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.

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

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.

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

Modelling of three-dimensional structures of cytochromes P450 11B1 and 11B2.

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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.

In Situ Proteolysis for Crystallization of Membrane Bound Cytochrome P450 17A1 and 17A2 Proteins from Zebrafish.

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Lei, Li; Egli, Martin

2016-04-01

Fish and human cytochrome P450 (P450) 17A1 catalyze both steroid 17α-hydroxylation and 17α,20-lyase reactions. Fish P450 17A2 catalyzes only 17α-hydroxylation. Both enzymes are microsomal-type P450s, integral membrane proteins that bind to the membrane through their N-terminal hydrophobic segment, the signal anchor sequence. The presence of this N-terminal region renders expression of full-length proteins challenging or impossible. For some proteins, variable truncation of the signal anchor sequence precludes expression or results in poor expression levels. To crystallize P450 17A1 and 17A2 in order to gain insight into their different activities, we used an alternative N-terminal sequence to boost expression together with in situ proteolysis. Key features of our approach to identify crystallizable P450 fragments were the use of an N-terminal leader sequence, a screen composed of 12 proteases to establish optimal cleavage, variations of protease concentration in combination with an SDS-PAGE assay, and analysis of the resulting fragments using Edman sequencing. Described in this unit are protocols for vector preparation, expression, purification, and in situ proteolytic crystallization of two membrane-bound P450 proteins. Copyright © 2016 John Wiley & Sons, Inc.

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

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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.

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...

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

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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.

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

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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

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

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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.

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.

Inter- and intramolecular deuterium isotope effects on the cytochrome P-450-catalyzed oxidative dehalogenation of 1,1,2,2-tetrachloroethane

International Nuclear Information System (INIS)

Hales, D.B.; Ho, B.; Thompson, J.A.

1987-01-01

The oxidation of 1,1,2,2-tetrachloroethane to dichloroacetic acid was investigated with rat liver microsomes and purified cytochrome P-450. Deuterium substitution had no effect on Km values, but both the inter- and intramolecular isotope effects (kH/kD) on Vmax were in the range 5.7-6.1. The equivalence of the inter- and intramolecular values indicates that 6.0 may be a good estimate of the intrinsic isotope effect. The intermolecular kH/kD value for the conversion of 1,1,2,2-trichloroethane and its 1- 2 H analog to chloroacetic acid was 5.5. These data, and the finding that 1 atom of 18 O was incorporated into the product when TCEA was oxidized in an 18 O 2 atmosphere, support an oxidative dechlorination mechanism that involves hydrogen atom abstraction by the P-450 intermediate oxo complex

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

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

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

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

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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.

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

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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.

{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.

NAD(P)H-dependent quinone oxidoreductase 1 (NQO1) and cytochrome P450 oxidoreductase (CYP450OR) differentially regulate menadione-mediated alterations in redox status, survival and metabolism in pancreatic β-cells.

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Gray, Joshua P; Karandrea, Shpetim; Burgos, Delaine Zayasbazan; Jaiswal, Anil A; Heart, Emma A

2016-11-16

NQO1 (NAD(P)H-quinone oxidoreductase 1) reduces quinones and xenobiotics to less-reactive compounds via 2-electron reduction, one feature responsible for the role of NQO1 in antioxidant defense in several tissues. In contrast, NADPH cytochrome P450 oxidoreductase (CYP450OR), catalyzes the 1-electron reduction of quinones and xenobiotics, resulting in enhanced superoxide formation. However, to date, the roles of NQO1 and CYP450OR in pancreatic β-cell metabolism under basal conditions and oxidant challenge have not been characterized. Using NQO1 inhibition, over-expression and knock out, we have demonstrated that, in addition to protection of β-cells from toxic concentrations of the redox cycling quinone menadione, NQO1 also regulates the basal level of reduced-to-oxidized nucleotides, suggesting other role(s) beside that of an antioxidant enzyme. In contrast, over-expression of NADPH cytochrome P450 oxidoreductase (CYP450OR) resulted in enhanced redox cycling activity and decreased cellular viability, consistent with the enhanced generation of superoxide and H 2 O 2 . Basal expression of NQO1 and CYP450OR was comparable in isolated islets and liver. However, NQO1, but not CYP450OR, was strongly induced in β-cells exposed to menadione. NQO1 and CYP450OR exhibited a reciprocal preference for reducing equivalents in β-cells: while CYP450OR preferentially utilized NADPH, NQO1 primarily utilized NADH. Together, these results demonstrate that NQO1 and CYP450OR reciprocally regulate oxidant metabolism in pancreatic β-cells. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

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.

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.

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.

Benzylic oxidation of gemfibrozil-1-O-beta-glucuronide by P450 2C8 leads to heme alkylation and irreversible inhibition.

Science.gov (United States)

Baer, Brian R; DeLisle, Robert Kirk; Allen, Andrew

2009-07-01

Gemfibrozil-1-O-beta-glucuronide (GEM-1-O-gluc), a major metabolite of the antihyperlipidemic drug gemfibrozil, is a mechanism-based inhibitor of P450 2C8 in vitro, and this irreversible inactivation may lead to clinical drug-drug interactions between gemfibrozil and other P450 2C8 substrates. In light of this in vitro finding and the observation that the glucuronide conjugate does not contain any obvious structural alerts, the current study was conducted to determine the potential site of GEM-1-O-gluc bioactivation and the subsequent mechanism of P450 2C8 inhibition (i.e., modification of apoprotein or heme). LC/MS analysis of a reaction mixture containing recombinant P450 2C8 and GEM-1-O-gluc revealed that the substrate was covalently linked to the heme prosthetic heme group during catalysis. A combination of mass spectrometry and deuterium isotope effects revealed that a benzylic carbon on the 2',5'-dimethylphenoxy group of GEM-1-O-gluc was covalently bound to the heme of P450 2C8. The regiospecificity of substrate addition to the heme group was not confirmed experimentally, but computational modeling experiments indicated that the gamma-meso position was the most likely site of modification. The metabolite profile, which consisted of two benzyl alcohol metabolites and a 4'-hydroxy-GEM-1-O-gluc metabolite, indicated that oxidation of GEM-1-O-gluc was limited to the 2',5'-dimethylphenoxy group. These results are consistent with an inactivation mechanism wherein GEM-1-O-gluc is oxidized to a benzyl radical intermediate, which evades oxygen rebound, and adds to the gamma-meso position of heme. Mechanism-based inhibition of P450 2C8 can be rationalized by the formation of the GEM-1-O-gluc-heme adduct and the consequential restriction of additional substrate access to the catalytic iron center.

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.

Buprofezin Is Metabolized by CYP353D1v2, a Cytochrome P450 Associated with Imidacloprid Resistance in Laodelphax striatellus

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Mohammed Esmail Abdalla Elzaki

2017-11-01

Full Text Available CYP353D1v2 is a cytochrome P450 related to imidacloprid resistance in Laodelphax striatellus. This work was conducted to examine the ability of CYP353D1v2 to metabolize other insecticides. Carbon monoxide difference spectra analysis indicates that CYP353D1v2 was successfully expressed in insect cell Sf9. The catalytic activity of CYP353D1v2 relating to degrading buprofezin, chlorpyrifos, and deltamethrin was tested by measuring substrate depletion and analyzing the formation of metabolites. The results showed the nicotinamide–adenine dinucleotide phosphate (NADPH-dependent depletion of buprofezin (eluting at 8.7 min and parallel formation of an unknown metabolite (eluting 9.5 min. However, CYP353D1v2 is unable to metabolize deltamethrin and chlorpyrifos. The recombinant CYP353D1v2 protein efficiently catalyzed the model substrate p-nitroanisole with a maximum velocity of 9.24 nmol/min/mg of protein and a Michaelis constant of Km = 6.21 µM. In addition, imidacloprid was metabolized in vitro by the recombinant CYP353D1v2 microsomes (catalytic constant Kcat 0.064 pmol/min/pmol P450, Km = 6.41 µM. The mass spectrum of UPLC-MS analysis shows that the metabolite was a product of buprofezin, which was buprofezin sulfone. This result provided direct evidence that L. striatellus cytochrome P450 CYP353D1v2 is capable of metabolizing imidacloprid and buprofezin.

Buprofezin Is Metabolized by CYP353D1v2, a Cytochrome P450 Associated with Imidacloprid Resistance in Laodelphax striatellus.

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Elzaki, Mohammed Esmail Abdalla; Miah, Mohammad Asaduzzaman; Han, Zhaojun

2017-11-29

CYP353D1v2 is a cytochrome P450 related to imidacloprid resistance in Laodelphax striatellus . This work was conducted to examine the ability of CYP353D1v2 to metabolize other insecticides. Carbon monoxide difference spectra analysis indicates that CYP353D1v2 was successfully expressed in insect cell Sf9. The catalytic activity of CYP353D1v2 relating to degrading buprofezin, chlorpyrifos, and deltamethrin was tested by measuring substrate depletion and analyzing the formation of metabolites. The results showed the nicotinamide-adenine dinucleotide phosphate (NADPH)-dependent depletion of buprofezin (eluting at 8.7 min) and parallel formation of an unknown metabolite (eluting 9.5 min). However, CYP353D1v2 is unable to metabolize deltamethrin and chlorpyrifos. The recombinant CYP353D1v2 protein efficiently catalyzed the model substrate p -nitroanisole with a maximum velocity of 9.24 nmol/min/mg of protein and a Michaelis constant of Km = 6.21 µM. In addition, imidacloprid was metabolized in vitro by the recombinant CYP353D1v2 microsomes (catalytic constant Kcat) 0.064 pmol/min/pmol P450, Km = 6.41 µM. The mass spectrum of UPLC-MS analysis shows that the metabolite was a product of buprofezin, which was buprofezin sulfone. This result provided direct evidence that L. striatellus cytochrome P450 CYP353D1v2 is capable of metabolizing imidacloprid and buprofezin.

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

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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.

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

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.

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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.

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

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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.

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

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

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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

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

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

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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.

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…

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,

Role of cytochrome P450 IA2 in acetanilide 4-hydroxylation as determined with cDNA expression and monoclonal antibodies.

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Liu, G; Gelboin, H V; Myers, M J

1991-02-01

The role of P450 IA2 in the hydroxylation of acetanilide was examined using an inhibitory monoclonal antibody (MAb) 1-7-1 and vaccinia cDNA expression producing murine P450 IA1 (mIA1), murine P450 IA2 (mIA2), or human P450 IA2 (hIA2). Acetanilide hydroxylase (AcOH) activity was measured using an HPLC method with more than 500-fold greater sensitivity than previously described procedures. This method, which does not require the use of radioactive acetanilide, was achieved by optimizing both the gradient system and the amount of enzyme needed to achieve detection by uv light. MAb 1-7-1 inhibits up to 80% of the AcOH activity in both rat liver microsomes and cDNA expressed mouse and human P450 IA2. MAb 1-7-1, which recognizes both P450 IA1 and P450 IA2, completely inhibits the aryl hydrocarbon hydroxylase (AHH) activity of cDNA expressed in IA1. The inhibition of only 80% of the AHH activity present in MC liver microsomes by MAb 1-7-1 suggests that additional P450 forms are contributing to the overall AHH activity present in methylcholanthrene (MC)-liver microsomes as MAb 1-7-1 almost completely inhibits the AHH activity of expressed mIA1. Maximal inhibition of IA2 by 1-7-1 results in an 80% decrease in acetanilide hydroxylase activity in both liver microsomes and expressed mouse and human IA2. The capacity of MAb 1-7-1 to produce identical levels of inhibition of acetanilide hydroxylase activity in rat MC microsomes (80%) and in expressed mouse (81%) and human P450 IA2 (80%) strongly suggests that P450 IA2 is the major and perhaps the only enzyme responsible for the metabolism of acetanilide. These results demonstrate the complementary utility of monoclonal antibodies and cDNA expression for defining the contribution of specific P450 enzymes to the metabolism of a given substrate. This complementary approach allows for a more precise determination of the inhibitory capacity of MAb with respect to the metabolic capacity of the target P450.

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)

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

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.

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.

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,

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

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.

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

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

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.

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.

Trapping of cis-2-butene-1,4-dial to measure furan metabolism in human liver microsomes by cytochrome P450 enzymes.

Science.gov (United States)

Gates, Leah A; Lu, Ding; Peterson, Lisa A

2012-03-01

Furan is a liver toxicant and carcinogen in rodents. It is classified as a possible human carcinogen, but the human health effects of furan exposure remain unknown. The oxidation of furan by cytochrome P450 (P450) enzymes is necessary for furan toxicity. The product of this reaction is the reactive α,β-unsaturated dialdehyde, cis-2-butene-1,4-dial (BDA). To determine whether human liver microsomes metabolize furan to BDA, a liquid chromatography/tandem mass spectrometry method was developed to detect and quantify BDA by trapping this reactive metabolite with N-acetyl-l-cysteine (NAC) and N-acetyl-l-lysine (NAL). Reaction of NAC and NAL with BDA generates N-acetyl-S-[1-(5-acetylamino-5-carboxypentyl)-1H-pyrrol-3-yl]-l-cysteine (NAC-BDA-NAL). Formation of NAC-BDA-NAL was quantified in 21 different human liver microsomal preparations. The levels of metabolism were comparable to that observed in F-344 rat and B6C3F1 mouse liver microsomes, two species known to be sensitive to furan-induced toxicity. Studies with recombinant human liver P450s indicated that CYP2E1 is the most active human liver furan oxidase. The activity of CYP2E1 as measured by p-nitrophenol hydroxylase activity was correlated to the extent of NAC-BDA-NAL formation in human liver microsomes. The formation of NAC-BDA-NAL was blocked by CYP2E1 inhibitors but not other P450 inhibitors. These results suggest that humans are capable of oxidizing furan to its toxic metabolite, BDA, at rates comparable to those of species sensitive to furan exposure. Therefore, humans may be susceptible to furan's toxic effects.

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...

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

New superhindered polydentate polyphosphine ligands P(CH2CH2P(t)Bu2)3, PhP(CH2CH2P(t)Bu2)2, P(CH2CH2CH2P(t)Bu2)3, and their ruthenium(II) chloride complexes.

Science.gov (United States)

Gilbert-Wilson, Ryan; Field, Leslie D; Bhadbhade, Mohan M

2012-03-05

The synthesis and characterization of the extremely hindered phosphine ligands, P(CH(2)CH(2)P(t)Bu(2))(3) (P(2)P(3)(tBu), 1), PhP(CH(2)CH(2)P(t)Bu(2))(2) (PhP(2)P(2)(tBu), 2), and P(CH(2)CH(2)CH(2)P(t)Bu(2))(3) (P(3)P(3)(tBu), 3) are reported, along with the synthesis and characterization of ruthenium chloro complexes RuCl(2)(P(2)P(3)(tBu)) (4), RuCl(2)(PhP(2)P(2)(tBu)) (5), and RuCl(2)(P(3)P(3)(tBu)) (6). The bulky P(2)P(3)(tBu) (1) and P(3)P(3)(tBu) (3) ligands are the most sterically encumbered PP(3)-type ligands so far synthesized, and in all cases, only three phosphorus donors are able to bind to the metal center. Complexes RuCl(2)(PhP(2)P(2)(tBu)) (5) and RuCl(2)(P(3)P(3)(tBu)) (6) were characterized by crystallography. Low temperature solution and solid state (31)P{(1)H} NMR were used to demonstrate that the structure of RuCl(2)(P(2)P(3)(tBu)) (4) is probably analogous to that of RuCl(2)(PhP(2)P(2)(tBu)) (5) which had been structurally characterized.

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

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

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.

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.

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...

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

Influence of paraoxonase-1 Q192R and cytochrome P450 2C19 polymorphisms on clopidogrel response

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Li L

2012-02-01

Full Text Available Rolf P Kreutz1,2, Perry Nystrom2, Yvonne Kreutz2, Jia Miao2, Zeruesenay Desta2, Jeffrey A Breall1, Lang Li2, ChienWei Chiang2, Richard Kovacs1, David A Flockhart2, Yan Jin21Krannert Institute of Cardiology, 2Division of Clinical Pharmacology, Indiana University School of Medicine, Indianapolis, IN, USABackground: The metabolic activation of clopidogrel is a two-step process. It has been suggested that paraoxonase-1 (PON1 is a rate-limiting enzyme in the conversion of 2-oxo-clopidogrel to an active thiol metabolite. Conflicting results have been reported in regard to (1 the association of a common polymorphism of PON1 (Q192R with reduced rates of coronary stent thrombosis in patients taking clopidogrel and (2 its effects on platelet inhibition in patient populations of European descent. Methods: Blood samples from 151 subjects of mixed racial background with established coronary artery disease and who received clopidogrel were analyzed. Platelet aggregation was determined with light transmittance aggregometry and VerifyNow® P2Y12 assay. Genotyping for cytochrome P450 2C19 (CYP2C19*2 and *3 and PON1 (Q192R polymorphisms was performed.Results: Carriers of CYP2C19*2 alleles exhibited lower levels of platelet inhibition and higher on-treatment platelet aggregation than noncarriers. There was no significant difference in platelet aggregation among PON1 Q192R genotypes. Homozygous carriers of the wild-type variant of PON1 (QQ192 had similar on-treatment platelet reactivity to carriers of increased-function variant alleles during maintenance clopidogrel dosing, as well as after administration of a clopidogrel 600 mg loading dose.Conclusion: CYP2C19*2 allele is associated with impaired platelet inhibition by clopidogrel and high on-treatment platelet aggregation. PON1 (Q192R polymorphism does not appear to be a significant determinant of clopidogrel response.Keywords: PON1, platelet, aggregation, cytochrome P450 enzymes

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.

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

Changes in the expression of Hepatic Cytochrome P450 Isoenzymes 2E1, 2B1/2, 4A, and 2C6 in mice infected with different levels of Schistosoma Mansoni Cercariae

International Nuclear Information System (INIS)

Sheweita, Salah A.

2005-01-01

Most xenobiotic agents are metabolized by cytochrome P450 system. In the present study, Western blotting was used to investigate the effect of different levels of Schistosoma Mansoni infection on the expression of somr cytochrome P450 isozymes (CYP 2E1, 2B1/2, 2C6, 4A) and to enzyme assay their related metabolic functions in mouse liver microsomes. Male mice were infected with 60, 120, 180, 300 and 600 Schistosoma Mansoni cercariae per mouse for 33 days and 60, 120, 180 and 300 cercariae/mouse with no change at the last level of Schistosoma Mansoni infection. Also the expression of CYP 4A was potentially induced at all levels of Schistosoma Mansoni infection. A significant induction of CYP 2B1/2 expression was observed at all levels of Schistosoma Mansoni infection with loss of signal at 180 cercariaea/mouse. In contrast, CYP 2C6 expression was induced at the first two levels and such expression was decreased at the last three levels. In addition, the infection of the mouse with 60, 120 and 180 cercariae/mouse decreased; [1] 7-methoxycoumarin O-demethylase activity by 36, 54 and 58% respectively; [2] 7-ethoxycoumarin O-deethylase activity by 33, 40 and 57% respectively; [3] coumarin hydroxlase activity by 33, 45 and 55% respectively. However, 300 and 600 cercariae/mouse induced: [1] 7-methoxycoumarin O-demethylase activity by 45 and 97% respectively: [2] 7-ethoxycoumarin O-deethylase activity by 26 and 90% respectively; [3] coumarin hydroxylase activity by 100 and 200% respectively. In addition, all levels of Schistosoma Mansoni infection decreased the sleeping time caused by hexobarital. It is concluded that different levels of Schistosoma Mansoni infection change the expression of different CYPisozymes and that these alterations could enhance the carcinogenicity of N-nitrosamines which is mainly dependent on CYP 2E1. The alterations in the expression of CYP 2E1, 4A and 2B1/2 isozymes as a result of Schistosoma Mansoni infection may change the therapeutic actions

Cytochrome b5 and epoxide hydrolase contribute to benzo[a]pyrene-DNA adduct formation catalyzed by cytochrome P450 1A1 under low NADPH:P450 oxidoreductase conditions

International Nuclear Information System (INIS)

Stiborová, Marie; Moserová, Michaela; Černá, Věra; Indra, Radek; Dračínský, Martin; Šulc, Miroslav; Henderson, Colin J.; Wolf, C. Roland; Schmeiser, Heinz H.; Phillips, David H.; Frei, Eva; Arlt, Volker M.

2014-01-01

In previous studies we had administered benzo[a]pyrene (BaP) to genetically engineered mice (HRN) which do not express NADPH:cytochrome P450 oxidoreductase (POR) in hepatocytes and observed higher DNA adduct levels in livers of these mice than in wild-type mice. To elucidate the reason for this unexpected finding we have used two different settings for in vitro incubations; hepatic microsomes from control and BaP-pretreated HRN mice and reconstituted systems with cytochrome P450 1A1 (CYP1A1), POR, cytochrome b 5 , and epoxide hydrolase (mEH) in different ratios. In microsomes from BaP-pretreated mice, in which Cyp1a1 was induced, higher levels of BaP metabolites were formed, mainly of BaP-7,8-dihydrodiol. At a low POR:CYP1A1 ratio of 0.05:1 in the reconstituted system, the amounts of BaP diones and BaP-9-ol formed were essentially the same as at an equimolar ratio, but formation of BaP-3-ol was ∼1.6-fold higher. Only after addition of mEH were BaP dihydrodiols found. Two BaP-DNA adducts were formed in the presence of mEH, but only one when CYP1A1 and POR were present alone. At a ratio of POR:CYP1A1 of 0.05:1, addition of cytochrome b 5 increased CYP1A1-mediated BaP oxidation to most of its metabolites indicating that cytochrome b 5 participates in the electron transfer from NADPH to CYP1A1 required for enzyme activity of this CYP. BaP-9-ol was formed even by CYP1A1 reconstituted with cytochrome b 5 without POR. Our results suggest that in livers of HRN mice Cyp1a1, cytochrome b 5 and mEH can effectively activate BaP to DNA binding species, even in the presence of very low amounts of POR

Cytochrome P450 1B1 and 2C9 genotypes and risk of ischemic vascular disease, cancer, and chronic obstructive pulmonary disease

DEFF Research Database (Denmark)

Kaur-Knudsen, Diljit; Bojesen, Stig E; Nordestgaard, Børge G

2012-01-01

The aim of this review is to summarize present knowledge of genetic variation in cytochrome P450 1B1 (CYP1B1) and 2C9 (CYP2C9) genes and risk of tobacco-related cancer, female cancer, chronic obstructive pulmonary disease and ischemic vascular disease. The CYP1B1 and CYP2C9 enzymes metabolize pol...

Buprofezin Is Metabolized by CYP353D1v2, a Cytochrome P450 Associated with Imidacloprid Resistance in Laodelphax striatellus

OpenAIRE

Mohammed Esmail Abdalla Elzaki; Mohammad Asaduzzaman Miah; Zhaojun Han

2017-01-01

CYP353D1v2 is a cytochrome P450 related to imidacloprid resistance in Laodelphax striatellus. This work was conducted to examine the ability of CYP353D1v2 to metabolize other insecticides. Carbon monoxide difference spectra analysis indicates that CYP353D1v2 was successfully expressed in insect cell Sf9. The catalytic activity of CYP353D1v2 relating to degrading buprofezin, chlorpyrifos, and deltamethrin was tested by measuring substrate depletion and analyzing the formation of metabolites. T...

Imidacloprid is degraded by CYP353D1v2, a cytochrome P450 overexpressed in a resistant strain of Laodelphax striatellus.

Science.gov (United States)

Elzaki, Mohammed Esmail Abdalla; Miah, Mohammad Asaduzzaman; Wu, Min; Zhang, Haomiao; Pu, Jian; Jiang, Ling; Han, Zhaojun

2017-07-01

Cytochrome P450s are associated with the metabolising of a wide range of compounds, including insecticides. CYP353D1v2 has been found to be overexpressed in an imidacloprid-resistant strain of Laodelphax striatellus. Thus, this study was conducted to express CYP353D1v2 in Sf9 cells as a recombinant protein, to assess its ability to metabolise imidacloprid. Western blot and carbon monoxide difference spectrum analysis indicated that the intact CYP353D1v2 protein had been successfully expressed in Sf9 insect cells. Catalytic activity tests with four traditional P450-activity-probing substrates found that the expressed CYP353D1v2 preferentially metabolised p-nitroanisole, ethoxycoumarin and ethoxyresorufin with specific activities of 32.70, 0.317 and 1.22 pmol min -1 pmol -1 protein respectively, but no activity to luciferin-H EGE. The enzyme activity for degrading imidacloprid was tested by measuring substrate depletion and formation of the metabolite. Kinetic parameters for imidacloprid were K m 5.99 ± 0.95 µm and k cat 0.03 ± 0.0004 min -1 . The chromatogram analysis showed clearly the NADPH-dependent depletion of imidacloprid and the formation of an unknown metabolite. The UPLC-MS mass spectrum demonstrated that the metabolite was an oxidative product of imidacloprid, 5-hydroxy-imidacloprid. These results suggest that CYP353D1v2 in L. striatellus is capable of degrading imidacloprid, and that enzyme activity can be evaluated well only by some traditional probing substrates. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

Alteration in the Expression of Cytochrome P450s (CYP1A1, CYP2E1, and CYP3A11 in the Liver of Mouse Induced by Microcystin-LR

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Bangjun Zhang

2015-03-01

Full Text Available Microcystins (MCs are cyclic heptapeptide toxins and can accumulate in the liver. Cytochrome P450s (CYPs play an important role in the biotransformation of endogenous substances and xenobiotics in animals. It is unclear if the CYPs are affected by MCs exposure. The objective of this study was to evaluate the effects of microcystin-LR (MCLR on cytochrome P450 isozymes (CYP1A1, CYP2E1, and CYP3A11 at mRNA level, protein content, and enzyme activity in the liver of mice the received daily, intraperitoneally, 2, 4, and 8 µg/kg body weight of MCLR for seven days. The result showed that MCLR significantly decreased ethoxyresorufin-O-deethylase (EROD (CYP1A1 and erythromycin N-demthylase (ERND (CYP3A11 activities and increased aniline hydroxylase (ANH activity (CYP2E1 in the liver of mice during the period of exposure. Our findings suggest that MCLR exposure may disrupt the function of CYPs in liver, which may be partly attributed to the toxicity of MCLR in mice.

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

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.

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

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.

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.

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 ...

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.

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.

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

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.

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

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

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

Promising Tools in Prostate Cancer Research: Selective Non-Steroidal Cytochrome P450 17A1 Inhibitors

Science.gov (United States)

Bonomo, Silvia; Hansen, Cecilie H.; Petrunak, Elyse M.; Scott, Emily E.; Styrishave, Bjarne; Jørgensen, Flemming Steen; Olsen, Lars

2016-07-01

Cytochrome P450 17A1 (CYP17A1) is an important target in the treatment of prostate cancer because it produces androgens required for tumour growth. The FDA has approved only one CYP17A1 inhibitor, abiraterone, which contains a steroidal scaffold similar to the endogenous CYP17A1 substrates. Abiraterone is structurally similar to the substrates of other cytochrome P450 enzymes involved in steroidogenesis, and interference can pose a liability in terms of side effects. Using non-steroidal scaffolds is expected to enable the design of compounds that interact more selectively with CYP17A1. Therefore, we combined a structure-based virtual screening approach with density functional theory (DFT) calculations to suggest non-steroidal compounds selective for CYP17A1. In vitro assays demonstrated that two such compounds selectively inhibited CYP17A1 17α-hydroxylase and 17,20-lyase activities with IC50 values in the nanomolar range, without affinity for the major drug-metabolizing CYP2D6 and CYP3A4 enzymes and CYP21A2, with the latter result confirmed in human H295R cells.

Oxidation reactivity of 1,2-bis(2,4,6-tribromophenoxy)ethane (BTBPE) by Compound I model of cytochrome P450s

Institute of Scientific and Technical Information of China (English)

Zhongyu Wang; Zhiqiang Fu; Qi Yu; Jingwen Chen

2017-01-01

Alternative brominated flame retardants (BFRs) have become prevalent as a consequence of restrictions on the use of polybrominated diphenyl ethers (PBDEs).For risk assessment of these alternatives,knowledge of their metabolism via cytochrome P450 enzymes is needed.We have previously proved that density functional theory (DFT) is able to predict the metabolism of PBDEs by revealing the molecular mechanisms.In the current study,the reactivity of 1,2-bis(2,4,6-tribromophenoxy)ethane and structurally similar chemicals with the Compound I model representing the active site of P450 enzymes was investigated.The DFT calculations delineated reaction pathways which lead to reasonable explanations for products that were detected by wet experiments,meanwhile intermediates which cannot be determined were also proposed.Results showed that alkyl hydrogen abstraction will lead to bis(2,4,6-tribromophenoxy)ethanol,which may undergo hydrolysis yielding 2,4,6-tribromophenol,a neurotoxic compound.In addition,a general pattern of oxidation reactivity regarding the 2,4,6-tribromophenyl moiety was observed among several model compounds.Our study has provided insights for convenient evaluation of the metabolism of other structurally similar BFRs.

Cytochrome P450 humanised mice

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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.

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

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

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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

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

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.

Two Arabidopsis cytochrome P450 monooxygenases, CYP714A1 and CYP714A2, function redundantly in plant development through gibberellin deactivation.

Science.gov (United States)

Zhang, Yingying; Zhang, Baichen; Yan, Dawei; Dong, Weixin; Yang, Weibing; Li, Qun; Zeng, Longjun; Wang, Jianjun; Wang, Linyou; Hicks, Leslie M; He, Zuhua

2011-07-01

The rice gene ELONGATED UPPERMOST INTERNODE1 (EUI1) encodes a P450 monooxygenase that epoxidizes gibberellins (GAs) in a deactivation reaction. The Arabidopsis genome contains a tandemly duplicated gene pair ELA1 (CYP714A1) and ELA2 (CYP714A2) that encode EUI homologs. In this work, we dissected the functions of the two proteins. ELA1 and ELA2 exhibited overlapping yet distinct gene expression patterns. We showed that while single mutants of ELA1 or ELA2 exhibited no obvious morphological phenotype, simultaneous elimination of ELA1 and ELA2 expression in ELA1-RNAi/ela2 resulted in increased biomass and enlarged organs. By contrast, transgenic plants constitutively expressing either ELA1 or ELA2 were dwarfed, similar to those overexpressing the rice EUI gene. We also discovered that overexpression of ELA1 resulted in a severe dwarf phenotype, while overexpression of ELA2 gave rise to a breeding-favored semi-dwarf phenotype in rice. Consistent with the phenotypes, we found that the ELA1-RNAi/ela2 plants increased amounts of biologically active GAs that were decreased in the internodes of transgenic rice with ELA1 and ELA2 overexpression. In contrast, the precursor GA(12) slightly accumulated in the transgenic rice, and GA(19) highly accumulated in the ELA2 overexpression rice. Taken together, our study strongly suggests that the two Arabidopsis EUI homologs subtly regulate plant growth most likely through catalyzing deactivation of bioactive GAs similar to rice EUI. The two P450s may also function in early stages of the GA biosynthetic pathway. Our results also suggest that ELA2 could be an excellent tool for molecular breeding for high yield potential in cereal crops. © 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.

Identification of SNPs involved in regulating a novel alternative transcript of P450 CYP6ER1 in the brown planthopper.

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Liang, Zhi-Kun; Pang, Rui; Dong, Yi; Sun, Zhong-Xiang; Ling, Yan; Zhang, Wen-Qing

2017-04-29

Cytochrome P450-mediated metabolic resistance is one of the major mechanisms involved in insecticide resistance. Although the up-regulation of cytochrome P450 plays a vital role in insecticide metabolism, the molecular basis for the transcriptional regulation of cytochrome P450 remains largely unknown. The P450 gene CYP6ER1, has been reported to confer imidacloprid resistance to the brown planthopper, Nilaparvata lugens. Here, we identified a novel alternative transcript of CYP6ER1 (transcript A2) that had different expression patterns between resistant and susceptible populations, and was more stable after insecticide induction. The promoter of this transcript was sequenced and multiple single nucleotide polymorphisms (SNPs) were detected in individuals from susceptible and resistant field-collected populations. Resistant alleles of four SNPs were found to significantly enhance the promoter activity of the CYP6ER1 transcript A2. Electrophoretic mobility shift assays (EMSAs) revealed that these SNPs might regulate the binding of transcription factors to the promoter. Our findings provide novel evidence regarding the transcriptional regulation of a metabolic resistance-related gene and may be useful to understand the resistance mechanism of N. lugens in the field. © 2017 Institute of Zoology, Chinese Academy of Sciences.

The Schistosoma mansoni Cytochrome P450 (CYP3050A1 Is Essential for Worm Survival and Egg Development.

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Peter D Ziniel

2015-12-01

Full Text Available Schistosomiasis affects millions of people in developing countries and is responsible for more than 200,000 deaths annually. Because of toxicity and limited spectrum of activity of alternatives, there is effectively only one drug, praziquantel, available for its treatment. Recent data suggest that drug resistance could soon be a problem. There is therefore the need to identify new drug targets and develop drugs for the treatment of schistosomiasis. Analysis of the Schistosoma mansoni genome sequence for proteins involved in detoxification processes found that it encodes a single cytochrome P450 (CYP450 gene. Here we report that the 1452 bp open reading frame has a characteristic heme-binding region in its catalytic domain with a conserved heme ligating cysteine, a hydrophobic leader sequence present as the membrane interacting region, and overall structural conservation. The highest sequence identity to human CYP450s is 22%. Double stranded RNA (dsRNA silencing of S. mansoni (SmCYP450 in schistosomula results in worm death. Treating larval or adult worms with antifungal azole CYP450 inhibitors results in worm death at low micromolar concentrations. In addition, combinations of SmCYP450-specific dsRNA and miconazole show additive schistosomicidal effects supporting the hypothesis that SmCYP450 is the target of miconazole. Treatment of developing S. mansoni eggs with miconazole results in a dose dependent arrest in embryonic development. Our results indicate that SmCYP450 is essential for worm survival and egg development and validates it as a novel drug target. Preliminary structure-activity relationship suggests that the 1-(2,4-dichlorophenyl-2-(1H-imidazol-1-ylethan-1-ol moiety of miconazole is necessary for activity and that miconazole activity and selectivity could be improved by rational drug design.

Oxidation of the carcinogenic non-aminoazo dye 1-phenylazo-2-hydroxynaphthalene (Sudan I) by cytochromes P450 and peroxidases: a comparative study

Czech Academy of Sciences Publication Activity Database

Stiborová, M.; Martínek, V.; Semanská, M.; Hodek, P.; Dračínský, Martin; Cvačka, Josef; Schmeiser, H. H.; Frei, E.

2009-01-01

Roč. 2, č. 3 (2009), s. 195-200 ISSN 1337-6853 Grant - others:GA MŠk(CZ) 1M0505; GA ČR(CZ) GA303/09/0472; GA ČR(CZ) GA203/09/0812 Program:1M Institutional research plan: CEZ:AV0Z40550506 Keywords : metabolism of xenobiotics * Sudan I * cytochrome P450 * peroxidase Subject RIV: CC - Organic Chemistry

Effects of chronic exposure to tributyltin on tissue-specific cytochrome P450 1 regulation in juvenile common carp.

Science.gov (United States)

Li, Zhi-Hua; Zhong, Li-Qiao; Mu, Wei-Na; Wu, Yan-Hua

2016-01-01

1. The purpose of this study was to compare tributyltin (TBT)-induced cytochrome P450 1 (CYP450 1) regulation in liver, gills and muscle of juvenile common carp (Cyprinus carpio). 2. Fish were exposed to sublethal concentrations of TBT (75, 0.75 and 7.5 μg/L) for 60 days. CYP450 1A was measured at the enzyme activity level as 7-ethoxyresorufin-O-deethylase (EROD) activity, as well as the mRNA expression of CYP450 1 family genes (CYP1A, CYP1B, CYP1C1 and CYP1C2) in fish tissues. 3. Based on the results, the liver displayed the highest absolute levels of EROD activity, both under nonexposed and exposed conditions. Additional, EROD activities and CYP1A gene levels showed a good correlation in all three organs. According to the mRNA expression of CYP450 1 family genes, it suggested that CYP1A was to accommodate most EROD activity in fish, but other CYP450 forms also involved in this proceeding. 4. Overall, the study revealed both similarities and differences in the concentration-dependent CYP450 1 responses of the three target organs, which could provide useful information to better understand the mechanisms of TBT-induced bio-toxicity.

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.

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.

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

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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.

Electrochemistry of Canis familiaris cytochrome P450 2D15 with gold nanoparticles: An alternative to animal testing in drug discovery.

Science.gov (United States)

Rua, Francesco; Sadeghi, Sheila J; Castrignanò, Silvia; Valetti, Francesca; Gilardi, Gianfranco

2015-10-01

This work reports for the first time the direct electron transfer of the Canis familiaris cytochrome P450 2D15 on glassy carbon electrodes to provide an analytical tool as an alternative to P450 animal testing in the drug discovery process. Cytochrome P450 2D15, that corresponds to the human homologue P450 2D6, was recombinantly expressed in Escherichia coli and entrapped on glassy carbon electrodes (GC) either with the cationic polymer polydiallyldimethylammonium chloride (PDDA) or in the presence of gold nanoparticles (AuNPs). Reversible electrochemical signals of P450 2D15 were observed with calculated midpoint potentials (E1/2) of −191 ± 5 and −233 ± 4 mV vs. Ag/AgCl for GC/PDDA/2D15 and GC/AuNPs/2D15, respectively. These experiments were then followed by the electro-catalytic activity of the immobilized enzyme in the presence of metoprolol. The latter drug is a beta-blocker used for the treatment of hypertension and is a specific marker of the human P450 2D6 activity. Electrocatalysis data showed that only in the presence of AuNps the expected α-hydroxy-metoprolol product was present as shown by HPLC. The successful immobilization of the electroactive C. familiaris cytochrome P450 2D15 on electrode surfaces addresses the ever increasing demand of developing alternative in vitromethods for amore detailed study of animal P450 enzymes' metabolism, reducing the number of animals sacrificed in preclinical tests.

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 ...

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...

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.

Cloning, expression and characterisation of P450-Hal1 (CYP116B62) from Halomonas sp. NCIMB 172: A self-sufficient P450 with high expression and diverse substrate scope.

Science.gov (United States)

Porter, Joanne L; Sabatini, Selina; Manning, Jack; Tavanti, Michele; Galman, James L; Turner, Nicholas J; Flitsch, Sabine L

2018-06-01

Cytochrome P450 monooxygenases are able to catalyse a range of synthetically challenging reactions ranging from hydroxylation and demethylation to sulfoxidation and epoxidation. As such they have great potential for biocatalytic applications but are underutilised due to often-poor expression, stability and solubility in recombinant bacterial hosts. The use of self-sufficient P450 s with fused haem and reductase domains has already contributed heavily to improving catalytic efficiency and simplifying an otherwise more complex multi-component system of P450 and redox partners. Herein, we present a new addition to the class VII family with the cloning, sequencing and characterisation of the self-sufficient CYP116B62 Hal1 from Halomonas sp. NCIMB 172, the genome of which has not yet been sequenced. Hal1 exhibits high levels of expression in a recombinant E. coli host and can be utilised from cell lysate or used in purified form. Hal1 favours NADPH as electron donor and displays a diverse range of activities including hydroxylation, demethylation and sulfoxidation. These properties make Hal1 suitable for future biocatalytic applications or as a template for optimisation through engineering. Copyright © 2018 Elsevier Inc. All rights reserved.

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

[μ-1,2-Bis(diphenylphosphinoethane-κ2P:P′]bis{[1,2-bis(diphenylphosphinoethane-κ2P,P′]cyanidocopper(I} methanol disolvate

Directory of Open Access Journals (Sweden)

Rong Wang

2010-08-01

Full Text Available The title centrosymmetric complex, [Cu2(CN2(C26H24P23]·2CH3OH, consists of two five-membered [Cu(dppeCN] rings [dppe is 1,2-bis(diphenylphosphinoethane] bridged by one μ2-dppe ligand, and two methanol solvent molecules. The angles around the central metal atom indicate that each CuI atom is located in the center of a distorted tetrahedron. The coordination sphere of each CuI atom is formed by three P atoms from two dppe ligands, and one C atom from the cyanide ligand. The crystal structure is stabilized by O—H...N hydrogen bonds, which are formed by the O—H donor group from methanol and the N-atom acceptor from a cyanide ligand.

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 [RR = 1.51; 95%CI = 0.08-90.06] or Chinese [RR = 1.99; 95%CI = 0.39-10.87]. The ethnicity-adjusted odds ratio is 2.39 with 95%CI, 0.72-7.89. Though our finding was not statistically significant due to the moderate sample size of the study, similarity to the study in Taiwan with only a slight loss in precision was demonstrated. The higher RR found for the same genotype in distinct populations confirmed that CYP2E1 is one of several NPC susceptibility genes and that the RsaI minus variant is one mutation that affects phenotype

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.

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.

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.

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

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

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

Suicidal gene therapy with rabbit cytochrome P450 4B1/4-ipomeanol, 2-aminoanthracene system in glioma cell

International Nuclear Information System (INIS)

Jang, Su Jin; Kang, Joo Hyun; Kim, Kwang Il; Lee, Tae Sup; Lee, Yong Jin; Woo, Kwang Sun; Chung, Wee Sup; Cheon, Gi Jeong; Choi, Chang Woon; Lim, Sang Moo

2010-01-01

Suicidal gene therapy is based on the transduction of tumor cells with 'suicide' genes encoding for prodrugactivating enzymes that render target cells susceptible to prodrug treatment. Suicidal gene therapy results in the death of tumor with the expression of gene encoding enzyme that converts non-toxic prodrug into cytotoxic product. Cytochrome P450 4B1 (CYP4B1) activates 4- ipomeanol (4-ipo) and 2-aminoanthracene (2-AA) to cytotoxic furane epoxide and unsaturated dialdehyde intermediate. In this study, therapeutic effects of suicidal gene therapy with rabbit CYP4B1/4-ipo or CYP4B1/2-AA system

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

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.

CYP2J2 and CYP2C19 are the major enzymes responsible for metabolism of albendazole and fenbendazole in human liver microsomes and recombinant P450 assay systems.

Science.gov (United States)

Wu, Zhexue; Lee, Doohyun; Joo, Jeongmin; Shin, Jung-Hoon; Kang, Wonku; Oh, Sangtaek; Lee, Do Yup; Lee, Su-Jun; Yea, Sung Su; Lee, Hye Suk; Lee, Taeho; Liu, Kwang-Hyeon

2013-11-01

Albendazole and fenbendazole are broad-spectrum anthelmintics that undergo extensive metabolism to form hydroxyl and sulfoxide metabolites. Although CYP3A and flavin-containing monooxygenase have been implicated in sulfoxide metabolite formation, the enzymes responsible for hydroxyl metabolite formation have not been identified. In this study, we used human liver microsomes and recombinant cytochrome P450s (P450s) to characterize the enzymes involved in the formation of hydroxyalbendazole and hydroxyfenbendazole from albendazole and fenbendazole, respectively. Of the 10 recombinant P450s, CYP2J2 and/or CYP2C19 was the predominant enzyme catalyzing the hydroxylation of albendazole and fenbendazole. Albendazole hydroxylation to hydroxyalbendazole is primarily mediated by CYP2J2 (0.34 μl/min/pmol P450, which is a rate 3.9- and 8.1-fold higher than the rates for CYP2C19 and CYP2E1, respectively), whereas CYP2C19 and CYP2J2 contributed to the formation of hydroxyfenbendazole from fenbendazole (2.68 and 1.94 μl/min/pmol P450 for CYP2C19 and CYP2J2, respectively, which are rates 11.7- and 8.4-fold higher than the rate for CYP2D6). Correlation analysis between the known P450 enzyme activities and the rate of hydroxyalbendazole and hydroxyfenbendazole formation in samples from 14 human liver microsomes showed that albendazole hydroxylation correlates with CYP2J2 activity and fenbendazole hydroxylation correlates with CYP2C19 and CYP2J2 activities. These findings were supported by a P450 isoform-selective inhibition study in human liver microsomes. In conclusion, our data for the first time suggest that albendazole hydroxylation is primarily catalyzed by CYP2J2, whereas fenbendazole hydroxylation is preferentially catalyzed by CYP2C19 and CYP2J2. The present data will be useful in understanding the pharmacokinetics and drug interactions of albendazole and fenbendazole in vivo.

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

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

Improvement in luminance of light-emitting diode using InP/ZnS quantum dot with 1-dodecanethiol ligand

Science.gov (United States)

Fukuda, Takeshi; Sasaki, Hironao

2018-03-01

We present the synthesis protocol of a red emissive InP/ZnS quantum dot with a 1-dodecanthiol ligand and its application to a quantum dot light-emitting diode. The ligand change from oleylamine to 1-dodecanthiol, which were connected around the InP/ZnS quantum dot, was confirmed by Fourier-transform infrared spectroscopy and thermal analysis. The absorption peak was blue-shifted by changing 1-dodecanthiol ligands from oleylamine ligands to prevent the unexpected nucleation of the InP core. In addition, the luminance of the light-emitting device was improved by using the InP/ZnS quantum dot with 1-dodecanthiol ligands, and the maximum current efficiency of 7.2 × 10-3 cd/A was achieved. The 1-dodecanthiol ligand is often used for capping to reduce the number of surface defects and/or prevent unexpected core growth, resulting in reduced Auger recombination. This result indicates that 1-dodecanthiol ligands prevent the deactivation of excitons while injecting carriers by applying a voltage, resulting in a high luminance efficiency.

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

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.

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...

Suicidal gene therapy with rabbit cytochrome P450 4B1/4-ipomeanol, 2-aminoanthracene system in glioma cell

Energy Technology Data Exchange (ETDEWEB)

Jang, Su Jin; Kang, Joo Hyun; Kim, Kwang Il; Lee, Tae Sup; Lee, Yong Jin; Woo, Kwang Sun; Chung, Wee Sup; Cheon, Gi Jeong; Choi, Chang Woon; Lim, Sang Moo [Korea Institute of Radiological and Medical Sciences, Seoul (Korea, Republic of)

2010-10-15

Suicidal gene therapy is based on the transduction of tumor cells with 'suicide' genes encoding for prodrugactivating enzymes that render target cells susceptible to prodrug treatment. Suicidal gene therapy results in the death of tumor with the expression of gene encoding enzyme that converts non-toxic prodrug into cytotoxic product. Cytochrome P450 4B1 (CYP4B1) activates 4- ipomeanol (4-ipo) and 2-aminoanthracene (2-AA) to cytotoxic furane epoxide and unsaturated dialdehyde intermediate. In this study, therapeutic effects of suicidal gene therapy with rabbit CYP4B1/4-ipo or CYP4B1/2-AA system

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

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

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

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

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

Occupational Toluene Exposure Induces Cytochrome P450 2E1 mRNA Expression in Peripheral Lymphocytes

Science.gov (United States)

Mendoza-Cantú, Ania; Castorena-Torres, Fabiola; de León, Mario Bermúdez; Cisneros, Bulmaro; López-Carrillo, Lizbeth; Rojas-García, Aurora E.; Aguilar-Salinas, Alberto; Manno, Maurizio; Albores, Arnulfo

2006-01-01

Print workers are exposed to organic solvents, of which the systemic toxicant toluene is a main component. Toluene induces expression of cytochrome P450 2E1 (CYP2E1), an enzyme involved in its own metabolism and that of other protoxicants, including some procarcinogens. Therefore, we investigated the association between toluene exposure and the CYP2E1 response, as assessed by mRNA content in peripheral lymphocytes or the 6-hydroxychlorzoxazone (6OH-CHZ)/chlorzoxazone (CHZ) quotient (known as CHZ metabolic ratio) in plasma, and the role of genotype (5′-flanking region RsaI/PstI polymorphic sites) in 97 male print workers. The geometric mean (GM) of toluene concentration in the air was 52.80 ppm (10–760 ppm); 54% of the study participants were exposed to toluene concentrations that exceeded the maximum permissible exposure level (MPEL). The GM of urinary hippuric acid at the end of a work shift (0.041 g/g creatinine) was elevated relative to that before the shift (0.027 g/g creatinine; p < 0.05). The GM of the CHZ metabolic ratio was 0.33 (0–9.3), with 40% of the subjects having ratios below the GM. However, the average CYP2E1 mRNA level in peripheral lymphocytes was 1.07 (0.30–3.08), and CYP2E1 mRNA levels within subjects correlated with the toluene exposure ratio (environmental toluene concentration:urinary hippuric acid concentration) (p = 0.014). Genotype did not alter the association between the toluene exposure ratio and mRNA content. In summary, with further validation, CYP2E1 mRNA content in peripheral lymphocytes could be a sensitive and noninvasive biomarker for the continuous monitoring of toluene effects in exposed persons. PMID:16581535

MicroRNA-450a-3p represses cell proliferation and regulates embryo development by regulating Bub1 expression in mouse.

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Min Luo

Full Text Available Bub1 is a critical component of the spindle assembly checkpoint (SAC and closely linked to cell proliferation and differentiation. We previously found that spontaneous abortion embryos contained a low level of Bub1 protein but normal mRNA level, while the knockdown of Bub1 leads to abnormal numerical chromosomes in embryonic cells. Here, we investigated the mechanism through which governs the post-transcriptional regulation of Bub1 protein expression level. We first conducted bioinformatics analysis and identified eight putative miRNAs that may target Bub1. Luciferase reporter assay confirmed that miR-450a-3p can directly regulate Bub1 by binding to the 3'-untranslated region of Bub1 mRNA. We found that the overexpression of miR-450a-3p in mouse embryonic fibroblast (MEF cells down-regulated Bub1 protein level, repressed cell proliferation, increased apoptosis and restricted most cells in G1 phase of the cell cycle. Furthermore, when the fertilized eggs were microinjected with miR-450a-3p mimics, the cleavage of zygotes was effectively suppressed. Our results strongly suggest that an abnormally decreased Bub1 level regulated by miRNAs may be implicated in the pathogenesis of spontaneous miscarriage. Therefore, the blockade of miR-450a-3p may be explored as a novel therapeutic strategy for preventing spontaneous miscarriages.

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

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

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...

Cytochrome P450 1D1: A novel CYP1A-related gene that is not transcriptionally activated by PCB126 or TCDD

DEFF Research Database (Denmark)

Goldstone, J.V.; Jönsson, M.E.; Behrendt, Lars

2009-01-01

Enzymes in the cytochrome P450 1 family oxidize many common environmental toxicants. We identified a new CYP1, termed CYP1D1, in zebrafish. Phylogenetically, CYP1D1 is paralogous to CYP1A and the two share 45% amino acid identity and similar gene structure. In adult zebrafish, CYP1D1 is most high...

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.

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.

Triterpene Structural Diversification by Plant Cytochrome P450 Enzymes

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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.

Aminotriazole alleviates acetaminophen poisoning via downregulating P450 2E1 and suppressing inflammation.

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Yuping Jing

Full Text Available Aminotriazole (ATZ is commonly used as a catalase (CAT inhibitor. We previously found ATZ attenuated oxidative liver injury, but the underlying mechanisms remain unknown. Acetaminophen (APAP overdose frequently induces life-threatening oxidative hepatitis. In the present study, the potential hepatoprotective effects of ATZ on oxidative liver injury and the underlying mechanisms were further investigated in a mouse model with APAP poisoning. The experimental data indicated that pretreatment with ATZ dose- and time-dependently suppressed the elevation of plasma aminotransferases in APAP exposed mice, these effects were accompanied with alleviated histological abnormality and improved survival rate of APAP-challenged mice. In mice exposed to APAP, ATZ pretreatment decreased the CAT activities, hydrogen peroxide (H2O2 levels, malondialdehyde (MDA contents, myeloperoxidase (MPO levels in liver and reduced TNF-α levels in plasma. Pretreatment with ATZ also downregulated APAP-induced cytochrome P450 2E1 (CYP2E1 expression and JNK phosphorylation. In addition, posttreatment with ATZ after APAP challenge decreased the levels of plasma aminotransferases and increased the survival rate of experimental animals. Posttreatment with ATZ had no effects on CYP2E1 expression or JNK phosphorylation, but it significantly decreased the levels of plasma TNF-α. Our data indicated that the LD50 of ATZ in mice was 5367.4 mg/kg body weight, which is much higher than the therapeutic dose of ATZ in the present study. These data suggested that ATZ might be effective and safe in protect mice against APAP-induced hepatotoxicity, the beneficial effects might resulted from downregulation of CYP2E1 and inhibiton of inflammation.

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

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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.

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

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.

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

Science.gov (United States)

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.

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.

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...

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.

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

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

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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.

Co-expression of human cytochrome P4501A1 (CYP1A1) variants and human NADPH-cytochrome P450 reductase in the baculovirus/insect cell system.

Science.gov (United States)

Schwarz, D; Kisselev, P; Honeck, H; Cascorbi, I; Schunck, W H; Roots, I

2001-06-01

1. Three human cytochrome P4501A1 (CYP1A1) variants, wild-type (CYP1A1.1), CYP1A1.2 (1462V) and CYP1A1.4 (T461N), were co-expressed with human NADPH-P450 reductase (OR) in Spodoptera frugiperda (Sf9) insect cells by baculovirus co-infection to elaborate a suitable system for studying the role of CYPA1 polymorphism in the metabolism of exogenous and endogenous substrates. 2. A wide range of conditions was examined to optimize co-expression with regard to such parameters as relative multiplicity of infection (MOI), time of harvest, haem precursor supplementation and post-translational stabilization. tinder optimized conditions, almost identical expression levels and molar OR/CYP1A1 ratios (20:1) were attained for all CYP1A1 variants. 3. Microsomes isolated from co-infected cells demonstrated ethoxyresorufin deethlylase activities (nmol/min(-1) nmol(-1) CYP1A1) of 16.0 (CYP1A1.1), 20.5 (CYP1A1.2) and 22.5 (CYP1A1.4). Pentoxyresorufin was dealkylated approximately 10-20 times slower with all enzyme variants. 4. All three CYP1A1 variants were active in metabolizing the precarcinogen benzo[a]pyrene (B[a]P), with wild-type enzyme showing the highest activity, followed by CYP1A1.4 (60%) and CYP1A1.2 (40%). Each variant produced all major metabolites including B[a]P-7,8-dihydrodiol, the precursor of the ultimate carcinogenic species. 5. These studies demonstrate that the baculovirus-mediated co-expression-by-co-infection approach all CYP1A1 variants yields functionally active enzyme systems with similar molar OR/CYP1A1 ratios, thus providing suitable preconditions to examine the metabolism of and environmental chemicals by the different CY1A1 variants.

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

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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...

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.

[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].

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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

Overexpression of a cytochrome P450 monooxygenase, CYP6ER1, is associated with resistance to imidacloprid in the brown planthopper, Nilaparvata lugens.

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Bass, C; Carvalho, R A; Oliphant, L; Puinean, A M; Field, L M; Nauen, R; Williamson, M S; Moores, G; Gorman, K

2011-12-01

The brown planthopper, Nilaparvata lugens, is an economically significant pest of rice throughout Asia and has evolved resistance to many insecticides including the neonicotinoid imidacloprid. The resistance of field populations of N. lugens to imidacloprid has been attributed to enhanced detoxification by cytochrome P450 monooxygenases (P450s), although, to date, the causative P450(s) has (have) not been identified. In the present study, biochemical assays using the model substrate 7-ethoxycoumarin showed enhanced P450 activity in several resistant N. lugens field strains when compared with a susceptible reference strain. Thirty three cDNA sequences encoding tentative unique P450s were identified from two recent sequencing projects and by degenerate PCR. The mRNA expression level of 32 of these was examined in susceptible, moderately resistant and highly resistant N. lugens strains using quantitative real-time PCR. A single P450 gene (CYP6ER1) was highly overexpressed in all resistant strains (up to 40-fold) and the level of expression observed in the different N. lugens strains was significantly correlated with the resistance phenotype. These results provide strong evidence for a role of CYP6ER1 in the resistance of N. lugens to imidacloprid. © 2011 The Authors. Insect Molecular Biology © 2011 The Royal Entomological Society.

(+-{1,2-Bis[(2R,5R-2,5-diethylphospholan-1-yl]ethane-κ2P,P′}(η4-cycloocta-1,5-dienerhodium(I tetrafluoridoborate

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Stefan Schulz

2010-11-01

Full Text Available The title compound, [Rh(C8H12(C18H36P2]BF4, exhibits a rhodium(I complex cation with a bidentate bisphosphine ligand and a bidentate η2,η2-coordinated cycloocta-1,5-diene ligand. The ligands form a slightly distorted square-planar coordination environment for the Rh(I atom. An intramolecular P–Rh–P bite angle of 83.91 (2° is observed. The dihedral angle between the P—Rh—P and the X—Rh—X planes (X is the centroid of a double bond is 14.0 (1°. The BF4 anion is disordered over two positions in a 0.515 (7:0.485 (7 ratio.

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.

R-warfarin clearances from plasma associated with polymorphic cytochrome P450 2C19 and simulated by individual physiologically based pharmacokinetic models for 11 cynomolgus monkeys.

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Utoh, Masahiro; Kusama, Takashi; Miura, Tomonori; Mitsui, Marina; Kawano, Mirai; Hirano, Takahiro; Shimizu, Makiko; Uno, Yasuhiro; Yamazaki, Hiroshi

2018-02-01

1. Cynomolgus monkey cytochrome P450 2C19 (formerly known as P450 2C75), homologous to human P450 2C19, has been identified as R-warfarin 7-hydroxylase. In this study, simulations of R-warfarin clearance in individual cynomolgus monkeys genotyped for P450 2C19 p.[(Phe100Asn; Ala103Val; Ile112Leu)] were performed using individual simplified physiologically based pharmacokinetic (PBPK) modeling. 2. Pharmacokinetic parameters and absorption rate constants, volumes of the systemic circulation, and hepatic intrinsic clearances for individual PBPK models were estimated for eleven cynomolgus monkeys. 3. One-way ANOVA revealed significant effects of the genotype (p warfarin among the homozygous mutant, heterozygous mutant, and wild-type groups. R-Warfarin clearances in individual cynomolgus monkeys genotyped for P450 2C19 were simulated by simplified PBPK modeling. The modeled hepatic intrinsic clearances were significantly associated with the P450 2C19 genotypes. The liver microsomal elimination rates of R-warfarin for individual animals after in vivo administration showed significant reductions associated with the genotype (p warfarin and related medicines associated with polymorphic P450 2C19 in individual cynomolgus monkeys, thereby facilitating calculation of the fraction of hepatic clearance.

Pregnane X receptor-dependent induction of the CYP3A4 gene by o,p'-1,1,1,-trichloro-2,2-bis (p-chlorophenyl)ethane.

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Medina-Díaz, Irma M; Arteaga-Illán, Georgina; de León, Mario Bermudez; Cisneros, Bulmaro; Sierra-Santoyo, Adolfo; Vega, Libia; Gonzalez, Frank J; Elizondo, Guillermo

2007-01-01

CYP3A4, the predominant cytochrome P450 (P450) expressed in human liver and intestine, contributes to the metabolism of approximately half the drugs in clinical use today. CYP3A4 catalyzes the 6beta-hydroxylation of a number of steroid hormones and is involved in the bioactivation of environmental procarcinogens. The expression of CYP3A4 is affected by several stimuli, including environmental factors such as insecticides and pesticides. The o,p'-1,1,1,-trichloro-2,2-bis (p-chlorophenyl)ethane (DDT) isomer of DDT comprises approximately 20% of technical grade DDT, which is an organochloride pesticide. We have recently shown that o,p'-DDT exposure increases CYP3A4 mRNA levels in HepG2 cells. To determine the mechanism by which o,p'-DDT induces CYP3A4 expression, transactivation and electrophoretic mobility shift assays were carried out, revealing that o,p'-DDT activates the CYP3A4 gene promoter through the pregnane X receptor (PXR). CYP3A4 gene promoter activation resulted in both an increase in CYP3A4 mRNA levels and an increase in the total CYP3A4 activity in HepG2 cells. We also observed induction of CYP3A4 and mouse Cyp3a11 mRNA in the intestine of CYP3A4-transgenic mice after exposure to 1 mg/kg o,p'-DDT. At higher doses, a decrease of CYP3A4 inducibility was observed together with an increase in levels of interleukin 6 mRNA, a proinflammatory cytokine that strongly represses CYP3A4 transcription. The present study indicates that regulation of other genes under PXR control may be altered by o,p'-DDT exposure.

Ascorbic acid deficiency decreases hepatic cytochrome P-450, especially CYP2B1/2B2, and simultaneously induces heme oxygenase-1 gene expression in scurvy-prone ODS rats.

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Kobayashi, Misato; Hoshinaga, Yukiko; Miura, Natsuko; Tokuda, Yuki; Shigeoka, Shigeru; Murai, Atsushi; Horio, Fumihiko

2014-01-01

The mechanisms underlying the decrease in hepatic cytochrome P-450 (CYP) content in ascorbic acid deficiency was investigated in scurvy-prone ODS rats. First, male ODS rats were fed a diet containing sufficient ascorbic acid (control) or a diet without ascorbic acid (deficient) for 18 days, with or without the intraperitoneal injection of phenobarbital. Ascorbic acid deficiency decreased hepatic microsomal total CYP content, CYP2B1/2B2 protein, and mitochondrial cytochrome oxidase (COX) complex IV subunit I protein, and simultaneously increased heme oxygenase-1 protein in microsomes and mitochondria. Next, heme oxygenase-1 inducers, that is lipopolysaccharide and hemin, were administered to phenobaribital-treated ODS rats fed sufficient ascorbic acid. The administration of these inducers decreased hepatic microsomal total CYP content, CYP2B1/2B2 protein, and mitochondrial COX complex IV subunit I protein. These results suggested that the stimulation of hepatic heme oxygenase-1 expression by ascorbic acid deficiency caused the decrease in CYP content in liver.

Inhibition selectivity of grapefruit juice components on human cytochromes P450.

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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

The reaction of TcCl3(Me2PhP)3 with dithio ligands. Synthesis, characterization and X-ray crystal structures of [TcCl2(Me2PhP)2(Me2dtp)]and [Tc(Me2PhP)(Etxan)3](Me2PhP=dimethylphenylphosphine, Me2dtp-=O,O'-dimethyldithiophosphate, Etxan-=ethylxanthate)

International Nuclear Information System (INIS)

Lorenz, B.; Schmidt, K.; Hiller, W.; Abram, U.; Huebener, R.

1993-01-01

Trichlorotris (dimethylphenylphosphine)technetium(III); [TcCl 3 (Me 2 PhP) 3 ], reacts with dithio ligands to form chelate complexes of different compositions and coordinations geometries. The reaction with ethylxanthate (Etxan - ) yields the diamagnetic seven-coordinate [Tc(Me 2 PhP)(Etxan) 3 ], which crystallizes monoclinic in the space group P2 2 /c with Z = 4 (a = 18.44(5), b = 9.2(1), c = 15.36(6) A, β = 104.3(2) ). The final R value is 0.029. The metal has a pentagonal-bipyramidal environment. With ammonium dimethyldithiophosphate, (NH 4 )Me 2 dtp, [TcCl 3 (Me 2 PhP) 3 ] forms the paramagnetic [TcCl 2 (Me 2 PhP) 2 (Me 2 dtp) in which the technetium atom has a distorted octahedral coordination sphere. The compound crystallizes orthorhombic, space group Pbcn, with Z = 4 (a = 16.20(1), b = 10.445(1), c 14.878(1)). The final R value is 0.031. The chloro ligands are in trans arrangement. (orig.)

Antibodies to P450IID6, SLA, PDH-E2 and BCKD-E2 in Japanese patients with chronic hepatitis.

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Nishioka, M; Morshed, S A; Parveen, S; Kono, K; Matsuoka, H; Manns, M P

1997-12-01

Auto-antibodies specific to various antigens in chronic hepatitis (CH) have been detected but their specificities and implications were uncertain. The aims of the present study were to investigate the frequency and the significance of seropositivity of antibodies to P450IID6 or liver/kidney microsome 1 (LKM1), soluble liver antigen (SLA), pyruvate dehydrogenase (PDH) and branched-chain keto acid dehydrogenase (BCKD) in 188 Japanese patients with different forms of CH by western blot or enzyme immunoassay (EIA). Anti-LKM1 was also measured by indirect immunofluorescent test. Anti-P450IID6 was found in 6/188 (3.2%) CH patients including 5/104 (4.8%) with hepatitis C virus (C) infection and 1/12 (8.3%) CH-C patients with antibodies to nuclear and smooth muscle antigens and hypergammaglobulinaemia (> 2.5 g/dL). This patient was the only one diagnosed with autoimmune hepatitis (AIH). All CH patients with hepatitis B (B), hepatitis non-B non-C (NBNC) and AIH were seronegative for anti-LKM1. Antibodies to soluble liver antigen were found in two of 188 (1%) patients, one with AIH and one with CH-B. Anti-BCKD-E2 but not anti-PDH-E2 was found in four patients (2.5%), one with AIH, two with CH-C, and one with NBNC. There was no obvious difference in age, sex ratio and laboratory findings in patients with or without anti-SLA and anti-BCKD-E2. Antibodies to P450IID6, SLA, PDH-E2 and BCKD-E2 are uncommon in adult CH-C, CH-B, CH-NBNC and AIH patients in Japan. Some of these patients positive for auto-antibodies appear to have autoimmune features and might require a careful follow up. The heterogeneity of these antibodies in CH preclude further justification for subtyping of AIH by the presence of the distinct auto-antibodies.

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.

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

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

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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.

μ-1,2-Bis(diphenylphosphinoethane-κ2P:P′-bis{[1,2-bis(diphenylphosphinoethane-κ2P,P′]bromidocopper(I} acetone disolvate

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Wen-Juan Shi

2008-10-01

Full Text Available In the crystal structure of the title compound, [Cu2Br2(dppe3]·2CH3COCH3 [dppe is 1,2-bis(diphenylphosphinoethane, C26H24P2], the two Cu centers are bridged by a dppe ligand and each metal center carries one chelating dppe unit, with the fourth coordination site available for the Br− anion. The molecule is centrosymmetric, with the center of symmetry located between the methylene C atoms of the bridging dppe ligand. The crystal structure is stabilized by intramolecular C—H...Br hydrogen bonds and intermolecular π–π interactions, with a centroid-to-centroid distance of 3.2055 (1 Å.

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

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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

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.

Expression and characterization of truncated human heme oxygenase (hHO-1) and a fusion protein of hHO-1 with human cytochrome P450 reductase.

Science.gov (United States)

Wilks, A; Black, S M; Miller, W L; Ortiz de Montellano, P R

1995-04-04

A human heme oxygenase (hHO-1) gene without the sequence coding for the last 23 amino acids has been expressed in Escherichia coli behind the pho A promoter. The truncated enzyme is obtained in high yields as a soluble, catalytically-active protein, making it available for the first time for detailed mechanistic studies. The purified, truncated hHO-1/heme complex is spectroscopically indistinguishable from that of the rat enzyme and converts heme to biliverdin when reconstituted with rat liver cytochrome P450 reductase. A self-sufficient heme oxygenase system has been obtained by fusing the truncated hHO-1 gene to the gene for human cytochrome P450 reductase without the sequence coding for the 20 amino acid membrane binding domain. Expression of the fusion protein in pCWori+ yields a protein that only requires NADPH for catalytic turnover. The failure of exogenous cytochrome P450 reductase to stimulate turnover and the insensitivity of the catalytic rate toward changes in ionic strength establish that electrons are transferred intramolecularly between the reductase and heme oxygenase domains of the fusion protein. The Vmax for the fusion protein is 2.5 times higher than that for the reconstituted system. Therefore, either the covalent tether does not interfere with normal docking and electron transfer between the flavin and heme domains or alternative but equally efficient electron transfer pathways are available that do not require specific docking.

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

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.

Acetaldehyde and parkinsonism: role of CYP450 2E1

Directory of Open Access Journals (Sweden)

Francesca eVaglini

2013-06-01

Full Text Available The present review update the relationship between acetaldehyde and parkinsonism with a specific focus on the role of P450 system and CYP 2E1 isozyme particularly.We have indicated that acetaldehyde is able to enhance the parkinsonism induced in mice by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, a neurotoxin able to damage the nigrostriatal dopaminergic pathway. Similarly diethyldithiocarbamate, the main metabolite of disulfiram, a drug widely used to control alcoholism, diallylsulfide and phenylisothiocyanate also markedly enhance the toxin-related parkinsonism. All these compounds are substrate/inhibitors of CYP450 2E1 isozyme. The presence of CYP 2E1 has been detected in the dopamine neurons of rodent Substantia Nigra, but a precise function of the enzyme has not been elucidated yet. By treating CYP 2E1 knockout mice with the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, the Substantia Nigra induced lesion was significantly reduced when compared with the lesion observed in wild-type animals. Several in vivo and in vitro studies led to the conclusion that CYP 2E1 may enhance the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine toxicity in mice by increasing free radical production inside the dopaminergic neurons. Acetaldehyde is a good substrate for CYP 2E1 enzyme as the other substrate-inhibitors and by this way may facilitate the susceptibility of dopaminergic neurons to toxic events. The literature suggests that ethanol and/or disulfiram may be responsible for toxic parkinsonism in human and it indicates that basal ganglia are the major targets of disulfiram toxicity. A very recent study reports that there are a decreased methylation of the CYP 2E1 gene and increased expression of CYP 2E1 mRNA in Parkinson’s Disease patient brains. This study suggests that epigenetic variants of this cytochrome contribute to the susceptibility, thus confirming multiples lines of evidence which indicate a link between environmental toxins and

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

[1,3-Bis(diphenylphosphinopentane-κ2P,P′]tetracarbonylchromium(0

Directory of Open Access Journals (Sweden)

2009-03-01

Full Text Available In the title compound, [Cr(C29H30P2(CO4], the Cr atom is octahedrally coordinated by four carbonyl ligands and one bidentate phosphine ligand, which is bounded as a chelate in a cis position. The average Cr—P and Cr—C bond lengths are 2.377 and 1.865 Å, respectively.

"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...

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...

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.

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.

Cytochrome P450 2E1 participation in the pathogenesis of experimental metabolic syndrome in guinea pigs

Directory of Open Access Journals (Sweden)

V. V. Rushchak

2016-04-01

Full Text Available In this work the experimental metabolic syndrome on the basis of protamine sulfate modeling in guinea pigs was reproduced and pathological processes in the liver of experimental animals were studied. We determined the level of free radicals and markers of liver damage in the blood of experimental animals. We investigated the liver glycogen content and K+,Na+-ATPase activity in the liver of experimental animals as well as measured the cytochrome P450 2E1 (CYP2E1 expression – one of the main factors of oxidative stress. Evidence of development of hepatotoxic processes, increasing of the CYP2E1 level as well as of the free radical level in the animals with metabolic syndrome were found. Using of CYP2E1 inhibitors had shown that the free radical level in the blood of experimental animals depended on the level of the enzyme expression and activity. The obtained results suggest that the changes in the CYP2E1 expression play an important role in the development of hepatotoxic processes upon experimental metabolic syndrome. It was assumed that pharmacological correction of the enzyme expression may be an important mechanism for the influence on the metabolic syndrome clinical course.

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

Piperine activates human pregnane X receptor to induce the expression of cytochrome P450 3A4 and multidrug resistance protein 1

Energy Technology Data Exchange (ETDEWEB)

Wang, Yue-Ming; Lin, Wenwei; Chai, Sergio C.; Wu, Jing; Ong, Su Sien [Department of Chemical Biology and Therapeutics, St. Jude Children' s Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105 (United States); Schuetz, Erin G. [Department of Pharmaceutical Sciences, St. Jude Children' s Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105 (United States); Chen, Taosheng, E-mail: taosheng.chen@stjude.org [Department of Chemical Biology and Therapeutics, St. Jude Children' s Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105 (United States)

2013-10-01

Activation of the pregnane X receptor (PXR) and subsequently its target genes, including those encoding drug transporters and metabolizing enzymes, while playing substantial roles in xenobiotic detoxification, might cause undesired drug-drug interactions. Recently, an increased awareness has been given to dietary components for potential induction of diet–drug interactions through activation of PXR. Here, we studied, whether piperine (PIP), a major component extracted from the widely-used daily spice black pepper, could induce PXR-mediated expression of cytochrome P450 3A4 (CYP3A4) and multidrug resistance protein 1 (MDR1). Our results showed that PIP activated human PXR (hPXR)-mediated CYP3A4 and MDR1 expression in human hepatocytes, intestine cells, and a mouse model; PIP activated hPXR by recruiting its coactivator SRC-1 in both cellular and cell-free systems; PIP bound to the hPXR ligand binding domain in a competitive ligand binding assay in vitro. The dichotomous effects of PIP on induction of CYP3A4 and MDR1 expression observed here and inhibition of their activity reported elsewhere challenges the potential use of PIP as a bioavailability enhancer and suggests that caution should be taken in PIP consumption during drug treatment in patients, particularly those who favor daily pepper spice or rely on certain pepper remedies. - Highlights: • Piperine induces PXR-mediated CYP3A4 and MDR1 expression. • Piperine activates PXR by binding to PXR and recruiting coactivator SRC-1. • Piperine induces PXR activation in vivo. • Caution should be taken in piperine consumption during drug treatment.

Targeting P-selectin glycoprotein ligand-1/P-selectin interactions as a novel therapy for metabolic syndrome.

Science.gov (United States)

Patel, Madhukar S; Miranda-Nieves, David; Chen, Jiaxuan; Haller, Carolyn A; Chaikof, Elliot L

2017-05-01

Obesity-induced insulin resistance and metabolic syndrome continue to pose an important public health challenge worldwide as they significantly increase the risk of type 2 diabetes and atherosclerotic cardiovascular disease. Advances in the pathophysiologic understanding of this process has identified that chronic inflammation plays a pivotal role. In this regard, given that both animal models and human studies have demonstrated that the interaction of P-selectin glycoprotein ligand-1 (PSGL-1) with P-selectin is not only critical for normal immune response but also is upregulated in the setting of metabolic syndrome, PSGL-1/P-selectin interactions provide a novel target for preventing and treating resultant disease. Current approaches of interfering with PSGL-1/P-selectin interactions include targeted antibodies, recombinant immunoglobulins that competitively bind P-selectin, and synthetic molecular therapies. Experimental models as well as clinical trials assessing the role of these modalities in a variety of diseases have continued to contribute to the understanding of PSGL-1/P-selectin interactions and have demonstrated the difficulty in creating clinically relevant therapeutics. Most recently, however, computational simulations have further enhanced our understanding of the structural features of PSGL-1 and related glycomimetics, which are responsible for high-affinity selectin interactions. Leveraging these insights for the design of next generation agents has thus led to development of a promising synthetic method for generating PSGL-1 glycosulfopeptide mimetics for the treatment of metabolic syndrome. Copyright © 2016 Elsevier Inc. All rights reserved.

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.

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.