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1

INHIBITION OF AROMATASE CYTOCHROME P-450 (ESTROGEN SYNTHETASE) BY DERIVATIVES OF ALPHA-NAPHTHOFLAVONE  

EPA Science Inventory

alpha-Naphthoflavone (ANF; 7,8-benzoflavone) is a potent competitive inhibitor of human aromatase cytochrome P-450 (J.T. Kellis, Jr. and L.E. Vickery, Science 225, 1032 (1984)). The authors have further investigated inhibition of aromatase by several derivatives of ANF. Using hum...

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

Placental expression and molecular characterization of aromatase cytochrome P450 in the spotted hyena (Crocuta crocuta).  

PubMed

At birth, the external genitalia of female spotted hyenas (Crocuta crocuta) are the most masculinized of any known mammal, but are still sexually differentiated. Placental aromatase cytochrome P450 (P450arom) is an important route of androgen metabolism protecting human female fetuses from virilization in utero. Therefore, placental P450arom expression was examined in spotted hyenas to determine levels during genital differentiation, and to compare molecular characteristics between the hyena and human placental enzymes. Hyena placental P450arom activity was determined at gestational days (GD) 31, 35, 45, 65 and 95 (term, 110), and the relative sensitivity of hyena and human placental enzyme to inhibition by the specific inhibitor, Letrozole, was also examined. Expression of hyena P450arom in placenta was localized by immuno-histochemistry, and a full-length cDNA was cloned for phylogenetic analysis. Aromatase activity increased from GD31 to a peak at 45 and 65, apparently decreasing later in gestation. This activity was more sensitive to inhibition by Letrozole than was human placental aromatase activity. Expression of P450arom was localized to syncytiotrophoblast and giant cells of mid-gestation placentas. The coding sequence of hyena P450arom was 94% and 86% identical to the canine and human enzymes respectively, as reflected by phylogenetic analyses. These data demonstrate for the first time that hyena placental aromatase activity is comparable to that of human placentas when genital differentiation is in progress. This suggests that even in female spotted hyenas clitoral differentiation is likely protected from virilization by placental androgen metabolism. Decreased placental aromatase activity in late gestation may be equally important in allowing androgen to program behaviors at birth. Although hyena P450arom is closely related to the canine enzyme, both placental anatomy and P450arom expression differ. Other hyaenids and carnivores must be investigated to determine the morphological and functional ancestral state of their placentas, as it relates to evolutionary relationships among species in this important taxonomic group. PMID:17198727

Conley, A J; Corbin, C J; Browne, P; Mapes, S M; Place, N J; Hughes, A L; Glickman, S E

2007-07-01

4

Structural Basis for the Functional Roles of Critical Residues in Human Cytochrome P450 Aromatase  

PubMed Central

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

2013-01-01

5

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...

6

Hormonal regulation of cytochrome P450 aromatase mRNA stability in non-luteinizing bovine granulosa cells in vitro.  

PubMed

In the present study, we determined the potential for post-transcriptional regulation of cytochrome P450 aromatase (Cyp19), cytochrome P450 side-chain cleavage (Cyp11a) and 17beta-hydroxysteroid dehydrogenase I (Hsd17b1) mRNA. Bovine granulosa cells were cultured in non-luteinizing conditions that permit long-term oestradiol secretion. Half-lives of mRNA were measured by Northern and/or reverse transcriptase (RT)-PCR after inhibition of gene transcription. In FSH-stimulated cells, the Cyp11a and Hsd17b1 mRNAs had half-lives greater than 12 h. The half-life of Cyp19 mRNA was significantly shorter at 3 h. The addition of the translation inhibitor cycloheximide to FSH-stimulated cells significantly increased Cyp19 mRNA half-life to approximately 12 h. Stimulation of cells with insulin resulted in Cyp19 mRNA half-life that was double (P<0.05) that in FSH-stimulated cells. We conclude that bovine Cyp19 mRNA is very labile under physiological conditions, and that Cyp19 expression is under hormonal control at a post-transcriptional level. PMID:16837615

Sahmi, Malha; Nicola, Edmir S; Price, Christopher A

2006-07-01

7

Brain cytochrome P450 aromatase activity in roach (Rutilus rutilus): seasonal variations and impact of environmental contaminants.  

PubMed

P450 aromatase catalyses the conversion of C19 androgens to C18 estrogens which is thought to be essential for the regulation of the reproductive function. In this study, brain aromatase activity (AA) was measured monthly over a reproductive cycle in wild roach (Rutilus rutilus) sampled in a reference site in Normandy. AA peaked during the breeding season, reaching 35 fmol mg(-1)min(-1) in both male and female fish, and was low during the rest of the year except for a significant rise in October. AA was correlated with ovary maturation (measured either as gonado-somatic index or by histological analysis of the gonads) and plasma sex-steroid levels (11-ketotestosterone in males and 17-?-estradiol in females). Measurements of AA in polluted sites showed that activity was significantly upregulated in sites with fish showing high levels of plasma vitellogenin and large proportion of intersexuality (20-50%) thus suggesting the occurrence of estrogenic compounds and their involvement in AA modulation. PMID:21820384

Geraudie, Perrine; Hinfray, Nathalie; Gerbron, Marie; Porcher, Jean-Marc; Brion, François; Minier, Christophe

2011-10-01

8

Mechanism of the Third Oxidative Step in the Conversion of Androgens to Estrogens by Cytochrome P450 19A1 Steroid Aromatase  

PubMed Central

Aromatase is the cytochrome P450 enzyme that cleaves the C10–C19 carbon–carbon bond of androgens to form estrogens, in a three-step process. Compound I (FeO3+) and ferric peroxide (FeO2–) have both been proposed in the literature as the active iron species in the third step, yielding an estrogen and formic acid. Incubation of purified aromatase with its 19-deutero-19-oxo androgen substrate was performed in the presence of 18O2, and the products were derivatized using a novel diazo reagent. Analysis of the products by high-resolution mass spectrometry showed a lack of 18O incorporation in the product formic acid, supporting only the Compound I pathway. Furthermore, a new androgen 19-carboxylic acid product was identified. The rates of nonenzymatic hydration of the 19-oxo androgen and dehydration of the 19,19-gem-diol were shown to be catalytically competent. Thus, the evidence supports Compound I and not ferric peroxide as the active iron species in the third step of the steroid aromatase reaction. PMID:25252141

2014-01-01

9

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

10

Cytochrome P450-activated prodrugs  

PubMed Central

A prodrug is a compound that has negligible, or lower, activity against a specified pharmacological target than one of its major metabolites. Prodrugs can be used to improve drug delivery or pharmacokinetics, to decrease toxicity, or to target the drug to specific cells or tissues. Ester and phosphate hydrolysis are widely used in prodrug design because of their simplicity, but such approaches are relatively ineffective for targeting drugs to specific sites. The activation of prodrugs by the cytochrome P450 system provides a highly versatile approach to prodrug design that is particularly adaptable for targeting drug activation to the liver, to tumors or to hypoxic tissues. PMID:23360144

Ortiz de Montellano, Paul R

2013-01-01

11

Cytochrome P450 aromatases: Impact on gonadal development, recrudescence and effect of hCG in the catfish, Clarias gariepinus.  

PubMed

Present study analyzed the importance of two forms of aromatases during ovarian development and recrudescence of north African/air-breathing catfish. We cloned both CYP19A1 (1941bp; ovarian form) and CYP19A2 (1786bp; brain form), which showed 47% homology between the two forms. Characterization of encoded proteins in non-steroidogenic COS-7 cells illustrated that both isoforms efficiently catalyzed the aromatization reaction by producing estradiol-17beta (E(2)) from testosterone. Tissue distribution pattern revealed preferential expression of CYP19A2 in brain while CYP19A1 predominated in ovary with trace amounts detected in other tissues including brain. Relative real-time PCR analysis revealed high transcript levels of both isoforms in the prespawning phase of ovarian cycle, which is in accordance with serum E(2) level. Aromatase activity in brain was comparatively lower than ovary, indicating the predominant requirement of aromatase in ovary. Ontogeny studies displayed sexual dimorphism, with early expression of CYP19A1 and CYP19A2 in ovary and brain, respectively. Phase-dependent rise of expression and enzyme activity of aromatase after hCG treatment revealed the stimulatory role of gonadotropin during preparatory and prespawning phases, preferentially to promote vitellogenesis. Lack of influence of hCG treatment during spawning phase endorses it further. A good correlation of expression, enzyme activity and serum E(2) levels suggests a crucial role of CYP19A1 during ovarian differentiation and ovarian cycle of catfish. Likewise, CYP19A2 might also be involved in these processes either indirectly or directly. PMID:20303968

Rasheeda, M K; Sridevi, P; Senthilkumaran, B

2010-06-01

12

Reactive Intermediates in Cytochrome P450 Catalysis*  

PubMed Central

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.

2013-01-01

13

Forty years of cytochrome P450.  

PubMed

The term "cytochrome P450" first appeared in literature in 1962. It was a microsomal membrane-bound hemoprotein without known physiological functions at that time and was characterized by a unique 450-nm optical absorption peak of its carbon monoxide-bound form, which was originally reported as the spectrum of a novel "microsomal carbon monoxide-binding pigment" in 1958. Elucidation of its function as the oxygenase in 1963 triggered a rapid expansion of research on this hemoprotein. Annual numbers of the published papers dealing with cytochrome P450, which were listed in Biological Abstracts, increased from 60 in 1970 to 500 in 1980, 900 in 1990, and 1500 in 1997. Cytochrome P450 is now regarded as the collective name of a large family of hemoproteins, "cytochrome P450 superfamily, "which seems to have diversified from a single ancestral protein to many forms during the course of biological evolution and is distributed widely among various forms of life from animals and plants to fungi and bacteria. Multicellular eukaryotic organisms including animals and plants have about 100 or more P450 genes in their genomes, and those many P450 genes are expressed tissue specifically and developmental stage specifically, indicating their diverse physiological functions. In mammals, various P450s participate in the biosynthesis and metabolism of sterols and steroid hormones and the metabolism of various lipid biofactors including eicosanoids, vitamin D3, and retinoids. Oxidative metabolism of foreign hydrophobic compounds as the first step of their excretion from the animal body is apparently another major function of cytochrome P450, which protects animals from noxious foreign compounds, man-created and natural. PMID:10603307

Omura, T

1999-12-29

14

The active site of aromatase cytochrome P-450. Differential effects of cyanide provide evidence for proximity of heme-iron and carbon-19 in the enzyme-substrate complex.  

PubMed

19-Norandrostenedione and androstenedione are shown to be metabolized by purified, reconstituted human placental aromatase cytochrome P-450. Kinetic evidence indicates that both steroids share a common catalytic site: 19-norandrostenedione is a competitive inhibitor of androstenedione aromatization, and the Ki value for its inhibition (120 nM) is similar to the Km value for its metabolism (132 nM). The two substrates differ, however, in their sensitivity to inhibition by the heme-iron ligand cyanide; 19-norandrostenedione is approximately 3-fold more sensitive to cyanide inhibition. Spectroscopic studies show that this differential inhibition by cyanide occurs because androstenedione competes with cyanide, whereas 19-norandrostenedione promotes cyanide binding to the heme-iron. It is proposed that these opposite effects on cyanide-iron coordination are due to the proximity of the heme-iron and C-19 of androstenedione in the enzyme-substrate complex, which results in steric exclusion of cyanide from the active site by the C-19 methyl group of androstenedione. Dioxygen is not excluded from binding to the heme-iron during catalysis, presumably because it bonds at an angle, in contrast to the linear bond of iron-cyanide complexes. A model for the active site of aromatase cytochrome P-450 is presented. PMID:3597396

Kellis, J T; Vickery, L E

1987-06-25

15

Oxygen Activation by Cytochrome P450 Monooxygenase  

PubMed Central

Unlike photosystem II (PSII) that catalyzes formation of the O-O bond, the cytochromes P450 (P450), members of a superfamily of hemoproteins, catalyze the scission of the O-O bond of dioxygen molecules and insert a single oxygen atom into unactivated hydrocarbons through a hydrogen abstraction-oxygen rebound mechanism. Hydroxylation of the unactivated hydrocarbons at physiological temperatures is vital for many cellar processes such as the biosynthesis of many endogenous compounds and the detoxification of xenobiotics in humans and plants. Even though it carries out the opposite of the water splitting reaction, P450 may share similarities to PSII in proton delivery networks, oxygen and water access channels, and consecutive electron transfer processes. In this article, we review recent advances in understanding the molecular mechanisms by which P450 activates dioxygen. PMID:18600471

Hamdane, Djemel; Zhang, Haoming; Hollenberg, Paul

2009-01-01

16

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. PMID:20846929

2010-01-01

17

Structure conservation in cytochromes P450  

Microsoft Academic Search

The recent availability of crystal structures for several diverse cytochromes P450 (CYPs) offers the possibility to perform an up-to- date comparative analysis to identify the degree of structure conservation among this superfamily of enzymes specially relevant for their involvement in drug metabolism and toxicity. A set of 9 CYPs shar- ing between 10% and 27% sequence identity was selected, including

Jordi Mestres

2005-01-01

18

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

2013-04-01

19

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

20

Cytochrome P-450 isozymes and monooxygenase activity in aquatic animals.  

PubMed Central

The roles of different forms of cytochrome P-450 in activation and deactivation of toxic chemicals, synthesis and breakdown of steroid hormones, and other functions, indicate the significance of these enzymes. Monooxygenase systems have been studied in species from several phyla of aquatic organisms. However, cytochrome P-450, the dominant catalyst in xenobiotic monooxygenase activity, is best studied in fish. Forms of cytochrome P-450 have been purified from several teleost species, including scup (Stenotomus chrysops), rainbow trout (Salmo gairdneri), and cod (Gadus morhua). Cytochrome P-450E from scup, cytochrome P-450 LM4b from trout, and cytochrome P-450c from cod have properties similar to each other and appear to be homologous hydrocarbon or BNF-inducible isozymes. Partially purified cytochrome DBA-P-450-I from little skate, Raja erinacea, is possibly an elasmobranch counterpart of these teleost forms. Cytochrome P-450E from scup is immunochemically related to the major BNF-inducible isozyme (cytochrome P-450c or BNF-B) in rats, indicating homology between the fish and mammalian BNF-inducible isozymes. Several other cytochrome P-450 forms with interesting or unusual properties have been purified from aquatic species. Mammalian homologs are not yet known for these isozymes. Further studies of cytochrome P-450 forms in aquatic species should establish additional homologies and the regulation of these forms by chemical and biological variables, possibly providing fundamental insights into the function and evolution of these proteins. Images FIGURE 1. PMID:3297668

Stegeman, J J; Kloepper-Sams, P J

1987-01-01

21

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. PMID:21553396

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

2011-01-01

22

Flower colour and cytochromes P450.  

PubMed

Cytochromes P450 play important roles in biosynthesis of flavonoids and their coloured class of compounds, anthocyanins, both of which are major floral pigments. The number of hydroxyl groups on the B-ring of anthocyanidins (the chromophores and precursors of anthocyanins) impact the anthocyanin colour, the more the bluer. The hydroxylation pattern is determined by two cytochromes P450, flavonoid 3'-hydroxylase (F3'H) and flavonoid 3',5'-hydroxylase (F3'5'H) and thus they play a crucial role in the determination of flower colour. F3'H and F3'5'H mostly belong to CYP75B and CYP75A, respectively, except for the F3'5'Hs in Compositae that were derived from gene duplication of CYP75B and neofunctionalization. Roses and carnations lack blue/violet flower colours owing to the deficiency of F3'5'H and therefore lack the B-ring-trihydroxylated anthocyanins based upon delphinidin. Successful redirection of the anthocyanin biosynthesis pathway to delphinidin was achieved by expressing F3'5'H coding regions resulting in carnations and roses with novel blue hues that have been commercialized. Suppression of F3'5'H and F3'H in delphinidin-producing plants reduced the number of hydroxyl groups on the anthocyanidin B-ring resulting in the production of monohydroxylated anthocyanins based on pelargonidin with a shift in flower colour to orange/red. Pelargonidin biosynthesis is enhanced by additional expression of a dihydroflavonol 4-reductase that can use the monohydroxylated dihydrokaempferol (the pelargonidin precursor). Flavone synthase II (FNSII)-catalysing flavone biosynthesis from flavanones is also a P450 (CYP93B) and contributes to flower colour, because flavones act as co-pigments to anthocyanins and can cause blueing and darkening of colour. However, transgenic plants expression of a FNSII gene yielded paler flowers owing to a reduction of anthocyanins because flavanones are precursors of anthocyanins and flavones. PMID:23297355

Tanaka, Yoshikazu; Brugliera, Filippa

2013-02-19

23

Flower colour and cytochromes P450  

PubMed Central

Cytochromes P450 play important roles in biosynthesis of flavonoids and their coloured class of compounds, anthocyanins, both of which are major floral pigments. The number of hydroxyl groups on the B-ring of anthocyanidins (the chromophores and precursors of anthocyanins) impact the anthocyanin colour, the more the bluer. The hydroxylation pattern is determined by two cytochromes P450, flavonoid 3?-hydroxylase (F3?H) and flavonoid 3?,5?-hydroxylase (F3?5?H) and thus they play a crucial role in the determination of flower colour. F3?H and F3?5?H mostly belong to CYP75B and CYP75A, respectively, except for the F3?5?Hs in Compositae that were derived from gene duplication of CYP75B and neofunctionalization. Roses and carnations lack blue/violet flower colours owing to the deficiency of F3?5?H and therefore lack the B-ring-trihydroxylated anthocyanins based upon delphinidin. Successful redirection of the anthocyanin biosynthesis pathway to delphinidin was achieved by expressing F3?5?H coding regions resulting in carnations and roses with novel blue hues that have been commercialized. Suppression of F3?5?H and F3?H in delphinidin-producing plants reduced the number of hydroxyl groups on the anthocyanidin B-ring resulting in the production of monohydroxylated anthocyanins based on pelargonidin with a shift in flower colour to orange/red. Pelargonidin biosynthesis is enhanced by additional expression of a dihydroflavonol 4-reductase that can use the monohydroxylated dihydrokaempferol (the pelargonidin precursor). Flavone synthase II (FNSII)-catalysing flavone biosynthesis from flavanones is also a P450 (CYP93B) and contributes to flower colour, because flavones act as co-pigments to anthocyanins and can cause blueing and darkening of colour. However, transgenic plants expression of a FNSII gene yielded paler flowers owing to a reduction of anthocyanins because flavanones are precursors of anthocyanins and flavones. PMID:23297355

Tanaka, Yoshikazu; Brugliera, Filippa

2013-01-01

24

Noninducibility of cytochrome P-450 in the earthworm Dendrobaena veneta.  

PubMed

Cytochrome P-450 has been measured in the earthworm Dendrobaena veneta (Rosa) in a direct spectrophotometric procedure. The P-450 was found not in the dense microsomal fraction, but in the less dense overlying fraction often referred to as buffy coat. Earthworm P-450 was not induced by 3-methylcholanthrene or phenobarbitol. PMID:2877809

Milligan, D L; Babish, J G; Neuhauser, E F

1986-01-01

25

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

2013-11-15

26

Induced synthesis of P450 aromatase and 17?-estradiol by D-aspartate in frog brain.  

PubMed

D-Aspartic acid is an endogenous amino acid occurring in the endocrine glands as well as in the nervous system of various animal phyla. Our previous studies have provided evidence that D-aspartate plays a role in the induction of estradiol synthesis in gonads. Recently, we have also demonstrated that D-aspartic acid induces P450 aromatase mRNA expression in the frog (Pelophylax esculentus) testis. P450 aromatase is the key enzyme in the estrogen synthetic pathway and irreversibly converts testosterone into 17?-estradiol. In this study, we firstly investigated the immunolocalisation of P450 aromatase in the brain of P. esculentus, which has never previously been described in amphibians. Therefore, to test the hypothesis that d-aspartate mediates a local synthesis of P450 aromatase in the frog brain, we administered D-aspartate in vivo to male frogs and then assessed brain aromatase expression, sex hormone levels and sex hormone receptor expression. We found that D-aspartate enhances brain aromatase expression (mRNA and protein) through the CREB pathway. Then, P450 aromatase induces 17?-estradiol production from testosterone, with a consequent increase of its receptor. Therefore, the regulation of d-aspartate-mediated P450 aromatase expression could be an important step in the control of neuroendocrine regulation of the reproductive axis. Accordingly, we found that the sites of P450 aromatase immunoreactivity in the frog brain correspond to the areas known to be involved in neurosteroid synthesis. PMID:22771744

Burrone, Lavinia; Santillo, Alessandra; Pinelli, Claudia; Baccari, Gabriella Chieffi; Di Fiore, Maria Maddalena

2012-10-15

27

Engineering Cytochrome P450 Biocatalysts for Biotechnology, Medicine, and Bioremediation  

PubMed Central

Importance of the field: Cytochrome P450 enzymes comprise a superfamily of heme monooxygenases that are of considerable interest for the: 1) synthesis of novel drugs and drug metabolites, 2) targeted cancer gene therapy, 3) biosensor design, and 4) bioremediation. However, their applications are limited because cytochrome P450, especially mammalian P450 enzymes, show a low turnover rate and stability, and require a complex source of electrons through cytochrome P450 reductase and NADPH. Areas covered in this review: In this review, we discuss the recent progress towards the use of P450 enzymes in a variety of above-mentioned applications. We also present alternate and cost-effective ways to perform P450-mediated reaction, especially using peroxides. Furthermore, we expand upon the current progress in P450 engineering approaches describing several recent examples that are utilized to enhance heterologous expression, stability, catalytic efficiency, and utilization of alternate oxidants. What the reader will gain: The review will provide a comprehensive knowledge in the design of P450 biocatalysts for potentially practical purposes. Finally, we provide a prospective on the future aspects of P450 engineering and its applications in biotechnology, medicine, and bioremediation. Take home message: Because of its wide applications, academic and pharmaceutical researchers, environmental scientists, and health care providers are expected to gain current knowledge and future prospects of the practical use of P450 biocatalysts. PMID:20064075

Kumar, Santosh

2009-01-01

28

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. PMID:21371610

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

2011-01-01

29

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

2015-01-06

30

African variation at Cytochrome P450 genes  

PubMed Central

The genomics revolution has provided a plethora of data from many previously uncharacterized populations. The increase in the amount of genetic data has improved our understanding of why individuals and populations differ in their susceptibility to multiple diseases. It has also enabled researchers to identify how genomic variation, including at the Cytochrome P450 (CYP450) super-family, affects the safety and efficacy of therapeutic drugs. CYP450 metabolize ?90% of clinically administered drugs. Variability in CYP450 expression is known to affect the safety and efficacy of therapeutic drugs, including many used in the treatment and control of infectious diseases. There are inter-ethnic differences in the frequencies of clinically relevant CYP450 variants which affect CYP450 expression. Comparative studies of African populations have identified population structuring at CYP450 genes. This is associated with intra-African differences in the success of drug therapies used in the treatment of infectious diseases. Therapeutic drugs dominate control strategies for infectious diseases and are widely administered through mass drug administration campaigns. However, resistance to chemotherapy is spreading across endemic regions. The most common response has been to increase chemotherapeutic dosages, and administer combination therapies. However, there are few pharmacovigilance data examining how these changes influence adverse drug reactions. This review provides an overview of current knowledge of intra-Africa CYP450 variation, and the known associations with sub-optimal clinical outcomes in the treatment of infectious diseases. In addition, the potential for evolutionary approaches in the study of CYP450 variation is discussed to examine their potential in preventative medicine and intervention strategies within Africa. PMID:24481193

Bains, Ripudaman K.

2013-01-01

31

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

2013-05-01

32

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

PubMed Central

There is an increasing demand for versatile and ubiquitous Cytochrome P450 (CYP) biocatalysts for biotechnology, medicine, and bioremediation. In the last decade there has been an increase in realization of the power of CYP biocatalysts for detoxification of soil and water contaminants using transgenic plants. However, the major limitations of mammalian CYP enzymes are that they require CYP reductase (CPR) for their activity, and they show relatively low activity, stability, and expression. On the other hand, bacterial CYP enzymes show limited substrate diversity and usually do not metabolize herbicides and industrial contaminants. Therefore, there has been a considerable interest for biotechnological industries and the scientific community to design CYP enzymes to improve their catalytic efficiency, stability, expression, substrate diversity, and the suitability of P450-CPR fusion enzymes. Engineered CYP enzymes have potential for transgenic plants-mediated phytoremediation of herbicides and environmental contaminants. In this review we discuss: 1) the role of CYP enzymes in phytoremediation using transgenic plants, 2) problems associated with wild-type CYP enzymes in phytoremediation, and 3) examples of engineered CYP enzymes and their potential role in transgenic plant-mediated phytoremediation. PMID:25298920

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

2013-01-01

33

Downregulation of male-specific cytochrome P450 by profenofos.  

PubMed

The health hazards of individual organophosphorus insecticides have been characterized by their acute toxicity, mainly by investigating their cholinesterase inhibition. However, the chronic effects of most of these toxicants on the drug-metabolizing enzymes have not been investigated. Profenofos (O-4-bromo-2-chlorophenyl O-ethyl S-propyl phosphorothioate) is an organophosphorus pesticide widely used in cotton cultivation. In the present study, we investigated the effect of profenofos on male-specific cytochrome P450 (CYP) enzymes in adult Wistar rats. We orally administered 17.8 mg/kg body weight, twice weekly for 65 days. Profenofos downregulated levels of hepatic and testicular CYP2C11 and CYP3A2 mRNA and protein expression. Testicular aromatase (CYP19A) mRNA was decreased in the profenofos-treated rats compared to controls. Overall, the present study suggests that profenofos acts as an endocrine disruptor of male-specific CYP enzymes and affects testosterone concentration, which implicates its deleterious effects on animal or human males chronically exposed to organophosphorus pesticide. PMID:18828448

Moustafa, Gihan G; Ibrahim, Zein S; Ahmed, Mohamed M; Ghoneim, Mervat H; Sakamoto, Kentaro Q; Ishizuka, Mayumi; Fujita, Shoichi

2008-08-01

34

The cytochrome P450 genesis locus: the origin and evolution of animal cytochrome P450s  

PubMed Central

The neighbourhoods of cytochrome P450 (CYP) genes in deuterostome genomes, as well as those of the cnidarians Nematostella vectensis and Acropora digitifera and the placozoan Trichoplax adhaerens were examined to find clues concerning the evolution of CYP genes in animals. CYP genes created by the 2R whole genome duplications in chordates have been identified. Both microsynteny and macrosynteny were used to identify genes that coexisted near CYP genes in the animal ancestor. We show that all 11 CYP clans began in a common gene environment. The evidence implies the existence of a single locus, which we term the ‘cytochrome P450 genesis locus’, where one progenitor CYP gene duplicated to create a tandem set of genes that were precursors of the 11 animal CYP clans: CYP Clans 2, 3, 4, 7, 19, 20, 26, 46, 51, 74 and mitochondrial. These early CYP genes existed side by side before the origin of cnidarians, possibly with a few additional genes interspersed. The Hox gene cluster, WNT genes, an NK gene cluster and at least one ARF gene were close neighbours to this original CYP locus. According to this evolutionary scenario, the CYP74 clan originated from animals and not from land plants nor from a common ancestor of plants and animals. The CYP7 and CYP19 families that are chordate-specific belong to CYP clans that seem to have originated in the CYP genesis locus as well, even though this requires many gene losses to explain their current distribution. The approach to uncovering the CYP genesis locus overcomes confounding effects because of gene conversion, sequence divergence, gene birth and death, and opens the way to understanding the biodiversity of CYP genes, families and subfamilies, which in animals has been obscured by more than 600 Myr of evolution. PMID:23297357

Nelson, David R.; Goldstone, Jared V.; Stegeman, John J.

2013-01-01

35

Phosphorylation of hepatic phenobarbital-inducible cytochrome P-450.  

PubMed Central

The major phenobarbital-inducible cytochrome P-450 purified from rat liver, a member of family II of the cytochrome P-450 gene superfamily, is rapidly phosphorylated by cAMP-dependent protein kinase. The phosphorylation reaches greater than 0.5 mol phosphate/mol P-450 after 5 min and is accompanied by a decrease in enzyme activity. The serine residue in position 128 was shown to be the sole phosphorylation site and a conformational change of the protein was indicated by a shift of the carbon monoxide difference spectrum of the reduced cytochrome from 450 to 420 nm. Comparison of amino acid sequences of various cytochrome P-450 families revealed a highly conserved arginine residue in the immediate vicinity of the phosphorylated serine residue which constitutes the kinase recognition sequence. It also revealed that only the members of the cytochrome P-450 family II carry this kinase recognition sequence. To find out whether this phosphorylation also occurs in vivo, the exchangeable phosphate pool of intact hepatocytes derived from phenobarbital-pretreated rats was labeled with 32Pi followed by an incubation of the cells with the membrane-permeating dibutyryl-cAMP or with the adenylate cyclase stimulator glucagon to activate endogenous kinase. As a result, a microsomal polypeptide with the same electrophoretic mobility as cytochrome P-450 became strongly labeled. Peptide mapping and immunoprecipitation with monospecific antibodies identified this protein as the major phenobarbital-inducible cytochrome P-450. It becomes phosphorylated at the same serine residues as in the cell-free phosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS) Images PMID:2583091

Pyerin, W; Taniguchi, H

1989-01-01

36

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

37

Cytochrome P450-mediated metabolism of vitamin D  

PubMed Central

The vitamin D signal transduction system involves a series of cytochrome P450-containing sterol hydroxylases to generate and degrade the active hormone, 1?,25-dihydroxyvitamin D3, which serves as a ligand for the vitamin D receptor-mediated transcriptional gene expression described in companion articles in this review series. This review updates our current knowledge of the specific anabolic cytochrome P450s involved in 25- and 1?-hydroxylation, as well as the catabolic cytochrome P450 involved in 24- and 23-hydroxylation steps, which are believed to initiate inactivation of the vitamin D molecule. We focus on the biochemical properties of these enzymes; key residues in their active sites derived from crystal structures and mutagenesis studies; the physiological roles of these enzymes as determined by animal knockout studies and human genetic diseases; and the regulation of these different cytochrome P450s by extracellular ions and peptide modulators. We highlight the importance of these cytochrome P450s in the pathogenesis of kidney disease, metabolic bone disease, and hyperproliferative diseases, such as psoriasis and cancer; as well as explore potential future developments in the field. PMID:23564710

Jones, Glenville; Prosser, David E.; Kaufmann, Martin

2014-01-01

38

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

39

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

Technology Transfer Automated Retrieval System (TEKTRAN)

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

40

Cytochrome P450 arachidonic acid metabolism in bovine corneal epithelium  

SciTech Connect

The presence of the cytochrom P450 system and its involvement in the metabolism of AA was studied in the corneal epithelium. This tissue contains cytochrome P450 as assessed directly by measurement of the carbon monoxide reduced spectrum (specific activity of 161 pmol/10 mg protein) and indirectly by measuring the activity of aryl hydrocarbon hydroxylase (AHH) - a cytochrome P450-dependent enzyme (11-39 pmol 3-OH benzopyrene/mg protein/10 min). When corneal epithelial microsomes were incubated with /sup 14/C-arachidonic acid, 30-50% of the total radioactivity was converted to two peaks, I and II. Further separation using high performance liquid chromatography has shown that each peak contains two metabolites, A,B and C,D. Metabolite formation was dependent on the addition of NADPH (1 mM) and inhibited by carbon monoxide and SKF-525A (100 ..mu..M) suggesting a cytochrome P450-dependent mechanism. Compound C (5-10 ..mu..M) inhibited the activity of corneal epithelial Na-K-ATPase by 30-60%, being 100-fold more potent than ouabain. Compound D (10-100 ng) induced a dose dependent relaxation of the rat caudal artery. Compound D also inhibited corneal Na-K-ATPase activity but less potently than compound C. These compounds may be important to transport processes of ocular epithelia and participate in the control of the ocular circulation and aqueous humor dynamics.

Masferrer, J.; Schwartzman, M.L.; Abraham, N.G.; Dunn, M.W.; McGiff, J.C.

1986-03-01

41

Cytochrome P450-Dependent Lipid Metabolism in Preovulatory Follicles  

E-print Network

Cytochrome P450-Dependent Lipid Metabolism in Preovulatory Follicles J. W. NEWMAN, J. E. STOK, J. D. However, CYP-dependent lipid metabolism has not been characterized with respect to follicular matura- tion, and concentra- tions of epoxy, hydroxy, and dihydroxy lipids were measured by liquid chromatography tandem mass

Hammock, Bruce D.

42

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

43

Aromatase P450 expression in human pituitary adenomas.  

PubMed

Estrogen has been shown to play an important role in pituitary tumor pathogenesis. In humans, this biosynthesis is mediated by aromatase, an enzyme that converts androgens to estrogens. Just a few studies about aromatase expression in human pituitary gland, both in normal and pathological ones, are found in the literature. This study aimed to assess aromatase enzyme expression in human pituitary adenomas and associate it with gender, tumor size and tumor subtype. We conducted a cross-sectional study, reviewed clinical data and surgical specimens of consecutive 65 patients (35 women and 30 men) with anatomopathologic diagnosis of pituitary adenoma who underwent adenomectomy at a neurosurgical referral center in southern Brazil. Immunohistochemistry was performed to assess aromatase expression and define tumor subtype, and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) to estimate aromatase gene expression. Mean patient age was 45.6 (±13.3) years (range, 18 to 73 years), 86.2% of our samples were macroadenomas while 13.8% were classified as microadenomas. Based on clinical and immunohistochemical data, 23 (35.4%) patients had non-functioning adenomas, 19 (29.2%) had somatotroph adenomas (acromegaly), 12 (18.5%) had lactotroph adenomas (hyperprolactinemic syndrome), and 11 (16.9%) had corticotroph adenomas (Cushing's disease). Immunohistochemical analysis was performed in 59 cases, and 58 (98.3%) showed no aromatase expression. Quantification by qRT-PCR was performed in 43 samples, and 36 (83.7%) revealed no gene expression. Among tumor specimens examined by both techniques (37 cases), 30 showed no gene or protein expression (concordance index, 0.81). It is possible to mention that aromatase expression was lost in most pituitary adenomas, regardless of gender, tumor subtype, or tumor size. PMID:25410472

Gonzales, Paulo Henrique; Mezzomo, Lisiane Cervieri; Ferreira, Nelson Pires; Roehe, Adriana Vial; Kohek, Maria Beatriz Fonte; Oliveira, Miriam da Costa

2014-11-20

44

Unusual properties of the cytochrome P450 superfamily  

PubMed Central

During the early years of cytochrome P450 research, a picture of conserved properties arose from studies of mammalian forms of these monooxygenases. They included the protohaem prosthetic group, the cysteine residue that coordinates to the haem iron and the reduced CO difference spectrum. Alternatively, the most variable feature of P450s was the enzymatic activities, which led to the conclusion that there are a large number of these enzymes, most of which have yet to be discovered. More recently, studies of these enzymes in other eukaryotes and in prokaryotes have led to the discovery of unexpected P450 properties. Many are variations of the original properties, whereas others are difficult to explain because of their unique nature relative to the rest of the known members of the superfamily. These novel properties expand our appreciation of the broad view of P450 structure and function, and generate curiosity concerning the evolution of P450s. In some cases, structural properties, previously not found in P450s, can lead to enzymatic activities impacting the biological function of organisms containing these enzymes; whereas, in other cases, the biological reason for the variations are not easily understood. Herein, we present particularly interesting examples in detail rather than cataloguing them all. PMID:23297356

Lamb, David C.; Waterman, Michael R.

2013-01-01

45

Rational redesign of the biodegradative enzyme cytochrome P450 cam:  

SciTech Connect

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

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

1991-03-01

46

Coding nucleotide, 5' regulatory, and deduced amino acid sequences of P-450BM-3, a single peptide cytochrome P-450:NADPH-P-450 reductase from Bacillus megaterium.  

PubMed

Cytochrome P-450BM-3 (P-450BM-3) from Bacillus megaterium incorporates both a P-450 and an NADPH:P-450 reductase in proteolytically separable domains of a single, 119-kDa polypeptide and functions as a fatty acid monooxygenase independently of any other protein. A 5-kilobase DNA fragment which contains the gene encoding P-450BM-3 was sequenced. A single continuous open reading frame starting at nucleotide 1541 of the 5-kilobase fragment correctly predicted the previously determined NH2-terminal protein sequences of the trypsin-generated P-450 and reductase domains and, in toto, predicted a mature polypeptide of 1,048-amino acid residues with Mr = 117,641. The trypsin site was found at arginine residue 471. The P-450 domain is most similar (about 25%) to the fatty acid omega-hydroxylases of P-450 family IV, while the reductase domain exhibits some 33% sequence similarity with the NADPH:P-450 reductases of mammalian liver. Both the P-450 and reductase domains of P-450BM-3 define new gene families but contain highly conserved segments which display as much as 50% sequence similarity with P-450s and reductases of eukaryotic origin. The mRNA for P-450BM-3 was found by S1 mapping to be 3,339 +/- 10 nucleotides in length. In the accompanying paper, two regions in the 1.5 kilobases 5' to the P-450BM-3 coding region have been implicated in the regulation of P-450BM-3 gene expression. PMID:2544578

Ruettinger, R T; Wen, L P; Fulco, A J

1989-07-01

47

Multiple Structural and Functional Abnormalities in the P450 Aromatase Expressing Transgenic Male Mice Are Ameliorated by a P450 Aromatase Inhibitor  

PubMed Central

The present study was undertaken to analyze the effect of a P450 aromatase inhibitor (finrozole) on 4-month-old transgenic mice expressing human P450 aromatase (P450arom) under the human ubiquitin C promoter (AROM+). AROM+ mice present several dysfunctions, such as adrenal and pituitary hyperplasia, cryptorchidism, Leydig cell hypertrophy and hyperplasia, and gynecomastia. The present study demonstrates that these abnormalities were efficiently treated by administration of a P450arom inhibitor, finrozole. The treatment normalized the reduced intratesticular and serum testosterone levels, while those of estradiol were decreased. The body weight and several affected organ weights were normalized with the treatment. Histological analysis revealed that both the pituitary and adrenal hyperplasia were diminished. Furthermore, the cryptorchid testes present in the untreated AROM+ males descended to scrotum, 4 to 15 days after inhibitor treatment. In addition, the disrupted spermatogenesis was recovered and qualitatively complete spermatogenesis appeared with the inhibitor treatment. This was associated with normalized structure of the interstitial tissue, as analyzed by immunohistochemical staining for Leydig cells and macrophages. One of the features was that the Leydig cell hypertrophy was markedly diminished in the treated mice. AROM+ mice also present with severe gynecomastia, while the development and differentiation of the mammary gland in AROM+ males was markedly diminished with the inhibitor treatment. Interestingly, the mammary gland involution was associated with the induction of androgen receptor in the epithelial cells, while estrogen receptors were still detectable in the epithelium. The data show that AROM+ mouse model is a novel tool to further analyze the use of P450arom inhibitors in the treatment of the dysfunctions in males associated with misbalanced estrogen to androgen ratio, such as pituitary adenoma, testicular dysfunction, and gynecomastia. PMID:14982857

Li, Xiangdong; Strauss, Leena; Mäkelä, Sari; Streng, Tomi; Huhtaniemi, Ilpo; Santti, Risto; Poutanen, Matti

2004-01-01

48

Cloning and molecular characterization of an ovarian-derived (brain-like) P450 aromatase cDNA and development of a competitive RT-PCR assay to quantify its expression in the fathead minnow (Pimephales promelas)  

Microsoft Academic Search

In higher vertebrates, estrogens are key players in sexual differentiation and development and their production is essential for normal sexual development in both females and males. Cytochrome P450 aromatase (P450arom) is the steroidogenic enzyme responsible for the production of estrogens from aromatizable androgens. In this study P450arom cDNA was cloned in the fathead minnow (Pimephales promelas) and a RT-PCR assay

S. Halm; M. Rand-Weaver; J. P. Sumpter; C. R. Tyler

2001-01-01

49

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...

50

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...

51

Modulation by phytochemicals of cytochrome P450-linked enzyme activity  

Microsoft Academic Search

Compounds derived from plant sources with putative anticancer properties were studied for their effects on alkoxyresorufin O-dealkylase activity, a measure of cytochrome P450 activity. The phytochemicals investigated included benzyl isothiocyanate, caffeic acid, chlorogenic acid, diosmin, ferulic acid, indole-3-carbinol, phenethyl isothiocyanate and resveratrol. Each phytochemical at concentrations of 0.25 and 0.5 ?M was incubated with 0.2 mg hamster liver microsomal protein

Robert W. Teel; Huong Huynh

1998-01-01

52

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. PMID:16347120

Sutherland, John B.

1986-01-01

53

Investigating the Structural Plasticity of a Cytochrome P450  

PubMed Central

Cytochrome P450s are heme-containing proteins that catalyze the oxidative metabolism of many physiological endogenous compounds. Because of their unique oxygen chemistry and their key role in drug and xenobiotic metabolism, particular attention has been devoted in elucidating their mechanism of substrate recognition. In this work, we analyzed the three-dimensional structures of a monomeric cytochrome P450 from Saccharopolyspora erythraea, commonly called EryK, and the binding kinetics to its physiological ligand, erythromycin D. Three different structures of EryK were obtained: two ligand-free forms and one in complex with its substrate. Analysis of the substrate-bound structure revealed the key structural determinants involved in substrate recognition and selectivity. Interestingly, the ligand-free structures of EryK suggested that the protein may explore an open and a closed conformation in the absence of substrate. In an effort to validate this hypothesis and to investigate the energetics between such alternative conformations, we performed stopped-flow absorbance experiments. Data demonstrated that EryK binds erythromycin D via a mechanism involving at least two steps. Contrary to previously characterized cytochrome P450s, analysis of double jump mixing experiments confirmed that this complex scenario arises from a pre-existing equilibrium between the open and closed subpopulations of EryK, rather than from an induced-fit type mechanism. PMID:19625248

Savino, Carmelinda; Montemiglio, Linda C.; Sciara, Giuliano; Miele, Adriana E.; Kendrew, Steven G.; Jemth, Per; Gianni, Stefano; Vallone, Beatrice

2009-01-01

54

Bioreactor Systems in Drug Metabolism: Synthesis of Cytochrome P450-Generated Metabolites  

Microsoft Academic Search

In this communication, we report that suspension cultures of Sf 21 insect cells, co-infected with baculovirus containing the cDNA for a single cytochrome P450 and NADPH–cytochrome P450 oxidoreductase, can be employed successfully as “bioreactors” for the synthesis of milligram quantities of cytochrome P450-generated metabolite(s). Three standard or probe substrates for the human P450s were chosen for the initial biosynthetic experiments:

Thomas H. Rushmore; Paul J. Reider; Don Slaughter; Carol Assang; Magang Shou

2000-01-01

55

Epoxidation activities of human cytochromes p450c17 and p450c21.  

PubMed

Some cytochrome P450 enzymes epoxidize unsaturated substrates, but this activity has not been described for the steroid hydroxylases. Physiologic steroid substrates, however, lack carbon-carbon double bonds in the parts of the pregnane molecules where steroidogenic hydroxylations occur. Limited data on the reactivity of steroidogenic P450s toward olefinic substrates exist, and the study of occult activities toward alternative substrates is a fundamental aspect of the growing field of combinatorial biosynthesis. We reasoned that human P450c17 (steroid 17-hydroxylase/17,20-lyase, CYP17A1), which 17- and 16?-hydroxylates progesterone, might catalyze the formation of the 16?,17-epoxide from 16,17-dehydroprogesterone (pregna-4,16-diene-3,20-dione). CYP17A1 catalyzed the novel 16?,17-epoxidation and the ordinarily minor 21-hydroxylation of 16,17-dehydroprogesterone in a 1:1 ratio. CYP17A1 mutation A105L, which has reduced progesterone 16?-hydroxylase activity, gave a 1:5 ratio of epoxide:21-hydroxylated products. In contrast, human P450c21 (steroid 21-hydroxylase, CYP21A2) converted 16,17-dehydroprogesterone to the 21-hydroxylated product and only a trace of epoxide. CYP21A2 mutation V359A, which has significant 16?-hydroxylase activity, likewise afforded the 21-hydroxylated product and slightly more epoxide. CYP17A1 wild-type and mutation A105L do not 21- or 16?-hydroxylate pregnenolone, but the enzymes 21-hydroxylated and 16?,17-epoxidized 16,17-dehydropregnenolone (pregna-5,16-diene-3?-ol-20-one) in 4:1 or 12:1 ratios, respectively. Catalase and superoxide dismutase did not prevent epoxide formation. The progesterone epoxide was not a time-dependent, irreversible CYP17A1 inhibitor. Our substrate modification studies have revealed occult epoxidase and 21-hydroxylase activities of CYP17A1, and the fraction of epoxide formed correlated with the 16?-hydroxylase activity of the enzymes. PMID:25386927

Yoshimoto, Francis K; Peng, Hwei-Ming; Zhang, Haoming; Anderson, Sean M; Auchus, Richard J

2014-12-01

56

Histidine residues in rabbit liver microsomal cytochrome P-450 2B4 control electron transfer from NADPH-cytochrome P-450 reductase and cytochrome b5.  

PubMed Central

Treatment of cytochrome P-450 2B4 (P-450 2B4) with diethylpyrocarbonate to introduce 10-11 equivalents of acylating agent per polypeptide chain resulted in the selective derivatization of histidine residues characterized by differential susceptibility toward the modifier. Second-derivative spectral analysis as well as fluorescence measurements disproved gross alterations in P-450 2B4 structure as a consequence of labelling. The modified haemoprotein retained its ability to bind hexobarbital and catalyse cumene hydroperoxide-sustained N-demethylation of the barbiturate. However, there was a steady attenuation of NAD(P)H-driven electron flux with increasing extent of P-450 2B4 carbethoxylation in reconstituted systems fortified with either NADPH-cytochrome P-450 reductase or NADH-cytochrome b5 reductase/cytochrome b5 as the redox partners, with 50% inhibition occurring when 6-7 histidines were blocked. Hampered P-450 2B4 reductase activities recovered to differing degrees upon treatment of the acylated mono-oxygenase with neutral hydroxylamine. Spectral data indicated that docking of the redox components to derivatized P-450 2B4 was not perturbed, so that disruption of the electron flows most likely resulted from some injury of the electron-transfer mechanisms. PMID:8836129

Hlavica, P; Lehnerer, M; Eulitz, M

1996-01-01

57

Catalysis by cytochrome P-450 of an oxidative reaction in xenobiotic aldehyde metabolism: deformylation with olefin formation.  

PubMed Central

As we have briefly described elsewhere, cytochrome P-450 catalyzes the oxidative deformylation of cyclohexane carboxaldehyde to yield cyclohexene and formic acid in a reaction believed to involve a peroxyhemiacetal-like adduct formed between the substrate and molecular oxygen-derived hydrogen peroxide. This reaction is a useful model for the demethylation reactions catalyzed by the steroidogenic P-450s, aromatase, and lanosterol demethylase. In the present study, the cytochrome P-450-catalyzed formation of olefinic products from a series of xenobiotic aldehydes has been demonstrated. Isobutyraldehyde and trimethylacetaldehyde, but not propionaldehyde, are converted to the predicted olefinic products, suggesting a requirement for branching at the alpha carbon. In addition, the four C5 aldehydes of similar hydrophobicity were compared for their ability to undergo the reaction. The straight-chain valeraldehyde gave no olefinic products with five different rabbit liver microsomal P-450 isozymes. However, increasing activity was seen with the other isomers in the order of isovaleraldehyde, 2-methylbutyraldehyde, and trimethylacetaldehyde, with all of the P-450 cytochromes. The catalytic rate with trimethylacetaldehyde is highest with antibiotic-inducible P-450 form 3A6, followed by phenobarbital-inducible form 2B4 and ethanol-inducible form 2E1. Citronellal, a beta-branched aldehyde that is found in many essential oils and is widely used as an odorant and a flavorant, was found to undergo the oxidative deformylation reaction to yield 2,6-dimethyl-1,5-heptadiene, but only with P-450 2B4. The oxidative cleavage reaction with olefin formation appears to be widespread, as judged by the variety of aldehydes that serve as substrates and of P-450 cytochromes that serve as catalysts. PMID:1924356

Roberts, E S; Vaz, A D; Coon, M J

1991-01-01

58

Catalysis by cytochrome P-450 of an oxidative reaction in xenobiotic aldehyde metabolism: deformylation with olefin formation.  

PubMed

As we have briefly described elsewhere, cytochrome P-450 catalyzes the oxidative deformylation of cyclohexane carboxaldehyde to yield cyclohexene and formic acid in a reaction believed to involve a peroxyhemiacetal-like adduct formed between the substrate and molecular oxygen-derived hydrogen peroxide. This reaction is a useful model for the demethylation reactions catalyzed by the steroidogenic P-450s, aromatase, and lanosterol demethylase. In the present study, the cytochrome P-450-catalyzed formation of olefinic products from a series of xenobiotic aldehydes has been demonstrated. Isobutyraldehyde and trimethylacetaldehyde, but not propionaldehyde, are converted to the predicted olefinic products, suggesting a requirement for branching at the alpha carbon. In addition, the four C5 aldehydes of similar hydrophobicity were compared for their ability to undergo the reaction. The straight-chain valeraldehyde gave no olefinic products with five different rabbit liver microsomal P-450 isozymes. However, increasing activity was seen with the other isomers in the order of isovaleraldehyde, 2-methylbutyraldehyde, and trimethylacetaldehyde, with all of the P-450 cytochromes. The catalytic rate with trimethylacetaldehyde is highest with antibiotic-inducible P-450 form 3A6, followed by phenobarbital-inducible form 2B4 and ethanol-inducible form 2E1. Citronellal, a beta-branched aldehyde that is found in many essential oils and is widely used as an odorant and a flavorant, was found to undergo the oxidative deformylation reaction to yield 2,6-dimethyl-1,5-heptadiene, but only with P-450 2B4. The oxidative cleavage reaction with olefin formation appears to be widespread, as judged by the variety of aldehydes that serve as substrates and of P-450 cytochromes that serve as catalysts. PMID:1924356

Roberts, E S; Vaz, A D; Coon, M J

1991-10-15

59

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. PMID:10749660

Honkakoski, P; Negishi, M

2000-01-01

60

Regulation of cytochrome P450 expression in Drosophila: Genomic insights  

PubMed Central

Genomic tools such as the availability of the Drosophila genome sequence, the relative ease of stable transformation, and DNA microarrays have made the fruit fly a powerful model in insecticide toxicology research. We have used transgenic promoter-GFP constructs to document the detailed pattern of induced Cyp6a2 gene expression in larval and adult Drosophila tissues. We also compared various insecticides and xenobiotics for their ability to induce this cytochrome P450 gene, and show that the pattern of Cyp6a2 inducibility is comparable to that of vertebrate CYP2B genes, and different from that of vertebrate CYP1A genes, suggesting a degree of evolutionary conservation for the “phenobarbital-type” induction mechanism. Our results are compared to the increasingly diverse reports on P450 induction that can be gleaned from whole genome or from “detox” microarray experiments in Drosophila. These suggest that only a third of the genomic repertoire of CYP genes is inducible by xenobiotics, and that there are distinct subsets of inducers / induced genes, suggesting multiple xenobiotic transduction mechanisms. A relationship between induction and resistance is not supported by expression data from the literature. The relative abundance of expression data now available is in contrast to the paucity of studies on functional expression of P450 enzymes, and this remains a challenge for our understanding of the toxicokinetic aspects of insecticide action. PMID:20582327

Giraudo, Maeva; Unnithan, G. Chandran; Le Goff, Gaëlle; Feyereisen, René

2009-01-01

61

Purification and characterization of three constitutive cytochrome P-450 isoforms from bovine olfactory epithelium.  

PubMed Central

Three constitutive forms of cytochrome P-450 (P-450s) were isolated from olfactory microsomes of cattle. The purified P-450s, designated P-450bov1, P-450bov2 and P-450bov3, were electrophoretically nearly homogeneous by SDS/PAGE and their apparent relative molecular masses were estimated to be 50000, 53000 and 51000 respectively. As indicated by several criteria including the N-terminal sequence and absorption spectra, the three olfactory forms of P-450 were distinct from each other and from all the other P-450s currently known in cattle. P-450bov1 and P-450bov2 were purified in the low-spin state, whereas P-450bov3 was in the high-spin state. Studies to evaluate, by Western blot analysis, the reactivity of these purified P-450s with antibodies raised against rat hepatic P-450 2E1, 2B, 1A and 3A and rabbit olfactory P-450NMa and P-450NMb showed that P-450bov3 strongly cross-reacted with anti-P-450NMb IgG, and P-450bov1 moderately with anti-P-450NMa IgG. As determined by immunoblots, P-450bov1 and P-450bov3 represented a great portion of the total olfactory P-450. In a reconstituted system with NADPH:cytochrome P-450 reductase and phospholipids, P-450bov1 was more active in the metabolism of xenobiotic compounds (i.e. O-de-ethylation of ethoxycoumarin and N-demethylation of hexamethylphosphoramide) than towards endogenous substrates (testosterone and progesterone). Conversely, P-450bov3 metabolized the xenobiotics at lower rates but exhibited total oxidation rates of the above sex hormones higher than those of P-450bov1. From the comparison of the catalytic, immunochemical and structural properties, it was inferred that P-450bov1 and P-450bov3 are the bovine orthologues of P-450NMa (2A) and P-450NMb (2G1) respectively, the only two olfactory P-450s previously purified from rabbit. P-450bov2, which showed low activity toward some exogenous and endogenous compounds, represents a novel purified olfactory hemoprotein possibly belonging to the 3A subfamily. These results are consistent with a specific presence of catalytically and structurally similar P-450s, at least for the major ones, in the olfactory mucosa of mammals. PMID:9173903

Longo, V; Amato, G; Santucci, A; Gervasi, P G

1997-01-01

62

Therapeutic Doses of SkQ1 Do Not Induce Cytochromes P450 in Rat Liver.  

PubMed

The effect of SkQ1 (a mitochondria-targeted antioxidant) on the level of cytochromes P450 in rat liver was studied. It was found that administration of therapeutic dose of SkQ1 with drinking water for 5 days (250 nmol/kg of body weight per day) did not alter the level of cytochromes P450. Under the same conditions, the standard dose of phenobarbital used for the induction of cytochromes P450 caused the 2.7-fold increase in the content of these cytochromes. We conclude that therapeutic doses of SkQ1 do not induce cytochromes P450 in rats. PMID:25519071

Myasoedova, K N; Silachev, D N

2014-10-01

63

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

PubMed

1. The role of cytochrome P-450 in the one-electron reduction of mitomycin c was studied in rat hepatic microsomal systems and in reconstituted systems of purified cytochrome P-450. Formation of H2O2 from redox cycling of the reduced mitomycin c in the presence of O2 and the alkylation of p-nitrobenzylpyridine (NBP) in the absence of O2 were taken as parameters. 2. With liver microsomes from both 3-methylcholanthrene (MC)- and phenobarbital (PB)-pretreated rats, reverse type I difference spectra were observed, indicative of a weak interaction between mitomycin c and the substrate binding site of cytochrome P-450. Mitomycin c inhibited the oxidative dealkylation of aminopyrine and ethoxyresorufin in both microsomal systems. 3. Under aerobic conditions the H2O2 production in the microsomal systems was dependent on NADPH, O2 and mitomycin c, and was inhibited by the cytochrome P-450 inhibitors, metyrapone and SKF-525A. 4. Although purified NADPH-cytochrome P-450 reductase was also effective in reduction of mitomycin c and the concomitant reduction of O2, complete microsomal systems and fully reconstituted systems of cytochrome P-450b or P-450c and the reductase were much more efficient. 5. Under anaerobic conditions in the microsomal systems both reduction of mitomycin c (measured as the rate of substrate disappearance) and the reductive alkylation of NBP were dependent on cytochrome P-450. 6. The relative rate of reduction of mitomycin c by purified NADPH-cytochrome P-450 reductase was lower than that by a complete microsomal system containing both cytochrome P-450 and a similar amount of NADPH-cytochrome P-450 reductase. 7. It is concluded that although NADPH-cytochrome P-450 reductase is active in the one-electron reduction of mitomycin c, the actual metabolic locus for the reduction of this compound in liver microsomes under a relatively low O2 tension is more likely the haem site of cytochrome P-450. PMID:2122607

Vromans, R M; van de Straat, R; Groeneveld, M; Vermeulen, N P

1990-09-01

64

Engineering of daidzein 3’-hydroxylase P450 enzyme into catalytically self-sufficient cytochrome P450  

PubMed Central

A cytochrome P450 (CYP) enzyme, 3’-daidzein hydroxylase, CYP105D7 (3’-DH), responsible for daidzein hydroxylation at the 3’-position, was recently reported. CYP105D7 (3’-DH) is a class I type of CYP that requires electrons provided through electron transfer proteins such as ferredoxin and ferredoxin reductase. Presently, we constructed an artificial CYP in order to develop a reaction host for the production of a hydroxylated product. Fusion-mediated construction with the reductase domain from self-sufficient CYP102D1 was done to increase electron transfer efficiency and coupling with the oxidative process. An artificial self-sufficient daidzein hydroxylase (3’-ASDH) displayed distinct spectral properties of both flavoprotein and CYP. The fusion enzyme catalyzed hydroxylation of daidzein more efficiently, with a kcat/Km value of 16.8??M-1?min-1, which was about 24-fold higher than that of the 3’-DH-camA/B reconstituted enzyme. Finally, a recombinant Streptomyces avermitilis host for the expression of 3’-ASDH and production of the hydroxylated product was developed. The conversion that was attained (34.6%) was 5.2-fold higher than that of the wild-type. PMID:22697884

2012-01-01

65

Nanodiscs in the studies of membrane-bound cytochrome P450 enzymes.  

PubMed

Cytochromes P450 from eukaryotes and their native redox partners cytochrome P450 reductases both belong to the class of monotopic membrane proteins containing one transmembrane anchor. Incorporation into the lipid bilayer significantly affects their equilibrium and kinetic properties and plays an important role in their interactions. We describe here the detailed protocols developed in our group for the functional self-assembly of mammalian cytochromes P450 and cytochrome P450 reductases into Nanodiscs with controlled lipid composition. The resulting preparations are fully functional, homogeneous in size, composition and oligomerization state of the heme enzyme, and show an improved stability with respect to P420 formation. We provide a brief overview of applications of Nanodisc technology to the biophysical and biochemical mechanistic studies of cytochromes P450 involved in steroidogenesis, and of the most abundant xenobiotic-metabolizing human cytochrome P450 CYP3A4. PMID:23475672

Luthra, A; Gregory, M; Grinkova, Y V; Denisov, I G; Sligar, S G

2013-01-01

66

Oxidation of pesticides by purified cytochrome P-450 isozymes from mouse liver.  

PubMed

5 cytochrome P-450 isozymes were purified from the livers of uninduced mice and reconstituted with purified NADPH cytochrome P-450 reductase and phospholipid. The pesticides parathion, fonofos, DEF, Mocap and profenofos were oxidized by the reconstituted monooxygenase system to form acetylcholinesterase (AChE) inhibitors. The bioactivation varied with the pesticide substrate and the cytochrome P-450 isozyme. Aldrin epoxidation occurred with all 5 isozymes, with cytochrome P-450 A1 being the most active. All fraction metabolized the pesticide synergist piperonyl butoxide (PBO) to form an inhibitory cytochrome P-450-PBO-metabolite complex. The reduced complex produced a spectrum in the Soret region which was characteristic for each of the cytochrome P-450 isozymes. Inhibition of aldrin epoxidation by PBO was found to be unrelated to the nature of the Soret spectrum. PMID:3983973

Levi, P E; Hodgson, E

1985-01-01

67

Bioavailable flavonoids: cytochrome P450-mediated metabolism of methoxyflavones.  

PubMed

Methoxylated flavones were recently shown to be promising cancer chemopreventive agents. Their high metabolic stability compared with the hydroxylated analogs was shown in our laboratory using the human hepatic S9 fraction with cofactors for glucuronidation, sulfation, and oxidation. In the present study, the resistance of methoxylated flavones toward oxidative metabolism was investigated with human liver microsomes and recombinant cytochrome P450 (P450) isoforms. Among 15 methoxylated flavones investigated, the two partially methylated compounds, tectochrysin and kaempferide, were among the most susceptible to microsomal oxidation (Cl(int) 283 and 82 ml/min/kg). Of the fully methylated compounds, 5,7-dimethoxyflavone and 5-methoxyflavone were the most stable (Cl(int) 13 and 18 ml/min/kg, respectively), whereas 4'-methoxyflavone, 3'-methoxyflavone, 5,4'-dimethoxyflavone, and 7,3'-dimethoxyflavone were the least stable (Cl(int) 161, 140, 119, and 92 ml/min/kg, respectively), emphasizing the importance of the positions of the methoxy substituents in the flavone ring system. Among the five P450 isoforms tested, CYP1A1 showed the highest rate of metabolism of fully methylated compounds, followed by CYP1A2 and CYP3A4. CYP2C9 and CYP2D6 gave minimal disappearance of the parent compound. Finally, in incubations with hepatic S9 fraction with cofactors for oxidation and both conjugation reactions, partially methylated flavones, as expected, were much less metabolically stable than fully methylated flavones, confirming that oxidative demethylation is the rate-limiting metabolic reaction for fully methylated flavones only. In summary, the rate of oxidative metabolism of methoxylated flavones, mainly involving CYP1A1 and CYP1A2, varied widely, even between compounds with very similar structures. PMID:17709371

Walle, U Kristina; Walle, Thomas

2007-11-01

68

Impact of liver P450 reductase suppression on cyclophosphamide activation, pharmacokinetics and antitumoral activity in a cytochrome P450-based cancer gene therapy model  

Microsoft Academic Search

The effect of the antithyroid drug methimazole (MMI) on cytochrome P450\\/P450 reductase-dependent activation of the anti-cancer prodrug cyclophosphamide (CPA) was investigated in a rat model of P450 prodrug activation-based cancer gene therapy. MMI treatment decreased the expression of hepatic P450 reductase by ?75% but did not alter P450 reductase levels in a 9L gliosarcoma growing in vivo as a subcutaneous

Zeqi Huang; Malay K Raychowdhury; David J Waxman

2000-01-01

69

Cholesterol ester oxidation by mycobacterial cytochrome P450.  

PubMed

Mycobacteria share a common cholesterol degradation pathway initiated by oxidation of the alkyl side chain by enzymes of cytochrome P450 (CYP) families 125 and 142. Structural and sequence comparisons of the two enzyme families revealed two insertions into the N-terminal region of the CYP125 family (residues 58-67 and 100-109 in the CYP125A1 sequence) that could potentially sterically block the oxidation of the longer cholesterol ester molecules. Catalytic assays revealed that only CYP142 enzymes are able to oxidize cholesteryl propionate, and although CYP125 enzymes could oxidize cholesteryl sulfate, they were much less efficient at doing so than the CYP142 enzymes. The crystal structure of CYP142A2 in complex with cholesteryl sulfate revealed a substrate tightly fit into a smaller active site than was previously observed for the complex of CYP125A1 with 4-cholesten-3-one. We propose that the larger CYP125 active site allows for multiple binding modes of cholesteryl sulfate, the majority of which trigger the P450 catalytic cycle, but in an uncoupled mode rather than one that oxidizes the sterol. In contrast, the more unhindered and compact CYP142 structure enables enzymes of this family to readily oxidize cholesteryl esters, thus providing an additional source of carbon for mycobacterial growth. PMID:25210044

Frank, Daniel J; Madrona, Yarrow; Ortiz de Montellano, Paul R

2014-10-31

70

Ab initio dynamics of the cytochrome P450 hydroxylation reaction.  

PubMed

The iron(IV)-oxo porphyrin ?-cation radical known as Compound I is the primary oxidant within the cytochromes P450, allowing these enzymes to affect the substrate hydroxylation. In the course of this reaction, a hydrogen atom is abstracted from the substrate to generate hydroxyiron(IV) porphyrin and a substrate-centered radical. The hydroxy radical then rebounds from the iron to the substrate, yielding the hydroxylated product. While Compound I has succumbed to theoretical and spectroscopic characterization, the associated hydroxyiron species is elusive as a consequence of its very short lifetime, for which there are no quantitative estimates. To ascertain the physical mechanism underlying substrate hydroxylation and probe this timescale, ab initio molecular dynamics simulations and free energy calculations are performed for a model of Compound I catalysis. Semiclassical estimates based on these calculations reveal the hydrogen atom abstraction step to be extremely fast, kinetically comparable to enzymes such as carbonic anhydrase. Using an ensemble of ab initio simulations, the resultant hydroxyiron species is found to have a similarly short lifetime, ranging between 300 fs and 3600 fs, putatively depending on the enzyme active site architecture. The addition of tunneling corrections to these rates suggests a strong contribution from nuclear quantum effects, which should accelerate every step of substrate hydroxylation by an order of magnitude. These observations have strong implications for the detection of individual hydroxylation intermediates during P450 catalysis. PMID:25681906

Elenewski, Justin E; Hackett, John C

2015-02-14

71

[Cytochrome P450 3A4 and Benzodiazepines].  

PubMed

Cytochrome P450(CYP)3A4 is one of the CYP enzymes catalyzing oxidative metabolism, and involved in the metabolism of many drugs. Among benzodiazepines, alprazolam, triazolam, brotizolam and midazolam are mainly metabolished by CYP3A4, and quazepam, diazepam and flunitrazepam are partly metabolised by this enzyme. Azole antifungals, macrolide antibiotics, calcium antagonists and grapefruit juice inhibit CYP3A4 activity, while antiepileptics and rifampicin induce the activity. The drugs affecting CYP3A4 activity inhibit or induce the metabolism of the benzodiazepines metabolised by this enzyme, and induce side effects or reduce therapeutic effects of these drugs. Therefore, the combination of the two groups of drugs should be avoided, and if it is unavoidable the dose of benzodiazepines should be adjusted. PMID:12875231

Otani, Koichi

2003-01-01

72

Induction and repression of the major phenobarbital-induced cytochrome P-450 measured by radioimmunoassay.  

PubMed Central

Two independent radioimmunoassay techniques for the major phenobarbital-inducible cytochrome P-450 (PB P-450) of rat liver microsomal membranes are described. The first technique employs as the source of radiolabelled antigen the products of translation in vitro labelled with [35S]methionine. The second technique employs purified antigen labelled with 125I and is quicker, less expensive and more precise. Both assays are highly specific for PB P-450 and can detect quantities of this variant as small as 1 ng. This is several orders of magnitude more sensitive than any method described previously for the quantification of cytochromes P-450, and consequently the technique is particularly well suited for the quantification of so-called constitutive cytochrome P-450 variants that are present in very low amounts. The results of the radioimmunoassays demonstrate that the apparent 2.6-fold induction of total cytochromes P-450 after phenobarbital treatment is due to a 43-fold increase in Pb P-450. Although beta-naphthoflavone increases the total content of cytochrome P-450 of microsomal membranes 1.4-fold, it actually causes a 55% decrease in the amount of PB P-450. Thus different xenobiotics can have differential effects on the expression of the genes for specific cytochrome P-450 variants. Images Fig. 1. Fig. 2. PMID:6870856

Phillips, I R; Shephard, E A; Bayney, R M; Pike, S F; Rabin, B R; Heath, R; Carter, N

1983-01-01

73

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

PubMed Central

LKM-1 autoantibodies, which are associated with autoimmune chronic active hepatitis, recognize P450IID6, a cytochrome P-450 monooxygenase. The reactivities of 26 LKM-1 antisera were tested with a panel of deletion mutants of P450IID6 expressed in Escherichia coli. 22 sera recognize a 33-amino acid segment of P450IID6, and 11 of these recognize a shorter segment, DPAQPPRD. PAQPPR is also found in IE175 of herpes simplex virus type 1 (HSV-1). Antibodies for HSV-1 proteins were detected by ELISA in 17 of 20 LKM-1 sera tested. An immobilized, synthetic peptide, DPAQPPRDC, was used to purify LKM-1 antibodies. Affinity purified LKM-1 autoantibodies react on immunoblots with a protein in BHK cells after infection with HSV-1. 11 of 24 LKM-1 sera, including 3 that recognize DPAQPPRD, also exhibit antibodies to the hepatitis C virus (HCV) protein, C100-3. Affinity purified LKM-1 antibodies did not recognize C100-3. However, partial sequence identity was evident between portions of the immunopositive 33-amino acid segment of P450IID6 and other portions of the putative HCV polyprotein. Immune cross-recognition of P450IID6 and HCV or HSV-1 proteins may contribute to the occurrence of LKM-1 autoantibodies. Images PMID:1717511

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

1991-01-01

74

Constitutive expression of hepatic cytochrome P450 genes  

Microsoft Academic Search

One of the more challenging areas for study of the regulation of P450s is understanding the constitutive regulation of hepatic P450s. In this article, Gonzalez and Lee provide insight into how unique tissue-en- riched transcription factors regulate expression of many of the hepatic P450s that are not under direct control of the \\

FRANK J. GONZALEZ; YING-HUE LEE

1996-01-01

75

Bacterial Cytochrome P450 System Catabolizing the Fusarium Toxin Deoxynivalenol  

PubMed Central

Deoxynivalenol (DON) is a natural toxin of fungi that cause Fusarium head blight disease of wheat and other small-grain cereals. DON accumulates in infected grains and promotes the spread of the infection on wheat, posing serious problems to grain production. The elucidation of DON-catabolic genes and enzymes in DON-degrading microbes will provide new approaches to decrease DON contamination. Here, we report a cytochrome P450 system capable of catabolizing DON in Sphingomonas sp. strain KSM1, a DON-utilizing bacterium newly isolated from lake water. The P450 gene ddnA was cloned through an activity-based screening of a KSM1 genomic library. The genes of its redox partner candidates (flavin adenine dinucleotide [FAD]-dependent ferredoxin reductase and mitochondrial-type [2Fe-2S] ferredoxin) were not found adjacent to ddnA; the redox partner candidates were further cloned separately based on conserved motifs. The DON-catabolic activity was reconstituted in vitro in an electron transfer chain comprising the three enzymes and NADH, with a catalytic efficiency (kcat/Km) of 6.4 mM?1 s?1. The reaction product was identified as 16-hydroxy-deoxynivalenol. A bioassay using wheat seedlings revealed that the hydroxylation dramatically reduced the toxicity of DON to wheat. The enzyme system showed similar catalytic efficiencies toward nivalenol and 3-acetyl deoxynivalenol, toxins that frequently cooccur with DON. These findings identify an enzyme system that catabolizes DON, leading to reduced phytotoxicity to wheat. PMID:23275503

Ito, Michihiro; Sato, Ikuo; Ishizaka, Masumi; Yoshida, Shin-ichiro; Koitabashi, Motoo; Yoshida, Shigenobu

2013-01-01

76

Transcriptional Regulation of Grape Cytochrome P450 Gene Expression in Response to Xylella fastidiosa  

Technology Transfer Automated Retrieval System (TEKTRAN)

Plant cytochrome P450 monooxygenases are versatile redox proteins that mediate biosynthesis of lignins, terpenes, alkaloids, and a variety of other secondary compounds as plant defense agents against a range of pathogens and insects. To determine if cytochrome P450 monooxygenases are involved in the...

77

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

SciTech Connect

Highlights: {yields} Mutations in human POR cause congenital adrenal hyperplasia. {yields} We are reporting a novel 3 amino acid deletion mutation in POR P399{sub E}401del. {yields} POR mutation P399{sub E}401del decreased P450 activities by 60-85%. {yields} Impairment of steroid metabolism may be caused by multiple hits. {yields} Severity of aromatase inhibition is related to degree of in utero virilization. -- Abstract: P450 oxidoreductase (POR) is the electron donor for all microsomal P450s including steroidogenic enzymes CYP17A1, CYP19A1 and CYP21A2. We found a novel POR mutation P399{sub E}401del in two unrelated Turkish patients with 46,XX disorder of sexual development. Recombinant POR proteins were produced in yeast and tested for their ability to support steroid metabolizing P450 activities. In comparison to wild-type POR, the P399{sub E}401del protein was found to decrease catalytic efficiency of 21-hydroxylation of progesterone by 68%, 17{alpha}-hydroxylation of progesterone by 76%, 17,20-lyase action on 17OH-pregnenolone by 69%, aromatization of androstenedione by 85% and cytochrome c reduction activity by 80%. Protein structure analysis of the three amino acid deletion P399{sub E}401 revealed reduced stability and flexibility of the mutant. In conclusion, P399{sub E}401del is a novel mutation in POR that provides valuable genotype-phenotype and structure-function correlation for mutations in a different region of POR compared to previous studies. Characterization of P399{sub E}401del provides further insight into specificity of different P450s for interaction with POR as well as nature of metabolic disruptions caused by more pronounced effect on specific P450s like CYP17A1 and aromatase.

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

2011-09-09

78

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. PMID:17544277

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

2007-01-01

79

Survey of Human Oxidoreductases and Cytochrome P450 Enzymes Involved in the Metabolism of Chemicals.  

PubMed

Analyzing the literature resources used in our previous reports, we calculated the fractions of the oxidoreductase enzymes FMO (microsomal flavin-containing monooxygenase), AKR (aldo-keto reductase), MAO (monoamine oxidase), and cytochrome P450 participating in metabolic reactions. The calculations show that the fractions of P450s involved in metabolism of all chemicals (general chemicals, natural and physiological compounds, and drugs) are rather consistent in the findings that > 90% of enzymatic reactions are catalyzed by P450s. Regarding drug metabolism, three-fourths of the human P450 reactions can be accounted for by a set of five P450s: 1A2, 2C9, 2C19, 2D6, and 3A4, and the largest fraction of the P450 reactions is catalyzed by P450 3A enzymes. P450 3A4 participation in metabolic reactions of drugs varied from 13% for general chemicals to 27% for drugs. PMID:25485457

Rendic, Slobodan Petar; Guengerich, F Peter

2014-12-01

80

Pharmacogenetic biomarkers: cytochrome P450 3A5.  

PubMed

The immunosuppressive drugs used for solid organ transplantation all have a narrow therapeutic index with wide variation between individuals in the blood concentration achieved by a given dose. Therapeutic drug monitoring is employed routinely but may not allow optimisation of drug exposure during the critical period two to three days following transplantation. A key factor in the inter-individual variability for tacrolimus, and probably sirolimus, is whether an individual is genetically predicted to express the drug metabolising enzyme cytochrome P450 3A5 (CYP3A5). Individuals predicted to express CYP3A5 by possession of at least one wild-type CYP3A5*1 allele require 1.5-2 times higher doses of tacrolimus to achieve target blood concentrations than individuals homozygous for the CYP3A5*3 allele who are functional non-expressers of CYP3A5. Planning the initial tacrolimus dose based on the CYP3A5 genotype has been shown to allow more rapid achievement of target blood concentrations after transplantation than a standard dose given to all patients. However, it remains to be demonstrated that use of this approach as an adjunct to therapeutic drug monitoring can reduce either efficacy failure (transplant rejection) or toxicity. Use of a pharmacogenetic approach to dosing sirolimus awaits testing and it is unlikely to be useful for ciclosporin or everolimus. PMID:22037511

MacPhee, Iain A M

2012-09-01

81

The role of cytochrome b5 structural domains in interaction with cytochromes P450.  

PubMed

To understand the role of the structural elements of cytochrome b5 in its interaction with cytochrome P450 and the catalysis performed by this heme protein, we carried out comparative structural and functional analysis of the two major mammalian forms of membrane-bound cytochrome b5 - microsomal and mitochondrial, designed chimeric forms of the heme proteins in which the hydrophilic domain of one heme protein is replaced by the hydrophilic domain of another one, and investigated the effect of the highly purified native and chimeric heme proteins on the enzymatic activity of recombinant cytochromes P4503A4 and P45017A1 (CYP3A4 and CYP17A1). We show that the presence of a hydrophobic domain in the structure of cytochrome b5 is necessary for its effective interaction with its redox partners, while the nature of the hydrophobic domain has no significant effect on the ability of cytochrome b5 to stimulate the activity of cytochrome P450-catalyzed reactions. Thus, the functional properties of cytochrome b5 are mainly determined by the structure of the heme-binding domain. PMID:24954591

Sergeev, G V; Gilep, A A; Usanov, S A

2014-05-01

82

Chlormethiazole inhibition of cytochrome P450 2E1 as assessed by chlorzoxazone hydroxylation in humans  

Microsoft Academic Search

Chlormethiazole is a sedative and anticonvulsive drug used in the treatment of alcohol withdrawal. Because it had been reported that chlormethiazole inhibits the alcohol-inducible cytochrome P450 2E1 in rat liver, we investigated the in vivo and in vitro effect of this drug on cytochrome P450 2E1 in human beings. The activity of this cytochrome was assessed using chlorzoxazone as a

AC Gebhardt; D Lucas; J Menez; HK Seitz

1997-01-01

83

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

PubMed Central

The interactions of protein components of the xenobiotic-metabolizing cytochrome P450 system, CYP6A1, P450 reductase, and cytochrome b5 from the house fly (Musca domestica) have been characterized. CYP6A1 activity is determined by the concentration of the CYP6A1-P450 reductase complex, regardless of which protein is present in excess. Both holo- and apo-b5 stimulated CYP6A1 heptachlor epoxidase and steroid hydroxylase activities and influenced the regioselectivity of testosterone hydroxylation. The conversion of CYP6A1 to its P420 form was decreased by the addition of apo-b5. The effects of cytochrome b5 may involve allosteric modification of the P450 enzyme that modify the conformation of the active site. The overall stoichiometry of the P450 reaction was substrate-dependent. High uncoupling of CYP6A1 was observed with generation of hydrogen peroxide, in excess over the concomitant testosterone hydroxylation or heptachlor epoxidation. Inclusion of cytochrome b5 in the reconstituted system improved efficiency of oxygen consumption and electron utilization from NADPH, or coupling of the P450 reaction. Depending on the reconstitution conditions, coupling efficiency varied from 8 to 25% for heptachlor epoxidation, and from 11 to 70% for testosterone hydroxylation. Because CYP6A1 is a P450 involved in insecticide resistance, this suggests that xenobiotic metabolism by constitutively overexpressed P450s may be linked to significant oxidative stress in the cell that may carry a fitness cost. PMID:18930820

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

2008-01-01

84

Hepatic Cytochrome P-450Dependent Metabolism and Enzymatic Conjugation of Foreign Compounds in Vitamin A-Deficient Rats  

Microsoft Academic Search

The temporal effects of vitamin A deficiency on hepatic cytochrome P-450-de pendent and conjugation reactions were studied in the rat. Cytochrome P-450 levels and N-methyl-p-chloroaniline N-demethylase activity were significantly reduced in the deficient animals. No other changes in parameters dependent on cytochrome P-450 were observed in vitro. Decreases in hepatic cytochrome P450 were accompanied by a prolongation in hexobarbital sleeping

Zahid H. Siddik; Roger Drew; Charles L. Litterst; Edward G. Mimnaugh; Branimir I. Sikic; Theodore E. Gram

1980-01-01

85

Effect of kanglaite on rat cytochrome P450.  

PubMed

Abstract Context: Kanglaite (KLT) is an oily substance extracted from Coix lacryma-jobi Linn. (Cramineae) and has been proved to significantly improve the life span and quality of life of patients, when combined with chemotherapy, radiotherapy, or surgery. Objective: The purpose of this study was to find out whether KLT influences the effect on rat cytochrome P450 (CYP) enzymes (CYP1A2, CYP2B6, CYP2C9, CYP2C19, and CYP3A4) by using cocktail probe drugs in vivo. Materials and methods: A cocktail solution at a dose of 5?mL/kg, which contained phenacetin (20?mg/kg), bupropion (20?mg/kg), tolbutamide (5?mg/kg), omeprazole (20?mg/kg), and midazolam (10?mg/kg), was given as oral administration to rats treated with 7?d intraperitoneal injection of KLT. Blood samples were collected at a series of time-points and the concentrations of probe drugs in plasma were determined by HPLC-MS/MS. The corresponding pharmacokinetic parameters were calculated by the software of DAS 2.0 (SPPS Inc., Chicago, IL). Results: In the experiment, there was a statistically significant difference in the t1/2, Cmax, AUC(0-?), and CL for phenacetin, bupropion, tolbutamide, omeprazole, and midazolam. Our study showed that treatment with multiple doses of KLT had induction effect on rat CYP1A2, while CYP2B6, CYP2C9, CYP2C19, and CYP3A4 enzyme activities had been inhibited after multiple doses of KLT treatment. Conclusions: KLT can either induce or inhibit activities of CYP. Therefore, caution is needed when KLT is co-administration with some CYP substrates in clinic, which may result in herb-drug interactions. PMID:25472767

Du, Xiaoxiang; Ye, Huizhou; Zhang, Chunxia; Ye, Lei; Lin, Guanyang

2014-12-01

86

Cytochrome P450-Dependent Metabolism of Caffeine in Drosophila melanogaster  

PubMed Central

Caffeine (1, 3, 7-trimethylxanthine), an alkaloid produced by plants, has antioxidant and insecticide properties that can affect metabolism and cognition. In vertebrates, the metabolites derived from caffeine have been identified, and their functions have been characterized. However, the metabolites of caffeine in insects remain unknown. Thus, using radiolabelled caffeine, we have identified some of the primary caffeine metabolites produced in the body of Drosophila melanogaster males, including theobromine, paraxanthine and theophylline. In contrast to mammals, theobromine was the predominant metabolite (paraxanthine in humans; theophylline in monkeys; 1, 3, 7-trimethyluric acid in rodents). A transcriptomic screen of Drosophila flies exposed to caffeine revealed the coordinated variation of a large set of genes that encode xenobiotic-metabolizing proteins, including several cytochromes P450s (CYPs) that were highly overexpressed. Flies treated with metyrapone—an inhibitor of CYP enzymes—showed dramatically decreased caffeine metabolism, indicating that CYPs are involved in this process. Using interference RNA genetic silencing, we measured the metabolic and transcriptomic effect of three candidate CYPs. Silencing of CYP6d5 completely abolished theobromine synthesis, whereas CYP6a8 and CYP12d1 silencing induced different consequences on metabolism and gene expression. Therefore, we characterized several metabolic products and some enzymes potentially involved in the degradation of caffeine. In conclusion, this pioneer approach to caffeine metabolism in insects opens novel perspectives for the investigation of the physiological effects of caffeine metabolites. It also indicates that caffeine could be used as a biomarker to evaluate CYP phenotypes in Drosophila and other insects. PMID:25671424

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

2015-01-01

87

Cytochrome P450-Dependent Metabolism of Caffeine in Drosophila melanogaster.  

PubMed

Caffeine (1, 3, 7-trimethylxanthine), an alkaloid produced by plants, has antioxidant and insecticide properties that can affect metabolism and cognition. In vertebrates, the metabolites derived from caffeine have been identified, and their functions have been characterized. However, the metabolites of caffeine in insects remain unknown. Thus, using radiolabelled caffeine, we have identified some of the primary caffeine metabolites produced in the body of Drosophila melanogaster males, including theobromine, paraxanthine and theophylline. In contrast to mammals, theobromine was the predominant metabolite (paraxanthine in humans; theophylline in monkeys; 1, 3, 7-trimethyluric acid in rodents). A transcriptomic screen of Drosophila flies exposed to caffeine revealed the coordinated variation of a large set of genes that encode xenobiotic-metabolizing proteins, including several cytochromes P450s (CYPs) that were highly overexpressed. Flies treated with metyrapone-an inhibitor of CYP enzymes-showed dramatically decreased caffeine metabolism, indicating that CYPs are involved in this process. Using interference RNA genetic silencing, we measured the metabolic and transcriptomic effect of three candidate CYPs. Silencing of CYP6d5 completely abolished theobromine synthesis, whereas CYP6a8 and CYP12d1 silencing induced different consequences on metabolism and gene expression. Therefore, we characterized several metabolic products and some enzymes potentially involved in the degradation of caffeine. In conclusion, this pioneer approach to caffeine metabolism in insects opens novel perspectives for the investigation of the physiological effects of caffeine metabolites. It also indicates that caffeine could be used as a biomarker to evaluate CYP phenotypes in Drosophila and other insects. PMID:25671424

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

2015-01-01

88

Uroporphyrinogen oxidation catalyzed by human cytochromes P450.  

PubMed

Porphyria cutanea tarda is associated with excess hepatic production of uroporphyrin. Oxidation of uroporphyrinogen to uroporphyrin was previously demonstrated to be specifically catalyzed by cytochrome P450 (CYP) 1A2. Here, we investigated the ability of human CYP1A2 to catalyze uroporphyrinogen oxidation (UROX). UROX activity in human liver microsomes was maximally only 10% of the activity in microsomes from livers of untreated mice. There was a poor correlation of UROX activity with methoxyresorufin demethylation, an activity catalyzed predominantly by CYP1A2 and strongly correlated with immunodetectable CYP1A2. With CYP forms expressed in HepG2 cells, the methoxyresorufin demethylation and (ethoxyresorufin deethylation) activities of murine and human CYP1A2 forms were similar, but UROX activity catalyzed by human CYP1A2 was only 15-20% of the activity catalyzed by murine CYP1A2. Human CYP1A1, CYP1A2, and CYP3A4 expressed in lymphoblastoid cells all catalyzed UROX. In insect cells, CYP1A2 was more active in catalyzing UROX than was CYP1A1, CYP2E, CYP3A4, or CYP3A5. Human CYP1A2 expressed in Escherichia coli as a fusion protein with rat CYP oxidoreductase also catalyzed UROX. Reconstituted human CYP1A2 and CYP3A4 were active in catalyzing UROX, with reconstituted CYP1A2 having the highest specific activity obtained in this study. From inhibitor studies, it was concluded that some of the UROX activity in the insect cell microsomes was attributable to expressed CYP and some to an unidentified source. These results indicate that human CYP1A2 is active in catalyzing UROX but has lower activity than the murine orthologue. The results also indicate that most of the UROX activity found in human liver microsomes is not due to CYP1A2. PMID:9763408

Sinclair, P R; Gorman, N; Tsyrlov, I B; Fuhr, U; Walton, H S; Sinclair, J F

1998-10-01

89

Chemical regulation of abscisic acid catabolism in plants by cytochrome P450 inhibitors  

Microsoft Academic Search

Plant hormone abscisic acid (ABA) is an important factor for conferring drought stress resistance on plants. Therefore, small molecules that regulate ABA levels in plants can be useful both for investigating functions of ABA and for developing new plant growth regulators. Abscisic acid (ABA) catabolism in plants is primarily regulated by ABA 8?-hydroxylase, which is a cytochrome P450 (P450). We

Nobutaka Kitahata; Shigeki Saito; Yutaka Miyazawa; Taishi Umezawa; Yukihisa Shimada; Yong Ki Min; Masaharu Mizutani; Nobuhiro Hirai; Kazuo Shinozaki; Shigeo Yoshida; Tadao Asami

2005-01-01

90

PROTEINS FROM EIGHT EUKARYOTIC CYTOCHROME P-450 FAMILIES SHARE A SEGMENTED REGION OF SEQUENCE SIMILARITY  

EPA Science Inventory

Proteins from eight eukaryotic families in the cytochrome P-450 superfamily share one region of sequence similarity. his region begins 275-310 amino acids from the amino terminus of each P-450, continues for 170 residues, and ends 35-50 amino acids before the carboxyl terminus. h...

91

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...

92

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

PubMed Central

In a subgroup of children with chronic active hepatitis, circulating autoantibodies occur that bind to liver and kidney endoplasmic reticulum (anti-liver/kidney microsome antibody type I or anti-LKM1). Anti-LKM1 titers follow the severity of the disease and the presence of these antibodies serves as a diagnostic marker for this autoimmune hepatitis type II. We demonstrate that anti-LKM1 IgGs specifically inhibit the hydroxylation of bufuralol in human liver microsomes. Using two assay systems with different selectivity for the two cytochrome P-450 isozymes catalyzing bufuralol metabolism in human liver, we show that anti-LKM1 exclusively recognizes cytochrome P-450db1. Immunopurification of the LKM1 antigen from solubilized human liver microsomes resulted in an electrophoretically homogenous protein that had the same molecular mass (50 kDa) as purified P-450db1 and an identical N-terminal amino acid sequence. Recognition of both purified P-450db1 and the immunoisolated protein on western blots by several monoclonal antibodies confirmed the identity of the LKM1 antigen with cytochrome P-450db1. Cytochrome P-450db1 has been identified as the target of a common genetic polymorphism of drug oxidation. However, the relationship between the polymorphic cytochrome P-450db1 and the appearance of anti-LKM1 autoantibodies as well as their role in the pathogenesis of chronic active hepatitis remains speculative. Images PMID:3186722

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

1988-01-01

93

Steroid transformation by Rhodococcus strains and bacterial cytochrome P450 enzymes.  

E-print Network

??The thesis describes activities of four bacterial steroid-induced cytochrome P450 enzymes in terms of their potential biotechnological use for steroid transformations by engineered Rhodococcus strains… (more)

Du Plessis-Rosloniec, Kamila Zofia

2011-01-01

94

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

95

Optical detection of cytochrome P450 by sensitizer-linked substrates  

PubMed Central

The ability to detect, characterize, and manipulate specific biomolecules in complex media is critical for understanding metabolic processes. Particularly important targets are oxygenases (cytochromes P450) involved in drug metabolism and many disease states, including liver and kidney dysfunction, neurological disorders, and cancer. We have found that Ru photosensitizers linked to P450 substrates specifically recognize submicromolar cytochrome P450cam in the presence of other heme proteins. In the P450:Ru-substrate conjugates, energy transfer to the heme dramatically accelerates the Ru-luminescence decay. The crystal structure of a P450cam:Ru-adamantyl complex reveals access to the active center via a channel whose depth (Ru-Fe distance is 21 ?) is virtually the same as that extracted from an analysis of the energy-transfer kinetics. Suitably constructed libraries of sensitizer-linked substrates could be employed to probe the steric and electronic properties of buried active sites. PMID:10557259

Dmochowski, Ivan J.; Crane, Brian R.; Wilker, Jonathan J.; Winkler, Jay R.; Gray, Harry B.

1999-01-01

96

Stimulation by paraquat of microsomal and cytochrome P-450-dependent oxidation of glycerol to formaldehyde.  

PubMed Central

Glycerol can be oxidized to formaldehyde by microsomes in a reaction that is dependent on cytochrome P-450. An oxidant derived from the interaction of H2O2 with iron was responsible for oxidizing the glycerol, with P-450 suggested to be necessary to produce H2O2 and reduce non-haem iron. The effect of paraquat on formaldehyde production from glycerol and whether paraquat could replace P-450 in supporting this reaction were studied. Paraquat increased NADPH-dependent microsomal oxidation of glycerol; the stimulation was inhibited by glutathione, catalase, EDTA and desferrioxamine, but not by superoxide dismutase or hydroxyl-radical scavengers. The paraquat stimulation was also inhibited by inhibitors, substrate and ligand for P-4502E1 (pyrazole-induced P-450 isozyme), as well as by anti-(P-4502E1) IgG. These results suggest that P-450 still played an important role in glycerol oxidation, even in the presence of paraquat. Purified NADPH-cytochrome P-450 reductase did not oxidize glycerol to formaldehyde; some oxidation, however, did occur in the presence of paraquat. Reductase plus P-4502E1 oxidized glycerol, and a large stimulation was observed in the presence of paraquat. Rates in the presence of P-450, reductase and paraquat were more than additive than the sums from the reductase plus P-450 and reductase plus paraquat rates, suggesting synergistic interactions between paraquat and P-450. These results indicate that paraquat increases oxidation of glycerol to formaldehyde by microsomes and reconstituted systems, that H2O2 and iron play a role in the overall reaction, and that paraquat can substitute, in part, for P-450 in supporting oxidation of glycerol. However, cytochrome P-450 is required for elevated rates of formaldehyde production even in the presence of paraquat. PMID:8240292

Clejan, L A; Cederbaum, A I

1993-01-01

97

Functional consequences of active hepatic uptake on cytochrome P450 inhibition in rat and human hepatocytes.  

PubMed

A series of cytochrome P450 (P450) inhibition experiments were conducted with four hepatic uptake substrates (AZ3, AZ25, atorvastatin, and pitavastatin) using hepatocytes and recombinant P450s. The uptake was shown to be temperature-dependent and was inhibited by estrone sulfate, signifying an active component. At the lowest concentrations tested, the inhibitors concentrated up to 1000-fold in rat hepatocytes, but demonstrated only 5-fold greater P450 inhibition relative to recombinant rat P450s, indicating high intracellular binding. Inhibitor accumulation was considerably lower in human hepatocytes and an increase in inhibitory potency relative to recombinant human P450s was not obvious. This study highlights several technical and conceptual issues in the study of P450 inhibition mediated by compounds actively transported across the basolateral hepatocyte membrane. Primarily, the incubation medium concentration once the inhibitor has fully accumulated into the hepatocytes rather than the starting medium concentration, along with the extent of intracellular binding, must be considered as a foundation for in vitro-in vivo extrapolations. Additionally, it is suggested that if the K(m) value for the active uptake process is close to the P450 inhibition K(i), hepatocytes may be used only to establish the free drug accumulation ratio at a clinically relevant drug concentration, and this information, along with the (recombinant P450) K(i) value, may be used to simulate the likely impact of active hepatic uptake on P450 inhibition in vivo. PMID:18474678

Grime, Ken; Webborn, Peter J H; Riley, Robert J

2008-08-01

98

Distinct organization of methylcholanthrene- and phenobarbital-inducible cytochrome P-450 genes in the rat.  

PubMed Central

The complete nucleotide sequence of the methylcholanthrene-inducible cytochrome P-450c gene was determined by sequence analysis of cloned genomic DNA and the sequence, consisting of 524 amino acids, of the protein was deduced therefrom. The gene for the cytochrome was approximately 6.0 kilobases long and was split into seven exons. Comparison of the gene with that of the phenobarbital-inducible cytochrome P-450e showed that the gene structures for the two types of cytochrome P-450 differ greatly; the location, number, and size of intervening sequences are very dissimilar. However, the sequence homology between the two types of cytochrome suggests that the two genes have evolved from a common ancestor. Images PMID:6089174

Sogawa, K; Gotoh, O; Kawajiri, K; Fujii-Kuriyama, Y

1984-01-01

99

Cofactor-Free Light-Driven Whole-Cell Cytochrome P450 Catalysis.  

PubMed

Cytochromes P450 can catalyze various regioselective and stereospecific oxidation reactions of non-functionalized hydrocarbons. Here, we have designed a novel light-driven platform for cofactor-free, whole-cell P450 photo-biocatalysis using eosin Y (EY) as a photosensitizer. EY can easily enter into the cytoplasm of Escherichia coli and bind specifically to the heme domain of P450. The catalytic turnover of P450 was mediated through the direct transfer of photoinduced electrons from the photosensitized EY to the P450 heme domain under visible light illumination. The photoactivation of the P450 catalytic cycle in the absence of cofactors and redox partners is successfully conducted using many bacterial P450s (variants of P450 BM3) and human P450s (CYPs 1A1, 1A2, 1B1, 2A6, 2E1, and 3A4) for the bioconversion of different substrates, including marketed drugs (simvastatin, lovastatin, and omeprazole) and a steroid (17?-estradiol), to demonstrate the general applicability of the light-driven, cofactor-free system. PMID:25430544

Park, Jong Hyun; Lee, Sahng Ha; Cha, Gun Su; Choi, Da Som; Nam, Dong Heon; Lee, Jae Hyung; Lee, Jung-Kul; Yun, Chul-Ho; Jeong, Ki Jun; Park, Chan Beum

2014-11-27

100

Biomonitoring environmental contamination with pipping black-crowned night heron embryos: Induction of cytochrome P450  

USGS Publications Warehouse

Cytochrome P450-associated monooxygenase activities and cytochrome P450 proteins were measured in pipping black-crowned night heron (Nycticorax nycticorax) embryos collected from a reference site (next to the Chincoteague National Wildlife Refuge, VA) and three polluted sites (Cat Island, Green Bay, Lake Michigan, WI; Bair Island, San Francisco Bay, CA; West Marin Island, San Francisco Bay, CA). In a laboratory study, artificially incubated night heron embryos from the reference site were treated with 3-methylcholanthrene (200 mu g administered into the air cell 2 d before pipping) or phenobarbital (2 mg daily for 2 d before pipping). Compared to controls (untreated + vehicle-treated embryos), 3-methylcholanthrene induced a greater than fivefold increase in activities of several monooxygenases (arylhydrocarbon hydroxylase, AHH; benzyloxyresorufin-O-dealkylase, BROD; ethoxyresorufin-O-dealkylase, EROD; pentoxyresorufin-O- dealkylase, PROD) and a greater than 100-fold increase in the concentration of immunodetected cytochrome P450 1A (CYP1A). Phenobarbital treatment resulted in only a slight increase in BROD activity but induced proteins recognized by antibodies to cytochrome P450 2B (CYP2B) by 2,000-fold. In a field study, activities of AHH, BROD, EROD, and ethoxycoumarin-O-dealkylase (ECOD) were up to 85-fold higher in pipping black- crowned night herons collected from Cat Island compared to other sites. Hepatic CYP1A and CYP2B cross- reactive proteins were detected in significantly more individuals from Cat Island than from the reference site. Greatest burdens of total PCBs and p,p'-DDE were detected in embryos from Cat Island. Cytochrome P450- associated monooxygenase activities and cytochrome P450 proteins (AHH, BROD, EROD, ECOD, CYP1A, CYP2B) were significantly associated with total PCB burdens (r = 0.50-0.72). These data indicate that cytochrome P450 may be a useful biomarker of exposure to some PCB mixtures in black-crowned night heron embryos.

Rattner, B.A.; Melancon, M.J.; Custer, T.W.; Hothem, R.L.; King, K.A.; LeCaptain, L.J.; Spann, J.W.; Woodin, B.R.; Stegeman, J.J.

1993-01-01

101

Biomonitoring environmental contamination with pipping black-crowned night heron embryos: Induction of cytochrome P450  

USGS Publications Warehouse

Cytochrome P450-associated monooxygenase activities and cytochrome P450 proteins were measured in pipping black-crowned night heron (Nycticorax nycticorax) embryos collected from a reference site (next to the Chincoteague National Wildlife Refuge, VA) and three polluted sites (Cat Island, Green Bay, Lake Michigan, WI; Bair Island, San Francisco Bay, CA; West Marin Island, San Francisco Bay, CA). In a laboratory study, artificially incubated night heron embryos from the reference site were treated with 3-methylcholanthrene (200 mu-g administered into the air cell 2 d before pipping) or phenobarbital (2 mg daily for 2 d before pipping). Compared to controls (untreated + vehicle-treated embryos), 3-methylcholanthrene induced a greater than five-fold increase in activities of several monooxygenases (arylhydrocarbon hydroxylase, AHH; benzyloxyresorufin-O-dealkylase, BROD; ethoxyresorufin-O-dealkylase, EROD; pentoxyresorufin-O-dealkylase, PROD) and a greater than 100-fold increase in the concentration of immunodetected cytochrome P450 1A (CYP1A). Phenobarbital treatment resulted in only a slight increase in BROD activity but induced proteins recognized by antibodies to cytochrome P450 2B (CYP2B) by 2,000-fold. In a field study, activities of AHH, BROD, EROD, and ethoxycoumarin-O-dealkylase (ECOD) were up to 85-fold higher in pipping black-crowned night herons collected from Cat Island compared to other sites. Hepatic CYP1A and CYP2B cross-reactive proteins were detected in significantly more individuals from Cat Island than from the reference site. Greatest burdens of total PCBs and p, p'-DDE were detected in embryos from Cat Island. Cytochrome P450-associated monooxygenase activities and cytochrome P450 proteins (AHH, BROD, EROD, ECOD, CYP1A, CYP2B) were significantly associated with total PCB burdens (r = 0.50-0.72). These data indicate that cytochrome P450 may be a useful biomarker of exposure to some PCB mixtures in black-crowned night heron embryos.

Rattner, B.A.; Melancon, M.J.; Custer, T.W.; Hothem, R.L.; King, K.A.; LeCaptain, L.J.; Spann, J.W.; Woodin, B.R.; Stegeman, J.J.

1993-01-01

102

The rabbit pulmonary cytochrome P450 arachidonic acid metabolic pathway: characterization and significance.  

PubMed Central

Cytochrome P450 metabolizes arachidonic acid to several unique and biologically active compounds in rabbit liver and kidney. Microsomal fractions prepared from rabbit lung homogenates metabolized arachidonic acid through cytochrome P450 pathways, yielding cis-epoxyeicosatrienoic acids (EETs) and their hydration products, vic-dihydroxyeicosatrienoic acids, mid-chain cis-trans conjugated dienols, and 19- and 20-hydroxyeicosatetraenoic acids. Inhibition studies using polyclonal antibodies prepared against purified CYP2B4 demonstrated 100% inhibition of arachidonic acid epoxide formation. Purified CYP2B4, reconstituted in the presence of NADPH-cytochrome P450 reductase and cytochrome b5, metabolized arachidonic acid, producing primarily EETs. EETs were detected in lung homogenate using gas chromatography/mass spectroscopy, providing evidence for the in vivo pulmonary cytochrome P450 epoxidation of arachidonic acid. Chiral analysis of these lung EETs demonstrated a preference for the 14(R),15(S)-, 11(S),12(R)-, and 8(S),9(R)-EET enantiomers. Both EETs and vic-dihydroxyeicosatrienoic acids were detected in bronchoalveolar lavage fluid. At micromolar concentrations, methylated 5,6-EET and 8,9-EET significantly relaxed histamine-contracted guinea pig hilar bronchi in vitro. In contrast, 20-hydroxyeicosatetraenoic acid caused contraction to near maximal tension. We conclude that CYP2B4, an abundant rabbit lung cytochrome P450 enzyme, is the primary constitutive pulmonary arachidonic acid epoxygenase and that these locally produced, biologically active eicosanoids may be involved in maintaining homeostasis within the lung. Images PMID:7738183

Zeldin, D C; Plitman, J D; Kobayashi, J; Miller, R F; Snapper, J R; Falck, J R; Szarek, J L; Philpot, R M; Capdevila, J H

1995-01-01

103

Cytochrome P450 inhibition assay for standardized extract of Terminalia chebula Retz.  

PubMed

The hydroalcoholic extract of fruit pulp of Terminalia chebula Retz. was standardized and evaluated for its safety through cytochrome P450 (CYP 450) inhibition assay. Standardization was performed through high performance thin layer chromatography (HPTLC) using gallic acid (GA) standard. Cytochrome P450-CO complex microplate assay was performed using rat liver microsomes. The effect of standardized extract, its fraction and bioactive marker compound were comparatively evaluated for its effect on CYP P450 enzymes. The extract of fruit pulp was used for HPTLC, where the R(f) value of the marker was found to be 0.43. The calibration plot was linear in the range of 2-14?µg of GA and correlation co-efficient of 0.99965. The mean quantity of GA was found to be 2.5% w/w. The CYP P450 concentration of the rat liver microsome sample used in the study was found to be 0.417?nmol/mg protein. The in vitro effect of various concentrations of extracts and fractions showed a linear concentration-dependent inhibition of cytochrome P450 up to 60?µL. The study showed more inhibition of fraction when compared to the extract and GA. Still, the inhibition showed by fraction is less when compared with standard Ketoconazole. Thus, this study indicated the in vitro cytochrome P450 inhibition potential of T. Chebula. PMID:20623819

Ponnusankar, S; Pandit, S; Venkatesh, M; Bandyopadhyay, A; Mukherjee, Pulok K

2011-01-01

104

THE PLANETARY BIOLOGY OF CYTOCHROME P450 AROMATASES  

Technology Transfer Automated Retrieval System (TEKTRAN)

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 p...

105

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

106

Cytochrome P450-encoding genes from the Heliconius genome as candidates for cyanogenesis.  

PubMed

Cytochrome P450s are important both in the metabolism of xenobiotics and the production of compounds such as cyanogenic glucosides, which insects use in their defence. In the present study, we use transcriptomic and genomic information to isolate and name P450-encoding genes from the butterfly Heliconius melpomene. We classify each of the putative genes into its appropriate superfamily and compare the distribution of P450s across sequenced insects. We also identify homologues of two P450s known to be involved in cyanogenesis in the six-spot Burnet moth, Zygaena filipendulae. Classification of Heliconius?P450s should be an important step in the dissection of their role in the exploitation of their host plant, the passion vine Passiflora. PMID:23834845

Chauhan, R; Jones, R; Wilkinson, P; Pauchet, Y; Ffrench-Constant, R H

2013-10-01

107

Regulation of Intestinal Cytochrome P450 Expression by Hepatic Cytochrome P450: Possible Involvement of Fibroblast Growth Factor 15 and Impact on Systemic Drug Exposure  

PubMed Central

Tissue-specific deletion of the gene for NADPH-cytochrome P450 (P450) reductase (CPR), the essential electron donor to all microsomal P450 enzymes, in either liver or intestine, leads to upregulation of many P450 genes in the tissue with the Cpr deletion. Here, by studying the liver-specific Cpr-null (LCN) mouse, we examined whether an interorgan regulatory pathway exists, such that a loss of hepatic CPR would cause compensatory changes in intestinal P450 expression and capacity for first-pass metabolism of oral drugs. We show for the first time that intestinal expression of CYP2B, 2C, and 3A proteins was increased in LCN mice by 2- to 3-fold compared with wild-type (WT) mice, accompanied by significant increases in small intestinal microsomal lovastatin-hydroxylase activity and systemic clearance of oral lovastatin (at 5 mg/kg). Additional studies showed that the hepatic Cpr deletion, which caused large decreases in bile acid (BA) levels in the liver, intestine, plasma, and intestinal content, led to drastic decreases in the mRNA levels of intestinal fibroblast growth factor 15 (FGF15), a target gene of the BA receptor farnesoid X receptor. Furthermore, treatment of mice with FGF19 (the human counterpart of mouse FGF15) abolished the difference between WT and LCN mice in small intestinal (SI) CYP3A levels at 6 hours after the treatment. Our findings reveal a previously unrecognized direct role of intestinal FGF15/19 in the regulation of SI P450 expression and may have profound implications for the prediction of drug exposure in patients with compromised hepatic P450 function. PMID:24184963

Zhu, Yi; Ding, Xinxin; Fang, Cheng

2014-01-01

108

The cytochrome P450 gene superfamily in Drosophila melanogaster: Annotation, intron-exon organization and phylogeny  

Microsoft Academic Search

The cytochrome P450 gene superfamily is represented by 90 sequences in the Drosophila melanogaster genome. Of these 90 P450 sequences, 83 code for apparently functional genes whereas seven are apparent pseudogenes. More than half of the genes belong to only two families, CYP4 and CYP6. The CYP6 family is insect specific whereas the CYP4 family includes sequences from vertebrates. There

Nathalie Tijet; Christian Helvig; René Feyereisen

2001-01-01

109

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

110

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

Microsoft Academic Search

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

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

2003-01-01

111

Bell pepper fruit fatty acid hydroperoxide lyase is a cytochrome P450 (CYP74B)  

Microsoft Academic Search

Fatty acid hydroperoxide lyases cleave a C?C bond adjacent to a hydroperoxide group in lipoxygenase derived lipid hydroperoxides to form short-chain aldehydes and oxo-acids. Previously, we showed that fatty acid hydroperoxide lyase from bell pepper fruits is a heme protein whose spectrophotometric properties greatly resemble a cytochrome P450. In order to ascertain the relationship of it to the P450 gene

Kenji Matsui; Mizuyoshi Shibutani; Toshiharu Hase; Tadahiko Kajiwara

1996-01-01

112

Crystal structure of cytochrome P-450cam complexed with the (1S)-camphor enantiomer  

Microsoft Academic Search

The crystal structure of cytochrome P-450cam complexed with the enantiomer (1S)-camphor has been solved to 1.8 Å resolution and compared with the structure of the (1R)-camphor P-450cam complex. The overall protein structure is the same for both enantiomer complexes. However, the orientation of the substrates in the heme pocket differs. In contrast to (1R)-camphor, the (1S)-enantiomer binds in at least

Ilme Schlichting; Christiane Jung; Heike Schulze

1997-01-01

113

Spectroscopic characterization of the iron-oxo intermediate in cytochrome P450.  

PubMed

From analogy to chloroperoxidase from Caldariomyces fumago, it is believed that the electronic structure of the intermediate iron-oxo species in the catalytic cycle of cytochrome P450 corresponds to an iron(IV) porphyrin-pi-cation radical (compound I). However, our recent studies on P450cam revealed that after 8 ms a tyrosine radical and iron(IV) were formed in the reaction of ferric P450 with external oxidants in the shunt pathway. The present study on the heme domain of P450BM3 (P450BMP) shows a similar result. In addition to a tyrosine radical, a contribution from a tryptophan radical was found in the electron paramagnetic resonance (EPR) spectra of P450BMP. Here we present comparative multi-frequency EPR (9.6, 94 and 285 GHz) and Mössbauer spectroscopic studies on freeze-quenched intermediates produced using peroxy acetic acid as oxidant for both P450 cytochromes. After 8 ms in both systems, amino acid radicals occurred instead of the proposed iron(IV) porphyrin-pi-cation radical, which may be transiently formed on a much faster time scale. These findings are discussed with respect to other heme thiolate proteins. Our studies demonstrate that intramolecular electron transfer from aromatic amino acids is a common feature in these enzymes. The electron transfer quenches the presumably transiently formed porphyrin-pi-cation radical, which makes it extremely difficult to trap compound I. PMID:16218876

Jung, Christiane; Schünemann, Volker; Lendzian, Friedhelm; Trautwein, Alfred X; Contzen, Jörg; Galander, Marcus; Böttger, Lars H; Richter, Matthias; Barra, Anne-Laure

2005-10-01

114

Cytochrome P450 Family 1 Inhibitors and Structure-Activity Relationships  

PubMed Central

With the widespread use of O-alkoxyresorufin dealkylation assays since the 1990’s, thousands of inhibitors of cytochrome P450 family 1 enzymes (P450s 1A1, 1A2, and 1B1) have been identified and studied. Generally, planar polycyclic molecules such as polycyclic aromatic hydrocarbons, stilbenoids, and flavonoids are considered to potentially be effective inhibitors of these enzymes. However, the details of structure-activity relationships and selectivity of these inhibitors are still ambiguous. In this review, we thoroughly discuss the selectivity of many representative P450 family 1 inhibitors reported in the past 20 years through a meta-analysis. PMID:24287985

Liu, Jiawang; Sridhar, Jayalakshmi; Foroozesh, Maryam

2014-01-01

115

Evaluation of recombinant cytochrome P450 enzymes as an in vitro system for metabolic clearance predictions.  

PubMed

The aim of this study was to explore the potential of recombinant cytochrome P450 (P450) enzymes for human metabolic clearance prediction. The relative abundance and relative activity approaches were compared as methods to bridge the gap between catalytic activities in recombinant P450 enzymes and human liver microsomes (HLMs). Relative activity factors were measured by determining the intrinsic clearance (CL(int)) of probe substrates (bufuralol-CYP2D6, diclofenac-CYP2C9, midazolam-CYP3A4, and phenacetin-CYP1A2) in recombinant P450s and 16 HLM donors. Simultaneous determination of drug depletion and metabolite formation profiles has enabled a direct comparison of these methods for CL(int) determination. Of the 110 drugs tested, 66% were metabolized by one or more P450 enzymes; of these 44% of were metabolized by CYP3A4 (0.3-21 microl/min/pmol of P450), 41% by CYP2D6 (0.6-60 microl/min/pmol of P450), 26% by CYP2C19 (0.4-8.1 microl/min/pmol of P450), 9% by CYP1A2 (0.4-2.5 microl/min/pmol of P450), and 4% by CYP2C9 (0.9-6.4 microl/min/pmol of P450). Recombinant enzymes demonstrated improved prediction reliability relative to HLMs and hepatocytes. The most reliable correlations in terms of lowest bias and highest precision were observed by comparing in vivo CL(int), calculated using the parallel-tube model and incorporating fraction unbound in blood, with in vitro CL(int) determined using relative activity factors and adjusted for nonspecific binding. Predictions were less reliable using the relative abundance approach. For these drugs, recombinant P450 enzymes offer improved assay sensitivity compared with HLMs and cryopreserved hepatocytes for CL(int) determination using the drug depletion method. PMID:19196847

Stringer, Rowan A; Strain-Damerell, Claire; Nicklin, Paul; Houston, J Brian

2009-05-01

116

Human Cytochrome P450 Oxidation of 5-Hydroxythalidomide and Pomalidomide, an Amino Analog of Thalidomide  

PubMed Central

The sedative and antiemetic drug thalidomide [?-(N-phthalimido)glutarimide] was withdrawn in the early 1960s due to its potent teratogenic effects but was approved for the treatment of lesions associated with leprosy in 1998 and multiple myeloma in 2006. The mechanism of teratogenicity of thalidomide still remains unclear, but it is well established that metabolism of thalidomide is important for both teratogenicity and cancer treatment outcome. Thalidomide is oxidized by various cytochrome P450 (P450) enzymes, the major being P450 2C19, to 5-hydroxy-, 5’-hydroxy-, and dihydroxythalidomide. We previously reported that P450 3A4 oxidizes thalidomide to the 5-hydroxy and dihydroxy metabolites, with the second oxidation step involving a reactive intermediate, possible an arene oxide, that can be trapped by glutathione (GSH) to GSH adducts. We now show that the dihydroxythalidomide metabolite can be further oxidized to a quinone intermediate. Human P450s 2J2, 2C18, and 4A11 were also found to oxidize 5-hydroxythalidomide to dihydroxy products. Unlike P450s 2C19 and 3A4, neither P450 2J2, 2C18, nor 4A11 oxidized thalidomide itself. A recently approved amino analog of thalidomide, pomalidomide (CC-4047, Actimid™), was also oxidized by human liver microsomes and P450s 2C19, 3A4, and 2J2 to the corresponding phthalimide ring-hydroxylated product. PMID:24350712

Chowdhury, Goutam; Shibata, Norio; Yamazaki, Hiroshi; Guengerich, F. Peter

2014-01-01

117

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

PubMed

Pyrethroid insecticides are used to control diseases spread by arthropods. We have developed a suite of pyrethroid mimetic activity-based probes (PyABPs) to selectively label and identify P450s associated with pyrethroid metabolism. The probes were screened against pyrethroid-metabolizing and nonmetabolizing mosquito P450s, as well as rodent microsomes, to measure labeling specificity, plus cytochrome P450 oxidoreductase and b5 knockout mouse livers to validate P450 activation and establish the role for b5 in probe activation. Using PyABPs, we were able to profile active enzymes in rat liver microsomes and identify pyrethroid-metabolizing enzymes in the target tissue. These included P450s as well as related detoxification enzymes, notably UDP-glucuronosyltransferases, suggesting a network of associated pyrethroid-metabolizing enzymes, or "pyrethrome." Considering the central role P450s play in metabolizing insecticides, we anticipate that PyABPs will aid in the identification and profiling of P450s associated with insecticide pharmacology in a wide range of species, improving understanding of P450-insecticide interactions and aiding the development of unique tools for disease control. PMID:24248381

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

2013-12-01

118

Human hepatic cytochrome P450-specific metabolism of the organophosphorus pesticides methyl parathion and diazinon.  

PubMed

Organophosphorus pesticides (OPs) are a public health concern due to their worldwide use and documented human exposures. Phosphorothioate OPs are metabolized by cytochrome P450s (P450s) through either a dearylation reaction to form an inactive metabolite, or through a desulfuration reaction to form an active oxon metabolite, which is a potent cholinesterase inhibitor. This study investigated the rate of desulfuration (activation) and dearylation (detoxification) of methyl parathion and diazinon in human liver microsomes. In addition, recombinant human P450s were used to determine the P450-specific kinetic parameters (K(m) and V(max)) for each compound for future use in refining human physiologically based pharmacokinetic/pharmacodynamic (PBPK/PD) models of OP exposure. The primary enzymes involved in bioactivation of methyl parathion were CYP2B6 (K(m) = 1.25 ?M; V(max) = 9.78 nmol · min(-1) · nmol P450(-1)), CYP2C19 (K(m) = 1.03 ?M; V(max) = 4.67 nmol · min(-1) · nmol P450(-1)), and CYP1A2 (K(m) = 1.96 ?M; V(max) = 5.14 nmol · min(-1) · nmol P450(-1)), and the bioactivation of diazinon was mediated primarily by CYP1A1 (K(m) = 3.05 ?M; V(max) = 2.35 nmol · min(-1) · nmol P450(-1)), CYP2C19 (K(m) = 7.74 ?M; V(max) = 4.14 nmol · min(-1) · nmol P450(-1)), and CYP2B6 (K(m) = 14.83 ?M; V(max) = 5.44 nmol · min(-1) · nmol P450(-1)). P450-mediated detoxification of methyl parathion only occurred to a limited extent with CYP1A2 (K(m) = 16.8 ?M; V(max) = 1.38 nmol · min(-1) · nmol P450(-1)) and 3A4 (K(m) = 104 ?M; V(max) = 5.15 nmol · min(-1) · nmol P450(-1)), whereas the major enzyme involved in diazinon detoxification was CYP2C19 (K(m) = 5.04 ?M; V(max) = 5.58 nmol · min(-1) · nmol P450(-1)). The OP- and P450-specific kinetic values will be helpful for future use in refining human PBPK/PD models of OP exposure. PMID:21969518

Ellison, Corie A; Tian, Yuan; Knaak, James B; Kostyniak, Paul J; Olson, James R

2012-01-01

119

Model studies in cytochrome P-450-mediated toxicity of halogenated compounds: radical processes involving iron porphyrins.  

PubMed Central

Haloalkane toxicity originates from attack on biological targets by reactive intermediates derived from haloalkane metabolism by a hemoprotein, cytochrome P-450. Carbon-centered radicals and their peroxyl derivatives are most likely involved. The reactions of iron porphyrin--a model for cytochrome P-450--with various carbon-centered and peroxyl radicals generated by pulse radiolysis are examined. Competition between iron porphyrin and unsaturated fatty acids for attack by peroxyl radicals is pointed out. These kinetic data are used to derive a model for toxicity of haloalkanes with particular attention to carbon tetrachloride and halothane. The importance of local oxygen concentration and structural arrangement of fatty acids around cytochrome P-450 is emphasized. PMID:3007100

Brault, D

1985-01-01

120

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 PMID:16668136

Halkier, Barbara Ann; Møller, Birger Lindberg

1991-01-01

121

An investigation into the stress relieving properties of Sutherlandia frutescens : inhibition of steroidogenic cytochrome P450 enzymes.  

E-print Network

??ENGLISH ABSTRACT: This study: T. Investigates the influence of S. frutescens on the binding properties of cytochrome P450-dependent enzymes in ovine adrenocortical mitochondria and microsomes,… (more)

Prevoo, Désirée

2005-01-01

122

Two-step purification of cytochrome P-450 from rat liver microsomes using high-performance liquid chromatography.  

PubMed

Cytochrome P-450 from rat liver microsomes treated with phenobarbital (PB) was separated into six fractions, as was cytochrome P-450 treated with 3-methylcholanthrene (MC), by high-performance liquid chromatography (HPLC) with an anion-exchange column. PB and MC induced three forms and one form of cytochrome P-450, respectively. The major forms induced by PB and by MC were further purified to apparent homogeneity based on sodium dodecyl(lauryl)sulphate--polyacrylamide gel electrophoresis by HPLC using a hydroxyapatite column. These new HPLC techniques are simple, rapid and useful for the purification of major forms of cytochrome P-450 from solubilized microsomes. PMID:3007551

Funae, Y; Seo, R; Imaoka, S

1986-01-24

123

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. PMID:21087487

2010-01-01

124

The Cytochrome P450 Superfamily Complement (CYPome) in the Annelid Capitella teleta  

PubMed Central

The Cytochrome P450 super family (CYP) is responsible for a wide range of functions in metazoans, having roles in both exogenous and endogenous substrate metabolism. Annelids are known to metabolize polycyclic aromatic hydrocarbons (PAHs) and produce estrogen. CYPs are postulated to be key enzymes in these processes in annelids. In this study, the CYP complement (CYPome) of the annelid Capitella teleta has been robustly identified and annotated with the genome assembly available. Phylogenetic analyses were performed to understand the evolutionary relationships between CYPs in C. teleta and other species. Predictions of which CYPs are potentially involved in both PAH metabolism and steroidogensis were made based on phylogeny. Annotation of 84 full length and 12 partial CYP sequences predicted a total of 96 functional CYPs in C. teleta. A further 13 CYP fragments were found but these may be pseudogenes. The C. teleta CYPome contained 24 novel CYP families and seven novel CYP subfamilies within existing families. A phylogenetic analysis identified that the C. teleta sequences were found in 9 of the 11 metazoan CYP clans. Two CYPs, CYP3071A1 and CYP3072A1, did not cluster with any metazoan CYP clans. We found xenobiotic response elements (XREs) upstream of C. teleta CYPs related to vertebrate CYP1 (CYP3060A1, CYP3061A1) and from families with reported transcriptional upregulation in response to PAH exposure (CYP4, CYP331). C. teleta had a CYP51A1 with ?65% identity to vertebrate CYP51A1 sequences and has been predicted to have lanosterol 14 ?-demethylase activity. CYP376A1, CYP3068A1, CYP3069A1, and CYP3070A1 were the most appropriate candidates for steroidogenesis genes based on their phylogeny and warrant further analyses, though no specific aromatase (estrogen synthesis) candidates were found. Presence of XREs upstream of C. teleta CYPs may indicate a functional aryl hydrocarbon receptor in C. teleta and candidate CYPs for studies of PAH metabolism. PMID:25390889

Dejong, Chris A.; Wilson, Joanna Y.

2014-01-01

125

Control of constitutively-expressed developmentally-activated rat hepatic cytochrome P450 genes.  

PubMed

Cytochromes P450 (P450) collectively refer to a superfamily of heme-containing enzymes that use O2 and electrons from NADPH to insert a single atom of oxygen into any one of a large number of substrates. Two general classes of P450s exist; a relatively limited number of P450 forms, expressed in specialized tissues that are associated with pathways of steroidogenesis and a large number of forms responsible for metabolism of foreign compounds. Most of the latter P450s are expressed in liver, the primary site for metabolism of drugs, unusual dietary compounds and environmental pollutants. Numerous forms of P450 are expressed in liver of untreated animals and these are regulated quite differently. Both developmentally-programmed and sex-specific expressions have been observed. In this review, I will summarize recent findings on the mechanisms by which two P450 genes are regulated in livers of developing rats. The CYP2E1 gene is transcriptionally activated within a few hours after birth while the CYP2C6 gene is activated just prior to rats reaching puberty. These genes are under control of two transcription factors, HNF-1 alpha and DBP, respectively, that themselves are developmentally controlled. PMID:1619850

Gonzalez, F J

1992-06-01

126

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 ...

127

Immobilized cytochrome P-450LM2. Dissociation and reassociation of oligomers.  

PubMed

Subunit interactions in the purified hexameric cytochrome P-450LM2 have been studied using covalent binding of one of the 6 promoters to an insoluble matrix. High ionic strength, large-scale pH changes, guanidine chloride and sodium cholate taken at membrane-solubilizing concentrations, had no effect on the aggregation state of the immobilized hemoprotein. SDS caused a 6-fold decrease in the amount of the bound cytochrome. Non-ionic detergents (Emulgen 913, octylglucoside, Tritons) induced hexamer dissociation. In the presence of Emulgen 913 (greater than 0.2%), monomers and immobilized dimers were obtained as cytochrome P-450 was studied in an aqueous medium and in the immobilized state, respectively. Immobilized dimers could be reconstituted to hexamers by treatment with an excess of solubilized monomers after removal of the detergent. In the presence of various phospholipids, which increased the immobilized cytochrome P-450LM2 demethylase activity and induced characteristic spectral changes, no hexamer dissociation was shown. The data obtained are thus in agreement with the suggestion that hexameric arrangement is inherent in the cytochrome P-450 when it is bound to the native membranes. PMID:1699791

Myasoedova, K N; Berndt, P

1990-09-17

128

Probing the open state of cytochrome P450cam with ruthenium-linker substrates  

PubMed Central

Cytochromes P450 play key roles in drug metabolism and disease by oxidizing a wide variety of natural and xenobiotic compounds. High-resolution crystal structures of P450cam bound to ruthenium sensitizer-linked substrates reveal an open conformation of the enzyme that allows substrates to access the active center via a 22-? deep channel. Interactions of alkyl and fluorinated biphenyl linkers with the channel demonstrate the importance of exploiting protein dynamics for specific inhibitor design. Large changes in peripheral enzyme structure (F and G helices) couple to conformational changes in active center residues (I helix) implicated in proton pumping and dioxygen activation. Common conformational states among P450cam and homologous enzymes indicate that static and dynamic variability in the F/G helix region allows the 54 human P450s to oxidize thousands of substrates. PMID:11606730

Dunn, Alexander R.; Dmochowski, Ivan J.; Bilwes, Alexandrine M.; Gray, Harry B.; Crane, Brian R.

2001-01-01

129

Testosterone-mediated regulation of mouse renal cytochrome P-450 isoenzymes.  

PubMed Central

We have studied the extent to which mouse renal cytochrome P-450 isoenzymes are sexually differentiated, and the factor(s) regulating this dimorphism. Intriguingly, sex differences were not seen in the expression of a single cytochrome P-450 enzyme, but were observed in the expression of all P-450 isoenzymes detectable, encoded by six gene families or sub-families. This effect was mediated by testosterone, which had the capacity to both induce and repress P-450 gene expression, and which was independent of growth hormone. The changes in protein content were mirrored in all but one case by changes in the levels of mRNA, indicating that these genes contain hormone-responsive elements. These findings are consistent with numerous reports of sex differences in the susceptibility of the mouse kidney to the toxic and carcinogenic effects of drugs and environmental chemicals, many of which are metabolized to cytotoxic products by the cytochrome P-450-dependent mono-oxygenases. These data imply that circulating androgen levels will be an important factor in susceptibility of the kidney to toxic or carcinogenic compounds which require metabolic activation. Images Fig. 2. Fig. 3. Fig. 4. Fig. 5. PMID:2327956

Henderson, C J; Scott, A R; Yang, C S; Wolf, C R

1990-01-01

130

Kinetic analysis of lauric acid hydroxylation by human cytochrome P450 4A11.  

PubMed

Cytochrome P450 (P450) 4A11 is the only functionally active subfamily 4A P450 in humans. P450 4A11 catalyzes mainly ?-hydroxylation of fatty acids in liver and kidney; this process is not a major degradative pathway, but at least one product, 20-hydroxyeicosatetraenoic acid, has important signaling properties. We studied catalysis by P450 4A11 and the issue of rate-limiting steps using lauric acid ?-hydroxylation, a prototypic substrate for this enzyme. Some individual reaction steps were studied using pre-steady-state kinetic approaches. Substrate and product binding and release were much faster than overall rates of catalysis. Reduction of ferric P450 4A11 (to ferrous) was rapid and not rate-limiting. Deuterium kinetic isotope effect (KIE) experiments yielded low but reproducible values (1.2-2) for 12-hydroxylation with 12-(2)H-substituted lauric acid. However, considerable "metabolic switching" to 11-hydroxylation was observed with [12-(2)H3]lauric acid. Analysis of switching results [Jones, J. P., et al. (1986) J. Am. Chem. Soc. 108, 7074-7078] and the use of tritium KIE analysis with [12-(3)H]lauric acid [Northrop, D. B. (1987) Methods Enzymol. 87, 607-625] both indicated a high intrinsic KIE (>10). Cytochrome b5 (b5) stimulated steady-state lauric acid ?-hydroxylation ?2-fold; the apoprotein was ineffective, indicating that electron transfer is involved in the b5 enhancement. The rate of b5 reoxidation was increased in the presence of ferrous P450 mixed with O2. Collectively, the results indicate that both the transfer of an electron to the ferrous·O2 complex and C-H bond-breaking limit the rate of P450 4A11 ?-oxidation. PMID:25203493

Kim, Donghak; Cha, Gun-Su; Nagy, Leslie D; Yun, Chul-Ho; Guengerich, F Peter

2014-10-01

131

Highly Miniaturized Formats for In Vitro Drug Metabolism Assays Using Vivid® Fluorescent Substrates and Recombinant Human Cytochrome P450 Enzymes  

Microsoft Academic Search

Highly miniaturized P450 screening assays designed to enable facile analysis of P450 drug interactions in a 1536-well plate format with the principal human cytochrome P450 enzymes (CYP3A4, 2D6, 2C9, 2C19, and 1A2) and Vivid® fluorogenic substrates were developed. The detailed characterization of the assays included stability, homogeneity, and reproducibility of the recombinant P450 enzymes and the kinetic parameters of their

Olga V. Trubetskoy; Jasmin R. Gibson; Bryan D. Marks

2005-01-01

132

Chlormethiazole inhibition of cytochrome P450 2E1 as assessed by chlorzoxazone hydroxylation in humans.  

PubMed

Chlormethiazole is a sedative and anticonvulsive drug used in the treatment of alcohol withdrawal. Because it had been reported that chlormethiazole inhibits the alcohol-inducible cytochrome P450 2E1 in rat liver, we investigated the in vivo and in vitro effect of this drug on cytochrome P450 2E1 in human beings. The activity of this cytochrome was assessed using chlorzoxazone as a probe. The 6-hydroxychlorzoxazone-chlorzoxazone blood concentration ratio, reflecting the cytochrome P450 2E1 activity, was determined in 10 controls and in 24 alcoholic patients who had entered a hospital for detoxification. Alcoholic patients were administered either chlormethiazole (1.3-2.3 g/d) or chlorazepate (100-300 mg/d) as a sedative. Cytochrome P450 2E1 activity was significantly increased in alcoholic patients treated with chlorazepate (1.16 +/- 0.40 vs. 0.27 +/- 0.03, P < .05). In contrast, chlormethiazole treatment inhibited chlorzoxazone hydroxylation almost totally (0.046 +/- 0.03, P < .001). After 7-14 days of ethanol withdrawal, alcoholic patients treated with chlorazepate had ratio values similar to those of controls (0.31 +/- 0.05), whereas values from alcoholic patients treated with chlormethiazole remained low (0.049 +/- 0.01) even though chlormethiazole doses were gradually decreased. Pharmacokinetic studies in controls showed that chlormethiazole-mediated inhibition was present even when chlormethiazole was not detectable in the blood. In addition, the effect of chlormethiazole on cytochrome P450 2E1 was studied in vitro using human liver microsomes. Dixon plot analyses showed a noncompetitive inhibition (Ki = 12 micromol/L). These data clearly show that chlormethiazole is an efficient inhibitor of chlorzoxazone metabolism and thus of cytochrome P450 2E1 activity in human beings. Because cytochrome P450 2E1 induction after chronic ethanol consumption has detrimental effects on the liver through free radical formation, treatment of alcohol detoxification with chlormethiazole may be beneficial. PMID:9328319

Gebhardt, A C; Lucas, D; Ménez, J F; Seitz, H K

1997-10-01

133

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. PMID:21737533

Cheng, Qian

2011-01-01

134

HEPATIC CYTOCHROME P450 UBIQUITINATION: CONFORMATIONAL PHOSPHODEGRONS FOR E2/E3 RECOGNITION?  

PubMed Central

Hepatic endoplasmic reticulum (ER) integral cytochromes P450 (P450s) are monooxygenases engaged in the biotransformation and elimination of endo- as well as xenobiotics. Of the human liver P450s, CYP3A4 is the major and most dominant catalyst, responsible for the biotransformation of over 50% of clinically prescribed drugs. CYP2E1 metabolizes smaller molecular weight compounds (EtOH), carcinogens, environmental toxins and endobiotics, and is justly implicated in various toxigenic/pathogenic mechanisms of human disease. Both P450s are notorious for their potential to generate pathogenic reactive oxygen species (ROS) during futile oxidative cycling and/or oxidative uncoupling. Such ROS not only oxidatively damage the P450 catalytic cage, but on their escape into the cytosol, also the P450 outer surface and any surrounding cell organelles. Given their ER-monotopic topology coupled with this high potential to acquire oxidative lesions in their cytosolic (C) domain, not surprisingly these P450 proteins exhibit shorter lifespans and are excellent prototype substrates of ER-associated degradation (“ERAD-C”) pathway. Indeed, we have shown that both CYP3A4 and CYP2E1 incur ERAD-C, during which they are first phosphorylated by protein kinases A and C, which greatly enhance/accelerate their ubiquitination by UBC7/gp78 and UbcH5a/CHIP/Hsp70/Hsp40 E2/E3 ubiquitin ligase complexes. Such P450 phosphorylation occurs on Ser/Thr residues within linear sequences as well as spatially clustered acidic (Asp/Glu) residues. We propose that such S/T phosphorylation within these clusters creates a negatively charged patch i.e. conformational phosphodegrons, for interaction with positively charged E2/E3 domains. Such P450 S/T phosphorylation we posit serves as a switch to turn on its ubiquitination and ERAD-C. PMID:24488826

Correia, Maria Almira; Wang, YongQiang; Kim, Sung-Mi; Guan, Shenheng

2014-01-01

135

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

2013-10-01

136

Immunochemical cross-reactivity of?-naphthoflavone-inducible cytochrome P450 (P450IA) in liver microsomes from different fish species and rat.  

PubMed

Antibodies prepared against the major ?-naphthoflavone (BNF)-inducible cytochrome P450 (P450) forms from three species of fish (rainbow trout, Atlantic cod, and scup) well separated in teleost phylogeny, were used to investigate the immunochemical relatedness of liver microsomal P450 in different species of BNF-treated fish and rat. Rabbit polyclonal IgG against all three P450s and mouse monoclonal antibodies prepared against scup P450E were employed in this study. Liver microsomes were prepared from BNF-treated specimens of hagfish, herring, rainbow trout, cod, scup, perch, plaice and rat. With Western blotting it was shown that the various antibodies cross-reacted with a protein band in liver microsomes in the P450-region of each of the BNF-treated fish species. The apparent molecular weight of the cross-reacting proteins showed differences within the range 54,000-59,000 daltons. The effects of the different antibodies on the microsomal BNF-inducible 7-ethoxyresorufin O-deethylase (EROD) activity gave inhibition patterns that reflected to a certain extent the phylogenetic relationship of the species investigated. In rat microsomes a protein band of relative molecular mass similar to rat P450c (Mr=54,000) was recognized by all antibodies. In addition, a second band of lower molecular mass was strongly recognized by anti-cod P450c antibodies, and faintly stained with anti-rainbow trout P450LM4b IgG and anti-scup P450E MAb 1-12-3. This band could correspond to rat P450d, the isosafrole-inducible rat isoenzyme. Considering the long separate evolutionary history of some of these fishes (50-200 million years), the results demonstrate that certain antigenic epitopes in the BNF-inducible P450 isoenzymes have been strongly conserved during the evolution of fish species. These conserved epitopes seem however not to be directly involved in the measured EROD activities. Furthermore, the results suggest that the BNF-inducible P450s in fish contain regions with structural similarity to the homologous counterpart that has evolved through gene duplication into a P450 family in mammals containing at least two gene products (the P450IA gene family). PMID:24214604

Goksøyr, A; Andersson, T; Buhler, D R; Stegeman, J J; Williams, D E; Förlin, L

1991-03-01

137

Interaction of azole antifungal antibiotics with cytochrome P-450-dependent 14 alpha-sterol demethylase purified from Candida albicans.  

PubMed Central

The interaction of azole antifungal antibiotics with purified Candida albicans cytochrome P-450-dependent 14 alpha-sterol demethylase (P-450DM) was measured spectrophotometrically and by inhibition of enzyme activity. Ketoconazole and ICI 153066 (a triazole derivative) formed low-spin complexes with the ferric cytochrome and induced type II difference spectra. These spectra are indicative of an interaction between the azole moiety and the sixth co-ordination position of P-450DM haem. Both azoles inhibited the binding of CO to the sodium dithionite-reduced ferrous cytochrome, and inhibited reconstituted P-450DM activity by binding to the cytochrome with a one-to-one stoichiometry. Similarly, total inhibition of enzyme activity occurred when equimolar amounts of clotrimazole, miconazole or fluconazole were added to reconstituted P-450DM. These results correlated with the inhibition of P-450DM in broken cell preparations, confirming that all five azoles are potent inhibitors of ergosterol biosynthesis in C. albicans. PMID:2180400

Hitchcock, C A; Dickinson, K; Brown, S B; Evans, E G; Adams, D J

1990-01-01

138

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. PMID:23911089

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

2013-01-01

139

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

140

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

141

QUANTITATIVE EVALUATION OF BROMODICHLOROMETHANE METABOLISM BY RECOMBINANT RAT AND HUMAN CYTOCHROME P450S  

EPA Science Inventory

ABSTRACT We report quantitative estimates of the parameters for metabolism of bromodichloromethane (BDCM) by recombinant preparations of hepatic cytochrome P450s (CYPs) from rat and human. BDCM is a drinking water disinfectant byproduct that has been implicated in liver, kidn...

142

PRIMARY STRUCTURE OF THE CYTOCHROME P450 LANOSTEROL 14A-DEMETHYLASE GENE FROM CANDIDA TROPIALIS  

EPA Science Inventory

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

143

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

EPA Science Inventory

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

144

Helitron mediated amplification of cytochrome P450 monooxygenase gene in maize  

E-print Network

Helitron mediated amplification of cytochrome P450 monooxygenase gene in maize Natalie Jameson Ã? reported between different maize inbred lines. However, Helitron captured-genes reported to date repre- sent truncated versions of their progenitor genes. In this report, we provide evidence that maize CYP72

145

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...

146

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...

147

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...

148

Comparison of random mutagenesis and semi-rational designed libraries for improved cytochrome P450  

E-print Network

Comparison of random mutagenesis and semi-rational designed libraries for improved cytochrome P450 January 10, 2012 Edited by Alan Berry Three semi-rational approaches, combinatorial site- saturation, these semi-rational approaches allowed for large jumps in sequence space to variants with the desired

Arnold, Frances H.

149

Cytochrome P450 gene diversity and function in marine animals: past, present, and future  

Microsoft Academic Search

The biotransformation of xenobiotics by microsomal cytochromes P450 is known to be pivotal in the effects of some compounds, and thought to be so for many. A knowledge of CYP gene diversity and CYP function and regulation in aquatic species is pursued, expecting that it will disclose mechanisms, allow predictions regarding species differences in susceptibility, and provide markers for exposure

J. J. Stegeman

2000-01-01

150

EVIDENCE FOR BROMODICHLOROMETHANE METABOLISM BY CYTOCHROME P-450 1A2  

EPA Science Inventory

EVIDENCE FOR BROMODICHLOROMETHANE METABOLISM BY CYTOCHROME P-450 1A2. T M Ross1, B P Anderson1, G Zhao2, R A Pegram1 and J W Allis1. 1U.S. EPA, ORD, NHEERL, Research Triangle Park, NC; 2University of North Carolina, Chapel Hill, NC. Sponsor: H Barton Bromodichlorometh...

151

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

152

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...

153

INCREASED BLOOD PRESSURE IN MICE LACKING CYTOCHROME P450 2J5  

Technology Transfer Automated Retrieval System (TEKTRAN)

The cytochrome P450 (CYP) enzymes participate in a wide range of biochemical functions including metabolism of arachidonic acid and steroid hormones. Mouse CYP2J5 is abundant in the kidney where its products, the cis-epoxyeicosatrienoic acids (EETs), modulate sodium transport and vascular tone. To d...

154

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 ...

155

Evidence for cytochrome P-450 as a source of catalytic iron in myoglobinuric acute renal failure  

Microsoft Academic Search

Evidence for cytochrome P-450 as a source of catalytic iron in myoglobinuric acute renal failure. Iron has been implicated to play an important role in several models of tissue injury, including myoglobinuric acute renal failure. In this model, myoglobin released from the injured muscle is generally accepted as a source of iron. In the present study we measured the bleomycin-detectable

Radhakrishna Baliga; Zhiwei Zhang; Mithra Baliga; Sudhir V Shah

1996-01-01

156

Phyllanthus urinaria extract attenuates acetaminophen induced hepatotoxicity: involvement of cytochrome P450 CYP2E1.  

PubMed

Acetaminophen is a commonly used drug for the treatment of patients with common cold and influenza. However, an overdose of acetaminophen may be fatal. In this study we investigated whether mice, administered intraperitoneally with a lethal dose of acetaminophen, when followed by oral administration of Phyllanthus urinaria extract, may be prevented from death. Histopathological analysis of mouse liver sections showed that Phyllanthus urinaria extract may protect the hepatocytes from acetaminophen-induced necrosis. Therapeutic dose of Phyllanthus urinaria extract did not show any toxicological phenomenon on mice. Immunohistochemical staining with the cytochrome P450 CYP2E1 antibody revealed that Phyllanthus urinaria extract reduced the cytochrome P450 CYP2E1 protein level in mice pre-treated with a lethal dose of acetaminophen. Phyllanthus urinaria extract also inhibited the cytochrome P450 CYP2E1 enzymatic activity in vitro. Heavy metals, including arsenic, cadmium, mercury and lead, as well as herbicide residues were not found above their detection limits. High performance liquid chromatography identified corilagin and gallic acid as the major components of the Phyllanthus urinaria extract. We conclude that Phyllanthus urinaria extract is effective in attenuating the acetaminophen induced hepatotoxicity, and inhibition of cytochrome P450 CYP2E1 enzyme may be an important factor for its therapeutic mechanism. PMID:19386480

Hau, Desmond Kwok Po; Gambari, Roberto; Wong, Raymond Siu Ming; Yuen, Marcus Chun Wah; Cheng, Gregory Yin Ming; Tong, Cindy Sze Wai; Zhu, Guo Yuan; Leung, Alexander Kai Man; Lai, Paul Bo San; Lau, Fung Yi; Chan, Andrew Kit Wah; Wong, Wai Yeung; Kok, Stanton Hon Lung; Cheng, Chor Hing; Kan, Chi Wai; Chan, Albert Sun Chi; Chui, Chung Hin; Tang, Johnny Cheuk On; Fong, David Wang Fun

2009-08-01

157

Regio-and Enantioselective Alkane Hydroxylation with Engineered Cytochromes P450 BM-3  

E-print Network

Regio- and Enantioselective Alkane Hydroxylation with Engineered Cytochromes P450 BM-3 Matthew W alkanes regio- and enantioselectively using atmospheric dioxygen as an oxidant. BM-3 variant 9-10A-A328V alkanes larger than hexane primarily at the 2-position but forms R-2-alcohols (40-55% ee

Arnold, Frances H.

158

Directed Evolution of a Cytochrome P450 Monooxygenase for Alkane Oxidation  

E-print Network

Directed Evolution of a Cytochrome P450 Monooxygenase for Alkane Oxidation Edgardo T. Farinas = slight affinity) accurately reflects the nature of these com- pounds: alkanes are notoriously inert unsaturated fatty acids.[7] The enzyme is reported to be inactive towards alkanes and methyl esters lacking

Arnold, Frances H.

159

Transcription of Cholesterol Side-Chain Cleavage Cytochrome P450 in the Placenta: Activating  

E-print Network

Transcription of Cholesterol Side-Chain Cleavage Cytochrome P450 in the Placenta: Activating initiates when cholesterol is converted in the mitochondria to the first steroid, pregnenolone. This reaction is cata- lyzed by a specialized enzyme complex that includes the cholesterol side-chain cleavage

Lebendiker, Mario

160

Cytochromes P450: Inhibition of CYP2A Enzymes Involved in Xenobiotic Metabolism and Generation of CYP26 Enzymes Involved in Retinoic Acid Metabolism  

E-print Network

The cytochrome P450 (P450) superfamily of mixed function oxidase enzymes catalyze the metabolism of a variety of endogenous and exogenous biochemicals, including steroids, fatty acids, vitamins, eicosanoids, drugs, pesticides, and toxins. P450...

Stephens, Eva Susanne

2012-08-31

161

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

EPA Science Inventory

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

162

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

EPA Science Inventory

The P450ALK gene, which is inducible by the assimilation of alkane in Candida tropicalis, was sequenced and characterized. tructural features described in promoter and terminator regions of Saccharomyces yeast genes are present in the P450alk gene and some particular structures a...

163

Estimation of drug-metabolizing capacity by cytochrome P450 genotyping and expression.  

PubMed

Many undesired side effects or therapeutic failures of drugs are the result of differences or changes in drug metabolism, primarily depending on the levels and activities of cytochrome P450 (P450) enzymes. To assess whether P450 expression profiles can reflect hepatic drug metabolism, we compared P450 mRNA levels in the liver or peripheral leukocytes with the corresponding hepatic P450 activities. A preliminary P450 genotyping for the most frequent polymorphisms in white populations (CYP2C9*2, CYP2C9*3, CYP2C19*2, CYP2C19*3, CYP2D6*3, CYP2D6*4, CYP2D6*6, and CYP3A5*3) was carried out before P450 phenotyping, excluding the donors with nonfunctional alleles of CYP2C9, CYP2C19, and CYP2D6 and those with a functional CYP3A5*1 allele from a correlation analysis. The hepatic mRNA levels of CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 displayed a strong association with P450 activities in the liver, whereas the expression of CYP1A2, CYP2C9, CYP2C19, and CYP3A4 in leukocytes was proven to reflect the hepatic activities of these P450 species. The leukocytes were found to be inappropriate cells for the assessment of hepatic CYP2B6 and CYP2D6 activities. Combining the results of P450 genotyping and phenotyping analyses, patients' drug-metabolizing capacities can be estimated by the P450 expression in the liver and in leukocytes with some limitations. Patients' genetic and nongenetic variations in P450 status can guide the appropriate selection of drugs and the optimal dose, minimizing the risk of harmful side effects and ensuring a successful outcome of drug therapy. PMID:22262920

Temesvári, Manna; Kóbori, László; Paulik, József; Sárváry, Eniko; Belic, Ales; Monostory, Katalin

2012-04-01

164

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

165

Selective Usage of Transcription Initiation and Polyadenylation Sites in Grape Cytochrome P450 Monooxygenase Gene CYP736B Expression  

Technology Transfer Automated Retrieval System (TEKTRAN)

Plant cytochrome P450 monooxygenases are versatile redox proteins that mediate biosynthesis of lignins, terpenes, alkaloids, and a variety of other secondary compounds as plant defense agents against a range of pathogens and insects. To determine if cytochrome P450 monooxygenases are involved in the...

166

Role of hepatic and intestinal cytochrome P450 3A and 2B6 in the metabolism,  

E-print Network

Role of hepatic and intestinal cytochrome P450 3A and 2B6 in the metabolism, disposition), catalyzed in vitro by intestinal, hepatic, and expressed cytochrome P450 (CYP) 3A4. However, the role of CYP-labeled and IV unlabeled methadone after pretreatment with rifampin (INN, rifampicin) (hepatic/intestinal CYP3A

Steinbach, Joe Henry

167

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

2013-01-01

168

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

SciTech Connect

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

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

1994-12-31

169

Unsaturated fatty acid regulation of cytochrome P450 expression via a CAR-dependent pathway  

PubMed Central

The liver is responsible for key metabolic functions, including control of normal homoeostasis in response to diet and xenobiotic metabolism/detoxification. We have shown previously that inactivation of the hepatic cytochrome P450 system through conditional deletion of POR (P450 oxidoreductase) induces hepatic steatosis, liver growth and P450 expression. We have exploited a new conditional model of POR deletion to investigate the mechanism underlying these changes. We demonstrate that P450 induction, liver growth and hepatic triacylglycerol (triglyceride) homoeostasis are intimately linked and provide evidence that the observed phenotypes result from hepatic accumulation of unsaturated fatty acids, which mediate these phenotypes by activation of the nuclear receptor CAR (constitutive androstane receptor) and, to a lesser degree, PXR (pregnane X receptor). To our knowledge this is the first direct evidence that P450s play a major role in controlling unsaturated fatty acid homoeostasis via CAR. The regulation of P450s involved in xenobiotic metabolism by this mechanism has potentially significant implications for individual responses to drugs and environmental chemicals. PMID:18778245

Finn, Robert D.; Henderson, Colin J.; Scott, Claire L.; Wolf, C. Roland

2008-01-01

170

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

PubMed

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

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

1995-12-01

171

Structural and Thermodynamic Basis of (+)-?-Pinene Binding to Human Cytochrome P450 2B6  

PubMed Central

Despite recent advances in atomic level understanding of drug and inhibitor interactions with human cytochromes P450, the decades-old questions of chemical and structural determinants of hydrocarbon binding are still unanswered. (+)-?-Pinene is a monoterpene hydrocarbon that is widely distributed in the environment and a potent P450 2B inhibitor. Therefore, a combined biophysical and structural analysis of human P450 2B6 interactions with (+)-?-pinene was undertaken to elucidate the basis of the very high affinity binding. Binding of (+)-?-pinene to the P450 active site was demonstrated by a Type I spectral shift. Thermodynamics of ligand binding were explored using isothermal titration calorimetry and compared to those of P450 2A6, which is much less flexible than 2B6 based on comparison of multiple X-ray crystal structures. Consistent with expectation, entropy is the major driving force for hydrocarbon binding to P450 2A6, as evidenced by the calorimetric results. However, formation of the 2B6-(+)-?-pinene complex has a significant enthalpic component. A 2.0 Å resolution crystal structure of this enzyme ligand complex reveals that the highly plastic 2B6 utilizes previously unrecognized rearrangements of protein motifs. The results indicate that the specific components of enthalpic contribution to ligand binding are closely tied to the degree of enzyme flexibility. PMID:23786449

Wilderman, P. Ross; Shah, Manish B.; Jang, Hyun-Hee; Stout, C. David; Halpert, James R.

2013-01-01

172

Evaluation of ?-cyano ethers as fluorescent substrates for assay of cytochrome P450 enzyme activity  

PubMed Central

We have previously reported the synthesis of four ?-cyano-containing ethers based on 2-naphthaldehyde (2-NA) as cytochrome P450 (P450) fluorescent substrates. Activity detection was based on the formation of fluorescent 2-NA following substrate hydrolysis. A major limitation of these substrates was the need to remove NADPH, a required cofactor for P450 oxidation, before measuring 2-NA fluorescence. In this article, we report the synthesis of a new series of novel P450 substrates using 6-dimethylamino-2-naphthaldehyde (6-DMANA), which has a green fluorescent emission that is well separated from the NADPH spectrum. A major advantage of the 6-DMANA substrates is that NADPH removal is not required before fluorescence detection. We used eight ?-cyano ether-based substrates to determine the O-dealkylation activity of human, mouse, and rat liver microsomes. In addition, substrate activities were compared with the commercial substrate 7-ethoxyresorufin (7-ER). The catalytic turnover rates of both the 6-DMANA- and 2-NA-based substrates were in some cases threefold faster than the catalytic turnover rate of 7-ER. The 2-NA-based substrates had greater turnover than did the 6-DMANA-based substrates. Murine and rat liver microsomes prepared from animals that had been treated with various P450 inducers were used to examine for isozyme-selective turnover of the substrates. The vastly improved optical properties and synthetic flexibility of the ?-cyano ether compounds suggest that they are possibly good general P450 substrates. PMID:16083846

Kang, Kyung-Don; Jones, Paul D.; Huang, Huazhang; Zhang, Rong; Mostovich, Lyudmila A.; Wheelock, Craig E.; Watanabe, Takaho; Gulyaeva, Lyudmila F.; Hammock, Bruce D.

2006-01-01

173

Proteins from eight eukaryotic cytochrome P-450 families share a segmented region of sequence similarity.  

PubMed Central

Proteins from eight eukaryotic families in the cytochrome P-450 superfamily share one region of sequence similarity. This region begins 275-310 amino acids from the amino terminus of each P-450, continues for approximately 170 residues, and ends 35-50 amino acids before the carboxyl terminus. The region can be divided into four domains of sequence similarity, each possessing its own pattern of invariant, conserved, and variable amino acids. The four domains are 56, 20, 59, and 28 residues long and are connected by three shorter segments of limited sequence similarity. The number of residues in these short segments varies with the P-450 protein but ranges from 0 to 20 residues. Consensus sequences based on these similarities can be used to determine whether the sequence of an unidentified peptide resembles that expected for a P-450. Sequence similarities between proteins sometimes reflect constraints imposed by the requirements of a common function. The fourth domain of the P-450s, for example, contains an invariant cysteine that provides the axial thiolate ligand to the heme iron. Other relationships between the four domains and P-450 function can be examined by in vitro mutagenic procedures that alter the conserved amino acids or modify the distance between domains. PMID:3050990

Kalb, V F; Loper, J C

1988-01-01

174

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. PMID:20446763

Kazanis, Sophia; Dang, Marina

2010-01-01

175

The evolutionary history of Cytochrome P450 genes in four filamentous Ascomycetes  

PubMed Central

Background The Cytochrome P450 system is important in fungal evolution for adapting to novel ecological niches. To elucidate the evolutionary process of cytochrome P450 genes in fungi with different life styles, we studied the patterns of gene gains and losses in the genomes of four filamentous Ascomycetes, including two saprotrophs (Aspergillus nidulans (AN) and Neurospora crassa (NC)) and two plant pathogens (Fusarium graminearum (FG) and Magnaporthe grisea (MG)). Results A total of 376 P450 genes were assigned to 168 families according to standard nomenclature. On average, only 1 to 2 genes per family were in each genome. To resolve conflicting results between different clustering analyses and standard family designation, a higher order relationship was formulated. 376 genes were clustered into 115 clans. Subsequently a novel approach based on parsimony was developed to build the evolutionary models. Based on these analyses, a core of 30 distinct clans of P450s was defined. The core clans experienced contraction in all four fungal lineages while new clans expanded in all with exception of NC. MG experienced more genes and clans gains compared to the other fungi. Parsimonious analyses unanimously supported one species topology for the four fungi. Conclusion The four studied fungi exhibit unprecedented diversity in their P450omes in terms of coding sequence, intron-exon structures and genome locations, suggesting a complicated evolutionary history of P450s in filamentous Ascomycetes. Clan classification and a novel strategy were developed to study evolutionary history. Contraction of core clans and expansion of novel clans were identified. The exception was the NC lineage, which exhibited pure P450 gene loss. PMID:17324274

Deng, Jixin; Carbone, Ignazio; Dean, Ralph A

2007-01-01

176

The B' helix determines cytochrome P450nor specificity for the electron donors NADH and NADPH.  

PubMed

Nitric oxide reductase (Nor) cytochrome P450nor (P450nor) is unique because it is catalytically self-sufficient, receiving electrons directly from NADH or NADPH. However, little is known about the direct binding of NADH to cytochrome. Here, we report that oxidized pyridine nucleotides (NAD(+) and NADP(+)) and an analogue induce a spectral perturbation in bound heme when mixed with P450nor. The P450nor isoforms are classified according to electron donor specificity for NADH or NADPH. One type (Fnor, a P450nor of Fusarium oxysporum) utilizes only NADH. We found that NAD(+) induced a type I spectral change in Fnor, whereas NADP(+) induced a reverse type I spectral change, although the K(d) values for both were comparable. In contrast, NADP(+) as well as NAD(+) caused a type I spectral change in Tnor, a P450nor isozyme from Trichosporon cutaneum that utilizes both NADH and NADPH as electron donors. The B' helix region of Tnor ((73)SAGGKAAA(80)) contains some Ala and Gly residues, whereas the sequence is replaced at a few sites with more bulky amino acid residues in Fnor ((73)SASGKQAA(80)). A single mutation (S75G) significantly improved the NADPH- dependent Nor activity of Fnor, and the overall activity was accelerated via the NADPH-enhanced reduction step. These results showed that pyridine nucleotide cofactors can bind P450nor and that only a few residues in the B' helix region determine cofactor specificity. We further showed that a poor electron donor (NADPH) could also bind Fnor, but an appropriate configuration for electron transfer is blocked by steric hindrance mainly by Ser(75) against the 2'-phosphate moiety. The present results along with previous observations together revealed a novel motif for cofactor binding. PMID:12105197

Zhang, Li; Kudo, Takashi; Takaya, Naoki; Shoun, Hirofumi

2002-09-13

177

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

178

Update on allele nomenclature for human cytochromes P450 and the Human Cytochrome P450 Allele (CYP-allele) Nomenclature Database.  

PubMed

Interindividual variability in xenobiotic metabolism and drug response is extensive and genetic factors play an important role in this variation. A majority of clinically used drugs are substrates for the cytochrome P450 (CYP) enzyme system and interindividual variability in expression and function of these enzymes is a major factor for explaining individual susceptibility for adverse drug reactions and drug response. Because of the existence of many polymorphic CYP genes, for many of which the number of allelic variants is continually increasing, a universal and official nomenclature system is important. Since 1999, all functionally relevant polymorphic CYP alleles are named and published on the Human Cytochrome P450 Allele (CYP-allele) Nomenclature Web site (http://www.cypalleles.ki.se). Currently, the database covers nomenclature of more than 660 alleles in a total of 30 genes that includes 29 CYPs as well as the cytochrome P450 oxidoreductase (POR) gene. On the CYP-allele Web site, each gene has its own Webpage, which lists the alleles with their nucleotide changes, their functional consequences, and links to publications identifying or characterizing the alleles. CYP2D6, CYP2C9, CYP2C19, and CYP3A4 are the most important CYPs in terms of drug metabolism, which is also reflected in their corresponding highest number of Webpage hits at the CYP-allele Web site.The main advantage of the CYP-allele database is that it offers a rapid online publication of CYP-alleles and their effects and provides an overview of peer-reviewed data to the scientific community. Here, we provide an update of the CYP-allele database and the associated nomenclature. PMID:23475683

Sim, Sarah C; Ingelman-Sundberg, Magnus

2013-01-01

179

Immunochemical evidence for multiple steroid-inducible hepatic cytochromes P-450 in the rat.  

PubMed Central

It has been established that there are glucocorticoid-inducible hepatic cytochromes P-450 in the rat (P-450p), the rabbit (LM3c) and man (HLp) which share extensive structural, functional and regulatory features. We prepared immunochemical probes to P-450p and identified a unique monoclonal antibody, 1G8, that recognizes purified P-450p, but neither purified LM3c nor HLp, on immunoblot analysis. The N-terminal amino acid sequence of purified samples of P-450p was identical with that previously reported for P-450PCN1 [Gonzalez, Nebert, Hardwick & Kasper (1985) J. Biol. Chem. 260, 7435-7441]. Immunoblot analyses of liver microsomes from untreated male rats revealed two 1G8-reactive proteins, whereas liver microsomes from untreated females contained none. Another monoclonal antibody, 13-7-10, reacted specifically with LM3c and HLp, but not with P-450p. A single 13-7-10-reactive microsomal protein was detected in untreated male and female rats, the latter protein exhibiting a greater apparent Mr. 1G8-reactive proteins were induced to the greatest extent by triacetyloleandomycin, followed by dexamethasone, chlordane, pregnenolone-16 alpha-carbonitrile and 2,4,2',4'-tetrachlorobiphenyl. In contrast, 13-7-10-reactive proteins were most strongly induced by dexamethasone, only moderately by triacetyloleandomycin and pregnenolone-16 alpha-carbonitrile, weakly by chlordane and not at all by 2,4,2',4'-tetrachlorobiphenyl. We conclude that the P-450p family in rat liver consists of three or more proteins that are structurally related and yet appear to be under distinct regulatory control. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. PMID:3311032

Hostetler, K A; Wrighton, S A; Kremers, P; Guzelian, P S

1987-01-01

180

Spectral characterization and chiral interactions of plant microsomal cytochrome P450 with metolachlor and herbicide safeners.  

PubMed

The content and spectral characteristics of cytochrome P450 (Cyt P450) and cytochrome b(5) (Cyt b(5)) extracted from shoots of etiolated maize and rice seedlings were studied by using ultraviolet (UV) difference spectrophotometry. The results showed that fenclorim, rac-metolachlor and S-metolachlor may induce the same P450 isoenzyme with lambda(max) at 453 nm, while naphthalic anhydride (NA) induced another one with lambda(max) at 447 nm. The microsomal Cyt P450 and Cyt b(5) content of maize seedlings was higher than that of rice, and the Cyt b(5) content was higher than that of Cyt P450. Maize and rice microsomal Cyt P450 and Cyt b(5) were induced at different levels by the four chemicals, with the order as follows: NA > fenclorim > rac-metolachlor > S-metolachlor with p < 0.05. When induced by NA, fenclorim, rac-metolachlor and S-metolachlor, the maize Cyt P450 content was, respectively, 5.63-, 3.30-, 3.02- and 2.48-fold that of the control, the rice Cyt P450 content was 8.54-, 2.20-, 1.91- and 1.33-fold that of the control, the maize Cyt b(5) content was 9.89-, 5.49-, 4.69- and 3.40-fold that of the control, and the rice Cyt b(5) content was 7.76-, 4.56-, 2.60- and 1.82-fold that of the control. An enantio-difference existed when rac- and S-metolachlor combined with plant Cyt P450. The interaction of microsomal Cyt P450 with S-metolachlor is higher than that with rac-metolachlor, which may be one of the reasons why S-metolachlor is superior at killing weeds compared with rac-metolachlor. These results will help to develop an understanding of the tolerance for and selectivity of rac- and S-metolachlor. PMID:20390928

Liu, Huijun

2010-01-01

181

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

SciTech Connect

The major objective of personalized medicine is to select optimized drug therapies and to a large degree such mission is determined by the expression profiles of cytochrome(s) P450 (CYP). Accordingly, a proteomic case study in personalized medicine is provided by the superfamily of cytochromes P450. Our knowledge about CYP isozyme expression on a protein level is very limited and based exclusively on DNA/mRNA derived data. Such information is not sufficient because transcription and translation events do not lead to correlated levels of expressed proteins. Here we report expression profiles of CYPs in human liver obtained by mass spectrometry (MS)-based proteomic approach. We analyzed 32 samples of human liver microsomes (HLM) of different sexes, ages and ethnicity along with samples of recombinant human CYPs. We have experimentally confirmed that each CYP isozyme can be effectively differentiated by their unique isozyme-specific tryptic peptide(s). Trypsin digestion patterns for almost 30 human CYP isozymes were established. Those findings should assist in selecting tryptic peptides suitable for MS-based quantitation. The data obtained demonstrate remarkable differences in CYP expression profiles. CYP2E1, CYP2C8 and CYP4A11 were the only isozymes found in all HLM samples. Female and pediatric HLM samples revealed much more diverse spectrum of expressed CYPs isozymes compared to male HLM. We have confirmed expression of a number of “rare” CYP (CYP2J2, CYP4B1, CYP4V2, CYP4F3, CYP4F11, CYP8B1, CYP19A1, CYP24A1 and CYP27A1) and obtained first direct experimental data showing expression of such CYPs as CYP2F1, CYP2S1, CYP2W1, CYP4A22, CYP4X1, and CYP26A1 on a protein level. - Highlights: ? First detailed proteomic analysis of CYP isozymes expression in human liver ? Trypsin digestion patterns for almost 30 human CYP isozymes established ? The data obtained demonstrate remarkable differences in CYP expression profiles. ? Female HLM samples revealed more diverse spectrum of CYP isozymes than male. ? First data showing expression of 2F1, 2S1, 2W1, 4A22, 4X1, 26A1 on a protein level.

Shrivas, Kamlesh; Mindaye, Samuel T.; Getie-Kebtie, Melkamu; Alterman, Michail A., E-mail: Michail.Alterman@fda.hhs.gov

2013-02-15

182

Assessing orally bioavailable commercial silver nanoparticle product on human cytochrome P450 enzyme activity.  

PubMed

Abstract Nanotechnology produces a wide range of medicinal compounds, including nanoparticulate silver, which are increasingly introduced in various forms for consumer use. As with all medicinal compounds, potential drug interactions are an important consideration for ingested silver nanoparticles. Nanoparticulate silver-drug interactions may be mediated through induced oxidative stress in liver tissue where the majority of systemically bioavailable silver nanoparticles is found. To investigate whether an orally ingested commercially available colloidal silver nanoproduct produces pharmacokinetic interference on select cytochrome P450 enzymes, a prospective, single-blind, controlled in vivo human study using simultaneous administration of standardized probes for P450 enzyme classes CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 was conducted. Oral ingestion of a commercial colloidal silver nanoproduct produces detectable silver in human serum after 14 days of dosing. This silver, however, elicits no demonstrable clinically significant changes in metabolic, hematologic, urinary, physical findings or cytochrome P450 enzyme inhibition or induction activity. Given their increasingly broad, diverse human exposures, future characterization of human cytochrome P450 enzyme activity for other systemically bioavailable nanotechnology products are warranted. PMID:25137296

Munger, Mark A; Hadlock, Greg; Stoddard, Greg; Slawson, Matthew H; Wilkins, Diana G; Cox, Nicholas; Rollins, Doug

2014-08-19

183

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

184

Structural and Kinetic Basis of Steroid 17?,20-Lyase Activity in Teleost Fish Cytochrome P450 17A1 and Its Absence in Cytochrome P450 17A2.  

PubMed

Cytochrome P450 (P450) 17A enzymes play a critical role in the oxidation of the steroids progesterone (Prog) and pregnenolone (Preg) to glucocorticoids and androgens. In mammals, a single enzyme, P450 17A1, catalyzes both 17?-hydroxylation and a subsequent 17?,20-lyase reaction with both Prog and Preg. Teleost fish contain two 17A P450s; zebrafish P450 17A1 catalyzes both 17?-hydroxylation and lyase reactions with Prog and Preg, and P450 17A2 is more efficient in pregnenolone 17?-hydroxylation but does not catalyze the lyase reaction, even in the presence of cytochrome b5. P450 17A2 binds all substrates and products, although more loosely than P450 17A1. Pulse-chase and kinetic spectral experiments and modeling established that the two-step P450 17A1 Prog oxidation is more distributive than the Preg reaction, i.e. 17?-OH product dissociates more prior to the lyase step. The drug orteronel selectively blocked the lyase reaction of P450 17A1 but only in the case of Prog. X-ray crystal structures of zebrafish P450 17A1 and 17A2 were obtained with the ligand abiraterone and with Prog for P450 17A2. Comparison of the two fish P450 17A-abiraterone structures with human P450 17A1 (DeVore, N. M., and Scott, E. E. (2013) Nature 482, 116-119) showed only a few differences near the active site, despite only ?50% identity among the three proteins. The P450 17A2 structure differed in four residues near the heme periphery. These residues may allow the proposed alternative ferric peroxide mechanism for the lyase reaction, or residues removed from the active site may allow conformations that lead to the lyase activity. PMID:25533464

Pallan, Pradeep S; Nagy, Leslie D; Lei, Li; Gonzalez, Eric; Kramlinger, Valerie M; Azumaya, Caleigh M; Wawrzak, Zdzislaw; Waterman, Michael R; Guengerich, F Peter; Egli, Martin

2015-02-01

185

Preparation of human metabolites of propranolol using laboratory-evolved bacterial cytochromes P450.  

PubMed

Testing the toxicities and biological activities of the human metabolites of drugs is important for development of safe and effective pharmaceuticals. Producing these metabolites using human cytochrome P450s is difficult, however, because the human enzymes are costly, poorly stable, and slow. We have used directed evolution to generate variants of P450 BM3 from Bacillus megaterium that function via the "peroxide shunt" pathway, using hydrogen peroxide in place of the reductase domain, oxygen and NADPH. Here, we report further evolution of the P450 BM3 heme domain peroxygenase to enhance production of the authentic human metabolites of propranolol by this biocatalytic route. This system offers a versatile, cost-effective, and scaleable route to the synthesis of drug metabolites. PMID:16224788

Otey, Christopher R; Bandara, Geethani; Lalonde, James; Takahashi, Katsuyuki; Arnold, Frances H

2006-02-20

186

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

187

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

2011-03-01

188

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

189

Identification of the main human cytochrome P450 enzymes involved in safrole 1'-hydroxylation.  

PubMed

Safrole is a natural plant constituent, found in sassafras oil and certain other essential oils. The carcinogenicity of safrole is mediated through 1'-hydroxysafrole formation, followed by sulfonation to an unstable sulfate that reacts to form DNA adducts. To identify the main cytochrome P450 (P450) involved in human hepatic safrole 1'-hydroxylation (SOH), we determined the SOH activities of human liver microsomes and Escherichia coli membranes expressing bicistronic human P450s. Human liver (n = 18) microsomal SOH activities were in the range of 3.5-16.9 nmol/min/mg protein with a mean value of 8.7 +/- 0.7 nmol/min/mg protein. In human liver (n = 3) microsomes, the mean K(m) and V(max) values of SOH were 5.7 +/- 1.2 mM and 0.14 +/- 0.03 micromol/min/nmol P450, respectively. The mean intrinsic clearance (V(max)/K(m)) was 25.3 +/- 2.3 microL/min/nmol P450. SOH was sensitive to the inhibition by a CYP2C9 inhibitor, sulfaphenazole, and CYP2E1 inhibitors, 4-methylpyrazole and diethyldithiocarbamate. The liver microsomal SOH activity showed significant correlations with tolbutamide hydroxylation (r = 0.569) and chlorzoxazone hydroxylation (r = 0.770) activities, which were the model reactions catalyzed by CYP2C9 and CYP2E1, respectively. Human CYP2C9 and CYP2E1 showed SOH activities at least 2-fold higher than the other P450s. CYP2E1 showed an intrinsic clearance 3-fold greater than CYP2C9. These results demonstrated that CYP2C9 and CYP2E1 were the main P450s involved in human hepatic SOH. PMID:15310247

Ueng, Yune-Fang; Hsieh, Chih-Hang; Don, Ming-Jaw; Chi, Chin-Wen; Ho, Li-Kang

2004-08-01

190

Toxicological significance of mechanism-based inactivation of cytochrome p450 enzymes by drugs.  

PubMed

Cytochrome P450 (P450) enzymes oxidize xenobiotics into chemically reactive metabolites or intermediates as well as into stable metabolites. If the reactivity of the product is very high, it binds to a catalytic site or sites of the enzyme itself and inactivates it. This phenomenon is referred to as mechanism-based inactivation. Many clinically important drugs are mechanism-based inactivators that include macrolide antibiotics, calcium channel blockers, and selective serotonin uptake inhibitors, but are not always structurally and pharmacologically related. The inactivation of P450s during drug therapy results in serious drug interactions, since irreversibility of the binding allows enzyme inhibition to be prolonged after elimination of the causal drug. The inhibition of the metabolism of drugs with narrow therapeutic indexes, such as terfenadine and astemizole, leads to toxicities. On the other hand, the fate of P450s after the inactivation and the toxicological consequences remains to be elucidated, while it has been suggested that P450s modified and degraded are involved in some forms of tissue toxicity. Porphyrinogenic drugs, such as griseofulvin, cause mechanism-based heme inactivation, leading to formation of ferrochelatase-inhibitory N-alkylated protoporphyrins and resulting in porphyria. Involvement of P450-derived free heme in halothane-induced hepatotoxicity and catalytic iron in cisplatin-induced nephrotoxicity has also been suggested. Autoantibodies against P450s have been found in hepatitis following administration of tienilic acid and dihydralazine. Tienilic acid is activated by and covalently bound to CYP2C9, and the neoantigens thus formed activate immune systems, resulting in the formation of an autoantibodydirected against CYP2C9, named anti-liver/kidney microsomal autoantibody type 2, whereas the pathological role of the autoantibodies in drug-induced hepatitis remains largely unknown. PMID:17612953

Masubuchi, Yasuhiro; Horie, Toshiharu

2007-06-01

191

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

192

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

PubMed

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

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

2014-01-01

193

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

PubMed

Spodoptera frugiperda is a polyphagous lepidopteran pest that encounters a wide range of toxic plant metabolites in its diet. The ability of this insect to adapt to its chemical environment might be explained by the action of major detoxification enzymes such as cytochrome P450s (or CYP). Forty-two sequences coding for P450s were identified and most of the transcripts were found to be expressed in the midgut, Malpighian tubules and fat body of S.?frugiperda larvae. Relatively few P450s were expressed in the established cell line Sf9. In order to gain information on how these genes respond to different chemical compounds, larvae and Sf9 cells were exposed to plant secondary metabolites (indole, indole-3-carbinol, quercetin, 2-tridecanone and xanthotoxin), insecticides (deltamethrin, fipronil, methoprene, methoxyfenozide) or model inducers (clofibrate and phenobarbital). Several genes were induced by plant chemicals such as P450s from the 6B, 321A and 9A subfamilies. Only a few genes responded to insecticides, belonging principally to the CYP9A family. There was little overlap between the response in vivo measured in the midgut and the response in vitro in Sf9 cells. In addition, regulatory elements were detected in the promoter region of these genes. In conclusion, several P450s were identified that could potentially be involved in the adaptation of S.?frugiperda to its chemical environment. PMID:25315858

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

2015-02-01

194

Die Bedeutung genetischer Polymorphismen im Enzym Cytochrom P450 2C9 für Pharmakokinetik und Wirkungen der nichtsteroidalen Antiphlogistika Diclofenac und Ibuprofen.  

E-print Network

??Die Bedeutung genetischer Polymorphismen im Enzym Cytochrom P450 2C9 für Pharmakokinetik und Wirkungen von Diclofenac und Ibuprofen Es wird angenommen, dass Cytochrom-P450 2C9 die 4’-Hydroxylierung… (more)

Freytag, Georg Tobias Heinrich

2005-01-01

195

Pungent ginger components modulates human cytochrome P450 enzymes in vitro  

PubMed Central

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

2013-01-01

196

Chemical modification and inactivation of rat liver microsomal cytochrome P-450c by 2-bromo-4'-nitroacetophenone  

SciTech Connect

The alkylating agent 2-bromo-4'-nitroacetophenone (BrNAP) binds covalently to each of 10 isozymes of purified rat liver microsomal cytochrome P-450 (P-450a-P-450j) but substantially inhibits the catalytic activity of only cytochrome P-450c. Regardless of pH, incubation time, presence of detergents, or concentration of BrNAP, treatment of cytochrome P-450c with BrNAP resulted in no more than 90% inhibition of catalytic activity. Alkylation with BrNAP did not cause the release of heme from the holoenzyme or alter the spectral properties of cytochrome P-450c, data that exclude the putative heme-binding cysteine, Cys-460, as the major site of alkylation. Two residues in cytochrome P-450c reacted rapidly with BrNAP, for which reason maximal loss of catalytic activity was invariably associated with the incorporation of approximately 1.5 mol of BrNAP/mol of cytochrome P-450c. Two major radio-labeled peptides were isolated from a tryptic digest of (/sup 14/CC)BrNAP-treated cytochrome P-450c by reverse-phase high performance liquid chromatography. The amino acid sequence of each peptide was determined by microsequence analysis, but the identification of the residues alkylated by BrNAP was complicated by the tendency of the adducts to decompose when subjected to automated Edman degradation. However, results of competitive binding experiments with the sulfhydryl reagent 4,4'-dithiodipyridine identified Cys-292 as the major site of alkylation and Cys-160 as the minor site of alkylation by BrNAP in cytochrome P-450c.

Parkinson, A.; Ryan, D.E.; Thomas, P.E.; Jerina, D.M.; Sayer, J.M.; van Bladeren, P.J.; Haniu, M.; Shively, J.E.; Levin, W.

1986-09-05

197

The role of cytochrome P450s in polycyclic aromatic hydrocarbon carcinogenesis  

SciTech Connect

Metabolic activation of polycyclic aromatic hydrocarbons (PAH) to carcinogenic diol epoxides has been determined to be a critical step in tumor initiation by PAH. The key enzyme(s) involved in the metabolic activation are members of the cytochrome P450 superfamily. Two distinct isoforms of cytochrome P450 have been determined to be induced upon treatment of cells in culture with benzo(a)pyrene (B(a)P) by use of Immobilized Artificial Membrane Column High Performance Liquid Chromatography, Western blotting, Northern blotting, and in vitro metabolism studies. Cytochrome P4501A is involved in the metabolism of PAH in the human hepatoma cell line, HepG2; the human mammary carcinoma cell line, MCF-7; and the mouse hepatoma cell line; Hepa-1; whereas cytochrome P450EF is involved in this metabolism in both secondary hamster and mouse embryo cell cultures. Induction of cytochrome P450s by B(a)P generally leads to an increased metabolism of tritiated B(a)P, DMBA, and DB(a,1)P to water-soluble metabolities and to the formation of PAH-DNA adducts, suggesting that induction by B(a)P alters the metabolism of PAH to metabolic activation. DMBA induction of cytochrome P450s leads to various changes in metabolism and PAH-DNA binding and these changes were both cell and PAH specific. These results suggest that DMBA can shift metabolism of certain PAH towards metabolic activation in some cells, while in other cells DMBA or one of its metabolities can compete with other PAH for metabolic activation. UDP-glucuronosyl-transferase and epoxide hydrase do not have significant roles in detoxifying proximate or ultimate carcinogenic PAH metabolites, however, sulfotransferase and glutathione-S-transferase do detoxify proximate and ultimate carcinogenic metabolities in the HepG2 cell line. Finally, attempts to inhibit B(a)P metabolism and DNA-binding in intact cells in culture through conjugation of inhibitory cytochrome P4501A1 antibodies to insulin or folic acid were examined.

Polzer, R.J.

1993-01-01

198

Cytochrome P450 Drives a HIF-regulated Behavioral Response to Reoxygenation by C. elegans  

PubMed Central

Oxygen deprivation followed by reoxygenation causes pathological responses in many disorders, including ischemic stroke, heart attacks and reperfusion injury. Key aspects of ischemia-reperfusion can be modeled by a C. elegans behavior, the O2-ON response, which is suppressed by hypoxic preconditioning or inactivation of the O2-sensing HIF (hypoxia-inducible-factor) hydroxylase EGL-9. From a genetic screen, we found that the cytochrome P450 oxygenase CYP-13A12 acts in response to the EGL-9/HIF-1 pathway to facilitate the O2-ON response. CYP-13A12 promotes oxidation of polyunsaturated fatty acids into eicosanoids, signaling molecules that can strongly affect inflammatory pain and ischemia-reperfusion injury responses in mammals. We propose that roles of the EGL-9/HIF-1 pathway and cytochrome P450 in controlling responses to anoxia-reoxygenation are evolutionarily conserved. PMID:23811225

Ma, Dengke K.; Rothe, Michael; Zheng, Shu; Bhatla, Nikhil; Pender, Corinne L.; Menzel, Ralph; Horvitz, H. Robert

2014-01-01

199

Cytochrome P450 drives a HIF-regulated behavioral response to reoxygenation by C. elegans.  

PubMed

Oxygen deprivation followed by reoxygenation causes pathological responses in many disorders, including ischemic stroke, heart attacks, and reperfusion injury. Key aspects of ischemia-reperfusion can be modeled by a Caenorhabditis elegans behavior, the O2-ON response, which is suppressed by hypoxic preconditioning or inactivation of the O2-sensing HIF (hypoxia-inducible factor) hydroxylase EGL-9. From a genetic screen, we found that the cytochrome P450 oxygenase CYP-13A12 acts in response to the EGL-9-HIF-1 pathway to facilitate the O2-ON response. CYP-13A12 promotes oxidation of polyunsaturated fatty acids into eicosanoids, signaling molecules that can strongly affect inflammatory pain and ischemia-reperfusion injury responses in mammals. We propose that roles of the EGL-9-HIF-1 pathway and cytochrome P450 in controlling responses to reoxygenation after anoxia are evolutionarily conserved. PMID:23811225

Ma, Dengke K; Rothe, Michael; Zheng, Shu; Bhatla, Nikhil; Pender, Corinne L; Menzel, Ralph; Horvitz, H Robert

2013-08-01

200

Fungal cytochrome P450s catalyzing hydroxylation of substituted toluenes to form their hydroxymethyl derivatives.  

PubMed

The degradation of a series of nitroaromatic compounds by the lignin-degrading fungus Phanerochaete chrysosporium was examined. From 4-nitrotoluene (4-NT), several metabolic intermediates were identified. Initially, 4-NT was converted to 4-nitrobenzyl alcohol (4-NBA), followed by the oxidation reactions to form 4-nitrobenzaldehyde and 4-nitrobenzoic acid, albeit slowly. Exogenously added 4-nitrobenzaldehyde and 4-nitrobenzoic acid were predominantly reduced to 4-NBA. The fungal formation of 4-NBA was inhibited by piperonyl butoxide, a cytochrome P450 inhibitor, suggesting the involvement of cytochrome P450 in the hydroxylation of the methyl group. Similarly, 2-, and 3-nitrotoluenes and 4-chlorotoluene were converted to the corresponding arylalcohols by P. chrysosporium. On the other hand, toluene and 4-methoxytoluene were not converted. Thus, P. chrysosporium possesses an alkyl hydroxylation activity against aromatic compounds substituted with a strong electron-withdrawing group. PMID:15135530

Teramoto, Hiroshi; Tanaka, Hiroo; Wariishi, Hiroyuki

2004-05-15

201

[The induction of Guerin's carcinoma cytochrome p450 hydroxylase activity by retinoids].  

PubMed

The interconnection of tumor growth process and the provision of the body with vitamin A was studied. The replenishment of vitamin A stores of vitamin-deficient tumor bearing animals modulated Guerin's carcinoma growth rate in a dose dependent manner (r = 0,83). The morphological parameters of tumor growth at different provision with vitamin A positively correlated with hydroxylase (r = 0,81) and demethylase (r = 0,49) activities of the Guerin's carcinoma cytochrome P450 system. The induction of hydroxylase and demethylase activities of cytochrome P450 in Guerin's carcinoma microsomal fraction, observed either under conditions of overdose supplementation, or selective liposomal form of all-trans-retinoic acid, suggests the stimulatory effect of retinoids on tumor growth. PMID:23289295

Shmarakov, I A; Katan, N V

2012-01-01

202

Cytochrome P-450-Dependent ?-Hydroxylation of Lauric Acid by Microsomes from Pea Seedlings 1  

PubMed Central

Microsomes from apical buds of pea (Pisum sativum L. var. Téléphone à rames) seedlings hydroxylate lauric acid at the ?-position. This oxidation is catalyzed by a cytochrome P-450 enzyme which differs from laurate hydroxylases previously described in microorganisms and mammals by its strict substrate specificity and the ability of low NADH concentrations to support unusually high oxidation rates. The apparent Km for lauric acid was 20 micromolar. NADPH- and NADH-dependent laurate hydroxylation followed non-Michaelian kinetics with apparent Km values ranging from 0.2 to 28 micromolar for NADPH, and 0.2 to 318 micromolar for NADH. When induced by the photomorphogenic photoreceptor phytochrome, the time course for the enhancement of laurate ?-hydroxylase was totally different from that of the cinnamic acid 4-hydroxylase, providing evidence for the existence of multiple cytochrome P-450 species in pea microsomes. PMID:16662431

Benveniste, Irène; Salaün, Jean-Pierre; Simon, Annick; Reichhart, Danièle; Durst, Francis

1982-01-01

203

A Model of the Membrane-bound Cytochrome b5-Cytochrome P450 Complex from NMR and Mutagenesis Data*  

PubMed Central

Microsomal cytochrome b5 (cytb5) is a membrane-bound protein that modulates the catalytic activity of its redox partner, cytochrome P4502B4 (cytP450). Here, we report the first structure of full-length rabbit ferric microsomal cytb5 (16 kDa), incorporated in two different membrane mimetics (detergent micelles and lipid bicelles). Differential line broadening of the cytb5 NMR resonances and site-directed mutagenesis data were used to characterize the cytb5 interaction epitope recognized by ferric microsomal cytP450 (56 kDa). Subsequently, a data-driven docking algorithm, HADDOCK (high ambiguity driven biomolecular docking), was used to generate the structure of the complex between cytP4502B4 and cytb5 using experimentally derived restraints from NMR, mutagenesis, and the double mutant cycle data obtained on the full-length proteins. Our docking and experimental results point to the formation of a dynamic electron transfer complex between the acidic convex surface of cytb5 and the concave basic proximal surface of cytP4502B4. The majority of the binding energy for the complex is provided by interactions between residues on the C-helix and ?-bulge of cytP450 and residues at the end of helix ?4 of cytb5. The structure of the complex allows us to propose an interprotein electron transfer pathway involving the highly conserved Arg-125 on cytP450 serving as a salt bridge between the heme propionates of cytP450 and cytb5. We have also shown that the addition of a substrate to cytP450 likely strengthens the cytb5-cytP450 interaction. This study paves the way to obtaining valuable structural, functional, and dynamic information on membrane-bound complexes. PMID:23709268

Ahuja, Shivani; Jahr, Nicole; Im, Sang-Choul; Vivekanandan, Subramanian; Popovych, Nataliya; Le Clair, Stéphanie V.; Huang, Rui; Soong, Ronald; Xu, Jiadi; Yamamoto, Kazutoshi; Nanga, Ravi P.; Bridges, Angela; Waskell, Lucy; Ramamoorthy, Ayyalusamy

2013-01-01

204

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

SciTech Connect

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

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

2013-01-25

205

Identification and characterization of a bacterial cytochrome P450 for the metabolism of diclofenac  

Microsoft Academic Search

The bacterium Actinoplanes sp. ATCC 53771 is known to perform drug metabolism of several xenobiotics similarly to humans. We identified a cytochrome\\u000a P450 enzyme from this strain, CYP107E4, and expressed it in Escherichia coli using the pET101 vector. The purified enzyme showed the characteristic reduced-CO difference spectra with a peak at 450 nm,\\u000a indicating the protein is produced in the active

Jamie E. Prior; Touraj Shokati; Uwe Christians; Ryan T. Gill

2010-01-01

206

Concomitant use of cytochrome P450 3A4 inhibitors and simvastatin  

Microsoft Academic Search

The long-term safety profile of simvastatin, established over 10 years of clinical use, is excellent. The principal adverse effect of all inhibitors of hydroxymethylglutarate co-enzyme A (HMG-CoA) reductase, myopathy, is infrequent. Simvastatin is a substrate for cytochrome P450 3A4 (CYP3A4). CYP3A4 inhibitors can elevate the plasma concentration of HMG-CoA reductase inhibitory activity derived from simvastatin. Clinical experience has shown that

Peter J. K Gruer; Jose M Vega; Michele F Mercuri; Michael R Dobrinska; Jonathan A Tobert

1999-01-01

207

Pesticides and Essential Minerals Modify Endogenous Antioxidants and Cytochrome P450 in Tissues of Rats  

Microsoft Academic Search

Two experiments were conducted in male Sprague Dawley (SD) rats (175–200 g) to determine changes in the activities of endogenous antioxidants superoxide dismutase (SOD), glutathione peroxidase (GPX), cytochrome P450 (ethoxyresorufin deethylase; EROD) and concentrations of glutathione (GSH) in the blood, liver, and small intestinal mucosa (IM). In both experiments, six rats\\/group were fed diets based on the AIN-93M diet (Control)

FREDERICK N. BEBE; MYNA PANEMANGALORE

2005-01-01

208

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

209

Cytochrome P-450 complex formation by dirithromycin and other macrolides in rat and human livers.  

PubMed Central

Some macrolide antibiotics cause clinical drug interactions, resulting in altered metabolism of concomitantly administered drugs, via formation of an inactive cytochrome P-450 complex. In the present study, the formation of a cytochrome P-450 type I binding spectrum and a metabolic intermediate complex by troleandomycin and dirithromycin was assessed in liver microsomes obtained from untreated rats and phenobarbital- or dexamethasone-pretreated rats. Troleandomycin produced a type I binding spectrum and metabolic intermediate complex in microsomes from dexamethasone- and phenobarbital-pretreated rats. Dirithromycin did not produce a detectable type I binding spectrum but formed a small cytochrome P-450 metabolic intermediate complex (6% of that formed by troleandomycin) in microsomes from dexamethasone-pretreated rats only. The formation of a cytochrome P-450 type I binding spectrum and a metabolic intermediate complex by troleandomycin, erythromycin, dirithromycin, and erythromycylamine was also assessed in microsomes prepared from human livers. Troleandomycin and erythromycin formed a type I binding spectrum and a metabolic intermediate complex which were larger in microsomes from subjects on barbiturate therapy than in microsomes from subjects with no recent barbiturate exposure. Erythromycylamine did not form a detectable type I binding spectrum with any of the human microsomal samples, but a small metabolic intermediate complex was formed with microsomes from a subject on phenobarbital, phenytoin, and propranolol therapy. Dirithromycin did not form a detectable type I binding spectrum or a metabolic intermediate complex in any human liver sample. Preclinical quantitation of the human metabolic intermediate complex may be helpful in predicting the possibility of clinical drug interactions of new drug candidates. PMID:8452357

Lindstrom, T D; Hanssen, B R; Wrighton, S A

1993-01-01

210

Phylogenetic Analysis of the Cytochrome P450 3 (CYP3) Gene Family  

Microsoft Academic Search

Cytochrome P450 genes (CYP) constitute a superfamily with members known from the Bacteria, Archaea, and Eukarya. The CYP3 gene family includes the CYP3A and CYP3B subfamilies. Members of the CYP3A subfamily represent the dominant CYP forms expressed in the digestive and respiratory tracts of vertebrates. The CYP3A enzymes metabolize a wide variety of chemically diverse lipophilic organic compounds. To understand

Andrew G. McArthur; Tove Hegelund; Rachel L. Cox; John J. Stegeman; Mette Liljenberg; Urban Olsson; Per Sundberg; Malin C. Celander

2003-01-01

211

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

212

Biosynthesis of cyanogenic glucosides in Triglochin maritima and the involvement of cytochrome P450 enzymes.  

PubMed

The biosynthesis of the two cyanogenic glucosides, taxiphyllin and triglochinin, in Triglochin maritima (seaside arrow grass) has been studied using undialyzed microsomal preparations from flowers and fruits. Tyrosine was converted to p-hydroxymandelonitrile with V(max) and K(m) values of 36 nmol mg(-1) g(-1) fresh weight and 0.14 mM, respectively. p-Hydroxyphenylacetaldoxime and p-hydroxyphenylacetonitrile accumulated as intermediates in the reaction mixtures. Using radiolabeled tyrosine as substrate, the radiolabel was easily trapped in p-hydroxyphenylacetaldoxime and p-hydroxyphenylacetonitrile when these were added as unlabeled compounds. p-Hydroxyphenylacetaldoxime was the only product obtained using microsomes prepared from green leaves or dialyzed microsomes prepared from flowers and fruits. These data contrast earlier reports (Hösel and Nahrstedt, Arch. Biochem. Biophys. 203, 753-757, 1980; and Cutler et al., J. Biol. Chem. 256, 4253-4258, 1981) where p-hydroxyphenylacetaldoxime was found not to accumulate. All steps in the conversion of tyrosine to p-hydroxymandelonitrile were found to be catalyzed by cytochrome P450 enzymes as documented by photoreversible carbon monoxide inhibition, inhibition by antibodies toward NADPH-cytochrome P450 oxidoreductase, and by cytochrome P450 inhibitors. We hypothesize that cyanogenic glucoside synthesis in T. maritima is catalyzed by multifunctional cytochrome P450 enzymes similar to CYP79A1 and CYP71E1 in Sorghum bicolor except that the homolog to CYP71E1 in T. maritima exhibits a less tight binding of p-hydroxyphenylacetonitrile, thus permitting the release of this intermediate and its conversion into triglochinin. PMID:10415119

Nielsen, J S; Moller, B L

1999-08-01

213

Quantitative evaluation of bromodichloromethane metabolism by recombinant rat and human cytochrome P450s  

Microsoft Academic Search

We report quantitative estimates of the parameters for metabolism of bromodichloromethane (BDCM) by recombinant preparations of hepatic cytochrome P450s (CYPs) from rat and human. Earlier work identified CYP2E1, CYP2B1\\/2 and CYP1A2 as activating enzymes necessary for hepatotoxicity in rat. In order to extend an existing PBPK model for rat to include a capability for extrapolation to humans, it is necessary

John W Allis; Guangyu Zhao

2002-01-01

214

Kinetics of bromodichloromethane metabolism by cytochrome P450 isoenzymes in human liver microsomes  

Microsoft Academic Search

The kinetic constants for the metabolism of bromodichloromethane (BDCM) by three cytochrome P450 (CYP) isoenzymes have been measured in human liver microsomes. The three CYP isoenzymes, CYP2E1, CYP1A2 and CYP3A4, have been identified previously as important in the metabolism of this compound. To measure the constants for each isoenzyme, enzyme-specific inhibitory antibodies were used to block the activities for two

Guangyu Zhao; John W Allis

2002-01-01

215

Species differences in the toxicity and cytochrome P450 IIIA-dependent metabolism of digitoxin.  

PubMed

In rats, cytochrome P450 (P450) IIIA enzymes are an important determinant of digitoxin toxicity. Induction of these liver microsomal enzymes decreases the toxicity of digitoxin by increasing its oxidative cleavage to digitoxigenin bis- and monodigitoxoside (dt2 and dt1). The present study shows that the susceptibility of different mammalian species to digitoxin toxicity is inversely related to liver microsomal P450 IIIA activity (measured as testosterone 6 beta-hydroxylase activity). Based on this correlation, we correctly predicted that hamsters, which have the highest P450 IIIA activity, are extremely resistant to digitoxin toxicity. To further examine the relationship between digitoxin toxicity and P450 IIIA activity, the pathways of digitoxin metabolism catalyzed by liver microsomes from nine mammalian species were examined by high performance liquid chromatography. The overall rate of digitoxin metabolism varied approximately 90-fold and followed the rank order: hamster greater than rat greater than guinea pig greater than dog greater than mouse approximately monkey greater than rabbit approximately cat greater than human. The qualitative differences in digitoxin metabolism were as striking as the quantitative differences. Formation of 16- and/or 17-hydroxydigitoxin was the major pathway of digitoxin oxidation catalyzed by liver microsomes from hamster, guinea pig, rabbit, cat, dog, and cynomolgus monkey. Guinea pig and, to a lesser extent, hamster liver microsomes also converted digitoxin to an unknown metabolite, the formation of which was catalyzed by P450. None of the species examined catalyzed the 12-hydroxylation of digitoxin to digoxin at a high rate. Similarly, none of the species examined catalyzed a high rate of conversion of digitoxin to dt2, with the notable exception of the rat. However, dt2 formation was the major pathway of digitoxin metabolism catalyzed by human liver microsomes, although humans were much less active (approximately 2%) than rats in this regard. The rate of dt2 formation varied approximately 41-fold among 22 samples of human liver microsomes, which was highly correlated (r = 0.841) with the rate of testosterone 6 beta-hydroxylation. Antibody against rat P450 IIIA1 inhibited the high rate of dt2 formation by rat liver microsomes and the low rate catalyzed by mouse, guinea pig, dog, monkey, and human liver microsomes. In contrast, anti-P450 IIIA1 did not inhibit the 12-, 16-, or 17-hydroxylation of digitoxin (or the formation of the unknown metabolite), despite the fact that anti-P450 IIIA1 strongly inhibited (greater than 70%) the 6 beta-hydroxylation of testosterone by liver microsomes from each of the species examined (except rabbit liver microsomes, which were inhibited only approximately 30%).(ABSTRACT TRUNCATED AT 400 WORDS) PMID:1944247

Eberhart, D C; Gemzik, B; Halvorson, M R; Parkinson, A

1991-11-01

216

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

2010-05-01

217

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

USGS Publications Warehouse

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

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

1999-01-01

218

Dechlorination of halocarbons by microsomes and vesicular reconstituted cytochrome P-450 systems under reductive conditions.  

PubMed Central

A spectrophotometric assay of the reductive dechlorination of halocarbons was developed and used to determine the kinetic characteristics of dechlorination of a range of haloethanes catalysed by microsomes from rat and rabbit liver. Analysis of the typical reaction of hexachloroethane shows that the reaction is catalysed by cytochrome P-450 and results in the formation of olefinic products as well as less chlorinated alkanes: in other respects the reaction resembles that known to occur with carbon tetrachloride. The dechlorination of haloethanes catalysed by a vesicular reconstituted system of cytochrome P-450 enzymes from rabbit liver was also studied and found to be similar to that catalysed by microsomes: both reductase and a phenobarbital inducible form of cytochrome P-450 were essential. There is no substantial dependence of maximum dechlorination rate on compound structure, suggesting that the reduction of substrate is not the rate limiting step in the overall reaction. The main factor in determining the apparent binding constant to the enzyme is the partition coefficient into the lipid membrane, as assessed by calculated log P values. PMID:3986141

Salmon, A G; Nash, J A; Walklin, C M; Freedman, R B

1985-01-01

219

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

220

Electrochemistry of mammalian cytochrome P450 2B4 indicates tunable thermodynamic parameters in surfactant films  

PubMed Central

Electrochemical methods continue to present an attractive means for achieving in vitro biocatalysis with cytochromes P450; however fully understanding the nature of electrode-bound P450 remains elusive. Herein we report thermodynamic parameters using electrochemical analysis of full-length mammalian microsomal cytochrome P450 2B4 (CYP 2B4) in didodecyldimethylammonium bromide (DDAB) surfactant films. Electronic absorption spectra of CYP 2B4-DDAB films on silica slides reveal an absorption maximum at 418 nm, characteristic of low-spin, six-coordinate, water-ligated FeIII heme in P450. The FeIII/II and FeII/I redox couples (E1/2) of substrate-free CYP 2B4 measured by cyclic voltammetry are ?0.23 V and ?1.02 V (vs. SCE, or 14 mV and ?776 mV vs. NHE) at 21°C. The standard heterogeneous rate constant for electron transfer from the electrode to the heme for the FeIII/II couple was estimated at 170 s?1. Experiments indicate that the system is capable of catalytic reduction of dioxygen, however substrate oxidation was not observed. From the variation of E1/2 with temperature (18 – 40 °C), we have measured entropy and enthalpy changes that accompany heme reduction, ?151 J mol?1 K?1 and ?46 kJ mol?1, respectfully. The corresponding entropy and enthalpy values are less for the six-coordinate low-spin, imidazole-ligated enzyme (?59 J mol?1 K?1 and ?18 kJ mol?1), consistent with limited conformational changes upon reduction. These thermodynamic parameters are comparable to those measured for bacterial P450 from Bacillus megaterium (CYP BM3), confirming our prior reports that the surfactant environment exerts a strong influence on the redox properties of the heme. PMID:24013063

Hagen, Katharine D.; Gillan, James M.; Im, Sang-Choul; Landefeld, Sally; Mead, Griffin; Hiley, Megan; Waskell, Lucy A.; Hill, Michael G.; Udit, Andrew K.

2013-01-01

221

Drug Interactions between Nine Antifungal Agents and Drugs Metabolized by Human Cytochromes P450.  

PubMed

This article reviews in vitro metabolic and in vivo pharmacokinetic drug-drug interactions of nine antifungal agents: six azoles (fluconazole, itraconazole, ketoconazole, miconazole, posaconazole, and voriconazole) and three echinocandins (anidulafungin, caspofungin, and micafungin). In in vitro interaction studies, itraconazole, ketoconazole, and miconazole were found to have higher inhibitory effects on cytochrome P450 (P450 or CYP) 3A4 and 3A5 activities than the other azoles or echinocandins did. Fluconazole, itraconazole, and voriconazole were relatively less potent inhibitors of CYP3A5 than of CYP3A4. The inhibitory effects of fluconazole, itraconazole, ketoconazole, and voriconazole against CYP3A4 and CYP3A5 seemed to be correlated with their dissociation constants for CYP51 (lanosterol 14a-demethylase) from Candida albicans. In in vivo pharmacokinetic studies, itraconazole was found to be a potent clinically important inhibitor of CYP3A4/5 substrates, and fluconazole and voriconazole increased the blood/plasma concentrations of not only CYP3A4/5 substrates but also CYP2C9 substrates.Miconazole was a potent inhibitor of all P450s investigated in vitro, although there are few detailed studies on the clinical significance of this except for CYP2C9. For the echinocandins, no marked inhibition of P450 activities, except for some inhibition of CYP3A4/5 activity, was observed in vitro. The blood/plasma concentrations of concomitant drugs were not markedly affected by coadministration of echinocandins in vivo, suggesting that echinocandins do not cause clinically significant interactions with drugs that are metabolized by P450s via the inhibition of metabolism. The differential effects of these antifungal agents on P450 activities must be considered when clinicians select antifungal agents for patients also receiving other drugs. PMID:25429674

Niwa, Toshiro; Imagawa, Yurie; Yamazaki, Hiroshi

2014-11-25

222

Synergistic Effects of Mutations in Cytochrome P450cam Designed to Mimic CYP101D1  

PubMed Central

A close ortholog to the cytochrome P450cam (CYP101A1) that catalyzes the same hydroxylation of camphor to 5-exo hydroxycamphor is CYP101D1. There are potentially important differences in and around the active site that could contribute to subtle functional differences. Adjacent to the heme iron ligand, Cys357, is Leu358 in P450cam while this residue is Ala in CYP101D1. Leu358 plays a role in binding of the P450cam redox partner, putidaredoxin (Pdx). On the opposite side of the heme about 15 – 20 Å away Asp251 in P450cam plays a critical role in a proton relay network required for O2 activation but forms strong ion pairs with Arg186 and Lys178. In CYP101D1 a Gly replaces Lys178. Thus, the local electrostatic environment and ion pairing is substantially different in CYP101D1. These sites have been systematically mutated in P450cam to the corresponding residues in CYP101D1 and the mutants analyzed by crystallography, kinetics, and UV/Vis spectroscopy. Individually the mutants have little effect on activity or structure but in combination there is a major drop in enzyme activity. This loss in activity is due the mutants being locked in the low-spin state which prevents electron transfer from the P450cam redox partner, Pdx. These studies illustrate the strong synergistic effects on well separated parts of the structure in controlling the equilibrium between the open (low-spin) and closed (high-spin) conformational states. PMID:23865948

Batabyal, Dipanwita; Li, Huiying; Poulos, Thomas L.

2013-01-01

223

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. PMID:23634216

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

2013-01-01

224

Malathion bioactivation in the human liver: the contribution of different cytochrome p450 isoforms.  

PubMed

Among organophosphorothioate (OPT) pesticides, malathion is considered relatively safe for use in mammals. Its rapid degradation by carboxylesterases competes with the cytochrome P450 (P450)-catalyzed formation of malaoxon, the toxic metabolite. However, impurities in commercial formulations are potent inhibitors of carboxylesterase, allowing a dramatic increase in malaoxon formation. Malathion desulfuration has been characterized in human liver microsomes (HLMs) with a method based on acetylcholinesterase inhibition that is able to detect nanomolar levels of oxon. The active P450 isoforms have been identified by means of a multifaceted strategy, including the use of cDNA-expressed human P450s and correlation, immunoinhibition, and chemical inhibition studies in a panel of phenotyped HLMs. HLMs catalyzed malaoxon formation with a high level of variability (>200-fold). One or two components (K(mapp1) = 53-67 microM; K(mapp2) = 427-1721 microM) were evidenced, depending on the relative specific P450 content. Results from different approaches indicated that, at low malathion concentration, malaoxon formation is catalyzed by CYP1A2 and, to a lesser extent, 2B6, whereas the role of 3A4 is relevant only at high malathion levels. These results are in line with those found with chlorpyrifos, diazinon, azynphos-methyl, and parathion, characterized by the presence of an aromatic ring in the molecule. Since malathion has linear chains as substituents at the thioether sulfur, it can be hypothesized that, independently from the chemical structure, OPTs are bioactivated by the same P450s. These results also suggest that CYP1A2 and 2B6 can be considered as possible metabolic biomarkers of susceptibility to OPT-induced toxic effects at actual human exposure levels. PMID:15557345

Buratti, Franca M; D'Aniello, Alessandra; Volpe, Maria Teresa; Meneguz, Annarita; Testai, Emanuela

2005-03-01

225

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

PubMed

Genome sequencing of basidiomycetes, a group of fungi capable of degrading/mineralizing plant material, revealed the presence of numerous cytochrome P450 monooxygenases (P450s) in their genomes, with some exceptions. Considering the large repertoire of P450s found in fungi, it is difficult to identify P450s that play an important role in fungal metabolism and the adaptation of fungi to diverse ecological niches. In this study, we followed Sir Charles Darwin's theory of natural selection to identify such P450s in model basidiomycete fungi showing a preference for different types of plant components degradation. Any P450 family comprising a large number of member P450s compared to other P450 families indicates its natural selection over other P450 families by its important role in fungal physiology. Genome-wide comparative P450 analysis in the basidiomycete species, Phanerochaete chrysosporium, Phanerochaete carnosa, Agaricus bisporus, Postia placenta, Ganoderma sp. and Serpula lacrymans, revealed enrichment of 11 P450 families (out of 68 P450 families), CYP63, CYP512, CYP5035, CYP5037, CYP5136, CYP5141, CYP5144, CYP5146, CYP5150, CYP5348 and CYP5359. Phylogenetic analysis of the P450 family showed species-specific alignment of P450s across the P450 families with the exception of P450s of Phanerochaete chrysosporium and Phanerochaete carnosa, suggesting paralogous evolution of P450s in model basidiomycetes. P450 gene-structure analysis revealed high conservation in the size of exons and the location of introns. P450s with the same gene structure were found tandemly arranged in the genomes of selected fungi. This clearly suggests that extensive gene duplications, particularly tandem gene duplications, led to the enrichment of selective P450 families in basidiomycetes. Functional analysis and gene expression profiling data suggest that members of the P450 families are catalytically versatile and possibly involved in fungal colonization of plant material. To our knowledge, this is the first report on the identification and comparative-evolutionary analysis of P450 families enriched in model basidiomycetes. PMID:24466198

Syed, Khajamohiddin; Shale, Karabo; Pagadala, Nataraj Sekhar; Tuszynski, Jack

2014-01-01

226

Systematic Identification and Evolutionary Analysis of Catalytically Versatile Cytochrome P450 Monooxygenase Families Enriched in Model Basidiomycete Fungi  

PubMed Central

Genome sequencing of basidiomycetes, a group of fungi capable of degrading/mineralizing plant material, revealed the presence of numerous cytochrome P450 monooxygenases (P450s) in their genomes, with some exceptions. Considering the large repertoire of P450s found in fungi, it is difficult to identify P450s that play an important role in fungal metabolism and the adaptation of fungi to diverse ecological niches. In this study, we followed Sir Charles Darwin’s theory of natural selection to identify such P450s in model basidiomycete fungi showing a preference for different types of plant components degradation. Any P450 family comprising a large number of member P450s compared to other P450 families indicates its natural selection over other P450 families by its important role in fungal physiology. Genome-wide comparative P450 analysis in the basidiomycete species, Phanerochaete chrysosporium, Phanerochaete carnosa, Agaricus bisporus, Postia placenta, Ganoderma sp. and Serpula lacrymans, revealed enrichment of 11 P450 families (out of 68 P450 families), CYP63, CYP512, CYP5035, CYP5037, CYP5136, CYP5141, CYP5144, CYP5146, CYP5150, CYP5348 and CYP5359. Phylogenetic analysis of the P450 family showed species-specific alignment of P450s across the P450 families with the exception of P450s of Phanerochaete chrysosporium and Phanerochaete carnosa, suggesting paralogous evolution of P450s in model basidiomycetes. P450 gene-structure analysis revealed high conservation in the size of exons and the location of introns. P450s with the same gene structure were found tandemly arranged in the genomes of selected fungi. This clearly suggests that extensive gene duplications, particularly tandem gene duplications, led to the enrichment of selective P450 families in basidiomycetes. Functional analysis and gene expression profiling data suggest that members of the P450 families are catalytically versatile and possibly involved in fungal colonization of plant material. To our knowledge, this is the first report on the identification and comparative-evolutionary analysis of P450 families enriched in model basidiomycetes. PMID:24466198

Syed, Khajamohiddin; Shale, Karabo; Pagadala, Nataraj Sekhar; Tuszynski, Jack

2014-01-01

227

Expression of a Soybean Cytochrome P450 Monooxygenase cDNA in Yeast and Tobacco Enhances the Metabolism of Phenylurea Herbicides  

Microsoft Academic Search

A strategy based on the random isolation and screening of soybean cDNAs encoding cytochrome P450 monooxygenases (P450s) was used in an attempt to identify P450 isozymes involved in herbicide metabolism. Nine full-length (or near-full-length) P450 cDNAs representing eight distinct P450 families were isolated by using PCR-based technologies. Five of the soybean P450 cDNAs were expressed successfully in yeast, and microsomal

Balazs Siminszky; Frederick T. Corbin; Eric R. Ward; Thomas J. Fleischmann; Ralph E. Dewey

1999-01-01

228

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

PubMed Central

Pulmonary toxicity of styrene is initiated by cytochromes P450-dependent metabolic activation. P450 2E1 and P450 2F2 are considered to be two main cytochrome P450 (CYP) enzymes responsible for styrene metabolism in mice. The objective of the current study was to determine the correlation between the formation of styrene metabolites (i.e. styrene oxide and 4-vinylphenol) and pulmonary toxicity of styrene, using Cyp2e1- and Cyp2f2-null mouse models. Dramatic decrease in the formation of styrene glycol and 4-vinylphenol was found in Cyp2f2-null mouse lung microsomes, relative to that in the wild-type mouse lung microsomes. However, no significant difference in the production of the styrene metabolites was observed between lung microsomes obtained from Cyp2e1-null and the wild-type mice. The knock–out and wild-type mice were treated with styrene (6.0 mmol/kg, ip), and cell counts and LDH activity in bronchoalveolar lavage fluids were monitored to evaluate the pulmonary toxicity induced by styrene. Cyp2e1-null mice displayed similar susceptibility to lung toxicity of styrene as the wild-type animals. However, Cyp2f2-null mice were resistant to styrene-induced pulmonary toxicity. In conclusion, both P450 2E1 and P450 2F2 are responsible for the metabolic activation of styrene. The latter enzyme plays an important role in styrene-induced pulmonary toxicity. Both styrene oxide and 4-vinylphenol are suggested to participate in the development of lung injury induced by styrene. PMID:24320693

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

2014-01-01

229

Cytochrome P450 mRNA Expression in the Rodent Brain: Species-, Sex-, and Region-Dependent Differences  

PubMed Central

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

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

2014-01-01

230

The cytochrome P450 system of atlantic salmon (Salmo salar): I. Basal properties and induction of P450 1A1 in liver of immature and mature fish.  

PubMed

The major components of the cytochrome P450 (P450) system in liver microsomes of Atlantic salmon were studied using spectrophotometric, catalytic and immunochemical techniques. In juvenile fish sampled during the winter season, high basal activities of 7-ethoxyresorufin O-deethylase (EROD) were found. The Km for 7-ethoxyresorufin was 0.4 µM, and Vmax 1.23 nmol/min/mg protein in juvenile fish. In mature fish sampled from the same group of fish in December, EROD activity was barely detectable (20-30 pmol/min/mg protein). Treatment with the P450 1A1 inducer ?-naphthoflavone (BNF) resulted in almost 2-fold induction of total P450, and 30-40-fold induction of EROD activity in immature fish. A similar fold increase was seen in mature fish. The differences in EROD activity between untreated and BNF-treated fish, was accompanied by similar differences in a P450 1A1 cross-reacting protein (Mr=58,000 D) in immunochemical studies using rabbit anti-cod P450 1A1 IgG. However, judging from these studies, the levels of P450 1A1-protein in mature salmon far exceeded those accounted for by the measured EROD activity in comparison to immature fish (both before and after BNF-treatment), indicating inhibiting effects of sex steroids on the measured activity. This effect was not seen on 7-ethoxycoumarin O-deethylase activity. A long-term storage experiment indicated that Atlantic salmon liver microsomes can be stored for 2 years at -80°C in 20% glycerol without losing more than 20-40% of its catalytic activity. PMID:24213730

Goksøyr, A; Larsen, H E

1991-12-01

231

The evolution of cytochrome P450 enzymes as biocatalysts in drug discovery and development.  

PubMed

Engineered biocatalysts offer the opportunity to introduce modifications into complex lead molecules and drug candidates in a chemo-, regio- and stereoselective manner that is difficult to accomplish using traditional synthetic organic chemistry. As candidate biocatalysts, the cytochrome P450 enzymes that metabolize drugs and other xenobiotics are amongst the most versatile agents known. Not only can they mediate an exceptional range of biotransformation reactions, but they act on an unparalleled diversity of substrates. However, this versatility comes at the cost of relatively poor catalytic efficiency and low rates of coupling of cofactor consumption to product formation. Directed evolution is being used to redefine the substrate specificity of P450 enzymes while at the same time improving their efficiency, thermostability and other properties. This review will outline the key successes with bacterial P450s used as biocatalysts, examine the studies done to date with mammalian forms, and assess the prospects for exploiting xenobiotic-metabolizing P450s for applications in medicinal chemistry. PMID:24047135

Gillam, Elizabeth M J; Hayes, Martin A

2013-01-01

232

Inhibition of human cytochrome P450 enzymes by the natural hepatotoxin safrole.  

PubMed

The hepatotoxin, safrole is a methylenedioxy phenyl compound, found in sassafras oil and certain other essential oils. Recombinant cytochrome P450 (CYP, P450) and human liver microsomes were studied to investigate the selective inhibitory effects of safrole on human P450 enzymes and the mechanisms of action. Using Escherichia coli-expressed human P450, our results demonstrated that safrole was a non-selective inhibitor of CYP1A2, CYP2A6, CYP2D6, CYP2E1, and CYP3A4 in the IC(50) order CYP2E1 < CYP1A2 < CYP2A6 < CYP3A4 < CYP2D6. Safrole strongly inhibited CYP1A2, CYP2A6, and CYP2E1 activities with IC(50) values less than 20 microM. Safrole caused competitive, non-competitive, and non-competitive inhibition of CYP1A2, CYP2A6 and CYP2E1 activities, respectively. The inhibitor constants were in the order CYP1A2 < CYP2E1 < CYP2A6. In human liver microsomes, 50 microM safrole strongly inhibited 7-ethoxyresorufin O-deethylation, coumarin hydroxylation, and chlorzoxazone hydroxylation activities. These results revealed that safrole was a potent inhibitor of human CYP1A2, CYP2A6, and CYP2E1. With relatively less potency, CYP2D6 and CYP3A4 were also inhibited. PMID:15778010

Ueng, Yune-Fang; Hsieh, Chih-Hang; Don, Ming-Jaw

2005-05-01

233

Freeze-quenched iron-oxo intermediates in cytochromes P450.  

PubMed

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 Mössbauer 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 Mössbauer 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. PMID:16143295

Jung, Christiane; Schünemann, Volker; Lendzian, Friedhelm

2005-12-01

234

Cytochrome P450 compound I: capture, characterization, and C-H bond activation kinetics.  

PubMed

Cytochrome P450 enzymes are responsible for the phase I metabolism of approximately 75% of known pharmaceuticals. P450s perform this and other important biological functions through the controlled activation of C-H bonds. Here, we report the spectroscopic and kinetic characterization of the long-sought principal intermediate involved in this process, P450 compound I (P450-I), which we prepared in approximately 75% yield by reacting ferric CYP119 with m-chloroperbenzoic acid. The Mössbauer spectrum of CYP119-I is similar to that of chloroperoxidase compound I, although its electron paramagnetic resonance spectrum reflects an increase in |J|/D, the ratio of the exchange coupling to the zero-field splitting. CYP119-I hydroxylates the unactivated C-H bonds of lauric acid [D(C-H) ~ 100 kilocalories per mole], with an apparent second-order rate constant of k(app) = 1.1 × 10(7) per molar per second at 4°C. Direct measurements put a lower limit of k ? 210 per second on the rate constant for bound substrate oxidation, whereas analyses involving kinetic isotope effects predict a value in excess of 1400 per second. PMID:21071661

Rittle, Jonathan; Green, Michael T

2010-11-12

235

Evaluation of six proton pump inhibitors as inhibitors of various human cytochromes P450: focus on cytochrome P450 2C19.  

PubMed

Six proton pump inhibitors (PPIs), omeprazole, lansoprazole, esomeprazole, dexlansoprazole, pantoprazole, and rabeprazole, were shown to be weak inhibitors of cytochromes P450 (CYP3A4, -2B6, -2D6, -2C9, -2C8, and -1A2) in human liver microsomes. In most cases, IC?? values were greater than 40 ?M, except for dexlansoprazole and lansoprazole with CYP1A2 (IC?? = ?8 ?M) and esomeprazole with CYP2C8 (IC?? = 31 ?M). With the exception of CYP2C19 inhibition by omeprazole and esomeprazole (IC?? ratio, 2.5 to 5.9), there was no evidence for a marked time-dependent shift in IC?? (IC?? ratio, ? 2) after a 30-min preincubation with NADPH. In the absence of preincubation, lansoprazole (IC?? = 0.73 ?M) and esomeprazole (IC?? = 3.7 ?M) were the most potent CYP2C19 inhibitors, followed by dexlansoprazole and omeprazole (IC?? = ?7.0 ?M). Rabeprazole and pantoprazole (IC?? = ? 25 ?M) were the weakest. A similar ranking was obtained with recombinant CYP2C19. Despite the IC?? ranking, after consideration of plasma levels (static and dynamic), protein binding, and metabolism-dependent inhibition, it is concluded that omeprazole and esomeprazole are the most potent CYP2C19 inhibitors. This was confirmed after the incubation of the individual PPIs with human primary hepatocytes (in the presence of human serum) and by monitoring their impact on diazepam N-demethylase activity at a low concentration of diazepam (2 ?M). Data described herein are consistent with reports that PPIs are mostly weak inhibitors of cytochromes P450 in vivo. However, two members of the PPI class (esomeprazole and omeprazole) are more likely to serve as clinically relevant inhibitors of CYP2C19. PMID:22648560

Zvyaga, Tatyana; Chang, Shu-Ying; Chen, Cliff; Yang, Zheng; Vuppugalla, Ragini; Hurley, Jeremy; Thorndike, Denise; Wagner, Andrew; Chimalakonda, Anjaneya; Rodrigues, A David

2012-09-01

236

SEASONAL HEPATIC CYTOCHROME P-450 INDUCTION IN COTTON RATS (SIGMODON HISPIDUS) INHABITING PETROCHEMICAL WASTE SITES. (R826242)  

EPA Science Inventory

Abstract Wildlife species inhabiting contaminated sites are often exposed to complex mixtures of chemicals that have known effects on physiological and biochemical function. We evaluated the induction of major hepatic cytochrome P-450 isoenzymes through O -dealky...

237

Production and X-ray crystallographic analysis of fully deuterated cytochrome P450cam  

SciTech Connect

Neutron protein crystallography allows H-atom positions to be located in biological structures at the relatively modest resolution of 1.5-2.0 {angstrom}. A difficulty of this technique arises from the incoherent scattering from hydrogen, which considerably reduces the signal-to-noise ratio of the data. This can be overcome by preparing fully deuterated samples. Efficient protocols for routine and low-cost production of in vivo deuterium-enriched proteins have been developed. Here, the overexpression and crystallization of highly (>99%) deuterium-enriched cytochrome P450cam for neutron analysis is reported. Cytochrome P450cam from Pseudomonas putida catalyses the hydroxylation of camphor from haem-bound molecular O{sub 2} via a mechanism that is thought to involve a proton-shuttle pathway to the active site. Since H atoms cannot be visualized in available X-ray structures, neutron diffraction is being used to determine the protonation states and water structure at the active site of the enzyme. Analysis of both hydrogenated and perdeuterated P450cam showed no significant changes between the X-ray structures determined at 1.4 and 1.7 {angstrom}, respectively. This work demonstrates that the fully deuterated protein is highly isomorphous with the native (hydrogenated) protein and is appropriate for neutron protein crystallographic analysis.

Meilleur, Flora [Institut Laue-Langevin (ILL); Dauvergne, M. T. [European Molecular Biology Laboratory (EMBL), France; Schlichting, Ilme [Max-Planck Institut fur Medizinische Forschung; Myles, Dean A A [ORNL

2005-03-01

238

EFFECT OF METHACRYLONITRILE ON CYTOCHROME P-450 2E1 (CYP2E1) EXPRESSION IN MALE F344 RATS  

Microsoft Academic Search

Tissue-specific induction of cytochrome P-450s (CYP) followed by increased in situ bioactivation may contribute to chemical-induced site-specific toxicity. In rats, methacrylonitrile (MAN) is metabolized by cytochrome P-450 2E1 (CYP2E1) to acetone, which is eliminated along with parent MAN in breath. Gavage administration of MAN to rats causes olfactory epithelial damage and liver enlargement. It was hypothesized that treatment of rats

Hongbing Wang; Brian Chanas; Burhan I. Ghanayem

2002-01-01

239

Cytochrome P450 2B6 is a growth-inhibitory and prognostic factor for prostate cancer  

Microsoft Academic Search

BACKGROUND: Cytochrome P450s (CYPs) influence the biological effects of carcinogens, drugs and hormones including testosterones. Among them, Cytochrome P450 2B6 (CYP2B6) plays a critical role in the deactivation of testosterone. In the present study, we examined CYP2B6 expression in human prostate tissues and prostate cancer. METHODS: Immunohistochemical analysis was performed in 98 benign and 106 malignant prostate tissues and patients'

Jinpei Kumagai; Tetsuya Fujimura; Satoru Takahashi; Tomohiko Urano; Tetsuo Ogushi; Kuniko Horie-Inoue; Yasuyoshi Ouchi; Tadaichi Kitamura; Masami Muramatsu; Bruce Blumberg; Satoshi Inoue

2007-01-01

240

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

SciTech Connect

Highlights: {yields} Humans with severe forms of cytochrome P450 oxidoreductase (CYPOR) mutations show bone defects as observed in Antley-Bixler Syndrome. {yields} First report showing knockdown of CYPOR in osteoblasts decreased Connexin 43 (Cx43) protein levels. Cx43 is known to play an important role in bone modeling. {yields} Knockdown of CYPOR decreased Gap Junctional Intercellular Communication and hemichannel activity. {yields} Knockdown of CYPOR decreased Cx43 in mouse primary calvarial osteoblasts. {yields} Decreased Cx43 expression was observed at the transcriptional level. -- Abstract: Cytochrome P450 oxidoreductase (CYPOR) is a microsomal electron-transferring enzyme containing both FAD and FMN as co-factors, which provides the reducing equivalents to various redox partners, such as cytochromes P450 (CYPs), heme oxygenase (HO), cytochrome b{sub 5} and squalene monooxygenase. Human patients with severe forms of CYPOR mutation show bone defects such as cranio- and humeroradial synostoses and long bone fractures, known as Antley-Bixler-like Syndrome (ABS). To elucidate the role of CYPOR in bone, we knocked-down CYPOR in multiple osteoblast cell lines using RNAi technology. In this study, knock-down of CYPOR decreased the expression of Connexin 43 (Cx43), known to play a critical role in bone formation, modeling, and remodeling. Knock-down of CYPOR also decreased Gap Junction Intercellular Communication (GJIC) and hemichannel activity. Promoter luciferase assays revealed that the decrease in expression of Cx43 in CYPOR knock-down cells was due to transcriptional repression. Primary osteoblasts isolated from bone specific Por knock-down mice calvariae confirmed the findings in the cell lines. Taken together, our study provides novel insights into the regulation of gap junction function by CYPOR and suggests that Cx43 may play an important role(s) in CYPOR-mediated bone defects seen in patients.

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

2011-08-05

241

DESIGN, SYNTHESIS, AND EVALUATION OF CARBAZOLE ANALOGS AS POTENTIAL CYTOCHROME P450 INHIBITORS  

PubMed Central

Carbazoles are a class of nitrogen-containing aromatic heterocyclic compounds. They not only have various biological activities (e.g. antibacterial, anti-inflammatory, antitumor), but also exhibit useful properties as organic materials due to their special structures. Cytochrome P450 enzymes are a superfamily of hemoproteins involved in the metabolism of endogenous and exogenous compounds including many drugs and environmental chemicals. Some aryl and arylalkyl acetylenes, and propargyl ethers have been shown to act as inhibitors of certain P450s. In an attempt to improve the potency and selectivity of inhibition, we have focused our attention on the design and synthesis of a new series of carbazole analogs, a few of which contain a propargyl ether functional group. For this project, eight carbazole analogs have been synthesized and characterized.

Foroozesh, Maryam; Jiang, Quan; Sridhar, Jayalakshmi; Liu, Jiawang; Dotson, Brandan; McClain, Erika

2014-01-01

242

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

DOEpatents

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

Marrone, Babetta L. (Los Alamos, NM); Simpson, Daniel J. (Los Alamos, NM); Unkefer, Clifford J. (Los Alamos, NM); Whaley, Thomas W. (Santa Fe, NM)

1992-01-01

243

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

DOEpatents

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

Marrone, Babetta L. (Los Alamos, NM); Simpson, Daniel J. (Los Alamos, NM); Unkefer, Clifford J. (Los Alamos, NM); Whaley, Thomas W. (Santa Fe, NM)

1993-01-01

244

Dysregulation of cytochrome P450c 17 alpha as the cause of polycystic ovarian syndrome.  

PubMed

Polycystic ovarian syndrome (PCOS) appears to be due to a previously unrecognized type of steroidogenic abnormality, one in which hyperandrogenism arises from a regulatory abnormality (dysregulation) rather than from enzyme deficiency. It appears that PCOS typically arises from masculinized regulation of the androgen-forming enzyme (cytochrome P450c17 alpha) within ovarian thecal cells. This may arise by either excessive stimulation by luteinizing hormone (LH) or by escape from desensitization to LH. We review evidence which is compatible with the concept that the latter situation may result from an intrinsic intraovarian flaw in the paracrine feedback mechanism by which thecal androgen biosynthesis is inhibited and that coexistent adrenal 17-ketosteroid hyper-responsiveness to corticotropin (ACTH) may be due to a similar type of dysregulation of adrenocortical P450c17 alpha. PMID:2185040

Rosenfield, R L; Barnes, R B; Cara, J F; Lucky, A W

1990-05-01

245

Differentially regulated NADPH:cytochrome P450 oxidoreductases in?parsley  

PubMed Central

Two NADPH:cytochrome P450 oxidoreductases (CPRs) from parsley (Petroselinum crispum) were cloned, and the complete proteins were expressed and functionally identified in yeast. The two enzymes, designated CPR1 and CPR2, are 80% identical in amino acid sequence with one another and about 75% identical with CPRs from several other plant species. The mRNA accumulation patterns for CPR1 and CPR2 in fungal elicitor-treated or UV-irradiated cultured parsley cells and in developing or infected parsley plants were compared with those for cinnamate 4-hydroxylase (C4H), one of the most abundant CPR-dependent P450 enzymes in plants. All treatments strongly induced the mRNAs for C4H and CPR1 but not for CPR2, suggesting distinct metabolic roles of CPR1 and CPR2 and a functional relationship between CPR1 and C4H. PMID:9405720

Koopmann, Edda; Hahlbrock, Klaus

1997-01-01

246

Cytochrome P450 enzyme-mediated degradation of Echinacea alkylamides in human liver microsomes.  

PubMed

Echinacea preparations are widely used herbal remedies for the prevention and treatment of colds. In this study we have investigated the metabolism by human liver microsomes of the alkylamide components from an Echinacea preparation as well as that of pure synthetic alkylamides. No significant degradation of alkylamides was evident in cytosolic fractions. Time- and NADPH-dependent degradation of alkylamides was observed in microsomal fractions suggesting they are metabolised by cytochrome P450 (P450) enzymes in human liver. There was a difference in the susceptibility of 2-ene and 2,4-diene pure synthetic alkylamides to microsomal degradation with (2E)-N-isobutylundeca-2-ene-8,10-diynamide (1) metabolised to only a tenth the extent of (2E,4E,8Z,10Z)-N-isobutyldodeca-2,4,8,10-tetraenamide (3) under identical incubation conditions. Markedly less degradation of 3 was evident in the mixture of alkylamides present in an ethanolic Echinacea extract, suggesting that metabolism by liver P450s was dependent both on their chemistry and the combination present in the incubation. Co-incubation of 1 with 3 at equimolar concentrations resulted in a significant decrease in the metabolism of 3 by liver microsomes. This inhibition by 1, which has a terminal alkyne moiety, was found to be time- and concentration-dependent, and due to a mechanism-based inactivation of the P450s. Alkylamide metabolites were detected and found to be the predicted epoxidation, hydroxylation and dealkylation products. These findings suggest that Echinacea may effect the P450-mediated metabolism of other concurrently ingested pharmaceuticals. PMID:15885678

Matthias, A; Gillam, E M J; Penman, K G; Matovic, N J; Bone, K M; De Voss, J J; Lehmann, R P

2005-06-30

247

Oxidation of N-Nitrosoalkylamines by Human Cytochrome P450 2A6  

PubMed Central

Cytochrome P450 (P450) 2A6 activates nitrosamines, including N,N-dimethylnitrosamine (DMN) and N,N-diethylnitrosamine (DEN), to alkyl diazohydroxides (which are DNA-alkylating agents) and also aldehydes (HCHO from DMN and CH3CHO from DEN). The N-dealkylation of DMN had a high intrinsic kinetic deuterium isotope effect (Dkapp ? 10), which was highly expressed in a variety of competitive and non-competitive experiments. The Dkapp for DEN was ?3 and not expressed in non-competitive experiments. DMN and DEN were also oxidized to HCO2H and CH3CO2H, respectively. In neither case was a lag observed, which was unexpected considering the kcat and Km parameters measured for oxidation of DMN and DEN to the aldehydes and for oxidation of the aldehydes to the carboxylic acids. Spectral analysis did not indicate strong affinity of the aldehydes for P450 2A6, but pulse-chase experiments showed only limited exchange with added (unlabeled) aldehydes in the oxidations of DMN and DEN to carboxylic acids. Substoichiometric kinetic bursts were observed in the pre-steady-state oxidations of DMN and DEN to aldehydes. A minimal kinetic model was developed that was consistent with all of the observed phenomena and involves a conformational change of P450 2A6 following substrate binding, equilibrium of the P450-substrate complex with a non-productive form, and oxidation of the aldehydes to carboxylic acids in a process that avoids relaxation of the conformation following the first oxidation (i.e. of DMN or DEN to an aldehyde). PMID:20061389

Chowdhury, Goutam; Calcutt, M. Wade; Guengerich, F. Peter

2010-01-01

248

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

2008-01-01

249

Initial purification and characterization of hepatic microsomal cytochrome P-450 from BNF-treated perch (Perca fluviatilis).  

PubMed

1. A procedure was developed for isolating and purifying cytochrome P-450 from hepatic microsomes of BNF-treated perch, using modified versions of the methods of Williams and Buhler (1982. Biochim. biophys. Acta 717, 398-404) and Goksøyr (1985. Biochim. biophys. Acta 850, 409-417). 2. Following chromatography on phenyl-Sepharose CL 4B and DEAE-Sepharose CL-6B, the major peaks, fractions b and c, were resolved into five fractions, possibly representing different isoenzymes, by a FPLC with a strong anion exchange column (Mono Q). 3. These fractions have been characterized on the basis of their spectral, electrophoretic and immunological properties. 4. The purified form of cytochrome P-450 in fraction V from perch liver showed a number of similarities to cytochrome P-450c, the major BNF-inducible cytochrome P-450 in cod liver. 5. Therefore we suggest that this purified form of cytochrome P-450 is a BNF-induced form in perch and that it is closely related to the gene subfamily cytochrome P-450 IA1. PMID:2060284

Zhang, Y S; Goksøyr, A; Andersson, T; Förlin, L

1991-01-01

250

Cytochrome P450 1A2 Detoxicates Aristolochic Acid in the Mouse  

PubMed Central

Aristolochic acids (AAs) are plant-derived nephrotoxins and carcinogens responsible for chronic renal failure and associated urothelial cell cancers in several clinical syndromes known collectively as aristolochic acid nephropathy (AAN). Mice provide a useful model for study of AAN because the renal histopathology of AA-treated mice is strikingly similar to that of humans. AA is also a potent carcinogen in mice with a tissue spectrum somewhat different from that in humans. The toxic dose of AA in mice is higher than that in humans; this difference in susceptibility has been postulated to reflect differing rates of detoxication between the species. Recent studies in mice have shown that the hepatic cytochrome P450 system detoxicates AA, and inducers of the arylhydrocarbon response protect mice from the nephrotoxic effects of AA. The purpose of this study was to determine the role of specific cytochrome P450 (P450) enzymes in AA metabolism in vivo. Of 18 human P450 enzymes we surveyed only two, CYP1A1 and CYP1A2, which were effective in demethylating 8-methoxy-6-nitro-phenanthro-(3,4-d)-1,3-dioxolo-5-carboxylic acid (AAI) to the nontoxic derivative 8-hydroxy-6-nitro-phenanthro-(3,4-d)-1,3-dioxolo-5-carboxylic acid (AAIa). Kinetic analysis revealed similar efficiencies of formation of AAIa by human and rat CYP1A2. We also report here that CYP1A2-deficient mice display increased sensitivity to the nephrotoxic effects of AAI. Furthermore, Cyp1a2 knockout mice accumulate AAI-derived DNA adducts in the kidney at a higher rate than control mice. Differences in bioavailability or hepatic metabolism of AAI, expression of CYP1A2, or efficiency of a competing nitroreduction pathway in vivo may explain the apparent differences between human and rodent sensitivity to AAI. PMID:20164109

Einolf, Heidi J.; Dickman, Kathleen G.; Wang, Lai; Smith, Amanda; Grollman, Arthur P.

2010-01-01

251

Purification and Characterization of an NADPH-Cytochrome P450 (Cytochrome c) Reductase from Spearmint ( Mentha spicata) Glandular Trichomes  

Microsoft Academic Search

Solubilized NADPH-cytochrome c (P450) reductase was purified to homogeneity from an extract of spearmint (Mentha spicata) glandular trichomes by dye-ligand interaction chromatography on Matrex-Gel Red A and affinity chromatography on 2?,5?-adenosine diphosphate agarose. SDS–PAGE of the purified enzyme preparation revealed the presence of two similar proteins with masses of 82 kDa (major) and 77 kDa (minor) that crossreacted on immunoblot

Krishan Ponnamperuma; Rodney Croteau

1996-01-01

252

Ecologically Appropriate Xenobiotics Induce Cytochrome P450s in Apis mellifera  

PubMed Central

Background Honey bees are exposed to phytochemicals through the nectar, pollen and propolis consumed to sustain the colony. They may also encounter mycotoxins produced by Aspergillus fungi infesting pollen in beebread. Moreover, bees are exposed to agricultural pesticides, particularly in-hive acaricides used against the parasite Varroa destructor. They cope with these and other xenobiotics primarily through enzymatic detoxificative processes, but the regulation of detoxificative enzymes in honey bees remains largely unexplored. Methodology/Principal Findings We used several approaches to ascertain effects of dietary toxins on bee susceptibility to synthetic and natural xenobiotics, including the acaricide tau-fluvalinate, the agricultural pesticide imidacloprid, and the naturally occurring mycotoxin aflatoxin. We administered potential inducers of cytochrome P450 enzymes, the principal biochemical system for Phase 1 detoxification in insects, to investigate how detoxification is regulated. The drug phenobarbital induces P450s in many insects, yet feeding bees with phenobarbital had no effect on the toxicity of tau-fluvalinate, a pesticide known to be detoxified by bee P450s. Similarly, no P450 induction, as measured by tau-fluvalinate tolerance, occurred in bees fed xanthotoxin, salicylic acid, or indole-3-carbinol, all of which induce P450s in other insects. Only quercetin, a common pollen and honey constituent, reduced tau-fluvalinate toxicity. In microarray comparisons no change in detoxificative gene expression was detected in phenobarbital-treated bees. However, northern blot analyses of guts of bees fed extracts of honey, pollen and propolis showed elevated expression of three CYP6AS P450 genes. Diet did not influence tau-fluvalinate or imidacloprid toxicity in bioassays; however, aflatoxin toxicity was higher in bees consuming sucrose or high-fructose corn syrup than in bees consuming honey. Conclusions/Significance These results suggest that regulation of honey bee P450s is tuned to chemicals occurring naturally in the hive environment and that, in terms of toxicological capacity, a diet of sugar is not equivalent to a diet of honey. PMID:22319603

Johnson, Reed M.; Mao, Wenfu; Pollock, Henry S.; Niu, Guodong; Schuler, Mary A.; Berenbaum, May R.

2012-01-01

253

Characterization of NADPH-cytochrome P450 reductase gene from the cotton bollworm, Helicoverpa armigera.  

PubMed

A complete cDNA encoding the NADPH-cytochrome P450 reductase (haCPR) and its genomic sequence from the cotton bollworm Helicoverpa armigera were cloned and sequenced. The open reading frame of haCPR codes for a protein of 687 amino acid residues with a calculated molecular mass of 77.4kDa. The haCPR gene spans over 11 kb and its coding region is interrupted by 11 introns. haCPR is ubiquitously expressed in various tissues and at various stages of development. Escherichia coli produced haCPR enzyme exhibited catalytic activity for NADPH-dependent reduction of cytochrome c, following Michaelis-Menten kinetics. The functionality of CPR was further demonstrated by its capacity to support cytochrome P450 (e.g. haCYP9A14 and chicken CYP1A5) mediated O-dealkylation activity of alkoxyresorufins. The flavoprotein-specific inhibitor (diphenyleneiodonium chloride, DPI) showed a potent inhibition to haCPR activity (IC50=1.69 ?M). Inhibitory effect of secondary metabolites in the host plants (tannic acid, quercetin and gossypol) on CPR activity (with an IC50 value ranged from 15 to 90 ?M) was also observed. PMID:24768738

Liu, Dong; Zhou, Xiaojie; Li, Mei; Zhu, Shunyi; Qiu, Xinghui

2014-07-25

254

Distribution of cytochrome P-450 monoxygenase enzymes in the nasal mucosa of hamster and rat.  

PubMed

Deposition of inhaled particulates onto the respiratory mucosa is relatively great in that portion of the nasal cavity unprotected by ciliated, goblet, or keratinized superficial cells. The cytochrome P-450 system is an important enzyme system involved in the biotransformation of xenobiotics into metabolites that are more readily absorbed. To examine the transitional region caudal to the nasal vestibule, nasal tissues of hamster and rat were prepared for immunocytochemistry. Blocks of tissue representing four levels along the long axis of the nasal cavity were examined. Paraffin sections were processed through the avidin-biotin peroxidase procedure, with diaminobenzidine tetrahydrochloride as the chromagen. Enzyme localization was accomplished through the use of antibodies for three rabbit cytochrome P-450 isozymes; 2, 5, and 6 (subfamilies IIB, IVB, and IA, respectively); and for rabbit NADPH-cytochrome P-450 reductase. Enzyme distribution was similar in both hamster and rat nasal tissues except in cells of striated and intercalated ducts of nasal glands and in cells of the nasolacrimal duct where immunoreactivity was greater in the hamster. Immunoreactivity for reductase and isozyme 2 was intense in nonciliated cells lining the nonolfactory epithelium, in sustentacular cells of the olfactory epithelium, and in acinar cells of olfactory glands. Distribution of reaction products to isozyme 5 and 6 were similar to but not so intense as those of reductase and isozyme 2. Reaction products for reductase and isozyme 2 occurred generally in the same cellular and intracellular regions with the following exceptions: isozyme 2 was more concentrated in cells of striated ducts and of the nasolacrimal duct, and reductase was more abundant in intercalated ducts of nasal glands.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2048556

Adams, D R; Jones, A M; Plopper, C G; Serabjit-Singh, C J; Philpot, R M

1991-03-01

255

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

PubMed

Combustion processes generate particulate matter that affects human health. When incineration fuels include components that are highly enriched in aromatic hydrocarbons (especially halogenated varieties) and redox-active metals, ultrafine particulate matter containing air-stable, environmentally persistent free radicals (EPFRs) is generated. The exposure to fine EPFRs (less than 2.5 ?m in diameter) has been shown to negatively influence pulmonary and cardiovascular functions in living organisms. The goal of this study was to determine if these EPFRs have a direct effect on cytochrome P450 function. This was accomplished by direct addition of the EPFRs to rat liver microsomal preparations and measurement of several P450 activities using form-selective substrates. The EPFRs used in this study were formed by heating vapors from an organic compound (either monochlorophenol (MCP230) or 1,2-dichlorobenzene (DCB230)) and 5% copper oxide supported on silica (approximately 0.2 ?m in diameter) to 230°C under vacuum. Both types of EPFRs (but not silica, physisorbed silica, or silica impregnated with copper oxide) dramatically inhibited the activities of CYP1A, CYP2B, CYP2E1, CYP2D2 and CYP3A when incubated at concentrations less than 0.1 mg/ml with microsomes and NADPH. Interestingly, at the same concentrations, the EPFRs did not inhibit HO-1 activity or the reduction of cytochrome c by NADPH-cytochrome P450 reductase. CYP2D2-selective metabolism by rat liver microsomes was examined in more detail. The inhibition of CYP2D2-selective metabolism by both DCB230- and MCP230-EPFRs appeared to be largely noncompetitive and was attenuated in the presence of catalase suggesting that reactive oxygen species may be involved in the mechanism of inhibition. PMID:24713513

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

2014-06-01

256

Quantitatively interpreted enhanced inhibition of cytochrome P450s by heteroaromatic rings containing nitrogen.  

PubMed

It has been known for a long time that certain substructures bind to the heme iron in cytochromes P450. Detection of spectroscopic changes and crystal structures of protein ligand complexes have provided qualitative evidence, including for aromatic nitrogen-containing ligands. Compounds containing these same substructures are more likely to inhibit cytochrome P450s than expected due to lipophilicity. These two sets of observations are not easily linked by experiment, because binding to the iron atom alone is not readily measured. Quantum mechanical (density functional) calculations of binding energies for a number of different aromatic heterocycles to heme iron in a range of oxidation and spin states can provide a quantitative link between the observed structures and the biochemical inhibition that is measured. The studies reported here for a set of heteroaromatic rings containing nitrogen begin with quantum mechanical calculations which provide geometries and binding energies. Subsequently, AstraZeneca's database of cytochrome P450 inhibition assays has been searched to find data that are relevant to the same set of heteroaromatic compounds. These data have been analyzed in a number of fashions to account for both the narrow dynamic range of the assays and the lipophilicity dependence of this kind of inhibition. Finally, crystal structures have provided experimental geometric information. Taken together these different sources suggest that binding to the metal in our inhibition assays is dominated by Fe(III) in its doublet state, most likely occurring when the iron is pentavalent. Computed binding energies to this state contrast with the hydrogen-bond acceptor ability and basicity of the compounds, neither of which are able to correctly account for the effect of the particular environment in which the iron is found. This highlights the value of modeling biochemical events as closely as can be computationally afforded. The computational protocol devised was used to make predictions about a set of as yet unknown heteroaromatic compounds suggested by Pitt et al. PMID:21542637

Leach, Andrew G; Kidley, Nathan J

2011-05-23

257

Arylacetamide kappa opioid receptor agonists with reduced cytochrome P450 2D6 inhibitory activity.  

PubMed

Some kappa opioid receptor agonists of the arylacetamide class, for example, ICI 199441 (1), were found to strongly inhibit the activity of cytochrome P450 2D6 (CYP2D6) (1: CYP2D6 IC50=26 nM). Certain analogs bearing a substituted sulfonylamino group, for example, 13, were discovered to have significantly reduced CYP2D6 inhibitory activity (13: CYP2D6 IC50>10 microM) while displaying high affinity toward the cloned human kappa opioid receptor, good kappa/delta and kappa/mu selectivity, and potent in vitro and in vivo agonist activity. PMID:15863335

Le Bourdonnec, Bertrand; Ajello, Christopher W; Seida, Pamela R; Susnow, Roberta G; Cassel, Joel A; Belanger, Serge; Stabley, Gabriel J; DeHaven, Robert N; DeHaven-Hudkins, Diane L; Dolle, Roland E

2005-05-16

258

Isolation and characterization of cDNA clones for cytochromes P-450 immunochemically related to rat hepatic P-450 form PB-1.  

PubMed

Rat hepatic cytochrome P-450 PB-1 is a prominent constitutive P-450 form whose levels increase approximately 2-3 fold upon phenobarbital administration. Antibodies raised against this protein recognized two major proteins in immunoblots of rat liver microsomal proteins and precipitated comparable amounts of two electrophoretically separable hepatic mRNA translation products. The levels of the two mRNAs encoding these polypeptides were increased substantially upon phenobarbital administration. The anti-PB-1 antibodies were used to screen a cDNA library, and two distinct cDNA clones, pTF-1 and pTF-2, were isolated. These clones contain inserts of 1227 and 410 base pairs, respectively, and show 80% nucleic acid sequence homology in their region of overlap. The DNA sequences of these clones show 54% sequence homology to the corresponding portions of the mRNA encoding P-450 PB-4, a major phenobarbital-inducible form of rat liver P-450, and can be optimally aligned with the PB-4 sequence without introducing insertions or deletions. The level of hepatic mRNA which hybridizes to clone pTF-2 increases approximately 2-4-fold after phenobarbital treatment, whereas mRNA which hybridizes to pTF-1 does not change in concentration after this treatment. mRNA, which hybridizes to pTF-1, is, however, 4-fold more abundant in livers of female rats than in livers of male rats.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:3801454

Friedberg, T; Waxman, D J; Atchison, M; Kumar, A; Haaparanta, T; Raphael, C; Adesnik, M

1986-12-01

259

Substrate-modulated Cytochrome P450 17A1 and Cytochrome b5 Interactions Revealed by NMR*  

PubMed Central

The membrane heme protein cytochrome b5 (b5) can enhance, inhibit, or have no effect on cytochrome P450 (P450) catalysis, depending on the specific P450, substrate, and reaction conditions, but the structural basis remains unclear. Here the interactions between the soluble domain of microsomal b5 and the catalytic domain of the bifunctional steroidogenic cytochrome P450 17A1 (CYP17A1) were investigated. CYP17A1 performs both steroid hydroxylation, which is unaffected by b5, and an androgen-forming lyase reaction that is facilitated 10-fold by b5. NMR chemical shift mapping of b5 titrations with CYP17A1 indicates that the interaction occurs in an intermediate exchange regime and identifies charged surface residues involved in the protein/protein interface. The role of these residues is confirmed by disruption of the complex upon mutagenesis of either the anionic b5 residues (Glu-48 or Glu-49) or the corresponding cationic CYP17A1 residues (Arg-347, Arg-358, or Arg-449). Cytochrome b5 binding to CYP17A1 is also mutually exclusive with binding of NADPH-cytochrome P450 reductase. To probe the differential effects of b5 on the two CYP17A1-mediated reactions and, thus, communication between the superficial b5 binding site and the buried CYP17A1 active site, CYP17A1/b5 complex formation was characterized with either hydroxylase or lyase substrates bound to CYP17A1. Significantly, the CYP17A1/b5 interaction is stronger when the hydroxylase substrate pregnenolone is present in the CYP17A1 active site than when the lyase substrate 17?-hydroxypregnenolone is in the active site. These findings form the basis for a clearer understanding of this important interaction by directly measuring the reversible binding of the two proteins, providing evidence of communication between the CYP17A1 active site and the superficial proximal b5 binding site. PMID:23620596

Estrada, D. Fernando; Laurence, Jennifer S.; Scott, Emily E.

2013-01-01

260

Inhibition of NADPH cytochrome P450 reductase by the model sulfur mustard vesicant 2-chloroethyl ethyl sulfide is associated with increased production of reactive oxygen species  

SciTech Connect

Inhalation of vesicants including sulfur mustard can cause significant damage to the upper airways. This is the result of vesicant-induced modifications of proteins important in maintaining the integrity of the lung. Cytochrome P450s are the major enzymes in the lung mediating detoxification of sulfur mustard and its metabolites. NADPH cytochrome P450 reductase is a flavin-containing electron donor for cytochrome P450. The present studies demonstrate that the sulfur mustard analog, 2-chloroethyl ethyl sulfide (CEES), is a potent inhibitor of human recombinant cytochrome P450 reductase, as well as native cytochrome P450 reductase from liver microsomes of saline and {beta}-naphthoflavone-treated rats, and cytochrome P450 reductase from type II lung epithelial cells. Using rat liver microsomes from {beta}-naphthoflavone-treated rats, CEES was found to inhibit CYP 1A1 activity. This inhibition was overcome by microsomal cytochrome P450 reductase from saline-treated rats, which lack CYP 1A1 activity, demonstrating that the CEES inhibitory activity was selective for cytochrome P450 reductase. Cytochrome P450 reductase also generates reactive oxygen species (ROS) via oxidation of NADPH. In contrast to its inhibitory effects on the reduction of cytochrome c and CYP1A1 activity, CEES was found to stimulate ROS formation. Taken together, these data demonstrate that sulfur mustard vesicants target cytochrome P450 reductase and that this effect may be an important mechanism mediating oxidative stress and lung injury.

Gray, Joshua P. [Department of Science, United States Coast Guard Academy, New London, CT (United States); Department of Pharmacology and Toxicology, Rutgers University, Piscataway, NJ (United States); Mishin, Vladimir [Department of Pharmacology and Toxicology, Rutgers University, Piscataway, NJ (United States); Heck, Diane E. [Department of Environmental Health Science, New York Medical College, Valhalla, NY (United States); Laskin, Debra L. [Department of Pharmacology and Toxicology, Rutgers University, Piscataway, NJ (United States); Laskin, Jeffrey D., E-mail: jlaskin@eohsi.rutgers.ed [Environmental and Occupational Medicine, UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ (United States)

2010-09-01

261

Inhibition of cytochrome P450-mediated metabolism enhances ex vivo susceptibility of Fasciola hepatica to triclabendazole.  

PubMed

A study has been carried out to investigate whether the action of triclabendazole (TCBZ) against Fasciola hepatica is altered by inhibition of drug metabolism. The cytochrome P450 (CYP P450) system was inhibited using piperonyl butoxide (PB). The Oberon TCBZ-resistant and Cullompton TCBZ-susceptible isolates were used for these experiments. The CYP P450 system was inhibited by a 2 h pre-incubation in PB (100 mum). Flukes were then incubated for a further 22 h in NCTC medium containing either PB; PB+nicotinamide adenine dinucleotide phosphate (NADPH) (1 nm); PB+NADPH+TCBZ (15 microg/ml); or PB+NADPH+TCBZ.SO (15 microg/ml). Morphological changes resulting from drug treatment and following metabolic inhibition were assessed using scanning electron microscopy. After treatment with either TCBZ or TCBZ.SO alone, there was greater disruption to the TCBZ-susceptible than the resistant isolate. However, co-incubation with PB and TCBZ/TCBZ.SO lead to more severe surface changes to the TCBZ-resistant Oberon isolate than with each drug on its own. With the TCBZ-susceptible Cullompton isolate, there was limited potentiation of drug action, and only with TCBZ.SO. The results support the concept of altered drug metabolism in TCBZ-resistant flukes and this process may play a role in the development of drug resistance. PMID:20025819

Devine, C; Brennan, G P; Lanusse, C E; Alvarez, L I; Trudgett, A; Hoey, E; Fairweather, I

2010-04-01

262

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

SciTech Connect

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

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

2011-01-01

263

Stereoselective inhibition of cytochrome P450 forms by lansoprazole and omeprazole in vitro.  

PubMed

The stereoselectivity of the inhibitory interaction potential of lansoprazole and omeprazole isomers on six human cytochrome P450 forms was evaluated using human liver microsomes. Lansoprazole enantiomers showed stereoselective inhibition of CYP2C9-catalysed tolbutamide 4-methylhydroxylation, CYP2C19-catalysed S-mephenytoin 4'-hydroxylation, CYP2D6-catalysed dextromethorphan O-demethylation, CYP2E1-catalysed chlorzoxazone 6-hydroxylation and CYP3A4-catalysed midazolam 1-hydroxylation, whereas omeprazole only inhibited CYP2C19 stereoselectively. Of the P450 forms tested, CYP2C19-catalysed S-mephenytoin 4'-hydroxylation was extensively inhibited by both the lansoprazole and omeprazole enantiomers in a competitive and stereoselective manner; the S-enantiomers of both drugs inhibited the hydroxylation more than the R-enantiomers. The estimated K(i) values determined for CYP2C19-catalysed S-mephenytoin 4'-hydroxylation were 0.6, 6.1, 3.4 and 5.7 microM for S-lansoprazole, R-lansoprazole, S-omeprazole and R-omeprazole, respectively. The results indicate that although both lansoprazole and omeprazole are strong inhibitors of CYP2C19, the inhibition of CYP2C19 by lansoprazole is highly stereoselective, whereas the inhibition by omeprazole is less stereoselective. In addition, S-lansoprazole, the most potent CYP2C19 inhibitor, is not a good CYP2C19-selective inhibitor owing to its inhibition of other P450 forms. PMID:15788366

Liu, K H; Kim, M J; Shon, J H; Moon, Y S; Seol, S Y; Kang, W; Cha, I J; Shin, J G

2005-01-01

264

Differential hepatotoxicity and cytochrome P450 responses of Fischer-344 rats to the three isomers of dichlorobenzene  

SciTech Connect

The acute hepatotoxicity and response of hepatic cytochrome P450 to treatment with the three isomers of dichlorobenzene (DCB) have been investigated. The objectives were to estimate the onset of toxicity and to further elucidate the role of cytochrome P450 in the metabolism and toxicity of these compounds. In a study design employing one animal per dose level, Fischer-344 rats were gavaged with up to 25 different dosages, then evaluated 24 h later. Hepatic necrosis, serum alanine aminotransferase, and serum aspartate aminotransferase exhibited similar patterns demonstrating that ortho-DCB (o-DCB) was the most toxic in terms of both earliest onset and degree of response at higher dosages. For these three endpoints, meta-DCB (m-DCB) exhibited a lesser toxicity. Para-DCB (p-DCB) did not cause changes in these three endpoints, but hepatic degenerative changes were found. Total hepatic cytochrome P450 responses were also different after treatment with each isomer. The o-DCB produced a dose-dependent decrease in P450 beginning at dosages lower than the onset of necrosis and appeared to be a suicide substrate for P450. The m-DCB treatment increased P450 at dosages below the onset of necrosis and decreased P450 at higher dosages, with the decline preceding the onset of hepatocyte death.

Allis, J.W.; Simmons, J.E.; House, D.E.; Robinson, B.L.; Berman, E.

1992-01-01

265

Cytochrome P450 responses and PCB congeners in pipping heron embryos from Virginia, the Great Lakes and San Francisco Bay  

USGS Publications Warehouse

Pipping black-crowned night-heron (Nvcticorax nvcticorax) embryos were collected from undisturbed (Chincoteague National Wildlife Refuge VA; CNWR) and industrialized (Cat Island, Green Bay WI and San Francisco Bay, CA; SFB) locations. Hepatic monooxygenases (AHH, EROD, BROD, ECOD) were induced up to 100-fold, and were correlated (r=0.50 to 0.72) with total PCB burdens (N =61 embryos). A subset of 30 embryos have now been analyzed by GC/MS for 12 AHH-active PCB congeners and by Western blot for cytochromes P450lA and P450llB. At Cat Island, concentrations of 8 congeners were greater (P <0.05) than at CNWR. P450lA and P450llB were detected in 44% and 100% of the Cat Island embryos compared to 8% and 33% of the CNWR + SFB embryos. Cytochrome P450 parameters were correlated with the total PCBs (r =0.44 to 0.67) and with at least 9 PCB congeners (r =0.39 to 0.77). Since P450 responses might be affected by other contaminants, sample extract potency in the H411E rat hepatoma bioassay is being determined to study relationships among dioxin equivalents and cytochrome P450 parameters.

Rattner, B.A.; Melancon, M.J.; Custer, T.W.; Tillett, D.E.; Woodin, Bruce R.; Stegeman, John J.

1992-01-01

266

Oxidase uncoupling in heme monoxygenases: Human cytochrome P450 CYP3A4 in Nanodiscs  

PubMed Central

The normal reaction mechanism of cytochrome P450 operates by utilizing two reducing equivalents to reduce atmospheric dioxygen, producing one molecule of water and an oxygenated product in an overall stoichiometry of 2 electrons : 1 dioxygen : 1 product. However, three alternate unproductive pathways exist where the intermediate iron-oxygen states in the catalytic cycle can yield reduced oxygen products without substrate metabolism. The first involves release of superoxide from the oxygenated intermediate while the second occurs after input of the second reducing equivalent. Superoxide rapidly dismutates and hence both processes produce hydrogen peroxide that can be cytotoxic to the organism. In both cases, the formation of hydrogen peroxide involves the same overall stoichiometry as oxygenases catalysis. The key step in the catalytic cycle of cytochrome P450 involves scission of the oxygen-oxygen bond of atmospheric dioxygen to produce a higher valent iron-oxo state termed “Compound I”. This intermediate initiates a radical reaction in the oxygenase pathway but also can uptake two additional reducing equivalents from reduced pyridine nucleotide (NADPH) and the flavoprotein reductase to produce a second molecule of water. This non-productive decay of Compound I thus yields an overall oxygen to NADPH ratio of 1:2 and does not produce hydrocarbon oxidation. This water uncoupling reaction provides one of a limited means to study the reactivity of the critical Compound I intermediate in P450 catalysis. We measured simultaneously the rates of NADPH and oxygen consumption as a function of substrate concentration during the steady-state hydroxylation of testosterone catalyzed by human P450 CYP3A4 reconstituted in Nanodiscs. We discovered that the “oxidase” uncoupling pathway is also operating in the substrate free form of the enzyme with rate of this pathway substantially increasing with the first substrate binding event. Surprisingly, a large fraction of the reducing equivalents used by the P450 system is wasted in this oxidase pathway. In addition, the overall coupling with testosterone and bromocryptine as substrates is significantly higher in the presence of anionic lipids, which is attributed to the changes in the redox potential of CYP3A4 and reductase. PMID:23266608

Grinkova, Yelena V.; Denisov, Ilia G.; McLean, Mark A.; Sligar, Stephen G.

2014-01-01

267

Metabolism of nicotine by rat liver cytochromes P-450. Assessment utilizing monoclonal antibodies to nicotine and cotinine.  

PubMed

Because of the prevalence of cigarette smoking in the general population and because studies suggest that a large percentage of nicotine is metabolized to cotinine in humans, it is important to study the enzymes responsible for nicotine metabolism. The cytochromes P-450 have long been implicated in the first step in the conversion of nicotine to nicotine delta 1'(5')-iminium ion. We demonstrate here that rat liver P-450IIB1 is able to convert nicotine to cotinine in the presence of cytosol with a Km of 5-7 microM. A constitutive form of P-450 is also implicated in nicotine metabolism, while purified P-450IA1 and P-450IIC6 show no detectable activity. The lack of P-450IA1 activity substantiates work by others who also failed to observe an increase in the efficiency of nicotine metabolism to cotinine by microsomes from rats that had been pretreated with benzanthracene. This result is in contrast to work with purified rabbit liver enzymes, in which P-450IA1 exhibited low but measurable activity. Our results support the notion that nicotine metabolism to cotinine by P-450 enzymes is highly species dependent. Thus, it is unwise in some cases to extrapolate results obtained by animal model study to the possible role of specific forms of the P-450 enzymes in nicotine metabolism in humans. PMID:1680658

Hammond, D K; Bjercke, R J; Langone, J J; Strobel, H W

1991-01-01

268

Quantitation of cytochrome P450 mRNA levels in human skin.  

PubMed

There is considerable interindividual variation in man's ability to metabolize drugs and foreign compounds. These differences can partly be attributed to genetic polymorphisms that result in the generation of multiple phenotypes with different drug-metabolizing capabilities. Genetically derived differences can easily be assessed by genotyping assays in cases where the polymorphism has been identified. However, many of the polymorphisms that result in these are not known, secondly not all the differences can be attributed to genetic polymorphisms, hence genotyping methods cannot be employed. We have therefore, developed real-time (Taqman) PCR assays to quantitate levels of P450 mRNAs in human tissues. These assays are highly sensitive, reproducible, and specific and will allow quantitation of P450 mRNA levels in various human tissues. We have applied these assays to quantitate cytochrome P450 mRNA levels in human skin samples from 27 healthy volunteers. The expression of 13 P450s was assessed. The major enzymes detected were CYP1B1, CYP2B6, CYP2D6, and CYP3A4 with mean values of 2.5, 2.6, 2.7, and 1.1 fg/18S rRNA in 50ng total RNA, respectively. Lower levels of CYP2C18, CYP2C19, and CYP3A5 were also detected while CYP1A2, 2A6, and 2C8 were below limits of detection. There was interindividual variation in the levels of mRNA among the 27 subjects studied although Poisson analysis showed data to be normally distributed, except for CYP2B6, as some individuals completely lacked CYP2B6 mRNA. PMID:12694732

Yengi, Lilian G; Xiang, Qian; Pan, Jinmei; Scatina, JoAnn; Kao, John; Ball, Simon E; Fruncillo, Richard; Ferron, Geraldine; Roland Wolf, C

2003-05-01

269

Activity, Inhibition, and Induction of Cytochrome P450 2J2 in Adult Human Primary Cardiomyocytes  

PubMed Central

Cytochrome P450 2J2 plays a significant role in the epoxidation of arachidonic acid to signaling molecules important in cardiovascular events. CYP2J2 also contributes to drug metabolism and is responsible for the intestinal clearance of ebastine. However, the interaction between arachidonic acid metabolism and drug metabolism in cardiac tissue, the main expression site of CYP2J2, has not been examined. Here we investigate an adult-derived human primary cardiac cell line as a suitable model to study metabolic drug interactions (inhibition and induction) of CYP2J2 in cardiac tissue. The primary human cardiomyocyte cell line demonstrated similar mRNA-expression profiles of P450 enzymes to adult human ventricular tissue. CYP2J2 was the dominant isozyme with minor contributions from CYP2D6 and CYP2E1. Both terfenadine and astemizole oxidation were observed in this cell line, whereas midazolam was not metabolized suggesting lack of CYP3A activity. Compared with recombinant CYP2J2, terfenadine was hydroxylated in cardiomyocytes at a similar Km value of 1.5 ?M. The Vmax of terfenadine hydroxylation in recombinant enzyme was found to be 29.4 pmol/pmol P450 per minute and in the cells 6.0 pmol/pmol P450 per minute. CYP2J2 activity in the cell line was inhibited by danazol, astemizole, and ketoconazole in submicromolar range, but also by xenobiotics known to cause cardiac adverse effects. Of the 14 compounds tested for CYP2J2 induction, only rosiglitazone increased mRNA expression, by 1.8-fold. This cell model can be a useful in vitro model to investigate the role of CYP2J2-mediated drug metabolism, arachidonic acid metabolism, and their association to drug induced cardiotoxicity. PMID:24021950

Evangelista, Eric A.; Kaspera, Rüdiger; Mokadam, Nahush A.; Jones, J. P.

2013-01-01

270

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

PubMed Central

Dihydroxyphenylalanine (DOPA) melanins formed from tyrosine by tyrosinases are found in microorganisms, plants, and animals. Most species in the soil-dwelling, gram-positive bacterial genus Streptomyces produce DOPA melanins and melanogenesis is one of the characteristics used for taxonomy. Here we report a novel melanin biosynthetic pathway involving a type III polyketide synthase (PKS), RppA, and a cytochrome P-450 enzyme, P-450mel, in Streptomyces griseus. In vitro reconstitution of the P-450mel catalyst with spinach ferredoxin-NADP+ reductase/ferredoxin revealed that it catalyzed oxidative biaryl coupling of 1,3,6,8-tetrahydroxynaphthalene (THN), which was formed from five molecules of malonyl-coenzyme A by the action of RppA to yield 1,4,6,7,9,12-hexahydroxyperylene-3,10-quinone (HPQ). HPQ readily autopolymerized to generate HPQ melanin. Disruption of either the chromosomal rppA or P-450mel gene resulted in abolishment of the HPQ melanin synthesis in S. griseus and a decrease in the resistance of spores to UV-light irradiation. These findings show that THN-derived melanins are not exclusive in eukaryotic fungal genera but an analogous pathway is conserved in prokaryotic streptomycete species as well. A vivid contrast in THN melanin biosynthesis between streptomycetes and fungi is that the THN synthesized by the action of a type III PKS is used directly for condensation in the former, while the THN synthesized by the action of type I PKSs is first reduced and the resultant 1,8-dihydroxynaphthalene is then condensed in the latter. PMID:16291687

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

2005-01-01

271

1-Ethynylpyrene, a suicide inhibitor of cytochrome P-450 dependent benzo(a)pyrene hydroxylase activity in liver microsomes  

SciTech Connect

The preparation of 1-ethynylpyrene (EP) by incubation of EP with liver microsomes in the presence of NADPH yields fluorescent products briefly. Addition of microsomes restores the original rate. The metabolism of EP is initially more rapid in microsomes from 5,6-benzoflavone- (BF) pretreated rats than in those from phenobarbital (PB) pretreated rats or controls. Ep inhibits the hydroxylation of benzo(a)pyrene (BP) by liver microsomes. Ep more effectively inhibits the oxidation of BP in liver microsomes from BF rats than from PB rats or from controls. The inhibition of BP hydroxylation activity due to EP is dependent upon NADPH and is apparently irreversible. Kinetic analyses show that the inhibition of BP hydroxylation is due to loss of the activity by a process that is first order in EP and that reaches a limiting value at infinite EP concentrations. A self-catalyzed inhibition of the cytochrome P-450 dependent BP hydroxylation may occur in the presence of EP. Incubation with EP under conditions that result in loss of BP hydroxylase activity in microsomes from BF rats and 66% of the activity from PB rats causes the loss of 6 and 12% of the cytochrome P-450, respectively. Thus the loss of P-450 content is an insensitive measure of the effect of this inhibitor upon this cytochrome P-450 dependent enzyme activity. Selectivity of the loss of P-450 due to the incubation of the different microsomal preparations with EP is observed to be different than the selectivity for loss of BP hydroxylase activity. It is proposed that the inhibition of cytochrome P-450 dependent enzymes by alkynes need not involve heme alkylation and a resulting loss of P-450 content. In vivo EP does not cause a significant change in the cytochrome P-450 content in the microsomes isolated, or result in the change in BP hydroxylation.

Gan, L.S.L.; Acebo, A.L.; Alworth, W.L.

1984-08-14

272

Molecular dynamics simulations of norcamphor-cytochrome P-450 sub cam and mutations of P-450 sub cam designed to alter the product specificity  

SciTech Connect

Molecular dynamics simulations were performed to investigate the possibility of altering the product specificity of cytochrome P-450{sub cam}. The design aims were to alter the product ratios of norcamphor to yield predominately 3-hydroxynorcamphor and to increase the coupling efficiency between NADH consumption and product formation. Two simulations of P-450{sub cam} mutations, Y96F and V247A, were conducted to test the predictive ability of the molecular dynamics simulations with this enzyme-substrate system. The theoretical results agree with the experimental results. Two further mutations were simulated: F87W and the double mutant F87W/Y96F. The results from these novel mutations suggest that the coupling of electron consumption to substrate hydroxylation can be increased. Further these mutations lead to substrate orientations which are consistent with substrate hydroxylation at the 3-position. 8 refs., 2 figs., 2 tabs.

Bass, M.B.; Paulsen, M.D.; Ornstein, R.L.

1991-07-01

273

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

PubMed

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

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

2011-06-01

274

Isotopically labeled chlorobenzenes as probes for the mechanism of cytochrome P-450 catalyzed aromatic hydroxylation  

SciTech Connect

Noncompetitive and competitive intermolecular deuterium isotope effects were measured for the cytochrome P-450 catalyzed hydroxylation of a series of selectively deuterated chlorobenzenes. An isotope effect of 1.27 accompanied the meta hydroxylation of chlorobenzene-2H5 as determined by two totally independent methods (EC-LC and GC-MS assays). All isotope effects associated with the meta hydroxylation of chlorobenzenes-3,5-2H2 and -2,4,6-2H3 were approximately 1.1. In contrast, competitive isotope studies on the ortho and para hydroxylation of chlorobenzenes-4-2H1, -3,5-2H2, and -2,4,6-2H3 resulted in significant inverse isotope effects (approximately 0.95) when deuterium was substituted at the site of oxidation whereas no isotope effect was observed for the oxidation of protio sites. These results eliminate initial epoxide formation and initial electron abstraction (charge transfer) as viable mechanisms for the cytochrome P-450 catalyzed hydroxylation of chlorobenzene. The results, however, can be explained by a mechanism in which an active triplet-like oxygen atom adds to the pi system in a manner analogous to that for olefin oxidation. The resulting tetrahedral intermediate can then rearrange to phenol directly or via epoxide or ketone intermediates.

Korzekwa, K.R.; Swinney, D.C.; Trager, W.F. (National Heart, Lung and Blood Institute, Bethesda, MD (USA))

1989-11-14

275

Inter-relation of cytochrome P450 and contaminants burdens in sibling heron embryos and nestlings  

USGS Publications Warehouse

Hepatic cytochrome P450-associated monooxygenase activities were measured in 11-day-old nestling black-crowned night-herons (Nycticorax nycticorax) collected from a reference site (next to the Chincoteague National Wildlife Refuge, Virginia) and three polluted sites (Cat Island, Green Bay, Lake Michigan, Wisconsin; Bair Island, San Francisco Bay, California; West Marin Island, San Francisco Bay, California). Activities of arylhydrocarbon hydroxylase (AHH) and benzyl-oxyresorufin-O-dealkylase (BROD) weremodestly elevated (cytochrome P450-associated monooxygenase activity of heron nestlings may have only limited value as a biomarker of exposure at this rapid-growth life stage.

Rattner, B.; Melancon, M.; Custer, T.; Hothem, R.

1995-01-01

276

Functional expression of a bark beetle cytochrome P450 that hydroxylates myrcene to ipsdienol.  

PubMed

The final steps in the pheromone-biosynthetic pathway of the pine engraver beetle, Ips pini (Say) (Coleoptera: Scolytidae) are unknown, but likely involve myrcene (7-methyl-3-methylene-1,6-octadiene) hydroxylation to produce the aggregation pheromone component, ipsdienol (2-methyl-6-methylene-2,7-octadien-4-ol). We have isolated a full-length I. pini cDNA encoding a cytochrome P450, CYP9T2. The recovered cDNA is 1.83kb and the open reading frame encodes a 532 amino acid protein. CYP9T2 is regulated by the same physiological factors that induce pheromone production. Quantitative real-time PCR experiments showed that feeding on host phloem induced CYP9T2 expression in males, but not females, and that basal expression levels are highest in male midguts, similar to other I. pini pheromone-biosynthetic genes. Microsomes prepared from Sf9 cells co-expressing baculoviral-mediated recombinant CYP9T2 and housefly (Musca domestica) NADPH-cytochrome P450 reductase converted myrcene to ipsdienol. The product identified by coupled GC-MS was mostly (4R)-(-)-ipsdienol, an important aggregation pheromone component for western North American I. pini. These results are consistent with CYP9T2 encoding a myrcene hydroxylase that functions near the end of the pheromone-biosynthetic pathway. PMID:17046597

Sandstrom, Pamela; Welch, William H; Blomquist, Gary J; Tittiger, Claus

2006-11-01

277

Cytochrome P-450 metabolic activity in embryonic and extraembryonic tissue lineages of mouse embryos.  

PubMed Central

Mouse morulae, blastocysts, and embryonic and extraembryonic tissue layers were examined for benzo[a]-pyrene metabolism by cytochrome P-450, using the sister chromatid exchange assay. Benzo[a]pyrene exposure in vitro increased sister chromatid exchanges in blastocysts of all genetically responsive mice examined [BALB/cDub, C3H/AnfCum, and outbred Dub:(ICR) strains] but not blastocysts of the nonresponsive AKR/J strain. Benzo[a]pyrene treatment of responsive 7 1/2- and 8 1/2-day (postimplantation-stage) embryos, either intact or as separate tissue layers, increased sister chromatid exchanges in tissues of both embryonic and extraembryonic lineages--i.e., in the embryo proper, in isolated embryonic ectoderm, and in yolk sac, chorion, extraembryonic ectoderm, and extraembryonic endoderm layers. These results indicate that cytochrome P-450 is active in most or all tissues of the early mammalian embryo. It could metabolize xenobiotic molecules reaching the conceptus near the onset of morphogenesis and organogenesis, or it could have another as yet undefined role in normal development. PMID:3858824

Pedersen, R A; Meneses, J; Spindle, A; Wu, K; Galloway, S M

1985-01-01

278

Effects of Cytochrome P450 Inhibitors on Itraconazole and Fluconazole Induced Cytotoxicity in Hepatocytes  

PubMed Central

Itraconazole and fluconazole have been reported to induce hepatotoxicity in patients. The present study was designed to investigate the role of cytochrome P450 inhibitors, SKF 525A, and curcumin pretreatment on the cytotoxicity of antifungal drugs fluconazole and itraconazole. For 3 consecutive days, female rats were administered daily SKF 525A or curcumin (5 and 25?mg/kg). Control rats received an equivalent amount of dosed vehicle. The animals were anaesthetized 24 hours after receiving the last dose for liver perfusion. Hepatocytes were then exposed to various concentrations of antifungal drugs. In vitro incubation of hepatocytes with itraconazole revealed significantly lower viability when compared to fluconazole as assessed by lactate dehydrogenase, aspartate aminotransferase and alanine aminotransferase activities. The cytotoxicity of itraconazole was enhanced when incubated with hepatocytes pretreated with SKF 525A. SKF 525A had no effects on the cytotoxicity of fluconazole. Curcumin failed to either increase or decrease the cytotoxicity of both antifungal drugs. ATP levels also showed significant decrease in both itraconazole and fluconazole incubated hepatocytes. However, SKF 525A pretreated hepatocytes had significantly lower ATP levels after itraconazole incubations. Collectively, these results confirm the involvement of cytochrome P450 in the cytoprotection in itraconazole induced hepatocyte toxicity. Differences of the effects of SKF 525A on the cytotoxicity induced by itraconazole and fluconazole may be due to the differences on the metabolism of each antifungal drug in vivo. PMID:20130764

Somchit, Nhareet; Ngee, Chong Sock; Yaakob, Azhar; Ahmad, Zuraini; Zakaria, Zainul Amiruddin

2009-01-01

279

Metalloporphyrins as biomimetic models for cytochrome p-450 in the oxidation of atrazine.  

PubMed

The aim of this work was to evaluate whether metalloporphyrin models could mimic the action of cytochrome P-450 in the oxidation of atrazine, a herbicide. The commercially available second-generation metalloporphyrins 5,10,15,20-tetrakis(2,6-dichlorophenyl)porphyrin metal(III) chloride [M(TDCPP)Cl] and 5,10,15,20-tetrakis(pentafluorophenyl)porphyrin metal(III) chloride [M(TFPP)Cl] (metal = Fe or Mn) and the oxidants iodosylbenzene and metachloroperbenzoic acid were employed in this study. Results showed that the metalloporphyrins used here can oxidize atrazine. Yields as high as 32% were obtained for the Mn(TFPP)Cl/PhIO system, which shows that these catalysts can mimic both the in vivo and the in vitro action of cytochrome P-450, with formation of the metabolites DEA and DIA. The formation of five other unknown products was also detected, but only one of them could be identified, since the other four were present in very low concentrations. The compound COA, identified by mass spectrometry, was the main product in most of the oxidation reactions. PMID:17177535

Gotardo, Maria C A F; Moraes, Luiz A B de; Assis, Marilda D

2006-12-27

280

Acyclic nucleoside phosphonates: a study on cytochrome P450 gene expression.  

PubMed

1.?Nucleotide analogues comprise an important class of drugs used in treatment of viral infections but also cancer. These drugs affect the structural integrity of DNA and activate different pathways and processes in the cell and may directly or indirectly influence the drug metabolizing system. Adefovir dipivoxil (AD) and tenofovir disoproxil (TD) are nucleotide analogues approved for the treatment of chronic hepatitis B and/or HIV/AIDS infection. 2.?To evaluate the risk of their drug-drug interactions on the level of drug metabolism, an effect of both compounds on cytochromes P450 expression was studied using cDNA microarrays, real-time RT-PCR and immunoblotting. Mice were given intraperitoneally 25?mg/kg of AD or TD, respectively. As a positive control, a combination of prototypic cytochromes P450 (CYP) inducers, phenobarbital and ?-naphthoflavone was chosen. 3.?The data obtained showed a significant CYP induction in the positive control group, but no clinically significant induction of CYP genes by AD or TD was observed. Our results support the evidence of safety of AD and TD with respect to drug-drug interactions based on enzyme induction. These findings are important as a plethora of new antivirals of different types are being tested and introduced to clinical practice, mostly to be used in combinations. PMID:24593268

Nekvindova, Jana; Contreras, Juan Antonio; Juvan, Peter; Fon Tacer, Klementina; Anzenbacher, Pavel; Zidek, Zdenek; Kopecna Zapletalova, Michaela; Rozman, Damjana; Anzenbacherova, Eva

2014-08-01

281

Human Cytochrome P450 1A1 Structure and Utility in Understanding Drug and Xenobiotic Metabolism*  

PubMed Central

Cytochrome P450 (CYP) 1A1 is an extrahepatic monooxygenase involved in the metabolism of endogenous substrates and drugs, as well as the activation of certain toxins and environmental pollutants. CYP1A1 is particularly well known for its ability to biotransform polycyclic aromatic hydrocarbons, such as benzo[a]pyrene in tobacco smoke, into carcinogens. CYP1A1 possesses functional similarities and differences with human CYP1A2 and CYP1B1 enzymes, but the structural basis for this has been unclear. We determined a 2.6 ? structure of human CYP1A1 with the inhibitor ?-naphthoflavone. ?-Naphthoflavone binds within an enclosed active site, with the planar benzochromen-4-one core packed flat against the I helix that composes one wall of the active site, and the 2-phenyl substituent oriented toward the catalytic heme iron. Comparisons with previously determined structures of the related cytochrome P450 1A2 and 1B1 enzymes reveal distinct features among the active sites that may underlie the functional variability of these enzymes. Finally, docking studies probed the ability of CYP1A structures to assist in understanding their known in vitro interactions with several typical substrates and inhibitors. PMID:23508959

Walsh, Agnes A.; Szklarz, Grazyna D.; Scott, Emily E.

2013-01-01

282

Comparative functional characterization of a novel benzoate hydroxylase cytochrome P450 of Fusarium oxysporum.  

PubMed

FoCYP53A19, a novel cytochrome P450 capable of performing benzoate hydroxylation, was identified and characterized from the ascomycete Fusarium oxysporum f.sp. lycopersici. Comparative functional analysis of FoCYP53A19 with the heterologous and homologous cytochrome P450 reductases (CPR) such as Saccharomyces cerevisiae (ScCPR), Candida albicans (CaCPR) and F. oxysporum (FoCPR) revealed novel catalytic properties. The catalytic efficiency and substrate specificity of FoCYP53A19 were significantly influenced and altered by the source of the reductase employed. The yeast reconstitution system of FoCYP53A19 with ScCPR performed the hydroxylation of benzoic acid (BA) and demethylation of 3-methoxybenzoic acid (3-MBA); but when reconstituted with CaCPR, FoCYP53A19 performed only the essential hydroxylation of fungal benzoate catabolism. Remarkably, FoCYP53A19 with its homologous reductase FoCPR, not only demonstrated the improved conversion rates of BA and 3-MBA, but also exhibited activity toward the hydroxylation of 3-hydroxybenzoic acid. The electron transfer compatibility and the coupling efficiency between the homologous FoCYP-FoCPR system are significant and it favored enhanced monooxygenase activity with broader substrate specificity. PMID:25659633

Durairaj, Pradeepraj; Jung, Eunok; Park, Hyun Ho; Kim, Byung-Gee; Yun, Hyungdon

2015-03-01

283

Cytochrome P450 gene CYP337 and heritability of fitness traits in the Glanville fritillary butterfly.  

PubMed

Fitness-related life history traits often show substantial heritable genetic variation in natural populations, but knowledge of the genetic architecture of these traits is limited. In the Glanville fritillary butterfly, we measured the heritability of key life history traits in a large outdoor population cage during 2 years and generations and combined this experiment with an association study of a set of candidate genes. The genes were selected on the basis of previous genomic and transcriptomic studies and have been linked to the physiology and life history of this or other arthropod species. Heritability was high and significant for two traits, post-diapause larval development time (h(2) = 0.37) and lifetime egg (and larval) production (h(2) = 0.62); the latter is closely related to lifetime reproductive success and therefore fitness. We discovered a strong association between genetic polymorphism in the cytochrome P450 gene CYP337 and lifetime egg production, which accounted for 14% of the additive variance in egg production. This gene belongs to a group of cytochrome P450 genes that have a well-documented role in host plant adaptations in Lepidoptera and other insects and is likely to play an important role in the ecology and microevolution of the Glanville fritillary. This study provides a prime example of a gene associated with heritable fitness variation, measured under semi-natural ecological conditions. PMID:24552294

de Jong, M A; Wong, S C; Lehtonen, R; Hanski, I

2014-04-01

284

The beet R locus encodes a new cytochrome P450 required for red betalain production.  

PubMed

Anthocyanins are red and violet pigments that color flowers, fruits and epidermal tissues in virtually all flowering plants. A single order, Caryophyllales, contains families in which an unrelated family of pigments, the betalains, color tissues normally pigmented by anthocyanins. Here we show that CYP76AD1 encoding a novel cytochrome P450 is required to produce the red betacyanin pigments in beets. Gene silencing of CYP76AD1 results in loss of red pigment and production of only yellow betaxanthin pigment. Yellow betalain mutants are complemented by transgenic expression of CYP76AD1, and an insertion in CYP76AD1 maps to the R locus that is responsible for yellow versus red pigmentation. Finally, expression of CYP76AD1 in yeast verifies its position in the betalain biosynthetic pathway. Thus, this cytochrome P450 performs the biosynthetic step that provides the cyclo-DOPA moiety of all red betacyanins. This discovery will contribute to our ability to engineer this simple, nutritionally valuable pathway into heterologous species. PMID:22660548

Hatlestad, Gregory J; Sunnadeniya, Rasika M; Akhavan, Neda A; Gonzalez, Antonio; Goldman, Irwin L; McGrath, J Mitchell; Lloyd, Alan M

2012-07-01

285

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

PubMed Central

Heme is vital to our aerobic universe. Heme cellular content is finely tuned through an exquisite control of synthesis and degradation. Heme deficiency is deleterious to cells, whereas excess heme is toxic. Most of the cellular heme serves as the prosthetic moiety of functionally diverse hemoproteins, including cytochromes P450 (P450s). In the liver, P450s are its major consumers with >50% of hepatic heme committed to their synthesis. Prosthetic heme is the sine qua non of P450 catalytic biotransformation of both endo- and xenobiotics. This well-recognized functional role notwithstanding, heme also regulates P450 protein synthesis, assembly, repair and disposal. These less well-appreciated aspects are reviewed herein. PMID:20860521

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

2011-01-01

286

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

PubMed

Heme is vital to our aerobic universe. Heme cellular content is finely tuned through an exquisite control of synthesis and degradation. Heme deficiency is deleterious to cells, whereas excess heme is toxic. Most of the cellular heme serves as the prosthetic moiety of functionally diverse hemoproteins, including cytochromes P450 (P450s). In the liver, P450s are its major consumers, with >50% of hepatic heme committed to their synthesis. Prosthetic heme is the sine qua non of P450 catalytic biotransformation of both endo- and xenobiotics. This well-recognized functional role notwithstanding, heme also regulates P450 protein synthesis, assembly, repair, and disposal. These less well-appreciated aspects are reviewed herein. PMID:20860521

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

2011-02-01

287

Modeling of Anopheles minimus Mosquito NADPH-Cytochrome P450 Oxidoreductase (CYPOR) and Mutagenesis Analysis.  

PubMed

Malaria is one of the most dangerous mosquito-borne diseases in many tropical countries, including Thailand. Studies in a deltamethrin resistant strain of Anopheles minimus mosquito, suggest cytochrome P450 enzymes contribute to the detoxification of pyrethroid insecticides. Purified A. minimus CYPOR enzyme (AnCYPOR), which is the redox partner of cytochrome P450s, loses ?avin-adenosine di-nucleotide (FAD) and FLAVIN mono-nucleotide (FMN) cofactors that affect its enzyme activity. Replacement of leucine residues at positions 86 and 219 with phenylalanines in FMN binding domain increases FMN binding, enzyme stability, and cytochrome c reduction activity. Membrane-Bound L86F/L219F-AnCYPOR increases A. minimus P450-mediated pyrethroid metabolism in vitro. In this study, we constructed a comparative model structure of AnCYPOR using a rat CYPOR structure as a template. Overall model structure is similar to rat CYPOR, with some prominent differences. Based on primary sequence and structural analysis of rat and A. minimus CYPOR, C427R, W678A, and W678H mutations were generated together with L86F/L219F resulting in three soluble ?55 triple mutants. The C427R triple AnCYPOR mutant retained a higher amount of FAD binding and increased cytochrome c reduction activity compared to wild-type and L86F/L219F-?55AnCYPOR double mutant. However W678A and W678H mutations did not increase FAD and NAD(P)H bindings. The L86F/L219F double and C427R triple membrane-bound AnCYPOR mutants supported benzyloxyresorufin O-deakylation (BROD) mediated by mosquito CYP6AA3 with a two- to three-fold increase in efficiency over wild-type AnCYPOR. The use of rat CYPOR in place of AnCYPOR most efficiently supported CYP6AA3-mediated BROD compared to all AnCYPORs. PMID:23325047

Sarapusit, Songklod; Lertkiatmongkol, Panida; Duangkaew, Panida; Rongnoparut, Pornpimol

2013-01-01

288

[Role of antioxidants in electro catalytic activity of cytochrome P450 3A4].  

PubMed

The electrochemical analysis of cytochrome ?450 3?4 catalytic activity has shown that vitamins C, A and ? influence on electron transfer and Fe3+/Fe2+ reduction process of cytochrome ?450 3?4. These data allow to assume possibility of cross effects and interference of vitamins-antioxidants with drugs metabolised by cytochrome ?450 3?4, at carrying out of complex therapy. This class of vitamins shows antioxidant properties that lead to increase of the cathodic current corresponding to heme reduction of this functionally significant haemoprotein. Ascorbic acid of 0.028-0.56 mM concentration stimulates cathodic peak (an electrochemical signal) of cytochrome ?450 3?4. At the presence of diclofenac (Voltaren) - a typical substrate of cytochrome ?450 3?4 - the increase growth of a catalytic current testifying to an electrocatalysis and stimulating action of ascorbic acid is observed. In the presence of vitamins A and ? also is registered dose-dependent (in a range of 10-100 M) increase in a catalytic current of cytochrome ?450 3?4: the maximum increase corresponds to 229 ± 20% for 100 M of vitamin A, and 162±10% for 100 M of vitamin E. Vitamin E in the presence of P450's inhibitor itraconazole doesn't give essential increase in a reductive current, unlike retinol (vitamin A). This effect can manifest substrate properties of tocopherol (vitamin E). The electrochemical approach for the analysis of catalytic activity of cytochrome ?450 3?4 and studies of influence of biologically active compounds on an electrocatalysis is the sensitive and effective sensor approach, allowing to use low concentration of protein on an electrode (till 10-15 mol/electrode), to carry out the analysis without participation of protein redox partners, and to reveal drug-drug or drug-vitamins interaction in pre-clinical experiments. PMID:24837311

Shumiantseva, V V; Makhova, A A; Bulko, T V; Shikh, E V; Kukes, V G; Usanov, S A; Archakov, A I

2014-01-01

289

Cytochrome P450 and Glutathione S-Transferase mRNA Expression in Human Fetal Liver Hematopoietic Stem Cells  

Microsoft Academic Search

During fetal development, the liver serves as the primary hemato- poietic organ in which hematopoietic stem cells (HSC) capable of initiating long-term hematopoiesis comprise a large proportion of the hepatic cell population. Although HSC are potential targets for transplacental chemicals, little is known regarding their xenobiotic biotransformation ability. We quantitated the steady-state mRNA expression of six cytochrome P450 (P450) and

Jing Shao; Patricia L. Stapleton; Yvonne S. Lin; Evan P. Gallagher

2006-01-01

290

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

PubMed

Caffeine (1,3,7-trimethylxanthine) is a phenotyping substrate for human cytochrome P450 1A2. 3-N-Demethylation of caffeine is the main human metabolic pathway, whereas monkeys extensively mediate the 7-N-demethylation of caffeine to form pharmacological active theophylline. Roles of monkey P450 enzymes in theophylline formation from caffeine were investigated using individual monkey liver microsomes and 14 recombinantly expressed monkey P450 enzymes, and the results were compared with those for human P450 enzymes. Caffeine 7-N-demethylation activity in microsomes from 20 monkey livers was not strongly inhibited by ?-naphthoflavone, quinidine or ketoconazole, and was roughly correlated with diclofenac 4'-hydroxylation activities. Monkey P450 2C9 had the highest activity for caffeine 7-N-demethylation. Kinetic analysis revealed that monkey P450 2C9 had a high Vmax/Km value for caffeine 7-N-demethylation, comparable to low Km value for monkey liver microsomes. Caffeine could dock favorably with monkey P450 2C9 modeled for 7-N-demethylation and with human P450 1A2 for 3-N-demethylation. The primary metabolite theophylline was oxidized to 8-hydroxytheophylline in similar ways by liver microsomes and by recombinant P450s in both humans and monkeys. These results collectively suggest a high activity for monkey liver P450 2C9 toward caffeine 7-N-demethylation, whereas, in humans, P450 1A2-mediated caffeine 3-N-demethylation is dominant. PMID:23679834

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

2013-12-01

291

Defining the in Vivo Role for Cytochrome b5 in Cytochrome P450 Function through the Conditional Hepatic Deletion of Microsomal Cytochrome b5*S?  

PubMed Central

In vitro, cytochrome b5 modulates the rate of cytochrome P450-dependent mono-oxygenation reactions. However, the role of this enzyme in determining drug pharmacokinetics in vivo and the consequential effects on drug absorption distribution, metabolism, excretion, and toxicity are unclear. In order to resolve this issue, we have carried out the conditional deletion of microsomal cytochrome b5 in the liver to create the hepatic microsomal cytochrome b5 null mouse. These mice develop and breed normally and have no overt phenotype. In vitro studies using a range of substrates for different P450 enzymes showed that in hepatic microsomal cytochrome b5 null NADH-mediated metabolism was essentially abolished for most substrates, and the NADPH-dependent metabolism of many substrates was reduced by 50–90%. This reduction in metabolism was also reflected in the in vivo elimination profiles of several drugs, including midazolam, metoprolol, and tolbutamide. In the case of chlorzoxazone, elimination was essentially unchanged. For some drugs, the pharmacokinetics were also markedly altered; for example, when administered orally, the maximum plasma concentration for midazolam was increased by 2.5-fold, and the clearance decreased by 3.6-fold in hepatic microsomal cytochrome b5 null mice. These data indicate that microsomal cytochrome b5 can play a major role in the in vivo metabolism of certain drugs and chemicals but in a P450- and substrate-dependent manner. PMID:18805792

Finn, Robert D.; McLaughlin, Lesley A.; Ronseaux, Sebastien; Rosewell, Ian; Houston, J. Brian; Henderson, Colin J.; Wolf, C. Roland

2008-01-01

292

Human cytochrome P450 4F11: Heterologous expression in bacteria, purification, and characterization of catalytic function  

PubMed Central

Human cytochrome P450 (P450) 4F11 is still considered an “orphan” because its function is not well characterized. A bacterial expression system was developed for human P450 4F11, producing ~230 nmol P450 from a 3-liter culture of Escherichia coli. P450 4F11 was purified and utilized for untargeted substrate searches in human liver extract using a liquid chromatography/mass spectrometry-based metabolomic and isotopic labeling approach (Z. Tang et al., Anal. Chem. 81, 3071–3078, 2009). Four fatty acids—palmitic, oleic, arachidonic, and docosahexaenoic—were identified in human liver and verified as substrates of P450 4F11. The products were characterized as ?-hydroxylated fatty acids by gas chromatography-mass spectrometry analysis of their trimethylsilyl derivatives. Kinetic analysis of the oxidation products confirmed that the fatty acids are substrates oxidized by P450 4F11. P450 4F11 also exhibited low activity for some drug N-demethylation reactions but none for activation of several procarcinogens. PMID:19932081

Tang, Zhongmei; Salamanca-Pinzón, Sandra Giovanna; Wu, Zhong-Liu; Xiao, Yi; Guengerich, F. Peter

2009-01-01

293

Inactivation of phenobarbital-inducible rabbit-liver microsomal cytochrome P-450 by allylisopropylacetamide: impact on electron transfer.  

PubMed

Application of a single dose of allylisopropylacetamide (AIA) to phenobarbital-pretreated rabbits resulted in partial destruction of the heme moiety of liver microsomal cytochrome P-450. A minor fraction of chromophore loss was accounted for by heme-derived product(s) covalently attached to microsomal proteins. Interestingly, cytochrome P-450 appeared to have undergone significant drug-mediated alkylation of the apohemoprotein. The modified species was purified to apparent homogeneity and shown to arise from AIA-induced blockage of about 2 histidines in the cytochrome P-450LM2 molecule located close to the heme edge. AIA administration to the animals caused inhibition of hexobarbital-promoted electron flow from NADPH-cytochrome P-450 reductase to phenobarbital-inducible ferricytochrome P-450 both in microsomal particles and reconstituted systems. The impaired interaction between the proteins was shown not to originate from decreased capacity to bind each other but more likely to be due to some defect in a step subsequent to complex formation. In contrast, treatment with the porphyrogenic agent did not affect microsomal electron transmission from cytochrome b5 to the ferric monooxygenase. However, when the intermediate carrier was to donate reducing equivalents to the ferrous oxycytochrome in the presence of benzphetamine, there was a pronounced deceleration of the electron flux observable. These findings were interpreted to mean that there exist multiple reductase- and cytochrome-b5-binding domains in phenobarbital-inducible cytochrome P-450, some of which seem to be common to the two redox proteins. This sheds interesting light on the molecular organization of the catalytic electron transfer complexes. PMID:8457584

Golly, I; Hlavica, P

1993-04-01

294

Preparative synthesis of drug metabolites using human cytochrome P450s 3A4, 2C9 and 1A2 with NADPH-P450 reductase expressed in Escherichia coli  

Microsoft Academic Search

Three human cytochrome P450s, 3A4, 2C9 and 1A2, were each co-expressed with NADPH-P450 reductase in Escherichia coli and used in the preparative synthesis of drug metabolites. Low dissolved oxygen (DO) concentration (<1%) during expression was found to be critical for producing active P450s. Control of temperature, pH and glycerol supplementation in 10-L fermentations enhanced enzyme expression 31–86%. Additional improvements were

Robert B. Vail; Michael J. Homann; Imad Hanna; Aleksey Zaks

2005-01-01

295

Significance of Cytochrome P450 System Responses and Levels of Bile Fluorescent Aromatic Compounds in Marine Wildlife Following Oil Spills  

SciTech Connect

The relationships among cytochrome P450 induction in marine wildlife species, levels of fluorescent aromatic compounds (FAC) in their bile, the chemical composition of the inducing compounds, the significance of the exposure pathway, and any resulting injury, as a consequence of exposure to crude oil following a spill, are reviewed. Fish collected after oil spills often show increases in cytochrome P450 system activity, cytochrome P4501A (CYP1A) and bile fluorescent aromatic compounds (FAC), that are correlated with exposure to polycyclic aromatic hydrocarbons (PAH) in the oil. There is also some evidence for increases in bile FAC and induction of cytochrome P450 in marine birds and mammals after oil spills. However, when observed, increases in these exposure indicators are transitory and generally decrease to background levels within one year after the exposure. Laboratory studies have shown induction of cytochrome P450 systems occurs after exposure of fish to crude oil in water, sediment or food. Most of the PAH found in crude oil (dominantly 2- and 3-ring PAH) are not strong inducers of cytochrome P450. Exposure to the 4-ring chrysenes or the photooxidized products of the PAH may account for the cytochrome P450 responses in fish collected from oil-spill sites. The contribution of non-spill background PAH, particularly combustion-derived (pyrogenic) PAH, to bile FAC and cytochrome P450 system responses can be confounding and needs to be considered when evaluating oil spill effects. The ubiquity of pyrogenic PAH makes it important to fully characterize all sources of PAH, including PAH from natural resources, e.g. retene, in oil spill studies. In addition, such parameters as species, sex, age, ambient temperature and season need to be taken into account. While increases in fish bile FAC and cytochrome P450 system responses, can together, be sensitive general indicators of PAH exposure after an oil spill, there is little unequivocal evidence to suggest a linkage to higher order biological effects, e.g. toxicity, lesions, reproductive failure.

Lee, Richard F.; Anderson, Jack W.

2005-07-01

296

Significance of cytochrome P450 system responses and levels of bile fluorescent aromatic compounds in marine wildlife following oil spills.  

PubMed

The relationships among cytochrome P450 induction in marine wildlife species, levels of fluorescent aromatic compounds (FAC) in their bile, the chemical composition of the inducing compounds, the significance of the exposure pathway, and any resulting injury, as a consequence of exposure to crude oil following a spill, are reviewed. Fish collected after oil spills often show increases in cytochrome P450 system activity, cytochrome P4501A (CYP1A) and bile fluorescent aromatic compounds (FAC), that are correlated with exposure to polycyclic aromatic hydrocarbons (PAH) in the oil. There is also some evidence for increases in bile FAC and induction of cytochrome P450 in marine birds and mammals after oil spills. However, when observed, increases in these exposure indicators are transitory and generally decrease to background levels within one year after the exposure. Laboratory studies have shown induction of cytochrome P450 systems occurs after exposure of fish to crude oil in water, sediment or food. Most of the PAH found in crude oil (dominantly 2- and 3-ring PAH) are not strong inducers of cytochrome P450. Exposure to the 4-ring chrysenes or the photooxidized products of the PAH may account for the cytochrome P450 responses in fish collected from oil-spill sites. The contribution of non-spill background PAH, particularly combustion-derived (pyrogenic) PAH, to bile FAC and cytochrome P450 system responses can be confounding and needs to be considered when evaluating oil spill effects. The ubiquity of pyrogenic PAH makes it important to fully characterize all sources of PAH, including PAH from natural resources, e.g. retene, in oil spill studies. In addition, such parameters as species, sex, age, ambient temperature and season need to be taken into account. While increases in fish bile FAC and cytochrome P450 system responses, can together, be sensitive general indicators of PAH exposure after an oil spill, there is little unequivocal evidence to suggest a linkage to higher order biological effects, e.g. toxicity, lesions, reproductive failure. PMID:15946701

Lee, Richard F; Anderson, Jack W

2005-07-01

297

Induction by phenobarbital in McA-RH7777 rat hepatoma cells of a polycyclic hydrocarbon inducible cytochrome P450  

SciTech Connect

The metabolism of 2-acetylaminofluorene (AAF) to its six oxidative metabolites has been used to study cytochrome P-450 monooxygenase activity in two rat hepatoma cell lines, McA-RH7777 and Reuber H4-II-E. McA-RH7777 cells exhibited considerably higher basal activities than H4-II-E cells for all metabolic pathways studied. Phenobarbital induced AAF metabolite formation in McA-RH7777 cells to a similar extent as 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD), but was only a weak inducer of these activities in H4-II-E cells. Northern blot analysis utilizing specific phenobarbital or 3-methylcholanthrene inducible cytochrome P-450 cDNA probes indicated that there was at least a 10-fold increase in a 3-methylcholanthrene inducible cytochrome P-450 transcript in phenobarbital treated McA-RH7777 cells.

McManus, M.E.; Minchin, R.F.; Schwartz, D.M.; Wirth, P.J.; Huber, B.E.

1986-05-29

298

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

PubMed Central

Background Yarrowia lipolytica efficiently metabolizes and assimilates hydrophobic compounds such as n-alkanes and fatty acids. Efficient substrate uptake is enabled by naturally secreted emulsifiers and a modified cell surface hydrophobicity and protrusions formed by this yeast. We were examining the potential of recombinant Y. lipolytica as a biocatalyst for the oxidation of hardly soluble hydrophobic steroids. Furthermore, two-liquid biphasic culture systems were evaluated to increase substrate availability. While cells, together with water soluble nutrients, are maintained in the aqueous phase, substrates and most of the products are contained in a second water-immiscible organic solvent phase. Results For the first time we have co-expressed the human cytochromes P450 2D6 and 3A4 genes in Y. lipolytica together with human cytochrome P450 reductase (hCPR) or Y. lipolytica cytochrome P450 reductase (YlCPR). These whole-cell biocatalysts were used for the conversion of poorly soluble steroids in biphasic systems. Employing a biphasic system with the organic solvent and Y. lipolytica carbon source ethyl oleate for the whole-cell bioconversion of progesterone, the initial specific hydroxylation rate in a 1.5?L stirred tank bioreactor was further increased 2-fold. Furthermore, the product formation was significantly prolonged as compared to the aqueous system. Co-expression of the human CPR gene led to a 4-10-fold higher specific activity, compared to the co-overexpression of the native Y. lipolytica CPR gene. Multicopy transformants showed a 50-70-fold increase of activity as compared to single copy strains. Conclusions Alkane-assimilating yeast Y. lipolytica, coupled with the described expression strategies, demonstrated its high potential for biotransformations of hydrophobic substrates in two-liquid biphasic systems. Especially organic solvents which can be efficiently taken up and/or metabolized by the cell might enable more efficient bioconversion as compared to aqueous systems and even enable simple, continuous or at least high yield long time processes. PMID:22876969

2012-01-01

299

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

SciTech Connect

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

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

2009-05-14

300

Evaluation of inhibition selectivity for human cytochrome P450 2A enzymes.  

PubMed

Cytochrome P450 (P450) enzymes are mixed-function oxidases that catalyze the metabolism of xenobiotics and endogenous biochemicals. Selective inhibitors are needed to accurately distinguish the contributions of individual P450 enzymes in the metabolism of drugs and the activation of procarcinogens in human tissues, but very frequently these enzymes have substantial overlapping selectivity. We evaluated a chemically diverse set of nine previously identified CYP2A6 inhibitors to determine which are able to discriminate between human CYP2A enzymes CYP2A6 and the 94%-identical CYP2A13 enzyme. Inhibitor binding to recombinant purified enzyme was evaluated, and affinities were determined. K(i) values were determined for inhibition of p-nitrophenol 2-hydroxylation, a reaction accomplished by CYP2A13 and CYP2A6 with more similar catalytic efficiencies (k(cat)/K(m) 0.19 and 0.12 ?M?¹ · min?¹, respectively) than hydroxylation of the classic substrate coumarin (0.11 and 0.53 ?M?¹ · min?¹, respectively). Of the nine compounds assayed, only tranylcypromine and (R)-(+)-menthofuran had a greater than 10-fold preference for CYP2A6 inhibition versus CYP2A13 inhibition. Most compounds evaluated [tryptamine, 4-dimethylaminobenzaldehyde, phenethyl isothiocyanate, ?-nicotyrine, (S)-nicotine, and pilocarpine] demonstrated only moderate or no preference for inhibition of one CYP2A enzyme over the other. However, 8-methoxypsoralen has a 6-fold lower K(i) for CYP2A13 than for CYP2A6. This information is useful to inform reinterpretation of previous data with these inhibitors and to guide future studies seeking to determine which human CYP2A enzyme is responsible for the in vivo metabolism of compounds in human tissues expressing both enzymes. PMID:22696418

Stephens, Eva S; Walsh, Agnes A; Scott, Emily E

2012-09-01

301

Genetic polymorphism and toxicology--with emphasis on cytochrome p450.  

PubMed

Individual susceptibility to environmental, chemical, and drug toxicity is to some extent determined by polymorphism in drug-metabolizing enzymes, in particular the cytochromes P450 (CYPs). This polymorphism is in particular translated into risk differences concerning drugs metabolized by the highly polymorphic enzymes CYP2C9, CYP2C19, and CYP2D6, whereas CYP enzymes active in procarcinogen activation are relatively well conserved without important functional polymorphisms. Examples of drug toxicities that can be predicted by P450 polymorphism include those exerted by codeine, tramadol, warfarin, acenocoumarol, and clopidogrel. The polymorphic CYP2A6 has a role in nicotine metabolism and smoking behavior. Besides this genetic variation, genome-wide association studies now allow for the identification of an increasing number of predictive genetic biomarkers among, e.g., human leukocyte antigens and to some extent drug transporters that provide useful information regarding the choice of the drug and drug dosage in order to avoid toxicity. The translation of this information into the clinical practice has been slow; however, an increasing number of pharmacogenomic drug labels are assigned, where the predictive genotyping before drug treatment can be mandatory, recommended, or only for informational purposes. In this review, we provide an update of the field with emphasis on CYP polymorphism. PMID:21149643

Johansson, Inger; Ingelman-Sundberg, Magnus

2011-03-01

302

Electrochemistry of cytochrome P450 BM3 in sodium dodecyl sulfate films.  

PubMed

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

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

2006-12-01

303

A Cytochrome P450 Serves as an Unexpected Terpene Cyclase during Fungal Meroterpenoid Biosynthesis  

PubMed Central

Viridicatumtoxin (1) is a tetracycline-like fungal meroterpenoid with a unique, fused spirobicyclic ring system. Puzzlingly, no dedicated terpene cyclase is found in the gene cluster identified in Penicillium aethiopicum. The two cytochrome P450 enzymes VrtE and VrtK in the vrt gene cluster were shown to catalyze C5-hydroxylation and spirobicyclic ring formation, respectively. Feeding of acyclic previridicatumtoxin (2) to Saccharomyces cerevisiae expressing VrtK confirmed that VrtK is the sole enzyme required for cyclization of the geranyl moiety. Thus, VrtK is the first example of a P450 that can catalyze terpene cyclization, most likely via the initial oxidation of C17 to an allylic carbocation. Quantum chemical modeling revealed a possible new tertiary carbocation intermediate E that forms after the allylic carbocation formation. The intermediate E can readily undergo concerted 1,2-alkyl shift/1,3-hydride shift, either spontaneously or further aided by the active site configuration of VrtK, followed by C7 Friedel-Crafts alkylation to afford 1,. The most likely stereochemical course of the reaction was proposed based on the results of our computations. PMID:24161266

Chooi, Yit-Heng; Hong, Young J.; Cacho, Ralph A.; Tantillo, Dean J.; Tang, Yi

2013-01-01

304

Using Molecular Dynamics to Probe the Structural Basis for Enhanced Stability in Thermal Stable Cytochromes P450  

PubMed Central

High temperature molecular dynamics (MD) has been used to assess if MD can be employed as a useful tool for probing the structural basis for enhanced stability in thermal stable cytochromes P450. CYP119, the most thermal stable P450 known, unfolds more slowly during 500K MD simulations compared to P450s that melt at lower temperatures, P450cam and P450cin. A comparison of the 500K MD trajectories shows that the Cys ligand loop, a critically important structural feature just under the heme, in both P450cin and P450cam completely unfolds while this region is quite stable in CYP119. In CYP119 this region is stabilized by tight nonpolar interactions involving Tyr26 and Leu308. The corresponding residues in P450cam are Gly and Thr, respectively. The in silico generated Y26A/L308A CYP119 double mutant is substantially less stable than wild type CYP119 and the Cys ligand loop unfolds similar to P450cam. The MD thus has identified a potential “hot spot” important for stability. As an experimental test of the MD results, the Y26A/L308A double mutant was prepared and thermal melting curves show that the double mutant exhibits a melting temperature (Tm) 16°C lower than wild type CYP119. Control mutations that were predicted by MD not to destabilize the protein were also generated and the experimental melting temperature was not significantly different from the wild type enzyme. Therefore, high temperature MD is a useful tool in predicting the structural underpinnings of thermal stability in P450s. PMID:20593793

Meharenna, Yergalem T.; Poulos, Thomas L.

2011-01-01

305

[Elimination of caffeine and metamizol in in vivo characterization of cytochrome P-450 dependent biotransformation reactions in aged humans].  

PubMed

Elimination of caffeine from plasma and excretion of main metabolites of metamizol (noramidopyrine methanesulphonate sodium) in urine were determined in young healthy (age 18-27 years) and in old aged volunteers (older than 65 years). From the elimination velocity of these model substances conclusions concerning the activity of 3-methylcholanthrene inducible (caffeine elimination) and of phenobarbital inducible (metamizol elimination) isoenzymes of cytochrome P-450 are drawn. Whereas in old age caffeine metabolism is unchanged, there is a strong delay in renal excretion of main metabolites of metamizol in old volunteers. It is concluded that different hepatic cytochrome P-450 isoenzymes are differentially influenced with increasing age in men. PMID:4095131

Ortweiler, W; Stein, G; Simon, H U; Siegert, C; Splinter, F K; Traeger, A

1985-11-01

306

Kinetic Isotope Effects in Hydroxylation Reactions Effected by Cytochrome P450 Compounds I Implicate Multiple Electrophilic Oxidants for P450-Catalyzed Oxidations†  

PubMed Central

Kinetic isotope effects were measured for oxidations of (S,S)-2-(p-trifluoromethylphenyl)cyclopropylmethane containing zero, two, and three deuterium atoms on the methyl group by Compounds I from the cytochrome P450 enzymes CYP119 and CYP2B4 at 22 °C. The oxidations displayed saturation kinetics, which permitted solution of both binding constants (Kbind) and first-order oxidation rate constants (kox) for both enzymes with the three substrates. The binding constant for CYP2B4 Compound I was about one order of magnitude greater than that for CYP119 Compound I, but the oxidation rate constants were similar for the two. In oxidations of 1-d0, kox = 10.4 s?1 for CYP119 Compound I, and kox = 12.4 s?1 for CYP2B4 Compound I. Primary kinetic isotope effects (P) and secondary kinetic isotope effects (S) were obtained from the results with the three isotopomers. The primary KIEs were large, P = 9.8 and P = 8.9 for CYP119 and CYP2B4 Compounds I, respectively, and the secondary KIEs were small and normal, S = 1.07 and S = 1.05, respectively. Large intermolecular KIEs for 1-d0 and 1-d3 of kH/kD = 11.2 and 9.8 found for the two Compounds I contrast with small intermolecular KIEs obtained previously for the same substrate in P450-catalyzed oxidations; these differences suggest that a second electrophilic oxidant, presumably iron-complexed hydrogen peroxide, is important in cytochromes P450 oxidations under turnover conditions. PMID:19182902

Sheng, Xin; Zhang, Haoming; Hollenberg, Paul F.; Newcomb, Martin

2009-01-01

307

Identification of a Cyclosporine-Specific P450 Hydroxylase Gene through Targeted Cytochrome P450 Complement (CYPome) Disruption in Sebekia benihana  

PubMed Central

It was previously proposed that regio-specific hydroxylation of an immunosuppressive cyclosporine (CsA) at the 4th N-methyl leucine is mediated by cytochrome P450 hydroxylase (CYP) in the rare actinomycete Sebekia benihana. This modification is thought to be the reason for the hair growth-promoting side effect without the immunosuppressive activity of CsA. Through S. benihana genome sequencing and in silico analysis, we identified the complete cytochrome P450 complement (CYPome) of S. benihana, including 21 CYPs and their electron transfer partners, consisting of 7 ferredoxins (FDs) and 4 ferredoxin reductases (FDRs). Using Escherichia coli conjugation-based S. benihana CYPome-targeted disruption, all of the identified CYP, FD, and FDR genes in S. benihana were individually inactivated. Among the 32 S. benihana exconjugant mutants tested, only a single S. benihana CYP mutant, ?CYP-sb21, failed to exhibit CsA hydroxylation activity. The hydroxylation was restored by CYP-sb21 gene complementation. Since all S. benihana FD and FDR disruption mutants maintained CsA hydroxylation activity, it can be concluded that CYP-sb21, a new member of the bacterial CYP107 family, is the only essential component of the in vivo regio-specific CsA hydroxylation process in S. benihana. Moreover, expression of an extra copy of the CYP-sb21 gene increased CsA hydroxylation in wild-type S. benihana and an NADPH-enriched Streptomyces coelicolor mutant, by 2-fold and 1.5-fold, respectively. These results show for the first time that regio-specific hydroxylation of CsA is carried out by a specific P450 hydroxylase present in S. benihana, and they set the stage for the biotechnological application of regio-specific CsA hydroxylation through heterologous CYP-sb21 expression. PMID:23354713

Lee, Mi-Jin; Kim, Hyun-Bum; Yoon, Yeo Joon; Han, Kyuboem

2013-01-01

308

Chemical modification of lysine residues in cytochrome P450LM2 (P450IIB4): influence on heme liganding of arylamines.  

PubMed

Treatment of cytochrome P450LM2 with fluorescein isothiocyanate to introduce up to two equivalents of fluorophore per polypeptide chain resulted in the selective derivatization of lysine residues. CD-spectral measurements revealed the overall conformation as well as the immediate heme environment of the hemoprotein to remain unaffected by attachment of the label. Modification caused decreased affinity of p-phenylenediamine and other 4-substituted anilines for the heme site, whereas there was a rise in the extent of substrate interaction. Experiments with pigment containing acetylated lysines gave analogous results, suggesting that the observed phenomenon was due to charge neutralization. There was linear correlation between the Hammett sigma P values and both the optical dissociation constants for arylamine binding to intact enzyme and the dipole moments of the anilines, indicating that basicity along with electronic factors controlled heme liganding; lipophilicity appeared to be of minor importance. Introduction of fluorescein isothiocyanate into the oxygenase was found to influence the bond-making process through modulating basicity of the nitrogenous compounds, but perturbation of optimal spacial orientation of the amine nitrogen toward the heme iron also might have been operative. The lysines studied seem to represent metabolically inactive elements of the substrate channel located on the cytosolic surface of the aggregates, as evidenced by steady-state fluorescence measurements. A hydrophilic segment in the cytochrome P450LM2 molecule that would accommodate the critical residues is discussed. PMID:1727644

Golly, I; Hlavica, P

1992-01-01

309

Cryoradiolysis and cryospectroscopy for studies of heme-oxygen intermediates in cytochromes p450.  

PubMed

Cryogenic radiolytic reduction is one of the most straightforward and convenient methods of generation and stabilization of reactive iron-oxygen intermediates for mechanistic studies in chemistry and biochemistry. The method is based on one-electron reduction of the precursor complex in frozen solution via exposure to the ionizing radiation at cryogenic temperatures. Such approach allows for accumulation of the fleeting reactive complexes which otherwise could not be generated at sufficient amount for structural and mechanistic studies. Application of this method allowed for characterizing of peroxo-ferric and hydroperoxo-ferric intermediates, which are common for the oxygen activation mechanism in cytochromes P450, heme oxygenases, and nitric oxide synthases, as well as for the peroxide metabolism by peroxidases and catalases. PMID:22573452

Denisov, I G; Grinkova, Y V; Sligar, S G

2012-01-01

310

Pharmacokinetic Interactions of Herbs with Cytochrome P450 and P-Glycoprotein  

PubMed Central

The concurrent use of drugs and herbal products is becoming increasingly prevalent over the last decade. Several herbal products have been known to modulate cytochrome P450 (CYP) enzymes and P-glycoprotein (P-gp) which are recognized as representative drug metabolizing enzymes and drug transporter, respectively. Thus, a summary of knowledge on the modulation of CYP and P-gp by commonly used herbs can provide robust fundamentals for optimizing CYP and/or P-gp substrate drug-based therapy. Herein, we review ten popular medicinal and/or dietary herbs as perpetrators of CYP- and P-gp-mediated pharmacokinetic herb-drug interactions. The main focus is placed on previous works on the ability of herbal extracts and their phytochemicals to modulate the expression and function of CYP and P-gp in several in vitro and in vivo animal and human systems. PMID:25632290

Cho, Hyun-Jong

2015-01-01

311

AFM study of the interaction of cytochrome P450 2C9 with phospholipid bilayers.  

PubMed

Cytochromes P450 (CYP) are key enzymes involved in the metabolism of drugs and other lipophilic xenobiotics and endogenous compounds. In this study, atomic force microscopy was applied to characterise the association of CYP2C9 to dimyristoylphosphatidylcholine (DMPC) supported phospholipid bilayers. CYP2C9 was found to exclusively localise in the gel domains of partially melted DMPC bilayers. Despite lacking the N-terminus transmembrane spanning domain, the CYP2C9 protein appeared to partially embed into the membrane bilayer, as evidenced by an increase in melting temperature of surrounding phospholipids. Reversible binding of CYP2C9 via an engineered His tag to a phospholipid bilayer was facilitated using nickel-chelating lipids, presenting potential applications for biosensor technologies. PMID:19925783

Nussio, Matthew R; Voelcker, Nicolas H; Miners, John O; Lewis, Benjamin C; Sykes, Matthew J; Shapter, Joseph G

2010-02-01

312

Experimentally restrained molecular dynamics simulations for characterizing the open states of cytochrome P450cama, b  

PubMed Central

Residual dipolar couplings were used as restraints in fully solvated molecular dynamics simulations of reduced substrate- and carbonmonoxy-bound cytochrome P450cam (CYP101A1), a 414-residue soluble monomeric heme-containing camphor monooxygenase from the soil bacterium Pseudomonas putida. The 1DNH residual dipolar couplings used as restraints were measured in two independent alignment media. A soft annealing protocol was used to heat the starting structures while incorporating the RDC restraints. After production dynamics, structures with the lowest total violation energies for RDC restraints were extracted to identify ensembles of conformers accessible to the enzyme in solution. The simulations result in substrate orientations different from that seen in crystallographic structures and a more open and accessible enzyme active site, and largely support previously reported differences between the open and closed states of CYP101A1. PMID:21265500

Asciutto, Eliana K.; Dang, Marina; Pochapsky, Susan Sondej; Madura, Jeffry D.; Pochapsky, Thomas C.

2011-01-01

313

Identification and characterization of a bacterial cytochrome P450 for the metabolism of diclofenac.  

PubMed

The bacterium Actinoplanes sp. ATCC 53771 is known to perform drug metabolism of several xenobiotics similarly to humans. We identified a cytochrome P450 enzyme from this strain, CYP107E4, and expressed it in Escherichia coli using the pET101 vector. The purified enzyme showed the characteristic reduced-CO difference spectra with a peak at 450 nm, indicating the protein is produced in the active form with proper heme incorporation. The CYP107E4 enzyme was found to bind the drug diclofenac. Using redox enzymes from spinach, the reconstituted system is able to produce hydroxylated metabolites of diclofenac. Production of the human 4'-hydroxydiclofenac metabolite by CYP107E4 was confirmed, and a second hydroxylated metabolite was also produced. PMID:19636551

Prior, Jamie E; Shokati, Touraj; Christians, Uwe; Gill, Ryan T

2010-01-01

314

Cytochrome P450 epoxygenase CYP2J2 attenuates nephropathy in streptozotocin-induced diabetic mice  

PubMed Central

Cytochrome P450 (CYP) epoxygenases metabolize arachidonic acid into epoxyeicosatrienoic acids (EETs), which play important and diverse roles in the cardiovascular system. The anti-inflammatory, anti-apoptotic, pro-angiogenic, and anti-hypertensive properties of EETs in the cardiovascular system suggest a beneficial role for EETs in diabetic nephropathy. This study investigated the effects of endothelial specific overexpression of the CYP2J2 epoxygenase on diabetic nephropathy in streptozotocin induced diabetic mice. Endothelial CYP2J2 overexpression attenuated renal damage as measured by urinary microalbumin and glomerulosclerosis. These effects were associated with inhibition of TGF-?/Smad signaling in the kidney. Indeed, overexpression of CYP2J2 prevented TGF-?1 induced renal tubular epithelial-mesenchymal transition in vitro. These findings highlight the bene cial roles of the CYP epoxygenase-EET system in the pathogenesis of diabetic nephropathy. PMID:21742052

Chen, Guangzhi; Wang, Peihua; Zhao, Gang; Xu, Gang; Gruzdev, Artiom; Lee, Craig R.; Zeldin, Darryl C.; Wang, Dao Wen

2011-01-01

315

Hepatocyte circadian clock controls acetaminophen bioactivation through NADPH-cytochrome P450 oxidoreductase.  

PubMed

The diurnal variation in acetaminophen (APAP) hepatotoxicity (chronotoxicity) reportedly is driven by oscillations in metabolism that are influenced by the circadian phases of feeding and fasting. To determine the relative contributions of the central clock and the hepatocyte circadian clock in modulating the chronotoxicity of APAP, we used a conditional null allele of brain and muscle Arnt-like 1 (Bmal1, aka Mop3 or Arntl) allowing deletion of the clock from hepatocytes while keeping the central and other peripheral clocks (e.g., the clocks controlling food intake) intact. We show that deletion of the hepatocyte clock dramatically reduces APAP bioactivation and toxicity in vivo and in vitro because of a reduction in NADPH-cytochrome P450 oxidoreductase gene expression, protein, and activity. PMID:25512522

Johnson, Brian P; Walisser, Jacqueline A; Liu, Yan; Shen, Anna L; McDearmon, Erin L; Moran, Susan M; McIntosh, Brian E; Vollrath, Aaron L; Schook, Andrew C; Takahashi, Joseph S; Bradfield, Christopher A

2014-12-30

316

Roles of renal cytochrome P450-dependent arachidonic acid metabolites in hypertension.  

PubMed

Cytochrome P450 represents the third metabolic pathway of arachidonic acid giving rise to several biologically active compounds, such as 19-HETE, 20-HETE and EETs and their corresponding DHETs. The kidney is the rich source of these metabolites which have some important biologic actions within the kidney. These metabolites have a wide and contrasting spectrum of biological and renal effects, from vasodilation to vasoconstriction and from inhibition to stimulation of Na-K-ATPase, their relative production rates may influence not only renal hemodynamics but also pro- and anti-hypertensive mechanisms of hypertension. There is increasing evidence that the abnormality of these metabolites in animal models of hypertension. However, sufficient evidence of the physiological and pathophysiological roles of hypertension in man is still lacking. PMID:1412450

Omata, K; Abe, K; Sheu, H L; Yoshida, K; Tsutsumi, E; Yoshinaga, K; Abraham, N G; Laniado-Schwartzman, M

1992-01-01

317

Effective cytochrome P450 (CYP) inhibitors isolated from tarragon (Artemisia dracunculus).  

PubMed

Two effective cytochrome P450 (CYP) inhibitors were isolated from tarragon, Artemisia dracunculus. Their structures were spectroscopically identified as 2E,4E-undeca-2,4-diene-8,10-diynoic acid isobutylamide (1) and 2E,4E-undeca-2,4-diene-8,10-diynoic acid piperidide (2). Both compounds had dose-dependent inhibitory effects on CYP3A4 activity with IC50 values of 10.0 ± 1.3 µM for compound 1 and 3.3 ± 0.2 µM for compound 2, and exhibited mechanism-based inhibition. This is the first reported isolation of effective CYP inhibitors from tarragon (Artemisia dracunculus) purchased from a Japanese market. PMID:22738981

Brahmi, Zeineb; Katho, Tatsuya; Hatsumata, Rie; Hiroi, Asako; Miyakawa, Nami; Yakou, Emi; Sugaya, Kouichi; Onose, Jun-ichi; Abe, Naoki

2012-01-01

318

Activation of promutagenic chemicals by Streptomyces griseus containing cytochrome P-450soy.  

PubMed

Streptomyces griseus cells containing cytochrome P-450soy oxidize a diverse array of xenobiotic compounds. This metabolic capability was exploited for activation of promutagenic chemicals such as polycyclic aromatic hydrocarbons, aromatic amines and small aliphatics in a modified Salmonella/Ames plate incorporation assay using tester strains TA98 and TA1538. In this assay promutagens such as 3,3'-dimethylbenzidine, 3,3'-dimethoxybenzidine, benzidine, 2-acetylaminofluorene, 2-aminoanthracene, 2,4-diaminotoluene, 4-aminobiphenyl, benzo(a)pyrene, chloropicrin and N-nitrosodimethylamine were oxidized to mutagenic metabolites by S. griseus intact cells which mutated Salmonella tester strains (TA98 and TA1538). S. griseus failed to activate 7,12-dimethylbenzanthracene and 4-chloro-2-nitroaniline. In parallel tests performed with rat liver homogenate (S9), N-nitrosodimethylamine was not activated. PMID:2105727

Sariaslani, F S; Stahl, R G

1990-01-30

319

Biotransformation of methyl tert-butyl ether by human cytochrome P450 2A6.  

PubMed

Methyl tert-butyl ether (MTBE) is widely used as gasoline oxygenate and octane number enhancer for more complete combustion in order to reduce the air pollution caused by motor vehicle exhaust. The possible adverse effects of MTBE on human health are of major public concern. However, information on the metabolism of MTBE in human tissues is scarce. The present study demonstrates that human cytochrome P450 2A6 is able to metabolize MTBE to tert-butyl alcohol (TBA), a major circulating metabolite and marker for exposure to MTBE. As CYP2A6 is known to be constitutively expressed in human livers, we infer that it may play a significant role in metabolism of gasoline ethers in liver tissue. PMID:21915685

Shamsipur, Mojtaba; Miran Beigi, Ali Akbar; Teymouri, Mohammad; Poursaberi, Tahereh; Mostafavi, S Mojtaba; Soleimani, Parviz; Chitsazian, Fereshteh; Tash, Shahram Abolhassan

2012-04-01

320

Peripheral heme substituents control the hydrogen-atom abstraction chemistry in cytochromes P450  

PubMed Central

We elucidate the hydroxylation of camphor by cytochrome P450 with the use of density functional and mixed quantum mechanics/molecular mechanics methods. Our results reveal that the enzyme catalyzes the hydrogen-atom abstraction step with a remarkably low free-energy barrier. This result provides a satisfactory explanation for the experimental failure to trap the proposed catalytically competent high-valent heme Fe(IV) oxo (oxyferryl) species responsible for this hydroxylation chemistry. The primary and previously unappreciated contribution to stabilization of the transition state is the interaction of positively charged residues in the active-site cavity with carboxylate groups on the heme periphery. A similar stabilization found in dioxygen binding to hemerythrin, albeit with reversed polarity, suggests that this mechanism for controlling the relative energetics of redox-active intermediates and transition states in metalloproteins may be widespread in nature. PMID:12771375

Guallar, Victor; Baik, Mu-Hyun; Lippard, Stephen J.; Friesner, Richard A.

2003-01-01

321

Hepatocyte circadian clock controls acetaminophen bioactivation through NADPH-cytochrome P450 oxidoreductase  

PubMed Central

The diurnal variation in acetaminophen (APAP) hepatotoxicity (chronotoxicity) reportedly is driven by oscillations in metabolism that are influenced by the circadian phases of feeding and fasting. To determine the relative contributions of the central clock and the hepatocyte circadian clock in modulating the chronotoxicity of APAP, we used a conditional null allele of brain and muscle Arnt-like 1 (Bmal1, aka Mop3 or Arntl) allowing deletion of the clock from hepatocytes while keeping the central and other peripheral clocks (e.g., the clocks controlling food intake) intact. We show that deletion of the hepatocyte clock dramatically reduces APAP bioactivation and toxicity in vivo and in vitro because of a reduction in NADPH-cytochrome P450 oxidoreductase gene expression, protein, and activity. PMID:25512522

Johnson, Brian P.; Walisser, Jacqueline A.; Liu, Yan; Shen, Anna L.; McDearmon, Erin L.; Moran, Susan M.; McIntosh, Brian E.; Vollrath, Aaron L.; Schook, Andrew C.; Takahashi, Joseph S.; Bradfield, Christopher A.

2014-01-01

322

Whole-cell biotransformation with recombinant cytochrome P450 for the selective oxidation of Grundmann's ketone.  

PubMed

25-Hydroxy-Grundmann's ketone is a key building block in the chemical synthesis of vitamin D3 and its derivatives through convergent routes. Generally, the chemical synthesis of this compound involves tedious procedures and results in a mixture of several products. Recently, the selective hydroxylation of Grundmann's ketone at position C25 by cytochrome P450 (CYP) 154E1 from Thermobifida fusca YX was described. In this study a recombinant whole-cell biocatalyst was developed and applied for hydroxylation of Grundmann's ketone. Biotransformation was performed by Escherichia coli cells expressing CYP154E1 along with two redox partner systems, Pdx/PdR and YkuN/FdR. The system comprising CYP154E1/Pdx/PdR showed the highest production of 25-hydroxy-Grundmann's ketone and resulted in 1.1mM (300mgL(-1)) product concentration. PMID:25023538

Hernández-Martín, Alba; von Bühler, Clemens J; Tieves, Florian; Fernández, Susana; Ferrero, Miguel; Urlacher, Vlada B

2014-10-15

323

Heterologous expression and functional characterization of the NADPH-cytochrome P450 reductase from Capsicum annuum.  

PubMed

Two NADPH-cytochrome P450 reductase (CPR) genes (CaCPR1 and CaCPR2) were isolated from hot pepper (Capsicum annuum L. cv. Bukang). At the red ripe stage, the expression level of CaCPR1 was more than 6-fold greater than that in leaves or flowers. It gradually increased during fruit ripening. The CaCPR2 gene seemed to be expressed constitutively in all of the tested tissues. To investigate the enzymatic properties of CaCPR1, the cDNA of CaCPR1 was heterologously expressed in Escherichia coli without any modification of amino acid sequences, and CaCPR1 was purified. The enzymatic properties of CaCPR1 were confirmed using cytochrome c and cytochrome b5 as protein substrates. The CaCPR1 could support human CYP1A2-catalyzed reaction. It also reduced tetrazolium salts and ferricyanide. These results show that CaCPR1 is the major CPR in most hot pepper tissues. It is suggested that the CaCPR1 can be used a prototype for studying biological functions and biotechnological applications of plant CPRs. PMID:24935229

Lee, Ga-Young; Kim, Hyun Min; Ma, Sang Hoon; Park, Se Hee; Joung, Young Hee; Yun, Chul-Ho

2014-09-01

324

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

PubMed

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

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

2010-12-01

325

Increased blood pressure in mice lacking cytochrome P450 2J5  

PubMed Central

The cytochrome P450 (CYP) enzymes participate in a wide range of biochemical functions, including metabolism of arachidonic acid and steroid hormones. Mouse CYP2J5 is abundant in the kidney where its products, the cis-epoxyeicosatrienoic acids (EETs), modulate sodium transport and vascular tone. To define the physiological role of CYP2J5 in the kidney, knockout mice were generated using a conventional gene targeting approach. Cyp2j5 (?/?) mice develop normally and exhibit no overt renal pathology. While renal EET biosynthesis was apparently unaffected by the absence of CYP2J5, deficiency of this CYP in female mice was associated with increased blood pressure, enhanced proximal tubular transport rates, and exaggerated afferent arteriolar responses to angiotensin II and endothelin I. Interestingly, plasma 17?-estradiol levels were reduced in female Cyp2j5 (?/?) mice and estrogen replacement restored blood pressure and vascular responsiveness to normal levels. There was no evidence of enhanced estrogen metabolism, or altered expression or activities of steroidogenic enzymes in female Cyp2j5 (?/?) mice, but their plasma levels of luteinizing hormone and follicle stimulating hormone were inappropriately low. Together, our findings illustrate a sex-specific role for CYP2J5 in regulation of blood pressure, proximal tubular transport, and afferent arteriolar responsiveness via an estrogen-dependent mechanism.—Athirakul, K., Bradbury, J. A., Graves, J. P., DeGraff, L. M., Ma, J., Zhao, Y., Couse, J. F., Quigley, R., Harder, D. R., Zhao, X., Imig, J. D., Pedersen, T. L., Newman, J. W., Hammock, B. D., Conley, A. J., Korach, K. S., Coffman, T. M., Zeldin, D. C. Increased blood pressure in mice lacking cytochrome P450 2J5. PMID:18716027

Athirakul, Krairerk; Bradbury, J. Alyce; Graves, Joan P.; DeGraff, Laura M.; Ma, Jixiang; Zhao, Yun; Couse, John F.; Quigley, Raymond; Harder, David R.; Zhao, Xueying; Imig, John D.; Pedersen, Theresa L.; Newman, John W.; Hammock, Bruce D.; Conley, Alan J.; Korach, Kenneth S.; Coffman, Thomas M.; Zeldin, Darryl C.

2008-01-01

326

Mechanism and structure-reactivity relationships for aromatic hydroxylation by cytochrome P450.  

PubMed

Cytochrome P450 enzymes play a central role in drug metabolism, and models of their mechanism could contribute significantly to pharmaceutical research and development of new drugs. The mechanism of cytochrome P450 mediated hydroxylation of aromatics and the effects of substituents on reactivity have been investigated using B3LYP density functional theory computations in a realistic porphyrin model system. Two different orientations of substrate approach for addition of Compound I to benzene, and also possible subsequent rearrangement pathways have been explored. The rate-limiting Compound I addition to an aromatic carbon atom proceeds on the doublet potential energy surface via a transition state with mixed radical and cationic character. Subsequent formation of epoxide, ketone and phenol products is shown to occur with low barriers, especially starting from a cation-like rather than a radical-like tetrahedral adduct of Compound I with benzene. Effects of ring substituents were explored by calculating the activation barriers for Compound I addition in the meta and para-position for a range of monosubstituted benzenes and for more complex polysubstituted benzenes. Two structure-reactivity relationships including 8 and 10 different substituted benzenes have been determined using (i) experimentally derived Hammett sigma-constants and (ii) a theoretical scale based on bond dissociation energies of hydroxyl adducts of the substrates, respectively. In both cases a dual-parameter approach that employs a combination of radical and cationic electronic descriptors gave good relationships with correlation coefficients R2 of 0.96 and 0.82, respectively. These relationships can be extended to predict the reactivity of other substituted aromatics, and thus can potentially be used in predictive drug metabolism models. PMID:15480465

Bathelt, Christine M; Ridder, Lars; Mulholland, Adrian J; Harvey, Jeremy N

2004-10-21

327

Proton coupling in the ligand-binding reaction of ferric cytochrome P-450 from Pseudomonas putida  

SciTech Connect

Effects of pH on the ligand-binding reactions of ferric heme in cytochrome P-450 from Pseudomonas putida (camphor 5-monooxygenase, EC 1.14.15.1) were studied by using cyanide, N-methylimidazole, pyridine, and ethylisocyanide as ligands. In all cases, affinity of the ferric heme for the ligand was found to increase as pH of the medium was raised from around 6 to 9. Depending on the ligand, the increase was 10- to 1000-fold and the shapes of their pH-affinity curves were remarkably different. Analyses such pH profiles disclosed the presence of a dissociable group in the enzyme with a pK value of approximately 9.5 and that its ionization greatly enhanced the affinity of the heme for ligands. When a dissociable ligand such as hydrogen cyanide and N-methylimidazole was used, the dissociated form of the ligand had a higher affinity toward the heme than the undissociated form. The shapes of the pH-affinity curves were successfully simulated as overlapping curves of ionization reactions of the ligand and the dissociable group. In addition, size of the ligand molecule was shown to be also important in the binding reaction: relatively large molecules such as pyridine, ethylisocyanide, and N-methylimidazole bound to the enzyme in a competitive manner against d-camphor concentration, whereas the binding of a smaller molecule such as cyanide was inhibited by the substrate in a noncompetitive manner. On the basis of these findings, control mechanisms for the ligand-binding reactions of the cytochrome P-450 from P. putida are discussed.

Totani, K.; Iizuka, T.; Shimada, H.; Makino, R.; Ishimura, Y.

1983-04-01

328

Reductive dehalogenation by cytochrome P450CAM: Substrate binding and catalysis  

SciTech Connect

Biological reductive dehalogenation reactions are important in environmental detoxification of organohalides. Only scarce information is available on the enzymology underlying these reactions. Cytochrome P450CAM with a known X-ray structure and well-studied oxygenase reaction cycle, has been studied for its ability to reduce carbon-halogen bonds under anaerobic conditions. The reductive reactions functioned with NADH and the physiological electron-transfer proteins or by using artificial electron donors to reduce cytochrome P450CAM. Halogenated methane and ethane substrates were transformed by a two-electron reduction and subsequent protonation, beta-elimination, or alpha-elimination to yield alkanes, alkene, or carbene-derived products, respectively. Halogenated substrates bound to the camphor binding site as indicated by saturable changes in the Fe(III)-heme spin state upon substrate addition. Hexachloromethane was bound with a dissociation constant (KD) of 0.7 microM and caused > 95% shift from low- to high-spin iron. Ethanes bearing fewer chlorine substituents were bound with increasing dissociation constants and gave lesser degrees of iron spin-state change. Camphor competitively inhibited hexachloroethane reduction with an inhibitor constant (KI) similar to the dissociation constant for camphor (KI = KD = 0.9 microM). Rate determinations with pentachloroethane indicated a 100-fold higher enzyme V/K compared to the second-order rate constant for hematin free in solution. These studies on substrate binding and catalysis will help reveal how biological systems enzymatically reduce carbon-halogen bonds in the environment.

Li, S.; Wackett, L.P. (Univ. of Minnesota, St. Paul (United States))

1993-09-14

329

Mice deficient in intestinal epithelium cytochrome P450 reductase are prone to acute toxin-induced mucosal damage.  

PubMed

Cytochrome P450 (P450) enzymes are a superfamily of heme-containing enzymes involved in the metabolism of various endogenous compounds, including retinoids, glucocorticoids, and eicosanoids, that are postulated to participate in the maintenance and/or development of inflammatory and immune reactions in the intestinal mucosa. To investigate the role of P450 enzymes in intestinal inflammation and immunity, we took advantage of IE-Cpr-null mice, which are deficient in intestinal epithelium of NADPH-cytochrome P450 reductase (CPR), the obligate redox partner of all microsomal P450 enzymes. We report that IE-Cpr-null mice, following an acute toxin challenge, had higher levels of pro-inflammatory chemokines and increased tissue damage compared to wild-type mice. IE-Cpr-null mice had normal Peyer's patch numbers and elicited normal secretory IgA (SIgA) responses. However, SIgA baseline levels in IE-Cpr-null mice were consistently elevated over WT littermates. While neither retinoic acid nor glucocorticoid levels in serum and intestinal homogenates were altered in IE-Cpr-null mice, basal levels of arachidonic acid metabolites (11,12-DiHETE and 14,15-DiHETE) with known anti-inflammatory property were significantly lower compared to WT controls. Overall, these findings reveal immunological and metabolic changes resulting from a genetic deficiency in CPR expression in the intestine, and support a role for microsomal P450 enzymes in mucosal homeostasis and immunity. PMID:24989705

Ahlawat, Sarita; Xie, Fang; Zhu, Yi; D'Hondt, Rebecca; Ding, Xinxin; Zhang, Qing-Yu; Mantis, Nicholas J

2014-01-01

330

The mitochondrial environment is required for activity of the cholesterol side-chain cleavage enzyme, cytochrome P450scc.  

PubMed Central

Steroidogenesis is initiated by the conversion of cholesterol to pregnenolone by mitochondrial cytochrome P450scc [cholesterol, reduced-adrenal-ferredoxin:oxygen oxidoreductase (side-chain-cleaving); EC 1.14.15.6]. Several subsequent steroidal conversions occur in the endoplasmic reticulum (ER), but the last step in the production of glucocorticoids and mineralocorticoids again occurs in the mitochondria. Although cellular compartmentalization of steroidogenic enzymes appears to be a feature of all steroidogenic pathways, some reports indicate that cholesterol can be converted to pregnenolone outside the mitochondria. To investigate whether P450scc can function outside the mitochondria, we constructed vectors producing P450scc and various fusion enzymes of P450scc with electron-transport proteins and directed their expression to either the ER or the mitochondria. Whether targeted to mitochondria or to the ER, plasmid vectors encoding P450scc and fusion proteins of P450scc with either mitochondrial or microsomal electron-transport proteins produced immunodetectable protein. When expressed in mitochondria, all of these constructions converted 22-hydroxycholesterol to pregnenolone, but when expressed in the ER none of them produced pregnenolone. These results show that P450scc can function only in the mitochondria. Furthermore, it appears to be the mitochondrial environment that is required, rather than the specific mitochondrial electron-transport intermediates. Images PMID:8041774

Black, S M; Harikrishna, J A; Szklarz, G D; Miller, W L

1994-01-01

331

Denitrification by the fungus Cylindrocarpon tonkinense: anaerobic cell growth and two isozyme forms of cytochrome P-450nor.  

PubMed Central

We examined the denitrification system of the fungus Cylindrocapon tonkinense and found several properties distinct from those of the denitrification system of Fusarium oxysporum. C. tonkinense could form N2O from nitrite under restricted aeration but could not reduce nitrate by dissimilatory metabolism. Nitrite-dependent N2O formation and/or cell growth during the anaerobic culture was not affected by further addition of ammonium ions but was suppressed by respiration inhibitors such as rotenone or antimycin, suggesting that denitrification plays a physiological role in respiration. Dissimilatory nitrite reductase and nitric oxide reductase (Nor) activities could not be detected in cell extracts of the denitrifying cells. The Nor activity was purified and found to depend upon two isoenzymes of Cytochrome P-450nor (P-450nor), which were designated P-450nor1 and P-450nor2. These isozymes differed in the N-terminal amino acid sequence, isoelectric point, specificity to the reduced pyridine nucleotide (NADH or NADPH), and the reactivity to the antibody to P-450nor of F. oxysporum. the difference between the specificities to NADH and NADPH suggests that P-450nor1 and P-450nor2 play different roles in anaerobic energy acquisition. PMID:7793922

Usuda, K; Toritsuka, N; Matsuo, Y; Kim, D H; Shoun, H

1995-01-01

332

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

PubMed Central

What is already known about this subject Gliclazide is a widely used oral hypoglycaemic agent. The major metabolites of gliclazide formed in vivo have been identified. However, the cytochrome P450 enzymes catalysing the rate-limiting pathways of gliclazide elimination are unknown. What this study adds CYP2C9 is the major enzyme involved in the various hydroxylation pathways of gliclazide, although a contribution of CYP2C19 to tolymethylhydroxylation, the major metabolic route, cannot be discounted. Factors known to influence CYP2C9 activity will provide the main source of variability in gliclazide pharmacokinetics. Aims To identify the human cytochrome P450 (CYP) enzymes responsible for the formation of the 6?-hydroxy (6?-OHGz), 7?-hydroxy (7?-OHGz) and hydroxymethyl (MeOH-Gz) metabolites of gliclizide (Gz). Methods 6?-OHGz, 7?-OHGz and MeOH-Gz formation by human liver microsomes and a panel of recombinant human P450s was measured using a high-performance liquid chromatography procedure, and the kinetics of metabolite formation was determined for each pathway. Effects of prototypic CYP enzyme selective inhibitors were characterized for each of the microsomal metabolic pathways. Results Microsomes from six human livers converted Gz to its 6?-OHGz, 7?-OHGz, and MeOH-Gz metabolites, with respective mean (± SD) Km values of 461 ± 139, 404 ± 143 and 334 ± 75 µm and mean Vmax values of 130 ± 55, 82 ± 31 and 268 ± 115 pmol min?1 mg?1, respectively. Vmax/Km ratios for the microsomal reactions parallelled relative metabolite formation in vivo. Sulfaphenazole inhibited microsomal 6?-OHGz, 7?-OHGz and MeOH-Gz formation by 87, 83 and 64%, respectively, whereas S-mephenytoin caused significant inhibition (48%) of only MeOH-Gz formation. Recombinant CYP2C9, CYP2C18 and CYP2C19 catalysed all hydroxylation pathways, whereas CYP2C8 formed only 6?-OHGz and 7?-OHGz. Conclusion Taken together, the results indicate that CYP2C9 is the major contributor to Gz metabolic clearance, although CYP2C19 may also be involved in MeOH-Gz formation (the major metabolic pathway). Factors known to influence CYP2C9 activity will provide the main source of variability in Gz pharmacokinetics. PMID:17517049

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

2007-01-01

333

Induction and Characterization of a Cytochrome P-450-Dependent Camphor Hydroxylase in Tissue Cultures of Common Sage (Salvia officinalis).  

PubMed Central

(+)-Camphor, a major monoterpene of the essential oil of common sage (Salvia officinalis), is catabolized in senescent tissue, and the pathway for the breakdown of this bicyclic ketone has been previously elucidated in sage cell-suspension cultures. In the initial step of catabolism, camphor is oxidized to 6-exo-hydroxycamphor, and the corresponding NADPH- and O2-dependent hydroxylase activity was demonstrated in microsomal preparations of sage cells. Several well-established inhibitors of cytochrome P-450-dependent reactions, including cytochrome c, clotrimazole, and CO, inhibited the hydroxylation of camphor, and CO-dependent inhibition was partially reversed by blue light. Upon treatment of sage suspension cultures with 30 mM MnCl2, camphor-6-hydroxylase activity was induced up to 7-fold. A polypeptide with estimated molecular mass of 58 kD from sage microsomal membranes exhibited antigenic cross-reactivity in western blot experiments with two heterologous polyclonal antibodies raised against cytochrome P-450 camphor-5-exo-hydroxylase from Pseudomonas putida and cytochrome P-450 limonene-6S-hydroxylase from spearmint (Mentha spicata). Dot blotting indicated that the concentration of this polypeptide increased with camphor hydroxylase activity in microsomes of Mn2+-induced sage cells. These results suggest that camphor-6-exo-hydroxylase from sage is a microsomal cytochrome P-450 monooxygenase that may share common properties and epitopes with bacterial and other plant monoterpene hydroxylases. PMID:12231778

Funk, C.; Croteau, R.

1993-01-01

334

Potential effects of environmental contaminants on P450 aromatase activity and DNA damage in swallows from the Rio Grande and Somerville, Texas  

USGS Publications Warehouse

Cliff swallows (Petrochelidon pyrrhonota) and cave swallows (P. fulva) were sampled during the breeding season at several locations in the Rio Grande, Texas, to evaluate the potential effects of environmental contaminants on P450 aromatase activity in brain and gonads and DNA damage in blood cells. The tritiated water-release aromatase assay was used to measure aromatase activity and flow cytometry was used to measure DNA damage in nucleated blood cells. There were no significant differences in brain and gonadal aromatase activities or in estimates of DNA damage (HPCV values) among cave swallow colonies from the Lower Rio Grande Valley (LRGV) and Somerville. However, both brain and gonadal aromatase activities were significantly higher (P < 0.05) in male cliff swallows from Laredo than in those from Somerville. Also, DNA damage estimates were significantly higher (P < 0.05) in cliff swallows (males and females combined) from Laredo than in those from Somerville. Contaminants of current high use in the LRGV, such as atrazine, and some of the highly persistent organochlorines, such as toxaphene and DDE, could be potentially associated with modulation of aromatase activity in avian tissues. Previous studies have indicated possible DNA damage in cliff swallows. We did not observe any differences in aromatase activity or DNA damage in cave swallows that could be associated with contaminant exposure. Also, the differences in aromatase activity and DNA damage between male cliff swallows from Laredo and Somerville could not be explained by contaminants measured at each site in previous studies. Our study provides baseline information on brain and gonadal aromatase activity in swallows that could be useful in future studies. ?? 2008 Springer Science+Business Media, LLC.

Sitzlar, M.A.; Mora, M.A.; Fleming, J.G.W.; Bazer, F.W.; Bickham, J.W.; Matson, C.W.

2009-01-01

335

Local Transformations of Androgens into Estradiol by Aromatase P450 Is Involved in the Regulation of Prolactin and the Proliferation of Pituitary Prolactin-Positive Cells  

PubMed Central

In previous studies we demonstrated the immunohistochemical expression of aromatase in pituitary cells. In order to determine whether pituitary aromatase is involved in the paracrine regulation of prolactin-producing pituitary cells and the physiological relevance of pituitary aromatase in the control of these cells, an in vivo and in vitro immunocytochemical and morphometric study of prolactin-positive pituitary cells was carried out on the pituitary glands of adult male rats treated with the aromatase antagonist fadrozole. Moreover, we analyzed the expression of mRNA for the enzyme in pituitary cells of male adult rats by in situ hybridization. The aromatase-mRNA was seen to be located in the cytoplasm of 41% of pituitary cells and was well correlated with the immunocytochemical staining. After in vivo treatment with fadrozole, the size (cellular and nuclear areas) of prolactin cells, as well as the percentage of prolactin-positive cells and the percentage of proliferating-prolactin cells, was significantly decreased. Moreover, fadrozole decreased serum prolactin levels. In vitro, treatment with fadrozole plus testosterone induced similar effects on prolactin-positive cells, inhibiting their cellular proliferation. Our results suggest that under physiological conditions aromatase P450 exerts a relevant control over male pituitary prolactin-cells, probably transforming testosterone to estradiol in the pituitary gland. PMID:24978194

Carretero Hernández, Marta; Iglesias Osma, María Carmen; Carretero, Manuel; Herrero, Julio J.

2014-01-01

336

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

PubMed

Electron transfer between membrane spanning oxidoreductase enzymes controls vital metabolic processes. Here we studied for the first time with single molecule resolution the function of P450 oxidoreductase (POR), the canonical membrane spanning activator of all microsomal cytochrome P450 enzymes. Measurements and statistical analysis of individual catalytic turnover cycles shows POR to sample at least two major functional states. This phenotype may underlie regulatory interactions with different cytochromes P450 but to date has remained masked in bulk kinetics. To ensure that we measured the inherent behavior of POR, we reconstituted the full length POR in "native like" membrane patches, nanodiscs. Nanodisc reconstitution increased stability by ?2-fold as compared to detergent solubilized POR and showed significantly increased activity at biologically relevant ionic strength conditions, highlighting the importance of studying POR function in a membrane environment. This assay paves the way for studying the function of additional membrane spanning oxidoreductases with single molecule resolution. PMID:24359083

Laursen, Tomas; Singha, Aparajita; Rantzau, Nicolai; Tutkus, Marijonas; Borch, Jonas; Hedegård, Per; Stamou, Dimitrios; Møller, Birger Lindberg; Hatzakis, Nikos S

2014-03-21

337

7-Ethynylcoumarins: Selective Inhibitors of Human Cytochrome P450s 1A1 and 1A2  

PubMed Central

To discover new selective mechanism-based P450 inhibitors, eight 7-ethynylcoumarin derivatives were prepared through a facile two-step synthetic route. Cytochrome P450 activity assays indicated that introduction of functional groups in the backbone of coumarin could enhance the inhibition activities toward P450s 1A1 and 1A2, providing good selectivity against P450s 2A6 and 2B1. The most potent product 7-ethynyl-3,4,8-trimethylcoumarin (7ETMC) showed IC50 values of 0.46 ?M and 0.50 ?M for P450s 1A1 and 1A2 in the first six minutes, respectively, and did not show any inhibition activity for P450s 2A6 and 2B1 even at the dose of 50 ?M. All of the inhibitors except 7-ethynyl-3-methyl-4-phenylcoumarin (7E3M4PC) showed mechanism-based inhibition of P450s 1A1 and 1A2. In order to explain this mechanistic difference in inhibitory activities, X-ray crystallography data were used to study the difference in conformation between 7E3M4PC and the other compounds studied. Docking simulations indicated that the binding orientations and affinities resulted in different behaviors of the inhibitors on P450 1A2. Specifically, 7E3M4PC with its two-plane structure fits into the P450 1A2’s active site cavity with an orientation leading to no reactive binding, causing it to act as a competitive inhibitor. PMID:22443586

Liu, Jiawang; Nguyen, Thong T.; Dupart, Patrick S.; Sridhar, Jayalakshmi; Zhang, Xiaoyi; Zhu, Naijue; Stevens, Cheryl L. Klein; Foroozesh, Maryam

2012-01-01

338

2,2?,3,3?,6,6?-Hexachlorobiphenyl (PCB 136) Atropisomers Interact Enantioselectively with Hepatic Microsomal Cytochrome P450 Enzymes  

PubMed Central

2,2?,3,3?,6,6?-Hexachlorobiphenyl (PCB 136) is a chiral and highly neurotoxic PCB congener of environmental relevance. (+)-PCB 136 was previously shown to be enriched in tissues from mice treated with racemic PCB 136. We investigated the spectral interactions of (+)-, (-)- and (±)-PCB 136 with mouse and rat hepatic microsomal cytochrome P450 (P450) enzymes to test the hypothesis that enantioselective binding to specific P450 enzymes causes the enrichment of (+)-PCB 136 in vivo. Hepatic microsomes prepared from C57BL/6 mice or Long Evans rats treated with ?-naphthoflavone or 3-methylcholanthrene, phenobarbital and dexamethasone (prototypical inducers of CYP1A, CYP2B and CYP3A, respectively) were used to determine first, if the (+)-PCB 136 atropisomer binds to hepatic microsomal P450 enzymes to a greater extent than does the (-)-PCB 136 atropisomer; and second, if P450 enzymes of one subfamily bind the two PCB 136 atropisomers more efficiently than do P450 enzymes of other subfamilies. Increasing concentrations of (+)-, (-)- or (±)-PCB 136 were added to hepatic microsomes and the difference spectrum and maximal absorbance change, a measure of PCB binding to P450 enzymes, were measured. A significantly larger absorbance change was observed with (+)-PCB 136 than with (-)-PCB 136 with all four hepatic microsomal preparations in mice and rats, indicating that (+)-PCB 136 interacted with microsomal P450 enzymes to a greater degree than did (-)-PCB 136. In addition, binding of the PCB 136 atropisomers was greatest in microsomes from PB-treated mice and rats, and was inhibited by CYP2B antibodies, indicating the involvement of CYP2B enzymes. Together these results suggest preferential binding of (+)-PCB 136 to P450 enzymes (such as CYP2B and CYP3A) in hepatic microsomes, an observation that may explain the enantioselective enrichment of the (+)-PCB 136 atropisomer in tissues of mice. PMID:18494506

Kania-Korwel, Izabela; Hrycay, Eugene G.; Bandiera, Stelvio M.; Lehmler, Hans-Joachim

2008-01-01

339

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

PubMed

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

Hlavica, P; Golly, I; Wolf, J

1987-09-01

340

Metazoan cytochrome P450 evolution 1 This article was invited by Guest Editors Dr John J. Stegeman and Dr David R. Livingstone to be part of a special issue of CBP on Cytochrome P450 (Comp. Biochem. Physiol. 121 C, pages 1–412, 1998). 1  

Microsoft Academic Search

There are 37 cytochrome P450 families currently identified in animals. The concept of higher order groupings of P450 families called P450 CLANS is introduced. The mammalian CYP3 and CYP5 families belong to the same clan as insect CYP6 and CYP9. All mitochondrial P450s seem to belong to the same clan. Lack of mitochondrial P450s in C. elegans suggests that mitochondrial

David R Nelson

1998-01-01

341

Fungal lactone ring opening of 6', 7'-dihydroxybergamottin diminishes cytochrome P450 3A4 inhibitory activity  

Technology Transfer Automated Retrieval System (TEKTRAN)

Furanocoumarins (FCs) are a class of aromatic compounds in grapefruit that inhibit human intestinal cytochrome P450 3A4 (CYP3A4). Since fungi metabolize polycyclic aromatic hydrocarbons, we hypothesized that certain fungi might also metabolize FCs into forms that may be inactive as CYP3A4 inhibitors...

342

Relative Contributions of Cyclooxygenase and Cytochrome P450 ?-Hydroxylase-Dependent Pathways to Hypoxic Dilation of Skeletal Muscle Resistance Arteries  

Microsoft Academic Search

This study determined the contribution of prostanoids, cytochrome P450 (CP450) 4A enzyme metabolites of arachidonic acid, and other potential mediators of hypoxic dilation of isolated rat skeletal muscle resistance arteries. Gracilis arteries (GA) were viewed via television microscopy and dilator responses to hypoxia (reduction in superfusate and perfusate PO2 from ?145 to ?40 mm Hg) were measured with a video

Jefferson C. Frisbee; Richard J. Roman; U. Murali Krishna; John R. Falck; Julian H. Lombard

2001-01-01

343

ISOLATION AND CHARACTERIZATION OF THE ALKANE-INDUCIBLE NADPH-CYTOCHROME P-450 OXIDOREDUCTASE GENE FROM CANDIDA TROPICALIS  

EPA Science Inventory

The gene coding for the Candida tropicalis NADPH-cytochrome P-450 oxidoreductase (CPR, NADPH: ferricytochrome oxidoreductase, EC 1.6.2.4) was isolated by immunoscreening of a C. tropicalis gtll expression library and colony hybridization of a C. tropicalis genomic library. he C. ...

344

Chemical modification of Tyr34 and Tyr129 in rabbit liver microsomal cytochrome b5 affects interaction with cytochrome P-450 2B4.  

PubMed

Rabbit liver microsomal cytochrome b5 was allowed to react with tetranitromethane. Up to three tyrosine residues in each cytochrome b5 molecule were found to be accessible to the nitrating agent. Co-modification of tryptophan and histidine residues could be disregarded. CD-spectral measurements disproved gross changes in cytochrome b5 structure as a consequence of derivatization. Introduction of 1.6 nitro groups/polypeptide chain resulted in a fivefold increase in binding affinity for cytochrome P-450 2B4 (P-450 2B4), whereas spectral interaction with cytochrome c remained unaffected. Furthermore, the capacity of nitrated cytochrome b5 to shift the spin equilibrium to the high-spin conformer of P-4502B4 was diminished by 44% compared with the control. This corresponded with the partial disruption of NADH-dependent electron flow to ferric P-450 2B4. Changes in the redox potential of cytochrome b5 could be discounted as being responsible for this effect. The overall oxidative turnover of 4-nitroanisole did not respond to cytochrome b5 modification. MS analysis and sequencing of peptide fragments produced by tryptic digestion of modified cytochrome b5 permitted the detection of three nitrated tyrosine residues located at positions 11, 34 and 129. Derivatization of cytochrome b5 in the presence of a protective amount of P-450 2B4 provided evidence of the involvement of Tyr34 and Tyr129 in complexation of the two hemoproteins. It is proposed that Tyr129 might control docking of cytochrome b5 to P-450 2B4, whereas Tyr34 could be of functional importance in electron transfer. PMID:7925401

Hlavica, P; Kellermann, J; Golly, I; Lehnerer, M

1994-09-15

345

Substrate and Reaction Specificity of Mycobacterium tuberculosis Cytochrome P450 CYP121  

PubMed Central

Cytochrome P450 CYP121 is essential for the viability of Mycobacterium tuberculosis. Studies in vitro show that it can use the cyclodipeptide cyclo(l-Tyr-l-Tyr) (cYY) as a substrate. We report an investigation of the substrate and reaction specificities of CYP121 involving analysis of the interaction between CYP121 and 14 cYY analogues with various modifications of the side chains or the diketopiperazine (DKP) ring. Spectral titration experiments show that CYP121 significantly bound only cyclodipeptides with a conserved DKP ring carrying two aryl side chains in l-configuration. CYP121 did not efficiently or selectively transform any of the cYY analogues tested, indicating a high specificity for cYY. The molecular determinants of this specificity were inferred from both crystal structures of CYP121-analog complexes solved at high resolution and solution NMR spectroscopy of the analogues. Bound cYY or its analogues all displayed a similar set of contacts with CYP121 residues Asn85, Phe168, and Trp182. The propensity of the cYY tyrosyl to point toward Arg386 was dependent on the presence of the DKP ring that limits the conformational freedom of the ligand. The correct positioning of the hydroxyl of this tyrosyl was essential for conversion of cYY. Thus, the specificity of CYP121 results from both a restricted binding specificity and a fine-tuned P450 substrate relationship. These results document the catalytic mechanism of CYP121 and improve our understanding of its function in vivo. This work contributes to progress toward the design of inhibitors of this essential protein of M. tuberculosis that could be used for antituberculosis therapy. PMID:23620594

Fonvielle, Matthieu; Le Du, Marie-Hélène; Lequin, Olivier; Lecoq, Alain; Jacquet, Mickaël; Thai, Robert; Dubois, Steven; Grach, Guillaume; Gondry, Muriel; Belin, Pascal

2013-01-01

346

Selective steroid oxyfunctionalisation by CYP154C5, a bacterial cytochrome P450  

PubMed Central

Background Cytochrome P450 monooxygenases – able to regio- and stereoselectively hydroxylate non-activated carbon atoms – are important enzymes for the synthesis of valuable intermediates in the production of steroid hormones in the pharmaceutical industry. However, up to now only a few bacterial enzymes able to hydroxylate steroids have been reported. CYP154C5 from Nocardia farcinica IFM 10152, a bacterial P450 monooxygenase, was previously shown to convert testosterone to 16?-hydroxytestosterone. Since the hydroxylation at 16?-position is of special interest for the pharmaceutical industry, we have studied this enzyme in more detail to investigate its activity and selectivity in bioconversions of further steroids. Results CYP154C5 was coexpressed in Escherichia coli together with putidaredoxin and putidaredoxin reductase from Pseudomonas putida as redox partners for electron transfer and applied in bioconversions of various pregnanes and androstanes [pregnenolone (1), dehydroepiandrosterone (2), progesterone (3), androstenedione (4), testosterone (5) and nandrolone (6)]. Structure elucidation of the formed products revealed an exclusive regio- and stereoselectivity of CYP154C5, always yielding the corresponding 16?-hydroxylated steroids. Application of whole cells expressing the three components, P450, Pdx and PdR, in steroid biotransformations resulted in significantly higher conversions and total turnover numbers (TTN) compared to reactions using cell-free extracts. Additionally, considerably higher substrate loads (up to 15 mM) were tolerated by the whole-cell system. Furthermore, turnover numbers (TON) were determined for the six different steroids using whole cells. Thus, testosterone was found to be the worst substrate with a TON of only 0.8 ?mol substrate consumed min-1 ?mol-1 CYP154C5, while progesterone and pregnenolone were converted the fastest resulting in TON of 3.3 ?mol substrate consumed min-1 ?mol-1 CYP154C5. Conclusion CYP154C5 from N. farcinica constitutes a promising catalyst due to its high regio- and stereoselectivity in the hydroxylation of different steroids as well as its efficient expression in E. coli at high yields. Using this enzyme, 16?-hydroxylated steroids, which are important precursors for the synthesis of high value steroidal drugs in the pharmaceutical industry, can be selectively produced on preparative scale with TTN (?mol substrate consumed ?mol-1 CYP154C5) exceeding 2000. PMID:24134652

2013-01-01

347

Relationship between paracetamol binding to and its oxidation by two cytochromes P-450 isozymes--a proton nuclear magnetic resonance and spectrophotometric study.  

PubMed

From the hepatic cytochrome P-450 isozymes b and c isolated from rats treated with phenobarbital and 3-methylcholanthrene respectively, only cytochrome P-450c was found to be active in the oxidation of paracetamol, in the presence of glutathione ultimately leading to the formation of the 3-glutathionyl conjugate. Paracetamol interacted with both cytochrome P-450b and c, as shown by difference spectrophotometry. Cytochrome P-450b was found to have a higher affinity for paracetamol than cytochrome P-450c and demonstrated a type I spectral change, whereas in the case of cytochrome P-450c a reverse type I spectral change was observed. Proton n.m.r. longitudinal relaxation rate measurements revealed that in the case of cytochrome P-450c, paracetamol was orientated with its phenolic hydroxyl group in closest proximity to the central haem iron ion. In the case of cytochrome P-450b, the acetylamino group of paracetamol most closely approached the haem iron ion. PMID:3825173

van de Straat, R; de Vries, J; de Boer, H J; Vromans, R M; Vermeulen, N P

1987-01-01

348

Immunohistochemical detection of human placental cytochrome P-450-associated mono-oxygenase system inducible by maternal cigarette smoking.  

PubMed

Several xenobiotic-metabolizing mono-oxygenase activities, in conjunction with the immunohistochemical localization of the respective cytochrome P-450 forms, were investigated in placentae from smoking and non-smoking women. The antibodies used in the immunohistochemical analyses were monoclonal antibodies (MAbs) 1-7-1 and 2-66-3, prepared against the 3-methylcholanthrene-induced and phenobarbital-induced rat liver P-450, respectively. The mono-oxygenase activities were higher in placental microsomes from smokers, although no difference was observed in microsomal P-450 content. A distinct positive staining with MAb 1-7-1 was detectable in the trophoblastic layer of all placentae from smokers. In placentae from non-smokers, minimal cytoplasmic staining was observed in large villi in a few trophoblastic cells. The staining reaction was negative when MAb 2-66-3 or non-specific antibody were used. These results demonstrate that maternal cigarette smoking induces in the trophoblastic layer of the placenta a cytochrome P-450 form which is detectable immunohistochemically with the monoclonal antibody to 3-methylcholanthrene-inducible P-450 in rat liver. PMID:3050970

Pasanen, M; Stenbäck, F; Park, S S; Gelboin, H V; Pelkonen, O

1988-01-01

349

Over-Expression of a Cytochrome P450 Is Associated with Resistance to Pyriproxyfen in the Greenhouse Whitefly Trialeurodes vaporariorum  

PubMed Central

Background The juvenile hormone mimic, pyriproxyfen is a suppressor of insect embryogenesis and development, and is effective at controlling pests such as the greenhouse whitefly Trialeurodes vaporariorum (Westwood) which are resistant to other chemical classes of insecticides. Although there are reports of insects evolving resistance to pyriproxyfen, the underlying resistance mechanism(s) are poorly understood. Results Bioassays against eggs of a German (TV8) population of T. vaporariorum revealed a moderate level (21-fold) of resistance to pyriproxyfen. This is the first time that pyriproxyfen resistance has been confirmed in this species. Sequential selection of TV8 rapidly generated a strain (TV8pyrsel) displaying a much higher resistance ratio (>4000-fold). The enzyme inhibitor piperonyl butoxide (PBO) suppressed this increased resistance, indicating that it was primarily mediated via metabolic detoxification. Microarray analysis identified a number of significantly over-expressed genes in TV8pyrsel as candidates for a role in resistance including cytochrome-P450 dependent monooxygenases (P450s). Quantitative PCR highlighted a single P450 gene (CYP4G61) that was highly over-expressed (81.7-fold) in TV8pyrsel. Conclusion Over-expression of a single cytochrome P450 gene (CYP4G61) has emerged as a strong candidate for causing the enhanced resistance phenotype. Further work is needed to confirm the role of the encoded P450 enzyme CYP4G61 in detoxifying pyriproxyfen. PMID:22347432

Karatolos, Nikos; Williamson, Martin S.; Denholm, Ian; Gorman, Kevin; ffrench-Constant, Richard H.; Bass, Chris

2012-01-01

350

New platform for cytochrome p450 reaction combining in situ immobilization on biopolymer.  

PubMed

We describe an efficienct chemical conversion platform with in situ immobilization of P450-BM3 on poly(3-hydroxybutyrate) granules. Through fusion with phasin, P450-BM3 is easily immobilized on poly(3-hydroxybutyrate) granules in Escherichia coli. In our work, the immobilized P450 exhibited higher stability and catalytic activity compared to free P450 against changes of pH, temperature, and concentrations of urea and ions. Through quick recovery of immobilized enzyme, the P450-P(3HB) complex successfully catalyzed an O-dealkylation reaction several times with maintained activity. Using the robust P450-P(3HB) complex, we performed a P450-catalyzed reaction on a preparative reactor scale (100 mL) and high-level production (12.3 ?M) of 7-hydroxycoumarine from 7-ethoxycoumarin could be achieved. PMID:25322062

Lee, Jae Hyung; Nam, Dong Heon; Lee, Sahng Ha; Park, Jong Hyun; Park, Si Jae; Lee, Seung Hwan; Park, Chan Beum; Jeong, Ki Jun

2014-12-17

351

The imidazoacridinone antitumor drug, C-1311, is metabolized by flavin monooxygenases but not by cytochrome P450s.  

PubMed

5-Diethylaminoethylamino-8-hydroxyimidazoacridinone (C-1311) is an antitumor agent that is also active against autoimmune diseases. The intention of the present studies was to elucidate the role of selected liver enzymes in metabolism of C-1311 and the less active 8-methyl derivative, 5-diethylaminoethylamino-8-methoxyimidazoacridinone (C-1330). Compounds were incubated with rat liver microsomal fraction, with a set of 16 human liver protein samples, and with human recombinant isoenzymes of cytochrome P450, flavin monooxygenases (FMO), and UDP-glucuronosyltransferase (UGT). Our results showed that C-1311 and C-1330 were metabolized with human liver microsomal enzymes but not with any tested human recombinant cytochromes P450 (P450s). Two of these, CYP1A2 and CYP3A4, were inhibited by both compounds. In addition, results of C-1311 elimination from hepatic reductase-null mice, in which liver NADPH-P450 oxidoreductase has been deleted indicated that liver P450s were slightly engaged in drug transformation. In contrast, both compounds were good substrates for human recombinant FMO1 and FMO3 but not for FMO5. The product of FMO metabolism, P(FMO), which is identified as an N-oxide derivative, was identical to P3(R) of liver microsomes. P3(R) was observed even in the presence of the P450 inhibitor, 1-aminobenzotriazole, and it disappeared after heating. Therefore, FMO enzymes could be responsible for microsomal metabolism to P3(R) = P(FMO). Glucuronidation on the 8-hydroxyl group of C-1311 was observed with liver microsomes supported by UDP-glucuronic acid and with recombinant UGT1A1, but it was not the case with UGT2B7. Summing up, we showed that, whereas liver P450 isoenzymes were involved in the metabolism of C-1311 to a limited extent, FMO plays a significant role in the microsomal transformations of this compound, which is also a specific substrate of UGT1A1. PMID:21555506

Potega, Agnieszka; Dabrowska, Emilia; Niemira, Magdalena; Kot-Wasik, Agata; Ronseaux, Sebastien; Henderson, Colin J; Wolf, C Roland; Mazerska, Zofia

2011-08-01

352

Immunochemical evidence for an ethanol-inducible form of liver microsomal cytochrome P-450 in rodents and primates  

SciTech Connect

Polyclonal antibodies against cytochrome P-450-4, a major liver microsomal P-450 isozyme purified from ethanol (E)-treated hamsters, were used to probe for immunochemically-related hemeproteins in other species. Liver microsomes (LM) were obtained from naive and E-treated rats, deermice, hamsters, and baboons. Baboon liver 9000 x g supernatant (S-9) was prepared from needle biopsy samples. LM and S-9 proteins were resolved by SDS-PAGE, then transferred to nylon membranes. Immunodetection was performed on the Western blots using rabbit anti P-450-4 IgG, anti-rabbit IgG-alk. phos., and an appropriate chromagen. Control LM from all species contained a cross-reacting protein of mol. wt. similar to P-450-4 (54k). The amount of this cross-reacting protein as reflected by staining intensity, was much higher in LM from E-treated animals. This protein was also detected in S-9 from E-treated baboons. In contrast, no increase in phenobarbital-inducible P-450-2 related LM protein (assessed using anti P-450-2) was observed after E treatment. Increased P-450-4 related protein in LM from E-treated animals was associated with enhanced oxidation of ethanol and aniline by these LM when compared to controls. In conclusion, LM from rats, deermice, and baboons contain a protein immunochemically homologous to hamster liver P-450-4. As observed in hamsters, the amount of this hepatic protein increases in these other species after E treatment.

Lasker, J.M.; Ardies, C.M.; Bloswick, B.P.; Lieber, C.S.

1986-05-01

353

Fenofibrate modulates cytochrome P450 and arachidonic acid metabolism in the heart and protects against isoproterenol-induced cardiac hypertrophy.  

PubMed

It has been previously shown that the cytochrome P450 (P450) modulator, fenofibrate, protects against cardiovascular diseases. P450 and their metabolites, epoxyeicosatrienoic acids (EETs) and 20-hydroxyeicosatetraenoic acid (20-HETE) were found to play an important role in cardiovascular diseases. Therefore, it is important to examine whether fenofibrate would modulate the cardiac P450 and its associated arachidonic acid metabolites and whether this modulation protects against isoproterenol-induced cardiac hypertrophy. For this purpose, male Sprague-Dawley rats were treated with fenofibrate (30 mg·kg·d), isoproterenol (4.2 mg·kg·d), or the combination of both. The expression of hypertrophic markers and different P450s along with their metabolites was determined. Our results showed that fenofibrate significantly induced the cardiac P450 epoxygenases, such as CYP2B1, CYP2B2, CYP2C11, and CYP2C23, whereas it decreased the cardiac ?-hydroxylase, CYP4A3. Moreover, fenofibrate significantly increased the formation of 14,15-EET, 11,12-EET, and 8,9-EET, whereas it decreased the formation of 20-HETE in the heart. Furthermore, fenofibrate significantly decreased the hypertrophic markers and the increase in heart-to-body weight ratio induced by isoproterenol. This study demonstrates that fenofibrate alters the expression of cardiac P450s and their metabolites and partially protects against isoproterenol-induced cardiac hypertrophy, which further confirms the role of P450s, EETs, and 20-HETE in the development of cardiac hypertrophy. PMID:24157956

Althurwi, Hassan N; Elshenawy, Osama H; El-Kadi, Ayman O S

2014-02-01

354

Role of metabolism by esterase and cytochrome P-450 in cocaine-induced suppression of the antibody response.  

PubMed

To investigate the role of metabolism in cocaine-induced immunosuppression, diazinon and beta-ionone were administered as an esterase inhibitor and a cytochrome P-450 (P-450) inducer, respectively, to B6C3F1 female mice. When 10 or 30 mg/kg of diazinon was administered 30 min before cocaine (30 mg/kg) was administered i.p. for 7 consecutive days, the suppression of the T-dependent antibody response to sheep red blood cells was potentiated greatly when compared to the suppression by cocaine alone. Spleen and thymus weights were decreased significantly and serum glutamate-pyruvate transaminase activities were elevated dramatically when cocaine and diazinon were administered together. beta-Ionone was administered s.c. for 7 consecutive days and the P-450 activities were determined 3 days after the last administration. beta-Ionone induced cocaine N-demethylation, which is the first step in the activation of cocaine to the metabolites capable of producing hepatotoxicity, as well as P-450IA1- and P-450IIB1-specific monooxygenases. The inductive effects of beta-ionone on P-450IA1/2 and P-450IIB1/2 proteins were confirmed by using Western immunoblotting with selective monoclonal antibodies. In addition, when beta-ionone (600 mg/kg) was administered with cocaine for 7 days, the suppression of the antibody response was potentiated greatly, thymus weight was decreased significantly and serum glutamate-pyruvate transaminase was elevated. Our present results suggest that inhibition of the esterase pathway of cocaine shunts the metabolism of cocaine into an immunotoxic pathway, and that the metabolism of cocaine by P-450 may be the critical pathway for the generation of the metabolites capable of suppressing the antibody response. PMID:7815357

Jeong, T C; Jordan, S D; Matulka, R A; Stanulis, E D; Kaminski, E J; Holsapple, M P

1995-01-01

355

Biochemical analysis of a multifunctional cytochrome P450 (CYP51) enzyme required for synthesis of antimicrobial triterpenes in plants  

PubMed Central

Members of the cytochromes P450 superfamily (P450s) catalyze a huge variety of oxidation reactions in microbes and higher organisms. Most P450 families are highly divergent, but in contrast the cytochrome P450 14?-sterol demethylase (CYP51) family is one of the most ancient and conserved, catalyzing sterol 14?-demethylase reactions required for essential sterol synthesis across the fungal, animal, and plant kingdoms. Oats (Avena spp.) produce antimicrobial compounds, avenacins, that provide protection against disease. Avenacins are synthesized from the simple triterpene, ?-amyrin. Previously we identified a gene encoding a member of the CYP51 family of cytochromes P450, AsCyp51H10 (also known as Saponin-deficient 2, Sad2), that is required for avenacin synthesis in a forward screen for avenacin-deficient oat mutants. sad2 mutants accumulate ?-amyrin, suggesting that they are blocked early in the pathway. Here, using a transient plant expression system, we show that AsCYP51H10 is a multifunctional P450 capable of modifying both the C and D rings of the pentacyclic triterpene scaffold to give 12,13?-epoxy-3?,16?-dihydroxy-oleanane (12,13?-epoxy-16?-hydroxy-?-amyrin). Molecular modeling and docking experiments indicate that C16 hydroxylation is likely to precede C12,13 epoxidation. Our computational modeling, in combination with analysis of a suite of sad2 mutants, provides insights into the unusual catalytic behavior of AsCYP51H10 and its active site mutants. Fungal bioassays show that the C12,13 epoxy group is an important determinant of antifungal activity. Accordingly, the oat AsCYP51H10 enzyme has been recruited from primary metabolism and has acquired a different function compared to other characterized members of the plant CYP51 family—as a multifunctional stereo- and regio-specific hydroxylase in plant specialized metabolism. PMID:23940321

Geisler, Katrin; Hughes, Richard K.; Sainsbury, Frank; Lomonossoff, George P.; Rejzek, Martin; Fairhurst, Shirley; Olsen, Carl-Erik; Motawia, Mohammed Saddik; Melton, Rachel E.; Hemmings, Andrew M.; Bak, Søren; Osbourn, Anne

2013-01-01

356

Identification of the cytochrome P450 enzymes involved in the N-demethylation of sildenafil  

PubMed Central

Aims To characterize the cytochrome P450 (CYP) enzymes responsible for the N-demethylation of sildenafil to its main metabolite, UK-103 320, to investigate the potential inhibitory effects of sildenafil on CYP enzymes and to evaluate the potential of selected drugs to affect sildenafil metabolism. Methods The metabolic pathways of sildenafil N-demethylation were studied using human liver microsomes, as well as microsomes expressing individual human CYP enzymes. Further studies to identify the individual enzymes were performed at 2.5 and 250 µm sildenafil, and employed a combination of chemical inhibition, correlation analysis, and metabolism by expressed recombinant CYP enzymes. In addition, the effect of sildenafil on the activity of the six major drug metabolizing enzymes was investigated. Results Sildenafil conversion was found to be mediated by at least two CYP enzymes, for which the mean kinetic parameters were Km1 = 6(±3 µm), Km2 = 81(±45 µm), Vmax1 = 22(±9 pmol) and Vmax2 = 138(±77 pmol) UK-103 320 formed min?1 mg?1. At 250 µm sildenafil, N-demethylation was primarily mediated through the low-affinity, high-Km enzyme (approximately 83%), whilst at 2.5 µm there was a greater role for the high-affinity, low-Km enzyme (approximately 61%). Ketoconazole strongly inhibited metabolism at both sildenafil concentrations and was the only significant inhibitor at 250 µm sildenafil. At the lower sildenafil concentration, sulphaphenazole and quinidine also inhibited formation of UK-103 320. Overall, 75% or more of the N-demethylation of sildenafil at any concentration is probably attributable to CYP3A4. These results were supported by experiments using expressed human CYP enzymes, in which only CYP3A4 and CYP2C9 exhibited substantial sildenafil N-demethylase activity (respective Km values of 221 µm and 27 µm). Sildenafil metabolism was inhibited by potent CYP3A4 inhibitors which are used clinically, but was found to be only a weak inhibitor of drug metabolizing enzymes itself, the strongest inhibition occurring against CYP2C9 (Ki = 80 µm). Conclusions Evidence is provided for CYP3A4 and to a lesser extent CYP2C9-mediated metabolism of sildenafil. There is the possibility that elevated plasma concentrations of sildenafil could occur with coadministration of known inhibitors of CYP2C9 or CYP3A4. Since peak plasma concentrations of clinical doses of sildenafil are only 200 ng ml?1 (?0.4 µm) it is very unlikely that sildenafil will significantly alter the plasma concentration of other compounds metabolized by cytochrome P450 enzymes. PMID:11298070

Hyland, R; Roe, E G H; Jones, B C; Smith, D A

2001-01-01

357

Inhibitory effect of mitragynine on human cytochrome P450 enzyme activities  

PubMed Central

Context: To date, many findings reveal that most of the modern drugs have the ability to interact with herbal drugs. Aims: This study was conducted to determine the inhibitory effects of mitragynine on cytochrome P450 2C9, 2D6 and 3A4 activities. Methods and Material: The in vitro study was conducted using a high-throughput luminescence assay. Statistical Analysis: Statistical analysis was conducted using one-way ANOVA and Dunnett's test with P < 0.05 vs. control. The IC50 values were calculated using the GraphPad Prism® 5 (Version 5.01, GraphPad Software, Inc., USA). Results: Assessment using recombinant enzymes showed that mitragynine gave the strongest inhibitory effect on CYP2D6 with an IC50 value of 0.45±0.33 mM, followed by CYP2C9 and CYP3A4 with IC50 values of 9.70±4.80 and 41.32±6.74 ?M respectively. Positive inhibitors appropriate for CYP2C9, CYP2D6, and CYP3A4 which are sulfaphenazole, quinidine and ketoconazole were used respectively. Vmax values of CYP2C9, CYP2D6 and CYP3A4 were 0.0005, 0.01155 and 0.0137 ?M luciferin formed/pmol/min respectively. Km values of CYP2C9, CYP2D6, and CYP3A4 were 32.65, 56.01, and 103.30 ?M respectively. Mitragynine noncompetitively inhibits CYP2C9 and CYP2D6 activities with the Ki values of 61.48 and 12.86 ?M respectively. On the other hand, mitragynine inhibits CYP3A4 competitively with a Ki value of 379.18 ?M. Conclusions: The findings of this study reveal that mitragynine might inhibit cytochrome P450 enzyme activities, specifically CYP2D6. Therefore, administration of mitragynine together with herbal or modern drugs which follow the same metabolic pathway may contribute to herb-drug interactions. PMID:24174816

Hanapi, N. A.; Ismail, S.; Mansor, S. M.

2013-01-01

358

A novel microplate-based HPLC-fluorescence assay for determination of NADPH-cytochrome P450 reductase activity.  

PubMed

A 96-well microplate-based HPLC endpoint assay is described for the determination of NADPH-cytochrome P450 reductase (CPR) activity. Novel sampling of NADPH into microplates was optimized. Separation was performed on a Zorbax Eclipse XDB-C?? analytical 4.6?×?150?mm, 5?µm column. To validate the method, recombinant human NADPH-P450 reductase and microsomes with cytochrome P450 CYP1A1 were used. The mobile phase consisted of 80% acetonitrile and 20% water at a flow-rate of 0.8?mL/min. The CPR activity was quantified using NADPH fluorescence at ?(Ex) ?=?340?nm and ?(Em) ?=?450?nm. Enzymatic activity was directly proportional to the decrease in NADPH fluorescence. This analytical process enables a highly sensitive endpoint determination for reductase activity in vitro and monitoring of the consumption of NADPH in enzymatic reactions. The method avoids the use of substrates and of organic solvents that may affect CPR and cytochrome P450 activity. In the reaction, molecular oxygen served as a proton source. The method can substitute spectrophotometric detection methods for its accuracy, high reproducibility (~100%) and sensitivity. The lower limit of detection, shown using the Agilent 1200 aparatus, is in the 250?nmol range. In addition, using this method it is possible to set up reactions in a high-throughput format. PMID:22120726

Erban, Tomas; Poltronieri, Palmiro; Stara, Jitka

2012-09-01

359

Hormonal perturbation as a possible mechanism for the alteration of cytochrome P450 by cyclophosphamide.  

PubMed

The mechanism by which the cytotoxic agent cyclophosphamide (CP) alters cytochrome P450 and some associated enzymes in the male rat has been investigated. CP, administered as a high single dose, decreases the activity of the enzymes CYP2C6, CYP2C11, CYP3A2 and CYP2E1 and NADPH P450 reductase and increases the activity of steroid 5 alpha-reductase. CP appears to exert its effect via an indirect mechanism that reaches its maximal effect 7 days after administration. The decreases in the activity of the enzymes CYP2C11, CYP2E1 and CYP3A2 are accompanied via a corresponding change in the amount of enzyme protein indicating that the alteration in expression of these enzymes occurs via changes in transcription and/or translation. Michaelis-Menten analysis confirmed this conclusion for the enzymes CYP2C11 and CYP3A2. The change in enzyme profile is accompanied by a reduction in the hormones, testosterone, TSH, T4 and T3. The reduction in hormone levels is also maximal 7 days after CP administration. To determine whether CP alters enzyme expression in the male rat via perturbation of hormonal regulation, daily replacement doses of hCG and/or T3 were administered for 7 days after a single dose of CP and hepatic CYP and associated enzyme activities assessed. Results indicated that daily hormone replacement with either hCG and/or T3 prevented the changes in expression of the hormone dependent enzymes NADPH P450 reductase, steroid 5 alpha-reductase and CYP3A2 following a single dose of CP. In contrast for other enzymes including the male sex-specific enzyme CYP2C11, the female predominant enzyme CYP2E1 and the sex-independent enzyme CYP2C6, daily replacement of hCG and/or T3 did not prevent the changes that occur 7 days following CP administration. As rats appeared anorexic and dehydrated and significant weight losses were recorded following CP treatment, blood was collected at the time of killing and subjected to biochemical analysis and a complete blood picture to identify any changes in such parameters that may have contributed to the changes in hormones and/or enzyme expression that occurred. However, significant variation in the treatment groups compared with controls for all parameters was not observed to occur except for an anticipated leukopoenia. We have concluded that CP alters the enzymes NADPH P450 reductase, steroid 5 alpha-reductase and CYP3A2 via perturbation of the regulation of these enzymes by testosterone and/or thyroid hormones. However, while interference with regulation by testosterone and/or thyroid hormones may be part of the mechanism by which CP alters CYP2C11 and CYP2E1, other factors are contributing.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:7598745

McClure, M T; Stupans, I

1995-06-16

360

Cytochrome-P450-Cytochrome-b5 Interaction in a Membrane Environment Changes 15N Chemical Shift Anisotropy Tensors  

PubMed Central

It has been well realized that the dependence of chemical shift anisotropy (CSA) tensors on the amino acid sequence, secondary structure, dynamics and electrostatic interactions can be utilized in the structural and dynamic studies of proteins by NMR spectroscopy. In addition, CSA tensors could also be utilized to measure the structural interactions between proteins in a protein-protein complex. To this end, here we report the experimentally measured backbone amide-15N CSA tensors for a membrane-bound 16.7-kDa full-length rabbit cytochrome-b5 (cytb5), in complexation with a 55.8-kDa microsomal rabbit cytochrome P450 2B4 (cytP4502B4). The 15N-CSAs, determined using the 15N CSA/15N-1H dipolar coupling transverse cross-correlated rates, for free cytb5 are compared with that for the cytb5 bound to cytP4502B4. An overall increase in backbone amide-15N transverse cross-correlated rates for the cytb5 residues in the cytb5-cytP450 complex was observed as compared to the free cytb5 residues. Due to fast spin-spin relaxation (T2) and subsequent broadening of the signals in the complex, we were able to measure amide-15N CSAs only for 48 residues of cytb5 as compared to 84 residues of free cytb5. We observed a change in 15N CSA for most residues of cytb5 in the complex, when compared to free cytb5, suggesting a dynamic interaction between the oppositely charged surfaces of anionic cytb5 and cationic cytP450. The mean values of 15N CSA determined for residues in helical, sheet and turn regions of cytb5 in the complex are ?184.5, ?146.8, and ?146.2 ppm, respectively, with an overall average value of ?165.5 ppm (excluding the values from residues in more flexible termini). The measured CSA value for residues in helical conformation is slightly larger as compared to previously reported values. This may be attributed to the paramagnetic effect from Fe(III) of the heme in cytb5, which is similar to our previously reported values for the free cytb5. PMID:24107224

Pandey, Manoj Kumar; Vivekanandan, Subramanian; Ahuja, Shivani; Huang, Rui; Im, Sang-Choul; Waskell, Lucy; Ramamoorthy, Ayyalusamy

2013-01-01

361

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

SciTech Connect

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

Koch, J.A.; Waxman, D.J. (Harvard Medical School, Boston, MA (USA))

1989-04-18

362

Identification of two Nereis virens (Annelida: Polychaeta) cytochromes P450 and induction by xenobiotics.  

PubMed

Cytochrome P450 (CYP) enzyme catalysed metabolism of xenobiotics such as polycyclic aromatic hydrocarbons (PAHs) are known to occur in polychaetes. Yet specific polychaete CYP enzymes have so far not been identified. Here, we report two partial CYP cDNA sequences, both of 453 bp, characterised from Nereis virens. These are the first CYP sequences reported in annelids. The deduced amino acid sequences both share highest identities to mammalian CYP4F enzymes (61% and 58%), indicating membership of the CYP4 family (accordingly, referred to as CYP41 and CYP42, respectively). The CYP42 gene expression was significantly higher in vehicle controls (corn oil) compared to untreated controls. Clofibrate increased the expression of the CYP42 genes. The induction by clofibrate and corn oil indicates regulatory similarities to vertebrate CYP4 enzymes, which are primarily involved in the metabolism of endogenous compounds such as fatty acids. Crude oil and benz(a)anthracene significantly induced CYP42 gene expression 2.6-fold, and because CYP enzymes often are induced by their own substrates, this induction may indicate involvement of N. virens CYP4 enzymes in the detoxification of environmental contaminants such as PAHs. The present study demonstrates that these N. virens CYP genes are transcriptionally inducible, and suggests that N. virens CYP4 enzymes may be involved in the metabolism of both exogenous and endogenous compounds. PMID:15313451

Rewitz, K F; Kjellerup, C; Jørgensen, A; Petersen, C; Andersen, O

2004-05-01

363

Cytotoxicity of bisphenol A glycidyl methacrylate on cytochrome P450-producing cells.  

PubMed

Of the cytochrome P450 (CYP) family of carcinogen-activating enzymes, CYP3A is the major form found in human livers. The purpose of this study was to investigate the cytotoxic effects of dental resin monomers after being metabolized by CYP3A4 and CYP3A7, using a colony formation assay and a neutral red assay. Specimen wells were plated with transfected cells derived from the Chinese hamster lung at 100 cells well(-1). The experimental group consisted of CYP-producing 3A4-10 and 3A7-40 cells, while the control group consisted of non-CYP-producing CR-119 cells. Bisphenol A (BPA) and bisphenol A glycidyl methacrylate (Bis-GMA) and a positive control (Aflatoxine Bl) were added separately to each well and cultured for 7 days. After cultivation, the number of the colonies was counted and IC50 values were determined. The data were statistically analysed by a Student's t-test. The resultant of IC50 values indicated that the monomers were not metabolically activated by CYP3A4 or CYP3A7 as compared with the control (P < 0.05). We also confirmed that these monomers act neither as activators nor as inhibitors of CYP3A4 and CYP3A7. PMID:12752938

Hikage, S; Nakayama, K; Saito, T; Takahashi, Y; Kamataki, T; Suzuki, S; Hongo, T; Sato, A

2003-05-01

364

Role of rat cytochromes P450 in the oxidation of 17?-ethinylestradiol.  

PubMed

17?-Ethinylestradiol (EE2) is an endocrine disruptor (ED) used as an ingredient of oral contraceptives. Rat hepatic microsomes metabolize EE2 to three products; two of them are hydroxylated EE2 derivatives. Of the hydroxylation reactions, 2-hydroxylation, is the major reaction. Cytochrome P450 (CYP) plays a major role in EE2 hydroxylation. To resolve which rat CYPs are responsible for EE2 oxidation, three approaches were used: induction of specific CYPs, selective inhibition of CYPs, and recombinant rat CYPs. The results demonstrate that EE2 is hydroxylated by several rat CYPs, among them CYP2C6 and 2C11 are most efficient in 2-hydroxy-EE2 formation, while CYP2A and 3A catalyze EE2 hydroxylation to the second product. EE2 is also an inhibitor of CYP2C- and CYP3A-catalyzed hydroxylation of endogenous EDs progesterone and testosterone. EE2 acts as a reversible inhibitor of CYP3A-mediated progesterone 6?-hydroxylation and inactivates CYP3A- and CYP2C-catalyzed testosterone 6?-hydroxylation and progesterone 21- or 16?-hydroxylation, respectively, in a mechanism-based manner. PMID:25461545

Bo?ek-Dohalská, Lucie; Valášková, Petra; Cerná, V?ra; Stiborová, Marie

2014-11-01

365

Geneva Cocktail for Cytochrome P450 and P-Glycoprotein Activity Assessment Using Dried Blood Spots  

PubMed Central

The suitability of the capillary dried blood spot (DBS) sampling method was assessed for simultaneous phenotyping of cytochrome P450 (CYP) enzymes and P-glycoprotein (P-gp) using a cocktail approach. Ten volunteers received an oral cocktail capsule containing low doses of the probes bupropion (CYP2B6), flurbiprofen (CYP2C9), omeprazole (CYP2C19), dextromethorphan (CYP2D6), midazolam (CYP3A), and fexofenadine (P-gp) with coffee/Coke (CYP1A2) on four occasions. They received the cocktail alone (session 1), and with the CYP inhibitors fluvoxamine and voriconazole (session 2) and quinidine (session 3). In session 4, subjects received the cocktail after a 7-day pretreatment with the inducer rifampicin. The concentrations of probes/metabolites were determined in DBS and plasma using a single liquid chromatography–tandem mass spectrometry method. The pharmacokinetic profiles of the drugs were comparable in DBS and plasma. Important modulation of CYP and P-gp activities was observed in the presence of inhibitors and the inducer. Minimally invasive one- and three-point (at 2, 3, and 6?h) DBS-sampling methods were found to reliably reflect CYP and P-gp activities at each session. PMID:24722393

Bosilkovska, M; Samer, C F; Déglon, J; Rebsamen, M; Staub, C; Dayer, P; Walder, B; Desmeules, J A; Daali, Y

2014-01-01

366

Sex difference in the principal cytochrome P-450 for tributyltin metabolism in rats  

SciTech Connect

Tributyltin is metabolized by cytochrome P-450 (CYP) system enzymes, and its metabolic fate may contribute to the toxicity of the chemical. In the present study, it is examined whether sex differences in the metabolism of tributyltin exist in rats. In addition, the in vivo and in vitro metabolism of tributyltin was investigated using rat hepatic CYP systems to confirm the principal CYP involved. A significant sex difference in metabolism occurred both in vivo and in vitro, suggesting that one of the CYPs responsible for tributyltin metabolism in rats is male specific or predominant at least. Eight cDNA-expressed rat CYPs, including typical phenobarbital (PB)-inducible forms and members of the CYP2C subfamily, were tested to determine their capability for tributyltin metabolism. Among the enzymes studied, a statistically significant dealkylation of tributyltin was mediated by CYP2C6 and 2C11. Furthermore, the sex difference in metabolism disappeared in vitro after anti-rat CYP2C11 antibody pretreatment because CYP2C11 is a major male-specific form in rats. These results indicate that CYP2C6 is the principal CYP for tributyltin metabolism in female rats, whereas CYP2C11 as well as 2C6 is involved in tributyltin metabolism in male rats, and it is suggested that CYP2C11 is responsible for the significant sex difference in the metabolism of tributyltin observed in rats.

Ohhira, Shuji [Department of Hygiene, Dokkyo University School of Medicine, Mibu-machi, Tochigi 321-0293 (Japan)]. E-mail: s-ohhira@dokkyomed.ac.jp; Enomoto, Mitsunori [Department of Hygiene, Dokkyo University School of Medicine, Mibu-machi, Tochigi 321-0293 (Japan); Matsui, Hisao [Department of Hygiene, Dokkyo University School of Medicine, Mibu-machi, Tochigi 321-0293 (Japan)

2006-01-15

367

Geneva cocktail for cytochrome p450 and P-glycoprotein activity assessment using dried blood spots.  

PubMed

The suitability of the capillary dried blood spot (DBS) sampling method was assessed for simultaneous phenotyping of cytochrome P450 (CYP) enzymes and P-glycoprotein (P-gp) using a cocktail approach. Ten volunteers received an oral cocktail capsule containing low doses of the probes bupropion (CYP2B6), flurbiprofen (CYP2C9), omeprazole (CYP2C19), dextromethorphan (CYP2D6), midazolam (CYP3A), and fexofenadine (P-gp) with coffee/Coke (CYP1A2) on four occasions. They received the cocktail alone (session 1), and with the CYP inhibitors fluvoxamine and voriconazole (session 2) and quinidine (session 3). In session 4, subjects received the cocktail after a 7-day pretreatment with the inducer rifampicin. The concentrations of probes/metabolites were determined in DBS and plasma using a single liquid chromatography-tandem mass spectrometry method. The pharmacokinetic profiles of the drugs were comparable in DBS and plasma. Important modulation of CYP and P-gp activities was observed in the presence of inhibitors and the inducer. Minimally invasive one- and three-point (at 2, 3, and 6 h) DBS-sampling methods were found to reliably reflect CYP and P-gp activities at each session. PMID:24722393

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