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Sample records for p450 1a2 ligands

  1. [Modeling of a three-dimensional structure of cytochrome P-450 1A2 and search for its new ligands].

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    Belkina, N V; Skvortsov, V S; Ivanov, A S; Archakov, A I

    1998-01-01

    The substances inhibiting cytochrome P450 1A2 (CYP1A2) represent a perspective class of new drugs, which application in clinical practice can become the important part in preventive maintenance in oncology. The present work is devoted to computer modelling of 3-D structure of CYP1A2 and searching of new inhibitors by database mining. The modelling of CYP1A2 was done based on homology with 4 bacterial cytochromes P450 with known 3-D structure. For optimization of CYP1A2 active site structure the models of its complexes with characteristic substrates (caffeine and 7-ethoxyresorufin) were designed. These complexes were optimized by molecular dynamics simulation in water. The models of 24 complexes of CYP1A2 with known ligands with known Kd were designed by means of DockSearch and LeapFrog programs. 3D-QSAR model with good predictive force was created based on these complexes. On a final stage the search of knew CYP1A2 ligands in testing database (more than 23.000 substances from database Maybridge and 112 known CYP1A2 ligands from database Metabolite, MDL) was executed. 680 potential ligands of CYP1A2 with Kd values, comparable with known ones were obtained. This number has included 73 compounds from 112 known ligands, introduced in tested database as the internal control.

  2. Scutellarin inhibits cytochrome P450 isoenzyme 1A2 (CYP1A2) in rats.

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    Jian, Tun-Yu; He, Jian-Chang; He, Gong-Hao; Feng, En-Fu; Li, Hong-Liang; Bai, Min; Xu, Gui-Li

    2012-08-01

    Scutellarin is the most important flavone glycoside in the herbal drug Erigeron breviscapus (Vant.) Hand.-Mazz. It is used frequently in the clinic to treat ischemic vascular diseases in China. However, the direct relationship between scutellarin and cytochrome P450 (CYP450) is unclear. The present study investigated the in vitro and in vivo effects of scutellarin on cytochrome P450 1A2 (CYP 1A2) metabolism. According to in vitro experiments, scutellarin (10-250 µM) decreased the formation of 4-acetamidophenol in a concentration-dependent manner, with an IC₅₀ value of 108.20 ± 0.657 µM. Furthermore, scutellarin exhibited a weak mixed-type inhibition against the activity of CYP1A2 in rat liver microsomes, with a K(i) value of 95.2 µM. Whereas in whole animal studies, scutellarin treatment for 7 days (at 5, 15, 30 mg/kg, i.p.) decreased the clearance (CL), and increased the T(1/2) (at 15, 30 mg/kg, i.p.), it did not affect the V(d) of phenacetin. Scutellarin treatment (at 5, 15, 30 mg/kg, i.p.) increased the AUC(0-∞) by 14.3%, 67.3% and 159.2%, respectively. Scutellarin at 30 mg/kg also weakly inhibited CYP1A2 activity, in accordance with our in vitro study. Thus, the results indicate that CYP1A2 is inhibited directly, but weakly, by scutellarin in vivo, and provide useful information on the safe and effective use of scutellarin in clinical practice. Copyright © 2012 John Wiley & Sons, Ltd.

  3. Genetic polymorphisms of cytochrome P450-1A2 (CYP1A2 among Emiratis.

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    Mohammad M Al-Ahmad

    Full Text Available Cytochrome P450 1A2 (CYP1A2 is one of the CYP450 mixed-function oxidase system that is of clinical importance due to the large number of drug interactions associated with its induction and inhibition. In addition, significant inter-individual differences in the elimination of drugs metabolized by CYP1A2 enzyme have been observed which are largely due to the highly polymorphic nature of CYP1A2 gene. However, there are limited studies on CYP1A2 phenotypes and CYP1A2 genotypes among Emiratis and thus this study was carried out to fill this gap. Five hundred and seventy six non-smoker Emirati subjects were asked to consume a soft drink containing caffeine (a non-toxic and reliable probe for predicting CYP1A2 phenotype and then provide a buccal swab along with a spot urine sample. Taq-Man Real Time PCR was used to determine the CYP1A2 genotype of each individual. Phenotyping was carried out by analyzing the caffeine metabolites using High Performance Liquid Chromatography (HPLC analysis. We found that 1.4%, 16.3% and 82.3% of the Emirati subjects were slow, intermediate and rapid CYP1A2 metabolizers, respectively. In addition, we found that 1.4% of the subjects were homozygote for derived alleles while 16.1% were heterozygote and 82.5% were homozygote for the ancestral allele. The genotype frequency of the ancestral allele, CYP1A2*1A/*1A, is the highest in this population, followed by CYP1A2 *1A/*1C and CYP1A2 *1A/*1K genotypes, with frequencies of 0.825, 0.102 and 0.058, respectively. The degree of phenotype/genotype concordance was equal to 81.6%. The CYP1A2*1C/*1C and CYP1A2*3/*3 genotypes showed significantly the lowest enzyme phenotypic activity. The frequency of slow activity CYP1A2 enzyme alleles is very low among Emiratis which correlates with the presence of low frequencies of derived alleles in CYP1A2 gene.

  4. Ligand Access Channels in Cytochrome P450 Enzymes: A Review

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    Philippe Urban

    2018-05-01

    Full Text Available Quantitative structure-activity relationships may bring invaluable information on structural elements of both enzymes and substrates that, together, govern substrate specificity. Buried active sites in cytochrome P450 enzymes are connected to the solvent by a network of channels exiting at the distal surface of the protein. This review presents different in silico tools that were developed to uncover such channels in P450 crystal structures. It also lists some of the experimental evidence that actually suggest that these predicted channels might indeed play a critical role in modulating P450 functions. Amino acid residues at the entrance of the channels may participate to a first global ligand recognition of ligands by P450 enzymes before they reach the buried active site. Moreover, different P450 enzymes show different networks of predicted channels. The plasticity of P450 structures is also important to take into account when looking at how channels might play their role.

  5. Classification of cytochrome P450 1A2 inhibitors and noninhibitors by machine learning techniques

    NARCIS (Netherlands)

    Vasanthanathan, P.; Taboureau, O.; Oostenbrink, C.; Vermeulen, N.P.; Olsen, L.; Jorgensen, F.S.

    2009-01-01

    The cytochrome P450 (P450) superfamily plays an important role in the metabolism of drug compounds, and it is therefore highly desirable to have models that can predict whether a compound interacts with a specific isoform of the P450s. In this work, we provide in silico models for classification of

  6. Linear Interaction Energy Based Prediction of Cytochrome P450 1A2 Binding Affinities with Reliability Estimation.

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    Luigi Capoferri

    Full Text Available Prediction of human Cytochrome P450 (CYP binding affinities of small ligands, i.e., substrates and inhibitors, represents an important task for predicting drug-drug interactions. A quantitative assessment of the ligand binding affinity towards different CYPs can provide an estimate of inhibitory activity or an indication of isoforms prone to interact with the substrate of inhibitors. However, the accuracy of global quantitative models for CYP substrate binding or inhibition based on traditional molecular descriptors can be limited, because of the lack of information on the structure and flexibility of the catalytic site of CYPs. Here we describe the application of a method that combines protein-ligand docking, Molecular Dynamics (MD simulations and Linear Interaction Energy (LIE theory, to allow for quantitative CYP affinity prediction. Using this combined approach, a LIE model for human CYP 1A2 was developed and evaluated, based on a structurally diverse dataset for which the estimated experimental uncertainty was 3.3 kJ mol-1. For the computed CYP 1A2 binding affinities, the model showed a root mean square error (RMSE of 4.1 kJ mol-1 and a standard error in prediction (SDEP in cross-validation of 4.3 kJ mol-1. A novel approach that includes information on both structural ligand description and protein-ligand interaction was developed for estimating the reliability of predictions, and was able to identify compounds from an external test set with a SDEP for the predicted affinities of 4.6 kJ mol-1 (corresponding to 0.8 pKi units.

  7. CW EPR parameters reveal cytochrome P450 ligand binding modes.

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    Lockart, Molly M; Rodriguez, Carlo A; Atkins, William M; Bowman, Michael K

    2018-06-01

    Cytochrome P450 (CYP) monoxygenses utilize heme cofactors to catalyze oxidation reactions. They play a critical role in metabolism of many classes of drugs, are an attractive target for drug development, and mediate several prominent drug interactions. Many substrates and inhibitors alter the spin state of the ferric heme by displacing the heme's axial water ligand in the resting enzyme to yield a five-coordinate iron complex, or they replace the axial water to yield a nitrogen-ligated six-coordinate iron complex, which are traditionally assigned by UV-vis spectroscopy. However, crystal structures and recent pulsed electron paramagnetic resonance (EPR) studies find a few cases where molecules hydrogen bond to the axial water. The water-bridged drug-H 2 O-heme has UV-vis spectra similar to nitrogen-ligated, six-coordinate complexes, but are closer to "reverse type I" complexes described in older liteature. Here, pulsed and continuous wave (CW) EPR demonstrate that water-bridged complexes are remarkably common among a range of nitrogenous drugs or drug fragments that bind to CYP3A4 or CYP2C9. Principal component analysis reveals a distinct clustering of CW EPR spectral parameters for water-bridged complexes. CW EPR reveals heterogeneous mixtures of ligated states, including multiple directly-coordinated complexes and water-bridged complexes. These results suggest that water-bridged complexes are under-represented in CYP structural databases and can have energies similar to other ligation modes. The data indicates that water-bridged binding modes can be identified and distinguished from directly-coordinated binding by CW EPR. Copyright © 2018 Elsevier Inc. All rights reserved.

  8. Environmentally persistent free radical-containing particulate matter competitively inhibits metabolism by cytochrome P450 1A2

    Energy Technology Data Exchange (ETDEWEB)

    Reed, James R., E-mail: rreed@lsuhsc.edu [Department of Pharmacology and Experimental Therapeutics and Stanley S. Scott Cancer Center, Louisiana State University Health Sciences Center, 533 Bolivar St., New Orleans, LA 70112 (United States); Cruz, Albert Leo N. dela, E-mail: adelac2@tigers.lsu.edu [Department of Environmental Sciences and LSU Superfund Research Center, Louisiana State University A& M College, Baton Rouge, LA 70803 (United States); Lomnicki, Slawo M., E-mail: slomni1@lsu.edu [Department of Environmental Sciences and LSU Superfund Research Center, Louisiana State University A& M College, Baton Rouge, LA 70803 (United States); Backes, Wayne L., E-mail: wbacke@lsuhsc.edu [Department of Pharmacology and Experimental Therapeutics and Stanley S. Scott Cancer Center, Louisiana State University Health Sciences Center, 533 Bolivar St., New Orleans, LA 70112 (United States)

    2015-12-01

    Combustion processes generate different types of particulate matter (PM) that can have deleterious effects on the pulmonary and cardiovascular systems. Environmentally persistent free radicals (EPFRs) represent a type of particulate matter that is generated after combustion of environmental wastes in the presence of redox-active metals and aromatic hydrocarbons. Cytochromes P450 (P450/CYP) are membrane-bound enzymes that are essential for the phase I metabolism of most lipophilic xenobiotics. The EPFR formed by chemisorption of 2-monochlorophenol to silica containing 5% copper oxide (MCP230) has been shown to generally inhibit the activities of different forms of P450s without affecting those of cytochrome P450 reductase and heme oxygenase-1. The mechanism of inhibition of rat liver microsomal CYP2D2 and purified rabbit CYP2B4 by MCP230 has been shown previously to be noncompetitive with respect to substrate. In this study, MCP230 was shown to competitively inhibit metabolism of 7-benzyl-4-trifluoromethylcoumarin and 7-ethoxyresorufin by the purified, reconstituted rabbit CYP1A2. MCP230 is at least 5- and 50-fold more potent as an inhibitor of CYP1A2 than silica containing 5% copper oxide and silica, respectively. Thus, even though PM generally inhibit multiple forms of P450, PM interacts differently with the forms of P450 resulting in different mechanisms of inhibition. P450s function as oligomeric complexes within the membrane. We also determined the mechanism by which PM inhibited metabolism by the mixed CYP1A2–CYP2B4 complex and found that the mechanism was purely competitive suggesting that the CYP2B4 is dramatically inhibited when bound to CYP1A2. - Highlights: • Combustion of organic pollutants generates long-lived particulate radicals (EPFRs). • Particulate matter (PM) competitively inhibited CYP1A2 activity. • EPFRs were much more potent CYP1A2 inhibitors than other types of PM. • PM interacts differently with different forms of P450. • PM

  9. The effect of lycopene on the total cytochrome P450, CYP1A2 and CYP2E1

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    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

  10. Phytoremediation of the organic Xenobiotic simazine by p450-1a2 transgenic Arabidopsis thaliana plants.

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    Azab, Ehab; Hegazy, Ahmad K; El-Sharnouby, Mohamed E; Abd Elsalam, Hassan E

    2016-01-01

    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.

  11. Development of an on-line high performance liquid chromatography detection system for human cytochrome P450 1A2 inhibitors in extracts of natural products

    NARCIS (Netherlands)

    Jeurissen, S.M.F.; Claassen, F.W.; Havlik, J.; Bouwmans, E.E.; Cnubben, N.H.P.; Sudhölter, E.J.R.; Rietjens, I.M.C.M.; Beek, T.A. van

    2007-01-01

    An on-line HPLC screening method for detection of inhibitors of human cytochrome P450 1A2 in extracts was developed. HPLC separation of extracts is connected to a continuous methoxyresorufin-O-demethylation (MROD) assay in which recombinant human P450 1A2 converts methoxyresorufin to its fluorescent

  12. Dietary effect on mixed function P450 1A2 activity assayed by estimation of caffeine metabolism in man.

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    Kall, M A; Clausen, J

    1995-10-01

    Two studies were performed in order to evaluate cytochrome P450 1A2 mediated caffeine metabolism during different nutritional conditions. 1. In the first study, 23 healthy male non-smokers, mean age 25, changed from a customary mixed diet to a standard diet in 6 days. The 6 day's standard diet was based on bread, potatoes, rice and boiled meat. Thus, broccoli, cabbage and other cruciferous vegetables, spinach, leeks, onion, parsley, grapefruit, toasted bread, fried and charcoal grilled food, smoked fish and meat, ham and sausages were avoided. 2. In the second study, 33 healthy non-smoking subjects, 24 men and nine women mean age 25 years, volunteered. The study was designed to compare a customary home dietary period with the 6 day period of low dietary P450 induction and with a 5 day supplementary dietary period, i.e. ingestion of known dietary inducers. None of the women were using oral contraceptives or were pregnant during the experimental period. In the period of diet supplementation, the volunteers received charcoal grilled hamburger as a supplement to the standard low induction diet for lunch for 5 days. The hamburgers were made with 150 g beef (18-20% fat) and were grilled on charcoal for 10 min on each side until they were 'well done'. In the present study P450 1A2 activity was estimated from the caffeine metabolic ratio, the so-called CYP 1A2 index:(AFMU + 1-MX + 1-MU/ 17 -DMU) of the caffeine metabolites formed after oral ingestion of 200 mg caffeine. Urine was collected 4-8 h after caffeine ingestion in study 1 and 5 h after caffeine ingestion in study 2. In study 1 the CYP 1A2 index decreased from 4.28 +/- 0.98 in the customary home dietary period to 3.87 +/- 0.69 in the standard dietary period corresponding to 10.6% (P hamburgers as a dietary supplement, the CYP 1A2 index increased to almost the same level as in the customary home dietary period. The index increased to 4.45 +/- 1.57 in the whole group of volunteers, corresponding to a 14.1% (P < 0

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

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    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…

  14. Theoretical study on the N-demethylation mechanism of theobromine catalyzed by P450 isoenzyme 1A2.

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    Tao, Jing; Kang, Yuan; Xue, Zhiyu; Wang, Yongting; Zhang, Yan; Chen, Qiu; Chen, Zeqin; Xue, Ying

    2015-09-01

    Theobromine, a widely consumed pharmacological active substance, can cause undesirable muscle stiffness, nausea and anorexia in high doses ingestion. The main N-demethylation metabolic mechanism of theobromine catalyzed by P450 isoenzyme 1A2 (CYP1A2) has been explored in this work using the unrestricted hybrid density functional method UB3LYP in conjunction with the LACVP(Fe)/6-31G (H, C, N, O, S, Cl) basis set. Single-point calculations including empirical dispersion corrections were carried out at the higher 6-311++G** basis set. Two N-demethylation pathways were characterized, i.e., 3-N and 7-N demethylations, which involve the initial N-methyl hydroxylation to form carbinolamines and the subsequent carbinolamines decomposition to yield monomethylxanthines and formaldehydes. Our results have shown that the rate-limiting N-methyl hydroxylation occurs via a hydrogen atom transfer (HAT) mechanism, which proceeds in a spin-selective mechanism (SSM) in the gas phase. The carbinolamines generated are prone to decomposition via the contiguous heteroatom-assisted proton-transfer. Strikingly, 3-N demethylation is more favorable than 7-N demethylation due to its lower free energy barrier and 7-methylxanthine therefore is the optimum product reported for the demethylation of theobromine catalyzed by CYP1A2, which are in good agreement with the experimental observation. This work has first revealed the detail N-demethylation mechanisms of theobromine at the theoretical level. It can offer more significant information for the metabolism of purine alkaloid. Copyright © 2015 Elsevier Inc. All rights reserved.

  15. Quantum Mechanics/Molecular Mechanics Modeling of Drug Metabolism: Mexiletine N-Hydroxylation by Cytochrome P450 1A2.

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    Lonsdale, Richard; Fort, Rachel M; Rydberg, Patrik; Harvey, Jeremy N; Mulholland, Adrian J

    2016-06-20

    The mechanism of cytochrome P450(CYP)-catalyzed hydroxylation of primary amines is currently unclear and is relevant to drug metabolism; previous small model calculations have suggested two possible mechanisms: direct N-oxidation and H-abstraction/rebound. We have modeled the N-hydroxylation of (R)-mexiletine in CYP1A2 with hybrid quantum mechanics/molecular mechanics (QM/MM) methods, providing a more detailed and realistic model. Multiple reaction barriers have been calculated at the QM(B3LYP-D)/MM(CHARMM27) level for the direct N-oxidation and H-abstraction/rebound mechanisms. Our calculated barriers indicate that the direct N-oxidation mechanism is preferred and proceeds via the doublet spin state of Compound I. Molecular dynamics simulations indicate that the presence of an ordered water molecule in the active site assists in the binding of mexiletine in the active site, but this is not a prerequisite for reaction via either mechanism. Several active site residues play a role in the binding of mexiletine in the active site, including Thr124 and Phe226. This work reveals key details of the N-hydroxylation of mexiletine and further demonstrates that mechanistic studies using QM/MM methods are useful for understanding drug metabolism.

  16. Inhibition of the human liver microsomal and human cytochrome P450 1A2 and 3A4 metabolism of estradiol by deployment-related and other chemicals.

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    Usmani, Khawja A; Cho, Taehyeon M; Rose, Randy L; Hodgson, Ernest

    2006-09-01

    Cytochromes P450 (P450s) are major catalysts in the metabolism of xenobiotics and endogenous substrates such as estradiol (E2). It has previously been shown that E2 is predominantly metabolized in humans by CYP1A2 and CYP3A4 with 2-hydroxyestradiol (2-OHE2) the major metabolite. This study examines effects of deployment-related and other chemicals on E2 metabolism by human liver microsomes (HLM) and individual P450 isoforms. Kinetic studies using HLM, CYP3A4, and CYP1A2 showed similar affinities (Km) for E2 with respect to 2-OHE2 production. Vmax and CLint values for HLM are 0.32 nmol/min/mg protein and 7.5 microl/min/mg protein; those for CYP3A4 are 6.9 nmol/min/nmol P450 and 291 microl/min/nmol P450; and those for CYP1A2 are 17.4 nmol/min/nmol P450 and 633 microl/min/nmol P450. Phenotyped HLM use showed that individuals with high levels of CYP1A2 and CYP3A4 have the greatest potential to metabolize E2. Preincubation of HLM with a variety of chemicals, including those used in military deployments, resulted in varying levels of inhibition of E2 metabolism. The greatest inhibition was observed with organophosphorus compounds, including chlorpyrifos and fonofos, with up to 80% inhibition for 2-OHE2 production. Carbaryl, a carbamate pesticide, and naphthalene, a jet fuel component, inhibited ca. 40% of E2 metabolism. Preincubation of CYP1A2 with chlorpyrifos, fonofos, carbaryl, or naphthalene resulted in 96, 59, 84, and 87% inhibition of E2 metabolism, respectively. Preincubation of CYP3A4 with chlorpyrifos, fonofos, deltamethrin, or permethrin resulted in 94, 87, 58, and 37% inhibition of E2 metabolism. Chlorpyrifos inhibition of E2 metabolism is shown to be irreversible.

  17. Benzo[alpyrene induction of cytochrome P450 1A1/1A2 in the lymph nodes of rats.

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    Borodin, Yu I; Safina, A F; Maiborodin, I V; Grishanova, A Yu

    2003-12-01

    Studies of mesenteric lymph nodes of rats by indirect immunoperoxidase method using monoclonal antibodies to cytochrome P450 1A/1A2 after oral dose of benzo[a]pyrene showed the presence of these cytochrome forms in monocytes, macrophages, reticular and litoral cells, cell detritus, and liquid contents of the paracortical zone and medullary substance sinuses. Oxidation of various exo- and endogenous toxins in the lymph nodes was revealed.

  18. Classification of Cytochrome P450 1A2 Inhibitors and Non-Inhibitors by Machine Learning Techniques

    DEFF Research Database (Denmark)

    Vasanthanathan, Poongavanam; Taboureau, Olivier; Oostenbrink, Chris

    2009-01-01

    of CYP1A2 inhibitors and non-inhibitors. Training and test sets consisted of about 400 and 7000 compounds, respectively. Various machine learning techniques, like binary QSAR, support vector machine (SVM), random forest, kappa nearest neighbors (kNN), and decision tree methods were used to develop...

  19. Polymorphism in cytochrome P450 1A2 and their interaction with risk factors in determining risk of squamous cell lung carcinoma in men.

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    Singh, Arvind P; Pant, Mohan C; Ruwali, Munindra; Shah, Parag P; Prasad, Rajendra; Mathur, Neeraj; Parmar, Devendra

    The present case-control study was carried out to investigate the association of functionally important polymorphisms of cytochrome P450 1A2 (CYP1A2) involved in the metabolic activation of tobacco derived procarcinogens with squamous cell carcinoma (SCC) of lung in North Indian men. The study consisted of 200 male cases with SCC of lung and an equal number of age and sex matched healthy controls. Our data showed that variant genotype of CYP1A2*1D and CYP1A2*1F were significantly associated with increased susceptibility to SCC of lung. Likewise, GSTM1 null genotype was found to be over represented in patients when compared to controls. Haplotype analysis revealed that haplotype, G-Tdel-T-C was significantly associated with risk to SCC of lung. Moreover, a significant increase in the risk to SCC of lung in the cases carrying combination of variant genotype of CYP1A2 with either CYP1A1 or GSTM1 have shown that gene-gene interactions may play an important role in squamous cell lung cancer risk. Our data also revealed that smokers or tobacco chewers carrying variant alleles of either CYP1A2*1D or CYP1A2*1F were at increased risk to SCC of lung, further demonstrating that CYP1A2 genotypes interact with environmental risk factors in enhancing the risk to squamous cell lung carcinoma.

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

    DEFF Research Database (Denmark)

    Vasanthanathan, P.; Hritz, Jozef; Taboureau, Olivier

    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...

  1. Induction of cytochromes P450 1A1 and 1A2 suppresses formation of DNA adducts by carcinogenic aristolochic acid I in rats in vivo

    International Nuclear Information System (INIS)

    Dračínská, Helena; Bárta, František; Levová, Kateřina; Hudecová, Alena; Moserová, Michaela; Schmeiser, Heinz H.; Kopka, Klaus; Frei, Eva; Arlt, Volker M.; Stiborová, Marie

    2016-01-01

    Highlights: • Oxidation and reduction of aristolochic acid I (AAI) dictate its (geno)toxicity in vivo. • Cytochrome P450 (CYP) 1A1 and 1A2 are induced in rats treated with Sudan I and AAI. • Induced CYP1A enzyme activity resulted in decreased AAI-DNA adduct levels in vivo. • CYP1A1 and 1A2 mainly detoxify AAI and attenuate its genotoxicity in vivo. - Abstract: Aristolochic acid I (AAI) is a natural plant alkaloid causing aristolochic acid nephropathy, Balkan endemic nephropathy and their associated urothelial malignancies. One of the most efficient enzymes reductively activating AAI to species forming AAI-DNA adducts is cytosolic NAD(P)H:quinone oxidoreductase 1. AAI is also either reductively activated or oxidatively detoxified to 8-hydroxyaristolochic acid (AAIa) by microsomal cytochrome P450 (CYP) 1A1 and 1A2. Here, we investigated which of these two opposing CYP1A1/2-catalyzed reactions prevails in AAI metabolism in vivo. The formation of AAI-DNA adducts was analyzed in liver, kidney and lung of rats treated with AAI, Sudan I, a potent inducer of CYP1A1/2, or AAI after pretreatment with Sudan I. Compared to rats treated with AAI alone, levels of AAI-DNA adducts determined by the 32 P-postlabeling method were lower in liver, kidney and lung of rats treated with AAI after Sudan I. The induction of CYP1A1/2 by Sudan I increased AAI detoxification to its O-demethylated metabolite AAIa, thereby reducing the actual amount of AAI available for reductive activation. This subsequently resulted in lower AAI-DNA adduct levels in the rat in vivo. Our results demonstrate that CYP1A1/2-mediated oxidative detoxification of AAI is the predominant role of these enzymes in rats in vivo, thereby suppressing levels of AAI-DNA adducts.

  2. Caffeine raises the serum melatonin level in healthy subjects: an indication of melatonin metabolism by cytochrome P450(CYP)1A2.

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    Ursing, C; Wikner, J; Brismar, K; Röjdmark, S

    2003-05-01

    Caffeine is metabolized in the liver by cytochrome P450(CYP)1A2. Recent findings imply that this enzyme may also be of importance for the metabolism of human melatonin (MT). If caffeine and MT are metabolized by the same enzyme, one may expect to find different serum MT levels after ingestion of coffee compared with placebo. Although coffee is consumed by people all over the world, few studies have focused on whether caffeine actually affects serum MT levels in normal subjects. We decided to study that particular topic. For that purpose 12 healthy individuals were tested on two occasions, one week apart. On one of these occasions they were given a capsule containing 200 mg caffeine in the evening. On the other, they received placebo. The experimental order was randomized. Serum MT levels were determined every second hour between 22:00 h and 08:00 h, and the melatonin areas under the curve (MT-AUCs) were calculated. After caffeine the serum MT level rose from 0.09 +/- 0.03 nmol/l at 22:00 h to 0.48 +/- 0.07 nmol/l at 04:00 h. The corresponding rise after placebo was less prominent (from 0.06 +/- 0.01 to 0.35 +/- 0.06 nmol/l). This was reflected by the MT-AUC which was 32% larger after ingestion of caffeine compared with placebo (MT-AUC(caffeine) 3.16 +/- 0.44 nmol/l x h vs MT-AUC(placebo) 2.39 +/- 0.40 nmol/l x h; p coffee, augments the nocturnal serum MT level, which in turn supports the notion that cytochrome P450(CYP)1A2 is involved in the hepatic metabolism of human MT.

  3. Machine Learning Based Dimensionality Reduction Facilitates Ligand Diffusion Paths Assessment: A Case of Cytochrome P450cam.

    Science.gov (United States)

    Rydzewski, J; Nowak, W

    2016-04-12

    In this work we propose an application of a nonlinear dimensionality reduction method to represent the high-dimensional configuration space of the ligand-protein dissociation process in a manner facilitating interpretation. Rugged ligand expulsion paths are mapped into 2-dimensional space. The mapping retains the main structural changes occurring during the dissociation. The topological similarity of the reduced paths may be easily studied using the Fréchet distances, and we show that this measure facilitates machine learning classification of the diffusion pathways. Further, low-dimensional configuration space allows for identification of residues active in transport during the ligand diffusion from a protein. The utility of this approach is illustrated by examination of the configuration space of cytochrome P450cam involved in expulsing camphor by means of enhanced all-atom molecular dynamics simulations. The expulsion trajectories are sampled and constructed on-the-fly during molecular dynamics simulations using the recently developed memetic algorithms [ Rydzewski, J.; Nowak, W. J. Chem. Phys. 2015 , 143 ( 12 ), 124101 ]. We show that the memetic algorithms are effective for enforcing the ligand diffusion and cavity exploration in the P450cam-camphor complex. Furthermore, we demonstrate that machine learning techniques are helpful in inspecting ligand diffusion landscapes and provide useful tools to examine structural changes accompanying rare events.

  4. SKF 525-A and cytochrome P-450 ligands inhibit with high affinity the binding of [3H]dextromethorphan and σligands to guinea pig brain

    International Nuclear Information System (INIS)

    Klein, M.; Canoll, P.D.; Musacchio, J.M.

    1991-01-01

    The DM 1 /σ 1 site binds dextromethorphan (DM) and σ receptor ligands. The broad binding specificity of this site and its peculiar subcellular distribution prompted us to explore the possibility that this site is a member of the cytochrome P-450 superfamily of enzymes. We tested the effects of the liver microsomal monooxygenase inhibitor SKF 525-A (Proadifen), and other P-450 substrates on the binding of [ 3 H]dextromethorphan, [ 3 H]3-(3-Hydroxyphenyl)-N-(1-propyl)piperidine and (+)-[ 3 H]1,3-Di-o-tolyl-guanidine ([ 3 H]DTG) to the guinea pig brain. SKF 525-A, l-lobeline and GBR-12909 inhibited the binding of the three labeled ligands with nM affinity. Each drug has identical nM K i values for the high-affinity site labeled by the three ligands. This indicated that they displaced the labeled ligands from the common DM 1 σ 1 site. Debrisoquine and sparteine, prototypical substrates for liver debrisoquine 4-hydroxylase, displayed K i values of 9-13 and 3-4 μM respectively against the three labeled ligands. These results, the broad specificity of the DM 1 /σ 1 binding site, and its peculiar subcellular distribution, raises the possibility that this binding site is a member of the cytochrome P-450 superfamily of isozymes, rather than a neurotransmitter receptor

  5. Evaluation of toxic equivalency factors for induction of cytochromes P450 CYP1A1 and CYP1A2 enzyme activity by dioxin-like compounds

    International Nuclear Information System (INIS)

    Toyoshiba, Hiroyoshi; Walker, Nigel J.; Bailer, A. John; Portier, Christopher J.

    2004-01-01

    The toxic equivalency factor (TEF) method has been used to characterize the toxicity of human mixtures of dioxin-like compounds and is being considered for use with other classes of potentially toxic agents. TEFs are estimated by examining the relative potencies of the various congeners for a series of biological and toxicological effects. In this paper, we consider changes in activity for two enzymes, cytochrome P450 1A1 (CYP1A1)-associated 7-ethoxyresorufin-O-deethylase (EROD) and CYP1A2-associated acetanilide-4-hydroxylase (A4H) activity, resulting from exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 3,3',4,4',5-pentachlorobiphenyl (PCB), 2,3,4,7,8-pentachlorodibenzofuran (PeCDF) or a mixture of these agents. The ratio of median effective dose (ED 50 ) is one way to estimate the relative potencies, especially for gene expression and protein endpoints. ED 50 's were estimated with a nonlinear regression model in which dose-related changes in mean responses are described by a Hill function. ED 50 's along with other model parameters were estimated by fitting this model to a given data set. Significant differences in estimated model parameters were tested by likelihood ratio methods. The estimated parameters indicated that congener-specific dose-response shapes were significantly different, that additivity failed for these congeners, and that the ratios of ED 50 's did not predict the response seen for the mixture. These results indicate that for some biological responses, the use of a single relative potency factor (RPF) is not appropriate for the comparison of the dose response behavior of different dioxin-like congeners

  6. 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

  7. Analysis of cellular responses to aflatoxin B1 in yeast expressing human cytochrome P450 1A2 using cDNA microarrays

    International Nuclear Information System (INIS)

    Guo Yingying; Breeden, Linda L.; Fan, Wenhong; Zhao Lueping; Eaton, David L.; Zarbl, Helmut

    2006-01-01

    Aflatoxin B1 (AFB 1 ) is a potent human hepatotoxin and hepatocarcinogen produced by the mold Aspergillus flavus. In human, AFB 1 is bioactivated by cytochrome P450 (CYP450) enzymes, primarily CYP1A2, to the genotoxic epoxide that forms N 7 -guanine DNA adducts. To characterize the transcriptional responses to genotoxic insults from AFB 1 , a strain of Saccharomyces cerevisiae engineered to express human CYP1A2 was exposed to doses of AFB 1 that resulted in minimal lethality, but substantial genotoxicity. Flow cytometric analysis demonstrated a dose and time dependent S phase delay under the same treatment conditions, indicating a checkpoint response to DNA damage. Replicate cDNA microarray analyses of AFB 1 treated cells showed that about 200 genes were significantly affected by the exposure. The genes activated by AFB 1 -treatment included RAD51, DUN1 and other members of the DNA damage response signature reported in a previous study with methylmethane sulfonate and ionizing radiation [A.P. Gasch, M. Huang, S. Metzner, D. Botstein, S.J. Elledge, P.O. Brown, Genomic expression responses to DNA-damaging agents and the regulatory role of the yeast ATR homolog Mec1p, Mol. Biol. Cell 12 (2001) 2987-3003]. However, unlike previous studies using highly cytotoxic doses, environmental stress response genes [A.P. Gasch, P.T. Spellman, C.M. Kao, O. Carmel-Harel, M.B. Eisen, G. Storz, D. Botstein, P.O. Brown, Genomic expression programs in the response of yeast cells to environmental changes, Mol. Biol. Cell 11 (2000) 4241-4257] were largely unaffected by our dosing regimen. About half of the transcripts affected are also known to be cell cycle regulated. The most strongly repressed transcripts were those encoding the histone genes and a group of genes that are cell cycle regulated and peak in M phase and early G1. These include most of the known daughter-specific genes. The rapid and coordinated repression of histones and M/G1-specific transcripts cannot be explained by

  8. Analysis of cellular responses to aflatoxin B{sub 1} in yeast expressing human cytochrome P450 1A2 using cDNA microarrays

    Energy Technology Data Exchange (ETDEWEB)

    Guo Yingying [Departmental of Environmental and Occupational Health Sciences, University of Washington, Seattle, WA (United States); Fred Hutchinson Cancer Research Center, Seattle, WA (United States); Breeden, Linda L. [Fred Hutchinson Cancer Research Center, Seattle, WA (United States); Fan, Wenhong [Fred Hutchinson Cancer Research Center, Seattle, WA (United States); Zhao Lueping [Fred Hutchinson Cancer Research Center, Seattle, WA (United States); Eaton, David L. [Departmental of Environmental and Occupational Health Sciences, University of Washington, Seattle, WA (United States); Fred Hutchinson Cancer Research Center, Seattle, WA (United States); Zarbl, Helmut [Departmental of Environmental and Occupational Health Sciences, University of Washington, Seattle, WA (United States) and Fred Hutchinson Cancer Research Center, Seattle, WA (United States)]. E-mail: hzarbl@fhcrc.org

    2006-01-29

    Aflatoxin B1 (AFB{sub 1}) is a potent human hepatotoxin and hepatocarcinogen produced by the mold Aspergillus flavus. In human, AFB{sub 1} is bioactivated by cytochrome P450 (CYP450) enzymes, primarily CYP1A2, to the genotoxic epoxide that forms N{sup 7}-guanine DNA adducts. To characterize the transcriptional responses to genotoxic insults from AFB{sub 1}, a strain of Saccharomyces cerevisiae engineered to express human CYP1A2 was exposed to doses of AFB{sub 1} that resulted in minimal lethality, but substantial genotoxicity. Flow cytometric analysis demonstrated a dose and time dependent S phase delay under the same treatment conditions, indicating a checkpoint response to DNA damage. Replicate cDNA microarray analyses of AFB{sub 1} treated cells showed that about 200 genes were significantly affected by the exposure. The genes activated by AFB{sub 1}-treatment included RAD51, DUN1 and other members of the DNA damage response signature reported in a previous study with methylmethane sulfonate and ionizing radiation [A.P. Gasch, M. Huang, S. Metzner, D. Botstein, S.J. Elledge, P.O. Brown, Genomic expression responses to DNA-damaging agents and the regulatory role of the yeast ATR homolog Mec1p, Mol. Biol. Cell 12 (2001) 2987-3003]. However, unlike previous studies using highly cytotoxic doses, environmental stress response genes [A.P. Gasch, P.T. Spellman, C.M. Kao, O. Carmel-Harel, M.B. Eisen, G. Storz, D. Botstein, P.O. Brown, Genomic expression programs in the response of yeast cells to environmental changes, Mol. Biol. Cell 11 (2000) 4241-4257] were largely unaffected by our dosing regimen. About half of the transcripts affected are also known to be cell cycle regulated. The most strongly repressed transcripts were those encoding the histone genes and a group of genes that are cell cycle regulated and peak in M phase and early G1. These include most of the known daughter-specific genes. The rapid and coordinated repression of histones and M/G1-specific

  9. Computational prediction of binding affinity for CYP1A2-ligand complexes using empirical free energy calculations

    DEFF Research Database (Denmark)

    Poongavanam, Vasanthanathan; Olsen, Lars; Jørgensen, Flemming Steen

    2010-01-01

    , and methods based on statistical mechanics. In the present investigation, we started from an LIE model to predict the binding free energy of structurally diverse compounds of cytochrome P450 1A2 ligands, one of the important human metabolizing isoforms of the cytochrome P450 family. The data set includes both...... substrates and inhibitors. It appears that the electrostatic contribution to the binding free energy becomes negligible in this particular protein and a simple empirical model was derived, based on a training set of eight compounds. The root mean square error for the training set was 3.7 kJ/mol. Subsequent......Predicting binding affinities for receptor-ligand complexes is still one of the challenging processes in computational structure-based ligand design. Many computational methods have been developed to achieve this goal, such as docking and scoring methods, the linear interaction energy (LIE) method...

  10. 15N NMR study on cyanide (C15N-) complex of cytochrome P-450cam. Effects of d-camphor and putidaredoxin on the iron-ligand structure

    International Nuclear Information System (INIS)

    Shiro, Yoshitsugu; Iizuka, Tetsutaro; Makino, Ryu; Ishimura, Yuzuru; Morishima, Isao

    1989-01-01

    The cyanide (C 15 N - ) complex of Pseudomonas putida cytochrome P-450 (P-450 cam ) exhibited well-resolved and hyperfine-shifted 15 N NMR resonances arising from the iron-bound C 15 N - at 423 and 500 ppm in the absence and presence of the substrate, d-camphor, respectively. The values were smaller than those for cyanide complexes of myoglobin and hemoglobin (∼ 1000 ppm) but fell into the same range as those for the cyanide complexes of peroxidases (∼ 500 ppm). The 15 N shift values of P-450 cam were not incompatible with the existence of anionic ligand, such as cysteinyl thiolate anion, at the fifth coordination site of heme iron. The difference in the 15 N chemical shift values between camphor-free and bound enzymes was inferred by the increase in the steric constraint to the Fe-C-N bond upon substrate binding

  11. Polymorphisms in the cytochrome P450 genes CYP1A2, CYP1B1, CYP3A4, CYP3A5, CYP11A1, CYP17A1, CYP19A1 and colorectal cancer risk

    Directory of Open Access Journals (Sweden)

    Withey Laura

    2007-07-01

    Full Text Available Abstract Background Cytochrome P450 (CYP enzymes have the potential to affect colorectal cancer (CRC risk by determining the genotoxic impact of exogenous carcinogens and levels of sex hormones. Methods To investigate if common variants of CYP1A2, CYP1B1, CYP3A4, CYP3A5, CYP11A1, CYP17A1 and CYP19A1 influence CRC risk we genotyped 2,575 CRC cases and 2,707 controls for 20 single nucleotide polymorphisms (SNPs that have not previously been shown to have functional consequence within these genes. Results There was a suggestion of increased risk, albeit insignificant after correction for multiple testing, of CRC for individuals homozygous for CYP1B1 rs162558 and heterozygous for CYP1A2 rs2069522 (odds ratio [OR] = 1.36, 95% confidence interval [CI]: 1.03–1.80 and OR = 1.34, 95% CI: 1.00–1.79 respectively. Conclusion This study provides some support for polymorphic variation in CYP1A2 and CYP1B1 playing a role in CRC susceptibility.

  12. Induction of CYP1A1, CYP1A2, and CYP1B1 mRNAs by nitropolycyclic aromatic hydrocarbons in various human tissue-derived cells: chemical-, cytochrome P450 isoform-, and cell-specific differences

    Energy Technology Data Exchange (ETDEWEB)

    Iwanari, M.; Nakajima, M.; Yokoi, T. [Div. of Drug Metabolism, Kanazawa Univ., Kanazawa (Japan); Kizu, R.; Hayakawa, K. [Lab. of Hygienic Chemistry, Kanazawa Univ., Kanazawa (Japan)

    2002-06-01

    Nitropolycyclic aromatic hydrocarbons (NPAHs) are found in diesel exhaust and ambient air. NPAHs as well as polycyclic aromatic hydrocarbons (PAHs) are known to have mutagenicity, carcinogenicity, and endocrine-disruptive effects. In the present study, the inducibility of the human cytochrome P450-1 (CYP1) family by NPAHs was compared with those produced by their parent PAHs and some reductive metabolites, amino-PAHs. Furthermore, to investigate the differences in the inducibility of the CYP1 family in human tissues, various human tissue-derived cell lines, namely HepG2 (hepatocellular carcinoma), ACHN (renal carcinoma), A549 (lung carcinoma), MCF-7 (breast carcinoma), LS-180 (colon carcinoma), HT-1197 (bladder carcinoma), HeLa (cervix of uterus adenocarcinoma), OMC-3 (ovarian carcinoma), and NEC14 (testis embryonal carcinoma), were treated with NPAHs, PAHs, or amino-PAHs. The mRNA levels of CYP1A1, CYP1A2, and CYP1B1 were determined with reverse transcription-polymerase chain reaction (RT-PCR). The cell lines were classified into two groups: CYP1 inducible cell lines, comprising HepG2, MCF-7, LS-180, and OMC-3 cells, and CYP1 non-inducible cell lines, comprising ACHN, A549, HT-1197, HeLa, and NEC14 cells. In inducible cell lines, the induction profile of chemical specificity was similar for CYP1A1, CYP1A2, and CYP1B1, although the extent of induction differed among the cell lines and for the CYP isoforms. Pyrene, 1-nitropyrene, 1-aminopyrene, 1,3-, 1,6-, and 1,8-dinitropyrenes slightly induced CYP1 mRNAs, but 1,3-dinitropyrene produced a 6-fold induction of CYP1A1 mRNA in MCF-7 cells. 2-Nitrofluoranthene and 3-nitrofluoranthene exhibited stronger inducibility than fluoranthene in the inducible cell lines. 6-Nitrochrysene induced CYP1 mRNAs to the same extent or more potently than chrysene. The induction potencies of 6-nitrobenzo[a]pyrene and 7-nitrobenz[a]anthracene were weaker than those of their parents benzo[a]pyrene and benz[a]anthracene, respectively. This

  13. Probing Ligand Exchange in the P450 Enzyme CYP121 from Mycobacterium tuberculosis: Dynamic Equilibrium of the Distal Heme Ligand as a Function of pH and Temperature.

    Science.gov (United States)

    Fielding, Andrew J; Dornevil, Kednerlin; Ma, Li; Davis, Ian; Liu, Aimin

    2017-12-06

    CYP121 is a cytochrome P450 enzyme from Mycobacterium tuberculosis that catalyzes the formation of a C-C bond between the aromatic groups of its cyclodityrosine substrate (cYY). The crystal structure of CYP121 in complex with cYY reveals that the solvent-derived ligand remains bound to the ferric ion in the enzyme-substrate complex. Whereas in the generally accepted P450 mechanism, binding of the primary substrate in the active-site triggers the release of the solvent-derived ligand, priming the metal center for reduction and subsequent O 2 binding. Here we employed sodium cyanide to probe the metal-ligand exchange of the enzyme and the enzyme-substrate complex. The cyano adducts were characterized by UV-vis, EPR, and ENDOR spectroscopies and X-ray crystallography. A 100-fold increase in the affinity of cyanide binding to the enzyme-substrate complex over the ligand-free enzyme was observed. The crystal structure of the [CYP121(cYY)CN] ternary complex showed a rearrangement of the substrate in the active-site, when compared to the structure of the binary [CYP121(cYY)] complex. Transient kinetic studies showed that cYY binding resulted in a lower second-order rate constant (k on (CN) ) but a much more stable cyanide adduct with 3 orders of magnitude slower k off (CN) rate. A dynamic equilibrium between multiple high- and low-spin species for both the enzyme and enzyme-substrate complex was also observed, which is sensitive to changes in both pH and temperature. Our data reveal the chemical and physical properties of the solvent-derived ligand of the enzyme, which will help to understand the initial steps of the catalytic mechanism.

  14. 7,12-Dimethylbenzanthracene induces apoptosis in RL95-2 human endometrial cancer cells: Ligand-selective activation of cytochrome P450 1B1

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Ji Young [Department of Anatomy and Cell Biology, College of Medicine, Dong-A University, Busan 602-714 (Korea, Republic of); Medical Research Science Center, Dong-A University, Busan 602-714 (Korea, Republic of); Lee, Seung Gee [Department of Anatomy and Cell Biology, College of Medicine, Dong-A University, Busan 602-714 (Korea, Republic of); Mitochondria Hub Regulation Center, Dong-A University, Busan 602-714 (Korea, Republic of); Chung, Jin-Yong [Department of Anatomy and Cell Biology, College of Medicine, Dong-A University, Busan 602-714 (Korea, Republic of); Medical Research Science Center, Dong-A University, Busan 602-714 (Korea, Republic of); Kim, Yoon-Jae [Department of Anatomy and Cell Biology, College of Medicine, Dong-A University, Busan 602-714 (Korea, Republic of); Mitochondria Hub Regulation Center, Dong-A University, Busan 602-714 (Korea, Republic of); Park, Ji-Eun [Department of Anatomy and Cell Biology, College of Medicine, Dong-A University, Busan 602-714 (Korea, Republic of); Medical Research Science Center, Dong-A University, Busan 602-714 (Korea, Republic of); Oh, Seunghoon [Department of Physiology, College of Medicine, Dankook University, Cheonan 330-714 (Korea, Republic of); Lee, Se Yong [Department of Obstetrics and Gynecology, Busan Medical Center, Busan 611-072 (Korea, Republic of); Choi, Hong Jo [Department of General Surgery, College of Medicine, Dong-A University, Busan 602-714 (Korea, Republic of); Yoo, Young Hyun, E-mail: yhyoo@dau.ac.kr [Department of Anatomy and Cell Biology, College of Medicine, Dong-A University, Busan 602-714 (Korea, Republic of); Mitochondria Hub Regulation Center, Dong-A University, Busan 602-714 (Korea, Republic of); Medical Research Science Center, Dong-A University, Busan 602-714 (Korea, Republic of); and others

    2012-04-15

    7,12-Dimethylbenzanthracene (DMBA), a polycyclic aromatic hydrocarbon, exhibits mutagenic, carcinogenic, immunosuppressive, and apoptogenic properties in various cell types. To achieve these functions effectively, DMBA is modified to its active form by cytochrome P450 1 (CYP1). Exposure to DMBA causes cytotoxicity-mediated apoptosis in bone marrow B cells and ovarian cells. Although uterine endometrium constitutively expresses CYP1A1 and CYP1B1, their apoptotic role after exposure to DMBA remains to be elucidated. Therefore, we chose RL95-2 endometrial cancer cells as a model system for studying DMBA-induced cytotoxicity and cell death and hypothesized that exposure to DMBA causes apoptosis in this cell type following CYP1A1 and/or CYP1B1 activation. We showed that DMBA-induced apoptosis in RL95-2 cells is associated with activation of caspases. In addition, mitochondrial changes, including decrease in mitochondrial potential and release of mitochondrial cytochrome c into the cytosol, support the hypothesis that a mitochondrial pathway is involved in DMBA-induced apoptosis. Exposure to DMBA upregulated the expression of AhR, Arnt, CYP1A1, and CYP1B1 significantly; this may be necessary for the conversion of DMBA to DMBA-3,4-diol-1,2-epoxide (DMBA-DE). Although both CYP1A1 and CYP1B1 were significantly upregulated by DMBA, only CYP1B1 exhibited activity. Moreover, knockdown of CYP1B1 abolished DMBA-induced apoptosis in RL95-2 cells. Our data show that RL95-2 cells are susceptible to apoptosis by exposure to DMBA and that CYP1B1 plays a pivotal role in DMBA-induced apoptosis in this system. -- Highlights: ► Cytotoxicity-mediated apoptogenic action of DMBA in human endometrial cancer cells. ► Mitochondrial pathway in DMBA-induced apoptosis of RL95-2 endometrial cancer cells. ► Requirement of ligand-selective activation of CYP1B1 in DMBA-induced apoptosis.

  15. The cytochrome p450 homepage.

    Science.gov (United States)

    Nelson, David R

    2009-10-01

    The Cytochrome P450 Homepage is a universal resource for nomenclature and sequence information on cytochrome P450 ( CYP ) genes. The site has been in continuous operation since February 1995. Currently, naming information for 11,512 CYPs are available on the web pages. The P450 sequences are manually curated by David Nelson, and the nomenclature system conforms to an evolutionary scheme such that members of CYP families and subfamilies share common ancestors. The organisation and content of the Homepage are described.

  16. The impact of Cytochrome P450 CYP1A2, CYP2C9, CYP2C19 and CYP2D6 genes on suicide attempt and suicide risk-a European multicentre study on treatment-resistant major depressive disorder.

    Science.gov (United States)

    Höfer, Peter; Schosser, Alexandra; Calati, Raffaella; Serretti, Alessandro; Massat, Isabelle; Kocabas, Neslihan Aygun; Konstantinidis, Anastasios; Linotte, Sylvie; Mendlewicz, Julien; Souery, Daniel; Zohar, Joseph; Juven-Wetzler, Alzbeta; Montgomery, Stuart; Kasper, Siegfried

    2013-08-01

    Recently published data have reported associations between cytochrome P450 metabolizer status and suicidality. The aim of our study was to investigate the role of genetic polymorphisms of the cytochrome P450 genes on suicide risk and/or a personal history of suicide attempts. Two hundred forty-three major depressive disorder patients were collected in the context of a European multicentre resistant depression study and treated with antidepressants at adequate doses for at least 4 weeks. Suicidality was assessed using the Mini International Neuropsychiatric Interview and the Hamilton Rating Scale for Depression (HAM-D). Treatment response was defined as HAM-D ≤ 17 and remission as HAM-D ≤ 7 after 4 weeks of treatment with antidepressants at adequate dose. Genotyping was performed for all relevant variations of the CYP1A2 gene (*1A, *1F, *1C, *1 J, *1 K), the CYP2C9 gene (*2, *3), the CYP2C19 gene (*2, *17) and the CYP2D6 gene (*3, *4, *5, *6, *9, *19, *XN). No association between both suicide risk and personal history of suicide attempts, and the above mentioned metabolic profiles were found after multiple testing corrections. In conclusion, the investigated cytochrome gene polymorphisms do not seem to be associated with suicide risk and/or a personal history of suicide attempts, though methodological and sample size limitations do not allow definitive conclusions.

  17. 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.

  18. Cytochrome P450 humanised mice

    Science.gov (United States)

    2004-01-01

    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. PMID:15588489

  19. 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 rationalize what metabolites these enzymes generate. In recent years, many different in silico approaches have been developed to predict binding or regioselective product formation for the different CYP isoforms. These comprise ligand-based methods that are trained on experimental CYP data and structure...

  20. Phytomonitoring and phytoremediation of agrochemicals and related compounds based on recombinant cytochrome P450s and aryl hydrocarbon receptors (AhRs).

    Science.gov (United States)

    Shimazu, Sayuri; Inui, Hideyuki; Ohkawa, Hideo

    2011-04-13

    Molecular mechanisms of metabolism and modes of actions of agrochemicals and related compounds are important for understanding selective toxicity, biodegradability, and monitoring of biological effects on nontarget organisms. It is well-known that in mammals, cytochrome P450 (P450 or CYP) monooxygenases metabolize lipophilic foreign compounds. These P450 species are inducible, and both CYP1A1 and CYP1A2 are induced by aryl hydrocarbon receptor (AhR) combined with a ligand. Gene engineering of P450 and NADPH cytochrome P450 oxidoreductase (P450 reductase) was established for bioconversion. Also, gene modification of AhRs was developed for recombinant AhR-mediated β-glucronidase (GUS) reporter assay of AhR ligands. Recombinant P450 genes were transformed into plants for phytoremediation, and recombinant AhR-mediated GUS reporter gene expression systems were each transformed into plants for phytomonitoring. Transgenic rice plants carrying CYP2B6 metabolized the herbicide metolachlor and remarkably reduced the residues in the plants and soils under paddy field conditions. Transgenic Arabidopsis plants carrying recombinant guinea pig (g) AhR-mediated GUS reporter genes detected PCB126 at the level of 10 ng/g soils in the presence of biosurfactants MEL-B. Both phytomonitoring and phytoremediation plants were each evaluated from the standpoint of practical uses.

  1. Cytochrome P450s and molecular epidemiology

    Science.gov (United States)

    Gonzalez, Frank J.; Gelboin, Harry V.

    1993-03-01

    Cytochrome P450 (P450) represent a superfamily of heme-containing monooxygenases that are found throughout the animal and plant kingdoms and in many microorganisms. A number of these enzymes are involved in biosynthetic pathways of steroid synthesis but in mammals the vast majority of P450s function to metabolize foreign chemicals or xenobiotics. In the classical phase I reactions on the latter, a membrane-bound P450 will hydroxylate a compound, usually hydrophobic in nature, and the hydroxyl group will serve as a substrate for the various transferases or phase II enzymes that attach hydrophilic substituents such as glutathione, sulfate or glucuronic acid. Some chemicals, however, are metabolically-activated by P450s to electrophiles capable of reacting with cellular macromolecules. The cellular concentrations of the chemical and P450, reactivity of the active metabolite with nucleic acid and the repairability of the resultant adducts, in addition to the nature of the cell type, likely determines whether a chemical will be toxic and kill the cell or will transform the cell. Immunocorrelative and cDNA-directed expression have been used to define the substrate specificities of numerous human P450s. Levels of expression of different human P450 forms have been measured by both in vivo and in vitro methodologies leading to the realization that a large degree of interindividual differences occur in P450 expression. Reliable procedures for measuring P450 expression in healthy and diseased subjects will lead to prospective and case- cohort studies to determine whether interindividual differences in levels of P450 are associated with susceptibility or resistance to environmentally-based disease.

  2. 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,

  3. Cytochrome P450-mediated metabolic engineering

    DEFF Research Database (Denmark)

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

    2014-01-01

    for the engineered bioproduction of such compounds. Two ground-breaking developments of commercial products driven by the engineering of P450s are the antimalarial drug precursor artemisinic acid and blue roses or carnations. Tedious optimizations were required to generate marketable products. Hurdles encountered...... in P450 engineering and their potential solutions are summarized here. Together with recent technical developments and novel approaches to metabolic engineering, the lessons from this pioneering work should considerably boost exploitation of the amazing P450 toolkit emerging from accelerated sequencing...

  4. Oxygen and xenobiotic reductase activities of cytochrome P450.

    NARCIS (Netherlands)

    Goeptar, A.R.; Scheerens, H.; Vermeulen, N.P.E.

    1995-01-01

    The oxygen reductase and xenobiotic reductase activities of cytochrome P450 (P450) are reviewed. During the oxygen reductase activity of P450, molecular oxygen is reduced to superoxide anion radicals (O

  5. 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

    2005-01-07

    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.

  6. Novel extrahepatic cytochrome P450s

    International Nuclear Information System (INIS)

    Karlgren, Maria; Miura, Shin-ichi; Ingelman-Sundberg, Magnus

    2005-01-01

    The cytochrome P450 enzymes are highly expressed in the liver and are involved in the metabolism of xenobiotics. Because of the initiatives associated with the Human Genome Project, a great progress has recently been seen in the identification and characterization of novel extrahepatic P450s, including CYP2S1, CYP2R1, CYP2U1 and CYP2W1. Like the hepatic enzymes, these P450s may play a role in the tissue-specific metabolism of foreign compounds, but they may also have important endogenous functions. CYP2S1 has been shown to metabolize all-trans retinoic acid and CYP2R1 is a major vitamin D 25-hydroxylase. Regarding their metabolism of xenobiotics, much remains to be established, but CYP2S1 metabolizes naphthalene and it is likely that these P450s are responsible for metabolic activation of several different kinds of xenobiotic chemicals and contribute to extrahepatic toxicity and carcinogenesis

  7. 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.

  8. 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

  9. Flower colour and cytochromes P450.

    Science.gov (United States)

    Tanaka, Yoshikazu; Brugliera, Filippa

    2013-02-19

    Cytochromes P450 play important roles in biosynthesis of flavonoids and their coloured class of compounds, anthocyanins, both of which are major floral pigments. The number of hydroxyl groups on the B-ring of anthocyanidins (the chromophores and precursors of anthocyanins) impact the anthocyanin colour, the more the bluer. The hydroxylation pattern is determined by two cytochromes P450, flavonoid 3'-hydroxylase (F3'H) and flavonoid 3',5'-hydroxylase (F3'5'H) and thus they play a crucial role in the determination of flower colour. F3'H and F3'5'H mostly belong to CYP75B and CYP75A, respectively, except for the F3'5'Hs in Compositae that were derived from gene duplication of CYP75B and neofunctionalization. Roses and carnations lack blue/violet flower colours owing to the deficiency of F3'5'H and therefore lack the B-ring-trihydroxylated anthocyanins based upon delphinidin. Successful redirection of the anthocyanin biosynthesis pathway to delphinidin was achieved by expressing F3'5'H coding regions resulting in carnations and roses with novel blue hues that have been commercialized. Suppression of F3'5'H and F3'H in delphinidin-producing plants reduced the number of hydroxyl groups on the anthocyanidin B-ring resulting in the production of monohydroxylated anthocyanins based on pelargonidin with a shift in flower colour to orange/red. Pelargonidin biosynthesis is enhanced by additional expression of a dihydroflavonol 4-reductase that can use the monohydroxylated dihydrokaempferol (the pelargonidin precursor). Flavone synthase II (FNSII)-catalysing flavone biosynthesis from flavanones is also a P450 (CYP93B) and contributes to flower colour, because flavones act as co-pigments to anthocyanins and can cause blueing and darkening of colour. However, transgenic plants expression of a FNSII gene yielded paler flowers owing to a reduction of anthocyanins because flavanones are precursors of anthocyanins and flavones.

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

    International Nuclear Information System (INIS)

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

    1987-01-01

    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

  11. Monkey liver cytochrome P450 2C9 is involved in caffeine 7-N-demethylation to form theophylline.

    Science.gov (United States)

    Utoh, Masahiro; Murayama, Norie; Uno, Yasuhiro; Onose, Yui; Hosaka, Shinya; Fujino, Hideki; Shimizu, Makiko; Iwasaki, Kazuhide; Yamazaki, Hiroshi

    2013-12-01

    Caffeine (1,3,7-trimethylxanthine) is a phenotyping substrate for human cytochrome P450 1A2. 3-N-Demethylation of caffeine is the main human metabolic pathway, whereas monkeys extensively mediate the 7-N-demethylation of caffeine to form pharmacological active theophylline. Roles of monkey P450 enzymes in theophylline formation from caffeine were investigated using individual monkey liver microsomes and 14 recombinantly expressed monkey P450 enzymes, and the results were compared with those for human P450 enzymes. Caffeine 7-N-demethylation activity in microsomes from 20 monkey livers was not strongly inhibited by α-naphthoflavone, quinidine or ketoconazole, and was roughly correlated with diclofenac 4'-hydroxylation activities. Monkey P450 2C9 had the highest activity for caffeine 7-N-demethylation. Kinetic analysis revealed that monkey P450 2C9 had a high Vmax/Km value for caffeine 7-N-demethylation, comparable to low Km value for monkey liver microsomes. Caffeine could dock favorably with monkey P450 2C9 modeled for 7-N-demethylation and with human P450 1A2 for 3-N-demethylation. The primary metabolite theophylline was oxidized to 8-hydroxytheophylline in similar ways by liver microsomes and by recombinant P450s in both humans and monkeys. These results collectively suggest a high activity for monkey liver P450 2C9 toward caffeine 7-N-demethylation, whereas, in humans, P450 1A2-mediated caffeine 3-N-demethylation is dominant.

  12. P450 oxidoreductase deficiency: a disorder of steroidogenesis with multiple clinical manifestations.

    Science.gov (United States)

    Miller, Walter L

    2012-10-23

    Cytochrome P450 enzymes catalyze the biosynthesis of steroid hormones and metabolize drugs. There are seven human type I P450 enzymes in mitochondria and 50 type II enzymes in endoplasmic reticulum. Type II enzymes, including both drug-metabolizing and some steroidogenic enzymes, require electron donation from a two-flavin protein, P450 oxidoreductase (POR). Although knockout of the POR gene causes embryonic lethality in mice, we discovered human POR deficiency as a disorder of steroidogenesis associated with the Antley-Bixler skeletal malformation syndrome and found mild POR mutations in phenotypically normal adults with infertility. Assay results of mutant forms of POR using the traditional but nonphysiologic assay (reduction of cytochrome c) did not correlate with patient phenotypes; assays based on the 17,20 lyase activity of P450c17 (CYP17) correlated with clinical phenotypes. The POR sequence in 842 normal individuals revealed many polymorphisms; amino acid sequence variant A503V is encoded by ~28% of human alleles. POR A503V has about 60% of wild-type activity in assays with CYP17, CYP2D6, and CYP3A4, but nearly wild-type activity with P450c21, CYP1A2, and CYP2C19. Activity of a particular POR variant with one P450 enzyme will not predict its activity with another P450 enzyme: Each POR-P450 combination must be studied individually. Human POR transcription, initiated from an untranslated exon, is regulated by Smad3/4, thyroid receptors, and the transcription factor AP-2. A promoter polymorphism reduces transcription to 60% in liver cells and to 35% in adrenal cells. POR deficiency is a newly described disorder of steroidogenesis, and POR variants may account for some genetic variation in drug metabolism.

  13. Molecular characterization of cytochrome P450 1B1 and effect of ...

    African Journals Online (AJOL)

    CYP1B which belongs to the cytochrome P450 superfamily of genes, is involved in the oxidation of endogenous and exogenous compounds, and could potentially be a useful biomarker in fish for exposure to arylhydrocarbon receptors (AhR) ligands. In this study, a new complementary DNA (cDNA) of the CYP1B subfamily ...

  14. Expression of cytochrome P450 regulators in cynomolgus macaque.

    Science.gov (United States)

    Uno, Yasuhiro; Yamazaki, Hiroshi

    2017-09-11

    1. Cytochrome P450 (P450) regulators including nuclear receptors and transcription factors have not been fully investigated in cynomolgus macaques, an important species used in drug metabolism studies. In this study, we analyzed 17 P450 regulators by sequence and phylogenetic analysis, and tissue expression. 2. Gene and genome structures of 17 P450 regulators were similar to the human orthologs, and the deduced amino acid sequences showed high sequence identities (92-95%) and more closely clustered in a phylogenetic tree, with the human orthologs. 3. Many of the P450 regulator mRNAs were preferentially expressed in the liver, kidney, and/or jejunum. Among the P450 regulator mRNAs, PXR was most abundant in the liver and jejunum, and HNF4α in the kidney. In the liver, the expression of most P450 regulator mRNAs did not show significant differential expression (>2.5-fold) between cynomolgus macaques bred in Cambodia, China, and Indonesia, or rhesus macaques. 4. By correlation analysis, most of the P450 regulators were significantly (p < 0.05) correlated to other P450 regulators, and many of them were also significantly (p < 0.05) correlated with P450s. 5. These results suggest that 17 P450 regulators of cynomolgus macaques had similar molecular characteristics to the human orthologs.

  15. Pharmacokinetics and Differential Regulation of Cytochrome P450 Enzymes in Type 1 Allergic Mice.

    Science.gov (United States)

    Tanino, Tadatoshi; Komada, Akira; Ueda, Koji; Bando, Toru; Nojiri, Yukie; Ueda, Yukari; Sakurai, Eiichi

    2016-12-01

    Type 1 allergic diseases are characterized by elevated production of specific immunoglobulin E (IgE) for each antigen and have become a significant health problem worldwide. This study investigated the effect of IgE-mediated allergy on drug pharmacokinetics. To further understand differential suppression of hepatic cytochrome P450 (P450) activity, we examined the inhibitory effect of nitric oxide (NO), a marker of allergic conditions. Seven days after primary sensitization (PS7) or secondary sensitization (SS7), hepatic CYP1A2, CYP2C, CYP2E1, and CYP3A activities were decreased to 45%-75% of the corresponding control; however, CYP2D activity was not downregulated. PS7 and SS7 did not change the expression levels of five P450 proteins. Disappearance of CYP1A2 and CYP2D substrates from the plasma was not significantly different between allergic mice and control mice. In contrast, the area under the curve of a CYP1A2-mediated metabolite in PS7 and SS7 mice was reduced by 50% of control values. Total clearances of a CYP2E1 substrate in PS7 and SS7 mice were significantly decreased to 70% and 50% respectively, of the control without altering plasma protein binding. Hepatic amounts of CYP1A2 and CYP2E1 substrates were enhanced by allergic induction, being responsible for each downregulated activity. NO scavenger treatment completely improved the downregulated P450 activities. Therefore, our data suggest that the onset of IgE-mediated allergy alters the pharmacokinetics of major P450-metabolic capacity-limited drugs except for CYP2D drugs. NO is highly expected to participate in regulatory mechanisms of the four P450 isoforms. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

  16. CHARACTERIZATION OF THE ALKANE-INDUCIBLE CYTOCHROME P450 (P450ALK) GENE FROM THE YEAST CANDIDA TROPICALIS: IDENTIFICATION OF A NEW P450 FAMILY

    Science.gov (United States)

    The P450alk gene, which is inducible by the assimilation of alkane in Candida tropicalis, was sequenced and characterized. Structural features described in promoter and terminator regions of Saccharomyces yeast genes are present in the P450alk gene and some particular structures ...

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

    DEFF Research Database (Denmark)

    Bavishi, Krutika; Laursen, Tomas; Martinez, Karen Laurence

    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...... 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...

  18. 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...

  19. The biodiversity of microbial cytochromes P450.

    Science.gov (United States)

    Kelly, Steven L; Lamb, David C; Jackson, Colin J; Warrilow, Andrew G; Kelly, Diane E

    2003-01-01

    The cytochrome P450 (CYP) superfamily of genes and proteins are well known for their involvement in pharmacology and toxicology, but also increasingly for their importance and diversity in microbes. The extent of diversity has only recently become apparent with the emergence of data from whole genome sequencing projects and the coming years will reveal even more information on the diversity in microbial eukaryotes. This review seeks to describe the historical development of these studies and to highlight the importance of the genes and proteins. CYPs are deeply involved in the development of strategies for deterrence and attraction as well as detoxification. As such, there is intense interest in pathways of secondary metabolism that include CYPs in oxidative tailoring of antibiotics, sometimes influencing potency as bioactive compounds. Further to this is interest in CYPs in metabolism of xenobiotics for use as carbon sources for microbial growth and as biotransformation agents or in bioremediation. CYPs are also current and potential drug targets; compounds inhibiting CYP are antifungal and anti-protozoan agents, and potentially similar compounds may be useful against some bacterial diseases such as tuberculosis. Of note is the diversity of CYP requirements within an organism, ranging from Escherichia coli that has no CYPs as in many bacteria, to Mycobacterium smegmatis that has 40 representing 1% of coding genes. The basidiomycete fungus Phanerochaete chrysosporium surprised all when it was found to contain a hundred or more CYPs. The functional genomic investigation of these orphan CYPs is a major challenge for the future.

  20. Epidermal CYP2 family cytochromes P450

    International Nuclear Information System (INIS)

    Du Liping; Hoffman, Susan M.G.; Keeney, Diane S.

    2004-01-01

    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

  1. 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...

  2. Insect P450 inhibitors and insecticides: challenges and opportunities.

    Science.gov (United States)

    Feyereisen, René

    2015-06-01

    P450 enzymes are encoded by a large number of genes in insects, often over a hundred. They play important roles in insecticide metabolism and resistance, and growing numbers of P450 enzymes are now known to catalyse important physiological reactions, such as hormone metabolism or cuticular hydrocarbon synthesis. Ways to inhibit P450 enzymes specifically or less specifically are well understood, as P450 inhibitors are found as drugs, as fungicides, as plant growth regulators and as insecticide synergists. Yet there are no P450 inhibitors as insecticides on the market. As new modes of action are constantly needed to support insecticide resistance management, P450 inhibitors should be considered because of their high potential for insect selectivity, their well-known mechanisms of action and the increasing ease of rational design and testing. © 2014 Society of Chemical Industry.

  3. Vitamin K3 (menadione) redox cycling inhibits cytochrome P450-mediated metabolism and inhibits parathion intoxication

    Energy Technology Data Exchange (ETDEWEB)

    Jan, Yi-Hua [Department of Environmental and Occupational Medicine, Rutgers Robert Wood Johnson Medical School, Piscataway, NJ (United States); Richardson, Jason R., E-mail: jricha3@eohsi.rutgers.edu [Department of Environmental and Occupational Medicine, Rutgers Robert Wood Johnson Medical School, Piscataway, NJ (United States); Baker, Angela A. [Department of Environmental and Occupational Medicine, Rutgers Robert Wood Johnson Medical School, Piscataway, NJ (United States); Mishin, Vladimir [Department of Pharmacology and Toxicology, Rutgers University, Piscataway, NJ (United States); Heck, Diane E. [Department of Environmental Health Science, New York Medical College, Valhalla, NY (United States); Laskin, Debra L. [Department of Pharmacology and Toxicology, Rutgers University, Piscataway, NJ (United States); Laskin, Jeffrey D., E-mail: jlaskin@eohsi.rutgers.edu [Department of Environmental and Occupational Medicine, Rutgers Robert Wood Johnson Medical School, Piscataway, NJ (United States)

    2015-10-01

    Parathion, a widely used organophosphate insecticide, is considered a high priority chemical threat. Parathion toxicity is dependent on its metabolism by the cytochrome P450 system to paraoxon (diethyl 4-nitrophenyl phosphate), a cytotoxic metabolite. As an effective inhibitor of cholinesterases, paraoxon causes the accumulation of acetylcholine in synapses and overstimulation of nicotinic and muscarinic cholinergic receptors, leading to characteristic signs of organophosphate poisoning. Inhibition of parathion metabolism to paraoxon represents a potential approach to counter parathion toxicity. Herein, we demonstrate that menadione (methyl-1,4-naphthoquinone, vitamin K3) is a potent inhibitor of cytochrome P450-mediated metabolism of parathion. Menadione is active in redox cycling, a reaction mediated by NADPH-cytochrome P450 reductase that preferentially uses electrons from NADPH at the expense of their supply to the P450s. Using human recombinant CYP 1A2, 2B6, 3A4 and human liver microsomes, menadione was found to inhibit the formation of paraoxon from parathion. Administration of menadione bisulfite (40 mg/kg, ip) to rats also reduced parathion-induced inhibition of brain cholinesterase activity, as well as parathion-induced tremors and the progression of other signs and symptoms of parathion poisoning. These data suggest that redox cycling compounds, such as menadione, have the potential to effectively mitigate the toxicity of organophosphorus pesticides including parathion which require cytochrome P450-mediated activation. - Highlights: • Menadione redox cycles with cytochrome P450 reductase and generates reactive oxygen species. • Redox cycling inhibits cytochrome P450-mediated parathion metabolism. • Short term administration of menadione inhibits parathion toxicity by inhibiting paraoxon formation.

  4. Vitamin K3 (menadione) redox cycling inhibits cytochrome P450-mediated metabolism and inhibits parathion intoxication

    International Nuclear Information System (INIS)

    Jan, Yi-Hua; Richardson, Jason R.; Baker, Angela A.; Mishin, Vladimir; Heck, Diane E.; Laskin, Debra L.; Laskin, Jeffrey D.

    2015-01-01

    Parathion, a widely used organophosphate insecticide, is considered a high priority chemical threat. Parathion toxicity is dependent on its metabolism by the cytochrome P450 system to paraoxon (diethyl 4-nitrophenyl phosphate), a cytotoxic metabolite. As an effective inhibitor of cholinesterases, paraoxon causes the accumulation of acetylcholine in synapses and overstimulation of nicotinic and muscarinic cholinergic receptors, leading to characteristic signs of organophosphate poisoning. Inhibition of parathion metabolism to paraoxon represents a potential approach to counter parathion toxicity. Herein, we demonstrate that menadione (methyl-1,4-naphthoquinone, vitamin K3) is a potent inhibitor of cytochrome P450-mediated metabolism of parathion. Menadione is active in redox cycling, a reaction mediated by NADPH-cytochrome P450 reductase that preferentially uses electrons from NADPH at the expense of their supply to the P450s. Using human recombinant CYP 1A2, 2B6, 3A4 and human liver microsomes, menadione was found to inhibit the formation of paraoxon from parathion. Administration of menadione bisulfite (40 mg/kg, ip) to rats also reduced parathion-induced inhibition of brain cholinesterase activity, as well as parathion-induced tremors and the progression of other signs and symptoms of parathion poisoning. These data suggest that redox cycling compounds, such as menadione, have the potential to effectively mitigate the toxicity of organophosphorus pesticides including parathion which require cytochrome P450-mediated activation. - Highlights: • Menadione redox cycles with cytochrome P450 reductase and generates reactive oxygen species. • Redox cycling inhibits cytochrome P450-mediated parathion metabolism. • Short term administration of menadione inhibits parathion toxicity by inhibiting paraoxon formation.

  5. Cytochrome P450 monooxygenases and insecticide resistance in insects.

    OpenAIRE

    Bergé, J B; Feyereisen, R; Amichot, M

    1998-01-01

    Cytochrome P450 monooxygenases are involved in many cases of resistance of insects to insecticides. Resistance has long been associated with an increase in monooxygenase activities and with an increase in cytochrome P450 content. However, this increase does not always account for all of the resistance. In Drosophila melanogaster, we have shown that the overproduction of cytochrome P450 can be lost by the fly without a corresponding complete loss of resistance. These results prompted the seque...

  6. Vitamin K3 (menadione) redox cycling inhibits cytochrome P450-mediated metabolism and inhibits parathion intoxication.

    Science.gov (United States)

    Jan, Yi-Hua; Richardson, Jason R; Baker, Angela A; Mishin, Vladimir; Heck, Diane E; Laskin, Debra L; Laskin, Jeffrey D

    2015-10-01

    Parathion, a widely used organophosphate insecticide, is considered a high priority chemical threat. Parathion toxicity is dependent on its metabolism by the cytochrome P450 system to paraoxon (diethyl 4-nitrophenyl phosphate), a cytotoxic metabolite. As an effective inhibitor of cholinesterases, paraoxon causes the accumulation of acetylcholine in synapses and overstimulation of nicotinic and muscarinic cholinergic receptors, leading to characteristic signs of organophosphate poisoning. Inhibition of parathion metabolism to paraoxon represents a potential approach to counter parathion toxicity. Herein, we demonstrate that menadione (methyl-1,4-naphthoquinone, vitamin K3) is a potent inhibitor of cytochrome P450-mediated metabolism of parathion. Menadione is active in redox cycling, a reaction mediated by NADPH-cytochrome P450 reductase that preferentially uses electrons from NADPH at the expense of their supply to the P450s. Using human recombinant CYP 1A2, 2B6, 3A4 and human liver microsomes, menadione was found to inhibit the formation of paraoxon from parathion. Administration of menadione bisulfite (40mg/kg, ip) to rats also reduced parathion-induced inhibition of brain cholinesterase activity, as well as parathion-induced tremors and the progression of other signs and symptoms of parathion poisoning. These data suggest that redox cycling compounds, such as menadione, have the potential to effectively mitigate the toxicity of organophosphorus pesticides including parathion which require cytochrome P450-mediated activation. Copyright © 2015 Elsevier Inc. All rights reserved.

  7. LKM-1 autoantibodies recognize a short linear sequence in P450IID6, a cytochrome P-450 monooxygenase.

    OpenAIRE

    Manns, M P; Griffin, K J; Sullivan, K F; Johnson, E F

    1991-01-01

    LKM-1 autoantibodies, which are associated with autoimmune chronic active hepatitis, recognize P450IID6, a cytochrome P-450 monooxygenase. The reactivities of 26 LKM-1 antisera were tested with a panel of deletion mutants of P450IID6 expressed in Escherichia coli. 22 sera recognize a 33-amino acid segment of P450IID6, and 11 of these recognize a shorter segment, DPAQPPRD. PAQPPR is also found in IE175 of herpes simplex virus type 1 (HSV-1). Antibodies for HSV-1 proteins were detected by ELISA...

  8. Computational identification of putative cytochrome P450 genes in ...

    African Journals Online (AJOL)

    Chattha

    Economically, legumes represent the second most important family of crop plants after Poacea (grass family), accounting for ... further characterization of P450 genes with both known and unknown functions. MATERIALS AND METHODS ..... Cytochrome P450. In: Somerville CR, Meyerowitz EM (eds) .The Arabidopsis book,.

  9. Identification of bottlenecks for P450 biotransformation processes

    DEFF Research Database (Denmark)

    Andersson, Marie Therese; Törnvall, Ulrika; Tufvesson, Pär

    Cytochrome P450 monooxygenases (P450 or CYP) is a group of heme-containing enzymes hydroxylating non-activated hydrocarbons in a stereospecific manner, something that is hard to achieve via classical chemistry. The importance of these reactions can be stressed by the hydroxylation of steroids, bu...... biotransformation process identifying the limiting parameters and defining relevant targets....

  10. 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....

  11. Cytochrome P450 isoform selectivity in human hepatic theobromine metabolism

    Science.gov (United States)

    Gates, Simon; Miners, John O

    1999-01-01

    Aims The plasma clearance of theobromine (TB; 3,7-dimethylxanthine) is known to be induced in cigarette smokers. To determine whether TB may serve as a model substrate for cytochrome P450 (CYP) 1A2, or possibly other isoforms, studies were undertaken to identify the individual human liver microsomal CYP isoforms responsible for the conversion of TB to its primary metabolites. Methods The kinetics of formation of the primary TB metabolites 3-methylxanthine (3-MX), 7-methylxanthine (7-MX) and 3,7-dimethyluric acid (3,7-DMU) by human liver microsomes were characterized using a specific hplc procedure. Effects of CYP isoform-selective xenobiotic inhibitor/substrate probes on each pathway were determined and confirmatory studies with recombinant enzymes were performed to define the contribution of individual isoforms to 3-MX, 7-MX and 3,7-DMU formation. Results The CYP1A2 inhibitor furafylline variably inhibited (0–65%) 7-MX formation, but had no effect on other pathways. Diethyldithiocarbamate and 4-nitrophenol, probes for CYP2E1, inhibited the formation of 3-MX, 7-MX and 3,7-DMU by ≈55–60%, 35–55% and 85%, respectively. Consistent with the microsomal studies, recombinant CYP1A2 and CYP2E1 exhibited similar apparent Km values for 7-MX formation and CYP2E1 was further shown to have the capacity to convert TB to both 3-MX and 3,7-DMU. Conclusions Given the contribution of multiple isoforms to 3-MX and 7-MX formation and the negligible formation of 3,7-DMU in vivo, TB is of little value as a CYP isoform-selective substrate in humans. PMID:10215755

  12. 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...

  13. Electrochemistry of cytochrome P450 17α-hydroxylase/17,20-lyase (P450c17).

    Science.gov (United States)

    Martin, Lisandra L; Kubeil, Clemens; Simonov, Alexandr N; Kuznetsov, Vladimir L; Corbin, C Jo; Auchus, Richard J; Conley, Alan J; Bond, Alan M; Rodgers, Raymond J

    2017-02-05

    Within the superfamily of cytochrome P450 enzymes (P450s), there is a small class which is functionally employed for steroid biosynthesis. The enzymes in this class appear to have a small active site to accommodate the steroid substrates specifically and snuggly, prior to the redox transformation or hydroxylation to form a product. Cytochrome P450c17 is one of these and is also a multi-functional P450, with two activities, the first 17α-hydroxylation of pregnenolone is followed by a subsequent 17,20-lyase transformation to dehydroepiandrosterone (DHEA) as the dominant pathways to cortisol precursors or androgens in humans, respectively. How P450c17 regulates these two redox reactions is of special interest. There is a paucity of direct electrochemical studies on steroidogenic P450s, and in this mini-review we provide an overview of these studies with P450c17. Historical consideration as to the difficulties in obtaining reliable electrochemistry due to issues of handling proteins on an electrode, together with advances in the electrochemical techniques are addressed. Recent work using Fourier transformed alternating current voltammetry is highlighted as this technique can provide both catalytic information simultaneously with the underlying redox transfer with the P450 haem. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  14. {sup 13}C-Methyl isocyanide as an NMR probe for cytochrome P450 active sites

    Energy Technology Data Exchange (ETDEWEB)

    McCullough, Christopher R.; Pullela, Phani Kumar [Marquette University, Chemical Proteomics Facility at Marquette, Department of Chemistry (United States); Im, Sang-Choul; Waskell, Lucy [University of Michigan and VA Medical Center, Department of Anesthesiology (United States); Sem, Daniel S. [Marquette University, Chemical Proteomics Facility at Marquette, Department of Chemistry (United States)], E-mail: Daniel.sem@marquette.edu

    2009-03-15

    The cytochromes P450 (CYPs) play a central role in many biologically important oxidation reactions, including the metabolism of drugs and other xenobiotic compounds. Because they are often assayed as both drug targets and anti-targets, any tools that provide: (a) confirmation of active site binding and (b) structural data, would be of great utility, especially if data could be obtained in reasonably high throughput. To this end, we have developed an analog of the promiscuous heme ligand, cyanide, with a {sup 13}CH{sub 3}-reporter attached. This {sup 13}C-methyl isocyanide ligand binds to bacterial (P450cam) and membrane-bound mammalian (CYP2B4) CYPs. It can be used in a rapid 1D experiment to identify binders, and provides a qualitative measure of structural changes in the active site.

  15. 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

  16. Inactivation of Cytochrome P450 (P450) 3A4 but not P450 3A5 by OSI-930, a Thiophene-Containing Anticancer DrugS⃞

    Science.gov (United States)

    Lin, Hsia-lien; Zhang, Haoming; Medower, Christine; Johnson, William W.

    2011-01-01

    An investigational anticancer agent that contains a thiophene moiety, 3-[(quinolin-4-ylmethyl)-amino]-N-[4-trifluoromethox)phenyl] thiophene-2-carboxamide (OSI-930), was tested to investigate its ability to modulate the activities of several cytochrome P450 enzymes. Results showed that OSI-930 inactivated purified, recombinant cytochrome P450 (P450) 3A4 in the reconstituted system in a mechanism-based manner. The inactivation was dependent on cytochrome b5 and required NADPH. Catalase did not protect against the inactivation. No inactivation was observed in studies with human 2B6, 2D6, or 3A5 either in the presence or in the absence of b5. The inactivation of 3A4 by OSI-930 was time- and concentration-dependent. The inactivation of the 7-benzyloxy-4-(trifluoromethyl)coumarin catalytic activity of 3A4 was characterized by a KI of 24 μM and a kinact of 0.04 min−1. This KI is significantly greater than the clinical OSI-930 Cmax of 1.7 μM at the maximum tolerated dose, indicating that clinical drug interactions of OSI-930 via this pathway are not likely. Spectral analysis of the inactivated protein indicated that the decrease in the reduced CO spectrum at 450 nm was comparable to the amount of inactivation, thereby suggesting that the inactivation was primarily due to modification of the heme. High-pressure liquid chromatography (HPLC) analysis with detection at 400 nm showed a loss of heme comparable to the activity loss, but a modified heme was not detected. This result suggests either that the heme must have been modified enough so as not to be observed in a HPLC chromatograph or, possibly, that it was destroyed. The partition ratio for the inactivation of P450 3A4 was approximately 23, suggesting that this P450 3A4-mediated pathway occurs with approximately 4% frequency during the metabolism of OSI-930. Modeling studies on the binding of OSI-930 to the active site of the P450 3A4 indicated that OSI-930 would be oriented properly in the active site for oxidation

  17. 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...

  18. 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.

    1993-01-01

    The role of metabolism of tumour promoters on the inhibition of intercellular communication was investigated in a modified V79 metabolic cooperation system. V79 cells, which stably express different rat cytochrome P450 enzymes (CYP1A1, CYP1A2 or CYP2B1), were used in the metabolic cooperation assay...... 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 communication of some tumour...... promoters. The modified metabolic cooperation assay presented here is valuable for detecting some inhibitory chemicals which have been 'false negative' in previous assays for gap junctional intercellular communication. The assay also discloses that cytochrome P450 metabolism alters intercellular...

  19. Reduction of Aromatic and Heterocyclic Aromatic N-Hydroxylamines by Human Cytochrome P450 2S1

    Science.gov (United States)

    Wang, Kai; 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 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 present study, 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

  20. Identifying Cytochrome P450 Functional Networks and Their Allosteric Regulatory Elements

    Science.gov (United States)

    2013-12-03

    based on physiochemical features not captured by residue co-evolution. In all the networks we characterized, it was evident that some residues were...corresponding iron and sulphur related parameters, were obtained from Bathelt et al. [46]. These parameters are based on QM/MM calculations and have been...2007) Adaptations for the oxidation of polycyclic aromatic hydrocarbons exhibited by the structure of human P450 1A2. J Biol Chem 282: 14348-14355. doi

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

    International Nuclear Information System (INIS)

    Muerhoff, A.S.; Williams, D.E.; Jackson, V.; Leithauser, M.T.; Waterman, M.R.; Johnson, E.F.; Masters, B.S.S.

    1987-01-01

    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 PGE 1 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 35 S-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 PGE 1 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. Characterisation of the cytochrome P450 enzymes involved in the in vitro metabolism of granisetron.

    Science.gov (United States)

    Bloomer, J C; Baldwin, S J; Smith, G J; Ayrton, A D; Clarke, S E; Chenery, R J

    1994-01-01

    1. The metabolism of granisetron was investigated in human liver microsomes to identify the specific forms of cytochrome P450 responsible. 2. 7-hydroxy and 9'-desmethyl granisetron were identified as the major products of metabolism following incubation of granisetron with human liver microsomes. At low, clinically relevant, concentrations of granisetron the 7-hydroxy metabolite predominated. Rates of granisetron 7-hydroxylation varied over 100-fold in the human livers investigated. 3. Enzyme kinetics demonstrated the involvement of at least two enzymes contributing to the 7-hydroxylation of granisetron, one of which was a high affinity component with a Km of 4 microM. A single, low affinity, enzyme was responsible for the 9'-desmethylation of granisetron. 4. Granisetron caused no inhibition of any of the cytochrome P450 activities investigated (CYP1A2, CYP2A6, CYP2B6, CYP2C9/8, CYP2C19, CYP2D6, CYP2E1 and CYP3A), at concentrations up to 250 microM. 5. Studies using chemical inhibitors selective for individual P450 enzymes indicated the involvement of cytochrome P450 3A (CYP3A), both pathways of granisetron metabolism being very sensitive to ketoconazole inhibition. Correlation data were consistent with the role of CYP3A3/4 in granisetron 9'-desmethylation but indicated that a different enzyme was involved in the 7-hydroxylation. PMID:7888294

  3. 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.

  4. Triterpene Structural Diversification by Plant Cytochrome P450 Enzymes

    Directory of Open Access Journals (Sweden)

    Sumit Ghosh

    2017-11-01

    Full Text Available Cytochrome P450 monooxygenases (P450s represent the largest enzyme family of the plant metabolism. Plants typically devote about 1% of the protein-coding genes for the P450s to execute primary metabolism and also to perform species-specific specialized functions including metabolism of the triterpenes, isoprene-derived 30-carbon compounds. Triterpenes constitute a large and structurally diverse class of natural products with various industrial and pharmaceutical applications. P450-catalyzed structural modification is crucial for the diversification and functionalization of the triterpene scaffolds. In recent times, a remarkable progress has been made in understanding the function of the P450s in plant triterpene metabolism. So far, ∼80 P450s are assigned biochemical functions related to the plant triterpene metabolism. The members of the subfamilies CYP51G, CYP85A, CYP90B-D, CYP710A, CYP724B, and CYP734A are generally conserved across the plant kingdom to take part in plant primary metabolism related to the biosynthesis of essential sterols and steroid hormones. However, the members of the subfamilies CYP51H, CYP71A,D, CYP72A, CYP81Q, CYP87D, CYP88D,L, CYP93E, CYP705A, CYP708A, and CYP716A,C,E,S,U,Y are required for the metabolism of the specialized triterpenes that might perform species-specific functions including chemical defense toward specialized pathogens. Moreover, a recent advancement in high-throughput sequencing of the transcriptomes and genomes has resulted in identification of a large number of candidate P450s from diverse plant species. Assigning biochemical functions to these P450s will be of interest to extend our knowledge on triterpene metabolism in diverse plant species and also for the sustainable production of valuable phytochemicals.

  5. Identification of human cytochrome P450s as autoantigens.

    Science.gov (United States)

    Manns, M P; Johnson, E F

    1991-01-01

    Antimicrosomal antibodies in inflammatory liver diseases all seem to be directed against members of the cytochrome P450 family of proteins. These autoantigens seem to be genetically polymorphic, the autoantibodies are inhibitory, and the autoepitopes are generally conserved among species. Anti-P450 autoantibodies share these characteristics with other autoantibodies, for example, antinuclear antibodies in systemic lupus erythematosus. The identification of P450s as human autoantigens is clinically important. Diagnostic tests will be developed on the basis of cloned antigen, facilitating a better diagnosis of drug-induced and idiopathic autoimmune hepatitis. It is unknown what triggers autoantibody production against cytochrome P450 proteins. Furthermore, their pathogenetic role and thus their involvement in tissue destruction is unclear. In this context LKM1 autoantibodies may serve as a model. Although LKM1 antibodies are inhibitory, all LKM1 antibody-positive patients tested so far are extensive metabolizers for drug metabolism mediated by P450IID6 and express this protein in their livers. Thus, the inhibitory LKM1 autoantibody does not sufficiently penetrate through the intact liver cell membrane to inhibit enzyme function in vivo. Presumably, tissue destruction in autoimmune hepatitis is mediated by liver-infiltrating T lymphocytes. T lymphocytes have been cloned from liver tissue that specifically proliferate in the presence of recombinant cytochrome P450IID6. The construction of overlapping cDNA subclones is also valuable to identify immunodominant B cell as well as relevant T cell epitopes.

  6. Fungal Cytochrome P450s and the P450 Complement (CYPome of Fusarium graminearum

    Directory of Open Access Journals (Sweden)

    Jiyoung Shin

    2018-03-01

    Full Text Available Cytochrome P450s (CYPs, heme-containing monooxygenases, play important roles in a wide variety of metabolic processes important for development as well as biotic/trophic interactions in most living organisms. Functions of some CYP enzymes are similar across organisms, but some are organism-specific; they are involved in the biosynthesis of structural components, signaling networks, secondary metabolisms, and xenobiotic/drug detoxification. Fungi possess more diverse CYP families than plants, animals, or bacteria. Various fungal CYPs are involved in not only ergosterol synthesis and virulence but also in the production of a wide array of secondary metabolites, which exert toxic effects on humans and other animals. Although few studies have investigated the functions of fungal CYPs, a recent systematic functional analysis of CYP genes in the plant pathogen Fusarium graminearum identified several novel CYPs specifically involved in virulence, asexual and sexual development, and degradation of xenobiotics. This review provides fundamental information on fungal CYPs and a new platform for further metabolomic and biochemical studies of CYPs in toxigenic fungi.

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

    International Nuclear Information System (INIS)

    Wang Jianping; Xu Dexiang; Sun Meifang; Chen Yuanhua; Wang Hua; Wei Wei

    2005-01-01

    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 GdCl 3 , a selective Kupffer cell toxicant. GdCl 3 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

  8. Cytochromes P450: History, Classes, Catalytic Mechanism, and Industrial Application.

    Science.gov (United States)

    Cook, D J; Finnigan, J D; Cook, K; Black, G W; Charnock, S J

    Cytochromes P450, a family of heme-containing monooxygenases that catalyze a diverse range of oxidative reactions, are so-called due to their maximum absorbance at 450nm, ie, "Pigment-450nm," when bound to carbon monoxide. They have appeal both academically and commercially due to their high degree of regio- and stereoselectivity, for example, in the area of active pharmaceutical ingredient synthesis. Despite this potential, they often exhibit poor stability, low turnover numbers and typically require electron transport protein(s) for catalysis. P450 systems exist in a variety of functional domain architectures, organized into 10 classes. P450s are also divided into families, each of which is based solely on amino acid sequence homology. Their catalytic mechanism employs a very complex, multistep catalytic cycle involving a range of transient intermediates. Mutagenesis is a powerful tool for the development of improved biocatalysts and has been used extensively with the archetypal Class VIII P450, BM3, from Bacillus megaterium, but with the increasing scale of genomic sequencing, a huge resource is now available for the discovery of novel P450s. © 2016 Elsevier Inc. All rights reserved.

  9. Cytochrome P450 enzyme mediated herbal drug interactions (Part 2)

    Science.gov (United States)

    Wanwimolruk, Sompon; Phopin, Kamonrat; Prachayasittikul, Virapong

    2014-01-01

    To date, a number of significant herbal drug interactions have their origins in the alteration of cytochrome P450 (CYP) activity by various phytochemicals. Among the most noteworthy are those involving St. John's wort and drugs metabolized by human CYP3A4 enzyme. This review article is the continued work from our previous article (Part 1) published in this journal (Wanwimolruk and Prachayasittikul, 2014[ref:133]). This article extends the scope of the review to six more herbs and updates information on herbal drug interactions. These include black cohosh, ginseng, grape seed extract, green tea, kava, saw palmetto and some important Chinese medicines are also presented. Even though there have been many studies to determine the effects of herbs and herbal medicines on the activity of CYP, most of them were in vitro and in animal studies. Therefore, the studies are limited in predicting the clinical relevance of herbal drug interactions. It appeared that the majority of the herbal medicines have no clear effects on most of the CYPs examined. For example, the existing clinical trial data imply that black cohosh, ginseng and saw palmetto are unlikely to affect the pharmacokinetics of conventional drugs metabolized by human CYPs. For grape seed extract and green tea, adverse herbal drug interactions are unlikely when they are concomitantly taken with prescription drugs that are CYP substrates. Although there were few clinical studies on potential CYP-mediated interactions produced by kava, present data suggest that kava supplements have the ability to inhibit CYP1A2 and CYP2E1 significantly. Therefore, caution should be taken when patients take kava with CYP1A2 or CYP2E1 substrate drugs as it may enhance their therapeutic and adverse effects. Despite the long use of traditional Chinese herbal medicines, little is known about the potential drug interactions with these herbs. Many popularly used Chinese medicines have been shown in vitro to significantly change the

  10. 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. © 2014 Society of Chemical Industry.

  11. 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.

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

    International Nuclear Information System (INIS)

    Ornstein, R.; Paulsen, M.; Bass, M.; Arnold, G.

    1991-03-01

    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

  13. Computational identification of putative cytochrome P450 genes in ...

    African Journals Online (AJOL)

    In this work, a computational study of expressed sequence tags (ESTs) of soybean was performed by data mining methods and bio-informatics tools and as a result 78 putative P450 genes were identified, including 57 new ones. These genes were classified into five clans and 20 families by sequence similarities and among ...

  14. Assembly of dynamic P450-mediated metabolons - order versus chaos

    DEFF Research Database (Denmark)

    Bassard, Jean-Étienne André; Møller, Birger Lindberg; Laursen, Tomas

    2017-01-01

    PURPOSE OF REVIEW: We provide an overview of the current knowledge on cytochrome P450-mediated metabolism organized as metabolons and factors that facilitate their stabilization. Essential parameters will be discussed including those that are commonly disregarded using the dhurrin metabolon from ...

  15. Multivariate Modeling of Cytochrome P450 Enzymes for 4 ...

    African Journals Online (AJOL)

    Conclusion: Apart from insights into important molecular properties for CYP inhibition, the findings may also guide further investigations of novel drug candidates that are unlikely to inhibit multiple CYP sub-types. Keywords: Antimalarial, Chloroquine, Cytochrome P450, Genetic algorithm-based multiple linear regression, ...

  16. Homology modelling of Drosophila cytochrome P450 enzymes associated with insecticide resistance.

    Science.gov (United States)

    Jones, Robert T; Bakker, Saskia E; Stone, Deborah; Shuttleworth, Sally N; Boundy, Sam; McCart, Caroline; Daborn, Phillip J; ffrench-Constant, Richard H; van den Elsen, Jean M H

    2010-10-01

    Overexpression of the cytochrome P450 gene Cyp6g1 confers resistance against DDT and a broad range of other insecticides in Drosophila melanogaster Meig. In the absence of crystal structures of CYP6G1 or complexes with its substrates, structural studies rely on homology modelling and ligand docking to understand P450-substrate interactions. Homology models are presented for CYP6G1, a P450 associated with resistance to DDT and neonicotinoids, and two other enzymes associated with insecticide resistance in D. melanogaster, CYP12D1 and CYP6A2. The models are based on a template of the X-ray structure of the phylogenetically related human CYP3A4, which is known for its broad substrate specificity. The model of CYP6G1 has a much smaller active site cavity than the template. The cavity is also 'V'-shaped and is lined with hydrophobic residues, showing high shape and chemical complementarity with the molecular characteristics of DDT. Comparison of the DDT-CYP6G1 complex and a non-resistant CYP6A2 homology model implies that tight-fit recognition of this insecticide is important in CYP6G1. The active site can accommodate differently shaped substrates ranging from imidacloprid to malathion but not the pyrethroids permethrin and cyfluthrin. The CYP6G1, CYP12D1 and CYP6A2 homology models can provide a structural insight into insecticide resistance in flies overexpressing P450 enzymes with broad substrate specificities.

  17. High-Throughput Cytochrome P450 Cocktail Inhibition Assay for Assessing Drug-Drug and Drug-Botanical Interactions.

    Science.gov (United States)

    Li, Guannan; Huang, Ke; Nikolic, Dejan; van Breemen, Richard B

    2015-11-01

    Detection of drug-drug interactions is essential during the early stages of drug discovery and development, and the understanding of drug-botanical interactions is important for the safe use of botanical dietary supplements. Among the different forms of drug interactions that are known, inhibition of cytochrome P450 (P450) enzymes is the most common cause of drug-drug or drug-botanical interactions. Therefore, a rapid and comprehensive mass spectrometry-based in vitro high-throughput P450 cocktail inhibition assay was developed that uses 10 substrates simultaneously against nine CYP isoforms. Including probe substrates for CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and two probes targeting different binding sites of CYP3A4/5, this cocktail simultaneously assesses at least as many P450 enzymes as previous assays while remaining among the fastest due to short incubation times and rapid analysis using ultrahigh pressure liquid chromatography-tandem mass spectrometry. The method was validated using known inhibitors of each P450 enzyme and then shown to be useful not only for single-compound testing but also for the evaluation of potential drug-botanical interactions using the botanical dietary supplement licorice (Glycyrrhiza glabra) as an example. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

  18. In vitro investigation of cytochrome P450-mediated metabolism of dietary flavonoids

    DEFF Research Database (Denmark)

    Breinholt, Vibeke; Offord, E.A.; Brouwer, C.

    2002-01-01

    Human and mouse liver microsomes And membranes isolated from Escherichia coli, which expressed cytochrome P450 (CYP) 1A2, 3A4 2C9 or 2D6, were used to investigate CYP-mediated metabolism of five selected dietary flavonoids. In human and mouse liver microsomes kaempferol, apigenin and naringenin...... were hydroxylated at the 3'-position to yield their corresponding analogs quercetin, luteolin and eriodietyol, whereas hesperetin and tamarixetin were demethylated at the 4'-position to yield eriodictyol and quercetin. respectively, Microsomal flavonoid metabolism as potently inhibited by the CYP1A2...... inhibitors. fluvoxamine and alpha-naphthoflavone. Recombinant CYP1A2 as capable of metabolizing all five investigated flavonoids. CYP3A4 recombinant protein did not catalyze hesperetin demethylation. but showed similar metabolic profiles for the remaining compounds, as did human microsomes and recombinant...

  19. Role of cytochrome P450 in drug interactions

    Directory of Open Access Journals (Sweden)

    Bibi Zakia

    2008-10-01

    Full Text Available Abstract Drug-drug interactions have become an important issue in health care. It is now realized that many drug-drug interactions can be explained by alterations in the metabolic enzymes that are present in the liver and other extra-hepatic tissues. Many of the major pharmacokinetic interactions between drugs are due to hepatic cytochrome P450 (P450 or CYP enzymes being affected by previous administration of other drugs. After coadministration, some drugs act as potent enzyme inducers, whereas others are inhibitors. However, reports of enzyme inhibition are very much more common. Understanding these mechanisms of enzyme inhibition or induction is extremely important in order to give appropriate multiple-drug therapies. In future, it may help to identify individuals at greatest risk of drug interactions and adverse events.

  20. Expanding P450 catalytic reaction space through evolution and engineering

    Science.gov (United States)

    McIntosh, John A.; Farwell, Christopher C.; Arnold, Frances H.

    2014-01-01

    Advances in protein and metabolic engineering have led to wider use of enzymes to synthesize important molecules. However, many desirable transformations are not catalyzed by any known enzyme, driving interest in understanding how new enzymes can be created. The cytochrome P450 enzyme family, whose members participate in xenobiotic metabolism and natural products biosynthesis, catalyzes an impressive range of difficult chemical reactions that continues to grow as new enzymes are characterized. Recent work has revealed that P450-derived enzymes can also catalyze useful reactions previously accessible only to synthetic chemistry. The evolution and engineering of these enzymes provides an excellent case study for how to genetically encode new chemistry and expand biology’s reaction space. PMID:24658056

  1. Differentially regulated NADPH: cytochrome p450 oxidoreductases in parsely

    International Nuclear Information System (INIS)

    Koopmann, E.; Hahlbrock, K.

    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 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. Guidelines for development and implementation of biocatalytic P450 processes

    DEFF Research Database (Denmark)

    Lundemo, Marie Therese; Woodley, John

    2015-01-01

    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.......Biocatalytic reactions performed by cytochrome P450 monooxygenases are interesting in pharmaceutical research since they are involved in human drug metabolism. Furthermore, they are potentially interesting as biocatalysts for synthetic chemistry because of the exquisite selectivity of the chemistry...... they undertake. For example, selective hydroxylation can be undertaken on a highly functionalized molecule without the need for functional group protection. Recent progress in the discovery of novel P450s as well as protein engineering of these enzymes strongly encourages further development of their application...

  3. Interaction of rocuronium with human liver cytochromes P450

    OpenAIRE

    Anzenbacherova, Eva; Spicakova, Alena; Jourova, Lenka; Ulrichova, Jitka; Adamus, Milan; Bachleda, Petr; Anzenbacher, Pavel

    2015-01-01

    Rocuronium is a neuromuscular blocking agent acting as a competitive antagonist of acetylcholine. Results of an inhibition of eight individual liver microsomal cytochromes P450 (CYP) are presented. As the patients are routinely premedicated with diazepam, possible interaction of diazepam with rocuronium has been also studied. Results indicated that rocuronium interacts with human liver microsomal CYPs by binding to the substrate site. Next, concentration dependent inhibition of liver micro...

  4. 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

  5. Influence of polyhalogenated aromatic hydrocarbons on the induction, activity, and stabilization of cytochrome P450

    International Nuclear Information System (INIS)

    Voorman, R.

    1987-01-01

    In the course of experiments evaluating the metabolism of polybrominated biphenyls by cytochrome P450 isozymes induced by 3,4,5,3',4',5'-hexabromobiphenyl (HBB), it was discovered that the inducer remained closely associated with cytochrome P450d. Subsequent purification of cytochromes from HBB treated rates revealed a 0.5:1 association of HBB to cytochrome P450d but virtually none with cytochrome P450c or cytochrome b5. Immunochemical quantitation of cytochrome P450d in the same microsomes yielded a ratio of P450d:HBB that approached unity. Measurement of cytochrome P450d estradiol 2-hydroxylase indicated non-competitive or mixed type inhibition caused by HBB at a concentration of 10-1000 nM. Inhibition was specific to cytochrome P450d since estradiol 2-hydroxylase catalyzed by cytochrome P450h was unaffected by HBB. The ability of HCB and isosafrole to stabilize cytochrome P450d, and thus indirectly influence regulation of the enzyme, was evaluated by treating rats with a dose of TCDD sufficient to produce maximum induction of cytochromes P450c and P450d via the Ah receptor, yet insufficient to bind to the enzyme. Subsequent treatment of these animals with HCB or isosafrole and a radiolabeled amino acid, revealed a significant increase in cytochrome P450d specific content relative to cytochrome P450c and significant retention of the radiolabel in P450d relative to rats treated only with TCDD

  6. A web-based resource for the Arabidopsis P450, cytochromes b5, NADPH-cytochrome P450 reductases, and family 1 glycosyltransferases (http://www.P450.kvl.dk).

    Science.gov (United States)

    Paquette, Suzanne M; Jensen, Kenneth; Bak, Søren

    2009-12-01

    Gene and genome duplication is a key driving force in evolution of plant diversity. This has resulted in a number of large multi-gene families. Two of the largest multi-gene families in plants are the cytochromes P450 (P450s) and family 1 glycosyltransferases (UGTs). These two families are key players in evolution, especially of plant secondary metabolism, and in adaption to abiotic and biotic stress. In the model plant Arabidopsis thaliana there are 246 and 112 cytochromes P450 and UGTs, respectively. The Arabidopsis P450, cytochromes b(5), NADPH-cytochrome P450 reductases, and family 1 glycosyltransferases website (http://www.P450.kvl.dk) is a sequence repository of manually curated sequences, multiple sequence alignments, phylogenetic trees, sequence motif logos, 3D structures, intron-exon maps, and customized BLAST datasets.

  7. Regulation of rat liver cytochrome P450j, a high affinity N-nitrosodimethylamine demethylase (NDMAD)

    International Nuclear Information System (INIS)

    Thomas, P.E.; Bandiera, S.; Maines, S.L.; Ryan, D.E.; Levin, W.

    1987-01-01

    Purified IgG from sera of rabbits immunized with homogeneous P450j was absorbed to produce monospecific anti-P450j. Results using anti-P450j in ELISA show that rat liver microsomal P450j content decreases between 3 and 6 wks of age in both sexes. Several xenobiotics (Aroclor 1254, mirex and 3-methylcholanthrene) repressed P450j levels when administered to male rats. In contrast, hepatic levels of P450j were induced by isoniazid, dimethylsulfoxide, pyrazole, 4-methylpyrazole, ethanol and chemically-induced diabetes. P450j levels were measurable in kidney, whereas this isozyme was barely detectable in lung, ovaries and testes; however, extra-hepatic P450j was inducible by isoniazid. Between 80-90% of microsomal NDMAD was inhibited by anti-P450j whether the microsomes were isolated from untreated rats or animals administered inducers or repressors of P450j. Results obtained with the reconstituted system suggest that the remaining microsomal NDMAD resistant to antibody inhibition is the result of the inaccessibility of a certain proportion of P450j due to interference by NADPH-P450 reductase. P450j content and NDMAD activity correlated well in microsomes from rats of all treatment groups. The evidence indicates that P450j is the primary, and possibly only, microsomal catalyst of NDMAD at substrate concentrations relevant to hepatocarcinogenesis induced by NDMA

  8. Metabolism of ethylbenzene by human liver microsomes and recombinant human cytochrome P450s (CYP).

    Science.gov (United States)

    Sams, Craig; Loizou, George D; Cocker, John; Lennard, Martin S

    2004-03-07

    The enzyme kinetics of the initial hydroxylation of ethylbenzene to form 1-phenylethanol were determined in human liver microsomes. The individual cytochrome P450 (CYP) forms catalysing this reaction were identified using selective inhibitors and recombinant preparations of hepatic CYPs. Production of 1-phenylethanol in hepatic microsomes exhibited biphasic kinetics with a high affinity, low Km, component (mean Km = 8 microM; V(max) = 689 pmol/min/mg protein; n = 6 livers) and a low affinity, high Km, component (Km = 391 microM; V(max) = 3039 pmol/min/mg protein; n = 6). The high-affinity component was inhibited 79%-95% (mean 86%) by diethyldithiocarbamate, and recombinant CYP2E1 was shown to metabolise ethylbenzene with low Km (35 microM), but also low (max) (7 pmol/min/pmol P450), indicating that this isoform catalysed the high-affinity component. Recombinant CYP1A2 and CYP2B6 exhibited high V(max) (88 and 71 pmol/min/pmol P450, respectively) and high Km (502 and 219 microM, respectively), suggesting their involvement in catalysing the low-affinity component. This study has demonstrated that CYP2E1 is the major enzyme responsible for high-affinity side chain hydroxylation of ethylbenzene in human liver microsomes. Activity of this enzyme in the population is highly variable due to induction or inhibition by physiological factors, chemicals in the diet or some pharmaceuticals. This variability can be incorporated into the risk assessment process to improve the setting of occupational exposure limits and guidance values for biological monitoring.

  9. The Role of Cytochromes P450 in Infection

    Directory of Open Access Journals (Sweden)

    Elisavet Stavropoulou

    2018-01-01

    Full Text Available Cytochromes are expressed in many different tissues of the human body. They are found mostly in intestinal and hepatic tissues. Cytochromes P450 (CYPs are enzymes that oxidize substances using iron and are able to metabolize a large variety of xenobiotic substances. CYP enzymes are linked to a wide array of reactions including and O-dealkylation, S-oxidation, epoxidation, and hydroxylation. The activity of the typical P450 cytochrome is influenced by a variety of factors, such as genus, environment, disease state, herbicide, alcohol, and herbal medications. However, diet seems to play a major role. The mechanisms of action of dietary chemicals, macro- and micronutrients on specific CYP isoenzymes have been extensively studied. Dietary modulation has effects upon the metabolism of xenobiotics. Cytochromes harbor intra- or interindividual and intra- or interethnic genetic polymorphisms. Bacteria were shown to express CYP-like genes. The tremendous metabolic activity of the microbiota is associated to its abundant pool of CYP enzymes, which catalyze phase I and II reactions in drug metabolism. Disease states, intestinal disturbances, aging, environmental toxic effects, chemical exposures or nutrition modulate the microbial metabolism of a drug before absorption. A plethora of effects exhibited by most of CYP enzymes can resemble those of proinflammatory cytokines and IFNs. Moreover, they are involved in the initiation and persistence of pathologic pain by directly activating sensory neurons and inflammatory cytokines.

  10. Novel approaches to mitigating parathion toxicity: targeting cytochrome P450–mediated metabolism with menadione

    Science.gov (United States)

    Jan, Yi-Hua; Richardson, Jason R.; Baker, Angela A.; Mishin, Vladimir; Heck, Diane E.; Laskin, Debra L.; Laskin, Jeffrey D.

    2016-01-01

    Accidental or intentional exposures to parathion, an organophosphorus (OP) pesticide, can cause severe poisoning in humans. Parathion toxicity is dependent on its metabolism by the cytochrome P450 (CYP) system to paraoxon (diethyl 4-nitrophenyl phosphate), a highly poisonous nerve agent and potent inhibitor of acetylcholinesterase (AChE). We have been investigating inhibitors of CYP-mediated bioactivation of OPs as a method of preventing or reversing progressive parathion toxicity. It is well recognized that NADPH–cytochrome P450 reductase, an enzyme required for the transfer of electrons to CYPs, mediates chemical redox cycling. In this process, the enzyme diverts electrons from CYPs to support chemical redox cycling, which results in inhibition of CYP-mediated biotransformation. Using menadione as the redox-cycling chemical, we discovered that this enzymatic reaction blocks metabolic activation of parathion in rat and human liver microsomes and in recombinant CYPs important to parathion metabolism, including CYP1A2, CYP2B6, and CYP3A4. Administration of menadione to rats reduces metabolism of parathion, as well as parathion-induced inhibition of brain cholinesterase activity. This resulted in inhibition of parathion neurotoxicity. Menadione has relatively low toxicity and is approved by the FDA for other indications. Its ability to block parathion metabolism makes it an attractive therapeutic candidate to mitigate parathion-induced neurotoxicity. PMID:27441453

  11. Novel approaches to mitigating parathion toxicity: targeting cytochrome P450-mediated metabolism with menadione.

    Science.gov (United States)

    Jan, Yi-Hua; Richardson, Jason R; Baker, Angela A; Mishin, Vladimir; Heck, Diane E; Laskin, Debra L; Laskin, Jeffrey D

    2016-08-01

    Accidental or intentional exposures to parathion, an organophosphorus (OP) pesticide, can cause severe poisoning in humans. Parathion toxicity is dependent on its metabolism by the cytochrome P450 (CYP) system to paraoxon (diethyl 4-nitrophenyl phosphate), a highly poisonous nerve agent and potent inhibitor of acetylcholinesterase. We have been investigating inhibitors of CYP-mediated bioactivation of OPs as a method of preventing or reversing progressive parathion toxicity. It is well recognized that NADPH-cytochrome P450 reductase, an enzyme required for the transfer of electrons to CYPs, mediates chemical redox cycling. In this process, the enzyme diverts electrons from CYPs to support chemical redox cycling, which results in inhibition of CYP-mediated biotransformation. Using menadione as the redox-cycling chemical, we discovered that this enzymatic reaction blocks metabolic activation of parathion in rat and human liver microsomes and in recombinant CYPs important to parathion metabolism, including CYP1A2, CYP2B6, and CYP3A4. Administration of menadione to rats reduces metabolism of parathion, as well as parathion-induced inhibition of brain cholinesterase activity. This resulted in inhibition of parathion neurotoxicity. Menadione has relatively low toxicity and is approved by the Food and Drug Administration for other indications. Its ability to block parathion metabolism makes it an attractive therapeutic candidate to mitigate parathion-induced neurotoxicity. © 2016 New York Academy of Sciences.

  12. The Flavin-Containing Reductase Domain of Cytochrome P450 BM3 Acts as a Surrogate for Mammalian NADPH-P450 Reductase.

    Science.gov (United States)

    Park, Seon-Ha; Kang, Ji-Yeon; Kim, Dong-Hyun; Ahn, Taeho; Yun, Chul-Ho

    2012-11-01

    Cytochrome P450 BM3 (CYP102A1) from Bacillus megaterium is a self-sufficient monooxygenase that consists of a heme domain and FAD/FMN-containing reductase domain (BMR). In this report, the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) by BMR was evaluated as a method for monitoring BMR activity. The electron transfer proceeds from NADPH to BMR and then to BMR substrates, MTT and CTC. MTT and CTC are monotetrazolium salts that form formazans upon reduction. The reduction of MTT and CTC followed classical Michaelis-Menten kinetics (kcat =4120 min(-1), Km =77 μM for MTT and kcat =6580 min(-1), Km =51 μM for CTC). Our continuous assay using MTT and CTC allows the simple, rapid measurement of BMR activity. The BMR was able to metabolize mitomycin C and doxorubicin, which are anticancer drug substrates for CPR, producing the same metabolites as those produced by CPR. Moreover, the BMR was able to interact with CYP1A2 and transfer electrons to promote the oxidation reactions of substrates by CYP1A2 and CYP2E1 in humans. The results of this study suggest the possibility of the utilization of BMR as a surrogate for mammalian CPR.

  13. Effect of carbon source on the accumulation of cytochrome P-450 in the yeast Saccharomyces cerevisiae.

    OpenAIRE

    Kärenlampi, S O; Marin, E; Hänninen, O O

    1981-01-01

    The appearance of cytochrome P-450 in the yeast Saccharomyces cerevisiae depended on the substrate supporting growth. Cytochrome P-450 was apparent in yeast cells grown on a strongly fermentable sugar such as D-glucose, D-fructose or sucrose. When yeast was grown on D-galactose, D-mannose or maltose, where fermentation and respiration occurred concomitantly, cytochrome P-450 was also formed. The cytochrome P-450 concentration was maximal at the beginning of the stationary phase of the culture...

  14. Cytochrome P450 polymorphism and postoperative cognitive dysfunction

    DEFF Research Database (Denmark)

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

    2012-01-01

    neuropsychological testing at one week had POCD, and 24 out of 307 (7.8%) had POCD at three months. None of the examined CYP2C19, 2D6 alleles, or various phenotypes were significantly associated with POCD. CONCLUSION: Polymorphisms in CYP2C19, or 2D6 genes do not seem to be related to the occurrence of cognitive......BACKGROUND:The etiology of postoperative cognitive dysfunction (POCD) remains unclear but toxicity of anesthetic drugs and their metabolites could be important. We aimed to assess the possible association between POCD after propofol anesthesia and various phenotypes owing to polymorphisms...... in 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...

  15. Advances in molecular modeling of human cytochrome P450 polymorphism.

    Science.gov (United States)

    Martiny, Virginie Y; Miteva, Maria A

    2013-11-01

    Cytochrome P450 (CYP) is a supergene family of metabolizing enzymes involved in the phase I metabolism of drugs and endogenous compounds. CYP oxidation often leads to inactive drug metabolites or to highly toxic or carcinogenic metabolites involved in adverse drug reactions (ADR). During the last decade, the impact of CYP polymorphism in various drug responses and ADR has been demonstrated. Of the drugs involved in ADR, 56% are metabolized by polymorphic phase I metabolizing enzymes, 86% among them being CYP. Here, we review the major CYP polymorphic forms, their impact for drug response and current advances in molecular modeling of CYP polymorphism. We focus on recent studies exploring CYP polymorphism performed by the use of sequence-based and/or protein-structure-based computational approaches. The importance of understanding the molecular mechanisms related to CYP polymorphism and drug response at the atomic level is outlined. © 2013.

  16. P450 Pharmacogenetics in Indigenous North American Populations

    Directory of Open Access Journals (Sweden)

    Lindsay M. Henderson

    2018-02-01

    Full Text Available Indigenous North American populations, including American Indian and Alaska Native peoples in the United States, the First Nations, Métis and Inuit peoples in Canada and Amerindians in Mexico, are historically under-represented in biomedical research, including genomic research on drug disposition and response. Without adequate representation in pharmacogenetic studies establishing genotype-phenotype relationships, Indigenous populations may not benefit fully from new innovations in precision medicine testing to tailor and improve the safety and efficacy of drug treatment, resulting in health care disparities. The purpose of this review is to summarize and evaluate what is currently known about cytochrome P450 genetic variation in Indigenous populations in North America and to highlight the importance of including these groups in future pharmacogenetic studies for implementation of personalized drug therapy.

  17. 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.

  18. 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 und...... to serve as an effective electron transferring "nano-machine"....

  19. One-electron reduction of mitomycin c by rat liver : role of cytochrome P-450 and NADPH-cytochrome P-450 reductase

    NARCIS (Netherlands)

    Vromans, R M; Van de Straat, R; Groeneveld, M.; Vermeulen, N P

    1. The role of cytochrome P-450 in the one-electron reduction of mitomycin c was studied in rat hepatic microsomal systems and in reconstituted systems of purified cytochrome P-450. Formation of H2O2 from redox cycling of the reduced mitomycin c in the presence of O2 and the alkylation of

  20. Isolation of insecticide resistance-related forms of cytochrome P-450 from Drosophila melanogaster.

    OpenAIRE

    Sundseth, S S; Nix, C E; Waters, L C

    1990-01-01

    Significant purification of the ubiquitous cytochrome P-450-A and the strain-specific P-450-B from Drosophila melanogaster has been achieved by sequential chromatography on octylamino-agarose, DEAE-cellulose and hydroxyapatite. Preparations of P-450-A (specific contents of 7-9 nmol/mg) were homogeneous as determined by SDS/polyacrylamide-gel electrophoresis (PAGE) analysis. Preparations enriched for P-450-B (specific contents of 4-7 nmol/mg) contained significant amounts of P-450-A but were e...

  1. Reverse Conservation Analysis Reveals the Specificity Determining Residues of Cytochrome P450 Family 2 (CYP 2

    Directory of Open Access Journals (Sweden)

    Tai-Sung Lee

    2008-01-01

    Full Text Available The concept of conservation of amino acids is widely used to identify important alignment positions of orthologs. The assumption is that important amino acid residues will be conserved in the protein family during the evolutionary process. For paralog alignment, on the other hand, the opposite concept can be used to identify residues that are responsible for specificity. Assuming that the function-specific or ligand-specific residue positions will have higher diversity since they are under evolutionary pressure to fit the target specificity, these function-specific or ligand-specific residues positions will have a lower degree of conservation than other positions in a highly conserved paralog alignment. This study assessed the ability of reverse conservation analysis to identify function-specific and ligand-specific residue positions in closely related paralog. Reverse conservation analysis of paralog alignments successfully identified all six previously reported substrate recognition sites (SRSs in cytochrome P450 family 2 (CYP 2. Further analysis of each subfamily identified the specificity-determining residues (SDRs that have been experimentally found. New potential SDRs were also predicted and await confirmation by further experiments or modeling calculations. This concept may be also applied to identify SDRs in other protein families.

  2. Heterogeneous binding of sigma radioligands in the rat brain and liver; Possible relationship to subforms of cytochrome P-450

    Energy Technology Data Exchange (ETDEWEB)

    Ross, S B [Research Laboratories, Astra Research Centre AB, Soedertaejle (Sweden)

    1991-01-01

    The binding of four sigma receptor ligands, {sup 3}H-(+)-N-allyl-N-normetazocine ({sup 3}H-(+)-SKF 10,047), {sup 3}H-(+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine ({sup 3}H-(+)-3-PPP), {sup 3}H-haloperidol and {sup 3}H-N,N'-di(o-totyl)guanidine ({sup 3}H-DTG), and the cytochrome P450IID6 ligand and dopamine uptake inhibitor {sup 3}H-1-(2-(diphenylmethoxy)ethyl)-4-(3-phenylpropyl)piperazine ({sup 3}H-GBR 12935) to membranal preparations of rat liver or whole rat brain was examined regarding kinetical properties and inhibition by various compounds with affinity for sigma binding sites or cytochrome P-450. In rat brain the density of binding sites was increased in order (+)-SKF 10,047<(+)-3-PPPligand was similar in brain and liver. With the exception of {sup 3}H-(+)-SKF 10,047 there were quite marked differences between the ligands studied. Multiple binding sites were also indicated by the low Hill coefficients found for most of the compounds studied. It was found that the cytochrome P-450 inhibitor proadifen (SKF 525A), like haloperidol, was a potent inhibitor of the binding of {sup 3}H-(+)-SKF 10,047, {sup 3}H-(+)-3-PPP and {sup 3}H-haloperidol to the liver and brain preparations, less active in inhibiting the binding of {sup 3}H-DTG and least effective on the binding of {sup 3}H-GBR 12935. Another cytochrome P-450 inhibitor, L-lobeline, was particularly potent in inhibiting the binding of {sup 3}H-DTG but was also quite potent inhibitor of the binding of the other sigma ligands. It was less potent in inhibiting the binding of {sup 3}H-GBR 12935. The binding of the latter ligand was potently inhibited by the analogous compound GBR 12909 but of the other compounds examined only L-lobeline, proadifen, haloperidol, DTG and (+)-3-PPP had IC50 values below 10 {mu}M. (Abstract Truncated)

  3. Clinical Pharmacogenetics of Cytochrome P450-Associated Drugs in Children

    Directory of Open Access Journals (Sweden)

    Ida Aka

    2017-11-01

    Full Text Available Cytochrome P450 (CYP enzymes are commonly involved in drug metabolism, and genetic variation in the genes encoding CYPs are associated with variable drug response. While genotype-guided therapy has been clinically implemented in adults, these associations are less well established for pediatric patients. In order to understand the frequency of pediatric exposures to drugs with known CYP interactions, we compiled all actionable drug–CYP interactions with a high level of evidence using Clinical Pharmacogenomic Implementation Consortium (CPIC data and surveyed 10 years of electronic health records (EHR data for the number of children exposed to CYP-associated drugs. Subsequently, we performed a focused literature review for drugs commonly used in pediatrics, defined as more than 5000 pediatric patients exposed in the decade-long EHR cohort. There were 48 drug–CYP interactions with a high level of evidence in the CPIC database. Of those, only 10 drugs were commonly used in children (ondansetron, oxycodone, codeine, omeprazole, lansoprazole, sertraline, amitriptyline, citalopram, escitalopram, and risperidone. For these drugs, reports of the drug–CYP interaction in cohorts including children were sparse. There are adequate data for implementation of genotype-guided therapy for children for three of the 10 commonly used drugs (codeine, omeprazole and lansoprazole. For the majority of commonly used drugs with known CYP interactions, more data are required to support pharmacogenomic implementation in children.

  4. Regulation of the cytochrome P450 2A genes

    International Nuclear Information System (INIS)

    Su Ting; Ding Xinxin

    2004-01-01

    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

  5. Interaction of rocuronium with human liver cytochromes P450.

    Science.gov (United States)

    Anzenbacherova, Eva; Spicakova, Alena; Jourova, Lenka; Ulrichova, Jitka; Adamus, Milan; Bachleda, Petr; Anzenbacher, Pavel

    2015-02-01

    Rocuronium is a neuromuscular blocking agent acting as a competitive antagonist of acetylcholine. Results of an inhibition of eight individual liver microsomal cytochromes P450 (CYP) are presented. As the patients are routinely premedicated with diazepam, possible interaction of diazepam with rocuronium has been also studied. Results indicated that rocuronium interacts with human liver microsomal CYPs by binding to the substrate site. Next, concentration dependent inhibition of liver microsomal CYP3A4 down to 42% (at rocuronium concentration 189 μM) was found. This effect has been confirmed with two CYP3A4 substrates, testosterone (formation of 6β-hydroxytestosterone) and diazepam (temazepam formation). CYP2C9 and CYP2C19 activities were inhibited down to 75-80% (at the same rocuronium concentration). Activities of other microsomal CYPs have not been inhibited by rocuronium. To prove the possibility of rocuronium interaction with other drugs (diazepam), the effect of rocuronium on formation of main diazepam metabolites, temazepam (by CYP3A4) and desmethyldiazepam, (also known as nordiazepam; formed by CYP2C19) in primary culture of human hepatocytes has been examined. Rocuronium has caused inhibition of both reactions by 20 and 15%, respectively. The results open a possibility that interactions of rocuronium with drugs metabolized by CYP3A4 (and possibly also CYP2C19) may be observed. Copyright © 2014 Japanese Pharmacological Society. Production and hosting by Elsevier B.V. All rights reserved.

  6. Polycyclic aromatic hydrocarbons and cytochrome P450 in HIV pathogenesis

    Science.gov (United States)

    Rao, P. S. S.; Kumar, Santosh

    2015-01-01

    High prevalence of cigarette smoking in HIV patients is associated with increased HIV pathogenesis and disease progression. While the effect of smoking on the occurrence of lung cancer has been studied extensively, the association between smoking and HIV pathogenesis is poorly studied. We have recently shown the possible role of cytochrome P450 (CYP) in smoking/nicotine-mediated viral replication. In this review, we focus on the potential role of CYP pathway in polycyclic aromatic hydrocarbons (PAH), important constituents of cigarette smoke, mediated HIV pathogenesis. More specifically, we will discuss the role of CYP1A1 and CYP1B1, which are the major PAH-activating CYP enzymes. Our results have shown that treatment with cigarette smoke condensate (CSC) increases viral replication in HIV-infected macrophages. CSC contains PAH, which are known to be activated by CYP1A1 and CYP1B1 into procarcinogens/toxic metabolites. The expression of these CYPs is regulated by aryl hydrocarbon receptors (AHR), the cellular target of PAH, and an important player in various diseases including cancer. We propose that PAH/AHR-mediated CYP pathway is a novel target to develop new interventions for HIV positive smokers. PMID:26082767

  7. 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)

    Skoda, R.C.; Gonzalez, F.L.; Demierre, A.; Meyer, R.A.

    1988-01-01

    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

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

    International Nuclear Information System (INIS)

    Hernandez, J.P.; Mota, L.C.; Huang, W.; Moore, D.D.; Baldwin, W.S.

    2009-01-01

    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

  9. Cytochrome P450s--Their expression, regulation, and role in insecticide resistance.

    Science.gov (United States)

    Liu, Nannan; Li, Ming; Gong, Youhui; Liu, Feng; Li, Ting

    2015-05-01

    P450s are known to be critical for the detoxification and/or activation of xenobiotics such as drugs and pesticides and overexpression of P450 genes can significantly affect the disposition of xenobiotics in the tissues of organisms, altering their pharmacological/toxicological effects. In insects, P450s play an important role in detoxifying exogenous compounds such as insecticides and plant toxins and their overexpression can result in increased levels of P450 proteins and P450 activities. This has been associated with enhanced metabolic detoxification of insecticides and has been implicated in the development of insecticide resistance in insects. Multiple P450 genes have been found to be co-overexpressed in individual insect species via several constitutive overexpression and induction mechanisms, which in turn are co-responsible for high levels of insecticide resistance. Many studies have also demonstrated that the transcriptional overexpression of P450 genes in resistant insects is regulated by trans and/or cis regulatory genes/factors. Taken together, these earlier findings suggest not only that insecticide resistance is conferred via multi-resistance P450 genes, but also that it is mediated through the interaction of regulatory genes/factors and resistance genes. This chapter reviews our current understanding of how the molecular mechanisms of P450 interaction/gene regulation govern the development of insecticide resistance in insects and our progress along the road to a comprehensive characterization of P450 detoxification-mediated insecticide resistance. Copyright © 2015 Elsevier Inc. All rights reserved.

  10. Certain tryptophan photoproducts are inhibitors of cytochrome P450-dependent mutagenicity

    International Nuclear Information System (INIS)

    Rannug, U.; Agurell, E.; Cederberg, H.; Rannug, A.

    1992-01-01

    Two photoproducts, derived from UV-irradiation of the amino acid L-tryptophan and with high Ah (TCDD) receptor binding affinity, were tested for genotoxic and antimutagenic effects. The two indolo[3,2-b]carbazole derivatives, with the molecular weights of 284 and 312, respectively, were tested in Saccharomyces cerevisiae strain D7 for mitotic gene conversion and reverse mutation and in strain RS112 for sister chromatid conversion and gene conversion. No significant (P > 0.05) genotoxic effects were found in strain D7, while strain RS112 showed a small but significant increase in the frequency of sister chromatid conversions. In Chinese hamster ovary (CHO) cells the two compounds induced a statistically significant but less than twofold increase in the frequency of sister chromatid exchanges (SCE). No mutations were detected when the compounds were tested in Salmonella tphimurium strains TA98 and TA100. However, both 284 and 312 acted as antimutagens on strain TA100+S9 in the presence of benzo(a)pyrene. The decrease in mutagenicity by the most potent compound 284 was 20 revertants/nmol. This effect could be explained by an inhibitory effect on the cytochrome P450-dependent ethoxyresorufin O-deethylase (EROD) activity as seen in rat hepatocytes. The two compounds were also tested with hamster cells expressing rat cytochrome P-4501A1. The results support the conclusion that this cytochrome P-450 isozyme is inhibited by the tryptophan photoproducts. Similar results were also seen with two other high affinity Ah receptor ligands the quinazolinocarboline alkaloids rutaecapine and dehydrorutaecarpine. 20 refs., 3 figs., 4 tabs

  11. Relative contribution of rat cytochrome P450 isoforms to the metabolism of caffeine: the pathway and concentration dependence.

    Science.gov (United States)

    Kot, Marta; Daniel, Władysława A

    2008-04-01

    The aim of the present study was to estimate the relative contribution of rat P450 isoforms to the metabolism of caffeine and to assess the usefulness of caffeine as a marker substance for estimating the activity of P450 in rat liver and its potential for pharmacokinetic interactions in pharmacological experiments. The results obtained using rat cDNA-expressed P450s indicated that 8-hydroxylation was the main oxidation pathway of caffeine (70%) in the rat. CYP1A2 was found to be a key enzyme catalyzing 8-hydroxylation (72%) and substantially contributing to 3-N-demethylation (47%) and 1-N-demethylation (37.5%) at a caffeine concentration of 0.1mM (relevant to "the maximum therapeutic concentration in humans"). Furthermore, CYP2C11 considerably contributed to 3-N-demethylation (31%). The CYP2C subfamily (66%) - mainly CYP2C6 (27%) and CYP2C11 (29%) - played a major role in catalyzing 7-N-demethylation. At higher substrate concentrations, the contribution of CYP1A2 to the metabolism of caffeine decreased in favor of CYP2C11 (N-demethylations) and CYP3A2 (mainly 8-hydroxylation). The obtained results were confirmed with liver microsomes (inhibition and correlation studies). Therefore, caffeine may be used as a marker substance for assessing the activity of CYP1A2 in rats, using 8-hydroxylation (but not 3-N-demethylation-like in humans); moreover, caffeine may also be used to simultaneously, preliminarily estimate the activity of CYP2C using 7-N-demethylation as a marker reaction. Hence caffeine pharmacokinetics in rats may be changed by drugs affecting the activity of CYP1A2 and/or CYP2C, e.g. by some antidepressants.

  12. P450 reductase and cytochrome b5 interactions with cytochrome P450: Effects on house fly CYP6A1 catalysis

    OpenAIRE

    Murataliev, Marat B.; Guzov, Victor M.; Walker, F. Ann; Feyereisen, René

    2008-01-01

    The interactions of protein components of the xenobiotic-metabolizing cytochrome P450 system, CYP6A1, P450 reductase, and cytochrome b5 from the house fly (Musca domestica) have been characterized. CYP6A1 activity is determined by the concentration of the CYP6A1-P450 reductase complex, regardless of which protein is present in excess. Both holo- and apo-b5 stimulated CYP6A1 heptachlor epoxidase and steroid hydroxylase activities and influenced the regioselectivity of testosterone hydroxylatio...

  13. Site-Specific Characterization of Cytochrome P450cam Conformations by Infrared Spectroscopy.

    Science.gov (United States)

    Basom, Edward J; Maj, Michał; Cho, Minhaeng; Thielges, Megan C

    2016-06-21

    Conformational changes are central to protein function but challenging to characterize with both high spatial and temporal precision. The inherently fast time scale and small chromophores of infrared (IR) spectroscopy are well-suited for characterization of potentially rapidly fluctuating environments, and when frequency-resolved probes are incorporated to overcome spectral congestion, enable characterization of specific sites in proteins. We selectively incorporated p-cyanophenylalanine (CNF) as a vibrational probe at five distinct locations in the enzyme cytochrome P450cam and used IR spectroscopy to characterize the environments in substrate and/or ligand complexes reflecting those in the catalytic cycle. Molecular dynamics (MD) simulations were performed to provide a structural basis for spectral interpretation. Together the experimental and simulation data suggest that the CN frequencies are sensitive to both long-range influences, resulting from the particular location of a residue within the enzyme, as well as short-range influences from hydrogen bonding and packing interactions. The IR spectra demonstrate that the environments and effects of substrate and/or ligand binding are different at each position probed and also provide evidence that a single site can experience multiple environments. This study illustrates how IR spectroscopy, when combined with the spectral decongestion and spatial selectivity afforded by CNF incorporation, provides detailed information about protein structural changes that underlie function.

  14. Characterization of the human cytochrome P450 enzymes involved in the metabolism of dihydrocodeine

    Science.gov (United States)

    Kirkwood, L. C.; Nation, R. L.; Somogyi, A. A.

    1997-01-01

    Aims Using human liver microsomes from donors of the CYP2D6 poor and extensive metabolizer genotypes, the role of individual cytochromes P-450 in the oxidative metabolism of dihydrocodeine was investigated. Methods The kinetics of formation of N- and O-demethylated metabolites, nordihydrocodeine and dihydromorphine, were determined using microsomes from six extensive and one poor metabolizer and the effects of chemical inhibitors selective for individual P-450 enzymes of the 1A, 2A, 2C, 2D, 2E and 3A families and of LKM1 (anti-CYP2D6) antibodies were studied. Results Nordihydrocodeine was the major metabolite in both poor and extensive metabolizers. Kinetic constants for N-demethylation derived from the single enzyme Michaelis-Menten model did not differ between the two groups. Troleandomycin and erythromycin selectively inhibited N-demethylation in both extensive and poor metabolizers. The CYP3A inducer, α-naphthoflavone, increased N-demethylation rates. The kinetics of formation of dihydromorphine in both groups were best described by a single enzyme Michaelis-Menten model although inhibition studies in extensive metabolizers suggested involvement of two enzymes with similar Km values. The kinetic constants for O-demethylation were significantly different in extensive and poor metabolizers. The extensive metabolizers had a mean intrinsic clearance to dihydromorphine more than ten times greater than the poor metabolizer. The CYP2D6 chemical inhibitors, quinidine and quinine, and LKM1 antibodies inhibited O-demethylation in extensive metabolizers; no effect was observed in microsomes from a poor metabolizer. Conclusions CYP2D6 is the major enzyme mediating O-demethylation of dihydrocodeine to dihydromorphine. In contrast, nordihydrocodeine formation is predominantly catalysed by CYP3A. PMID:9431830

  15. Cytochrome P450-Dependent Metabolism of Caffeine in Drosophila melanogaster

    Science.gov (United States)

    Coelho, Alexandra; Fraichard, Stephane; Le Goff, Gaëlle; Faure, Philippe; Artur, Yves; Ferveur, Jean-François; Heydel, Jean-Marie

    2015-01-01

    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. PMID:25671424

  16. Regulation of cytochrome P-450 monooxygenases in the mouse

    International Nuclear Information System (INIS)

    Kelley, M.F.

    1986-01-01

    Recently, the compound 1,4-bis[2-(3,4-dichloropyridyloxy)] benzene (TCPOBOP) has been identified as a highly potent phenobabital-like agonist in mice. This finding has led to the suggestion that a receptor-mediated process may govern the induction of cytochrome P-450 monooxygenases by phenobarbital and phenobarbital-like agonists. This dissertation examines: (1) the effects of structural alterations of the TCPOBOP molecule on enzyme induction activity, (2) the induction response to phenobarbital and TCPOBOP among inbred mouse strains, (3) the spectrum of monooxygenase activities induced by phenobarbital and TCPOBOP compared to 3-methylcholanthrene, isosafrole and pregnenolone 16α-carbonitrile (PCN) and (4) the binding of [ 3 H] TCPOBOP in hepatic cytosol. Changes in the structure of the pyridyloxy or benzene rings markedly affect enzyme induction activity and provide additional indirect evidence for a receptor-mediated response. An evaluation of monooxygenase induction by TCPOBOP for 27 inbred mouse strains and by phenobarbital for 15 inbred mouse strains failed to identify a strain which was completely nonresponsive to these compounds, although several strains exhibited decreased responsiveness for select monooxygenase reactions. TCPOBOP, PCN and phenobarbital were all found to significantly increase the rate of hydroxylation of testosterone at the 2α-, 6β- and 15β- positions but only TCPOBOP and phenobarbital dramatically increased the rate of pentoxyresorufin O-dealkylation. The results demonstrates that TCPOBOP most closely resembles phenobarbital in its mode of monooxygenase induction in mice. Sucrose density gradient analysis of [ 3 H] TCPOBOP-hepatic cytosol incubations failed to identify specific, saturable binding of [ 3 H] TCPOBOP to cytosolic marcomolecular elements

  17. 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.

  18. Functional characterisation of an engineered multidomain human P450 2E1 by molecular Lego.

    Science.gov (United States)

    Fairhead, Michael; Giannini, Silva; Gillam, Elizabeth M J; Gilardi, Gianfranco

    2005-12-01

    The human cytochrome P450s constitute an important family of monooxygenase enzymes that carry out essential roles in the metabolism of endogenous compounds and foreign chemicals. We present here results of a fusion between a human P450 enzyme and a bacterial reductase that for the first time is shown does not require the addition of lipids or detergents to achieve wild-type-like activities. The fusion enzyme, P450 2E1-BMR, contains the N-terminally modified residues 22-493 of the human P450 2E1 fused at the C-terminus to residues 473-1049 of the P450 BM3 reductase (BMR). The P450 2E1-BMR enzyme is active, self-sufficient and presents the typical marker activities of the native human P450 2E1: the hydroxylation of p-nitrophenol (KM=1.84+/-0.09 mM and kcat of 2.98+/-0.04 nmol of p-nitrocatechol formed per minute per nanomole of P450) and chlorzoxazone (KM=0.65+/-0.08 mM and kcat of 0.95+/-0.10 nmol of 6-hydroxychlorzoxazone formed per minute per nanomole of P450). A 3D model of human P450 2E1 was generated to rationalise the functional data and to allow an analysis of the surface potentials. The distribution of charges on the model of P450 2E1 compared with that of the FMN domain of BMR provides the ground for the understanding of the interaction between the fused domains. The results point the way to successfully engineer a variety of catalytically self-sufficient human P450 enzymes for drug metabolism studies in solution.

  19. 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)

    Liu, Senyan; Yao, Yunyi; Lu, Shijun; Aldous, Kenneth; Ding, Xinxin; Mei, Changlin; Gu, Jun

    2013-01-01

    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

  20. Immunohistochemical detection of cytochrome P450 isoenzymes in cultured human epidermal cells.

    Science.gov (United States)

    Van Pelt, F N; Meierink, Y J; Blaauboer, B J; Weterings, P J

    1990-12-01

    We used specific monoclonal antibodies (MAb) to human cytochrome P450 isoenzymes to determine the presence of these proteins in human epidermal cells. Two MAb (P450-5 and P450-8) recognize major forms of hepatic cytochrome P450 involved in biotransformation of xenobiotics. A third MAb, to cytochrome P450-9, is not fully characterized. The proteins were determined by the indirect immunoperoxidase technique after fixation with methanol and acetone. Biopsy materials for cultured keratinocytes, i.e., foreskin and hair follicles, contained the two major forms of cytochrome P450. In cultured keratinocytes derived from hair follicles the proteins were undetectable, whereas the keratinocytes derived from foreskin continued to express the two major forms of hepatic cytochrome P450. Cultured human fibroblasts and a human keratinocyte cell line (SVK14) showed staining similar to that of the foreskin keratinocytes. Cytochrome P450-9 was detectable only in human hepatocytes. The results indicate that, under the culture conditions applied, cultured human foreskin cells and the cell line SVK14 continue to express specific cytochrome P450 isoenzymes in culture, in contrast to hair follicle keratinocytes.

  1. 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.

  2. Inhibitory effects of kale ingestion on metabolism by cytochrome P450 enzymes in rats.

    Science.gov (United States)

    Yamasaki, Izumi; Yamada, Masayoshi; Uotsu, Nobuo; Teramoto, Sachiyuki; Takayanagi, Risa; Yamada, Yasuhiko

    2012-01-01

    Kale (Brassica oleracea L. var acephala DC) is a leafy green vegetable belonging to the cabbage family (Brassicaceae) that contains a large amount of health-promoting phytochemicals. There are any reports about the effects of kale ingestion on the chemoprevention function and mechanism, but the interactions between kale and drugs have not been researched. We investigated the effects of kale intake on cytochrome P450 (CYP) metabolism by using cocktail probe drugs, including midazolam (for CYP3A4), caffeine (for CYP1A2), dextromethorphan (for CYP2D6), tolbutamide (for CYP2C9), omeprazole (for CYP2C19), and chlorzoxazone (for CYP2E1). Cocktail drugs were administered into rats treated with kale and cabbage (2000 mg/kg) for a week. The results showed that kale intake induced a significant increase in plasma levels and the AUC of midazolam, caffeine, and dextromethorphan. In addition, the plasma concentration and AUC of omeprazole tended to increase. Additionally, no almost differences in the mRNA expression levels of CYP enzymes in the liver were observed. In conclusion, kale ingestion was considered to have an inhibitory effect on the activities of CYP3A4, 1A2, 2D6, and 2C19 for a reason competitive inhibition than inhibitory changes in the mRNA expressions.

  3. Regional specificity in deltamethrin induced cytochrome P450 expression in rat brain

    International Nuclear Information System (INIS)

    Yadav, Sanjay; Johri, Ashu; Dhawan, Alok; Seth, Prahlad K.; Parmar, Devendra

    2006-01-01

    Oral administration of deltamethrin (5 mg/kg x 7 or 15 or 21 days) was found to produce a time-dependent increase in the mRNA expression of xenobiotic metabolizing cytochrome P450 1A1 (CYP1A1), 1A2 and CYP2B1, 2B2 isoenzymes in rat brain. RT-PCR studies further showed that increase in the mRNA expression of these CYP isoenzymes observed after 21 days of exposure was region specific. Hippocampus exhibited maximum increase in the mRNA expression of CYP1A1, which was followed by pons-medulla, cerebellum and hypothalamus. The mRNA expression of CYP2B1 also exhibited maximum increase in the hypothalamus and hippocampus followed by almost similar increase in midbrain and cerebellum. In contrast, mRNA expression of CYP1A2 and CYP2B2, the constitutive isoenzymes exhibited relatively higher increase in pons-medulla, cerebellum and frontal cortex. Immunoblotting studies carried out with polyclonal antibody raised against rat liver CYP1A1/1A2 or CYP2B1/2B2 isoenzymes also showed increase in immunoreactivity comigrating with CYP1A1/1A2 or 2B1/2B2 in the microsomal fractions isolated from hippocampus, hypothalamus and cerebellum of rat treated with deltamethrin. Though the exact relationship of the xenobiotic metabolizing CYPs with the physiological function of the brain is yet to be clearly understood, the increase in the mRNA expression of the CYPs in the brain regions that regulate specific brain functions affected by deltamethrin have further indicated that modulation of these CYPs could be associated with the various endogenous functions of the brain

  4. Inhibitory and inductive effects of Phikud Navakot extract on human cytochrome P450.

    Science.gov (United States)

    Chiangsom, Abhiruj; Lawanprasert, Somsong; Oda, Shingo; Kulthong, Kornphimol; Luechapudiporn, Rataya; Yokoi, Tsuyoshi; Maniratanachote, Rawiwan

    2016-06-01

    Effects of the hydroethanolic extract of Phikud Navakot (PN), a Thai traditional remedy, on human cytochrome P450s (CYPs) were investigated in vitro. Selective substrates of CYPs were used to investigate the effects and kinetics of PN on CYP inhibition using human liver microsomes. Primary human hepatocytes were used to assess the inductive effects of PN on CYP enzyme activities and protein expressions. The results showed that PN inhibited the activities of CYP1A2, CYP2C9, CYP2D6, and CYP3A4 with half maximal inhibitory concentration (IC50) values of 13, 62, 67, and 88 μg/mL, respectively. Meanwhile, it had no effect on the activities of CYP2C19 and CYP2E1 (IC50 > 1 mg/mL). PN exhibited competitive inhibition of CYP1A2 (Ki = 34 μg/mL), mixed type inhibition of CYP2C9 and CYP2D6 (Ki = 80 and 12 μg/mL, respectively), and uncompetitive inhibition of CYP3A4 (Ki = 150 μg/mL). PN did not have an inductive effect on CYP1A2, CYP2C9, CYP2C19 and CYP3A4 in primary human hepatocytes, which is an advantageous characteristic of the extract. However the extract may cause herb-drug interactions via inhibition of CYP1A2, CYP2C9, CYP2D6 and CYP3A4, and precautions should be taken when PN is coadministered with drugs that are metabolized by these CYP enzymes. Copyright © 2016 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.

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

    Science.gov (United States)

    Sun, Jie; Jin, Chunlan; Wu, Huangan; Zhao, Jimeng; Cui, Yunhua; Liu, Huirong; Wu, Lingxiang; Shi, Yin; Zhu, Bing

    2013-01-01

    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 and

  6. Effects of Electro-Acupuncture on Ovarian P450arom, P450c17α and mRNA Expression Induced by Letrozole in PCOS Rats

    Science.gov (United States)

    Wu, Huangan; Zhao, Jimeng; Cui, Yunhua; Liu, Huirong; Wu, Lingxiang; Shi, Yin; Zhu, Bing

    2013-01-01

    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 and

  7. 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

  8. Roles of Human CYP2A6 and Monkey CYP2A24 and 2A26 Cytochrome P450 Enzymes in the Oxidation of 2,5,2',5'-Tetrachlorobiphenyl.

    Science.gov (United States)

    Shimada, Tsutomu; Kakimoto, Kensaku; Takenaka, Shigeo; Koga, Nobuyuki; Uehara, Shotaro; Murayama, Norie; Yamazaki, Hiroshi; Kim, Donghak; Guengerich, F Peter; Komori, Masayuki

    2016-12-01

    2,5,2',5'-Tetrachlorobiphenyl (TCB) induced type I binding spectra with cytochrome P450 (P450) 2A6 and 2A13, with K s values of 9.4 and 0.51 µM, respectively. However, CYP2A6 oxidized 2,5,2',5'-TCB to form 4-hydroxylated products at a much higher rate (∼1.0 minute -1 ) than CYP2A13 (∼0.02 minute -1 ) based on analysis by liquid chromatography-tandem mass spectrometry. Formation of 4-hydroxy-2,5,2',5'-TCB by CYP2A6 was greater than that of 3-hydroxy-2,5,2',5'-TCB and three other hydroxylated products. Several human P450 enzymes, including CYP1A1, 1A2, 1B1, 2B6, 2D6, 2E1, 2C9, and 3A4, did not show any detectable activities in oxidizing 2,5,2',5'-TCB. Cynomolgus monkey CYP2A24, which shows 95% amino acid identity to human CYP2A6, catalyzed 4-hydroxylation of 2,5,2',5'-TCB at a higher rate (∼0.3 minute -1 ) than CYP2A26 (93% identity to CYP2A6, ∼0.13 minute -1 ) and CYP2A23 (94% identity to CYP2A13, ∼0.008 minute -1 ). None of these human and monkey CYP2A enzymes were catalytically active in oxidizing other TCB congeners, such as 2,4,3',4'-, 3,4,3',4'-, and 3,5,3',5'-TCB. Molecular docking analysis suggested that there are different orientations of interaction of 2,5,2',5'-TCB with the active sites (over the heme) of human and monkey CYP2A enzymes, and that ligand interaction energies (U values) of bound protein-ligand complexes show structural relationships of interaction of TCBs and other ligands with active sites of CYP2A enzymes. Catalytic differences in human and monkey CYP2A enzymes in the oxidation of 2,5,2',5'-TCB are suggested to be due to amino acid changes at substrate recognition sites, i.e., V110L, I209S, I300F, V365M, S369G, and R372H, based on the comparison of primary sequences. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

  9. Effects of curcumin on cytochrome P450 and glutathione S-transferase activities in rat liver.

    NARCIS (Netherlands)

    Oetari, S.; Sudibyo, M.; Commandeur, J.N.M.; Samhoedi, R.; Vermeulen, N.P.E.

    1996-01-01

    The stability of curcumin, as well as the interactions between curcumin and cytochrome P450s (P450s) and glutathione S-transferases (GSTs) in rat liver, were studied. Curcumin is relatively unstable in phosphate buffer at pH 7.4. The stability of curcumin was strongly improved by lowering the pH or

  10. CYTOCHROME P450-DEPENDENT METABOLISM OF TRICHLOROETHYLENE IN THE RAT KIDNEY

    Science.gov (United States)

    The metabolism of trichloroethylene (Tri) by cytochrome P450 (P450) was studied in microsomes from liver and kidney homogenates and from isolated renal proximal tubular (PT) and distal tubular (DT) cells from male Fischer 344 rats. Chloral hydrate (CH) was the only metabolite con...

  11. Effect of carbon source on the accumulation of cytochrome P-450 in the yeast Saccharomyces cerevisiae.

    Science.gov (United States)

    Kärenlampi, S O; Marin, E; Hänninen, O O

    1981-02-15

    The appearance of cytochrome P-450 in the yeast Saccharomyces cerevisiae depended on the substrate supporting growth. Cytochrome P-450 was apparent in yeast cells grown on a strongly fermentable sugar such as D-glucose, D-fructose or sucrose. When yeast was grown on D-galactose, D-mannose or maltose, where fermentation and respiration occurred concomitantly, cytochrome P-450 was also formed. The cytochrome P-450 concentration was maximal at the beginning of the stationary phase of the culture. Thereafter the concentration decreased, reaching zero at a late-stationary phase. When the yeast was grown on a medium that contained lactose or pentoses (L-arabinose, L-rhamnose, D-ribose and D-xylose), cytochrome P-450 did not occur. When a non-fermentable energy source (glycerol, lactate or ethanol) was used, no cytochrome P-450 was detectable. Transfer of cells from D-glucose medium to ethanol medium caused a slow disappearance of cytochrome P-450, although the amount of the haemoprotein still continued to increase in the control cultures. Cytochrome P-450 appeared thus to accumulate in conditions where the rate of growth was fast and fermentation occurred. Occurrence of this haemoprotein is not necessarily linked, however, with the repression of mitochondrial haemoprotein synthesis.

  12. Pyrethroid activity-based probes for profiling cytochrome P450 activities associated with insecticide interactions.

    Science.gov (United States)

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

    2013-12-03

    Pyrethroid insecticides are used to control 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 nonmetabolizing mosquito P450s, as well as rodent microsomes, to measure labeling specificity, plus cytochrome P450 oxidoreductase and b5 knockout mouse livers to validate P450 activation and establish the role for b5 in probe activation. Using PyABPs, we were able to profile active enzymes in rat liver microsomes and identify pyrethroid-metabolizing enzymes in the target tissue. These included P450s as well as related detoxification enzymes, notably UDP-glucuronosyltransferases, suggesting a network of associated pyrethroid-metabolizing enzymes, or "pyrethrome." Considering the central role P450s play in metabolizing insecticides, we anticipate that PyABPs will aid in 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 unique tools for disease control.

  13. Cytochromes P450 for natural product biosynthesis in Streptomyces: sequence, structure, and function.

    Science.gov (United States)

    Rudolf, Jeffrey D; Chang, Chin-Yuan; Ma, Ming; Shen, Ben

    2017-08-30

    Covering: up to January 2017Cytochrome P450 enzymes (P450s) are some of the most exquisite and versatile biocatalysts found in nature. In addition to their well-known roles in steroid biosynthesis and drug metabolism in humans, P450s are key players in natural product biosynthetic pathways. Natural products, the most chemically and structurally diverse small molecules known, require an extensive collection of P450s to accept and functionalize their unique scaffolds. In this review, we survey the current catalytic landscape of P450s within the Streptomyces genus, one of the most prolific producers of natural products, and comprehensively summarize the functionally characterized P450s from Streptomyces. A sequence similarity network of >8500 P450s revealed insights into the sequence-function relationships of these oxygen-dependent metalloenzymes. Although only ∼2.4% and structurally characterized, respectively, the study of streptomycete P450s involved in the biosynthesis of natural products has revealed their diverse roles in nature, expanded their catalytic repertoire, created structural and mechanistic paradigms, and exposed their potential for biomedical and biotechnological applications. Continued study of these remarkable enzymes will undoubtedly expose their true complement of chemical and biological capabilities.

  14. Relationship between hydrocarbon structure and induction of P450: effects on protein levels and enzyme activities.

    Science.gov (United States)

    Backes, W L; Sequeira, D J; Cawley, G F; Eyer, C S

    1993-12-01

    1. Treatment of male rat with the small aromatic hydrocarbons, benzene, toluene, ethylbenzene, n-propylbenzene, m-xylene, and p-xylene increased several P450-dependent activities, with ethylbenzene, m-xylene, and n-propylbenzene producing the greatest response. Hydrocarbon treatment differentially affected toluene metabolism, producing a response dependent on the metabolite monitored. In untreated rats, benzyl alcohol was the major hydroxylation product of toluene metabolism, comprising > 99% of the total metabolites formed. Hydrocarbon treatment increased the overall rate of toluene metabolism by dramatically increasing the amount of aromatic hydroxylation. Ethylbenzene, n-propylbenzene and m-xylene were the most effective inducers of aromatic hydroxylation of toluene. In contrast, production of the major toluene metabolite benzyl alcohol was increased only after treatment with m-xylene. 2. P450 2B1/2B2 levels were induced by each of the hydrocarbons examined, with the magnitude of induction increasing with increasing hydrocarbon size. P450 1A1 was also induced after hydrocarbon exposure; however, the degree of induction was smaller than that observed for P450 2B1/2B2. P450 2C11 levels were suppressed after treatment with benzene, ethylbenzene and n-propylbenzene. 3. Taken together these results display two induction patterns. The first generally corresponds to changes in the P450 2B subfamily, where activities (e.g. the aromatic hydroxylations of toluene) were most effectively induced by ethylbenzene, n-propylbenzene and m-xylene. In the second, induction was observed only after m-xylene treatment, a pattern that was found when the metabolism of the substrate was catalysed by both the P450 2B subfamily and P450 2C11. Hydrocarbons that both induced P450 2B1/2B2 and suppressed P450 2C11 (such as ethylbenzene and n-propylbenzene) showed little change in activities catalysed by both isozymes (e.g. aliphatic hydroxylation of toluene, and aniline hydroxylation

  15. 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 11-fold higher in 766b than in WHO-SRS. Significant differences between expression of P450 genes between F1 flies from 845b and established laboratory strains were shown. In general, P450 gene expression in 845b was 2–14-fold higher than in the reference strain (P

  16. 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.

  17. Cytochrome P450 CYP1A1: wider roles in cancer progression and prevention

    International Nuclear Information System (INIS)

    Androutsopoulos, Vasilis P; Tsatsakis, Aristidis M; Spandidos, Demetrios A

    2009-01-01

    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

  18. Catalytic diversity and homotropic allostery of two Cytochrome P450 monooxygenase like proteins from Trichoderma brevicompactum.

    Science.gov (United States)

    Hussain, Razak; Kumari, Indu; Sharma, Shikha; Ahmed, Mushtaq; Khan, Tabreiz Ahmad; Akhter, Yusuf

    2017-12-01

    Trichothecenes are the secondary metabolites produced by Trichoderma spp. Some of these molecules have been reported for their ability to stimulate plant growth by suppressing plant diseases and hence enabling Trichoderma spp. to be efficiently used as biocontrol agents in modern agriculture. Many of the proteins involved in the trichothecenes biosynthetic pathway in Trichoderma spp. are encoded by the genes present in the tri cluster. Tri4 protein catalyzes three consecutive oxygenation reaction steps during biosynthesis of isotrichodiol in the trichothecenes biosynthetic pathway, while tri11 protein catalyzes the C4 hydroxylation of 12, 13-epoxytrichothec-9-ene to produce trichodermol. In the present study, we have homology modelled the three-dimensional structures of tri4 and tri11 proteins. Furthermore, molecular dynamics simulations were carried out to elucidate the mechanism of their action. Both tri4 and tri11 encode for cytochrome P450 monooxygenase like proteins. These data also revealed effector-induced allosteric changes on substrate binding at an alternative binding site and showed potential homotropic negative cooperativity. These analyses also showed that their catalytic mechanism relies on protein-ligand and protein-heme interactions controlled by hydrophobic and hydrogen-bonding interactions which orient the complex in optimal conformation within the active sites.

  19. Virtual screening for cytochromes p450: successes of machine learning filters.

    Science.gov (United States)

    Burton, Julien; Ijjaali, Ismail; Petitet, François; Michel, André; Vercauteren, Daniel P

    2009-05-01

    Cytochromes P450 (CYPs) are crucial targets when predicting the ADME properties (absorption, distribution, metabolism, and excretion) of drugs in development. Particularly, CYPs mediated drug-drug interactions are responsible for major failures in the drug design process. Accurate and robust screening filters are thus needed to predict interactions of potent compounds with CYPs as early as possible in the process. In recent years, more and more 3D structures of various CYP isoforms have been solved, opening the gate of accurate structure-based studies of interactions. Nevertheless, the ligand-based approach still remains popular. This success can be explained by the growing number of available data and the satisfying performances of existing machine learning (ML) methods. The aim of this contribution is to give an overview of the recent achievements in ML applications to CYP datasets. Particularly, popular methods such as support vector machine, decision trees, artificial neural networks, k-nearest neighbors, and partial least squares will be compared as well as the quality of the datasets and the descriptors used. Consensus of different methods will also be discussed. Often reaching 90% of accuracy, the models will be analyzed to highlight the key descriptors permitting the good prediction of CYPs binding.

  20. 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.

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

    International Nuclear Information System (INIS)

    Kaspera, Rüdiger; Sahele, Tariku; Lakatos, Kyle; Totah, Rheem A.

    2012-01-01

    Highlights: ► Cytochrome P450BM-3 reduced aldehydes to alcohols efficiently (k cat ∼ 25 min −1 ). ► Reduction is a direct hydride transfer from R-NADP 2 H to the carbonyl moiety. ► P450 domain variants enhance reduction through potential allosteric/redox interactions. ► Novel reaction will have implications for metabolism of xenobiotics. -- Abstract: Cytochrome P450BM-3 catalyzed the reduction of lipophilic aldehydes to alcohols efficiently. A k cat of ∼25 min −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 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 2 H but not D 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.

  2. 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. Copyright © 2012 Elsevier Inc. All rights reserved.

  3. 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.

  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.

  5. Effects of Pristane on Cytochrome P450 Isozyme Expression in Rat Tissues

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    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. Carbonated soft drinks alter hepatic cytochrome P450 isoform expression in Wistar rats.

    Science.gov (United States)

    Alkhedaide, Adel; Soliman, Mohamed Mohamed; Ibrahim, Zein Shaban

    2016-11-01

    The aim of the current study was to examine the effects of chronic consumption of soft drinks (SDs) on hepatic oxidative stress and cytochrome P450 enzymes (CYPs) expression in the livers of Wistar rats. For 3 consecutive months, the rats had free access to three different soft drinks, Coca-Cola, Pepsi-Cola and 7-UP. The rats were subsequently compared with control group rats that had consumed water. Blood and hepatic tissue samples were assayed for the changes in antioxidants, liver function biomarkers and hepatic gene expression for different isoforms of hepatic CYP. The results indicated that SD consumption (SDC) decreased serum antioxidant levels and increased malondialdehyde secretion, and increased liver biomarkers (glutamate pyruvate transaminase and glutamate oxaloacetate). SD induced alterations in mRNA expression of hepatic antioxidants and cytochrome isoforms. The expression of peroxidase, catalase, CYP1A2, CYP3A2 and CYP2C11 in the liver were upregulated following SDC. By contrast, CYP2B1 was downregulated after 3 months of SDC in liver tissue samples. Thus, the present findings indicate that SDs induced oxidative stress in the liver of Wistar rats and for the first time, to the best of our knowledge, indicate that SDC disrupts hepatic CYP enzymes that may affect drug metabolism. Therefore, drug-dosing programs should be carefully designed to take these novel findings into consideration for the treatment of diseases.

  7. Metabolism of styrene to styrene oxide and vinylphenols in cytochrome P450 2F2- and P450 2E1-knockout mouse liver and lung microsomes.

    Science.gov (United States)

    Shen, Shuijie; Li, Lei; Ding, Xinxin; Zheng, Jiang

    2014-01-21

    Pulmonary toxicity of styrene is initiated by cytochromes P450-dependent metabolic activation. P450 2E1 and P450 2F2 are considered to be two main cytochrome P450 enzymes responsible for styrene metabolism in mice. The objective of the current study was to determine the correlation between the formation of styrene metabolites (i.e., styrene oxide and 4-vinylphenol) and pulmonary toxicity of styrene, using Cyp2e1- and Cyp2f2-null mouse models. A dramatic decrease in the formation of styrene glycol and 4-vinylphenol was found in Cyp2f2-null mouse lung microsomes relative to that in the wild-type mouse lung microsomes; however, no significant difference in the production of the styrene metabolites was observed between lung microsomes obtained from Cyp2e1-null and the wild-type mice. The knockout and wild-type mice were treated with styrene (6.0 mmol/kg, ip), and cell counts and LDH activity in bronchoalveolar lavage fluids were monitored to evaluate the pulmonary toxicity induced by styrene. Cyp2e1-null mice displayed a susceptibility to lung toxicity of styrene similar to that of the wild-type animals; however, Cyp2f2-null mice were resistant to styrene-induced pulmonary toxicity. In conclusion, both P450 2E1 and P450 2F2 are responsible for the metabolic activation of styrene. The latter enzyme plays an important role in styrene-induced pulmonary toxicity. Both styrene oxide and 4-vinylphenol are suggested to participate in the development of lung injury induced by styrene.

  8. 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.

  9. Anti-liver-kidney microsome antibody type 1 recognizes human cytochrome P450 db1.

    Science.gov (United States)

    Gueguen, M; Yamamoto, A M; Bernard, O; Alvarez, F

    1989-03-15

    Anti-liver-kidney microsome antibody type 1 (LKM1), present in the sera of a group of children with autoimmune hepatitis, was recently shown to recognize a 50 kDa protein identified as rat liver cytochromes P450 db1 and db2. High homology between these two members of the rat P450 IID subfamily and human P450 db1 suggested that anti-LKM1 antibody is directed against this human protein. To test this hypothesis, a human liver cDNA expression library in phage lambda GT-11 was screened using rat P450 db1 cDNA as a probe. Two human cDNA clones were found to be identical to human P450 db1 by restriction mapping. Immunoblot analysis using as antigen, the purified fusion protein from one of the human cDNA clones showed that only anti-LKM1 with anti-50 kDa reactivity recognized the fusion protein. This fusion protein was further used to develop an ELISA test that was shown to be specific for sera of children with this disease. These results: 1) identify the human liver antigen recognized by anti-LKM1 auto-antibodies as cytochrome P450 db1, 2) allow to speculate that mutation on the human P450 db1 gene could alter its expression in the hepatocyte and make it auto-antigenic, 3) provide a simple and specific diagnostic test for this disease.

  10. Antibodies against human cytochrome P-450db1 in autoimmune hepatitis type II.

    Science.gov (United States)

    Zanger, U M; Hauri, H P; Loeper, J; Homberg, J C; Meyer, U A

    1988-11-01

    In a subgroup of children with chronic active hepatitis, circulating autoantibodies occur that bind to liver and kidney endoplasmic reticulum (anti-liver/kidney microsome antibody type I or anti-LKM1). Anti-LKM1 titers follow the severity of the disease and the presence of these antibodies serves as a diagnostic marker for this autoimmune hepatitis type II. We demonstrate that anti-LKM1 IgGs specifically inhibit the hydroxylation of bufuralol in human liver microsomes. Using two assay systems with different selectivity for the two cytochrome P-450 isozymes catalyzing bufuralol metabolism in human liver, we show that anti-LKM1 exclusively recognizes cytochrome P-450db1. Immunopurification of the LKM1 antigen from solubilized human liver microsomes resulted in an electrophoretically homogenous protein that had the same molecular mass (50 kDa) as purified P-450db1 and an identical N-terminal amino acid sequence. Recognition of both purified P-450db1 and the immunoisolated protein on western blots by several monoclonal antibodies confirmed the identity of the LKM1 antigen with cytochrome P-450db1. Cytochrome P-450db1 has been identified as the target of a common genetic polymorphism of drug oxidation. However, the relationship between the polymorphic cytochrome P-450db1 and the appearance of anti-LKM1 autoantibodies as well as their role in the pathogenesis of chronic active hepatitis remains speculative.

  11. 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.

  12. Differential Expression of Cytochrome P450 Enzymes in Normal and Tumor Tissues from Childhood Rhabdomyosarcoma

    Science.gov (United States)

    Molina-Ortiz, Dora; Camacho-Carranza, Rafael; González-Zamora, José Francisco; Shalkow-Kalincovstein, Jaime; Cárdenas-Cardós, Rocío; Ností-Palacios, Rosario; Vences-Mejía, Araceli

    2014-01-01

    Intratumoral expression of genes encoding Cytochrome P450 enzymes (CYP) might play a critical role not only in cancer development but also in the metabolism of anticancer drugs. The purpose of this study was to compare the mRNA expression patterns of seven representative CYPs in paired tumor and normal tissue of child patients with rabdomyosarcoma (RMS). Using real time quantitative RT-PCR, the gene expression pattern of CYP1A1, CYP1A2, CYP1B1, CYP2E1, CYP2W1, CYP3A4, and CYP3A5 were analyzed in tumor and adjacent non-tumor tissues from 13 child RMS patients. Protein concentration of CYPs was determined using Western blot. The expression levels were tested for correlation with the clinical and pathological data of the patients. Our data showed that the expression levels of CYP1A1 and CYP1A2 were negligible. Elevated expression of CYP1B1 mRNA and protein was detected in most RMS tumors and adjacent normal tissues. Most cancerous samples exhibit higher levels of both CYP3A4 and CYP3A5 compared with normal tissue samples. Expression of CYP2E1 mRNA was found to be significantly higher in tumor tissue, however no relation was found with protein levels. CYP2W1 mRNA and/or protein are mainly expressed in tumors. In conclusion, we defined the CYP gene expression profile in tumor and paired normal tissue of child patients with RMS. The overexpression of CYP2W1, CYP3A4 and CYP3A5 in tumor tissues suggests that they may be involved in RMS chemoresistance; furthermore, they may be exploited for the localized activation of anticancer prodrugs. PMID:24699256

  13. 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.

  14. Expression of a Human Cytochrome P450 in Yeast Permits Analysis of Pathways for Response to and Repair of Aflatoxin-Induced DNA Damage†

    OpenAIRE

    Guo, Yingying; Breeden, Linda L.; Zarbl, Helmut; Preston, Bradley D.; Eaton, David L.

    2005-01-01

    Aflatoxin B1 (AFB1) is a human hepatotoxin and hepatocarcinogen produced by the mold Aspergillus flavus. In humans, AFB1 is primarily bioactivated by cytochrome P450 1A2 (CYP1A2) and 3A4 to a genotoxic epoxide that forms N7-guanine DNA adducts. A series of yeast haploid mutants defective in DNA repair and cell cycle checkpoints were transformed with human CYP1A2 to investigate how these DNA adducts are repaired. Cell survival and mutagenesis following aflatoxin B1 treatment was assayed in str...

  15. 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

  16. "Possible involvement of the long terminal repeat of transposable element 17.6 in regulating expression of an insecticide resistance-associated P450 gene in Drosophila.".

    OpenAIRE

    Waters, L C; Zelhof, A C; Shaw, B J; Ch'ang, L Y

    1992-01-01

    P450-A and P450-B are electrophoretically defined subsets of cytochrome P450 enzymes in Drosophila melanogaster. P450-A is present among all strains tested, whereas expression of P450-B is associated with resistance to insecticides. Monoclonal antibodies were used to obtain cDNA clones for an enzyme from each P450 subset (i.e., P450-A1 and P450-B1). The P450-B1 cDNA was sequenced and shown to code for a P450 of 507 amino acids. Its gene has been named CYP6A2. Comparative molecular analyses of...

  17. Vinclozolin modulates hepatic cytochrome P450 isoforms during pregnancy.

    Science.gov (United States)

    de Oca, Félix Genoveva García-Montes; López-González, Ma de Lourdes; Escobar-Wilches, Derly Constanza; Chavira-Ramírez, Roberto; Sierra-Santoyo, Adolfo

    2015-06-01

    Vinclozolin (V) is classified as a potent endocrine disruptor. The aim of the present study was to determine the effects of V on rat liver CYP regulation and on serum levels of testosterone and estradiol during pregnancy. Pregnancy decreased the liver total CYP content by 65%, enzyme activities of MROD, PROD, and PNPH, and testosterone hydroxylation activities, as well as the protein content of CYP2A and 3A. V exposure remarkably induced the protein content and enzyme activities of CYP1A, 2A, 2B and 3A subfamilies. Testosterone and estradiol were affected in an opposite manner, provoking a 3.5-fold increase in the estradiol/testosterone ratio. These results suggest that V could regulate the hepatic CYP expression through interaction with receptors and coactivators involved in its expression and may play an important role in hormonal balance during pregnancy. In addition, the results may also contribute to understanding the toxicity of V by in utero exposure. Copyright © 2015 Elsevier Inc. All rights reserved.

  18. 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.

  19. An expression tag toolbox for microbial production of membrane bound plant cytochromes P450

    DEFF Research Database (Denmark)

    Vazquez Albacete, Dario; Cavaleiro, Mafalda; Christensen, Ulla

    2017-01-01

    of the intermediate and the final product of the pathway. Finally, the effect of a robustly performing expression tag was explored with a library of 49 different P450s from medicinal plants and nearly half of these were improved in expression by more than 2-fold. The developed toolbox serves as platform to tune P450...... tag chimeras of the model plant P450 CYP79A1 in different Escherichia coli strains. Using a high-throughput screening platform based on C-terminal GFP fusions, we identify several highly expressing and robustly performing chimeric designs. Analysis of long-term cultures by flow cytometry showed...... homogeneous populations for some of the conditions. Three chimeric designs were chosen for a more complex combinatorial assembly of a multigene pathway consisting of two P450s and a redox partner. Cells expressing these recombinant enzymes catalysed the conversion of the substrate to highly different ratios...

  20. Functional evolution and structural conservation in chimeric cytochromes p450: calibrating a structure-guided approach.

    Science.gov (United States)

    Otey, Christopher R; Silberg, Jonathan J; Voigt, Christopher A; Endelman, Jeffrey B; Bandara, Geethani; Arnold, Frances H

    2004-03-01

    Recombination generates chimeric proteins whose ability to fold depends on minimizing structural perturbations that result when portions of the sequence are inherited from different parents. These chimeric sequences can display functional properties characteristic of the parents or acquire entirely new functions. Seventeen chimeras were generated from two CYP102 members of the functionally diverse cytochrome p450 family. Chimeras predicted to have limited structural disruption, as defined by the SCHEMA algorithm, displayed CO binding spectra characteristic of folded p450s. Even this small population exhibited significant functional diversity: chimeras displayed altered substrate specificities, a wide range in thermostabilities, up to a 40-fold increase in peroxidase activity, and ability to hydroxylate a substrate toward which neither parent heme domain shows detectable activity. These results suggest that SCHEMA-guided recombination can be used to generate diverse p450s for exploring function evolution within the p450 structural framework.

  1. Cytochrome P450-mediated metabolic engineering: current progress and future challenges.

    Science.gov (United States)

    Renault, Hugues; Bassard, Jean-Etienne; Hamberger, Björn; Werck-Reichhart, Danièle

    2014-06-01

    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 the engineered bioproduction of such compounds. Two ground-breaking developments of commercial products driven by the engineering of P450s are the antimalarial drug precursor artemisinic acid and blue roses or carnations. Tedious optimizations were required to generate marketable products. Hurdles encountered in P450 engineering and their potential solutions are summarized here. Together with recent technical developments and novel approaches to metabolic engineering, the lessons from this pioneering work should considerably boost exploitation of the amazing P450 toolkit emerging from accelerated sequencing of plant genomes. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

  2. 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

  3. [Immunomodulators with an 8-azasteroid structure as inducers of liver cytochrome P-450].

    Science.gov (United States)

    Kuz'mitskiĭ, B B; Dad'kov, I G; Mashkovich, A E; Stoma, O V; Slepneva, L M

    1990-01-01

    Two structural analogues of D-homo-8-azasteroids, both an immunostimulant and an immunodepressant, are inductors of the liver cytochrome P-450 in animals. This capability was shown by means of both a decrease of the hexenal sleep duration in the pharmacological test and an increase of the quantity of cytochrome P-450 and the rate of N-demethylation of aminopyrine in the biochemical assays.

  4. Export of Cytochrome P450 105D1 to the Periplasmic Space of Escherichia coli

    OpenAIRE

    Kaderbhai, Mustak A.; Ugochukwu, Cynthia C.; Kelly, Steven L.; Lamb, David C.

    2001-01-01

    CYP105D1, a cytochrome P450 from Streptomyces griseus, was appended at its amino terminus to the secretory signal of Escherichia coli alkaline phosphatase and placed under the transcriptional control of the native phoA promoter. Heterologous expression in E. coli phosphate-limited medium resulted in abundant synthesis of recombinant CYP105D1 that was translocated across the bacterial inner membrane and processed to yield authentic, heme-incorporated P450 within the periplasmic space. Cell ext...

  5. Cytochrome P-450 complex formation in rat liver by the antibiotic tiamulin.

    Science.gov (United States)

    Witkamp, R F; Nijmeijer, S M; van Miert, A S

    1996-01-01

    Tiamulin is a semisynthetic diterpene antibiotic frequently used in farm animals. The drug has been shown to produce clinically important--often lethal--interactions with other compounds. It has been suggested that this is caused by a selective inhibition of oxidative drug metabolism via the formation of a cytochrome P-450 metabolic intermediate complex. In the present study, rats were treated orally for 6 days with tiamulin at two different doses: 40 and 226 mg/kg of body weight. For comparison, another group received 300 mg of triacetyloleandomycin (TAO) per kg, which is equivalent to the 226-mg/kg tiamulin group. Subsequently, microsomal P-450 contents, P-450 enzyme activities, metabolic intermediate complex spectra, and P-450 apoprotein concentrations were assessed. In addition, effects on individual microsomal P-450 activities were studied in control microsomes at different tiamulin and substrate concentrations. In the rats treated with tiamulin, a dose-dependent complex formation as evidenced by its absorption spectrum and an increase in cytochrome P-4503A1/2 contents as assessed by Western blotting (immunoblotting) were found. The effects were comparable to those of TAO. Tiamulin induced microsomal P-450 content, testosterone 6 beta-hydroxylation rate, erythromycin N-demethylation rate, and the ethoxyresorufin O-deethylation activity. Other activities were not affected or decreased. When tiamulin was added to microsomes of control rats, the testosterone 6 beta-hydroxylation rate and the erythromycin N-demethylation were strongly inhibited. It is concluded that tiamulin is a potent and selective inducer-inhibitor of cytochrome P-450. Though not belonging to the macrolides, the compound produces an effect on P-450 similar to those of TAO and related compounds.

  6. Chemoenzymatic elaboration of monosaccharides using engineered cytochrome P450_(BM3) demethylases

    OpenAIRE

    Lewis, Jared C.; Bastian, Sabine; Bennett, Clay S.; Fu, Yu; Mitsuda, Yuuichi; Chen, Mike M.; Greenberg, William A.; Wong, Chi-Huey; Arnold, Frances H.

    2009-01-01

    Polysaccharides comprise an extremely important class of biopolymers that play critical roles in a wide range of biological processes, but the synthesis of these compounds is challenging because of their complex structures. We have developed a chemoenzymatic method for regioselective deprotection of monosaccharide substrates using engineered Bacillus megaterium cytochrome P450 (P450_(BM3)) demethylases that provides a highly efficient means to access valuable intermediate...

  7. 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 ...... (available from the SRA at NCBI). All three genes were shown to be functional upon reconstitution into nanodiscs, confirming that none of the isoforms are pseudogenes....

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

    International Nuclear Information System (INIS)

    Ekstroem, G.; Norsten, C.; Cronholm, T.; Ingelman-Sundberg, M.

    1987-01-01

    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- 2 H 2 ] ethanol at various concentrations, and a competitive method, where 1:1 mixtures of [1- 13 C]- and [ 2 H 6 ] ethanol or [2,2,2- 2 H 3 ]- and [1,1- 2 H 2 ] 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 LM 2 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- 2 H] 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 C 1 -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

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

    Science.gov (United States)

    Antony, Tresa Remya Thomas; Nagarajan, Shanthi

    2006-11-14

    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. www.bicpu.edu.in/DrugMetZDB/

  10. Microbial P450 Enzymes in Bioremediation and Drug Discovery: Emerging Potentials and Challenges.

    Science.gov (United States)

    Bhattacharya, Sukanta S; Yadav, Jagjit S

    2018-01-01

    Cytochrome P450 enzymes are a structurally conserved but functionally diverse group of heme-containing mixed function oxidases found across both prokaryotic and eukaryotic forms of the microbial world. Microbial P450s are known to perform diverse functions ranging from the synthesis of cell wall components to xenobiotic/drug metabolism to biodegradation of environmental chemicals. Conventionally, many microbial systems have been reported to mimic mammalian P450-like activation of drugs and were proposed as the in-vitro models of mammalian drug metabolism. Recent reports suggest that native or engineered forms of specific microbial P450s from these and other microbial systems could be employed for desired specific biotransformation reactions toward natural and synthetic (drug) compounds underscoring their emerging potential in drug improvement and discovery. On the other hand, microorganisms particularly fungi and actinomycetes have been shown to possess catabolic P450s with unusual potential to degrade toxic environmental chemicals including persistent organic pollutants (POPs). Wood-rotting basidiomycete fungi in particular have revealed the presence of exceptionally large P450 repertoire (P450ome) in their genomes, majority of which are however orphan (with no known function). Our pre- and post-genomic studies have led to functional characterization of several fungal P450s inducible in response to exposure to several environmental toxicants and demonstration of their potential in bioremediation of these chemicals. This review is an attempt to summarize the postgenomic unveiling of this versatile enzyme superfamily in microbial systems and investigation of their potential to synthesize new drugs and degrade persistent pollutants, among other biotechnological applications. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  11. The molecular evolution of cytochrome P450 genes within and between drosophila species.

    Science.gov (United States)

    Good, Robert T; Gramzow, Lydia; Battlay, Paul; Sztal, Tamar; Batterham, Philip; Robin, Charles

    2014-04-20

    We map 114 gene gains and 74 gene losses in the P450 gene family across the phylogeny of 12 Drosophila species by examining the congruence of gene trees and species trees. Although the number of P450 genes varies from 74 to 94 in the species examined, we infer that there were at least 77 P450 genes in the ancestral Drosophila genome. One of the most striking observations in the data set is the elevated loss of P450 genes in the Drosophila sechellia lineage. The gain and loss events are not evenly distributed among the P450 genes-with 30 genes showing no gene gains or losses whereas others show as many as 20 copy number changes among the species examined. The P450 gene clades showing the fewest number of gene gain and loss events tend to be those evolving with the most purifying selection acting on the protein sequences, although there are exceptions, such as the rapid rate of amino acid replacement observed in the single copy phantom (Cyp306a1) gene. Within D. melanogaster, we observe gene copy number polymorphism in ten P450 genes including multiple cases of interparalog chimeras. Nonallelic homologous recombination (NAHR) has been associated with deleterious mutations in humans, but here we provide a second possible example of an NAHR event in insect P450s being adaptive. Specifically, we find that a polymorphic Cyp12a4/Cyp12a5 chimera correlates with resistance to an insecticide. Although we observe such interparalog exchange in our within-species data sets, we have little evidence of it between species, raising the possibility that such events may occur more frequently than appreciated but are masked by subsequent sequence change. © The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  12. Characterization and expression of the cytochrome P450 gene family in diamondback moth, Plutella xylostella (L.).

    Science.gov (United States)

    Yu, Liying; Tang, Weiqi; He, Weiyi; Ma, Xiaoli; Vasseur, Liette; Baxter, Simon W; Yang, Guang; Huang, Shiguo; Song, Fengqin; You, Minsheng

    2015-03-10

    Cytochrome P450 monooxygenases are present in almost all organisms and can play vital roles in hormone regulation, metabolism of xenobiotics and in biosynthesis or inactivation of endogenous compounds. In the present study, a genome-wide approach was used to identify and analyze the P450 gene family of diamondback moth, Plutella xylostella, a destructive worldwide pest of cruciferous crops. We identified 85 putative cytochrome P450 genes from the P. xylostella genome, including 84 functional genes and 1 pseudogene. These genes were classified into 26 families and 52 subfamilies. A phylogenetic tree constructed with three additional insect species shows extensive gene expansions of P. xylostella P450 genes from clans 3 and 4. Gene expression of cytochrome P450s was quantified across multiple developmental stages (egg, larva, pupa and adult) and tissues (head and midgut) using P. xylostella strains susceptible or resistant to insecticides chlorpyrifos and fiprinol. Expression of the lepidopteran specific CYP367s predominantly occurred in head tissue suggesting a role in either olfaction or detoxification. CYP340s with abundant transposable elements and relatively high expression in the midgut probably contribute to the detoxification of insecticides or plant toxins in P. xylostella. This study will facilitate future functional studies of the P. xylostella P450s in detoxification.

  13. Third international symposium: Cytochrome P450 biodiversity. Final report, January 1, 1995--December 31, 1995

    Energy Technology Data Exchange (ETDEWEB)

    Loper, J.C.

    1997-03-01

    The Symposium was held on October 8-12, 1995 at the Marine Biological Laboratory in Woods Hole Massachusetts. Other international symposia promote cytochrome P450 research but have a primary focus on mammalian systems. This symposium is exclusively devoted to research in other organisms, and major topics reflect the distribution and dominance of non-mammalian species in the biosphere. The five sessions focused on basic mechanism, regulation, biodiversity, host-parasite interactions, and practical applications. 170 Scientists contributed 38 oral presentations and 91 posters, with a truly international composition of the symposium. Practical applications were a recurring feature, linking reports on mechanism and regulation to studies on the engineering of substrate specificity, microorganisms to degrade halogenated hydrocarbons and herbicides, and the production of in vitro P450 electrochemical bioreactors. At the time of the symposium there were 477 cytochrome P450 sequences in the database. Expansion of the known plant P450 genes was reported, with 20 new plant P450 families added in the last 3 years. Of these only 5 families have a physiological function associated with them. A growing number of identified invertebrate P450s was documented, where in insects, the forms identified are primarily involved in inducible xenobiotic metabolism and detoxification of toxic plant substances.

  14. The binding sites on human heme oxygenase-1 for cytochrome p450 reductase and biliverdin reductase.

    Science.gov (United States)

    Wang, Jinling; de Montellano, Paul R Ortiz

    2003-05-30

    Human heme oxygenase-1 (hHO-1) catalyzes the NADPH-cytochrome P450 reductase-dependent oxidation of heme to biliverdin, CO, and free iron. The biliverdin is subsequently reduced to bilirubin by biliverdin reductase. Earlier kinetic studies suggested that biliverdin reductase facilitates the release of biliverdin from hHO-1 (Liu, Y., and Ortiz de Montellano, P. R. (2000) J. Biol. Chem. 275, 5297-5307). We have investigated the binding of P450 reductase and biliverdin reductase to truncated, soluble hHO-1 by fluorescence resonance energy transfer and site-specific mutagenesis. P450 reductase and biliverdin reductase bind to truncated hHO-1 with Kd = 0.4 +/- 0.1 and 0.2 +/- 0.1 microm, respectively. FRET experiments indicate that biliverdin reductase and P450 reductase compete for binding to truncated hHO-1. Mutation of surface ionic residues shows that hHO-1 residues Lys18, Lys22, Lys179, Arg183, Arg198, Glu19, Glu127, and Glu190 contribute to the binding of cytochrome P450 reductase. The mutagenesis results and a computational analysis of the protein surfaces partially define the binding site for P450 reductase. An overlapping binding site including Lys18, Lys22, Lys179, Arg183, and Arg185 is similarly defined for biliverdin reductase. These results confirm the binding of biliverdin reductase to hHO-1 and define binding sites of the two reductases.

  15. An improved microphotometry system for measurement of cytochrome P-450 in hepatocyte cytoplasm.

    Science.gov (United States)

    Watanabe, J; Kanamura, S

    1991-05-01

    To measure cytochrome P-450 (P-450) content in hepatocyte cytoplasm, we developed a dual monochromator-equipped microphotometry system (KWSP-1). Simultaneous measurements of absorbance at 450 and 490 nm with narrow band width (0.5 nm) and small spot size (2 microns) were accomplished by this system. Corresponding fields in serial sections could be easily and rapidly identified under the Nomarski imaging mode of KWSP-1. Photometric accuracy and repeatability of wavelength setting of KWSP-1 were also satisfactory for measurement of P-450. With this system, it is thus possible to measure the extinction of P-450 from many small measuring areas and to precisely determine P-450 content in the cytoplasm of rat hepatocytes. A microphotometric method was developed using cuvette slides and two serial 10-microns thick sections (mapping method). The intracellular distribution of P-450 in individual hepatocytes could be visualized by the mapping method with KWSP-1. However, this method was not applicable to tissue sections containing hemoglobin larger than 4 microM.

  16. 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

  17. An in-vitro cocktail assay for assessing compound-mediated inhibition of six major cytochrome P450 enzymes

    Directory of Open Access Journals (Sweden)

    Jing-Jing Wang

    2014-08-01

    Full Text Available An efficient screening assay was developed and validated for simultaneous assessment of compound-mediated inhibition of six major human cytochrome P450 (CYP enzymes. This method employed a cocktail of six probe substrates (i.e., phenacetin, amodiaquine, diclofenac, S-mephenytoin, dextromethorphan and midazolam for CYP1A2, 2C8, 2C9, 2C19, 2D6 and 3A4, respectively as well as individual prototypical inhibitors of the six CYP enzymes in human liver microsomes under optimized incubation conditions. The corresponding marker metabolites (i.e., acetaminophen, N-desethylamodiaquine, 4-OH-diclofenac, 4-OH-S-mephenytoin, dextrorphan and 1-OH-midazolam in the incubates were quantified using LC–MS/MS methods either by an internal standard (IS calibration curve or a simplified analyte-to-IS peak area ratio approach. The results showed that the IC50 values determined by the cocktail approach were in good agreement with those obtained by the individual substrate approach as well as those reported in the literature. Besides, no remarkable difference was observed between the two quantification approaches. In conclusion, this new cocktail assay can be used for reliable screening of compound-mediated CYP inhibition. Keywords: LC–MS/MS, Cytochrome P450, Cocktail-probe, Inhibition assessment, Drug screenning

  18. Expression induction of P450 genes by imidacloprid in Nilaparvata lugens: A genome-scale analysis.

    Science.gov (United States)

    Zhang, Jianhua; Zhang, Yixi; Wang, Yunchao; Yang, Yuanxue; Cang, Xinzhu; Liu, Zewen

    2016-09-01

    The overexpression of P450 monooxygenase genes is a main mechanism for the resistance to imidacloprid, a representative neonicotinoid insecticide, in Nilaparvata lugens (brown planthopper, BPH). However, only two P450 genes (CYP6AY1 and CYP6ER1), among fifty-four P450 genes identified from BPH genome database, have been reported to play important roles in imidacloprid resistance until now. In this study, after the confirmation of important roles of P450s in imidacloprid resistance by the synergism analysis, the expression induction by imidacloprid was determined for all P450 genes. In the susceptible (Sus) strain, eight P450 genes in Clade4, eight in Clade3 and two in Clade2 were up-regulated by imidacloprid, among which three genes (CYP6CS1, CYP6CW1 and CYP6ER1, all in Clade3) were increased to above 4.0-fold and eight genes to above 2.0-fold. In contrast, no P450 genes were induced in Mito clade. Eight genes induced to above 2.0-fold were selected to determine their expression and induced levels in Huzhou population, in which piperonyl butoxide showed the biggest effects on imidacloprid toxicity among eight field populations. The expression levels of seven P450 genes were higher in Huzhou population than that in Sus strain, with the biggest differences for CYP6CS1 (9.8-fold), CYP6ER1 (7.7-fold) and CYP6AY1 (5.1-fold). The induction levels for all tested genes were bigger in Sus strain than that in Huzhou population except CYP425B1. Screening the induction of P450 genes by imidacloprid in the genome-scale will provide an overall view on the possible metabolic factors in the resistance to neonicotinoid insecticides. The further work, such as the functional study of recombinant proteins, will be performed to validate the roles of these P450s in imidacloprid resistance. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. Curcumin Prevents Aflatoxin B1 Hepatoxicity by Inhibition of Cytochrome P450 Isozymes in Chick Liver

    Directory of Open Access Journals (Sweden)

    Ni-Ya Zhang

    2016-11-01

    Full Text Available This study was designed to establish if Curcumin (CM alleviates Aflatoxin B1 (AFB1-induced hepatotoxic effects and to determine whether alteration of the expression of cytochrome P450 (CYP450 isozymes is involved in the regulation of these effects in chick liver. One-day-old male broilers (n = 120 were divided into four groups and used in a two by two factorial trial in which the main factors included supplementing AFB1 (< 5 vs. 100 μg/kg and CM (0 vs. 150 mg/kg in a corn/soybean-based diet. Administration of AFB1 induced liver injury, significantly decreasing albumin and total protein concentrations and increasing alanine aminotransferase and aspartate aminotransferase activities in serum, and induced hepatic histological lesions at week 2. AFB1 also significantly decreased hepatic glutathione peroxidase, catalase, and glutathione levels, while increasing malondialdehyde, 8-hydroxydeoxyguanosine, and exo-AFB1-8,9-epoxide (AFBO-DNA concentrations. In addition, the mRNA and/or activity of enzymes responsible for the bioactivation of AFB1 into AFBO—including CYP1A1, CYP1A2, CYP2A6, and CYP3A4—were significantly induced in liver microsomes after 2-week exposure to AFB1. These alterations induced by AFB1 were prevented by CM supplementation. Conclusively, dietary CM protected chicks from AFB1-induced liver injury, potentially through the synergistic actions of increased antioxidant capacities and inhibition of the pivotal CYP450 isozyme-mediated activation of AFB1 to toxic AFBO.

  20. Responsiveness of cerebral and hepatic cytochrome P450s in rat offspring prenatally exposed to lindane

    International Nuclear Information System (INIS)

    Johri, Ashu; Yadav, Sanjay; Dhawan, Alok; Parmar, Devendra

    2008-01-01

    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 LD 50 ; 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

  1. Differences in hepatic microsomal cytochrome P-450 isoenzyme induction by pyrazole, chronic ethanol, 3-methylcholanthrene, and phenobarbital in high alcohol sensitivity (HAS) and low alcohol sensitivity (LAS) rats.

    Science.gov (United States)

    Lucas, D; Ménez, J F; Berthou, F; Cauvin, J M; Deitrich, R A

    1992-10-01

    High and low alcohol sensitivity (HAS and LAS) rats have been selected for their differences in ethanol-induced sleep time. Liver monooxygenase activities were studied in HAS and LAS rats before and after treatments with known inducers such as chronic ethanol, pyrazole, 3-methylcholanthrene (3-MC) and phenobarbital (PB) to determine whether the selection procedure also selected for differences in the cytochrome P-450 (P-450) inducibility. This previously has been shown with long sleep (LS) and short sleep (SS) mice, which were selected using a similar criterion. 3-MC and PB, in conjunction with chronic ethanol treatment, were used in order to evaluate the interactions of ethanol with these inducers. Prior to treatment, total P-450 content was slightly lower in LAS than in HAS rats. However, both lines displayed the same microsomal monooxygenase activities related to different P-450 isozymes. This was demonstrated by ethoxyresorufin deethylation (EROD) for cytochrome P-450 1A1 (CYP1A1), acetanilide hydroxylation (ACET) for CYP1A2, pentoxyresorufin dealkylation (PROD) for CYP2B, 1-butanol oxidation (BUTAN) and N-nitrosodimethylamine demethylation (NDMA) for CYP2E1. After the different treatments, HAS rats did not differ from LAS rats in their CYP2E1 inducibility. However, pyrazole, PB and 3-MC treatment led to differences in CYP1A and CYP2B monooxygenase activities between the two lines. The enhancement of PROD by pyrazole treatment was less prominent in LAS (1.7-fold of the control value) than in HAS rats (3.8-fold).(ABSTRACT TRUNCATED AT 250 WORDS)

  2. 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

  3. Steroid hydroxylations: A paradigm for cytochrome P450 catalyzed mammalian monooxygenation reactions

    International Nuclear Information System (INIS)

    Estabrook, Ronald W.

    2005-01-01

    The present article reviews the history of research on the hydroxylation of steroid hormones as catalyzed by enzymes present in mammalian tissues. The report describes how studies of steroid hormone synthesis have played a central role in the discovery of the monooxygenase functions of the cytochrome P450s. Studies of steroid hydroxylation reactions can be credited with showing that: (a) the adrenal mitochondrial enzyme catalyzing the 11β-hydroxylation of deoxycorticosterone was the first mammalian enzyme shown by O 18 studies to be an oxygenase; (b) the adrenal microsomal enzyme catalyzing the 21-hydroxylation of steroids was the first mammalian enzyme to show experimentally the proposed 1:1:1 stoichiometry (substrate:oxygen:reduced pyridine nucleotide) of a monooxygenase reaction; (c) application of the photochemical action spectrum technique for reversal of carbon monoxide inhibition of the 21-hydroxylation of 17α-OH progesterone was the first demonstration that cytochrome P450 was an oxygenase; (d) spectrophotometric studies of the binding of 17α-OH progesterone to bovine adrenal microsomal P450 revealed the first step in the cyclic reaction scheme of P450, as it catalyzes the 'activation' of oxygen in a monooxygenase reaction; (e) purified adrenodoxin was shown to function as an electron transport component of the adrenal mitochondrial monooxygenase system required for the activity of the 11β-hydroxylase reaction. Adrenodoxin was the first iron-sulfur protein isolated and purified from mammalian tissues and the first soluble protein identified as a reductase of a P450; (f) fractionation of adrenal mitochondrial P450 and incubation with adrenodoxin and a cytosolic (flavoprotein) fraction were the first demonstration of the reconstitution of a mammalian P450 monooxygenase reaction

  4. Metabolic imidacloprid resistance in the brown planthopper, Nilaparvata lugens, relies on multiple P450 enzymes.

    Science.gov (United States)

    Zhang, Yixi; Yang, Yuanxue; Sun, Huahua; Liu, Zewen

    2016-12-01

    Target insensitivity contributing to imidacloprid resistance in Nilaparvata lugens has been reported to occur either through point mutations or quantitative change in nicotinic acetylcholine receptors (nAChRs). However, the metabolic resistance, especially the enhanced detoxification by P450 enzymes, is the major mechanism in fields. From one field-originated N. lugens population, an imidacloprid resistant strain G25 and a susceptible counterpart S25 were obtained to analyze putative roles of P450s in imidacloprid resistance. Compared to S25, over-expression of twelve P450 genes was observed in G25, with ratios above 5.0-fold for CYP6AY1, CYP6ER1, CYP6CS1, CYP6CW1, CYP4CE1 and CYP425B1. RNAi against these genes in vivo and recombinant tests on the corresponding proteins in vitro revealed that four P450s, CYP6AY1, CYP6ER1, CYP4CE1 and CYP6CW1, played important roles in imidacloprid resistance. The importance of the four P450s was not equal at different stages of resistance development based on their over-expression levels, among which CYP6ER1 was important at all stages, and that the others might only contribute at certain stages. The results indicated that, to completely reflect roles of P450s in insecticide resistances, their over-expression in resistant individuals, expression changes at the stages of resistance development, and catalytic activities against insecticides should be considered. In this study, multiple P450s, CYP6AY1, CYP6ER1, CYP4CE1 and CYP6CW1, have proven to be important in imidacloprid resistance. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. Functional characterization of cytochromes P450 2B from the desert woodrat Neotoma lepida

    International Nuclear Information System (INIS)

    Wilderman, P. Ross; Jang, Hyun-Hee; Malenke, Jael R.; Salib, Mariam; Angermeier, Elisabeth; Lamime, Sonia; Dearing, M. Denise; Halpert, James R.

    2014-01-01

    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 2 B substrate recognition remains to be clarified. • Reported N. lepida gene

  6. 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

  7. Effect of zolpidem on human cytochrome P450 activity, and on transport mediated by P-glycoprotein.

    Science.gov (United States)

    von Moltke, Lisa L; Weemhoff, James L; Perloff, Michael D; Hesse, Leah M; Harmatz, Jerold S; Roth-Schechter, Barbara F; Greenblatt, David J

    2002-12-01

    The influence of high concentrations of zolpidem (100 microM, corresponding to approximately 200 times maximum therapeutic concentrations) on the activity of six human Cytochrome P450 (CYP) enzymes was evaluated in a model system using human liver microsomes. Zolpidem produced negligible or weak inhibition of human CYP1A2, 2B6, 2C9, 2C19, 2D6, and 3A. Transport of rhodamine 123, presumed to be mediated mainly by the energy-dependent efflux transport protein P-glycoprotein, was studied in a cell culture system using a human intestinal cell line. High concentrations of zolpidem (100 microM), exceeding the usual therapeutic range by more than 100-fold, produced only modest impairment of rhodamine 123 transport. The findings indicate that zolpidem is very unlikely to cause clinical drug interactions attributable to impairment of CYP activity or P-gp mediated transport. Copyright 2002 John Wiley & Sons, Ltd.

  8. Permethrin induction of multiple cytochrome P450 genes in insecticide resistant mosquitoes, Culex quinquefasciatus.

    Science.gov (United States)

    Gong, Youhui; Li, Ting; Zhang, Lee; Gao, Xiwu; Liu, Nannan

    2013-01-01

    The expression of some insect P450 genes can be induced by both exogenous and endogenous compounds and there is evidence to suggest that multiple constitutively overexpressed P450 genes are co-responsible for the development of resistance to permethrin in resistant mosquitoes. This study characterized the permethrin induction profiles of P450 genes known to be constitutively overexpressed in resistant mosquitoes, Culex quinquefasciatus. The gene expression in 7 of the 19 P450 genes CYP325K3v1, CYP4D42v2, CYP9J45, (CYP) CPIJ000926, CYP325G4, CYP4C38, CYP4H40 in the HAmCqG8 strain, increased more than 2-fold after exposure to permethrin at an LC50 concentration (10 ppm) compared to their acetone treated counterpart; no significant differences in the expression of these P450 genes in susceptible S-Lab mosquitoes were observed after permethrin treatment. Eleven of the fourteen P450 genes overexpressed in the MAmCqG6 strain, CYP9M10, CYP6Z12, CYP9J33, CYP9J43, CYP9J34, CYP306A1, CYP6Z15, CYP9J45, CYPPAL1, CYP4C52v1, CYP9J39, were also induced more than doubled after exposure to an LC50 (0.7 ppm) dose of permethrin. No significant induction in P450 gene expression was observed in the susceptible S-Lab mosquitoes after permethrin treatment except for CYP6Z15 and CYP9J39, suggesting that permethrin induction of these two P450 genes are common to both susceptible and resistant mosquitoes while the induction of the others are specific to insecticide resistant mosquitoes. These results demonstrate that multiple P450 genes are co-up-regulated in insecticide resistant mosquitoes through both constitutive overexpression and induction mechanisms, providing additional support for their involvement in the detoxification of insecticides and the development of insecticide resistance.

  9. Cytochrome P450s from the fall armyworm (Spodoptera frugiperda): responses to plant allelochemicals and pesticides.

    Science.gov (United States)

    Giraudo, M; Hilliou, F; Fricaux, T; Audant, P; Feyereisen, R; Le Goff, G

    2015-02-01

    Spodoptera frugiperda is a polyphagous lepidopteran pest that encounters a wide range of toxic plant metabolites in its diet. The ability of this insect to adapt to its chemical environment might be explained by the action of major detoxification enzymes such as cytochrome P450s (or CYP). Forty-two sequences coding for P450s were identified and most of the transcripts were found to be expressed in the midgut, Malpighian tubules and fat body of S. frugiperda larvae. Relatively few P450s were expressed in the established cell line Sf9. In order to gain information on how these genes respond to different chemical compounds, larvae and Sf9 cells were exposed to plant secondary metabolites (indole, indole-3-carbinol, quercetin, 2-tridecanone and xanthotoxin), insecticides (deltamethrin, fipronil, methoprene, methoxyfenozide) or model inducers (clofibrate and phenobarbital). Several genes were induced by plant chemicals such as P450s from the 6B, 321A and 9A subfamilies. Only a few genes responded to insecticides, belonging principally to the CYP9A family. There was little overlap between the response in vivo measured in the midgut and the response in vitro in Sf9 cells. In addition, regulatory elements were detected in the promoter region of these genes. In conclusion, several P450s were identified that could potentially be involved in the adaptation of S. frugiperda to its chemical environment. © 2014 The Royal Entomological Society.

  10. New reactions and products resulting from alternative interactions between the P450 enzyme and redox partners.

    Science.gov (United States)

    Zhang, Wei; Liu, Yi; Yan, Jinyong; Cao, Shaona; Bai, Fali; Yang, Ying; Huang, Shaohua; Yao, Lishan; Anzai, Yojiro; Kato, Fumio; Podust, Larissa M; Sherman, David H; Li, Shengying

    2014-03-05

    Cytochrome P450 enzymes are capable of catalyzing a great variety of synthetically useful reactions such as selective C-H functionalization. Surrogate redox partners are widely used for reconstitution of P450 activity based on the assumption that the choice of these auxiliary proteins or their mode of action does not affect the type and selectivity of reactions catalyzed by P450s. Herein, we present an exceptional example to challenge this postulate. MycG, a multifunctional biosynthetic P450 monooxygenase responsible for hydroxylation and epoxidation of 16-membered ring macrolide mycinamicins, is shown to catalyze the unnatural N-demethylation(s) of a range of mycinamicin substrates when partnered with the free Rhodococcus reductase domain RhFRED or the engineered Rhodococcus-spinach hybrid reductase RhFRED-Fdx. By contrast, MycG fused with the RhFRED or RhFRED-Fdx reductase domain mediates only physiological oxidations. This finding highlights the larger potential role of variant redox partner protein-protein interactions in modulating the catalytic activity of P450 enzymes.

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

    International Nuclear Information System (INIS)

    Yoshida, Nobutaka; Osawa, Yoshio

    1991-01-01

    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- 3 H,4- 14 C]androstenedione as substrate. The total recovery of purified aromatase activity was 32.2%, and P-450 recovery was 17.6%. The very high K m 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

  12. Transcriptome analysis and identification of P450 genes relevant to imidacloprid detoxification in Bradysia odoriphaga.

    Science.gov (United States)

    Chen, Chengyu; Wang, Cuicui; Liu, Ying; Shi, Xueyan; Gao, Xiwu

    2018-02-07

    Pesticide tolerance poses many challenges for pest control, particularly for destructive pests such as Bradysia odoriphaga. Imidacloprid has been used to control B. odoriphaga since 2013, however, imidacloprid resistance in B. odoriphaga has developed in recent years. Identifying actual and potential genes involved in detoxification metabolism of imidacloprid could offer solutions for controlling this insect. In this study, RNA-seq was used to explore differentially expressed genes in B. odoriphaga that respond to imidacloprid treatment. Differential expression data between imidacloprid treatment and the control revealed 281 transcripts (176 with annotations) showing upregulation and 394 transcripts (235 with annotations) showing downregulation. Among them, differential expression levels of seven P450 unigenes were associated with imidacloprid detoxification mechanism, with 4 unigenes that were upregulated and 3 unigenes that were downregulated. The qRT-PCR results of the seven differential expression P450 unigenes after imidacloprid treatment were consistent with RNA-Seq data. Furthermore, oral delivery mediated RNA interference of these four upregulated P450 unigenes followed by an insecticide bioassay significantly increased the mortality of imidacloprid-treated B. odoriphaga. This result indicated that the four upregulated P450s are involved in detoxification of imidacloprid. This study provides a genetic basis for further exploring P450 genes for imidacloprid detoxification in B. odoriphaga.

  13. NADPH–Cytochrome P450 Oxidoreductase: Roles in Physiology, Pharmacology, and Toxicology

    Science.gov (United States)

    Ding, Xinxin; Wolf, C. Roland; Porter, Todd D.; Pandey, Amit V.; Zhang, Qing-Yu; Gu, Jun; Finn, Robert D.; Ronseaux, Sebastien; McLaughlin, Lesley A.; Henderson, Colin J.; Zou, Ling; Flück, Christa E.

    2013-01-01

    This is a report on a symposium sponsored by the American Society for Pharmacology and Experimental Therapeutics and held at the Experimental Biology 2012 meeting in San Diego, California, on April 25, 2012. The symposium speakers summarized and critically evaluated our current understanding of the physiologic, pharmacological, and toxicological roles of NADPH–cytochrome P450 oxidoreductase (POR), a flavoprotein involved in electron transfer to microsomal cytochromes P450 (P450), cytochrome b5, squalene mono-oxygenase, and heme oxygenase. Considerable insight has been derived from the development and characterization of mouse models with conditional Por deletion in particular tissues or partial suppression of POR expression in all tissues. Additional mouse models with global or conditional hepatic deletion of cytochrome b5 are helping to clarify the P450 isoform- and substrate-specific influences of cytochrome b5 on P450 electron transfer and catalytic function. This symposium also considered studies using siRNA to suppress POR expression in a hepatoma cell–culture model to explore the basis of the hepatic lipidosis phenotype observed in mice with conditional deletion of Por in liver. The symposium concluded with a strong translational perspective, relating the basic science of human POR structure and function to the impacts of POR genetic variation on human drug and steroid metabolism. PMID:23086197

  14. Engineering human cytochrome P450 enzymes into catalytically self-sufficient chimeras using molecular Lego.

    Science.gov (United States)

    Dodhia, Vikash Rajnikant; Fantuzzi, Andrea; Gilardi, Gianfranco

    2006-10-01

    The membrane-bound human cytochrome P450s have essential roles in the metabolism of endogenous compounds and drugs. Presented here are the results on the construction and characterization of three fusion proteins containing the N-terminally modified human cytochrome P450s CYP2C9, CY2C19 and CYP3A4 fused to the soluble NADPH-dependent oxidoreductase domain of CYP102A1 from Bacillus megaterium. The constructs, CYP2C9/BMR, CYP2C19/BMR and CYP3A4/BMR are well expressed in Escherichia coli as holo proteins. The chimeras can be purified in the absence of detergent and the purified enzymes are both active and correctly folded in the absence of detergent, as demonstrated by circular dichroism and functional studies. Additionally, in comparison with the parent P450 enzyme, these chimeras have greatly improved solubility properties. The chimeras are catalytically self-sufficient and present turnover rates similar to those reported for the native enzymes in reconstituted systems, unlike previously reported mammalian cytochrome P450 fusion proteins. Furthermore the specific activities of these chimeras are not dependent on the enzyme concentration present in the reaction buffer and they do not require the addition of accessory proteins, detergents or phospholipids to be fully active. The solubility, catalytic self-sufficiency and wild-type like activities of these chimeras would greatly simplify the studies of cytochrome P450 mediated drug metabolism in solution.

  15. Molecular LEGO by domain-imprinting of cytochrome P450 BM3.

    Science.gov (United States)

    Jetzschmann, K J; Yarman, A; Rustam, L; Kielb, P; Urlacher, V B; Fischer, A; Weidinger, I M; Wollenberger, U; Scheller, F W

    2018-04-01

    Electrosynthesis of the MIP nano-film after binding of the separated domains or holo-cytochrome BM3 via an engineered anchor should result in domain-specific cavities in the polymer layer. Both the two domains and the holo P450 BM3 have been bound prior polymer deposition via a N-terminal engineered his6-anchor to the electrode surface. Each step of MIP preparation was characterized by cyclic voltammetry of the redox-marker ferricyanide. Rebinding after template removal was evaluated by quantifying the suppression of the diffusive permeability of the signal for ferricyanide and by the NADH-dependent reduction of cytochrome c by the reductase domain (BMR). The working hypothesis is verified by the discrimination of the two domains by the respective MIPs: The holoenzyme P450 BM3 was ca. 5.5 times more effectively recognized by the film imprinted with the oxidase domain (BMO) as compared to the BMR-MIP or the non-imprinted polymer (NIP). Obviously, a cavity is formed during the imprinting process around the his 6 -tag-anchored BMR which cannot accommodate the broader BMO or the P450 BM3. The affinity of the MIP towards P450 BM3 is comparable with that to the monomer in solution. The his 6 -tagged P450 BM3 binds (30 percent) stronger which shows the additive effect of the interaction with the MIP and the binding to the electrode. Copyright © 2018. Published by Elsevier B.V.

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

    Science.gov (United States)

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

    2014-08-01

    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). 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 11-fold higher in 766b than in WHO-SRS. Significant differences between expression of P450 genes between F1 flies from 845b and established laboratory strains were shown. In general, P450 gene expression in 845b was 2-14-fold higher than in the reference strain (P resistance. There is a strong indication that CYP6G4 is a major insecticide resistance gene involved in neonicotinoid resistance. © 2013 Society of Chemical Industry.

  17. Inhibitory effects of cytostatically active 6-aminobenzo[c]phenanthridines on cytochrome P450 enzymes in human hepatic microsomes.

    Science.gov (United States)

    Zebothsen, Inga; Kunze, Thomas; Clement, Bernd

    2006-07-01

    Besides assays for the evaluation of efficacy new drug candidates have to undergo extensive testings for enhancement of pharmaceutical drug safety and optimization of application. The objective of the present work was to investigate the pharmacokinetic drug drug interaction potential for the cytostatically active 6-aminobenzo[c]phenanthridines BP-11 (6-amino-11,12-dihydro-11-(4-hydroxy-3,5-dimethoxyphenyl)benzo[c]phenanthridine) and BP-D7 (6-amino-11-(3,4,5-trimethoxyphenyl)benzo[c]phenanthridine) in vitro through incubation with human hepatic microsomes and marker substrates. For these studies the cytochrome P-450 isoenzymes and corresponding marker substrates recommended by the EMEA (The European Agency for the Evaluation of Medicinal Products) were chosen. In detail these selective substrates were caffeine (CYP1A2), coumarin (CYP2A6), tolbutamide (CYP2C9), S-(+)-mephenytoin (CYP2C19), dextromethorphane (CYP2D6), chlorzoxazone (CYP2E1) and testosterone (CYP3A4). Incubations with each substrate were carried out without a possible inhibitor and in the presence of a benzo[c]phenanthridine or a selective inhibitor at varying concentrations. Marker activities were determined by HPLC (high performance liquid chromatography). For the isoenzymes showing more than 50% inhibition by the addition of 20 microM BP-11 or BP-D7 additional concentrations of substrate and inhibitor were tested for a characterization of the inhibition. The studies showed a moderate risk for BP-11 for interactions with the cytochrome P-450 isoenzymes CYP1A2, CYP2C9, CYP2D6 and CYP3A4. BP-D7, the compound with the highest cytotstatic efficacy, showed only a moderate risk for interactions with drugs, also metabolized by CYP3A4.

  18. Improving Delivery of Photosynthetic Reducing Power to Cytochrome P450s

    DEFF Research Database (Denmark)

    Mellor, Silas Busck

    at sustainable production of high-value and commodity products. Cytochrome P450 enzymes play key roles in the biosynthesis of important natural products. The electron carrier ferredoxin can couple P450s non-natively to photosynthetic electron supply, providing ample reducing power for catalysis. However......, photosynthetic reducing power feeds into both central and specialized metabolism, which leads to a fiercely competitive system from which to siphon reductant. This thesis explores the optimization of light-driven P450 activity, and proposes strategies to overcome the limitations imposed by competition...... for photosynthetic reducing power. Photosynthetic electron carrier proteins interact with widely different partners because they use relatively non-specific interactions. The mechanistic basis of these interactions and its impact on natural electron transfer complexes is discussed. This particular type...

  19. Why there is no cookbook approach to palliative care: implications of the P450 enzyme system.

    Science.gov (United States)

    Kuebler, Kim K; Varga, James; Mihelic, Ronald A

    2003-01-01

    A plethora of literature describes the impact of the P450 enzyme system, but this information is limited regarding its relevancy to nursing practice. However, oncology nurses providing palliative symptom management must have a working knowledge of the P450 enzyme system to recognize the variability that exists among individual medication reactions or why a "cookbook approach" to symptom management is not always effective and appropriate. This article describes the variations associated with medication metabolism with reference to ethnic differences. Having a basic understanding of the P450 enzyme system and, more specifically, the CYP2D6 influence on the metabolism of common medications used in palliative symptom management can help to prevent medication toxicity or underdosing, which interferes with patients' quality of life.

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

    International Nuclear Information System (INIS)

    Roccatano, Danilo

    2015-01-01

    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. (topical review)

  1. 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.

  2. Analysis of the oxidation of short chain alkynes by flavocytochrome P450 BM3.

    Science.gov (United States)

    Waltham, Timothy N; Girvan, Hazel M; Butler, Christopher F; Rigby, Stuart R; Dunford, Adrian J; Holt, Robert A; Munro, Andrew W

    2011-04-01

    Bacillus megaterium flavocytochrome P450 BM3 (BM3) is a high activity fatty acid hydroxylase, formed by the fusion of soluble cytochrome P450 and cytochrome P450 reductase modules. Short chain (C6, C8) alkynes were shown to be substrates for BM3, with productive outcomes (i.e. alkyne hydroxylation) dependent on position of the carbon-carbon triple bond in the molecule. Wild-type P450 BM3 catalyses ω-3 hydroxylation of both 1-hexyne and 1-octyne, but is suicidally inactivated in NADPH-dependent turnover with non-terminal alkynes. A F87G mutant of P450 BM3 also undergoes turnover-dependent heme destruction with the terminal alkynes, pointing to a key role for Phe87 in controlling regioselectivity of alkyne oxidation. The terminal alkynes access the BM3 heme active site led by the acetylene functional group, since hydroxylated products are not observed near the opposite end of the molecules. For both 1-hexyne and 1-octyne, the predominant enantiomeric product formed (up to ∼90%) is the (S)-(-)-1-alkyn-3-ol form. Wild-type P450 BM3 is shown to be an effective oxidase catalyst of terminal alkynes, with strict regioselectivity of oxidation and potential biotechnological applications. The absence of measurable octanoic or hexanoic acid products from oxidation of the relevant 1-alkynes is also consistent with previous studies suggesting that removal of the phenyl group in the F87G mutant does not lead to significant levels of ω-oxidation of alkyl chain substrates.

  3. Biodegradation of Cosmetics Products: A Computational Study of Cytochrome P450 Metabolism of Phthalates

    Directory of Open Access Journals (Sweden)

    Fabián G. Cantú Reinhard

    2017-11-01

    Full Text Available Cytochrome P450s are a broad class of enzymes in the human body with important functions for human health, which include the metabolism and detoxification of compounds in the liver. Thus, in their catalytic cycle, the P450s form a high-valent iron(IV-oxo heme cation radical as the active species (called Compound I that reacts with substrates through oxygen atom transfer. This work discusses the possible degradation mechanisms of phthalates by cytochrome P450s in the liver, through computational modelling, using 2-ethylhexyl-phthalate as a model substrate. Phthalates are a type of compound commonly found in the environment from cosmetics usage, but their biodegradation in the liver may lead to toxic metabolites. Experimental studies revealed a multitude of products and varying product distributions among P450 isozymes. To understand the regio- and chemoselectivity of phthalate activation by P450 isozymes, we focus here on the mechanisms of phthalate activation by Compound I leading to O-dealkylation, aliphatic hydroxylation and aromatic hydroxylation processes. We set up model complexes of Compound I with the substrate and investigated the reaction mechanisms for products using the density functional theory on models and did a molecular mechanics study on enzymatic structures. The work shows that several reaction barriers in the gas-phase are close in energy, leading to a mixture of products. However, when we tried to dock the substrate into a P450 isozyme, some of the channels were inaccessible due to unfavorable substrate positions. Product distributions are discussed under various reaction conditions and rationalized with valence bond and thermodynamic models.

  4. 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

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

    International Nuclear Information System (INIS)

    Marinello, A.J.; Bansal, S.K.; Paul, B.; Koser, P.L.; Love, J.; Struck, R.F.; Gurtoo, H.L.

    1984-01-01

    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

  6. Active site diversification of P450cam with indole generates catalysts for benzylic oxidation reactions

    Directory of Open Access Journals (Sweden)

    Paul P. Kelly

    2015-09-01

    Full Text Available Cytochrome P450 monooxygenases are useful biocatalysts for C–H activation, and there is a need to expand the range of these enzymes beyond what is naturally available. A panel of 93 variants of active self-sufficient P450cam[Tyr96Phe]-RhFRed fusion enzymes with a broad diversity in active site amino acids was developed by screening a large mutant library of 16,500 clones using a simple, highly sensitive colony-based colorimetric screen against indole. These mutants showed distinct fingerprints of activity not only when screened in oxidations of substituted indoles but also for unrelated oxidations such as benzylic hydroxylations.

  7. Induction of P450 genes in Nilaparvata lugens and Sogatella furcifera by two neonicotinoid insecticides.

    Science.gov (United States)

    Yang, Yuan-Xue; Yu, Na; Zhang, Jian-Hua; Zhang, Yi-Xi; Liu, Ze-Wen

    2018-06-01

    Nilaparvata lugens and Sogatella furcifera are two primary planthoppers on rice throughout Asian countries and areas. Neonicotinoid insecticides, such as imidacloprid (IMI), have been extensively used to control rice planthoppers and IMI resistance consequently occurred with an important mechanism from the over-expression of P450 genes. The induction of P450 genes by IMI may increase the ability to metabolize this insecticide in planthoppers and increase the resistance risk. In this study, the induction of P450 genes was compared in S. furcifera treated with IMI and nitromethyleneimidazole (NMI), in two planthopper species by IMI lethal dose that kills 85% of the population (LD 85 ), and in N. lugens among three IMI doses (LD 15 , LD 50 and LD 85 ). When IMI and NMI at the LD 85 dose were applied to S. furcifera, the expression changes in most P450 genes were similar, including the up-regulation of nine genes and down-regulation of three genes. In terms of the expression changes in 12 homologous P450 genes between N. lugens and S. furcifera treated with IMI at the LD 85 dose, 10 genes had very similar patterns, such as up-regulation in seven genes, down-regulation in one gene and no significant changes in two genes. When three different IMI doses were applied to N. lugens, the changes in P450 gene expression were much different, such as up-regulation in four genes at all doses and dose-dependent regulation of the other nine genes. For example, CYP6AY1 could be induced by all IMI doses, while CYP6ER1 was only up-regulated by the LD 50 dose, although both genes were reported important in IMI resistance. In conclusion, P450 genes in two planthopper species showed similar regulation patterns in responding to IMI, and the two neonicotinoid insecticides had similar effects on P450 gene expression, although the regulation was often dose-dependent. © 2017 Institute of Zoology, Chinese Academy of Sciences.

  8. Radical Intermediates in the Catalytic Oxidation of Hydrocarbons by Bacterial and Human Cytochrome P450 Enzymes†

    OpenAIRE

    Jiang, Yongying; He, Xiang; Ortiz de Montellano, Paul R.

    2006-01-01

    Cytochromes P450cam and P450BM3 oxidize α- and β-thujone into multiple products, including 7-hydroxy-α-(or β-)thujone, 7,8-dehydro-α-(or β-)thujone, 4-hydroxy-α-(or β-)thujone, 2-hydroxy α-(or β-)thujone, 5-hydroxy-5-isopropyl-2-methyl-2-cyclohexen-1-one, 4,10-dehydrothujone, and carvacrol. Quantitative analysis of the 4-hydroxylated isomers and the ring opened product indicates that the hydroxylation proceeds via a radical mechanism with a radical recombination rate ranging from 0.7 ± 0.3 × ...

  9. 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

    . In the present study, we examine the phylogenetic relationships of these P450 genes in holometabolous insects belonging to the orders Hymenoptera, Coleoptera, Lepidoptera and Diptera. The analyzed insect genomes each contains single orthologs of Phantom (CYP306A1), Disembodied (CYP302A1), Shadow (CYP315A1...... of orthologous Halloween genes indicates selective constraint on these residues to prevent functional divergence. The results suggest that duplications of ancestral P450 genes that acquired novel functions may have been an important mechanism for evolving the ecdysteroidogenic pathway. © 2007 Elsevier B.V. All...

  10. Plant Expression of a Bacterial Cytochrome P450 That Catalyzes Activation of a Sulfonylurea Pro-Herbicide.

    Science.gov (United States)

    O'Keefe, D. P.; Tepperman, J. M.; Dean, C.; Leto, K. J.; Erbes, D. L.; Odell, J. T.

    1994-01-01

    The Streptomyces griseolus gene encoding herbicide-metabolizing cytochrome P450SU1 (CYP105A1) was expressed in transgenic tobacco (Nicotiana tabacum). Because this P450 can be reduced by plant chloroplast ferredoxin in vitro, chloroplast-targeted and nontargeted expression were compared. Whereas P450SU1 antigen was found in the transgenic plants regardless of the targeting, only those with chloroplast-directed enzyme performed P450SU1-mediated N-dealkylation of the sulfonylurea 2-methylethyl-2,3-dihydro-N-[(4,6-dimethoxypyrimidin-2-yl)aminocarbonyl]-1, 2-benzoisothiazole- 7-sulfonamide-1,1-dioxide (R7402). Chloroplast targeting appears to be essential for the bacterial P450 to function in the plant. Because the R7402 metabolite has greater phytotoxicity than R7402 itself, plants bearing active P450SU1 are susceptible to injury from R7402 treatment that is harmless to plants without P450SU1. Thus, P450SU1 expression and R7402 treatment can be used as a negative selection system in plants. Furthermore, expression of P450SU1 from a tissue-specific promoter can sequester production of the phytotoxic R7402 metabolite to a single plant tissue. In tobacco expressing P450SU1 from a tapetum-specific promoter, treatment of immature flower buds with R7402 caused dramatically lowered pollen viability. Such treatment could be the basis for a chemical hybridizing agent. PMID:12232216

  11. Resonance Raman study on the structure of the active sites of microsomal cytochrome P-450 isozymes LM2 and LM4.

    Science.gov (United States)

    Hildebrandt, P; Greinert, R; Stier, A; Taniguchi, H

    1989-12-08

    The isozymes 2 and 4 of rabbit microsomal cytochrome P-450 (LM2, LM4) have been studied by resonance Raman spectroscopy. Based on high quality spectra, a vibrational assignment of the porphyrin modes in the frequency range between 100-1700 cm-1 is presented for different ferric states of cytochrome P-450 LM2 and LM4. The resonance Raman spectra are interpreted in terms of the spin and ligation state of the heme iron and of heme-protein interactions. While in cytochrome P-450 LM2 the six-coordinated low-spin configuration is predominantly occupied, in the isozyme LM4 the five-coordinated high-spin form is the most stable state. The different stability of these two spin configurations in LM2 and LM4 can be attributed to the structures of the active sites. In the low-spin form of the isozymes LM4 the protein matrix forces the heme into a more rigid conformation than in LM2. These steric constraints are removed upon dissociation of the sixth ligand leading to a more flexible structure of the active site in the high-spin form of the isozyme LM4. The vibrational modes of the vinyl groups were found to be characteristic markers for the specific structures of the heme pockets in both isozymes. They also respond sensitively to type-I substrate binding. While in cytochrome P-450 LM4 the occupation of the substrate-binding pocket induces conformational changes of the vinyl groups, as reflected by frequency shifts of the vinyl modes, in the LM2 isozyme the ground-state conformation of these substituents remain unaffected, suggesting that the more flexible heme pocket can accommodate substrates without imposing steric constraints on the porphyrin. The resonance Raman technique makes structural changes visible which are induced by substrate binding in addition and independent of the changes associated with the shift of the spin state equilibrium: the high-spin states in the substrate-bound and substrate-free enzyme are structurally different. The formation of the inactive form

  12. A glycine insertion in the estrogen-related receptor (ERR is associated with enhanced expression of three cytochrome P450 genes in transgenic Drosophila melanogaster.

    Directory of Open Access Journals (Sweden)

    Weilin Sun

    Full Text Available Insecticide-resistant Drosophila melanogaster strains represent a resource for the discovery of the underlying molecular mechanisms of cytochrome P450 constitutive over-expression, even if some of these P450s are not directly involved in the resistance phenotype. For example, in select 4,4'-dichlorodiphenyltrichloroethane (DDT resistant strains the glucocorticoid receptor-like (GR-like potential transcription factor binding motifs (TFBMs have previously been shown to be associated with constitutively differentially-expressed cytochrome P450s, Cyp12d1, Cyp6g2 and Cyp9c1. However, insects are not known to have glucocorticoids. The only ortholog to the mammalian glucocorticoid receptor (GR in D. melanogaster is an estrogen-related receptor (ERR gene, which has two predicted alternative splice isoforms (ERRa and ERRb. Sequencing of ERRa and ERRb in select DDT susceptible and resistant D. melanogaster strains has revealed a glycine (G codon insertion which was only observed in the ligand binding domain of ERR from the resistant strains tested (ERR-G. Transgenic flies, expressing the ERRa-G allele, constitutively over-expressed Cyp12d1, Cyp6g2 and Cyp9c1. Only Cyp12d1 and Cyp6g2 were over-expressed in the ERRb-G transgenic flies. Phylogenetic studies show that the G-insertion appeared to be located in a less conserved domain in ERR and this insertion is found in multiple species across the Sophophora subgenera.

  13. Posttranslational modification of hepatic cytochrome P-450. Phosphorylation of phenobarbital-inducible P-450 forms PB-4 (IIB1) and PB-5 (IIB2) in isolated rat hepatocytes and in vivo

    International Nuclear Information System (INIS)

    Koch, J.A.; Waxman, D.J.

    1989-01-01

    Phosphorylation of hepatic cytochrome P-450 was studied in isolated hepatocytes incubated in the presence of agents known to stimulate protein kinase activity. Incubation of hepatocytes isolated from phenobarbital-induced adult male rats with [ 32 P]orthophosphate in the presence of N 6 , O 2' -dibutyryl-cAMP (diBtcAMP) or glucagon resulted in the phosphorylation of microsomal proteins that are immunoprecipitable by polyclonal antibodies raised to the phenobarbital-induced P-450 form PB-4 (P-450 gene IIB1). Two-dimensional gel electrophoresis revealed that these 32 P-labeled microsomal proteins consist of a mixture of P-450 PB-4 and the closely related P-450 PB-5 (gene IIB2), both of which exhibited heterogeneity in the isoelectric focusing dimension. Phosphorylation of both P-450 forms was markedly enhanced by diBtcAMP at concentrations as low as 5 μM. Phosphoamino acid analysis of the 32 P-labeled P-450 PB-4 + PB-5 immunoprecipitate revealed that these P-450s are phosphorylated on serine in the isolated hepatocytes. Peptide mapping indicated that the site of phosphorylation in hepatocytes is indistinguishable from the site utilized by cAMP-dependent protein kinase in vitro, which was previously identified as serine-128 for the related rabbit protein P-450 LM2. In vitro analyses revealed that phosphorylation of P-450 PB-4 leads to a loss of monooxygenase activity, suggesting that the posttranslational modification of this P-450 enzyme by cAMP-dependent protein kinase may play a role in the modulation of P-450-dependent monooxygenase activity in vivo

  14. Potent inhibition of cytochrome P450 2B6 by sibutramine in human liver microsomes.

    Science.gov (United States)

    Bae, Soo Hyeon; Kwon, Min Jo; Choi, Eu Jin; Zheng, Yu Fen; Yoon, Kee Dong; Liu, Kwang-Hyeon; Bae, Soo Kyung

    2013-09-05

    The present study was performed to evaluate the potency and specificity of sibutramine as an inhibitor of the activities of nine human CYP isoforms in liver microsomes. Using a cocktail assay, the effects of sibutramine on specific marker reactions of the nine CYP isoforms were measured in human liver microsomes. Sibutramine showed potent inhibition of CYP2B6-mediated bupropion 6-hydroxylation with an IC50 value of 1.61μM and Ki value of 0.466μM in a competitive manner at microsomal protein concentrations of 0.25mg/ml; this was 3.49-fold more potent than the typical CYP2B6 inhibitor thio-TEPA (Ki=1.59μM). In addition, sibutramine slightly inhibited CYP2C19 activity (Ki=16.6μM, noncompetitive inhibition) and CYP2D6 activity (Ki=15.7μM, noncompetitive inhibition). These observations indicated 35.6- and 33.7-fold decreases in inhibition potency, respectively, compared with that of CYP2B6 by sibutramine. However, no inhibition of CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2D6, or CYP2E1 activities was observed. In addition, the CYP2B6 inhibitory potential of sibutramine was enhanced at a lower microsomal protein concentration of 0.05mg/ml. After 30min preincubation of human liver microsomes with sibutramine in the presence of NADPH, no shift in IC50 was observed in terms of inhibition of the activities of the nine CYPs, suggesting that sibutramine is not a time-dependent inactivator. These observations suggest that sibutramine is a selective and potent inhibitor of CYP2B6 in vitro, whereas inhibition of other CYPs is substantially lower. These in vitro data support the use of sibutramine as a well-known inhibitor of CYP2B6 for routine screening of P450 reversible inhibition when human liver microsomes are used as the enzyme source. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  15. 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

    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...

  16. 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

  17. INTERACTION OF AROMATIC CYTOKININS WITH HUMAN LIVER MICROSOMAL CYTOCHROMES P450

    Czech Academy of Sciences Publication Activity Database

    Anzenbacherová, E.; Janalík, J.; Popa, Igor; Strnad, Miroslav; Anzenbacher, P.

    2005-01-01

    Roč. 149, č. 2 (2005), s. 349-351 ISSN 1213-8118 Institutional research plan: CEZ:AV0Z50380511 Keywords : Cytokinins * Cyclin dependent kinase inhibitor * Cytochrome P450 Subject RIV: CE - Biochemistry http://publib.upol.cz/~obd/fulltext/Biomed/2005/2/349.pdf

  18. Rat liver microsomal cytochrome P450-dependent oxidation of 3,5-disubstituted analogues of paracetamol

    NARCIS (Netherlands)

    Bessems, J.G.M.; Koppele, J.M. te; Dijk, P.A. van; Stee, L.L.P. van; Commandeur, J.N.M.; Vermeulen, N.P.E.

    1996-01-01

    1. The cytochrome P450-dependent binding of paracetamol and a series of 3,5-disubstituted paracetamol analogues (R = -F, -Cl, -Br, -I, -C(H)3, -C2H5, -iC3H7) have been determined with β-naphthoflavone (βNF)-induced rat liver microsomes and produced reverse type I spectral changes. K(s,app) varied

  19. Effect of fumonisin B1 on rat hepatic P450 system

    NARCIS (Netherlands)

    Spotti, M.; Maas, R.F.M.; Nijs, C.M. de; Fink-Gremmels, J.

    2000-01-01

    The effects of the mycotoxin fumonisin B1 (FB1) on the hepatic cytochrome P450 system were investigated in male rats dosed daily by oral gavage with 3 mg FB1 per kg body weight for 9 consecutive days. FB1 treatment resulted in a reduced weight gain. At the same time, CYP2E activity was increased,

  20. Study on the cytochrome P-450- and glutathione-dependent biotransformation of trichloroethylene in humans

    NARCIS (Netherlands)

    Bloemen, L. J.; Monster, A. C.; Kezic, S.; Commandeur, J. N.; Veulemans, H.; Vermeulen, N. P.; Wilmer, J. W.

    2001-01-01

    To investigate in humans the contribution of the cytochrome P-450- and glutathione-dependent biotransformation of trichloroethylene (TRI) under controlled repeated exposure in volunteers, and under occupational conditions. Volunteers were exposed to TRI, using repeated 15 min exposures at 50 and 100

  1. PRIMARY STRUCTURE OF THE CYTOCHROME P450 LANOSTEROL 14A-DEMETHYLASE GENE FROM CANDIDA TROPICALIS

    Science.gov (United States)

    We report the nucleotide sequence of the gene and flanking DNA for the cytochrome P450 lanosterol 14 alpha-demethylase (14DM) from the yeast Candida tropicalis ATCC750. An open reading frame (ORF) of 528 codons encoding a 60.9-kD protein is identified. This ORF includes a charact...

  2. Inhibitors of steroidal cytochrome p450 enzymes as targets for drug development.

    Science.gov (United States)

    Baston, Eckhard; Leroux, Frédéric R

    2007-01-01

    Cytochrome P450's are enzymes which catalyze a large number of biological reactions, for example hydroxylation, N-, O-, S- dealkylation, epoxidation or desamination. Their substrates include fatty acids, steroids or prostaglandins. In addition, a high number of various xenobiotics are metabolized by these enzymes. The enzyme 17alpha-hydroxylase-C17,20-lyase (P450(17), CYP 17, androgen synthase), a cytochrome P450 monooxygenase, is the key enzyme for androgen biosynthesis. It catalyzes the last step of the androgen biosynthesis in the testes and adrenal glands and produces androstenedione and dehydroepiandrosterone from progesterone and pregnenolone. The microsomal enzyme aromatase (CYP19) transforms these androgens to estrone and estradiol. Estrogens stimulate tumor growth in hormone dependent breast cancer. In addition, about 80 percent of prostate cancers are androgen dependent. Selective inhibitors of these enzymes are thus important alternatives to treatment options like antiandrogens or antiestrogens. The present article deals with recent patents (focus on publications from 2000 - 2006) concerning P450 inhibitor design where steroidal substrates are involved. In this context a special focus is provided for CYP17 and CYP19. Mechanisms of action will also be discussed. Inhibitors of CYP11B2 (aldosterone synthase) will also be dealt with.

  3. 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...

  4. Short-term fasting alters cytochrome P450-mediated drug metabolism in humans

    NARCIS (Netherlands)

    Lammers, Laureen A.; Achterbergh, Roos; de Vries, Emmely M.; van Nierop, F. Samuel; Klümpen, Heinz-Josef; Soeters, Maarten R.; Boelen, Anita; Romijn, Johannes A.; Mathôt, Ron A. A.

    2015-01-01

    Experimental studies indicate that short-term fasting alters drug metabolism. However, the effects of short-term fasting on drug metabolism in humans need further investigation. Therefore, the aim of this study was to evaluate the effects of short-term fasting (36 h) on P450-mediated drug

  5. Occupation of the cytochrome P450 substrate pocket by diverse compounds at general anesthesia concentrations.

    Science.gov (United States)

    LaBella, F S; Stein, D; Queen, G

    1998-10-02

    Each of a diverse array of compounds, at concentrations reported to effect general anesthesia, when added to liver microsomes, forms a complex with cytochromes P450 to generate, with reference to a cuvette containing microsomes only, a characteristic absorbance-difference spectrum. This spectrum results from a change in the electron-spin state of the heme iron atom induced upon entry by the anesthetic molecule into the enzyme catalytic pocket. The difference spectrum, representing the anesthetic-P450 complex, is characteristic of substances that are substrates for the enzyme. For the group of compounds as a whole, the magnitudes of the absorbance-difference spectra vary only about twofold, although the anesthetic potencies vary by several orders of magnitude. The dissociation constants (Ks), calculated from absorbance data and representing affinities of the anesthetics for P450, agree closely with the respective EC50 (concentration that effects anesthesia in 50% of individuals) values, and with the respective Ki (concentration that inhibits P450 catalytic activities half-maximally) values reported by us previously. The absorbance complex resulting from the occupation of the catalytic pocket by endogenous substrates, androstenedione and arachidonic acid, is inhibited, competitively, by anesthetics. Occupation of and perturbation of the heme catalytic pocket by anesthetic, as monitored by the absorbance-difference spectrum, is rapidly reversible. The presumed in vivo consequences of perturbation by general anesthetics of heme proteins is suppression of the generation of chemical signals that determine cell sensitivity and response.

  6. Substrate binding in the active site of cytochrome P450cam

    NARCIS (Netherlands)

    Swart, M.; Groenhof, A.R.; Ehlers, A.W.; Lammertsma, K.

    2005-01-01

    We have studied the binding of camphor in the active site of cytochrome P450cam with density functional theory (DFT) calculations. A strong hydrogen bond (>6 kcal/mol) to a tyrosine residue (Tyr96) is observed, that may account for the high specificity of the reaction taking place. The DFT

  7. Heterotropic and homotropic cooperativity by a drug-metabolising mutant of cytochrome P450 BM3

    NARCIS (Netherlands)

    van Vugt-Lussenburg, B.M.A.; Damsten, M.C.; Maasdijk, D.M.; Vermeulen, N.P.E.; Commandeur, J.N.M.

    2006-01-01

    Recently, we described a triple mutant of the bacterial cytochrome P450 BM3 as the first mutant with affinity for drug-like compounds. In this paper, we show that this mutant, but not wild-type BM3, is able to metabolise testosterone and several drug-like molecules such as amodiaquine,

  8. Correlates of Cytochrome P450 1A1 Expression in Bottlenose Dolphin (Tursiops truncatus) Integument Biopsies

    NARCIS (Netherlands)

    Wilson, J.Y.; Wells, R.; Anguilar, A.; Borrell, A.; Tornero, V.; Reijnders, P.J.H.; Moore, M.

    2007-01-01

    Integument biopsy is a nondestructive method for sampling free-ranging cetaceans, which allows for the determination of both contaminant concentrations and biomarker responses. Cytochrome P450 1A1 (CYP1A1) expression is induced by polycyclic aromatic hydrocarbons and planar halogenated aromatic

  9. 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 in Escherichia 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) in E. coli. Altogether, this study establishes E. coli as 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.

  10. 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

  11. 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

  12. Effect of p-amino-diphenyl ethers on hepatic microsomal cytochrome P450.

    Science.gov (United States)

    Jiang, Huidi; Xuan, Guida

    2003-09-01

    The present paper aims to investigate whether p-amino-2',4'-dichlorodiphenyl ether and p-amino-4'-methyldiphenyl ether are inhibitors as well as inducers of P450. Mice were given daily intraperitoneal (ip) injections of p-amino-2',4'-dichlorodiphenyl ether (0.25 mmol/kg) or p-amino-4'-methyldiphenyl ether (0.25 mmol/kg) for 4 days and tested at 24 h and 48 h after the last dose injection. The results showed the mice pentobarbital sleeping time was shorter and the P450 content of hepatic microsome increased significantly in the group pretreated with p-amino-4'-methyldiphenyl ether when compared with the control group, while in mice pretreated with p-amino-2',4'-dichlorodiphenyl ether the hepatic microsome P450 content increased but the pentobarbital sleeping time was extended in clear contrast to the control group. The sleeping time of the phenobarbital group (80 mg/kg daily ip injection for 4 days) was shortened at 24 h after the last injection with increased P450 content of hepatic microsome, but it showed no difference at 48 h. The zoxazolamine-paralysis times of mice treated with p-amino-2',4'-dichlorodiphenyl ether were longer than those of the control mice, while the same dose of zoxazolamine did not lead to paralysis in mice pretreated with BNF. p-Amino-2',4'-dichlorodiphenyl ether and p-amino-4'-methyldiphenyl ether inhibited the activity of 7-ethoxyresorufin O-deethylase from rat hepatic microsome induced by BNF in vitro by 70.0% and 50.1% respectively. These results suggest that p-amino-2',4'-dichlorodiphenyl ether and p-amino-4'-methyldiphenyl ether are inhibitors as well as inducers of P450.

  13. An indole-deficient Escherichia coli strain improves screening of cytochromes P450 for biotechnological applications.

    Science.gov (United States)

    Brixius-Anderko, Simone; Hannemann, Frank; Ringle, Michael; Khatri, Yogan; Bernhardt, Rita

    2017-05-01

    Escherichia coli has developed into an attractive organism for heterologous cytochrome P450 production, but, in some cases, was restricted as a host in view of a screening of orphan cytochromes P450 or mutant libraries in the context of molecular evolution due to the formation of the cytochrome P450 inhibitor indole by the enzyme tryptophanase (TnaA). To overcome this effect, we disrupted the tnaA gene locus of E. coli C43(DE3) and evaluated the new strain for whole-cell substrate conversions with three indole-sensitive cytochromes P450, myxobacterial CYP264A1, and CYP109D1 as well as bovine steroidogenic CYP21A2. For purified CYP264A1 and CYP21A2, the half maximal inhibitory indole concentration was determined to be 140 and 500 μM, which is within the physiological concentration range occurring during cultivation of E. coli in complex medium. Biotransformations with C43(DE3)_∆tnaA achieved a 30% higher product formation in the case of CYP21A2 and an even fourfold increase with CYP264A1 compared with C43(DE3) cells. In whole-cell conversion based on CYP109D1, which converts indole to indigo, we could successfully avoid this reaction. Results in microplate format indicate that our newly designed strain is a suitable host for a fast and efficient screening of indole-influenced cytochromes P450 in complex medium. © 2016 International Union of Biochemistry and Molecular Biology, Inc.

  14. 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. Copyright © 2013 Elsevier Inc. All rights reserved.

  15. Similar substrate specificity of cynomolgus monkey cytochrome P450 2C19 to reported human P450 2C counterpart enzymes by evaluation of 89 drug clearances.

    Science.gov (United States)

    Hosaka, Shinya; Murayama, Norie; Satsukawa, Masahiro; Uehara, Shotaro; Shimizu, Makiko; Iwasaki, Kazuhide; Iwano, Shunsuke; Uno, Yasuhiro; Yamazaki, Hiroshi

    2015-12-01

    Cynomolgus monkeys are used widely in preclinical studies as non-human primate species. The amino acid sequence of cynomolgus monkey cytochrome P450 (P450 or CYP) 2C19 is reportedly highly correlated to that of human CYP2C19 (92%) and CYP2C9 (93%). In the present study, 89 commercially available compounds were screened to find potential substrates for cynomolgus monkey CYP2C19. Of 89 drugs, 34 were metabolically depleted by cynomolgus monkey CYP2C19 with relatively high rates. Among them, 30 compounds have been reported as substrates or inhibitors of, either or both, human CYP2C19 and CYP2C9. Several compounds, including loratadine, showed high selectivity to cynomolgus monkey CYP2C19, and all of these have been reported as human CYP2C19 and/or CYP2C9 substrates. In addition, cynomolgus monkey CYP2C19 formed the same loratadine metabolite as human CYP2C19, descarboethoxyloratadine. These results suggest that cynomolgus monkey CYP2C19 is generally similar to human CYP2C19 and CYP2C9 in its substrate recognition functionality. Copyright © 2015 John Wiley & Sons, Ltd.

  16. Identification of putative substrates for cynomolgus monkey cytochrome P450 2C8 by substrate depletion assays with 22 human P450 substrates and inhibitors.

    Science.gov (United States)

    Hosaka, Shinya; Murayama, Norie; Satsukawa, Masahiro; Uehara, Shotaro; Shimizu, Makiko; Iwasaki, Kazuhide; Iwano, Shunsuke; Uno, Yasuhiro; Yamazaki, Hiroshi

    2016-07-01

    Cynomolgus monkeys are widely used in drug developmental stages as non-human primate models. Previous studies used 89 compounds to investigate species differences associated with cytochrome P450 (P450 or CYP) function that reported monkey specific CYP2C76 cleared 19 chemicals, and homologous CYP2C9 and CYP2C19 metabolized 17 and 30 human CYP2C9 and/or CYP2C19 substrates/inhibitors, respectively. In the present study, 22 compounds selected from viewpoints of global drug interaction guidances and guidelines were further evaluated to seek potential substrates for monkey CYP2C8, which is highly homologous to human CYP2C8 (92%). Amodiaquine, montelukast, quercetin and rosiglitazone, known as substrates or competitive inhibitors of human CYP2C8, were metabolically depleted by recombinant monkey CYP2C8 at relatively high rates. Taken together with our reported findings of the slow eliminations of amodiaquine and montelukast by monkey CYP2C9, CYP2C19 and CYP2C76, the present results suggest that these at least four chemicals may be good marker substrates for monkey CYP2C8. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

  17. Engineering and improvement of the efficiency of a chimeric [P450cam-RhFRed reductase domain] enzyme.

    Science.gov (United States)

    Robin, Aélig; Roberts, Gareth A; Kisch, Johannes; Sabbadin, Federico; Grogan, Gideon; Bruce, Neil; Turner, Nicholas J; Flitsch, Sabine L

    2009-05-14

    A chimeric oxygenase, in which the P450cam domain was fused to the reductase host domains of a P450RhF from Rhodococcus sp. strain NCIMB 9784 was optimised to allow for a biotransformation at 30 mM substrate in 80% overall yield, with the linker region between P450 and FMN domain proving to be important for the effective biotransformation of (+)-camphor to 5-exo-hydroxycamphor.

  18. Wpływ pojedynczej dawki paracetamolu i/lub N-acetylocysteiny na szczury przewlekle eksponowane na trichloroetylen. III. Wpływ na wybrane izoformy cytochromu P450 w wątrobie

    Directory of Open Access Journals (Sweden)

    Andrzej Plewka

    2012-12-01

    Full Text Available Background: In case of overdose of paracetamol the ability of hepatic biotransformation is saturated and accumulation of toxic metabolite – NAPQI takes place. Main CYP isoforms considered to be responsible for bioactivation of APAP and promoting the same liver intoxication are CYP2E1, CYP1A2, CYP3A4 and in animals 2B1/2 isoforms additionally. Purpose of this work was examination of paracetamol influence and/or trichloroethylene on the composition of hepatic cytochrome P450 isoforms. Materials and method: Tests were carried out on rats which were treated with trichloroethylene, paracetamol and/or N-acetylcysteine. In the microsomal fraction content of three isoforms of cytochrome P450 i.e. CYP2E1, CYP2B1/2 and CYP1A2 were determined. Results: Paracetamol slightly stimulated CYP2B1/2 lowering simultaneously level of CYP1A2. Trichloroethylene stimulated CYP2B1/2. N-acetylcysteine stimulated all tested P450 isoforms. N-acetylcysteine given together with examinated xenobiotics induced studied P450 isoforms. Conclusions: N-acetylcysteine demonstrated a protective effect on studied CYP isoforms especially when was given upon termination of xenobiotics exposure

  19. 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...

  20. Mangifera indica L. extract and mangiferin modulate cytochrome P450 and UDP-glucuronosyltransferase enzymes in primary cultures of human hepatocytes.

    Science.gov (United States)

    Rodeiro, Idania; José Gómez-Lechón, M; Perez, Gabriela; Hernandez, Ivones; Herrera, José Alfredo; Delgado, Rene; Castell, José V; Teresa Donato, M

    2013-05-01

    The aqueous stem bark extract of Mangifera indica L. (MSBE) has been reported to have antioxidant, anti-inflammatory and analgesic properties. In previous studies, we showed that MSBE and mangiferin, its main component, lower the activity of some cytochrome P-450 (P450) enzymes in rat hepatocytes and human liver microsomes. In the present study, the effects of MSBE and mangiferin on several P450 enzymes and UDP-glucuronosyltransferases (UGTs) in human-cultured hepatocytes have been examined. After hepatocytes underwent a 48-h treatment with sub-cytotoxic concentrations of the products (50-250 µg/mL), a concentration-dependent decrease of the activity of the five P450 enzymes measured (CYP1A2, 2A6, 2C9, 2D6 and 3A4) was observed. For all the activities, a reduction of at least 50% at the highest concentration (250 µg/mL) was observed. In addition, UGT activities diminished. MSBE considerably reduced UGT1A9 activity (about 60% at 250 µg/mL) and lesser effects on the other UGTs. In contrast, 250 µg/mL mangiferin had greater effects on UGT1A1 and 2B7 than on UGT1A9 (about 55% vs. 35% reduction, respectively). Quantification of specific mRNAs revealed reduced CYP3A4 and 3A5 mRNAs content, and an increase in CYP1A1, CYP1A2, UGT1A1 and UGT1A9 mRNAs. No remarkable effects on the CYP2A6, 2B6, 2C9, 2C19, 2D6 and 2E1 levels were observed. Our results suggest that the activity and/or expression of major P450 and UGT enzymes is modulated by MSBE and that potential herb-drugs interactions could arise after a combined intake of this extract with conventional medicines. Therefore, the potential safety risks of this natural product derived by altering the ADMET properties of co-administered drugs should be examined. Copyright © 2012 John Wiley & Sons, Ltd.

  1. Protective effect of tea polyphenols against paracetamol-induced hepatotoxicity in mice is significantly correlated with cytochrome P450 suppression.

    Science.gov (United States)

    Chen, Xia; Sun, Chang-Kai; Han, Guo-Zhu; Peng, Jin-Yong; Li, Ying; Liu, Yan-Xia; Lv, Yuan-Yuan; Liu, Ke-Xin; Zhou, Qin; Sun, Hui-Jun

    2009-04-21

    To investigate the hepatoprotective activity of tea polyphenols (TP) and its relation with cytochrome P450 (CYP450) expression in mice. Hepatic CYP450 and CYPb(5) levels were measured by UV-spectrophotometry in mice 2 d after intraperitoneal TP (25, 50 and 100 mg/kg per day). Then the mice were intragastricly pre-treated with TP (100, 200 and 400 mg/kg per day) for six days before paracetamol (1000 mg/kg) was given. Their acute mortality was compared with that of control mice. The mice were pre-treated with TP (100, 200, and 400 mg/kg per day) for five days before paracetamol (500 mg/kg) was given. Hepatic CYP2E1 and CYP1A2 protein and mRNA expression levels were evaluated by Western blotting, immunohistochemical staining and transcriptase-polymerase chain reaction. The hepatic CYP450 and CYPb(5) levels in mice of TP-treated groups (100, 200 and 400 mg/kg per day) were decreased in a dose-dependent manner compared with those in the negative control mice. TP significantly attenuated the paracetamol-induced hepatic injury and dramatically reduced the mortality of paracetamol-treated mice. Furthermore, TP reduced CYP2E1 and CYP1A2 expression at both protein and mRNA levels in a dose-dependent manner. TP possess potential hepatoprotective properties and can suppress CYP450 expression.

  2. Protection by Nigella sativa against carbon tetrachloride-induced downregulation of hepatic cytochrome P450 isozymes in rats.

    Science.gov (United States)

    Ibrahim, Zein S; Ishizuka, Mayumi; Soliman, Mohamed; ElBohi, Khlood; Sobhy, Wageh; Muzandu, Kaampwe; Elkattawy, Azza M; Sakamoto, Kentaro Q; Fujita, Shoichi

    2008-11-01

    Nigella sativa (family Ranunculaceae) is an annual plant that has been traditionally used on the Indian subcontinent and in Middle Eastern countries. In this study, we investigated the effect of N. sativa oil on the drug-metabolizing cytochrome P450 (CYP) enzymes and whether it has a protective effect against the acute hepatotoxicity of CCl4. Intraperitoneal injection of rats with CCl4 drastically decreased CYP2E1, CYP2B, CYP3A2, CYP2C11, and CYP1A2 mRNA and protein expressions. Oral administration of 1 ml/kg N. sativa oil every day for one week prior to CCl4 injection alleviated CCl4-induced suppression of CYP2B, CYP3A2, CYP2C11, and CYP1A2. Moreover, CCl4 increased iNOS and TNFalpha mRNA, while N. sativa oil administration for one week prior to CCl4 injection downregulated the CCl4-induced iNOS mRNA and up-regulated IL-10 mRNA. These results indicate that N. sativa oil administration has a protective effect against the CCl4-mediated suppression of hepatic CYPs and that this protective effect is partly due to the downregulation of NO production and up-regulation of the anti-inflammatory IL-10.

  3. Evaluation of the Effects of Mitragyna speciosa Alkaloid Extract on Cytochrome P450 Enzymes Using a High Throughput Assay

    Directory of Open Access Journals (Sweden)

    Raja Elina Raja Aziddin

    2011-08-01

    Full Text Available 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 has the most potent inhibitory effect on CYP3A4 and CYP2D6, with apparent half-maximal inhibitory concentration (IC50 values of 0.78 µg/mL and 0.636 µg/mL, respectively. In addition, moderate inhibition was observed for CYP1A2, with an IC50 of 39 µg/mL, and weak inhibition was detected for CYP2C19. The IC50 of CYP2C19 could not be determined, however, because inhibition was < 50%. Competitive inhibition was found for the MSE-treated CYP2D6 inhibition assay, whereas non-competitive inhibition was shown in inhibition assays using CYP3A4, CYP1A2 and CYP2C19. Quinidine (CYP2D6, ketoconazole (CYP3A4, tranylcypromine (CYP2C19 and furafylline (CYP1A2 were used as positive controls throughout the experiments. This study shows that MSE may contribute to an herb-drug interaction if administered concomitantly with drugs that are substrates for CYP3A4, CYP2D6 and CYP1A2.

  4. Inhibition of Human Cytochrome P450 Enzymes by Allergen Removed Rhus verniciflua Stoke Standardized Extract and Constituents

    Directory of Open Access Journals (Sweden)

    Hyunsik Jung

    2014-01-01

    Full Text Available Objective. Potential interactions between herbal extracts and the cytochrome P450 (CYP system lead to serious adverse events or decreased drug efficacy. Rhus verniciflua stoke (RVS and its constituents have been reported to have various pharmacological properties. We evaluated the inhibitory potential of RVS and its constituents on the major CYP isoforms. Methods. The effects of allergen removed RVS (aRVS standardized extract and major components, fustin and fisetin isolated from aRVS, were evaluated on CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 isoenzyme activity by a luminescent CYP recombinant human enzyme assay. Results. The aRVS extract showed relative potent inhibitory effects on the CYP2C9 (IC50, <0.001 μg/mL, CYP2C19 (IC50, 9.68 μg/mL, and CYP1A2 (IC50, 10.0 μg/mL. However, it showed weak inhibition on CYP3A4 and CYP2D6. Fustin showed moderate inhibitory effects on the CYP2C19 (IC50, 64.3 μg/mL and weak inhibition of the other CYP isoforms similar to aRVS. Fisetin showed potent inhibitory effects on CYP2C9, CYP2C19, and CYP1A2. Fisetin showed moderate inhibition of CYP2D6 and weak inhibition of CYP3A4. Conclusions. These results indicate that aRVS, a clinically available herbal medicine, could contribute to herb-drug interactions when orally coadministered with drugs metabolized by CYP2C9, CYP2C19, and CYP1A2.

  5. Pharmacokinetic Effects of Isavuconazole Coadministration With the Cytochrome P450 Enzyme Substrates Bupropion, Repaglinide, Caffeine, Dextromethorphan, and Methadone in Healthy Subjects

    OpenAIRE

    Yamazaki, Takao; Desai, Amit; Goldwater, Ronald; Han, David; Howieson, Corrie; Akhtar, Shahzad; Kowalski, Donna; Lademacher, Christopher; Pearlman, Helene; Rammelsberg, Diane; Townsend, Robert

    2016-01-01

    Abstract This report describes phase 1 clinical trials performed to assess interactions of oral isavuconazole at the clinically targeted dose (200 mg, administered as isavuconazonium sulfate 372 mg, 3 times a day for 2 days; 200 mg once daily [QD] thereafter) with single oral doses of the cytochrome P450 (CYP) substrates: bupropion hydrochloride (CYP2B6; 100?mg; n = 24), repaglinide (CYP2C8/CYP3A4; 0.5 mg; n = 24), caffeine (CYP1A2; 200 mg; n = 24), dextromethorphan hydrobromide (CYP2D6/CYP3A...

  6. Identification of rabbit cytochromes P450 2C1 and 2C2 as arachidonic acid epoxygenases.

    Science.gov (United States)

    Laethem, R M; Koop, D R

    1992-12-01

    Microsomes prepared from COS-1 cells transiently expressing rabbit cytochromes P450 2C1 and 2C2 catalyzed the metabolism of arachidonic acid to predominantly 11,12- and 14,15-epoxyeicosatrienoic acids (EETs) when microsomal epoxide hydrolase activity was inhibited by 0.2 mM 1,2-epoxy-3,3,3-trichloropropane. P450 2C2 catalyzed the formation of 11,12-EET and 14,15-EET at a ratio of 3.0 and also produced 19-hydroxyeicosatetraenoic acid (19-HETE). The 11,12-EET, 14,15-EET, and 19-HETE represented 48.3, 15.9, and 12.8%, respectively, of the total metabolites formed. P450 2C1 produced a similar but distinct ratio of 11,12-EET to 14,15-EET (2.0) and did not produce any detectable 19-HETE. The 11,12-EET and 14,15-EET represented 63.0 and 31.1%, respectively, of the total metabolites formed. The 8,9- and 5,6-EETs were not detected with either enzyme. The ratio of the 11,12-EET to 14,15-EET was 1.5 with P450 2CAA, a P450 arachidonic acid epoxygenase (P450 2CAA) that had an amino-terminal sequence identical to that of P450 2C2 [J. Biol. Chem. 267:5552-5559 (1992)]. P450 2C1, 2C2, and 2CAA metabolized lauric acid. The ratio of omega-1- to omega-hydroxylated laurate was 3.6, 3.4, and 2.4 for P450 2CAA, P450 2C2, and P450 2C1, respectively. Purified P450 2CAA had a slightly greater apparent molecular weight than expressed P450 2C2 on sodium dodecyl sulfate-polyacrylamide gels. The results clearly establish that rabbit P450 2C1 and 2C2 are arachidonic acid epoxygenases, and they suggest that P450 2CAA and 2C2 are very similar but may not be identical isoforms.

  7. Differentially regulated NADPH:cytochrome P450 oxidoreductases in parsley

    Science.gov (United States)

    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 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. PMID:9405720

  8. El citocromo P-450 y la respuesta terapéutica a los antimaláricos Cytochrome P-450 and the response to antimalarial drugs

    Directory of Open Access Journals (Sweden)

    Valentina Guzmán

    2006-01-01

    Full Text Available OBJETIVOS: Evaluar la relación entre los factores genéticos y fenotípicos del sistema enzimático del citocromo P-450 y la respuesta terapéutica antimalárica a la cloroquina, la amodiaquina, la mefloquina y el proguanil, así como determinar la influencia de algunos factores biológicos y sociales del hospedero en el comportamiento de este complejo enzimático. MÉTODOS: Revisión sistemática de las bases de literatura biomédica PubMed, Excerpta Medica, LILACS y SciELO mediante descriptores en español e inglés. Se usaron los siguientes descriptores: "CYP-450" y "citocromo P-450" y sus combinaciones con "proguanil" (y lo mismo con "mefloquina", "cloroquina" y "amodiaquina", "farmacocinética de proguanil" (y lo mismo con "mefloquina", "cloroquina" y "amodiaquina", "resistencia a proguanil" (y lo mismo con "mefloquina", "cloroquina" y "amodiaquina", "metabolismo", "farmacogenética", "enfermedad", "inflamación", "infección", "enfermedad hepática", "malaria", "nutrición" y "desnutrición". Estos mismos términos se usaron en inglés. La búsqueda se limitó a los artículos publicados en español, inglés y portugués hasta el 30 de junio de 2005 y a cuatro medicamentos antimaláricos: amodiaquina, cloroquina, mefloquina y proguanil. RESULTADOS: Algunos factores genéticos del citocromo P-450 humano (principalmente su polimorfismo, así como otros de tipo biológico y social (la propia presencia de enfermedad, inflamación o infección, la administración de medicamentos antimaláricos y su combinación, y el estado nutricional del paciente, influyen en la actividad de ese complejo enzimático. Solo en la última década se ha abordado el estudio de las bases genéticas de los citocromos y se han podido dilucidar los mecanismos de algunas interacciones entre fármacos y del metabolismo de estos, lo que ha permitido caracterizar el proceso de biotransformación de la amodiaquina y de la cloroquina. Se espera que nuevas investigaciones

  9. Cloning of cDNA encoding steroid 11β-hydroxylase (P450c11)

    International Nuclear Information System (INIS)

    Chua, S.C.; Szabo, P.; Vitek, A.; Grzeschik, K.H.; John, M.; White, P.C.

    1987-01-01

    The authors have isolated bovine and human adrenal cDNA clones encoding the adrenal cytochrome P-450 specific for 11β-hydroxylation (P450c11). A bovine adrenal cDNA library constructed in the bacteriophage λ vector gt10 was probed with a previously isolated cDNA clone corresponding to part of the 3' untranslated region of the 4.2-kilobase (kb) mRNA encoding P450c11. Several clones with 3.2-kb cDNA inserts were isolated. Sequence analysis showed that they overlapped the original probe by 300 base pairs (bp). Combined cDNA and RNA sequence data demonstrated a continuous open reading frame of 1509 bases. P450c11 is predicted to contain 479 amino acid residues in the mature protein in addition to a 24-residue amino-terminal mitochondrial signal sequence. A bovine clone was used to isolate a homologous clone with a 3.5-kb insert from a human adrenal cDNA library. A region of 1100 bp was 81% homologous to 769 bp of the coding sequence of the bovine cDNA except for a 400-bp segment presumed to be an unprocessed intron. Hybridization of the human cDNA to DNA from a panel of human-rodent somatic cell hybrid lines and in situ hybridization to metaphase spreads of human chromosomes localized the gene to the middle of the long arm of chromosome 8. These data should be useful in developing reagents for heterozygote detection and prenatal diagnosis of 11β-hydroxylase deficiency, the second most frequent cause of congenital adrenal hyperplasia

  10. Study on the interaction of chemopreventive compounds and food born carcinogens with cytochrome P450 enzymes

    OpenAIRE

    Brabencová, Eliška

    2013-01-01

    The use of food supplements containing natural chemopreventive compounds increased in recent years. Some of the most popular chemopreventive compounds are flavonoids. Due to their natural origin, flavonoids are generally accepted as safe compounds. They exert antioxidant, anti-cancer and anti-inflammatory properties. However, flavonoids should be considered as foreign compounds (xenobiotics). Flavonoids interact with many enzymes, among the most important belong cytochromes P450 (CYPs), key e...

  11. Characterisation of the cytochrome P450 enzymes involved in the in vitro metabolism of granisetron.

    OpenAIRE

    Bloomer, J C; Baldwin, S J; Smith, G J; Ayrton, A D; Clarke, S E; Chenery, R J

    1994-01-01

    1. The metabolism of granisetron was investigated in human liver microsomes to identify the specific forms of cytochrome P450 responsible. 2. 7-hydroxy and 9'-desmethyl granisetron were identified as the major products of metabolism following incubation of granisetron with human liver microsomes. At low, clinically relevant, concentrations of granisetron the 7-hydroxy metabolite predominated. Rates of granisetron 7-hydroxylation varied over 100-fold in the human livers investigated. 3. Enzyme...

  12. Control by substrate of the cytochrome p450-dependent redox machinery: mechanistic insights.

    Science.gov (United States)

    Hlavica, Peter

    2007-08-01

    Based on initial studies with bacterial CYP101A1, a popular concept emerged predicting that substrate-induced low-to-high spin conversion of P450s is universally associated with shifts of the midpoint potential to a more positive value to maximize rates of electron transfer and metabolic turnover. However, evaluation of the plethora of observations with pro- and eukaryotic hemoproteins suggests a caveat as to generalization of this principle. Thus, some P450s are inherently high-spin, so that there is no need for a supportive substrate-triggered impulse to electron flow. With other enzymes, high-spin content is not consonant with reductive activity, and spin transition as such is not essential to sustaining substrate oxidation. Also, with certain proteins the low-spin conformer is reduced as swift as the high-spin entity. Moreover, there is not regularly a linear relationship between high-spin level and anodic shift of the reduction potential. Similarly, in given cases turnover may proceed despite insignificant or even lacking substrate-provoked alterations in the redox behaviour. Thus, folding of the disparate and sometimes conflicting data into a harmonized overall picture is a lingering problem. Apart from direct perturbation of the electrochemical properties, substrate docking may entail changes in enzyme conformation such as to favour productive complexation with redox partners or modulate electron transfer conduits within preformed donor/acceptor adducts, resulting in elevated ease of flow of reducing equivalents. Substrate-steered ordering of the oligomeric aggregation state of P450s is likely to impose steric constraints on heterodimers, causing one component to more readily align with electron carriers. Careful uncovering of electrochemical mechanisms in these systems will be fruitful to tailoring of novel bioenergetic machines and redox chains via redox-inspired protein engineering or molecular Lego, capable of generating products of interest or degrading

  13. Cytochrome P-450 complex formation in rat liver by the antibiotic tiamulin.

    OpenAIRE

    Witkamp, R F; Nijmeijer, S M; van Miert, A S

    1996-01-01

    Tiamulin is a semisynthetic diterpene antibiotic frequently used in farm animals. The drug has been shown to produce clinically important--often lethal--interactions with other compounds. It has been suggested that this is caused by a selective inhibition of oxidative drug metabolism via the formation of a cytochrome P-450 metabolic intermediate complex. In the present study, rats were treated orally for 6 days with tiamulin at two different doses: 40 and 226 mg/kg of body weight. For compari...

  14. Cloning and tissue expression of cytochrome P450 1B1 and 1C1 ...

    African Journals Online (AJOL)

    Cytochrome P450 1 (CYP1) is widely used as an indicator of exposure to environmental contaminants. In the study, two full-length complementary DNAs encode for CYP1B1 and CYP1C1 were cloned from medaka liver exposed to 500 ppb β-naphthoflavone for 24 h. CYP1B1, having 1984 bp, contains an open reading ...

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

    DEFF Research Database (Denmark)

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

    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,...... of the less demanding methods are applied to study the CYP3A4 metabolism of progesterone and dextromethorphan....

  16. In vivo cytochrome P450 activity alterations in diabetic nonalcoholic steatohepatitis mice

    OpenAIRE

    Li, Hui; Clarke, John D.; Dzierlenga, Anika L.; Bear, John; Goedken, Michael J.; Cherrington, Nathan J.

    2016-01-01

    Nonalcoholic steatohepatitis (NASH) has been identified as a source of significant interindividual variation in drug metabolism. A previous ex vivo study demonstrated significant changes in hepatic Cytochrome P450 (CYP) activity in human NASH. This study evaluated the in vivo activities of multiple CYP isoforms simultaneously in prominent diabetic NASH mouse models. The pharmacokinetics of CYP selective substrates: caffeine, losartan, and omeprazole changed significantly in a diabetic NASH mo...

  17. 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...... nitrogen atom seems to be of vital importance for the amount of N-oxidized product formed relative to dealkylation and hydroxylation products....

  18. Characterization of cytochrome P450 monooxygenase CYP154H1 from the thermophilic soil bacterium Thermobifida fusca

    NARCIS (Netherlands)

    Schallmey, Anett; den Besten, Gijs; Teune, Ite G. P.; Kembaren, Roga F.; Janssen, Dick B.

    Cytochrome P450 monooxygenases are valuable biocatalysts due to their ability to hydroxylate unactivated carbon atoms using molecular oxygen. We have cloned the gene for a new cytochrome P450 monooxygenase, named CYP154H1, from the moderately thermophilic soil bacterium Thermobifida fusca. The

  19. A theoretical study on the metabolic activation of paracetamol by cytochrome P-450 : indications for a uniform oxidation mechanism

    NARCIS (Netherlands)

    Koymans, L.; Lenthe, J.H.; Van de Straat, R; Donné-Op den Kelder, G M; Vermeulen, N P

    1989-01-01

    The cytochrome P-450 mediated activation of paracetamol (PAR) to the reactive electrophilic intermediate N-acetyl-p-benzoquinone imine (NAPQI) has been studied by use of SV 6-31G ab initio energy calculations and spin distributions. A simplified model for cytochrome P-450 has been used by

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

    NARCIS (Netherlands)

    Cankar, K.; van Houwelingen, A.; Bosch, H.J.; Sonke, T.; Bouwmeester, H.; Beekwilder, J.P.

    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

  1. Molecular cloning of P450 aromatase from the leopard gecko and its expression in the ovary.

    Science.gov (United States)

    Endo, Daisuke; Park, Min Kyun

    2005-07-01

    In this study, we identified the cDNA of P450 aromatase in the leopard gecko, a lizard with temperature-dependent sex determination. The cDNA encodes a putative protein of 505 amino acids. The deduced amino acid sequence of leopard gecko aromatase cDNA showed 80% identity with that of turtles, 70% with humans and 77% with chickens. This is the first report of the identification of P450 aromatase cDNA in squamata species. It has been reported that this gene is expressed in different layers of cells in the ovary of mammalian species and avian species. Thus, we also investigated cells expressing the mRNA of this gene in the ovary of the leopard gecko by RT-PCR and in situ hybridization. The mRNA expression of leopard gecko P450 aromatase was localized in both the thecal and granulosa cell layers in the ovary. The expression in thecal and granulosa cell layers was examined in the largest follicle, second largest follicle and third largest follicle by RT-PCR. A higher level of mRNA expression was observed in the granulosa cell layer of the second largest follicle than in other cell layers. This result may reflect the characteristics of follicles in species with automonochronic ovulation.

  2. Preferential hydroxylation over epoxidation catalysis by a horseradish peroxidase mutant: a cytochrome P450 mimic.

    Science.gov (United States)

    de Visser, Sam P

    2007-10-25

    Density functional theory calculations are presented on the catalytic properties of a horseradish peroxidase mutant whereby the axial nitrogen atom is replaced by phosphorus. This mutant has never been studied experimentally and only one theoretical report on this system is known (de Visser, S. P. J. Phys. Chem. B 2006, 110, 20759-20761). Thus, a one-atom substitution in horseradish peroxidase changes the properties of the catalytic center of the enzyme to more cytochrome P450-type qualities. In particular, the phosphorus-substituted horseradish peroxidase mutant reacts with substrates via a unique reactivity pattern, whereby alkanes are regioselectively hydroxylated even in the presence of a double bond. Reaction barriers of propene epoxidation and hydroxylation are almost identical to ones observed for a cytochrome P450 catalyst and significantly higher than those obtained for a horseradish peroxidase catalyst. It is shown that the regioselectivity difference is entropy and thermally driven and that the electron-transfer processes that occur during the reaction mechanism follow cytochrome P450-type patterns in the hydroxylation reaction.

  3. Food Polyphenol Apigenin Inhibits the Cytochrome P450 Monoxygenase Branch of the Arachidonic Acid Cascade.

    Science.gov (United States)

    Steuck, Maryvonne; Hellhake, Stefan; Schebb, Nils Helge

    2016-11-30

    The product of cytochrome P450 monooxygenase (P450) ω-hydroxylation of arachidonic acid (AA), 20- hydroxyeicosatetraenoic acid (HETE), is a potent vasoconstrictor. Utilizing microsomes as well as individual CYP4 isoforms we demonstrate here that flavonoids can block 20-HETE formation. Apigenin inhibits CYP4F2 with an IC 50 value of 4.6 μM and 20-HETE formation in human liver and kidney microsomes at 2.4-9.8 μM. Interestingly, the structurally similar naringenin shows no relevant effect on the formation of 20-HETE. Based on these in vitro data, it is impossible to evaluate if a relevant blockade of 20-HETE formation can result in humans from intake of polyphenols with the diet. However, the potency of apigenin is comparable to those of P450 inhibitors such as ketoconazole. Moreover, an IC 50 value in the micromolar range is also described for the inhibition of CYP-mediated drug metabolism leading to food-drug interactions. The modulation of the arachidonic acid cascade by food polyphenols therefore warrants further investigation.

  4. Construction and engineering of a thermostable self-sufficient cytochrome P450

    Energy Technology Data Exchange (ETDEWEB)

    Mandai, Takao; Fujiwara, Shinsuke [Nanobiotechnology Research Center and Department of Bioscience, School of Science and Technology, Kwansei Gakuin University, 2-1 Gakuen, Sanda 669-1337 (Japan); Imaoka, Susumu, E-mail: imaoka@kwansei.ac.jp [Nanobiotechnology Research Center and Department of Bioscience, School of Science and Technology, Kwansei Gakuin University, 2-1 Gakuen, Sanda 669-1337 (Japan)

    2009-06-19

    CYP175A1 is a thermophilic cytochrome P450 and hydroxylates {beta}-carotene. We previously identified a native electron transport system for CYP175A1. In this report, we constructed two fusion proteins consisting of CYP175A1, ferredoxin (Fdx), and ferredoxin-NADP{sup +} reductase (FNR): H{sub 2}N-CYP175A1-Fdx-FNR-COOH (175FR) and H{sub 2}N-CYP175A1-FNR-Fdx-COOH (175RF). Both 175FR and 175RF were expressed in Escherichia coli and purified. The V{sub max} value for {beta}-carotene hydroxylation was 25 times higher with 175RF than 175FR and 9 times higher with 175RF than CYP175A1 (non-fused protein), although the k{sub m} values of these enzymes were similar. 175RF retained 50% residual activity even at 80 {sup o}C. Furthermore, several mutants of the CYP175A1 domain of 175RF were prepared and one mutant (Q67G/Y68I) catalyzed the hydroxylation of an unnatural substrate, testosterone. Thus, this is the first report of a thermostable self-sufficient cytochrome P450 and the engineering of a thermophilic cytochrome P450 for the oxidation of an unnatural substrate.

  5. Construction and engineering of a thermostable self-sufficient cytochrome P450

    International Nuclear Information System (INIS)

    Mandai, Takao; Fujiwara, Shinsuke; Imaoka, Susumu

    2009-01-01

    CYP175A1 is a thermophilic cytochrome P450 and hydroxylates β-carotene. We previously identified a native electron transport system for CYP175A1. In this report, we constructed two fusion proteins consisting of CYP175A1, ferredoxin (Fdx), and ferredoxin-NADP + reductase (FNR): H 2 N-CYP175A1-Fdx-FNR-COOH (175FR) and H 2 N-CYP175A1-FNR-Fdx-COOH (175RF). Both 175FR and 175RF were expressed in Escherichia coli and purified. The V max value for β-carotene hydroxylation was 25 times higher with 175RF than 175FR and 9 times higher with 175RF than CYP175A1 (non-fused protein), although the k m values of these enzymes were similar. 175RF retained 50% residual activity even at 80 o C. Furthermore, several mutants of the CYP175A1 domain of 175RF were prepared and one mutant (Q67G/Y68I) catalyzed the hydroxylation of an unnatural substrate, testosterone. Thus, this is the first report of a thermostable self-sufficient cytochrome P450 and the engineering of a thermophilic cytochrome P450 for the oxidation of an unnatural substrate.

  6. Comparison of basal and induced cytochromes P450 in 6 species of waterfowl

    Science.gov (United States)

    Melancon, M.J.; Rattner, B.A.; Hoffman, D.J.; Beeman, D.; Day, D.; Custer, T.

    1999-01-01

    Cytochrome P450-associated monooxygenase activities were measured in control and prototype inducer-treated mallard duck, black duck, wood duck, lesser scaup, Canada goose and mute swan. Ages of the birds ranged from pipping embryos (that were treated approximately 3 days before pipping) to adults. Three or more of the following hepatic microsomal monooxygenases were assayed in each species: Benzyloxyresorufin-O-dealkylase (BROD), Ethoxyresorufin-O-dealkylase (EROD), methoxyresorufin-O-dealkylase (MROD), and pentoxyresorufin-O-dealkylase (PROD). Baseline activities differed between species, but because of differences in ages, sources of the eggs or birds, and diets, these cannot be viewed as absolute differences. The cytochrome P450 inducers utilized were beta-naphthoflavone (BNF), 3-methylcholanthrene (3MC) and phenobarbital (PB). In general, there was little response to PB; only lesser scaup were induced to greater than three times control level and most species were well under this. Responses to BNF and 3MC occurred in each species studied, but differed in which of the monooxygenases was most induced (absolute values and ratios to control values) and in relative induction between species. BROD frequently had an induction ratio EROD. Overall, lesser scaup were the most responsive, canada geese the least responsive, and the other species intermediate in responsiveness to the cytochrome P450 inducers studied.

  7. 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.

  8. Electroactive cytochrome P450BM3 cast polyion films on graphite electrodes

    International Nuclear Information System (INIS)

    Pardo-Jacques, Aurelie; Basseguy, Regine; Bergel, Alain

    2006-01-01

    Films of electrochemically active cytochrome P450 BM 3 were constructed on graphite electrodes using alternate assembly with polyethyleneimine (PEI). The original layer-by-layer adsorption method was slightly modified here to form so-called 'cast polyion' films. The cast polyion films were elaborated by immobilizing two successive layers of PEI and protein in very large excess with respect to a monolayer, without any intermediate washing step. Following the immobilization steps by SEM showed that uniform films of a few micrometers were deposited on the graphite surface. The electrochemically activity of the immobilized cytP450 was tested with regard to the reduction of oxygen and the one-electron reduction of the heme. Cyclic voltammetry indicated surface concentration of electrochemically active cytP450 around 0.6nmol/cm 2 , which corresponded to 5% of the total amount of protein that was consumed by the immobilisation process. Adapting the procedure to a graphite felt electrode with the view of scaling up porous electrodes for large scale synthesis increased the concentration to 0.9nmol/cm 2 . Cast polyion films may represent a simple technique to immobilize high amount of electrochemically active protein, keeping the advantage of the electrostatic interactions of the regular layer-by-layer method

  9. In vitro inhibitory mechanisms and molecular docking of 1'-S-1'-acetoxychavicol acetate on human cytochrome P450 enzymes.

    Science.gov (United States)

    Haque, A K M Mahmudul; Leong, Kok Hoong; Lo, Yoke Lin; Awang, Khalijah; Nagoor, Noor Hasima

    2017-07-15

    The compound, 1'-S-1'-acetoxychavicol acetate (ACA), isolated from the rhizomes of a Malaysian ethno-medicinal plant, Alpinia conchigera Griff. (Zingiberaceae), was previously shown to have potential in vivo antitumour activities. In the development of a new drug entity, potential interactions of the compound with the cytochrome P450 superfamily metabolizing enzymes need to be ascertain. The concomitant use of therapeutic drugs may cause potential drug-drug interactions by decreasing or increasing plasma levels of the administered drugs, leading to a suboptimal clinical efficacy or a higher risk of toxicity. Thus, evaluating the inhibitory potential of a new chemical entity, and to clarify the mechanism of inhibition and kinetics in the various CYP enzymes is an important step to predict drug-drug interactions. This study was designed to assess the potential inhibitory effects of Alpinia conchigera Griff. rhizomes extract and its active constituent, ACA, on nine c-DNA expressed human cytochrome P450s (CYPs) enzymes using fluorescent CYP inhibition assay. The half maximal inhibitory concentration (IC 50 ) of Alpinia conchigera Griff. rhizomes extract and ACA was determined for CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C19, CYP2D6, CYP2E1, CYP3A4 and CYP3A5. A. conchigera extract only moderately inhibits on CYP3A4 (IC 50 = 6.76 ± 1.88µg/ml) whereas ACA moderately inhibits the activities of CYP1A2 (IC 50 = 4.50 ± 0.10µM), CYP2D6 (IC 50 = 7.50 ± 0.17µM) and CYP3A4 (IC 50 = 9.50 ± 0.57µM) while other isoenzymes are weakly inhibited. In addition, mechanism-based inhibition studies reveal that CYP1A2 and CYP3A4 exhibited non-mechanism based inhibition whereas CYP2D6 showed mechanism-based inhibition. Lineweaver-Burk plots depict that ACA competitively inhibited both CYP1A2 and CYP3A4, with a K i values of 2.36 ± 0.03 µM and 5.55 ± 0.06µM, respectively, and mixed inhibition towards CYP2D6 with a K i value of 4.50 ± 0.08µ

  10. Cytochrome P450 regulation: the interplay between its heme and apoprotein moieties in synthesis, assembly, repair, and disposal.

    Science.gov (United States)

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

    2011-02-01

    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 biotransformation of both endo- and xenobiotics. This well-recognized functional role notwithstanding, heme also regulates P450 protein synthesis, assembly, repair, and disposal. These less well-appreciated aspects are reviewed herein.

  11. A Panel of Cytochrome P450 BM3 Variants To Produce Drug Metabolites and Diversify Lead Compounds

    Science.gov (United States)

    Sawayama, Andrew M.; Chen, Michael M. Y.; Kulanthaivel, Palaniappan; Kuo, Ming-Shang; Hemmerle, Horst; Arnold, Frances H.

    2011-01-01

    Here we demonstrate that a small panel of variants of cytochrome P450 BM3 from Bacillus megaterium covers the breadth of reactivity of human P450s by producing 12 of 13 mammalian metabolites for two marketed drugs, verapamil and astemizole, and one research compound. The most active enzymes support preparation of individual metabolites for preclinical bioactivity and toxicology evaluations. Underscoring their potential utility in drug lead diversification, engineered P450 BM3 variants also produce novel metabolites by catalyzing reactions at carbon centers beyond those targeted by animal and human P450s. Production of a specific metabolite can be improved by directed evolution of the enzyme catalyst. Some variants are more active on the more hydrophobic parent drug than on its metabolites, which limits production of multiply-hydroxylated species, a preference that appears to depend on the evolutionary history of the P450 variant. PMID:19774562

  12. Effect of strychnine hydrochloride on liver cytochrome P450 mRNA expression and monooxygenase activities in rat

    Directory of Open Access Journals (Sweden)

    Qian Gao

    2011-08-01

    Full Text Available Strychnos nux-vomica L. has been frequently used in traditional Chinese medicine but has high acute toxicity. It is commonly taken with Glycyrrhizae radix to decrease its toxicity but the mechanism of this interaction is unknown. In this work, the mRNA expression and the activity of four cytochrome P450 (CYP enzymes representative of four subfamilies (CYP1A, CYP3A, CYP2C and CYP2E were determined ex vivo in rat livers from groups of Wistar rats orally administered strychnine hydrochloride (SH at three doses (0.1, 0.3 and 0.9 mg/kg/day alone and, at the highest dose, in combination with glycyrrhetinic acid (GA, 25 mg/kg/day or liquiritin (LQ, 20 mg/kg/day once a day for 7 consecutive days. Compared to control, the mRNA expressions of CYP3A1, 1A2 and 2E1 were higher in rats receiving the highest dose of SH but lower for CYP3A1 and CYP2E1 in rats receiving the SH+GA and SH+LQ combinations. CYP2E1 activity was higher and CYP2C, CYP3A and CYP1A2 activities were lower in rats receiving the highest dose of SH. In contrast CYP1A2 and CYP2C activities were higher and CYP2E1 and CYP3A activities lower in rats receiving the SH+GA combination. CYP2E1 and CYP3A activities were also lower in rats receiving the SH+LQ combination. The results show that treatment with SH for 7 days affects the expression and the activity of CYP enzymes and that coadministration of GA and LQ modulates these effects. This modulation may explain the role of Glycyrrhizae radix in reducing the acute toxicity of Strychnos nux-vomica L.CYPs enzymes.

  13. El sistema citocromo P450 y el metabolismo de xenobióticos

    Directory of Open Access Journals (Sweden)

    Julio César Rodríguez González

    Full Text Available Los organismos están constantemente expuestos a una gran variedad de xenobióticos. Las enzimas citocromo P450 participan en la fase I del metabolismo de xenobióticos, incluyendo los fármacos, y en funciones biosintéticas endógenas por reacciones de oxidación, reducción e hidrólisis. En el hombre se estima que pueden metabolizar hasta dos tercios de las drogas y la mayor parte de estas reacciones ocurre en el hígado. Estas enzimas se encuentran en todos los reinos biológicos. Actualmente se conocen más de 18 000 genes citocromo P450 organizados en familias y subfamilias según el porcentaje de identidad de secuencia de sus aminoácidos, y este número aumenta cada año con el hallazgo de nuevas secuencias del genoma. Ellas son una superfamilia de hemoproteínas monooxidasas del sistema oxidasa de función mixta localizadas en las membranas del retículo endoplasmático liso y mitocondrial interna. La diversidad de reacciones que cataliza y su amplia especificidad de sustrato lo destacan como uno de los catalizadores más diversos y versátiles conocidos y juega un papel crítico en la bioquímica, farmacología y toxicología. Se realizó una búsqueda por palabras clave en las bases de datos Pubmed y Medscape en los últimos diez años. También se consultaron sitios de Internet relacionados con investigaciones del citocromo P450 como bases de datos. Esta revisión es una actualización sobre aspectos generales del citocromo P450 y comprende una breve historia de la investigación del citocromo P450, su sistema de nomenclatura estándar; y describe su multiplicidad, la distribución a nivel de órgano y localización subcelular, estructura y función.

  14. Emergence of Function in P450-Proteins: A Combined Quantum Mechanical/Molecular Mechanical and Molecular Dynamics Study of the Reactive Species in the H2O2-Dependent Cytochrome P450SPα and Its Regio- and Enantioselective Hydroxylation of Fatty Acids.

    Science.gov (United States)

    Ramanan, Rajeev; Dubey, Kshatresh Dutta; Wang, Binju; Mandal, Debasish; Shaik, Sason

    2016-06-01

    This work uses combined quantum mechanical/molecular mechanical and molecular dynamics simulations to investigate the mechanism and selectivity of H2O2-dependent hydroxylation of fatty acids by the P450SPα class of enzymes. H2O2 is found to serve as the surrogate oxidant for generating the principal oxidant, Compound I (Cpd I), in a mechanism that involves homolytic O-O bond cleavage followed by H-abstraction from the Fe-OH moiety. Our results rule out a substrate-assisted heterolytic cleavage of H2O2 en route to Cpd I. We show, however, that substrate binding stabilizes the resultant Fe-H2O2 complex, which is crucial for the formation of Cpd I in the homolytic pathway. A network of hydrogen bonds locks the HO· radical, formed by the O-O homolysis, thus directing it to exclusively abstract the hydrogen atom from Fe-OH, thereby forming Cpd I, while preventing the autoxoidative reaction, with the porphyrin ligand, and the substrate oxidation. The so formed Cpd I subsequently hydroxylates fatty acids at their α-position with S-enantioselectivity. These selectivity patterns are controlled by the active site: substrate's binding by Arg241 determines the α-regioselectivity, while the Pro242 residue locks the prochiral α-CH2, thereby leading to hydroxylation of the pro-S C-H bond. Our study of the mutant Pro242Ala sheds light on potential modifications of the enzyme's active site in order to modify reaction selectivity. Comparisons of P450SPα to P450BM3 and to P450BSβ reveal that function has evolved in these related metalloenzymes by strategically placing very few residues in the active site.

  15. Development of an in vitro cytochrome P450 cocktail inhibition assay for assessing the inhibition risk of drugs of abuse.

    Science.gov (United States)

    Dinger, Julia; Meyer, Markus R; Maurer, Hans H

    2014-10-01

    Drugs of abuse are not tested for cytochrome P450 (CYP) inhibition potential before distribution. Therefore, a cocktail assay should be developed for testing the inhibition potential for all relevant CYPs. The following CYP test substrates and selective inhibitors were incubated in pooled human liver microsomes: phenacetin (alpha-naphthoflavone for CYP1A2), coumarin (tranylcypromine, CYP2A6), bupropion (sertraline, CYP2B6), amodiaquine (trimethoprim, CYP2C8), diclofenac (sulfaphenazole, CYP2C9), omeprazole (fluconazole, CYP2C19), dextromethorphan (quinidine, CYP2D6), chlorzoxazone (clomethiazole, CYP2E1), testosterone (verapamil, CYP3A). Samples were analyzed after protein precipitation using a Thermo Fisher Q-Exactive LC-high-resolution-MS/MS. The IC50 values were calculated by plotting the concentration of the formed metabolite, relative to the control sample, over the logarithm of the inhibitor concentration. They were determined either for single substrate or the cocktail incubation. Unfortunately, the cocktail assay had to be split because of interferences during incubation caused by substrates or metabolites, but the mixture of both incubates could be analyzed in one analytical run. The IC50 values determined in the single substrate or both cocktail incubations were comparable among themselves and with published data. In conclusion, the new inhibition cocktail assay was reproducible and applicable for testing the inhibition potential of drugs of abuse as exemplified for 2,5-dimethoxy-4-iodo-amfetamine (DOI). Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  16. Changes in cytochrome P450 gene expression and enzyme activity induced by xenobiotics in rabbits in vivo and in vitro

    Directory of Open Access Journals (Sweden)

    Orsolya Palócz

    2017-06-01

    Full Text Available As considerable inter-species differences exist in xenobiotic metabolism, developing new pharmaceutical therapies for use in different species is fraught with difficulties. For this reason, very few medicines have been registered for use in rabbits, despite their importance in inter alia meat and fur production. We have developed a rapid and sensitive screening system for drug safety in rabbits based on cytochrome P450 enzyme assays, specifically CYP1A1, CYP1A2 and CYP3A6, employing an adaptation of the luciferin-based clinical assay currently used in human drug screening. Short-term (4-h cultured rabbit primary hepatocytes were treated with a cytochrome inducer (phenobarbital and 2 inhibitors (alpha-naphthoflavone and ketoconazole. In parallel, and to provide verification, New Zealand white rabbits were dosed with 80 mg/kg phenobarbital or 40 mg/kg ketoconazole for 3 d. Ketoconazole significantly increased CYP3A6 gene expression and decreased CYP3A6 activity both in vitro and in vivo. CYP1A1 activity was decreased by ketoconazole in vitro and increased in vivo. This is the first report of the inducer effect of ketoconazole on rabbit cytochrome isoenzymes in vivo. Our data support the use of a luciferin-based assay in short-term primary hepatocytes as an appropriate tool for xenobiotic metabolism assays and short-term toxicity testing in rabbits.

  17. Traditional Preparations and Methanol Extracts of Medicinal Plants from Papua New Guinea Exhibit Similar Cytochrome P450 Inhibition

    Directory of Open Access Journals (Sweden)

    Erica C. Larson

    2016-01-01

    Full Text Available The hypothesis underlying this current work is that fresh juice expressed from Papua New Guinea (PNG medicinal plants (succus will inhibit human Cytochrome P450s (CYPs. The CYP inhibitory activity identified in fresh material was compared with inhibition in methanol extracts of dried material. Succus is the most common method of traditional medicine (TM preparation for consumption in PNG. There is increasing concern that TMs might antagonize or complicate drug therapy. We have previously shown that methanol extracts of commonly consumed PNG medicinal plants are able to induce and/or inhibit human CYPs in vitro. In this current work plant succus was prepared from fresh plant leaves. Inhibition of three major CYPs was determined using human liver microsomes and enzyme-selective model substrates. Of 15 species tested, succus from 6/15 was found to inhibit CYP1A2, 7/15 inhibited CYP3A4, and 4/15 inhibited CYP2D6. Chi-squared tests determined differences in inhibitory activity between succus and methanol preparations. Over 80% agreement was found. Thus, fresh juice from PNG medicinal plants does exhibit the potential to complicate drug therapy in at risk populations. Further, the general reproducibility of these findings suggests that methanol extraction of dried material is a reasonable surrogate preparation method for fresh plant samples.

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

    International Nuclear Information System (INIS)

    Polusani, Srikanth R.; Kar, Rekha; Riquelme, Manuel A.; Masters, Bettie Sue; Panda, Satya P.

    2011-01-01

    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 b 5 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.

  19. Molecular cloning and functional characterization of multiple NADPH-cytochrome P450 reductases from Andrographis paniculata.

    Science.gov (United States)

    Lin, Huixin; Wang, Jian; Qi, Mengdie; Guo, Juan; Rong, Qixian; Tang, Jinfu; Wu, Yisheng; Ma, Xiaojing; Huang, Luqi

    2017-09-01

    Andrographis paniculata (Burm.f.) Wall. ex Nees is widely used as medicinal herb in Southern and Southeastern Asia and andrographolide is its main medicinal constituent. Based on the structure of andrographolide, it has been proposed that cytochrome P450 enzymes play vital roles on its biosynthesis. NADPH:cytochrome P450 reductase (CPR) is the most important redox partner of multiple P450s. In this study, three CPRs were identified in the genomic data of A. paniculata (namely ApCPR1, ApCPR2, and ApCPR3), and their coding regions were cloned. They varied from 62% to 70% identities to each other at the amino acid sequence level. ApCPR1 belongs to Class I of dicotyledonous CPR while both ApCPR2 and ApCPR3 are grouped to Class II. The recombinant enzymes ApCPR1 and ApCPR2 reduced cytochrome c and ferricyanide in an NADPH-dependent manner. In yeast, they supported the activity of CYP76AH1, a ferruginol-forming enzyme. However, ApCPR3 did not show any enzymatic activities either in vitro or in vivo. Quantitative real-time PCR analysis showed that both ApCPR1 and ApCPR2 expressed in all tissues examined, but ApCPR2 showed higher expression in leaves. Expression of ApCPR2 was inducible by MeJA and its pattern matched with andrographolide accumulation. Present investigation suggested ApCPR2 involves in the biosynthesis of secondary metabolites including andrographolide. Copyright © 2017. Published by Elsevier B.V.

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

    Energy Technology Data Exchange (ETDEWEB)

    Polusani, Srikanth R.; Kar, Rekha; Riquelme, Manuel A.; Masters, Bettie Sue [The University of Texas Health Science Center at San Antonio, Department of Biochemistry, San Antonio, TX 78229 (United States); Panda, Satya P., E-mail: panda@uthscsa.edu [The University of Texas Health Science Center at San Antonio, Department of Biochemistry, San Antonio, TX 78229 (United States)

    2011-08-05

    Highlights: {yields} Humans with severe forms of cytochrome P450 oxidoreductase (CYPOR) mutations show bone defects as observed in Antley-Bixler Syndrome. {yields} 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. {yields} Knockdown of CYPOR decreased Gap Junctional Intercellular Communication and hemichannel activity. {yields} Knockdown of CYPOR decreased Cx43 in mouse primary calvarial osteoblasts. {yields} 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 b{sub 5} 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.

  1. N-Heterocyclic Carbene Capture by Cytochrome P450 3A4

    Science.gov (United States)

    Jennings, Gareth K.; Ritchie, Caroline M.; Shock, Lisa S.; Lyons, Charles E.

    2016-01-01

    Cytochrome P450 3A4 (CYP3A4) is the dominant P450 enzyme involved in human drug metabolism, and its inhibition may result in adverse interactions or, conversely, favorably reduce the systemic elimination rates of poorly bioavailable drugs. Herein we describe a spectroscopic investigation of the interaction of CYP3A4 with N-methylritonavir, an analog of ritonavir, widely used as a pharmacoenhancer. In contrast to ritonavir, the binding affinity of N-methylritonavir for CYP3A4 is pH-dependent. At pH UV-visible spectroscopy binding studies with molecular fragments narrows the source of this pH dependence to its N-methylthiazolium fragment. The C2 proton of this group is acidic, and variable-pH resonance Raman spectroscopy tentatively assigns it a pKa of 7.4. Hence, this fragment of N-methylritonavir is expected to be readily deprotonated under physiologic conditions to yield a thiazol-2-ylidene, which is an N-heterocyclic carbene that has high-affinity for and is presumed to be subsequently captured by the heme iron. This mechanism is supported by time-dependent density functional theory with an active site model that accurately reproduces distinguishing features of the experimental UV-visible spectra of N-methylritonavir bound to CYP3A4. Finally, density functional theory calculations support that this novel interaction is as strong as the tightest-binding azaheterocycles found in P450 inhibitors and could offer new avenues for inhibitor development. PMID:27126611

  2. Metabolism of 7-ethoxycoumarin, flavanone and steroids by cytochrome P450 2C9 variants.

    Science.gov (United States)

    Uno, Tomohide; Nakano, Ryosuke; Kanamaru, Kengo; Takenaka, Shinji; Uno, Yuichi; Imaishi, Hiromasa

    2017-11-01

    CYP2C9 is a human microsomal cytochrome P450c (CYP). Much of the variation in CYP2C9 levels and activity can be attributed to polymorphisms of this gene. Wild-type CYP2C9 and mutants were coexpressed with NADPH-cytochrome P450 reductase in Escherichia coli. The hydroxylase activities toward 7-ethoxycoumarin, flavanone and steroids were examined. Six CYP2C9 variants showed Soret peaks (450 nm) typical of P450 in reduced CO-difference spectra. CYP2C9.38 had the highest 7-ethoxycoumarin de-ethylase activity. All the CYP2C9 variants showed lower flavanone 6-hydroxylation activities than CYP2C9.1 (the wild-type). CYP2C9.38 showed higher activities in testosterone 6β-hydroxylation, progesterone 6β-/16α-hydroxylation, estrone 11α-hydroxylation and estradiol 6α-hydroxylation than CYP2C9.1. CYP2C9.40 showed higher testosterone 17-oxidase activity than CYP2C9.1; CYP2C9.8 showed higher estrone 16α-hydroxylase activity and CYP2C9.12 showed higher estrone 11α-hydroxylase activity. CYP2C9.9 and CYP2C9.10 showed similar activities to CYP2C9.1. These results indicate that the substrate specificity of CYP2C9.9 and CYP2C9.10 was not changed, but CYP2C9.8, CYP2C9.12 and CYP2C9.40 showed different substrate specificity toward steroids compared with CYP2C9.1; and especially CYP2C9.38 displayed diverse substrate specificities towards 7-ethoxycoumarin and steroids. Copyright © 2017 John Wiley & Sons, Ltd.

  3. Cytochrome P450 associated with insecticide resistance catalyzes cuticular hydrocarbon production in Anopheles gambiae.

    Science.gov (United States)

    Balabanidou, Vasileia; Kampouraki, Anastasia; MacLean, Marina; Blomquist, Gary J; Tittiger, Claus; Juárez, M Patricia; Mijailovsky, Sergio J; Chalepakis, George; Anthousi, Amalia; Lynd, Amy; Antoine, Sanou; Hemingway, Janet; Ranson, Hilary; Lycett, Gareth J; Vontas, John

    2016-08-16

    The role of cuticle changes in insecticide resistance in the major malaria vector Anopheles gambiae was assessed. The rate of internalization of (14)C deltamethrin was significantly slower in a resistant strain than in a susceptible strain. Topical application of an acetone insecticide formulation to circumvent lipid-based uptake barriers decreased the resistance ratio by ∼50%. Cuticle analysis by electron microscopy and characterization of lipid extracts indicated that resistant mosquitoes had a thicker epicuticular layer and a significant increase in cuticular hydrocarbon (CHC) content (∼29%). However, the CHC profile and relative distribution were similar in resistant and susceptible insects. The cellular localization and in vitro activity of two P450 enzymes, CYP4G16 and CYP4G17, whose genes are frequently overexpressed in resistant Anopheles mosquitoes, were analyzed. These enzymes are potential orthologs of the CYP4G1/2 enzymes that catalyze the final step of CHC biosynthesis in Drosophila and Musca domestica, respectively. Immunostaining indicated that both CYP4G16 and CYP4G17 are highly abundant in oenocytes, the insect cell type thought to secrete hydrocarbons. However, an intriguing difference was indicated; CYP4G17 occurs throughout the cell, as expected for a microsomal P450, but CYP4G16 localizes to the periphery of the cell and lies on the cytoplasmic side of the cell membrane, a unique position for a P450 enzyme. CYP4G16 and CYP4G17 were functionally expressed in insect cells. CYP4G16 produced hydrocarbons from a C18 aldehyde substrate and thus has bona fide decarbonylase activity similar to that of dmCYP4G1/2. The data support the hypothesis that the coevolution of multiple mechanisms, including cuticular barriers, has occurred in highly pyrethroid-resistant An gambiae.

  4. 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.

  5. In vitro inhibitory activities of the extract of Hibiscus sabdariffa L. (family Malvaceae) on selected cytochrome P450 isoforms.

    Science.gov (United States)

    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-in-one assay method. The ethanolic extract of Hibiscus sabdariffa showed inhibitory activities against nine selected CYP isoforms: CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4. The concentrations of the extract which produced 50% inhibition of the CYP isoforms ranged from 306 µg/ml to 1660 µg/ml, and the degree of inhibition based on the IC50 values for each CYP isoform was in the following order: CYP1A2 > CYP2C8 > CYP2D6 > CYP2B6 > CYP2E1 > CYP2C19 > CYP3A4 > CYP2C9 > CYP2A6. Ethanolic extract of Hibiscus sabdariffa caused inhibition of CYP isoforms in vitro. These observed inhibitions may not cause clinically significant herb-drug interactions; however, caution may need to be taken in co-administering the water extract of Hibiscus sabdariffa with other drugs until clinical studies are available to further clarify these findings.

  6. Role of brain cytochrome P450 mono-oxygenases in bilirubin oxidation-specific induction and activity.

    Science.gov (United States)

    Gambaro, Sabrina E; Robert, Maria C; Tiribelli, Claudio; Gazzin, Silvia

    2016-02-01

    In the Crigler-Najjar type I syndrome, the genetic absence of efficient hepatic glucuronidation of unconjugated bilirubin (UCB) by the uridine 5'-diphospho-glucuronosyltransferase1A1 (UGT1A1) enzyme produces the rise of UCB level in blood. Its entry to central nervous system could generate toxicity and neurological damage, and even death. In the past years, a compensatory mechanism to liver glucuronidation has been indicated in the hepatic cytochromes P450 enzymes (Cyps) which are able to oxidize bilirubin. Cyps are expressed also in the central nervous system, the target of bilirubin toxicity, thus making them theoretically important to confer a protective activity toward bilirubin accumulation and neurotoxicity. We therefore investigated the functional induction (mRNA, EROD/MROD) and the ability to oxidize bilirubin of Cyp1A1, 1A2, and 2A3 in primary astrocytes cultures obtained from two rat brain region (cortex: Cx and cerebellum: Cll). We observed that Cyp1A1 was the Cyp isoform more easily induced by beta-naphtoflavone (βNF) in both Cx and Cll astrocytes, but oxidized bilirubin only after uncoupling by 3, 4,3',4'-tetrachlorobiphenyl (TCB). On the contrary, Cyp1A2 was the most active Cyp in bilirubin clearance without uncoupling, but its induction was confined only in Cx cells. Brain Cyp2A3 was not inducible. In conclusion, the exposure of astrocytes to βNF plus TCB significantly enhanced Cyp1A1 mediating bilirubin clearance, improving cell viability in both regions. These results may be a relevant groundwork for the manipulation of brain Cyps as a therapeutic approach in reducing bilirubin-induced neurological damage.

  7. In vitro cytochrome P450 inhibition potential of methylenedioxy-derived designer drugs studied with a two-cocktail approach.

    Science.gov (United States)

    Dinger, Julia; Meyer, Markus R; Maurer, Hans H

    2016-02-01

    In vitro cytochrome P450 (CYP) inhibition assays are common approaches for testing the inhibition potential of drugs for predicting potential interactions. In contrast to marketed medicaments, drugs of abuse, particularly the so-called novel psychoactive substances, were not tested before distribution and consumption. Therefore, the inhibition potential of methylenedioxy-derived designer drugs (MDD) of different drug classes such as aminoindanes, amphetamines, benzofurans, cathinones, piperazines, pyrrolidinophenones, and tryptamines should be elucidated. The FDA-preferred test substrates, split in two cocktails, were incubated with pooled human liver microsomes and analysed after protein precipitation using LC-high-resolution-MS/MS. IC50 values were determined of MDD showing more than 50 % inhibition in the prescreening. Values were calculated by plotting the relative metabolite concentration formed over the logarithm of the inhibitor concentration. All MDD showed inhibition against CYP2D6 activity and most of them in the range of the clinically relevant CYP2D6 inhibitors quinidine and fluoxetine. In addition, the beta-keto compounds showed inhibition of the activity of CYP2B6, 5,6-MD-DALT of CYP1A2 and CYP3A, and MDAI of CYP2A6, all in the range of clinically relevant inhibitors. In summary, all MDD showed inhibition of the activity of CYP2D6, six of CYP1A2, three of CYP2A6, 13 of CYP2B6, two of CYP2C9, six of CYP2C19, one of CYP2E1, and six of CYP3A. These results showed that the CYP inhibition by MDD might be clinically relevant, but further studies are needed for final conclusions.

  8. AM-2201 Inhibits Multiple Cytochrome P450 and Uridine 5′-Diphospho-Glucuronosyltransferase Enzyme Activities in Human Liver Microsomes

    Directory of Open Access Journals (Sweden)

    Ju-Hyun Kim

    2017-03-01

    Full Text Available AM-2201 is a synthetic cannabinoid that acts as a potent agonist at cannabinoid receptors and its abuse has increased. However, there are no reports of the inhibitory effect of AM-2201 on human cytochrome P450 (CYP or uridine 5′-diphospho-glucuronosyltransferase (UGT enzymes. We evaluated the inhibitory effect of AM-2201 on the activities of eight major human CYPs (1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4 and six major human UGTs (1A1, 1A3, 1A4, 1A6, 1A9, and 2B7 enzymes in pooled human liver microsomes using liquid chromatography–tandem mass spectrometry to investigate drug interaction potentials of AM-2201. AM-2201 potently inhibited CYP2C9-catalyzed diclofenac 4′-hydroxylation, CYP3A4-catalyzed midazolam 1′-hydroxylation, UGT1A3-catalyzed chenodeoxycholic acid 24-acyl-glucuronidation, and UGT2B7-catalyzed naloxone 3-glucuronidation with IC50 values of 3.9, 4.0, 4.3, and 10.0 μM, respectively, and showed mechanism-based inhibition of CYP2C8-catalyzed amodiaquine N-deethylation with a Ki value of 2.1 μM. It negligibly inhibited CYP1A2, CYP2A6, CYP2B6, CYP2C19, CYP2D6, UGT1A1, UGT1A4, UGT1A6, and UGT1A9 activities at 50 μM in human liver microsomes. These in vitro results indicate that AM-2201 needs to be examined for potential pharmacokinetic drug interactions in vivo due to its potent inhibition of CYP2C8, CYP2C9, CYP3A4, UGT1A3, and UGT2B7 enzyme activities.

  9. Monkey liver cytochrome P450 2C19 is involved in R- and S-warfarin 7-hydroxylation.

    Science.gov (United States)

    Hosoi, Yoshio; Uno, Yasuhiro; Murayama, Norie; Fujino, Hideki; Shukuya, Mitsunori; Iwasaki, Kazuhide; Shimizu, Makiko; Utoh, Masahiro; Yamazaki, Hiroshi

    2012-12-15

    Cynomolgus monkeys are widely used as primate models in preclinical studies. However, some differences are occasionally seen between monkeys and humans in the activities of cytochrome P450 enzymes. R- and S-warfarin are model substrates for stereoselective oxidation in humans. In this current research, the activities of monkey liver microsomes and 14 recombinantly expressed monkey cytochrome P450 enzymes were analyzed with respect to R- and S-warfarin 6- and 7-hydroxylation. Monkey liver microsomes efficiently mediated both R- and S-warfarin 7-hydroxylation, in contrast to human liver microsomes, which preferentially catalyzed S-warfarin 7-hydroxylation. R-Warfarin 7-hydroxylation activities in monkey liver microsomes were not inhibited by α-naphthoflavone or ketoconazole, and were roughly correlated with P450 2C19 levels and flurbiprofen 4-hydroxylation activities in microsomes from 20 monkey livers. In contrast, S-warfarin 7-hydroxylation activities were not correlated with the four marker drug oxidation activities used. Among the 14 recombinantly expressed monkey P450 enzymes tested, P450 2C19 had the highest activities for R- and S-warfarin 7-hydroxylations. Monkey P450 3A4 and 3A5 slowly mediated R- and S-warfarin 6-hydroxylations. Kinetic analysis revealed that monkey P450 2C19 had high V(max) and low K(m) values for R-warfarin 7-hydroxylation, comparable to those for monkey liver microsomes. Monkey P450 2C19 also mediated S-warfarin 7-hydroxylation with V(max) and V(max)/K(m) values comparable to those for recombinant human P450 2C9. R-warfarin could dock favorably into monkey P450 2C19 modeled. These results collectively suggest high activities for monkey liver P450 2C19 toward R- and S-warfarin 6- and 7-hydroxylation in contrast to the saturation kinetics of human P450 2C9-mediated S-warfarin 7-hydroxylation. Copyright © 2012 Elsevier Inc. All rights reserved.

  10. FAME 2: Simple and Effective Machine Learning Model of Cytochrome P450 Regioselectivity

    Czech Academy of Sciences Publication Activity Database

    Sicho, M.; Kops, C.B.; Stork, C.; Svozil, Daniel; Kirchmair, J.

    2017-01-01

    Roč. 57, č. 8 (2017), s. 1832-1846 ISSN 1549-9596 R&D Projects: GA MŠk LO1220; GA MŠk LM2015063 Institutional support: RVO:68378050 Keywords : cyp-mediated sites * predicting drug-metabolism * xenobiotic metabolism * population analysis * pathway prediction * wave-functions * rs-predictor * p450 * reactivity * smartcyp Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Computer sciences, information science, bioinformathics (hardware development to be 2.2, social aspect to be 5.8) Impact factor: 3.760, year: 2016

  11. 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

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

    International Nuclear Information System (INIS)

    Reed, James R.; Cawley, George F.; Ardoin, Taylor G.; Dellinger, Barry; Lomnicki, Slawomir M.; Hasan, Farhana; Kiruri, Lucy W.; Backes, Wayne L.

    2014-01-01

    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

    Energy Technology Data Exchange (ETDEWEB)

    Schuenemann, V. [University of Luebeck, Institute of Physics (Germany); Jung, C. [Max-Delbrueck-Center for Molecular Medicine (Germany); Lendzian, F. [Technical University, PC 14, Max-Volmer Laboratory for Biophysical Chemistry (Germany); Barra, A.-L. [Grenoble High Magnetic Field Laboratory (France); Teschner, T.; Trautwein, A. X. [University of Luebeck, Institute of Physics (Germany)

    2004-12-15

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

    International Nuclear Information System (INIS)

    Schuenemann, V.; Jung, C.; Lendzian, F.; Barra, A.-L.; Teschner, T.; Trautwein, A. X.

    2004-01-01

    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.

  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...... 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....

  16. Mechanism of the N-Hydroxylation of Primary and Secondary Amines by Cytochrome P450

    DEFF Research Database (Denmark)

    Seger, Signe T.; Rydberg, Patrik; Olsen, Lars

    2015-01-01

    Cytochrome P450 enzymes (CYPs) metabolize alkyl- and arylamines, generating several different products. For the primary and secondary amines, some of these reactions result in hydroxylated amines, which may be toxic. Thus, when designing new drugs containing amine groups, it is important to be able...... to predict if a given compound will be a substrate for CYPs, in order to avoid toxic metabolites, and hence to understand the mechanism that is utilized by CYPs. Two possible mechanisms, for the N-hydroxylation of primary and secondary amines mediated by CYPs, are studied by density functional theory (DFT...

  17. 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 reproduced by computationally less demanding methods. The best methods (a B3LYP calculation of the activation energy of a methoxy-radical model or a partial least-squares model of the semiempirical AM1 bond dissociation energies and spin densities of the tetrahedral intermediate for both a hydroxyl...

  18. Engineering soluble insect and plant cytochromes P450 for biochemical characterization

    DEFF Research Database (Denmark)

    Jensen, Mikael Kryger

    specificity, unlike many mammalian cytochromes P450. CYP405A2 from Zygaena filipendulae and CYP79D3 from Lotus corniculatus both convert isoleucine and valine into their corresponding oximes, but neither will convert leucine neatly illustrating the high degree of specificity the enzymes possess. Previous work...... of substrate specificity, although possibly not the only determinants. The results obtained in this PhD, represent an advance in our understanding of how these enzymes function and have achieved their high degree of specificity. Furthermore, the accumulated knowledge this thesis represents regarding expression...

  19. Nitrous oxide-forming codenitrification catalyzed by cytochrome P450nor.

    Science.gov (United States)

    Su, Fei; Takaya, Naoki; Shoun, Hirofumi

    2004-02-01

    Intact cells of the denitrifying fungus Fusarium oxysporum were previously shown to catalyze codenitrification to form a hybrid nitrous oxide (N2O) species from nitrite and other nitrogen compounds such as azide and ammonia. Here we show that cytochrome P450nor can catalyze the codenitrification reaction to form N2O from nitric oxide (NO) but not nitrite, and azide or ammonia. The results show that the direct substrate of the codenitrification by intact cells should not be nitrite but NO, which is formed from nitrite by the reaction of a dissimilatory nitrite reductase.

  20. Human HepaRG Cells can be Cultured in Hanging-drop Plates for Cytochrome P450 Induction and Function Assays.

    Science.gov (United States)

    Murayama, Norie; Usui, Takashi; Slawny, Nicky; Chesné, Christophe; Yamazaki, Hiroshi

    2015-01-01

    Recent guidance/guidelines for industry recommend that cytochrome P450 induction can be assessed using human hepatocyte enzyme activity and/or mRNA levels to evaluate potential drug- drug interactions. To evaluate time-dependent cytochrome P450 induction precisely, induction of CYP1A2, CYP2B6, and CYP3A4 mRNA was confirmed (>2-fold) by the treatment with omeprazole, phenobarbital, and rifampicin, respectively, for 24 or 48 h on day 3 from the start of culture. After 24 h, the fold induction of CYP1A2 with 3.6 and 1.8x10(4) HepaRG cells per well was lower than that for 7.2x10(4) cells. CYP1A2 induction levels at 24 h were higher than those after 48 h. In contrast, higher CYP2B6 inductions were confirmed after 48 h exposure than after 24 h, independent of the number of cells per well. To help reduce the use of human cryopreserved hepatocytes, typical P450-dependent enzyme activities were investigated in human HepaRG cells cultured in commercial hanging-drop plates. Newly designed 96-well hanging-drop plates were capable of maintaining human CYP3A-dependent midazolam hydroxylation activities for up to 4 days using only 10% of the recommended initial 7.2x10(4) cells per well. Favorable HepaRG function using hanging-drop plates was confirmed by detecting 1'- hydroxymidazolam O-glucuronide on day 3, suggesting an improvement over traditional control plates in which this metabolite can be detected for 24-well plates. These results suggest that the catalytic function and/or induction of CYP1A2, CYP2B6, and CYP3A4 can be readily assessed with reduced numbers of starting HepaRG cells cultured in three-dimensional cultures in drops prepared with hanging-drop plates.

  1. In vitro inhibitory effects of plumbagin, the promising antimalarial candidate, on human cytochrome P450 enzymes.

    Science.gov (United States)

    Sumsakul, Wiriyaporn; Chaijaroenkul, Wanna; Na-Bangchang, Kesara

    2015-11-01

    To investigate the propensity of plumbagin to inhibit the three isoforms of human cytochrome P450 (CYP), i.e., CYP1A2, CYP2C19, and CYP3A4 using human liver microsomes in vitro. Inhibitory effects of plumbagin on the three human CYP isoforms were investigated using pooled human liver microsomes. Phenacetin O-deethylation, omeprazole hydroxylation and nifedipine oxidation were used as selective substrates for CYP1A2, CYP2C19 and CYP3A4 activities, respectively. Concentrations of paracetamol, 5-hydroxyomeprazole, and oxidized nifedipine were determined in microsomal incubation mixture using high-performance liquid chromatography. Plumbagin showed significant inhibitory effects on all CYP isoforms, but with the most potent activity on CYP2C19-mediated omeprazole hydroxylation. The IC50 (concentration that inhibits enzyme activity by 50%) values of plumbagin and nootkatone (selective inhibitor) for CYP2C19 were (0.78 ± 0.01) and (27.31 ± 0.66) μM, respectively. The inhibitory activities on CYP1A2-mediated phenacetin O-deethylation and CYP3A4-mediated nifedipine oxidation were moderate. The IC50 values of plumbagin and α-naphthoflavone (selective inhibitor) for CYP1A2 were (1.39 ± 0.01) and (0.02 ± 0.36) μM, respectively. The corresponding IC50 values of plumbagin and ketoconazole (selective inhibitor) for CYP3A4 were (2.37 ± 0.10) and (0.18 ± 0.06) μM, respectively. Clinical relevance of the interference of human drug metabolizing enzymes should be aware of for further development scheme of plumbagin as antimalarial drug when used in combination with other antimalarial drugs which are metabolized by these CYP isoforms. Copyright © 2015 Hainan Medical College. Production and hosting by Elsevier B.V. All rights reserved.

  2. 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.

  3. Evidence for induction of cytochrome P-450I in patients with tropical chronic pancreatitis.

    Science.gov (United States)

    Chaloner, C; Sandle, L N; Mohan, V; Snehalatha, C; Viswanathan, M; Braganza, J M

    1990-06-01

    Theophylline kinetics, as an in vivo probe for the potentially toxic cytochrome P-450I pathway of drug metabolism, were studied in 11 healthy volunteers and 11 patients with calcific chronic pancreatitis at Madras, South India. Theophylline clearance was faster in the patients than controls [median 69 (range 39-114) vs 45 (33-56) ml h-1 kg-1, p = 0.003]. In keeping with this finding, detailed social histories identified a higher exposure level in the patients to xenobiotics that are inducers of cytochrome P-450I and/or yield reactive metabolites upon processing thereby (score 7, 4-11 vs 3, 2-9, p = 0.002). However, the concentration of D-glucaric acid in urine, as a marker of phase II conjugating pathways of drug metabolism, was similar in patients and controls. This pattern of drug metabolism could predispose to oxidant stress: hence micronutrient antioxidant supplements may have therapeutic (or even prophylactic) value in tropical chronic pancreatitis.

  4. Effects of Cytochrome P 450 Inhibitors on Itraconazole and Fluconazole Induced Cytotoxicity in Hepatocytes

    International Nuclear Information System (INIS)

    Somchit, N.; Ngee, C.S.; Yaakob, A.; Ahmad, Z.; Zakaria, Z.A.

    2009-01-01

    Itraconazole and fluconazole have been reported to induce hepatotoxicity in patients. The present study was designed to investigate the role of cytochrome P450 inhibitors, SKF 525A, and curcumin pretreatment on the cytotoxicity of antifungal drugs fluconazole and itraconazole. For 3 consecutive days, female rats were administered daily SKF 525A or curcumin (5 and 25?mg/kg). Control rats received an equivalent amount of dosed vehicle. The animals were anaesthetised 24 hours after receiving the last dose for liver perfusion. Hepatocytes were then exposed to various concentrations of antifungal drugs. In vitro incubation of hepatocytes with itraconazole revealed significantly lower viability when compared to fluconazole as assessed by lactate dehydrogenase, aspartate aminotransferase and alanine aminotransferase activities. The cytotoxicity of itraconazole was enhanced when incubated with hepatocytes pretreated with SKF 525A. SKF 525A had no effects on the cytotoxicity of fluconazole. Curcumin failed to either increase or decrease the cytotoxicity of both antifungal drugs. ATP levels also showed significant decrease in both itraconazole and fluconazole incubated hepatocytes. However, SKF 525A pretreated hepatocytes had significantly lower ATP levels after itraconazole incubations. Collectively, these results confirm the involvement of cytochrome P450 in the cytoprotection in itraconazole induced hepatocyte toxicity. Differences of the effects of SKF 525A on the cytotoxicity induced by itraconazole and fluconazole may be due to the differences on the metabolism of each antifungal drug in vivo.

  5. The human cytochrome P450 3A locus. Gene evolution by capture of downstream exons.

    Science.gov (United States)

    Finta, C; Zaphiropoulos, P G

    2000-12-30

    Using a bacterial artificial chromosome (BAC) clone, we have mapped the human cytochrome P450 3A (CYP3A) locus containing the genes encoding for CYP3A4, CYP3A5 and CYP3A7. The genes lie in a head-to-tail orientation in the order of 3A4, 3A7 and 3A5. In both intergenic regions (3A4-3A7 and 3A7-3A5), we have detected several additional cytochrome P450 3A exons, forming two CYP3A pseudogenes. These pseudogenes have the same orientation as the CYP3A genes. To our surprise, a 3A7 mRNA species has been detected in which the exons 2 and 13 of one of the pseudogenes (the one that is downstream of 3A7) are spliced after the 3A7 terminal exon. This results in an mRNA molecule that consists of the 13 3A7 exons and two additional exons at the 3' end. The additional two exons originating from the pseudogene are in an altered reading frame and consequently have the capability to code a completely different amino acid sequence than the canonical CYP3A exons 2 and 13. These findings may represent a generalized evolutionary process with genes having the potential to capture neighboring sequences and use them as functional exons.

  6. Understanding uncoupling in the multiredox centre P450 3A4-BMR model system.

    Science.gov (United States)

    Degregorio, Danilo; Sadeghi, Sheila J; Di Nardo, Giovanna; Gilardi, Gianfranco; Solinas, Sandro P

    2011-01-01

    Understanding the uncoupling at the haem active site and/or at the level of multidomain electron transfer is an important element in cytochrome P450 chemistry. Here a chimeric model system consisting of human cytochrome P450 3A4 and the soluble reductase domain of CYP102A1 from Bacillus megaterium (BMR) is used to study the relationship between electron transfer and the coupling efficiency in substrate monoxygenation. Several regulatory features were considered. FAD and FMN added to apoenzyme in oversaturating concentrations influence neither formaldehyde production nor coupling efficiency. The optimal conditions of coupling efficiency depended only on the NADPH concentration. The pH (8.0) and ionic strength (50 mM potassium phosphate) were found to modulate the level of coupling, indicating an influence over the formation of a productive interaction between the BMR and the haem domain. Overall, uncoupling is found to be an intrinsic property of the haem domain, and the covalent linkage of the reductase in a single polypeptide chain has little influence over the activity coupled to product formation.

  7. Bioactivation and Regioselectivity of Pig Cytochrome P450 3A29 towards Aflatoxin B1

    Directory of Open Access Journals (Sweden)

    Jun Wu

    2016-09-01

    Full Text Available Due to unavoidable contaminations in feedstuff, pigs are easily exposed to aflatoxin B1 (AFB1 and suffer from poisoning, thus the poisoned products potentially affect human health. Heretofore, the metabolic process of AFB1 in pigs remains to be clarified, especially the principal cytochrome P450 oxidases responsible for its activation. In this study, we cloned CYP3A29 from pig liver and expressed it in Escherichia coli, and its activity has been confirmed with the typical P450 CO-reduced spectral characteristic and nifedipine-oxidizing activity. The reconstituted membrane incubation proved that the recombinant CYP3A29 was able to oxidize AFB1 to form AFB1-exo-8,9-epoxide in vitro. The structural basis for the regioselective epoxidation of AFB1 by CYP3A29 was further addressed. The T309A mutation significantly decreased the production of AFBO, whereas F304A exhibited an enhanced activation towards AFB1. In agreement with the mutagenesis study, the molecular docking simulation suggested that Thr309 played a significant role in stabilization of AFB1 binding in the active center through a hydrogen bond. In addition, the bulk phenyl group of Phe304 potentially imposed steric hindrance on the binding of AFB1. Our study demonstrates the bioactivation of pig CYP3A29 towards AFB1 in vitro, and provides the insight for understanding regioselectivity of CYP3A29 to AFB1.

  8. Cycle affects imidacloprid efficiency by mediating cytochrome P450 expression in the brown planthopper Nilaparvata lugens.

    Science.gov (United States)

    Kang, K; Yang, P; Pang, R; Yue, L; Zhang, W

    2017-10-01

    Circadian clocks influence most behaviours and physiological activities in animals, including daily fluctuations in metabolism. However, how the clock gene cycle influences insects' responses to pesticides has rarely been reported. Here, we provide evidence that cycle affects imidacloprid efficacy by mediating the expression of cytochrome P450 genes in the brown planthopper (BPH) Nilaparvata lugens, a serious insect pest of rice. Survival bioassays showed that the susceptibility of BPH adults to imidacloprid differed significantly between the two time points tested [Zeitgeber Time 8 (ZT8) and ZT4]. After cloning the cycle gene in the BPH (Nlcycle), we found that Nlcycle was expressed at higher levels in the fat body and midgut, and its expression was rhythmic with two peaks. Knockdown of Nlcycle affected the expression levels and rhythms of cytochrome P450 genes as well as susceptibility to imidacloprid. The survival rates of BPH adults after treatment with imidacloprid did not significantly differ between ZT4 and ZT8 after double-stranded Nlcycle treatment. These findings can be used to improve pesticide use and increase pesticide efficiency in the field. © 2017 The Royal Entomological Society.

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

    International Nuclear Information System (INIS)

    Anzenbacherova, Eva; Hudecek, Jiri; Murgida, Daniel; Hildebrandt, Peter; Marchal, Stephane; Lange, Reinhard; Anzenbacher, Pavel

    2005-01-01

    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

  10. Engineered P450 biocatalysts show improved activity and regio-promiscuity in aromatic nitration.

    Science.gov (United States)

    Zuo, Ran; Zhang, Yi; Jiang, Chao; Hackett, John C; Loria, Rosemary; Bruner, Steven D; Ding, Yousong

    2017-04-12

    Nitroaromatics are among the most important and commonly used chemicals but their production often suffers from multiple unsolved challenges. We have previously described the development of biocatalytic nitration processes driven by an engineered P450 TxtE fusion construct. Herein we report the creation of improved nitration biocatalysts through constructing and characterizing fusion proteins of TxtE with the reductase domain of CYP102A1 (P450BM3, BM3R). The majority of constructs contained variable linker length while one was rationally designed for optimizing protein-protein interactions. Detailed biochemical characterization identified multiple active chimeras that showed improved nitration activity, increased coupling efficiency and higher total turnover numbers compared with TxtE. Substrate promiscuity of the most active chimera was further assessed with a substrate library. Finally, a biocatalytic nitration process was developed to nitrate 4-Me-DL-Trp. The production of both 4-Me-5-NO 2 -L-Trp and 4-Me-7-NO 2 -L-Trp uncovered remarkable regio-promiscuity of nitration biocatalysts.

  11. Phorate can reverse P450 metabolism-based herbicide resistance in Lolium rigidum.

    Science.gov (United States)

    Busi, Roberto; Gaines, Todd Adam; Powles, Stephen

    2017-02-01

    Organophosphate insecticides can inhibit specific cytochrome P450 enzymes involved in metabolic herbicide resistance mechanisms, leading to synergistic interactions between the insecticide and the herbicide. In this study we report synergistic versus antagonistic interactions between the organophosphate insecticide phorate and five different herbicides observed in a population of multiple herbicide-resistant Lolium rigidum. Phorate synergised with three different herbicide modes of action, enhancing the activity of the ALS inhibitor chlorsulfuron (60% LD 50 reduction), the VLCFAE inhibitor pyroxasulfone (45% LD 50 reduction) and the mitosis inhibitor trifluralin (70% LD 50 reduction). Conversely, phorate antagonised the two thiocarbamate herbicides prosulfocarb and triallate with a 12-fold LD 50 increase. We report the selective reversal of P450-mediated metabolic multiple resistance to chlorsulfuron and trifluralin in the grass weed L. rigidum by synergistic interaction with the insecticide phorate, and discuss the putative mechanistic basis. This research should encourage diversity in herbicide use patterns for weed control as part of a long-term integrated management effort to reduce the risk of selection of metabolism-based multiple herbicide resistance in L. rigidum. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  12. Cytochrome P450 Bioconjugate as a Nanovehicle for Improved Chemotherapy Treatment.

    Science.gov (United States)

    Quester, Katrin; Juarez-Moreno, Karla; Secundino, Isamel; Roseinstein, Yvonne; Alejo, Karla P; Huerta-Saquero, Alejandro; Vazquez-Duhalt, Rafael

    2017-05-01

    Cancer is still a growing public health problem, especially breast cancer that is one of the most important cancers in women. Chemotherapy, even though a successful treatment, is accompanied by severe side effects. Moreover, most of the drugs used for chemotherapy are administered as prodrugs and need to be transformed to the active form by cytochromes P450 (CYPs). In addition, increasing numbers of cancer tissues show lower CYP activity than the surrounding healthy tissues in which prodrugs are preferentially activated causing cytotoxicity. Here, the design of a functionalized cytochrome P450 bioconjugate is reported as nanovehicle for the enzyme direct delivery to the tumor tissue in order to improve the local drug activation. MCF-7 breast cancer cells are treated with CYP-polyethylene glycol bioconjugate functionalized folic acid, where it activates the prodrug tamoxifen and significantly reduces the dose of tamoxifen needed to kill the tumor cells. The CYP bioconjugate covered with polyethylene glycol shows no immunogenic activity. The advantages of increasing the site-specific CYP activity in tumor tissues are discussed. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Electrochemistry of Cytochrome P450 BM3 in Sodium Dodecyl Sulfate Films

    Science.gov (United States)

    Udit, Andrew K.; Hill, Michael G.; Gray, Harry B.

    2008-01-01

    Direct electrochemistry of the cytochrome P450 BM3 heme domain (BM3) was achieved by confining the protein within sodium dodecyl sulfate (SDS) films on the surface of basal-plane graphite (BPG) electrodes. Cyclic voltammetry revealed the heme FeIII/II redox couple at −330 mV (vs. Ag/AgCl, pH 7.4). Up to 10 V/s, the peak current was linear with scan rate, allowing us to treat the system as surface-confined within this regime. The standard heterogeneous rate constant determined at 10 V/s was estimated to be 10 s−1. Voltammograms obtained for the BM3-SDS-BPG system in the presence of dioxygen exhibited catalytic waves at the onset of FeIII reduction. The altered heme reduction potential of the BM3-SDS-graphite system indicates that SDS is likely bound in the enzyme active-site region. Compared to other P450-surfactant systems, we find redox potentials and electron transfer rates that differ by ~ 100 mV and > 10-fold, respectively, indicating that the nature of the surfactant environment has a significant effect on the observed heme redox properties. PMID:17129070

  14. Combinatorial Alanine Substitution Enables Rapid Optimization of Cytochrome P450BM3 for Selective Hydroxylation of Large Substrates

    KAUST Repository

    Lewis, Jared C.; Mantovani, Simone M.; Fu, Yu; Snow, Christopher D.; Komor, Russell S.; Wong , Chi-Huey; Arnold, Frances H.

    2010-01-01

    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

  15. Protein and DNA technologies for functional expression of membrane-associated cytochromes P450 in bacterial cell factories

    DEFF Research Database (Denmark)

    Vazquez Albacete, Dario

    450 engineering guidelines and serves as platform to improve performance of microbial cells, thereby boosting recombinant production of complex plant P450-derived biochemicals. The knowledge generated, could guide future reconstruction of functional plant metabolic pathways leading to high valuable...... potential as medicines, fuels or food for humans. Plants conquered different environments thereby developing adaptation strategies based on the biosynthesis of a myriad of compounds. Unfortunately they are present in small amounts in plants and are too complex and to produce by organic chemical synthesis....... In most of biosynthetic pathways leading to these chemicals the cytochrome P450 enzyme family (P450s) is responsible for their final functionalization. However, the membrane-bound nature of P450s, makes their expression in microbial hosts a challenge. In order to meet the global demand for these natural...

  16. Time course for the modulation of hepatic cytochrome P450 after administration of ethylbenzene and its correlation with toluene metabolism.

    Science.gov (United States)

    Yuan, W; Sequeira, D J; Cawley, G F; Eyer, C S; Backes, W L

    1997-03-01

    The goal of the present study was to examine the time course for changes in P450 expression and hydrocarbon metabolism after acute treatment with the simple aromatic hydrocarbon ethylbenzene (EB) and to correlate these alterations with the changes observed in alkylbenzene metabolism. Male Holtzman rats were treated with a single intraperitoneal injection of EB, and the effects on specific P450-dependent activities, immunoreactive P450 isozyme levels, and RNA levels were measured at various times after injection. Toluene was used as the test alkylbenzene for examination of the EB-mediated changes on in vitro hydrocarbon metabolism. In untreated rats, toluene was metabolized almost entirely by aliphatic hydroxylation (to benzyl alcohol); however, in EB-treated rats, significant quantities of benzyl alcohol, o-cresol, and p-cresol were produced. Interestingly, 5-10 h after EB treatment, there was a 40% decrease in benzyl alcohol production. By 24 h, rates of benzyl alcohol formation returned to control levels, whereas there was a 7-fold increase in o-cresol and a greater that 50-fold increase in p-cresol production. The changes in the disposition of toluene were then correlated with changes in particular P450 isozymes. Several P450 isozymes were induced after EB administration. P450 2B1/2-dependent testosterone 16 beta-hydroxylation and P450 2B1/2-immunoreactive protein were elevated 30-fold after EB administration, reaching maxima by 24 h and remaining elevated 48 h after exposure. Changes in P450 2B1 and 2B2 RNA preceded those of the proteins. Similar results were observed with P450 1A1. P450 2E1 RNA levels were elevated after a single EB injection. However, the elevation in P450 2E1-dependent activities and immunoreactive protein levels preceded the changes in RNA, suggesting that multiple steps are affected by EB exposure. In contrast to the increases in some isozymes, P450 2C11 protein was rapidly suppressed (within the first 2-10 h) after hydrocarbon exposure

  17. A human cytochrome P-450 is recognized by anti-liver/kidney microsome antibodies in autoimmune chronic hepatitis.

    Science.gov (United States)

    Kiffel, L; Loeper, J; Homberg, J C; Leroux, J P

    1989-02-28

    1- Anti-liver/kidney microsome autoantibodies type 1 (anti-LKM1), observed in some children with chronic active hepatitis, were used to isolate their antigen in human liver microsomes. A protein, called P-LKM1 was thus purified. This protein was recognized by a rabbit antiserum directed against the related human cytochromes P-450 bufI and P-450 bufII. 2- A human liver microsomal protein immunoprecipitated with anti-LKM1 sera was also recognized by anti cytochromes P-450 bufI/II antibodies. 3- Anti-LKM1 antibodies potently inhibited microsomal bufuralol 1'-hydroxylation. These results displayed the possible identity between cytochrome P-450 bufI/II and LKM1 antigen.

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

    Science.gov (United States)

    Mahmood, Khalid; Højland, Dorte H; Asp, Torben; Kristensen, Michael

    2016-01-01

    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. 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. 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 in xenobiotic detoxification.

  19. 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.

  20. Genomic and transcriptomic insights into the cytochrome P450 monooxygenase gene repertoire in the rice pest brown planthopper, Nilaparvata lugens.

    Science.gov (United States)

    Lao, Shu-Hua; Huang, Xiao-Hui; Huang, Hai-Jian; Liu, Cheng-Wen; Zhang, Chuan-Xi; Bao, Yan-Yuan

    2015-11-01

    The cytochrome P450 monooxygenase (P450) gene family is one of the most abundant eukaryotic gene families that encode detoxification enzymes. In this study, we identified an abundance of P450 gene repertoire through genome- and transcriptome-wide analysis in the brown planthopper (Nilaparvata lugens), the most destructive rice pest in Asia. Detailed gene information including the exon-intron organization, size, transcription orientation and distribution in the genome revealed that many P450 loci were closely situated on the same scaffold, indicating frequent occurrence of gene duplications. Insecticide-response expression profiling revealed that imidacloprid significantly increased NlCYP6CS1v2, NLCYP4CE1v2, NlCYP4DE1, NlCYP417A1v2 and NlCYP439A1 expression; while triazophos and deltamethrin notably enhanced NlCYP303A1 expression. Expression analysis at the developmental stage showed the egg-, nymph-, male- and female-specific expression patterns of N. lugens P450 genes. These novel findings will be helpful for clarifying the P450 functions in physiological processes including development, reproduction and insecticide resistance in this insect species. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. Prototype Systems Containing Human Cytochrome P450 for High-Throughput Real-Time Detection of DNA Damage by Compounds That Form DNA-Reactive Metabolites.

    Science.gov (United States)

    Brito Palma, Bernardo; Fisher, Charles W; Rueff, José; Kranendonk, Michel

    2016-05-16

    The formation of reactive metabolites through biotransformation is the suspected cause of many adverse drug reactions. Testing for the propensity of a drug to form reactive metabolites has increasingly become an integral part of lead-optimization strategy in drug discovery. DNA reactivity is one undesirable facet of a drug or its metabolites and can lead to increased risk of cancer and reproductive toxicity. Many drugs are metabolized by cytochromes P450 in the liver and other tissues, and these reactions can generate hard electrophiles. These hard electrophilic reactive metabolites may react with DNA and may be detected in standard in vitro genotoxicity assays; however, the majority of these assays fall short due to the use of animal-derived organ extracts that inadequately represent human metabolism. The current study describes the development of bacterial systems that efficiently detect DNA-damaging electrophilic reactive metabolites generated by human P450 biotransformation. These assays use a GFP reporter system that detects DNA damage through induction of the SOS response and a GFP reporter to control for cytotoxicity. Two human CYP1A2-competent prototypes presented here have appropriate characteristics for the detection of DNA-damaging reactive metabolites in a high-throughput manner. The advantages of this approach include a short assay time (120-180 min) with real-time measurement, sensitivity to small amounts of compound, and adaptability to a microplate format. These systems are suitable for high-throughput assays and can serve as prototypes for the development of future enhanced versions.

  2. Simultaneous quantification of the abundance of several cytochrome P450 and uridine 5'-diphospho-glucuronosyltransferase enzymes in human liver microsomes using multiplexed targeted proteomics.

    Science.gov (United States)

    Achour, Brahim; Russell, Matthew R; Barber, Jill; Rostami-Hodjegan, Amin

    2014-04-01

    Cytochrome P450 (P450) and uridine 5'-diphospho-glucuronosyltransferase (UGT) enzymes mediate a major proportion of phase I and phase II metabolism of xenobiotics. In vitro-in vivo extrapolation (IVIVE) of hepatic clearance in conjunction with physiologically-based pharmacokinetics (PBPK) has become common practice in drug development. However, prediction of xenobiotic kinetics in virtual populations requires knowledge of both enzyme abundances and the extent to which these correlate. A multiplexed quantification concatemer (QconCAT) strategy was used in this study to quantify the expression of several P450 and UGT enzymes simultaneously and to establish correlations between various enzyme abundances in 24 individual liver samples (ages 27-66, 14 male). Abundances were comparable to previously reported values, including CYP2C9 (40.0 ± 26.0 pmol mg(-1)), CYP2D6 (11.9 ± 13.2 pmol mg(-1)), CYP3A4 (68.1 ± 52.3 pmol mg(-1)), UGT1A1 (33.6 ± 34.0 pmol mg(-1)), and UGT2B7 (82.9 ± 36.1 pmol mg(-1)), expressed as mean ± S.D. Previous reports of correlations in expression of various P450 (CYP3A4/CYP3A5*1/*3, CYP2C8/CYP2C9, and CYP3A4/CYP2B6) were confirmed. New correlations were demonstrated between UGTs [including UGT1A6/UGT1A9 (r(s) = 0.82, P enzymes were shown to be correlated [including CYP1A2/UGT2B4 (r(s) = 0.67, P = 0.0002)]. The expression of CYP3A5 in individuals with *1/*3 genotype (n = 11) was higher than those with *3/*3 genotype (n = 10) (P history of smoking or alcohol use on enzyme expression was observed; however, expression of several enzymes declined with age. The correlation matrix produced for the first time by this study can be used to generate more realistic virtual populations with respect to abundance of various enzymes.

  3. Cytochrome P450IID6 recognized by LKM1 antibody is not exposed on the surface of hepatocytes.

    Science.gov (United States)

    Yamamoto, A M; Mura, C; De Lemos-Chiarandini, C; Krishnamoorthy, R; Alvarez, F

    1993-06-01

    LKM1 autoantibody, directed against P450IID6, is accepted as a marker of a particular type of autoimmune hepatitis, but its role in the pathogenesis of the disease is controversial. Localization of P450IID6 on the cell surface of rat hepatocytes was previously reported, suggesting that membrane-bound P450IID6 could be the target of LKM1 antibodies, thus allowing immune lysis of hepatocytes. The objective of the present study was to determine, using various methods, the cell localization of P450IID6 in human and rat hepatocytes. Incubation of rat and human hepatocytes with LKM1-positive serum showed slight, if any, cell membrane staining using immunofluorescence, immunoperoxidase and immunoelectron microscopic studies. No staining of the plasma membrane of human hepatocytes was observed when incubations were carried out with immunoaffinity-purified antibody directed against peptide 254-271, the main epitope of P450IID6 recognized by all LKM1 sera tested. Chinese hamster ovary cells, transfected with the complete P450IID6 cDNA and incubated with the supernatant from a B cell lymphoblastoid cell line prepared with the lymphocytes of a LKM1-positive patient, did not show any staining of the cell surface by immunofluorescence. Incubation of rat microsomal fraction vesicles with LKM1-positive serum, followed by protein A-gold immunoelectron microscopy, displayed a staining of almost all vesicles, confirming that P450IID6 is present on the cytoplasmic side of the microsomal membrane, which makes it unable to be expressed on the cell surface even if it were transported from the endoplasmic reticulum (ER). Sulpho NHS Biotin labelling of rat hepatocyte cell membranes did not show the presence of a 50-kD molecule that could have reacted with LKM1 antibody. DNA sequencing of exon 1 of the CYP2D6 gene of a patient positive for LKM1 antibody did not show any difference from that of the normal published sequence of the gene. This does not favour an alteration of the NH2 terminal

  4. Generalized cytochrome P450-mediated oxidation and oxygenation reactions in aromatic substrates with activated N-H, O-H, C-H, or S-H substituents

    NARCIS (Netherlands)

    Koymans, L.; Donné-Op den Kelder, G M; te Koppele, J.M.; Vermeulen, N P

    1. The general mechanism of metabolic oxidation of substrates by cytochromes P450 (P450s) appears to consist of sequential one-electron oxidation steps rather than of a single concerted transfer of activated oxygen species from P450 to substrates. 2. In case of the acetanilides paracetamol (PAR),

  5. 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...

  6. Menadione Suppresses Benzo(αpyrene-Induced Activation of Cytochromes P450 1A: Insights into a Possible Molecular Mechanism.

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    Yulia A Sidorova

    Full Text Available Oxidative reactions that are catalyzed by cytochromes P450 1A (CYP1A lead to formation of carcinogenic derivatives of arylamines and polycyclic aromatic hydrocarbons (PAHs, such as the widespread environmental pollutant benzo(αpyrene (BP. These compounds upregulate CYP1A at the transcriptional level via an arylhydrocarbon receptor (AhR-dependent signaling pathway. Because of the involvement of AhR-dependent genes in chemically induced carcinogenesis, suppression of this signaling pathway could prevent tumor formation and/or progression. Here we show that menadione (a water-soluble analog of vitamin K3 inhibits BP-induced expression and enzymatic activity of both CYP1A1 and CYP1A2 in vivo (in the rat liver and BP-induced activity of CYP1A1 in vitro. Coadministration of BP and menadione reduced DNA-binding activity of AhR and increased DNA-binding activity of transcription factors Oct-1 and CCAAT/enhancer binding protein (C/EBP, which are known to be involved in negative regulation of AhR-dependent genes, in vivo. Expression of another factor involved in downregulation of CYP1A-pAhR repressor (AhRR-was lower in the liver of the rats treated with BP and menadione, indicating that the inhibitory effect of menadione on CYP1A is not mediated by this protein. Furthermore, menadione was well tolerated by the animals: no signs of acute toxicity were detected by visual examination or by assessment of weight gain dynamics or liver function. Taken together, our results suggest that menadione can be used in further studies on animal models of chemically induced carcinogenesis because menadione may suppress tumor formation and possibly progression.

  7. Menadione Suppresses Benzo(α)pyrene-Induced Activation of Cytochromes P450 1A: Insights into a Possible Molecular Mechanism.

    Science.gov (United States)

    Sidorova, Yulia A; Perepechaeva, Maria L; Pivovarova, Elena N; Markel, Arkady L; Lyakhovich, Vyacheslav V; Grishanova, Alevtina Y

    2016-01-01

    Oxidative reactions that are catalyzed by cytochromes P450 1A (CYP1A) lead to formation of carcinogenic derivatives of arylamines and polycyclic aromatic hydrocarbons (PAHs), such as the widespread environmental pollutant benzo(α)pyrene (BP). These compounds upregulate CYP1A at the transcriptional level via an arylhydrocarbon receptor (AhR)-dependent signaling pathway. Because of the involvement of AhR-dependent genes in chemically induced carcinogenesis, suppression of this signaling pathway could prevent tumor formation and/or progression. Here we show that menadione (a water-soluble analog of vitamin K3) inhibits BP-induced expression and enzymatic activity of both CYP1A1 and CYP1A2 in vivo (in the rat liver) and BP-induced activity of CYP1A1 in vitro. Coadministration of BP and menadione reduced DNA-binding activity of AhR and increased DNA-binding activity of transcription factors Oct-1 and CCAAT/enhancer binding protein (C/EBP), which are known to be involved in negative regulation of AhR-dependent genes, in vivo. Expression of another factor involved in downregulation of CYP1A-pAhR repressor (AhRR)-was lower in the liver of the rats treated with BP and menadione, indicating that the inhibitory effect of menadione on CYP1A is not mediated by this protein. Furthermore, menadione was well tolerated by the animals: no signs of acute toxicity were detected by visual examination or by assessment of weight gain dynamics or liver function. Taken together, our results suggest that menadione can be used in further studies on animal models of chemically induced carcinogenesis because menadione may suppress tumor formation and possibly progression.

  8. Inhibitory Effects of Trapping Agents of Sulfur Drug Reactive Intermediates against Major Human Cytochrome P450 Isoforms

    Directory of Open Access Journals (Sweden)

    Jasleen K. Sodhi

    2017-07-01

    Full Text Available In some cases, the formation of reactive species from the metabolism of xenobiotics has been linked to toxicity and therefore it is imperative to detect potential bioactivation for candidate drugs during drug discovery. Reactive species can covalently bind to trapping agents in in vitro incubations of compound with human liver microsomes (HLM fortified with β-nicotinamide adenine dinucleotide phosphate (NADPH, resulting in a stable conjugate of trapping agent and reactive species, thereby facilitating analytical detection and providing evidence of short-lived reactive metabolites. Since reactive metabolites are typically generated by cytochrome P450 (CYP oxidation, it is important to ensure high concentrations of trapping agents are not inhibiting the activities of CYP isoforms. Here we assessed the inhibitory properties of fourteen trapping agents against the major human CYP isoforms (CYP1A2, 2C9, 2C19, 2D6 and 3A. Based on our findings, eleven trapping agents displayed inhibition, three of which had IC50 values less than 1 mM (2-mercaptoethanol, N-methylmaleimide and N-ethylmaleimide (NEM. Three trapping agents (dimedone, N-acetyl-lysine and arsenite did not inhibit CYP isoforms at concentrations tested. To illustrate effects of CYP inhibition by trapping agents on reactive intermediate trapping, an example drug (ticlopidine and trapping agent (NEM were chosen for further studies. For the same amount of ticlopidine (1 μM, increasing concentrations of the trapping agent NEM (0.007–40 mM resulted in a bell-shaped response curve of NEM-trapped ticlopidine S-oxide (TSO-NEM, due to CYP inhibition by NEM. Thus, trapping studies should be designed to include several concentrations of trapping agent to ensure optimal trapping of reactive metabolites.

  9. Modulation of expression and activity of cytochrome P450s and alteration of praziquantel kinetics during murine schistosomiasis

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    Mara A Gotardo

    2011-03-01

    Full Text Available In this study, we investigated the expression and activity of liver cytochrome P450s (CYPs and praziquantel (PZQ kinetics in mice infected with Schistosoma mansoni. Swiss Webster (SW mice of both genders were infected (100 cercariae on postnatal day 10 and killed on post-infection days (PIDs 30 or 55. Non-infected mice of the same age and sex served as controls. Regardless of mouse sex, infection depressed the activities of CYP1A [ethoxy/methoxy-resorufin-O-dealkylases (EROD/MROD], 2B9/10 [pentoxy/benzyloxy-resorufin-O-dealkylases (PROD, BROD], 2E1 [p-nitrophenol-hydroxylase (PNPH] and 3A11 [erythromycin N-demethylase (END] on PID 55 but not on PID 30. On PID 55, infection decreased liver CYP mRNA levels (real-time reverse transcription-polymerase chain reaction. On PID 30, whereas mRNA levels remained unaltered in males, they were depressed in females. Plasma PZQ (200 and 400 mg/kg body weight intraperitoneally levels were measured (high-performance liquid chromatography at different post-treatment intervals. In males and females, infection delayed the PZQ clearance on PID 55, but not on PID 30. Therefore, it can be concluded that schistosomiasis down-modulated CYP expression and activity and delayed PZQ clearance on PID 55, when a great number of parasite eggs were lodged in the liver. On PID 30, when egg-laying was initiated by the worms, no change of CYP expression and activity was found, except for a depression of CYP1A2 and 3A11 mRNAs in female mice.

  10. The role of P450 metabolism in the estrogenic activity of bifenthrin in fish.

    Science.gov (United States)

    DeGroot, Breanna C; Brander, Susanne M

    2014-11-01

    Bifenthrin, a pyrethroid pesticide, is estrogenic in vivo in fishes. However, bifenthrin is documented to be anti-estrogenic in vitro, in the ER-CALUX (estrogen receptor) cell line. We investigated whether metabolite formation is the reason for this incongruity. We exposed Menidia beryllina (inland silversides) to 10ng/l bifenthrin, 10ng/l 4-hydroxy bifenthrin, and 10ng/l bifenthrin with 25μg/l piperonyl butoxide (PBO) - a P450 inhibitor. Metabolite-exposed juveniles had significantly higher estrogen-mediated protein levels (choriogenin) than bifenthrin/PBO-exposed, while bifenthrin alone was intermediate (not significantly different from either). This suggests that metabolites are the main contributors to bifenthrin's in vivo estrogenicity. Copyright © 2014 Elsevier B.V. All rights reserved.

  11. Mechanism of Cytochrome P450 17A1-Catalyzed Hydroxylase and Lyase Reactions

    DEFF Research Database (Denmark)

    Bonomo, Silvia; Jorgensen, Flemming Steen; Olsen, Lars

    2017-01-01

    Cytochrome P450 17A1 (CYP17A1) catalyzes C17 hydroxylation of pregnenolone and progesterone and the subsequent C17–C20 bond cleavage (lyase reaction) to form androgen precursors. Compound I (Cpd I) and peroxo anion (POA) are the heme-reactive species underlying the two reactions. We have characte...... the concept that the selectivity of the steroidogenic CYPs is ruled by direct interactions with the enzyme, in contrast to the selectivity of drug-metabolizing CYPs, where the reactivity of the substrates dominates....... characterized the reaction path for both the hydroxylase and lyase reactions using density functional theory (DFT) calculations and the enzyme–substrate interactions by molecular dynamics (MD) simulations. Activation barriers for positions subject to hydroxylase reaction have values close to each other and span...

  12. Comparative study of hops-containing products on human cytochrome P450-mediated metabolism.

    Science.gov (United States)

    Foster, Brian C; Arnason, John T; Saleem, Ammar; Tam, Teresa W; Liu, Rui; Mao, Jingqin; Desjardins, Suzanne

    2011-05-11

    The potential for 15 different ales (6), ciders (2 apple and 1 pear), and porters (6) and 2 non-alcoholic products to affect cytochrome P450 (CYP)-mediated biotransformation and P-glycoprotein-mediated efflux of rhodamine was examined. As in our previous study, a wide range of recovered nonvolatile suspended solids dry weights were noted. Aliquots were also found to have varying effects on biotransformation and efflux. Distinct differences in product ability to affect the safety and efficacy of therapeutic products confirmed our initial findings that some porters (stouts) have a potential to affect the safety and efficacy of health products metabolized by CYP2D6 and CYP3A4 isozymes. Most products, except 2 of the ciders and the 2 non-alcoholic products, also have the potential to affect the safety of CYP2C9 metabolized medications and supplements. Further studies are required to determine the clinical significance of these findings.

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

    DEFF Research Database (Denmark)

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

    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.......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...

  14. Water Complexes of Cytochrome P450: Insights from Energy Decomposition Analysis

    Directory of Open Access Journals (Sweden)

    Hajime Hirao

    2013-06-01

    Full Text Available Water is a small molecule that nevertheless perturbs, sometimes significantly, the electronic properties of an enzyme’s active site. In this study, interactions of a water molecule with the ferric heme and the compound I (Cpd I intermediate of cytochrome P450 are studied. Energy decomposition analysis (EDA schemes are used to investigate the physical origins of these interactions. Localized molecular orbital EDA (LMOEDA implemented in the quantum chemistry software GAMESS and the EDA method implemented in the ADF quantum chemistry program are used. EDA reveals that the electrostatic and polarization effects act as the major driving force in both of these interactions. The hydrogen bonding in the Cpd I•••H2O complex is similar to that in the water dimer; however, the relative importance of the electrostatic effect is somewhat larger in the water dimer.

  15. The Effect of 5-Aminolevulinic Acid on Cytochrome P450-Mediated Prodrug Activation.

    Directory of Open Access Journals (Sweden)

    Mai Miura

    Full Text Available Of late, numerous prodrugs are widely used for therapy. The hemeprotein cytochrome P450 (CYP catalyzes the activation of prodrugs to form active metabolites. Therefore, the activation of CYP function might allow the use of lower doses of prodrugs and decrease toxicity. We hypothesized that the addition of 5-aminolevulinic acid (ALA, a precursor in the porphyrin biosynthetic pathway, enhances the synthesis of heme, leading to the up-regulation of CYP activity. To test this hypothesis, we treated a human gastric cancer cell line with ALA and determined the effect on CYP-dependent prodrug activation. For this purpose, we focused on the anticancer prodrug tegafur, which is converted to its active metabolite 5-fluorouracil (5-FU mainly by CYP2A6. We show here that ALA increased CYP2A6-dependent tegafur activation, suggesting that ALA elevated CYP activity and potentiated the activation of the prodrug.

  16. Drug-enhanced carbon monoxide production from heme by cytochrome P450 reductase

    Directory of Open Access Journals (Sweden)

    Dragic Vukomanovic

    2017-01-01

    Full Text Available Carbon monoxide (CO formed endogenously is considered to be cytoprotective, and the vast majority of CO formation is attributed to the degradation of heme by heme oxygenases-1 and -2 (HO-1, HO-2. Previously, we observed that brain microsomes containing HO-2 produced many-fold more CO in the presence of menadione and its congeners; herein we explored these observations further. We determined the effects of various drugs on CO production of rat brain microsomes and recombinant human cytochrome P450 reductase (CPR; CO was measured by gas chromatography with reductive detection. Brain microsomes of Sprague-Dawley rats or recombinant human cytochrome P450 reductase (CPR were incubated with NADPH and various drugs in closed vials in phosphate buffer at pH 7.4 and 37°C. After 15 minutes, the reaction was stopped by cooling in dry ice, and the headspace gas was analyzed for CO production using gas chromatography with reductive (mercuric oxide detection. We observed drug-enhanced CO production in the presence of both microsomes and recombinant CPR alone; the presence of HO was not required. A range of structurally diverse drugs were capable of amplifying this CO formation; these molecules had structures consistent with redox cycling capability. The addition of catalase to a reaction mixture, that contained activating drugs, inhibited the production of CO. Drug-enhanced CO formation can be catalyzed by CPR. The mechanism of CPR activation was not through classical drug-receptor mediation. Redox cycling may be involved in the drug-induced amplification of CO production by CPR through the production of reactive oxygen species.

  17. Expression of Xanthophyllomyces dendrorhous cytochrome-P450 hydroxylase and reductase in Mucor circinelloides.

    Science.gov (United States)

    Csernetics, Árpád; Tóth, Eszter; Farkas, Anita; Nagy, Gábor; Bencsik, Ottó; Vágvölgyi, Csaba; Papp, Tamás

    2015-02-01

    Carotenoids are natural pigments that act as powerful antioxidants and have various beneficial effects on human and animal health. Mucor circinelloides (Mucoromycotina) is a carotenoid producing zygomycetes fungus, which accumulates β-carotene as the main carotenoid but also able to produce the hydroxylated derivatives of β-carotene (i.e. zeaxanthin and β-cryptoxanthin) in low amount. These xanthophylls, together with the ketolated derivatives of β-carotene (such as canthaxanthin, echinenone and astaxanthin) have better antioxidant activity than β-carotene. In this study our aim was to modify and enhance the xanthophyll production of the M. circinelloides by expression of heterologous genes responsible for the astaxanthin biosynthesis. The crtS and crtR genes, encoding the cytochrome-P450 hydroxylase and reductase, respectively, of wild-type and astaxanthin overproducing mutant Xanthophyllomyces dendrorhous strains were amplified from cDNA and the nucleotide and the deduced amino acid sequences were compared to each other. Introduction of the crtS on autonomously replicating plasmid in the wild-type M. circinelloides resulted enhanced zeaxanthin and β-cryptoxanthin accumulation and the presence of canthaxanthin, echinenone and astaxanthin in low amount; the β-carotene hydroxylase and ketolase activity of the X. dendrorhous cytochrome-P450 hydroxylase in M. circinelloides was verified. Increased canthaxanthin and echinenone production was observed by expression of the gene in a canthaxanthin producing mutant M. circinelloides. Co-expression of the crtR and crtS genes led to increase in the total carotenoid and slight change in xanthophyll accumulation in comparison with transformants harbouring the single crtS gene.

  18. 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.

  19. Neofunctionalization of Duplicated P450 Genes Drives the Evolution of Insecticide Resistance in the Brown Planthopper.

    Science.gov (United States)

    Zimmer, Christoph T; Garrood, William T; Singh, Kumar Saurabh; Randall, Emma; Lueke, Bettina; Gutbrod, Oliver; Matthiesen, Svend; Kohler, Maxie; Nauen, Ralf; Davies, T G Emyr; Bass, Chris

    2018-01-22

    Gene duplication is a major source of genetic variation that has been shown to underpin the evolution of a wide range of adaptive traits [1, 2]. For example, duplication or amplification of genes encoding detoxification enzymes has been shown to play an important role in the evolution of insecticide resistance [3-5]. In this context, gene duplication performs an adaptive function as a result of its effects on gene dosage and not as a source of functional novelty [3, 6-8]. Here, we show that duplication and neofunctionalization of a cytochrome P450, CYP6ER1, led to the evolution of insecticide resistance in the brown planthopper. Considerable genetic variation was observed in the coding sequence of CYP6ER1 in populations of brown planthopper collected from across Asia, but just two sequence variants are highly overexpressed in resistant strains and metabolize imidacloprid. Both variants are characterized by profound amino-acid alterations in substrate recognition sites, and the introduction of these mutations into a susceptible P450 sequence is sufficient to confer resistance. CYP6ER1 is duplicated in resistant strains with individuals carrying paralogs with and without the gain-of-function mutations. Despite numerical parity in the genome, the susceptible and mutant copies exhibit marked asymmetry in their expression with the resistant paralogs overexpressed. In the primary resistance-conferring CYP6ER1 variant, this results from an extended region of novel sequence upstream of the gene that provides enhanced expression. Our findings illustrate the versatility of gene duplication in providing opportunities for functional and regulatory innovation during the evolution of an adaptive trait. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

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

    International Nuclear Information System (INIS)

    Matteson, K.J.; Phillips, J.A. III; Miller, W.L.; Chung, B.C.; Orlando, P.J.; Frisch, H.; Ferrandez, A.; Burr, I.M.

    1987-01-01

    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

  1. Transposable elements are enriched within or in close proximity to xenobiotic-metabolizing cytochrome P450 genes

    Directory of Open Access Journals (Sweden)

    Li Xianchun

    2007-03-01

    Full Text Available Abstract Background Transposons, i.e. transposable elements (TEs, are the major internal spontaneous mutation agents for the variability of eukaryotic genomes. To address the general issue of whether transposons mediate genomic changes in environment-adaptation genes, we scanned two alleles per each of the six xenobiotic-metabolizing Helicoverpa zea cytochrome P450 loci, including CYP6B8, CYP6B27, CYP321A1, CYP321A2, CYP9A12v3 and CYP9A14, for the presence of transposon insertions by genome walking and sequence analysis. We also scanned thirteen Drosophila melanogaster P450s genes for TE insertions by in silico mapping and literature search. Results Twelve novel transposons, including LINEs (long interspersed nuclear elements, SINEs (short interspersed nuclear elements, MITEs (miniature inverted-repeat transposable elements, one full-length transib-like transposon, and one full-length Tcl-like DNA transpson, are identified from the alleles of the six H. zea P450 genes. The twelve transposons are inserted into the 5'flanking region, 3'flanking region, exon, or intron of the six environment-adaptation P450 genes. In D. melanogaster, seven out of the eight Drosophila P450s (CYP4E2, CYP6A2, CYP6A8, CYP6A9, CYP6G1, CYP6W1, CYP12A4, CYP12D1 implicated in insecticide resistance are associated with a variety of transposons. By contrast, all the five Drosophila P450s (CYP302A1, CYP306A1, CYP307A1, CYP314A1 and CYP315A1 involved in ecdysone biosynthesis and developmental regulation are free of TE insertions. Conclusion These results indicate that TEs are selectively retained within or in close proximity to xenobiotic-metabolizing P450 genes.

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

    Science.gov (United States)

    Liu, Nannan; Li, Ting; Reid, William R; Yang, Ting; Zhang, Lee

    2011-01-01

    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. Gender-specific induction of cytochrome P450s in nonylphenol-treated FVB/NJ mice

    International Nuclear Information System (INIS)

    Hernandez, Juan P.; Chapman, Laura M.; Kretschmer, Xiomara C.; Baldwin, William S.

    2006-01-01

    Nonylphenol (NP) is a breakdown product of nonylphenol ethoxylates, which are used in a variety of industrial, agricultural, household cleaning, and beauty products. NP is one of the most commonly found toxicants in the United States and Europe and is considered a toxicant of concern because of its long half-life. NP is an environmental estrogen that also activates the pregnane X-receptor (PXR) and in turn induces P450s. No study to date has examined the gender-specific effects of NP on hepatic P450 expression. We provided NP at 0, 50 or 75 mg/kg/day for 7 days to male and female FVB/NJ mice and compared their P450 expression profiles. Q-PCR was performed on hepatic cDNA using primers to several CYP isoforms regulated by PXR or its relative, the constitutive androstane receptor (CAR). In female mice, NP induced Cyp2b10 and Cyp2b13, and downregulated the female-specific P450s, Cyp3a41 and Cyp3a44. In contrast, male mice treated with NP showed increased expression of Cyp2a4, Cyp2b9, and Cyp2b10. Western blots confirmed induction of Cyp2b subfamily members in both males and females. Consistent with the Q-PCR data, Western blots showed dose-dependent downregulation of Cyp3a only in females and induction of Cyp2a only in males. The overall increase in female-predominant P450s in males (Cyp2a4, 2b9) and the decrease in female-predominant P450s in females (Cyp3a41, 3a44) suggest that NP is in part feminizing the P450 profile in males and masculinizing the P450 profile in females. Testosterone hydroxylation was also altered in a gender-specific manner, as testosterone 16α-hydroxylase activity was only induced in NP-treated males. In contrast, NP-treated females demonstrated a greater propensity for metabolizing zoxazolamine probably due to greater Cyp2b induction in females. In conclusion, NP causes gender-specific P450 induction and therefore exposure to NP may cause distinct pharmacological and toxicological effects in males compared to females

  4. 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 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.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.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.

  5. Induction of P450 3A1/2 and 2C6 by gemfibrozil in Sprague-Dawley rats.

    Science.gov (United States)

    Liu, Aiming; Yang, Julin; Zhao, Xin; Jiao, Xiaolan; Zhao, Weihong; Ma, Qing; Tang, Zhiyuan; Dai, Renke

    2011-01-01

    Fibrates are a group of peroxisome proliferator-activated receptor α agonists used in the treatment of dyslipidemia; however, they have been reported to cause species-related hepatocarcinogenesis and clinical myotoxicity. Gemfibrozil is one of the most commonly used fibrates, and it shows the highest risk for myotoxicity among the fibrates. The inhibitory drug-drug interaction mechanism associated with gemfibrozil has been explored recently, and the induction of human P450 3A4 and 2C8 has been reported. In this study, in vivo induction of rat P450 by gemfibrozil was studied in Sprague-Dawley rats. After the rats were dosed with gemfibrozil by oral gavage, microsomes were prepared. The metabolic activities of P450 3A1/2, 2C6, and 2D2 were assayed using probe substrates, and the systemic concentration of gemfibrozil during its administration was determined. P450 3A1/2 and 2C6 activities were induced 32-77% in the rats by gemfibrozil when the exposure concentration was in the clinical range. These data indicate that the inducibility of homologous P450 isoforms by gemfibrozil is similar in Sprague-Dawley rats and in humans. Inductive drug-drug interactions and inhibitory actions are involved in the co-administration of gemfibrozil with other drugs, which suggests the relevance for a fibrate-toxicology investigation.

  6. The catalytic function of cytochrome P450 is entwined with its membrane-bound nature [version 1; referees: 4 approved

    Directory of Open Access Journals (Sweden)

    Carlo Barnaba

    2017-05-01

    Full Text Available Cytochrome P450, a family of monooxygenase enzymes, is organized as a catalytic metabolon, which requires enzymatic partners as well as environmental factors that tune its complex dynamic. P450 and its reducing counterparts—cytochrome P450-reductase and cytochrome b5—are membrane-bound proteins located in the cytosolic side of the endoplasmic reticulum. They are believed to dynamically associate to form functional complexes. Increasing experimental evidence signifies the role(s played by both protein-protein and protein-lipid interactions in P450 catalytic function and efficiency. However, the biophysical challenges posed by their membrane-bound nature have severely limited high-resolution understanding of the molecular interfaces of these interactions. In this article, we provide an overview of the current knowledge on cytochrome P450, highlighting the environmental factors that are entwined with its metabolic function. Recent advances in structural biophysics are also discussed, setting up the bases for a new paradigm in the study of this important class of membrane-bound enzymes.

  7. Evaluation of herb-drug interaction of a polyherbal Ayurvedic formulation through high throughput cytochrome P450 enzyme inhibition assay.

    Science.gov (United States)

    Pandit, Subrata; Kanjilal, Satyajyoti; Awasthi, Anshumali; Chaudhary, Anika; Banerjee, Dipankar; Bhatt, B N; Narwaria, Avinash; Singh, Rahul; Dutta, Kakoli; Jaggi, Manu; Singh, Anu T; Sharma, Neena; Katiyar, Chandra Kant

    2017-02-02

    Arishtas are Ayurvedic formulation made with decoction of herbs. Arjunarishta formulation is being used in Ayurveda for cardio-protective activity. Ashwagandharishta formulation possesses antioxidant, anti-atherosclerotic and anti-stress properties. Ridayarishta, a novel empirical formulation was prepared using combination of selected ingredients from these two formulations to support healthy heart functions and to reduce stress. Aim of the Study was to investigate herb-drug interaction (HDI) of Ridayarishta formulation through human hepatic cytochrome P450 (CYP450) enzyme inhibition assay. Ridayarishta formulation was phyto-chemically standardized against arjunolic acid, arjunetin, berberine, piperine, resveratrol and withaferin-A using high performance thin layer chromatography (HPTLC) analysis. The formulation was standardized with respect to ethanol by gas chromatographic (GC) analysis. HDI was evaluated with Ridayarishta formulation and amlodipine besilate, atenolol, atorvastatin, metformin, glipizide glimepiride cocktail using high throughput CYP450 enzyme inhibition assay; against CYP1A2, 2C19, 2D6 and 3A4 isozymes. Contents of arjunolic acid, arjunetin, berberine, piperine, resveratrol and withaferin-A in Ridayarishta formulation were found to be 1.76±0.12, 1.51±0.09, 1.85±0.05, 3.2±0.12, 1.21±0.08, and 2.16±0.09ppm, respectively. Quantity of ethanol in Ridayarishta was found to be 7.95±0.023% (V/V). Ridayarishta showed significantly higher (Pdrugs showed significantly (P<0.001and P<0.01) less or negligible HDI. Ridayarishta formulation alone and cocktail with amlodipine besilate, atenolol, atorvastatin, metformin, glipizide, glimepiride had negligible or insignificant effect on CYP450 inhibition. It may be concluded that consumption of Ridayarishta along with selective cardio protective, antihypertensive and anti-diabetic conventional medicine is safe with negligible or without any significant CYP450 (CYP1A2, 2C19, 2D6 and 3A4) inhibition mediated

  8. Inhibitory Effects of Dimethyllirioresinol, Epimagnolin A, Eudesmin, Fargesin, and Magnolin on Cytochrome P450 Enzyme Activities in Human Liver Microsomes

    Directory of Open Access Journals (Sweden)

    Ju-Hyun Kim

    2017-05-01

    Full Text Available Magnolin, epimagnolin A, dimethyllirioresinol, eudesmin, and fargesin are pharmacologically active tetrahydrofurofuranoid lignans found in Flos Magnoliae. The inhibitory potentials of dimethyllirioresinol, epimagnolin A, eudesmin, fargesin, and magnolin on eight major human cytochrome P450 (CYP enzyme activities in human liver microsomes were evaluated using liquid chromatography–tandem mass spectrometry to determine the inhibition mechanisms and inhibition potency. Fargesin inhibited CYP2C9-catalyzed diclofenac 4’-hydroxylation with a Ki value of 16.3 μM, and it exhibited mechanism-based inhibition of CYP2C19-catalyzed [S]-mephenytoin 4’-hydroxylation (Ki, 3.7 μM; kinact, 0.102 min−1, CYP2C8-catalyzed amodiaquine N-deethylation (Ki, 10.7 μM; kinact, 0.082 min−1, and CYP3A4-catalyzed midazolam 1’-hydroxylation (Ki, 23.0 μM; kinact, 0.050 min−1 in human liver microsomes. Fargesin negligibly inhibited CYP1A2-catalyzed phenacetin O-deethylation, CYP2A6-catalyzed coumarin 7-hydroxylation, CYP2B6-catalyzed bupropion hydroxylation, and CYP2D6-catalyzed bufuralol 1’-hydroxylation at 100 μM in human liver microsomes. Dimethyllirioresinol weakly inhibited CYP2C19 and CYP2C8 with IC50 values of 55.1 and 85.0 μM, respectively, without inhibition of CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2D6, and CYP3A4 activities at 100 μM. Epimagnolin A, eudesmin, and magnolin showed no the reversible and time-dependent inhibition of eight major CYP activities at 100 μM in human liver microsomes. These in vitro results suggest that it is necessary to investigate the potentials of in vivo fargesin-drug interaction with CYP2C8, CYP2C9, CYP2C19, and CYP3A4 substrates.

  9. Pyrethroid insecticide lambda-cyhalothrin induces hepatic cytochrome P450 enzymes, oxidative stress and apoptosis in rats.

    Science.gov (United States)

    Martínez, María-Aránzazu; Ares, Irma; Rodríguez, José-Luis; Martínez, Marta; Roura-Martínez, David; Castellano, Victor; Lopez-Torres, Bernardo; Martínez-Larrañaga, María-Rosa; Anadón, Arturo

    2018-08-01

    This study aimed to examine in rats the effects of the Type II pyrethroid lambda-cyhalothrin on hepatic microsomal cytochrome P450 (CYP) isoform activities, oxidative stress markers, gene expression of proinflammatory, oxidative stress and apoptosis mediators, and CYP isoform gene expression and metabolism phase I enzyme PCR array analysis. Lambda-cyhalothrin, at oral doses of 1, 2, 4 and 8mg/kg bw for 6days, increased, in a dose-dependent manner, hepatic activities of ethoxyresorufin O-deethylase (CYP1A1), methoxyresorufin O-demethylase (CYP1A2), pentoxyresorufin O-depentylase (CYP2B1/2), testosterone 7α- (CYP2A1), 16β- (CYP2B1), and 6β-hydroxylase (CYP3A1/2), and lauric acid 11- and 12-hydroxylase (CYP4A1/2). Similarly, lambda-cyhalothrin (4 and 8mg/kg bw, for 6days), in a dose-dependent manner, increased significantly hepatic CYP1A1, 1A2, 2A1, 2B1, 2B2, 2E1, 3A1, 3A2 and 4A1 mRNA levels and IL-1β, NFκB, Nrf2, p53, caspase-3 and Bax gene expressions. PCR array analysis showed from 84 genes examined (P1.5), changes in mRNA levels in 18 genes: 13 up-regulated and 5 down-regulated. A greater fold change reversion than 3-fold was observed on the up-regulated ALDH1A1, CYP2B2, CYP2C80 and CYP2D4 genes. Ingenuity Pathway Analysis (IPA) groups the expressed genes into biological mechanisms that are mainly related to drug metabolism. In the top canonical pathways, Oxidative ethanol degradation III together with Fatty Acid α-oxidation may be significant pathways for lambda-cyhalothrin. Our results may provide further understanding of molecular aspects involved in lambda-cyhalothrin-induced liver injury. Copyright © 2018. Published by Elsevier B.V.

  10. Genetic polymorphism of human cytochrome P-450 (S)-mephenytoin 4-hydroxylase. Studies with human autoantibodies suggest a functionally altered cytochrome P-450 isozyme as cause of the genetic deficiency

    International Nuclear Information System (INIS)

    Meier, U.T.; Meyer, U.A.

    1987-01-01

    The metabolism of the anticonvulsant mephenytoin is subject to a genetic polymorphism. In 2-5% of Caucasians and 18-23% of Japanese subjects a specific cytochrome P-450 isozyme, P-450 meph, is functionally deficient or missing. The authors have accumulated evidence that autoimmune antibodies observed in sera of patients with tienilic acid induced hepatitis (anti-liver kidney microsome 2 or anti-LKM2 antibodies) specifically recognize the cytochrome P-450 involved in the mephrenytoin hydroxylation polymorphism. This is demonstrated by immunoinhibition and immunoprecipitation of microsomal (S)-mephenytoin 4-hydroxylation activity and by the recognition by anti-LKM2 antibodies of a single [ 125 I]-protein band on immunoblots of human liver microsomes after sodium dodecyl sulfate-polyacrylamide gel electrophoresis or isoelectric focusing. The cytochrome P-450 recognized by anti-LKM2 antibodies was immunopurified from microsomes derived from livers of extensive (EM) or poor metabolizers (PM) of (S)-mephenytoin. Comparison of the EM-type cytochrome P-450 to that isolated from PM livers revealed no difference in regard to immuno-cross-reactivity, molecular weight, isoelectric point, relative content in microsomes, two-dimensional tryptic peptide maps, one-dimensional peptide maps with three proteases, amino acid composition, and amino-terminal protein sequence. Finally, the same protein was precipitated from microsomes prepared from the liver biopsy of a subject phenotyped in vivo as a poor metabolizer of mephenytoin. These data strongly suggest that the mephenytoin hydroxylation deficiency is caused by a minor structural change leading to a functionally altered cytochrome P-450 isozyme

  11. [Effects of electroacupuncture of "Guanyuan" (CV 4)-"Zhongji" (CV 3) on ovarian P450 arom and P450c 17alpha expression and relevant sex hormone levels in rats with polycystic ovary syndrome].

    Science.gov (United States)

    Sun, Jie; Zhao, Ji-meng; Ji, Rong; Liu, Hui-rong; Shi, Yin; Jin, Chun-lan

    2013-12-01

    To observe the effect of electroacupuncture (EA) on ovarian P 450 arom and P 450 c 17 alpha (aromatases) expression and related sex hormone levels in polycystic ovary syndrome (PCOS) rats. Thirty SD rats were randomly divided into normal control group, model group and EA group (10 rats/group). PCOS model was made by intragastric administration of letrozole at 1 mg/kg per day for consecutive 21 days. "Guanyuan" (CV 4) and "Zhongji" (CV 3) acupoints were stimulated 20 min by EA (2 mA, 2 Hz), once daily for consecutive 14 days. The damp ovarian weight was weighed and the pathological changes of the ovarian tissue were observed after H. E. staining. Ultrastructural changes of the ovarian tissue were observed by transmission electron microscope. Immunohistochemical staining was adopted to detect ovarian follicle granulosa cell P 450 arom and follicle membrane cell P 450 c 17 alpha expression. The contents of estradiol (E 2), estrone (E 1), androstenedione (ASD), testosterone (T), follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in the ovarian tissue were measured by ELISA. Compared with the normal group, there was a significant increase in the damp weight of both left and right ovarian tissues in the model group (P ovarian weight was remarkably reduced (P ovarian tissue such as thickening of the superficial albugineous coat of the ovary, thinning of the granular cell layer, and disappearance of the intraovular oocytes and coronaradiata under light microscope, and mitochondrion swelling, fracture or disappearance of mitochondrial cristae, and enlargement of the endoplasmic reticulum, etc. after modeling were obviously improved in the EA group. In comparison to the control group, the expression of the follicle granulosa cell P450 arom was significantly down-regulated and that of follicle membrane cell P 450 c 17 alpha was significantly upregulated in the model group (P ovarian tissues (P ovarian E 1 and E2 (P ovarian ASD, T and LH levels were notably

  12. Oxygen Atom Transfer Using an Iron(IV)-Oxo Embedded in a Tetracyclic N-Heterocyclic Carbene System: How Does the Reactivity Compare to Cytochrome P450 Compound I?

    Science.gov (United States)

    Cantú Reinhard, Fabián G; de Visser, Sam P

    2017-02-24

    N-Heterocyclic carbenes (NHC) are commonly featured as ligands in transition metal catalysis. Recently, a cyclic system containing four NHC groups with a central iron atom was synthesized and its iron(IV)-oxo species, [Fe IV (O)(cNHC 4 )] 2+ , was characterized. This tetracyclic NHC ligand system may give the iron(IV)-oxo species unique catalytic properties as compared to traditional non-heme and heme iron ligand systems. Therefore, we performed a computational study on the structure and reactivity of the [Fe IV (O)(cNHC 4 )] 2+ complex in substrate hydroxylation and epoxidation reactions. The reactivity patterns are compared with cytochrome P450 Compound I and non-heme iron(IV)-oxo models and it is shown that the [Fe IV (O)(cNHC 4 )] 2+ system is an effective oxidant with oxidative power analogous to P450 Compound I. Unfortunately, in polar solvents, a solvent molecule will bind to the sixth ligand position and decrease the catalytic activity of the oxidant. A molecular orbital and valence bond analysis provides insight into the origin of the reactivity differences and makes predictions of how to further exploit these systems in chemical catalysis. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Ketamine and ketamine metabolites as novel estrogen receptor ligands: Induction of cytochrome P450 and AMPA glutamate receptor gene expression.

    Science.gov (United States)

    Ho, Ming-Fen; Correia, Cristina; Ingle, James N; Kaddurah-Daouk, Rima; Wang, Liewei; Kaufmann, Scott H; Weinshilboum, Richard M

    2018-04-03

    Major depressive disorder (MDD) is the most common psychiatric illness worldwide, and it displays a striking sex-dependent difference in incidence, with two thirds of MDD patients being women. Ketamine treatment can produce rapid antidepressant effects in MDD patients, effects that are mediated-at least partially-through glutamatergic neurotransmission. Two active metabolites of ketamine, (2R,6R)-hydroxynorketamine (HNK) and (2S,6S)-HNK, also appear to play a key role in ketamine's rapid antidepressant effects through the activation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptors. In the present study, we demonstrated that estrogen plus ketamine or estrogen plus active ketamine metabolites displayed additive effects on the induction of the expression of AMPA receptor subunits. In parallel, the expression of estrogen receptor alpha (ERα) was also significantly upregulated. Even more striking, radioligand binding assays demonstrated that [ 3 H]-ketamine can directly bind to ERα (K D : 344.5 ± 13 nM). Furthermore, ketamine and its (2R,6R)-HNK and (2S,6S)-HNK metabolites displayed similar affinity for ERα (IC 50 : 2.31 ± 0.1, 3.40 ± 0.2, and 3.53 ± 0.2 µM, respectively) as determined by [ 3 H]-ketamine displacement assays. Finally, induction of AMPA receptors by either estrogens or ketamine and its metabolites was lost when ERα was knocked down or silenced pharmacologically. These results suggest a positive feedback loop by which estrogens can augment the effects of ketamine and its (2R,6R)-HNK and (2S,6S)-HNK metabolites on the ERα-induced transcription of CYP2A6 and CYP2B6, estrogen inducible enzymes that catalyze ketamine's biotransformation to form the two active metabolites. These observations provide novel insight into ketamine's molecular mechanism(s) of action and have potential implications for the treatment of MDD. Copyright © 2018 Elsevier Inc. All rights reserved.

  14. Identification of cytochrome P450 differentiated expression related to developmental stages in bromadiolone resistance in rats (Rattus norvegicus)

    DEFF Research Database (Denmark)

    Markussen, Mette; Heiberg, Ann-Charlotte; Fredholm, Merete

    2008-01-01

    over-express the Cyp2a1 gene. TGhe altered gene expression has been suggested to be involved in the bromadiolone resistance by facilitating enhanced anticoagulant metabolism. To investigate the gene expression of these cytochrome P450 genes in rats of different developmental stages we compared...... expression profiles, from 8-, 12- and 20-week-old resistant rats of the Danish strain to profiles of anticoagulant-susceptible rats of same ages. The three age-groups were selected to represent a group of pre-pubertal, pubertal and adult rats. We found expression profiles of the pre-pubertal and pubertal...... resistant rats to concur with profiles of the adults suggesting that cytochrome P450 enzymes are involved in the Danish bromadiolone resistance regardless of developmental stage. We also investigated the relative importance of the six cytochrome P450s in the different development stages of the resistant...

  15. 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

    2011-01-03

    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. Copyright © 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  16. 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.

  17. 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

  18. 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

  19. 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

  20. Beyond Iron: Iridium-Containing P450 Enzymes for Selective Cyclopropanations of Structurally Diverse Alkenes

    International Nuclear Information System (INIS)

    Key, Hanna M.; Dydio, Paweł; Liu, Zhennan

    2017-01-01

    Enzymes catalyze organic transformations with exquisite levels of selectivity, including chemoselectivity, stereoselectivity, and substrate selectivity, but the types of reactions catalyzed by enzymes are more limited than those of chemical catalysts. Thus, the convergence of chemical catalysis and biocatalysis can enable enzymatic systems to catalyze abiological reactions with high selectivity. Recently, we disclosed artificial enzymes constructed from the apo form of heme proteins and iridium porphyrins that catalyze the insertion of carbenes into a C-H bond. Here, we postulated that the same type of Ir(Me)-PIX enzymes could catalyze the cyclopropanation of a broad range of alkenes with control of multiple modes of selectivity. Here, we report the evolution of artificial enzymes that are highly active and highly stereoselective for the addition of carbenes to a wide range of alkenes. These enzymes catalyze the cyclopropanation of terminal and internal, activated and unactivated, electron-rich and electron-deficient, conjugated and nonconjugated alkenes. In particular, Ir(Me)-PIX enzymes derived from CYP119 catalyze highly enantio- and diastereoselective cyclopropanations of styrene with ±98% ee, > 70:1 dr, > 75% yield, and ~10,000 turnovers (TON), as well as 1,2-disubstituted styrenes with up to 99% ee, 35:1 dr, and 54% yield. Moreover, Ir(Me)-PIX enzymes catalyze cyclopropanation of internal, unactivated alkenes with up to 99% stereoselectivity, 76% yield, and 1300 TON. They also catalyze cyclopropanation of natural products with diastereoselectivities that are complementary to those attained with standard transition metal catalysts. Finally, Ir(Me)-PIX P450 variants react with substrate selectivity that is reminiscent of natural enzymes; they react preferentially with less reactive internal alkenes in the presence of more reactive terminal alkenes. Altogether, the studies reveal the suitability of Ir-containing P450s to combine the broad reactivity and

  1. Mass spectrometry-based proteomic analysis of human liver cytochrome(s) P450

    International Nuclear Information System (INIS)

    Shrivas, Kamlesh; Mindaye, Samuel T.; Getie-Kebtie, Melkamu; Alterman, Michail A.

    2013-01-01

    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

  2. Autoantibodies against Cytochrome P450 Side-Chain Cleavage Enzyme in Dogs (Canis lupus familiaris) Affected with Hypoadrenocorticism (Addison's Disease).

    Science.gov (United States)

    Boag, Alisdair M; Christie, Michael R; McLaughlin, Kerry A; Syme, Harriet M; Graham, Peter; Catchpole, Brian

    2015-01-01

    Canine hypoadrenocorticism likely arises from immune-mediated destruction of adrenocortical tissue, leading to glucocorticoid and mineralocorticoid deficiency. In humans with autoimmune Addison's disease (AAD) or autoimmune polyendocrine syndrome (APS), circulating autoantibodies have been demonstrated against enzymes associated with adrenal steroid synthesis. The current study investigates autoantibodies against steroid synthesis enzymes in dogs with spontaneous hypoadrenocorticism. Coding regions of canine CYP21A2 (21-hydroxylase; 21-OH), CYP17A1 (17-hydroxylase; 17-OH), CYP11A1 (P450 side-chain cleavage enzyme; P450scc) and HSD3B2 (3β hydroxysteroid dehydrogenase; 3βHSD) were amplified, cloned and expressed as 35S-methionine radiolabelled recombinant protein. In a pilot study, serum samples from 20 dogs with hypoadrenocorticism and four unaffected control dogs were screened by radio-immunoprecipitation assay. There was no evidence of reactivity against 21-OH, 17-OH or 3βHSD, but five dogs with hypoadrenocorticism showed immunoreactivity to P450scc compared with controls. Serum samples were subsequently obtained from 213 dogs diagnosed with hypoadrenocorticism and 110 dogs from a hospital control population. Thirty control dogs were randomly selected to establish a threshold for antibody positivity (mean + 3 × standard deviation). Dogs with hypoadrenocorticism were more likely to be P450scc autoantibody positive than hospital controls (24% vs. 1.2%, respectively; p = 0.0016). Sex was significantly associated with the presence of P450scc autoantibodies in the case population, with 30% of females testing positive compared with 17% of males (p = 0.037). Significant associations with breed (p = 0.015) and DLA-type (DQA1*006:01 allele; p = 0.017) were also found. This cross-sectional study indicates that P450scc autoantibodies are present in a proportion of dogs affected with hypoadrenocorticism.

  3. Autoantibodies against Cytochrome P450 Side-Chain Cleavage Enzyme in Dogs (Canis lupus familiaris Affected with Hypoadrenocorticism (Addison's Disease.

    Directory of Open Access Journals (Sweden)

    Alisdair M Boag

    Full Text Available Canine hypoadrenocorticism likely arises from immune-mediated destruction of adrenocortical tissue, leading to glucocorticoid and mineralocorticoid deficiency. In humans with autoimmune Addison's disease (AAD or autoimmune polyendocrine syndrome (APS, circulating autoantibodies have been demonstrated against enzymes associated with adrenal steroid synthesis. The current study investigates autoantibodies against steroid synthesis enzymes in dogs with spontaneous hypoadrenocorticism. Coding regions of canine CYP21A2 (21-hydroxylase; 21-OH, CYP17A1 (17-hydroxylase; 17-OH, CYP11A1 (P450 side-chain cleavage enzyme; P450scc and HSD3B2 (3β hydroxysteroid dehydrogenase; 3βHSD were amplified, cloned and expressed as 35S-methionine radiolabelled recombinant protein. In a pilot study, serum samples from 20 dogs with hypoadrenocorticism and four unaffected control dogs were screened by radio-immunoprecipitation assay. There was no evidence of reactivity against 21-OH, 17-OH or 3βHSD, but five dogs with hypoadrenocorticism showed immunoreactivity to P450scc compared with controls. Serum samples were subsequently obtained from 213 dogs diagnosed with hypoadrenocorticism and 110 dogs from a hospital control population. Thirty control dogs were randomly selected to establish a threshold for antibody positivity (mean + 3 × standard deviation. Dogs with hypoadrenocorticism were more likely to be P450scc autoantibody positive than hospital controls (24% vs. 1.2%, respectively; p = 0.0016. Sex was significantly associated with the presence of P450scc autoantibodies in the case population, with 30% of females testing positive compared with 17% of males (p = 0.037. Significant associations with breed (p = 0.015 and DLA-type (DQA1*006:01 allele; p = 0.017 were also found. This cross-sectional study indicates that P450scc autoantibodies are present in a proportion of dogs affected with hypoadrenocorticism.

  4. Modulation of the interaction between human P450 3A4 and B. megaterium reductase via engineered loops.

    Science.gov (United States)

    Castrignanò, Silvia; D'Avino, Serena; Di Nardo, Giovanna; Catucci, Gianluca; Sadeghi, Sheila J; Gilardi, Gianfranco

    2018-01-01

    Chimerogenesis involving cytochromes P450 is a successful approach to generate catalytically self-sufficient enzymes. However, the connection between the different functional modules should allow a certain degree of flexibility in order to obtain functional and catalytically efficient proteins. We previously applied the molecular Lego approach to develop a chimeric P450 3A4 enzyme linked to the reductase domain of P450 BM3 (BMR). Three constructs were designed with the connecting loop containing no glycine, 3 glycine or 5 glycine residues and showed a different catalytic activity and coupling efficiency. Here we investigate how the linker affects the ability of P450 3A4 to bind substrates and inhibitors. We measure the electron transfer rates and the catalytic properties of the enzyme also in the presence of ketoconazole as inhibitor. The data show that the construct 3A4-5GLY-BMR with the longest loop better retains the binding ability and cooperativity for testosterone, compared to P450 3A4. In both 3A4-3GLY-BMR and 3A4-5GLY-BMR, the substrate induces an increase in the first electron transfer rate and a shorter lag phase related to a domain rearrangements, when compared to the construct without Gly. These data are consistent with docking results and secondary structure predictions showing a propensity to form helical structures in the loop of the 3A4-BMR and 3A4-3GLY-BMR. All three chimeras retain the ability to bind the inhibitor ketoconazole and show an IC 50 comparable with those reported for the wild type protein. This article is part of a Special Issue entitled: Cytochrome P450 biodiversity and biotechnology, edited by Erika Plettner, Gianfranco Gilardi, Luet Wong, Vlada Urlacher, Jared Goldstone. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Deletion of P399{sub E}401 in NADPH cytochrome P450 oxidoreductase results in partial mixed oxidase deficiency

    Energy Technology Data Exchange (ETDEWEB)

    Flueck, Christa E., E-mail: christa.flueck@dkf.unibe.ch [Pediatric Endocrinology, Diabetology and Metabolism, University Children' s Hospital, Bern (Switzerland); Mallet, Delphine [Service d' Endocrinologie Moleculaire et Maladies Rares, Hospices Civils de Lyon, Bron (France); Hofer, Gaby [Pediatric Endocrinology, Diabetology and Metabolism, University Children' s Hospital, Bern (Switzerland); Samara-Boustani, Dinane [Hopital Necker-Enfants malades, Paris (France); Leger, Juliane [Hopital Robert Debre, Paris (France); Polak, Michel [Hopital Necker-Enfants malades, Paris (France); Morel, Yves [Service d' Endocrinologie Moleculaire et Maladies Rares, Hospices Civils de Lyon, Bron (France); Pandey, Amit V., E-mail: amit@pandeylab.org [Pediatric Endocrinology, Diabetology and Metabolism, University Children' s Hospital, Bern (Switzerland)

    2011-09-09

    Highlights: {yields} Mutations in human POR cause congenital adrenal hyperplasia. {yields} We are reporting a novel 3 amino acid deletion mutation in POR P399{sub E}401del. {yields} POR mutation P399{sub E}401del decreased P450 activities by 60-85%. {yields} Impairment of steroid metabolism may be caused by multiple hits. {yields} 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 P399{sub E}401del 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 P399{sub E}401del protein was found to decrease catalytic efficiency of 21-hydroxylation of progesterone by 68%, 17{alpha}-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 P399{sub E}401 revealed reduced stability and flexibility of the mutant. In conclusion, P399{sub E}401del 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 P399{sub E}401del 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.

  6. Heme exporter FLVCR1a regulates heme synthesis and degradation and controls activity of cytochromes P450.

    Science.gov (United States)

    Vinchi, Francesca; Ingoglia, Giada; Chiabrando, Deborah; Mercurio, Sonia; Turco, Emilia; Silengo, Lorenzo; Altruda, Fiorella; Tolosano, Emanuela

    2014-05-01

    The liver has one of the highest rates of heme synthesis of any organ. More than 50% of the heme synthesized in the liver is used for synthesis of P450 enzymes, which metabolize exogenous and endogenous compounds that include natural products, hormones, drugs, and carcinogens. Feline leukemia virus subgroup C cellular receptor 1a (FLVCR1a) is plasma membrane heme exporter that is ubiquitously expressed and controls intracellular heme content in hematopoietic lineages. We investigated the role of Flvcr1a in liver function in mice. We created mice with conditional disruption of Mfsd7b, which encodes Flvcr1a, in hepatocytes (Flvcr1a(fl/fl);alb-cre mice). Mice were analyzed under basal conditions, after phenylhydrazine-induced hemolysis, and after induction of cytochromes P450 synthesis. Livers were collected and analyzed by histologic, quantitative real-time polymerase chain reaction, and immunoblot analyses. Hepatic P450 enzymatic activities were measured. Flvcr1a(fl/fl);alb-cre mice accumulated heme and iron in liver despite up-regulation of heme oxygenase 1, ferroportin, and ferritins. Hepatic heme export activity of Flvcr1a was closely associated with heme biosynthesis, which is required to sustain cytochrome induction. Upon cytochromes P450 stimulation, Flvcr1a(fl/fl);alb-cre mice had reduced cytochrome activity, associated with accumulation of heme in hepatocytes. The expansion of the cytosolic heme pool in these mice was likely responsible for the early inhibition of heme synthesis and increased degradation of heme, which reduced expression and activity of cytochromes P450. In livers of mice, Flvcr1a maintains a free heme pool that regulates heme synthesis and degradation as well as cytochromes P450 expression and activity. These findings have important implications for drug metabolism. Copyright © 2014 AGA Institute. Published by Elsevier Inc. All rights reserved.

  7. Heme Exporter FLVCR1a Regulates Heme Synthesis and Degradation and Controls Activity of Cytochromes P450

    Science.gov (United States)

    Vinchi, Francesca; Ingoglia, Giada; Chiabrando, Deborah; Mercurio, Sonia; Turco, Emilia; Silengo, Lorenzo; Altruda, Fiorella; Tolosano, Emanuela

    2014-01-01

    Background & Aims The liver has one of the highest rates of heme synthesis of any organ. More than 50% of the heme synthesized in the liver is used for synthesis of P450 enzymes, which metabolize exogenous and endogenous compounds that include natural products, hormones, drugs, and carcinogens. Feline leukemia virus subgroup C cellular receptor 1a (FLVCR1a) is plasma membrane heme exporter that is ubiquitously expressed and controls intracellular heme content in hematopoietic lineages. We investigated the role of Flvcr1a in liver function in mice. Methods We created mice with conditional disruption of Mfsd7b, which encodes Flvcr1a, in hepatocytes (Flvcr1afl/fl;alb-cre mice). Mice were analyzed under basal conditions, after phenylhydrazine-induced hemolysis, and after induction of cytochromes P450 synthesis. Livers were collected and analyzed by histologic, quantitative real-time polymerase chain reaction, and immunoblot analyses. Hepatic P450 enzymatic activities were measured. Results Flvcr1afl/fl;alb-cre mice accumulated heme and iron in liver despite up-regulation of heme oxygenase 1, ferroportin, and ferritins. Hepatic heme export activity of Flvcr1a was closely associated with heme biosynthesis, which is required to sustain cytochrome induction. Upon cytochromes P450 stimulation, Flvcr1afl/fl;alb-cre mice had reduced cytochrome activity, associated with accumulation of heme in hepatocytes. The expansion of the cytosolic heme pool in these mice was likely responsible for the early inhibition of heme synthesis and increased degradation of heme, which reduced expression and activity of cytochromes P450. Conclusions In livers of mice, Flvcr1a maintains a free heme pool that regulates heme synthesis and degradation as well as cytochromes P450 expression and activity. These findings have important implications for drug metabolism. PMID:24486949

  8. RNA interference of NADPH-cytochrome P450 reductase results in reduced insecticide resistance in the bed bug, Cimex lectularius.

    Science.gov (United States)

    Zhu, Fang; Sams, Sarah; Moural, Tim; Haynes, Kenneth F; Potter, Michael F; Palli, Subba R

    2012-01-01

    NADPH-cytochrome P450 reductase (CPR) plays a central role in cytochrome P450 action. The genes coding for P450s are not yet fully identified in the bed bug, Cimex lectularius. Hence, we decided to clone cDNA and knockdown the expression of the gene coding for CPR which is suggested to be required for the function of all P450s to determine whether or not P450s are involved in resistance of bed bugs to insecticides. The full length Cimex lectularius CPR (ClCPR) cDNA was isolated from a deltamethrin resistant bed bug population (CIN-1) using a combined PCR strategy. Bioinformatics and in silico modeling were employed to identify three conserved binding domains (FMN, FAD, NADP), a FAD binding motif, and the catalytic residues. The critical amino acids involved in FMN, FAD, NADP binding and their putative functions were also analyzed. No signal peptide but a membrane anchor domain with 21 amino acids which facilitates the localization of ClCPR on the endoplasmic reticulum was identified in ClCPR protein. Phylogenetic analysis showed that ClCPR is closer to the CPR from the body louse, Pediculus humanus corporis than to the CPRs from the other insect species studied. The ClCPR gene was ubiquitously expressed in all tissues tested but showed an increase in expression as immature stages develop into adults. We exploited the traumatic insemination mechanism of bed bugs to inject dsRNA and successfully knockdown the expression of the gene coding for ClCPR. Suppression of the ClCPR expression increased susceptibility to deltamethrin in resistant populations but not in the susceptible population of bed bugs. These data suggest that P450-mediated metabolic detoxification may serve as one of the resistance mechanisms in bed bugs.

  9. Deletion of P399E401 in NADPH cytochrome P450 oxidoreductase results in partial mixed oxidase deficiency

    International Nuclear Information System (INIS)

    Flueck, Christa E.; Mallet, Delphine; Hofer, Gaby; Samara-Boustani, Dinane; Leger, Juliane; Polak, Michel; Morel, Yves; Pandey, Amit V.

    2011-01-01

    Highlights: → Mutations in human POR cause congenital adrenal hyperplasia. → We are reporting a novel 3 amino acid deletion mutation in POR P399 E 401del. → POR mutation P399 E 401del 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 P399 E 401del 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 P399 E 401del 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 P399 E 401 revealed reduced stability and flexibility of the mutant. In conclusion, P399 E 401del 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 P399 E 401del 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.

  10. Significance of cytochrome P450 system responses and levels of bile fluorescent aromatic compounds in marine wildlife following oil spills

    International Nuclear Information System (INIS)

    Lee, R.F.; Anderson, J.W.

    2005-01-01

    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

  11. Polymerase chain reaction amplification fails to detect aromatase cytochrome P450 transcripts in normal human endometrium or decidua.

    Science.gov (United States)

    Bulun, S E; Mahendroo, M S; Simpson, E R

    1993-06-01

    It has been proposed that the biosynthesis of estrogens by the human endometrium may be of physiological significance during the menstrual cycle. Local estrogen production was also suggested to be important in the development of endometrial cancer; however, the presence or absence of aromatase enzyme activity in normal human endometrium is controversial. To address this issue, we used a sensitive technique capable of detecting mRNA transcripts present in only very low copy number. The polymerase chain reaction linked to reverse transcription (RT-PCR) was used to evaluate the presence or absence of aromatase cytochrome P450 (P450arom) transcripts in endometrial tissues (n = 7) and endometrial stromal cells (n = 9) under various culture conditions. RNA was isolated from four proliferative and three secretory tissue samples and from cultured endometrial stromal cells isolated from seven proliferative and two secretory endometria. Five sets of cultures were treated with medroxyprogesterone acetate (MPA), estradiol (E2), and forskolin. Additionally, RNA was isolated from decidualized endometrium obtained from a patient with tubal pregnancy. A single stranded cDNA was synthesized from total RNA using Moloney murine leukemia virus reverse transcriptase and a P450arom-specific oligonucleotide. The single stranded cDNA was used as a template for PCR and was amplified for 20-35 cycles using P450arom-specific primers. RNA from adipose tissue and placenta was amplified to provide positive controls, whereas myometrial RNA was used as a negative control. In two experiments involving two endometrial tissues and three sets of cells in culture, a rat P450arom cRNA was coamplified in each sample as an internal control to demonstrate that the remote possibility of RT-PCR failures in individual test samples cannot account for our negative results. By Southern or slot blot hybridization of the amplified fragments using human and rat P450arom-specific probes, we found no evidence for

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

    OpenAIRE

    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 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...

  13. Influence of sex hormones on relative quantities of multiple species of cytochrome P-450 in rat liver microsomes

    International Nuclear Information System (INIS)

    Fujita, S.; Peisach, J.; Chevion, M.; Hebrew Univ., Jerusalem

    1981-01-01

    EPR spectra of rat liver microsomes from male, female and hormonally-treated castrated hepatectomized rats were studied. The spectra, especially in the region of gsub(max) suggested a heterogeneity of local environments of the low spin ferric heme indicative of multiple structures for cytochrome P-450. Certain features in the spectrum correlated with sexual differences. It is suggested that the changes in the relative amplitudes of the EPR features represent differences in the relative abundance of the individual proteins in the mixture that, in turn, are related to the sexual differences of metabolic patterns for reactions catalyzed by cytochrome P-450. (author)

  14. 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.

  15. 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.

  16. Genome-Wide Analysis, Classification, Evolution, and Expression Analysis of the Cytochrome P450 93 Family in Land Plants

    OpenAIRE

    Du, Hai; Ran, Feng; Dong, Hong-Li; Wen, Jing; Li, Jia-Na; Liang, Zhe

    2016-01-01

    Cytochrome P450 93 family (CYP93) belonging to the cytochrome P450 superfamily plays important roles in diverse plant processes. However, no previous studies have investigated the evolution and expression of the members of this family. In this study, we performed comprehensive genome-wide analysis to identify CYP93 genes in 60 green plants. In all, 214 CYP93 proteins were identified; they were specifically found in flowering plants and could be classified into ten subfamilies?CYP93A?K, with t...

  17. Expression of xenobiotic metabolizing cytochrome P450 genes in a spinosad-resistant Musca domestica L. strain.

    Directory of Open Access Journals (Sweden)

    Dorte H Højland

    Full Text Available Spinosad is important in pest management strategies of multiple insect pests. However, spinosad resistance is emerging in various pest species. Resistance has in some species been associated with alterations of the target-site receptor, but in others P450s seems to be involved. We test the possible importance of nine cytochrome P450 genes in the spinosad-resistant housefly strain 791spin and investigate the influence of spinosad on P450 expression in four other housefly strains.Significant differences in P450 expression of the nine P450 genes in the four strains after spinosad treatment were identified in 40% of cases, most of these as induction. The highly expressed CYP4G2 was induced 6.6-fold in the insecticide susceptible WHO-SRS females, but decreased 2-fold in resistant 791spin males. CYP6G4 was constitutively higher expressed in the resistant strain compared to the susceptible strain. Furthermore, CYP6G4 gene expression was increased in susceptible WHO-SRS flies by spinosad while the expression level did not alter significantly in resistant fly strains. Expression of CYP6A1 and male CYP6D3 was constitutively higher in the resistant strain compared to the susceptible. However, in both cases male expression was higher than female expression.CYP4G2, CYP6A1, CYP6D3 and CYP6G4 have expressions patterns approaching the expectations of a hypothesized sex specific spinosad resistance gene. CYP4G2 fit requirements of a spinosad resistance gene best, making it the most likely candidate. The overall high expression level of CYP4G2 throughout the strains also indicates importance of this gene. However, the data on 791spin are not conclusive concerning spinosad resistance and small contributions from multiple P450s with different enzymatic capabilities could be speculated to do the job in 791spin. Differential expression of P450s between sexes is more a rule than an exception. Noteworthy differences between spinosad influenced expression of P450 genes

  18. Lack of evidence for metabolism of p-phenylenediamine by human hepatic cytochrome P450 enzymes

    International Nuclear Information System (INIS)

    Stanley, Lesley A.; Skare, Julie A.; Doyle, Edward; Powrie, Robert; D'Angelo, Diane; Elcombe, Clifford R.

    2005-01-01

    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 14 C-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

  19. Comparison of xenobiotic-metabolising human, porcine, rodent, and piscine cytochrome P450

    International Nuclear Information System (INIS)

    Burkina, Viktoriia; Rasmussen, Martin Krøyer; Pilipenko, Nadezhda; Zamaratskaia, Galia

    2017-01-01

    Highlights: • The percent identity of porcine, murine and piscine CYPs was compared with human CYPs. • Main similarities and differences were reviewed. • Understanding of molecular mechanisms of CYP system will provide further insights into the CYP regulatory processes, and responses to different factors. - Abstract: Cytochrome P450 proteins (CYP450s) are present in most domains of life and play a critical role in the metabolism of endogenous compounds and xenobiotics. The effects of exposure to xenobiotics depend heavily on the expression and activity of drug-metabolizing CYP450s, which is determined by species, genetic background, age, gender, diet, and exposure to environmental pollutants. Numerous reports have investigated the role of different vertebrate CYP450s in xenobiotic metabolism. Model organisms provide powerful experimental tools to investigate Phase I metabolism. The aim of the present review is to compare the existing data on human CYP450 proteins (1–3 families) with those found in pigs, mice, and fish. We will highlight differences and similarities and identify research gaps which need to be addressed in order to use these species as models that mimic human traits. Moreover, we will discuss the roles of nuclear receptors in the cellular regulation of CYP450 expression in select organisms.

  20. Degradation of Diuron by Phanerochaete chrysosporium: Role of Ligninolytic Enzymes and Cytochrome P450

    Directory of Open Access Journals (Sweden)

    Jaqueline da Silva Coelho-Moreira

    2013-01-01

    Full Text Available The white-rot fungus Phanerochaete chrysosporium was investigated for its capacity to degrade the herbicide diuron in liquid stationary cultures. The presence of diuron increased the production of lignin peroxidase in relation to control cultures but only barely affected the production of manganese peroxidase. The herbicide at the concentration of 7 μg/mL did not cause any reduction in the biomass production and it was almost completely removed after 10 days. Concomitantly with the removal of diuron, two metabolites, DCPMU [1-(3,4-dichlorophenyl-3-methylurea] and DCPU [(3,4-dichlorophenylurea], were detected in the culture medium at the concentrations of 0.74 μg/mL and 0.06 μg/mL, respectively. Crude extracellular ligninolytic enzymes were not efficient in the in vitro degradation of diuron. In addition, 1-aminobenzotriazole (ABT, a cytochrome P450 inhibitor, significantly inhibited both diuron degradation and metabolites production. Significant reduction in the toxicity evaluated by the Lactuca sativa L. bioassay was observed in the cultures after 10 days of cultivation. In conclusion, P. chrysosporium can efficiently metabolize diuron without the accumulation of toxic products.

  1. Degradation of diuron by Phanerochaete chrysosporium: role of ligninolytic enzymes and cytochrome P450.

    Science.gov (United States)

    Coelho-Moreira, Jaqueline da Silva; Bracht, Adelar; de Souza, Aline Cristine da Silva; Oliveira, Roselene Ferreira; de Sá-Nakanishi, Anacharis Babeto; de Souza, Cristina Giatti Marques; Peralta, Rosane Marina

    2013-01-01

    The white-rot fungus Phanerochaete chrysosporium was investigated for its capacity to degrade the herbicide diuron in liquid stationary cultures. The presence of diuron increased the production of lignin peroxidase in relation to control cultures but only barely affected the production of manganese peroxidase. The herbicide at the concentration of 7 μ g/mL did not cause any reduction in the biomass production and it was almost completely removed after 10 days. Concomitantly with the removal of diuron, two metabolites, DCPMU [1-(3,4-dichlorophenyl)-3-methylurea] and DCPU [(3,4-dichlorophenyl)urea], were detected in the culture medium at the concentrations of 0.74 μ g/mL and 0.06 μ g/mL, respectively. Crude extracellular ligninolytic enzymes were not efficient in the in vitro degradation of diuron. In addition, 1-aminobenzotriazole (ABT), a cytochrome P450 inhibitor, significantly inhibited both diuron degradation and metabolites production. Significant reduction in the toxicity evaluated by the Lactuca sativa L. bioassay was observed in the cultures after 10 days of cultivation. In conclusion, P. chrysosporium can efficiently metabolize diuron without the accumulation of toxic products.

  2. Endocrine disruptors induce cytochrome P450 by affecting transcriptional regulation via pregnane X receptor

    International Nuclear Information System (INIS)

    Mikamo, Eriko; Harada, Shingo; Nishikawa, Jun-ichi; Nishihara, Tsutomu

    2003-01-01

    Pregnane X receptor (PXR) is a nuclear receptor that regulates the expression of genes for cytochrome P450 3A (CYP3A), multidrug resistance 1 (MDR1), and organic anion-transporting peptide 2 (OATP2). These genes control the metabolism (CYP3A subfamily) and aspects of the pharmacokinetics (MDR1 and OATP2) of both endogenous and xenobiotic compounds. Since PXR is important in understanding the actions of endocrine disruptors (EDs), we determined the ability of suspected EDs to interact with PXR. In our study, 7 of 54 xenobiotics compounds interacted with PXR, including methoxychlor and benzophenone. All of the chemicals activated PXR in vitro and induced CYP3A mRNA in the male rat liver. In addition, CYP2C11 was also induced by some PXR agonists and converted methoxychlor into xenoestrogen. These findings suggest that some EDs affect sex hormone receptor indirectly by induction of metabolic enzyme via PXR, to produce rapidly higher concentrations of effective metabolites, leading to disturbance of the endocrine system

  3. Structural organization and classification of cytochrome P450 genes in flax (Linum usitatissimum L.).

    Science.gov (United States)

    Babu, Peram Ravindra; Rao, Khareedu Venkateswara; Reddy, Vudem Dashavantha

    2013-01-15

    Flax CYPome analysis resulted in the identification of 334 putative cytochrome P450 (CYP450) genes in the cultivated flax genome. Classification of flax CYP450 genes based on the sequence similarity with Arabidopsis orthologs and CYP450 nomenclature, revealed 10 clans representing 44 families and 98 subfamilies. CYP80, CYP83, CYP92, CYP702, CYP705, CYP708, CYP728, CYP729, CYP733 and CYP736 families are absent in the flax genome. The subfamily members exhibited conserved sequences, length of exons and phasing of introns. Similarity search of the genomic resources of wild flax species Linum bienne with CYP450 coding sequences of the cultivated flax, revealed the presence of 127 CYP450 gene orthologs, indicating amplification of novel CYP450 genes in the cultivated flax. Seven families CYP73, 74, 75, 76, 77, 84 and 709, coding for enzymes associated with phenylpropanoid/fatty acid metabolism, showed extensive gene amplification in the flax. About 59% of the flax CYP450 genes were present in the EST libraries. Copyright © 2012 Elsevier B.V. All rights reserved.

  4. Progesterone Metabolites Produced by Cytochrome P450 3A Modulate Uterine Contractility in a Murine Model

    Science.gov (United States)

    Patil, Avinash S.; Swamy, Geeta K.; Murtha, Amy P.; Heine, R. Phillips; Zheng, Xiaomei; Grotegut, Chad A.

    2015-01-01

    Objective: We seek to characterize the effect of progesterone metabolites on spontaneous and oxytocin-induced uterine contractility. Study Design: Spontaneous contractility was studied in mouse uterine horns after treatment with progesterone, 2α-hydroxyprogesterone, 6β-hydroxyprogesterone (6β-OHP), 16α-hydroxyprogesterone (16α-OHP), or 17-hydroxyprogesterone caproate (17-OHPC) at 10−9 to 10−6 mol/L. Uterine horns were exposed to progestins (10−6 mol/L), followed by increasing concentrations of oxytocin (1-100 nmol/L) to study oxytocin-induced contractility. Contraction parameters were compared for each progestin and matched vehicle control using repeated measures 2-way analysis of variance. In vitro metabolism of progesterone by recombinant cytochrome P450 3A (CYP3A) microsomes (3A5, 3A5, and 3A7) identified major metabolites. Results: Oxytocin-induced contractile frequency was decreased by 16α-OHP (P = .03) and increased by 6β-OHP (P = .05). Progesterone and 17-OHPC decreased oxytocin-induced contractile force (P = .02 and P = .04, respectively) and frequency (P = .02 and P = .03, respectively). Only progesterone decreased spontaneous contractile force (P = .02). Production of 16α-OHP and 6β-OHP metabolites were confirmed in all CYP3A isoforms tested. Conclusion: Progesterone metabolites produced by maternal or fetal CYP3A enzymes influence uterine contractility. PMID:26037300

  5. Studies on the toxic interaction between monensin and tiamulin in rats: effects on P450 activities.

    Science.gov (United States)

    Szücs, G; Laczay, P; Bajnógel, J; Móra, Z

    2000-01-01

    Studies were carried out to investigate the effects of monensin and tiamulin, and the simultaneous administration of both compounds on microsomal enzymes in rats. In Phase I of the experiments the effects of monensin and tiamulin were studied separately (monensin 10, 30, and 50 mg/kg or tiamulin 40, 120, and 200 mg/kg body weight, respectively), while in Phase II the two compounds were administered simultaneously (monesin 10 mg/kg and tiamulin 40 mg/kg b.w., respectively). When monensin was administered by itself, it exerted no significant effect on microsomal liver enzymes. In a few cases, slight inhibition of certain enzyme activities was seen. Tiamulin provoked a dose-dependent hepatic enzyme induction. The combined administration of monensin and tiamulin at low doses (10 and 40 mg/kg, respectively) resulted in marked elevation of P450-related enzyme activities. The enzyme induction was more pronounced in females than in males. The results suggest that the simultaneous administration of tiamulin may influence the biotransformation of monensin, possibly increasing the amount of reactive metabolite(s) of the ionophore antibiotic.

  6. Functional characterization of cytochrome P450-derived epoxyeicosatrienoic acids in adipogenesis and obesity.

    Science.gov (United States)

    Zha, Weibin; Edin, Matthew L; Vendrov, Kimberly C; Schuck, Robert N; Lih, Fred B; Jat, Jawahar Lal; Bradbury, J Alyce; DeGraff, Laura M; Hua, Kunjie; Tomer, Kenneth B; Falck, John R; Zeldin, Darryl C; Lee, Craig R

    2014-10-01

    Adipogenesis plays a critical role in the initiation and progression of obesity. Although cytochrome P450 (CYP)-derived epoxyeicosatrienoic acids (EETs) have emerged as a potential therapeutic target for cardiometabolic disease, the functional contribution of EETs to adipogenesis and the pathogenesis of obesity remain poorly understood. Our studies demonstrated that induction of adipogenesis in differentiated 3T3-L1 cells (in vitro) and obesity-associated adipose expansion in high-fat diet (HFD)-fed mice (in vivo) significantly dysregulate the CYP epoxygenase pathway and evoke a marked suppression of adipose-derived EET levels. Subsequent in vitro experiments demonstrated that exogenous EET analog administration elicits potent anti-adipogenic effects via inhibition of the early phase of adipogenesis. Furthermore, EET analog administration to mice significantly mitigated HFD-induced weight gain, adipose tissue expansion, pro-adipogenic gene expression, and glucose intolerance. Collectively, these findings suggest that suppression of EET bioavailability in adipose tissue is a key pathological consequence of obesity, and strategies that promote the protective effects of EETs in adipose tissue offer enormous therapeutic potential for obesity and its downstream pathological consequences. Copyright © 2014 by the American Society for Biochemistry and Molecular Biology, Inc.

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

    Science.gov (United States)

    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 environmentally relevant doses of TCE (8 ppb and 800 ppb) and RNA was extracted from cardiac and extra-cardiac tissue (whole embryo without heart). Real time PCR showed upregulation of CYP2H1 transcripts in response to TCE exposure in the heart. No detectable cytochrome expression was found in extra-cardiac tissue. As seen previously, the dose response was non-monotonic and 8ppb elicited stronger upregulation than 800 ppb. Immunostaining for CYP2C subfamily expression confirmed protein expression and showed localization in both myocardium and endothelium. TCE exposure increased protein expression in both tissues. These data demonstrate that the earliest embryonic expression of phase I detoxification enzymes is in the developing heart. Expression of these CYPs is likely to be relevant to the susceptibility of the developing heart to environmental teratogens. PMID:22855351

  8. A Conserved Cytochrome P450 Evolved in Seed Plants Regulates Flower Maturation.

    Science.gov (United States)

    Liu, Zhenhua; Boachon, Benoît; Lugan, Raphaël; Tavares, Raquel; Erhardt, Mathieu; Mutterer, Jérôme; Demais, Valérie; Pateyron, Stéphanie; Brunaud, Véronique; Ohnishi, Toshiyuki; Pencik, Ales; Achard, Patrick; Gong, Fan; Hedden, Peter; Werck-Reichhart, Danièle; Renault, Hugues

    2015-12-07

    Global inspection of plant genomes identifies genes maintained in low copies across taxa and under strong purifying selection, which are likely to have essential functions. Based on this rationale, we investigated the function of the low-duplicated CYP715 cytochrome P450 gene family that appeared early in seed plants and evolved under strong negative selection. Arabidopsis CYP715A1 showed a restricted tissue-specific expression in the tapetum of flower buds and in the anther filaments upon anthesis. cyp715a1 insertion lines showed a strong defect in petal development, and transient alteration of pollen intine deposition. Comparative expression analysis revealed the downregulated expression of genes involved in pollen development, cell wall biogenesis, hormone homeostasis, and floral sesquiterpene biosynthesis, especially TPS21 and several key genes regulating floral development such as MYB21, MYB24, and MYC2. Accordingly, floral sesquiterpene emission was suppressed in the cyp715a1 mutants. Flower hormone profiling, in addition, indicated a modification of gibberellin homeostasis and a strong disturbance of the turnover of jasmonic acid derivatives. Petal growth was partially restored by the active gibberellin GA3 or the functional analog of jasmonoyl-isoleucine, coronatine. CYP715 appears to function as a key regulator of flower maturation, synchronizing petal expansion and volatile emission. It is thus expected to be an important determinant of flower-insect interaction. Copyright © 2015 The Author. Published by Elsevier Inc. All rights reserved.

  9. Sex difference in the principal cytochrome P-450 for tributyltin metabolism in rats

    International Nuclear Information System (INIS)

    Ohhira, Shuji; Enomoto, Mitsunori; Matsui, Hisao

    2006-01-01

    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

  10. 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.

  11. 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.

  12. The human genome project and novel aspects of cytochrome P450 research

    International Nuclear Information System (INIS)

    Ingelman-Sundberg, Magnus

    2005-01-01

    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

  13. 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.

  14. Rice cytochrome P450 MAX1 homologs catalyze distinct steps in strigolactone biosynthesis

    KAUST Repository

    Zhang, Yanxia; van Dijk, Aalt D J; Scaffidi, Adrian; Flematti, Gavin R.; Hofmann, Manuel; Charnikhova, Tatsiana; Verstappen, Francel; Hepworth, Jo; van der Krol, Sander; Leyser, Ottoline; Smith, Steven M.; Zwanenburg, Binne; Al-Babili, Salim; Ruyter-Spira, Carolien; Bouwmeester, Harro J.

    2014-01-01

    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.

  15. Stereochemical inversion of (S)-reticuline by a cytochrome P450 fusion in opium poppy.

    Science.gov (United States)

    Farrow, Scott C; Hagel, Jillian M; Beaudoin, Guillaume A W; Burns, Darcy C; Facchini, Peter J

    2015-09-01

    The gateway to morphine biosynthesis in opium poppy (Papaver somniferum) is the stereochemical inversion of (S)-reticuline since the enzyme yielding the first committed intermediate salutaridine is specific for (R)-reticuline. A fusion between a cytochrome P450 (CYP) and an aldo-keto reductase (AKR) catalyzes the S-to-R epimerization of reticuline via 1,2-dehydroreticuline. The reticuline epimerase (REPI) fusion was detected in opium poppy and in Papaver bracteatum, which accumulates thebaine. In contrast, orthologs encoding independent CYP and AKR enzymes catalyzing the respective synthesis and reduction of 1,2-dehydroreticuline were isolated from Papaver rhoeas, which does not accumulate morphinan alkaloids. An ancestral relationship between these enzymes is supported by a conservation of introns in the gene fusions and independent orthologs. Suppression of REPI transcripts using virus-induced gene silencing in opium poppy reduced levels of (R)-reticuline and morphinan alkaloids and increased the overall abundance of (S)-reticuline and its O-methylated derivatives. Discovery of REPI completes the isolation of genes responsible for known steps of morphine biosynthesis.

  16. Comparative study of hop-containing products on human cytochrome p450-mediated metabolism.

    Science.gov (United States)

    Foster, Brian C; Kearns, Nikia; Arnason, John T; Saleem, Ammar; Ogrodowczyk, Carolina; Desjardins, Suzanne

    2009-06-10

    Thirty-five national and international brands of beer were examined for their potential to affect human cytochrome P450 (CYP)-mediated metabolism. They represented the two main categories of beer, ales and lagers, and included a number of specialty products including bitter (porter, stout), coffee, ice, wheat, Pilsner, and hemp seed. Aliquots were examined for nonvolatile soluble solids, effect on CYP metabolism and P-glycoprotein (Pgp) transport, and major alpha- and beta-hop acids. Wide variance was detected in contents of alcohol, nonvolatile suspended solids, and hop acids and in the potential to affect CYP-mediated metabolism and Pgp-mediated efflux transport. Many of the products affected CYP2C9-mediated metabolism, and only two (NRP 306 and 307) markedly affected CYP3A4; hence, some products have the capacity to affect drug safety. CYP3A4, CYP3A5, CYP3A7, and CYP19 (aromatase) inhibition to the log concentration of beta-acid content was significant with r(2) > 0.37, suggesting that these components can account for some of the variation in inhibition of CYP metabolism.

  17. 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.

  18. Direct hydride shift mechanism and stereoselectivity of P450nor confirmed by QM/MM calculations.

    Science.gov (United States)

    Krámos, Balázs; Menyhárd, Dóra K; Oláh, Julianna

    2012-01-19

    Nitric oxide reductase (P450(nor)) found in Fusarium oxysporum catalyzes the reduction of nitric oxide to N(2)O in a multistep process. The reducing agent, NADH, is bound in the distal pocket of the enzyme, and direct hydride transfer occurs from NADH to the nitric oxide bound heme enzyme, forming intermediate I. Here we studied the possibility of hydride transfer from NADH to both the nitrogen and oxygen of the heme-bound nitric oxide, using quantum chemical and combined quantum mechanics/molecular mechanics (QM/MM) calculations, on two different protein models, representing both possible stereochemistries, a syn- and an anti-NADH arrangement. All calculations clearly favor hydride transfer to the nitrogen of nitric oxide, and the QM-only barrier and kinetic isotope effects are good agreement with the experimental values of intermediate I formation. We obtained higher barriers in the QM/MM calculations for both pathways, but hydride transfer to the nitrogen of nitric oxide is still clearly favored. The barriers obtained for the syn, Pro-R conformation of NADH are lower and show significantly less variation than the barriers obtained in the case of anti conformation. The effect of basis set and wide range of functionals on the obtained results are also discussed.

  19. Enhanced metabolism of halogenated hydrocarbons in transgenic plants containing mammalian cytochrome P450 2E1

    Science.gov (United States)

    Lafferty Doty, Sharon; Shang, Tanya Q.; Wilson, Angela M.; Tangen, Jeff; Westergreen, Aram D.; Newman, Lee A.; Strand, Stuart E.; Gordon, Milton P.

    2000-06-01

    Chlorinated solvents, especially trichloroethylene (TCE), are the most widespread groundwater contaminants in the United States. Existing methods of pumping and treating are expensive and laborious. Phytoremediation, the use of plants for remediation of soil and groundwater pollution, is less expensive and has low maintenance; however, it requires large land areas and there are a limited number of suitable plants that are known to combine adaptation to a particular environment with efficient metabolism of the contaminant. In this work, we have engineered plants with a profound increase in metabolism of the most common contaminant, TCE, by introducing the mammalian cytochrome P450 2E1. This enzyme oxidizes a wide range of important pollutants, including TCE, ethylene dibromide, carbon tetrachloride, chloroform, and vinyl chloride. The transgenic plants had a dramatic enhancement in metabolism of TCE of up to 640-fold as compared with null vector control plants. The transgenic plants also showed an increased uptake and debromination of ethylene dibromide. Therefore, transgenic plants with this enzyme could be used for more efficient remediation of many sites contaminated with halogenated hydrocarbons.

  20. 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 simulations, the cavities are completely filled with water molecules, although with approximately 20% lower density than in bulk water. The 2A6 protein differs from the other three in that it has a very small cavity with only two water molecules and no exchange with the surroundings. The other three proteins...... channels, through which there is a quite frequent exchange of water molecules (one molecule is exchanged every 30-200 ps), except in 2A6. Most of the channels are observed also in the crystal structures, but two to three channels in each protein open only during the simulations. There are no water...

  1. 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.

  2. Cytotoxic 1-deoxysphingolipids are metabolized by a cytochrome P450-dependent pathway.

    Science.gov (United States)

    Alecu, Irina; Othman, Alaa; Penno, Anke; Saied, Essa M; Arenz, Christoph; von Eckardstein, Arnold; Hornemann, Thorsten

    2017-01-01

    The 1-deoxysphingolipids (1-deoxySLs) are atypical sphingolipids (SLs) that are formed when serine palmitoyltransferase condenses palmitoyl-CoA with alanine instead of serine during SL synthesis. The 1-deoxySLs are toxic to neurons and pancreatic β-cells. Pathologically elevated 1-deoxySLs cause the inherited neuropathy, hereditary sensory autonomic neuropathy type 1 (HSAN1), and are also found in T2D. Diabetic sensory polyneuropathy (DSN) and HSAN1 are clinically very similar, suggesting that 1-deoxySLs may be implicated in both pathologies. The 1-deoxySLs are considered to be dead-end metabolites, as they lack the C1-hydroxyl group, which is essential for the canonical degradation of SLs. Here, we report a previously unknown metabolic pathway, which is capable of degrading 1-deoxySLs. Using a variety of metabolic labeling approaches and high-resolution high-accuracy MS, we identified eight 1-deoxySL downstream metabolites, which appear to be formed by cytochrome P450 (CYP)4F enzymes. Comprehensive inhibition and induction of CYP4F enzymes blocked and stimulated, respectively, the formation of the downstream metabolites. Consequently, CYP4F enzymes might be novel therapeutic targets for the treatment of HSAN1 and DSN, as well as for the prevention of T2D. Copyright © 2017 by the American Society for Biochemistry and Molecular Biology, Inc.

  3. Induction of rabbit lung cytochrome P450 prostaglandin in omega-hyroxylase during pregnancy: evidence for regulation at the genetic level

    International Nuclear Information System (INIS)

    Master, B.S.S.; Muerhoff, A.S.; Jackson, V.; Williams, D.E.; Waterman, M.R.; Johnson, E.F.

    1986-01-01

    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 35 S-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

  4. Genetic polymorphisms of drug-metabolizing cytochrome P450 enzymes in cynomolgus and rhesus monkeys and common marmosets in preclinical studies for humans.

    Science.gov (United States)

    Uno, Yasuhiro; Uehara, Shotaro; Yamazaki, Hiroshi

    2017-12-23

    Cynomolgus monkeys (Macaca fascicularis, Old World Monkeys) and common marmosets (Callithrix jacchus, New World Monkeys) have been widely, and expectedly, used as non-human primate models in drug development studies. Major drug-metabolizing cytochrome P450 (P450) enzymes information is now available that supports these primate species as animal models, and it is established that multiple forms of cynomolgus monkey and common marmoset P450 enzymes have generally similar substrate recognition functionality to human P450 enzymes. This research update provides information on genetic polymorphisms of P450 enzymes in cynomolgus monkey and common marmoset like human P450 enzymes. Information on rhesus monkeys (Macaca mulatta), another macaque species used in drug metabolism studies, is also included for comparison. Among a variety of cynomolgus monkey P450 variants investigated, typical examples include individual pharmacokinetic data for efavirenz and R-warfarin associated with cynomolgus monkey P450 2C9 (formerly 2C43) and 2C19 (2C75) variants, respectively, and for R-omeprazole and S-warfarin associated with marmoset P450 2C19 variants. These findings provide a foundation for understanding the individual pharmacokinetic and toxicological results in non-human primates as preclinical models and will help to further support understanding of molecular mechanisms of human P450 function. In addition to these polymorphic P450 enzymes, effects of aging on some drug clearances mediated by cynomolgus monkey and common marmoset P450 enzymes were found in elder animals or animals pretreated with rifampicin. This review describes genetic and acquired individual differences in cynomolgus monkey and common marmoset P450 enzymes involved in drug oxidation associated with pharmacological and/or toxicological effects. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Overexpression of cerebral and hepatic cytochrome P450s alters behavioral activity of rat offspring following prenatal exposure to lindane

    Energy Technology Data Exchange (ETDEWEB)

    Johri, Ashu; Yadav, Sanjay; Dhawan, Alok [Developmental Toxicology Division, Industrial Toxicology Research Centre, P. O. Box 80, M. G. Marg, Lucknow-226 001, U. P. (India); Parmar, Devendra [Developmental Toxicology Division, Industrial Toxicology Research Centre, P. O. Box 80, M. G. Marg, Lucknow-226 001, U. P. (India)

    2007-12-15

    Oral administration of different doses (0.0625, 0.125 or 0.25 mg/kg corresponding to 1/1400th, 1/700th or 1/350th of LD{sub 50}) of lindane to the pregnant Wistar rats from gestation days 5 to 21 were found to produce a dose-dependent increase in the activity of cytochrome P450 (CYP)-dependent 7-ethoxyresorufin-O-deethylase (EROD), 7-pentoxyresorufin-O-dealkylase (PROD) and N-nitrosodimethylamine demethylase (NDMA-d) in brain and liver of offspring postnatally at 3 weeks. The increase in the activity of CYP monooxygenases was found to be associated with the increase in the mRNA and protein expression of xenobiotic metabolizing CYP1A, 2B and 2E1 isoenzymes in the brain and liver of offspring. Dose-dependent alterations in the parameters of spontaneous locomotor activity in the offspring postnatally at 3 weeks have suggested that increase in CYP activity may possibly lead to the formation of metabolites to the levels that may be sufficient to alter the behavioral activity of the offspring. Interestingly, the inductive effect on cerebral and hepatic CYPs was found to persist postnatally up to 6 weeks in the offspring at the relatively higher doses (0.125 and 0.25 mg/kg) of lindane and up to 9 weeks at the highest dose (0.25 mg/kg), though the magnitude of induction was less than that observed at 3 weeks. Alterations in the parameters of spontaneous locomotor activity in the offspring postnatally at 6 and 9 weeks, though significant only in the offspring at 3 and 6-week of age, have further indicated that due to the reduced activity of the CYPs during the ontogeny, lindane and its metabolites may not be effectively cleared from the brain. The data suggest that low dose prenatal exposure to the pesticide has the potential to produce overexpression of xenobiotic metabolizing CYPs in brain and liver of the offspring which may account for the behavioral changes observed in the offspring.

  6. Overexpression of cerebral and hepatic cytochrome P450s alters behavioral activity of rat offspring following prenatal exposure to lindane

    International Nuclear Information System (INIS)

    Johri, Ashu; Yadav, Sanjay; Dhawan, Alok; Parmar, Devendra

    2007-01-01

    Oral administration of different doses (0.0625, 0.125 or 0.25 mg/kg corresponding to 1/1400th, 1/700th or 1/350th of LD 50 ) of lindane to the pregnant Wistar rats from gestation days 5 to 21 were found to produce a dose-dependent increase in the activity of cytochrome P450 (CYP)-dependent 7-ethoxyresorufin-O-deethylase (EROD), 7-pentoxyresorufin-O-dealkylase (PROD) and N-nitrosodimethylamine demethylase (NDMA-d) in brain and liver of offspring postnatally at 3 weeks. The increase in the activity of CYP monooxygenases was found to be associated with the increase in the mRNA and protein expression of xenobiotic metabolizing CYP1A, 2B and 2E1 isoenzymes in the brain and liver of offspring. Dose-dependent alterations in the parameters of spontaneous locomotor activity in the offspring postnatally at 3 weeks have suggested that increase in CYP activity may possibly lead to the formation of metabolites to the levels that may be sufficient to alter the behavioral activity of the offspring. Interestingly, the inductive effect on cerebral and hepatic CYPs was found to persist postnatally up to 6 weeks in the offspring at the relatively higher doses (0.125 and 0.25 mg/kg) of lindane and up to 9 weeks at the highest dose (0.25 mg/kg), though the magnitude of induction was less than that observed at 3 weeks. Alterations in the parameters of spontaneous locomotor activity in the offspring postnatally at 6 and 9 weeks, though significant only in the offspring at 3 and 6-week of age, have further indicated that due to the reduced activity of the CYPs during the ontogeny, lindane and its metabolites may not be effectively cleared from the brain. The data suggest that low dose prenatal exposure to the pesticide has the potential to produce overexpression of xenobiotic metabolizing CYPs in brain and liver of the offspring which may account for the behavioral changes observed in the offspring

  7. Pharmacophore modeling and in silico / in vitro screening for human cytochrome P450 11B1 & cytochrome P450 11B2 inhibitors

    Science.gov (United States)

    Akram, Muhammad; Waratchareeyakul, Watcharee; Haupenthal, Joerg; Hartmann, Rolf W.; Schuster, Daniela

    2017-12-01

    Cortisol synthase (CYP11B1) is the main enzyme for the endogenous synthesis of cortisol and its inhibition is a potential way for the treatment of diseases associated with increased cortisol levels, such as Cushing’s syndrome, metabolic diseases, and delayed wound healing. Aldosterone synthase (CYP11B2) is the key enzyme for aldosterone biosynthesis and its inhibition is a promising approach for the treatment of congestive heart failure, cardiac fibrosis, and certain forms of hypertension. Both CYP11B1 and CYP11B2 are structurally very similar and expressed in the adrenal cortex. To facilitate the identification of novel inhibitors of these enzymes, ligand-based pharmacophore models of CYP11B1 and CYP11B2 inhibition were developed. A virtual screening of the SPECS database was performed with our pharmacophore queries. Biological evaluation of the selected hits lead to the discovery of three potent novel inhibitors of both CYP11B1 and CYP11B2 in the submicromolar range (compounds 8-10), one selective CYP11B1 inhibitor (Compound 11, IC50 = 2.5 µM), and one selective CYP11B2 inhibitor (compound 12, IC50 = 1.1 µM), respectively. The overall success rate of this prospective virtual screening experiment is 20.8% indicating good predictive power of the pharmacophore models.

  8. Pharmacophore Modeling and in Silico/in Vitro Screening for Human Cytochrome P450 11B1 and Cytochrome P450 11B2 Inhibitors.

    Science.gov (United States)

    Akram, Muhammad; Waratchareeyakul, Watcharee; Haupenthal, Joerg; Hartmann, Rolf W; Schuster, Daniela

    2017-01-01

    Cortisol synthase (CYP11B1) is the main enzyme for the endogenous synthesis of cortisol and its inhibition is a potential way for the treatment of diseases associated with increased cortisol levels, such as Cushing's syndrome, metabolic diseases, and delayed wound healing. Aldosterone synthase (CYP11B2) is the key enzyme for aldosterone biosynthesis and its inhibition is a promising approach for the treatment of congestive heart failure, cardiac fibrosis, and certain forms of hypertension. Both CYP11B1 and CYP11B2 are structurally very similar and expressed in the adrenal cortex. To facilitate the identification of novel inhibitors of these enzymes, ligand-based pharmacophore models of CYP11B1 and CYP11B2 inhibition were developed. A virtual screening of the SPECS database was performed with our pharmacophore queries. Biological evaluation of the selected hits lead to the discovery of three potent novel inhibitors of both CYP11B1 and CYP11B2 in the submicromolar range (compounds 8 - 10 ), one selective CYP11B1 inhibitor (Compound 11 , IC 50 = 2.5 μM), and one selective CYP11B2 inhibitor (compound 12 , IC 50 = 1.1 μM), respectively. The overall success rate of this prospective virtual screening experiment is 20.8% indicating good predictive power of the pharmacophore models.

  9. Pharmacophore Modeling and in Silico/in Vitro Screening for Human Cytochrome P450 11B1 and Cytochrome P450 11B2 Inhibitors

    Directory of Open Access Journals (Sweden)

    Muhammad Akram

    2017-12-01

    Full Text Available Cortisol synthase (CYP11B1 is the main enzyme for the endogenous synthesis of cortisol and its inhibition is a potential way for the treatment of diseases associated with increased cortisol levels, such as Cushing's syndrome, metabolic diseases, and delayed wound healing. Aldosterone synthase (CYP11B2 is the key enzyme for aldosterone biosynthesis and its inhibition is a promising approach for the treatment of congestive heart failure, cardiac fibrosis, and certain forms of hypertension. Both CYP11B1 and CYP11B2 are structurally very similar and expressed in the adrenal cortex. To facilitate the identification of novel inhibitors of these enzymes, ligand-based pharmacophore models of CYP11B1 and CYP11B2 inhibition were developed. A virtual screening of the SPECS database was performed with our pharmacophore queries. Biological evaluation of the selected hits lead to the discovery of three potent novel inhibitors of both CYP11B1 and CYP11B2 in the submicromolar range (compounds 8–10, one selective CYP11B1 inhibitor (Compound 11, IC50 = 2.5 μM, and one selective CYP11B2 inhibitor (compound 12, IC50 = 1.1 μM, respectively. The overall success rate of this prospective virtual screening experiment is 20.8% indicating good predictive power of the pharmacophore models.

  10. Hydrogen atom abstraction of 3,5-disubstituted analogues of paracetamol by horseradish peroxidase and cytochrome P450

    NARCIS (Netherlands)

    Bessems, J.G.M.; Groot, M.J. de; Baede, E.J.; Koppele, J.M. te; Vermeulen, N.P.E.

    1998-01-01

    1. The formation of free radicals during enzyme catalysed oxidation of eight 3,5-disubstituted analogues of paracetamol (PAR) has been studied. A simple peroxidase system as well as cytochrome P450-containing systems were used. Radicals were detected by electron spin resonance (ESR) on incubation of

  11. Drug metabolism by cytochrome p450 enzymes: what distinguishes the pathways leading to substrate hydroxylation over desaturation?

    Science.gov (United States)

    Ji, Li; Faponle, Abayomi S; Quesne, Matthew G; Sainna, Mala A; Zhang, Jing; Franke, Alicja; Kumar, Devesh; van Eldik, Rudi; Liu, Weiping; de Visser, Sam P

    2015-06-15

    Cytochrome P450 enzymes are highly versatile biological catalysts in our body that react with a broad range of substrates. Key functions in the liver include the metabolism of drugs and xenobiotics. One particular metabolic pathway that is poorly understood relates to the P450 activation of aliphatic groups leading to either hydroxylation or desaturation pathways. A DFT and QM/MM study has been carried out on the factors that determine the regioselectivity of aliphatic hydroxylation over desaturation of compounds by P450 isozymes. The calculations establish multistate reactivity patterns, whereby the product distributions differ on each of the spin-state surfaces; hence spin-selective product formation was found. The electronic and thermochemical factors that determine the bifurcation pathways were analysed and a model that predicts the regioselectivity of aliphatic hydroxylation over desaturation pathways was established from valence bond and molecular orbital theories. Thus, the difference in energy of the OH versus the OC bond formed and the π-conjugation energy determines the degree of desaturation products. In addition, environmental effects of the substrate binding pocket that affect the regioselectivities were identified. These studies imply that bioengineering P450 isozymes for desaturation reactions will have to include modifications in the substrate binding pocket to restrict the hydroxylation rebound reaction. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Metabolic stereoselectivity of cytochrome P450 3A4 towards deoxypodophyllotoxin : In silico predictions and experimental validation

    NARCIS (Netherlands)

    Julsing, Mattijs K.; Vasilev, Nikolay P.; Schneidman-Duhovny, Dina; Muntendarn, Remco; Woerdenbag, Herman J.; Quax, Wim J.; Wolfson, Haim J.; Ionkova, Iliana; Kayser, Oliver

    Deoxypodophyllotoxin is stereoselectively converted into epipodophyllotoxin by recombinant human cytochrome P450 3A4 (CY-P3A4). Further kinetic analysis revealed that the Michaelis-Menten K(m) and V(max) for hydroxylation of deoxypodophyllotoxin by CYP3A4 at C7 position were 1.93 mu M and 1.48

  13. 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

    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...

  14. Purification, Reconstitution, and Inhibition of Cytochrome P-450 Sterol Δ22-Desaturase from the Pathogenic Fungus Candida glabrata

    Science.gov (United States)

    Lamb, David C.; Maspahy, Segula; Kelly, Diane E.; Manning, Nigel J.; Geber, Antonia; Bennett, John E.; Kelly, Steven L.

    1999-01-01

    Sterol Δ22-desaturase has been purified from a strain of Candida glabrata with a disruption in the gene encoding sterol 14α-demethylase (cytochrome P-45051; CYP51). The purified cytochrome P-450 exhibited sterol Δ22-desaturase activity in a reconstituted system with NADPH–cytochrome P-450 reductase in dilaurylphosphatidylcholine, with the enzyme kinetic studies revealing a Km for ergosta-5,7-dienol of 12.5 μM and a Vmax of 0.59 nmol of this substrate metabolized/min/nmol of P-450. This enzyme is encoded by CYP61 (ERG5) in Saccharomyces cerevisiae, and homologues have been shown in the Candida albicans and Schizosaccharomyces pombe genome projects. Ketoconazole, itraconazole, and fluconazole formed low-spin complexes with the ferric cytochrome and exhibited type II spectra, which are indicative of an interaction between the azole moiety and the cytochrome heme. The azole antifungal compounds inhibited reconstituted sterol Δ22-desaturase activity by binding to the cytochrome with a one-to-one stoichiometry, with total inhibition of enzyme activity occurring when equimolar amounts of azole and cytochrome P-450 were added. These results reveal the potential for sterol Δ22-desaturase to be an antifungal target and to contribute to the binding of drugs within the fungal cell. PMID:10390230

  15. 4-Alkyl radical extrusion in the cytochrome P-450-catalyzed oxidation of 4-alkyl-1,4-dihydropyridines

    International Nuclear Information System (INIS)

    Lee, J.S.; Jacobsen, N.E.; Ortiz de Montellano, P.R.

    1988-01-01

    Rat liver microsomal cytochrome P-450 oxidizes the 4-methyl, 4-ethyl (DDEP), and 4-isopropyl derivatives of 3,5-bis(carbethoxy)-2,6-dimethyl-1,4,-dihydropyridine to mixtures of the corresponding 4-alkyl and 4-dealkyl pyridines. A fraction of the total microsomal enzyme is destroyed in the process. The 4-dealkyl to 4-alkyl pyridine metabolite ratio, the extent of cytochrome P-450 destruction, and the rate of spin-trapped radical accumulation are correlated in a linear inverse manner with the homolytic or heterolytic bond energies of the 4-alkyl groups of the 4-alkyl-1,4-dihydropyridines. No isotope effects are observed on the pyridine matabolite ratio, the destruction of cytochrome P-450, or the formation of ethyl radicals when [4- 2 H]DDEP is used instead of DDEP. N-Methyl- and N-ethyl-DDEP undergo N-dealkylation rather than aromatization but N-phenyl-DDEP is oxidized to a mixture of the 4-ethyl and 4-deethyl N-phenylpyridinium metabolites. In contrast to the absence of an isotope effect in the oxidation of DDEP, the 4-deethyl to 4-ethyl N-phenylpyridinium metabolite ratio increases 6-fold when N-phenyl[4- 2 H]DDEP is used. The results support the hypothesis that cytochrome P-450 catalyzes the oxidation of dihydropyridines to radical cations and show that the radical cations decay to nonradical products by multiple, substituent-dependent, mechanisms

  16. ISOLATION OF THE CANDIDA TROPICALIS GENE FOR P450 LANOSTEROL DEMETHYLASE AND ITS EXPRESSION IN SACCAROMYCES CEREVISIAE

    Science.gov (United States)

    We have isolated the gene for cytochrome P450 lanosterol 14-demethylase (14DM) from the yeast Candida tropicalis. This was accomplished by screening genomic libraries of strain ATCC750 in E. coli using a DNA fragment containing the yeast Saccharomyces cerevisiae 14DM gene. Identi...

  17. Modelling of three-dimensional structures of cytochromes P450 11B1 and 11B2.

    Science.gov (United States)

    Belkina, N V; Lisurek, M; Ivanov, A S; Bernhardt, R

    2001-12-15

    The final steps of the biosynthesis of glucocorticoids and mineralocorticoids in the adrenal cortex require the action of two different cytochromes P450--CYP11B1 and CYP11B2. Homology modelling of the three-dimensional structures of these cytochromes was performed based on crystallographic coordinates of two bacterial P450s, CYP102 (P450BM-3) and CYP108 (P450terp). Principal attention was given to the modelling of the active sites and a comparison of the active site structures of CYP11B1 and CYP11B2 was performed. It can be demonstrated that key residue contacts within the active site appear to depend on the orientation of the heme. The obtained 3D structures of CYP11B1 and CYP11B2 were used for investigation of structure-function relationships of these enzymes. Previously obtained results on naturally occurring mutants and on mutants obtained by site-directed mutagenesis are discussed.

  18. Comprehensive and Automated Linear Interaction Energy Based Binding-Affinity Prediction for Multifarious Cytochrome P450 Aromatase Inhibitors

    NARCIS (Netherlands)

    van Dijk, Marc; Ter Laak, Antonius M; Wichard, Jörg D; Capoferri, Luigi; Vermeulen, Nico P E; Geerke, Daan P

    2017-01-01

    Cytochrome P450 aromatase (CYP19A1) plays a key role in the development of estrogen dependent breast cancer, and aromatase inhibitors have been at the front line of treatment for the past three decades. The development of potent, selective and safer inhibitors is ongoing with in silico screening

  19. Selective inhibition by chloramphenicol of pregnenolone-16 α-carbonitrile-inducible rat liver cytochrome P-450 isozymes

    International Nuclear Information System (INIS)

    Graves, P.E.; Kaminsky, L.S.; Halpert, J.

    1986-01-01

    Pregnenolone-16 α-carbonitrile (PCN) has been shown to induce, in male rats, cytochrome P-450 isozymes responsible for the formation of R-10-hydroxywarfarin and R-dehydrowarfarin. Antibodies to the major PCN-inducible isozyme (PB/PCN-E) inhibit both activities in microsomal preparations. Recently the authors have shown that PCN treatment of female rats also induces the formation of both R-warfarin metabolites. However, in both sexes chloramphenicol (CAP) treatment selectively inhibits only the rate of formation of the R-dehydrowarfarin. A decrease in microsomal P-450 content occurs after in vivo administration of CAP to PCN-treated rats of both sexes. This is in contrast to the lack of effect of CAP on P-450 levels in phenobarbital-treated rats. Covalent binding of 14 C-CAP to microsomal protein in vitro was increased 3 to 4-fold following PCN treatment. Chromatographic evidences suggests the presence of at least two PCN-induced isozymes of similar molecular weights in both male and female rat liver microsomes. These data are consistent with the multiplicity of PCN-inducible P-450 in rat liver

  20. 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

    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...

  1. The participation of human hepatic P450 isoforms, flavin-containing monooxygenases and aldehyde oxidase in the biotransformation of the insecticide fenthion

    International Nuclear Information System (INIS)

    Leoni, Claudia; Buratti, Franca M.; Testai, Emanuela

    2008-01-01

    Although fenthion (FEN) is widely used as a broad spectrum insecticide on various crops in many countries, very scant data are available on its biotransformation in humans. In this study the in vitro human hepatic FEN biotransformation was characterized, identifying the relative contributions of cytochrome P450 (CYPs) and/or flavin-containing monooxygenase (FMOs) by using single c-DNA expressed human enzymes, human liver microsomes and cytosol and CYP/FMO-specific inhibitors. Two major metabolites, FEN-sulfoxide and FEN-oxon (FOX), are formed by some CYPs although at very different levels, depending on the relative CYP hepatic content. Formation of further oxidation products and the reduction of FEN-sulfoxide back to FEN by the cytosolic aldehyde oxidase enzyme were ruled out. Comparing intrinsic clearance values, FOX formation seemed to be favored and at low FEN concentrations CYP2B6 and 1A2 are mainly involved in its formation. At higher levels, a more widespread CYP involvement was evident, as in the case of FEN-sulfoxide, although a higher efficiency of CYP2C family was suggested. Hepatic FMOs were able to catalyze only sulfoxide formation, but at low FEN concentrations hepatic FEN sulfoxidation is predominantly P450-driven. Indeed, the contribution of the hepatic isoforms FMO 3 and FMO 5 was generally negligible, although at high FEN concentrations FMO's showed activities comparable to the active CYPs, accounting for up to 30% of total sulfoxidation. Recombinant FMO 1 showed the highest efficiency with respect to CYPs and the other FMOs, but it is not expressed in the adult human liver. This suggests that FMO 1 -catalysed sulfoxidation may represent the major extra-hepatic pathway of FEN biotransformation

  2. Blarina brevicauda as a biological monitor of polychlorinated biphenyls: evaluation of hepatic cytochrome P450 induction.

    Science.gov (United States)

    Russell, Julie S; Halbrook, Richard S; Woolf, Alan; French, John B; Melancon, Mark J

    2004-08-01

    We assessed the value of short-tailed shrews (Blarina brevicauda) as a possible biomonitor for polychlorinated biphenyl pollution through measurement of the induction of hepatic cytochrome P450 and associated enzyme activities. First, we checked the inducibility of four monooxygenases (benzyloxyresorufin-O-dealkylase [BROD], ethoxyresorufin-O-dealkylase [EROD], methoxyresorufin-O-dealkylase [MROD], and pentoxyresorufin-O-dealkylase [PROD]) by measuring the activity of these enzymes in hepatic microsomes prepared from shrews injected with beta-naphthoflavone (betaNF) or phenobarbital (PB), typical inducers of cytochrome P4501A (CYP1A) and CYP2B enzyme families, respectively. Enzyme activity was induced in shrews that received betaNF but not in shrews that received PB; PROD was not induced by either exposure. Later, shrews were exposed to a mixture of polychlorinated biphenyls (PCBs) (Aroclor 1242:1254, in 1:2 ratio) at 0.6, 9.6, and 150 ppm in food, for 31 d. Induction in these shrews was measured by specific enzyme activity (BROD, EROD, and MROD) in hepatic microsomes, by western blotting of solubilized microsomes against antibodies to CYP1A or CYP2B, and by duration of sodium pentobarbital-induced sleep. These three CYP enzymes were induced in shrews by PCBs at similar levels of exposure as in cotton rat (Sigmodon hispidus). Neither sleep time nor the amount of CYP2B family protein were affected by PCB exposure. Blarina brevicauda can be a useful biomonitor of PCBs that induce CYP1A, especially in habitats where they are the abundant small mammal.

  3. 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.

  4. The Role of Drug Metabolites in the Inhibition of Cytochrome P450 Enzymes.

    Science.gov (United States)

    Mikov, Momir; Đanić, Maja; Pavlović, Nebojša; Stanimirov, Bojan; Goločorbin-Kon, Svetlana; Stankov, Karmen; Al-Salami, Hani

    2017-12-01

    Following the drug administration, patients are exposed not only to the parent drug itself, but also to the metabolites generated by drug-metabolizing enzymes. The role of drug metabolites in cytochrome P450 (CYP) inhibition and subsequent drug-drug interactions (DDIs) have recently become a topic of considerable interest and scientific debate. The list of metabolites that were found to significantly contribute to clinically relevant DDIs is constantly being expanded and reported in the literature. New strategies have been developed for better understanding how different metabolites of a drug candidate contribute to its pharmacokinetic properties and pharmacological as well as its toxicological effects. However, the testing of the role of metabolites in CYP inhibition is still not routinely performed during the process of drug development, although the evaluation of time-dependent CYP inhibition during the clinical candidate selection process may provide information on possible effects of metabolites in CYP inhibition. Due to large number of compounds to be tested in the early stages of drug discovery, the experimental approaches for assessment of CYP-mediated metabolic profiles are particularly resource demanding. Consequently, a large number of in silico or computational tools have been developed as useful complement to experimental approaches. In summary, circulating metabolites may be recognized as significant CYP inhibitors. Current data may suggest the need for an optimized effort to characterize the inhibitory potential of parent drugs metabolites on CYP, as well as the necessity to develop the advanced in vitro models that would allow a better quantitative predictive value of in vivo studies.

  5. 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

  6. Pyridine induction of cytochrome P450IIE1: Evidence for enhanced protein synthesis

    International Nuclear Information System (INIS)

    Kim, S.G.; Novak, R.F.

    1990-01-01

    The dose-, and time-dependent induction of P450IIE1 in the rat by pyridine (PY) has been characterized. A single injection of PY (100 mg/kg, i.p.) increased as the levels of IIE1 2-, 3- and 4-fold at 6, 10 and 24 hr, respectively, relative to controls based on p-nitrophenol hydroxylase activity and Western blot analysis. Induction of IIE1 was dose-dependent over the range 10 to 200 mg/kg. Cycloheximide administration completely prevented the induction of IIE1 by PY, while actinomycin D failed to affect PY induction of IIE1. The rate of IIE1 synthesis was examined by labelling of proteins with [ 14 C] leucine in vivo, followed by SDS-PAGE and autoradiographic analysis of isolated microsomes. Enhanced intensity of the IIE1 band was observed in microsomes isolated from rats treated with either PY or acetone relative to untreated rats. Slot and Northern blot analyses were employed to assess IIE1 mRNA levels in total RNA and poly(A + ) mRNA isolated from livers of rats at 1, 5 and 12 hr following a single dose of PY. No increase in IIE1 mRNA in total RNA was monitored. A time-dependent decrease in IIE1 poly(A + ) mRNA however, was observed with the maximal decrease occurring at ∼12 hr. These results suggest that induction of IIE1 by PY does not involve transcriptional activation but occurs by protein synthesis possibly through increased translational efficiency

  7. Developing and Evaluating the HRM Technique for Identifying Cytochrome P450 2D6 Polymorphisms.

    Science.gov (United States)

    Lu, Hsiu-Chin; Chang, Ya-Sian; Chang, Chun-Chi; Lin, Ching-Hsiung; Chang, Jan-Gowth

    2015-05-01

    Cytochrome P450 2D6 is one of the important enzymes involved in the metabolism of many widely used drugs. Genetic polymorphisms of CYP2D6 can affect its activity. Therefore, an efficient method for identifying CYP2D6 polymorphisms is clinically important. We developed a high-resolution melting (HRM) analysis to investigate CYP2D6 polymorphisms. Genomic DNA was extracted from peripheral blood samples from 71 healthy individuals. All nine exons of the CYP2D6 gene were sequenced before screening by HRM analysis. This method can detect the most genotypes (*1, *2, *4, *10, *14, *21 *39, and *41) of CYP2D6 in Chinese. All samples were successfully genotyped. The four most common mutant CYP2D6 alleles (*1, *2, *10, and *41) can be genotyped. The single nucleotides polymorphism (SNP) frequencies of 100C > T (rs1065852), 1039C > T (rs1081003), 1661G > C (rs1058164), 2663G > A (rs28371722), 2850C > T (rs16947), 2988G > A (rs28371725), 3181A > G, and 4180G > C (rs1135840) were 58%, 61%, 73%, 1%, 13%, 3%, 1%, 73%, respectively. We identified 100% of all heterozygotes without any errors. The two homozygous genotypes (1661G > C and 4180G > C) can be distinguished by mixing with a known genotype sample to generate an artificial heterozygote for HRM analysis. Therefore, all samples could be identified using our HRM method, and the results of HRM analysis are identical to those obtained by sequencing. Our method achieved 100% sensitivity, specificity, positive prediction value and negative prediction value. HRM analysis is a nongel resolution method that is faster and less expensive than direct sequencing. Our study shows that it is an efficient tool for typing CYP2D6 polymorphisms. © 2014 Wiley Periodicals, Inc.

  8. Cytochrome P450 2E1 polymorphism and nasopharyngeal carcinoma development in Thailand: a correlative study

    International Nuclear Information System (INIS)

    Kongruttanachok, Narisorn; Sukdikul, Sairoong; Setavarin, Surachai; Kerekhjanarong, Verachai; Supiyaphun, Pakpoom; Voravud, Narin; Poovorawan, Yong; Mutirangura, Apiwat

    2001-01-01

    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