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1

Purification and Characterization of Equine Testicular Cytochrome P-450 Aromatase: Comparison with the Human Enzyme  

Microsoft Academic Search

Cytochrome P-450 aromatase was purified by five chromatographic steps from adult stallion testis. It was first separated from NADPH-cytochrome P-450 reductase (reductase) on ?-aminohexyl-Sepharose 4B then purified to homogeneity on concanavalin A-Sepharose 4B, hydroxyapatite-Sepharose 4B, DEAE-Sepharose CL-6B and on a second hydroxyapatite-Sepharose 4B. On the other hand, purifications of the equine testicular and rat liver reductases, which allowed the reconstitution

Safa Moslemi; Alain Vibet; Vassilios Papadopoulos; Luc Camoin; Pierre Silberzahn; Jean-Luc Gaillard

1997-01-01

2

Aromatase Cytochrome P450 and Extragonadal Estrogen Play a Role in Ischemic Neuroprotection  

Microsoft Academic Search

Female animals are protected from many forms of neurological injury and degeneration relative to their male counterparts, in part attributable to their native estrogens. We hypothesized that estradiol aromatized from precursor androgens via the cytochrome P450 aromatase contributes to ischemic neuroprotection in the female. Female homozygous aromatase knock-out (ArKO) mice and randomly cycling, wild-type (WT) female littermates were treated with

Louise D. McCullough; Kathleen Blizzard; Evan R. Simpson; Orhan K. Oz; Patricia D. Hurn

2003-01-01

3

Hormonal and Molecular Regulation of the Cytochrome P450 Aromatase Gene Expression in the Ovary  

Microsoft Academic Search

The cytochrome P450 aromatase (aromatase), encoded by the Cyp19a1 (Cyp19) gene, is the enzyme that converts androgens into estrogens in the ovarian granulosa cells. Estradiol-17? (estradiol), the\\u000a most potent estrogen, is crucial for female and male fertility, as proved by the severe reproductive defects observed when\\u000a its synthesis [1] or actions [2] are blocked. In the ovary, locally produced estradiol

Carlos Stocco

4

Mutagenesis study at a postulated hydrophobic region near the active site of aromatase cytochrome P450.  

PubMed

Aromatase, a cytochrome P450, catalyzes three consecutive hydroxylation reactions converting C19 androgens to aromatic C18 estrogenic steroids. On the basis of a recent computer modeling of the active site of aromatase, a hydrophobic pocket, thought to be important for the binding of some aromatase inhibitors, was predicted to extend roughly in the plane of the steroid substrate, from the position that would be occupied by its C4 and C7 atoms. Four mutants, G121A, I125N, F235N, and I474F, were generated to test this model. Although the mutagenesis results have shown that the current model for the active site of aromatase almost certainly contains a number of errors, the results are in general very satisfactory in that they suggest how the model should be altered by local realignments of the aromatase sequence with that of cytochrome P450cam. Among the mutants, I474F is the most interesting one. Its Km value for androstenedione was found to be lower than the wild type enzyme, and the kinetic analysis exhibited a substrate inhibition-like kinetic profile through an "in-cell" assay. In addition, this mutation reduces the binding affinity of an aromatase inhibitor, 4-hydroxyandrostenedione, and increases the binding affinity of two aromatase inhibitors, aminoglutethimide and CGS 16949. This study demonstrates a useful approach, by a combination of computer modeling, site-directed mutagenesis, and inhibitor binding studies, to examine the structure of the active site of aromatase and the binding nature of various aromatase inhibitors. PMID:8034720

Zhou, D; Cam, L L; Laughton, C A; Korzekwa, K R; Chen, S

1994-07-29

5

Structural basis for the functional roles of critical residues in human cytochrome p450 aromatase.  

PubMed

Cytochrome P450 aromatase (CYP19A1) is the only enzyme known to catalyze the biosynthesis of estrogens from androgens. The crystal structure of human placental aromatase (pArom) has paved the way toward understanding the structure-function relationships of this remarkable enzyme. Using an amino terminus-truncated recombinant human aromatase (rArom) construct, we investigate the roles of key amino acids in the active site, at the intermolecular interface, inside the access channel, and at the lipid-protein boundary for their roles in enzyme function and higher-order organization. Replacing the active site residue D309 with an N yields an inactive enzyme, consistent with its proposed involvement in aromatization. Mutation of R192 at the lipid interface, pivotal to the proton relay network in the access channel, results in the loss of enzyme activity. In addition to the distal catalytic residues, we show that mutation of K440 and Y361 of the heme-proximal region critically interferes with substrate binding, enzyme activity, and heme stability. The D-E loop deletion mutant Del7 that disrupts the intermolecular interaction significantly reduces enzyme activity. However, the less drastic Del4 and point mutants E181A and E181K do not. Furthermore, native gel electrophoresis, size-exclusion chromatography, and analytical ultracentrifugation are used to show that mutations in the intermolecular interface alter the quaternary organization of the enzyme in solution. As a validation for interpretation of the mutational results in the context of the innate molecule, we determine the crystal structure of rArom to show that the active site, tertiary, and quaternary structures are identical to those of pArom. PMID:23899247

Lo, Jessica; Di Nardo, Giovanna; Griswold, Jennifer; Egbuta, Chinaza; Jiang, Wenhua; Gilardi, Gianfranco; Ghosh, Debashis

2013-08-16

6

Adult male rat hippocampus synthesizes estradiol from pregnenolone by cytochromes P45017 and P450 aromatase localized in neurons  

Microsoft Academic Search

In adult mammalian brain, occurrence of the synthesis of estradiol from endogenous cholesterol has been doubted because of the inability to detect dehydroepiandrosterone synthase, P45017. In adult male rat hippocampal formation, significant localization was demonstrated for both cytochromes P45017 and P450 aromatase, in pyramidal neurons in the CA1-CA3 regions, as well as in the granule cells in the dentate gyrus,

Yasushi Hojo; Taka-Aki Hattori; Taihei Enami; Aizo Furukawa; Kumiko Suzuki; Hiro-Taka Ishii; Hideo Mukai; John H. Morrison; William G. M. Janssen; Shiro Kominami; Nobuhiro Harada; Tetsuya Kimoto; Suguru Kawato

2004-01-01

7

Porcine Hypothalamic Aromatase Cytochrome P450: Isoform Characterization, Sex-Dependent Activity, Regional Expression, and Regulation by Enzyme Inhibition in Neonatal Boars  

Technology Transfer Automated Retrieval System (TEKTRAN)

Domestic pigs have three CYP19 genes encoding functional paralogues of the enzyme aromatase cytochrome P450 (P450arom) that are expressed in the gonads, placenta and pre-implantation blastocyst. All catalyze estrogen synthesis, but the “gonadal” type enzyme is unique in also synthesizing a nonaromat...

8

Cytochromes P450  

PubMed Central

There are 272 cytochrome P450 genes (including 26 pseudogenes) in the Arabidopsis genome. P450s thus form one of the largest families of proteins in higher plants. This explosion of the P450 family is thought to have occurred via gene duplication and conversion, and to result from the need of sessile plants to adapt to a harsh environment and to protect themselves from pathogens and predators. P450s sometimes share less than 20% identity and catalyze extremely diverse reactions. Their biological functions range from the synthesis of structural macromolecules such as lignin, cutin or suberin, to the synthesis or catabolism of all types of hormone or signaling molecules, the synthesis of pigments and defense compounds, and to the metabolism of xenobiotics. In despite of a huge acceleration in our understanding of plant P450 functions in the recent years, the vast majority of these functions remain completely unknown.

Werck-Reichhart, Daniele; Bak, S?ren; Paquette, Suzanne

2002-01-01

9

Cytochrome P450 enzymes  

Microsoft Academic Search

Cytochrome P450 enzymes are proving to be the stalwarts of the detoxication system in the human body, and in other biological systems. They are found in every type of cell within the body (except red blood cells and skeletal muscle cells), and they are found in every biological kingdom including bacteria. Cytochrome enzymes appear to defend the body against environmental

Guengerich

2009-01-01

10

The final catalytic step of cytochrome p450 aromatase: a density functional theory study.  

PubMed

B3LYP density functional theory calculations are used to unravel the mysterious third step of aromatase catalysis. The feasibility of mechanisms in which the reduced ferrous dioxygen intermediate mediates androgen aromatization is explored and determined to be unlikely. However, proton-assisted homolysis of the peroxo hemiacetal intermediate to produce P450 compound I and the C19 gem-diol likely proceeds with a low energetic barrier. Mechanisms for the aromatization and deformylation sequence which are initiated by 1beta-hydrogen atom abstraction by P450 compound I are considered. 1beta-Hydrogen atom abstraction from substrates in the presence of the 2,3-enol encounters strikingly low barriers (5.3-7.8 kcal/mol), whereas barriers for this same process rise to 17.0-27.1 kcal/mol in the keto tautomer. Transition states for 1beta-hydrogen atom abstraction from enolized substrates in the presence of the 19-gem-diol decayed directly to the experimentally observed products. If the C19 aldehyde remains unhydrated, aromatization occurs with concomitant decarbonylation and therefore does not support dehydration of the C19 aldehyde prior to the final catalytic step. On the doublet surface, the transition state connects to a potentially labile 1(10) dehydrogenated product, which may undergo rapid aromatization, as well as formic acid. Ab initio molecular dynamics confirmed that the 1beta-hydrogen atom abstraction and deformylation or decarbonylation occur in a nonsynchronous, coordinated manner. These calculations support a dehydrogenase behavior of aromatase in the final catalytic step, which can be summarized by 1beta-hydrogen atom abstraction followed by gem-diol deprotonation. PMID:15810858

Hackett, John C; Brueggemeier, Robert W; Hadad, Christopher M

2005-04-13

11

Effects of organochlorine compounds on cytochrome P450 aromatase activity in an immortal sea turtle cell line.  

PubMed

Many classes of environmental contaminants affect the reproductive function of animals through interactions with the endocrine system. The primary components affected by endocrine active compounds (EACs) are the steroid receptors and the enzymes responsible for steroidogenesis. This study sought to develop an in vitro model for assessing EAC effects in sea turtles by examining their ability to alter cytochrome P450 aromatase (CYP19) activity. Aromatase is the enzyme responsible for the conversion of testosterone to estradiol. This enzyme is critical in the sexual differentiation of reptiles which demonstrate temperature-dependent sex determination. An immortal testis cell line GST-TS from a green sea turtle was grown in culture at 30 degrees C in RPMI 1640 media. The cells were exposed to three known aromatase inducers; dexamethasone (Dex), 8Br-cyclic AMP, or human chronic gonadotropin (HCG) and one aromatase inhibitor 4-androstenol-dione (4-OHA). In addition, the GST-TS cells were exposed to 0.1-30 microM atrazine and 3-100 microM 4,4'-DDE. The inducing compounds that have been shown to increase aromatase activity in other systems failed to induce aromatase activity in the GST-TS cells, yet exposure to the inhibiting compound, 4-OHA, did result in a significant reduction. Atrazine (0.1, 1.0 and 10 microM) significantly induced aromatase activity following a 24 h exposure, and 4,4'-DDE inhibited the activity but only at cytotoxic concentrations (100 microM). Based on these results, this in vitro model can be useful in examining the endocrine effects of EACs in sea turtles. PMID:15178053

Keller, Jennifer M; McClellan-Green, Patricia

12

Isolation and characterization of two cytochrome P450 aromatase forms in killifish ( Fundulus heteroclitus): Differential expression in fish from polluted and unpolluted environments  

Microsoft Academic Search

Populations of killifish (Fundulus heteroclitus) persist in many different highly polluted environment indicative of adaptation or tolerance. In this study, we sought to determine whether long term, multigenerational exposures to environmental contaminants has affected reproductively relevant genes and biological processes. A homology cloning strategy was used to isolate the killifish cytochrome P450 aromatase (P450arom, estrogen synthetase) cDNAs. Consistent with previous

Sarah R. Greytak; Denise Champlin; Gloria V. Callard

2005-01-01

13

Equine cytochrome P450 aromatase exhibits an estrogen 2-hydroxylase activity in vitro  

Microsoft Academic Search

Aromatase (estrogen synthetase) is a steroidogenic enzyme complex which catalyzes the conversion of androgens to estrogens (termed aromatization). This enzyme was purified from adult equine testis to homogeneity by five chromatographic steps. The ability of purified and reconstituted equine aromatase to exhibit an estrogen 2-hydroxylase activity was tested and compared to testosterone aromatization. Enzymatic activities were assessed by tritiated water

Jamal Almadhidi; Safa Moslemi; Michel A. Drosdowsky; Gilles-Eric Séralini

1996-01-01

14

Towards a physiological role for cytochrome P450 aromatase in ejaculated human sperm  

Microsoft Academic Search

BACKGROUND: Advances in the definition of the function and the mechanism of estrogen action in different tis- sues have come from human and animal models of estrogen insufficiency. Recently we have demonstrated that aromatase is present and biologically active in human ejaculated sperm, suggesting that autonomous estradiol sperm production may influence sperm functions. In the present study we investigate a

Saveria Aquila; Diego Sisci; Mariaelena Gentile; Amalia Carpino; Emilia Middea; Stefania Catalano; Vittoria Rago

2003-01-01

15

The Cytochrome P450 Homepage  

PubMed Central

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.

2009-01-01

16

Effects of xenoestrogens on the expression of vitellogenin (vtg) and cytochrome P450 aromatase (cyp19a and b) genes in zebrafish (Danio rerio) larvae  

Microsoft Academic Search

In the present study, expression levels of vitellogenin (vtg) and cytochrome P450 aromatase genes (cyp19a and cyp19b) in zebrafish larvae during the early stages of development were investigated by quantitative real time-PCR assay. The results indicated that vtg gene transcription was induced seven days after zebrafish larvae fertilization, whereas the expression of cyp19a and cyp19b genes was detected as early

Jingxian Wang; Xiongjie Shi; Yongbin Du; Bingsheng Zhou

2011-01-01

17

Expression of two cytochrome P450 aromatase genes is regulated by endocrine disrupting chemicals in rare minnow Gobiocypris rarus juveniles  

Microsoft Academic Search

To elucidate the effects of endocrine disrupting chemicals (EDCs) on aromatase, the rare minnow ovarian and brain P450 aromatase (cyp19a1a and cyp19a1b) cDNA and their 5?-flanking regions were isolated and characterized. RT-PCR analysis revealed that the rare minnow cyp19a1a mRNA was predominantly expressed in ovary while cyp19a1b was predominantly expressed in brain. Sequences for binding sites of steroidogenic factor-1, peroxisome

Jingjing Wang; Xiaolin Liu; Houpeng Wang; Tingting Wu; Xiaoqi Hu; Fang Qin; Zaizhao Wang

2010-01-01

18

Fungal cytochrome P450 database  

PubMed Central

Background Cytochrome P450 enzymes play critical roles in fungal biology and ecology. To support studies on the roles and evolution of cytochrome P450 enzymes in fungi based on rapidly accumulating genome sequences from diverse fungal species, an efficient bioinformatics platform specialized for this super family of proteins is highly desirable. Results The Fungal Cytochrome P450 Database (FCPD) archives genes encoding P450s in the genomes of 66 fungal and 4 oomycete species (4,538 in total) and supports analyses of their sequences, chromosomal distribution pattern, and evolutionary histories and relationships. The archived P450s were classified into 16 classes based on InterPro terms and clustered into 141 groups using tribe-MCL. The proportion of P450s in the total proteome and class distribution in individual species exhibited certain taxon-specific characteristics. Conclusion The FCPD will facilitate systematic identification and multifaceted analyses of P450s at multiple taxon levels via the web. All data and functions are available at the web site .

Park, Jongsun; Lee, Seungmin; Choi, Jaeyoung; Ahn, Kyohun; Park, Bongsoo; Park, Jaejin; Kang, Seogchan; Lee, Yong-Hwan

2008-01-01

19

Molecular biology of channel catfish brain cytochrome P450 aromatase (CYP19A2): cloning, preovulatory induction of gene expression, hormonal gene regulation and analysis of promoter region  

Microsoft Academic Search

Cytochrome P450 aromatase (CYP19) converts androgens to estrogens. Unlike mammals, teleosts have two CYP19 genes, expressed differentially in ovary (CYP19A1) and neuronal tissues (CYP19A2). The primary purpose of this study was to demonstrate the potential involvement of CYP19A2 in the reproductive endocrinology of teleosts. Channel catfish CYP19A2 (ccCYP19A2) cDNAs were isolated from the brain using a PCR-based strategy. The ccCYP19A2

Y Kazeto; J M Trant

2005-01-01

20

Cytochrome P450 (CYP450) Tests  

MedlinePLUS

Cytochrome P450 (CYP450) tests Basics Resources Reprints A single copy of this article may be reprinted for personal, noncommercial use only. Cytochrome P450 (CYP450) tests By Mayo Clinic staff Original Article: ...

21

Characterization and expression profile of the ovarian cytochrome P-450 aromatase (cyp19A1) gene during thermolabile sex determination in Pejerrey, Odontesthes bonariensis  

USGS Publications Warehouse

Cytochrome P450 aromatase (cyp19) is an enzyme that catalyzes the conversion of androgens to estrogens and may play a role in temperature- dependent sex determination (TSD) of reptiles, amphibians, and fishes. In this study, the ovarian P450 aromatase form (cyp19A1) of pejerrey Odontesthes bonariensis, a teleost with marked TSD, was cloned and its expression profile evaluated during gonadal differentiation at feminizing (17??C, 100% females), mixed-sex producing (24 and 25??C, 73.3 and 26.7% females, respectively), and masculinizing (29??C, 0% females) temperatures. The deduced cyp19A1 amino acid sequence shared high identity (>77.8%) with that from other teleosts but had low identity (<61.8%) with brain forms (cyp19A2), including that of pejerrey itself. The tissue distribution analysis of cyp19A1 mRNA in adult fish revealed high expression in the ovary. Semi-quantitative reverse transcription polymerase chain reaction analysis of the bodies of larvae revealed that cyp19A1 expression increased before the appearance of the first histological signs of ovarian differentiation at the feminizing temperature but remained low at the masculinizing temperature. The expression levels at mixed-sex producing temperatures were bimodal rather than intermediate, showing low and high modal values similar to those at the feminizing and masculinizing temperatures, respectively. The population percentages of high and low expression levels at intermediate temperatures were proportional to the percentage of females and males, respectively, and high levels were first observed at about the time of sex differentiation of females. These results suggest that cyp19A1 is involved in the process of ovarian formation and possibly also in the TSD of pejerrey. ?? 2007 Wiley-Liss, Inc.

Karube, M.; Fernandino, J. I.; Strobl-Mazzulla, P.; Strussmann, C. A.; Yoshizaki, G.; Somoza, G. M.; Patino, R.

2007-01-01

22

Reactive intermediates in cytochrome p450 catalysis.  

PubMed

Recently, we reported the spectroscopic and kinetic characterizations of cytochrome P450 compound I in CYP119A1, effectively closing the catalytic cycle of cytochrome P450-mediated hydroxylations. In this minireview, we focus on the developments that made this breakthrough possible. We examine the importance of enzyme purification in the quest for reactive intermediates and report the preparation of compound I in a second P450 (P450ST). In an effort to bring clarity to the field, we also examine the validity of controversial reports claiming the production of P450 compound I through the use of peroxynitrite and laser flash photolysis. PMID:23632017

Krest, Courtney M; Onderko, Elizabeth L; Yosca, Timothy H; Calixto, Julio C; Karp, Richard F; Livada, Jovan; Rittle, Jonathan; Green, Michael T

2013-04-30

23

The Cytochrome P450 Superfamily of Monooxygenases  

Microsoft Academic Search

Cytochrome P450 monooxygenases (P450) are encoded by a superfamily of genes that is ubiquitously present in bacteria, animals\\u000a and plants. Plants have many different P450s and use them for biosynthesis and for detoxification. Plant P450s function in\\u000a primary and secondary metabolism and are involved in biosynthesis of hormons and signalling molecules.

Alfons Gierl

24

Cytochrome P450 aromatase in grey mullet: cDNA and promoter isolation; brain, pituitary and ovarian expression during puberty  

Microsoft Academic Search

In a study towards elucidating the role of aromatases during puberty in female grey mullet, the cDNAs of the brain (muCyp19b) and ovarian (muCyp19a) aromatase were isolated by RT-PCR and their relative expression levels were determined by quantitative real-time RT-PCR. The muCyp19a ORF of 1515bp encoded 505 predicted amino acid residues, while that of muCyp19b was 1485bp and encoded 495

Josephine N. Nocillado; Abigail Elizur; Ayelet Avitan; Frank Carrick; Berta Levavi-Sivan

2007-01-01

25

Cytochrome P450 Isoenzymes: Nursing Considerations  

Microsoft Academic Search

Recent studies in pharmacology have focused on liver enzyme activity in the biotransformation of drugs. Among the 34 hepatic cytochrome P450 isoenzymes identifed to date, several have been found to be important in the metabolism of many psychoactive drugs and other agents. This article describes the role of cytochrome P450 isoenzymes in drug metabolism and potential drug-drug interactions and addresses

Maureen Applegate

1999-01-01

26

Cytochrome P450 isoenzymes: Nursing considerations  

Microsoft Academic Search

Recent studies in pharmacology have focused on liver enzyme activity in the biotransformation of drugs. Among the 34 hepatic cytochrome P450 isoenzymes identified to date, several have been found to be important in the metabolism of many psychoactive drugs and other agents. This article describes the role of cytochrome P450 isoenzymes in drug metabolism and potential drug-drug interactions and addresses

Maureen Applegate

1999-01-01

27

Cloning and developmental expression of the cytochrome P450 aromatase gene (CYP19) in the European eel ( Anguilla anguilla)  

Microsoft Academic Search

To characterize the involvement of the aromatase gene during the process of sex determination in the European eel (Anguilla anguilla), the expression of its gonadal form was determined during various developmental stages. The cloned cDNA from the European eel gonad (EeCYP19) contains an open reading frame of 1539bp, encoding a deduced protein of 513 residues. The predicted amino acid sequence

Itai Tzchori; Gad Degani; Avshalom Hurvitz; Boaz Moav

2004-01-01

28

Cytochromes p450: roles in diseases.  

PubMed

The cytochrome P450 superfamily consists of a large number of heme-containing monooxygenases. Many human P450s metabolize drugs used to treat human diseases. Others are necessary for synthesis of endogenous compounds essential for human physiology. In some instances, alterations in specific P450s affect the biological processes that they mediate and lead to a disease. In this minireview, we describe medically significant human P450s (from families 2, 4, 7, 11, 17, 19, 21, 24, 27, 46, and 51) and the diseases associated with these P450s. PMID:23632021

Pikuleva, Irina A; Waterman, Michael R

2013-04-30

29

Unusual cytochrome p450 enzymes and reactions.  

PubMed

Cytochrome P450 enzymes primarily catalyze mixed-function oxidation reactions, plus some reductions and rearrangements of oxygenated species, e.g. prostaglandins. Most of these reactions can be rationalized in a paradigm involving Compound I, a high-valent iron-oxygen complex (FeO(3+)), to explain seemingly unusual reactions, including ring couplings, ring expansion and contraction, and fusion of substrates. Most P450s interact with flavoenzymes or iron-sulfur proteins to receive electrons from NAD(P)H. In some cases, P450s are fused to protein partners. Other P450s catalyze non-redox isomerization reactions. A number of permutations on the P450 theme reveal the diversity of cytochrome P450 form and function. PMID:23632016

Guengerich, F Peter; Munro, Andrew W

2013-04-30

30

Novel extrahepatic cytochrome P450s  

Microsoft Academic Search

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

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

2005-01-01

31

A world of cytochrome P450s.  

PubMed

The world we live in is a biosphere influenced by all organisms who inhabit it. It is also an ecology of genes, with some having rather startling effects. The premise put forth in this issue is cytochrome P450 is a significant player in the world around us. Life and the Earth itself would be visibly different and diminished without cytochrome P450s. The contributions to this issue range from evolution on the billion year scale to the colour of roses, from Darwin to Rachel Carson; all as seen through the lens of cytochrome P450. PMID:23297353

Nelson, David R

2013-01-06

32

Differentiation of chicken gonad as an endocrine organ: expression of LH receptor, FSH receptor, cytochrome P450c17 and aromatase genes.  

PubMed

The gonad is an endocrine organ secreting sex hormones and also a target of pituitary gonadotrophins. The expression of mRNAs encoding LH receptor (LHR), FSH receptor (FSHR), P450c17 and P450aromatase in the developing gonads of embryos between day 4 and day 6 of incubation was determined using a RT-PCR to elucidate the chicken gonad as a target organ of gonadotrophins. Although expression of mRNAs encoding LHR, FSHR and P450c17 was detected at day 4 of incubation in both sexes, mRNA encoding P450aromatase appeared at day 6 in female embryos only, indicating that mRNAs encoding gonadotrophin receptors can be identified before sexual differentiation. Quantitative PCR analysis revealed that expression of mRNA encoding LHR and FSHR remained low in male gonads from day 4 to day 6 of incubation, whereas they increased on day 6 in female gonads. The sexual dimorphism in the expression of mRNAs encoding LHR and FSHR was confirmed in the sexually differentiated gonads of embryos at day 12 of incubation (LHR in ovary ratio LHR in testis = 7 ratio 1; FSHR in ovary ratio FSHR in testis = 9 ratio 1). PMID:12006100

Akazome, Y; Abe, T; Mori, T

2002-05-01

33

Genetics Home Reference: Cytochrome P450 oxidoreductase deficiency  

MedlinePLUS

... Recent literature OMIM Genetic disorder catalog Conditions > Cytochrome P450 oxidoreductase deficiency On this page: Description Genetic changes ... Glossary definitions Reviewed December 2009 What is cytochrome P450 oxidoreductase deficiency? Cytochrome P450 oxidoreductase deficiency is a ...

34

Cytochrome P450 1A2  

Center for Drug Evaluation (CDER)

... Vaccines, Blood & Biologics; Animal & Veterinary; Cosmetics; Tobacco Products. Drugs. ... Cytochrome P450 1A2. Induced by smoking tobacco. ... More results from www.fda.gov/drugs/developmentapprovalprocess/developmentresources

35

Cytochrome P450 2C19  

Center for Drug Evaluation (CDER)

... Cytochrome P450 2C19. Absent in 20–30% of Asians, 3–5% Caucasians. Primary metabolism of: Diazepam; Phenytoin; Omeprazole. Inhibited by: ... More results from www.fda.gov/drugs/developmentapprovalprocess/developmentresources

36

Cytochrome P450 2S1 is Reduced by NADPH-Cytochrome P450 Reductase  

PubMed Central

Cytochrome P450 (P450) 2S1 is one of the orphan P450s without a clear physiological function. Controversy has arisen as to whether it can interact with NADPH-P450 reductase and accept electrons. The reduction of 1,4-bis{[2-(dimethylamino-N-oxide)ethyl]amino}-5,8-dihydroxyanthracene-9,10-dione (AQ4N) by P450 2S1 was confirmed, and the NADPH consumption rates were measured aerobically and anaerobically in the absence and presence of the drug. The reduction kinetics of P450 2S1 were rapid, as measured by stopped-flow kinetics. These results confirm that P450 2S1 can be reduced by NADPH-P450 reductase and suggest normal mixed-function oxidase roles of P450 2S1 to be revealed.

Xiao, Yi; Shinkyo, Raku

2011-01-01

37

Seasonal changes in spermatogenesis and immunolocalization of cytochrome P450 17alpha-hydroxylase/c17-20 lyase and cytochrome P450 aromatase in the wild male ground squirrel (Citellus dauricus Brandt).  

PubMed

The purpose of this study was to investigate seasonal changes of spermatogenesis and the cellular localization of P450c17 and P450arom in wild male ground squirrels during the breeding and non-breeding seasons. The testicular weight, testicular size and score count of spermatogenesis from April to September were measured, and histological and immunohistochemical observations of testicular tissues were performed in wild male ground squirrels. In addition, total proteins were extracted from testicular tissue in the breeding and non-breeding seasons and were used for Western blotting analysis for P450c17 and P450arom. There were marked variations in testicular weight, testicular size and score count of spermatogenesis from the breeding season (April) to the non-breeding season (September). Histologically, spermatogonia, primary spermatocytes, secondary spermatocytes and spermatozoa were identified in the breeding season (April). Immunolocalization of P450c17 was detected in Leydig cells and spermatozoa during the breeding season and was only found in Leydig cells during the non-breeding season. The positive signals of P450c17 by Western blotting were both observed in the breeding and non-breeding seasons. Immunolocalization of P450arom was observed in Leydig cells, Sertoli cells and all types of spermatogenic cells including mature-phase spermatozoa in the breeding season, while immunoreactivity for P450arom was not present in the testis of the non-breeding season. With P450arom antibody, a band was also only detected in the breeding season by Western blotting. These results suggest that the seasonal changes in testicular weight and size are correlated with spermatogenesis and immunolocalization of P450c17 and P450arom, and androgen and estrogen may play an important role in the spermatogenesis and testicular recrudescence and regression process. PMID:20197644

Zhang, Haolin; Sheng, Xia; Hu, Xiao; Li, Xiuwen; Xu, Hui; Zhang, Mengyuan; Li, Ben; Xu, Meiyu; Weng, Qiang; Zhang, Zhixiang; Taya, Kazuyoshi

2010-03-03

38

Cytochromes P450 and insecticide resistance  

Microsoft Academic Search

The cytochrome P450-dependent monooxygenases (monooxygenases) are an extremely important metabolic system involved in the catabolism and anabolism of xenobiotics and endogenous compounds. Monooxygenase-mediated metabolism is a common mechanism by which insects become resistant to insecticides as evidenced by the numerous insect species and insecticides affected. This review begins by presenting background information about P450s, the role of monooxygenases in insects,

Jeffrey G. Scott

1999-01-01

39

Cytochrome P450s and molecular epidemiology  

NASA Astrophysics Data System (ADS)

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.

Gonzalez, Frank J.; Gelboin, Harry V.

1993-03-01

40

Cooperative properties of cytochromes P450  

PubMed Central

Cytochromes P450 form a large and important class of heme monooxygenases with a broad spectrum of substrates and corresponding functions, from steroid hormone biosynthesis to the metabolism of xenobiotics. Despite decades of study, the molecular mechanisms responsible for the complex non-Michaelis behavior observed with many members of this super-family during metabolism, often termed ‘cooperativity,’ remain to be fully elucidated. Although there is evidence that oligomerization may play an important role in defining the observed cooperativity, some monomeric cytochromes P450, particularly those involved in xenobiotic metabolism, also display this behavior due to their ability to simultaneously bind several substrate molecules. As a result, formation of distinct enzyme-substrate complexes with different stoichiometry and functional properties can give rise to homotropic and heterotropic cooperative behavior. This review aims to summarize the current understanding of cooperativity in cytochromes P450, with a focus on the nature of cooperative effects in monomeric enzymes.

Denisov, Ilia G.; Frank, Daniel J.; Sligar, Stephen G.

2009-01-01

41

The biodiversity of microbial cytochromes P450  

Microsoft Academic Search

The cytochrome P450 (CYP) superfamily of genes and proteins are well known for their involvement in pharmacology and toxicology, but also increasingly for their importance and diversity in microbes. The extent of diversity has only recently become apparent with the emergence of data from whole genome sequencing projects and the coming years will reveal even more information on the diversity

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

2003-01-01

42

Intronic polymorphisms of cytochromes P450  

PubMed Central

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

2010-01-01

43

Structure and Function of Cytochrome P-450 Genes.  

National Technical Information Service (NTIS)

The cytochrome P450IIC2 (previously designated P-450PBc) subfamily of the phenobarbital-inducible family of cytrochrome P-450 contains several closely related members. The authors have reported previously the characterization of complementary deoxyribonuc...

B. Kemper

1988-01-01

44

THE HUMAN INTESTINAL CYTOCHROME P450 "PIE"  

PubMed Central

Cytochromes P450 (P450s) 3A, 2C, and 1A2 constitute the major “pieces” of the human liver P450 “pie” and account, on average, for 40, 25, and 18%, respectively, of total immunoquantified P450s (J Pharmacol Exp Ther 270:414–423, 1994). The P450 profile in the human small intestine has not been fully characterized. Therefore, microsomes prepared from mucosal scrapings from the duodenal/jejunal portion of 31 human donor small intestines were analyzed by Western blot using selective P450 antibodies. P450s 3A4, 2C9, 2C19, and 2J2 were detected in all individuals and ranged from 8.8 to 150, 2.9 to 27, <0.6 to 3.9, and <0.2 to 3.1 pmol/mg, respectively. CYP2D6 was detected in 29 individuals and ranged from <0.2 to 1.4 pmol/mg. CYP3A5 was detected readily in 11 individuals, with a range (average) of 4.9 to 25 (16) pmol/mg that represented from 3 to 50% of total CYP3A (CYP3A4 + CYP3A5) content. CYP1A1 was detected readily in three individuals, with a range (average) of 3.6 to 7.7 (5.6) pmol/mg. P450s 1A2, 2A6, 2B6, 2C8, and 2E1 were not or only faintly detected. As anticipated, average CYP3A content (50 pmol/mg) was the highest. Excluding CYP1A1, the remaining enzymes had the following rank order: 2C9 > 2C19 > 2J2 > 2D6 (8.4, 1.1, 0.9, and 0.5 pmol/mg, respectively). Analysis of a pooled preparation of the 31 donor specimens substantiated these results. In summary, as in the liver, large interindividual variation exists in the expression levels of individual P450s. On average, CYP3A and CYP2C9 represents the major pieces of the intestinal P450 pie, accounting for 80 and 15%, respectively, of total immunoquantified P450s.

Paine, Mary F.; Hart, Heather L.; Ludington, Shana S.; Haining, Robert L.; Rettie, Allan E.; Zeldin, Darryl C.

2007-01-01

45

Cytochrome P450 (CYP) in fish.  

PubMed

Cytochrome P450 (CYP) enzymes are members of the hemoprotein superfamily, and are involved in the mono-oxygenation reactions of a wide range of endogenous and exogenous compounds in mammals and plants. Characterization of CYP genes in fish has been carried out intensively over the last 20 years. In Japanese pufferfish (Takifugu rubripes), 54 genes encoding P450s have been identified. Across all species of fish, 137 genes encoding P450s have been identified. These genes are classified into 18 CYP families: namely, CYP1, CYP2, CYP3, CYP4, CYP5, CYP7, CYP8, CYP11, CYP17, CYP19, CYP20, CYP21, CYP24, CYP26, CYP27, CYP39, CYP46 and CYP51.We pinpointed eight CYP families: namely, CYP1, CYP2, CYP3, CYP4, CYP11, CYP17, CYP19 and CYP26 in this review because these CYP families are studied in detail. Studies of fish P450s have provided insights into the regulation of P450 genes by environmental stresses including water pollution. In this review, we present an overview of the CYP families in fish. PMID:22418068

Uno, Tomohide; Ishizuka, Mayumi; Itakura, Takao

2012-02-17

46

Flower colour and cytochromes P450  

Microsoft Academic Search

Flavonoids are major constituents of flower colour. Plants accumulate specific flavonoids and thus every species often exhibits a limited flower colour range. Three cytochromes P450 play critical roles in the flavonoid biosynthetic pathway. Flavonoid 3?-hydroxylase (F3?H, CYP75B) and flavonoid 3?,5?-hydroxylase (F3?5?H, CYP75A) catalyze the hydroxylation of the B-ring of flavonoids and are necessary to biosynthesize cyanidin-(red to magenta) and delphinidin-(violet

Yoshikazu Tanaka

2006-01-01

47

Bioluminescent assays for cytochrome P450 enzymes.  

PubMed

The cytochrome P450 (CYP) family contains 57 enzymes in humans. The activity of CYPs against xenobiotics is a primary consideration in drug optimization efforts. Here we describe a series of bioluminescent assays that enable the rapid profiling of CYP activity against compound collections. The assays employ a coupled-enzyme format where firefly luciferase is used to measure CYP enzyme activity through metabolism of pro-luciferase substrates. PMID:23475663

Auld, Douglas S; Veith, Henrike; Cali, James J

2013-01-01

48

Testosterone, cytochrome P450, and cardiac hypertrophy  

Microsoft Academic Search

Cytochrome P450 mono-oxygenases (CYP) play an essential role in steroid metabolism, and there is speculation that sex hormones might influence car- diac mass and physiology. As CYP mono-oxygenases activity is frequently altered during disease, we tested our hypothesis that CYP mono-oxygenase expression and testosterone metabolism are altered in cardiac hypertrophy. We investigate major CYP mono-oxygen- ase isoforms and other steroid-metabolizing

THOMAS THUM; JURGEN BORLAK

2002-01-01

49

Novel extrahepatic cytochrome P450s  

SciTech Connect

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.

Karlgren, Maria [Division of Molecular Toxicology, Institute of Environmental Medicine, Karolinska Institutet, SE-171 77 Stockholm (Sweden)]. E-mail: Maria.Karlgren@imm.ki.se; Miura, Shin-ichi [Division of Molecular Toxicology, Institute of Environmental Medicine, Karolinska Institutet, SE-171 77 Stockholm (Sweden); Ingelman-Sundberg, Magnus [Division of Molecular Toxicology, Institute of Environmental Medicine, Karolinska Institutet, SE-171 77 Stockholm (Sweden)

2005-09-01

50

Novel extrahepatic cytochrome P450s.  

PubMed

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. PMID:15987645

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

2005-09-01

51

Formation of Indigo by Recombinant Mammalian Cytochrome P450  

Microsoft Academic Search

The development of bicistronic systems for coexpression of recombinant human cytochrome P450 enzymes (P450s) with their redox partner, NADPH-cytochrome P450 reductase (NPR), has enabled P450 activity to be reconstituted within bacterial cells. During expression of recombinant P450 2E1 and some other forms, we observed the formation of a blue pigment in bacterial cultures. The pigment was extracted from cultures and

Elizabeth M. J. Gillam; Anna Marie A. Aguinaldo; Lisa M. Notley; Donghak Kim; Ralf G. Mundkowski; Alexander A. Volkov; Frances H. Arnold; Pavel Sou?ek; James J. DeVoss; F. Peter Guengerich

1999-01-01

52

Organization of Cytochrome P450 Enzymes Involved in Sex Steroid Synthesis  

PubMed Central

Mounting evidence underscores the importance of protein-protein interactions in the functional regulation of drug-metabolizing P450s, but few studies have been conducted in membrane environments, and none have examined P450s catalyzing sex steroid synthesis. Here we report specific protein-protein interactions for full-length, human, wild type steroidogenic cytochrome P450 (P450, CYP) enzymes: 17?-hydroxylase/17,20-lyase (P450c17, CYP17) and aromatase (P450arom, CYP19), as well as their electron donor NADPH-cytochrome P450 oxidoreductase (CPR). Fluorescence resonance energy transfer (FRET)3 in live cells, coupled with quartz crystal microbalance (QCM), and atomic force microscopy (AFM) studies on phosphatidyl choline ± cholesterol (mammalian) biomimetic membranes were used to investigate steroidogenic P450 interactions. The FRET results in living cells demonstrated that both P450c17 and P450arom homodimerize but do not heterodimerize, although they each heterodimerize with CPR. The lack of heteroassociation between P450c17 and P450arom was confirmed by QCM, wherein neither enzyme bound a membrane saturated with the other. In contrast, the CPR bound readily to either P450c17- or P450arom-saturated surfaces. Interestingly, N-terminally modified P450arom was stably incorporated and gave similar results to the wild type, although saturation was achieved with much less protein, suggesting that the putative transmembrane domain is not required for membrane association but for orientation. In fact, all of the proteins were remarkably stable in the membrane, such that high resolution AFM images were obtained, further supporting the formation of P450c17, P450arom, and CPR homodimers and oligomers in lipid bilayers. This unique combination of in vivo and in vitro studies has provided strong evidence for homodimerization and perhaps some higher order interactions for both P450c17 and P450arom.

Praporski, Slavica; Ng, Su May; Nguyen, Ann D.; Corbin, C. Jo; Mechler, Adam; Zheng, Jie; Conley, Alan J.; Martin, Lisandra L.

2009-01-01

53

Colocalization of p450 aromatase and oxytocin immunostaining in the rat hypothalamus.  

PubMed

With combined immunoperoxidase and immunofluorescence, we observed colocalization of cytochrome P450 aromatase with the posterior lobe peptide oxytocin and its associated neurophysin 1 in adult male rats. P450 was most abundant in the anterior hypothalamus. Colocalization of OT with P450 was observed in the preoptic region, the periventricular nucleus of the hypothalamus, the lateral subcommissural nucleus, and in the zona incerta. Magnocellular perikarya in the supraoptic and in the paraventricular nuclei contained only occasionally both antigens. P450 immunostaining overlapped to a great extent with known estrogen target regions. Oxytocinergic functions are controlled by estradiol while androgen receptors are mostly absent in neuroendocrine hypothalamic nuclei. Our findings suggest that systemic androgens may be aromatized to estrogens in male oxytocinergic neurons linked to the limbic system. PMID:23225240

El-Emam Dief, A; Caldwell, J D; Jirikowski, G F

2012-12-07

54

Aldehyde Reduction by Cytochrome P450  

PubMed Central

This protocol describes the procedure for measuring the relative rates of metabolism of the ?,?-unsaturated aldehydes, 9-anthracene aldehyde (9-AA) and 4-hydroxy-trans-2-nonenal (4-HNE); specifically the aldehyde reduction reactions of cytochrome P450s (CYPs). These assays can be performed using either liver microsomal or other tissue fractions, spherosome preparations of recombinant CYPs, or recombinant CYPs from other sources. The method used here to study the reduction of a model ?,?-unsaturated aldehyde, 9-AA, by CYPs was adapted from the assay used to investigate 9-anthracene oxidation as reported by Marini et al. (Marini et al., 2003). For experiments measuring reduction of the endogenous aldehyde, 4-HNE, the substrate was incubated with CYP in the presence of oxygen and NADPH and the metabolites were separated by High Pressure Liquid Chromatograpy (HPLC), using an adaptation of the method of Srivastava et al. (Srivastava et al., 2010). For study of 9-AA and 4-HNE reduction, the first step involves incubation of the substrate with the CYP in appropriate media, followed by quantification of metabolites through either spectrofluorimetry or analysis by HPLC coupled with a radiometric assay, respectively. Metabolite identification can be achieved by HPLC GC-mass spectrometric analysis. Inhibitors of cytochrome P450 function can be utilized to show the role of the hemoprotein or other enzymes in these reduction reactions. The reduction reactions for CYP’s were not inhibited by either anaerobiosis or inclusion of CO in the gaseous phase of the reaction mixture. These character of these reactions are similar to those reported for some cytochrome P450-catalyzed azo reduction reactions.

Amunom, Immaculate; Srivastava, Sanjay; Prough, Russell A.

2011-01-01

55

Distributions and properties of cytochromes P-450 and cytochrome P-450 reductase from rat colon mucosal cells.  

PubMed

Cytochrome P-450 reductase and cytochrome P-450 fractions have been separated and partially purified from colonic mucosal microsomes of rat pretreated with phenobarbital or beta-naphthoflavone. Colonic cytochrome P-450 reductase has a molecular weight of 76,000. The Km values of colonic cytochrome P-450 reductase for the artificial electron acceptors cytochrome c, ferricyanide, and dichlorophenolindophenol and the electron donor NADPH are 6, 50, 11 and 11 microM, respectively. Immunochemical techniques identified the presence of beta-naphthoflavone Forms 1, 4 and 5 after beta-naphthoflavone treatment but beta-naphthoflavone Forms 1 and 4 and phenobarbital Form 1 after phenobarbital treatment. PMID:3114017

Oshinsky, R J; Strobel, H W

1987-01-01

56

Monoclonal antibodies to drosophila cytochrome P-450's  

SciTech Connect

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.

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

1987-05-01

57

Plant cytochrome P450-mediated herbicide metabolism  

Microsoft Academic Search

In the last two decades it has become apparent that enzymes of the P450 monooxygenase (P450) superfamily are responsible for the Phase I metabolism of numerous herbicides representing several classes of organic compounds. The majority of experimental evidence for P450 involvement in herbicide metabolism has been derived from in vitro studies in which the catalytic activity of plant microsomes towards

Balazs Siminszky

2006-01-01

58

Immunochemical examinations of cytochrome P-450 in various tissues of human fetuses using antibodies to human fetal cytochrome P-450, P-450 HFLa.  

PubMed

P-450 HFLa is a form of cytochrome P-450 purified from human fetal livers. The amounts of P-450 HFLa in several fetal tissues were determined immunochemically. Detectable amounts presented in livers, kidneys, adrenals, lungs and some other tissues of human fetuses. The amounts were the highest in livers. Activities of 7-ethoxycoumarin O-deethylase and benzo(a)pyrene hydroxylase in livers but not in adrenals were inhibited by the anti-P-450 HFLa antibodies, probably suggesting that distinct forms of cytochrome P-450 are responsible for the oxidations in livers and adrenals. PMID:4052083

Kitada, M; Kamataki, T; Itahashi, K; Rikihisa, T; Kato, R; Kanakubo, Y

1985-09-30

59

Arachidonic acid cytochrome P450 epoxygenase pathway.  

PubMed

Cytochrome P450 (CYP) epoxygenases convert arachidonic acid to four epoxyeicosatrienoic acid (EET) regioisomers, 5,6-, 8,9-, 11,12-, and 14,15-EET, that function as autacrine and paracrine mediators. EETs produce vascular relaxation by activating smooth muscle large-conductance Ca2+-activated K+ channels (BKCa). In addition, they have anti-inflammatory effects on blood vessels and in the kidney, promote angiogenesis, and protect ischemic myocardium and brain. CYP epoxygenases also convert eicosapentaenoic acid to vasoactive epoxy-derivatives, and endocannabinoids containing 11,12- and 14,15-EET are formed. Many EET actions appear to be initiated by EET binding to a membrane receptor that activates ion channels and intracellular signal transduction pathways. However, EETs also are taken up by cells, are incorporated into phospholipids, and bind to cytosolic proteins and nuclear receptors, suggesting that some functions may occur through direct interaction of the EET with intracellular effector systems. Soluble epoxide hydrolase (sEH) converts EETs to dihydroxyeicosatrienoic acids (DHETs). Because this attenuates many of the functional effects of EETs, sEH inhibition is being evaluated as a mechanism for increasing and prolonging the beneficial actions of EETs. PMID:18952572

Spector, Arthur A

2008-10-23

60

Brain Cytochrome P450 Aromatase Gene Isoforms and Activity Levels in Atlantic Salmon After Waterborne Exposure to Nominal Environmental Concentrations of the Pharmaceutical Ethynylestradiol and Antifoulant Tributyltin  

Microsoft Academic Search

In this study, the effects of two environmental endocrine disruptors, the synthetic pharmaceutical estrogen (ethynylestra- diol, EE2) and antifoulant (tributyltin, TBT) representing two different modes of action on the endocrine system, were studied on brain steroidogenic pathway of juvenile Atlantic salmon (Salmo salar). Neurosteroidogenesis was studied using brain aromatase gene isoforms and enzyme activity, in parallel with typical xenoestrogen responses,

Angeliki Lyssimachou; Bjørn Munro Jenssen; Augustine Arukwe

2006-01-01

61

A Suite of Activity-Based Probes for Human Cytochrome P450 Enzymes  

PubMed Central

Cytochrome P450 (P450) enzymes regulate a variety of endogenous signaling molecules and play central roles in the metabolism of xenobiotics and drugs. We recently showed that an aryl alkyne serves as an effective activity-based probe for profiling mouse liver microsomal P450s in vitro and in vivo. However, individual P450s display distinct substrate and inhibitor specificities, indicating that multiple probe structures may be required to achieve comprehensive coverage of this large and diverse enzyme family. Here, we have synthesized a suite of P450-directed, activity-based protein profiling (ABPP) probes that contain: 1) varied chemical architectures validated as mechanism-based inhibitors of the P450 enzyme family, and 2) terminal alkyne groups for click chemistry conjugation of reporter tags. This set of probes was screened against a wide cross-section of human P450s, leading to the discovery of an optimal set of probes that provide broad coverage of this enzyme family. We used these probes to profile the effects on P450 activity of aromatase inhibitors in current clinical use for the treatment of breast cancer. We describe the surprising discovery that one of these aromatase inhibitors, anastrozole, significantly increases probe-labeling of P450 1A2, indicative of a heterotypic cooperativity effect on a central P450 isozyme involved in metabolizing numerous drugs and xenobiotics. The results presented herein greatly expand the suite of ABPP probes for profiling P450s and illuminate new applications for these tools to understand P450-drug interactions.

Wright, Aaron T.; Song, Joongyu D.; Cravatt, Benjamin F.

2009-01-01

62

Reinvestigation of the Synthesis and Evaluation of [N-methyl-11C]Vorozole, a Radiotracer Targeting Cytochrome P450 Aromatase  

PubMed Central

Introduction We reinvestigated the synthesis of [N-methyl-11C]vorozole, a radiotracer for aromatase, and discovered the presence of an N-methyl isomer which was not removed in the original purification method. Herein we report the preparation and PET studies of pure [N-methyl-11C]vorozole. Methods Norvorozole was alkylated with [11C]methyl iodide as previously described and also with unlabeled methyl iodide. A HPLC method was developed to separate the regioisomers. NMR spectroscopy (13C and 2D-NOESY NMR) was used to identify and assign structures to the N-methylated products. Pure [N-methyl-11C]vorozole and the contaminating isomer were compared by PET imaging in the baboon. Results Methylation of norvorozole resulted in a mixture of isomers (1:1:1 ratio) based on new HPLC analysis using a pentafluorophenylpropyl (PFPP) bonded silica column, in which vorozole co-eluted one of its isomers under the original HPLC conditions. Baseline separation of the three labeled isomers was achieved. The N-3 isomer was the contaminant of vorozole, thus correcting the original assignment of isomers. PET studies of pure [N-methyl-11C]vorozole with and without the contaminating N-3 isomer revealed that only [N-methyl-11C]vorozole binds to aromatase. [N-methyl-11C]Vorozole accumulated in all brain regions with highest accumulation in the aromatase-rich amygdala and preoptic area. Accumulation was blocked with vorozole and letrozole consistent with reports of some level of aromatase in many brain regions. Conclusions The discovery of a contaminating labeled isomer and the development of a method for isolating pure [N-methyl-11C]vorozole combine to provide a new scientific tool for PET studies of the biology of aromatase and for drug research and development.

Kim, Sung Won; Biegon, Anat; Katsamanis, Zachary E.; Ehrlich, Carolin W.; Hooker, Jacob M.; Shea, Colleen; Muench, Lisa; Xu, Youwen; King, Payton; Carter, Pauline; Alexoff, David L.; Fowler, Joanna S.

2009-01-01

63

Cytochrome P-450 epitope typing in animals and humans with monoclonal antibodies to ethanol induced rat liver microsomal cytochrome P-450 (P-450et)  

SciTech Connect

Hybridomas were prepared from mouse myeloma cells and spleen cells derived from BALB/c female mice that had been immunized with P-450et. The monoclonal antibody (MAb)-producing hybridomas were screened by RIA. Thirty one independent hybrid clones were isolated with each producing an MAb of a single immunoglobulin subclass. All of these MAbs had high affinities for P-450et but only one MAb had a strong inhibitory effect on aniline rho-hydroxylase and N-nitrosodimethylamine demethylase. Western blots and RIAs based on ten MAbs (C1-C10) were used to determine the epitope homology of purified cytochromes P-450 from rats, rabbits, and humans. All ten MAbs had high affinity for both P-450et and a rat P-450 which is induced by acetone (P-450ac). Classes of these MAbs were identified which crossreacted toward different forms of rat P-450. In addition, several MAbs (C3, C6, C9) recognized a P-450 form of human liver, while other MAbs (C7, C9) recognized P-450/sub LM2/ of rabbits. Three MAbs (C4, C5, C8) were specific for only P-450et and P-450ac. These results demonstrate the different degrees of epitope relatedness among the multiple forms of cytochrome P-450.

Park, S.S.; Ko, I.Y.; Yang, C.; Guengerich, F.G.; Schenkman, J.B.; Coon, M.J.; Gelboin, H.V.

1986-05-01

64

Rearrangement Reactions Catalyzed by Cytochrome P450s  

PubMed Central

Cytochrome P450s promote a variety of rearrangement reactions both as a consequence of the nature of the radical and other intermediates generated during catalysis, and of the neighboring structures in the substrate that can interact either with the initial radical intermediates or with further downstream products of the reactions. This article will review several kinds of previously published cytochrome P450-catalyzed rearrangement reactions, including changes in stereochemistry, radical clock reactions, allylic rearrangements, “NIH” and related shifts, ring contractions and expansions, and cyclizations that result from neighboring group interactions. Although most of these reactions can be carried out by many members of the cytochrome P450 superfamily, some have only been observed with select P450s, including some reactions that are catalyzed by specific endoperoxidases and cytochrome P450s found in plants.

Ortiz de Montellano, Paul R.; Nelson, Sidney D.

2010-01-01

65

Investigation of aryl halides as ketone bioisosteres: Refinement of potent and selective inhibitors of human cytochrome P450 19A1 (aromatase).  

PubMed

Bioisosteric replacement of cyclic ketone functionality with aryl halides was investigated on a centrally-flexible, five-component 1,2,3-triazole-containing pharmacophore, resulting in enhanced inhibition of aromatase (CYP450 19A1). Structure-activity data generated from both syn- and anti-aldol precursors provides significant insights into the requirements for enhanced potency, validating this novel ketone-to-aryl halide bioisostere hypothesis. PMID:24113062

McNulty, James; Nielsen, Alexander J; Brown, Carla E; Difrancesco, Benjamin R; Vurgun, Nesrin; Nair, Jerald J; Crankshaw, Denis J; Holloway, Alison C

2013-09-18

66

Discovery of a novel class of aldol-derived 1,2,3-triazoles: Potent and selective inhibitors of human cytochrome P450 19A1 (aromatase)  

Microsoft Academic Search

The discovery of a novel five-component 1,2,3-triazole-containing pharmacophore that exhibits potent and selective inhibition of aromatase (CYP 450 19A1) is described. All compounds are derived from an initial aldol reaction of a phenylacetate derivative with an aromatic aldehyde. Structure–activity data generated from both syn- and anti-aldol adducts provides initial insights into the requirements for both potency and selectivity.

James McNulty; Jerald J. Nair; Nesrin Vurgun; Benjamin R. DiFrancesco; Carla E. Brown; Bernice Tsoi; Denis J. Crankshaw; Alison C. Holloway

67

Differential response of Leydig cells in expressing 11beta-HSD type I and cytochrome P450 aromatase in male rats subjected to corticosterone deficiency.  

PubMed

Emerging evidence suggests that the glucocorticoid and estradiol are important for Leydig cell steroidogenesis and are regulated via aromatase for estradiol production and 11beta-HSD for oxidatively inactivating glucocorticoid. Although it is known that corticosterone deficiency impaired Leydig cell steroidogenesis, its effect on the expression of Leydig cell 11beta-HSD type I and aromatase are yet to be recognized. Following metyrapone-induced corticosterone deficiency, serum corticosterone and testosterone levels decrease, whereas serum estradiol remains unaltered. 11beta-HSD type I mRNA and its activity was decreased by corticosterone deficiency, whereas the activity and mRNA of aromatase remains unaltered. Simultaneous administration of corticosterone prevented its deficiency-induced changes of 11beta-HSD type I in Leydig cells. Our results show that metyrapone-induced corticosterone deficiency impairs Leydig cell 11beta-HSD enzyme activity and 11beta-HSD type I mRNA expression, and the Leydig cells need to maintain their intracellular concentration of corticosterone for a normal function. PMID:19583995

Parthasarathy, Chandrakesan; Yuvaraj, Sambandam; Ilangovan, Ramachandran; Janani, Panneerselvam; Kanagaraj, Palaniyandi; Balaganesh, Muthusamy; Natarajan, Bhaskaran; Sittadjody, Sivanandane; Balasubramanian, Karundevi

2009-07-05

68

Cytochrome P450 pharmacogenetics in African populations.  

PubMed

The Cytochrome P450 (CYP450) family of enzymes is involved in the oxidative metabolism of many therapeutic drugs, carcinogens and various endogenous substrates. These enzymes are highly polymorphic at an inter-individual and inter-ethnic level. Polymorphisms or genetic variations account for up to 30% of inter-individual differences seen in a variety of drug responses. The frequencies of the different metabolizer categories (slow, intermediate, extensive and ultra-rapid), the distribution of genetic variants, genotype-phenotype correlations and the clinical importance of the CYP450 enzymes have been extensively documented in Caucasian and Oriental populations. Limited data exists for African populations, despite the fact that this knowledge is critically important for these populations who experience a heavy burden of communicable and non-communicable diseases. In addition, the costs incurred through adverse drug reactions and non-responsiveness to therapy could be reduced through the wide-scale application of pharmacogenetics. This review provides an overview and investigation of CYP450 genotypic and phenotypic reports published from 1980 to present in African populations. Our findings confirm the high degree of variability that is expected when comparing individuals of African origin to other ethnic groups and also highlight the distribution of clinically relevant CYP450 alleles amongst the various African populations. The notable discordance in genotypic and phenotypic data amongst African populations exemplifies the need for in-depth and well-orchestrated molecular and pharmacological investigations of these populations in the future, for which whole genome sequencing and association studies will be critical. PMID:23590174

Alessandrini, Marco; Asfaha, Sahle; Dodgen, Tyren Mark; Warnich, Louise; Pepper, Michael Sean

2013-04-16

69

Cytochrome P-450 from the Mesocarp of Avocado (Persea americana)  

PubMed Central

The microsomal fraction from the mesocarp of avocado (Persea americana) is one of few identified rich sources of plant cytochrome P-450. Cytochrome P-450 from this tissue has been solubilized and purified. Enzymatic assays (p-chloro-N-methylaniline demethylase) and spectroscopic observations of substrate binding suggest a low spin form of the cytochrome, resembling that in the microsomal membrane, can be recovered. However, this preparation of native protein is a mixture of nearly equal proportions of two cytochrome P-450 polypeptides that have been resolved only under denaturing conditions. Overall similarities between these polypeptides include indistinguishable amino acid compositions, similar trypsin digest patterns, and cross reactivity with the same antibody. The amino terminal sequences of both polypeptides are identical, with the exception that one of them lacks a methionine residue at the amino terminus. This sequence exhibits some similarities with the membrane targeting signal found at the amino terminus of most mammalian cytochromes P-450. Images Figure 3

O'Keefe, Daniel P.; Leto, Kenneth J.

1989-01-01

70

Cytochrome P-450 expression in sudden infant death syndrome  

Microsoft Academic Search

In the human liver, the major rise of the cytochrome P-450 isoform content occurs during the first months following birth (e.g., the high vulnerability period to sudden infant death syndrome (SIDS), a syndrome frequently associated with viral infection and drug hypersensitivity. We examined the expression of individual P-450 isoforms in liver samples collected postmortem from SIDS infants and compared values

Jean Marc Treluyer; Gérard Cheron; Michelle Sonnier; Thierry Cresteil

1996-01-01

71

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

ClinicalTrials.gov

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

2013-09-16

72

Organization of cytochrome P450 enzymes involved in sex steroid synthesis: PROTEIN-PROTEIN INTERACTIONS IN LIPID MEMBRANES.  

PubMed

Mounting evidence underscores the importance of protein-protein interactions in the functional regulation of drug-metabolizing P450s, but few studies have been conducted in membrane environments, and none have examined P450s catalyzing sex steroid synthesis. Here we report specific protein-protein interactions for full-length, human, wild type steroidogenic cytochrome P450 (P450, CYP) enzymes: 17alpha-hydroxylase/17,20-lyase (P450c17, CYP17) and aromatase (P450arom, CYP19), as well as their electron donor NADPH-cytochrome P450 oxidoreductase (CPR). Fluorescence resonance energy transfer (FRET)(3) in live cells, coupled with quartz crystal microbalance (QCM), and atomic force microscopy (AFM) studies on phosphatidyl choline +/- cholesterol (mammalian) biomimetic membranes were used to investigate steroidogenic P450 interactions. The FRET results in living cells demonstrated that both P450c17 and P450arom homodimerize but do not heterodimerize, although they each heterodimerize with CPR. The lack of heteroassociation between P450c17 and P450arom was confirmed by QCM, wherein neither enzyme bound a membrane saturated with the other. In contrast, the CPR bound readily to either P450c17- or P450arom-saturated surfaces. Interestingly, N-terminally modified P450arom was stably incorporated and gave similar results to the wild type, although saturation was achieved with much less protein, suggesting that the putative transmembrane domain is not required for membrane association but for orientation. In fact, all of the proteins were remarkably stable in the membrane, such that high resolution AFM images were obtained, further supporting the formation of P450c17, P450arom, and CPR homodimers and oligomers in lipid bilayers. This unique combination of in vivo and in vitro studies has provided strong evidence for homodimerization and perhaps some higher order interactions for both P450c17 and P450arom. PMID:19805543

Praporski, Slavica; Ng, Su May; Nguyen, Ann D; Corbin, C Jo; Mechler, Adam; Zheng, Jie; Conley, Alan J; Martin, Lisandra L

2009-10-05

73

Role of colonic cytochrome P-450 in large bowel carcinogenesis.  

PubMed

Rat colon mucosa microsomes contain a competent mixed function oxidase system that hydroxylates the N-methyl drugs benzphetamine and ethylmorphine, the O-alkyl drugs p-nitroanisole and p-nitrophenetole and the polycyclic carcinogen benzo[alpha]pyrene. The colon system's hydroxylation activities can be selectively induced by pretreatment with phenobarbital or beta-naphthoflavone and can be selectively inhibited by SKF-525A or 7,8-benzoflavone. The colon microsomal system has been solubilized with the non-ionic detergent Renex 690 and resolved by column chromatography into its components cytochrome P-450 and cytochrome P-450 reductase. Colon cytochrome P-450 and cytochrome P-450 reductase can be recombined to reconstitute hydroxylation activity. The colon system is also able to activate carcinogens to mutagenic metabolites as demonstrated in the Ames test system. In addition, the activity of the colon system is markedly increased by pretreatment with gastrointestinal hormones. PMID:6766798

Strobel, H W; Fang, W F; Oshinsky, R J

1980-03-15

74

Cytochromes P450 Catalyze the Reduction of ?,?-Unsaturated Aldehydes  

PubMed Central

The metabolism of ?,?-unsaturated aldehydes, e.g. 4-hydroxynonenal, involves oxidation to carboxylic acids, reduction to alcohols, and glutathionylation to eventually form mercapturide conjugates. Recently we demonstrated that P450s can oxidize aldehydes to carboxylic acids, a reaction previously thought to involve aldehyde dehydrogenase. When recombinant cytochrome P450 3A4 was incubated with 4-hydroxynonenal, O2, and NADPH, several products were produced, including 1,4-dihydroxynonene (DHN), 4-hydroxy-2-nonenoic acid (HNA), and an unknown metabolite. Several P450s catalyzed the reduction reaction in the order (human) P450 2B6 ? P450 3A4 > P450 1A2 > P450 2J2 > (mouse) P450 2c29. Other P450s did not catalyze the reduction reaction (human P450 2E1 & rabbit P450 2B4). Metabolism by isolated rat hepatocytes showed that HNA formation was inhibited by cyanamide, while DHN formation was not affected. Troleandomycin increased HNA production 1.6-fold while inhibiting DHN formation, suggesting that P450 3A11 is a major enzyme involved in rat hepatic clearance of 4-HNE. A fluorescent assay was developed using 9-anthracenealdehyde to measure both reactions. Feeding mice diet containing t-butylated hydroxyanisole increased the level of both activities with hepatic microsomal fractions, but not proportionally. Miconazole (0.5 mM) was a potent inhibitor of these microsomal reduction reactions, while phenytoin and ?-naphthoflavone (both at 0.5 mM) were partial inhibitors, suggesting the role of multiple P450 enzymes. The oxidative metabolism of these aldehydes was inhibited >90% in an Ar or CO atmosphere, while the reductive reactions were not greatly affected. These results suggest that P450s are significant catalysts of reduction of ?,?-unsaturated aldehydes in liver.

Amunom, Immaculate; Dieter, Laura J.; Tamasi, Viola; Cai, Jan; Conklin, Daniel J.; Srivastava, Sanjay; Martin, Martha V.; Guengerich, F. Peter; Prough, Russell A.

2011-01-01

75

Effect of sinomenine on human cytochrome P450 activity  

Microsoft Academic Search

BackgroundCytochrome P450s superfamily expressed widely in organisms are known to play an important role in the biotransformation of many endogenous and exogenous substances. Inhibition or induction of cytochrome P450 isozymes is one of the major causes for clinical drug–drug interactions. Sinomenine can be metabolized to at least 2 metabolites in human, rat in vivo and in human liver microsomes. The

Yong-Mei Yao; Wei Cao; Ya-Jie Cao; Ze-Neng Cheng; Dong-Sheng Ou-Yang; Zhao-Qian Liu; Hong-Hao Zhou

2007-01-01

76

Mobility of Cytochrome P450 in the Endoplasmic Reticulum Membrane  

Microsoft Academic Search

Cytochrome P450 2C2 is a resident endoplasmic reticulum (ER) membrane protein that is excluded from the recycling pathway and contains redundant retention functions in its N-terminal transmembrane signal\\/anchor sequence and its large, cytoplasmic domain. Unlike some ER resident proteins, cytochrome P450 2C2 does not contain any known retention\\/retrieval signals. One hypothesis to explain exclusion of resident ER proteins from the

Elzbieta Szczesna-Skorupa; Ci-Di Chen; Steven Rogers; Byron Kemper

1998-01-01

77

Cytochrome P-450 2E1 in Rat Liver Peroxisomes  

Microsoft Academic Search

Cytochrome P-450 containing enzymes, known to be present in the endoplasmic reticulum and mitochondria, catalyze the oxidation of various compounds. In this study we have used highly purified peroxisomes (>95%) to provide evidence by analytical cell fractionation, enzyme activity, Western blot, and immunocytochemical analysis that cytochrome P-450 2E1 (Cyp 2E1) is present in peroxisomes. Similar specific activities of aniline hydroxylase,

Kalipada Pahan; Brian T Smith; Avtar K Singh; Inderjit Singh

1997-01-01

78

Effects of bisphenol A on the expression of cytochrome P450 aromatase (CYP19) in human fetal osteoblastic and granulosa cell-like cell lines.  

PubMed

The effects of bisphenol A (BPA), an endocrine disruptor, on aromatase (CYP19) expression in human osteoblastic (SV-HFO) and ovarian granulosa-like (KGN) cell lines were examined. CYP19 enzyme activity was suppressed in the presence of BPA in a dose-dependent fashion in both cell lines. CYP19 gene transcript expression, as well as activities of promoter I.4 in SV-HFO and promoter II in KGN, was down-regulated by BPA, suggesting that BPA affects CYP19 at the gene-expression level. These data and the previous finding that BPA induced the down-regulation of promoter I.1 activity within the human placental cell line suggest that there may be a conserved signaling pathway that down-regulates CYP19 expression in response to BPA in both cell lines. Additionally, differences between promoter I.4 and II suggest that there may be cell- and promoter-specific down-regulating mechanisms downstream from the actions of BPA. PMID:22327052

Watanabe, Masatada; Ohno, Shuji; Nakajin, Shizuo

2012-02-04

79

Effect of low dose exposure to the herbicide atrazine and its metabolite on cytochrome P450 aromatase and steroidogenic factor-1 mRNA levels in the brain of premetamorphic bullfrog tadpoles (Rana catesbeiana)  

PubMed Central

The transcriptional regulator steroidogenic factor 1 (SF-1) and the enzyme cytochrome P450 aromatase (CYP19) play a central role in modulation of a broad range of tissue-specific developmental processes associated with hormone homeostasis that includes differentiation of the central nervous system. SF-1 and CYP19 expression may be targeted by a variety of endocrine disruptive agents prevalent within the environment. In the present study, we cloned and characterized partial sequences for bullfrog (Rana catesbeiana) SF-1 and CYP19 and examined the effects of a 48 h exposure to 1 and 100 ?g/L of the herbicide atrazine (ATZ) and its major metabolite desethylatrazine (DEA), as well as 5 ng/L of the estrogenic chemical, 17?-ethynylestradiol (EE2), and 673 ng/L of the thyroid hormone, 3,5, 3?-triiodothyronine (T3), on SF-1 and CYP19 mRNA abundance in the brains of premetamorphic bullfrog tadpoles. Quantitative RT-PCR analysis showed an increase in CYP19 mRNA following a 48 h exposure to EE2 but not T3 while no significant changes in SF-1 transcript levels occurred. We observed a strong positive correlation between CYP19 and SF-1 transcript abundance in the ATZ-exposed animals which was not evident with DEA- or hormone-exposed tadpoles. Our results are intriguing in light of reported behavioral changes in ATZ-exposed frogs and suggest that further research is warranted to examine the relationship and role of CYP19 and SF-1 in amphibian brain development.

Gunderson, Mark P.; Veldhoen, Nik; Skirrow, Rachel C.; Macnab, Magnus K.; Ding, Wei; van Aggelen, Graham; Helbing, Caren C.

2011-01-01

80

Directed evolution of cytochrome P450 for sterol epoxidation.  

PubMed

16,17-Epoxysterol plays an important role in pharmaceutical steroid synthesis. To investigate the potential application of cytochrome P450 for epoxysterol synthesis, an approach to the epoxidation of 16,17-epoxysterol, based on directed evolution of cytochrome P450 BM-3, was developed. This comprised random gene mutagenesis for optimizing the activity of P450 BM-3 for epoxidation of hydrophobic sterol, followed by the 7-ethoxycoumarin de-ethylation assay for general enzyme activity detection and the modified picric acid assay for epoxidation activity screening. By the two-step screening, one mutant from 792 clones showed specific substrate activity of converting progesterone to 16,17-epoxysterol, which validated the possibility to evolve the cytochrome P450 for the synthesis of steroidal epoxides. PMID:23794050

Jiang, Dan; Tu, Ran; Bai, Peng; Wang, Qinhong

2013-06-21

81

Light-driven biocatalysis with cytochrome P450 peroxygenases.  

PubMed

The cytochrome P450 peroxygenases P450(Bs?) (CYP152A1) from Bacillus subtilis and P450(Cla) (CYP152A2) from Clostridium acetobutylicum belong to a unique group of P450s with high synthetic potential. They consume hydrogen peroxide via the peroxide shunt and therefore do not require additional electron transfer proteins for biocatalytic activity. Their high synthetic potential is, however, impaired by their rather poor operational stability in the presence of hydrogen peroxide. Herein, we report the use of a light-driven approach utilizing light-excited flavins (riboflavin, flavin mononucleotide, or flavin adenine dinucleotide) and the electron donor ethylenediaminetetraacetate as the electron source for the in situ generation of hydrogen peroxide. This approach represents a simple and easily applicable way to promote oxyfunctionalization reactions catalyzed by P450 peroxygenases and is useful for biocatalysis with these enzymes. PMID:23586998

Girhard, Marco; Kunigk, Elmar; Tihovsky, Svetlana; Shumyantseva, Victoria V; Urlacher, Vlada B

2013-01-25

82

Stable Expression of Human Cytochrome P450 3A4 in Conjunction with Human NADPH-Cytochrome P450 Oxidoreductase in V79 Chinese Hamster Cells  

Microsoft Academic Search

V79 Chinese hamster cells were constructed for stable expression of human cytochrome P450 3A4 with and without coexpression of human NADPH-cytochrome P450 oxidoreductase. Expression of the cDNAs was shown by Northern and Western analyses. Activity was tested by 6?-hydroxylation of testosterone for cytochrome P450 3A4 and by cytochrome c reduction for NADPH-cytochrome P450 reductase. Five V79 cell lines were obtained

Anneliese Schneider; Wolfgang A. Schmalix; Vasanthi Siruguri; Els M. de Groene; G. Jean Horbach; Britta Kleingeist; Dieter Lang; Ronald Böcker; Claire Belloc; Philippe Beaune; Helmut Greim; Johannes Doehmer

1996-01-01

83

Homotropic cooperativity of monomeric cytochrome P450 3A4  

SciTech Connect

Mechanistic studies of mammalian cytochrome P450s are often obscured by the phase heterogeneity of solubilized preparations of membrane enzymes. The various protein-protein aggregation states of microsomes, detergent solubilized cytochrome or a family of aqueous multimeric complexes can effect measured substrate binding events as well as subsequent steps in the reaction cycle. In addition, these P450 monooxygenases are normally found in a membrane environment and the bilayer composition and dynamics can also effect these catalytic steps. Here, we describe the structural and functional characterization of a homogeneous monomeric population of cytochrome P450 3A4 (CYP 3A4) in a soluble nanoscale membrane bilayer, or Nanodisc [Nano Lett. 2 (2002) 853]. Cytochrome P450 3A4:Nanodisc assemblies were formed and purified to yield a 1:1 ratio of CYP 3A4 to Nanodisc. Solution small angle X-ray scattering was used to structurally characterize this monomeric CYP 3A4 in the membrane bilayer. The purified CYP 3A4:Nanodiscs showed a heretofore undescribed high level of homotropic cooperativity in the binding of testosterone. Soluble CYP 3A4:Nanodisc retains its known function and shows prototypic hydroxylation of testosterone when driven by hydrogen peroxide. This represents the first functional characterization of a true monomeric preparation of cytochrome P450 monooxygenase in a phospholipid bilayer and elucidates new properties of the monomeric form.

Baas, Bradley J.; Denisov, Ilia G.; Sligar, Stephen G. (UIUC)

2010-11-16

84

Oxidation of Nonionic Detergents by Cytochrome P450 Enzymes  

Microsoft Academic Search

Nonionic phenolic detergents are commonly used in the purification of membrane-associated proteins. Triton N-101 was shown to be oxidized by NADPH-fortified human liver microsomes and recombinant human cytochromes P450 (P450). Oxidation was monitored using HPLC and the fluorescence properties of Triton N-101 and other alkylphenol ethoxylate detergents, which are similar to those of anisole. Human liver microsomes and recombinantly expressed

Natilie A. Hosea; F. Peter Guengerich

1998-01-01

85

Screening for cytochrome P450 reactivity with a reporter enzyme.  

PubMed

The identification of novel substrates of cytochrome P450 enzymes by high-throughput screening assays is of utmost importance to further increase the scope of these enzymes for future applications. Most screens are either confined to individual substrate analogues or hampered by low throughput due to elaborate analysis techniques. Here we describe a general high-throughput screening assay that interrogates the activity of P450 enzymes with the aid of catalase as a reporter enzyme. PMID:23475675

Rabe, Kersten S; Niemeyer, Christof M

2013-01-01

86

Role of cytochrome P450 in drug interactions  

Microsoft Academic Search

Drug-drug interactions have become an important issue in health care. It is now realized that many drug-drug interactions can be explained by alterations in the metabolic enzymes that are present in the liver and other extra-hepatic tissues. Many of the major pharmacokinetic interactions between drugs are due to hepatic cytochrome P450 (P450 or CYP) enzymes being affected by previous administration

Zakia Bibi

2008-01-01

87

Cytochrome P450: taming a wild type enzyme  

PubMed Central

Protein engineering of cytochrome P450 monooxygenases (P450s) has been very successful in generating valuable non-natural activities and properties, allowing these powerful catalysts to be used for the synthesis of drug metabolites and in biosynthetic pathways for the production of precursors of artemisinin and paclitaxel. Collected experience indicates that the P450s are highly 'evolvable'--they are particularly robust to mutation in their active sites and readily accept new substrates and exhibit new selectivities. Their ability to adapt to new challenges upon mutation may reflect the nonpolar nature of their active sites as well as their high degree of conformational variability.

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

2011-01-01

88

The Interaction of Microsomal Cytochrome P450 2B4 with its Redox Partners, Cytochrome P450 Reductase and Cytochrome b5  

PubMed Central

1 Cytochrome P450 2B4 is a microsomal protein with a multi-step reaction cycle similar to that observed in the majority of other cytochromes P450. The cytochrome P450 2B4-substrate complex is reduced from the ferric to the ferrous form by cytochrome P450 reductase. After binding oxygen, the oxyferrous protein accepts a second electron which is provided by either cytochrome P450 reductase or cytochrome b5. In both instances, product formation occurs. When the second electron is donated by cytochrome b5, catalysis (product formation) is ? 10 to 100-fold faster than in the presence of cytochrome P450 reductase. This allows less time for side product formation (hydrogen peroxide and superoxide) and improves by ? 15% the coupling of NADPH consumption to product formation. Cytochrome b5 has also been shown to compete with cytochrome P450 reductase for a binding site on the proximal surface of cytochrome P450 2B4. These two different effects of cytochrome b5 on cytochrome P450 2B4 reactivity can explain how cytochrome b5 is able to stimulate, inhibit, or have no effect on cytochrome P450 2B4 activity. At low molar ratios (<1) of cytochrome b5 to cytochrome P450 reductase, the more rapid catalysis results in enhanced substrate metabolism. In contrast, at high molar ratios (>1) of cytochome b5 to cytochrome P450 reductase, cytochrome b5 inhibits activity by binding to the proximal surface of cytochrome P450 and preventing the reductase from reducing ferric cytochrome P450 to the ferrous protein, thereby aborting the catalytic reaction cycle. When the stimulatory and inhibitory effects of cytochrome b5 are equal, it will appear to have no effect on the enzymatic activity. It is hypothesized that cytochrome b5 stimulates catalysis by causing a conformational change in the active site, which allows the active oxidizing oxyferryl species of cytochrome P450 to be formed more rapidly than in the presence of reductase.

Im, Sang-Choul; Waskell, Lucy

2010-01-01

89

Regioselectivity of metabolic activation of acetylenic steroids by hepatic cytochrome P450 isozymes.  

PubMed

Liver cytochrome P450 monooxygenases (P450), a group of isozymes that catalyze the reductive cleavage of molecular oxygen, dominate hepatic metabolism of xenobiotic lipophilic substances. These P450 enzymes exhibit broad and overlapping substrate specificities, in contrast to the P450 isozymes of the steroid biosynthetic pathways, which are highly substrate specific. Hepatic heme pigments, N-alkylated porphyrins, accumulate following the self-catalyzed destruction of P450 by the metabolic activation of 17 alpha-ethynyl steroids. Acetylenic substituted steroidal aromatase inactivators, norethisterone (NET), and 10-(2-propynyl)estr-4-ene-3,17-dione (MDL 18,962) were administered to rats to determine if the acetylenic substituent was activated by hepatic P450 mixed-function oxidases. This metabolism could result in the formation of a reactive species that would alkylate a pyrrole nitrogen atom of heme. Male Sprague-Dawley rats were treated with 0, 10, 30, or 100 mg/kg NET or MDL 18,962 intraperitoneally. Four hours later, these animals received 40 mg/kg sodium pentobarbital and their sleeping times were recorded. On arousal, the rats were killed and their livers were taken for determination of P450 content and formation of N-alkylated porphyrins (green pigments). Norethisterone inhibited hepatic P450 isozymes, resulting in a dose-related increased sleeping time (89.2 +/- 3.5 to 156.3 +/- 7.6 minutes) and decreased P450 levels (maximum 25% decrease at 100 mg/kg), and the amount of green pigments increased with doses of 10 to 100 mg/kg. In contrast, MDL 18,962 treatment did not increase sleeping time and caused only a 15% decrease in hepatic P450 content at 100 mg/kg, with no detectable green pigments.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1871782

Johnston, J O; Wright, C L; Leeson, G A

1991-04-01

90

Purification of the pyrazole-inducible cytochrome P-450 isozyme  

SciTech Connect

The alcohol dehydrogenase inhibitor, pyrazole, appears to induce a cytochrome P-450 isozyme with properties similar to the ethanol-inducible P-450. The pyrazole-inducible P-450 isozyme was purified from the liver microsomes of rats treated with pyrazole essentially by the procedure of Ryan et al and also by chromatofocussing. The final preparation appeared homogenous by SDS-PAGE with an apparent molecular weight of 52,000, had a specific content of 11 nmoles P-450 per mg protein, showed very high activity of low K/sub m/ dimethylnitrosamine demethylase and produced a type II binding spectrum with dimethylsulfoxide. The enzyme was also active with aniline and aminopyrine as substrates. Pyrazole itself served as an excellent substrate with 4-hydroxy pyrazole being the product. An antibody against the pyrazole-inducible P-450 raised in chickens recognized a protein with mol.wt of about 52,000 in control microsomes. This band was highly enriched in microsomes from rats treated with pyrazole, 4-methyl-pyrazole, ethanol or acetone, but not phenobarbital or 3-methylcholanthrene. In summary, the pyrazole-inducible P-450 has been purified and appears to be identical in its catalytic and immunological properties to the alcohol-inducible P-450.

Palakodety, R.; Clejan, L.; Krikun, G.; Feierman, D.; Cederbaum, A.I.

1987-05-01

91

The cytochrome P450 (CYP) gene superfamily in Daphnia pulex  

PubMed Central

Background Cytochrome P450s (CYPs) in animals fall into two categories: those that synthesize or metabolize endogenous molecules and those that interact with exogenous chemicals from the diet or the environment. The latter form a critical component of detoxification systems. Results Data mining and manual curation of the Daphnia pulex genome identified 75 functional CYP genes, and three CYP pseudogenes. These CYPs belong to 4 clans, 13 families, and 19 subfamilies. The CYP 2, 3, 4, and mitochondrial clans are the same four clans found in other sequenced protostome genomes. Comparison of the CYPs from D. pulex to the CYPs from insects, vertebrates and sea anemone (Nematostella vectensis) show that the CYP2 clan, and to a lesser degree, the CYP4 clan has expanded in Daphnia pulex, whereas the CYP3 clan has expanded in insects. However, the expansion of the Daphnia CYP2 clan is not as great as the expansion observed in deuterostomes and the nematode C. elegans. Mapping of CYP tandem repeat regions demonstrated the unusual expansion of the CYP370 family of the CYP2 clan. The CYP370s are similar to the CYP15s and CYP303s that occur as solo genes in insects, but the CYP370s constitute ~20% of all the CYP genes in Daphnia pulex. Lastly, our phylogenetic comparisons provide new insights into the potential origins of otherwise mysterious CYPs such as CYP46 and CYP19 (aromatase). Conclusion Overall, the cladoceran, D. pulex has a wide range of CYPs with the same clans as insects and nematodes, but with distinct changes in the size and composition of each clan.

Baldwin, William S; Marko, Peter B; Nelson, David R

2009-01-01

92

Steroid regulation of drug-metabolizing cytochromes P450.  

PubMed

Cytochrome P450 (P450) monooxygenases are capable of catalyzing metabolism of various endogenous and exogenous compounds, such as bile acids, fatty acids, retinoids, steroids, drugs and other xenobiotics. The enzymes, belonging to CYP1, CYP2 and CYP3 families are primarily involved in the metabolism of drugs and xenobiotics. P450-mediated defense mechanism protects organisms from the potentially toxic effects of xenobiotics to which they are exposed. The adaptive transcriptional induction of P450s by xenobiotics is mediated by aromatic hydrocarbon receptor of Per-ARNT-Sim family, and nuclear hormone receptors, including pregnane X receptor, constitutive androstane receptor and glucocorticoid receptor. In addition to the receptor-mediated induction, endogenous factors (developmental, sex or hormonal factors) can also modulate P450 expression. Steroid hormones are biologically active compounds, controlling many physiological processes via endocrine signaling pathways and contributing to the transcriptional regulation of drug metabolizing P450s. Any change in P450 activities influences the rate of activation or inactivation of drugs. Exposure to xenobiotics (drugs, environmental pollutants) can exert changes in endocrine function both directly as hormone agonists/antagonists or indirectly altering the rates of hormone metabolism and consequently the circulating levels of hormones. Modulation of P450 expression by xenobiotics can affect the subsequent metabolism of not only foreign chemicals, but also steroid hormones. Perturbation in hormone metabolism leads to the imbalance in sexual and reproductive development, and in glucose, lipid and salt/water homeostasis. The purpose of this review is to highlight the interplay between drug-metabolizing P450s and steroid hormones as well as the interactions of xenosensor with steroid signaling pathways. PMID:21395541

Monostory, Katalin; Dvorak, Zdenek

2011-02-01

93

Cytochrome P-450 Polymorphisms and Response to Clopidogrel  

Microsoft Academic Search

Background Clopidogrel requires transformation into an active metabolite by cytochrome P-450 (CYP) enzymes for its antiplatelet effect. The genes encoding CYP enzymes are poly- morphic, with common alleles conferring reduced function. Methods We tested the association between functional genetic variants in CYP genes, plasma concentrations of active drug metabolite, and platelet inhibition in response to clopi- dogrel in 162 healthy

Jessica L. Mega; Sandra L. Close; Stephen D. Wiviott; Lei Shen; Richard D. Hockett; John T. Brandt; Joseph R. Walker; Elliott M. Antman; William Macias; Eugene Braunwald; Marc S. Sabatine; Daiichi Sankyo Pharma

2010-01-01

94

Cytochrome P450s as genes for crop improvement.  

PubMed

In the past year, several cytochrome P450 genes have been identified that will be important for generating crop protectants and natural medicinal products. Among these are the 2-hydroxyisoflavone synthase (CYP93C) and the indole-3-acetaldoxime N-hydroxylase (CYP83B1) genes, which catalyze the formation of isoflavones and glucosinolates, respectively. PMID:11228441

Feldmann, K A

2001-04-01

95

The cytochrome P450 (CYP) gene superfamily in Daphnia pulex  

Microsoft Academic Search

BACKGROUND: Cytochrome P450s (CYPs) in animals fall into two categories: those that synthesize or metabolize endogenous molecules and those that interact with exogenous chemicals from the diet or the environment. The latter form a critical component of detoxification systems. RESULTS: Data mining and manual curation of the Daphnia pulex genome identified 75 functional CYP genes, and three CYP pseudogenes. These

William S Baldwin; Peter B Marko; David R Nelson

2009-01-01

96

Fungal denitrification and nitric oxide reductase cytochrome P450nor  

PubMed Central

We have shown that many fungi (eukaryotes) exhibit distinct denitrifying activities, although occurrence of denitrification was previously thought to be restricted to bacteria (prokaryotes), and have characterized the fungal denitrification system. It comprises NirK (copper-containing nitrite reductase) and P450nor (a cytochrome P450 nitric oxide (NO) reductase (Nor)) to reduce nitrite to nitrous oxide (N2O). The system is localized in mitochondria functioning during anaerobic respiration. Some fungal systems further contain and use dissimilatory and assimilatory nitrate reductases to denitrify nitrate. Phylogenetic analysis of nirK genes showed that the fungal-denitrifying system has the same ancestor as the bacterial counterpart and suggested a possibility of its proto-mitochondrial origin. By contrast, fungi that have acquired a P450 from bacteria by horizontal transfer of the gene, modulated its function to give a Nor activity replacing the original Nor with P450nor. P450nor receives electrons directly from nicotinamide adenine dinucleotide to reduce NO to N2O. The mechanism of this unprecedented electron transfer has been extensively studied and thoroughly elucidated. Fungal denitrification is often accompanied by a unique phenomenon, co-denitrification, in which a hybrid N2 or N2O species is formed upon the combination of nitrogen atoms of nitrite with a nitrogen donor (amines and imines). Possible involvement of NirK and P450nor is suggested.

Shoun, Hirofumi; Fushinobu, Shinya; Jiang, Li; Kim, Sang-Wan; Wakagi, Takayoshi

2012-01-01

97

Recombinant enzymes overexpressed in bacteria show broad catalytic specificity of human cytochrome P450 2W1 and limited activity of human cytochrome P450 2S1.  

PubMed

Human cytochromes P450 2S1 and 2W1 have received only limited attention with regard to characterization of function. Both cytochromes P450 have been reported to be overexpressed in human tumors, and cytochrome P450 2S1 is induced by carcinogenic polycyclic hydrocarbons. We report methods for high-level expression and purification of both cytochromes P450 from Escherichia coli, with the goal of establishing function. The level of expression of human cytochrome P450 2W1 achieved using codon optimization for E. coli was 1800 nmol of cytochrome P450 per liter of culture, the highest level achieved in this laboratory to date. Assays with a number of the typical cytochrome P450 substrates showed no detectable activity, including some for which qualitative reports have appeared in the literature. Cytochrome P450 2W1 catalyzed benzphetamine N-demethylation (k(cat), 3.8/min) and arachidonic acid oxidation, albeit at a very low rate (approximately 0.05/min). In a umu genotoxicity screen, cytochrome P450 2W1 catalyzed the activation of several procarcinogens, particularly polycyclic hydrocarbon diols, but cytochrome P450 2S1 did not. The bioactivation of procarcinogens by cytochrome P450 2W1 may be of significance in the context of reports of preferential expression of the enzyme in tumors, in that activation of procarcinogens could lead to the accumulation of mutations and enhance the carcinogenic process. PMID:16551781

Wu, Zhong-Liu; Sohl, Christal D; Shimada, Tsutomu; Guengerich, F Peter

2006-03-21

98

Cytochrome P450 gene clusters in Drosophila melanogaster  

Microsoft Academic Search

Twelve cytochrome P450 cDNA fragments were cloned fromDrosophila melanogaster by reverse transcriptase\\/PCR (RT\\/PCR) using degenerate oligonucleotide primers. The corresponding genes belong to several subfamilies of the CYP4 and CYP9 P450 families. Only two of these genes,Cyp4d1 andCyp4d2, have previously been described.In situ hybridization of each of the cDNA fragments showed two clusters of genes; one near the tip of theX

Boris C. Dunkov; Rosario Rodriguez-Arnaiz; Barry Pittendrigh; Richard H. ffrench-Constant; René Feyereisen

1996-01-01

99

Inactivation of rat liver cytochrome P450 (P450) by N, N-dimethylformamide and N, N-dimethylacetamide  

Microsoft Academic Search

N,N-dimethylformamide (DMF), an organic solvent widely used in industry, is bioactivated by cytochrome P450 (P450) to reactive metabolites which are believed to be responsible for the hepatotoxicity observed in animals and humans. A decrease of the activating enzyme has been reported in rats treated with DMF, although the specific P450 isoform(s) involved and the nature of the reactive species responsible

Roberto Tolando; Alberta Zanovello; Roberta Ferrara; Jim N. Iley; Maurizio Manno

2001-01-01

100

Spectroscopic features of cytochrome P450 reaction intermediates  

PubMed Central

Preface Cytochromes P450 constitute a broad class of heme monooxygenase enzymes with more than 11,500 isozymes which have been identified in organisms from all biological kingdoms [1]. These enzymes are responsible for catalyzing dozens chemical oxidative transformations such as hydroxylation, epoxidation, N-demethylation, etc., with very broad range of substrates [2-3]. Historically these enzymes received their name from ‘pigment 450’ due to the unusual position of the Soret band in UV-Vis absorption spectra of the reduced CO-saturated state [4-5]. Despite detailed biochemical characterization of many isozymes, as well as later discoveries of other ‘P450-like heme enzymes’ such as nitric oxide synthase and chloroperoxidase, the phenomenological term ‘cytochrome P450’ is still commonly used as indicating an essential spectroscopic feature of the functionally active protein which is now known to be due to the presence of a thiolate ligand to the heme iron [6]. Heme proteins with an imidazole ligand such as myoglobin and hemoglobin as well as an inactive form of P450 are characterized by Soret maxima at 420 nm [7]. This historical perspective highlights the importance of spectroscopic methods for biochemical studies in general, and especially for heme enzymes, where the presence of the heme iron and porphyrin macrocycle provides rich variety of specific spectroscopic markers available for monitoring chemical transformations and transitions between active intermediates of catalytic cycle.

Luthra, Abhinav; Denisov, Ilia G.; Sligar, Stephen G.

2010-01-01

101

ISOLATION OF THE ALKANE INDUCIBLE CYTOCHROME P450 (P450ALK) GENE FROM THE YEAST CANDIDA TROPICALIS  

EPA Science Inventory

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

102

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

EPA Science Inventory

The gene for the alkane-inducible cytochrome P450, P450alk, has been isolated from the yeast Candida tropicalis by immunoscreening a ¿gt11 library. Isolation of the gene has been identified on the basis of its inducibility and partial DNA sequence. Transcripts of this gene were i...

103

Isolation of the Alkane Inducible Cytochrome P450 (P450alk) Gene from the Yeast 'Candida tropicalis'.  

National Technical Information Service (NTIS)

The gene for the alkane-inducible cytochrome P450, P450alk, has been isolated from the yeast Candida tropicalis by immunoscreening a lambda gt11 library. Isolation of the gene has been identified on the basis of its inducibility and partial DNA sequence. ...

D. Sanglard C. Chen J. C. Loper

1987-01-01

104

Cytochrome P450 in fluke Opisthorchis felineus: identification and characterization.  

PubMed

Infection with the human liver fluke Opisthorchis felineus is a serious public health problem in Russia and other Eastern Europe countries. The aim of this work was to identify and sequence cytochrome P450 mRNA from O. felineus and to analyze its expression at different developmental stages. We found only one cytochrome P450 in O. felineus. It contains a conserved Pfam00067 domain which was typical of the CYP450 II eukaryotic microsomal type, and a putative transmembrane domain. Additionally, we identified a high degree of homology between a 3D model of O. felineus CYP450 and mammalian CYP2 structures. The level of O. felineus CYP mRNA expression in maritae (adult stage in definitive mammal host) is significantly higher than in metacercaria. This fact indicates an important role of this biotransformation enzyme in the biochemistry of the parasite at the maritae stage. PMID:22115821

Pakharukova, Maria Y; Ershov, Nikita I; Vorontsova, Elena V; Katokhin, Alexei V; Merkulova, Tatiana I; Mordvinov, Viatcheslav A

2011-11-18

105

The naturally occurring cytochrome P450 (P450) 2B6 K262R mutant of P450 2B6 exhibits alterations in substrate metabolism and inactivation.  

PubMed

The polymorphic human cytochrome P450 (P450) 2B6 is primarily responsible for the metabolism of several clinically relevant drugs including bupropion, cyclophosphamide, propofol, and efavirenz. Although a number of single nucleotide polymorphisms have been found in the P450 2B6 gene, the influence of these variants on the metabolism of substrates and on the response to known inactivators of P450 2B6 has not been examined. We have compared the metabolism of different substrates of P450 2B6 (P450 Delta2B6) and the effects of mechanism-based inactivators with that observed with the polymorphic P450 Delta2B6 K262R in a reconstituted monooxygenase system (reconstituted system). Metabolism of bupropion by P450 Delta2B6 K262R resulted in increased production of hydroxybupropion compared with P450 Delta2B6. However, production of formaldehyde from the metabolism of benzphetamine by the P450 Delta2B6 K262R mutant was significantly less than that of the wild-type isozyme. P450 Delta2B6 K262R formed fewer benzphetamine metabolites compared with the wild type. N,N',N''-Triethylenethiophosphoramide (tTEPA) and bergamottin decreased the ability of both enzymes to hydroxylate bupropion and to O-deethylate 7-hydroxy-4-(trifluoromethyl)coumarin (7-EFC). Incubation with 17-alpha-ethynylestradiol decreased bupropion hydroxylation and 7-EFC O-deethylation with the wild-type enzyme but had no effect on the mutant. The kinetics for inactivation of the variant by tTEPA and bergamottin were determined using 7-EFC. The KI values for inactivation of the variant were significantly greater than those determined for the wild-type enzyme. These data demonstrate a functional difference between P450 Delta2B6 and the allelic variant P450 Delta2B6 K262R. PMID:15769884

Bumpus, Namandjé N; Sridar, Chitra; Kent, Ute M; Hollenberg, Paul F

2005-03-15

106

Cytochrome P450 (CYP) inhibition screening: Comparison of three tests  

Microsoft Academic Search

There are several different experimental systems for screening of in vitro inhibitory potency of drugs under development. In this study we compared three different types of cytochrome P450 (CYP) inhibition tests: the traditional single substrate assays, the fluorescent probe method with recombinant human CYPs, and a novel n-in-one technique. All major hepatic drug-metabolizing CYPs were included (1A2, 2A6, 2B6, 2C8,

Miia Turpeinen; Laura E. Korhonen; Ari Tolonen; Jouko Uusitalo; Risto Juvonen; Hannu Raunio; Olavi Pelkonen

2006-01-01

107

Cytochrome P450Dependent Lipid Metabolism in Preovulatory Follicles  

Microsoft Academic Search

Estrogen biosynthesis and proteolysis are both important processes involved in ovarian follicular development, which may be influenced by cytochrome P450 (CYP)-dependent fatty acid metabolites. However, CYP-dependent lipid metabolism has not been characterized with respect to follicular matura- tion in vivo. Therefore, follicular fluid was collected in the hours before and after the LH surge in pigs, and concentra- tions of

J. W. Newman; J. E. STOK; J. D. VIDAL; C. J. CORBIN; Q. HUANG; B. D. HAMMOCK; A. J. CONLEY

2004-01-01

108

Classification of Cytochrome P-450 Activities Using Machine Learning Methods  

Microsoft Academic Search

The cytochrome P-450 (GYP) system plays an integral part in the metabolism of drugs and other xenobiotics. Knowledge of the structural features required for interaction with any of the different isoforms of the CYP system is therefore immensely valuable in early drug discovery. In this paper, we focus on three major isoforms (CYP 1 A2, CYP 2D6, and CYP 3A4)

Felix Hammann; Heike Gutmann; Ulli Baumann; Christoph Helma; Juergen Drewe

2009-01-01

109

Cytochrome P450 is regulated by noradrenergic and serotonergic systems  

Microsoft Academic Search

The aim of the present study was to ascertain whether the noradrenergic or serotonergic systems may affect the expression of liver cytochrome P450 (CYP). Rats were injected intraperitoneally with N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4, a noradrenergic neurotoxin) or p-chloroamphetamine (PCA, a serotonergic neurotoxin) or p-chlorophenylalanine (PCPA, an inhibitor of serotonin synthesis). One week after neurotoxin injection the levels of neurotransmitters (noradrenaline, dopamine, serotonin)

Marta Kot; W?adys?awa A. Daniel

2011-01-01

110

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

Microsoft Academic Search

In this study, we identified the cDNA of P450 aromatase in the leopard gecko, a lizard with temperature-dependent sex determination. The cDNA encodes a putative protein of 505 amino acids. The deduced amino acid sequence of leopard gecko aromatase cDNA showed 80% identity with that of turtles, 70% with humans and 77% with chickens. This is the first report of

Daisuke Endo; Min Kyun Park

2005-01-01

111

Demethylation of Veratrole by Cytochrome P-450 in Streptomyces setonii  

PubMed Central

The actinomycete Streptomyces setonii 75Vi2 demethylates vanillic acid and guaiacol to protocatechuic acid and catechol, respectively, and then metabolizes the products by the ?-ketoadipate pathway. UV spectroscopy showed that this strain could also metabolize veratrole (1,2-dimethoxybenzene). When grown in veratrole-containing media supplemented with 2,2?-dipyridyl to inhibit cleavage of the aromatic ring, S. setonii accumulated catechol, which was detected by both liquid chromatography and gas chromatography. Reduced cell extracts from veratrole-grown cultures, but not sodium succinate-grown cultures, produced a carbon monoxide difference spectrum with a peak at 450 nm that indicated the presence of soluble cytochrome P-450. Addition of veratrole or guaiacol to oxidized cell extracts from veratrole-grown cultures produced difference spectra that indicated that these compounds were substrates for cytochrome P-450. My results suggest that S. setonii produces a cytochrome P-450 that is involved in the demethylation of veratrole and guaiacol to catechol, which is then catabolized by the ?-ketoadipate pathway.

Sutherland, John B.

1986-01-01

112

Significance of cytochrome P-450 (P-450 HFLa) of human fetal livers in the steroid and drug oxidations.  

PubMed

The purpose of this study was to clarify the pharmacological and physiological significance of P-450 HFLa. Thus, correlations between cytochrome P-450 (P-450 HFLa) level and different monooxygenase activities were investigated in liver homogenates from human fetuses. Poor correlation was seen between P-450 HFLa level and the activity of benzphetamine N-demethylation or aniline hydroxylation. In contrast, the content of P-450 HFLa was highly correlated with the activity of benzo(a)pyrene hydroxylation, 7-ethoxycoumarin O-deethylation or testosterone 6 beta-hydroxylation. In microsomes from human adult livers, a moderate relationship was also observed between testosterone 6 beta-hydroxylation and P-450 HFLa level. Furthermore, antibodies to P-450 HFLa inhibited testosterone 6 beta-hydroxylase activity in fetal and adult livers to similar extents. We conclude that P-450 HFLa is a form of cytochrome P-450 which catalyzes testosterone 6 beta-hydroxylation and limited drug oxidations in human fetal and adult livers. PMID:3493777

Kitada, M; Kamataki, T; Itahashi, K; Rikihisa, T; Kanakubo, Y

1987-02-15

113

Progesterone Oxidation by Cytochrome P450 2D Isoforms in the Brain  

Microsoft Academic Search

The existence of cytochrome P450 2D isoforms in the brain has been demonstrated, although their physiological functions remain to be elucidated. In this study we demonstrated that recombinant rat cytochrome P450 2D1 and 2D4 and human cytochrome P450 2D6 possess progesterone 6- and 16- hydroxylation activities; 2- and 21-hydroxylation activities; and 2-, 6-, 16- and 21-hydroxylation activities, respectively. Cytochrome P450

TOYOKO HIROI; WATARU KISHIMOTO; TOSHIO CHOW; SUSUMU IMAOKA; TAKASHI IGARASHI; YOSHIHIKO FUNAE

2010-01-01

114

Oxidation and rearrangements of flavanones by mammalian cytochrome P450.  

PubMed

To clarify the metabolic pathways of flavanones in mammals, the metabolism of (+/-)-flavanone and (+/-)-4'-methoxyflavanone by rat liver microsomes and recombinant human P450s in which structural changes are readily identifiable were examined. The beta-nicotinamide adenine dinucleotide phosphate (NADPH)-dependent formation of flavone plus (+/-)-2,3-trans-flavanonol and of 4'-methoxyflavone plus (+/-)-2,3-trans-4'-methoxyflavanonol, respectively, by rat liver microsomes was observed. The same metabolites were generated by recombinant human P450s in addition to the formation of isoflavone from (+/-)-flavanone. The kinetic isotope effects in these reactions were examined using deuterated (+/-)-flavanone and (+/-)-4'-methoxyflavanone. There was a strong isotope effect in the production of flavanonols, but the isotope effect in the production of flavones was small. The results indicated that the P450-mediated conversion of (+/-)-flavanone and of (+/-)-4'-methoxyflavanone to the corresponding metabolites proceeded via abstraction of a hydrogen radical from the C-2- or C-3-position of the flavanone skeleton. The antioxidant properties of flavanone and its metabolites were examined by measuring superoxide-scavenging activity in a xanthine-xanthine oxidase-cytochrome c system. (+/-)-2,3-trans-Flavanonol had higher activity than that of other flavonoids. Flavanones are metabolized by mammalian P450s, providing important information relevant to the metabolism and pharmacological action of dietary flavanones. PMID:15742975

Kagawa, H; Takahashi, T; Ohta, S; Harigaya, Y

2004-09-01

115

Biochemical Markers of Aquatic Environment Contamination - Cytochrome P450 in Fish. A Review  

Microsoft Academic Search

iroká Z., J. Drastichová: Biochemical Markers of Aquatic Environment Contamination - Cytochrome P450 in Fish. A Review. Acta Vet. Brno 2004, 73: 123-132. The paper reviews the most recent data on the role of cytochrome P450 as a biochemical marker of aquatic environment pollution. The important chemical characteristics of cytochrome P450, classification of its isoforms, its functions and involvement in

J. DRASTICHOVÁ

116

Cytochrome P450-derived eicosanoids: the neglected pathway in cancer  

PubMed Central

Endogenously produced lipid autacoids are locally acting small molecule mediators that play a central role in the regulation of inflammation and tissue homeostasis. A well-studied group of autacoids are the products of arachidonic acid metabolism, among which the prostaglandins and leukotrienes are the best known. They are generated by two pathways controlled by the enzyme systems cyclooxygenase and lipoxygenase, respectively. However, arachidonic acid is also substrate for a third enzymatic pathway, the cytochrome P450 (CYP) system. This third eicosanoid pathway consists of two main branches: ?-hydroxylases convert arachidonic acid to hydroxyeicosatetraenoic acids (HETEs) and epoxygenases convert it to epoxyeicosatrienoic acids (EETs). This third CYP pathway was originally studied in conjunction with inflammatory and cardiovascular disease. Arachidonic acid and its metabolites have recently stimulated great interest in cancer biology; but, unlike prostaglandins and leukotrienes the link between cytochome P450 metabolites and cancer has received little attention. In this review, the emerging role in cancer of cytochrome P450 metabolites, notably 20-HETE and EETs, are discussed.

Kaipainen, Arja; Greene, Emily R.; Huang, Sui

2010-01-01

117

Cytochrome p450 epoxygenase metabolism of arachidonic acid inhibits apoptosis.  

PubMed

The ubiquitous cytochrome P450 hemoproteins play important functional roles in the metabolism and detoxification of foreign chemicals. However, other than established roles in cholesterol catabolism and steroid hormone biosynthesis, their cellular and/or organ physiological functions remain to be fully characterized. Here we show that the cytochrome P450 epoxygenase arachidonic acid metabolite 14,15-epoxyeicosatrienoic acid (14,15-EET) inhibits apoptosis induced by serum withdrawal, H(2)O(2), etoposide, or excess free arachidonic acid (AA), as determined by DNA laddering, Hoechst staining, and fluorescein isothiocyanate-labeled annexin V binding. In the stable transfectants (BM3 cells) expressing a mutant bacterial P450 AA epoxygenase, F87V BM3, which was genetically engineered to metabolize arachidonic acid only to 14,15-EET, AA did not induce apoptosis and protected against agonist-induced apoptosis. Ceramide assays demonstrated increased AA-induced ceramide production within 1 h and elevated ceramide levels for up to 48 h, the longest time tested, in empty-vector-transfected cells (Vector cells) but not in BM3 cells. Inhibition of cytochrome P450 activity by 17-octadecynoic acid restored AA-induced ceramide production in BM3 cells. Exogenous C2-ceramide markedly increased apoptosis in quiescent Vector cells as well as BM3 cells, and apoptosis was prevented by pretreatment of Vector cells with exogenous 14,15-EET and by pretreatment of BM3 cells with AA. The ceramide synthase inhibitor fumonisin B1 did not affect AA-induced ceramide production and apoptosis; in contrast, these effects of AA were blocked by the neutral sphingomyelinase inhibitor scyphostatin. The pan-caspase inhibitor Z-VAD-fmk had no effect on AA-induced ceramide generation but abolished AA-induced apoptosis. The antiapoptotic effects of 14,15-EET were blocked by two mechanistically and structurally distinct phosphatidylinositol-3 (PI-3) kinase inhibitors, wortmannin and LY294002, but not by the specific mitogen-activated protein kinase kinase inhibitor PD98059. Immunoprecipitation followed by an in vitro kinase assay revealed activation of Akt kinase within 10 min after 14,15-EET addition, which was completely abolished by either wortmannin or LY294002 pretreatment. In summary, the present studies demonstrated that 14,15-EET inhibits apoptosis by activation of a PI-3 kinase-Akt signaling pathway. Furthermore, cytochrome P450 epoxygenase promotes cell survival both by production of 14,15-EET and by metabolism of unesterified AA, thereby preventing activation of the neutral sphingomyelinase pathway and proapoptotic ceramide formation. PMID:11509673

Chen, J K; Capdevila, J; Harris, R C

2001-09-01

118

Role of cytochrome P450 in drug interactions  

PubMed Central

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

Bibi, Zakia

2008-01-01

119

Immunohistochemical detection of pulmonary cytochrome P450IA and metabolic activities associated with P450IA1 and P450IA2 isozymes in lung cancer patients.  

PubMed Central

The main polycyclic aromatic hydrocarbon-inducible cytochrome P450 was studied in lung tissue from 57 lung cancer patients by immunohistochemistry, using a monoclonal antibody (1-7-1) that recognizes P450IA1 and P450IA2 isozymes. The intensity of immunostaining was compared with the pulmonary activity of a P450IA1-dependent enzyme, aryl hydrocarbon hydroxylase (AHH), and with P450IA2-related metabolic activity estimated from the ratio of caffeine metabolites in urine. Immunostaining was not observed in peripheral lung tissue of nonsmokers or ex-smokers but was seen in the bronchiolar and alveolar epithelium of all patients who were smokers and had a peripheral carcinoma (16/16) and of 60% (10/17) of those who had a bronchial carcinoma. AHH activity was positively related to the intensity of immunostaining, and an almost 2-fold increase due to smoking was detected in the ratios of caffeine metabolites. These results demonstrate that tobacco smoke induces P450IA1 in the lung and probably P450IA2 in the liver, and suggest a role for certain metabolic phenotypes of P450IA1 in peripheral pulmonary carcinoma. Images FIGURE 1. A FIGURE 1. B

Anttila, S; Vainio, H; Hietanen, E; Camus, A M; Malaveille, C; Brun, G; Husgafvel-Pursiainen, K; Heikkila, L; Karjalainen, A; Bartsch, H

1992-01-01

120

Purification and properties of cytochrome P-450 from homogenates of human fetal livers.  

PubMed

A form of cytochrome P-450, namely P-450HFLa of human fetal livers, was purified to a specific content of 12.6 nmol/mg protein. The cytochrome P-450 preparation was electrophoretically homogeneous and had an apparent monomeric molecular weight of 51,000 as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The cytochrome showed catalytic activities as oxidations of N-methylaniline, ethylmorphine, N,N-dimethylaniline, N,N-dimethylnitrosamine, benzphetamine, aminopyrine, aniline, p-nitroanisole, and 7-ethoxycoumarin to various extents. In fetal liver homogenate, the amount of cytochrome P-450 that reacted with the antiserum to P-450HFLa accounted for more than 36% of the total cytochrome P-450 in three different fetal livers. On the other hand, the amount of P-450HFLa was less than 5% of the total cytochrome P-450 in adult liver microsomes. PMID:4026319

Kitada, M; Kamataki, T; Itahashi, K; Rikihisa, T; Kato, R; Kanakubo, Y

1985-08-15

121

Oxidation of Dihydrotestosterone by Human Cytochromes P450 19A1 and 3A4*  

PubMed Central

Dihydrotestosterone is a more potent androgen than testosterone and plays an important role in endocrine function. We demonstrated that, like testosterone, dihydrotestosterone can be oxidized by human cytochrome P450 (P450) 19A1, the steroid aromatase. The products identified include the 19-hydroxy- and 19-oxo derivatives and the resulting ?1,10-, ?5,10-, and ?9,10-dehydro 19-norsteroid products (loss of 19-methyl group). The overall catalytic efficiency of oxidation was ?10-fold higher than reported for 3?-reduction by 3?-hydroxysteroid dehydrogenase, the major enzyme known to deactivate dihydrotestosterone. These and other studies demonstrate the flexibility of P450 19A1 in removing the 1- and 2-hydrogens from 19-norsteroids, the 2-hydrogen from estrone, and (in this case) the 1-, 5?-, and 9?-hydrogens of dihydrotestosterone. Incubation of dihydrotestosterone with human liver microsomes and NADPH yielded the 18- and 19-hydroxy products plus the ?1,10-dehydro 19-nor product identified in the P450 19A1 reaction. The 18- and 19-hydroxylation reactions were attributed to P450 3A4, and 18- and 19-hydroxydihydrotestosterone were identified in human plasma and urine samples. The change in the pucker of the A ring caused by reduction of the ?4,5 bond is remarkable in shifting the course of hydroxylation from the 6?-, 2?-, 1?-, and 15?-methylene carbons (testosterone) to the axial methyl groups (18, 19) in dihydrotestosterone and demonstrates the sensitivity of P450 3A4, even with its large active site, to small changes in substrate structure.

Cheng, Qian; Sohl, Christal D.; Yoshimoto, Francis K.; Guengerich, F. Peter

2012-01-01

122

Update: clinically significant cytochrome P-450 drug interactions.  

PubMed

Recent technologies have resulted in an explosion of information concerning the cytochrome P-450 isoenzymes and increased awareness of life-threatening interactions with such commonly prescribed drugs as cisapride and some antihistamines. Knowledge of the substrates, inhibitors, and inducers of these enzymes assists in predicting clinically significant drug interactions. In addition to inhibition and induction, microsomal drug metabolism is affected by genetic polymorphisms, age, nutrition, hepatic disease, and endogenous chemicals. Of the more than 30 human isoenzymes identified to date, the major ones responsible for drug metabolism include CYP3A4, CYP2D6, CYP1A2, and the CYP2C subfamily. PMID:9469685

Michalets, E L

123

Regulation of cytochrome P450 (CYP) genes by nuclear receptors.  

PubMed Central

Members of the nuclear-receptor superfamily mediate crucial physiological functions by regulating the synthesis of their target genes. Nuclear receptors are usually activated by ligand binding. Cytochrome P450 (CYP) isoforms often catalyse both formation and degradation of these ligands. CYPs also metabolize many exogenous compounds, some of which may act as activators of nuclear receptors and disruptors of endocrine and cellular homoeostasis. This review summarizes recent findings that indicate that major classes of CYP genes are selectively regulated by certain ligand-activated nuclear receptors, thus creating tightly controlled networks.

Honkakoski, P; Negishi, M

2000-01-01

124

Human cytochromes P450 in health and disease.  

PubMed

There are 18 mammalian cytochrome P450 (CYP) families, which encode 57 genes in the human genome. CYP2, CYP3 and CYP4 families contain far more genes than the other 15 families; these three families are also the ones that are dramatically larger in rodent genomes. Most (if not all) genes in the CYP1, CYP2, CYP3 and CYP4 families encode enzymes involved in eicosanoid metabolism and are inducible by various environmental stimuli (i.e. diet, chemical inducers, drugs, pheromones, etc.), whereas the other 14 gene families often have only a single member, and are rarely if ever inducible or redundant. Although the CYP2 and CYP3 families can be regarded as largely redundant and promiscuous, mutations or other defects in one or more genes of the remaining 16 gene families are primarily the ones responsible for P450-specific diseases-confirming these genes are not superfluous or promiscuous but rather are more directly involved in critical life functions. P450-mediated diseases comprise those caused by: aberrant steroidogenesis; defects in fatty acid, cholesterol and bile acid pathways; vitamin D dysregulation and retinoid (as well as putative eicosanoid) dysregulation during fertilization, implantation, embryogenesis, foetogenesis and neonatal development. PMID:23297354

Nebert, Daniel W; Wikvall, Kjell; Miller, Walter L

2013-01-06

125

P450 camr , a cytochrome P450 catalysing the stereospecific 6- endo -hydroxylation of (1 R )-(+)-camphor  

Microsoft Academic Search

.   \\u000a Rhodococcus sp. NCIMB 9784 accumulated 6-endo-hydroxycamphor 3 when grown on (1R)-(+)-camphor 1 as sole carbon source. The structure of 3 has been unambiguously assigned for the first time using X-ray crystallography. A soluble cytochrome P450 hydroxylase, induced\\u000a by growth on (1R)-(+)-camphor and designated P450camr, has been isolated from the bacterium Rhodococcus sp. NCIMB 9784. Using authentic 6-endo hydroxycamphor as

G. Grogan; G. Roberts; S. Parsons; N. Turner; S. Flitsch

2002-01-01

126

The inhaled glucocorticoid fluticasone propionate efficiently inactivates cytochrome P450 3A5, a predominant lung P450 enzyme  

PubMed Central

Inhaled glucocorticoid (GC) therapy is a vital part of the management of chronic asthma. GCs are metabolized by members of the cytochrome P450 3A family in both liver and lung, but the enzymes are differentially expressed. Selective inhibition of one or more P450 3A enzymes could substantially modify target and systemic concentrations of GCs. In this study, we have evaluated the mechanism-based inactivation of P450 3A4, 3A5 and 3A7 enzymes by GCs. Among the five major inhaled GCs approved for clinical use in the United States, fluticasone propionate (FLT) was the most potent mechanism-based inactivator of P450 3A5, the predominant P450 enzyme in the lung. FLT inactivated P450 3A5 in a time- and concentration-dependent manner with KI, kinact and partition ratio of 16 ?M, 0.027 min-1 and 3, respectively. In contrast, FLT minimally inactivated P450 3A4 and did not inactivate 3A7, even with a concentration of 100 ?M. The inactivation of P450 3A5 by FLT was irreversible because dialysis did not restore enzyme activity. In addition, the exogenous nucleophilic scavenger GSH did not attenuate inactivation. The prosthetic heme of P450 3A5 was not modified by FLT. The loss of P450 3A5 activity in lung cells could substantially decrease the metabolism of FLT, which would increase the effective FLT concentration at its target site, the respiratory epithelium. Also, inactivation of lung P450 3A5 could increase the absorption of inhaled FLT, which could lead to high systemic concentrations and adverse effects, such as life-threatening adrenal crises or cataracts that have been documented in children receiving high doses of inhaled GCs.

Murai, Takahiro; Reilly, Christopher R.; Ward, Robert M.; Yost, Garold S.

2010-01-01

127

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

PubMed

In this study, we identified the cDNA of P450 aromatase in the leopard gecko, a lizard with temperature-dependent sex determination. The cDNA encodes a putative protein of 505 amino acids. The deduced amino acid sequence of leopard gecko aromatase cDNA showed 80% identity with that of turtles, 70% with humans and 77% with chickens. This is the first report of the identification of P450 aromatase cDNA in squamata species. It has been reported that this gene is expressed in different layers of cells in the ovary of mammalian species and avian species. Thus, we also investigated cells expressing the mRNA of this gene in the ovary of the leopard gecko by RT-PCR and in situ hybridization. The mRNA expression of leopard gecko P450 aromatase was localized in both the thecal and granulosa cell layers in the ovary. The expression in thecal and granulosa cell layers was examined in the largest follicle, second largest follicle and third largest follicle by RT-PCR. A higher level of mRNA expression was observed in the granulosa cell layer of the second largest follicle than in other cell layers. This result may reflect the characteristics of follicles in species with automonochronic ovulation. PMID:15893926

Endo, Daisuke; Park, Min Kyun

2005-07-01

128

New cytochrome P450 isoforms as cancer biomarkers and targets for chemopreventive and chemotherapeutic agents.  

PubMed

Cytochromes P450 (P450) are a multigene family of enzymes possessing a central role in the oxidative metabolism of a wide range of xenobiotics, including anticancer drugs, and endogenous compounds. The activity of different P450 isoforms varies within specific tissues and cell types and is selectively regulated together with their gene expression. Moreover, differential expression of certain P450 isoforms' genes in tumor cells compared to normal tissues can be observed. This creates the potential for the use of these isozymes as tumor markers or selective prodrug activators. This article discusses the characteristics and function of five isoforms of cytochrome P450 (P450 1B1, P450 2W1, P450 2S1, P450 2R1, P450 2U1) that could be potential targets for tumor therapeutic and preventive strategies. These isoforms have been chosen because their level of expression in tumor tissues is definitely higher than in normal tissues. PMID:24018436

Szaefer, Hanna; Cichocki, Micha?; Majchrzak-Celi?ska, Aleksandra

2013-08-02

129

Zonation of hepatic cytochrome P-450 expression and regulation.  

PubMed Central

The CYP genes encode enzymes of the cytochrome P-450 superfamily. Cytochrome P-450 (CYP) enzymes are expressed mainly in the liver and are active in mono-oxygenation and hydroxylation of various xenobiotics, including drugs and alcohols, as well as that of endogenous compounds such as steroids, bile acids, prostaglandins, leukotrienes and biogenic amines. In the liver the CYP enzymes are constitutively expressed and commonly also induced by chemicals in a characteristic zonated pattern with high expression prevailing in the downstream perivenous region. In the present review we summarize recent studies, mainly based on rat liver, on the factors regulating this position-dependent expression and induction. Pituitary-dependent signals mediated by growth hormone and thyroid hormone seem to selectively down-regulate the upstream periportal expression of certain CYP forms. It is at present unknown to what extent other hormones that also affect total hepatic CYP activities, i.e. insulin, glucagon, glucocorticoids and gonadal hormones, act zone-specifically. The expression and induction of CYP enzymes in the perivenous region probably have important toxicological implications, since many CYP-activated chemicals cause cell injury primarily in this region of the liver.

Oinonen, T; Lindros, K O

1998-01-01

130

Resveratrol Is a Selective Human Cytochrome P450 1A1 Inhibitor  

Microsoft Academic Search

Resveratrol (trans-3,4?,5-trihydroxystilbene) is a phytoalexin compound found in juice and wine produced from dark-skinned grape cultivars and reported to have anti-inflammatory and anticarcinogenic activities. To investigate the mechanism of anticarcinogenic activities of resveratrol, the effects on cytochrome P450 (P450) were determined in human liver microsomes and Escherichia coli membranes coexpressing human P450 1A1 or P450 1A2 with human NADPH-P450 reductase

Young Jin Chun; Mie Young Kim; F. Peter Guengerich

1999-01-01

131

Opioids activate brain analgesic circuits through cytochrome P450/epoxygenase signaling.  

PubMed

To assess the importance of brain cytochrome P450 (P450) activity in mu opioid analgesic action, we generated a mutant mouse with brain neuron-specific reductions in P450 activity; these mice showed highly attenuated morphine antinociception compared with controls. Pharmacological inhibition of brain P450 arachidonate epoxygenases also blocked morphine antinociception in mice and rats. Our findings indicate that a neuronal P450 epoxygenase mediates the pain-relieving properties of morphine. PMID:20139973

Conroy, Jennie L; Fang, Cheng; Gu, Jun; Zeitlin, Scott O; Yang, Weizhu; Yang, Jun; VanAlstine, Melissa A; Nalwalk, Julia W; Albrecht, Phillip J; Mazurkiewicz, Joseph E; Snyder-Keller, Abigail; Shan, Zhixing; Zhang, Shao-Zhong; Wentland, Mark P; Behr, Melissa; Knapp, Brian I; Bidlack, Jean M; Zuiderveld, Obbe P; Leurs, Rob; Ding, Xinxin; Hough, Lindsay B

2010-02-07

132

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

National Technical Information Service (NTIS)

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...

F. H. Arnold

2012-01-01

133

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

PubMed

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

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

2004-11-18

134

Cloning and expression of a member of a new cytochrome P-450 family: cytochrome P-450lin (CYP111) from Pseudomonas incognita.  

PubMed Central

Cytochrome P-450lin catalyzes the 8-methyl hydroxylation of linalool as the first committed step of its utilization by Pseudomonas incognita as the sole carbon source. By using a polymerase chain reaction-based cloning strategy, a 2.1-kb DNA fragment containing the cytochrome P-450lin gene (linC) was isolated. An open reading frame of 406 amino acids has been identified as that of P-450lin on the basis of amino acid sequence data from peptides of the native protein. Heterologous expression of functional holoprotein is exhibited by Escherichia coli transformed with pUC18 containing the subcloned linC gene under constitutive transcriptional control of the lac promoter. The G+C content of linC was found to be 55% overall and 58% in the third codon position. An optimized amino acid sequence alignment of P-450lin with cytochrome P-450cam shows that the two enzymes have only 25% identity. P-450lin was found to exhibit the expected conservation in the axial cysteine heme ligand-containing peptide and the threonine region postulated to form an O2-binding pocket (T. L. Poulos, B. C. Finzel, and A. J. Howard, J. Mol. Biol. 195:687-700, 1987). The low amino acid sequence identity between P-450lin and all other P-450 sequences has shown that P-450lin is the first member of the CYP111 P-450 gene family.

Ropp, J D; Gunsalus, I C; Sligar, S G

1993-01-01

135

Computational models for predicting interactions with cytochrome p450 enzyme.  

PubMed

Cytochrome p450 (CYP) enzymes are predominantly involved in Phase 1 metabolism of xenobiotics. As only 6 isoenzymes are responsible for approximately 90 % of known oxidative drug metabolism, a number of frequently prescribed drugs share the CYP-mediated metabolic pathways. Competing for a single enzyme by the co-administered therapeutic agents can substantially alter the plasma concentration and clearance of the agents. Furthermore, many drugs are known to inhibit certain p450 enzymes which they are not substrates for. Because some drug-drug interactions could cause serious adverse events leading to a costly failure of drug development, early detection of potential drug-drug interactions is highly desirable. The ultimate goal is to be able to predict the CYP specificity and the interactions for a novel compound from its chemical structure. Current computational modeling approaches, such as two-dimensional and three-dimensional quantitative structure-activity relationship (QSAR), pharmacophore mapping and machine learning methods have resulted in statistically valid predictions. Homology models have been often combined with 3D-QSAR models to impose additional steric restrictions and/or to identify the interaction site on the proteins. This article summarizes the available models, methods, and key findings for CYP1A2, 2A6, 2C9, 2D6 and 3A4 isoenzymes. PMID:16918472

Arimoto, Rieko

2006-01-01

136

Interactions of phospholipase D and cytochrome P450 protein stability  

SciTech Connect

Previous studies have suggested a relationship between cytochrome P450 (P450) 3A (CYP3A) conformation and the phospholipid composition of the associated membrane. In this study, we utilized a novel microsomal incubation system that mimics many of the characteristics of CYP3A degradation pathway that have been observed in vivo and in cultured cells to study the effects of phospholipid composition on protein stability. We found that addition of phosphatidylcholine-specific phospholipase D (PLD) stabilized CYP3A in this system, but that phosphatidylinositol-specific phospholipase C (PLC) was without effect. Addition of phosphatidic acid also stabilized CYP3A protein in the microsomes. The use of 1,10-phenanthroline (phenanthroline), an inhibitor of PLD activity, decreased CYP3A stability in incubated microsomes. Similarly, 6-h treatment of primary cultures of rat hepatocytes with phenanthroline resulted in nearly complete loss of CYP3A protein. Treatment of rats with nicardipine or dimethylsulfoxide (DMSO), which have been shown to affect CYP3A stability, altered the phospholipid composition of hepatic microsomes. It did not appear, though, that the changes in phospholipid composition that resulted from these in vivo treatments accounted for the change in CYP3A stability observed in hepatic microsomes from these animals.

Zangar, Richard C.; Fan, Yang-Yi; Chapkin, Robert S.

2004-08-01

137

Advances in molecular modeling of human cytochrome p450 polymorphism.  

PubMed

Cytochrome P450 (CYP) is a supergene family of metabolizing enzymes involved in the phase I metabolism of drugs and endogenous compounds. CYP oxidation often leads to inactive drug metabolites or to highly toxic or carcinogenic metabolites involved in adverse drug reactions (ADR). During the last decade, the impact of CYP polymorphism in various drug responses and ADR has been demonstrated. Of the drugs involved in ADR, 56% are metabolized by polymorphic phase I metabolizing enzymes, 86% among them being CYP. Here, we review the major CYP polymorphic forms, their impact for drug response and current advances in molecular modeling of CYP polymorphism. We focus on recent studies exploring CYP polymorphism performed by the use of sequence-based and/or protein-structure-based computational approaches. The importance of understanding the molecular mechanisms related to CYP polymorphism and drug response at the atomic level is outlined. PMID:23856621

Martiny, Virginie Y; Miteva, Maria A

2013-07-13

138

Advances in human cytochrome p450 and personalized medicine.  

PubMed

Among all the drug metabolic enzymes, cytochrome P450 (CYP450) superfamily acts as an important role responsible for the oxidation of almost 90% currently used drugs. As variations of Single Nucleotide Polymorphism (SNPs) in human CYP450 genes will cause different drug effects and even adverse effects, studies on SNPs of human CYP450 genes can be used for indicating the most possible genes associated with human diseases and relevant therapeutic targets, predicting the drug efficacy and adverse drug response, investigating individual gene specific properties and then providing personalized and optimal clinic therapies. Recently, some new bioinformatics methods are introduced in SNPs researches, which significantly facilitate the development of drug and medicine. The review will focus on a brief introduction of the SNPs of human drug metabolic enzymes and their relationships with personalized medicine. Besides, common bioinformatics analysis methods and some latest progresses and applications in this area will also be discussed. PMID:21453272

Chen, Qi; Zhang, Tao; Wang, Jing-Fang; Wei, Dong-Qing

2011-06-01

139

In silico prediction of cytochrome P450-mediated drug metabolism.  

PubMed

The application of combinatorial chemistry and high-throughput screening technique enables the large number of chemicals to be generated and tested simultaneously, which will facilitate the drug development and discovery. At the same time, it brings about a challenge of how to efficiently identify the potential drug candidates from thousands of compounds. A way used to deal with the challenge is to consider the drug pharmacokinetic properties, such as absorption, distribution, metabolism and excretion (ADME), in the early stage of drug development. Among ADME properties, metabolism is of importance due to the strong association with efficacy and safety of drug. The review will focus on in silico approaches for prediction of Cytochrome P450-mediated drug metabolism. We will describe these predictive methods from two aspects, structure-based and data-based. Moreover, the applications and limitations of various methods will be discussed. Finally, we provide further direction toward improving the predictive accuracy of these in silico methods. PMID:21470181

Zhang, Tao; Chen, Qi; Li, Li; Liu, Limin Angela; Wei, Dong-Qing

2011-06-01

140

Active site dynamics of toluene hydroxylation by cytochrome P-450  

SciTech Connect

Rat liver cytochrome P-450 hydroxylates toluene to benzyl alcohol plus o-, m-, and p-cresol. Deuterated toluenes were incubated under saturating conditions with liver microsomes from phenobarbital-pretreated rats, and product yields and ratios were measured. Stepwise deuteration of the methyl leads to stepwise decreases in the alcohol/cresol ratio without changing the cresol isomer ratios. Extensive deuterium retention in the benzyl alcohols from PhCH{sub 2}D and PhCHD{sub 2} suggests there is a large intrinsic isotope effect for benzylic hydroxylation. After replacement of the third benzylic H by D, the drop in the alcohol/cresol ratio was particularly acute, suggsting that metabolic switching from D to H within the methyl group was easier than switching from the methyl to the ring. Comparison of the alcohol/cresol ratio for PhCH{sub 3} vs PhCD{sub 3} indicated a net isotope effect of 6.9 for benzylic hydroxylation. From product yield data for PhCH{sub 3} and PhCD{sub 3}, {sup D}V for benzyl alcohol formation is only 1.92, whereas {sup D}V for total product formation is 0.67 (i.e., inverse). From competitive incubations of PhCH{sub 3}/PhCD{sub 3} mixtures {sup D}(V/K) isotope effects on benzyl alcohol formation and total product formation (3.6 and 1.23, respectively) are greatly reduced, implying strong commitment to catalysis. In contrast, {sup D}(V/K) for the alcohol/cresol ratio is 6.3, indicating that the majority of the intrinsic isotope effect is expressed through metabolic switching. Overall, these data are consistent with reversible formation of a complex between toluene and the active oxygen form of cytochrome P-450, which rearranges internally and reacts to form products faster than it dissociates back to release substrate.

Hanzlik, R.P.; Kahhiing John Ling (Univ. of Kansas, Lawrence (United States))

1990-06-22

141

Role of P-450 aromatase in sex determination of the diamondback terrapin, Malaclemys terrapin.  

PubMed

Sex determination in the diamondback terrapin, Malaclemys terrapin, is temperature-dependent. Eggs incubated at 31 degrees C, and above, hatch in approximately 45 days as females. Eggs incubated below 27 degrees C hatch in about 60 days as males. Sex is not reversible after hatching. Nest temperatures in the wild can be as low as 20 degrees C and as high as 37 degrees C with as much as a 10 degrees C diel cycle. The shortest incubation time measured in nature was 56 days and the longest approaching 120 days. Nests in our study site produced predominantly (> 95%) male hatchlings. Treatment of developing embryos with estrogen produces females at male producing temperatures while treatment with fadrozole (a nonsteroidal aromatase inhibitor) induces partial male-like gonads. Treatment with a steroidal aromatase inhibitor (4-hydroxyandrostenedione, 4-OHA) had no effect on sex determination. Both fadrozole and 4-OHA are potent competitive inhibitors (Ki approximately 40-50 nM) for terrapin in vitro aromatase activity. These findings are consistent with aromatase expression being a key step in sex determination of terrapins. We have cloned a partial single copy P-450 aromatase from the terrapin using a cDNA library constructed from ovarian mRNA. This partial clone is highly homologous to other vertebrate aromatases. PMID:7931130

Jeyasuria, P; Roosenburg, W M; Place, A R

1994-09-15

142

Synthesis and processing of mitochondrial steroid hydroxylases. In vivo maturation of the precursor forms of cytochrome P-450scc, cytochrome P-450(11)beta, and adrenodoxin  

SciTech Connect

The synthesis and maturation of the precursor forms of three mitochondrial enzymes involved in steroid hormone biosynthesis have been studied in vivo. Primary cultures of bovine adrenocortical cells were radiolabeled with (TVS) methionine and newly synthesized cholesterol side-chain cleavage cytochrome P-450 (P-450scc), 11 beta-hydroxylase cytochrome P-450 (P-450(11)beta), and adrenodoxin immunoisolated using specific antibodies. Both the precursor and mature forms of P-450scc and P-450(11)beta were detected during short periods of pulse labeling. Pulse-chase experiments showed that the precursor form of each cytochrome P-450 had an apparent half-life of 3.5 min. In contrast, the precursor form of adrenodoxin was not readily detected in pulse-labeling experiments until a substantial amount of its mature form had accumulated. The synthesis of the P-450scc and P-450(11)beta precursors was induced in cells maintained in the presence of adrenocorticotropin, and the rates of appearance of their processed forms were also increased. The mature forms of all three proteins were immunoisolated from a trypsinized mitochondrial fraction prepared from the radiolabeled cells, demonstrating that the mature proteins were localized within the organelle. These studies establish that the maturation of the precursor forms of the mitochondrial steroidogenic enzymes are characterized by steps similar to those reported for other mitochondrial precursor proteins.

Matocha, M.F.; Waterman, M.R.

1985-10-05

143

Involvement of cytochrome P450 in hydroxylation of propylbenzene by Fusarium moniliforme strain MS31.  

PubMed

Fusarium moniliforme strain MS31 can oxidize propylbenzene to (R)-1-phenylpropanol with what may be a cytochrome P450. Hydroxylation of propylbenzene needed molecular oxygen, and NADPH as a coenzyme gave a higher yield than NADH. The hydroxylation proceeded further when FAD and FMN were added than in their absence, suggesting that the enzyme was a flavo-protein. Carbon monoxide inhibited the hydroxylation, as did other cytochrome P450 inhibitors such as SKF 525A and miconazole. These characteristics matched those of a microsomal cytochrome P450 monooxygenase system that contained NADPH-cytochrome P450 reductase. PMID:16233150

Uzura, A; Suzuki, T; Katsuragi, T; Tani, Y

2001-01-01

144

Physical Incorporation of NADPH-cytochrome P450 Reductase and Cytochrome P450 into Phospholipid Vesicles using Glycocholate and Biobeads*  

PubMed Central

In a previous study from our lab (Drug Metab. Disp. (2006) 34, 660–666), we found several limitations with published methods (cholate gel filtration and cholate dialysis) for the incorporation of cytochromes P450 and P450 reductase into phospholipid vesicles. We found that a significant proportion of reductase was not incorporated in the vesicles when the amount of reductase was equal to or greater than that of CYP2B4 in the systems reconstituted with phosphatidylcholine. Furthermore, implementation of these methods compromised the ability of the CYP2B4 to form a ferrous carbon monoxy complex. In the current study, a comparison of results using the detergent-dialysis method with five similar detergents having the “bile salt” ring structure finds that glycocholate results in the greatest incorporation of reductase and the least loss in carbon monoxy ferrous CYP2B4 complex. The method is further improved by using Biobeads SM-2 to remove detergent instead of the lengthy dialysis procedure or the size exclusion chromatography which significantly dilutes the protein and lipid concentrations of the preparation. The method is shown to be applicable over a range of lipid to CYP2B4 ratios; and by employing assay methods for total lipid, reductase, and CYP2B4, this improved reconstitution method resulted in increased incorporation efficiencies while minimizing the protein degradation inherent with these procedures.

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

2009-01-01

145

Insecticide and Insecticide Metabolite Interactions with Cytochrome P450 Mediated Activities in Maize  

Microsoft Academic Search

In vitroassays were used to determine if organophosphate, carbamate, and synthetic pyrethroid insecticides affected the cytochrome P450 monooxygenase (P450) catalyzed hydroxylation of nicosulfuron, bentazon, cinnamic acid, or lauric acid in maize microsomes. All P450 activities were inhibited approximately 50% by carbaryl, and none were inhibited by permethrin. Hydroxylations of nicosulfuron, bentazon, lauric acid, and cinnamic acid were inhibited by malathion

Roger J. Baerg; Michael Barrett; Nicholas D. Polge

1996-01-01

146

Effects of aromatase inhibitor on sex differentiation and levels of P450 (17 alpha) and P450 arom messenger ribonucleic acid of gonads in chicken embryos.  

PubMed

On Day 5 of incubation fertilized eggs of single-comb White Leghorn hens were injected with an aromatase inhibitor (AI) and the sex reversal effect and levels of mRNA of P450(17alpha-hydroxylase) (P450(17alpha)) and 450(aromatase) (P450(arom)) were evaluated by observation of gonadal phenotype and by Northern and slot blot analysis. Individual genetic sex was evaluated by Southern blot analysis of red blood cells using a female sex chromosomal W-specific DNA probe. Saline injection had no sex reversal effect, whereas AI injection resulted in 50% sex reversal from genetic female but no effect on male. Levels of P450(17-alpha) mRNA were high in both ovary and testis in the control group but these levels were lowered significantly in ovary, testis, and sex-reversed gonad (testis) in the AI-treated group. On the other hand, levels of P450(arom) mRNA in the ovary were higher than those in the testis of the control group. AI treatment significantly suppressed ovarian levels of P450(arom) mRNA. Although estradiol alone failed to prevent the phenotypic male to female change, coadministration of estrogen suppressed the sex-reversal effect of AI and restored mRNA levels of P450(arom) in the ovary to control levels. These results suggest that expression of P450(arom) mRNA and estrogen plays an important role in sex differentiation of the female gonad of the chicken. PMID:8998968

Abinawanto; Shimada, K; Yoshida, K; Saito, N

1996-05-01

147

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

PubMed Central

Background Cytochrome P450 monooxygenases (P450s) catalyze oxidation of various substrates using oxygen and NAD(P)H. Plant P450s are involved in the biosynthesis of primary and secondary metabolites performing diverse biological functions. The recent availability of the soybean genome sequence allows us to identify and analyze soybean putative P450s at a genome scale. Co-expression analysis using an available soybean microarray and Illumina sequencing data provides clues for functional annotation of these enzymes. This approach is based on the assumption that genes that have similar expression patterns across a set of conditions may have a functional relationship. Results We have identified a total number of 332 full-length P450 genes and 378 pseudogenes from the soybean genome. From the full-length sequences, 195 genes belong to A-type, which could be further divided into 20 families. The remaining 137 genes belong to non-A type P450s and are classified into 28 families. A total of 178 probe sets were found to correspond to P450 genes on the Affymetrix soybean array. Out of these probe sets, 108 represented single genes. Using the 28 publicly available microarray libraries that contain organ-specific information, some tissue-specific P450s were identified. Similarly, stress responsive soybean P450s were retrieved from 99 microarray soybean libraries. We also utilized Illumina transcriptome sequencing technology to analyze the expressions of all 332 soybean P450 genes. This dataset contains total RNAs isolated from nodules, roots, root tips, leaves, flowers, green pods, apical meristem, mock-inoculated and Bradyrhizobium japonicum-infected root hair cells. The tissue-specific expression patterns of these P450 genes were analyzed and the expression of a representative set of genes were confirmed by qRT-PCR. We performed the co-expression analysis on many of the 108 P450 genes on the Affymetrix arrays. First we confirmed that CYP93C5 (an isoflavone synthase gene) is co-expressed with several genes encoding isoflavonoid-related metabolic enzymes. We then focused on nodulation-induced P450s and found that CYP728H1 was co-expressed with the genes involved in phenylpropanoid metabolism. Similarly, CYP736A34 was highly co-expressed with lipoxygenase, lectin and CYP83D1, all of which are involved in root and nodule development. Conclusions The genome scale analysis of P450s in soybean reveals many unique features of these important enzymes in this crop although the functions of most of them are largely unknown. Gene co-expression analysis proves to be a useful tool to infer the function of uncharacterized genes. Our work presented here could provide important leads toward functional genomics studies of soybean P450s and their regulatory network through the integration of reverse genetics, biochemistry, and metabolic profiling tools. The identification of nodule-specific P450s and their further exploitation may help us to better understand the intriguing process of soybean and rhizobium interaction.

2010-01-01

148

Chemical proteomic probes for profiling cytochrome P450 activities and drug interactions in vivo  

PubMed Central

The cytochrome P450 (P450) superfamily metabolizes many endogenous signaling molecules and drugs. P450 enzymes are regulated by post-translational mechanisms in vivo, which hinders their functional characterization by conventional genomic or proteomic methods. Here, we describe a chemical proteomic strategy to profile P450 activities directly in living systems. Derivatization of a mechanism-based inhibitor with a “clickable” handle provided an activity-based probe that labels multiple P450s both in proteomic extracts and in vivo. This probe was used to record alterations in liver P450 activities triggered by chemical agents, including inducers of P450 expression and direct P450 inhibitors. The chemical proteomic strategy described herein thus offers a versatile method to monitor P450 activities and small molecule interactions in any biological system and, through doing so, should facilitate the functional characterization of this large and diverse enzyme class.

Wright, Aaron T.; Cravatt, Benjamin F.

2007-01-01

149

Marine invertebrate cytochrome P450: Emerging insights from vertebrate and insect analogies  

Microsoft Academic Search

Cytochrome P450 enzymes (P450s) are responsible for the oxidative metabolism of a plethora of endogenous and exogenous substrates. P450s and associated activities have been demonstrated in numerous marine invertebrates belonging to the phyla Cnidaria, Annelida (Polychaeta), Mollusca, Arthropoda (Crustacea) and Echinodermata. P450s of marine invertebrates and vertebrates show considerable sequence divergence and the few orthologs reveal the selective constraint on

Kim F. Rewitz; Bjarne Styrishave; Anders Løbner-Olesen; Ole Andersen

2006-01-01

150

A novel class of self-sufficient cytochrome P450 monooxygenases in prokaryotes  

Microsoft Academic Search

The Bacillus cytochrome P450 BM3 integrates an entire P450 system in one polypeptide and represents a convenient prokaryotic model for microsomal P450s. This self-sufficient class II P450 is also present in actinomycetes and fungi. By genome analysis we have identified additional homologues in the pathogenic species Bacillus anthracis and Bacillus cereus, and in Ralstonia metallidurans. This analysis also revealed a

René De Mot; Annabel H. A Parret

2002-01-01

151

Cytochrome P450 3A9 catalyzes the metabolism of progesterone and other steroid hormones  

Microsoft Academic Search

The catalytic requirements and the role of P450 3A9, a female-specific isoform of CYP3A from rat brain, in the metabolism of several steroid hormones were studied using recombinant P450 3A9 protein. The optimal steroid hormone hydroxylase activities of P450 3A9 required cholate but not cytochrome b5. P450 3A9 was active in the hydroxylation reactions of testosterone, androstenedione, progesterone and dehydroepiandrosterone

Huamin Wang; Kimberly L. Napoli; Henry W. Strobel

2000-01-01

152

Ethynyl and Propynylpyrene Inhibitors of Cytochrome P450  

PubMed Central

The single-crystal X-ray structures and in vivo activities of three aryl acetylenic inhibitors of cytochromes P450 1A1, 1A2, 2A6, and 2B1 have been determined and are reported herein. These are 1-ethynylpyrene, 1-propy-nylpyrene, and 4-propynylpyrene. To investigate electronic influences on the mechanism of enzyme inhibition, the experimental electron density distribution of 1-ethynylpy-rene has been determined using low-temperature X-ray diffraction measurements, and the resulting net atomic charges compared with various theoretical calculations. A total of 82,390 reflections were measured with Mo K? radiation to a (sin?/?)max = 0.985 Å?1. Averaging symmetry equivalent reflections yielded 8,889 unique reflections. A least squares refinement procedure was used in which multipole parameters were added to describe the distortions of the atomic electron distributions from spherical symmetry. A map of the model electron density distribution of 1-ethynylpyrene was obtained. Net atomic charges calculated from refined monopole population parameters yielded charges that showed that the terminal acetylenic carbon atom (C18) is more negative than the internal carbon (C17). Net atomic charges calculated by ab initio, density functional theory, and semi-empirical methods are consistent with this trend suggesting that the terminal acetylenic carbon atom is more likely to be the site of oxidation. This is consistent with the inhibition mechanism pathway that results in the formation of a reactive ketene intermediate. This is also consistent with assay results that determined that 1-ethynylpyrene acts as a mechanism-based inhibitor of P450s 1A1 and 1A2 and as a reversible inhibitor of P450 2B1. Crystallographic data: 1-ethynylpyrene, C18H10, P21/c, a = 14.571(2) Å, b = 3.9094(5) Å, c = 20.242(3) Å, ? = 105.042(2)°, V = 1,113.5(2) Å3; 1-propynylpyrene, C19H12, P21/n, a = 8.970(2) Å, b = 10.136(1) Å, c = 14.080(3) Å, ? = 99.77(2)°, V = 1,261.5(4) Å3; 4-propynylpyrene, C19H12, Pbca, a = 9.904(1) Å, b = 13.174(2) Å, c = 19.401(1) Å, V = 2,531.4(5) Å3.

Zhu, Naijue; Lightsey, Danielle; Liu, Jiawang; Foroozesh, Maryam; Morgan, Kathleen M.; Stevens, Edwin D.

2010-01-01

153

Regulation of cytochrome P450 gene transcription by phenobarbital.  

PubMed

The ability of phenobarbital to induce levels of drug metabolism in mammals has been known for over 40 years. However, the molecular mechanisms underlying increased expression of the genes of the key enzyme in drug metabolism, cytochrome P450, have not been elucidated, primarily because in vitro model systems in which the induction could be studied were not available. Transfected primary cultured hepatocytes, transfection of liver in situ, and transgenic mice now provide suitable models for phenobarbital induction. In this review, progress toward understanding the mechanism of phenobarbital induction of gene expression is discussed with an emphasis on the mammalian genes, CYP2B1, CYP2B2, and Cyp2b10, which are most highly inducible by phenobarbital. Barbiturate induction of P450s in Bacillus megaterium, which is the system best understood, and its relevance to mammalian mechanisms of induction are also discussed. In B. megaterium, the binding of a repressor to several motifs is reversed by direct effects of barbiturates and by induction of positively acting factors. One of the repressor binding sites, the barbie box, is present in many mammalian phenobarbital-inducible genes, including the promimal promoter regions of CYP2B1, CYP2B2, and Cyp2B10. In the mammalian P450 genes, evidence has been proposed for phenobarbital-regulated elements both in the proximal promoter region and in a distal enhancer region. The role of the proximal region is controversial. A positively acting element that overlaps the barbie box sequence and a negative element have been proposed to mediate induction of CYP2B1/2, based primarily on protein binding and cell-free transcription assays. In contrast, other investigators have not found differences in phenobarbital-dependent protein binding in the proximal promoter region nor mediation of phenobarbital induction by this region. A distal gene fragment, at about -2000 kb in CYP2B1, CYP2B2, and Cyp2b10, has been shown to be a phenobarbital-responsive enhancer independent of proximal promoter elements. This fragment contains several binding sites for proteins and several functional elements, including an NF-1 site, and, therefore, has been designated as a phenobarbital-responsive unit. Possible models are presented in which phenobarbital treatment induces altered chromatin structure, which allows the binding of positively acting factors, or activates factors already bound, to the distal enhancer and the proximal promoter. PMID:9752718

Kemper, B

1998-01-01

154

POTENT INHIBITION OF RECOMBINANT HUMAN CYTOCHROME P-450 1A1 BY PENTAMETHOXYSTILBENE  

Microsoft Academic Search

Previously it was reported that various hydroxystilbene compounds strongly inhibit human cytochrome P-450 1 enzymes and were postulated as candidate chemopreventive agents. In this study, the inhibitory potential of P-450 1 enzyme activities by 3,5,3?,4?,5?-pentamethoxystilbene (PMS), a synthetic stilbene compound, was evaluated with the Escherichia coli (E. coli) membranes of recombinant human cytochrome P-450 1A1, 1A2, or 1B1 coexpressed with

Sang-Kwang Lee; Yongmo Kim; Mie Young Kim; Young-Jin Chun; Sanghee Kim

2004-01-01

155

Oxidative and reductive metabolism by cytochrome P450 2E1  

Microsoft Academic Search

We are constantly exposed to many poten- tially toxic chemicals. Most require metabolic activation to species responsible for cell injury. Although cytochrome P450 2E1 is only one of many different forms of cytochrome P450 that catalyze these reactions, it has an important role in human health as a result of being readily induced by acute and chronic alcohol ingestion. The

DENNIS R. KOOP

156

Gender differences in the regulation of P450 aromatase expression and activity in human adipose tissue  

Microsoft Academic Search

OBJECTIVE: To investigate the hormonal regulation of P450 aromatase activity (responsible for the conversion of C19 androgens to C18 oestrogens) in human adipose tissue from men and pre- and post-menopausal women.SUBJECTS: Subcutaneous abdominal adipose tissue was obtained from 19 subjects: six pre-menopausal females (mean age 41.8±(s.e.m.) 2.5; mean weight 76.01±5.6 kg), eight post-menopausal females (mean age 59.9±2.0; mean weight 63.5±2.6

PG McTernan; A Anwar; MC Eggo; AH Barnett; PM Stewart; S Kumar

2000-01-01

157

A key cytochrome P450 hydroxylase in pradimicin biosynthesis  

PubMed Central

Pradimicins A-C (1–3) are a group of antifungal and antiviral polyketides from Actinomadura hibisca. The sugar moieties in pradimicins are required for their biological activities. Consequently, the 5-OH that is used for glycosylation plays a critical role in pradimicin biosynthesis. A cytochrome P450 monooxygenase gene, pdmJ, was amplified from the genomic DNA of A. hibisca and expressed in Escherichia coli BL21(DE3). PdmJ introduced a hydroxyl group to G-2A (4), a key pradimicin biosynthetic intermediate, at C-5 to form JX134 (5). A D-Ala-containing pradimicin analog, JX137a (6) was tested as an alternative substrate, but no product was detected by LC-MS, indicating that PdmJ has strict substrate specificity. Kinetic studies revealed a typical substrate inhibition of PdmJ activity. The optimal substrate concentration for the highest velocity is 115 ?M under the test conditions. Moreover, the conversion rate of 4 to 5 was reduced by the presence of 6, likely due to competitive inhibition. Coexpression of PdmJ and a glucose 1-dehydrogenase in E. coli BL21(DE3) provides an efficient method to produce the important intermediate 5 from 4.

Napan, Kandy L.; Zeng, Jia; Takemoto, Jon Y.; Zhan, Jixun

2011-01-01

158

Cytochrome P450 eicosanoids and cerebral vascular function  

PubMed Central

The eicosanoids 20-hydroxyeicosatetraenoic acid (20-HETE) and epoxyeicosatrienoic acids (EETs), which are generated from the metabolism of arachidonic acid by cytochrome P450 (CYP) enzymes, possess a wide array of biological actions, including the regulation of blood flow to organs. 20-HETE and EETs are generated in various cell types in the brain and cerebral blood vessels, and contribute significantly to cerebral blood flow autoregulation and the coupling of regional brain blood flow to neuronal activity (neurovascular coupling). Investigations are beginning to unravel the molecular and cellular mechanisms by which these CYP eicosanoids regulate cerebral vascular function and the changes that occur in pathological states. Intriguingly, 20-HETE and the soluble epoxide hydrolase (sEH) enzyme that regulates EET levels have been explored as molecular therapeutic targets for cerebral vascular diseases. Inhibition of 20-HETE, or increasing EET levels by inhibiting the sEH enzyme, decreases cerebral damage following stroke. The improved outcome following cerebral ischaemia is a consequence of improving cerebral vascular structure or function and protecting neurons from cell death. Thus, the CYP eicosanoids are key regulators of cerebral vascular function and novel therapeutic targets for cardiovascular diseases and neurological disorders.

Imig, John D.; Simpkins, Alexis N.; Renic, Marija; Harder, David R.

2013-01-01

159

Cytochrome P450 is regulated by noradrenergic and serotonergic systems.  

PubMed

The aim of the present study was to ascertain whether the noradrenergic or serotonergic systems may affect the expression of liver cytochrome P450 (CYP). Rats were injected intraperitoneally with N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4, a noradrenergic neurotoxin) or p-chloroamphetamine (PCA, a serotonergic neurotoxin) or p-chlorophenylalanine (PCPA, an inhibitor of serotonin synthesis). One week after neurotoxin injection the levels of neurotransmitters (noradrenaline, dopamine, serotonin) and their metabolites were measured in brain structures, and the activity and protein levels of CYP isoforms were measured in the liver. In the brain, DSP-4 or PCA and PCPA selectively decreased noradrenaline or serotonin levels, respectively. In the liver, the applied neurotoxins evoked decrease in the activity of CYP2B, CYP2C11 and CYP3A (DSP-4, PCA, PCPA) and increase in the activity of CYP1A (PCA, PCPA), while the activity of CYP2A, CYP2C6 and CYP2D was not affected by the applied neurotoxins. Since the affected isoforms (CYP1A/2B/2C11/3A) are regulated by endogenous hormones (growth hormone, corticosterone, thyroid hormones), the latter being under control of the central nervous system, it is postulated that the brain noradrenergic and serotonergic systems are involved in the physiological regulation of liver CYP expression. PMID:21742037

Kot, Marta; Daniel, W?adys?awa A

2011-06-30

160

Nanoscale-engineered cytochrome p450 system with a branch structure.  

PubMed

Most of the bacterial cytochrome P450 s require two kinds of electron transfer proteins, ferredoxin and ferredoxin reductase, and thus P450 s do not show catalytic activity by themselves. A microbial transglutaminase-mediated site-specific cross-linking enables the formation of fusion P450 protein with a branched structure, which is generated from a genetic fusion protein of P450-ferredoxin reductase and ferredoxin, an interactive nanoscale protein structure. This fusion P450 system is self-sufficient due to intramolecular electron transfer, which means the system does not require additional electron-transferring proteins. Because some components of bacterial cytochrome P450 system are interchangeable, this self-sufficient system can be applied to non-natural combination of P450 and electron transfer proteins from different species of bacteria. PMID:21553178

Hirakawa, Hidehiko; Nagamune, Teruyuki

2011-01-01

161

Functional expression system for cytochrome P450 genes using the reductase domain of self-sufficient P450RhF from Rhodococcus sp. NCIMB 9784  

Microsoft Academic Search

Cytochrome P450RhF from Rhodococcus sp. NCIMB 9784 is a self-sufficient P450 monooxygenase. We report here a simple system for the functional expression of various P450 genes using the reductase domain of this P450RhF, which comprises flavin mononucleotide- and nicotinamide adenine dinucleotide phosphate binding motifs and a [2Fe2S] ferredoxin-like center. Vector pRED was constructed, which carried the T7 promoter, cloning sites

Miho Nodate; Mitsutoshi Kubota; Norihiko Misawa

2006-01-01

162

Suppression of P450 aromatase gene expression in sex-reversed males produced by rearing genetically female larvae at a high water temperature during a period of sex diVerentiation in the Japanese flounder (Paralichthys olivaceus)  

Microsoft Academic Search

The phenotypic sex of many teleost fishes including flounders can be experimentally altered by treating embryos or larvae with varied temperatures or sex-steroid hormones. To analyse the sex determi- nation mechanism, especially the role of cytochrome P450 aromatase (P450arom), an enzyme that catalyses the conversion of androgens to estrogens, in temperature-dependent gonadal sex diVeren- tiation in the Japanese flounder, we

T Kitano; K Takamune; T Kobayashi; Y Nagahama; S-I Abe

163

Homology modeling of plant cytochrome P450s  

Microsoft Academic Search

Plant P450 monooxygenases represent the largest family of plant proteins and the largest collection of P450s available for comparative studies and biotechnological applications. They have been shown to catalyze a diverse array of difficult chemical reactions and have demonstrated potential to be used in pharmacological, agronomic and phytoremediative applications. Central to our use of these catalytically competent enzymes is the

Sanjeewa Rupasinghe; Mary A. Schuler

2006-01-01

164

Isolation and functional analysis of cytochrome P450 CYP153A genes from various environments.  

PubMed

The cytochrome P450 CYP153 family is thought to mediate the terminal hydroxylation reactions of n-alkanes. We isolated 16 new P450 CYP153A genes (central region) from various environments such as petroleum-contaminated soil and groundwater, as well as one from the n-alkane-degrading bacterium Alcanivorax borkumensis SK2 (designated P450balk). The sequences of the new P450 genes were extended by PCR to generate full-length chimeric P450 genes, using the N- and C-terminal domains of P450balk. A differential CO-reduced P450 spectral analysis indicated that 8 P450 genes among the 16 chimeric genes were expressed in Escherichia coli to generate a soluble and functional enzyme. The several functional chimeric P450s and P450balk were further fused to the reductase domain of the self-sufficient P450 monooxygenase (P450RhF) at the C-terminus. E. coli cells expressing these self-sufficient P450 chimeric genes converted n-alkanes, cyclohexane, 1-octene, n-butylbenzene, and 4-phenyl-1-butene into 1-alkanols, cyclohexanol, 1,2-epoxyoctane, 1-phenyl-4-butanol, and 2-phenethyl-oxirane, respectively. PMID:16377903

Kubota, Mitsutoshi; Nodate, Miho; Yasumoto-Hirose, Mina; Uchiyama, Taku; Kagami, Osamu; Shizuri, Yoshikazu; Misawa, Norihiko

2005-12-01

165

Targeting Cytochrome P450 Enzymes: A New Approach in Anti-cancer Drug Development  

PubMed Central

Cytochrome P450s (CYPs) represent a large class of heme-containing enzymes that catalyze the metabolism of multitudes of substrates both endogenous and exogenous. Until recently, however, CYPs have been largely overlooked in cancer drug development, acknowledged only for their role in Phase I metabolism of chemotherapeutics. The first successful strategy targeting CYP enzymes in cancer therapy was the development of potent inhibitors of CYP19 (aromatase) for the treatment of breast cancer. Aromatase inhibitors ushered in a new era in hormone ablation therapy for estrogen dependent cancers, and have paved the way for similar strategies (i.e. inhibition of CYP17) that combat androgen dependent prostate cancer. Identification of CYPs involved in the inactivation of anti-cancer metabolites of Vitamin D3 and Vitamin A has triggered development of agents that target these enzymes as well. The discovery of the over-expression of exogenous metabolizing CYPs, such as CYP1B1, in cancer cells has roused interest in the development of inhibitors for chemoprevention and of prodrugs designed to be activated by CYPs only in cancer cells. Finally, the expression of CYPs within tumors has been utilized in the development of bioreductive molecules that are activated by CYPs only under hypoxic conditions. This review offers the first comprehensive analysis of strategies in drug development that either inhibit or exploit CYP enzymes for the treatment of cancer.

Bruno, Robert D.; Njar, Vincent C.O.

2007-01-01

166

Clofibrate-induced cytochrome P450-lauric acid omega hydroxylase(P450LA omega):purification, cDNA cloning, sequence and regulation  

SciTech Connect

A cytochrome P450 that hydroxylates lauric acid at the 12 position (P450LA omega) was isolated from liver microsomes of clofibrate treated rats. P450LA omega was immunologically distinct from P450s a,b,c,d,e,f,g,h,j,PB1, and PCN1. Polyclonal antibody against P450LA omega was utilized to screen a gt11 cDNA library. A clone (pP450LA omega), was isolated and its sequence determined. The P450LA omega mRNA is a minimum 2387 nts in length and codes for a P450 of Mr.58,222 daltons. This protein shares less than 35% amino acid similarity with P450s b,c,d,e,f,PB1, and PCN1; however, it does contain a hydrophobic amino terminal peptide and a conserved sequence surrounding the Cys residue at position 456, which is similar to other microsomal P450s. P450LA omega is present at high levels in untreated rat kidney and is induced by clofibrate in both kidney and liver. This induction is the result of an accumulation of mRNA through a rapid transcriptional activation of the P450LA gene. Southern blotting data suggest the presence of 2 or 3 genes in the P450LA omega family. This P450 gene family may be associated with arachidonic acid and prostraglandin metabolism in kidney and other tissues.

Hardwick, J.P.; Song, B.J.; Gonzalez, F.J.

1986-05-01

167

Conformational Adaptation of Human Cytochrome P450 2B6 and Rabbit Cytochrome P450 2B4 Revealed Upon Binding Multiple Amlodipine Molecules?  

PubMed Central

Structures of human cytochrome P450 2B6 and rabbit cytochrome P450 2B4 in complex with two molecules of the calcium channel blocker amlodipine have been determined by X-ray crystallography. The presence of two drug molecules suggests clear substrate access channels in each P450. According to a previously established nomenclature, amlodipine molecules were trapped in access pathway 2f in P450 2B6 and in pathway 2a or 2f in P450 2B4. These pathways overlap for part of the length and then diverge as they extend toward the protein surface. A previously described solvent channel was also found in each enzyme. The results indicate that key residues located on the surface and at the entrance of the substrate access channels in each of these P450s may play a crucial role in guiding substrate entry. In addition, the region of P450 2B6 and 2B4 involving helices B’, F, F’, G’ and part of helix G is substantially more open in the amlodipine complexes compared with the corresponding 4-(4-chlorophenyl)imidazole complexes. The increased active site volume observed results from the major retraction of helices F, F’ and B’ and the ?4 sheet region located close to the binding cavity to accommodate amlodipine. These structures demonstrate novel insight into distinct conformational states not observed with previous P450 2B structures and provide clear evidence of the substrate access channels in two drug metabolizing P450s. In addition, the structures exhibit the versatility that can be exploited in silico studies with other P450 2B6 ligands as large as raloxifene and itraconazole.

Shah, Manish B.; Wilderman, P. Ross; Pascual, Jaime; Zhang, Qinghai; Stout, C. David; Halpert, James R.

2012-01-01

168

Inhibitors of Cytochrome P450 4A Suppress Angiogenic Responses  

PubMed Central

Cytochrome P450 enzymes of the 4A family (CYP4A) convert arachidonic acid to 20-hydroxyeicosatetraenoic acid (20-HETE) in blood vessels of several vascular beds. The present study examined the effects of inhibiting the formation of 20-HETE with N-hydroxy-N?-(4-butyl-2-methylphenol) formamidine (HET0016) on the mitogenic response of vascular endothelial growth factor (VEGF) in human umbilical vein endothelial cells (HUVECs) in vitro, and on growth factor-induced angiogenesis in the cornea of rats in vivo. HET0016 (10 ?mol/L and 20 ?g, respectively) abolished the mitogenic response to VEGF in HUVECs and the angiogenic response to VEGF, basic fibroblast growth factor, and epidermal growth factor in vivo by 80 to 90% (P < 0.001). Dibromododecenyl methylsulfonimide (DDMS), a structurally and mechanistically different inhibitor of 20-HETE synthesis, also abolished angiogenic responses when tested with VEGF. Additionally, administration of the stable 20-HETE agonist, 20-hydroxyeicosa-6(Z) 15(Z)-dienoic acid (WIT003) induced mitogenesis in HUVECs and angiogenesis in the rat cornea in vivo. We studied the ability of HET0016 to alter the angiogenic response in the rat cornea to human glioblastoma cancer cells (U251). When administered locally into the cornea, HET0016 (20 ?g) reduced the angiogenic response to U251 cancer cells by 70%. These results suggest that a product of CYP4A product, possibly 20-HETE, plays a critical role in the regulation of angiogenesis and may provide a useful target for reduction of pathological angiogenesis.

Chen, Ping; Guo, Meng; Wygle, Dana; Edwards, Paul A.; Falck, John R.; Roman, Richard J.; Scicli, A. Guillermo

2005-01-01

169

Regulation of cytochrome P-450 monooxygenases in the mouse  

SciTech Connect

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

Kelley, M.F.

1986-01-01

170

Expression of P450(17 alpha) hydroxylase and P450 aromatase genes in the chicken gonad before and after sexual differentiation.  

PubMed

The onset and localization of P450(17-alpha)-hydroxylase (P450(17-alpha)) and P450 aromatase (P450arom) mRNA expression were studied in the gonads of chicken embryos at Days 4-9 of incubation by in situ hybridization analysis, and expression of both mRNAs was measured in the ovary and testis of the chicken after sexual differentiation by Northern and slot-blot analysis and was related to changes in concentration of 17-beta-estradiol (E2) in the gonad. In situ hybridization analysis showed the first detection of P450(17-alpha) mRNA at Days 5-6 of incubation in the genetic male and female gonads and the first detection of P450arom at Day 6.5 of incubation in the female gonad but none was detected in the male gonad. Both mRNAs were observed in the medullary cords of the gonad. Slot-blot analysis demonstrated that levels of P450(17-alpha) mRNA in the ovary remained high during the period between Day 10 of incubation and 7 days of posthatching, while those in the testis remained low at Days 10-14 of incubation but increased at Day 16 of incubation up to posthatching days. Levels of P450arom mRNA in the left ovary were invariably higher than those of the right ovary and testis throughout the period between Day 10 of incubation and 7 days of posthatching. These results suggest that expression of P450arom mRNA occurring in the genetic female gonad around Day 6 is associated, at least in a part, with sexual differentiation toward femaleness by increasing E2 production which may promote cellular proliferation of the left gonad. In contrast, in the genetic male gonad marked expression of P450(17-alpha) mRNA with little expression of P450arom mRNA may lead to production of androgen which promotes the gonadal development to the testis. PMID:8998967

Yoshida, K; Shimada, K; Saito, N

1996-05-01

171

Comparative defluorination and cytochrome P-450 loss by the microsomal metabolism of fluoro- and fluorochloroethenes.  

PubMed

Halogenated ethenes are oxidatively metabolized by cytochrome P-450 to intermediates which inactivate cytochrome P-450 by destroying heme and to epoxides which may react with cellular macromolecules or decompose to other products. To determine the relative capabilities of fluoroethenes to inactivate cytochrome P-450 and undergo metabolism, fluoride release, cytochrome P-450 loss, and heme loss due to the metabolism of trifluorochloroethene (TFCE), chlorodifluoroethene (CDE), difluoroethene (DFE), and trifluoroethene (TFE) were compared in rat hepatic microsomes. Fluoride release, in order of decreasing amounts of fluoride released, followed the order: CDE greater than TFCE much greater than TFE greater than DFE. In contrast, in order of each compound's decreasing effectiveness to destroy both cytochrome P-450 and heme, the following sequence was obtained: TFE greater than CDE greater than TFCE greater than DFE. In phenobarbital-induced hepatic microsomes, TFE inactivated up to 67% of the cytochrome P-450, whereas DFE inactivated only up to 17%. The results of this study indicate that chloro substituents enhance defluorination of the ethenes, and that cytochrome P-450 inactivation by the fluoroethenes is highly dependent on the degree and nature of the halogen substituents. PMID:2888623

Baker, M T; Bates, J N; Leff, S V

172

Disruption of the 'Saccharomyces cerevisiae' Gene for NADPH-Cytochrome P450 Reductase Causes Increased Sensitivity to Ketoconazole.  

National Technical Information Service (NTIS)

Strains of Saccharomyces cerevisiae deleted in the NADPH-cytochrome P450 reductase gene by transplacement are 200-fold more sensitive to ketoconazole, an inhibitor of the cytochrome P450 lanosterol 14alpha-demethylase. Resistance is restored through compl...

T. R. Sutter J. C. Loper

1989-01-01

173

Novel evidence of cytochrome P450-catalyzed oxidation of phenanthrene in Phanerochaete chrysosporium under ligninolytic conditions.  

PubMed

The presence of cytochrome P450 and P450-mediated phenanthrene oxidation in the white rot fungus Phanerochaete chrysosporium under ligninolytic condition was first demonstrated in this study. The carbon monoxide difference spectra indicated induction of P450 (130 pmol mg(-1) in the microsomal fraction) by phenanthrene. The microsomal P450 degraded phenanthrene with a NADPH-dependent activity of 0.44 ± 0.02 min(-1). One of major detectable metabolites of phenanthrene in the ligninolytic cultures and microsomal fractions was identified as phenanthrene trans-9,10-dihydrodiol. Piperonyl butoxide, a P450 inhibitor which had no effect on manganese peroxidase activity, significantly inhibited phenanthrene degradation and the trans-9,10-dihydrodiol formation in both intact cultures and microsomal fractions. Furthermore, phenanthrene was also efficiently degraded by the extracellular fraction with high manganese peroxidase activity. These results indicate important roles of both manganese peroxidase and cytochrome P450 in phenanthrene metabolism by ligninolytic P. chrysosporium. PMID:20333538

Ning, Daliang; Wang, Hui; Ding, Chang; Lu, Huijie

2010-03-24

174

Hippocampal cytochrome P450s synthesize brain neurosteroids which are paracrine neuromodulators of synaptic signal transduction  

Microsoft Academic Search

Hippocampal pyramidal neurons and granule neurons of adult male rats are equipped with a complete machinery for the synthesis of pregnenolone, dehydroepiandrosterone, 17?-estradiol and testosterone as well as their sulfate esters. These brain neurosteroids are synthesized by cytochrome P450s (P450scc, P45017? and P450arom) from endogenous cholesterol. Synthesis is acutely dependent on the Ca2+ influx attendant upon neuron–neuron communication via N-methyl-d-aspartate

Keisuke Shibuya; Norio Takata; Yasushi Hojo; Aizo Furukawa; Nobuaki Yasumatsu; Tetsuya Kimoto; Taihei Enami; Kumiko Suzuki; Nobuaki Tanabe; Hirotaka Ishii; Hideo Mukai; Taiki Takahashi; Taka-aki Hattori; Suguru Kawato

2003-01-01

175

Activation of Chemically Diverse Procarcinogens by Human Cytochrome P-450 IBI1  

Microsoft Academic Search

A human cytochrome P-450 (P450) IBI cDNA was expressed in Sat-- charomyces cerevisiae, and the microsomes containing P450 IBI were used to examine the selectivity of this enzyme in the activation of a variety of environmental carcinogens and mutagens in Salmonella typhimurium TA1535\\/pSK1002 or NM2009 tester strains, using the SOS response as an end point of DNA damage. We also

Tsutomu Shimada; Carrie L. Hayes; Hiroshi Yamazaki; Shantu Amin; Stephen S. Hecht; F. Peter Guengerich; Thomas R. Sutler

176

Identification of a New Class of Cytochrome P450 from a Rhodococcus sp  

Microsoft Academic Search

A degenerate set of PCR primers were used to clone a gene encoding a cytochrome P450 (the P450RhF gene) from Rhodococcus sp. strain NCIMB 9784 which is of unique primary structural organization. Surprisingly, analysis of the translation product revealed that the P450 is fused to a reductase domain at the C terminus which displays sequence conservation for dioxygenase reductase proteins.

Gareth A. Roberts; Gideon Grogan; Andy Greter; Sabine L. Flitsch; Nicholas J. Turner

2002-01-01

177

Iron and Salicylate Induction of Cytochrome P450 BM1 in Bacillus megaterium  

Microsoft Academic Search

.   The effects of iron and salicylate on the expression of cytochrome P450s in Bacillus megaterium were investigated in this report. Immunoblot analysis showed that the addition of 4 mM ferric iron or 10 mM salicylate to\\u000a the culture medium resulted in a significant increase in the P450BM-1 level, while the same condition had little effect on P450BM-3 expression. Substantial

Gwo-Chyuan Shaw; Hsun-Sheng Kao; Chi-Chang Sung; An-Na Chiang

1997-01-01

178

Immunochemical similarity of P-450 HFLa, a form of cytochrome P-450 in human fetal livers, to a form of rat liver cytochrome P-450 inducible by macrolide antibiotics.  

PubMed

A protein immunochemically related to P-450 HFLa, a form of cytochrome P-450 purified from human fetal livers, was detected in rat liver microsomes. The content of the immunoreactive protein in rat liver microsomes was increased by treatments with phenobarbital, pregnenolone 16 alpha-carbonitrile (PCN), erythromycin, erythromycin estolate, and oleandomycin but not with 3-methylcholanthrene, imidazole, ethanol, isosafrole, josamycin, midecamycin, or miocamycin. The activity of erythromycin N-demethylase correlated with the content of the immunoreactive protein in rat liver microsomes (r = 0.72). In addition, anti-P-450 HFLa IgG inhibited erythromycin N-demethylase in liver microsomes from erythromycin- or oleandomycin-pretreated rats. Furthermore, the content of the immunoreactive protein highly correlated with that of P-450 PB-1, which is distinct from Waxman's terminology, and is one of the forms of PCN-inducible cytochrome P-450s (r = 0.95). From these results and the results reported so far, it seems possible that P-450 HFLa is one of the forms of cytochrome P-450 inducible by glucocorticoids. PMID:3395131

Kitada, M; Igoshi, N; Kamataki, T; Itahashi, K; Imaoka, S; Komori, M; Funae, Y; Rikihisa, T; Kanakubo, Y

1988-07-01

179

Membrane insertion of cytochrome P450 1A2 promoted by anionic phospholipids.  

PubMed

The role of phospholipids in the membrane binding and subsequent insertion of the microsomal protein rabbit cytochrome P450 (P450) 1A2 into phospholipid bilayers was investigated. The insertion of P450 1A2 into phospholipid bilayers was measured by the quenching of Trp fluorescence of P450 1A2 by pyrene and brominated and doxyl-labeled phospholipids. When the phosphatidylcholine (PC) matrix was replaced with acidic phospholipids [phosphatidic acid (PA), phosphatidylserine, and phosphatidylinositol] and phosphatidylethanolamine (PE), the extent of insertion into lipid bilayers was strictly dependent on the type of acidic phospholipids. All anionic phospholipids caused the penetration of P450 1A2 into lipid bilayers, but PA was the most efficient in facilitating deep penetration of P450 1A2 into bilayers. On the other hand, binding of P450 1A2 to liposomes was increased by acidic phospholipids to the same degree regardless of the type of acidic phospholipids. PE was found to act as an inert matrix phospholipid, similar to PC, as it exerted very little effect on the insertion of P450 1A2 into lipid bilayers and the binding of P450 1A2 to membranes. It was also found that the phospholipid-dependent membrane insertion of P450 1A2 was associated with altered enzyme activity, increased alpha-helix content, and increased Trp fluorescence of P450 1A2. These results indicate that negative charges on the acidic phospholipids are important for the initial binding of P450 1A2 to membranes, but the penetration of P450 1A2 into lipid bilayers is regulated by the type of acidic phospholipids, and that phospholipid-dependent insertion of P450 1A2 is accompanied by a structural change of P450 1A2. PMID:9737864

Ahn, T; Guengerich, F P; Yun, C H

1998-09-15

180

Prediction of cytochrome P450 isoform responsible for metabolizing a drug molecule  

Microsoft Academic Search

BACKGROUND: Different isoforms of Cytochrome P450 (CYP) metabolized different types of substrates (or drugs molecule) and make them soluble during biotransformation. Therefore, fate of any drug molecule depends on how they are treated or metabolized by CYP isoform. There is a need to develop models for predicting substrate specificity of major isoforms of P450, in order to understand whether a

Nitish K Mishra; Sandhya Agarwal; Gajendra PS Raghava

2010-01-01

181

ISOLATION OF A CYTOCHROME P-450 STRUCTURAL GENE FROM SACCHAROMYCES CEREVISIAE  

EPA Science Inventory

We have transformed a Saccharomyces cerevisiae host with an S. cerevisiae genomic library contained in the shuttle vector YEp24 and screened the resultant transformants for resistance to ketoconazole (Kc), an inhibitor of the cytochrome P-450 (P-450) enzyme lanosterol 14-demethyl...

182

Functional expression system for cytochrome P450 genes using the reductase domain of self-sufficient P450RhF from Rhodococcus sp. NCIMB 9784.  

PubMed

Cytochrome P450RhF from Rhodococcus sp. NCIMB 9784 is a self-sufficient P450 monooxygenase. We report here a simple system for the functional expression of various P450 genes using the reductase domain of this P450RhF, which comprises flavin mononucleotide- and nicotinamide adenine dinucleotide phosphate binding motifs and a [2Fe2S] ferredoxin-like center. Vector pRED was constructed, which carried the T7 promoter, cloning sites for a P450, a linker sequence, and the P450RhF reductase domain, in this order. The known P450 genes, encoding P450cam from Pseudomonas putida (CYP101A) and P450bzo from an environmental metagenome library (CYP203A), were expressed on vector pRED as soluble fusion enzymes with their natural spectral features in Escherichia coli. These E. coli cells expressing the P450cam and P450bzo genes could convert (+)-camphor and 4-hydroxybenzoate into 5-exo-hydroxycamphor and protocatechuate (3,4-dihydroxybenzoate), respectively (the expected products). Using this system, we also succeeded in directly identifying the function of P450 CYP153A as alkane 1-monooxygenase for the first time, i.e., E. coli cells expressing a P450 CYP153A gene named P450balk, which was isolated form Alcanivorax borkumensis SK2, converted octane into 1-octanol with high efficiency (800 mg/l). The system presented here may be applicable to the functional identification of a wide variety of bacterial cytochromes P450. PMID:16195793

Nodate, Miho; Kubota, Mitsutoshi; Misawa, Norihiko

2005-09-30

183

P-450 HFLa, a form of cytochrome P-450 purified from human fetal livers, is the 16 alpha-hydroxylase of dehydroepiandrosterone 3-sulfate.  

PubMed

In a reconstituted system containing NADPH, dilauroyl-L-3-phosphatidylcholine, and NADPH-cytochrome P-450 reductase purified from rat liver microsomes, cytochrome P-450 (P-450 HFLa) purified from human fetal livers catalyzed the 16 alpha-hydroxylation of dehydroepiandrosterone 3-sulfate (DHEA-sulfate). Addition of cytochrome b5 purified from rat liver microsomes to the reconstituted system resulted in a remarkable increase in the hydroxylase activity. The level of P-450 HFLa in liver homogenates from human fetuses highly correlated with the activity of DHEA-sulfate 16 alpha-hydroxylase. Antibodies to P-450 HFLa inhibited the 16 alpha-hydroxylation of DHEA-sulfate in a dose-dependent manner. The NH2-terminal amino acid sequence of P-450 HFLa was similar to that of P-450NF (Beaune, P. H., Umbenhauer, D. R., Bork, R. W., Lloyd, R. S., and Guengerich, F. P. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 8064-8068). We conclude that P-450 HFLa is a form of cytochrome P-450 involved in the 16 alpha-hydroxylation of DHEA-sulfate. PMID:3654629

Kitada, M; Kamataki, T; Itahashi, K; Rikihisa, T; Kanakubo, Y

1987-10-01

184

Cytochrome P450 in the brain: neuroendocrine functions.  

PubMed

The effectiveness of steroid hormone metabolites as sedatives and anesthetics has been known for many years. More recently, their interaction with neurotransmitter receptors has helped to elucidate their mechanism of action, but their physiological functions and their role in disturbances of behavior, anxiety, and sleep/wakefulness have yet to be elucidated. Until 1981 it was assumed that metabolites of steroid hormones arose from the adrenals and gonads and that their action on neurotransmitter receptors was a mechanism of communication between the brain and the periphery. The evidence that the brain could accumulate steroids independently of the adrenals and gonads in 1981 and later the evidence for the presence of the cholesterol side chain cleavage enzyme (P450scc) in the brain have challenged this concept and stimulated a great deal of interest in the possibility that the brain could be making its own steroids from cholesterol for some as yet undefined purpose. In this review we examine the data pertaining to the role of brain P450 in the synthesis and degradation of neurosteroids. We summarize the data on the presence of P450scc in the brain and try to answer the following questions: (1) Does P450scc in the brain contribute significantly to the synthesis of GABAA receptor active steroids? (2) Can the P450scc in the brain account for the accumulation of pregnenolone in the brain? (3) Is there evidence for special functions of the pregnenolone synthesized in the brain? (4) Is there a role for other forms of brain P450 in neurosteroid action? PMID:7556851

Warner, M; Gustafsson, J A

1995-07-01

185

Ovarian P450 aromatase activity in the catfish Heteropneustes fossilis: seasonal changes and effects of catecholestrogens.  

PubMed

Ovarian microsomal aromatase (P450arom) activity was studied in relation to season and incubation of follicles with catecholestrogens [(2-hydroxyestradiol-17beta (2-OHE2) and 2-methoxyestradiol-17 beta (2-methoxyE2)] using a product (estradiol-17 beta) assay. Peak P450arom activity was noticed in late preparatory phase (April) and it decreased significantly in pre-spawning, spawning and post-spawning phases to give the lowest value in resting phase. Apparent Km and Vmax of the enzyme varied significantly and the values were high in the preparatory (vitellogenic) phase (Km 74.62+/-1.73 nM, Vmax 0.81+/-0.01 pmol/mg protein/min) and low in the spawning (post-vitellogenic) phase (Km 62.01+/-1.68 nM, Vmax 0.69+/-0.002 pmol/mg protein/min). The incubation of the ovarian microsomes with 2-OHE2 elicited significant biphasic effects on enzyme activity. In the vitellogenic phase, concentrations of the steroid up to 1 microM inhibited enzyme activity significantly with the highest inhibition at 10nM. However, in the post-vitellogenic ovary, the highest inhibition was registered at 100 nM. The higher concentrations (10 microM or 100 microM) did not elicit any significant change compared to the control groups. A comparison of the aromatase inhibition index (AI50, indicates 50% inhibition of aromatase activity) of fadrozole, a known aromatase inhibitor and 2-OHE2 shows that the AI50 was 4.4 nM for fadrozole and 0.864 nM (vitellogenic phase) and 1.31 nM (post-vitellogenic phase) for 2-OHE2 indicating higher potency of the latter. The incubation of the ovarian microsomes with 2-methoxyE2 increased enzyme activity only at the higher concentrations (1-100 microM). The results show seasonality in the potential of the ovary to synthesize E2 and the potent enzyme inhibiting activity of 2-OHE2, which is reported for the first time. PMID:18395205

Chourasia, T K; Joy, K P

2008-03-10

186

Approaches to Deorphanization of Human and Microbial Cytochrome P450 Enzymes  

PubMed Central

One of the general problems in biology today is that we are characterizing genomic sequences much faster than identifying the functions of the gene products, and the same problem exists with cytochromes P450 (P450). One-fourth of the human P450s are not well-characterized and therefore considered “orphans.” A number of approaches to deorphanization are discussed generally. Several liquid chromatography-mass spectrometry approaches have been applied to some of the human and Streptomyces coelicolor P450s. One current limitation is that too many fatty acid oxidations have been identified and we are probably missing more relevant substrates, possibly due to limits of sensitivity.

Guengerich, F. Peter; Tang, Zhongmei; Cheng, Qian; Salamanca-Pinzon, S. Giovanna

2010-01-01

187

Arabidopsis CYP72C1 is an atypical cytochrome P450 that inactivates brassinosteroids  

Microsoft Academic Search

Cytochrome P450 monooxygenases (P450s) are a diverse family of proteins that have specialized roles in secondary metabolism\\u000a and in normal cell development. Two P450s in particular, CYP734A1 and CYP72C1, have been identified as brassinosteroid-inactivating\\u000a enzymes important for steroid-mediated signal transduction in Arabidopsis thaliana. Genetic analyses have demonstrated that these P450s modulate growth throughout plant development. While members of the CYP734A

Leeann E. ThorntonSanjeewa; Sanjeewa G. Rupasinghe; Hao Peng; Mary A. Schuler; Michael M. Neff

2010-01-01

188

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

National Technical Information Service (NTIS)

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 devote...

J. C. Loper

1997-01-01

189

Role of cytochrome P-450 as a source of catalytic iron in cisplatin-induced nephrotoxicity  

Microsoft Academic Search

Role of cytochrome P-450 as a source of catalytic iron in cisplatin-induced nephrotoxicity.BackgroundIron plays a role in free radical-mediated tissue injury, including cisplatin-induced nephrotoxicity. However, the source of iron (catalyzing free radical reactions) is not known. We examined the role of cytochrome P-450 as a source of catalytic iron in cisplatin-induced nephrotoxicity both in vivo and in vitro.MethodsCisplatin-induced acute renal

Radhakrishna Baliga; Zhiwei Zhang; Mithra Baliga; Norishi Ueda; Sudhir V. Shah

1998-01-01

190

Theoretical study of the substrate specificity of cytochrome P-450 isoforms  

Microsoft Academic Search

The substrate specificity of cytochrome P-450 isoforms has been theoretically studied within the framework of a three-dimensional\\u000a (3D) QSAR algorithm (BiS). According to this approach, the substrates are classified into two groups — metabolized and non-metabolized\\u000a on a given isoform — depending on the molecular field characteristics. Interactions in the “substrate–cytochrome P-450 isoform”\\u000a complexes have been simulated for a detailed

M. A. Grishina; A. A. Pogrebnoi; V. A. Potemkin; T. Yu. Zrakova

2005-01-01

191

Iron and salicylate induction of cytochrome P450BM-1 in Bacillus megaterium.  

PubMed

The effects of iron and salicylate on the expression of cytochrome P450s in Bacillus megaterium were investigated in this report. Immunoblot analysis showed that the addition of 4 mM ferric iron or 10 mM salicylate to the culture medium resulted in a significant increase in the P450BM-1 level, while the same condition had little effect on P450BM-3 expression. Substantial induction of chloramphenicol acetyltransferase (CAT) activity by iron and salicylate in B. megaterium cells bearing a P450BM-1 promoter-cat transcriptional fusion vector suggests that the induction of P450BM-1 by iron and salicylate occurs at the transcriptional level. Unexpectedly, in contrast to the bm1P1-dependent induction of P450BM-1 by pentobarbital, disruption of bm1P1 gene did not affect induction of P450BM-1 by iron and salicylate. This result suggests that the induction of P450BM-1 by iron and salicylate occurs by a bm1P1-independent mechanism. To our knowledge, this is the first example of an iron-regulated cytochrome P450 gene in prokaryotes. PMID:9175556

Shaw, G C; Kao, H S; Sung, C C; Chiang, A N

1997-07-01

192

Forster Distances of Ligand-Heme Pairs in Cytochrome P450 3A4  

NASA Astrophysics Data System (ADS)

Cytochrome P450 3A4 is a protein in the human intestine and liver which oxidizes over half of drugs in use today. Cytochrome P450 3A4 has proven resistant to structure determination by NMR or x-ray crystallography. Fluorescence Resonance Energy Transfer (FRET) studies of P450 3A4 can be used to compute distances between fluorophores in the protein, providing information on the structure of the protein. For a ligand to be suitably used as a probe its fluorescence must not be completely quenched by the heme cofactor in P450 3A4. By using quantum yields, fluorescence, and the absorption spectra of six P450 ligands, the following Forster distances between each ligand and the P450 heme moiety were obtained: pyrene 4.6 nm, aflatoxin B2 5.7 nm, alpha-naphthoflavone 3.7 nm, indinavir 2.6 nm, quinidine 3.5 nm, and terfenadine 2.8 nm. Having these distances should yield a better low-resolution cytochrome P450 3A4 structure. Using the Forster distances, FRET experiments on inter-ligand placement in P450 3A4 will be undertaken soon.

Fern, Joel; Guengerich, F. Peter; Marsch, Glenn A.

2003-04-01

193

Identification of a novel cytochrome P-450 gene from the white rot fungus Phanerochaete chrysosporium.  

PubMed Central

A gene fragment belonging to the cytochrome P-450 superfamily has been cloned and identified from stationary cultures of the filamentous fungus Phanerochaete chrysosporium by reverse transcriptase (RT)-PCR. A set of degenerate primers homologous to highly conserved regions of known cytochrome P-450 sequences were used for initial RT-PCRs. Individual PCR products were cloned, sequenced, and identified as those belonging to the cytochrome P-450 superfamily based on amino acid sequence homologies and the presence of the highly conserved heme binding region. The nucleotide sequence of a single cDNA clone indicated the presence of an open reading frame encoding a partial cytochrome P-450 protein of 208 amino acids. Comparisons of the deduced amino acid sequence of the partial protein to other known cytochrome P-450 sequences indicate that it is the first member of a new family of cytochrome P-450s, designated CYP63-1A. Northern blot analysis suggests that CYP63-1A is expressed under both nitrogen-rich and nitrogen-deficient culture conditions and thus not under the same regulatory constraints as the well-studied lignin and manganese peroxidases. Western blot analyses using antibodies raised to the heme binding region of CYP63-1A indicate that the protein has a molecular mass of approximately 44,000 Da.

Kullman, S W; Matsumura, F

1997-01-01

194

Involvement of cytochrome P450 in pentachlorophenol transformation in a white rot fungus Phanerochaete chrysosporium.  

PubMed

The occurrence of cytochrome P450 and P450-mediated pentachlorophenol oxidation in a white rot fungus Phanerochaete chrysosporium was demonstrated in this study. The carbon monoxide difference spectra indicated induction of P450 (103±13 pmol P450 per mg protein in the microsomal fraction) by pentachlorophenol. The pentachlorophenol oxidation by the microsomal P450 was NADPH-dependent at a rate of 19.0±1.2 pmol min(-1) (mg protein)(-1), which led to formation of tetrachlorohydroquinone and was significantly inhibited by piperonyl butoxide (a P450 inhibitor). Tetrachlorohydroquinone was also found in the cultures, while the extracellular ligninases which were reported to be involved in tetrachlorohydroquinone formation were undetectable. The formation of tetrachlorohydroquinone was not detectable in the cultures added with either piperonyl butoxide or cycloheximide (an inhibitor of de novo protein synthesis). These results revealed the pentachlorophenol oxidation by induced P450 in the fungus, and it should be the first time that P450-mediated pentachlorophenol oxidation was demonstrated in a microorganism. Furthermore, the addition of the P450 inhibitor to the cultures led to obvious increase of pentachlorophenol, suggesting that the relationship between P450 and pentachlorophenol methylation is worthy of further research. PMID:23029295

Ning, Daliang; Wang, Hui

2012-09-20

195

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

SciTech Connect

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

Yasutake, Yoshiaki [Research Institute of Genome-based Biofactory, National Institute of Advanced Industrial Science and Technology (AIST), 2-17-2-1 Tsukisamu-Higashi, Toyohira-ku, Sapporo 062-8517 (Japan); Imoto, Noriko [Laboratory of Molecular Environmental Microbiology, Graduate School of Agriculture, Hokkaido University, Kita-9, Nishi-9, Kita-ku, Sapporo 060-8589 (Japan); Fujii, Yoshikazu; Fujii, Tadashi; Arisawa, Akira [Bioresource Laboratories, Mercian Corporation, 1808 Nakaizumi, Iwata, Shizuoka 438-0078 (Japan); Tamura, Tomohiro [Research Institute of Genome-based Biofactory, National Institute of Advanced Industrial Science and Technology (AIST), 2-17-2-1 Tsukisamu-Higashi, Toyohira-ku, Sapporo 062-8517 (Japan); Laboratory of Molecular Environmental Microbiology, Graduate School of Agriculture, Hokkaido University, Kita-9, Nishi-9, Kita-ku, Sapporo 060-8589 (Japan)], E-mail: t-tamura@aist.go.jp

2007-10-05

196

Involvement of Cytochrome P450 in Pentachlorophenol Transformation in a White Rot Fungus Phanerochaete chrysosporium  

PubMed Central

The occurrence of cytochrome P450 and P450-mediated pentachlorophenol oxidation in a white rot fungus Phanerochaete chrysosporium was demonstrated in this study. The carbon monoxide difference spectra indicated induction of P450 (103±13 pmol P450 per mg protein in the microsomal fraction) by pentachlorophenol. The pentachlorophenol oxidation by the microsomal P450 was NADPH-dependent at a rate of 19.0±1.2 pmol min?1 (mg protein)?1, which led to formation of tetrachlorohydroquinone and was significantly inhibited by piperonyl butoxide (a P450 inhibitor). Tetrachlorohydroquinone was also found in the cultures, while the extracellular ligninases which were reported to be involved in tetrachlorohydroquinone formation were undetectable. The formation of tetrachlorohydroquinone was not detectable in the cultures added with either piperonyl butoxide or cycloheximide (an inhibitor of de novo protein synthesis). These results revealed the pentachlorophenol oxidation by induced P450 in the fungus, and it should be the first time that P450-mediated pentachlorophenol oxidation was demonstrated in a microorganism. Furthermore, the addition of the P450 inhibitor to the cultures led to obvious increase of pentachlorophenol, suggesting that the relationship between P450 and pentachlorophenol methylation is worthy of further research.

Ning, Daliang; Wang, Hui

2012-01-01

197

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

ERIC Educational Resources Information Center

|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…

Furge, Laura Lowe; Fletke, Kyle J.

2007-01-01

198

Cytochromes P450 (CYP) in Tropical Fishes: Catalytic Activities, Expression of Multiple CYP Proteins and High Levels of Microsomal P450 in Liver of Fishes From Bermuda  

Microsoft Academic Search

Hepatic microsomes prepared from 10 fish species from Bermuda were studied to establish features of cytochrome P450 (CYP) systems in tropical marine fish. The majority (7\\/10) of the species had total P450 content between 0.1 and 0.5 nmol\\/mg, and cytochrome b5 content between 0.025 and 0.25 nmol\\/mg. Ethoxycoumarin O-deethylase (ECOD) and aminopyrine N-demethylase (APND) rates in these 7 species were

John J. Stegeman; Bruce R. Woodin; Hanuman Singh; Marjorie F. Oleksiak; Malin Celander

1997-01-01

199

The enantiomer of progesterone ( ent-progesterone) is a competitive inhibitor of human cytochromes P450c17 and P450c21  

Microsoft Academic Search

Human cytochrome P450c17 (17?-hydroxylase, 17,20-lyase) (CYP17) and cytochrome P450c21 (21-hydroxylase) (CYP21) differ by only 14 amino acids in length and share 29% amino acid identity. Both enzymes hydroxylate progesterone at carbon atoms that lie only 2.6Å apart, but CYP17 also metabolizes other steroids and demonstrates additional catalytic activities. To probe the active site topologies of these related enzymes, we synthesized

Richard J. Auchus; A. Sampath Kumar; C. Andrew Boswell; Manisha K. Gupta; Kristen Bruce; Nigam P. Rath; Douglas F. Covey

2003-01-01

200

A Fluorescence Spectroscopic Study of Cytochromes P450 1A2 and 3A4.  

NASA Astrophysics Data System (ADS)

Fluorescence spectroscopy was used to study cytochromes P450 1A2 and 3A4. Spectra of P450s were acquired in the presence and absence of acrylamide quencher. In both P450s, quenching revealed three distinguishable species of amino acid fluorescence, with maxima at 297, 323, and 345 nm. The 345 nm tryptophan fluorescence was quenched by low levels of acrylamide; the 297 nm tyrosine fluorescence was resistant to quenching. The 323 nm fluorescence was observed at intermediate concentrations of quencher. Stern-Volmer plots of P450 quenching were non-linear, but were well-fitted to a superposition of linear plots for each fluorophore species. The effect of P450 1A2 binding on pyrene fluorescence was also examined. Upon binding to P450 1A2, the intensity of the 383 nm pyrene vibronic band was decreased relative to the intensities of the 372 and 393 nm bands. Fluorescence quenching of pyrene and other ligands upon binding to P450s will be used to evaluate distances between ligands and the P450 heme moiety by fluorescence resonance energy transfer. Fluorescence quantum yields of ligands, overlap integrals, and Förster distances of many ligand-heme donor-acceptor pairs were calculated. Steady-state spectra and time-resolved data of bound ligand will be used to calculate substrate-heme distances in the P450 enzymes.

Marsch, Glenn; Guengerich, F. P.; Inks, Joshua

2006-03-01

201

Four forms of cytochrome P-450 in human fetal liver: purification and their capacity to activate promutagens.  

PubMed

Four forms of cytochrome P-450 were separated and purified to electrophoretic homogeneity from human fetal livers. These forms of cytochrome P-450, termed P-450HFLa, P-450HFLb, P-450HFLc and P-450HFLd, were distinguishable from each other in their molecular weights, spectral properties, immunochemical properties and mutagen-producing activities from promutagens. The molecular weights of P-450HFLa, b, c and d were estimated to be 51,500, 49,000, 51,500 and 50,000, respectively. Antibodies to P-450HFLa recognized P-450HFLc but not P-450HFLb or d, and antibodies to rat P-448-H (P-450IA2) cross-reacted with P-450HFLb but not with other forms of cytochrome P-450. The N-terminal amino acid sequence of P-450HFLc was highly homologous, but not identical, to that of P-450HFLa. Each form of cytochrome P-450 catalyzed mutagenic activation of aflatoxin B1 (AFB1), 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and 2-amino-6-methyldipyrido-[1,2-a:3',2'-d]imidazole (Glu-P-1) at different rates. P-450 HFLa showed activities to produce mutagen(s) from AFB1, IQ and to a lesser extent from Glu-P-1. P-450 HFLb activated IQ at a faster rate than did the other forms. P-450 HFLc produced a mutagen from AFB1 and Glu-P-1 but not from IQ. P-450 HFLd did not activate these promutagens at significant rates. PMID:1904422

Kitada, M; Taneda, M; Itahashi, K; Kamataki, T

1991-04-01

202

Evaluation of cytochrome P450{sub BS{beta}} reactivity against polycyclic aromatic hydrocarbons and drugs  

SciTech Connect

The oxidation of 10 polycyclic aromatic hydrocarbons (PAH) by cytochrome P450{sub BS{beta}} using three different electron acceptors is reported. Three PAH were found to be substrates for the oxidation by P450{sub BS{beta}}, namely anthracene, 9-methyl-anthracene and azulene. The respective oxidation products were identified by reversed-phase high-performance liquid chromatography coupled to electrospray ionization-mass spectrometry. In addition, 10 drug-like compounds were investigated for their effects on the catalytic activity of P450{sub BS{beta}} by carrying out inhibition studies. The stability of P450{sub BS{beta}} against hydrogen peroxide, cumene, and ter-butyl hydroperoxide was determined. Overall, the results of this study suggested that the P450{sub BS{beta}} enzyme represents a powerful catalyst in terms of the catalytic activity and operational stability.

Torres, Eduardo [Universitaet Dortmund, Fachbereich Chemie, Biologisch-Chemische Mikrostrukturtechnik, Otto-Hahn Str. 6, D-44227 Dortmund (Germany); Hayen, Heiko [ISAS-Institute for Analytical Sciences, Bunsen-Kirchhoff-Str. 11, 44139 Dortmund (Germany); Niemeyer, Christof M. [Universitaet Dortmund, Fachbereich Chemie, Biologisch-Chemische Mikrostrukturtechnik, Otto-Hahn Str. 6, D-44227 Dortmund (Germany); ISAS-Institute for Analytical Sciences, Bunsen-Kirchhoff-Str. 11, 44139 Dortmund (Germany); E-mail: christof.niemeyer@uni-dortmund.de

2007-03-30

203

A Cytochrome P450 Conserved in Insects Is Involved in Cuticle Formation  

PubMed Central

The sequencing of numerous insect genomes has revealed dynamic changes in the number and identity of cytochrome P450 genes in different insects. In the evolutionary sense, the rapid birth and death of many P450 genes is observed, with only a small number of P450 genes showing orthology between insects with sequenced genomes. It is likely that these conserved P450s function in conserved pathways. In this study, we demonstrate the P450 gene, Cyp301a1, present in all insect genomes sequenced to date, affects the formation of the adult cuticle in Drosophila melanogaster. A Cyp301a1 piggyBac insertion mutant and RNAi of Cyp301a1 both show a similar cuticle malformation phenotype, which can be reduced by 20-hydroxyecdysone, suggesting that Cyp301a1 is an important gene involved in the formation of the adult cuticle and may be involved in ecdysone regulation in this tissue.

Sztal, Tamar; Chung, Henry; Berger, Silke; Currie, Peter D.; Batterham, Philip; Daborn, Phillip J.

2012-01-01

204

Plant cytochromes P450: tools for pharmacology, plant protection and phytoremediation.  

PubMed

Cytochromes P450 catalyse extremely diverse and often complex regiospecific and/or stereospecific reactions in the biosynthesis or catabolism of plant bioactive molecules. Engineered P450 expression is needed for low-cost production of antineoplastic drugs such as taxol or indole alkaloids and offers the possibility to increase the content of nutraceuticals such as phytoestrogens and antioxidants in plants. Natural products may serve important functions in plant defence and metabolic engineering of P450s is a prime target to improve plant defence against insects and pathogens. Herbicides, pollutants and other xenobiotics are metabolised by some plant P450 enzymes. These P450s are tools to modify herbicide tolerance, as selectable markers and for bioremediation. PMID:12732316

Morant, Marc; Bak, Søren; Møller, Birger Lindberg; Werck-Reichhart, Danièle

2003-04-01

205

Mechanism of the plant cytochrome P450 for herbicide resistance: a modelling study.  

PubMed

Plant cytochrome P450 is a key enzyme responsible for the herbicide resistance but the molecular basis of the mechanism is unclear. To understand this, four typical plant P450s and a widely resistant herbicide chlortoluron were analysed by carrying out homology modelling, molecular docking, molecular dynamics simulations and binding free energy analysis. Our results demonstrate that: (i) the putative hydrophobic residues located in the F-helix and polar residues in I-helix are critical in the herbicide resistance; (ii) the binding mode analysis and binding free energy calculation indicate that the distance between catalytic site of chlortoluron and heme of P450, as well as the binding affinity are key elements affecting the resistance for plants. In conclusion, this work provides a new insight into the interactions of plant P450s with herbicide from a molecular level, offering valuable information for the future design of novel effective herbicides which also escape from the P450 metabolism. PMID:23057845

Li, Qinfan; Fang, Yupeng; Li, Xiuxiu; Zhang, Hong; Liu, Mengmeng; Yang, Huibin; Kang, Zhuo; Li, Yan; Wang, Yonghua

2012-10-11

206

Oxidation of arylcyclopropanes by rabbit liver cytochrome P-450. Mechanistic implications of a multiple oxidation.  

PubMed Central

The arylcyclopropanes (cyclopropylarenes) cyclopropylbenzene and diphenylcyclopropane are oxidized by rabbit liver microsomal cytochrome P-450, both by the microsomal fraction and by the purified cytochrome in a reconstituted system. The products formed, principally benzoic acid, are due to an unusual triple oxidation of the substrate, which probably remains attached to the active site during the several steps of the oxidation. Both substrates were found to be inhibitors of the cytochrome P-450-dependent O-de-ethylation of 7-ethoxycoumarin. Model oxidation studies with cumene hydroperoxide as oxidizing agent and rabbit liver microsomal fraction as source of enzyme gave similar products to the microsomal and reconstituted systems. The significance of these results in the mechanism of oxidation catalysed by cytochrome P-450 is discussed.

Suckling, C J; Nonhebel, D C; Brown, L; Suckling, K E; Seilman, S; Wolf, C R

1985-01-01

207

Oxidation of arylcyclopropanes by rabbit liver cytochrome P-450. Mechanistic implications of a multiple oxidation.  

PubMed

The arylcyclopropanes (cyclopropylarenes) cyclopropylbenzene and diphenylcyclopropane are oxidized by rabbit liver microsomal cytochrome P-450, both by the microsomal fraction and by the purified cytochrome in a reconstituted system. The products formed, principally benzoic acid, are due to an unusual triple oxidation of the substrate, which probably remains attached to the active site during the several steps of the oxidation. Both substrates were found to be inhibitors of the cytochrome P-450-dependent O-de-ethylation of 7-ethoxycoumarin. Model oxidation studies with cumene hydroperoxide as oxidizing agent and rabbit liver microsomal fraction as source of enzyme gave similar products to the microsomal and reconstituted systems. The significance of these results in the mechanism of oxidation catalysed by cytochrome P-450 is discussed. PMID:4084228

Suckling, C J; Nonhebel, D C; Brown, L; Suckling, K E; Seilman, S; Wolf, C R

1985-11-15

208

Regulation of 17,20 lyase activity by cytochrome b5 and by serine phosphorylation of P450c17  

Microsoft Academic Search

Cytochrome P450c17 catalyzes the 17alpha-hydroxylase activity required for glucocorticoid synthesis and the 17,20 lyase activity required for sex steroid synthesis. Most P450 enzymes have fixed ratios of their various activities, but the ratio of these two activities of P450c17 is regulated post-translationally. We have shown that serine phosphorylation of P450c17 and the allosteric action of cytochrome b5 increase 17,20 lyase

A. V. Pandey; W. L. Miller

2005-01-01

209

Activation of mitomycin C by NADPH:cytochrome P-450 reductase.  

PubMed

Mitomycin C is an alkylating agent used in cancer chemotherapy that shows some specificity towards hypoxic cells. The therapeutic effects of this compound are thought to result from its metabolic activation by enzymes such as NADPH:cytochrome P-450 reductase. In a previous report we described a Chinese hamster ovary cell line resistant to mitomycin C, which had a decreased NADPH:cytochrome P-450 reductase activity coupled with a lower rate of mitomycin C metabolism. In order to provide further evidence that the lower reductase activity is a factor in the resistance mechanism, we incorporated NADPH:cytochrome P-450 reductase into cytotoxicity assays and showed that it significantly sensitizes cells to mitomycin C. Also, the difference in drug sensitivity between the wild-type and drug-resistant Chinese hamster ovary cells was no longer observed. In addition to these studies, we expressed a rat liver NADPH:cytochrome P-450 reductase cDNA in a Salmonella typhimurium strain, LR5000. The bacteria expressing the rat NADPH: cytochrome P-450 reductase showed increased sensitivity to mitomycin C when incubated with this compound under aerobic conditions. However, under hypoxic conditions increased sensitivity was not observed. This parallels the previous finding with mitomycin C-resistant Chinese hamster ovary cells. These data provide direct evidence for the role of NADPH:cytochrome P-450 reductase in the cytotoxic action of this mitomycin C under aerobic but not hypoxic conditions and suggest that reduced levels of this enzyme can lead to drug resistance. P-450 reductase expressed in S. typhimurium may provide a valuable tool for evaluating the role of this enzyme in the toxicity of drugs activated through a one electron reduction pathway. PMID:2123741

Bligh, H F; Bartoszek, A; Robson, C N; Hickson, I D; Kasper, C B; Beggs, J D; Wolf, C R

1990-12-15

210

A rapid screening for cytochrome P450 catalysis on new chemical entities: cytochrome P450 BM3 and 1,2,5-oxadiazole derivatives.  

PubMed

This work presents the validation of a rapid screening procedure for the catalysis of cytochrome P450 on new chemical entities. The assay is tested on the prototypical, catalytically self-sufficient and soluble cytochrome P450 BM3 from Bacillus megaterium that shares a high degree of homology with mammalian counterparts. The so-called alkali assay developed in our laboratory is validated here also by product formation and molecular modeling on a number of derivatives sharing the molecular scaffold of the 1,2,5-oxadiazole ring, a class of molecules very different from the long-chain fatty acids known to be oxidized by cytochrome P450 BM3. The alkali assay reveals the ability of this cytochrome to oxidize NADPH in the presence of nine out of thirteen 1,2,5-oxadiazole derivatives tested. The enzyme shows high affinity and coupling efficiencies when incubated with four 1,2,5-oxadiazole derivatives. The presence of oxidation products deriving from catalysis was also confirmed by high-performance liquid chromatography (HPLC). Molecular docking suggests that a key factor for the 1,2,5-oxadiazole derivatives to enter the active site and induce catalysis is the presence of the -SO(2) moiety bridging the 1,2,5-oxadiazole and phenyl rings. These data indicate that the alkali assay is able to quickly and cheaply detect the recognition of new substrates by cytochrome P450. The assay is not intended to substitute HPLC-mass spectrometry analysis, but it is a preliminary screening that allows elimination of obvious nonsubstrates from the start. PMID:22983164

Tsotsou, Georgia E; Di Nardo, Giovanna; Sadeghi, Sheila J; Fruttero, Roberta; Lazzarato, Loretta; Bertinaria, Massimo; Gilardi, Gianfranco

2012-09-13

211

Phenotyping of cytochromes P-450 in human tissues with monoclonal antibodies  

PubMed Central

Cytochrome P-450 (P-450)-dependent aryl hydrocarbon hydroxylase (AHHase) and 7-ethoxycoumarin deethylase (ECDEtase) in human tissues were differentially inhibited by monoclonal antibodies (MAbs) that were prepared to inhibit and completely inhibited the activity of 3-methylcholanthrene-induced rat liver P-450. The AHHase and ECDEtase of placentas from individual women who smoked were inhibited by the MAbs by 83-90% and by 34-74%, respectively. Benz[a]anthracene (BaA)-induced AHHase and ECDEtase in lymphocytes were inhibited 18-65% and 30-78%, respectively. The enzymes in both control and BaA-induced human cells in culture were inhibited to different extents. Both the AHHase and ECDEtase in control and BaA-induced monocytes and in normal liver were largely unaffected by the MAb. Thus, we have with the MAbs: (i) identified P-450s with a common antigenic site in placenta, lymphocytes, and human cells in culture; (ii) identified two forms of hydrocarbon-induced P-450s in lymphocytes, at least one of which is common with the induced P-450s of placenta and with a P-450 form present in uninduced lymphocytes; (iii) identified two forms of P-450 responsible for smoking-induced ECDEtase activity in placenta, one of which is also responsible for AHHase activity; (iv) shown that the P-450s of liver, basal, and BaA-induced monocytes are different from the MAb-sensitive P-450s of placenta and lymphocytes; (v) quantitated in several human tissues the percentages of control and inducible AHHase and ECDEtase that are dependent on the MAb-sensitive P-450; and (vi) defined by HPLC the contribution of the MAb-sensitive P-450 to the formation of specific benzo[a]-pyrene metabolites. The results demonstrate the value of MAbs for defining antigenic site relatedness for different enzymatic functions of P-450s and for identifying and quantifying the amount of a specific enzyme activity in a tissue dependent on specific P-450s. This study may be a prototype for the use of MAbs for phenotyping and mapping of P-450s responsible for specific metabolic reactions and, thus, may be useful in determining the relationship of P-450 phenotype to individual differences in drug metabolism and carcinogen susceptibility.

Fujino, Tadahiko; Park, Sang Shin; West, Donna; Gelboin, Harry V.

1982-01-01

212

Coordinate modulation of murine hepatic xanthine oxidase activity and the cytochrome P-450 system by interferons.  

PubMed

The role of xanthine oxidase (XO) in the interferon (IFN)-dependent modulation of the hepatic cytochrome P-450 system was assessed in SENCAR mice. Intraperitoneal administration of 10(4)-10(5) units of IFN-gamma resulted in dose-dependent increases in hepatic XO activities. XO activity was significantly elevated within 12 h of IFN-gamma treatment, and reached a maximum between 24-48 h, and returned to basal levels within 72-96 h. Although the kinetics of increase and decline of XO activity correlated with the loss and subsequent recovery of hepatic P-450 levels, there was no quantitative correlation between hepatic XO activity and P-450 content. Comparable results were obtained in mice pretreated with the P-450 inducer Aroclor 1254 3 days prior to IFN-gamma administration. The increases in XO activity following IFN-gamma treatment were the consequence of increases in xanthine dehydrogenase (XD), and the conversion of XD to XO. The ad libitum administration of allopurinol to IFN-gamma-treated mice reduced XO specific activity to approximately 4% of the basal activity of control mice, but did not prevent reductions in cytochrome P-450 levels or the activities of two P-450 dependent monooxygenases. Collectively, these data suggest that the reductions in the hepatic P-450 system noted after IFN administration are not a consequence of elevated XO activities. PMID:1692864

Reiners, J J; Cantu, A R; Rupp, T A

1990-04-01

213

Taxol biosynthesis: Taxane 13?-hydroxylase is a cytochrome P450-dependent monooxygenase  

PubMed Central

A central feature in the biosynthesis of Taxol is oxygenation at multiple positions of the taxane core structure, reactions that are considered to be mediated by cytochrome P450-dependent monooxygenases. A PCR-based differential display-cloning approach, using Taxus (yew) cells induced for Taxol production, yielded a family of related cytochrome P450 genes, one of which was assigned as a taxane 10?-hydroxylase by functional expression in yeast. The acquired clones that did not function in yeast were heterologously expressed by using the Spodoptera fugiperda-baculovirus-based system and were screened for catalytic capability by using taxa-4(20),11(12)-dien-5?-ol and its acetate ester as test substrates. This approach allowed identification of one of the cytochrome P450 clones (which bore 63% deduced sequence identity to the aforementioned taxane 10?-hydroxylase) as a taxane 13?-hydroxylase by chromatographic and spectrometric characterization of the corresponding recombinant enzyme product. The demonstration of a second relevant hydroxylase from the induced family of cytochrome P450 genes validates this strategy for elucidating the oxygenation steps of taxane diterpenoid (taxoid) metabolism. Additionally, substrate specificity studies with the available cytochrome P450 hydroxylases now indicate that there is likely more than one biosynthetic route to Taxol in yew species.

Jennewein, Stefan; Rithner, Christopher D.; Williams, Robert M.; Croteau, Rodney B.

2001-01-01

214

Natural variation in the expression of cytochrome P-450 and dimethylnitrosamine demethylase in Drosophila  

SciTech Connect

Electrophoresis of Drosophila microsomes resolves two major heme-containing protein bands with apparent molecular weights of 59,290 (band a) and 55,750 (band b). The hemoproteins in these two bands can account for most of the cytochrome P-450 in the organism. Band a is present in all strains examined: band b is not. Dimethylnitrosamine demethylase, a P-450 enzyme, is a component of band b. 22 references, 2 figures, 1 table.

Waters, L.C.; Simms, S.I.; Nix, C.E.

1984-09-28

215

Plant activation of aromatic amines mediated by cytochromes P450 and flavin-containing monooxygenases  

Microsoft Academic Search

To know the mechanisms involved in the activation of promutagenic aromatic amines mediated by plants, we used Persea americana S117 system (S117) for the activation of 2-aminofluorene (2-AF) and m-phenylenediamine (m-PDA) in Ames assays. In these assays, the effect of the diphenylene iodonium (DPI), an inhibitor of flavin-containing monooxygenases (FMOs), of the 1-aminobenzotriazole (1-ABT), an inhibitor of cytochromes P450 (cyt-P450s)

Carles Chiapella; Rodrigo D Radovan; José Antonio Moreno; Lorena Casares; Jordi Barbé; Montserrat Llagostera

2000-01-01

216

Spin-State Changes in Cytochrome P-450cam on Binding of Specific Substrates  

Microsoft Academic Search

The electron paramagnetic resonance signals of the soluble P-450 cytochrome from Pseudomonas putida were observed at temperatures from 4.2 to 80 degrees K. As isolated, P-450 has a signal typical of a low spin ferric-heme compound with sulfur as one of the axial ligands (g = 2.45, 2.26, 1.915). We also detected a minor signal typical of high spin ferric

R. Tsai; C. A. Yu; I. C. Gunsalus; J. Peisach; W. Blumberg; W. H. Orme-Johnson; H. Beinert

1970-01-01

217

Helitron mediated amplification of cytochrome P450 monooxygenase gene in maize  

Microsoft Academic Search

The mass movement of gene sequences by Helitrons has significantly contributed to the lack of gene collinearity reported between different maize inbred lines. However, Helitron captured-genes reported to date represent truncated versions of their progenitor genes. In this report, we provide evidence\\u000a that maize CYP72A27-Zm gene represents a cytochrome P450 monooxygenase (P450) gene recently captured by a Helitron and transposed

Natalie Jameson; Nikolaos Georgelis; Eric Fouladbash; Sara Martens; L. Curtis Hannah; Shailesh Lal

2008-01-01

218

Bioconversion of vitamin D to its active form by bacterial or mammalian cytochrome P450  

Microsoft Academic Search

Bioconversion processes, including specific hydroxylations, promise to be useful for practical applications because chemical syntheses often involve complex procedures. One of the successful applications of P450 reactions is the bioconversion of vitamin D3 to 1?,25-dihydroxyvitamin D3. Recently, a cytochrome P450 gene encoding a vitamin D hydroxylase from the CYP107 family was cloned from Pseudonocardia autotrophica and is now applied in

Toshiyuki Sakaki; Hiroshi Sugimoto; Keiko Hayashi; Kaori Yasuda; Eiji Munetsuna; Masaki Kamakura; Shinichi Ikushiro; Yoshitsugu Shiro

2011-01-01

219

Identification of human liver cytochrome P450 enzymes that metabolize the nonsedating antihistamine loratadine  

Microsoft Academic Search

[3H]Loratadine was incubated with human liver microsomes to determine which cytochrome P450 (CYP) enzymes are responsible for its oxidative metabolism. Specific enzymes were identified by correlation analysis, by inhibition studies (chemical and immunoinhibition), and by incubation with various cDNA-expressed human P450 enzymes. Descarboethoxyloratadine (DCL) was the major metabolite of loratadine detected following incubation with pooled human liver microsomes. Although DCL

Nathan Yumibe; Keith Huie; Kwang-Jong Chen; Mark Snow; Robert P. Clement; Mitchell N. Cayen

1996-01-01

220

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

Microsoft Academic Search

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

S. Oetari; M. Sudibyo; Jan N. M. Commandeur; R. Samhoedi; Nico P. E. Vermeulen

1996-01-01

221

Induction of Hepatic Cytochrome P-450 by Phenobarbital in Semiaquatic Frog ( Rana pipiens)  

Microsoft Academic Search

Hepatic microsomal cytochrome P450 (equivalent to rat P4502B1 isozymic form, a CYPIIB gene product) can be induced by pentobarbital (PB) in the adults of the semiaquatic frog,Rana pipiens(as in other terrestrial vertebrates), but not in adults of the aquatic frogXenopus laevisor in bluegill sunfish (Lepomis macrochirus). The activity of PB-induced P450 (2B1) towards aldrin and pentoxyresorufin increases respectively by about

M. A. Q. Khan; Syed Yamin Qadri; Supriya Tomar; Debbie Fish; Lavanya Gururajan; Mahipal S. Poria

1998-01-01

222

Suppression of male-specific cytochrome P450 isoforms by bisphenol A in rat liver  

Microsoft Academic Search

We examined the effect of bisphenol A (BPA) on microsomal cytochrome P450 (P450) enzymes in rats. Rats were treated intraperitoneally\\u000a with BPA daily for 4 days, at doses of 10, 20, and 40 mg\\/kg. Among the P450-dependent monooxygenase activities, testosterone\\u000a 2?-hydroxylase (T2AH) and testosterone 6?-hydroxylase (T6BH) activities, which are associated with CYP2C11 and CYP3A2 respectively, were remarkably decreased by 40

Nobumitsu Hanioka; Hideto Jinno; Tetsuji Nishimura; Masanori Ando

1998-01-01

223

Efficient Bioelectronic Actuation of the Natural Catalytic Pathway of Human Metabolic Cytochrome P450s  

PubMed Central

Cytochrome (cyt) P450s comprise the enzyme superfamily responsible for human oxidative metabolism of a majority of drugs and xenobiotics. Electronic delivery of electrons to cyt P450s could be used to drive the natural catalytic cycle for fundamental investigations, stereo- and regioselective synthesis, and biosensors. We describe herein nm-thick films on electrodes featuring excess human cyt P450s and cyt P450 reductase (CPR) microsomes that efficiently mimic the natural catalytic pathway for the first time. Redox potentials, electron-transfer rates, CO-binding, and substrate conversion rates confirmed that electrons are delivered from the electrode to CPR, which transfers them to cyt P450. The film system enabled electrochemical probing of the interaction between cyt P450 and CPR for the first time. Agreement of film voltammetry data with theoretical simulations support a pathway featuring a key equilibrium redox reaction in the natural catalytic pathway between reduced CPR and cyt P450 occurring within a CPR-cyt P450 complex uniquely poised for substrate conversion.

Krishnan, Sadagopan; Wasalathanthri, Dhanuka; Zhao, Linlin; Schenkman, John B.; Rusling, James F

2011-01-01

224

Metabolism of the herbicide chlortoluron by human cytochrome P450 3A4.  

PubMed

Studies were carried out to investigate the metabolism of herbicide chlortoluron in the microsomal fractions and whole cells of Saccharomyces cerevisiae expressing human cytochrome P450 3A4. Both whole cells and microsomal fractions of yeast expressing human cytochrome P450 3A4 exhibited a typical dithionite-reduced, CO-difference absorbance spectrum with maximum absorbance at 448 nm. Chlortoluron produced a type I binding spectrum with cytochrome P450 3A4 with a Ks value of 200 microM. Chlortoluron was metabolised into four metabolites; hydroxylated-N-monodemethylated, hydroxylated ring methylated, N-didemethylated and N-monodemethylated products. Chlortoluron metabolism was absolutely dependent on NADPH and no metabolism was observed in control transformants. PMID:8574549

Mehmood, Z; Kelly, D E; Kelly, S L

1995-12-01

225

Human Cytochrome P450s: The Work of Frederick Peter Guengerich  

PubMed Central

Purification and Characterization of the Human Liver Cytochromes P-450 Involved in Debrisoquine 4-Hydroxylation and Phenacetin O-Deethylation, Two Prototypes for Genetic Polymorphism in Oxidative Drug Metabolism (Distlerath, L. M., Reilly, P. E. B., Martin, M. V., Davis, G. G., Wilkinson, G. R., and Guengerich, F. P. (1985) J. Biol. Chem. 260, 9057–9067) Human Liver Microsomal Cytochrome P-450 Mephenytoin 4-Hydroxylase, a Prototype of Genetic Polymorphism in Oxidative Drug Metabolism. Purification and Characterization of Two Similar Forms Involved in the Reaction (Shimada, T., Misono, K. S., and Guengerich, F. P. (1986) J. Biol. Chem. 261, 909–921) Characterization of Rat and Human Liver Microsomal Cytochrome P-450 Forms Involved in Nifedipine Oxidation, a Prototype for Genetic Polymorphism in Oxidative Drug Metabolism (Guengerich, F. P., Martin, M. V., Beaune, P. H., Kremers, P., Wolff, T., and Waxman, D. J. (1986) J. Biol. Chem. 261, 5051–5060)

Mukhopadhyay, Rajendrani

2012-01-01

226

Cytochrome P450 Initiates Degradation of cis-Dichloroethene by Polaromonas sp. Strain JS666  

PubMed Central

Polaromonas sp. strain JS666 grows on cis-1,2-dichoroethene (cDCE) as the sole carbon and energy source under aerobic conditions, but the degradation mechanism and the enzymes involved are unknown. In this study, we established the complete pathway for cDCE degradation through heterologous gene expression, inhibition studies, enzyme assays, and analysis of intermediates. Several lines of evidence indicate that a cytochrome P450 monooxygenase catalyzes the initial step of cDCE degradation. Both the transient accumulation of dichloroacetaldehyde in cDCE-degrading cultures and dichloroacetaldehyde dehydrogenase activities in cell extracts of JS666 support a pathway for degradation of cDCE through dichloroacetaldehyde. The mechanism minimizes the formation of cDCE epoxide. The molecular phylogeny of the cytochrome P450 gene and the organization of neighboring genes suggest that the cDCE degradation pathway recently evolved in a progenitor capable of degrading 1,2-dichloroethane either by the recruitment of the cytochrome P450 monooxygenase gene from an alkane catabolic pathway or by selection for variants of the P450 in a preexisting 1,2-dichloroethane catabolic pathway. The results presented here add yet another role to the broad array of productive reactions catalyzed by cytochrome P450 enzymes.

Nishino, Shirley F.; Shin, Kwanghee A.; Gossett, James M.

2013-01-01

227

Immunochemical studies for the presence of P-450 HFLa, a form of cytochrome P-450 in human fetal livers in human hepatocellular carcinoma cells.  

PubMed

Immunohistochemical localization of P-450 HFLa, a form of cytochrome P-450 in human fetal livers was investigated in human hepatocellular carcinoma. The cytoplasm of carcinoma cells positively reacted with anti-P-450 HFLa antibodies. It was found that the carcinoma cells showing a pseudoglandular pattern or poorly differentiated appearance exhibited a weaker reactivity with anti-P-450 HFLa antibodies than did relatively differentiated carcinoma cells. In the case of hepatoblastoma, the polygonal or round-shaped tumor cells which differentiated into epithelial structure exhibited a positive reaction with anti-P-450 HFLa antibodies, whereas the spindle-shaped tumor cells which showed a sarcomatous appearance did not react with the antibodies. PMID:2550607

Kitada, M; Tsukidate, K; Takeuchi, J; Taneda, M; Komori, M; Ohi, H; Itahashi, K; Kamataki, T

1989-06-01

228

Involvement of Cytochrome P-450 in the Biosynthesis of Dhurrin in Sorghum bicolor (L.) Moench 1  

PubMed Central

The biosynthesis of the tyrosine-derived cyanogenic glucoside dhurrin involves N-hydroxytyrosine, (E)- and (Z)-p-hydroxyphenylacetaldehyde oxime, p-hydroxyphenylacetonitrile, and p-hydroxymandelonitrile as intermediates and has been studied in vitro using a microsomal enzyme system obtained from etiolated sorghum (Sorghum bicolor [L.] Moench) seedlings. The biosynthesis is inhibited by carbon monoxide and the inhibition is reversed by 450 nm light demonstrating the involvement of cytochrome P-450. The combined use of two differently prepared microsomal enzyme systems and of tyrosine, p-hydroxyphenylacetaldehyde oxime, and p-hydroxyphenylacetonitrile as substrates identify two cytochrome P-450-dependent monooxygenases: the N-hydroxylase which converts tyrosine into N-hydroxytyrosine and the C-hydroxylase converting p-hydroxyphenylacetonitrile into p-hydroxymandelonitrile. The inhibitory effect of a number of putative cytochrome P-450 inhibitors confirms the involvement of cytochrome P-450. Monospecific polyclonal antibodies raised toward NADPH-cytochrome P-450-reductase isolated from sorghum inhibits the same metabolic conversions as carbon monoxide. No cytochrome P-450-dependent monooxygenase catalyzing an N-hydroxylation reaction has previously been reported in plants. The metabolism of p-hydroxyphenylacetaldehyde oxime is completely dependent on the presence of NADPH and oxygen and results in the production of p-hydroxymandelonitrile with no accumulation of the intermediate p-hydroxyphenylacetonitrile in the reaction mixture. The apparent NADPH and oxygen requirements of the oxime-metabolizing enzyme are identical to those of the succeeding C-hydroxylase converting p-hydroxyphenylacetonitrile to p-hydroxymandelonitrile. Due to the complex kinetics of the microsomal enzyme system, these requirements may not appertain to the oxime-metabolizing enzyme, which may convert p-hydroxyphenylacetaldehyde oxime to p-hydroxyacetonitrile by a simple dehydration. ImagesFigure 3Figure 4

Halkier, Barbara Ann; M?ller, Birger Lindberg

1991-01-01

229

Quantification of cytochrome P-450-dependent cyclohexane hydroxylase activity in normal and neoplastic reproductive tissues.  

PubMed Central

It is well established that liver microsomal cytochrome P-450 participates in steroid metabolism and probably also in the metabolism of anti-oestrogens such as tamoxifen (Nolvadex). Thus it is possible that variations in cytochrome P-450 levels may influence the responsiveness of human breast and endometrial carcinomas to endocrine therapy. Therefore a simple sensitive spectrophotometric assay for determining levels of cytochrome P-450-dependent cyclohexane hydroxylation activity in breast and uterine microsomes (microsomal fractions) has been developed. Cyclohexane was chosen as a substrate because of the relatively high levels of cyclohexane hydroxylase activity in tumour microsomes and because cyclohexane serves as a substrate for several forms of cytochrome P-450. As previously described [Senler, Dean, Pierce & Wittliff (1985) Anal. Biochem. 144, 152-158], a direct method utilizing isotope-dilution/gas chromatography-mass spectrometry was also developed in order to confirm the results of the spectrophotometric assay. The average activity (cyclohexane-dependent NADPH oxidation) for 139 human breast-tumour microsome preparations was 1.34 nmol/min per mg, which is in the range of that found in untreated mammalian liver (1-3 nmol/min per mg). Also, high enzyme activity was demonstrated in human ovary, normal uterus as well as uterine leiomyomas. Endocrine status appeared to influence enzyme levels, in that mammary tissue from virgin rats contained significantly (P less than 0.025) higher amounts of activity than did tissues from either pregnant or lactating rats. Furthermore, carbon monoxide, as well as an antibody against rat liver cytochrome P-450, completely inhibited NADPH oxidation by breast-carcinoma microsomes. These results strengthen our hypothesis that tumours with high levels of cytochrome P-450 may have a reduced response to additive endocrine therapy.

Senler, T I; Dean, W L; Murray, L F; Wittliff, J L

1985-01-01

230

Enhancement of DMNQ-induced hepatocyte toxicity by cytochrome P450 inhibition  

SciTech Connect

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

Ishihara, Yasuhiro [Department of Biology, Graduate School of Science, Osaka University, Osaka 532-8686 (Japan); Shiba, Dai [Department of Biology, Graduate School of Science, Osaka University, Osaka 532-8686 (Japan); Shimamoto, Norio [Faculty of Pharmaceutical Sciences at Kagawa Campus, Tokushima Bunri University, 1314-1, Shido, Sanuki, Kagawa 769-2193 (Japan)]. E-mail: n-shimamoto@kph.bunri-u.ac.jp

2006-07-15

231

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

PubMed

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 implications of this for drug efficacy and safety. PMID:23226058

Cavallari, Larisa H; Jeong, Hyunyoung; Bress, Adam

2011-11-07

232

Recombinant cytochrome P450 2D18 metabolism of dopamine and arachidonic acid.  

PubMed

The function of cytochrome P450 (P450) in the mammalian brain is not well understood. In an effort to further this understanding, this study identifies two endogenous substrates for P450 2D18. Previous reports have shown that this isoform is expressed in the rat brain, and the recombinant enzyme catalyzes the N-demethylation of the antidepressants imipramine and desipramine. By further examining the substrate profile of P450 2D18, inferences can be made as to potential endogenous P450 substrates. Herein we demonstrate the metabolism of the central nervous system-acting compounds chlorpromazine and chlorzoxazone with turnover numbers of 1.8 and 0. 9 nmol/min/nmol, respectively. Because the four aforementioned pharmaceutical substrates work by binding to neurotransmitter receptors, binding assays and oxidation reactions were performed to test whether dopamine is a substrate for P450 2D18. These data indicate a K(S) value of 678 microM and that P450 2D18 can support the oxidation of dopamine to aminochrome through a peroxide-shunt mechanism. We also report the P450 2D18-mediated omega-hydroxylation and epoxygenation of arachidonic acid, primarily leading to the formation of 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acids, compounds that have been shown to have vasoactive properties in brain, kidney, and heart tissues. The data presented herein suggest a possible role for P450 involvement in membrane and receptor regulation via epoxyeicosatrienoic acid formation and a potential involvement of P450 in the oxidation of dopamine to reactive oxygen species under aberrant physiological conditions where the sequestering of dopamine becomes compromised, such as in Parkinson's disease. PMID:10945868

Thompson, C M; Capdevila, J H; Strobel, H W

2000-09-01

233

Regulation of cytochrome P-450j in rat hepatocytes in vivo and in primry monolayer culture  

SciTech Connect

Cytochrome P-450j is of importance because it metabolically activates carcinogens and cytotoxic agents. They treated groups of 4 rats with 10% ethanol (EtOH) or 0.1% isoniazid (INH) and found that the amount of liver microsomal P-450j protein was increased 1.5- and 2.4 fold, respectively, over untreated control levels. When liver RNA samples were subjected to Northern and slot blot analyses, a 1.8 kb RNA species that hybridized with a cDNA probe which encodes for HLj (the human ortholog of P-450j) was increased 1.6- and 1.7-fold, respectively, in EtOH- and INH-treated rats as compared to controls. To investigate the P-450j induction mechanism, they isolated hepatocytes from untreated rats and found that when the cells were incubated on an extracellular biomatrix in serum free medium, the amount of P-450j immunoreactive protein decrease to < 50% of the level at time 0 after 72 h in culture and < 25% after 120h. Additions to the culture medium of pyrazole, hydrazine, or INH for 72 h increased P-450j immunoreactive protein up to 4.5-fold, 2.4-fold, and 1.6-fold, respectively, over control values. Moreover, P-450j mRNA decreased becoming undetectable within 24-48 h in culture. Additions of pyrazole but not other inducers increased P-450j mRNA to detectable levels. They conclude that at least some inducers of P-450j act directly on the hepatocyte by a mechanism(s) that involves both increases in P-450j mRNA and, apparently, other post-transcriptional events.

Hunt, C.M.; Molowa, D.T.; Thomas, P.E.; Levin, W.; Guzelian, P.S.; Wrighton, S.A.

1987-05-01

234

Transcriptional regulation of the grape cytochrome P450 monooxygenase gene CYP736B expression in response to Xylella fastidiosa infection  

Microsoft Academic Search

BACKGROUND: Plant cytochrome P450 monooxygenases (CYP) mediate synthesis and metabolism of many physiologically important primary and secondary compounds that are related to plant defense against a range of pathogenic microbes and insects. To determine if cytochrome P450 monooxygenases are involved in defense response to Xylella fastidiosa (Xf) infection, we investigated expression and regulatory mechanisms of the cytochrome P450 monooxygenase CYP736B

Davis W Cheng; Hong Lin; Yuri Takahashi; M Andrew Walker; Edwin L Civerolo; Drake C Stenger

2010-01-01

235

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

SciTech Connect

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

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

1984-10-01

236

Identification of human liver cytochromes P450 by using MALDI-TOF mass spectrometry  

Microsoft Academic Search

Proteomic approaches have been used for detection and identification of cytochromes P450 forms from highly purified membrane\\u000a preparations of human liver. These included the protein separation by 2D-and\\/or 1D-electrophoresis and molecular scanning\\u000a of a SDS-PAGE gel fragment in a range 45–66 kDa (this area corresponds molecular weights of cytochromes P450). The analysis\\u000a of protein content was statistically evaluated by means

N. A. Petushkova; A. V. Lisitsa; I. I. Karuzina; V. G. Zgoda; G. F. Sheremetyeva; N. F. Samenkova; I. P. Nikitin; T. A. Sakharova; A. T. Kopylov; A. I. Archakov

2008-01-01

237

Different pathways for mitogenic and enzyme induction signal transduction by cytochrome P450 inducers.  

PubMed

The correlation between enzyme induction and cell proliferation caused by inducers of xenobiotic metabolizing enzymes was studied using a cell culture expressing a constitutive level of cytochrome P450 (hepatoma McA RH 7777) and a cell culture in which cytochrome P450 was absent (hepatoma 27). In hepatoma 27 cells, the inducers did not induce the synthesis of xenobiotic metabolizing enzymes but stimulated cell proliferation. Thus, the processes of signal transduction for enzyme induction and for cell proliferation by the inducers are different. PMID:10498810

Gujaeva, E L; Ostashkina, N M; Kondalenko, V F; Koblyakov, V A

1999-08-01

238

The planetary biology of cytochrome P450 aromatases  

Microsoft Academic Search

BACKGROUND: Joining a model for the molecular evolution of a protein family to the paleontological and geological records (geobiology), and then to the chemical structures of substrates, products, and protein folds, is emerging as a broad strategy for generating hypotheses concerning function in a post-genomic world. This strategy expands systems biology to a planetary context, necessary for a notion of

Eric A Gaucher; Logan G Graddy; Tang Li; Rosalia CM Simmen; Frank A Simmen; David R Schreiber; David A Liberles; Christine M Janis; Steven A Benner

2004-01-01

239

Mammalian cytochrome P450 enzymes catalyze the phenol-coupling step in endogenous morphine biosynthesis.  

PubMed

A cytochrome P450 (P450) enzyme in porcine liver that catalyzed the phenol-coupling reaction of the substrate (R)-reticuline to salutaridine was previously purified to homogeneity (Amann, T., Roos, P. H., Huh, H., and Zenk, M. H. (1995) Heterocycles 40, 425-440). This reaction was found to be catalyzed by human P450s 2D6 and 3A4 in the presence of (R)-reticuline and NADPH to yield not a single product, but rather (-)-isoboldine, (-)-corytuberine, (+)-pallidine, and salutaridine, the para-ortho coupled established precursor of morphine in the poppy plant and most likely also in mammals. (S)-Reticuline, a substrate of both P450 enzymes, yielded the phenol-coupled alkaloids (+)-isoboldine, (+)-corytuberine, (-)-pallidine, and sinoacutine; none of these serve as a morphine precursor. Catalytic efficiencies were similar for P450 2D6 and P450 3A4 in the presence of cytochrome b(5) with (R)-reticuline as substrate. The mechanism of phenol coupling is not yet established; however, we favor a single cycle of iron oxidation to yield salutaridine and the three other alkaloids from (R)-reticuline. The total yield of salutaridine formed can supply the 10 nm concentration of morphine found in human neuroblastoma cell cultures and in brain tissues of mice. PMID:19561069

Grobe, Nadja; Zhang, Baichen; Fisinger, Ursula; Kutchan, Toni M; Zenk, Meinhart H; Guengerich, F Peter

2009-06-26

240

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

PubMed

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. PMID:12626517

Wang, Jinling; de Montellano, Paul R Ortiz

2003-03-06

241

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

SciTech Connect

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.

Loper, J.C.

1997-03-01

242

Mammalian Cytochrome P450 Enzymes Catalyze the Phenol-coupling Step in Endogenous Morphine Biosynthesis*  

PubMed Central

A cytochrome P450 (P450) enzyme in porcine liver that catalyzed the phenol-coupling reaction of the substrate (R)-reticuline to salutaridine was previously purified to homogeneity (Amann, T., Roos, P. H., Huh, H., and Zenk, M. H. (1995) Heterocycles 40, 425–440). This reaction was found to be catalyzed by human P450s 2D6 and 3A4 in the presence of (R)-reticuline and NADPH to yield not a single product, but rather (?)-isoboldine, (?)-corytuberine, (+)-pallidine, and salutaridine, the para-ortho coupled established precursor of morphine in the poppy plant and most likely also in mammals. (S)-Reticuline, a substrate of both P450 enzymes, yielded the phenol-coupled alkaloids (+)-isoboldine, (+)-corytuberine, (?)-pallidine, and sinoacutine; none of these serve as a morphine precursor. Catalytic efficiencies were similar for P450 2D6 and P450 3A4 in the presence of cytochrome b5 with (R)-reticuline as substrate. The mechanism of phenol coupling is not yet established; however, we favor a single cycle of iron oxidation to yield salutaridine and the three other alkaloids from (R)-reticuline. The total yield of salutaridine formed can supply the 10 nm concentration of morphine found in human neuroblastoma cell cultures and in brain tissues of mice.

Grobe, Nadja; Zhang, Baichen; Fisinger, Ursula; Kutchan, Toni M.; Zenk, Meinhart H.; Guengerich, F. Peter

2009-01-01

243

Characterization of the Alkane-Inducible Cytochrome P450 (P450alk) Gene from the Yeast 'Candida tropicalis': Identification of a New P450 Gene Family.  

National Technical Information Service (NTIS)

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 so...

D. Sanglard J. C. Loper

1989-01-01

244

Metabolism of tentoxin by hepatic cytochrome P-450 3A isozymes.  

PubMed

The interaction between rat and human liver cytochrome P-450 with tentoxin, a natural phytotoxic cyclotetrapeptide having chlorotic properties, was studied by difference ultraviolet visible spectroscopy. Tentoxin interacted with rat liver microsomes and the difference spectrum was characteristic of binding to a protein site close to the heme. The intensity of this spectrum was clearly dependent on the amounts of P-450 3A in the microsomes and was optimal in dexamethasone-treated rat microsomes. Tentoxin exhibited a high affinity for P-450 3A (Ks approximately 10 microM). Similar results were observed with human P-450 isozymes expressed in yeast. Only P-450 3A4 and 3A5 were able to give spectral interactions with tentoxin. Liver microsomes from rats pretreated with dexamethasone, a specific inducer of P-450 3A, were found to be particularly active for the oxidation of tentoxin, which occurs mainly on its Ala(Me) function leading to demethylation. Yeast-expressed P-450 3A also exhibited high activity to metabolize tentoxin. The metabolites were identified by their ultraviolet and mass spectra in fast atom bombardment and collision-activated dissociation modes. In addition to the major N-demethylated metabolite, other hydroxylated metabolites were formed. Preliminary analysis showed that as tentoxin, some metabolites were still efficient chloroplast ATPase inhibitors, while at least one of them exhibited even at low concentration stimulatory effects. PMID:9432003

Delaforge, M; Andre, F; Jaouen, M; Dolgos, H; Benech, H; Gomis, J M; Noel, J P; Cavelier, F; Verducci, J; Aubagnac, J L; Liebermann, B

1997-11-15

245

Characterization of Maize Cytochrome P450 Monooxygenases Induced in Response to Safeners and Bacterial Pathogens1  

PubMed Central

Plants use a diverse array of cytochrome P450 monooxygenases in their biosynthetic and detoxification pathways. To determine the extent to which various maize P450s are induced in response to chemical inducers, such as naphthalic anhydride (NA), triasulfuron (T), phenobarbital, and bacterial pathogens (Erwinia stuartii, Acidovorax avenae), we have analyzed the response patterns of seven P450 transcripts after treatment of seedlings with these inducers. Each of these P450 transcripts has distinct developmental, tissue-specific, and chemical cues regulating their expression even when they encode P450s within the same biosynthetic pathway. Most notably, the CYP71C1 and CYP71C3 transcripts, encoding P450s in the DIMBOA biosynthetic pathway, are induced to the same level in response to wounding and NA treatment of younger seedlings and differentially in response to NA/T treatment of younger seedlings and NA and NA/T treatment of older seedlings. NA and T induce expression of both CYP92A1 and CYP72A5 transcripts in older seedling shoots, whereas phenobarbital induces CYP92A1 expression in older seedling shoots and highly induces CYP72A5 expression in young and older seedling roots. Expressed sequence tag (EST) 6c06b11 transcripts, encoding an undefined P450 activity, are highly induced in seedling shoots infected with bacterial pathogens.

Persans, Michael W.; Wang, Jian; Schuler, Mary A.

2001-01-01

246

Cytochrome P450 107U1 is required for sporulation and antibiotic production in Streptomyces coelicolor.  

PubMed

The filamentous bacterium Streptomyces coelicolor has a complex life cycle involving the formation of hair-like aerial mycelia on the colony surface, which differentiate into chains of spores. Genes required for the initiation of aerial mycelium formation have been termed 'bld' (bald), describing the smooth, undifferentiated colonies of mutant strains. We report the identification of a new bld gene designated as sco3099 and biochemical analysis of its encoded enzyme, cytochrome P450 (P450, or CYP) 107U1. Deletion of sco3099 resulted in a mutant defective in aerial hyphae sporulation and sensitive to heat shock, indicating that P450 107U1 plays a key role in growth and development of S. coelicolor. This is the first P450 reported to participate in a sporulation process in Streptomycetes. The substrate and catalytic properties of P450 107U1 were further investigated in mass spectrometry-based metabolomic studies. Glycocholic acid (from the medium) was identified as a substrate of P450 107U1 and was oxidized to glyco-7-oxo-deoxycholic acid. Although this reaction is apparently not relevant to the observed sporulation deficiency, it suggests that P450 107U1 might exert its physiological function by oxidizing other steroid-like molecules. PMID:23357279

Tian, Zhenhua; Cheng, Qian; Yoshimoto, Francis K; Lei, Li; Lamb, David C; Guengerich, F Peter

2013-01-25

247

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

PubMed

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

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

2009-08-15

248

Identification and developmental expression of the full complement of Cytochrome P450 genes in Zebrafish  

PubMed Central

Background Increasing use of zebrafish in drug discovery and mechanistic toxicology demands knowledge of cytochrome P450 (CYP) gene regulation and function. CYP enzymes catalyze oxidative transformation leading to activation or inactivation of many endogenous and exogenous chemicals, with consequences for normal physiology and disease processes. Many CYPs potentially have roles in developmental specification, and many chemicals that cause developmental abnormalities are substrates for CYPs. Here we identify and annotate the full suite of CYP genes in zebrafish, compare these to the human CYP gene complement, and determine the expression of CYP genes during normal development. Results Zebrafish have a total of 94 CYP genes, distributed among 18 gene families found also in mammals. There are 32 genes in CYP families 5 to 51, most of which are direct orthologs of human CYPs that are involved in endogenous functions including synthesis or inactivation of regulatory molecules. The high degree of sequence similarity suggests conservation of enzyme activities for these CYPs, confirmed in reports for some steroidogenic enzymes (e.g. CYP19, aromatase; CYP11A, P450scc; CYP17, steroid 17a-hydroxylase), and the CYP26 retinoic acid hydroxylases. Complexity is much greater in gene families 1, 2, and 3, which include CYPs prominent in metabolism of drugs and pollutants, as well as of endogenous substrates. There are orthologous relationships for some CYP1 s and some CYP3 s between zebrafish and human. In contrast, zebrafish have 47 CYP2 genes, compared to 16 in human, with only two (CYP2R1 and CYP2U1) recognized as orthologous based on sequence. Analysis of shared synteny identified CYP2 gene clusters evolutionarily related to mammalian CYP2 s, as well as unique clusters. Conclusions Transcript profiling by microarray and quantitative PCR revealed that the majority of zebrafish CYP genes are expressed in embryos, with waves of expression of different sets of genes over the course of development. Transcripts of some CYP occur also in oocytes. The results provide a foundation for the use of zebrafish as a model in toxicological, pharmacological and chemical disease research.

2010-01-01

249

Engineering of Artificial Plant Cytochrome P450 Enzymes for Synthesis of Isoflavones by Escherichia coli?  

PubMed Central

Engineered microbes are becoming increasingly important as recombinant production platforms. However, the nonfunctionality of membrane-bound cytochrome P450 enzymes precludes the use of industrially relevant prokaryotes such as Escherichia coli for high-level in vivo synthesis of many functional plant-derived compounds. We describe the design of a series of artificial isoflavone synthases that allowed the robust production of plant estrogen pharmaceuticals by E. coli. Through this methodology, a plant P450 construct was assembled to mimic the architecture of a self-sufficient bacterial P450 and contained tailor-made membrane recognition signals. The specific in vivo production catalyzed by one identified chimera was up to 20-fold higher than that achieved by the native enzyme expressed in a eukaryotic host and up to 10-fold higher than production by plants. This novel biological device is a strategy for the utilization of laboratory bacteria to robustly manufacture high-value plant P450 products.

Leonard, Effendi; Koffas, Mattheos A. G.

2007-01-01

250

Recollection of the early years of the research on cytochrome P450  

PubMed Central

Since the publication of the first paper on “cytochrome P450” in 1962, the biochemical research on this novel hemoprotein expanded rapidly in the 1960s and the 1970s as its principal roles in various important metabolic processes including steroid hormone biosynthesis in the steroidogenic organs and drug metabolism in the liver were elucidated. Establishment of the purification procedures of microsomal and mitochondrial P450s in the middle of the 1970s together with the introduction of molecular biological techniques accelerated the remarkable expansion of the research on P450 in the following years. This review paper summarizes the important developments in the research on P450 in the early years, for about two decades from the beginning, together with my personal recollections.

OMURA, Tsuneo

2011-01-01

251

Cytochrome P450 Monooxygenases for Fatty Acids and Xenobiotics in Marine Macroalgae1  

PubMed Central

The metabolism of xenobiotics has mainly been investigated in higher plant species. We studied them in various marine macroalgae of the phyla Chlorophyta, Chromophyta, and Rhodophyta. Microsomes contained high oxidative activities for known cytochrome (Cyt) P450 substrates (fatty acids, cinnamic acid, 3- and 4-chlorobiphenyl, 2,3-dichlorobiphenyl, and isoproturon; up to 54 pkat/mg protein). The presence of Cyt P450 (approximately 50 pmol/mg protein) in microsomes of the three algal families was demonstrated by CO-difference absorption spectra. Intact algal tissue converted 3-chlorobiphenyl to the same monohydroxy-metabolite formed in vitro. This conversion was 5-fold stimulated upon addition of phenobarbital, and was abolished by the known P450 inhibitor, 1-aminobenzotriazole. It is concluded that marine macroalgae contain active species of Cyt P450 and could act as a metabolic sink for marine pollutants.

Pflugmacher, Stephan; Sandermann, Heinrich

1998-01-01

252

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

PubMed

The major objective of personalized medicine is to select optimized drug therapies and to a large degree such mission is determined by the expression profiles of cytochrome(s) P450 (CYP). Accordingly, a proteomic case study in personalized medicine is provided by the superfamily of cytochromes P450. Our knowledge about CYP isozyme expression on a protein level is very limited and based exclusively on DNA/mRNA derived data. Such information is not sufficient because transcription and translation events do not lead to correlated levels of expressed proteins. Here we report expression profiles of CYPs in human liver obtained by mass spectrometry (MS)-based proteomic approach. We analyzed 32 samples of human liver microsomes (HLM) of different sexes, ages and ethnicity along with samples of recombinant human CYPs. We have experimentally confirmed that each CYP isozyme can be effectively differentiated by their unique isozyme-specific tryptic peptide(s). Trypsin digestion patterns for almost 30 human CYP isozymes were established. Those findings should assist in selecting tryptic peptides suitable for MS-based quantitation. The data obtained demonstrate remarkable differences in CYP expression profiles. CYP2E1, CYP2C8 and CYP4A11 were the only isozymes found in all HLM samples. Female and pediatric HLM samples revealed much more diverse spectrum of expressed CYPs isozymes compared to male HLM. We have confirmed expression of a number of "rare" CYP (CYP2J2, CYP4B1, CYP4V2, CYP4F3, CYP4F11, CYP8B1, CYP19A1, CYP24A1 and CYP27A1) and obtained first direct experimental data showing expression of such CYPs as CYP2F1, CYP2S1, CYP2W1, CYP4A22, CYP4X1, and CYP26A1 on a protein level. PMID:23274569

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

2012-12-27

253

Structure of Genes in the Cytochrome P-450PBc Subfamily: Conservation of Intron Locations in the Phenobarbital-Inducible Family.  

National Technical Information Service (NTIS)

The structures of P-450 genes in the rabbit phenobarbital-inducible subfamily of P-450 genes, defined by the cytochrome P-450PBc cDNAs, have been determined by restriction mapping and nucleotide sequencing. A genomic clone corresponding to the 5-flanking ...

S. Govind P. A. Bell B. Kemper

1986-01-01

254

Cytochrome P450 monooxygenases as reporters for circadian-regulated pathways.  

PubMed

Cytochrome P450 monooxygenases (P450s) play important roles in the synthesis of diverse secondary compounds in Arabidopsis (Arabidopsis thaliana). Comparison of four data sets analyzing seedlings harvested over a 2-d period of constant conditions after growth with varying photoperiods and thermocycles recorded a total of 98 P450 loci as circadian regulated for at least one of the four conditions. Here, we further describe the circadian-regulated pathways using, as reporters, individual P450 loci that are likely to be rate limiting in secondary metabolic pathways. Reverse transcription-polymerase chain reaction gel blot analyses have confirmed circadian regulation of P450s in phenylpropanoid, carotenoid, oxylipin, glucosinolate, and brassinosteroid biosyntheses and have shown that both P450 and non-P450 genes in the many branches of the phenylpropanoid pathway have similar circadian patterns of expression. In silico analyses of the subsets of coregulated promoters have identified overrepresented promoter elements in various biosynthetic pathway genes, including MYB and MYB4 elements that are significantly more abundant in promoters for the core and lignin sections of phenylpropanoid metabolism. Interactions with these elements important for circadian regulation do not involve the MYB transcription factor PAP1, as previously proposed, since the expression patterns of circadian-regulated P450s are the same in pap1-D mutant seedlings as in wild-type seedlings. Further analysis of circadian-regulated promoters in other biochemical pathways provides us with the opportunity to identify novel promoter motifs that might be important in P450 circadian regulation. PMID:19386812

Pan, Yinghong; Michael, Todd P; Hudson, Matthew E; Kay, Steve A; Chory, Joanne; Schuler, Mary A

2009-04-22

255

Orphans in the Human Cytochrome P450 Superfamily: Approaches to Discovering Functions and Relevance in Pharmacology  

PubMed Central

As a result of technical advances in recombinant DNA technology and nucleotide sequencing, entire genome sequences have become available in the past decade and offer potential in understanding diseases. However, a central problem in the biochemical sciences is that the functions of only a fraction of the genes/proteins are known, and this is also an issue in pharmacology. This review is focused on issues related to the functions of cytochrome P450 (P450) enzymes. P450 functions can be categorized in several groups: 1) Some P450s have critical roles in the metabolism of endogenous substrates (e.g., sterols and fat-soluble vitamins). 2) Some P450s are not generally critical to normal physiology but function in relatively nonselective protection from the many xenobiotic chemicals to which mammals (including humans) are exposed in their diets [as well as more anthropomorphic chemicals (e.g., drugs, pesticides)]. 3) Some P450s have not been extensively studied and are termed “orphans” here. With regard to elucidation of any physiological functions of the orphan P450s, the major subject of this review, it is clear that simple trial-and-error approaches with individual substrate candidates will not be very productive in addressing questions about function. A series of liquid chromatography/mass spectrometry/informatics approaches are discussed, along with some successes with both human and bacterial P450s. Current information on what are still considered “orphan” P450s is presented. The potential for application of some of these approaches to other enzyme systems is also discussed.

Cheng, Qian

2011-01-01

256

Metabolic Intermediate Complex Formation of Human Cytochrome P450 3A4 by Lapatinib  

PubMed Central

Lapatinib, an oral breast cancer drug, has recently been reported to be a mechanism-based inactivator of cytochrome P450 (P450) 3A4 and also an idiosyncratic hepatotoxicant. It was suggested that formation of a reactive quinoneimine metabolite was involved in mechanism-based inactivation (MBI) and/or hepatotoxicity. We investigated the mechanism of MBI of P450 3A4 by lapatinib. Liquid chromatography-mass spectrometry analysis of P450 3A4 after incubation with lapatinib did not show any peak corresponding to irreversible modifications. The enzymatic activity inactivated by lapatinib was completely restored by the addition of potassium ferricyanide. These results indicate that the mechanism of MBI by lapatinib is quasi-irreversible and mediated via metabolic intermediate complex (MI complex) formation. This finding was verified by the increase in a signature Soret absorbance at approximately 455 nm. Two amine oxidation products of the metabolism of lapatinib by P450 3A4 were characterized: N-hydroxy lapatinib (M3) and the oxime form of N-dealkylated lapatinib (M2), suggesting that a nitroso or another related intermediate generated from M3 is involved in MI complex formation. In contrast, P450 3A5 was much less susceptible to MBI by lapatinib via MI complex formation than P450 3A4. In addition, P450 3A5 had a significantly lower ability than 3A4 to generate M3, consistent with N-hydroxylation as the initial step in the pathway to MI complex formation. In conclusion, our results demonstrate that the primary mechanism for MBI of P450 3A4 by lapatinib is not irreversible modification by the quinoneimine metabolite, but quasi-irreversible MI complex formation mediated via oxidation of the secondary amine group of lapatinib.

Takakusa, Hideo; Wahlin, Michelle D.; Zhao, Chunsheng; Hanson, Kelsey L.; New, Lee Sun; Chan, Eric Chun Yong

2011-01-01

257

Adrenodoxin supports reactions catalyzed by microsomal steroidogenic cytochrome P450s  

SciTech Connect

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

Pechurskaya, Tatiana A. [Institute of Bioorganic Chemistry, Academy of Sciences of Belarus, Kuprevicha st., 5/2, Minsk 220141 (Belarus); Harnastai, Ivan N. [Institute of Bioorganic Chemistry, Academy of Sciences of Belarus, Kuprevicha st., 5/2, Minsk 220141 (Belarus); Grabovec, Irina P. [Institute of Bioorganic Chemistry, Academy of Sciences of Belarus, Kuprevicha st., 5/2, Minsk 220141 (Belarus); Gilep, Andrei A. [Institute of Bioorganic Chemistry, Academy of Sciences of Belarus, Kuprevicha st., 5/2, Minsk 220141 (Belarus); Usanov, Sergey A. [Institute of Bioorganic Chemistry, Academy of Sciences of Belarus, Kuprevicha st., 5/2, Minsk 220141 (Belarus)]. E-mail: usanov@iboch.bas-net.by

2007-02-16

258

Modelling Species Selectivity in Rat and Human Cytochrome P450 2D Enzymes  

PubMed Central

Updated models of the Rat Cytochrome P450 2D enzymes are produced based on the recent x-ray structures of the Human P450 2D6 enzyme both with and without a ligand bound. The differences in species selectivity between the epimers quinine and quinidine are rationalised using these models and the results are discussed with regard to previous studies. A close approach to the heme is not observed in this study. The x-ray structure of the enzyme with a ligand bound is shown to be a better model for explaining the observed experimental binding of quinine and quinidine. Hence models with larger closed binding sites are recommended for comparative docking studies. This is consistent with molecular recognition in Cytochrome P450 enzymes being the result of a number of non-specific interactions in a large binding site.

Edmund, Grace H. C.; Howlin, Brendan J.

2013-01-01

259

Azole antifungals are potent inhibitors of cytochrome P450 mono-oxygenases and bacterial growth in mycobacteria and streptomycetes  

Microsoft Academic Search

The genome sequence of Mycobacterium tuberculosis has revealed the presence of 20 different cytochrome P450 mono-oxygenases (P450s) within this organism, and subsequent genome sequences of other mycobacteria and of Streptomyces coelicolor have indicated that these actinomycetes also have large complements of P450s, pointing to important physiological roles for these enzymes. The actinomycete P450s include homologues of 14a-sterol demethylases, the targets

Kirsty J. McLean; Ker R. Marshall; Alison Richmond; Iain S. Hunter; Kay Fowler; Tobias Kieser; Sudagar S. Gurcha; Gurydal S. Besra; Andrew W. Munro

260

Catalytic activity and immunochemical quantification of hepatic cytochrome P-450 in ?-naphthoflavone and isosafrol treated rainbow trout ( Oncorhynchus mykiss )  

Microsoft Academic Search

In addition to catalytical assays, immunochemical techniques have recently been employed to measure induction of the cytochrome P-450 (P450) monooxygenase system in fish with polyaromatic hydrocarbons (PAH). In the present study, polyclonal antibodies were raised against rainbow trout P450IA1. Levels of rainbow trout P450IA1 determined using protein blotting- and ELISA procedures were compared with levels of 7-ethoxyresorufin-O-deethylase (7-EROD) activity in

Malin Celander; Lars Förlin

1991-01-01

261

Structural Evidence: A Single Charged Residue Affects Substrate Binding in Cytochrome P450 BM-3.  

PubMed

Cytochrome P450 BM-3 is a bacterial enzyme with sequence similarity to mammalian P450s that catalyzes the hydroxylation of fatty acids with high efficiency. Enzyme-substrate binding and dynamics has been an important topic of study for cytochromes P450 because most of the crystal structures of substrate-bound structures show the complex in an inactive state. We have determined a new crystal structure for cytochrome P450 BM-3 in complex with N-palmitoylglycine (NPG), which unexpectedly showed a direct bidentate ion pair between NPG and arginine 47 (R47). We further explored the role of R47, the only charged residue in the binding pocket in cytochrome P450 BM-3, through mutagenesis and crystallographic studies. The mutations of R47 to glutamine (R47Q), glutamic acid (R47E), and lysine (R47K) were designed to investigate the role of its charge in binding and catalysis. The oppositely charged R47E mutation had the greatest effect on activity and binding. The crystal structure of R47E BMP shows that the glutamic acid side chain is blocking the entrance to the binding pocket, accounting for NPG's low binding affinity and charge repulsion. For R47Q and R47K BM-3, the mutations caused only a slight change in kcat and a large change in Km and Kd, which suggests that R47 mostly is involved in binding and that our crystal structure, 4KPA , represents an initial binding step in the P450 cycle. PMID:23829560

Catalano, Jaclyn; Sadre-Bazzaz, Kianoush; Amodeo, Gabriele A; Tong, Liang; McDermott, Ann

2013-09-16

262

Immunohistochemical and immunoelectron microscopic study of cytochrome P-450 of human fetal livers (P-450HFLa): implications for an onco-feto-placental enzyme.  

PubMed

Cytochrome P-450 of human fetal livers (P-450HFLa) was demonstrated by the avidin-biotin immunoperoxidase technique in tissue samples as follows: human fetal organs, adult livers, human and cynomolgus placenta, and gynecologic organs which were obtained from 40 patients with gynecologic malignancies and 32 patients with benign diseases. P-450HFLa was clearly localized in the cytoplasm and membranes of the hepatocytes, and the fact was confirmed by an immunoelectron microscopic examination. In addition, a semiquantitative assay of staining intensity demonstrated that this enzyme tended to decrease with advancing age. These findings suggest that hepatic P-450HFLa synthesis is inversely proportional to age, and that this enzyme is one of the differentiation antigens. P-450HFLa was also detected immunohistochemically in other fetal organs. The present study thus confirms that P-450HFLa is not specific to the liver and is ubiquitous even in the fetus. Marked positive staining for P-450HFLa was demonstrated in villous syncytiotrophoblasts. In contrast, no positive staining was found in the cynomolgus-monkey placenta, unlike the case for many other placental antigens. These findings lead to the tentative conclusion that P-450HFLa is a feto-placental enzyme peculiar to humans. P-450HFLa was demonstrated to occur very frequently in gynecologic malignancies. The mean positivity rate for all gynecologic malignancies was 85%, while the rate was below 25% for benign gynecologic diseases, indicating that P-450HFLa is one of the onco-feto-placental enzymes. The present study thus suggests that this enzyme could be a promising new tumor marker for gynecologic malignancies. PMID:8250768

Okajima, Y; Inaba, N; Fukazawa, I; Ota, Y; Hirai, Y; Sato, N; Yamamoto, G; Itahashi, K; Kitada, M; Kamataki, T

1993-09-01

263

Downregulation of male-specific cytochrome P450s 2C11 and 3A2 in bile duct-ligated male rats: Importance to reduced hepatic content of cytochrome P450 in cholestasis  

Microsoft Academic Search

The effects of bile duct ligation (BDL) on the activity and content of individual hepatic mixed-function oxidases (MFOs) was examined. Five days after BDL, hepatic microsomal total cytochrome P450 (CYP) content and NADPH-cytochrome P450-reductase (P450-reductase) activity were reduced to 56% and 57% of control, respectively. MFO activities attributable to the sexually undifferentiated CYPs 1A, 2A1, 2C6, and 2E1 were decreased

Jiezhong Chen; Michael Murray; Christopher Liddle; Xing-mai Jiang; Geoffrey C. Farrell

1995-01-01

264

CYTOCHROME P450 1B1 CONTRIBUTES TO ANGIOTENSIN II-INDUCED HYPERTENSION AND ASSOCIATED PATHOPHYSIOLOGY  

PubMed Central

Hypertension is the leading cause of cardiovascular diseases, and angiotensin II is one of the major components of the mechanisms that contribute to the development of hypertension. However, the precise mechanisms for the development of hypertension are unknown. Our recent study that angiotensin II-induced vascular smooth muscle cell growth is dependent on cytochrome P450 1B1 led us to investigate its contribution to hypertension caused by this peptide. Angiotensin II was infused via miniosmotic pump into rats (150 ng/kg/min) or mice (1000 ?g/kg/day) for 13 days resulting in increased blood pressure, increased cardiac and vascular hypertrophy, increased vascular reactivity to vasoconstrictor agents, increased reactive oxygen species production, and endothelial dysfunction in both species. The increase in blood pressure and associated pathophysiological changes were minimized by the cytochrome P450 1B1 inhibitor, 2,3?,4,5?-tetramethoxystilbene in both species and was markedly reduced in Cyp1b1-/- mice. These data suggest that cytochrome P450 1B1 contributes to angiotensin II-induced hypertension and associated pathophysiological changes. Moreover, 2,3?,4,5?-tetramethoxystilbene which prevents both cytochrome P450 1B1-dependent and independent components of angiotensin II-induced hypertension and inhibits associated pathophysiological changes could be clinically useful in the treatment of hypertension and associated cardiovascular and inflammatory diseases.

Jennings, Brett L.; Sahan-Firat, Seyhan; Estes, Anne M.; Das, Kanak; Farjana, Nasreen; Fang, Xiao R.; Gonzalez, Frank J.; Malik, Kafait U.

2010-01-01

265

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

Technology Transfer Automated Retrieval System (TEKTRAN)

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

266

FLUCONAZOLE-INDUCED HEPATIC CYTOCHROME P450 GENE EXPRESSION AND ENZYMATIC ACTIVITIES IN RATS AND MICE  

EPA Science Inventory

This study was undertaken to examine the effects of the triazole antifungal agent fluconazole on the expression of hepatic cytochrome P450 (Cyp) genes and the activities of Cyp enzymes in male Sprague-Dawley rats and male CD-1 mice. Alkoxyresorufin O-dealkylation (AROD) methods w...

267

Key Elements of the Chemistry of Cytochrome P-450: The Oxygen Rebound Mechanism.  

ERIC Educational Resources Information Center

|Discusses the structure and function of the liver protein cytochrome P-450, an important catalyst for a variety of detoxification reactions. Diagnostic substracts for this heme-containing monooxygenase, synthetic modes of the active site, and oxidations with synthetic metalloporphyrins are the major topic areas considered. (JN)|

Groves, John T.

1985-01-01

268

TRANSFECTION OF CYTOCHROME P450 CDNAS INTO MAMMALIAN CELLS USED IN MUTATION AND TRANSFORMATION ASSAYS  

EPA Science Inventory

The present work demonstrates that cDNAs coding for cytochrome P450 enzymes can be tranfected into mammalian cells and expressed, n the present studies, two different cell systems were used for transfection: 0T1/2 cells which can be used to study initiation and promotion (Diamond...

269

Oxygenation of Hydrocarbons by Cytochrome P-450 Model Compounds: Modification of Reactivity by Axial Ligands  

NASA Astrophysics Data System (ADS)

The rate of olefin oxygenation catalyzed by synthetic metalloporphyrins is examined, employing sodium hypochlorite as the oxygen atom source. The rate of epoxidation and the stability of the catalyst are shown to be dependent on the nature of the axial ligand employed. A rationale for this effect is presented and analogy is made to the role of the thiolate ligand in cytochrome P-450.

Collman, James P.; Kodadek, Thomas; Raybuck, Scott A.; Meunier, Bernard

1983-11-01

270

Identification and developmental expression of the full complement of Cytochrome P450 genes in Zebrafish  

Microsoft Academic Search

BACKGROUND: Increasing use of zebrafish in drug discovery and mechanistic toxicology demands knowledge of cytochrome P450 (CYP) gene regulation and function. CYP enzymes catalyze oxidative transformation leading to activation or inactivation of many endogenous and exogenous chemicals, with consequences for normal physiology and disease processes. Many CYPs potentially have roles in developmental specification, and many chemicals that cause developmental abnormalities

Jared V Goldstone; Andrew G McArthur; Akira Kubota; Juliano Zanette; Thiago Parente; Maria E Jönsson; David R Nelson; John J Stegeman

2010-01-01

271

Redox-Linked Domain Movements in the Catalytic Cycle of Cytochrome P450 Reductase  

PubMed Central

Summary NADPH-cytochrome P450 reductase is a key component of the P450 mono-oxygenase drug-metabolizing system. There is evidence for a conformational equilibrium involving large-scale domain motions in this enzyme. We now show, using small-angle X-ray scattering (SAXS) and small-angle neutron scattering, that delivery of two electrons to cytochrome P450 reductase leads to a shift in this equilibrium from a compact form, similar to the crystal structure, toward an extended form, while coenzyme binding favors the compact form. We present a model for the extended form of the enzyme based on nuclear magnetic resonance and SAXS data. Using the effects of changes in solution conditions and of site-directed mutagenesis, we demonstrate that the conversion to the extended form leads to an enhanced ability to transfer electrons to cytochrome c. This structural evidence shows that domain motion is linked closely to the individual steps of the catalytic cycle of cytochrome P450 reductase, and we propose a mechanism for this.

Huang, Wei-Cheng; Ellis, Jacqueline; Moody, Peter C.E.; Raven, Emma L.; Roberts, Gordon C.K.

2013-01-01

272

Intragraft iNOS induction during human liver allograft rejection depresses cytochrome p450 activity  

Microsoft Academic Search

Allograft function may become impaired during rejection after human liver transplantation. Cytokines induce nitric oxide (NO) production in hepatocytes, Kupffer cells and infiltrating mononuclear cells. NO inhibits cytoplasmatic cytochrome p450 (CYP) enzyme activity in vitro. It is not known whether this mechanism plays a role in vivo. In order to characterize the role of locally produced cytokines in the pathogenesis

Alexandra Westerholt; Sigrid Himpel; Birgit Hager-Gensch; Stefan Maier; Martin Werner; Josef Stadler; Johannes Doehmer; Claus-Dieter Heidecke

2004-01-01

273

Cytochrome P450 CYP307A1\\/Spook: A regulator for ecdysone synthesis in insects  

Microsoft Academic Search

The prothoracic gland (PG) has essential roles in synthesizing and secreting a steroid hormone called ecdysone that is critical for molting and metamorphosis of insects. However, little is known about the genes controlling ecdysteroidogenesis in the PG. To identify genes functioning in the PG of the silkworm, Bombyx mori, we used differential display PCR and focused on a cytochrome P450

Toshiki Namiki; Ryusuke Niwa; Takashi Sakudoh; Ken-ichi Shirai; Hideaki Takeuchi; Hiroshi Kataoka

2005-01-01

274

Cytochrome P450-mediated enzyme activities and polychlorinated biphenyl accumulation in harp seal ( Phoca groenlandica)  

Microsoft Academic Search

The presence and activities of hepatic cytochrome P450 (CYP) isoforms in Barents Sea harp seals (n=13) were studied using catalytic activities, selective inhibitors, and western blots. In addition the polychlorinated biphenyl (PCB) burden was measured in both the blubber and the food of the seals. The CYP activities and CYP isoforms present were related to the blubber PCB load and

J Wolkers; I. C Burkow; M Monshouwer; C Lydersen; S Dahle; R. F Witkamp

1999-01-01

275

Aryl Hydroxylation of the Herbicide Diclofop by a Wheat Cytochrome P-450 Monooxygenase 1  

PubMed Central

Wheat (Triticum aestivum L. cv Etoile de Choisy) microsomes catalyzed the cytochrome P-450-dependent oxidation of the herbicide diclofop to three hydroxy-diclofop isomers. Hydroxylation was predominant at carbon 4, with migration of chlorine to carbon 5 (67%) and carbon 3 (25%). The 2,4-dichloro-5-hydroxy isomer was identified as a minor reaction product (8%). Substrate-specificity studies showed that the activity was not inhibited or was weakly inhibited by a range of xenobiotic or physiological cytochrome P-450 substrates, with the exception of lauric acid. Wheat microsomes also catalyze the metabolism of the herbicides chlorsulfuron, chlortoluron, and 2,4-dichlorophenoxyacetic acid and of the model substrate ethoxycoumarin, as well as the hydroxylation of the endogenous substrates cinnamic and lauric acids. Treatments of wheat seedlings with phenobarbital or the safener naphthalic acid anhydride enhanced the cytochrome P-450 content of the microsomes and all related activities except that of cinnamic acid 4-hydroxylase, which was reduced. The stimulation patterns of diclofop aryl hydroxylase and lauric acid hydroxylase were similar, in contrast with the other activities tested. Lauric acid inhibited competitively (Ki = 9 ?m) the oxidation of diclofop and reciprocally. The similarity of diclofop aryl hydroxylase and lauric acid hydroxylase was further investigated by alternative substrate kinetics, autocatalytic inactivation, and computer-aided molecular modelisation studies, and the results suggest that both reactions are catalyzed by the same cytochrome P-450 isozyme.

Zimmerlin, Alfred; Durst, Francis

1992-01-01

276

ANTHONY Y. H. LU COMMEMORATIVE ISSUE PHYSIOLOGICAL AND PATHOPHYSIOLOGICAL REGULATION OF CYTOCHROME P450  

Microsoft Academic Search

This article is a report on a symposium sponsored by the American Society for Pharmacology and Experimental Therapeutics and held at the April 1998 Experimental Biology '98 meeting in San Fran- cisco. The presentations focused on the mechanisms of regulation of cytochrome P450 gene expression by developmental factors and by hormones and cytokines, as well as on the interplay be-

EDWARD T. MORGAN; MARION B. SEWER; HEINRICH IBER; FRANK J. GONZALEZ; YING-HUE LEE; ROBERT H. TUKEY; STEVE OKINO; TIEN VU; YUE-HWA CHEN; JASPREET S. SIDHU; CURTIS J. OMIECINSKI

277

Regioselective and stereoselective metabolism of ibuprofen by human cytochrome P450 2C  

Microsoft Academic Search

The cytochrome P450s responsible for the regio- and stereoselectivity in the 2- and 3-hydroxylation of the chiral non-steroidal antiinflammatory drug ibuprofen were characterized in human liver microsomes. The rates of formation of both the 2- and 3-hydroxy metabolites exhibited monophasic (N = 2; N is the number of microsomal preparations) and biphasic (N = 2) substrate concentration dependence for both

Mitchell A. Hamman; Gary A. Thompson; Stephen D. Hall

1997-01-01

278

Screening and identification of novel cytochrome P450s in ticks  

Technology Transfer Automated Retrieval System (TEKTRAN)

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

279

Key Elements of the Chemistry of Cytochrome P-450: The Oxygen Rebound Mechanism.  

ERIC Educational Resources Information Center

Discusses the structure and function of the liver protein cytochrome P-450, an important catalyst for a variety of detoxification reactions. Diagnostic substracts for this heme-containing monooxygenase, synthetic modes of the active site, and oxidations with synthetic metalloporphyrins are the major topic areas considered. (JN)

Groves, John T.

1985-01-01

280

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

EPA Science Inventory

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

281

INDUCTION OF CYTOCHROME P450 ISOFORMS IN RAT LIVER BY TWO CONAZOLES, TRIADIMEFON AND MYCLOBUTANIL  

EPA Science Inventory

1. This study was undertaken to examine the inductive effects of two triazole antifungal agents, myclobutanil and triadimefon on the expression of hepatic cytochrome P450 (CYP) genes and on the activities of CYP enzymes in male Sprague-Dawley rats. Rats were dosed by gavage for 1...

282

Oxygenation of hydrocarbons by cytochrome P-450 model compounds: modification of reactivity by axial ligands.  

PubMed Central

The rate of olefin oxygenation catalyzed by synthetic metalloporphyrins is examined, employing sodium hypochlorite as the oxygen atom source. The rate of epoxidation and the stability of the catalyst are shown to be dependent on the nature of the axial ligand employed. A rationale for this effect is presented and analogy is made to the role of the thiolate ligand in cytochrome P-450.

Collman, J P; Kodadek, T; Raybuck, S A; Meunier, B

1983-01-01

283

Prediction and analysis of the modular structure of cytochrome P450 monooxygenases  

Microsoft Academic Search

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

Demet Sirim; Michael Widmann; Florian Wagner; Jürgen Pleiss

2010-01-01

284

Primary Structure of the Cytochrome P450 Lanosterol 14 alpha-Demethylase Gene from 'Candida tropicalis'.  

National Technical Information Service (NTIS)

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

C. Chen V. F. Kalb T. G. Turi J. C. Loper

1988-01-01

285

Role of a cytochrome P450-dependent monooxygenase in the hydroxylation of 24- epi-brassinolide  

Microsoft Academic Search

24-epi-Brassinolide, exogenously applied to cell suspension cultures of Lycopersicon esculentum is hydroxylated at C-25 and C-26, respectively, followed by glucosylation of the newly formed hydroxyl group. Treatment of the cell cultures with the specific cytochrome P450 inhibitors, clotrimazole and ketoconazole, resulted in a strong decrease of only the C-25 hydroxylation, whereas hydroxylation at C-26 was not affected. The common cytochrome

Jochen Winter; Bernd Schneider; Dieter Strack; Günter Adam

1997-01-01

286

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

SciTech Connect

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 oxidants of various P450s directly using a chemical approach to generate the radical in situ. This resulted in the first report of direct methane to methanol conversion by a heme porphyrin catalyst using the soluble P450 from Mycobacterium sp, CYP153A6.

Arnold, Frances H.

2012-02-27

287

Ontogeny of Novel Cytochrome P450 Gene Isoforms during Postnatal Liver Maturation in Mice  

PubMed Central

The ontogeny of the first four families of cytochromes P450 (P450s) (i.e., Cyp1–Cyp4) can affect the biotransformation of drugs and dietary chemicals in liver, resulting in unique pharmacological reactions in children. Because genome-scale investigations have identified many novel P450 isoforms, it is critical to perform a systematic characterization of these P450s during liver development. In this study, livers were collected from C57BL/6 mice 2 days before birth and at various postnatal ages (0–45 days of age). The mRNA levels for 75 P450 isoforms (Cyp1–Cyp4) were quantified with branched DNA assays and reverse transcription-polymerase chain reaction assays. More than half of the mouse P450s are conserved in humans, but there are more isoforms in mice. The P450 mRNA levels increased after birth in mouse liver, forming four distinct ontogenic patterns. The majority of P450s form a total of eight genomic clusters, namely, Cyp1a1 and Cyp1a2 genes on chromosome 9 (cluster 1), Cyp2a, Cyp2b, Cyp2f, Cyp2g, and Cyp2t genes on chromosome 7 (cluster 2), Cyp2c genes on chromosome 19 (cluster 3), Cyp2d genes on chromosome 15 (cluster 4), Cyp2j genes on chromosome 4 (cluster 5), Cyp3a genes on chromosome 5 (cluster 6), Cyp4a, Cyp4b, and Cyp4x genes on chromosome 4 (cluster 7), and Cyp4f genes on chromosome 17 (cluster 8). Some P450 isoforms within the same genomic cluster showed similar ontogenic patterns. In conclusion, the present study revealed four patterns of ontogeny for P450s in liver and showed that many P450s within a genomic cluster exhibited similar ontogenic patterns, which suggests that some P450s within a cluster are likely regulated by a common pathway during liver development.

Cui, Julia Yue; Renaud, Helen J.

2012-01-01

288

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

SciTech Connect

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-/sup 2/H/sub 2/) ethanol at various concentrations, and a competitive method, where 1:1 mixtures of (1-/sup 13/C)- and (/sup 2/H/sub 6/) ethanol or (2,2,2-/sup 2/H/sub 3/)- and (1,1-/sup 2/H/sub 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/sub 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-/sup 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/sub 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.

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

1987-11-17

289

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

SciTech Connect

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.

Skoda, R.C.; Gonzalez, F.L.; Demierre, A.; Meyer, R.A. (Biocenter of the Univ. of Basel (Switzerland))

1988-07-01

290

Differences in adult and foetal human cytochrome P-450 forms recognized by monoclonal antibodies with specificity for the P450III family.  

PubMed Central

Six murine monoclonal antibodies raised against a major human adult liver cytochrome P-450 (P-450) of the PCN family (P450III) detected a protein in human foetal liver microsomes (microsomal fractions) which had an approx. 1 kDa higher molecular mass on SDS/polyacrylamide-gel electrophoresis than the protein recognized in human adult liver microsomes. Although each of the antibodies recognized both the adult and the foetal forms, antibody HL4 showed higher affinity for the foetal form. Recognition by the monoclonal antibodies of peptides generated by proteolytic cleavage of microsomal proteins showed different patterns for the adult and foetal forms. It is concluded that the foetal P-450 form recognized by antibodies to the major human adult liver form P450hA7, although structurally similar, is either a distinct P-450 isoenzyme or that the adult and foetal proteins have different covalent modification. Immunoquantification experiments showed comparable levels of the P-450 forms in adult and foetal liver, although there appeared to be less inter-individual variation in foetal livers. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5.

Barnes, T S; Burke, M D; Melvin, W T

1989-01-01

291

Conformational Plasticity and Structure/Function Relationships in Cytochromes P450  

PubMed Central

Abstract The cytochrome P450s are a superfamily of enzymes that are found in all kingdoms of living organisms, and typically catalyze the oxidative addition of atomic oxygen to an unactivated C-C or C-H bond. Over 8000 nonredundant sequences of putative and confirmed P450 enzymes have been identified, but three-dimensional structures have been determined for only a small fraction of these. While all P450 enzymes for which structures have been determined share a common global fold, the flexibility and modularity of structure around the active site account for the ability of P450 enzymes to accommodate a vast number of structurally dissimilar substrates and support a wide range of selective oxidations. In this review, known P450 structures are compared, and some structural criteria for prediction of substrate selectivity and reaction type are suggested. The importance of dynamic processes such as redox-dependent and effector-induced conformational changes in determining catalytic competence and regio- and stereoselectivity is discussed, and noncrystallographic methods for characterizing P450 structures and dynamics, in particular, mass spectrometry and nuclear magnetic resonance spectroscopy are reviewed. Antioxid. Redox Signal. 13, 1273–1296.

Kazanis, Sophia; Dang, Marina

2010-01-01

292

Water Oxidation by a Cytochrome P450: Mechanism and Function of the Reaction  

PubMed Central

P450cam (CYP101A1) is a bacterial monooxygenase that is known to catalyze the oxidation of camphor, the first committed step in camphor degradation, with simultaneous reduction of oxygen (O2). We report that P450cam catalysis is controlled by oxygen levels: at high O2 concentration, P450cam catalyzes the known oxidation reaction, whereas at low O2 concentration the enzyme catalyzes the reduction of camphor to borneol. We confirmed, using 17O and 2H NMR, that the hydrogen atom added to camphor comes from water, which is oxidized to hydrogen peroxide (H2O2). This is the first time a cytochrome P450 has been observed to catalyze oxidation of water to H2O2, a difficult reaction to catalyze due to its high barrier. The reduction of camphor and simultaneous oxidation of water are likely catalyzed by the iron-oxo intermediate of P450cam, and we present a plausible mechanism that accounts for the 1?1 borneol:H2O2 stoichiometry we observed. This reaction has an adaptive value to bacteria that express this camphor catabolism pathway, which requires O2, for two reasons: 1) the borneol and H2O2 mixture generated is toxic to other bacteria and 2) borneol down-regulates the expression of P450cam and its electron transfer partners. Since the reaction described here only occurs under low O2 conditions, the down-regulation only occurs when O2 is scarce.

Prasad, Brinda; Mah, Derrick J.; Lewis, Andrew R.; Plettner, Erika

2013-01-01

293

[Genome-wide analysis of cytochrome P450 monooxygenase genes in the tobacco].  

PubMed

Plant cytochrome P450 monooxygenases (CYP) constitute a large superfamily of heme-thiolate proteins, which are involved in a wide range of metabolic pathways. In this study, comparative genomic approaches were used to analyze tobacco CYP genes and their expression patterns. Based on analysis of the tobacco genomic DNA sequences that are currently available, 263 P450 genes that belong to 44 distinct clans were identified. EST evidence from 173 of the CYPs suggested that these genes are transcribed. Sequence features and secondary structures of the tobacco P450 genes were further analyzed through comparison with known P450 proteins. The expression profiles of 73 P450 genes were subsequently investigated by analyses of tobacco microarray data and RT-PCR. The results showed a variety of expression patterns of these genes in different tissues with a number of genes expressed in a tissue-specific manner. This study has set a foundation for further studies on functions of P450 genes in tobacco. PMID:23575545

Xie, Min-Min; Gong, Da-Ping; Li, Feng-Xia; Liu, Guan-Shan; Sun, Yu-He

2013-03-01

294

Characterization of a novel ACTH inducible cytochrome P-450 from rat adrenal microsomes  

SciTech Connect

In rat adrenal cortex 7,12 dimethylbenz(a)anthracene (DMBA) causes massive necrosis that is dependent of ACTH. This is related to an ACTH inducible adrenal microsomal cytochrome P-450 that catalyzes hydrocarbon metabolism. Rat adrenal microsomes, catalyze the formation of DMBA 3,4 diol a precursor of the bay region reactive electrophile DMBA 3,4 diol 1,2 oxide. Both DMBA metabolism and a 57Kd protein have disappeared from microsomes 30 days after hypophysectomy, but are restored by 14 days treatment with ACTH. Dexamethasone which fully suppresses ACTH only partially suppresses this activity. The 57 Kd protein was partially purified to a single major band in one step from solubilized microsomes by h.p.l.c. chromatography using detergent elution from a novel column that mimics phospholipid membranes. This preparation exhibits a specific content of 2 nm P-450/mg protein and a turnover number of 1,500pm DMBA/nm P-450/minutes. A polyclonal antisera raised against this preparation provides a single western blot corresponding to the 57Kd ACTH sensitive protein. This antibody did not blot microsomal P-450 c21, nor did selected antibodies from known families react with this adrenal P-450 protein, suggesting substantial sequence differences from known P-450's.

Otto, S.A.; Marcus, C.M.; Jefcoate, C.R. (Univ. of Wisconsin, Madison (United States))

1990-02-26

295

Bioconversion of vitamin D to its active form by bacterial or mammalian cytochrome P450.  

PubMed

Bioconversion processes, including specific hydroxylations, promise to be useful for practical applications because chemical syntheses often involve complex procedures. One of the successful applications of P450 reactions is the bioconversion of vitamin D? to 1?,25-dihydroxyvitamin D?. Recently, a cytochrome P450 gene encoding a vitamin D hydroxylase from the CYP107 family was cloned from Pseudonocardia autotrophica and is now applied in the bioconversion process that produces 1?,25-dihydroxyvitamin D?. In addition, the directed evolution study of CYP107 has significantly enhanced its activity. On the other hand, we found that Streptomyces griseolus CYP105A1 can convert vitamin D? to 1?,25-dihydroxyvitamin D?. Site-directed mutagenesis of CYP105A1 based on its crystal structure dramatically enhanced its activity. To date, multiple vitamin D hydroxylases have been found in bacteria, fungi, and mammals, suggesting that vitamin D is a popular substrate of the enzymes belonging to the P450 superfamily. A combination of these cytochrome P450s would produce a large number of compounds from vitamin D and its analogs. Therefore, we believe that the bioconversion of vitamin D and its analogs is one of the most promising P450 reactions in terms of practical application. PMID:20654743

Sakaki, Toshiyuki; Sugimoto, Hiroshi; Hayashi, Keiko; Yasuda, Kaori; Munetsuna, Eiji; Kamakura, Masaki; Ikushiro, Shinichi; Shiro, Yoshitsugu

2010-07-27

296

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

SciTech Connect

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 11{beta}-hydroxylation of deoxycorticosterone was the first mammalian enzyme shown by O{sup 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 17{alpha}-OH progesterone was the first demonstration that cytochrome P450 was an oxygenase; (d) spectrophotometric studies of the binding of 17{alpha}-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 11{beta}-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.

Estabrook, Ronald W. [Virginia Lazenby O'Hara Professor of Biochemistry, Ida and Cecil Green Chair in the Biomedical Sciences, Department of Biochemistry, Room Y7.206B, University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd., Dallas, TX 75390-9038 (United States)]. E-mail: Ronald.estabrook@utsouthwestern.edu

2005-12-09

297

Possibility of application of cytochrome P450 to bioremediation of dioxins.  

PubMed

Dioxins, including polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans, and coplanar polychlorinated biphenyls, are known to be metabolized by enzymes such as cytochrome (CYP) P450, angular dioxygenase, lignin peroxidase, and dehalogenase. It is noted that all of these enzymes have metal ions in their active centers, and the enzyme systems except for peroxidase each have a distinct electron transport chain. Among these enzyme systems, we have focused on cytochrome P450-dependent metabolism of dioxins from the viewpoint of practical use for bioremediation. Mammalian and fungal cytochromes P450 showed remarkable activity toward low-chlorinated PCDDs. In particular, mammalian cytochromes P450 belonging to the CYP1 family showed high activity. Rat CYP1A1 showed high activity toward 2,3,7-trichloro-dibenzo-p-dioxin but no detectable activity for 2,3,7,8-tetrachloro-dibenzo-p-dioxin (2,3,7,8-TCDD). On the basis of these results, we assumed that enlarging the space of the substrate-binding pocket of rat CYP1A1 might generate TCDD-metabolizing enzyme. Large-sized amino acids located at putative substrate-recognition sites and F-G loop were substituted for alanine by site-directed mutagenesis. Finally, we successfully generated 2,3,7,8-TCDD-metabolizing enzyme by site-directed mutagenesis of rat CYP1A1. We hope that recombinant microorganisms harboring genetically engineered cytochrome P450 will be used for bioremediation of soil contaminated with PCDDs, polychlorinated dibenzofurans, and coplanar polychlorinated biphenyls in the future. PMID:23586993

Sakaki, Toshiyuki; Yamamoto, Keiko; Ikushiro, Shinichi

2013-01-16

298

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

SciTech Connect

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

Stiborova, Marie [Department of Biochemistry, Faculty of Science, Charles University, Albertov 2030, 128 40 Prague 2 (Czech Republic)], E-mail: stiborov@natur.cuni.cz; Arlt, Volker M. [Section of Molecular Carcinogenesis, Institute of Cancer Research, Brookes Lawley Building, Sutton, Surrey SM2 5NG (United Kingdom); Henderson, Colin J.; Wolf, C. Roland [Cancer Research UK Molecular Pharmacology Unit, Biomedical Research Centre, Dundee DD1 9SY (United Kingdom); Kotrbova, Vera; Moserova, Michaela; Hudecek, Jiri [Department of Biochemistry, Faculty of Science, Charles University, Albertov 2030, 128 40 Prague 2 (Czech Republic); Phillips, David H. [Section of Molecular Carcinogenesis, Institute of Cancer Research, Brookes Lawley Building, Sutton, Surrey SM2 5NG (United Kingdom); Frei, Eva [Division of Molecular Toxicology, German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg (Germany)

2008-02-01

299

Sensitization of Human Breast Cancer Cells to Cyclophosphamide and Ifosfamide by Transfer of a Liver Cytochrome P450 Gene1  

Microsoft Academic Search

The cancer chemotherapeutic agent Cyclophosphamide (CPA) and its isomer ifosfamide (IFA) are alkylating agent prodrugs that require me tabolism by liver cytochrome P450 (P450) enzymes for antitumor activity. The therapeutic effectiveness of these oxazaphosphorines is limited by the hematopoietic, renal, and cardiac toxicity that accompanies the systemic distribution of liver-derived activated drug metabolites. Transfer of a liver cytochrome P450 gene,

Ling Chen; David J. Waxman; Dongshu Chen; Donald W. Kufe

1996-01-01

300

Characteristic properties of a retinoic acid synthetic cytochrome P-450 purified from liver microsomes of 3-methylcholanthrene-induced rats  

Microsoft Academic Search

An inducible cytochrome P-450 (P-450) catalyzing retinoic acid synthesis was purified from liver microsomes of 3-methylcholanthrene (3-MC)-treated rats, based on the activity of all-trans-retinoic acid formation from all-trans-retinal. We previously reported that the retinoic acid synthesis by microsomes was catalyzed by a cytochrome P-450-linked monooxygenase system (Tomita et al. (1993) Int. J. Biochem. 25, 1775–1784). This microsomal retinoic acid synthesis

Shuhei Tomita; Eisaku Okuyama; Taira Ohnishi; Yoshiyuki Ichikawa

1996-01-01

301

Human Cytochrome P450scc (CYP11A1) Catalyzes Epoxide Formation with Ergosterol  

PubMed Central

Cytochrome P450scc (P450scc) catalyzes the cleavage of the side chain of both cholesterol and the vitamin D3 precursor, 7-dehydrocholesterol. The aim of this study was to test the ability of human P450scc to metabolize ergosterol, the vitamin D2 precursor, and define the structure of the major products. P450scc incorporated into the bilayer of phospholipid vesicles converted ergosterol to two major and four minor products with a kcat of 53 mol · min?1 · mol P450scc?1 and a Km of 0.18 mol ergosterol/mol phospholipid, similar to the values observed for cholesterol metabolism. The reaction of ergosterol with P450scc was scaled up to make enough of the two major products for structural analysis. From mass spectrometry, NMR, and comparison of the NMR data to that for similar molecules, we determined the structures of the two major products as 20-hydroxy-22,23-epoxy-22,23-dihydroergosterol and 22-keto-23-hydroxy-22,23-dihydroergosterol. Molecular modeling and nuclear Overhauser effect (or enhancement) spectroscopy spectra analysis helped to establish the configurations at C20, C22, and C23 and determine the final structures of major products as 22R,23S-epoxyergosta-5,7-diene-3?,20?-diol and 3?,23S-dihydroxyergosta-5,7-dien-22-one. It is likely that the formation of the second product is through a 22,23-epoxy (oxirane) intermediate followed by C22 hydroxylation with the formation of strained 22-hydroxy-22,23-epoxide (oxiranol), which is immediately transformed to the more stable ?-hydroxyketone. Molecular modeling of ergosterol into the P450scc crystal structure positioned the ergosterol side chain consistent with formation of the above products. Thus, we have shown that P450scc efficiently catalyzes epoxide formation with ergosterol giving rise to novel epoxy, hydroxy, and keto derivatives, without causing cleavage of the side chain.

Nguyen, Minh N.; Chen, Jianjun; Slominski, Andrzej T.; Baldisseri, Donna M.; Tieu, Elaine W.; Zjawiony, Jordan K.; Li, Wei

2012-01-01

302

Identification of Novel Endogenous Cytochrome P450 Arachidonate Metabolites with High Affinity for Cannabinoid Receptors*  

PubMed Central

Arachidonic acid is an essential constituent of cell membranes that is esterified to the sn-2-position of glycerophospholipids and is released from selected lipid pools by phospholipase cleavage. The released arachidonic acid can be metabolized by three enzymatic pathways: the cyclooxygenase pathway forming prostaglandins and thromboxanes, the lipoxygenase pathway generating leukotrienes and lipoxins, and the cytochrome P450 (cP450) pathway producing epoxyeicosatrienoic acids and hydroxyeicosatetraenoic acids. The present study describes a novel group of cP450 epoxygenase-dependent metabolites of arachidonic acid, termed 2-epoxyeicosatrienoylglycerols (2-EG), including two regioisomers, 2-(11,12-epoxyeicosatrienoyl)glycerol (2-11,12-EG) and 2-(14,15-epoxyeicosatrienoyl)glycerol (2-14,15-EG), which are both produced in the kidney and spleen, whereas 2-11,12-EG is also detected in the brain. Both 2-11,12-EG and 2-14,15-EG activated the two cannabinoid (CB) receptor subtypes, CB1 and CB2, with high affinity and elicited biological responses in cultured cells expressing CB receptors and in intact animals. In contrast, the parental arachidonic acid and epoxyeicosatrienoic acids failed to activate CB1 or CB2 receptors. Thus, these cP450 epoxygenase-dependent metabolites are a novel class of endogenously produced, biologically active lipid mediators with the characteristics of endocannabinoids. This is the first evidence of a cytochrome P450-dependent arachidonate metabolite that can activate G-protein-coupled cell membrane receptors and suggests a functional link between the cytochrome P450 enzyme system and the endocannabinoid system.

Chen, Jian-Kang; Chen, Jianchun; Imig, John D.; Wei, Shouzuo; Hachey, David L.; Guthi, Jagadeesh Setti; Falck, John R.; Capdevila, Jorge H.; Harris, Raymond C.

2008-01-01

303

Transcriptional Regulation of the Grape Cytochrome P450 Monooxygenase Gene CYP736B Expression in Response to Xylella fastidiosa Infection  

Technology Transfer Automated Retrieval System (TEKTRAN)

Plant cytochrome P450 monooxygenases are a group of versatile redox proteins that mediate the biosynthesis of lignins, terpenes, alkaloids, and a variety of other secondary compounds which act as plant defense agents. To determine if cytochrome P450 monooxygenases are involved in defense response to...

304

DISRUPTION OF THE SACCHAROMYCES CEREVISIAE GENE FOR NADPH-CYTOCHROME P450-REDUCTASE CAUSES INCREASED SENSITIVITY TO KETOCONANZOLE  

EPA Science Inventory

Strains of Saccharomyces cerevisiae deleted in the NADPH-Cytochrome P450 reductase gene by transplacement are 200-fold more sensitive to ketoconazole, an inhibitor of the cytochrome P450 lanosterol 14a-demethylase. esistance is restored through complementation by the plasmid-born...

305

DISRUPTION OF THE SACCHAROMYCES CEREVISIAE GENE FOR NADPH-CYTOCHROME P450-REDUCTASE CAUSES INCREASED SENSITIVITY TO KETOCONAZOLE  

EPA Science Inventory

Strains of Saccharomyces cerevisiae deleted in the NADPH-cytochrome P450 reductase gene by transplacement are 200-fold more sensitive to ketoconazole, an inhibitor of the cytochrome P450 lanosterol 14-demethylase. Resistance is restored through complementation by the plasmid-born...

306

Ultraviolet-B Exposure of Human Skin Induces Cytochromes P450 1A1 and 1B1  

Microsoft Academic Search

The cytochromes P450 belong to a multigene superfamily and are responsible for the metabolic activation of both xenobiotics and endobiotics. The expression of cytochrome P450 genes in target cells is an important determinant of human susceptibility to cancers and other chemically initiated diseases. In this study using immunohistochemistry, reverse transcription polymerase chain reaction, and western blot analysis, we investigated the

Santosh K. Katiyar; Mary S. Matsui; Hasan Mukhtar

2000-01-01

307

Chemical inhibitors of cytochrome P450 isoforms in human liver microsomes: a re-evaluation of P450 isoform selectivity.  

PubMed

The majority of marketed small-molecule drugs undergo metabolism by hepatic Cytochrome P450 (CYP) enzymes (Rendic 2002). Since these enzymes metabolize a structurally diverse number of drugs, metabolism-based drug-drug interactions (DDIs) can potentially occur when multiple drugs are coadministered to patients. Thus, a careful in vitro assessment of the contribution of various CYP isoforms to the total metabolism is important for predicting whether such DDIs might take place. One method of CYP phenotyping involves the use of potent and selective chemical inhibitors in human liver microsomal incubations in the presence of a test compound. The selectivity of such inhibitors plays a critical role in deciphering the involvement of specific CYP isoforms. Here, we review published data on the potency and selectivity of chemical inhibitors of the major human hepatic CYP isoforms. The most selective inhibitors available are furafylline (in co-incubation and pre-incubation conditions) for CYP1A2, 2-phenyl-2-(1-piperidinyl)propane (PPP) for CYP2B6, montelukast for CYP2C8, sulfaphenazole for CYP2C9, (-)-N-3-benzyl-phenobarbital for CYP2C19 and quinidine for CYP2D6. As for CYP2A6, tranylcypromine is the most widely used inhibitor, but on the basis of initial studies, either 3-(pyridin-3-yl)-1H-pyrazol-5-yl)methanamine (PPM) and 3-(2-methyl-1H-imidazol-1-yl)pyridine (MIP) can replace tranylcypromine as the most selective CYP2A6 inhibitor. For CYP3A4, ketoconazole is widely used in phenotyping studies, although azamulin is a far more selective CYP3A inhibitor. Most of the phenotyping studies do not include CYP2E1, mostly because of the limited number of new drug candidates that are metabolized by this enzyme. Among the inhibitors for this enzyme, 4-methylpyrazole appears to be selective. PMID:21336516

Khojasteh, Siamak Cyrus; Prabhu, Saileta; Kenny, Jane R; Halladay, Jason S; Lu, Anthony Y H

2011-02-19

308

Efficient functional analysis system for cyanobacterial or plant cytochromes P450 involved in sesquiterpene biosynthesis  

Microsoft Academic Search

Tractable plasmids (pAC-Mv-based plasmids) for Escherichia coli were constructed, which carried a mevalonate-utilizing gene cluster, towards an efficient functional analysis of cytochromes\\u000a P450 involved in sesquiterpene biosynthesis. They included genes coding for a series of redox partners that transfer the electrons\\u000a from NAD(P)H to a P450 protein. The redox partners used were ferredoxin reductases (CamA and NsRED) and ferredoxins (CamB

Hisashi Harada; Kazutoshi Shindo; Kanoko Iki; Ayuko Teraoka; Sho Okamoto; Fengnian Yu; Jun-ichiro Hattan; Ryutaro Utsumi; Norihiko Misawa

2011-01-01

309

REGULATION OF MICROSOMAL P450, REDOX PARTNER PROTEINS AND STEROIDOGENESIS IN THE DEVELOPING TESTES OF THE NEONATAL PIG  

Technology Transfer Automated Retrieval System (TEKTRAN)

Testicular growth and plasma androgen concentrations increase markedly in the first weeks of neonatal life of pigs. The regulation of steroidogenesis through this period was examined by measuring total microsomal cytochromes P450 (P450), 17alpha-hydroxylase/17,20-lyase P450 (P450c17) and aromatase ...

310

Purification and characterization of a benzene hydroxylase: A cytochrome P-450 from rat liver mitochondria  

SciTech Connect

This laboratory previously demonstrated that incubation of ({sup 14}C)benzene with isolated mitochondria resulted in the formation of mtDNA adducts. Since benzene is incapable of spontaneously covalently binding to nuclei acids, it was hypothesized that enzyme(s) present in the organelle metabolized benzene to reactive derivatives. We have purified, to electrophoretic homogeneity, a 52 kDa cytochrome P-450 from liver mitoplasts which metabolizes benzene to phenol. The enzyme has a K{sub M} for benzene of 0.012 mM, and a V{sub MAX} of 22.6 nmol phenol/nmol P-450/10 min, and requires NADPH, adrenodoxin, and adrenodoxin reductase for activity. Activity also can be reconstituted with microsomal cytochrome P-450 reductase. Benzene hydroxylase activity could be inhibited by carbon monoxide and SKF-525A, and by specific inhibitors of microsomal benzene metabolism. The purified enzyme oxidized phenol, forming catechol; aminopyrine N-demethylase activity was also demonstrated. These data confirm that a cytochrome P-450 of mitochondrial origin is involved in benzene metabolism, and indicate a role for the mitochondrion in xenobiotic activation.

Karaszkiewicz, J.W.

1989-01-01

311

Protection against chemical-induced lung injury by inhibition of pulmonary cytochrome P-450  

SciTech Connect

Protection afforded by trialkyl phosphorothionates against the lung injury caused by trialkyl phosphorothiolates probably results from the inhibition by the P{double bond}S moiety of the thionates, of one or more pulmonary cytochrome P-450 isozymes. The aromatic hydrocarbons p-xylene and pseudocumene also protect against this injury and inhibit some P-450 isozymes, but by a different mechanism. OOS-Trimethylphosphorothionate and p-xylene were compared as protective agents against the effect of OOS-trimethylphosphorothiolate and two other lung toxins ipomeanol and 1-nitronaphthalene that are known to be activated by cytochrome P-450. The effects of these protective compounds, in vivo, on pulmonary cytochrome P-450 activity were also determined. Both compounds inhibited pentoxyresorufin O-deethylase activity, but not ethoxyresorufin O-deethylase. The phosphorothionate was most effective against lung injury caused by the phosphorothiolates and 1-nitronaphthalene, whereas p-xylene was much more effective against ipomeanol. {beta}-Naphthoflavone, which induces pulmonary ethoxyresorufin O-deethylase activity, did not protect against phosphorothiolate or 1-nitronaphthalene injury, and it was only marginally effective in decreasing the toxicity or ipomeanol.

Verschoyle, R.D.; Dinsdale, D. (Medical Research Council Laboratories, Carshalton Surrey (England))

1990-04-01

312

Preparation and characterization of monoclonal antibodies recognizing unique epitopes on sexually differentiated rat liver cytochrome P-450 isozymes.  

PubMed

Cytochrome P-450 isozymes P-450(16 alpha), P-450(15 beta), and P-450DEa are immunochemically related, as indicated by mutual cross-reactivity with polyclonal antibody preparations. We have isolated five monoclonal antibodies to P-450(15 beta) and one antibody to P-450(16 alpha) that show selectivity for the respective antigens. High frequencies of cross-reactivity were observed, indicating a high degree of homology among P-450(16 alpha), P-450(15 beta), and P-450DEa. All of the P-450(15 beta-specific antibodies bound to the same epitope, or closely grouped epitopes, supporting this conclusion. The specificity of each monoclonal antibody was characterized by enzyme-linked immunosorbent assay. Western immunoblotting, and antibody-Sepharose immunoadsorption of solubilized rat liver microsomes. Antibodies F22 and F23, which were apparently identical, were specific for P-450(15 beta) by these criteria. However, the apparent specificities of antibodies F3 and F20 for P-450(15 beta), and of M16 for P-450(16 alpha), were highly dependent on the analytical technique used. The five anti-P-450(15 beta) antibodies all inhibited the catalytic activity of microsomal P-450(15 beta), by a maximum of 70%. However, they also produced a similar inhibition of microsomal P-450(16 alpha-specific antibody M16 and F23 have a low-affinity interaction with an epitope on P-450(16 alpha). The P-450(16 alpha)-specific antibody M16 was not inhibitory. The results indicate that the apparent specificity of a monoclonal antibody for an antigen determined by, e.g., Western blotting does not allow the conclusive identification of a protein in another system, e.g., immunoprecipitation of in vitro translation reaction products.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2444248

Morgan, E T; Rönnholm, M; Gustafsson, J A

1987-07-14

313

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

USGS Publications Warehouse

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

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

1996-01-01

314

In vitro inhibition of multiple cytochrome P450 isoforms by xanthone derivatives from mangosteen extract.  

PubMed

Mangosteen is a xanthone-containing fruit found in Southeast Asia for which health claims include maintaining healthy immune and gastrointestinal systems to slowing the progression of tumor growth and neurodegenerative diseases. Previous studies have identified multiple xanthones in the pericarp of the mangosteen fruit. The aim of the current study was to assess the drug inhibition potential of mangosteen in vitro as well as the cytochrome P450 (P450) enzymes responsible for the metabolism of its individual components. The various xanthone derivatives were found to be both substrates and inhibitors for multiple P450 isoforms. Aqueous extracts of the mangosteen pericarp were analyzed for xanthone content as well as inhibition potency. Finally, in vivo plasma concentrations of alpha-mangostin, the most abundant xanthone derivative found in mangosteen, were predicted using Simcyp and found to be well above their respective in vitro K(i) values for CYP2C8 and CYP2C9. PMID:19541824

Foti, Robert S; Pearson, Josh T; Rock, Dan A; Wahlstrom, Jan L; Wienkers, Larry C

2009-06-18

315

Electrochemical generation of a high-valent state of cytochrome P450.  

PubMed

Cyclic voltammetry performed at rapid scan rates on cytochrome P450 from Pseudomonas putida (P450CAM) in didodecyldimethylammonium bromide (DDAB) films on graphite electrodes revealed a couple (E) at 830mV (vs Ag/AgCl). E was not significantly observed at scan rates less than 30V/s at room temperature, suggesting that the oxidized species is unstable. The lifetime of E could be prolonged at 4 degrees C, which allowed reversible access to E at scan rates as low as 1V/s. E was found to be sensitive to imidazole in solution and to variations in pH, suggesting that the redox reaction is occurring at the metal center (i.e., Fe(IV/III)). Electrolysis reactions with different P450 substrates revealed that the electrochemically generated high-valent species is able to convert thioanisole to methyl phenyl sulfoxide. PMID:16504300

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

2006-02-28

316

In Silico Docking of Ligands to Drug Oxidation Enzymes Cytochrome P450 3A4 and Cytochrome P450 1A2.  

NASA Astrophysics Data System (ADS)

Cytochrome P450 3A4 (CYP3A4) and Cytochrome P450 1A2 (CYP1A2) oxidize most drugs in humans. Protein modeling toolkits from OpenEye Scientific Software were used to examine the interaction of drug substrates with CYP3A4 and CYP1A2. Conformers and partial atomic charges were generated for each drug molecule. User-defined volumes were defined around CYP3A4 and CYP1A2 active sites. Ligands were docked assuming protein and substrates as rigid bodies. To assess rigid docking accuracy, x-ray diffraction coordinates of CYP3A4-erythromycin and CYP3A4-metyrapone complexes were obtained. Rigid re-docking of erythromycin and metyrapone into CYP3A4 yielded poses similar to the crystal structures. Rigid docking revealed two other energetically-favorable CYP3A4-metyrapone poses. The best poses were obtained by using all the Open Eye scoring functions. Optimization of protein-ligand interactions within 5-10 Angstroms of the docked ligand was then performed using the Merck Molecular Force Field in which the protein was assumed to be flexible and the ligand to be rigid. Nearby protein residues pulled slightly closer to the substrate, reducing the volume of the active site.

Smith, David; Guglielmon, Jonathan; Glenn, Marsch; Peter, Guengerich F.

2009-03-01

317

In situ estrogen synthesized by aromatase P450 in uterine leiomyoma cells promotes cell growth probably via an autocrine/intracrine mechanism.  

PubMed

In the present study we characterized in detail the expression of aromatase P450 in leiomyomas to determine the role of in situ estrogen in the growth advantage of leiomyomas. The levels of aromatase P450 transcripts were determined by quantitative RT-PCR to be significantly higher in leiomyomas than in corresponding myometrium. The overexpression of aromatase P450 in leiomyomas was also confirmed by Western blot analysis. The estimated size of immunoreactive aromatase was 58 kDa, similar to that in placenta. To identify a cell type that express aromatase P450 in leiomyomas, histological specimens were stained for aromatase P450 using a polyclonal antibody. Strong immunoreactivity was detected in the cytoplasm of leiomyoma cells, whereas surrounding normal myometrium displayed weak or negative staining. Smooth muscle-like cells in culture obtained from leiomyomas, positive for actin D fiber, possessed immunoreactive granules of aromatase in the cytoplasm. Conversion of androgen to estrogen was effectively stimulated by phorbol myristate acetate and dexamethasone plus interleukin-1beta and was completely abolished by selective inhibitors of aromatase P450 (fadrozole and TZA-2209), but not by inhibitors of 5alpha-reductase (finasteride and flutamide). The apparent Km of androstenedione was 3 nM in the presence of dexamethasone and interleukin-1beta, corresponding to the plasma concentration of androstenedione in women of reproductive age. To determine whether endogenous aromatase P450 plays a role in the growth promotion of leiomyoma cells, we evaluated the cell growth of smooth muscle-like cells treated with various concentrations of estrogen and androgen using a WST-1 assay. Treatment with testosterone (10(-8) and 10(-7) M) and androstenedione (10(-8) and 10(-7) M) stimulated the growth of smooth muscle-like cells obtained from leiomyomas to the same extent as estradiol (10(-10)-10(-7) M), whereas dihydrotestosterone (10(-11)-10(-8) M) did not. The stimulatory effect of testosterone on cell growth was again abolished by cotreatment with fadrozole. The level of estradiol in the medium of testosterone (10(-8) M)-treated smooth muscle-like cells was 10(-11) M, which was 1 order lower than the minimum concentration of estradiol necessary to promote cell growth (10(-10) M). This indicates that estradiol synthesized in leiomyomas promotes their growth via an autocrine/intracrine mechanism. We conclude that myometrial cells of leiomyomas overexpress aromatase P450 and are able to synthesize sufficient estrogen to accelerate their own cell growth. Overexpression of aromatase P450 may play a role in the growth advantage of leiomyoma tissue over surrounding myometrium via an autocrine/intracrine mechanism. PMID:11014242

Sumitani, H; Shozu, M; Segawa, T; Murakami, K; Yang, H J; Shimada, K; Inoue, M

2000-10-01

318

Role of intestinal cytochrome p450 enzymes in diclofenac-induced toxicity in the small intestine.  

PubMed

The aim of this study was to determine the role of small intestinal (SI) cytochrome P450 (P450) enzymes in the metabolic activation of diclofenac (DCF), a widely used nonsteroidal anti-inflammatory drug, and DCF-induced intestinal toxicity. DCF induces intestinal ulcers in humans and mice, but the underlying mechanisms, including the necessity for drug bioactivation in the target tissues and the sources and identities of reactive intermediates, are not fully understood. We found that the number of DCF-induced (at 50 mg/kg p.o.) intestinal ulcers was significantly smaller in an intestinal epithelium (IE)-specific P450 reductase (CPR) knockout (IE-Cpr-null) mouse model, which has little P450 activity in the IE, than in wild-type (WT) mice, determined at 14 h after DCF administration. The involvement of intestinal P450 enzymes was confirmed by large reductions (>80-90%) in the rates of in vitro formation, in SI microsomal reactions, of hydroxylated DCF metabolites and reactive intermediates, trapped as DCF-glutathione (GSH) conjugates, in the IE-Cpr-null, compared with WT mice. The SI levels of DCF-GSH conjugates (at 4 h after dosing) and DCF-protein adducts (at 14 h after dosing) were significantly lower in IE-Cpr-null than in WT mice. In additional experiments, we found that pretreatment of mice with grapefruit juice, which is known to inhibit SI P450 activity, ameliorated DCF-induced intestinal toxicity in WT mice. Our results not only strongly support the notion that SI P450 enzymes play an important role in DCF-induced intestinal toxicity, but also illustrate the possibility of preventing DCF-induced intestinal toxicity through dietary intervention. PMID:22892338

Zhu, Yi; Zhang, Qing-Yu

2012-08-14

319

Pulmonary oxygen toxicity in rats treated with cytochrome P-450 inducers  

SciTech Connect

Pulmonary oxygen toxicity is assumed to result from damage caused by superoxide (O/sub 2//sup -/) hydrogen peroxide (H/sub 2/O/sub 2/) and/or hydroxyl radical (OH) produced by the partial reduction of molecular oxygen (O/sub 2/). The microsomal cytochrome P-450 (P-450) monooxygenase system is known to produce O/sub 2//sup -/ and H/sub 2/O/sub 2/. They have studied the influence of monooxygenase induction using phenobarbital (PB) and ..beta..-naphthoflavone (..beta..-NF) on O/sub 2/ toxicity in the rat. PB- or ..beta..-NF induce hepatic P-450 but only ..beta..-NF induces pulmonary P-450. Pulmonary microsomes produced O/sub 2//sup -/ and H/sub 2/O/sub 2/ at rates (expressed per mg microsomal protein) which did not vary as a function of pretreatment. Rats were exposed to 100% O/sub 2/ for up to 3 days. After 3 days of O/sub 2/, lung weights were about 50% above controls regardless of pretreatment. The microsomal monooxygenase enzymes (P-450, b/sub 5/ and NADPH P-450 reductase) were quantified in liver and lung. Lung microsomal P-450 was reduced after 3 days of O/sub 2/ exposure regardless of pretreatment. The protective enzymes (catalase, superoxide dismutase (SOD) and glutathione (GSH) peroxidase) and non-protein sulfhydryl groups (NPSH) were also quantified in lung and liver samples. Lung NPSH and GSH peroxidase were increased after 3 days of O/sub 2/ exposure regardless of pretreatment while SOD was increased in controls and PB- but not ..beta..-NF-treated rats. Three of 14 ..beta..-NF-treated rats died during O/sub 2/ exposure while no animals in the control or PB-treated groups died.

Ebel, R.E.; Barlow, R.L.; Gregory, E.M.

1987-05-01

320

Efficient functional analysis system for cyanobacterial or plant cytochromes P450 involved in sesquiterpene biosynthesis.  

PubMed

Tractable plasmids (pAC-Mv-based plasmids) for Escherichia coli were constructed, which carried a mevalonate-utilizing gene cluster, towards an efficient functional analysis of cytochromes P450 involved in sesquiterpene biosynthesis. They included genes coding for a series of redox partners that transfer the electrons from NAD(P)H to a P450 protein. The redox partners used were ferredoxin reductases (CamA and NsRED) and ferredoxins (CamB and NsFER), which are derived from Pseudomonas putida and cyanobacterium Nostoc sp. strain PCC 7120, respectively, as well as three higher-plant NADPH-P450 reductases, the Arabidopsis thaliana ATR2 and two corresponding enzymes derived from ginger (Zingiber officinale), named ZoRED1 and ZoRED2. We also constructed plasmids for functional analysis of two P450s, ?-humulene-8-hydroxylase (CYP71BA1) from shampoo ginger (Zingiber zerumbet) and germacrene A hydroxylase (P450NS; CYP110C1) from Nostoc sp. PCC 7120, and co-transformed E. coli with each of the pAC-Mv-based plasmids. Production levels of 8-hydroxy-?-humulene with recombinant E. coli cells (for CYP71BA1) were 1.5- to 2.3-fold higher than that of a control strain without the mevalonate-pathway genes. Level of the P450NS product with the combination of NsRED and NsFER was 2.9-fold higher than that of the CamA and CamB. The predominant product of P450NS was identified as 1,2,3,5,6,7,8,8a-octahydro-6-isopropenyl-4,8a-dimethylnaphth-1-ol with NMR analyses. PMID:21229242

Harada, Hisashi; Shindo, Kazutoshi; Iki, Kanoko; Teraoka, Ayuko; Okamoto, Sho; Yu, Fengnian; Hattan, Jun-ichiro; Utsumi, Ryutaro; Misawa, Norihiko

2011-01-13

321

Characterization of human cytochrome p450 enzymes involved in the metabolism of cilostazol.  

PubMed

Cilostazol (OPC-13013; 6-[4-(1-cyclohexl-1H-tetrazol-5-yl)butoxy]-3,4-dihydro-2(1H)-quinolinone) is widely used as an antiplatelet vasodilator agent. In vitro, the hydroxylation of the quinone moiety of cilostazol to OPC-13326 [6-[4-(1-cyclohexyl-1H-tetrazol-5-yl)butoxy]-3,4-dihydro-4-hydroxy-2(1H)-quinolinone], is the predominant route, and the hydroxylation of the hexane moiety to OPC-13217 is the second most predominant route. This study was carried out to identify and kinetically characterize the human cytochrome P450 (P450) isozymes responsible for the formation of the two major metabolites of cilostazol, namely, OPC-13326 and OPC-13217 [3,4-dihydro-6-[4-[1-(cis-4-hydroxycyclohexyl)-1H-tetrazol-5-yl)butoxy]-2(1H)-quinolinone)]. In in vitro studies using 14 recombinant human P450 isozymes, CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP2J2, CYP3A4, CYP3A5, and CYP4A11, cilostazol was metabolized to OPC-13326 mainly by CYP3A4 (K(m) = 5.26 muM, intrinsic clearance (CL(int)) = 0.34 microl/pmol P450/min), CYP1B1 (K(m) = 11.2 microM, CL(int) = 0.03 microl/pmol P450/min), and CYP3A5 (K(m) = 2.89 microM, CL(int) = 0.05 microl/pmol P450/min) and to OPC-13217 mainly by CYP3A5 (K(m) = 1.60 microM, CL(int) = 0.57 microl/pmol P450/min), CYP2C19 (K(m) = 5.95 microM, CL(int) = 0.16 microl/pmol P450/min), CYP3A4 (K(m) = 5.35 microM, CL(int) = 0.10 microl/pmol P450/min), and CYP2C8 (K(m) = 33.8 microM, CL(int) = 0.009 microl/pmol P450/min). The present study showed that the two major metabolites of cilostazol in vitro, namely, OPC-13326 and OPC-13217, are mainly catalyzed by CYP3A4 and CYP3A5, respectively. PMID:17646278

Hiratsuka, Masahiro; Hinai, Yudai; Sasaki, Takamitsu; Konno, Yumiko; Imagawa, Kenichi; Ishikawa, Masaaki; Mizugaki, Michinao

2007-07-23

322

Cytochrome p450 architecture and cysteine nucleophile placement impact raloxifene-mediated mechanism-based inactivation.  

PubMed

The propensity for cytochrome P450 (P450) enzymes to bioactivate xenobiotics is governed by the inherent chemistry of the xenobiotic itself and the active site architecture of the P450 enzyme(s). Accessible nucleophiles in the active site or egress channels of the P450 enzyme have the potential of sequestering reactive metabolites through covalent modification, thereby limiting their exposure to other proteins. Raloxifene, a drug known to undergo CYP3A-mediated reactive metabolite formation and time-dependent inhibition in vitro, was used to explore the potential for bioactivation and enzyme inactivation of additional P450 enzymes (CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A5). Every P450 tested except CYP2E1 was capable of raloxifene bioactivation, based on glutathione adduct formation. However, raloxifene-mediated time-dependent inhibition only occurred in CYP2C8 and CYP3A4. Comparable inactivation kinetics were achieved with K(I) and k(inact) values of 0.26 ?M and 0.10 min(-1) and 0.81 ?M and 0.20 min(-1) for CYP2C8 and CYP3A4, respectively. Proteolytic digests of CYP2C8 and CYP3A4 Supersomes revealed adducts to Cys225 and Cys239 for CYP2C8 and CYP3A4, respectively. For each P450 enzyme, proposed substrate/metabolite access channels were mapped and active site cysteines were identified, which revealed that only CYP2C8 and CYP3A4 possess accessible cysteine residues near the active site cavities, a result consistent with the observed kinetics. The combined data suggest that the extent of bioactivation across P450 enzymes does not correlate with P450 inactivation. In addition, multiple factors contribute to the ability of reactive metabolites to form apo-adducts with P450 enzymes. PMID:22859722

VandenBrink, Brooke M; Davis, John A; Pearson, Josh T; Foti, Robert S; Wienkers, Larry C; Rock, Dan A

2012-08-02

323

Key mutations alter the cytochrome P450 BM3 conformational landscape and remove inherent substrate bias.  

PubMed

Cytochrome P450 monooxygenases (P450s) have enormous potential in the production of oxychemicals, due to their unparalleled regio- and stereoselectivity. The Bacillus megaterium P450 BM3 enzyme is a key model system, with several mutants (many distant from the active site) reported to alter substrate selectivity. It has the highest reported monooxygenase activity of the P450 enzymes, and this catalytic efficiency has inspired protein engineering to enable its exploitation for biotechnologically relevant oxidations with structurally diverse substrates. However, a structural rationale is lacking to explain how these mutations have such effects in the absence of direct change to the active site architecture. Here, we provide the first crystal structures of BM3 mutants in complex with a human drug substrate, the proton pump inhibitor omeprazole. Supported by solution data, these structures reveal how mutation alters the conformational landscape and decreases the free energy barrier for transition to the substrate-bound state. Our data point to the importance of such "gatekeeper" mutations in enabling major changes in substrate recognition. We further demonstrate that these mutants catalyze the same 5-hydroxylation reaction as performed by human CYP2C19, the major human omeprazole-metabolizing P450 enzyme. PMID:23828198

Butler, Christopher F; Peet, Caroline; Mason, Amy E; Voice, Michael W; Leys, David; Munro, Andrew W

2013-07-03

324

Functional diversity of cytochrome P450s of the white-rot fungus Phanerochaete chrysosporium.  

PubMed

The functional diversity of cytochrome P450s (P450s) of the white-rot basidiomycete, Phanerochaete chrysosporium, was studied. A series of compounds known to be P450 substrates of other organisms were utilized for metabolic studies of P. chrysosporium. Metabolic conversions of benzoic acid, camphor, 1,8-cineol, cinnamic acid, p-coumaric acid, coumarin, cumene, 1,12-dodecanediol, 1-dodecanol, 4-ethoxybenzoic acid, and 7-ethoxycoumarin were observed with P. chrysosporium for the first time. 1-Dodecanol was hydroxylated at seven different positions to form 1,12-, 1,11-, 1,10-, 1,9-, 1,8-, 1,7-, and 1,6-dodecandiols. The effect of piperonyl butoxide, a P450 inhibitor, on the fungal conversion of 1-dodecanol was also investigated, indicating that hydroxylation reactions of 1-dodecanol were inhibited by piperonyl butoxide in a concentration-dependent manner. With 11 substrates, 23 hydroxylation reactions and 2 deethylation reactions were determined and 6 products were new with the position of hydroxyl group incorporated. In conclusion, fungal P450s were shown to have diverse and unique functions. PMID:15465031

Matsuzaki, Fumiko; Wariishi, Hiroyuki

2004-11-01

325

In vitro activity of uva-ursi against cytochrome P450 isoenzymes and P-glycoprotein.  

PubMed

Some natural health products (NHPs) affect drug metabolism enzymes and transport proteins, potentially affecting the safety and efficacy of the drug or other NHPs. This study was undertaken to characterize the effect of uva-ursi (Arctostaphylos uva-ursi) on cytochrome P450 isozyme (3A4, 3A5, 3A7, 2C19, and 19)-mediated metabolism and P-glycoprotein (P-gp) transport. Three bulk and 2 capsulated uva-ursi samples were obtained from commercial outlets. The capsules were batched, and herbal samples were ground to a common consistency. Aqueous and methanol extracts were freshly prepared. Cytochrome P450 isozyme-mediated metabolism was determined by using in vitro bioassays. P-gp transport function was determined by using a rhodamine 123 (Rh123) uptake test in human (THP-1) monocytes and human Caco-2 cells. All products were analyzed by HPLC for arbutin, gallic acid, myricitrin, and isoquercetin. A large variation was observed in the biomarkers found between the bulk and capsulated samples. Our data indicate that both the aqueous and methanol extracts of all 5 uva-ursi products showed high cytochrome P450 isozyme inhibition, with the exception of the methanol extracts against cytochromes P3A4 and P19, which had low to moderate activity. The aqueous extracts of uva-ursi showed an inhibitory effect on Rh123 efflux by P-gp at 1 h and an inductive effect at 18 h for both cell lines. Our results show that the uva-ursi herbal products tested here have pharmacological properties, including the potential capacity to affect drug safety and efficacy. Further studies are warranted against a wider range of cytochrome P450 isozymes and to determine whether these effects are clinically significant. PMID:18066112

Chauhan, B; Yu, C; Krantis, A; Scott, I; Arnason, J T; Marles, R J; Foster, B C

2007-11-01

326

Pungent ginger components modulates human cytochrome P450 enzymes in vitro.  

PubMed

Aim:Ginger rhizome is used worldwide as a spicy flavor agent. This study was designed to explore the potential effects of pungent ginger components, 6-, 8-, and 10-gingerol, on human cytochrome P450 (CYP450) enzymes that are responsible for the metabolism of many prescription drugs.Methods:The activities of human CYP2C9, CYP2C19, CYP2D6, and CYP3A4 were analyzed using Vivid P450 assay kits. The mRNA expression of CYP3A4 in human hepatocellular carcinoma cell line HepG2 was measured using quantitative real-time PCR assay.Results:All three gingerols potently inhibited CYP2C9 activity, exerted moderate inhibition on CYP2C19 and CYP3A4, and weak inhibion on CYP2D6. 8-Gingerol was the most potent in inhibition of P450 enzymes with IC50 values of 6.8, 12.5, 8.7, and 42.7 ?mol/L for CYP2C9, CYP2C19, CYP3A4, and CYP2D6, respectively. By comparing the effects of gingerols on CYP3A4 with three different fluorescent substrate probes, it was demonstrated that the inhibition of gingerols on CYP3A4 had no substrate-dependence. In HepG2 cells, 8-gingerol and 10-gingerol inhibited, but 6-gingerol induced mRNA expression of CYP3A4.Conclusion:6-, 8-, and 10-gingerol suppress human cytochrome P450 activity, while 8- and 10-gingerol inhibit CYP3A4 expression. The results may have an implication for the use of ginger or ginger products when combined with therapeutic drugs that are metabolized by cytochrome P450 enzymes. PMID:23770984

Li, Mian; Chen, Pei-Zhan; Yue, Qing-Xi; Li, Jing-Quan; Chu, Rui-Ai; Zhang, Wei; Wang, Hui

2013-06-17

327

Genetic control of a cytochrome P450 metabolism-based herbicide resistance mechanism in Lolium rigidum  

PubMed Central

The dynamics of herbicide resistance evolution in plants are influenced by many factors, especially the biochemical and genetic basis of resistance. Herbicide resistance can be endowed by enhanced rates of herbicide metabolism because of the activity of cytochrome P450 enzymes, although in weedy plants the genetic control of cytochrome P450-endowed herbicide resistance is poorly understood. In this study we have examined the genetic control of P450 metabolism-based herbicide resistance in a well-characterized Lolium rigidum biotype. The phenotypic resistance segregation in herbicide resistant and susceptible parents, F1, F2 and backcross (BC) families was analyzed as plant survival following treatment with the chemically unrelated herbicides diclofop-methyl or chlorsulfuron. Dominance and nuclear gene inheritance was observed in F1 families when treated at the recommended field doses of both herbicides. The segregation values of P450 herbicide resistance phenotypic traits observed in F2 and BC families was consistent with resistance endowed by two additive genes in most cases. In obligate out-crossing species such as L. rigidum, herbicide selection can easily result in accumulation of resistance genes within individuals.

Busi, R; Vila-Aiub, M M; Powles, S B

2011-01-01

328

Olefin cyclopropanation via carbene transfer catalyzed by engineered cytochrome P450 enzymes.  

PubMed

Transition metal-catalyzed transfers of carbenes, nitrenes, and oxenes are powerful methods for functionalizing C=C and C-H bonds. Nature has evolved a diverse toolbox for oxene transfers, as exemplified by the myriad monooxygenation reactions catalyzed by cytochrome P450 enzymes. The isoelectronic carbene transfer to olefins, a widely used C-C bond-forming reaction in organic synthesis, has no biological counterpart. Here we report engineered variants of cytochrome P450(BM3) that catalyze highly diastereo- and enantioselective cyclopropanation of styrenes from diazoester reagents via putative carbene transfer. This work highlights the capacity to adapt existing enzymes for the catalysis of synthetically important reactions not previously observed in nature. PMID:23258409

Coelho, Pedro S; Brustad, Eric M; Kannan, Arvind; Arnold, Frances H

2012-12-20

329

Anti-inflammatory properties of cytochrome P450 epoxygenase-derived eicosanoids.  

PubMed

The epoxyeicosatrienoic acids (EETs) are products of cytochrome P450 epoxygenases that have vasodilatory properties similar to that of endothelium-derived hyperpolarizing factor. The cytochrome P450 isoform CYP2J2 was cloned and identified as a potential source of EETs in human endothelial cells. Physiological concentrations of EETs or overexpression of CYP2J2 decreased cytokine-induced endothelial cell adhesion molecule expression, and EETs prevented leukocyte adhesion to the vascular wall by a mechanism involving inhibition of transcription factor NF-kappaB and IkappaB kinase. The inhibitory effects of EETs were independent of their membrane-hyperpolarizing effects, suggesting that these molecules play an important nonvasodilatory role in vascular inflammation. PMID:10455056

Node, K; Huo, Y; Ruan, X; Yang, B; Spiecker, M; Ley, K; Zeldin, D C; Liao, J K

1999-08-20

330

Anti-inflammatory Properties of Cytochrome P450 Epoxygenase-Derived Eicosanoids  

PubMed Central

The epoxyeicosatrienoic acids (EETs) are products of cytochrome P450 epoxygenases that have vasodilatory properties similar to that of endothelium-derived hyperpolarizing factor. The cytochrome P450 isoform CYP2J2 was cloned and identified as a potential source of EETs in human endothelial cells. Physiological concentrations of EETs or overexpression of CYP2J2 decreased cytokine-induced endothelial cell adhesion molecule expression, and EETs prevented leukocyte adhesion to the vascular wall by a mechanism involving inhibition of transcription factor NF-?B and I?B kinase. The inhibitory effects of EETs were independent of their membrane-hyperpolarizing effects, suggesting that these molecules play an important nonvasodilatory role in vascular inflammation.

Node, Koichi; Huo, Yuqing; Ruan, Xiulu; Yang, Baichun; Spiecker, Martin; Ley, Klaus; Zeldin, Darryl C.; Liao, James K.

2009-01-01

331

The Role of Cytochrome b 5 in the Biosynthesis of Androgens by Human P450c17  

Microsoft Academic Search

Human cytochrome b5 has a profound effect on the 17,20-lyase activities catalyzed by purified, human cytochrome P450c17. It enhances the conversion of 17?-hydroxypregnenolone to dehydroepiandrosterone by 13-fold and the conversion of 17?-hydroxyprogesterone to androstenedione by at least 10-fold. This latter activity is virtually undetectable in the absence of cytochrome b5. Other activities catalyzed by P450c17 include 17?-hydroxylation of progesterone and

M. Katagiri; N. Kagawa; M. R. Waterman

1995-01-01

332

Endocrine disruptors induce cytochrome P450 by affecting transcriptional regulation via pregnane X receptor  

Microsoft Academic Search

Pregnane X receptor (PXR) is a nuclear receptor that regulates the expression of genes for cytochrome P450 3A (CYP3A), multidrug resistance 1 (MDR1), and organic anion-transporting peptide 2 (OATP2). These genes control the metabolism (CYP3A subfamily) and aspects of the pharmacokinetics (MDR1 and OATP2) of both endogenous and xenobiotic compounds. Since PXR is important in understanding the actions of endocrine

Eriko Mikamo; Shingo Harada; Jun-ichi Nishikawa; Tsutomu Nishihara

2003-01-01

333

Lack of evidence for metabolism of p-phenylenediamine by human hepatic cytochrome P450 enzymes  

Microsoft Academic Search

p-Phenylenediamine (PPD) is a widely used ingredient in permanent hair dyes; however, little has been published on its metabolism, especially with respect to hepatic cytochrome P450 (CYP)-mediated oxidation. This is regarded as a key step in the activation of carcinogenic arylamines that ultimately leads to the development of bladder cancer. Most epidemiology studies show no significant association between personal use

Lesley A. Stanley; Julie A. Skare; Edward Doyle; Robert Powrie; Diane D’Angelo; Clifford R. Elcombe

2005-01-01

334

Expression and characterization of two new alkane-inducible cytochrome P450s from Trichoderma harzianum  

Microsoft Academic Search

n-Dodecane and fatty acids were good inducers of cytochrome P450 (CYP) and the ?-hydroxylase of lauric acid, which is a marker\\u000a for ?-hydroxylation of n-alkanes, in Trichoderma harzianum. A cDNA, containing an ORF of 1520 bp, encoding a CYP52 of 520 amino acids, was isolated by RACE. Another n-alkane-inducible CYP was identified by LLC-MS\\/MS analysis of a microsomal protein band induced

R. Del Carratore; P. G. Gervasi; M. P. Contini; P. Beffy; B. E. Maserti; G. Giovannetti; A. Brondolo; V. Longo

2011-01-01

335

Inhibition of human recombinant cytochrome P450s by curcumin and curcumin decomposition products  

Microsoft Academic Search

Curcumin (diferuloylmethane) is a major yellow pigment and dietary component derived from Curcuma longa. It has potent anti-inflammatory, anticarcinogenic, antioxidant and chemoprotective activities among others. We studied the interactions of curcumin, a mixture of its decomposition products, and four of its individually identified decomposition products (vanillin, vanillic acid, ferulic aldehyde and ferulic acid) on five major human drug-metabolizing cytochrome P450s

Regina Appiah-Opong; Jan N. M. Commandeur; Barbara van Vugt-Lussenburg; Nico P. E. Vermeulen

2007-01-01

336

Human cytochromes P450 expressed in Escherichia coli: production of specific antibodies  

Microsoft Academic Search

Cytochromes P450 (CYP) constitute a superfamily of enzymes involved in the metabolism of xenobiotics. Within the same subfamily, the isoforms present strong similarities, making them difficult to characterize and differentiate. Using heterologous expression in bacteria, five pure human CYP (1A1, 1A2, 2C9, 2E1, 3A4) were easily obtained and used as antigens to raise specific antibodies. These antibodies were characterized for

Claire Belloc; Susan Baird; José Cosme; Sylvaine Lecoeur; Jean-Charles Gautier; Dominique Challine; Isabelle De Waziers; Jean-Pierre Flinois; Philippe H. Beaune

1996-01-01

337

Cytochrome P450 2E1 genetic polymorphism and gastric cancer in Changle, Fujian Province  

Microsoft Academic Search

AIM: Genetic polymorphism in enzymes of carcinogen metabolism has been found to have the influence on the susceptibility to cancer. Cytochrome P450 2E1 (CYP2E1) is considered to play an important role in the metabolic a ctivation of procarcinogens such as N- nitrosoamines and low molecular weight organi c compounds. The purpose of this study is to determine whether CYP450 2E1

Lin Cai; Shun-Zhang Yu; Zuo-Feng Zhang

2001-01-01

338

Comprehensive characterization of cytochrome P450 isozyme selectivity across chemical libraries  

Microsoft Academic Search

The cytochrome P450 (CYP) gene family catalyzes drug metabolism and bioactivation and is therefore relevant to drug development. We determined potency values for 17,143 compounds against five recombinant CYP isozymes (1A2, 2C9, 2C19, 2D6 and 3A4) using an in vitro bioluminescent assay. The compounds included libraries of US Food and Drug Administration (FDA)-approved drugs and screening libraries. We observed cross-library

Henrike Veith; Noel Southall; Ruili Huang; Tim James; Darren Fayne; Natalia Artemenko; Min Shen; James Inglese; Christopher P Austin; David G Lloyd; Douglas S Auld

2009-01-01

339

Expression and Induction of Cytochrome P450 Isoenzymes in Human Skin Equivalents  

Microsoft Academic Search

Organotypic skin models are frequently used for a wide range of applications and latterly also for dermatotoxicological studies. To evaluate their practicability for the investigation of xenobiotic metabolism in human skin we compared three types of organotypic skin models, acquired by purchase from different manufacturers, to a self-constructed in-house model with regard to cytochrome P450 (CYP) isoenzyme expression on mRNA

M. M. Neis; A. Wendel; T. Wiederholt; Y. Marquardt; S. Joussen; J. M. Baron; H. F. Merk

2010-01-01

340

Molecular Cloning, Expression and Characterization of an Endogenous Human Cytochrome P450 Arachidonic Acid Epoxygenase Isoform  

Microsoft Academic Search

A cDNA containing an open reading frame coding for a human cytochrome P450 arachidonic acid epoxygenase was isolated from a male human kidney cDNA library. Sequence analysis showed that, with few exceptions, this cDNA was nearly identical to the published sequence for human liver Cyp 2C8 (S. T. Okino et al., 1987, J. Biol. Chem. 262, 16072-16079) and encoded a

D. C. Zeldin; R. N. Dubois; J. R. Falck; J. H. Capdevila

1995-01-01

341

Cisplatin-Induced Hepatotoxicity Is Enhanced by Elevated Expression of Cytochrome P450 2E1  

Microsoft Academic Search

In this study, the possible potentiation of cisplatin-induced hepatotoxicity by cytochrome P450 2E1 (CYP2E1) was examined both in vitro and in vivo. Transfected HepG2 cells expressing CYP2E1 (E47 cells) and not expressing CYP2E1 (C34 cells) were used as an in vitro model, and mice drinking 2% acetone for 7 days to induce CYP2E1 were used as an in vivo model.

Yongke Lu; Arthur I. Cederbaum

2006-01-01

342

The impact of cytochrome P450 2D6 metabolism in women receiving adjuvant tamoxifen  

Microsoft Academic Search

Background  Tamoxifen is biotransformed to the potent anti-estrogen, endoxifen, by the cytochrome P450 (CYP) 2D6 enzyme. CYP2D6 genetic variation and inhibitors of the enzyme markedly reduce endoxifen plasma concentrations in tamoxifen-treated patients.\\u000a Using a North Central Cancer Treatment Group adjuvant tamoxifen trial, we performed a comprehensive evaluation of CYP2D6 metabolism\\u000a by assessing the combined effect of genetic variation and inhibition of

Matthew P. Goetz; Stacey K. Knox; Vera J. Suman; James M. Rae; Stephanie L. Safgren; Matthew M. Ames; Daniel W. Visscher; Carol Reynolds; Fergus J. Couch; Wilma L. Lingle; Richard M. Weinshilboum; Emily G. Barr Fritcher; Andrea M. Nibbe; Zeruesenay Desta; Anne Nguyen; David A. Flockhart; Edith A. Perez; James N. Ingle

2007-01-01

343

Cytochrome P450-specific human PBPK\\/PD models for the organophosphorus pesticides: Chlorpyrifos and parathion  

Microsoft Academic Search

Organophosphorus pesticides (OPs) remain a potential concern to human health because of their continuing use worldwide. Phosphororthioate OPs like chlorpyrifos and parathion are directly activated and detoxified by various cytochrome P450s (CYPs), with the primary CYPs involved being CYP2B6 and CYP2C19. The goal of the current study was to convert a previously reported human pharmacokinetic and pharmacodynamic (PBPK\\/PD) model for

Robert J. Foxenberg; Corie A. Ellison; James B. Knaak; Changxing Ma; James R. Olson

2011-01-01

344

Cytochrome P-450 Complex Formation in Rat Liver by the Antibiotic Tiamulin  

Microsoft Academic Search

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

RENGER F. WITKAMP; SANDRA M. NIJMEIJER

345

Cytochrome P450 epoxygenase CYP2J2 and the risk of coronary artery disease.  

PubMed

Cytochrome P450 (CYP) enzyme 2J2, an epoxygenase predominantly expressed in the heart, metabolizes arachidonic acid to biologically active eicosanoids. One of the CYP2J2 products, 11,12-epoxyeicosatrienoic acid, has several vasoprotective effects. A frequent promoter polymorphism of CYP2J2 decreases gene expression and is associated with coronary artery disease. This association supports the vascular protective role of CYP-derived eicosanoids in cardiovascular disease. PMID:16839864

Spiecker, Martin; Liao, Jamesk

2006-08-01

346

Acute hypoxia and cytochrome P450–mediated hepatic drug metabolism in humans  

Microsoft Academic Search

Objective: Our objective was to investigate the effect of acute hypoxia on the activity of hepatic cytochrome P450 (CYP) enzymes.Methods: Twelve healthy subjects who lived at sea level were exposed to altitude-induced hypoxia for 7 days at 4559 m above sea level. Hepatic CYP enzyme activity was measured before departure, at 24 and 96 hours after arrival to high-altitude location,

Gesche Jürgens; Hanne Rolighed Christensen; Kim Brøsen; Jesper Sonne; Steffen Loft; Niels Vidiendal Olsen

2002-01-01

347

The human genome project and novel aspects of cytochrome P450 research  

Microsoft Academic Search

Currently, 57 active cytochrome P450 (CYP) genes and 58 pseudogenes are known to be present in the human genome. Among the genes discovered by initiatives in the human genome project are CYP2R1, CYP2W1, CYP2S1, CYP2U1 and CYP3A43, the latter apparently encoding a pseudoenzyme. The function, polymorphism and regulation of these genes are still to be discovered to a great extent.

Magnus Ingelman-Sundberg

2005-01-01

348

Molecular modeling of human cytochrome P450 2W1 and its interactions with substrates  

Microsoft Academic Search

The human cytochrome P450 2W1 (CYP2W1) was categorized into the so-called “orphan” CYPs because of its unknown enzymatic function. However, recent studies showed that the recombinant CYP2W1 exhibited broad catalytic activity towards several chemicals. Furthermore, this enzyme was selectively expressed in some forms of cancers, whereas a very low expression was found in human normal issues. These render CYP2W1 as

Weihua Li; Yun Tang; Tyuji Hoshino; Saburo Neya

2009-01-01

349

Colorectal cancer-specific cytochrome P450 2W1: intracellular localization, glycosylation, and catalytic activity.  

PubMed

Cytochrome P450 2W1 (CYP2W1) is expressed at high levels in colorectal cancer cells. Moreover, we have shown previously that a higher tumor expression is associated with less survival. In this study, we characterize post-translational modification, inverted endoplasmic reticulum (ER) topology, and catalytic activity of CYP2W1. The analysis of colorectal normal and cancer tissues and CYP2W1 overexpressing human embryonic kidney (HEK) 293 cells showed that a fraction of CYP2W1 is modified by N-glycosylation. Bioinformatic analysis identified Asn177 as the only possible glycosylation site of CYP2W1, which was supported by the inability of an N177A mutant to be glycosylated in HEK 293 cells. Analysis of the membrane topology indicated that unlike other cytochromes P450, CYP2W1 in HEK 293-transfected cells and in nontransfected Caco2TC7 and HepG2 cells is oriented toward the lumen of the ER, a topology making CYP2W1 available to the ER glycosylation machinery. Immunofluorescence microscopy and cell surface biotinylation experiments revealed approximately 8% of the CYP2W1 on the cell surface. Despite the reverse orientation of CYP2W1 in the ER membrane, apparently making functional interactions with NADPH-cytochrome P450 reductase impossible, CYP2W1 in HEK 293 cells was active in the metabolism of indoline substrates and was able to activate aflatoxin B1 into cytotoxic products. The study identifies for the first time a cytochrome P450 enzyme with a luminal ER orientation and still retaining catalytic activity. Together, these results suggest the possibility of using CYP2W1 as a drug target in the treatment of colon cancer using antibodies and/or specific CYP2W1 activated prodrugs. PMID:20805301

Gomez, Alvin; Nekvindova, Jana; Travica, Sandra; Lee, Mi-Young; Johansson, Inger; Edler, David; Mkrtchian, Souren; Ingelman-Sundberg, Magnus

2010-08-30

350

Tumor-specific expression of the novel cytochrome P450 enzyme, CYP2W1  

Microsoft Academic Search

A novel human cytochrome P450, CYP2W1, was cloned and expressed heterologously. No or very low CYP2W1 mRNA levels were detected in fetal and adult human tissues, expression was however seen in 54% of human tumor samples investigated (n=37), in particular colon and adrenal tumors. Western blotting also revealed high expression of CYP2W1 in some human colon tumors. In rat tissues,

Maria Karlgren; Alvin Gomez; Katarina Stark; Jenny Svärd; Cristina Rodriguez-Antona; Ernst Oliw; Maria Luisa Bernal; Santiago Ramón y Cajal; Inger Johansson; Magnus Ingelman-Sundberg

2006-01-01

351

Ginseng Extracts Facilitate Cytochrome P450 Xenobiotic Metabolism in Primary Cultures of Rat Hepatocytes  

Microsoft Academic Search

A 70% methanol extract from red ginseng (steamed and dried roots of Panax ginseng C. A. Meyer, a kind of Ginseng Radix) has been shown to have various actions on physiological functions. We investigated whether the ginseng extract (Ext.) affected the mRNA expression of cytochrome P450 (CYP) CYP1A1, 2B1, 2C11, 2D1, 3A1, and 3A2 in rat primary hepatocytes. After treatment

Atsushi Kawase; Fumiaki Takeshita; Ayano Yamada; Kazuya Murat; Hideaki Matsuda; Keiichi Samukawa; Masahiro Iwaki

352

Immunochemical Analysis of Liver Microsomal Cytochromes P450 of the American Alligator, Alligator mississippiensis  

Microsoft Academic Search

Ten antibodies raised against various mammalian and fish cytochromes P450 (CYP) enzymes were used to probe the effects of xenobiotic pretreatment on liver microsomes of the American alligator, Alligator mississippiensis. Pretreatment with phenobarbital (PB), 3-methylcholanthrene (3MC), and PB plus 3MC elicited significant induction of multiple CYP enzymes in alligator, as detected by antibodies to CYP1A, CYP2B, CYP2C, CYP2E, CYP2K, and

Robin P. Ertl; Stelvio M. Bandiera; Donald R. Buhler; John J. Stegeman; Gary W. Winston

1999-01-01

353

Cloning and expression of koala ( Phascolarctos cinereus) liver cytochrome P450 CYP4A15  

Microsoft Academic Search

In the present study, the cloning, expression and characterization of hepatic cytochrome P450 (CYP) CYP4A from koala (Phascolarctos cinereus), an obligate eucalyptus feeder, is described. It has been previously reported that microsomal lauric acid hydroxylase activity (a CYP4A marker) and CYP content were higher in koala liver in comparison to that in human, rat or wallaby, species that do not

Suong Ngoc Thi Ngo; Ross Allan McKinnon; Ieva Stupans

2006-01-01

354

Acenocoumarol Pharmacokinetics in Relation to Cytochrome P450 2C9 Genotype  

Microsoft Academic Search

Background and objectives: Cytochrome P450 (CYP) 2C9 is one of the major CYP enzymes involved in the biotransformation of drugs, among others, the oral anticoagulant acenocoumarol. The enzyme has several polymorphisms, with the CYP2C9*2 and CYP2C9*3 variants most commonly present in white patients. Patients with the CYP2C9*3 variant are known to require a lower maintenance dose of racemic acenocoumarol. We

Henk H. W. Thijssen; Bas Ritzen

2003-01-01

355

Cytochrome P450 enzymes in aquatic invertebrates: recent advances and future directions  

Microsoft Academic Search

A variety of enzymes and other proteins are produced by organisms in response to xenobiotic exposures. Cytochrome P450s (CYP) are one of the major phase I-type classes of detoxification enzymes found in terrestrial and aquatic organisms ranging from bacteria to vertebrates. These enzymes metabolize a wide variety of substrates including endogenous molecules (e.g. fatty acids, eicosenoids, steroids) and xenobiotics (e.g.

Mark J Snyder

2000-01-01

356

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

Microsoft Academic Search

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 hydrocarbons such as the non-ortho and mono-ortho polychlorinated biphenyls (PCBs). CYP1A induction has been used extensively as a biomarker of exposure to such

Joanna Y. Wilson; Randall Wells; A. Anguilar; Asuncion Borrell; Victoria Tornero; Peter Reijnders; Michael Moore; John J. Stegeman

2007-01-01

357

The Effect of Echinacea (Echinacea purpurea Root) on Cytochrome P450 Activity in Vivo  

Microsoft Academic Search

Background: Echinacea is a widely available over-the-counter herbal remedy. Tinctures of echinacea have been shown to inhibit cytochrome P450 (CYP) in vitro. The effect of echinacea (Echinacea purpurea root) on CYP activity in vivo was assessed by use of the CYP probe drugs caffeine (CYP1A2), tolbutamide (CYP2C9), dextromethorphan (CYP2D6), and midazolam (hepatic and intestinal CYP3A).Methods: Twelve healthy subjects (6 men)

J. Christopher Gorski; Shiew-Mei Huang; Amar Pinto; Mitchell A. Hamman; Janna K. Hilligoss; Narjis A. Zaheer; Mehul Desai; Margaret Miller; Stephen D. Hall

2004-01-01

358

The effects of St John's wort (Hypericum perforatum) on human cytochrome P450 activity  

Microsoft Academic Search

Background: St John's wort(Hypericum perforatum) is a popular over-the-counter dietary supplement and herbal remedy that has been implicated in drug interactions with substrates of several cytochrome P450 (CYP) isozymes. The effect of St John's wort on CYP activity in vivo was examined with a probe drug cocktail.Methods: Twelve healthy subjects (5 female, 7 male) completed this 3-period, open-label, fixed schedule

Zaiqi Wang; J. Christopher Gorski; Mitchell A. Hamman; Shiew-Mei Huang; Lawrence J. Lesko; Stephen D. Hall

2001-01-01

359

Characterization of cytochrome P450-mediated bensulfuron-methyl O-demethylation in rice  

Microsoft Academic Search

The cytochrome P450-mediated metabolism of the herbicide bensulfuron-methyl (BSM) was investigated in rice (Oryza sativa L., cv. Lemont) seedlings. Shoots and roots of rice seedlings were harvested at 0, 4, 8, 12, 24, and 48h following treatment with 1?M [14C]BSM. BSM and its metabolites were extracted from plant tissues with aqueous methanol, purified by TLC, and identified by HPLC and

Fan Deng; Kriton K Hatzios

2002-01-01

360

Cytochrome P450 Pathway Contributes to Methanandamide-induced Vasorelaxation in Rat Aorta  

Microsoft Academic Search

Purpose  The generation of hyperpolarising vasorelaxant endothelial cytochrome P450 epoxygenase (CYP)—derived metabolites of arachidonic\\u000a may provide beneficial effects for the treatment of cardiovascular diseases in which the bioavailability of NO is impaired.\\u000a The cannabinoid methanandamide has vasodilatory properties linked to hyperpolarisation. The aim of the present work was to\\u000a investigate the vasorelaxant effects of methanandamide in rat aorta, focusing on the

Visitación López-Miranda; M. Teresa Dannert; Esperanza Herradón; Angela Alsasua; M. Isabel Martín

2010-01-01

361

Cytochrome P450-dependent hydroxylation in the biosynthesis of rosmarinic acid in Coleus  

Microsoft Academic Search

Three membrane-bound, cytochrome P450-dependent hydroxylases which are involved in the biosynthesis of rosmarinic acid have been characterized in microsomal preparations from cell cultures of Coleus blumei. Cinnamic acid 4-hydroxylase introduces the 4-hydroxyl group into cinnamic acid and forms 4-coumaric acid. This enzyme from Coleus blumei displayed saturation concentrations of 0.5 mM for both cinnamic acid and NADPH. The apparent Km-values

Maike Petersen

1997-01-01

362

Clinical efficacy of nebivolol in hypertensive patients based on cytochrome p-450 2d6 phenotype  

Microsoft Academic Search

Objectives: To evaluate the efficacy of Nebivolol (N), a beta-blocking agent, in hypertensive patients classified as extensive metabolizers (EM) or non-extensive metabolizers (non-EM) based on phenotype for cytochrome 2D6.Methods: Approximately 400 hypertensive patients (clinic DBP ? 95 mm Hg) were phenotyped for P-450 2D6 polymorphism. Each screened patient was instructed to take dextrometorphan 30 mg at bedtime and to collect

L. Poirier; J. Lefebvre

2000-01-01

363

5?-reduced C 21 steroids are substrates for human cytochrome P450c17  

Microsoft Academic Search

The 5?-reduction of testosterone in target tissues is a key step in androgen physiology; however, 5?-reduced C19 steroids are sometimes synthesized in testis via a pathway that does not involve testosterone as an intermediate. We studied the metabolism of 5?-reduced C21 steroids by human cytochrome P450c17 (hCYP17), the enzyme responsible for conversion of C21 steroids to C19 steroids via its

Manisha K. Gupta; Oleg L. Guryev; Richard J. Auchus

2003-01-01

364

Kinetic analysis of duodenal and testicular cytochrome P450c17 in the rat  

Microsoft Academic Search

In the adult rat, the duodenal tissue of both sexes can convert progesterone to 17?-hydroxyprogesterone, androstenedione and testosterone. The transition from C21 to C19 steroids is apparently controlled by the same cytochrome P450c17 expressed in the testis, which catalyzes both 17?-hydroxylation and C-17,20 bond scission at a single bifunctional active site. The kinetic parameters of this enzyme were measured at

L. Dalla Valle; A. Ramina; S. Vianello; P. Belvedere; L. Colombo

1996-01-01

365

Analysis of cytochrome P450 and phase II conjugating enzyme expression in adult male rat hepatocytes  

Microsoft Academic Search

Summary  The induction of cytochrome P450 (CYP450) and Phase II conjugating enzymes by prototypical hepatic enzyme inducers was studied\\u000a in adult male rat hepatocytes. Hepatocytes were suspended and cultured in diluted Matrigel® in a basal serum-free Dulbecco’s\\u000a modified Eagle medium and exposed to the prototypical liver enzyme inducers, 3-methylcholanthrene, phenobarbital, hydrocortisone,\\u000a and clofibrate for 48 h. Total RNA and microsomes were

Julio C. Davila; Dale L. Morris

1999-01-01

366

Prasugrel, a New Thienopyridine Antiplatelet Drug, Weakly Inhibits Cytochrome P450 2B6 in Humans  

Microsoft Academic Search

Prasugrel, a thienopyridine prodrug, is hydrolyzed in vivo by esterases to a thiolactone followed by a single cytochrome P450 (CYP)—dependent step to an active metabolite that is a potent inhibitor of adenosine diphosphate—induced platelet aggregation. This open-label, multiple-dose, 2-period, fixed-sequence study assessed CYP2B6 inhibition by prasugrel using bupropion as a probe substrate, where its active metabolite, hydroxybupropion, is almost exclusively

Nagy A. Farid; Christopher D. Payne; C. Steven Ernest; Y. Grace Li; Kenneth J. Winters; Daniel E. Salazar; David S. Small

2008-01-01

367

Inhibition of Cytochrome P450 3A: Relevant Drug Interactions in Gastroenterology  

Microsoft Academic Search

Cytochrome P450 3A (CYP3A) is involved in biotransformation of more than half of all drugs currently available. Drug interactions by inhibition of CYP3A are of major interest in patients receiving combinations of drugs. Some interactions with CYP3A inhibitors also involve inhibition of the multidrug export pump, P-glycoprotein. An increasing number of adverse drug reactions might be avoided on the basis

A. Sagir; M. Schmitt; K. Dilger; D. Häussinger

2003-01-01

368

Induction of functional cytochrome P450 and its involvement in degradation of benzoic acid by Phanerochaete chrysosporium.  

PubMed

The white rot fungus Phanerochaete chrysosporium has the largest cytochrome P450 contingent known to date in fungi, but the study on the function of these P450s is limited. In this study, induction of functional P450 in P. chrysosporium was first shown and P450-mediate degradation of benzoic acid was demonstrated in this fungus. Carbon monoxide difference spectra indicated significant induction of P450 by benzoic acid, m-chlorobenzoic acid, p-chlorobenzoic acid and n-hexane, and showed the effect of inducer concentration and nutrient condition on the induction of P450. The high contents of P450 in the microsomal fractions facilitated the study on the function of P450. While the n-hexane-induced P450 could not interact with benzoic acid, the microsomal P450 induced by benzoic acid produced type I substrate binding spectra upon the addition of benzoic acid. The benzoic acid degradation by the microsomal P450 was NADPH-dependent at a specific rate of 194 +/- 14 min(-1), and significantly inhibited by piperonyl butoxide (a P450 inhibitor). However, inhibition of benzoic acid degradation by piperonyl butoxide was slight or not detectable in the cultures of this fungus, suggesting presumable involvement of other enzyme in benzoic acid degradation. The extracellular ligninolytic enzymes, lignin peroxidase and manganese-dependent peroxidase, were not involved in initial metabolism of benzoic acid under the test conditions. PMID:19787435

Ning, Daliang; Wang, Hui; Zhuang, Yuan

2009-09-29

369

Alteration in surface structure of Clara cells and pulmonary cytochrome P-450b level in rats exposed to ozone.  

PubMed

Changes in the surface structure of Clara cells in the terminal bronchioles following exposure of rats to 0.4 ppm ozone (O3) for 14 days were evaluated and compared to the content of pulmonary cytochrome P-450, an enzyme active in xenobiotic metabolism. Exposure to O3 caused a striking alteration of Clara cells in the terminal bronchiole. After 6 h exposure apical protrusions of Clara cells enlarged and these Clara cells formed clusters. However after 24 h exposure, the Clara cells decreased in number and flattened. They increased in number and enlarged again during the subsequent period of exposure. By the 14th day of O3 exposure the number of Clara cells had increased significantly. The content of cytochrome P-450b (IIB1), a main isozyme of pulmonary cytochromes P-450 of rats, was determined by an immuno-blotting method using anti-cytochrome P-450b antibody. The cytochrome P-450b in the rats exposed to O3 increased significantly to 1.37- and 1.81-times that of the control on the 7th and 14th days, respectively. Immuno-electron microscopy demonstrated that cytochrome P-450b was localized abundantly in endoplasmic reticulum of Clara cells. Morphological alterations in Clara cells appear to be closely related with changes in the cytochrome P-450b content of the lung. PMID:1736414

Suzuki, E; Takahashi, Y; Aida, S; Kimula, Y; Ito, Y; Miura, T

1992-01-01

370

Cytochromes P450-mediated degradation of fuel oxygenates by environmental isolates.  

PubMed

The degradation of fuel oxygenates [methyl tert-butyl ether (MTBE), ethyl tert-butyl ether (ETBE) and tert-amyl methyl ether (TAME)] by Rhodococcus ruber IFP 2001, Rhodococcus zopfii IFP 2005 and Gordonia sp. IFP 2009 (formerly Mycobacterium sp.) isolated from different environments was compared. Strains IFP 2001, IFP 2005 and IFP 2009 grew on ETBE due in part to the activity of a cytochrome P450, CYP249. All of these strains were able to degrade ETBE to tert-butyl alcohol and are harboring the CYP249 cytochrome P450. They were also able to degrade MTBE and TAME, but ETBE was degraded in all cases most efficiently, with degradation rates measured after growth on ETBE of 2.1, 3.5 and 1.6 mmol ETBE g(-1) dry weight h(-1) for strains IFP 2001, IFP 2005 and IFP 2009, respectively. The phylogenetic relationships between the different ethR (encoding the regulator) and ethB (encoding the cytochrome P450) genes were determined and showed high identity between different ethB genes (>99%). Only ETBE was able to induce the expression of ethB in strains IFP 2001 and IFP 2005 as measured by reverse transcriptase quantitative PCR. Our results are a first indication of the possible role played by the ethB gene in the ecology of ETBE degradation. PMID:20337704

Malandain, Cédric; Fayolle-Guichard, Françoise; Vogel, Timothy M

2010-02-11

371

Inhibition of cytochrome P-450 attenuates hypoxemia of acute lung injury in dogs.  

PubMed

The intravenous administration of ethchlorvynol (ECV), in dogs, resulted in an acute lung injury (ALI) characterized by a 200 +/- 80% increase in venous admixture and a 142 +/- 30% increase in extravascular lung water (EVLW). Pretreatment with the cytochrome P-450 inhibitor 8-methoxypsoralen prevented the ECV-induced increase in venous admixture but not the increased EVLW. These findings parallel those reported for cyclooxygenase inhibition in ECV-induced ALI and suggest that an arachidonic acid (AA) metabolite of pulmonary cytochrome P-450 activity may mediate the increase in venous admixture of ALI. We demonstrate that canine pulmonary microsomes metabolize [1-(14)C]AA to a variety of products, including the cytochrome P-450 metabolites 5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acid (EET). In prostaglandin F2 alpha-contracted, isolated pulmonary venous rings, 5,6-EET induced relaxation in a concentration-dependent manner. This action of 5,6-EET was prevented by indomethacin (10(-5) M). These results suggest that may serve as the cyclooxygenase-dependent endogenous pulmonary vasodilator responsible for the increase in venous admixture of ECV-induced ALI. PMID:8967376

Stephenson, A H; Sprague, R S; Weintraub, N L; McMurdo, L; Lonigro, A J

1996-04-01

372

Novel Cytochrome P450, cyp6a17, Is Required for Temperature Preference Behavior in Drosophila  

PubMed Central

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

Kang, Jongkyun; Kim, Jaeseob; Choi, Kwang-Wook

2011-01-01

373

[Alcohol-xenobiotic interactions. Role of cytochrome P450 2E1].  

PubMed

Alcohol and xenobiotics share the same oxidative microsomal pathway, which is mainly located in the endoplasmic reticulum of hepatocytes. This pathway involves enzymes that belong to the super family of cytochrome P450 and allows to explain a lot of pharmacokinetic or toxic interactions between alcohol and xenobiotics. Cytochrome P450 2E1 (CYP2E1) is the key enzyme of the microsomal pathway of ethanol oxidation. It is inducible by chronic ethanol consumption and its activity is increased by three to five fold in liver from alcoholics subjects. This induction involves to a lesser extent cytochromes P450 3A4 and 1A2 and contributes to the metabolic tolerance of alcohol and drugs observed in alcoholics. The metabolic tolerance persits several days after ethanol withdrawal. Furthermore, CYP2E1 has a high capacity to activate numerous xenobiotics into toxic or carcinogenic compounds. Drugs currently used such as paracetamol, anesthetics (enflurane, halothane), industrial solvents (benzene or its derivatives), halogenated solvents (CCl4, trichlorethylene) and nitrosamines which are present in food or tobacco smoke are included. Therefore, heavy consumption of alcohol, which results in CYP2E1 induction, increases individual susceptibility to the toxic or carcinogenic effects of these xenobiotics. PMID:11762131

Meskar, A; Plee-Gautier, E; Amet, Y; Berthou, F; Lucas, D

2001-11-01

374

Distinct forms of cytochrome P-450 are responsible for 6 beta-hydroxylation of bile acids and of neutral steroids.  

PubMed Central

Cytochrome P-450-dependent 6 beta-hydroxylation of bile acids in rat liver contributes to the synthesis of the quantitatively important pool of 6-hydroxylated bile acids, as well as to the detoxification of hydrophobic bile acids. The lithocholic acid 6 beta-hydroxylation reaction was investigated and compared with androstenedione 6 beta-hydroxylation. Differential responses of these two activities to inducers and inhibitors of microsomal P-450 enzymes, lack of mutual inhibition by the two substrates and differential inhibition by antibodies raised against several purified hepatic cytochromes P-450 were observed. From these results it was concluded that 6 beta-hydroxylation of lithocholic acid is catalysed by P-450 form(s) different from the subfamily IIIA cytochromes P-450 which are responsible for the bulk of microsomal androstenedione 6 beta-hydroxylation. Similar, but more tentative, results revealed that the 7 alpha-hydroxylation of lithocholic acid and of androstenedione may be also catalysed by distinct P-450 enzymes. The results indicate that cytochromes P-450 hydroxylating bile acids are distinct from analogous enzymes that carry out reactions of the same regio- and stereo-specificity on neutral steroids (steroid hormones). A comparison of pairs of cytochromes P-450 that catalyse the same reaction on closely related steroid molecules will help to define those structural elements in the proteins that determine the recognition of their respective substrates.

Zimniak, P; Holsztynska, E J; Radominska, A; Iscan, M; Lester, R; Waxman, D J

1991-01-01

375

Time course for induction of cytochrome P450 - Dependent activities by ethylbenzene  

SciTech Connect

The goal of this study was to examine the time course for ethylbenzene-mediated induction of cytochrome P450-dependent activities. Male Holtzman rats were treated with a single i.p. injection of ethylbenzene (EB) suspended in corn oil. In this study, the rats were injected at different times so all animals were killed at the same time P450 levels were transiently elevated at 1 hr after EB treatment, and returned to control levels by 2 hrs. Levels increased again at 10 hrs to a maximum at 24 hrs, returning to control after 48 hrs. Cytochrome b{sub 5} levels reached a minimum at 5 hrs and returned to controls after 10 hrs. In general, P450-dependent activities produced maximal induction after 24 hrs. Most of the P450-dependent activities examined returned to controls by 48 hrs; however, p-nitroanisole demethylation remained elevated after 48 hrs. Toluene metabolism was also induced by EB treatment, with each of the three metabolites exhibiting its own pattern of induction. Benzyl alcohol formation dropped to a minimum at 5-10 hrs, returning to controls by 24 hrs. Production of o-cresol was elevated more than 10 fold at 24 hrs and remained elevated after 48 hrs. Production of p-cresol followed a biphasic patter of induction increasing about 7 fold after 1 hr, and further increasing to a maximum between 10 and 24 hrs. P-cresol levels remained elevated at 48 hrs. Western blotting showed induction of both P450 1A1 and 2B1 at 1 and 2 hr, respectively, reaching a maximum at 24 hrs.

Sequeira, D.J.; Eyer, C.S.; Cawley, G.F.; Backes, W.L. (Louisiana State Univ., New Orleans, LA (United States))

1992-02-26

376

Molecular evolution of the insect Halloween family of cytochrome P450s: Phylogeny, gene organization and functional conservation  

Microsoft Academic Search

The insect molting hormone, 20-hydroxyecdysone (20E), is a major modulator of the developmental processes resulting in molting and metamorphosis. During evolution selective forces have preserved the Halloween genes encoding cytochrome P450 (P450) enzymes that mediate the biosynthesis of 20E. In the present study, we examine the phylogenetic relationships of these P450 genes in holometabolous insects belonging to the orders Hymenoptera,

Kim F. Rewitz; Michael B. O’Connor; Lawrence I. Gilbert

2007-01-01

377

Five of 12 Forms of Vaccinia Virus-Expressed Human Hepatic Cytochrome P450 Metabolically Activate Aflatoxin B_1  

Microsoft Academic Search

Twelve forms of human hepatic cytochrome P450 were expressed in hepatoma cells by means of recombinant vaccinia viruses. The expressed P450s were analyzed for their abilities to activate the potent hepatocarcinogen aflatoxin B_1 to metabolites having mutagenic or DNA-binding properties. Five forms, P450s IA2, IIA3, IIB7, IIIA3, and IIIA4, activated aflatoxin B_1 to mutagenic metabolites as assessed by the production

Toshifumi Aoyama; Shigeru Yamano; Philip S. Guzelian; Harry V. Gelboin; Frank J. Gonzalez

1990-01-01

378

A recombinant Escherichia coli whole cell biocatalyst harboring a cytochrome P450cam monooxygenase system coupled with enzymatic cofactor regeneration  

Microsoft Academic Search

A cytochrome P450cam monooxygenase (P450cam) system from the soil bacterium Pseudomonas putida requires electron transfer among three different proteins and a cofactor, nicotinamide adenine dinucleotide (NADH), for oxygenation of its natural substrate, camphor. Herein, we report a facile way to significantly enhance the catalytic efficiency of the P450cam system by the coupling of its native electron transfer system with enzymatic

Tsuyoshi Mouri; Junji Michizoe; Hirofumi Ichinose; Noriho Kamiya; Masahiro Goto

2006-01-01

379

Mössbauer and EPR Study of Reaction Intermediates of Cytochrome P450  

NASA Astrophysics Data System (ADS)

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

Schünemann, V.; Trautwein, A. X.; Jung, C.; Terner, J.

2002-06-01

380

Spectroscopic characterization of a newly isolated cytochrome P450 from Rhodococcus rhodochrous.  

PubMed Central

Cytochrome P450 (P450) from Rhodococcus rhodochrous have been characterized through circular dichroism and nuclear magnetic resonance (NMR) spectroscopy, both in the substrate-free and substrate-bound forms. The data are compared with those of P450cam and indicate a close similarity of the structure of the active site in the two proteins. The substrate-free species contains low-spin iron(III), while the 2-ethoxyphenol bound species contains high-spin iron(III). The substrate is in slow exchange on the NMR time scale. The binding of CN- has been investigated and the final adduct characterized through NMR spectra. Nuclear relaxation times of the isotropically shifted signals turn out to be shorter than in other heme proteins, both in the high- and in the low-spin species. This is the result of longer electron relaxation times in P450s than in peroxidases and metmyoglobin. This property, as well as the electron paramagnetic resonance (EPR) spectrum of the substrate-free form, are discussed in terms of the presence of the cysteine as the fifth ligand of the iron ion instead of a histidine as it occurs in peroxidases and myoglobin.

Banci, L; Bertini, I; Eltis, L D; Pierattelli, R

1993-01-01

381

Differential induction of cytochrome P450-mediated triasulfuron metabolism by naphthalic anhydride and triasulfuron.  

PubMed Central

Cytochrome P450 monooxygenases play paramount roles in the detoxification of herbicides as well as in the synthesis of lignins, flavonoids, and phenolic acids. Biochemical analysis of triasulfuron metabolism in maize (Zea mays) seedlings has demonstrated that the P450(s) responsible for detoxification of this herbicide is induced by naphthalic anhydride (NA), a plant safener, and by triasulfuron, the herbicide itself. Induction studies conducted with seedlings of different ages suggest that two separate response pathways modulate this P-450 activity. Induction by NA is independent of the developmental age of the seedlings up to 6.5 d; induction by triasulfuron is tightly modulated with respect to developmental age in that triasulfuron metabolism can be induced by triasulfuron in young (2.5 d) but not older (6.5 d) seedlings. Induction by NA administered in combination with triasulfuron synergistically enhances triasulfuron metabolism in younger seedlings to levels substantially above that obtained with either herbicide or safener treatment alone. In older seedlings, NA plus triasulfuron treatment induces triasulfuron metabolism to only the level of NA treatment alone, indicating again that the induction cascade responding to triasulfuron is nonfunctional in later development. MnCl2 studies indicate that the triasulfuron insensitivity of older seedlings does not result from a general limitation in the inducibility of this P-450 detoxification system but rather from specific limitations in the triasulfuron-response pathway.

Persans, M W; Schuler, M A

1995-01-01

382

Incorporation of an oxygen from water into troglitazone quinone by cytochrome P450 and myeloperoxidase.  

PubMed

Troglitazone (TGZ) was the first glitazone used for the treatment of type II diabetes mellitus. TGZ undergoes an oxidative chroman ring-opening reaction to form a quinone product. Recently, cytochrome P450 (P450) was shown to be able to catalyze the formation of TGZ quinone. TGZ quinone was the major metabolite formed by dexamethasone-induced rat liver microsomes or myeloperoxidase (MPO) incubated with TGZ. The ultimate source for the quinone carbonyl oxygen atom of TGZ quinone was investigated using (18)O water in both enzyme reaction systems followed by liquid chromatography/tandem mass spectometry analysis of the TGZ quinone product. The resultant TGZ quinone formed by either liver microsomes or MPO contained a single atom of (18)O. The (18)O atom was determined to be the quinone carbonyl oxygen by collision-induced dissociation fragmentation of the (18)O-labeled TGZ quinone. The formation of TGZ quinone was inhibited approximately 90% by coincubation with ascorbic acid or cysteine in the MPO reaction system but only 10 to 20% in liver microsomes, which might reflect the difference in the mechanism by which TGZ quinone is formed by P450 and peroxidase. These results suggest that P450 catalyze an atypical reaction to form TGZ quinone, involving the incorporation of an oxygen from water into the quinone carbonyl position. PMID:15039298

He, Kan; Talaat, Rasmy E; Woolf, Thomas F

2004-04-01

383

Silybin is metabolized by cytochrome P450 2C8 in vitro.  

PubMed

Silybin (a flavonolignan, the main component of silymarin, an extract from the seeds of Silybum marianum) has been used to date mostly as a hepatoprotectant. However, it also has other interesting activities, e.g., anticancer and hypocholesterolemic effects. It is also known that silybin can inhibit the activities of the cytochrome P450 (P450) enzymes. In this study, a weak interaction of silybin with human microsomal CYP2E1, 2A6, 2B6, 2C19, and 2D6 (IC(50) > or = 250 microM) was found; a moderate inhibition was observed for CYP1A2 and 2C8. The most prominent inhibition effect was found with CYP3A4 and CYP2C9 (IC(50) < or = 50 microM). Using mass spectometry detection, production of O-demethylated (the main metabolite) as well as hydroxylated derivatives of silybin formed by P450 enzymes was detected. The effect of different P450 inhibitors on the formation of O-demethylated product was also studied. In particular, a relatively specific inhibitor of CYP2C8 (quercetin) markedly inhibited the formation of this metabolite. With the help of recombinant enzymes (bactosomes), it was confirmed that the CYP2C8 enzyme is responsible for the reaction leading to O-demethylated silybin. PMID:17670841

Jancová, Petra; Anzenbacherová, Eva; Papousková, Barbora; Lemr, Karel; Luzná, Pavla; Veinlichová, Alena; Anzenbacher, Pavel; Simánek, Vilím

2007-08-01

384

Freeze-quenched iron-oxo intermediates in cytochromes P450  

SciTech Connect

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

Jung, Christiane [Max-Delbrueck-Center for Molecular Medicine, 13125 Berlin (Germany)]. E-mail: christiane_jung@hotmail.com; Schuenemann, Volker [Technical University of Kaiserslautern, Department of Physics, 67663 Kaiserslautern (Germany); Lendzian, Friedhelm [Max-Volmer Laboratory for Biophysical Chemistry, Technical University Berlin, PC 14, 10623 Berlin (Germany)

2005-12-09

385

Characterization of benidipine and its enantiomers' metabolism by human liver cytochrome P450 enzymes.  

PubMed

Benidipine is a dihydropyridine calcium antagonist that has been used clinically as an antihypertensive and antianginal agent. It is used clinically as a racemate, containing the (-)-alpha and (+)-alpha isomers of benidipine. This study was performed to elucidate the metabolism of benidipine and its enantiomers in human liver microsomes (HLMs) and to characterize the cytochrome P450 (P450) enzymes that are involved in the metabolism of benidipine. Human liver microsomal incubation of benidipine in the presence of NADPH resulted in the formation of two metabolites, N-desbenzylbenidipine and dehydrobenidipine. The intrinsic clearance (CL(int)) of the formation of N-desbenzylbenidipine and dehydrobenidipine metabolites from (-)-alpha isomer was similar to those from the (+)-alpha isomer (1.9 +/- 0.1 versus 2.3 +/- 2.3 microl/min/pmol P450 and 0.5 +/- 0.2 versus 0.6 +/- 0.6 microl/min/pmol P450, respectively). Correlation analysis between the known P450 enzyme activities and the rate of the formation of benidipine metabolites in the 15 HLMs showed that benidipine metabolism is correlated with CYP3A activity. The P450 isoform-selective inhibition study in liver microsomes and the incubation study of cDNA-expressed enzymes also showed that theN-debenzylation and dehydrogenation of benidipine are mainly mediated by CYP3A4 and CYP3A5. The total CL(int) values of CYP3A4-mediated metabolite formation from (-)-alpha isomer were similar to those from (+)-alpha isomer (17.7 versus 14.4 microl/min/pmol P450, respectively). The total CL(int) values of CYP3A5-mediated metabolite formation from (-)-alpha isomer were also similar to those from (+)-alpha isomer (8.3 versus 11.0 microl/min/pmol P450, respectively). These findings suggest that CYP3A4 and CYP3A5 isoforms are major enzymes contributing to the disposition of benidipine, but stereoselective disposition of benidipine in vivo may be influenced not by stereoselective metabolism but by other factors. PMID:17537876

Yoon, Yune-Jung; Kim, Kwon-Bok; Kim, Hyunmi; Seo, Kyung-Ah; Kim, Ho-Sook; Cha, In-June; Kim, Eun-Young; Liu, Kwang-Hyeon; Shin, Jae-Gook

2007-05-30

386

Immunolocalization of steroidogenic enzymes (P450scc, P450c17, P450arom, and 3beta-HSD) in immature and mature testes of rainbow trout (Oncorhynchus mykiss).  

PubMed

We examined the localization of steroidogenic cells in rainbow trout (Oncorhynchus mykiss) testis during spermatogenesis by using polyclonal antibodies generated against rainbow trout cholesterol side-chain cleavage enzyme cytochrome P450 (P450scc), 3beta-hydroxysteroid dehydrogenase (3beta-HSD), 17alpha-hydroxylase/C17,21 lyase (P450c17), and aromatase cytochrome P450 (P450arom) as markers of steroid production. Since we had previously produced specific antibodies against 3beta-HSD and P450arom, antibodies against oligopeptides corresponding to C-terminal sequences of P450scc and P450c17, predicted from rainbow trout P450scc and P450c17 cDNAs, were produced in this study. These two antibodies recognized 54-kDa (P450scc) and 59-kDa (P450c17) bands specifically in several steroidogenic organs, i.e., testis, ovary, and interrenal tissue (head kidney) in Western blots. Immunohistochemically, immunoreactive P450scc, P450c17, and 3beta-HSD, but not P450arom, were found only in interstitial Leydig cells of immature and mature testes. Immunoreactive P450arom was not detected in either testis. This study suggests that Sertoli cells and germ cells of rainbow trout testis do not contain P450scc, P450c17, P450arom, or 3beta-HSD. PMID:9582414

Kobayashi, T; Nakamura, M; Kajiura-Kobayashi, H; Young, G; Nagahama, Y

1998-06-01

387

Cytochrome P450 during ontogenic development: occurrence in the ductus arteriosus and other tissues.  

PubMed

Our previous investigations have implicated a cytochrome P450 mechanism in the oxygen contraction of the ductus arteriosus and, by extension, in the closure of the vessel at birth. This study was undertaken in fetal and newborn sheep to characterize the ductal cytochrome and gather insight into its operation. Other tissues, vascular and extravascular, were used as a reference. Benzo[a]pyrene hydroxylation (a marker for the 3-methylcholanthrene-inducible isozyme) and aminopyrine N-demethylation (a marker for the glucocorticoid-inducible isozyme) had insignificant activity in the ductus and aorta from fetal sheep, and no increase was noted after exposing the animal in utero to beta-naphthoflavone or dexamethasone, both alone and in combination with phenobarbital. However, dexamethasone and, particularly, dexamethasone plus phenobarbital produced a variable constriction of the fetal ductus. No monooxygenase activity was found in the naturally closing ductus of the newborn. Conversely, both enzyme reactions were measurable in the fetal liver, and they became more active after treatment with the inducers or at birth. Scanning immunoelectron microscopy of cultured endothelial and muscle cells from both ductus and aorta showed specific gold labelling for the glucocorticoid-inducible cytochrome P450 only in ductal muscle. By transmission electron microscopy, this immunoreactivity was located along the sarcolemma and in the sarcoplasmic reticulum. These findings indicate the presence in the ductus arteriosus of a cytochrome P450 belonging to the 3A subfamily. However, considering the uneven action of the inducers on enzyme activity in ductal tissue and muscle tone, the role of this cytochrome in closure of the vessel at birth remains to be ascertained. PMID:8069768

Coceani, F; Kelsey, L; Ackerley, C; Rabinovitch, M; Gelboin, H

1994-03-01

388

Solubilization and reconstitution of pisatin demethylase, a cytochrome P-450 from the pathogenic fungus Nectria haematococca  

SciTech Connect

Some isolates of the fungus Nectria haematococca Berk. and Br. can demethylate pisatin, a phytoalexin from pea (Pisum sativum L.). Pisatin demethylation appears to be necessary for tolerance to pisatin and virulence on pea, and is catalyzed by a microsomal cytochrome P-450. We now report solubilization of this enzyme from N. haematococca microsomes. Pisatin demethylase activity was obtained in the high speed supernatant of detergent treated microsomes, if detergent was removed before assay. The CO-binding spectrum of the soluble enzyme preparation indicated the presence of cytochrome P-450. Cholic acids were the most effective of the detergents tested for solubilizing enzyme activity. Loss of enzyme activity during solubilization was reduced by certain protease inhibitors, but not by substrate, reducing agents, antioxidants, or phospholipids. The most effective solubilization medium tests was 1% sodium cholate, 100 millimolar potassium phosphate, 500 millimolar sucrose, 1 millimolar phenylmethylsulfonyl fluoride, pH 7.5, which yielded approximately 30% of the pisatin demethylase and over 95% of the NADPH-cytochrome c reductase in the soluble fraction. Demethylase activity was lost when the reductase was removed by adsorption on 2',5'-ADP-agarose. The demethylase activity of reductase-free fractions could be restored by adding a reductase preparation purified approximately 100-fold from microsomes of N. haematococca isolate 74-8-1, which does not demethylate pisatin. We conclude that pisatin demethylase requires NADPH-cytochrome c reductase for activity. The inability of some isolates to demethylate pisatin appears to be due to the absence of a suitable cytochrome P-450, rather than to a lack of functional reductase. 24 references, 4 figures, 4 tables.

Desjardins, A.E.; Matthews, D.E.; VanEtten, H.D.

1984-07-01

389

Inhibitory metabolite complex formation of methylenedioxymethamphetamine with rat and human cytochrome P450. Particular involvement of CYP 2D  

Microsoft Academic Search

Methylenedioxymethamphetamine (MDMA or ecstasy) is a common recreational drug used at rave parties. Unfortunately, MDMA may have neurological effects and in some cases causes hepatotoxicity. MDMA binds to cytochrome P450 in rat and human hepatic microsomal preparations. Upon metabolic transformation of either the methylenedioxy or the methylamino function, it forms an inhibitory P450-metabolite complex. This inhibitory complex is formed predominantly

Marcel Delaforge; Maryse Jaouen; Geneviève Bouille

1999-01-01

390

DIFFERENTIAL EXPRESSION OF TWO CYTOCHROME P450 GENES IN COMPATIBLE AND INCOMPATIBLE HESSIAN FLY/WHEAT INTERACTIONS  

Technology Transfer Automated Retrieval System (TEKTRAN)

We have recovered two Hessian fly cytochrome P450 cDNAs from an ongoing midgut EST project. CYP6AZ1 and CYP6BA1 represent two new subfamilies within the CYP6 family. The deduced amino acid sequences for CYP6AZ1 and CYP6BA1 show conserved structural and functional domains of insect P450s. A phylog...

391

Marked detergents effects on safranine T-mediated photo-induced electron transfer in cytochrome P-450 1A2  

Microsoft Academic Search

Cytochrome P-450 accepts electrons from electron transfer proteins to facilitate monooxidation reactions. It is suggested that basic amino acids such as Lys and Arg on the P-450 molecular surface interact with acidic amino acids such as Gin and Asp of the electron transfer protein. Safranine T is a basic compound which mediates electron transfer with illumination. It was found with

Ryosuke Nakano; Hideo Konami; Hideaki Sato; Osamu Ito; Toru Shimizu

1995-01-01

392

Microarray-based analysis of gene expression in very large gene families: the cytochrome P450 gene superfamily of Arabidopsis thaliana  

Microsoft Academic Search

Cytochrome P450 (P450s) are heme-thiolate protein products of a very large gene superfamily, present in all kingdoms and involved in a variety of metabolic reactions. P450s are classified according to the degree of amino acid sequence identity, with P450s of the same family defined as having >40% identity, and P450s of the same subfamily having >55% identity. Currently, 273 P450

Wenying Xu; Søren Bak; Adria Decker; Suzanne M Paquette; René Feyereisen; David W Galbraith

2001-01-01

393

Role of human cytochrome P450 (CYP) in the metabolic activation of N-alkylnitrosamines: application of genetically engineered Salmonella typhimurium YG7108 expressing each form of CYP together with human NADPH-cytochrome P450 reductase  

Microsoft Academic Search

The role of human cytochrome P450 (CYP) in the metabolic activation of N-alkylnitrosamines was examined by Ames test using genetically engineered Salmonella typhimurium (S. typhimurium)YG7108 cells expressing each form of human CYP together with human NADPH-cytochrome P450 reductase (OR). The relationship between the structure of N-alkylnitrosamines and CYP form(s) involved in the activation was evaluated. Eleven strains of S. typhimurium

Ken-ichi Fujita; Tetsuya Kamataki

2001-01-01

394

Enhancement of 7-methoxyresorufin O-demethylation activity of human cytochrome P450 1A2 by molecular breeding  

Microsoft Academic Search

Alkylresorufins are model substrates for cytochrome P450 (P450) 1A2. The ability of human P450 1A2 to catalyze 7-methoxyresorufin O -demethylation was improved by screening of random mutant libraries (expressed in Escherichia coli ) on the basis of 7-methoxyresorufin O-demethylation. After three rounds of mutagenesis and screening, the triple mutant E163K\\/V193M\\/K170Q yielded a kcat>five times faster than wild type P450 1A2

Donghak Kim; F. Peter Guengerich

2004-01-01

395

Inhibition of cytochrome P450 2E1 expression by 2-(allylthio)Pyrazine, a Potential chemoprotective agent: hepatoprotective Effects  

Microsoft Academic Search

Cytochrome P450 2E1 (P450 2E1) is active in both the detoxification and activation of small organic molecules. The effects of 2-(allylthio)pyrazine (2-AP) on P450 2El-catalytic activity and the expression of rat hepatic P450 2E1 were examined. 2-AP competitively inhibited 4-nitrophenol hydroxylase activity in vitro (Ki, 12 ?M). 2-AP treatment of rats (200 mg\\/kg\\/day, po, 1–3 days old) resulted in 20–30%

Nak Doo Kim; Mi Kyong Kwak; Sang Geon Kim

1997-01-01

396

Caffeine as a marker substrate for testing cytochrome P450 activity in human and rat.  

PubMed

The current knowledge on the involvement of cytochrome P450 (P450, CYP) isoforms in the metabolism of caffeine in rat and human liver is reviewed. Attention is also paid to species- and concentration-dependent metabolism of caffeine. Finally, we discuss the P450-mediated metabolism of caffeine in relation to coffee addiction and drug interactions. Due to its safety, favorable pharmacokinetic properties, and P450 isoform-selective metabolism, caffeine has great potential as a metabolic marker substance in both humans and rats, and as a more universal metabolic tool in the latter species. However, the qualitative and relative quantitative contribution of P450 isoforms to the metabolism of caffeine is species- and concentration-dependent. While 3-N-demethylation is quantitatively the main oxidation pathway in human, 8-hydroxylation is the dominant metabolic pathway in rat. Both of these main reactions in the two species are specifically catalyzed by CYP1A2. Caffeine may be applied as a marker substance for assessing the activity of CYP1A2 in human and rat liver, but by using different reactions: 3-N-demethylation in humans and C-8-hydroxylation in rats. In addition, caffeine can be used to preliminarily and simultaneously estimate CYP2C activity in rat liver using 7-N-demethylation as a marker reaction. On the other hand, CYP3A4-catalyzed 8-hydroxylation in humans is not sufficiently isoform-specific to mark the activity of CYP3A4. Caffeine pharmacokinetics may be changed by drugs affecting the activity of CYP1A2 (human and rat) or CYP2C (rat), e.g. via autoinduction or by treatment with certain antidepressants or neuroleptics. Therefore, patients taking caffeine-containing medicine or coffee drinkers taking drugs that interact with CYP1A2 may require proper dosage adjustments upon caffeine ingestion and cessation. PMID:19211970

Kot, Marta; Daniel, W?adys?awa A

397

Light-induced cytochrome P450-dependent enzyme in indole alkaloid biosynthesis: tabersonine 16-hydroxylase.  

PubMed

Vinblastine and vincristine are two medically important bisindole alkaloids from Catharanthus roseus (Madagascar periwinkle). Attempts at production in cell cultures failed because a part of the complex pathway was not active, i.e. from tabersonine to vindoline. It starts with tabersonine 16-hydroxylase (T16H), a cytochrome P450-dependent enzyme. We now show that T16H is induced in the suspension culture by light and we report the cloning of the cDNA. The enzyme was expressed in Escherichia coli as translational fusion with the P450 reductase from C. roseus, and the reaction product was identified by mass spectrometry. The protein (CYP71D12) shares 47-52% identity with other members of the CYP71D subfamily with unknown function. The induction by light was strongly enhanced by a nutritional downshift (transfer into 8% aqueous sucrose). We discuss the possibility that the entire pathway to bisindoles can be expressed in suspension cultures. PMID:10481044

Schröder, G; Unterbusch, E; Kaltenbach, M; Schmidt, J; Strack, D; De Luca, V; Schröder, J

1999-09-17

398

Immobilization and activity assay of cytochrome P450 on patterned lipid membranes  

SciTech Connect

We report on a methodology for immobilizing cytochrome P450 on the surface of micropatterned lipid bilayer membranes and measuring the enzymatic activity. The patterned bilayer comprised a matrix of polymeric lipid bilayers and embedded fluid lipid bilayers. The polymeric lipid bilayer domains act as a barrier to confine fluid lipid bilayers in defined areas and as a framework to stabilize embedded membranes. The fluid bilayer domains, on the other hand, can contain lipid compositions that facilitate the fusion between lipid membranes, and are intended to be used as the binding agent of microsomes containing rat CYP1A1. By optimizing the membrane compositions of the fluid bilayers, we could selectively immobilize microsomal membranes on these domains. The enzymatic activity was significantly higher on lipid bilayer substrates compared with direct adsorption on glass. Furthermore, competitive assay experiment between two fluorogenic substrates demonstrated the feasibility of bioassays based on immobilized P450s.

Ueda, Yoshihiro [Research Center for Environmental Genomics, Kobe University, Rokkodaicho 1-1, Nada, Kobe 657-8501 (Japan); Research Institute for Cell Engineering, National Institute of Advanced Industrial Science and Technology (AIST), Midorigaoka, Ikeda 563-8577 (Japan); Morigaki, Kenichi [Research Institute for Cell Engineering, National Institute of Advanced Industrial Science and Technology (AIST), Midorigaoka, Ikeda 563-8577 (Japan)]. E-mail: morigaki-kenichi@aist.go.jp; Tatsu, Yoshiro [Research Institute for Cell Engineering, National Institute of Advanced Industrial Scien