. The purpose of this study is to determine whether CYP450 2Elpolymorphisms are associated with risk s of gastric cancer. METHODS: We conducted a population based case-control study in Changle county, Fujian Province, a high-risk region of gastric cancer in China. Ninety-one incident gastric cancer patients and ninety-four healthy controls were included in our study. Datas including dsmographic characteristcs, diet intake, and alcohol and tobacco consumption of indivduals in our study were completed by a standardized questionnaire. PCR-RFLP revealed three genotypes: heterozygote (C1/C2) and two homozygotes (C1/C1 and C2/C2) in CYP2E1. RESULTS: The frequency of variant genotypes (C1/C2 and C2/C2) in gastric cancer cases and controls was 36.3% and 24.5%, respectively. The rare homozygous C2/C2 genotype was found in 6 indivduals in gastric cancer group(6.6%),whereas there was only one in the control group (1.1%).However, there was no statistically significan difference between the two groups (two-tailed Fisher′s exact test, P =0.066). Indivduals in gastric cancer group were more likely to carry genotype C1/C2 (odds ratio, OR = 1.50) and C2/C2(OR = 7.34) than indivduals in control group (X2 = 4.597, for trend P=0.032). The frequencies of genofypes with the C2allele ( C1/C2 and C2/C2 genotypes) were compared with those of genotypes without C2 allele ( C1/C1 genotype )among indivduals in gastric cancer group and control group according to the pattern of gastric cancer risk factors. The results show that indivduals who exposed to these gastric cancer risk factors and carry the C2 allele seemed to have a higher risk of developing gastric cancer. CONCLUSION

  8. Metabolism of bilirubin by human cytochrome P450 2A6

    International Nuclear Information System (INIS)

    The mouse cytochrome P450 (CYP) 2A5 has recently been shown to function as hepatic “Bilirubin Oxidase” (Abu-Bakar, A., et al., 2011. Toxicol. Appl. Pharmacol. 257, 14–22). To date, no information is available on human CYP isoforms involvement in bilirubin metabolism. In this paper we provide novel evidence for human CYP2A6 metabolising the tetrapyrrole bilirubin. Incubation of bilirubin with recombinant yeast microsomes expressing the CYP2A6 showed that bilirubin inhibited CYP2A6-dependent coumarin 7-hydroxylase activity to almost 100% with an estimated Ki of 2.23 μM. Metabolite screening by a high-performance liquid chromatography/electrospray ionisation mass spectrometry indicated that CYP2A6 oxidised bilirubin to biliverdin and to three other smaller products with m/z values of 301, 315 and 333. Molecular docking analyses indicated that bilirubin and its positively charged intermediate interacted with key amino acid residues at the enzyme's active site. They were stabilised at the site in a conformation favouring biliverdin formation. By contrast, the end product, biliverdin was less fitting to the active site with the critical central methylene bridge distanced from the CYP2A6 haem iron facilitating its release. Furthermore, bilirubin treatment of HepG2 cells increased the CYP2A6 protein and activity levels with no effect on the corresponding mRNA. Co-treatment with cycloheximide (CHX), a protein synthesis inhibitor, resulted in increased half-life of the CYP2A6 compared to cells treated only with CHX. Collectively, the observations indicate that the CYP2A6 may function as human “Bilirubin Oxidase” where bilirubin is potentially a substrate and a regulator of the enzyme. -- Highlights: ► Human CYP2A6 interacts with bilirubin with a high affinity. ► Bilirubin docking to the CYP2A6 active site is more stable than biliverdin docking. ► Recombinant CYP2A6 microsomes metabolised bilirubin to biliverdin. ► Bilirubin increased the hepatic CYP2A6

  9. Functional characterization of cytochromes P450 2B from the desert woodrat Neotoma lepida

    Energy Technology Data Exchange (ETDEWEB)

    Wilderman, P. Ross, E-mail: pwilderman@ucsd.edu [Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA (United States); Jang, Hyun-Hee [Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA (United States); Malenke, Jael R. [Department of Biology, University of Utah, Salt Lake City, UT (United States); Salib, Mariam; Angermeier, Elisabeth; Lamime, Sonia [Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA (United States); Dearing, M. Denise [Department of Biology, University of Utah, Salt Lake City, UT (United States); Halpert, James R. [Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California, San Diego, La Jolla, CA (United States)

    2014-02-01

    Mammalian detoxification processes have been the focus of intense research, but little is known about how wild herbivores process plant secondary compounds, many of which have medicinal value or are drugs. cDNA sequences that code for three enzymes of the cytochrome P450 (CYP) 2B subfamily, here termed 2B35, 2B36, and 2B37 have been recently identified from a wild rodent, the desert woodrat (Malenke et al., 2012). Two variant clones of each enzyme were engineered to increase protein solubility and to facilitate purification, as reported for CYP2B enzymes from multiple species. When expressed in Escherichia coli each of the woodrat proteins gave the characteristic maximum at 450 nm in a reduced carbon monoxide difference spectrum but generally expressed at lower levels than rat CYP2B1. Two enzymes, 2B36 and 2B37, showed dealkylation activity with the model substrates 7-ethoxy-4-(trifluoromethyl)coumarin and 7-benzyloxyresorufin, whereas 2B35 was inactive. Binding of the monoterpene (+)-α-pinene produced a Type I shift in the absorbance spectrum of each enzyme. Mutation of 2B37 at residues 114, 262, or 480, key residues governing ligand interactions with other CYP2B enzymes, did not significantly change expression levels or produce the expected functional changes. In summary, two catalytic and one ligand-binding assay are sufficient to distinguish among CYP2B35, 2B36, and 2B37. Differences in functional profiles between 2B36 and 2B37 are partially explained by changes in substrate recognition site residue 114, but not 480. The results advance our understanding of the mechanisms of detoxification in wild mammalian herbivores and highlight the complexity of this system. - Highlights: • Three CYP2B enzymes from Neotoma lepida were cloned, engineered, and expressed. • A mix of catalytic and binding assays yields unique results for each enzyme. • Mutational analysis indicates CYP{sub 2}B substrate recognition remains to be clarified. • Reported N. lepida gene

  10. Pharmacogenomics In Drug Therapy And Interaction: The Role Of Cytochrome P450

    Directory of Open Access Journals (Sweden)

    Peter Pook

    2008-12-01

    Full Text Available Pharmacogenomics (or pharmacogenetics,the study of the effects of genetic differences on aperson’s response to drugs, can help in optimizing drugefficacy and minimizing adverse drug reactions.Interperson difference in drug metabolism is one of theimportant consequences of such genetic variation. Thisvariation is determined in part by mutations incytochrome P450 enzymes (CYPs. IMU is part of amajor collaborative research project in the area ofphamacogenetics and drug metabolism. Workingtogether with USM and UiTM, our group has, since2000, generated useful population database on geneticpolymorphism of various CYP isoforms. We havesuccessfully genotyped three major ethnic groups,Malay, Indian and Chinese for their allelic frequency ofimportant isoforms. These include CYP2D6, CYP2C9,CYP2C8 and CYP2A6. Data generated so farcollectively have contributed to our effort in mappingand constructing genomic database for Malaysianpopulation.Since early 2002, our research has been focusing ondeveloping in vitro methods in studying the functionalconsequences of genetic polymorphism of CYP enzymes.Using site-directed mutagenesis, CYP mutants, carryingnucleotide changes as reported in known alleles inhuman populations, were generated and expressed inE. coli system, and the expressed recombinant proteinswere characterized using enzyme assays to determinethe functional consequences of mutations. We haveestablished a series of HPLC (high performance liquidchromatography-based and fluorescence-based assaysto investigate CYP activities. Assays that have beendeveloped include tolbutamide methylhydroxylase,paclitaxel 6α-hydroxylase, dextromethorphanO-demethylation, testosterone 6β-hydroxylation andcoumarin 7-hydroxylase assays. These assays serve asactivity markers allowing comparison of catalyticactivities of mutant proteins generated. Another focusof our work is to use the developed assays as a screeningtool to investigate drug-herb interactions. This wasachieved

  11. Opposing regulation of cytochrome P450 expression by CAR and PXR in hypothyroid mice

    International Nuclear Information System (INIS)

    Clinical hypothyroidism affects various metabolic processes including drug metabolism. CYP2B and CYP3A are important cytochrome P450 drug metabolizing enzymes that are regulated by the xenobiotic receptors constitutive androstane receptor (CAR, NR1I3) and pregnane X receptor (PXR, NR1I2). We evaluated the regulation of the hepatic expression of CYPs by CAR and PXR in the hypothyroid state induced by a low-iodine diet containing 0.15% propylthiouracil. Expression of Cyp3a11 was suppressed in hypothyroid C57BL/6 wild type (WT) mice and a further decrement was observed in hypothyroid CAR−/− mice, but not in hypothyroid PXR−/− mice. In contrast, expression of Cyp2b10 was induced in both WT and PXR−/− hypothyroid mice, and this induction was abolished in CAR−/− mice and in and CAR−/− PXR−/− double knockouts. CAR mRNA expression was increased by hypothyroidism, while PXR expression remained unchanged. Carbamazepine (CBZ) is a commonly used antiepileptic that is metabolized by CYP3A isoforms. After CBZ treatment of normal chow fed mice, serum CBZ levels were highest in CAR−/− mice and lowest in WT and PXR−/− mice. Hypothyroid WT or PXR−/− mice survived chronic CBZ treatment, but all hypothyroid CAR−/− and CAR−/− PXR−/− mice died, with CAR−/−PXR−/− mice surviving longer than CAR−/− mice (12.3 ± 3.3 days vs. 6.3 ± 2.1 days, p = 0.04). All these findings suggest that hypothyroid status affects xenobiotic metabolism, with opposing responses of CAR and PXR and their CYP targets that can cancel each other out, decreasing serious metabolic derangement in response to a xenobiotic challenge. -- Highlights: ► Hypothyroid status activates CAR in mice and induces Cyp2b10 expression. ► Hypothyroid status suppresses PXR activity in mice and represses Cyp3a11 expression. ► These responses balance each other out in normal mice. ► Hypothyroidism sensitizes CAR null mice to toxic effects of carbamazepine.

  12. Metabolism of bilirubin by human cytochrome P450 2A6

    Energy Technology Data Exchange (ETDEWEB)

    Abu-Bakar, A' edah, E-mail: a.abubakar@uq.edu.au [The University of Queensland, National Research Centre for Environmental Toxicology (Entox), 4072 Brisbane, Queensland (Australia); Arthur, Dionne M. [The University of Queensland, National Research Centre for Environmental Toxicology (Entox), 4072 Brisbane, Queensland (Australia); Cooperative Research Centre for Contamination Assessment and Remediation of the Environment, Adelaide (Australia); Wikman, Anna S. [The University of Queensland, National Research Centre for Environmental Toxicology (Entox), 4072 Brisbane, Queensland (Australia); Department of Pharmaceutical Biosciences, Uppsala University, SE-75123 Uppsala (Sweden); Rahnasto, Minna; Juvonen, Risto O.; Vepsäläinen, Jouko; Raunio, Hannu [School of Pharmacy, Faculty of Health Sciences, University of Eastern Finland, POB 1627, 70211 Kuopio (Finland); Ng, Jack C. [The University of Queensland, National Research Centre for Environmental Toxicology (Entox), 4072 Brisbane, Queensland (Australia); Cooperative Research Centre for Contamination Assessment and Remediation of the Environment, Adelaide (Australia); Lang, Matti A. [The University of Queensland, National Research Centre for Environmental Toxicology (Entox), 4072 Brisbane, Queensland (Australia)

    2012-05-15

    The mouse cytochrome P450 (CYP) 2A5 has recently been shown to function as hepatic “Bilirubin Oxidase” (Abu-Bakar, A., et al., 2011. Toxicol. Appl. Pharmacol. 257, 14–22). To date, no information is available on human CYP isoforms involvement in bilirubin metabolism. In this paper we provide novel evidence for human CYP2A6 metabolising the tetrapyrrole bilirubin. Incubation of bilirubin with recombinant yeast microsomes expressing the CYP2A6 showed that bilirubin inhibited CYP2A6-dependent coumarin 7-hydroxylase activity to almost 100% with an estimated K{sub i} of 2.23 μM. Metabolite screening by a high-performance liquid chromatography/electrospray ionisation mass spectrometry indicated that CYP2A6 oxidised bilirubin to biliverdin and to three other smaller products with m/z values of 301, 315 and 333. Molecular docking analyses indicated that bilirubin and its positively charged intermediate interacted with key amino acid residues at the enzyme's active site. They were stabilised at the site in a conformation favouring biliverdin formation. By contrast, the end product, biliverdin was less fitting to the active site with the critical central methylene bridge distanced from the CYP2A6 haem iron facilitating its release. Furthermore, bilirubin treatment of HepG2 cells increased the CYP2A6 protein and activity levels with no effect on the corresponding mRNA. Co-treatment with cycloheximide (CHX), a protein synthesis inhibitor, resulted in increased half-life of the CYP2A6 compared to cells treated only with CHX. Collectively, the observations indicate that the CYP2A6 may function as human “Bilirubin Oxidase” where bilirubin is potentially a substrate and a regulator of the enzyme. -- Highlights: ► Human CYP2A6 interacts with bilirubin with a high affinity. ► Bilirubin docking to the CYP2A6 active site is more stable than biliverdin docking. ► Recombinant CYP2A6 microsomes metabolised bilirubin to biliverdin. ► Bilirubin increased the hepatic

  13. Benzene metabolism by human liver microsomes in relation to cytochrome P450 2E1 activity.

    Science.gov (United States)

    Seaton, M J; Schlosser, P M; Bond, J A; Medinsky, M A

    1994-09-01

    Low levels of benzene from sources including cigarette smoke and automobile emissions are ubiquitous in the environment. Since the toxicity of benzene probably results from oxidative metabolites, an understanding of the profile of biotransformation of low levels of benzene is critical in making a valid risk assessment. To that end, we have investigated metabolism of a low concentration of [14C]benzene (3.4 microM) by microsomes from human, mouse and rat liver. The extent of phase I benzene metabolism by microsomal preparations from 10 human liver samples and single microsomal preparations from both mice and rats was then related to measured activities of cytochrome P450 (CYP) 2E1. Measured CYP 2E1 activities, as determined by hydroxylation of p-nitrophenol, varied 13-fold (0.253-3.266 nmol/min/mg) for human samples. The fraction of benzene metabolized in 16 min ranged from 10% to 59%. Also at 16 min, significant amounts of oxidative metabolites were formed. Phenol was the main metabolite formed by all but two human microsomal preparations. In those samples, both of which had high CYP 2E1 activity, hydroquinone was the major metabolite formed. Both hydroquinone and catechol formation showed a direct correlation with CYP 2E1 activity over the range of activities present. A simulation model was developed based on a mechanism of competitive inhibition between benzene and its oxidized metabolites, and was fit to time-course data for three human liver preparations. Model calculations for initial rates of benzene metabolism ranging from 0.344 to 4.442 nmol/mg/min are directly proportional to measured CYP 2E1 activities. The model predicted the dependence of benzene metabolism on the measured CYP 2E1 activity in human liver samples, as well as in mouse and rat liver samples. These results suggest that differences in measured hepatic CYP 2E1 activity may be a major factor contributing to both interindividual and interspecies variations in hepatic metabolism of benzene

  14. Classification of cytochrome P450 inhibitors and noninhibitors using combined classifiers.

    Science.gov (United States)

    Cheng, Feixiong; Yu, Yue; Shen, Jie; Yang, Lei; Li, Weihua; Liu, Guixia; Lee, Philip W; Tang, Yun

    2011-05-23

    Adverse side effects of drug-drug interactions induced by human cytochrome P450 (CYP) inhibition is an important consideration, especially, during the research phase of drug discovery. It is highly desirable to develop computational models that can predict the inhibitive effect of a compound against a specific CYP isoform. In this study, inhibitor predicting models were developed for five major CYP isoforms, namely 1A2, 2C9, 2C19, 2D6, and 3A4, using a combined classifier algorithm on a large data set containing more than 24,700 unique compounds, extracted from PubChem. The combined classifiers algorithm is an ensemble of different independent machine learning classifiers including support vector machine, C4.5 decision tree, k-nearest neighbor, and naïve Bayes, fused by a back-propagation artificial neural network (BP-ANN). All developed models were validated by 5-fold cross-validation and a diverse validation set composed of about 9000 diverse unique compounds. The range of the area under the receiver operating characteristic curve (AUC) for the validation sets was 0.764 to 0.815 for CYP1A2, 0.837 to 0.861 for CYP2C9, 0.793 to 0.842 for CYP2C19, 0.839 to 0.886 for CYP2D6, and 0.754 to 0.790 for CYP3A4, respectively, using the new developed combined classifiers. The overall performance of the combined classifiers fused by BP-ANN was superior to that of three classic fusion techniques (Mean, Maximum, and Multiply). The chemical spaces of data sets were explored by multidimensional scaling plots, and the use of applicability domain improved the prediction accuracies of models. In addition, some representative substructure fragments differentiating CYP inhibitors and noninhibitors were characterized by the substructure fragment analysis. These classification models are applicable for virtual screening of the five major CYP isoforms inhibitors or can be used as simple filters of potential chemicals in drug discovery. PMID:21491913

  15. Equine cytochrome P450 2B6--genomic identification, expression and functional characterization with ketamine.

    Science.gov (United States)

    Peters, L M; Demmel, S; Pusch, G; Buters, J T M; Thormann, W; Zielinski, J; Leeb, T; Mevissen, M; Schmitz, A

    2013-01-01

    Ketamine is an anesthetic and analgesic regularly used in veterinary patients. As ketamine is almost always administered in combination with other drugs, interactions between ketamine and other drugs bear the risk of either adverse effects or diminished efficacy. Since cytochrome P450 enzymes (CYPs) play a pivotal role in the phase I metabolism of the majority of all marketed drugs, drug-drug interactions often occur at the active site of these enzymes. CYPs have been thoroughly examined in humans and laboratory animals, but little is known about equine CYPs. The characterization of equine CYPs is essential for a better understanding of drug metabolism in horses. We report annotation, cloning and heterologous expression of the equine CYP2B6 in V79 Chinese hamster fibroblasts. After computational annotation of all CYP2B genes, the coding sequence (CDS) of equine CYP2B6 was amplified by RT-PCR from horse liver total RNA and revealed an amino acid sequence identity of 77% and a similarity of 93.7% to its human ortholog. A non-synonymous variant c.226G>A in exon 2 of the equine CYP2B6 was detected in 97 horses. The mutant A-allele showed an allele frequency of 82%. Two further variants in exon 3 were detected in one and two horses of this group, respectively. Transfected V79 cells were incubated with racemic ketamine and norketamine as probe substrates to determine metabolic activity. The recombinant equine CYP2B6 N-demethylated ketamine to norketamine and produced metabolites of norketamine, such as hydroxylated norketamines and 5,6-dehydronorketamine. V(max) for S-/and R-norketamine formation was 0.49 and 0.45nmol/h/mg cellular protein and K(m) was 3.41 and 2.66μM, respectively. The N-demethylation of S-/R-ketamine was inhibited concentration-dependently with clopidogrel showing an IC(50) of 5.63 and 6.26μM, respectively. The functional importance of the recorded genetic variants remains to be explored. Equine CYP2B6 was determined to be a CYP enzyme involved in

  16. Discovery of a regioselectivity switch in nitrating P450s guided by molecular dynamics simulations and Markov models

    Science.gov (United States)

    Dodani, Sheel C.; Kiss, Gert; Cahn, Jackson K. B.; Su, Ye; Pande, Vijay S.; Arnold, Frances H.

    2016-05-01

    The dynamic motions of protein structural elements, particularly flexible loops, are intimately linked with diverse aspects of enzyme catalysis. Engineering of these loop regions can alter protein stability, substrate binding and even dramatically impact enzyme function. When these flexible regions are unresolvable structurally, computational reconstruction in combination with large-scale molecular dynamics simulations can be used to guide the engineering strategy. Here we present a collaborative approach that consists of both experiment and computation and led to the discovery of a single mutation in the F/G loop of the nitrating cytochrome P450 TxtE that simultaneously controls loop dynamics and completely shifts the enzyme's regioselectivity from the C4 to the C5 position of L-tryptophan. Furthermore, we find that this loop mutation is naturally present in a subset of homologous nitrating P450s and confirm that these uncharacterized enzymes exclusively produce 5-nitro-L-tryptophan, a previously unknown biosynthetic intermediate.

  17. The effect of celery and parsley juices on pharmacodynamic activity of drugs involving cytochrome P450 in their metabolism.

    Science.gov (United States)

    Jakovljevic, V; Raskovic, A; Popovic, M; Sabo, J

    2002-01-01

    Celery (Apium graveolens) and parsley (Petroselinum sativum), plants used worldwide in human nutrition, are the natural sources of methoxsalen. In this study we investigated the effect of mice pretreatment with juices of this plants on the hypnotic action of pentobarbital and analgesic action of paracetamol and aminopyrine, the drugs involving cytochrome P450 superfamily in their metabolism. In mice pretreated with celery and parsley juices a prolonged action of pentobarbital with respect to control was observed, statistical significance being attained only with parsley-pretreated animals. Both pretreatments increased and prolonged the analgesic action of aminopyrine and paracetamol, pretreatment with parsley being again more effective. Celery and parsley juices given to animals two hours before their decapitation caused a significant decrease of cytochrome P450 in the liver homogenate as compared to control. PMID:12365194

  18. Electrochemiluminescent Arrays for Cytochrome P450-Activated Genotoxicity Screening. DNA Damage from Benzo[a]pyrene Metabolites

    Science.gov (United States)

    Hvastkovs, Eli G.; So, Minjeong; Krishnan, Sadagopan; Bajrami, Besnik; Tarun, Maricar; Jansson, Ingela; Schenkman, John B.; Rusling, James F.

    2007-01-01

    Arrays suitable for genotoxicity screening are reported that generate metabolites from cytochrome P450 enzymes (CYPs) in thin-film spots. Array spots containing DNA, various human cyt P450s, and electrochemiluminescence (ECL) generating metallopolymer [Ru(bpy)2PVP10]2+ were exposed to H2O2 to activate the enzymes. ECL from all spots was visualized simultaneously using a CCD camera. Using benzo[a]pyrene as a test substrate, enzyme activity for producing DNA damage in the arrays was found in the order CYP1B1 > CYP1A2 > CYP1A1 > CYP2E1 > myoglobin, the same as the order of their metabolic activity. Thus, these arrays estimate the relative propensity of different enzymes to produce genotoxic metabolites. This is the first demonstration of ECL arrays for high-throughput in vitro genotoxicity screening. PMID:17261025

  19. Discovery of a regioselectivity switch in nitrating P450s guided by molecular dynamics simulations and Markov models.

    Science.gov (United States)

    Dodani, Sheel C; Kiss, Gert; Cahn, Jackson K B; Su, Ye; Pande, Vijay S; Arnold, Frances H

    2016-05-01

    The dynamic motions of protein structural elements, particularly flexible loops, are intimately linked with diverse aspects of enzyme catalysis. Engineering of these loop regions can alter protein stability, substrate binding and even dramatically impact enzyme function. When these flexible regions are unresolvable structurally, computational reconstruction in combination with large-scale molecular dynamics simulations can be used to guide the engineering strategy. Here we present a collaborative approach that consists of both experiment and computation and led to the discovery of a single mutation in the F/G loop of the nitrating cytochrome P450 TxtE that simultaneously controls loop dynamics and completely shifts the enzyme's regioselectivity from the C4 to the C5 position of L-tryptophan. Furthermore, we find that this loop mutation is naturally present in a subset of homologous nitrating P450s and confirm that these uncharacterized enzymes exclusively produce 5-nitro-L-tryptophan, a previously unknown biosynthetic intermediate. PMID:27102675

  20. Renal cytochrome P450 ω-hydroxylase and epoxygenase activity are differentially modified by nitric oxide and sodium chloride

    OpenAIRE

    Oyekan, A.O.; Youseff, T.; Fulton, D.; Quilley, J.; McGiff, J. C.

    1999-01-01

    Renal function is perturbed by inhibition of nitric oxide synthase (NOS). To probe the basis of this effect, we characterized the effects of nitric oxide (NO), a known suppressor of cytochrome P450 (CYP) enzymes, on metabolism of arachidonic acid (AA), the expression of ω-hydroxylase, and the efflux of 20-hydroxyeicosatetraenoic acid (20-HETE) from the isolated kidney. The capacity to convert [14C]AA to HETEs and epoxides (EETs) was greater in cortical microsomes than in medullary microsomes....

  1. Recombinant Saccharomyces cerevisiae Expressing P450 in Artificial Digestive Systems: a Model for Biodetoxication in the Human Digestive Environment

    OpenAIRE

    Blanquet, S; Meunier, J. P.; Minekus, M.; Marol-Bonnin, S.; Alric, M.

    2003-01-01

    The use of genetically engineered microorganisms such as bacteria or yeasts as live vehicles to carry out bioconversion directly in the digestive environment is an important challenge for the development of innovative biodrugs. A system that mimics the human gastrointestinal tract was combined with a computer simulation to evaluate the survival rate and cinnamate 4-hydroxylase activity of a recombinant model of Saccharomyces cerevisiae expressing the plant P450 73A1. The yeasts showed a high ...

  2. Effects of probiotic Escherichia coli Nissle 1917 on expression of cytochromes P450 along the gastrointestinal tract of male rats

    Czech Academy of Sciences Publication Activity Database

    Matušková, Z.; Tunková, A.; Anzenbacherová, E.; Večeřa, R.; Šiller, M.; Tlaskalová-Hogenová, Helena; Zídek, Zdeněk; Anzenbacher, P.

    2010-01-01

    Roč. 31, č. 2 (2010), s. 46-50. ISSN 0172-780X R&D Projects: GA ČR GA305/08/0535 Institutional research plan: CEZ:AV0Z50200510; CEZ:AV0Z50390512 Keywords : probiotic * Escherichia coli * cytochrome P450 Subject RIV: FB - Endocrinology, Diabetology, Metabolism, Nutrition Impact factor: 1.621, year: 2010

  3. N-Hydroxylation of dapsone by multiple enzymes of cytochrome P450: implications for inhibition of haemotoxicity.

    OpenAIRE

    Gill, H J; Tingle, M D; Park, B.K.

    1995-01-01

    1. The adverse reactions associated with the administration of dapsone are believed to be caused by metabolism to its hydroxylamine. Previous reports suggest that CYP3A4 is responsible for this biotransformation [1]. 2. Data presented in this paper illustrate the involvement of more than one cytochrome P450 enzyme in dapsone hydroxylamine formation using human liver microsomes. Eadie-Hofstee plots demonstrated bi-phasic kinetics in several livers. No correlation could be established between h...

  4. The Oxidized Linoleic Acid Metabolite-Cytochrome P450 System is Active in Biopsies from Patients with Inflammatory Dental Pain

    OpenAIRE

    Ruparel, Shivani; Hargreaves, Kenneth. M.; Eskander, Michael; Rowan, Spencer; Almeida, Jose F. A.; Roman, Linda; Henry, Michael A.

    2013-01-01

    Endogenous TRPV1 agonists such as oxidized linoleic acid metabolites (OLAMs) and the enzymes releasing them [e.g., cytochrome P450 (CYP)], are up-regulated following inflammation in the rat. However, it is not known if such agonists are elevated in human inflammatory pain conditions. Since TRPV1 is expressed in human dental pulp nociceptors, we hypothesized that OLAM-CYP machinery is active in this tissue type and is increased under painful inflammatory conditions such as irreversible pulpiti...

  5. Cimetidine-associated patent ductus arteriosus is mediated via a cytochrome P450 mechanism independent of H2 receptor antagonism

    OpenAIRE

    Cotton, Robert B.; Shah, Lisa P.; Poole, Stanley D.; Ehinger, Noah J.; Brown, Naoko; Shelton, Elaine L.; Slaughter, James C.; Baldwin, H. Scott; Paria, Bibhash C.; Reese, Jeff

    2013-01-01

    Persistent patency of the ductus arteriosus (PDA) is a common problem in preterm infants. The antacid cimetidine is a potent antagonist of the H2 histamine receptor but also inhibits certain cytochrome P450 enzymes (CYPs), which may affect DA patency. We examined whether cimetidine contributes to PDA and is mediated by CYP inhibition rather than H2 blockade. Analysis of a clinical trial to prevent lung injury in premature infants revealed a significant association between cimetidine treatment...

  6. Effect of Ketoconazole, a Cytochrome P450 Inhibitor, on the Efficacy of Quinine and Halofantrine against Schistosoma mansoni in Mice

    OpenAIRE

    Seif el-Din, Sayed Hassan; Sabra, Abdel-Nasser Abdel-Aal; Hammam, Olfat Ali; El-Lakkany, Naglaa Mohamed

    2013-01-01

    The fear that schistosomes will become resistant to praziquantel (PZQ) motivates the search for alternatives to treat schistosomiasis. The antimalarials quinine (QN) and halofantrine (HF) possess moderate antischistosomal properties. The major metabolic pathway of QN and HF is through cytochrome P450 (CYP) 3A4. Accordingly, this study investigates the effects of CYP3A4 inhibitor, ketoconazole (KTZ), on the antischistosomal potential of these quinolines against Schistosoma mansoni infection by...

  7. Sensitivity of the system of cytochrome P-450 of poultry liver to the action of red laser light

    International Nuclear Information System (INIS)

    It is detected that irradiation of poultry embryos by red laser light (λ = 633 nm) at a doze of 1 - 6 mJ can influence a level of the cytochrome P-450 oxidized form in poultry liver. It is supposed that this level is changed due to variations in the content of lipid peroxide compounds in tissue under low-level red laser radiation and is one of the factors of regulation of the intensity of peroxide processes in tissues

  8. Allelic Variation of Cytochrome P450s Drives Resistance to Bednet Insecticides in a Major Malaria Vector

    OpenAIRE

    Sulaiman S Ibrahim; Riveron, Jacob M.; Bibby, Jaclyn; Irving, Helen; Yunta, Cristina; Paine, Mark J. I.; Wondji, Charles S.

    2015-01-01

    Scale up of Long Lasting Insecticide Nets (LLINs) has massively contributed to reduce malaria mortality across Africa. However, resistance to pyrethroid insecticides in malaria vectors threatens its continued effectiveness. Deciphering the detailed molecular basis of such resistance and designing diagnostic tools is critical to implement suitable resistance management strategies. Here, we demonstrated that allelic variation in two cytochrome P450 genes is the most important driver of pyrethro...

  9. Allelic Variation of Cytochrome P450s Drives Resistance to Bednet Insecticides in a Major Malaria Vector.

    OpenAIRE

    Sulaiman S Ibrahim; Riveron, Jacob M.; Jaclyn Bibby; Helen Irving; Cristina Yunta; Paine, Mark J. I.; Wondji, Charles S.

    2015-01-01

    Scale up of Long Lasting Insecticide Nets (LLINs) has massively contributed to reduce malaria mortality across Africa. However, resistance to pyrethroid insecticides in malaria vectors threatens its continued effectiveness. Deciphering the detailed molecular basis of such resistance and designing diagnostic tools is critical to implement suitable resistance management strategies. Here, we demonstrated that allelic variation in two cytochrome P450 genes is the most important driver of pyrethro...

  10. Mechanism-Based Inhibition of Recombinant Human Cytochrome P450 3A4 by Tomato Juice Extract

    OpenAIRE

    須永, 克佳; 大川, 健一; 中村, 健一; 大久保, 温子; 原田, 園子; 津田, 整

    2012-01-01

    This study investigates whether tomato juice can inhibit cytochrome P450 (CYP) 3A4-mediated drug metabolism. Three commercially available, additive-free tomato juices, along with homogenized fresh tomato, were analyzed for their ability to inhibit testosterone 6β-hydroxylation activity using human recombinant CYP3A4. Results were compared to that of grapefruit juice. Ethyl acetate extracts of the tomato juices moderately reduced residual activity of CYP3A4 testosterone 6β-hydroxylation activi...

  11. Cytochrome P450 2E1 inhibition prevents hepatic carcinogenesis induced by diethylnitrosamine in alcohol-fed rats

    OpenAIRE

    Ye, Qinyuan; Lian, Fuzhi; Chavez, Pollyanna R. G.; Chung, Jayong; Ling, Wenhua; Qin, Hua; Seitz, Helmut K.; Wang, Xiang-Dong

    2012-01-01

    Chronic alcohol ingestion increases hepatic cytochrome P450 2E1 (CYP2E1), which is associated with hepatocarcinogenesis. We investigated whether treatment with chlormethiazole (CMZ), a CYP2E1 inhibitor, protects against alcohol-associated hepatic carcinogenesis in rats. Rats were fed either an ethanol liquid diet or a non-ethanol liquid diet, with or without CMZ for one and ten months. A single intraperitoneal injection of diethylnitrosamine (DEN, 20 mg/kg) was given to initiate hepatic carci...

  12. An improved substrate cocktail for assessing direct inhibition and time-dependent inhibition of multiple cytochrome P450s

    OpenAIRE

    Chen, Zhong-hua; Zhang, Su-xing; Long, Na; Lin, Li-shan; Tao CHEN; Zhang, Fei-peng; Lv, Xue-Qin; Ye, Pei-zhen; Li, Ning; Zhang, Ke-Zhi

    2016-01-01

    Aim: The substrate cocktail is frequently used to evaluate cytochrome P450 (CYP) enzyme-mediated drug interactions and potential interactions among the probe substrates. Here, we re-optimized the substrate cocktail method to increase the reliability and accuracy of screening for candidate compounds and expanded the method from a direct CYP inhibition assay to a time-dependent inhibition (TDI) assay. Methods: In the reaction mixtures containing human liver microsome (0.1 mg/mL), both the conce...

  13. Evaluation of the Effects of Mitragyna speciosa Alkaloid Extract on Cytochrome P450 Enzymes Using a High Throughput Assay

    OpenAIRE

    Raja Elina Raja Aziddin; Zahurin Mohamed; Umarani Subramaniam; Murali Ramachandra; Zamri Chik; Wai Mun Kong

    2011-01-01

    The extract from Mitragyna speciosa has been widely used as an opium substitute, mainly due to its morphine-like pharmacological effects. This study investigated the effects of M. speciosa alkaloid extract (MSE) on human recombinant cytochrome P450 (CYP) enzyme activities using a modified Crespi method. As compared with the liquid chromatography-mass spectrometry method, this method has shown to be a fast and cost-effective way to perform CYP inhibition studies. The results indicated that MSE...

  14. Molecular evolution and population genetics of two Drosophila mettleri cytochrome P450 genes involved in host plant utilization

    OpenAIRE

    Jeremy M Bono; Matzkin, Luciano M.; Castrezana, Sergio; Therese A Markow

    2008-01-01

    Understanding the genetic basis of adaptation is one of the primary goals of evolutionary biology. The evolution of xenobiotic resistance in insects has proven to be an especially suitable arena for studying the genetics of adaptation, and resistant phenotypes are known to result from both coding and regulatory changes. In this study, we examine the evolutionary history and population genetics of two Drosophila mettleri cytochrome P450 genes that are putatively involved in the detoxification ...

  15. Insights into electron leakage in the reaction cycle of cytochrome P450 BM3 revealed by kinetic modeling and mutagenesis.

    Science.gov (United States)

    Lim, Joseph B; Barker, Kimberly A; Eller, Kristen A; Jiang, Linda; Molina, Veronica; Saifee, Jessica F; Sikes, Hadley D

    2015-11-01

    As a single polypeptide, cytochrome P450 BM3 fuses oxidase and reductase domains and couples each domain's function to perform catalysis with exceptional activity upon binding of substrate for hydroxylation. Mutations introduced into the enzyme to change its substrate specificity often decrease coupling efficiency between the two domains, resulting in unproductive consumption of cofactors and formation of water and/or reactive species. This phenomenon can correlate with leakage, in which P450 BM3 uses electrons from NADPH to reduce oxygen to water and/or reactive species even without bound substrate. The physical basis for leakage is not yet well understood in this particular member of the cytochrome P450 family. To clarify the relationship between leakage and coupling, we used simulations to illustrate how different combinations of kinetic parameters related to substrate-free consumption of NADPH and substrate hydroxylation can lead to either minimal effects on coupling or a dramatic decrease in coupling as a result of leakage. We explored leakage in P450 BM3 by introducing leakage-enhancing mutations and combining these mutations to assess whether doing so increases leakage further. The variants in this study provide evidence that while a transition to high spin may be vital for coupled hydroxylation, it is not required for enhanced leakage; substrate binding and the consequent shift in spin state are not necessary as a redox switch for catalytic oxidation of NADPH. Additionally, the variants in this study suggest a tradeoff between leakage and stability and thus evolvability, as the mutations we investigated were far more deleterious than other mutations that have been used to change substrate specificity. PMID:26311413

  16. In Vitro Inhibitory Activities of the Extract of Hibiscus Sabdariffa L. (Family Malvaceae) on Selected Cytochrome P450 Isoforms

    OpenAIRE

    Johnson, Showande Segun; Oyelola, Fakeye Titilayo; Ari, Tolonen; Juho, Hokkanen

    2013-01-01

    Literature is scanty on the interaction potential of Hibiscus sabdariffa L., plant extract with other drugs and the affected targets. This study was conducted to investigate the cytochrome P450 (CYP) isoforms that are inhibited by the extract of Hibiscus sabdariffa L. in vitro. The inhibition towards the major drug metabolizing CYP isoforms by the plant extract were estimated in human liver microsomal incubations, by monitoring the CYP-specific model reactions through previously validated N-i...

  17. Transcriptional regulation of the grape cytochrome P450 monooxygenase gene CYP736B expression in response to Xylella fastidiosa infection

    Directory of Open Access Journals (Sweden)

    Walker M Andrew

    2010-07-01

    Full Text Available Abstract Background Plant cytochrome P450 monooxygenases (CYP mediate synthesis and metabolism of many physiologically important primary and secondary compounds that are related to plant defense against a range of pathogenic microbes and insects. To determine if cytochrome P450 monooxygenases are involved in defense response to Xylella fastidiosa (Xf infection, we investigated expression and regulatory mechanisms of the cytochrome P450 monooxygenase CYP736B gene in both disease resistant and susceptible grapevines. Results Cloning of genomic DNA and cDNA revealed that the CYP736B gene was composed of two exons and one intron with GT as a donor site and AG as an acceptor site. CYP736B transcript was up-regulated in PD-resistant plants and down-regulated in PD-susceptible plants 6 weeks after Xf inoculation. However, CYP736B expression was very low in stem tissues at all evaluated time points. 5'RACE and 3'RACE sequence analyses revealed that there were three candidate transcription start sites (TSS in the upstream region and three candidate polyadenylation (PolyA sites in the downstream region of CYP736B. Usage frequencies of each transcription initiation site and each polyadenylation site varied depending on plant genotype, developmental stage, tissue, and treatment. These results demonstrate that expression of CYP736B is regulated developmentally and in response to Xf infection at both transcriptional and post-transcriptional levels. Multiple transcription start and polyadenylation sites contribute to regulation of CYP736B expression. Conclusions This report provides evidence that the cytochrome P450 monooxygenase CYP736B gene is involved in defense response at a specific stage of Xf infection in grapevines; multiple transcription initiation and polyadenylation sites exist for CYP736B in grapevine; and coordinative and selective use of transcription initiation and polyadenylation sites play an important role in regulation of CYP736B expression

  18. Cytochrome P450 or high pH induce structural and functional changes of Coenzyme Q10

    OpenAIRE

    Gulaboski, Rubin; Bogeski, Ivan; Mirceski, Valentin; Reinhard, Kappl; Hoth, Markus

    2010-01-01

    Coenzyme Q10 is a lipid soluble compound indispensable for optimal functioning of all living organisms, and it is the only non-protein in the mitochondrial electron transfer chain. Using voltammetry, UV-VIS spectrophotometry, EPR and NMR we have shown that Cytochrome P450 and high pH's induce structural and functional changes in Coenzyme Q10. The new derivative has much greater antioxidative potential and ability to bind Ca2+ and other earth-alkaline cations.

  19. Discovery of a P450-catalyzed step in vindoline biosynthesis: a link between the aspidosperma and eburnamine alkaloids.

    Science.gov (United States)

    Kellner, Franziska; Geu-Flores, Fernando; Sherden, Nathaniel H; Brown, Stephanie; Foureau, Emilien; Courdavault, Vincent; O'Connor, Sarah E

    2015-05-01

    Here we report the discovery of a cytochrome P450 that is required for the biosynthesis of vindoline, a plant-derived natural product used for semi-synthesis of several anti-cancer drugs. This enzyme catalyzes the formation of an epoxide that can undergo rearrangement to yield the vincamine-eburnamine backbone, thereby providing evidence for the long-standing hypothesis that the aspidosperma- and eburnamine-type alkaloids are biosynthetically related. PMID:25850027

  20. Combinatorial Alanine Substitution Enables Rapid Optimization of Cytochrome P450BM3 for Selective Hydroxylation of Large Substrates

    KAUST Repository

    Lewis, Jared C.

    2010-11-24

    Made for each other: Combinatorial alanine substitution of active site residues in a thermostable cytochrome P450BM3 variant was used to generate an enzyme that is active with large substrates. Selective hydroxylation of methoxymethylated monosaccharides, alkaloids, and steroids was thus made possible (see Scheme). This approach could be useful for improving the activity of enzymes that show only limited activity with larger substrates. © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Effects of prior oral contraceptive use and soy isoflavonoids on estrogen-metabolizing cytochrome P450 enzymes

    OpenAIRE

    Scott, LM; Durant, P; Leone-Kabler, S; Wood, CE; Register, TC; Townsend, A.; Cline, JM

    2008-01-01

    Estrogen exposure and metabolism may play an important role in the development of estrogen-sensitive cancers in postmenopausal women. In this study we investigated whether past oral contraceptive (OC) administration or current dietary isoflavonoids (IF) affected expression and/or activity of steroid hormone-metabolizing cytochrome P450 (CYP) enzymes using complementary primate and cell culture models. One-hundred-eighty-one female cynomolgus macaques were randomized to receive OC or nothing f...

  2. Using resonance Raman cross-section data to estimate the spin state populations of Cytochromes P450.

    Science.gov (United States)

    Mak, Piotr J; Zhu, Qianhong; Kincaid, James R

    2013-12-01

    The cytochromes P450 (CYPs) are heme proteins responsible for the oxidation of xenobiotics and pharmaceuticals and the biosynthesis of essential steroid products. In all cases, substrate binding initiates the enzymatic cycle, converting ferric low spin (LS) to high-spin (HS), with the efficiency of the conversion varying widely for different substrates, so documentation of this conversion for a given substrate is an important objective. Resonance Raman (rR) spectroscopy can effectively yield distinctive frequencies for the ν3 "spin state marker" bands. Here, employing a reference cytochrome P450 (CYP101), the intensities of the ν3 modes (ILS) and (IHS) relative to an internal standard (sodium sulfate) yield relative populations for the two spin states; i.e., a value of 1.24 was determined for the ratio of the relative cross sections for the ν3 modes. Use of this value was then shown to permit a reliable calculation of relative populations of the two spin states from rR spectra of several other Cytochromes P450. The importance of this work is that, using this information, it is now possible to conveniently document by rR the spin state population without conducting separate experiments requiring different analytical methods, instrumentation and additional sample. PMID:24443630

  3. Expression of P450arom and Estrogen Receptor Alpha in the Oviduct of Chinese Brown Frog (Rana dybowskii during Prehibernation

    Directory of Open Access Journals (Sweden)

    Ji Weng

    2015-01-01

    Full Text Available One specific physiological phenomenon of Chinese brown frog (Rana dybowskii is that its oviduct expands prior to hibernation instead of expanding during the breeding period. In this study, we investigated the expression of P450arom and estrogen receptors α and β (ERα and ERβ in the oviduct of Rana dybowskii during the breeding period and prehibernation. The results of the present study showed that there were significant differences in both oviductal weight and size with values markedly higher in prehibernation than in the breeding period. P450arom was observed in stromal tissue in both the breeding period and prehibernation. ERα was expressed in stromal tissue and epithelial cells in both periods, whereas ERβ could not be detected. The mean protein and mRNA levels of P450arom and ERα were significantly higher in prehibernation as compared to the breeding period. Besides, oviductal content of 17β-estradiol was also higher in prehibernation than in the breeding period. These results suggested that estrogen may play autocrine/paracrine roles mediated by ERα in regulating the oviductal hypertrophy during prehibernation.

  4. An artificial self-sufficient cytochrome P450 directly nitrates fluorinated tryptophan analogs with a different regio-selectivity.

    Science.gov (United States)

    Zuo, Ran; Zhang, Yi; Huguet-Tapia, Jose C; Mehta, Mishal; Dedic, Evelina; Bruner, Steven D; Loria, Rosemary; Ding, Yousong

    2016-05-01

    Aromatic nitration is an immensely important industrial process to produce chemicals for a variety of applications, but it often suffers from multiple unsolved challenges. Enzymes as biocatalysts have been increasingly used for organic chemistry synthesis due to their high selectivity and environmental friendliness, but nitration has benefited minimally from the development of biocatalysis. In this work, we aimed to develop TxtE as practical biocatalysts for aromatic nitration. TxtE is a unique class I cytochrome P450 enzyme that nitrates the indole of l-tryptophan. To develop cost-efficient nitration processes, we fused TxtE with the reductase domains of CYP102A1 (P450BM3) and of P450RhF to create class III self-sufficient biocatalysts. The best engineered fusion protein was comparable with wild type TxtE in terms of nitration performance and other key biochemical properties. To demonstrate the application potential of the fusion enzyme, we nitrated 4-F-dl-tryptophan and 5-F-l-tryptophan in large scale enzymatic reactions. Tandem MS/MS and NMR analyses of isolated products revealed altered nitration sites. To our knowledge, these studies represent the first practice in developing biological nitration approaches and lay a solid basis to the use of TxtE-based biocatalysts for the production of valuable nitroaromatics. PMID:26743860

  5. Phytoremediation of the organic Xenobiotic simazine by p450-1a2 transgenic Arabidopsis thaliana plants.

    Science.gov (United States)

    Azab, Ehab; Hegazy, Ahmad K; El-Sharnouby, Mohamed E; Abd Elsalam, Hassan E

    2016-07-01

    The potential use of human P450-transgenic plants for phytoremediation of pesticide contaminated soils was tested in laboratory and greenhouse experiments. The transgenic P450 CYP1A2 gene Arabidopsis thaliana plants metabolize number of herbicides, insecticides and industrial chemicals. The P450 isozymes CYP1A2 expressed in A. thaliana were examined regarding the herbicide simazine (SIM). Transgenic A. thaliana plants expressing CYP1A2 gene showed significant resistance to SIM supplemented either in plant growth medium or sprayed on foliar parts. The results showed that SIM produces harmful effect on both rosette diameter and primary root length of the wild type (WT) plants. In transgenic A. thaliana lines, the rosette diameter and primary root length were not affected by SIM concentrations used in this experiment. The results indicate that CYP1A2 can be used as a selectable marker for plant transformation, allowing efficient selection of transgenic lines in growth medium and/or in soil-grown plants. The transgenic A. thaliana plants exhibited a healthy growth using doses of up to 250 μmol SIM treatments, while the non-transgenic A. thaliana plants were severely damaged with doses above 50 μmol SIM treatments. The transgenic A. thaliana plants can be used as phytoremediator of environmental SIM contaminants. PMID:26771455

  6. Influence of some anti-inflammatory drugs on the activity of aryl hydrocarbon hydroxylase and the cytochrome P450 content

    Energy Technology Data Exchange (ETDEWEB)

    Mostafa, M.H.; Sheweita, S.A.; Abdel-Moneam, N.M. (Alexandria Univ. (Egypt))

    1990-06-01

    The metabolism of benzo({alpha})pyrene is mediated by the mixed function oxidase system including the cytochrome P450-dependent aryl hydrocarbon hydroxylase. The data of the present study revealed the ability of various commonly used anti-inflammatory drugs to alter the activity of this enzyme system, where all the tested drugs, namely phenyl butazone, ketoprofen, piroxicam, and acetaminophen, caused an increase in both the activity of aryl hydrocarbon hydroxylase and the cytochrome P450 content whether administered as a single dose or as a repeated dose for 6 consecutive days. The percentage of change for all drugs except phenyl butazone was proportional to the duration of drug administration. On the other hand, pyrazole which is chemically related to phenyl butazone, had no significant effect when administered as a single dose but caused a decrease in both studied parameters when administered as a repeated dose for 6 consecutive days. The mechanisms by which these commonly used drugs modify the aryl hydrocarbon hydroxylase activity and the cytochrome p450 content are discussed in the text.

  7. The hepatoprotective cytochrome P-450 enzyme inhibitor isolated from the Nigerian medicinal plant Cochlospermum planchonii is a zinc salt.

    Science.gov (United States)

    Aliyu, R; Okoye, Z S; Shier, W T

    1995-10-01

    Aqueous extracts of Cochlospermum planchonii Hook.f. (Cochlospermaceae) rhizomes are used by native medical practitioners in northern Nigeria to treat jaundice. An extract prepared by a laboratory adaptation of their method was hepatoprotective in carbon tetrachloride-treated rats (CCl4), and it inhibited cytochrome P-450 enzymes, which constitutes a plausible hepatoprotective mechanism. A crystalline inhibitor (0.3% of dry weight of rhizomes) was isolated using inhibition of two rat cytochrome P-450 enzymes, aminopyrine-N-demethylase and aniline hydroxylase, as bioassays to guide fractionation by solvent partitioning, polyamdie column chromatography, preparative thin layer chromatography and fractional crystallization. The inhibitor was identified as zinc formate by inductively coupled plasma atomic emission spectroscopy, nuclear magnetic resonance spectroscopy and comparison with synthetic material by power X-ray diffraction crystallography. Synthetic and plant-derived zinc formate were equally effective as inhibitors of cytochrome P-450 enzymes and as hepatoprotective agents in carbon tetrachloride-treated rats. Cochlospermum planchonii rhizomes contain unusually high levels of manganese and zinc, although much higher levels have been observed in plants considered to be hyperaccumulators of these metals. PMID:8583799

  8. Genetic polymorphisms in the Cytochrome P450 family and squamous cell carcinoma of the oral cavity, pharynx and larynx

    Directory of Open Access Journals (Sweden)

    Celso Muller Bandeira

    2014-10-01

    Full Text Available Objective: To analyze the genetic polymorphisms of the cytochrome P450 family and their relationship with squamous cell carcinoma of the oral cavity, pharynx and larynx. Methods: We present a narrative literature review, conducted in Pubmed, Lilacs and Cochrane Databases of articles published in the last five years correlating genetic polymorphisms of the cytochrome P450 family and cancer risk in different populations worldwide. Results: We initially found 65 articles and, after selection criteria, 20 case-control studies with various populations worldwide were eligible. The most studied polymorphisms were those of CYP2E1 and CYP1A1 subfamilies. There is little about the other subfamilies. The association found between polymorphisms and cancer risk amounted to a countless number of variables, amongst them: population, selection methods, racial factors and different modes of exposure to carcinogens, genotyping methods, and nomenclature of the polymorphisms. Conclusion: so far, there is no proven link between genetic polymorphisms of cytochrome P450 family and squamous cell carcinoma of the oral cavity, pharynx and larynx relationship.

  9. Cloning, purification, crystallization and preliminary X-ray analysis of a chimeric NADPH-cytochrome P450 reductase

    International Nuclear Information System (INIS)

    A 2.5 Å resolution data set was collected from a crystal of a soluble chimeric form of NADPH-cytochrome P450 reductase (CPR) produced using a fusion gene composed of the yeast FMN and the human FAD domains. The chimeric protein was crystallized in a modified conformation compared with the previously solved structures. NADPH-cytochrome P450 reductase (CPR) is the favoured redox partner of microsomal cytochromes P450. This protein is composed of two flavin-containing domains (FMN and FAD) connected by a structured linker. An active CPR chimera consisting of the yeast FMN and human FAD domains has been produced, purified and crystallized. The crystals belonged to the monoclinic space group C2 and contained one molecule per asymmetric unit. Molecular replacement was performed using the published rat and yeast structures as search models. The initial electron-density maps revealed that the chimeric enzyme had crystallized in a conformation that differed from those of previously solved structures

  10. Equine cytochrome P450 2B6 — Genomic identification, expression and functional characterization with ketamine

    Energy Technology Data Exchange (ETDEWEB)

    Peters, L.M.; Demmel, S. [Division Veterinary Pharmacology and Toxicology, Vetsuisse Faculty, University Bern, Laenggassstr. 124, 3012 Bern (Switzerland); Pusch, G.; Buters, J.T.M. [ZAUM — Center of Allergy and Environment, Helmholtz Zentrum München/Technische Universität München, Biedersteiner Str. 29, 80802 München (Germany); Thormann, W. [Clinical Pharmacology Laboratory, Institute for Infectious Diseases, University of Bern, Murtenstrasse 35, 3010 Bern (Switzerland); Zielinski, J. [Division Veterinary Pharmacology and Toxicology, Vetsuisse Faculty, University Bern, Laenggassstr. 124, 3012 Bern (Switzerland); Leeb, T. [Institute of Genetics, Vetsuisse Faculty, University Bern, Bremgartenstr. 109, 3012 Bern (Switzerland); Mevissen, M. [Division Veterinary Pharmacology and Toxicology, Vetsuisse Faculty, University Bern, Laenggassstr. 124, 3012 Bern (Switzerland); Schmitz, A., E-mail: andrea.schmitz@vetsuisse.unibe.ch [Division Veterinary Pharmacology and Toxicology, Vetsuisse Faculty, University Bern, Laenggassstr. 124, 3012 Bern (Switzerland)

    2013-01-01

    Ketamine is an anesthetic and analgesic regularly used in veterinary patients. As ketamine is almost always administered in combination with other drugs, interactions between ketamine and other drugs bear the risk of either adverse effects or diminished efficacy. Since cytochrome P450 enzymes (CYPs) play a pivotal role in the phase I metabolism of the majority of all marketed drugs, drug–drug interactions often occur at the active site of these enzymes. CYPs have been thoroughly examined in humans and laboratory animals, but little is known about equine CYPs. The characterization of equine CYPs is essential for a better understanding of drug metabolism in horses. We report annotation, cloning and heterologous expression of the equine CYP2B6 in V79 Chinese hamster fibroblasts. After computational annotation of all CYP2B genes, the coding sequence (CDS) of equine CYP2B6 was amplified by RT-PCR from horse liver total RNA and revealed an amino acid sequence identity of 77% and a similarity of 93.7% to its human ortholog. A non-synonymous variant c.226G>A in exon 2 of the equine CYP2B6 was detected in 97 horses. The mutant A-allele showed an allele frequency of 82%. Two further variants in exon 3 were detected in one and two horses of this group, respectively. Transfected V79 cells were incubated with racemic ketamine and norketamine as probe substrates to determine metabolic activity. The recombinant equine CYP2B6 N-demethylated ketamine to norketamine and produced metabolites of norketamine, such as hydroxylated norketamines and 5,6-dehydronorketamine. V{sub max} for S-/and R-norketamine formation was 0.49 and 0.45 nmol/h/mg cellular protein and K{sub m} was 3.41 and 2.66 μM, respectively. The N-demethylation of S-/R-ketamine was inhibited concentration-dependently with clopidogrel showing an IC{sub 50} of 5.63 and 6.26 μM, respectively. The functional importance of the recorded genetic variants remains to be explored. Equine CYP2B6 was determined to be a CYP

  11. Equine cytochrome P450 2B6 — Genomic identification, expression and functional characterization with ketamine

    International Nuclear Information System (INIS)

    Ketamine is an anesthetic and analgesic regularly used in veterinary patients. As ketamine is almost always administered in combination with other drugs, interactions between ketamine and other drugs bear the risk of either adverse effects or diminished efficacy. Since cytochrome P450 enzymes (CYPs) play a pivotal role in the phase I metabolism of the majority of all marketed drugs, drug–drug interactions often occur at the active site of these enzymes. CYPs have been thoroughly examined in humans and laboratory animals, but little is known about equine CYPs. The characterization of equine CYPs is essential for a better understanding of drug metabolism in horses. We report annotation, cloning and heterologous expression of the equine CYP2B6 in V79 Chinese hamster fibroblasts. After computational annotation of all CYP2B genes, the coding sequence (CDS) of equine CYP2B6 was amplified by RT-PCR from horse liver total RNA and revealed an amino acid sequence identity of 77% and a similarity of 93.7% to its human ortholog. A non-synonymous variant c.226G>A in exon 2 of the equine CYP2B6 was detected in 97 horses. The mutant A-allele showed an allele frequency of 82%. Two further variants in exon 3 were detected in one and two horses of this group, respectively. Transfected V79 cells were incubated with racemic ketamine and norketamine as probe substrates to determine metabolic activity. The recombinant equine CYP2B6 N-demethylated ketamine to norketamine and produced metabolites of norketamine, such as hydroxylated norketamines and 5,6-dehydronorketamine. Vmax for S-/and R-norketamine formation was 0.49 and 0.45 nmol/h/mg cellular protein and Km was 3.41 and 2.66 μM, respectively. The N-demethylation of S-/R-ketamine was inhibited concentration-dependently with clopidogrel showing an IC50 of 5.63 and 6.26 μM, respectively. The functional importance of the recorded genetic variants remains to be explored. Equine CYP2B6 was determined to be a CYP enzyme involved in

  12. Alkane-induced expression, substrate binding profile, and immunolocalization of a cytochrome P450 encoded on the nifD excision element of Anabaena 7120

    Directory of Open Access Journals (Sweden)

    Fjetland Conrad R

    2005-03-01

    Full Text Available Abstract Background Alkanes have been hypothesized to act as universal inducers of bacterial cytochrome P450 gene expression. We tested this hypothesis on an unusual P450 gene (cyp110 found on a conserved 11 kilobase episomal DNA element of unknown function found in filamentous cyanobacteria. We also monitored the binding of potential substrates to the P450 protein and explored the distribution of P450 protein in vegetative cells and nitrogen-fixing heterocysts using immuno-electron microscopy. Results Hexadecane treatments resulted in a two-fold increase in mRNA, and a four-fold increase in P450 protein levels relative to control cultures. Hexane, octane and dodecane were toxic and induced substantial changes in membrane morphology. Long-chain saturated and unsaturated fatty acids were shown to bind the CYP110 protein using a spectroscopic spin-shift assay, but alkanes did not bind. CYP110 protein was detected in vegetative cells but not in differentiated heterocysts where nitrogen fixation occurs. Conclusion Hexadecane treatment was an effective inducer of CYP110 expression in cyanobacteria. Based on substrate binding profiles and amino acid sequence similarities it is hypothesized that CYP110 is a fatty acid ω-hydroxylase in photosynthetic cells. CYP110 was found associated with membrane fractions unlike other soluble microbial P450 proteins, and in this regard CYP110 more closely resembles eukarytotic P450s. Substrate stablization is an unlikely mechanism for alkane induction because alkanes did not bind to purified CYP110 protein.

  13. Individual Differences in Metabolic Clearance of S-Warfarin Efficiently Mediated by Polymorphic Marmoset Cytochrome P450 2C19 in Livers.

    Science.gov (United States)

    Uehara, Shotaro; Uno, Yasuhiro; Inoue, Takashi; Kawano, Mirai; Shimizu, Makiko; Toda, Akiko; Utoh, Masahiro; Sasaki, Erika; Yamazaki, Hiroshi

    2016-07-01

    Marmoset cytochrome P450 2C19, highly homologous to human P450 2C9 and 2C19, has been identified in common marmosets (Callithrix jacchus), a nonhuman primate species used in drug metabolism studies. Although genetic variants in human and macaque P450 2C genes account for the interindividual variability in drug metabolism, genetic variants have not been investigated in the marmoset P450 2C19 In this study, sequencing of P450 2C19 in 24 marmosets identified three variants p.[(Phe7Leu; Ser254Leu; Ile469Thr)], which showed substantially reduced metabolic capacity of S-warfarin compared with the wild-type group in vivo and in vitro. Although mean plasma concentrations of R-warfarin in marmosets determined after chiral separation were similar between the homozygous mutant and wild-type groups up to 24 hours after the intravenous and oral administrations of racemic warfarin, S-warfarin depletion from plasma was significantly faster in the three wild-type marmosets compared with the three homozygous mutant marmosets. These variants, cosegregating in the marmosets analyzed, influenced metabolic activities in 18 marmoset liver microsomes because the homozygotes and heterozygotes showed significantly reduced catalytic activities in liver microsomes toward S-warfarin 7-hydroxylation compared with the wild-type group. Kinetic analysis for S-warfarin 7-hydroxylation indicated that the recombinant P450 2C19 Ser254Leu variant would change the metabolic capacity. These results indicated that the interindividual variability of P450 2C-dependent drug metabolism such as S-warfarin clearance is at least partly accounted for by P450 2C19 variants in marmosets, suggesting that polymorphic P450 2C-dependent catalytic functions are relatively similar between marmosets and humans. PMID:27098744

  14. Phenobarbital induction of cytochromes P-450. High-level long-term responsiveness of primary rat hepatocyte cultures to drug induction, and glucocorticoid dependence of the phenobarbital response.

    Science.gov (United States)

    Waxman, D J; Morrissey, J J; Naik, S; Jauregui, H O

    1990-01-01

    The induction of hepatic cytochromes P-450 by phenobarbital (PB) was studied in rat hepatocytes cultured for up to 5 weeks on Vitrogen-coated plates in serum-free modified Chee's medium then exposed to PB (0.75 mM) for an additional 4 days. Immunoblotting analysis indicated that P-450 forms PB4 (IIB1) and PB5 (IIB2) were induced dramatically (greater than 50-fold increase), up to levels nearly as high as those achieved in PB-induced rat liver in vivo. The newly synthesized cytochrome P-450 was enzymically active, as shown by the major induction of the P-450 PB4-dependent steroid 16 beta-hydroxylase and pentoxyresorufin O-dealkylase activities in the PB-induced hepatocyte microsomes (up to 90-fold increase). PB induction of these P-450s was markedly enhanced by the presence of dexamethasone (50 nM-1 microM), which alone was not an affective inducing agent, and was inhibited by greater than 90% by 10% fetal bovine serum. The PB response was also inhibited (greater than 85%) by growth hormone (250 ng/ml), indicating that this hormone probably acts directly on the hepatocyte when it antagonizes the induction of P-450 PB4 in intact rats. In untreated hepatocytes, P-450 RLM2 (IIA2), P-450 3 (IIA1) and NADPH P-450 reductase levels were substantially maintained in the cultures for 10-20 days. The latter two enzymes were also inducible by PB to an extent (3-4 fold elevation) that is comparable with that observed in the liver in vivo. Moreover, P-450c (IA1) and P-450 3 (IIA1) were highly inducible by 3-methylcholanthrene (5 microM; 48 h exposure) even after 3 weeks in culture. In contrast, the male-specific pituitary-regulated P-450 form 2c (IIC11) was rapidly lost upon culturing the hepatocytes, suggesting that supplementation of appropriate hormonal factors may be necessary for its expression. The present hepatocyte culture system exhibits a responsiveness to drug inducers that is qualitatively and quantitatively comparable with that observed in vivo, and should prove

  15. How Does the Reductase Help To Regulate the Catalytic Cycle of Cytochrome P450 3A4 Using the Conserved Water Channel?

    OpenAIRE

    Fishelovitch, Dan; Shaik, Sason; Wolfson, Haim J.; Nussinov, Ruth

    2010-01-01

    Water molecules play a major role in the P450 catalytic cycle. Here, we locate the preferred water pathways and their gating mechanisms for the human cytochrome P450 3A4 (CYP3A4) and elucidate the role of the cytochrome P450 reductase (CPR) in turning on and activating these water channels. We perform explicit solvent molecular dynamic simulations of CYP3A4, unbound and bound to two substrates, and with and without the flavin mononucleotide (FMN)-binding domain of CPR. We observe in/out passa...

  16. 细胞色素P450 BM-3羟基化吲哚能力的半理性改造%Semi-rational directed evolution in improving indole-hydroxylation ability of cytochrome P450 BM-3

    Institute of Scientific and Technical Information of China (English)

    胡升; 虞青; 梅乐和; 姚善泾; 金志华

    2009-01-01

    Guided by postulations in the relationship between structure and functionality, a saturation mutagenesis was performed on the D168 position of a P450 BM-3 (A74G/F87V/L188Q/E435T) mutant which originally transformed indole largely into indigo. Two novel mutants have been obtained based on the differences in color and absorption spectrum of the indole-hydroxylation products followed by product composition analysis using HPLC. One mutant, P450 BM-3 (A74G/F87V/L188Q/E435T/D168W) catalyzes indole into indirubin in 90% with a decrease of k_(cat)/K_m in 80%, mainly owing to its K_m is increased by 4.8 times as compared with the parent. The other one, P450 BM-3 (A74G/F87V/L188Q/ E435T/D168R) transforms indole into indigo in about 87%, which is higher than the parent and its k_(cat)/ K_m has an increase in over 1. 37 times as that of the parent. Such results indicate that the amino acid residue substitution on the D168 position combined with the mutation of E435T can influence the hydroxylation regioselectivity of P450 BM-3 as well as catalytic activity, which determines indole-hydroxylation product as indigo or indirubin. Such influence could not be caused by the mutation on a single amino acid residue site, neither E435 nor D168, because of the lack in some synergistic effect. In conclusion, the directed evolution guided by rational hypothesis for engineering the catalytic properties of P450 BM-3 has succeeded in getting two mutants exhibiting novel characteristics for indole-hydroxylation.%为进一步改造细胞色素P450 BM-3酶对吲哚的羟基化能力,以P450 BM-3结构与功能关系的推测为指导,选择突变酶P450 BM-3 (A74G/F87V/L188Q/E435T)为父本,在可能影响P450 BM-3催化吲哚羟基化区域选择性的D168位点进行定点饱和突变,根据全细胞催化产物颜色及组成进行筛选,得到了产物组成、酶动力学性质与父本不同的两个突变酶.突变酶D168W的吲哚羟基化产物中90%是靛

  17. Characterization and evolution of furanocoumarin-inducible cytochrome P450s in the parsnip webworm, Depressaria pastinacella.

    Science.gov (United States)

    Li, W; Zangerl, A R; Schuler, M A; Berenbaum, M R

    2004-12-01

    Depressaria pastinacella, the parsnip webworm, a specialist on two genera in the Apiaceae, routinely consumes plant tissues high in furanocoumarin content and is capable of rapid cytochrome P450-mediated detoxification of these compounds. In this study, four cDNAs were cloned from the larval midgut of this insect: two full-length CYP6AB3 and CYP6AE1 cDNAs are closely related to members of the furanocoumarin-metabolizing CYP6B subfamily and two partial CYP9A6 and CYP9A7 cDNAs are related to members of the CYP9A subfamily that have also been linked to the detoxification of xenobiotics. At least one of these P450s (CYP6AB3) is inducible by dietary furanocoumarins, indicating its potential involvement in furanocoumarin metabolism. A homology model of CYP6AB3 was constructed and compared to models of CYP6B1 from the specialist species, Papilio polyxenes, and CYP6B4 from the generalist species, P. glaucus. Structural superpositioning of these models has revealed very high spatial similarity of elements, including the B helix, B'-C loop, I helix and C-terminal domain, within the catalytic sites of these proteins. Most importantly, key amino acid residues that can potentially come into contact with furanocoumarin substrates display conservation in their spatial positioning and side chain polarities. Three of these residues, Val103, Leu113 and Phe118 (numbered according to CYP6AB3), are conserved in all three of these proteins, further implicating CYP6AB3 in furanocoumarin metabolism by parsnip webworms. Characterization of these P450 cDNAs will allow for functional analyses aimed at elucidating the molecular mechanisms underlying the coevolutionary interactions between this herbivore and its principal host plant. PMID:15606809

  18. Cytochrome P-450-catalyzed desaturation of valproic acid in vitro. Species differences, induction effects, and mechanistic studies

    International Nuclear Information System (INIS)

    The cytochrome P-450-mediated desaturation of valproic acid (VPA) to its hepatotoxic metabolite, 2-n-propyl-4-pentenoic acid (4-ene-VPA), was examined in liver microsomes from rats, mice, rabbits and humans. The highest substrate turnover was found with microsomes from rabbits (44.2 +/- 2.7 pmol of product/nmol P-450/15 min), while lower activities were observed in preparations from human, mouse, and rat liver, in that order. Pretreatment of animals with phenobarbital led to enhanced rates of formation of 4-ene-VPA in vitro and yielded induction ratios for desaturation ranging from 2.5 to 8.4, depending upon the species. Comparative studies in the rat showed that phenobarbital is a more potent inducer of olefin formation than either phenytoin or carbamazepine. The mechanism of the desaturation reaction was studied by inter- and intramolecular deuterium isotope effect experiments, which demonstrated that removal of a hydrogen atom from the subterminal C-4 position of VPA is rate limiting in the formation of both 4-ene- and 4-hydroxy-VPA. Hydroxylation at the neighboring C-5 position, on the other hand, was highly sensitive to deuterium substitution at that site, but not to deuteration at C-4. Based on these findings, it is proposed that 4-ene- and 4-hydroxy-VPA are products of a common P-450-dependent metabolic pathway, in which a carbon-centered free radical at C-4 serves as the key intermediate. 5-Hydroxy-VPA, in contrast, derives from an independent hydroxylation reaction

  19. Suicidal gene therapy with rabbit cytochrome P450 4B1/4-ipomeanol, 2-aminoanthracene system in glioma cell

    Energy Technology Data Exchange (ETDEWEB)

    Jang, Su Jin; Kang, Joo Hyun; Kim, Kwang Il; Lee, Tae Sup; Lee, Yong Jin; Woo, Kwang Sun; Chung, Wee Sup; Cheon, Gi Jeong; Choi, Chang Woon; Lim, Sang Moo [Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of)

    2010-10-15

    Suicidal gene therapy is based on the transduction of tumor cells with 'suicide' genes encoding for prodrugactivating enzymes that render target cells susceptible to prodrug treatment. Suicidal gene therapy results in the death of tumor with the expression of gene encoding enzyme that converts non-toxic prodrug into cytotoxic product. Cytochrome P450 4B1 (CYP4B1) activates 4- ipomeanol (4-ipo) and 2-aminoanthracene (2-AA) to cytotoxic furane epoxide and unsaturated dialdehyde intermediate. In this study, therapeutic effects of suicidal gene therapy with rabbit CYP4B1/4-ipo or CYP4B1/2-AA system

  20. Hepatic cytochrome P450s play a major role in monocrotaline-induced renal toxicity in mice

    OpenAIRE

    Yao, Jun; Li, Cheng-Gang; Gong, Li-kun; FENG, CHEN-CHEN; Li, Chun-zhu; Gao, Man; Luan, Yang; Qi, Xin-ming; Ren, Jin

    2013-01-01

    Aim: Monocrotaline (MCT) in plants of the genus Crotalaria induces significant toxicity in multiple organs including the liver, lung and kidney. Metabolic activation of MCT is required for MCT-induced toxicity. In this study, we attempted to determine whether the toxicity of MCT in kidney was a consequence of the metabolic activation of MCT in the liver. Methods: Liver-specific cytochrome P450 reductase-null (Null) mice, wild-type (WT) mice and CYP3A inhibitor ketoconazole-pretreated WT (KET-...