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Sample records for aromatase cytochrome p450

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

    SciTech Connect

    Yoshida, Nobutaka; Osawa, Yoshio )

    1991-03-26

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

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

    PubMed

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

    2007-07-01

    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

  4. Stereochemistry of estrogen biosynthesis by a reconstituted aromatase cytochrome P-450 preparation from human placenta

    SciTech Connect

    Muto, N.; Tan, L.

    1986-04-29

    According to the literature, the multistep reaction mechanism of estrogen biosynthesis proceeds with stereospecific loss of the equatorial 1 beta-, and axial 2 beta-protons. These results were deduced from experiments carried out, either with crude microsomes, or at best with impure enzyme extracts. However, when (1,2- /sup 3/H)4-androstene-3,17-dione of known absolute /sup 3/H-label distribution was incubated with a reconstituted enzyme system, consisting of homogeneous NADPH-cytochrome P-450 reductase and highly purified aromatase, we obtained results that can only be logically explained by a trans- and antiparallel elimination reaction of both the axially oriented C-2 beta-, and C-1-alpha protons. We further demonstrate that the reconstituted enzyme has an aromatase activity optimum at pH 7.2, and an apparent Km of 0.66 microM for NADPH and of 0.24 microM for 4-androstene-3,17-dione. Also, the enzyme requires 3 nmoles of NADPH for each nmole of estrogen that is formed.

  5. Evolutionary comparisons predict that dimerization of human cytochrome P450 aromatase increases its enzymatic activity and efficiency.

    PubMed

    Martin, Lisandra L; Holien, Jessica K; Mizrachi, Dario; Corbin, C Jo; Conley, Alan J; Parker, Michael W; Rodgers, Raymond J

    2015-11-01

    Estrogen is an essential vertebrate hormone synthesized from androgens involving multiple hydroxylations, catalyzed by cytochrome P450 aromatase (P450arom or CYP19) enzymes. Despite their importance, very few comparative studies have been conducted on vertebrate and/or mammalian P450arom enzymes, either structurally or functionally. Here we directly compared the human (h-) and porcine gonadal (p(g)-) P450arom, as p(g)-P450arom has very low catalytic efficiency, with a ten-fold higher affinity (Km) for a substrate (androstenedione) and ten-fold reduction in turnover (Vmax). We recombinantly expressed these proteins and compared their interactions on a membrane using a quartz crystal microbalance (QCM) and also with the electron donor protein cytochrome P450 oxidoreductase (CPR). Changes in frequency and dissipation in the QCM supported the h-P450arom forming a homodimer that agreed with the FRET data, but not p(g)-P450arom. Analysis of the X-ray crystal structure of the h-P450arom suggested a likely site of homo-dimerization and found that certain key interacting residues were not conserved in pg-P450arom. Molecular dynamics simulations provide support for the importance of these residues in homo-dimerization. Here we propose that the lower affinity and higher activity with reduced release of intermediate metabolites by the h-P450arom is as a consequence of its ability to form homodimers. The functional implications of dimerization provide an important mechanistic step in the requirement for efficient aromatization. PMID:26361012

  6. 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...

  7. Cytochromes P450

    PubMed Central

    Werck-Reichhart, Danile; Bak, Sren; Paquette, Suzanne

    2002-01-01

    There are 272 cytochrome P450 genes (including 26 pseudogenes) in the Arabidopsis genome. P450s thus form one of the largest families of proteins in higher plants. This explosion of the P450 family is thought to have occurred via gene duplication and conversion, and to result from the need of sessile plants to adapt to a harsh environment and to protect themselves from pathogens and predators. P450s sometimes share less than 20% identity and catalyze extremely diverse reactions. Their biological functions range from the synthesis of structural macromolecules such as lignin, cutin or suberin, to the synthesis or catabolism of all types of hormone or signaling molecules, the synthesis of pigments and defense compounds, and to the metabolism of xenobiotics. In despite of a huge acceleration in our understanding of plant P450 functions in the recent years, the vast majority of these functions remain completely unknown. PMID:22303202

  8. Distinct cytochrome P450 aromatase isoforms in the common carp (Cyprinus carpio): sexual dimorphism and onset of ontogenic expression.

    PubMed

    Barney, Megan L; Patil, Jawahar G; Gunasekera, Rasanthi M; Carter, Chris G

    2008-05-01

    Cytochrome P450 aromatase (CYP19) is a key enzyme in the steroidogenic pathway that catalyses the conversion of testosterone to estrogen, and therefore is thought to influence gonadal sex differentiation. In an effort to understand the role of this enzyme in ovarian differentiation, we isolated cDNA encoding the two distinct isoforms, ovarian and brain (termed cyp19a and cyp19b, respectively) of adult common carp, Cyprinus carpio. The cloned cDNA for cyp19a had an open reading frame (ORF) of 518 amino acid residues, in contrast to cyp19b with an ORF of 511 amino acids. Sequence and phylogenetic analysis showed that these CYP19 isoforms were orthologous with previously described cyp19a and cyp19b from other teleosts. Quantitative real-time PCR indicated that both isoforms are expressed in adult ovary and brain, with predominant expression of cyp19a in the ovary and cyp19b in the brain. The major aromatase expressing tissue was found to be the brain, with greatest cyp19b expression in the anterior quarter (telencephalon) in both sexes. The gonad showed sexually dimorphic expression of both genes and dimorphic expression of cyp19a was observed in the cerebellum and the liver. Ontogenic expression showed that only the ovarian aromatase transcript is inherited maternally, with lower expression observed through early larval development under warmer rearing conditions. The differential and overlapping expression suggests these two aromatase genes have different roles in reproductive physiology. PMID:18430424

  9. Cytochromes p450.

    PubMed

    Bak, Sren; Beisson, Fred; Bishop, Gerard; Hamberger, Bjrn; Hfer, Ren; Paquette, Suzanne; Werck-Reichhart, Danile

    2011-01-01

    There are 244 cytochrome P450 genes (and 28 pseudogenes) in the Arabidopsis genome. P450s thus form one of the largest gene families in plants. Contrary to what was initially thought, this family diversification results in very limited functional redundancy and seems to mirror the complexity of plant metabolism. P450s sometimes share less than 20% identity and catalyze extremely diverse reactions leading to the precursors of structural macromolecules such as lignin, cutin, suberin and sporopollenin, or are involved in biosynthesis or catabolism of all hormone and signaling molecules, of pigments, odorants, flavors, antioxidants, allelochemicals and defense compounds, and in the metabolism of xenobiotics. The mechanisms of gene duplication and diversification are getting better understood and together with co-expression data provide leads to functional characterization. PMID:22303269

  10. Cytochromes P450

    PubMed Central

    Bak, Søren; Beisson, Fred; Bishop, Gerard; Hamberger, Björn; Höfer, René; Paquette, Suzanne; Werck-Reichhart, Danièle

    2011-01-01

    There are 244 cytochrome P450 genes (and 28 pseudogenes) in the Arabidopsis genome. P450s thus form one of the largest gene families in plants. Contrary to what was initially thought, this family diversification results in very limited functional redundancy and seems to mirror the complexity of plant metabolism. P450s sometimes share less than 20% identity and catalyze extremely diverse reactions leading to the precursors of structural macromolecules such as lignin, cutin, suberin and sporopollenin, or are involved in biosynthesis or catabolism of all hormone and signaling molecules, of pigments, odorants, flavors, antioxidants, allelochemicals and defense compounds, and in the metabolism of xenobiotics. The mechanisms of gene duplication and diversification are getting better understood and together with co-expression data provide leads to functional characterization. PMID:22303269

  11. Estrogen synthetase (aromatase) reconstitution using cytochrome P-450 and NADPH-cytochrome C reductase from human term placental microsomes

    SciTech Connect

    Lobo, J.O.; Bellino, F.L.

    1986-05-01

    The physiologically important cytochrome P-450 enzyme system responsible for the aromatization of androgen to form estrogen has not been systematically studied in reconstitution systems. This study utilized homogeneous NADPH-cytochrome c reductase and partially purified cytochrome P-450. Activity was stimulated several-fold by optimal concentrations of phosphatidylcholine, and demonstrated hyperbolic kinetics with increasing amounts of NADPH-cytochrome c reductase or cytochrome P-450. Aromatization was linear for 30-60 min, then under certain conditions terminated prematurely with up to 80% of the substrate remaining. This termination did not appear to be due to limiting cofactor, production of an inhibitor during aromatization, or lipid peroxidation. An identical effect was observed with placental microsomes when the activity to substrate ratio was decreased to the value in the reconstitution system. Examination of the steroid product and intermediates by 2-D tlc using /sup 3/H-androstenedione (A) as substrate gave 19-OH (2%) and 19-oxoA(3%) and estrone (50%). Radioactivity in areas corresponding to testosterone and estradiol was observed only in trace amounts (< 0.2%). Two as yet unidentified radioactive compounds with R/sub f/'s close to 19-OH and 19-oxo A (not corresponding to 2-OH estrone or 19-nor A) were formed in small amounts (1%).

  12. Lead reduces messenger RNA and protein levels of cytochrome p450 aromatase and estrogen receptor beta in human ovarian granulosa cells.

    PubMed

    Taupeau, Crystel; Poupon, Jol; Treton, Dominique; Brosse, Aurlie; Richard, Yolande; Machelon, Vronique

    2003-06-01

    Exposure to lead causes decreased fertility in women. In the present study, we examined the in vitro effects of lead on cytochrome p450 aromatase (p450 arom) and on estrogen receptor beta (ERbeta), two key proteins for the human ovary. Aromatase is required for the bioconversion of androgen to estradiol; ERbeta mediates estrogen effects in granulosa cells. Granulosa cells were collected from women undergoing in vitro fertilization and then cultured with 10 microM lead acetate. Using atomic absorption spectrometry, we showed that lead accumulated in cells. Aromatase activity as measured by a tritiated water production assay was significantly reduced. Using semiquantitative reverse transcription-polymerase chain reaction and Western blotting procedures, we showed that p450 arom and ERbeta mRNA and protein content were both significantly reduced. Adding 10 microg/ml of cycloheximide, a protein inhibitor, did not eliminate the effects of lead. The present results support the hypothesis that the action of lead on fertility in women may result, in part, from the down-regulation of p450 arom and ERbeta gene transcription in ovarian granulosa. PMID:12606482

  13. Insulin and IGF-I are necessary for FSH-induced cytochrome P450 aromatase but not cytochrome P450 side-chain cleavage gene expression in oestrogenic bovine granulosa cells in vitro.

    PubMed

    Silva, J M; Price, C A

    2002-09-01

    The earliest biochemical indicators of ovarian follicle deviation in cattle include lower oestradiol and free IGF concentrations in subordinate compared with dominant follicles. We determined if decreases in FSH, IGF-I or insulin cause decreased P450 aromatase (P450arom) or P450 cholesterol side-chain cleavage (P450scc) mRNA expression in oestrogenic bovine granulosa cells in vitro. In the first experiment, cells obtained from small follicles (2-5 mm diameter) were cultured in serum-free medium supplemented with physiological concentrations of FSH, IGF-I and insulin for 4 days. A decrease in specific hormone concentration was produced by replacing 70% of spent medium with medium devoid of FSH, insulin, or insulin and IGF-I on day 4 and again on day 5 of culture. Cultures were terminated on day 7. A reduction in FSH concentrations during the last 3 days of culture decreased P450arom and P450scc mRNA levels. A reduction in insulin reduced P450arom but not P450scc mRNA levels, and a reduction of both insulin and IGF-I concentrations further decreased P450arom mRNA levels and decreased P450scc mRNA levels. In a second experiment, cells obtained from small follicles (2-5 mm diameter) were cultured with insulin (100 ng/ml) without FSH for 4 days, and then insulin was withdrawn from the culture and FSH added for a further 3 days. The withdrawal of insulin decreased (P<0.02) oestradiol accumulation and reduced P450arom mRNA to below detectable levels, but did not affect P450scc mRNA levels. The addition of FSH transiently increased oestradiol secretion and P450arom mRNA levels, but P450arom mRNA levels were undetectable at the end of the culture period. The addition of FSH significantly enhanced P450scc mRNA levels and progesterone accumulation. These data demonstrated that a reduction of insulin-like activity reduced aromatase gene expression in bovine follicles without necessarily affecting progesterone synthetic capability, and thus may initiate follicle regression in cattle at the time of follicle divergence. PMID:12208671

  14. Modulation of ovarian cytochrome P450 17 alpha-hydroxylase and cytochrome aromatase messenger ribonucleic acid by prolactin in the domestic turkey.

    PubMed

    Tabibzadeh, C; Rozenboim, I; Silsby, J L; Pitts, G R; Foster, D N; el Halawani, M

    1995-03-01

    The effect of exogenous ovine prolactin (oPRL) on preovulatory follicle P450 17 alpha-hydroxylase (C17) and aromatase (ARO) mRNA abundance was investigated in turkeys. Ovine PRL (124 IU/hen per day) was injected i.m. into four sets (n = 8) of laying turkeys for 2, 4, 8, or 14 days. Vehicle was injected into control hens for 8 days (n = 8). Blood samples were collected and serum was assayed for LH, progesterone (P), testosterone (T), and estradiol (E). Theca layers from the largest (F1) and the third (F3), fifth (F5), and seventh (F7) largest preovulatory follicles and from small white follicles (SWF) were examined for C17 and ARO mRNA contents. The number of atretic follicles increased from 0 (vehicle-injected controls) to 9 (14-day-oPRL-injected hens). Serum E, T, and LH levels decreased, while P levels remained unchanged. There was a transient increase in theca C17 mRNA abundance of 2- and 4-day-oPRL-treated hen follicles. Cytochrome P450 ARO mRNA levels were reduced in SWF and F7 in response to oPRL. Thecal C17 and ARO mRNA content was reduced during follicular maturation in laying hens. ARO mRNA was not detectable in granulosa cells. The progressive decline in C17 and ARO mRNA content associated with follicular maturation as well as the absence of ARO mRNA in granulosa cells is consistent with the secretory activity of P, T, and E in preovulatory follicles. These findings suggest that reduced circulating E may be a consequence of suppressed ARO gene expression whereas the oPRL suppression of T secretion may not be coupled to C17 gene expression. PMID:7756453

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

    PubMed Central

    2014-01-01

    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

  16. Phenotyping cytochromes P450 with monoclonal antibodies.

    PubMed

    Friedman, F K; Park, S S; Fujino, T; Song, B J; Robinson, R C; West, D; Radkowsky, A K; Miller, H; Gelboin, H V

    1984-01-01

    Monoclonal antibodies (MAbs) to cytochrome P-450 isozymes can be used to phenotype tissues for epitope-specific cytochrome P-450 content. MAbs that inhibit specific cytochrome P-450 dependent drug or carcinogen reactions are useful tools for quantitative measurement of the individual or classes of cytochromes P-450 that catalyze these reactions. This method has been applied successfully to animal as well as human tissues. Radioimmunoassays based on MAbs have been developed and provide a rapid and efficient means for detecting cytochromes P-450 independent of functional enzyme activity. In addition, MAbs coupled to a Sepharose support can be used to immunopurify cytochromes P-450 in a procedure that is more rapid and efficient than conventional purification schemes. MAbs add a new dimension to analyses of cytochrome P-450 multiplicity and will find numerous applications in elucidation of the relationship between cytochrome P-450 phenotype and carcinogen or drug metabolism. PMID:11478317

  17. Cytochrome P450-activated prodrugs

    PubMed Central

    Ortiz de Montellano, Paul R

    2013-01-01

    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

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

    USGS Publications Warehouse

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

    2007-01-01

    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.

  19. [Cytochrome P450 inactivation on a cathode].

    PubMed

    Archakov, A I; Kuznetsov, B V; Izotov, M V; Karuzina, I I

    1981-01-01

    It was concluded on the grounds of spectrophotometric analysis of cytochrome P450 electrolysis products that cytochrome P450 unlike other hem-containing proteins is not reduced on the cathode. At the potential -1.5V it is reduced simultaneously with the transformation into cytochrome P420. Inactivation in the electric field on the electrode surface is irreversible. When the potential is above -1.4V no inactivation of cytochrome P450 takes place. PMID:7260142

  20. Cytochromes P450: Roles in Diseases*

    PubMed Central

    Pikuleva, Irina A.; Waterman, Michael R.

    2013-01-01

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

  1. Unusual Cytochrome P450 Enzymes and Reactions*

    PubMed Central

    Guengerich, F. Peter; Munro, Andrew W.

    2013-01-01

    Cytochrome P450 enzymes primarily catalyze mixed-function oxidation reactions, plus some reductions and rearrangements of oxygenated species, e.g. prostaglandins. Most of these reactions can be rationalized in a paradigm involving Compound I, a high-valent iron-oxygen complex (FeO3+), to explain seemingly unusual reactions, including ring couplings, ring expansion and contraction, and fusion of substrates. Most P450s interact with flavoenzymes or iron-sulfur proteins to receive electrons from NAD(P)H. In some cases, P450s are fused to protein partners. Other P450s catalyze non-redox isomerization reactions. A number of permutations on the P450 theme reveal the diversity of cytochrome P450 form and function. PMID:23632016

  2. A world of cytochrome P450s

    PubMed Central

    Nelson, David R.

    2013-01-01

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

  3. Measurement of cytochrome P450 and NADPH-cytochrome P450 reductase.

    PubMed

    Guengerich, F Peter; Martin, Martha V; Sohl, Christal D; Cheng, Qian

    2009-01-01

    Cytochrome P450 (P450) enzymes are important in the metabolism of steroids, vitamins, carcinogens, drugs and other compounds. Two of the commonly used assays in this field are the measurements of total P450 and NADPH-P450 reductase in biological preparations. A detailed protocol is presented for the measurement of P450 by its spectral properties, along with a protocol for measuring NADPH-P450 reductase by its NADPH-cytochrome c reduction activity. Each assay can be completed in 5-10 min. Detailed explanations for the rationale of particular sequences in the protocols are provided, along with potential confounding problems. PMID:19661994

  4. Pharmacophore modeling and in silico screening for new P450 19 (aromatase) inhibitors.

    PubMed

    Schuster, Daniela; Laggner, Christian; Steindl, Theodora M; Palusczak, Anja; Hartmann, Rolf W; Langer, Thierry

    2006-01-01

    Cytochrome P450 19 (P450 19, aromatase) constitutes a successful target for the treatment of breast cancer. This study analyzes chemical features common to P450 19 inhibitors to develop ligand-based, selective pharmacophore models for this enzyme. The HipHop and HypoRefine algorithms implemented in the Catalyst software package were employed to create both common feature and quantitative models. The common feature model for P450 19 includes two ring aromatic features in its core and two hydrogen bond acceptors at the ends. The models were used as database search queries to identify active compounds from the NCI database. PMID:16711749

  5. Cytochrome P450s and molecular epidemiology

    NASA Astrophysics Data System (ADS)

    Gonzalez, Frank J.; Gelboin, Harry V.

    1993-03-01

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

  6. Nerval influences on liver cytochrome P450.

    PubMed

    Klinger, W; Karge, E; Danz, M; Krug, M

    1995-09-01

    In male young adult Wistar rats the influences of nucleus raphe electrocoagulation, spinal cord dissection (cordotomy between C7 and Th1), vagotomy and denervation of liver hilus by phenol on liver cytochrome P450-system (cytochrome P450 concentration, ethylmorphine N-demethylation and ethoxycoumarin O-deethylation activities, hexobarbitone sleeping time) were investigated. In general the influences were small or negligible when compared with sham operated controls, only after vagotomy the depressing effect of sham operation was abolished. In all cases sham operation had a depressing effect until up to five weeks after operation. PMID:8855125

  7. Mapping of genes for cytochromes P-450b, P-450e, P-450g and P-450h in the rat

    SciTech Connect

    Rampersaud, A.; Walz, F.G. Jr.

    1987-05-01

    Inbred ACI, WF and RCS rats having characteristic markers for albino (c), hemoglobin ..beta..-chain (Hbb) and pink-eyed dilution (p) loci on chromosome l and expressing electrophoretic variants for hepatic cytochromes P-450b, P-450e and P-450h and a likely Cis-acting regulatory variant of P-450g were used in genetic mapping studies of these hemoproteins. Phenotypes for these microsomal cytochromes P-450 were analyzed using 2-D electrophoresis and the results of WF x (ACI x WF)fl and RCS x (WF x RCS)fl backcrosses revealed two gene clusters designated the P450-b,e and P450-g,h loci. The interval separating P450-b and P450-e was <1 centiMorgan (cM) and that separating P450-g from P450-h was, 3.7 cM at a 90% confidence level. P450-g,h is not linked with P450-b,e and the other markers tested on chromosome 1. The linkage map P450-b,e--p--c--Hbb on rat chromosome 1 was demonstrated and found to be congruent with Coh(P450-b,e)--p--c--Hbb on mouse chromosome 7. It appears that close genetic linkage, rather than common functional/regulatory properties, typify members of cytochrome P-450 subfamilies.

  8. Aldehyde reduction by cytochrome P450.

    PubMed

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

    2011-05-01

    This protocol describes the procedure for measuring the relative rates of metabolism of the α,β-unsaturated aldehydes 9-anthracene aldehyde (9-AA) and 4-hydroxy-trans-2-nonenal (4-HNE). More specifically, these assays measure the aldehyde reduction reactions of cytochrome P450s (CYPs). They can be performed using liver microsomal or other tissue fractions, spherosome preparations of recombinant CYPs, or recombinant CYPs from other sources. The method for reduction of 9-AA (a model α,β-unsaturated aldehyde) by CYPs was adapted from an assay for 9-anthracene oxidation published by Marini et al. (2003). For 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 HPLC, using an adaptation of the method by Srivastava et al. (2010). For both 9-AA and 4-HNE, the first step involves incubation of the substrate with the CYP in an appropriate medium. This is followed by quantification of metabolites through by spectrofluorometry (9-AA) or HPLC coupled with a radiometric assay (4-HNE). Metabolite identification can be achieved by HPLC GC/MS analysis. Inhibitors of cytochrome P450 can be utilized to show the role of the hemoprotein or other enzymes in these reduction reactions. The reduction of CYPs is not inhibited by either anaerobiosis or inclusion of CO in the gaseous phase of the reaction mixture. These characteristics are similar to those reported for some cytochrome P450-catalyzed azo reduction reactions. PMID:21553396

  9. Novel extrahepatic cytochrome P450s

    SciTech Connect

    Karlgren, Maria . E-mail: Maria.Karlgren@imm.ki.se; Miura, Shin-ichi; Ingelman-Sundberg, Magnus

    2005-09-01

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

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

    PubMed Central

    Stegeman, J J; Kloepper-Sams, P J

    1987-01-01

    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

  11. Domain Motion in Cytochrome P450 Reductase

    PubMed Central

    Ellis, Jacqueline; Gutierrez, Aldo; Barsukov, Igor L.; Huang, Wei-Cheng; Grossmann, J. Günter; Roberts, Gordon C. K.

    2009-01-01

    NADPH-cytochrome P450 reductase (CPR), a diflavin reductase, plays a key role in the mammalian P450 mono-oxygenase system. In its crystal structure, the two flavins are close together, positioned for interflavin electron transfer but not for electron transfer to cytochrome P450. A number of lines of evidence suggest that domain motion is important in the action of the enzyme. We report NMR and small-angle x-ray scattering experiments addressing directly the question of domain organization in human CPR. Comparison of the 1H-15N heteronuclear single quantum correlation spectrum of CPR with that of the isolated FMN domain permitted identification of residues in the FMN domain whose environment differs in the two situations. These include several residues that are solvent-exposed in the CPR crystal structure, indicating the existence of a second conformation in which the FMN domain is involved in a different interdomain interface. Small-angle x-ray scattering experiments showed that oxidized and NADPH-reduced CPRs have different overall shapes. The scattering curve of the reduced enzyme can be adequately explained by the crystal structure, whereas analysis of the data for the oxidized enzyme indicates that it exists as a mixture of approximately equal amounts of two conformations, one consistent with the crystal structure and one a more extended structure consistent with that inferred from the NMR data. The correlation between the effects of adenosine 2′,5′-bisphosphate and NADPH on the scattering curve and their effects on the rate of interflavin electron transfer suggests that this conformational equilibrium is physiologically relevant. PMID:19858215

  12. Flower colour and cytochromes P450

    PubMed Central

    Tanaka, Yoshikazu; Brugliera, Filippa

    2013-01-01

    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

  13. Diversity and evolution of cytochrome P450 monooxygenases in Oomycetes

    PubMed Central

    Sello, Mopeli Marshal; Jafta, Norventia; Nelson, David R; Chen, Wanping; Yu, Jae-Hyuk; Parvez, Mohammad; Kgosiemang, Ipeleng Kopano Rosinah; Monyaki, Richie; Raselemane, Seiso Caiphus; Qhanya, Lehlohonolo Benedict; Mthakathi, Ntsane Trevor; Sitheni Mashele, Samson; Syed, Khajamohiddin

    2015-01-01

    Cytochrome P450 monooxygenases (P450s) are heme-thiolate proteins whose role as drug targets against pathogens, as well as in valuable chemical production and bioremediation, has been explored. In this study we performed comprehensive comparative analysis of P450s in 13 newly explored oomycete pathogens. Three hundred and fifty-six P450s were found in oomycetes. These P450s were grouped into 15 P450 families and 84 P450 subfamilies. Among these, nine P450 families and 31 P450 subfamilies were newly found in oomycetes. Research revealed that oomycetes belonging to different orders contain distinct P450 families and subfamilies in their genomes. Evolutionary analysis and sequence homology data revealed P450 family blooms in oomycetes. Tandem arrangement of a large number of P450s belonging to the same family indicated that P450 family blooming is possibly due to its members’ duplications. A unique combination of amino acid patterns was observed at EXXR and CXG motifs for the P450 families CYP5014, CYP5015 and CYP5017. A novel P450 fusion protein (CYP5619 family) with an N-terminal P450 domain fused to a heme peroxidase/dioxygenase domain was discovered in Saprolegnia declina. Oomycete P450 patterns suggested host influence in shaping their P450 content. This manuscript serves as reference for future P450 annotations in newly explored oomycetes. PMID:26129850

  14. Phenomenon of activation of cytochrome P450 by nonionic detergents.

    PubMed

    Myasoedova, Ksenia N; Arutyunyan, Alexandr M; Magretova, Natalya N

    2006-02-01

    Mechanism of substitution of nonionic detergent Emulgen 913 for phospholipid as an activator of N-demethylase activity of cytochrome P450 form 2B4 (LM(2)) has been studied. It is shown that such an activation takes place at the detergent concentrations below values critical for micelle formation. Under these conditions, Emulgen does not affect the hexameric state of the cytochrome. The stimulating effect proved to be similar in reconstituted monooxygenase systems containing (a) cytochrome P450 2B4 and NADPH-cytochrome P-450 reductase and (b) cytochrome 2B4 and organic hydroperoxides. These results indicate that the activation is due to an effect of the detergent upon P450 2B4 per se rather than upon P450/flavoprotein complex formation. The above conclusion is supported by the sedimentation data and measurement of the CD spectra of cytochrome P450 2B4 at 380-450 nm. PMID:16779669

  15. Purification of cytochrome P450 from filamentous fungus Rhyzopus nigricans.

    PubMed

    Makovec, T; Breskvar, K

    2000-01-01

    It has been reported in our previous studies that steroid hydroxylation system of Rhizopus nigricans involves cytochrome P450 and NADPH-cytochrome P450 reductase in the electron transport system. Both enzymes are membrane bound and are located in the microsomal preparations of progesterone induced fungal mycelia. In order to identify and characterize the cytochrome P450 component of fungal monoxygenase system, microsomal proteins from induced mycelia were subjected to HIGH Q anion exchange and MONO P (FPLC) anion exchange chromatography. Four fractions containing cytochrome P450 have been resolved on MONO P column. They exhibit CO difference spectra and type II difference spectra with ketoconazole. PMID:10653160

  16. Endocannabinoid metabolism by cytochrome P450 monooxygenases.

    PubMed

    Zelasko, Susan; Arnold, William R; Das, Aditi

    2015-01-01

    The endogenous cannabinoid system was first uncovered following studies of the recreational drug Cannabis sativa. It is now recognized as a vital network of signaling pathways that regulate several physiological processes. Following the initial discovery of the cannabinoid receptors 1 (CB1) and 2 (CB2), activated by Cannabis-derived analogs, many endogenous fatty acids termed "endocannabinoids" are now known to be partial agonists of the CB receptors. At present, the most thoroughly studied endocannabinoid signaling molecules are anandamide (AEA) and 2-arachidonylglycerol (2-AG), which are both derived from arachidonic acid. Both AEA and 2-AG are also substrates for the eicosanoid-synthesizing pathways, namely, certain cyclooxygenase (COX), lipoxygenase (LOX), and cytochrome P450 (CYP) enzymes. In the past, research in the endocannabinoid field focused on the interaction of AEA and 2-AG with the COX and LOX enzymes, but accumulating evidence also points to the involvement of CYPs in modulating endocannabinoid signaling. The focus of this review is to explore the current understanding of CYP-mediated metabolism of endocannabinoids. PMID:25461979

  17. Engineering Cytochrome P450 Biocatalysts for Biotechnology, Medicine, and Bioremediation

    PubMed Central

    Kumar, Santosh

    2009-01-01

    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

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

    SciTech Connect

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

    1986-05-01

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

  19. Substrate Binding to Cytochromes P450

    PubMed Central

    Isin, Emre M.; Guengerich, F. Peter

    2016-01-01

    P450s have attracted tremendous attention due not only to their involvement in the metabolism of drug molecules and endogenous substrates but also the unusual nature of the reaction they catalyze, namely the oxidation of unactivated C-H bonds. The binding of substrates to P450s, which is usually viewed as the first step in the catalytic cycle, has been studied extensively via a variety of biochemical and biophysical approaches. These studies were directed towards answering different questions related to P450s including, mechanism of oxidation, substrate properties, unusual substrate oxidation kinetics, function, and active site features. Some of the substrate binding studies extending over a period of more than forty years of dedicated work has been summarized in this review and categorized by the techniques employed in the binding studies. PMID:18622598

  20. Theca cell cytochrome P450 17-hydroxylase and aromatase messenger ribonucleic acid abundance and serum steroid levels during follicular atresia associated with incubation behavior in the domestic turkey hen.

    PubMed

    Tabibzadeh, C; Silsby, J L; Rozenboim, I; Pitts, G R; Foster, D N; el Halawani, M E

    1994-10-01

    This study was designed to examine changes in cytochrome P450 17 alpha-hydroxylase (C17) and aromatase (ARO) mRNA contents in the theca layer of preovulatory follicles (POF) as turkey hens transit from egg laying to incubation. Hens were grouped into the following categories: 1) laying hens--laid one egg per day and nested 1-2 times per day; 2) transitional hens--laid one egg per day and nested > 4 times per day; and 3) Day 1, Day 3, and Day 5 incubating hens--laid no eggs for 2, 4, or 6 days, respectively, and nested > 4 times per day. Small white follicles (SWF) and the theca layer from the largest (F1) and the third (F3), fifth (F5), and seventh (F7) largest POF were dispersed and challenged with testosterone (T) for 5 h. Relative levels of C17 and ARO mRNA were examined from the theca layers of F1, F3, F5, F7, and SWF. The number of atretic follicles increased from 0 (layers) to 8 (Day 5 incubating hens). Serum LH, progesterone (P), and estradiol (E), but not T, declined on Day 1 of incubation. Basal levels of P, T, and E from theca and SWF cells declined in incubating hens. Both basal and T-stimulated theca and SWF production of E decreased in incubating hens. C17 and ARO mRNA declined in SWF, F7, and F5 during follicular atresia. It is suggested that reduced gene expression of ovarian steroidogenic enzymes may be a partial determinant of reduced circulating sex steroid levels in incubating hens.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7819456

  1. 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

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

    2011-01-01

    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

  2. Genetics Home Reference: Cytochrome P450 oxidoreductase deficiency

    MedlinePLUS

    ... and Families Resources for Health Professionals What glossary definitions help with understanding cytochrome P450 oxidoreductase deficiency? acne ; ... recessive ; reproduction ; stress ; syndrome ; testosterone You may find definitions for these and many other terms in the ...

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

    PubMed

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

    2013-02-19

    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

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

    PubMed Central

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

    2013-01-01

    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

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

    ClinicalTrials.gov

    2015-12-09

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

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

    PubMed Central

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

    2011-01-01

    The metabolism of ?,?-unsaturated aldehydes, e.g. 4-hydroxynonenal, involves oxidation to carboxylic acids, reduction to alcohols, and glutathionylation to eventually form mercapturide conjugates. Recently we demonstrated that P450s can oxidize aldehydes to carboxylic acids, a reaction previously thought to involve aldehyde dehydrogenase. When recombinant cytochrome P450 3A4 was incubated with 4-hydroxynonenal, O2, and NADPH, several products were produced, including 1,4-dihydroxynonene (DHN), 4-hydroxy-2-nonenoic acid (HNA), and an unknown metabolite. Several P450s catalyzed the reduction reaction in the order (human) P450 2B6 ? P450 3A4 > P450 1A2 > P450 2J2 > (mouse) P450 2c29. Other P450s did not catalyze the reduction reaction (human P450 2E1 & rabbit P450 2B4). Metabolism by isolated rat hepatocytes showed that HNA formation was inhibited by cyanamide, while DHN formation was not affected. Troleandomycin increased HNA production 1.6-fold while inhibiting DHN formation, suggesting that P450 3A11 is a major enzyme involved in rat hepatic clearance of 4-HNE. A fluorescent assay was developed using 9-anthracenealdehyde to measure both reactions. Feeding mice diet containing t-butylated hydroxyanisole increased the level of both activities with hepatic microsomal fractions, but not proportionally. Miconazole (0.5 mM) was a potent inhibitor of these microsomal reduction reactions, while phenytoin and ?-naphthoflavone (both at 0.5 mM) were partial inhibitors, suggesting the role of multiple P450 enzymes. The oxidative metabolism of these aldehydes was inhibited >90% in an Ar or CO atmosphere, while the reductive reactions were not greatly affected. These results suggest that P450s are significant catalysts of reduction of ?,?-unsaturated aldehydes in liver. PMID:21766881

  7. Dynamics of carbon monoxide binding with cytochromes P-450.

    PubMed

    Ttreau, C; Di Primo, C; Lange, R; Tourbez, H; Lavalette, D

    1997-08-19

    The dynamics of CO rebinding with cytochromes P-450cam, P-450scc, and P-450LM2 after laser flash photolysis have been investigated from 293 to 77 K, and the distribution functions of the rate parameters P(k) and of the activation enthalpy P(H) were determined using the maximum entropy method. In a fluid solvent, geminate rebinding is nonexponential, presumably because of a spectral shift induced by protein relaxation on the same time scale. Substrate binding increases the yield of the bimolecular process and decreases the bimolecular rate by 1 or 2 orders of magnitude. The amplitude of these effects seems to correlate with substrate specificity. In a rigid environment at low temperature, cytochromes P-450 exhibit a bimodal distribution of activation enthalpy; P(H) consists of two distinct bands which are in a thermal equilibrium even at 77 K. The results lead to a scheme in which a common structural perturbation splits the conformational substates of cytochromes P-450 into pairs of "doublet" substates with different dynamic properties. The hierarchy of conformational substates of cytochromes P-450 thus contrasts with that of oxygen-binding hemoproteins such as myoglobin. PMID:9254625

  8. Cytochrome P450-mediated metabolism of vitamin D.

    PubMed

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

    2014-01-01

    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

  9. Homotropic cooperativity of monomeric cytochrome P450 3A4

    SciTech Connect

    Baas, Bradley J.; Denisov, Ilia G.; Sligar, Stephen G.

    2010-11-16

    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.

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

    PubMed Central

    Im, Sang-Choul; Waskell, Lucy

    2010-01-01

    1 Cytochrome P450 2B4 is a microsomal protein with a multi-step reaction cycle similar to that observed in the majority of other cytochromes P450. The cytochrome P450 2B4-substrate complex is reduced from the ferric to the ferrous form by cytochrome P450 reductase. After binding oxygen, the oxyferrous protein accepts a second electron which is provided by either cytochrome P450 reductase or cytochrome b5. In both instances, product formation occurs. When the second electron is donated by cytochrome b5, catalysis (product formation) is ? 10 to 100-fold faster than in the presence of cytochrome P450 reductase. This allows less time for side product formation (hydrogen peroxide and superoxide) and improves by ? 15% the coupling of NADPH consumption to product formation. Cytochrome b5 has also been shown to compete with cytochrome P450 reductase for a binding site on the proximal surface of cytochrome P450 2B4. These two different effects of cytochrome b5 on cytochrome P450 2B4 reactivity can explain how cytochrome b5 is able to stimulate, inhibit, or have no effect on cytochrome P450 2B4 activity. At low molar ratios (<1) of cytochrome b5 to cytochrome P450 reductase, the more rapid catalysis results in enhanced substrate metabolism. In contrast, at high molar ratios (>1) of cytochome b5 to cytochrome P450 reductase, cytochrome b5 inhibits activity by binding to the proximal surface of cytochrome P450 and preventing the reductase from reducing ferric cytochrome P450 to the ferrous protein, thereby aborting the catalytic reaction cycle. When the stimulatory and inhibitory effects of cytochrome b5 are equal, it will appear to have no effect on the enzymatic activity. It is hypothesized that cytochrome b5 stimulates catalysis by causing a conformational change in the active site, which allows the active oxidizing oxyferryl species of cytochrome P450 to be formed more rapidly than in the presence of reductase. PMID:21055385

  11. Cytochrome P450s: creating novel ligand sets.

    PubMed

    Seward, Harriet E; Girvan, Hazel M; Munro, Andrew W

    2005-11-01

    Cytochromes P450 are a ubiquitous group of hemoproteins that perform vital cellular reactions in all lifeforms. Until recently, it was thought that P450s contained non-covalently bound heme. However, it was established that covalent linkage of the heme macrocycle occurs naturally in one major group of the P450 superfamily. The reaction involves heme linkage to a conserved amino acid and is autocatalytic, occurring as a consequence of P450 turnover. This finding presents opportunities to engineer biotechnologically important P450s to covalently link the heme, in order to stabilize cofactor binding and to enhance operational stability of these P450s. This opportunity has been taken in studies on two important bacterial P450s and has produced variants with intriguingly different properties. In this article we survey the developments in the field, the relationships with heme macrocycle ligations in other proteins and the important impact that recent studies of heme ligation have had on our general appreciation of P450 structure and mechanism. PMID:16234920

  12. 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...

  13. The cytochrome P450 (CYP) gene superfamily in Daphnia pulex

    PubMed Central

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

    2009-01-01

    Background Cytochrome P450s (CYPs) in animals fall into two categories: those that synthesize or metabolize endogenous molecules and those that interact with exogenous chemicals from the diet or the environment. The latter form a critical component of detoxification systems. Results Data mining and manual curation of the Daphnia pulex genome identified 75 functional CYP genes, and three CYP pseudogenes. These CYPs belong to 4 clans, 13 families, and 19 subfamilies. The CYP 2, 3, 4, and mitochondrial clans are the same four clans found in other sequenced protostome genomes. Comparison of the CYPs from D. pulex to the CYPs from insects, vertebrates and sea anemone (Nematostella vectensis) show that the CYP2 clan, and to a lesser degree, the CYP4 clan has expanded in Daphnia pulex, whereas the CYP3 clan has expanded in insects. However, the expansion of the Daphnia CYP2 clan is not as great as the expansion observed in deuterostomes and the nematode C. elegans. Mapping of CYP tandem repeat regions demonstrated the unusual expansion of the CYP370 family of the CYP2 clan. The CYP370s are similar to the CYP15s and CYP303s that occur as solo genes in insects, but the CYP370s constitute ~20% of all the CYP genes in Daphnia pulex. Lastly, our phylogenetic comparisons provide new insights into the potential origins of otherwise mysterious CYPs such as CYP46 and CYP19 (aromatase). Conclusion Overall, the cladoceran, D. pulex has a wide range of CYPs with the same clans as insects and nematodes, but with distinct changes in the size and composition of each clan. PMID:19383150

  14. Asymmetric distribution of cytochrome P-450 and NADPH–cytochrome P-450 (cytochrome c) reductase in vesicles from smooth endoplasmic reticulum of rat liver

    PubMed Central

    Cooper, Michael B.; Craft, John A.; Estall, Margaret R.; Rabin, Brian R.

    1980-01-01

    1. The topography of cytochrome P-450 in vesicles from smooth endoplasmic reticulum of rat liver has been examined. Approx. 50% of the cytochrome is directly accessible to the action of trypsin in intact vesicles whereas the remainder is inaccessible and partitioned between luminal-facing or phospholipid-embedded loci. Analysis by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis reveals three major species of the cytochrome. Of these, the variant with a mol.wt. of 52000 is induced by phenobarbitone and this species is susceptible to trypsin. 2. After trypsin treatment of smooth membrane, some NADPH–cytochrome P-450 (cytochrome c) reductase activity remains and this remaining activity is enhanced by treatment with 0.05% deoxycholate, which renders the membranes permeable to macromolecules. In non-trypsin-treated control membranes the reductase activity is increased to a similar extent. These observations suggest an asymmetric distribution of NADPH–cytochrome P-450 (cytochrome c) reductase in the membrane. 3. As compared with dithionite, NADPH reduces only 44% of the cytochrome P-450 present in intact membranes. After tryptic digestion, none of the remaining cytochrome P-450 is reducible by NADPH. 4. In the presence of both a superoxide-generating system (xanthine plus xanthine oxidase) and NADPH, all the cytochrome P-450 in intact membrane (as judged by dithionite reducibility) is reduced. The cytochrome P-450 remaining after trypsin treatment of smooth vesicles cannot be reduced by this method. 5. The superoxide-dependent reduction of cytochrome P-450 is prevented by treatment of the membranes with mersalyl, which inhibits NADPH–cytochrome P-450 (cytochrome c) reductase. Thus the effect of superoxide may involve NADPH–cytochrome P-450 reductase and cytosolically orientated membrane factor(s). ImagesFig. 3. PMID:6258576

  15. Effect of cannabidiol on cytochrome P-450 isozymes.

    PubMed

    Bornheim, L M; Correia, M A

    1989-09-01

    Cannabidiol (CBD) has been shown to inhibit mouse hepatic mixed-function oxidations of several drugs after acute treatment, whereas repetitive treatment resulted in the restoration of drug-metabolizing capabilities. We have found that acute CBD treatment modestly decreased cytochrome P-450 content but markedly decreased hexobarbital hydroxylase, erythromycin N-demethylase, and 6 beta-testosterone hydroxylase activities. Repetitive CBD treatment, on the other hand, resulted in the restoration of cytochrome P-450 content as well as hexobarbital hydroxylase and erythromycin N-demethylase activities. However, after such repeated treatments a fresh dose of CBD can once again inactivate erythromycin N-demethylase activity but not hexobarbital hydroxylase activity. The resistance of hexobarbital hydroxylase to re-inactivation by CBD was paralleled by stimulation of pentoxyresorufin O-dealkylase activity and the appearance of a 50 kD protein that was immunoreactive to an antibody raised against rat hepatic cytochrome P-450b. CBD metabolism in vitro by microsomes prepared from such CBD-"induced" animals, resulted in a pattern of metabolites different from that observed from comparable incubations with liver microsomes from either untreated or phenobarbital-treated animals. Thus, it appears that CBD initially inactivates at least one cytochrome P-450 isozyme, but after repetitive CBD treatment, an isozyme is induced that is resistant to further re-inactivation by CBD. This isozyme appears to be immunochemically similar to, but somewhat functionally distinct from, the isozyme induced by phenobarbital treatment in mice. PMID:2550010

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

    SciTech Connect

    Voorman, R.

    1987-01-01

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

  17. Unusual properties of the cytochrome P450 superfamily

    PubMed Central

    Lamb, David C.; Waterman, Michael R.

    2013-01-01

    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

  18. Unusual properties of the cytochrome P450 superfamily.

    PubMed

    Lamb, David C; Waterman, Michael R

    2013-02-19

    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

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

    PubMed

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

    2016-03-01

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

  20. Molecular cloning and characterization of brain and ovarian cytochrome P450 aromatase genes in the catfish Heteropneustes fossilis: Sex, tissue and seasonal variation in, and effects of gonadotropin on gene expression.

    PubMed

    Chaube, Radha; Rawat, Arpana; Joy, Keerrikkattil P

    2015-09-15

    Cytochrome P450 aromatase (Cyp19arom) is the rate-limiting enzyme controlling estrogen biosynthesis, coded by Cyp19a1 in most gnathostomes. Most teleosts have two forms expressed differentially in ovary (cyp19a1a) and neural tissue (cyp19a1b). In this study, full length cDNAs of 2006 bp and 1913 bp with ORFs of 1575 bp and 1488 bp were isolated from the brain and ovary, respectively, of the catfish Heteropneustes fossilis, an air-breathing species with high aquaculture potential. The ORFs encode predicted proteins of 495 and 524 amino acid residues, respectively. The proteins show 62% identity with each other and cluster in two distinct clades (the brain type and ovary type) in the teleost taxon, separated from the tetrapod type. In the in situ localization study, both cyp19a1a and cyp19a1b transcripts were localized in the brain but the signal intensity was higher for the brain type paralog. The transcript signals were observed in the radial glial cells and in neuronal populations of the dorso-lateral region of the telencephalon, pre-tectum, hypothalamus and medulla oblongata. In the ovary, both paralogs were expressed in the follicular layer with a high signal intensity of the ovarian type (cyp19a1a). The differential expression of the gene paralogs was evident from qPCR analysis. Cyp19a1b has relatively a high abundance in the female brain, followed by other peripheral tissues (gonads, liver, gill, kidney and muscle). On the other hand, cyp19a1a has relatively a high transcript abundance in the ovary and female brain, followed by the testis and male brain, and female liver and muscle. The expression was low in male liver and muscle, and the lowest in the gill and kidney. The expression of the two paralogs exhibit brain regional differences; both types have relatively a high transcript abundance in telencephalon-preoptic area with the cyp19a1b expression higher in females than males. In hypothalamus, the expression of both types is higher in males than females. In medulla, the expression of the cyp19a1b is higher than cyp19a1a, and the transcript abundance of the ovarian type is higher in females than males. The expression of the gene paralogs elicits significant seasonal variations in the ovary and brain. In both tissues, the expression increases from the resting to preparatory phases, and decreases through the prespawning phase to low levels in spawning phase. In vivo and/or in vitro treatments with human chorionic gonadotropin (hCG) stimulated the expression of the gene paralogs in the brain and ovary, time-dependently. In conclusion, both paralogs have an overlapping distribution at different levels of the brain-pituitary-gonad axis and may function as a single functional unit as far as the estrogen synthesis is concerned. PMID:26144886

  1. Induction of microsomal NADPH-cytochrome P-450 reductase and cytochrome P-450IVA1 (P-450LA omega) by dehydroepiandrosterone in rats: a possible peroxisomal proliferator.

    PubMed

    Wu, H Q; Masset-Brown, J; Tweedie, D J; Milewich, L; Frenkel, R A; Martin-Wixtrom, C; Estabrook, R W; Prough, R A

    1989-05-01

    Dehydroepiandrosterone (DHEA) is a naturally occurring C19-steroid that is found in the peripheral circulation of mammals, including humans. The feeding of DHEA to rodents has been shown to inhibit chemical carcinogenesis in colon, liver, and lung. Therefore, the effect of DHEA on hepatic enzyme activities that are associated with carcinogen metabolism was assessed. Microsomal NADPH-cytochrome P-450 reductase activity and the content of cytochrome b5 were induced 1.8- and 1.4-fold, respectively, upon feeding male Sprague-Dawley rats a synthetic diet containing 0.45% DHEA (w/w). No significant changes in total content of microsomal cytochrome P-450 or the activities of microsomal NADH-cytochrome b5 reductase and cytosolic or microsomal NAD(P)H-quinone oxidoreductase were noted at day 7 of feeding. Cytosolic glutathione S-transferase activity was decreased to 68% of control activity. Administration of DHEA p.o. or by i.p. injection for 5 days led to the same extent of induction of NADPH-cytochrome P-450 reductase activity. Maximal induction of this flavoprotein reductase was noted between days 3 and 4 of feeding or at a dose of 80-120 mg/kg i.p. A small but statistically significant increase in total microsomal cytochrome P-450 was observed after DHEA administration i.p. Rats fed DHEA had a slower growth rate compared with rats fed control diet, whereas rats treated with DHEA i.p. had growth rates identical to those of controls. The liver weights of rats given DHEA by p.o. or i.p. routes were increased significantly compared to those of control rats. Pair feeding of rats with DHA-containing or control diets served to demonstrate that the levels of induction of hepatic microsomal NADPH-cytochrome P-450 reductase and at least one form of cytochrome P450 (P-450IVA1) were the same as those seen in livers of rats fed DHEA ad libitum. This finding suggested that the induction of the flavoprotein and at least one form of the cytochrome was not due to caloric restriction. The increase in NADPH-cytochrome P-450 reductase content of liver microsomes prepared from rats either fed or treated i.p. with DHEA was also observed by Western blotting techniques. DHEA did not appear to induce any of the major forms of rat liver microsomal cytochrome P-450 that are normally increased by either phenobarbital, beta-naphthoflavone, or dexamethasone pretreatment of rats in vivo. However, the measurement of androstenedione and testosterone metabolism in vitro showed pronounced decreases in the 16 alpha-hydroxylase activities of liver microsomes following DHEA feeding.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:2523237

  2. Precocious development of cytochrome P-450 in neonatal rat liver after glucocorticoid treatment.

    PubMed Central

    Leakey, J E; Fouts, J R

    1979-01-01

    Intraperitoneal injection of neonatal rats with glucocorticoid hormones causes precocious development of hepatic cytochrome P-450. Glucagon injection fails to stimulate this cytochrome P-450 development. Adult liver cytochrome P-450 is less responsive to glucocorticoid stimulation than is that of neonatal rat liver. Adrenalectomy of prematurely delivered neonatal animals prevents the early postnatal development of cytochrome P-450. Glucocorticoids failed to increase cytochrome P-450 concentrations in foetal rat liver. These findings imply that, although glucocorticoids are mandatory regulatory factors controlling cytochrome P-450 development, they are not themselves the 'trigger' initiating onset of that development. PMID:40549

  3. Spectroscopic features of cytochrome P450 reaction intermediates.

    PubMed

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

    2011-03-01

    Cytochromes P450 constitute a broad class of heme monooxygenase enzymes with more than 11,500 isozymes which have been identified in organisms from all biological kingdoms [1]. These enzymes are responsible for catalyzing dozens chemical oxidative transformations such as hydroxylation, epoxidation, N-demethylation, etc., with very broad range of substrates [2,3]. Historically these enzymes received their name from 'pigment 450' due to the unusual position of the Soret band in UV-vis absorption spectra of the reduced CO-saturated state [4,5]. Despite detailed biochemical characterization of many isozymes, as well as later discoveries of other 'P450-like heme enzymes' such as nitric oxide synthase and chloroperoxidase, the phenomenological term 'cytochrome P450' is still commonly used as indicating an essential spectroscopic feature of the functionally active protein which is now known to be due to the presence of a thiolate ligand to the heme iron [6]. Heme proteins with an imidazole ligand such as myoglobin and hemoglobin as well as an inactive form of P450 are characterized by Soret maxima at 420nm [7]. This historical perspective highlights the importance of spectroscopic methods for biochemical studies in general, and especially for heme enzymes, where the presence of the heme iron and porphyrin macrocycle provides rich variety of specific spectroscopic markers available for monitoring chemical transformations and transitions between active intermediates of catalytic cycle. PMID:21167809

  4. Spectroscopic features of cytochrome P450 reaction intermediates

    PubMed Central

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

    2010-01-01

    Preface Cytochromes P450 constitute a broad class of heme monooxygenase enzymes with more than 11,500 isozymes which have been identified in organisms from all biological kingdoms [1]. These enzymes are responsible for catalyzing dozens chemical oxidative transformations such as hydroxylation, epoxidation, N-demethylation, etc., with very broad range of substrates [2-3]. Historically these enzymes received their name from pigment 450 due to the unusual position of the Soret band in UV-Vis absorption spectra of the reduced CO-saturated state [4-5]. Despite detailed biochemical characterization of many isozymes, as well as later discoveries of other P450-like heme enzymes such as nitric oxide synthase and chloroperoxidase, the phenomenological term cytochrome P450 is still commonly used as indicating an essential spectroscopic feature of the functionally active protein which is now known to be due to the presence of a thiolate ligand to the heme iron [6]. Heme proteins with an imidazole ligand such as myoglobin and hemoglobin as well as an inactive form of P450 are characterized by Soret maxima at 420 nm [7]. This historical perspective highlights the importance of spectroscopic methods for biochemical studies in general, and especially for heme enzymes, where the presence of the heme iron and porphyrin macrocycle provides rich variety of specific spectroscopic markers available for monitoring chemical transformations and transitions between active intermediates of catalytic cycle. PMID:21167809

  5. Inhibition of cytochrome p450 enzymes by quinones and anthraquinones.

    PubMed

    Sridhar, Jayalakshmi; Liu, Jiawang; Foroozesh, Maryam; Klein Stevens, Cheryl L

    2012-02-20

    In silico docking studies and quantitative structure-activity relationship analysis of a number of in-house cytochrome P450 inhibitors have revealed important structural characteristics that are required for a molecule to function as a good inhibitor of P450 enzymes 1A1, 1A2, 2B1, and/or 2A6. These insights were incorporated into the design of pharmacophores used for a 2D search of the Chinese medicine database. Emodin, a natural anthraquinone isolated from Rheum emodi and known to be metabolized by cytochrome P450 enzymes, was one of the hits and was used as the lead compound. Emodin was found to inhibit P450s 1A1, 1A2, and 2B1 with IC(50) values of 12.25, 3.73, and 14.89 ?M, respectively. On the basis of the emodin molecular structure, further similarity searches of the PubChem and ZINC chemical databases were conducted resulting in the identification of 12 emodin analogues for testing against P450s 1A1-, 1A2-, 2B1-, and 2A6-dependent activities. 1-Amino-4-chloro-2-methylanthracene-9,10-dione (compound 1) showed the best inhibition potency for P450 1A1 with an IC(50) value of 0.40 ?M. 1-Amino-4-chloro-2-methylanthracene-9,10-dione (compound 1) and 1-amino-4-hydroxyanthracene-9,10-dione (compound 2) both inhibited P450 1A2 with the same IC(50) value of 0.53 ?M. In addition, compound 1 acted as a mechanism-based inhibitor of cytochrome P450s 1A1 and 1A2 with K(I) and K(inactivation) values of 5.38 ?M and 1.57 min(-1) for P450 1A1 and 0.50 ?M and 0.08 min(-1) for P450 1A2. 2,6-Di-tert-butyl-5-hydroxynaphthalene-1,4-dione (compound 8) directly inhibited P450 2B1 with good selectivity and inhibition potency (IC(50) = 5.66 ?M). Docking studies using the 3D structures of the enzymes were carried out on all of the compounds. The binding modes of these compounds revealed the structural characteristics responsible for their potency and selectivity. Compound 1, which is structurally similar to compound 2 with the presence of an amino group at position 1, showed a difference in the mechanism of inhibition toward P450s 1A1 and 1A2. The mechanism-based inhibition seen for compound 1 may be attributed to the presence of the methyl group at the 2-position, in close proximity to the amino group. Compound 2, which is otherwise similar, lacks that methyl moiety and did not show mechanism-based inhibition. PMID:22185593

  6. Computer-aided design of aptamers for cytochrome p450.

    PubMed

    Shcherbinin, Dmitrii S; Gnedenko, Oksana V; Khmeleva, Svetlana A; Usanov, Sergey A; Gilep, Andrei A; Yantsevich, Aliaksei V; Shkel, Tatsiana V; Yushkevich, Ivan V; Radko, Sergey P; Ivanov, Alexis S; Veselovsky, Alexander V; Archakov, Alexander I

    2015-08-01

    Aptamers are short single-stranded DNA or RNA oligonucleotides that can bind to their targets with high affinity and specificity. Usually, they are experimentally selected using the SELEX method. Here, we describe an approach toward the in silico selection of aptamers for proteins. This approach involves three steps: finding a potential binding site, designing the recognition and structural parts of the aptamers and evaluating the experimental affinity. Using this approach, a set of 15-mer aptamers for cytochrome P450 51A1 was designed using docking and molecular dynamics simulation. An experimental evaluation of the synthesized aptamers using SPR biosensor showed that these aptamers interact with cytochrome P450 51A1 with Kd values in the range of 10(-6)-10(-7) M. PMID:26166326

  7. 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...

  8. 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 λgt11 library. Isolation of the gene has been identified on the basis of its inducibility and partial DNA sequence. Transcripts of this gene were i...

  9. 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...

  10. Epoxidation Activities of Human Cytochromes P450c17 and P450c21

    PubMed Central

    2015-01-01

    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 16α-hydroxylates progesterone, might catalyze the formation of the 16α,17-epoxide from 16,17-dehydroprogesterone (pregna-4,16-diene-3,20-dione). CYP17A1 catalyzed the novel 16α,17-epoxidation and the ordinarily minor 21-hydroxylation of 16,17-dehydroprogesterone in a 1:1 ratio. CYP17A1 mutation A105L, which has reduced progesterone 16α-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 16α-hydroxylase activity, likewise afforded the 21-hydroxylated product and slightly more epoxide. CYP17A1 wild-type and mutation A105L do not 21- or 16α-hydroxylate pregnenolone, but the enzymes 21-hydroxylated and 16α,17-epoxidized 16,17-dehydropregnenolone (pregna-5,16-diene-3β-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 16α-hydroxylase activity of the enzymes. PMID:25386927

  11. Cytochrome b5 increases the rate of product formation by cytochrome P450 2B4 and competes with cytochrome P450 reductase for a binding site on cytochrome P450 2B4.

    PubMed

    Zhang, Haoming; Im, Sang-Choul; Waskell, Lucy

    2007-10-12

    The kinetics of product formation by cytochrome P450 2B4 were compared in the presence of cytochrome b(5) (cyt b(5)) and NADPH-cyt P450 reductase (CPR) under conditions in which cytochrome P450 (cyt P450) underwent a single catalytic cycle with two substrates, benzphetamine and cyclohexane. At a cyt P450:cyt b(5) molar ratio of 1:1 under single turnover conditions, cyt P450 2B4 catalyzes the oxidation of the substrates, benzphetamine and cyclohexane, with rate constants of 18 +/- 2 and 29 +/- 4.5 s(-1), respectively. Approximately 500 pmol of norbenzphetamine and 58 pmol of cyclohexanol were formed per nmol of cyt P450. In marked contrast, at a cyt P450:CPR molar ratio of 1:1, cyt P450 2B4 catalyzes the oxidation of benzphetamine congruent with100-fold (k = 0.15 +/- 0.05 s(-1)) and cyclohexane congruent with10-fold (k = 2.5 +/- 0.35 s(-1)) more slowly. Four hundred picomoles of norbenzphetamine and 21 pmol of cyclohexanol were formed per nmol of cyt P450. In the presence of equimolar concentrations of cyt P450, cyt b(5), and CPR, product formation is biphasic and occurs with fast and slow rate constants characteristic of catalysis by cyt b(5) and CPR. Increasing the concentration of cyt b(5) enhanced the amount of product formed by cyt b(5) while decreasing the amount of product generated by CPR. Under steady-state conditions at all cyt b(5):cyt P450 molar ratios examined, cyt b(5) inhibits the rate of NADPH consumption. Nevertheless, at low cyt b(5):cyt P450 molar ratios P450 molar ratios, cyt b(5) progressively inhibits both NADPH consumption and the rate of metabolism. It is proposed that the ability of cyt b(5) to enhance substrate metabolism by cyt P450 is related to its ability to increase the rate of catalysis and that the inhibitory properties of cyt b(5) are because of its ability to occupy the reductase-binding site on cyt P450 2B4, thereby preventing reduction of ferric cyt P450 and initiation of the catalytic cycle. It is proposed that cyt b(5) and CPR compete for a binding site on cyt P450 2B4. PMID:17693640

  12. Cytochrome P-450 induction by clofibrate. Purification and properties of a hepatic cytochrome P-450 relatively specific for the 12- and 11-hydroxylation of dodecanoic acid (lauric acid)

    PubMed Central

    Gibson, G. Gordon; Orton, Terry C.; Tamburini, Paul P.

    1982-01-01

    Hypolipidaemic drugs induce peroxisomal proliferation in the liver and many induce the formation of the hepatic endoplasmic reticulum in general and the formation of cytochrome P-450 in particular. We have induced the formation of rat liver microsomal cytochrome P-450 by the administration of the hypolipidaemic drug clofibrate, isolated the endoplasmic reticulum, solubilized the cytochrome P-450 from these membranes and subdivided the cytochrome P-450 into four fractions by the use of hydrophobic, anionic, cationic and adsorption chromatography. One of these fractions (cytochrome P-450 fraction 1) was highly purified to a specific content of 17nmol of cytochrome P-450/mg of protein and the protein was active in a reconstituted enzyme system towards the 12- and 11-hydroxylation of the fatty acid, dodecanoic (lauric) acid, with preferential activity towards the 12-hydroxy metabolite. This reconstituted activity was absolutely dependent on NADPH, NADPH-cytochrome P-450 reductase and cytochrome P-450, indicating the role of the mixed-function oxidase system in the metabolism of lauric acid. Another fraction of the haemoprotein (cytochrome P-450 fraction 2) preferentially formed 11-hydroxylauric acid, whereas a third fraction (cytochrome P-450 fraction 3) exhibited only trace laurate oxidase activity and was similar to the phenobarbitone form of the haemoprotein in that these last two cytochromes rapidly turned-over the drug benzphetamine. The molecular weights and spectral properties of these cytochrome P-450 fractions are reported, along with the phenobarbitone-induced form of the enzyme and the nature of the cytochrome(s) induced by clofibrate pretreatment are discussed in the terms of possible haemoprotein heterogeneity. ImagesFig. 2. PMID:7103935

  13. Role of cytochrome P450 in drug interactions

    PubMed Central

    Bibi, Zakia

    2008-01-01

    Drug-drug interactions have become an important issue in health care. It is now realized that many drug-drug interactions can be explained by alterations in the metabolic enzymes that are present in the liver and other extra-hepatic tissues. Many of the major pharmacokinetic interactions between drugs are due to hepatic cytochrome P450 (P450 or CYP) enzymes being affected by previous administration of other drugs. After coadministration, some drugs act as potent enzyme inducers, whereas others are inhibitors. However, reports of enzyme inhibition are very much more common. Understanding these mechanisms of enzyme inhibition or induction is extremely important in order to give appropriate multiple-drug therapies. In future, it may help to identify individuals at greatest risk of drug interactions and adverse events. PMID:18928560

  14. Regulation of cytochrome P-450Ia1 gene expression

    SciTech Connect

    Kamps, C.A.

    1989-01-01

    The mechanism by which cytochrome P-450IA1 gene expression is induced by polycyclic aromatic hydrocarbons and various polychlorinated dibenzo-p-dioxins involves an intracellular protein known as the Ah receptor. Within the past few years, a second protein has been identified which binds to certain polycyclic aromatic hydrocarbons (PAHs) but not to the receptor ligand, 2,3,7,8-tetrachlorodibenzo-para-dioxin (TCDD). The protein, named the 4S PAH binding protein, has been reported to bind to a site on the DNA in the 5{prime} regulatory region for the cytochrome P-450IA1 gene. This finding led to the hypothesis that the 4S PAH binding protein may be involved in the trans-regulation of this gene. The work presented in this manuscript addressed this hypothesis by (1) screening animals and cell lines for the presence or absence of the Ah receptor and 4S PAH binding protein, (2) screening polycyclic aromatic hydrocarbons (PAHs) to identify ligands which specifically bind only the 4S protein, (3) determining dose-response curves for TCDD and 4S protein specific ligands in mammalian cell lines, (4) co-administering a 4S binding protein ligand and TCDD in mammalian cell lines to determine the effects of the 4S protein-ligand complex on TCDD-induced cytochrome P-450IA1 expression, and (5) co-administering TCDD and 6-methyl 1,3,8-trichlorodibenzofuran (MCDF), a compound reported to be an antagonist of TCDD-induced benzo(a)pyrene-3-hydroxylase (AHH) activity, to determine whether antagonism occurs at the transcriptional level. The results of gradient assays show that the Ah receptor and the 4S binding protein were expressed in the rat strains which were studied. In the cell lines, H4IIE cells (rat hepatoma expressed only the receptor whereas Hepa1c1c7 cells mouse hepatoma) expressed both proteins.

  15. Cytochromes P450 for terpene functionalisation and metabolic engineering.

    PubMed

    Pateraki, Irini; Heskes, Allison Maree; Hamberger, Bjrn

    2015-01-01

    Plants have evolved the capacity to produce a striking array of specialised metabolites. Terpenoids are the oldest and most diverse class of such compounds and have attracted interest for industrial and pharmaceutical applications. The development of biotechnological alternatives for their production is the focus of intense research. Photosynthetic systems provide new strategies for autotrophic metabolic engineering. Focusing on cytochromes P450, involved in the functionalisation of the core terpene molecules, this review highlights the latest approaches in this field and looks towards recent discoveries that have the potential to shape the future of terpenoid bioengineering. PMID:25636487

  16. Regulation of cytochrome P450 expression in Drosophila: Genomic insights

    PubMed Central

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

    2009-01-01

    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

  17. Regulation of cytochrome P450 expression in Drosophila: Genomic insights.

    PubMed

    Giraudo, Maeva; Unnithan, G Chandran; Le Goff, Galle; Feyereisen, Ren

    2010-06-01

    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

  18. Human cytochromes P450 in health and disease

    PubMed Central

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

    2013-01-01

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

  19. Zonation of hepatic cytochrome P-450 expression and regulation.

    PubMed Central

    Oinonen, T; Lindros, K O

    1998-01-01

    The CYP genes encode enzymes of the cytochrome P-450 superfamily. Cytochrome P-450 (CYP) enzymes are expressed mainly in the liver and are active in mono-oxygenation and hydroxylation of various xenobiotics, including drugs and alcohols, as well as that of endogenous compounds such as steroids, bile acids, prostaglandins, leukotrienes and biogenic amines. In the liver the CYP enzymes are constitutively expressed and commonly also induced by chemicals in a characteristic zonated pattern with high expression prevailing in the downstream perivenous region. In the present review we summarize recent studies, mainly based on rat liver, on the factors regulating this position-dependent expression and induction. Pituitary-dependent signals mediated by growth hormone and thyroid hormone seem to selectively down-regulate the upstream periportal expression of certain CYP forms. It is at present unknown to what extent other hormones that also affect total hepatic CYP activities, i.e. insulin, glucagon, glucocorticoids and gonadal hormones, act zone-specifically. The expression and induction of CYP enzymes in the perivenous region probably have important toxicological implications, since many CYP-activated chemicals cause cell injury primarily in this region of the liver. PMID:9405271

  20. Effect of cobalt protoporphyrin on hepatic drug-metabolizing enzymes. Specificity for cytochrome P-450.

    PubMed

    Spaethe, S M; Jollow, D J

    1989-06-15

    Cobaltic protoporphyrin IX (cobalt protoporphyrin) is known to cause an extensive and long-lasting depletion of hepatic cytochrome P-450 in rats, and it has been used to evaluate the role of hepatic cytochrome P-450 in xenobiotic metabolism and toxicity. To examine the specificity of cobalt protoporphyrin for hepatic cytochrome P-450, cobalt protoporphyrin was administered to rats and hamsters, and its effects on cytochrome P-450-dependent and non-P-450-dependent phase I and phase II metabolism were determined. Cobalt protoporphyrin pretreatment depleted hepatic cytochrome P-450 in both species and lowered their Vmax values for the hepatic microsomal metabolism of ethylmorphine, aminopyrine, ethoxyresorufin and ethoxycoumarin, without change in their Km values. In the rat, cobalt protoporphyrin treatment lowered both the Vmax and Km for microsomal metabolism of aniline. In vivo hepatic cytochrome P-450-dependent metabolism, as measured by antipyrine clearance, was decreased in both species. UDP-Glucuronyltransferase, phenolsulfotransferase and glutathione-S-transferase were unaffected, as was hepatic glutathione. Modest effects of cobalt protoporphyrin were seen on the hepatic microsomal flavoprotein mixed-function oxidase (hamster only), cytochrome P-450 reductase, cytochrome b5 (rat only), UDPGA (rat only), and glycogen, and on blood glucose (rat). In in vivo studies with hamsters given a low dose of acetaminophen, cobalt protoporphyrin suppressed the apparent rate constants for the cytochrome P-450-dependent pathways of acetaminophen metabolism but had no effect on acetaminophen glucuronidation and sulfation. Polyacrylamide gel electrophoresis analysis indicated that cobalt protoporphyrin markedly reduced the levels of the cytochrome P-450 holoenzyme but did not alter either the content or profile of the cytochrome P-450 apoenzyme. collectively, the data indicate that cobalt protoporphyrin shows relatively high selectivity for the hepatic cytochrome P-450 system, and support the use of this compound as a tool for resolution of the role of hepatic cytochrome P-450 in xenobiotic metabolism and toxicity. PMID:2742604

  1. Cholesterol ester oxidation by mycobacterial cytochrome P450.

    PubMed

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

    2014-10-31

    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

  2. Nanoscale electron transport measurements of immobilized cytochrome P450 proteins

    NASA Astrophysics Data System (ADS)

    Bostick, Christopher D.; Flora, Darcy R.; Gannett, Peter M.; Tracy, Timothy S.; Lederman, David

    2015-04-01

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

  3. Interactions of phospholipase D and cytochrome P450 protein stability

    SciTech Connect

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

    2004-08-01

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

  4. Nanoscale electron transport measurements of immobilized cytochrome P450 proteins.

    PubMed

    Bostick, Christopher D; Flora, Darcy R; Gannett, Peter M; Tracy, Timothy S; Lederman, David

    2015-04-17

    Gold nanopillars, functionalized with an organic self-assembled monolayer, can be used to measure the electrical conductance properties of immobilized proteins without aggregation. Measurements of the conductance of nanopillars with cytochrome P450 2C9 (CYP2C9) proteins using conducting probe atomic force microscopy demonstrate that a correlation exists between the energy barrier height between hopping sites and CYP2C9 metabolic activity. Measurements performed as a function of tip force indicate that, when subjected to a large force, the protein is more stable in the presence of a substrate. This agrees with the hypothesis that substrate entry into the active site helps to stabilize the enzyme. The relative distance between hopping sites also increases with increasing force, possibly because protein functional groups responsible for electron transport (ETp) depend on the structure of the protein. The inhibitor sulfaphenazole, in addition to the previously studied aniline, increased the barrier height for electron transfer and thereby makes CYP2C9 reduction more difficult and inhibits metabolism. This suggests that P450 Type II binders may decrease the ease of ETp processes in the enzyme, in addition to occupying the active site. PMID:25804257

  5. Ab initio dynamics of the cytochrome P450 hydroxylation reaction

    SciTech Connect

    Elenewski, Justin E.; Hackett, John C

    2015-02-14

    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.

  6. Cholesterol Ester Oxidation by Mycobacterial Cytochrome P450*

    PubMed Central

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

    2014-01-01

    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 5867 and 100109 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

  7. Spaceflight Effects on Cytochrome P450 Content in Mouse Liver

    PubMed Central

    Moskaleva, Natalia; Moysa, Alexander; Novikova, Svetlana; Tikhonova, Olga; Zgoda, Victor; Archakov, Alexander

    2015-01-01

    Hard conditions of long-term manned spaceflight can affect functions of many biological systems including a system of drug metabolism. The cytochrome P450 (CYP) superfamily plays a key role in the drug metabolism. In this study we examined the hepatic content of some P450 isoforms in mice exposed to 30 days of space flight and microgravity. The CYP content was established by the mass-spectrometric method of selected reaction monitoring (SRM). Significant changes in the CYP2C29, CYP2E1 and CYP1A2 contents were detected in mice of the flight group compared to the ground control group. Within seven days after landing and corresponding recovery period changes in the content of CYP2C29 and CYP1A2 returned to the control level, while the CYP2E1 level remained elevated. The induction of enzyme observed in the mice in the conditions of the spaceflight could lead to an accelerated biotransformation and change in efficiency of pharmacological agents, metabolizing by corresponding CYP isoforms. Such possibility of an individual pharmacological response to medication during long-term spaceflights and early period of postflight adaptation should be taken into account in space medicine. PMID:26561010

  8. Ab initio dynamics of the cytochrome P450 hydroxylation reaction

    PubMed Central

    Elenewski, Justin E.; Hackett, John C

    2015-01-01

    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

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

    PubMed

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

    1994-09-15

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

  10. Cytochrome P450 Enzyme Metabolites in Lead Discovery and Development

    PubMed Central

    Kandel, Sylvie E.; Wienkers, Larry C.; Lampe, Jed N.

    2015-01-01

    The cytochrome P450 (CYP) enzymes are a versatile superfamily of heme-containing monooxygenases, perhaps best known for their role in the oxidation of xenobiotic compounds. However, due to their unique oxidative chemistry, CYPs are also important in natural product drug discovery and in the generation of active metabolites with unique therapeutic properties. New tools for the analysis and production of CYP metabolites, including microscale analytical technologies and combinatorial biosynthesis, are providing medicinal chemists with the opportunity to use CYPs as a novel platform for lead discovery and development. In this review, we will highlight some of the recent examples of drug leads identified from CYP metabolites and the exciting possibilities of using CYPs as catalysts for future drug discovery. PMID:25797999

  11. Cholesterol-metabolizing cytochromes P450: implications for cholesterol lowering

    PubMed Central

    Pikuleva, Irina A.

    2010-01-01

    Cardiovascular disease (CVD) continues to be a leading cause of death worldwide. Elevated serum cholesterol is one of the classical risk factors for CVD which also include age, hypertension, smoking, diabetes mellitus, obesity and family history. A number of therapeutic drug classes have been developed to treat hypercholesterolemia, yet, an important percentage of patients do not reach their treatment goals. Therefore, new cholesterol-lowering medications, having a site of action different from that of currently available drugs need to be developed. This review summarizes new information about cytochrome P450 enzymes 7A1, 27A1, and 46A1, that play key roles in cholesterol elimination and that have potential to serve as targets for cholesterol-lowering. PMID:18950282

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

    PubMed

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

    2015-09-01

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

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

    SciTech Connect

    Hanzlik, R.P.; Kahhiing John Ling )

    1990-06-22

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

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

    SciTech Connect

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

    1987-05-01

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

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

    PubMed Central

    Simonov, Alexandr N.; Holien, Jessica K.; Yeung, Joyee Chun In; Nguyen, Ann D.; Corbin, C. Jo; Zheng, Jie; Kuznetsov, Vladimir L.; Auchus, Richard J.; Conley, Alan J.; Bond, Alan M.; Parker, Michael W.; Rodgers, Raymond J.; Martin, Lisandra L.

    2015-01-01

    Cytochrome P450c17 (P450 17A1, CYP17A1) is a critical enzyme in the synthesis of androgens and is now a target enzyme for the treatment of prostate cancer. Cytochrome P450c17 can exhibit either one or two physiological enzymatic activities differentially regulated by cytochrome b5. How this is achieved remains unknown. Here, comprehensive in silico, in vivo and in vitro analyses were undertaken. Fluorescence Resonance Energy Transfer analysis showed close interactions within living cells between cytochrome P450c17 and cytochrome b5. In silico modeling identified the sites of interaction and confirmed that E48 and E49 residues in cytochrome b5 are essential for activity. Quartz crystal microbalance studies identified specific protein-protein interactions in a lipid membrane. Voltammetric analysis revealed that the wild type cytochrome b5, but not a mutated, E48G/E49G cyt b5, altered the kinetics of electron transfer between the electrode and the P450c17. We conclude that cytochrome b5 can influence the electronic conductivity of cytochrome P450c17 via allosteric, protein-protein interactions. PMID:26587646

  16. Induction of cytochrome P450III and P450IV family proteins in streptozotocin-induced diabetes.

    PubMed Central

    Barnett, C R; Gibson, G G; Wolf, C R; Flatt, P R; Ioannides, C

    1990-01-01

    The effect of insulin-dependent diabetes on the hepatic microsomal activity of cytochrome P450III and P450IV family proteins was investigated in rats pretreated with streptozotocin. In order to discern between the effects of the diabetogen per se and those of the ensuing diabetes, streptozotocin-treated rats received in addition either nicotinamide to prevent the onset of diabetes or daily treatment with insulin to antagonize the effects of diabetes. Streptozotocin-treated rats displayed higher ethylmorphine and erythromycin N-demethylase activities and lauric acid hydroxylase activity. Increases were also detected immunologically by using monospecific polyclonal antibodies against the P450III and P450IV families. All effects were prevented by nicotinamide and effectively antagonized by insulin. In order to evaluate the role of the ketone bodies in the diabetes-induced increases in the above activities, rats were rendered hyperketonaemic by dietary administration of medium-chain triacylglycerols. These hyperketonaemic animals displayed high laurate hydroxylase activity and P450IV apoprotein levels, similar to those seen in the diabetic animals. Hyperketonaemia induced by dietary means caused a modest increase in the demethylation of erythromycin and had no significant effect on the N-demethylation of ethylmorphine. Furthermore, no marked increases were evident in the P450III apoprotein levels in the hyperketonaemic animals. It is concluded that insulin-dependent diabetes induces proteins of the P450III and P450IV families, and that the hyperketonaemia that accompanies diabetes is largely responsible for the changes in the latter family. Images Fig. 1. Fig. 2. PMID:2141978

  17. Crystal structure of human cytochrome P450 2D6.

    PubMed

    Rowland, Paul; Blaney, Frank E; Smyth, Martin G; Jones, Jo J; Leydon, Vaughan R; Oxbrow, Amanda K; Lewis, Ceri J; Tennant, Mike G; Modi, Sandeep; Eggleston, Drake S; Chenery, Richard J; Bridges, Angela M

    2006-03-17

    Cytochrome P450 2D6 is a heme-containing enzyme that is responsible for the metabolism of at least 20% of known drugs. Substrates of 2D6 typically contain a basic nitrogen and a planar aromatic ring. The crystal structure of human 2D6 has been solved and refined to 3.0A resolution. The structure shows the characteristic P450 fold as seen in other members of the family, with the lengths and orientations of the individual secondary structural elements being very similar to those seen in 2C9. There are, however, several important differences, the most notable involving the F helix, the F-G loop, the B'helix, beta sheet 4, and part of beta sheet 1, all of which are situated on the distal face of the protein. The 2D6 structure has a well defined active site cavity above the heme group, containing many important residues that have been implicated in substrate recognition and binding, including Asp-301, Glu-216, Phe-483, and Phe-120. The crystal structure helps to explain how Asp-301, Glu-216, and Phe-483 can act as substrate binding residues and suggests that the role of Phe-120 is to control the orientation of the aromatic ring found in most substrates with respect to the heme. The structure has been compared with published homology models and has been used to explain much of the reported site-directed mutagenesis data and help understand the metabolism of several compounds. PMID:16352597

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

    PubMed

    Cheng, Qian; Guengerich, F Peter

    2013-01-01

    Microtiter plate-based fluorescence assays allow rapid measurement of the catalytic activities of cytochrome P450 oxygenases (P450s). We describe a high-throughput fluorescence assay of P450 3A4, one of the key enzymes involved in xenobiotic metabolism. The assay involves the oxidative debenzylation of 7-hydroxy-4-trifluoromethyl coumarin, producing an increase in fluorescence. PMID:23475676

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

    PubMed Central

    Cheng, Qian; Guengerich, F. Peter.

    2014-01-01

    Summary Microtiter plate-based fluorescence assays allow rapid measurement of the catalytic activities of cytochrome P450 oxygenases (P450s). We describe a high-throughput fluorescence assay of P450 3A4, one of the key enzymes involved in xenobiotic metabolism. The assay involves the oxidative debenzylation of 7-hydroxy-4-trifluoromethyl coumarin, producing an increase in fluorescence. PMID:23475676

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

    PubMed Central

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

    1983-01-01

    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

  1. Polycyclic aromatic hydrocarbons and cytochrome P450 in HIV pathogenesis.

    PubMed

    Rao, P S S; Kumar, Santosh

    2015-01-01

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

  2. Polycyclic aromatic hydrocarbons and cytochrome P450 in HIV pathogenesis

    PubMed Central

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

    2015-01-01

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

  3. Cytochrome P450 ?-Hydroxylases in Inflammation and Cancer

    PubMed Central

    Johnson, Amanda L.; Edson, Katheryne Z.; Totah, Rheem A.; Rettie, Allan E.

    2015-01-01

    Cytochrome P450-dependent ?-hydroxylation is a prototypic metabolic reaction of CYP4 family members that is important for the elimination and bioactivation of not only therapeutic drugs, but also endogenous compounds, principally fatty acids. Eicosanoids, derived from arachidonic acid, are key substrates in the latter category. Human CYP4 enzymes, mainly CYP4A11, CYP4F2, and CYP4F3B, hydroxylate arachidonic acid at the omega position to form 20-HETE, which has important effects in tumor progression and on angiogenesis and blood pressure regulation in the vasculature and kidney. CYP4F3A in myeloid tissue catalyzes the ?-hydroxylation of leukotriene B4 to 20-hydroxy leukotriene B4, an inactivation process that is critical for the regulation of the inflammatory response. Here, we review the enzymology, tissue distribution, and substrate selectivity of human CYP4 ?-hydroxylases and their roles as catalysts for the formation and termination of the biological effects of key eicosanoid metabolites in inflammation and cancer progression. PMID:26233909

  4. A key cytochrome P450 hydroxylase in pradimicin biosynthesis

    PubMed Central

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

    2011-01-01

    Pradimicins A-C (13) are a group of antifungal and antiviral polyketides from Actinomadura hibisca. The sugar moieties in pradimicins are required for their biological activities. Consequently, the 5-OH that is used for glycosylation plays a critical role in pradimicin biosynthesis. A cytochrome P450 monooxygenase gene, pdmJ, was amplified from the genomic DNA of A. hibisca and expressed in Escherichia coli BL21(DE3). PdmJ introduced a hydroxyl group to G-2A (4), a key pradimicin biosynthetic intermediate, at C-5 to form JX134 (5). A D-Ala-containing pradimicin analog, JX137a (6) was tested as an alternative substrate, but no product was detected by LC-MS, indicating that PdmJ has strict substrate specificity. Kinetic studies revealed a typical substrate inhibition of PdmJ activity. The optimal substrate concentration for the highest velocity is 115 ?M under the test conditions. Moreover, the conversion rate of 4 to 5 was reduced by the presence of 6, likely due to competitive inhibition. Coexpression of PdmJ and a glucose 1-dehydrogenase in E. coli BL21(DE3) provides an efficient method to produce the important intermediate 5 from 4. PMID:22082563

  5. Association between cytochrome P450 promoter polymorphisms and ischemic stroke

    PubMed Central

    KIM, SU KANG; YIM, SUNG-VIN; LEE, BYUNG-CHEOL

    2012-01-01

    The human cytochrome P450 (CYP) superfamily includes at least 57 genes that encode enzymes with diverse metabolic and biosynthetic functions. This study was conducted in order to investigate the associations between polymorphisms in CYP superfamily genes (CYP11B2, CYP17A1, CYP2B6, CYP2C9, CYP2E1 and CYP7A1) and ischemic stroke (IS). Six single nucleotide polymorphisms (SNPs) of CYP superfamily genes were selected and genotyped by direct sequencing in 121 patients with IS and 321 control subjects. The genetic data were analyzed using SNPStats and SPSS 18.0. Multiple logistic regression models (codominant 1, codominant 2, dominant, recessive and log-additive) were used to evaluate odds ratios (ORs), 95% confidence intervals (CIs) and p-values. The rs179998 SNP of CYP11B2 was significantly associated with IS (p=0.0336 in a log-additive model). The rs3813867 SNP of CYP2E1 was significantly associated with smoking in IS (p=0.0336 in a log-additive model). The rs1799998 SNP of CYP11B2 and rs3808607 of CYP7A1 were related to diabetes mellitus in IS (p<0.05). CYP11B2, CYP2E1 and CYP7A1 SNPs were associated with IS in the population studied. Further study is required to confirm these associations and to determine their biological significance. PMID:22969879

  6. Cytochrome P450 genetic polymorphisms of Mexican indigenous populations.

    PubMed

    Sosa-Macías, Martha; Llerena, Adrián

    2013-01-01

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

  7. 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...

  8. KINETICS OF BROMODICHLOROMETHANE METABOLISM BY CYTOCHROME P450 ISOENZYMES IN HUMAN LIVER MICROSOMES

    EPA Science Inventory

    Kinetics of Bromodichloromethane Metabolism by
    Cytochrome P450 Isoenzymes in Human Liver Microsomes

    Guangyu Zhao and John W. Allis

    ABSTRACT
    The kinetic constants for the metabolism of bromodichloromethane (BDCM) by three cytochrome P450 (CYP) isoenzymes have ...

  9. Human Liver Cytochrome P450 3A4 Ubiquitination

    PubMed Central

    Wang, YongQiang; Kim, Sung-Mi; Trnka, Michael J.; Liu, Yi; Burlingame, A. L.; Correia, Maria Almira

    2015-01-01

    CYP3A4 is an abundant and catalytically dominant human liver endoplasmic reticulum-anchored cytochrome P450 enzyme engaged in the biotransformation of endo- and xenobiotics, including >50% of clinically relevant drugs. Alterations of CYP3A4 protein turnover can influence clinically relevant drug metabolism and bioavailability and drug-drug interactions. This CYP3A4 turnover involves endoplasmic reticulum-associated degradation via the ubiquitin (Ub)-dependent 26 S proteasomal system that relies on two highly complementary E2 Ub-conjugating-E3 Ub-ligase (UBC7-gp78 and UbcH5a-C terminus of Hsc70-interacting protein (CHIP)-Hsc70-Hsp40) complexes, as well as protein kinases (PK) A and C. We have documented that CYP3A4 Ser/Thr phosphorylation (Ser(P)/Thr(P)) by PKA and/or PKC accelerates/enhances its Lys ubiquitination by either of these E2-E3 systems. Intriguingly, CYP3A4 Ser(P)/Thr(P) and ubiquitinated Lys residues reside within the cytosol-accessible surface loop and/or conformationally assembled acidic Asp/Glu clusters, leading us to propose that such post-translational Ser/Thr protein phosphorylation primes CYP3A4 for ubiquitination. Herein, this possibility was examined through various complementary approaches, including site-directed mutagenesis, chemical cross-linking, peptide mapping, and LC-MS/MS analyses. Our findings reveal that such CYP3A4 Asp/Glu/Ser(P)/Thr(P) surface clusters are indeed important for its intermolecular electrostatic interactions with each of these E2-E3 subcomponents. By imparting additional negative charge to these Asp/Glu clusters, such Ser/Thr phosphorylation would generate P450 phosphodegrons for molecular recognition by the E2-E3 complexes, thereby controlling the timing of CYP3A4 ubiquitination and endoplasmic reticulum-associated degradation. Although the importance of phosphodegrons in the CHIP targeting of its substrates is known, to our knowledge this is the first example of phosphodegron involvement in gp78-substrate recruitment, an important step in CYP3A4 proteasomal degradation. PMID:25451919

  10. Hepatic metabolism of cyclodiene insecticides by constitutive forms of cytochrome P-450 from lower vertebrates.

    PubMed

    Ronis, M J; Walker, C H; Peakall, D

    1987-01-01

    1. Multiple forms of cytochrome P-450 were separated from the hepatic microsomes of untreated male rats, pigeons (Columbia livia), razorbills (Alca torda), puffins (Fratercula arctica), and rainbow trout (Salmo gairdnerii), using anion exchange chromatography and DEAE-cellulose. 2. In some cases cytochrome P-450 forms were further purified on hydroxylapatite and carboxymethyl-sephadex columns. 3. Considerable differences in the distribution of forms between these five species were evident from elution profiles on DEAE cellulose, and on analysis of the cytochrome P-450 containing pools by SDS-PAGE. 4. The metabolism of two organochlorine compounds, aldrin and the dieldrin analogue HCE, were studied in (a) intact microsomes and (b) reconstituted systems containing cytochrome P-450, from each of the five species. 5. In spite of their close structural similarity, significant differences were found between the two substrates in the distribution of catalytic activity between the cytochrome P-450 isozymes of each species. PMID:2888582

  11. Determination of the rate of reduction of oxyferrous cytochrome P450 2B4 by 5-deazariboflavin adenine dinucleotide T491V cytochrome P450 reductase.

    PubMed

    Zhang, Haoming; Gruenke, Larry; Arscott, Dave; Shen, Anna; Kasper, Charles; Harris, Danni L; Glavanovich, Michael; Johnson, Richard; Waskell, Lucy

    2003-10-14

    The use of 5-deazaFAD T491V cytochrome P450 reductase has made it possible to directly measure the rate of electron transfer to microsomal oxyferrous cytochrome (cyt) P450 2B4. In this reductase the FMN moiety can be reduced to the hydroquinone, FMNH(2), while the 5-deazaFAD moiety remains oxidized [Zhang, H., et al. (2003) Biochemistry 42, 6804-6813]. The rate of electron transfer from 5-deazaFAD cyt P450 reductase to oxyferrous cyt P450 was determined by rapidly mixing the ferrous cyt P450-2-electron-reduced 5-deazaFAD T491V reductase complex with oxygen in the presence of substrate. The 5-deazaFAD T491V reductase which can only donate a single electron reduces the oxyferrous cyt P450 and oxidizes to the air-stable semiquinone, with rate constants of 8.4 and 0.37 s(-1) at 15 degrees C. Surprisingly, oxyferrous cyt P450 turns over more slowly with a rate constant of 0.09 s(-1), which is the rate of catalysis under steady-state conditions at 15 degrees C (k(cat) = 0.08 s(-1)). In contrast, the rate constant for electron transfer from ferrous cyt b(5) to oxyferrous cyt P450 is 10 s(-1) with oxyferrous cyt P450 and cyt b(5) simultaneously undergoing spectral changes. Quantitative analyses by LC-MS/MS revealed that the product, norbenzphetamine, was formed with a coupling efficiency of 52% with cyt b(5) and 32% with 5-deazaFAD T491V reductase. Collectively, these results suggest that during catalysis a relatively stable reduced oxyferrous intermediate of cyt P450 is formed in the presence of cyt P450 reductase but not cyt b(5) and that the rate-limiting step in catalysis follows introduction of the second electron. PMID:14529269

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

    PubMed Central

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

    2007-01-01

    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

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

    SciTech Connect

    Flueck, Christa E.; Mallet, Delphine; Hofer, Gaby; Samara-Boustani, Dinane; Leger, Juliane; Polak, Michel; Morel, Yves; Pandey, Amit V.

    2011-09-09

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

  14. Cytochrome P450 isoform selectivity in human hepatic theobromine metabolism

    PubMed Central

    Gates, Simon; Miners, John O

    1999-01-01

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

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

    PubMed

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

    2015-01-01

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

  16. Differential cytochrome P450 2D metabolism alters tafenoquine pharmacokinetics.

    PubMed

    Vuong, Chau; Xie, Lisa H; Potter, Brittney M J; Zhang, Jing; Zhang, Ping; Duan, Dehui; Nolan, Christina K; Sciotti, Richard J; Zottig, Victor E; Nanayakkara, N P Dhammika; Tekwani, Babu L; Walker, Larry A; Smith, Philip L; Paris, Robert M; Read, Lisa T; Li, Qigui; Pybus, Brandon S; Sousa, Jason C; Reichard, Gregory A; Smith, Bryan; Marcsisin, Sean R

    2015-07-01

    Cytochrome P450 (CYP) 2D metabolism is required for the liver-stage antimalarial efficacy of the 8-aminoquinoline molecule tafenoquine in mice. This could be problematic for Plasmodium vivax radical cure, as the human CYP 2D ortholog (2D6) is highly polymorphic. Diminished CYP 2D6 enzyme activity, as in the poor-metabolizer phenotype, could compromise radical curative efficacy in humans. Despite the importance of CYP 2D metabolism for tafenoquine liver-stage efficacy, the exact role that CYP 2D metabolism plays in the metabolism and pharmacokinetics of tafenoquine and other 8-aminoquinoline molecules has not been extensively studied. In this study, a series of tafenoquine pharmacokinetic experiments were conducted in mice with different CYP 2D metabolism statuses, including wild-type (WT) (reflecting extensive metabolizers for CYP 2D6 substrates) and CYPmouse 2D knockout (KO) (reflecting poor metabolizers for CYP 2D6 substrates) mice. Plasma and liver pharmacokinetic profiles from a single 20-mg/kg of body weight dose of tafenoquine differed between the strains; however, the differences were less striking than previous results obtained for primaquine in the same model. Additionally, the presence of a 5,6-ortho-quinone tafenoquine metabolite was examined in both mouse strains. The 5,6-ortho-quinone species of tafenoquine was observed, and concentrations of the metabolite were highest in the WT extensive-metabolizer phenotype. Altogether, this study indicates that CYP 2D metabolism in mice affects tafenoquine pharmacokinetics and could have implications for human tafenoquine pharmacokinetics in polymorphic CYP 2D6 human populations. PMID:25870069

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed Central

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

    2012-01-01

    Structures of human cytochrome P450 2B6 and rabbit cytochrome P450 2B4 in complex with two molecules of the calcium channel blocker amlodipine have been determined by X-ray crystallography. The presence of two drug molecules suggests clear substrate access channels in each P450. According to a previously established nomenclature, amlodipine molecules were trapped in access pathway 2f in P450 2B6 and in pathway 2a or 2f in P450 2B4. These pathways overlap for part of the length and then diverge as they extend toward the protein surface. A previously described solvent channel was also found in each enzyme. The results indicate that key residues located on the surface and at the entrance of the substrate access channels in each of these P450s may play a crucial role in guiding substrate entry. In addition, the region of P450 2B6 and 2B4 involving helices B’, F, F’, G’ and part of helix G is substantially more open in the amlodipine complexes compared with the corresponding 4-(4-chlorophenyl)imidazole complexes. The increased active site volume observed results from the major retraction of helices F, F’ and B’ and the β4 sheet region located close to the binding cavity to accommodate amlodipine. These structures demonstrate novel insight into distinct conformational states not observed with previous P450 2B structures and provide clear evidence of the substrate access channels in two drug metabolizing P450s. In addition, the structures exhibit the versatility that can be exploited in silico studies with other P450 2B6 ligands as large as raloxifene and itraconazole. PMID:22909231

  19. Peroxo-iron and oxenoid-iron species as alternative oxygenating agents in cytochrome P450-catalyzed reactions: switching by threonine-302 to alanine mutagenesis of cytochrome P450 2B4.

    PubMed Central

    Vaz, A D; Pernecky, S J; Raner, G M; Coon, M J

    1996-01-01

    Among biological catalysts, cytochrome P450 is unmatched in its multiplicity of isoforms, inducers, substrates, and types of chemical reactions catalyzed. In the present study, evidence is given that this versatility extends to the nature of the active oxidant. Although mechanistic evidence from several laboratories points to a hypervalent iron-oxenoid species in P450-catalyzed oxygenation reactions, Akhtar and colleagues [Akhtar, M., Calder, M. R., Corina, D. L. & Wright, J. N. (1982) Biochem. J. 201, 569-580] proposed that in steroid deformylation effected by P450 aromatase an iron-peroxo species is involved. We have shown more recently that purified liver microsomal P450 cytochromes, including phenobarbital-induced P450 2B4, catalyze the analogous deformylation of a series of xenobiotic aldehydes with olefin formation. The investigation presented here on the effect of site-directed mutagenesis of threonine-302 to alanine on the activities of recombinant P450 2B4 with N-terminal amino acids 2-27 deleted [2B4 (delta2-27)] makes use of evidence from other laboratories that the corresponding mutation in bacterial P450s interferes with the activation of dioxygen to the oxenoid species by blocking proton delivery to the active site. The rates of NADPH oxidation, hydrogen peroxide production, and product formation from four substrates, including formaldehyde from benzphetamine N-demethylation, acetophenone from 1-phenylethanol oxidation, cyclohexanol from cyclohexane hydroxylation, and cyclohexene from cyclohexane carboxaldehyde deformylation, were determined with P450s 2B4, 2B4 (delta2-27), and 2B4 (delta2-27) T302A. Replacement of the threonine residue in the truncated cytochrome gave a 1.6- to 2.5-fold increase in peroxide formation in the presence of a substrate, but resulted in decreased product formation from benzphetamine (9-fold), cyclohexane (4-fold), and 1-phenylethanol (2-fold). In sharp contrast, the deformylation of cyclohexane carboxaldehyde by the T302A mutant was increased about 10-fold. On the basis of these findings and our previous evidence that aldehyde deformylation is supported by added H202, but not by artificial oxidants, we conclude that the iron-peroxy species is the direct oxygen donor. It remains to be established which of the many other oxidative reactions involving P450 utilize this species and the extent to which peroxo-iron and oxenoid-iron function as alternative oxygenating agents with the numerous isoforms of this versatile catalyst. PMID:8643457

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

    PubMed Central

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

    2008-01-01

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

  1. Comparison of intrinsic dynamics of cytochrome p450 proteins using normal mode analysis.

    PubMed

    Dorner, Mariah E; McMunn, Ryan D; Bartholow, Thomas G; Calhoon, Brecken E; Conlon, Michelle R; Dulli, Jessica M; Fehling, Samuel C; Fisher, Cody R; Hodgson, Shane W; Keenan, Shawn W; Kruger, Alyssa N; Mabin, Justin W; Mazula, Daniel L; Monte, Christopher A; Olthafer, Augustus; Sexton, Ashley E; Soderholm, Beatrice R; Strom, Alexander M; Hati, Sanchita

    2015-09-01

    Cytochrome P450 enzymes are hemeproteins that catalyze the monooxygenation of a wide-range of structurally diverse substrates of endogenous and exogenous origin. These heme monooxygenases receive electrons from NADH/NADPH via electron transfer proteins. The cytochrome P450 enzymes, which constitute a diverse superfamily of more than 8,700 proteins, share a common tertiary fold but?cytochrome P450 proteins are classified into six broad classes. Traditional methods of pro are based on the canonical paradigm that attributes proteins' function to their three-dimensional structure, which is determined by their primary structure that is the amino acid sequence. It is increasingly recognized that protein dynamics play an important role in molecular recognition and catalytic activity. As the mobility of a protein is an intrinsic property that is encrypted in its primary structure, we examined if different classes of cytochrome P450 enzymes display any unique patterns of intrinsic mobility. Normal mode analysis was performed to characterize the intrinsic dynamics of five classes of cytochrome P450 proteins. The present study revealed that cytochrome P450 enzymes share a strong dynamic similarity (root mean squared inner product?>?55% and Bhattacharyya coefficient?>?80%), despite the low sequence identity (< 25%) and sequence similarity (< 50%) across the cytochrome P450 superfamily. Noticeable differences in C? atom fluctuations of structural elements responsible for substrate binding were noticed. These differences in residue fluctuations might be crucial for substrate selectivity in these enzymes. PMID:26130403

  2. Identification of cytochrome P-450 isozymes involved in the hydroxylation of dantrolene by rat liver microsomes.

    PubMed

    Jayyosi, Z; Villoutreix, J; Ziegler, J M; Batt, A M; De Maack, F; Siest, G; Thomas, P E

    1993-01-01

    The role of individual rat liver cytochrome P-450 isozymes in the metabolism of the skeletal muscle relaxant, dantrolene, was studied. Following incubation of dantrolene with hepatic microsomes from 3-methylcholanthrene-treated rats, two major hydroxylated metabolites were identified. Using inhibitory antibodies specific for individual cytochrome P-450 isozymes, cytochromes P-450 1A1, 1A2, and 3A were identified to be involved in dantrolene hydroxylations. In liver microsomes from 3-methylcholanthrene-treated rats, antibodies specific for cytochrome P-450 1A1 and 1A2 inhibited hydroxylation of dantrolene by 60% and 20%, respectively. Kinetics studies using these microsomes showed that dantrolene hydroxylation was biphasic with a low KM (0.06-0.08 microM) and high KM (5-7 microM). Cytochrome P-450 1A1 was responsible for the low KM hydroxylation of dantrolene, whereas cytochrome P-450 1A2 was responsible for the high KM. In hepatic microsomes from pregnenolone-16 alpha-carbonitrile-treated rats, an antibody specific for cytochrome P-450 3A completely inhibited the formation of 5-hydroxydantrolene, the major metabolite formed by these microsomes. PMID:7902259

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

    PubMed

    Wanwimolruk, Sompon; Phopin, Kamonrat; Prachayasittikul, Virapong

    2014-01-01

    To date, a number of significant herbal drug interactions have their origins in the alteration of cytochrome P450 (CYP) activity by various phytochemicals. Among the most noteworthy are those involving St. John's wort and drugs metabolized by human CYP3A4 enzyme. This review article is the continued work from our previous article (Part 1) published in this journal (Wanwimolruk and Prachayasittikul, 2014[ref:133]). This article extends the scope of the review to six more herbs and updates information on herbal drug interactions. These include black cohosh, ginseng, grape seed extract, green tea, kava, saw palmetto and some important Chinese medicines are also presented. Even though there have been many studies to determine the effects of herbs and herbal medicines on the activity of CYP, most of them were in vitro and in animal studies. Therefore, the studies are limited in predicting the clinical relevance of herbal drug interactions. It appeared that the majority of the herbal medicines have no clear effects on most of the CYPs examined. For example, the existing clinical trial data imply that black cohosh, ginseng and saw palmetto are unlikely to affect the pharmacokinetics of conventional drugs metabolized by human CYPs. For grape seed extract and green tea, adverse herbal drug interactions are unlikely when they are concomitantly taken with prescription drugs that are CYP substrates. Although there were few clinical studies on potential CYP-mediated interactions produced by kava, present data suggest that kava supplements have the ability to inhibit CYP1A2 and CYP2E1 significantly. Therefore, caution should be taken when patients take kava with CYP1A2 or CYP2E1 substrate drugs as it may enhance their therapeutic and adverse effects. Despite the long use of traditional Chinese herbal medicines, little is known about the potential drug interactions with these herbs. Many popularly used Chinese medicines have been shown in vitro to significantly change the activity of human CYP. However, with little confirming evidence from clinical studies, precaution should be exercised when patients are taking Chinese herbal medicines concomitantly with drugs that are CYP substrates. Currently there is sufficient evidence to indicate that herbal drug interactions can occur and may lead to serious clinical consequence. Further clinical trial research should be conducted to verify these herbal drug interactions. Education on herbal drug interactions and communication with patients on their use of herbal products is also important. PMID:26417310

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

    PubMed Central

    Wanwimolruk, Sompon; Phopin, Kamonrat; Prachayasittikul, Virapong

    2014-01-01

    To date, a number of significant herbal drug interactions have their origins in the alteration of cytochrome P450 (CYP) activity by various phytochemicals. Among the most noteworthy are those involving St. John's wort and drugs metabolized by human CYP3A4 enzyme. This review article is the continued work from our previous article (Part 1) published in this journal (Wanwimolruk and Prachayasittikul, 2014[ref:133]). This article extends the scope of the review to six more herbs and updates information on herbal drug interactions. These include black cohosh, ginseng, grape seed extract, green tea, kava, saw palmetto and some important Chinese medicines are also presented. Even though there have been many studies to determine the effects of herbs and herbal medicines on the activity of CYP, most of them were in vitro and in animal studies. Therefore, the studies are limited in predicting the clinical relevance of herbal drug interactions. It appeared that the majority of the herbal medicines have no clear effects on most of the CYPs examined. For example, the existing clinical trial data imply that black cohosh, ginseng and saw palmetto are unlikely to affect the pharmacokinetics of conventional drugs metabolized by human CYPs. For grape seed extract and green tea, adverse herbal drug interactions are unlikely when they are concomitantly taken with prescription drugs that are CYP substrates. Although there were few clinical studies on potential CYP-mediated interactions produced by kava, present data suggest that kava supplements have the ability to inhibit CYP1A2 and CYP2E1 significantly. Therefore, caution should be taken when patients take kava with CYP1A2 or CYP2E1 substrate drugs as it may enhance their therapeutic and adverse effects. Despite the long use of traditional Chinese herbal medicines, little is known about the potential drug interactions with these herbs. Many popularly used Chinese medicines have been shown in vitro to significantly change the activity of human CYP. However, with little confirming evidence from clinical studies, precaution should be exercised when patients are taking Chinese herbal medicines concomitantly with drugs that are CYP substrates. Currently there is sufficient evidence to indicate that herbal drug interactions can occur and may lead to serious clinical consequence. Further clinical trial research should be conducted to verify these herbal drug interactions. Education on herbal drug interactions and communication with patients on their use of herbal products is also important. PMID:26417310

  5. Cytochrome P450 enzyme mediated herbal drug interactions (Part 1)

    PubMed Central

    Wanwimolruk, Sompon; Prachayasittikul, Virapong

    2014-01-01

    It is well recognized that herbal supplements or herbal medicines are now commonly used. As many patients taking prescription medications are concomitantly using herbal supplements, there is considerable risk for adverse herbal drug interactions. Such interactions can enhance the risk for an individual patient, especially with regard to drugs with a narrow therapeutic index such as warfarin, cyclosporine A and digoxin. Herbal drug interactions can alter pharmacokinetic or/and pharmacodynamic properties of administered drugs. The most common pharmacokinetic interactions usually involve either the inhibition or induction of the metabolism of drugs catalyzed by the important enzymes, cytochrome P450 (CYP). The aim of the present article is to provide an updated review of clinically relevant metabolic CYP-mediated drug interactions between selected herbal supplements and prescription drugs. The commonly used herbal supplements selected include Echinacea, Ginkgo biloba, garlic, St. John's wort, goldenseal, and milk thistle. To date, several significant herbal drug interactions have their origins in the alteration of CYP enzyme activity by various phytochemicals. Numerous herbal drug interactions have been reported. Although the significance of many interactions is uncertain but several interactions, especially those with St. John’s wort, may have critical clinical consequences. St. John’s wort is a source of hyperforin, an active ingredient that has a strong affinity for the pregnane xenobiotic receptor (PXR). As a PXR ligand, hyperforin promotes expression of CYP3A4 enzymes in the small intestine and liver. This in turn causes induction of CYP3A4 and can reduce the oral bioavailability of many drugs making them less effective. The available evidence indicates that, at commonly recommended doses, other selected herbs including Echinacea, Ginkgo biloba, garlic, goldenseal and milk thistle do not act as potent or moderate inhibitors or inducers of CYP enzymes. A good knowledge of the mechanisms of herbal drug interactions is necessary for assessing and minimizing clinical risks. These processes help prediction of interactions between herbal supplements and prescription drugs. Healthcare professionals should remain vigilant for potential interactions between herbal supplements/medicines and prescription drugs, especially for drugs with a narrow therapeutic index are used. PMID:26417265

  6. Cytochrome P450 enzyme mediated herbal drug interactions (Part 1).

    PubMed

    Wanwimolruk, Sompon; Prachayasittikul, Virapong

    2014-01-01

    It is well recognized that herbal supplements or herbal medicines are now commonly used. As many patients taking prescription medications are concomitantly using herbal supplements, there is considerable risk for adverse herbal drug interactions. Such interactions can enhance the risk for an individual patient, especially with regard to drugs with a narrow therapeutic index such as warfarin, cyclosporine A and digoxin. Herbal drug interactions can alter pharmacokinetic or/and pharmacodynamic properties of administered drugs. The most common pharmacokinetic interactions usually involve either the inhibition or induction of the metabolism of drugs catalyzed by the important enzymes, cytochrome P450 (CYP). The aim of the present article is to provide an updated review of clinically relevant metabolic CYP-mediated drug interactions between selected herbal supplements and prescription drugs. The commonly used herbal supplements selected include Echinacea, Ginkgo biloba, garlic, St. John's wort, goldenseal, and milk thistle. To date, several significant herbal drug interactions have their origins in the alteration of CYP enzyme activity by various phytochemicals. Numerous herbal drug interactions have been reported. Although the significance of many interactions is uncertain but several interactions, especially those with St. John's wort, may have critical clinical consequences. St. John's wort is a source of hyperforin, an active ingredient that has a strong affinity for the pregnane xenobiotic receptor (PXR). As a PXR ligand, hyperforin promotes expression of CYP3A4 enzymes in the small intestine and liver. This in turn causes induction of CYP3A4 and can reduce the oral bioavailability of many drugs making them less effective. The available evidence indicates that, at commonly recommended doses, other selected herbs including Echinacea, Ginkgo biloba, garlic, goldenseal and milk thistle do not act as potent or moderate inhibitors or inducers of CYP enzymes. A good knowledge of the mechanisms of herbal drug interactions is necessary for assessing and minimizing clinical risks. These processes help prediction of interactions between herbal supplements and prescription drugs. Healthcare professionals should remain vigilant for potential interactions between herbal supplements/medicines and prescription drugs, especially for drugs with a narrow therapeutic index are used. PMID:26417265

  7. Cytochrome P450 for Citreohybridonol Synthesis: Oxidative Derivatization of the Andrastin Scaffold.

    PubMed

    Matsuda, Yudai; Quan, Zhiyang; Mitsuhashi, Takaaki; Li, Chang; Abe, Ikuro

    2016-01-15

    A biosynthetic gene cluster similar to that for andrastin A (1) was discovered in Emericella variecolor NBRC 32302. Ctr-P450, a cytochrome P450 uniquely present in the cluster, was coexpressed with the andrastin A biosynthetic genes, leading to the production of the antifeedant agent citreohybridonol (4), along with four new andrastin derivatives. The results revealed the unusual multifunctionality of Ctr-P450 and indicated that this approach can be applied for further natural product diversification. PMID:26731665

  8. Inducible cytochrome P-450 from rat liver mitochondria

    SciTech Connect

    Raza, H.; Shayiq, F.M.; Avadhani, N.G.

    1987-05-01

    In the present study they have purified US -naphthoflavone (BNF, which induces isotypes similar to 3-MC) and PB induced mitochondrial isoforms. They have been able to purify two isoforms with molecular weights of 54 Kd and 52 Kd from BNF induced mitochondria. Only the 54 KD form, but not the 52 KD species reacts with the polyclonal antibody to microsomal P-450c, though, both show arylhydrocarbon hydroxylase activity in an in vitro system reconstituted with adrenodoxin and adrenodoxin-reductase. Fingerprint analyses, N-terminal sequencing and use of monoclonal antibody probes show that the two mitochondrial isoforms are different from the microsomal P-450c. Further, the 54 Kd mitochondrial isoform is not detected in control mitochondria indicating that it is truly an induced form. Similarly, a PB induced mitochondrial form which exhibits physical, immunochemical and enzymatic properties different from the microsomal P-450b has also been purified.

  9. Purification and characterization of NADPH-cytochrome P450 reductase from filamentous fungus Rhizopus nigricans.

    PubMed

    Makovec, T; Breskvar, K

    1998-09-15

    We report here the isolation and partial characterization of a flavoprotein, NADPH-cytochrome P450 (cytochrome c) reductase. The enzyme is a part of steroid 11 alpha-hydroxylating system and is associated with the microsomal fraction of the fungus Rhizopus nigricans. Fungal reductase was solubilized from microsomal membranes with Triton X-100 and purified to apparent homogeneity by affinity and high-performance ion-exchange chromatography. A 350-fold purification of the enzyme with specific activity of 37 mumol cytochrome c reduced/min/mg protein was achieved. A single protein band was obtained on SDS-PAGE analysis with an apparent molecular weight of 79 kDa. Purified reductase contained approximately equimolar quantities of flavin adenine dinucleotide and flavin mononucleotide per mole of the enzyme. Upon induction of the steroid hydroxylating system with progesterone the activity of microsomal NADPH-cytochrome c (P450) reductase increased 10-fold. This is in good correlation with the increase in content of fungal cytochrome P450. Purified fungal flavoprotein was active in a reconstituted system with cytochrome P450 C21 from adrenal gland but could not replace adrenodoxin reductase in the mitochondrial steroid 11 beta-hydroxylating system. We were able to confirm the role of the enzyme by reconstituting steroid 11 alpha-hydroxylating activity from the separated components NADPH-cytochrome P450 reductase and cytochrome P450, partly purified from fungal microsomes. PMID:9735172

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

    PubMed

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

    2011-01-01

    The NADPH-dependent cytochrome P450 reductase (CPR) is a key electron donor to eucaryotic cytochromes P450 (CYPs). CPR shuttles electrons from NADPH through the FAD and FMN-coenzymes into the iron of the prosthetic heme-group of the CYP. In the course of these electron transfer reactions, CPR undergoes large conformational changes. This mini-review discusses the new evidence provided for such conformational changes involving a combination of a "swinging" and "rotating" model and highlights the molecular mechanisms by which formation of these conformations are controlled and thereby enables CPR to serve as an effective electron transferring "nano-machine". PMID:20624491

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

    PubMed Central

    Bozak, K R; Yu, H; Sirevg, R; Christoffersen, R E

    1990-01-01

    The ripening of avocado fruit is associated with the expression of a number of mRNAs concomitant with overt changes in texture and flavor. Two overlapping cDNAs for a mRNA that accumulates during ripening were identified. Sequence analysis of these two cDNAs revealed a polypeptide of 471 amino acids with characteristics of a typical P-450: an N-terminal hydrophobic membrane anchor, a conserved heme-binding domain in the C-terminal region, and patches of similarity to various P-450 family members. Further evidence that this polypeptide represents a cytochrome P-450 oxidase comes from the recent isolation and characterization of a cytochrome P-450 from ripe avocado mesocarp [O'Keefe, D. P. & Leto, K. J. (1989) Plant Physiol. 89, 1141-1149]. The N terminus of the predicted polypeptide in the cDNAs is identical to the N terminus of the purified avocado P-450. Gel blot analysis of RNA from fruit at various stages of ripening showed the accumulation of an 1800-nucleotide P-450 mRNA that hybridized to the P-450 cDNA. The P-450 protein predicted by the avocado cDNA sequence shares less than 40% positional identity with any known P-450 gene family. We propose therefore that it be placed in a separate family, P450LXXI, and that the corresponding gene from avocado be named cyp71A1. Images PMID:1692626

  12. 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...

  13. 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...

  14. Interactions among cytochromes P450 in microsomal membranes: oligomerization of cytochromes P450 3A4, 3A5, and 2E1 and its functional consequences.

    PubMed

    Davydov, Dmitri R; Davydova, Nadezhda Y; Sineva, Elena V; Halpert, James R

    2015-02-01

    The body of evidence of physiologically relevant P450-P450 interactions in microsomal membranes continues to grow. Here we probe oligomerization of human CYP3A4, CYP3A5, and CYP2E1 in microsomal membranes. Using a technique based on luminescence resonance energy transfer, we demonstrate that all three proteins are subject to a concentration-dependent equilibrium between the monomeric and oligomeric states. We also observed the formation of mixed oligomers in CYP3A4/CYP3A5, CYP3A4/CYP2E1, and CYP3A5/CYP2E1 pairs and demonstrated that the association of either CYP3A4 or CYP3A5 with CYP2E1 causes activation of the latter enzyme. Earlier we hypothesized that the intersubunit interface in CYP3A4 oligomers is similar to that observed in the crystallographic dimers of some microsomal drug-metabolizing cytochromes P450 (Davydov, D. R., Davydova, N. Y., Sineva, E. V., Kufareva, I., and Halpert, J. R. (2013) Pivotal role of P450-P450 interactions in CYP3A4 allostery: the case of ?-naphthoflavone. Biochem. J. 453, 219-230). Here we report the results of intermolecular cross-linking of CYP3A4 oligomers with thiol-reactive bifunctional reagents as well as the luminescence resonance energy transfer measurements of interprobe distances in the oligomers of labeled CYP3A4 single-cysteine mutants. The results provide compelling support for the physiological relevance of the dimer-specific peripheral ligand-binding site observed in certain CYP3A4 structures. According to our interpretation, these results reveal an important general mechanism that regulates the activity and substrate specificity of the cytochrome P450 ensemble through interactions between multiple P450 species. As a result of P450-P450 cross-talk, the catalytic properties of the cytochrome P450 ensemble cannot be predicted by simple summation of the properties of the individual P450 species. PMID:25533469

  15. Purification and characterization of an acetone-inducible cytochrome P-450 from hamster liver microsomes.

    PubMed Central

    Puccini, P; Menicagli, S; Longo, V; Santucci, A; Gervasi, P G

    1992-01-01

    A form of cytochrome P-450 has been purified to electrophoretic homogeneity from the hepatic microsomes of Syrian golden hamsters treated with acetone. This P-450 form, designated ha P-450j, had an M(r) of approximately 55,000, bound dimethyl sulphoxide and exhibited a CO-reduced absorbance maximum at 451 nm. The absolute spectra of its oxidized form indicated that ha P-450j was predominantly in the low-spin state. In a reconstituted system, ha P-450j showed relatively low catalytic activities towards 7-ethoxycoumarin, 7-ethoxyresorufin, aminopyrine, ethylmorphine and benzphetamine, whereas it catalysed the oxidation of aniline, acetone and thiobenzamide with a high catalytic-centre activity. In addition, ha P-450j catalysed at a high rate the high-affinity component of dimethylnitrosamine N-demethylase; in contrast, only the low-affinity component of diethylnitrosamine N-de-ethylase was efficiently catalysed. The addition of cytochrome b5 to the reconstitution system decreased the Km value for dimethylnitrosamine N-demethylase by a factor of 5 and increased the Vmax. value, and slightly enhanced the other activities. Thiobenzamide and diethyldithiocarbamate were found to be the most effective inhibitors of the ha-P-450j-dependent aniline hydroxylation. Polyclonal antibodies against rat P-450j recognized ha P-450j in immunoblots of control and treated hamster liver microsomes. Treatment of hamsters with acetone increased the apparent abundance of ha P-450j in microsomes, whereas phenobarbital and beta-naphthoflavone did not induce it. Analysis of N-terminal amino acid sequences demonstrated that ha P-450j has a high degree of sequence identity with rat P-450j. All the evidence presented in this study indicates that ha P-450j could represent the hamster orthologue of the previously described CYP2E1(s) of other species. Images Fig. 1. Fig. 6. PMID:1445245

  16. Expression and purification of orphan cytochrome P450 4X1 and oxidation of anandamide

    PubMed Central

    Stark, Katarina; Dostalek, Miroslav; Guengerich, F. Peter

    2016-01-01

    Summary Cytochrome P450 (P450) 4X1 is one of the so-called orphan P450s without assigned biological function. Codon-optimized P450 4X1 and a number of N-terminal modified sequences were expressed in Escherichia coli. Native P450 4X1 showed a characteristic P450 spectrum but low expression in E. coli DH5? cells (<100 nmol P450/L). The highest level of expression (300-450 nmol P450/L culture) was achieved with a bicistronic P450 4X1 construct (N-terminal MAKKTSSKGKL, change of E2A, amino acids 3-44 truncated). Anandamide (arachidonoyl ethanolamide) has emerged as an important signaling molecule in the neurovascular cascade. Recombinant P450 4X1 protein, co-expressed with human NADPH-P450 reductase in E. coli, was found to convert the natural endocannabinoid anandamide to a single monooxygenated product, 14,15-epoxyeicosatrienoic (EET) ethanolamide. A stable anandamide analog (CD-25) was also converted to a monooxygenated product. Arachidonic acid was oxidized more slowly to 14,15- and 8,9-EETs but only in the presence of cytochrome b5. Other fatty acids were investigated as putative substrates but showed only little or minor oxidation. Real-time PCR analysis demonstrated extrahepatic mRNA expression, including several human brain structures (cerebellum, amygdala, and basal ganglia), in addition to expression in human heart, liver, prostate, and breast. The highest mRNA expression levels were detected in amygdala and skin. The ability of P450 4X1 to generate anandamide derivatives and the mRNA distribution pattern suggest a potential role for P450 4X1 in anandamide signaling in the brain. PMID:18549450

  17. Oxygenation mechanism in conversion of aldehyde to carboxylic acid catalyzed by a cytochrome P-450 isozyme.

    PubMed

    Watanabe, K; Narimatsu, S; Yamamoto, I; Yoshimura, H

    1991-02-15

    The oxygenation of an aldehyde, 11-oxo-delta 8-tetrahydrocannabinol to a carboxylic acid, delta 8-tetrahydrocannabinol-11-oic acid was catalyzed by cytochrome P-450 MUT-2 purified from hepatic microsomes of male ddN mice. The oxygenation mechanism was confirmed by the incorporation of oxygen-18 from molecular oxygen into the carboxylic acid formed. An aldehyde form but not a hydrated form of 11-oxo-delta 8-tetrahydrocannabinol may be a substrate for the cytochrome P-450. The oxygenation of aldehyde catalyzed by cytochrome P-450 might be a common metabolic reaction in biological systems, and should be considered as an additional role of cytochrome P-450 in biotransformation of endogenous compounds and xenobiotics. PMID:1847130

  18. [Nanoelectrochemistry of cytochrome P450s: direct electron transfer and electrocatalysis].

    PubMed

    Shumiantseva, V V; Bulko, T V; Rudakov, Iu O; Samenkova, N F; Lisitsa, A V; Karuzina, I I; Archakov, A I

    2006-01-01

    The present study demonstrates the direct electron transfer between cytochrome P450 2B4 (CYP2B4), P450 1A2 (CYP1A2), sterol 14alpha-demethylase (CYP51MT) and screen printed graphite electrodes, modified with gold nanoparticles and didodecyldimethylammonium bromide (DDAB). Electrodetection of heme proteins is possible when 2-200 pmol P450/electrode were adsorbed on the surface of nanostructured electrochemical interfaces. Electron transfer, direct electrochemical reduction and interaction with P450 substrates (oxygen, benzphetamine, lanosterol) and inhibitor ketoconazole were analyzed using cyclic voltammetry (CV), square wave (SWV) or differential pulse (DPV) voltammetry, amperometry. PMID:17180920

  19. The ratio of two isozyme groups in microsomal cytochrome P-450 under exogenous influence of carbon tetrachloride and cyclophosphamide.

    PubMed

    Izotov, M V; Shcherbakov, V M; Devichensky, V M; Spiridonova, S M; Lugovaja, L V; Benediktova, S A

    1988-12-01

    A method for measuring the content of two groups of microsomal cytochrome P-450 isozymes--cytochromes P-450W and P-450L--with the active sites directed into the water phase and membrane lipids, respectively, has been developed. The method is based on the ability of the xanthine oxidase-menadione complex to reduce microsomal cytochromes b5 and P-450 under anaerobic conditions by transferring electrons to hemoproteins with the active sites directed into the water phase. Cytochrome b5 is completely reduced (to the dithionite level) and cytochrome P-450 is reduced partially (only a group of cytochromes P-450W). The amount of cytochromes P-450L is estimated using the difference between the total content of cytochrome P-450 reduced by sodium dithionite and the content of cytochromes P-450W. The possibility of controlling the ratio of these two isozyme groups in cytochrome P-450 in vivo in membranes of the endoplasmic reticulum by pretreatment of animals with a variety of chemicals has been demonstrated. The ratio of cytochromes P-450W and P-450L has been shown to decrease two-fold 18 days after three injections of phenobarbital into mice. Carbon tetrachloride and cyclophosphamide also decrease this ratio in vivo. PMID:3233147

  20. [Preparation of NADPH-cytochrome P-450 reductase and the role in metabolism of xenobiotics].

    PubMed

    Wang, L E; Yang, M; Xie, G

    1997-07-01

    NADPH-Cytochrome P-450 Reductase is the major part of MFO (mixed function oxidase) system of liver. A simple and economic method of preparation of reductase was introduced in this paper and it was certified that xenobiotics benzene, bichloroethylene, and asbestos can be oxidized by reductase and reconstituted cytochrome P-450 2B1. But the metabolites of the 3 compounds can not cause the changes of plasmid PBR322 DNA. PMID:10325590

  1. Xenobiotic oxidation by cytochrome P-450-enriched extracts of Streptomyces griseus.

    PubMed

    Trower, M K; Sariaslani, F S; Kitson, F G

    1988-12-30

    Crude extracts of Streptomyces griseus grown on soybean flour-enriched medium contain high levels of cytochrome P-450. The cytochrome P-450-enriched fractions, obtained by ammonium sulfate fractionation (30-50% saturation), catalyze the NADPH-dependent oxidation of a variety of xenobiotics when complemented with both spinach ferredoxin:NADP+ oxidoreductase and spinach ferredoxin. Reactions observed are aromatic, benzylic and alicyclic hydroxylations, O-dealkylation, non-aromatic double bond epoxidation, N-oxidation and N-acetylation. PMID:3144975

  2. Marmoset cytochrome P450 2D8 in livers and small intestines metabolizes typical human P450 2D6 substrates, metoprolol, bufuralol and dextromethorphan.

    PubMed

    Uehara, Shotaro; Uno, Yasuhiro; Hagihira, Yuya; Murayama, Norie; Shimizu, Makiko; Inoue, Takashi; Sasaki, Erika; Yamazaki, Hiroshi

    2015-01-01

    1.?Although the New World non-human primate, the common marmoset (Callithrix jacchus), is a potentially useful animal model, comprehensive understanding of drug metabolizing enzymes is insufficient. 2.?A cDNA encoding a novel cytochrome P450 (P450) 2D8 was identified in marmosets. The amino acid sequence deduced from P450 2D8 cDNA showed a high sequence identity (83-86%) with other primate P450 2Ds. Phylogenetic analysis showed that marmoset P450 2D8 was closely clustered with human P450 2D6, unlike P450 2Ds of miniature pig, dog, rabbit, guinea pig, mouse or rat. 3.?Marmoset P450 2D8 mRNA was predominantly expressed in the liver and small intestine among the tissues types analyzed, whereas marmoset P450 2D6 mRNA was expressed predominantly in the liver where P450 2D protein was detected by immunoblotting. 4.?By metabolic assays using marmoset P450 2D8 protein heterologously expressed in Escherichia coli, although P450 2D8 exhibits lower catalytic efficiency compared to marmoset and human P450 2D6 enzymes, P450 2D8 mediated O-demethylations of metoprolol and dextromethorphan and bufuralol 1'-hydroxylation. 5.?These results suggest that marmoset P450 2D8 (also expressed in the extrahepatic tissues) has potential roles in drug metabolism in a similar manner to those of human and marmoset P450 2D6. PMID:25801057

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

    SciTech Connect

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

    1993-09-01

    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 or phenobarbital. Compared to controls, 3-methylcholanthrene induced a greater than fivefold increase in activities of several monooxygenases 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[prime]-DDE were detected in embryos from Cat Island. Cytochrome P450-associated monooxygenase activities and cytochrome P450 proteins (AHH, BROD, EROD, ECOD, CYP1A, CYP1B) were significantly associated with total PCB burdens.

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

    PubMed Central

    Kullman, S W; Matsumura, F

    1997-01-01

    A gene fragment belonging to the cytochrome P-450 superfamily has been cloned and identified from stationary cultures of the filamentous fungus Phanerochaete chrysosporium by reverse transcriptase (RT)-PCR. A set of degenerate primers homologous to highly conserved regions of known cytochrome P-450 sequences were used for initial RT-PCRs. Individual PCR products were cloned, sequenced, and identified as those belonging to the cytochrome P-450 superfamily based on amino acid sequence homologies and the presence of the highly conserved heme binding region. The nucleotide sequence of a single cDNA clone indicated the presence of an open reading frame encoding a partial cytochrome P-450 protein of 208 amino acids. Comparisons of the deduced amino acid sequence of the partial protein to other known cytochrome P-450 sequences indicate that it is the first member of a new family of cytochrome P-450s, designated CYP63-1A. Northern blot analysis suggests that CYP63-1A is expressed under both nitrogen-rich and nitrogen-deficient culture conditions and thus not under the same regulatory constraints as the well-studied lignin and manganese peroxidases. Western blot analyses using antibodies raised to the heme binding region of CYP63-1A indicate that the protein has a molecular mass of approximately 44,000 Da. PMID:9212420

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

    PubMed Central

    Ning, Daliang; Wang, Hui

    2012-01-01

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

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

    SciTech Connect

    Yasutake, Yoshiaki; Imoto, Noriko; Fujii, Yoshikazu; Fujii, Tadashi; Arisawa, Akira; Tamura, Tomohiro

    2007-10-05

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

  7. Substrate Selectivities and Catalytic Activities of Marmoset Liver Cytochrome P450 2A6 Differed from Those of Human P450 2A6.

    PubMed

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

    2015-07-01

    The common marmoset (Callithrix jacchus), a New World primate species, is potentially a useful animal model for preclinical studies in drug development. However, cytochrome P450 (P450) enzymes have not been fully identified and characterized in marmosets. In this study, we identified P450 2A6 cDNA with the sequence highly identical (91-94%) to human P450 2A6, 2A7, and 2A13 cDNA and cynomolgus monkey P450 2A23, 2A24, and 2A26 cDNA. Among the tissue types examined, marmoset P450 2A6 mRNA was most abundantly expressed in livers where P450 2A6 protein was also detected by immunoblotting. Phylogenetic analysis showed that marmoset P450 2A6 was more closely clustered with human and cynomolgus monkey P450 2As than P450 2As of dog, rat, and mouse (the species also used in drug metabolism). Marmoset P450 2A6 heterologously expressed in Escherichia coli membranes efficiently catalyzed 7-ethoxycoumarin O-deethylation, similar to human P450 2A6 and 2A13 and cynomolgus monkey P450 2A23, 2A24, and 2A26, but much less effectively coumarin 7-hydroxylation, showing some difference as well. Interestingly, marmoset P450 2A6 and cynomolgus monkey P450 2A23 catalyzed phenacetin O-deethylation, which is catalyzed by human P450 1A2 and 2A13, but not by P450 2A6. Marmoset P450 2A6 also exhibited catalytic activity toward testosterone by the multiple sites, but not rat P450 2A-specific testosterone 7α-hydroxylation activity. These results indicated that marmoset P450 2A6 had functional characteristics different from those of human and cynomolgus monkey P450 2As in terms of partially different substrate specificities and catalytic activities, indicating its importance of further studies for P450 2A-dependent drug metabolism in marmosets. PMID:25858611

  8. Copper deficiency increases cytochrome P450-dependent 7-ethoxyresorufin-O-deethylase activity in rat small intestine.

    PubMed

    Johnson, W T; Smith, T B

    1994-12-01

    Although impaired heme synthesis during copper deficiency may limit the production and function of hemoproteins, little is known about the effects of copper deficiency on the cytochromes P450, an important family of hemoproteins, in the small intestine. A series of experiments was conducted to examine the effects of copper deficiency on cytochrome P450 content, ethoxyresorufin-O-deethylase (EROD) activity, and NADPH-cytochrome P450 reductase activity in rat small intestine. Sixteen hours after a single oral administration of 5,6-benzoflavone (BF), an inducer of cytochromes P4501A1 and P4501A2, intestinal cytochrome P450 content was elevated as indicated by the CO-difference spectrum of the reduced cytochrome and by immunoblotting using anticytochrome P4501A1/1A2. However, cytochrome P450 content, measured by either method following BF induction, was not affected by copper deficiency. Thus, copper deficiency did not impair the availability of heme for cytochrome P450 synthesis in the small intestine. Even though copper deficiency did not affect intestinal cytochrome P450 content, EROD activity, which is a cytochrome P450-associated monooxygenase activity, was 9-fold higher in copper-deficient rats compared with controls following BF treatment. The finding that copper deficiency has no effect on intestinal cytochrome P450 content suggests that the increased EROD activity results from an effect of copper deficiency on the cytochrome P450 reductase component of the monooxygenase system. Measurement of cytochrome P450 reductase activity showed a 2-fold increase in the small intestines of copper-deficient rats compared with controls. It is possible, therefore, that increased intestinal cytochrome P450 reductase activity during copper deficiency increases EROD activity by facilitating the flow of electrons to cytochrome P450 during the redox cycle that cytochrome P450 undergoes during the O-deethylation of ethoxyresorufin. PMID:7800686

  9. Systematic genetic and genomic analysis of cytochrome P450 enzyme activities in human liver

    PubMed Central

    Yang, Xia; Zhang, Bin; Molony, Cliona; Chudin, Eugene; Hao, Ke; Zhu, Jun; Gaedigk, Andrea; Suver, Christine; Zhong, Hua; Leeder, J. Steven; Guengerich, F. Peter; Strom, Stephen C.; Schuetz, Erin; Rushmore, Thomas H.; Ulrich, Roger G.; Slatter, J. Greg; Schadt, Eric E.; Kasarskis, Andrew; Lum, Pek Yee

    2010-01-01

    Liver cytochrome P450s (P450s) play critical roles in drug metabolism, toxicology, and metabolic processes. Despite rapid progress in the understanding of these enzymes, a systematic investigation of the full spectrum of functionality of individual P450s, the interrelationship or networks connecting them, and the genetic control of each gene/enzyme is lacking. To this end, we genotyped, expression-profiled, and measured P450 activities of 466 human liver samples and applied a systems biology approach via the integration of genetics, gene expression, and enzyme activity measurements. We found that most P450s were positively correlated among themselves and were highly correlated with known regulators as well as thousands of other genes enriched for pathways relevant to the metabolism of drugs, fatty acids, amino acids, and steroids. Genome-wide association analyses between genetic polymorphisms and P450 expression or enzyme activities revealed sets of SNPs associated with P450 traits, and suggested the existence of both cis-regulation of P450 expression (especially for CYP2D6) and more complex trans-regulation of P450 activity. Several novel SNPs associated with CYP2D6 expression and enzyme activity were validated in an independent human cohort. By constructing a weighted coexpression network and a Bayesian regulatory network, we defined the human liver transcriptional network structure, uncovered subnetworks representative of the P450 regulatory system, and identified novel candidate regulatory genes, namely, EHHADH, SLC10A1, and AKR1D1. The P450 subnetworks were then validated using gene signatures responsive to ligands of known P450 regulators in mouse and rat. This systematic survey provides a comprehensive view of the functionality, genetic control, and interactions of P450s. PMID:20538623

  10. An artificial electron donor supported catalytic cycle of Pseudomonas putida cytochrome P450{sub cam}

    SciTech Connect

    Prasad, Swati . E-mail: swati@scripps.edu; Murugan, Rajamanickam; Mitra, Samaresh

    2005-09-23

    Putidaredoxin (PdX), the physiological effector of cytochrome P450{sub cam} (P450{sub cam}), serves to gate electron transfer into oxy-P450{sub cam} during the catalytic cycle of the enzyme. Redox-linked structural changes in PdX are necessary for the effective P450{sub cam} turnover reaction. PdX is believed to be difficult to be replaced by an artificial electron donor in the reaction pathway of P450{sub cam}. We demonstrate that the catalytic cycle of wild-type P450{sub cam} can be supported in the presence of an artificial reductant, potassium ferrocyanide. Upon rapid mixing of ferrocyanide ion with P450{sub cam}, we observed an intermediate with spectral features characteristic of compound I. The rate constant for the formation of compound I in the presence of ferrocyanide supported reaction cycle was found to be comparable to the ones observed for H{sub 2}O{sub 2} supported compound I formation in wild-type P450{sub cam}, but was much lower than those observed for classical peroxidases. The results presented in this paper form the first kinetic analysis of this intermediate for an artificial electron-driven P450{sub cam} catalytic pathway in solution.

  11. The diverse chemistry of cytochrome P450 17A1 (P450c17, CYP17A1).

    PubMed

    Yoshimoto, Francis K; Auchus, Richard J

    2015-07-01

    The steroid hydroxylation and carbon-carbon bond cleavage activities of cytochrome P450 17A1 (CYP17A1) are responsible for the production of glucocorticoids and androgens, respectively. The inhibition of androgen synthesis is an important strategy to treat androgen-dependent prostate cancer. We discuss the different enzymatic activities towards the various substrates of CYP17A1, demonstrating its promiscuity. Additionally, a novel interhelical interaction is proposed between the F-G loop and the B'-helix to explain the 16?-hydroxylase activity of human CYP17A1 with progesterone as the substrate. The techniques used by biochemists to study this important enzyme are also summarized. This article is part of a Special Issue entitled 'Steroid/Sterol signaling'. PMID:25482340

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

    USGS Publications Warehouse

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

    1993-01-01

    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.

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

    USGS Publications Warehouse

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

    1993-01-01

    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.

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

    ERIC Educational Resources Information Center

    Furge, Laura Lowe; Fletke, Kyle J.

    2007-01-01

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

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

    ERIC Educational Resources Information Center

    Furge, Laura Lowe; Fletke, Kyle J.

    2007-01-01

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

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

    PubMed Central

    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

    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

  17. Cytochromes P450 2C1/2 and P450 2E1 are retained in the endoplasmic reticulum membrane by different mechanisms.

    PubMed

    Szczesna-Skorupa, E; Chen, C D; Kemper, B

    2000-02-15

    Cytochrome P450 (P450) 2C1/2 contains redundant endoplasmic reticulum (ER) retention signals and is excluded from the recycling pathway. Other P450s, such as P450 2E1, have been detected in the plasma membrane and Golgi apparatus. To examine whether the mechanisms of ER retention might differ for P450 2C1/2 and P450 2E1, chimeras of green flourescent protein and the full-length proteins, N-terminal signal/anchor sequences, or the cytoplasmic catalytic domains from these proteins have been expressed in COS1 cells. Chimeras with either the N-terminal signal/anchor sequence or the cytoplasmic domain of P450 2C1/2 were retained in the ER and the distribution was not altered by treatment with nocodazole. A chimera with full-length P450 2E1 was located in the ER, but in contrast to P450 2C1/2, treatment with nocodazole resulted in redistribution to a vesicular pattern, which suggested that this protein was retained in the ER by a retrieval mechanism. In support of this possibility, the P450 2E1 chimera, but not the P450 2C1/2 chimera, was included in transport vesicles generated in an in vitro budding assay. A chimera with only the N-terminal signal/anchor sequence of P450 2E1 fused to green fluorescent protein was located in the ER and nocodazole treatment altered its distribution, whereas a chimera with only the cytoplasmic domain of P450 2E1 was not efficiently retained in the ER and accumulated primarily in the Golgi region. These results demonstrate that the mechanisms for retention in the ER of two closely related members of the P450 superfamily are different and that the N-terminal signal/anchor sequence contains the dominant retention signal. PMID:10666290

  18. Structural rationalization of novel drug metabolizing mutants of cytochrome P450 BM3.

    PubMed

    Stjernschantz, Eva; van Vugt-Lussenburg, Barbara M A; Bonifacio, Alois; de Beer, Stephanie B A; van der Zwan, Gert; Gooijer, Cees; Commandeur, Jan N M; Vermeulen, Nico P E; Oostenbrink, Chris

    2008-04-01

    Three newly discovered drug metabolizing mutants of cytochrome P450 BM3 (van Vugt-Lussenburg et al., Identification of critical residues in novel drug metabolizing mutants of Cytochrome P450 BM3 using random mutagenesis, J Med Chem 2007;50:455-461) have been studied at an atomistic level to provide structural explanations for a number of their characteristics. In this study, computational methods are combined with experimental techniques. Molecular dynamics simulations, resonance Raman and UV-VIS spectroscopy, as well as coupling efficiency and substrate-binding experiments, have been performed. The computational findings, supported by the experimental results, enable structural rationalizations of the mutants. The substrates used in this study are known to be metabolized by human cytochrome P450 2D6. Interestingly, the major metabolites formed by the P450 BM3 mutants differ from those formed by human cytochrome P450 2D6. The computational findings, supported by resonance Raman data, suggest a conformational change of one of the heme propionate groups. The modeling results furthermore suggest that this conformational change allows for an interaction between the negatively charged carboxylate of the heme substituent and the positively charged nitrogen of the substrates. This allows for an orientation of the substrates favorable for formation of the major metabolite by P450 BM3. PMID:17957765

  19. Polymer phase partition in the purification of cytochrome P-450 and cytochrome b5 from the yeast Brettanomyces anomalus.

    PubMed

    Krenlampi, S O; Nikkil, H; Hynninen, P H

    1986-02-01

    About 0.5% of the total cellular protein in the yeast Brettanomyces anomalus is membrane-bound cytochrome P-450, when this yeast is grown in the presence of 5% glucose as the main carbon and energy source. A partial purification of cytochrome P-450 by phase partition is described. Breakdown of yeast cell walls with microbial enzyme preparations led to extensive losses of this hemoprotein. Instead, by a carefully controlled mechanical breakage as much as 50% of the total cellular cytochrome P-450 could be recovered. During the solubilization of cytochrome P-450 from the cell homogenate with Triton X-100, the protective agents dithiothreitol, EDTA, and butylated hydroxytoluene prevented major losses of the hemoprotein. Applying a three-phase partition system (polyethylene glycol-Ficoll-dextran) to the solubilized whole cell homogenate in the presence of 1 M sodium chloride, followed by a precipitation of the top "oily layer" with 25% polyethylene glycol, a 25- to 60-fold enrichment of cytochrome P-450 was obtained. This corresponds to a specific content of 0.8-2.2 nmol of cytochrome P-450 per milligram of protein. Cytochrome b5 enriched (41%) to the PEG-Ficoll interphase, and NADPH-cytochrome c reductase and "cytochromes P-420" to the Ficoll and dextran phases. The polymer phase partition system thus serves as an excellent initial purification step of cytochrome P-450 without a need for the preparation of the microsomal fraction. Another advantage of the method is that it allows the simultaneous partial purification of cytochrome b5. PMID:3828082

  20. [Effect of allyl isothiocyanate on cytochrome P-450 of rat liver microsomes].

    PubMed

    Plass, R; Lewerenz, H J; Macholz, R M

    1983-01-01

    The thrice-repeated oral administration of graded doses (50, 100 and 150 mg/kg of body mass) of allyl mustard oil to rats resulted in a decrease of the cytochrome P-450 and cytochrome b5 contents of the liver microsomes. There occurred a dose-dependent increase in mass of this organ. PMID:6669172

  1. Electrochemical properties of cytochroms P450 using nanostructured electrodes: direct electron transfer and electro catalysis.

    PubMed

    Shumyantseva, V V; Bulko, T V; Rudakov, Yu O; Kuznetsova, G P; Samenkova, N F; Lisitsa, A V; Karuzina, I I; Archakov, A I

    2007-05-01

    The present study demonstrates direct electron transfer between cytochromes P450 2B4 (CYP2B4), P450 1A2 (CYP1A2), sterol 14alpha-demethylase (CYP51b1) on the one hand and screen-printed graphite electrodes, modified with gold nanoparticles and didodecyldimethylammonium bromide (DDAB) on the other. Electro detection of heme proteins was possible when 2-200 pmol P450/electrode were adsorbed on the surface of nanostructured electrochemical interfaces. Electron transfer, direct electrochemical reduction and interaction with P450 substrates (oxygen, benzphetamine, and lanosterol) and with P450 inhibitor (ketoconazole) were analyzed using cyclic voltammetry (CV), square wave voltammetry (SWV) differential pulse voltammetry (DPV), and amperometry. PMID:17376532

  2. A Cytochrome P450 Conserved in Insects Is Involved in Cuticle Formation

    PubMed Central

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

    2012-01-01

    The sequencing of numerous insect genomes has revealed dynamic changes in the number and identity of cytochrome P450 genes in different insects. In the evolutionary sense, the rapid birth and death of many P450 genes is observed, with only a small number of P450 genes showing orthology between insects with sequenced genomes. It is likely that these conserved P450s function in conserved pathways. In this study, we demonstrate the P450 gene, Cyp301a1, present in all insect genomes sequenced to date, affects the formation of the adult cuticle in Drosophila melanogaster. A Cyp301a1 piggyBac insertion mutant and RNAi of Cyp301a1 both show a similar cuticle malformation phenotype, which can be reduced by 20-hydroxyecdysone, suggesting that Cyp301a1 is an important gene involved in the formation of the adult cuticle and may be involved in ecdysone regulation in this tissue. PMID:22574182

  3. Effects of photoisomers of cyclodiene insecticides on liver and microsomal cytochrome P450 in rats.

    PubMed

    Khan, M A; Jovanovich, L V; Martin, L T; Qadri, S Y; Podowski, A A

    1998-01-01

    Threshold dosages of the photoisomers of cyclodiene insecticides, namely photochlordane, photodieldrin, and photoheptachlor, for the induction of hepatic microsomal cytochrome P450 (P450) and liver hypertrophy in male rats were at least one-quarter of those reported for corresponding parent cyclodienes. Maximum increase in total P450 concentration (30%) and demethylases activities (100%) was always respectively one-third or one-tenth of that reported for parent cyclodienes. The P450 isozymic form induced by photoheptachlor resembled that induced by pentobarbital (P4502B1) in its substrate specificity, spectral characteristics, and electrophoretic mobility. The induction of P450 was initially followed by hepatic hypertrophy. However, higher dosages of photoisomers caused wasting and lowered both the liver weight and the activity of aniline hydroxylase while those of mirex and endrin, which also caused wasting and lowered aniline hydroxylase activity, continued causing further hepatic hypertrophy. PMID:9456078

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

    PubMed

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

    2013-10-01

    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

  5. Steroid 11-hydroxylation by a fungal microsomal cytochrome P450.

    PubMed

    Jnig, G R; Pfeil, D; Mller-Frohne, M; Riemer, H; Henning, M; Schwarze, W; Ruckpaul, K

    1992-12-01

    The steroid 11-hydroxylase activity of the fungus Cochliobolus lunatus was increased about 100-fold by cultivation of mycelia for 4-5 h with 20-hydroxymethyl-1,4-pregnadien-3-one. Cell-free extracts revealed a maximum activity of 45 nmol 11-hydroxyprogesterone/hmg protein in the 100,000 g pellet fraction. The 11-hydroxylation was dependent on NADPH. The formation of 11-hydroxyprogesterone correlated linearly with the cytochrome P450 concentration. The fungal 11-hydroxylase transformed both 21-methyl and 21-hydroxymethyl steroids. The enzyme showed a broader substrate specificity and lower regioselectivity as compared with the adrenal cytochrome P45011 system. The fungal cytochrome P450 was partially purified to a specific content of 700 pmol P450/mg protein. Western blots showed that polyclonal antibodies against cytochrome P45011? from Rhizopus nigricans cross-react with a 60 kD protein of partially purified fractions. The NADPH-cytochrome c reductase was enriched up to a specific activity of 20 U/mg protein. Polyclonal antibodies against NADPH-cytochrome P450 reductases from Candida maltosa and rat liver cross-reacted with the fungal reductase. It is concluded that the 11-hydroxylase of Cochliobolus lunatus represents a microsomal two-component monooxygenase system which is composed of a cytochrome P450 (M(r) 60 kD) and a NADPH-cytochrome P450 reductase (M(r) 79 kD). PMID:22217857

  6. Structure of the open conformation of a functional chimeric NADPH cytochrome P450 reductase

    PubMed Central

    Aigrain, Louise; Pompon, Denis; Morra, Solange; Truan, Gilles

    2009-01-01

    Two catalytic domains, bearing FMN and FAD cofactors, joined by a connecting domain, compose the core of the NADPH cytochrome P450 reductase (CPR). The FMN domain of CPR mediates electron shuttling from the FAD domain to cytochromes P450. Together, both enzymes form the main mixed-function oxidase system that participates in the metabolism of endo- and xenobiotic compounds in mammals. Available CPR structures show a closed conformation, with the two cofactors in tight proximity, which is consistent with FAD-to-FMN, but not FMN-to-P450, electron transfer. Here, we report the 2.5 resolution crystal structure of a functionally competent yeasthuman chimeric CPR in an open conformation, compatible with FMN-to-P450 electron transfer. Comparison with closed structures shows a major conformational change separating the FMN and FAD cofactors from 86 . PMID:19483672

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

    SciTech Connect

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

    2012-02-17

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

  8. Inhalation of butanols: changes in the cytochrome P-450 enzyme system.

    PubMed

    Aarstad, K; Zahlsen, K; Nilsen, O G

    1985-01-01

    After inhalation of different butanol isomers for 3 days (2000 ppm) and 5 days (500 ppm), liver and kidney parameters of the microsomal cytochrome P-450 enzyme system were increased. sec-Butanol caused the highest increase in cytochrome P-450 concentration with a 47% rise in the kidneys (500 ppm for 5 days) and 33% in the liver (2000 ppm for 3 days). A concomitant increase of the in vitro n-hexane metabolism in liver microsomes was observed with a 77% increased formation of the preneurotoxic metabolite 2-hexanol compared with control. iso-Butanol did not alter total cytochrome P-450 concentration but caused a significant 30% decrease in the formation of 2-hexanol. Inhalation of all butanols slightly decreased the enzyme levels in the lung. Changes in microsomal enzymes did not correlate with measured serum concentrations of the different butanols showing different inducing capacities among the butanol isomers themselves or the participation of metabolites in the inducing process. As a conclusion sec-butanol, probably through its metabolite methyl-ethyl-ketone, is the most potent inducer of microsomal cytochrome P-450 in liver and kidney while iso-butanol does not alter total cytochrome P-450. PMID:3938271

  9. Incorporation of haemoglobin haem into the rat hepatic haemoproteins tryptophan pyrrolase and cytochrome P-450.

    PubMed Central

    Wyman, J F; Gollan, J L; Settle, W; Farrell, G C; Correia, M A

    1986-01-01

    After its administration to intact rats, haemoglobin haem was incorporated into hepatic tryptophan pyrrolase as shown by the marked increase in functional constitution of this enzyme. Incorporation of haemoglobin haem into cytochrome P-450 was demonstrated in intact rats and in the isolated rat liver perfused with haemoglobin-free medium. In both systems, haemoglobin haem restored cytochrome P-450 content and its dependent mixed-function-oxidase activity after substrate-induced destruction of the cytochrome P-450 haem moiety. Further confirmation that haemoglobin haem could be incorporated prosthetically into cytochrome P-450 was achieved by administration of [3H]haemoglobin to rats and subsequent isolation and characterization of radiolabelled substrate-alkylated products of cytochrome P-450 haem. Our findings indicate that, although hepatic uptake of parenteral haemoglobin is slower than that of haem, it appears to serve as an effective haem donor to the intrahepatic 'free' haem pool. Thus parenteral haemoglobin may warrant consideration as a therapeutic alternative to haem in the acute hepatic porphyrias. PMID:3800964

  10. Incorporation of haemoglobin haem into the rat hepatic haemoproteins tryptophan pyrrolase and cytochrome P-450

    SciTech Connect

    Wyman, J.F.; Gollan, J.L.; Settle, W.; Farrell, G.C.; Correia, M.A.

    1986-01-01

    After its administration to intact rats, haemoglobin haem was incorporated into hepatic tryptophan pyrrolase as shown by the marked increase in functional constitution of this enzyme. Incorporation of haemoglobin haem into cytochrome P-450 was demonstrated in intact rats and in the isolated rat liver perfused with haemoglogin-free medium. In both systems, haemoglobin haem restored cytochrome P-450 content and its dependent mixed-function-oxidase activity after substrate-induced destruction of the cytochrome P-450 haem moiety. Further confirmation that heamoglobin haem could be incorporated prosthetically into cytochrome P-450 was achieved by administration of (tritium) haemoglobin to rats and subsequent isolation and characterization of radiolabelled substrate-alkylated products of cytochrome P-450 haem. Findings indicate that, although hepatic uptake of parenteral haemoglobin is slower than that of haem, it appears to serve as an effective haem donor to the intrahepatic free haem pool. Thus parenteral haemoglobin may warrant consideration as a therapeutic alternative to haem in the acute hepatic porphyrias.

  11. NADPH-cytochrome P450 oxidoreductase: roles in physiology, pharmacology, and toxicology.

    PubMed

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

    2013-01-01

    This is a report on a symposium sponsored by the American Society for Pharmacology and Experimental Therapeutics and held at the Experimental Biology 2012 meeting in San Diego, California, on April 25, 2012. The symposium speakers summarized and critically evaluated our current understanding of the physiologic, pharmacological, and toxicological roles of NADPH-cytochrome P450 oxidoreductase (POR), a flavoprotein involved in electron transfer to microsomal cytochromes P450 (P450), cytochrome b(5), squalene mono-oxygenase, and heme oxygenase. Considerable insight has been derived from the development and characterization of mouse models with conditional Por deletion in particular tissues or partial suppression of POR expression in all tissues. Additional mouse models with global or conditional hepatic deletion of cytochrome b(5) are helping to clarify the P450 isoform- and substrate-specific influences of cytochrome b(5) on P450 electron transfer and catalytic function. This symposium also considered studies using siRNA to suppress POR expression in a hepatoma cell-culture model to explore the basis of the hepatic lipidosis phenotype observed in mice with conditional deletion of Por in liver. The symposium concluded with a strong translational perspective, relating the basic science of human POR structure and function to the impacts of POR genetic variation on human drug and steroid metabolism. PMID:23086197

  12. Purification and characterization of an anticonvulsant-induced human cytochrome P-450 catalysing cyclosporin metabolism.

    PubMed Central

    Shaw, P M; Barnes, T S; Cameron, D; Engeset, J; Melvin, W T; Omar, G; Petrie, J C; Rush, W R; Snyder, C P; Whiting, P H

    1989-01-01

    A form of human hepatic microsomal cytochrome P-450 (P450hA7) with subunit Mr 50,400 has been purified from an epileptic who had been receiving long-term treatment with anticonvulsant drugs. P450hA7 metabolized the immunosuppressant drug cyclosporin A and the dihydropyridine calcium channel antagonist nifedipine, but did not metabolize a similar dihydropyridine drug, nicardipine, nor a series of alkoxyresorufin model substrates. The hepatic microsomal concentration of P450hA7 was higher in five individuals who had been receiving long-term anticonvulsant treatment than in any of 21 individuals who had not been similarly treated. The mean P450hA7 concentration in the treated individuals was 5-fold higher than the mean concentration in the untreated individuals. It is concluded that P450hA7 is a member of the cytochrome P450III family which is induced by anticonvulsant drugs in man. Images Fig. 1. Fig. 4. Fig. 5. Fig. 6. PMID:2688634

  13. Cytochrome P450 Family 1 Inhibitors and Structure-Activity Relationships

    PubMed Central

    Liu, Jiawang; Sridhar, Jayalakshmi; Foroozesh, Maryam

    2014-01-01

    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

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

    PubMed

    Cheng, Qian; Sohl, Christal D; Guengerich, F Peter

    2009-01-01

    Cytochrome P450 mono-oxygenases (P450s) are the principal enzymes involved in the oxidative metabolism of drugs and other xenobiotics. In this protocol, we describe a fluorescence-based, high-throughput assay for measuring the activity of P450 3A4, one of the key enzymes involved in drug metabolism. The assay involves the oxidative debenzylation of a substituted coumarin, yielding an increase in fluorescence on reaction. The entire procedure can be accomplished in 1 h or less. PMID:19661996

  15. Identification of key residues in rabbit liver microsomal cytochrome P450 2B4: importance in interactions with NADPH-cytochrome P450 reductase.

    PubMed

    Lehnerer, M; Schulze, J; Achterhold, K; Lewis, D F; Hlavica, P

    2000-01-01

    A cytochrome P450 2B4 (CYP2B4) model was used to select key residues supposed to serve in interactions with NADPH-cytochrome P450 reductase (P450R). Eight amino acid residues located on the surface of the hemoprotein were chosen for mutagenesis experiments with CYP2B4(Delta2-27) lacking the NH(2)-terminal signal anchor sequence. The mutated proteins were expressed in Escherichia coli, purified, and characterized by EPR- and CD-spectral analysis. Replacement of histidine 226 with alanine caused a 3.8-fold fall in the affinity for P450R with undisturbed reductive capacity of the system. Similarly, the K225A, R232A, and R253A variants exhibited P450R-directed activity that was depressed to about half that of the control enzyme, suggesting that the deletion of positive charges on the surface of CYP2B4(Delta2-27) resulted in impaired electrostatic contacts with complementary amino acids on the P450R protein. While the Y235A mutant did not show appreciably perturbed reduction activity, the conservative substitution with alanine of the phenylalanine residues at positions 223 and 227 gave a 2.1- to 6. 1-fold increase in the K(m) values with unchanged V(max); this was attributed to the disruption of hydrophobic forces rather than to global structural rearrangement(s) of the engineered pigments. Measurement of the stoichiometry of aerobic NADPH consumption and H(2)O(2) formation revealed the oxyferrous forms of the F223A, H226A, and F227A mutants to autoxidize more readily owing to less efficient coupling of the systems. Noteworthy, the F244A enzyme did not exhibit significant reduction activity, suggesting a pivotal role of Phe-244 in the functional coupling of P450R. The residue was predicted to constitute part of an obligatory electron transfer conduit through pi-stacking with Phe-296 located close to the heme unit. All of the residues examined reside in the putative G helix of CYP2B4, so that this domain obviously defines part of the binding site for P450R. PMID:10731679

  16. Purification and characterization of an NADPH-cytochrome P450 (cytochrome c) reductase from spearmint (Mentha spicata) glandular trichomes.

    PubMed

    Ponnamperuma, K; Croteau, R

    1996-05-01

    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 analysis with polyclonal antibodies directed against NADPH-cytochrome P450 reductase from Jerusalem artichoke and from mung bean. Complete immunoinhibition of reductase activity was observed with both types of polyclonal antibodies, while only partial inhibition of activity resulted using a family of monoclonal antibodies directed against the Jerusalem artichoke cytochrome P450 reductase. Inhibition of the spearmint oil gland cytochrome c reductase was also observed with the diphenyliodonium ion. The K(m) values for the cosubstrates NADPH and cytochrome c were 6.2 and 3.7 microM, respectively, and the pH optimum for activity was at 8.5. The NADPH-cytochrome c reductase reconstituted NADPH-dependent (-)-4S-limonene-6-hydroxylase activity in the presence of cytochrome P450, purified from the microsomal fraction of spearmint oil gland cells and dilauroyl phosphatidyl choline. These characteristics establish the identity of the purified enzyme as a NADPH-cytochrome P450 reductase. PMID:8619640

  17. Expression of a Ripening-Related Avocado (Persea americana) Cytochrome P450 in Yeast 1

    PubMed Central

    Bozak, Kristin R.; O'Keefe, Daniel P.; Christoffersen, Rolf E.

    1992-01-01

    One of the mRNAs that accumulates during the ripening of avocado (Persea americana Mill. cv Hass) has been previously identified as a cytochrome P450 (P450) monooxygenase and the corresponding gene designated CYP71A1. In this report we demonstrate that during ripening the accumulation of antigenically detected CYP71A1 gene product (CYP71A1) correlates with increases in total P450 and two P450-dependent enzyme activities: para-chloro-N-methylaniline demethylase, and trans-cinnamic acid hydroxylase (tCAH). To determine whether both of these activities are derived from CYP71A1, we have expressed this protein in yeast (Saccharomyces cerevisiae) using a galactose-inducible yeast promoter. Following induction, the microsomal fraction of transformed yeast cells undergoes a large increase in P450 level, attributable almost exclusively to the plant CYP71A1 protein. These membranes exhibit NADPH-dependent para-chloro-N-methylaniline demethylase activity at a rate comparable to that in avocado microsomes but have no detectable tCAH. These results demonstrate both that the CYP71A1 protein is not a tCAH and that a plant P450 is fully functional upon heterologous expression in yeast. These findings also indicate that the heterologous P450 protein can interact with the yeast NADPH:P450 reductase to produce a functional complex. Images Figure 1 Figure 3 PMID:16653226

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

    PubMed

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

    2016-01-01

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

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

    PubMed

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

    2015-01-01

    Cytochrome P450 monooxygenases are present in almost all organisms and can play vital roles in hormone regulation, metabolism of xenobiotics and in biosynthesis or inactivation of endogenous compounds. In the present study, a genome-wide approach was used to identify and analyze the P450 gene family of diamondback moth, Plutella xylostella, a destructive worldwide pest of cruciferous crops. We identified 85 putative cytochrome P450 genes from the P. xylostella genome, including 84 functional genes and 1 pseudogene. These genes were classified into 26 families and 52 subfamilies. A phylogenetic tree constructed with three additional insect species shows extensive gene expansions of P. xylostella P450 genes from clans 3 and 4. Gene expression of cytochrome P450s was quantified across multiple developmental stages (egg, larva, pupa and adult) and tissues (head and midgut) using P. xylostella strains susceptible or resistant to insecticides chlorpyrifos and fiprinol. Expression of the lepidopteran specific CYP367s predominantly occurred in head tissue suggesting a role in either olfaction or detoxification. CYP340s with abundant transposable elements and relatively high expression in the midgut probably contribute to the detoxification of insecticides or plant toxins in P. xylostella. This study will facilitate future functional studies of the P. xylostella P450s in detoxification. PMID:25752830

  20. Apocytochrome P-450: reconstitution of functional cytochrome with hemin in vitro.

    PubMed Central

    Correia, M A; Meyer, U A

    1975-01-01

    Synthesis of microsomal cytochrome P-450 in rat liver requires synthesis of apoprotein in rough endoplasmic reticulum and of heme in mitochondria. Dissociation of apoprotein and heme synthesis by concomitant treatment of rats with inducers of cytochrome P-450 (i.e., phenobarbital) and inhibitors of heme synthesis (i.e., cobalt) resulted in a relative excess of apocytochrome P-450. Under these circumstances, it was possible to reconstitute the holocytochrome by addition of hemin in vitro. The holocytochrome was detected spectrophotometrically by its CO-binding properties and functionally by its increased oxidative activity. Heme-mediated reconstitution was most efficient in cell fractions rich in mitochondria-rough endoplasmic reticulum complexes (640 times g fraction), suggesting that the structural association of these two organelles may represent a functional unit essential for the synthesis of holocytochrome P-450. These findings indicate that phenobarbital-mediated induction of apocytochrome P-450 is independent of heme synthesis. It is suggested that synthesis of the apocytochrome may be the primary and rate-limiting event in the formation of cytochrome P-450. PMID:1054513

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

    PubMed Central

    Brault, D

    1985-01-01

    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

  2. Redox Potential Control by Drug Binding to Cytochrome P450 3A4

    PubMed Central

    Das, Aditi; Grinkova, Yelena V.; Sligar, Stephen G.

    2008-01-01

    The cytochrome P450s are ubiquitous heme proteins that utilize two reducing equivalents to cleave a ferrous iron - dioxygen complex to produce a single water molecule with the insertion of one oxygen atom into a bound substrate. For the case of soluble cytochrome P450 CYP101, it has been shown that there is a linear free energy relationship between heme redox potential and the spin state of the ferric protein. However, the universality of this relationship has been challenged in the case of mammalian enzymes. Most cytochrome P450s are integral membrane proteins, and detailed redox potential measurements have proved difficult due protein aggregation or the necessary presence of detergent. In this communication we utilize a soluble nanometer scale membrane bilayer disc (Nanodisc) to stabilize monomeric human cytochrome P450 CYP3A4. The Nanodisc system allows facile redox potential measurements to be made on substrate-free CYP3A4 as well as with several drug molecules bound at the active site. We show that substrate binding can dramatically effect the redox potential of the heme protein through modulation of the ferric spin state. A linear free energy relationship is observed, analogous to that noted for the soluble P450s, indicating a common mechanism for this linkage and providing a means for control of electron input in response to the presence of a metabolizable substrate, this potentially limiting the unwanted production of reduced oxygen species. PMID:17948999

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

    PubMed Central

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

    2013-01-01

    Polaromonas sp. strain JS666 grows on cis-1,2-dichoroethene (cDCE) as the sole carbon and energy source under aerobic conditions, but the degradation mechanism and the enzymes involved are unknown. In this study, we established the complete pathway for cDCE degradation through heterologous gene expression, inhibition studies, enzyme assays, and analysis of intermediates. Several lines of evidence indicate that a cytochrome P450 monooxygenase catalyzes the initial step of cDCE degradation. Both the transient accumulation of dichloroacetaldehyde in cDCE-degrading cultures and dichloroacetaldehyde dehydrogenase activities in cell extracts of JS666 support a pathway for degradation of cDCE through dichloroacetaldehyde. The mechanism minimizes the formation of cDCE epoxide. The molecular phylogeny of the cytochrome P450 gene and the organization of neighboring genes suggest that the cDCE degradation pathway recently evolved in a progenitor capable of degrading 1,2-dichloroethane either by the recruitment of the cytochrome P450 monooxygenase gene from an alkane catabolic pathway or by selection for variants of the P450 in a preexisting 1,2-dichloroethane catabolic pathway. The results presented here add yet another role to the broad array of productive reactions catalyzed by cytochrome P450 enzymes. PMID:23354711

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

    PubMed

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

    2015-10-01

    Parathion, a widely used organophosphate insecticide, is considered a high priority chemical threat. Parathion toxicity is dependent on its metabolism by the cytochrome P450 system to paraoxon (diethyl 4-nitrophenyl phosphate), a cytotoxic metabolite. As an effective inhibitor of cholinesterases, paraoxon causes the accumulation of acetylcholine in synapses and overstimulation of nicotinic and muscarinic cholinergic receptors, leading to characteristic signs of organophosphate poisoning. Inhibition of parathion metabolism to paraoxon represents a potential approach to counter parathion toxicity. Herein, we demonstrate that menadione (methyl-1,4-naphthoquinone, vitamin K3) is a potent inhibitor of cytochrome P450-mediated metabolism of parathion. Menadione is active in redox cycling, a reaction mediated by NADPH-cytochrome P450 reductase that preferentially uses electrons from NADPH at the expense of their supply to the P450s. Using human recombinant CYP 1A2, 2B6, 3A4 and human liver microsomes, menadione was found to inhibit the formation of paraoxon from parathion. Administration of menadione bisulfite (40mg/kg, ip) to rats also reduced parathion-induced inhibition of brain cholinesterase activity, as well as parathion-induced tremors and the progression of other signs and symptoms of parathion poisoning. These data suggest that redox cycling compounds, such as menadione, have the potential to effectively mitigate the toxicity of organophosphorus pesticides including parathion which require cytochrome P450-mediated activation. PMID:26212258

  5. Conformational heterogeneity of cytochrome P450 3A4 revealed by high pressure spectroscopy.

    PubMed

    Davydov, Dmitri R; Halpert, James R; Renaud, Jean-Paul; Hui Bon Hoa, Gaston

    2003-12-01

    We applied hydrostatic pressure perturbation to study substrate-induced transitions in human cytochrome P450 3A4 (CYP3A4) with bromocriptine (BCT) as a substrate. The barotropic behavior of the purified enzyme in solution was compared with that observed in recombinant microsomes of Saccharomyces cerevisiae coexpressing CYP3A4, cytochrome b(5), (b(5)) and NADPH-cytochrome P450 reductase (CPR). Important barotropic heterogeneity of CYP3A4 was detected in both cases. Only about 70% of CYP3A4 in solution and about 50% of the microsomal enzyme were susceptible to a pressure-induced P450-->P420 transition. The results suggest that both in solution and in the membrane CYP3A4 is represented by two conformers with different positions of spin equilibrium and different barotropic properties. No interconversion between these conformers was observed within the time frame of the experiment. Importantly, a pressure-induced spin shift, which is characteristic of all cytochromes P450 studied to date, was detected in CYP3A4 in solution only; the P450-->P420 transition was the sole pressure-induced process detected in microsomes. This fact suggests unusual stabilization of the high-spin state of CYP3A4, which is assumed to reflect decreased water accessibility of the heme moiety due to specific interactions of the hemoprotein with the protein partners (b(5) and CPR) and/or membrane lipids. PMID:14630029

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

    SciTech Connect

    Ishihara, Yasuhiro; Shiba, Dai; Shimamoto, Norio . E-mail: n-shimamoto@kph.bunri-u.ac.jp

    2006-07-15

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

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

    PubMed Central

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

    1985-01-01

    It is well established that liver microsomal cytochrome P-450 participates in steroid metabolism and probably also in the metabolism of anti-oestrogens such as tamoxifen (Nolvadex). Thus it is possible that variations in cytochrome P-450 levels may influence the responsiveness of human breast and endometrial carcinomas to endocrine therapy. Therefore a simple sensitive spectrophotometric assay for determining levels of cytochrome P-450-dependent cyclohexane hydroxylation activity in breast and uterine microsomes (microsomal fractions) has been developed. Cyclohexane was chosen as a substrate because of the relatively high levels of cyclohexane hydroxylase activity in tumour microsomes and because cyclohexane serves as a substrate for several forms of cytochrome P-450. As previously described [Senler, Dean, Pierce & Wittliff (1985) Anal. Biochem. 144, 152-158], a direct method utilizing isotope-dilution/gas chromatography-mass spectrometry was also developed in order to confirm the results of the spectrophotometric assay. The average activity (cyclohexane-dependent NADPH oxidation) for 139 human breast-tumour microsome preparations was 1.34 nmol/min per mg, which is in the range of that found in untreated mammalian liver (1-3 nmol/min per mg). Also, high enzyme activity was demonstrated in human ovary, normal uterus as well as uterine leiomyomas. Endocrine status appeared to influence enzyme levels, in that mammary tissue from virgin rats contained significantly (P less than 0.025) higher amounts of activity than did tissues from either pregnant or lactating rats. Furthermore, carbon monoxide, as well as an antibody against rat liver cytochrome P-450, completely inhibited NADPH oxidation by breast-carcinoma microsomes. These results strengthen our hypothesis that tumours with high levels of cytochrome P-450 may have a reduced response to additive endocrine therapy. PMID:4004771

  8. Acetate anion-triggered peroxygenation of non-native substrates by wild-type cytochrome P450s.

    PubMed

    Onoda, Hiroki; Shoji, Osami; Watanabe, Yoshihito

    2015-09-14

    Cytochrome P450SP? (P450SP?) and cytochrome P450BS? (P450BS?) belonging to the CYP152 family of enzymes (CYP152s) can utilize H2O2 efficiently as an oxidant for the generation of compound I. Although P450SP? and P450BS? have very high substrate specificity and catalyse hydroxylation of long-chain fatty acids exclusively, we found that they can oxidize non-native substrates such as styrene simply by including medium chain length n-alkyl carboxylic acids as "decoy molecules." Although we had assumed that acetic acid did not serve as a decoy molecule, P450SP? and P450BS? efficiently catalysed oxidation of non-native substrates when the reaction was carried out at a high concentration of acetate anion. The turnover rate for epoxidation of styrene catalysed by P450BS? in the presence of 1 M acetate anion reached 590 30 min(-1). PMID:26125329

  9. Effects of serum from patients with chronic renal failure on rat hepatic cytochrome P450

    PubMed Central

    Michaud, Jose; Dub, Pierre; Naud, Judith; Leblond, Francois A; Desbiens, Karine; Bonnardeaux, Alain; Pichette, Vincent

    2005-01-01

    In humans, chronic renal failure (CRF) is associated with decreased hepatic drug metabolism, particularly that mediated by the cytochrome P450 (P450). The mechanisms remain poorly understood. The present study aimed to investigate the effects of the serum of patients with CRF on liver P450, and to evaluate whether renal replacement therapies (dialysis or transplantation) impede the inhibition of CRF serum on P450. Rat hepatocytes were incubated for 24?h with serum from patients with severe CRF and from controls to measure (1) P450 level, (2) protein expression and mRNA levels of P450 isoforms and (3) metabolic activities of CYP3A and CYP1A. Similar experiments were performed with serum of patients once on chronic hemodialysis and after kidney transplantation. In rat hepatocytes incubated for 24?h with serum from patients with CRF, P450 level and protein expression, as well as mRNA levels of P450 isoforms (CYP1A2, 2C6, 2C11, 2D1/2D2, 3A2 and 4A1/4A3), were decreased by more than 45% (P<0.001) compared to control serum, while the levels of CYP2E1 were not modified. CYP3A and CYP1A activities were decreased by 51 and 59% (P<0.001), respectively. The inhibitory effect of serum obtained from patients before first dialysis was similar after 1 or 6 months on chronic hemodialysis but was lost after successful kidney transplantation. In CRF serum, the fraction containing proteins between 10 and 15?kDa decreases P450. Human uremic serum contains mediator(s) that decreases rat hepatic P450 activity and expression secondary to reduced gene expression. The inhibitory effect of serum persists even after initiation of dialysis, but disappears after normalization of renal function following kidney transplantation. PMID:15700027

  10. Metabolism of sesamin by cytochrome P450 in human liver microsomes.

    PubMed

    Yasuda, Kaori; Ikushiro, Shinichi; Kamakura, Masaki; Ohta, Miho; Sakaki, Toshiyuki

    2010-12-01

    Metabolism of sesamin by cytochrome P450 (P450) was examined using yeast expression system and human liver microsomes. Saccharomyces cerevisiae cells expressing each of human P450 isoforms (CYP1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, and 3A4) were cultivated with sesamin, and monocatechol metabolite was observed in most of P450s. Kinetic analysis using the microsomal fractions of the recombinant S. cerevisiae cells revealed that CYP2C19 had the largest k(cat)/K(m) value. Based on the kinetic data and average contents of the P450 isoforms in the human liver, the putative contribution of P450s for sesamin metabolism was large in the order of CYP2C9, 1A2, 2C19, and 2D6. A good correlation was observed between sesamin catecholization activity and CYP2C9-specific activity in in vitro studies using 10 individual human liver microsomes, strongly suggesting that CYP2C9 is the most important P450 isoform for sesamin catecholization in human liver. Inhibition studies using each anti-P450 isoform-specific antibody confirmed that CYP2C9 was the most important, and the secondary most important P450 was CYP1A2. We also examined the inhibitory effect of sesamin for P450 isoform-specific activities and found a mechanism-based inhibition of CYP2C9 by sesamin. In contrast, no mechanism-based inhibition by sesamin was observed in CYP1A2-specific activity. Our findings strongly suggest that further studies are needed to reveal the interaction between sesamin and therapeutic drugs mainly metabolized by CYP2C9. PMID:20851877

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

    SciTech Connect

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

    1984-10-01

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

  12. Opposing mechanisms of NADPH-cytochrome P450 oxidoreductase regulation by peroxisome proliferators.

    PubMed

    Fan, Li-Qun; Coley, Jackie; Miller, Richard T; Cattley, Russell C; Corton, J Christopher

    2003-03-15

    Peroxisome proliferators (PPs) regulate a battery of rodent P450 genes, including CYP2B, CYP2C, and CYP4A family members. We hypothesized that other components of the P450-metabolizing system are altered by exposure to PPs, including NADPH-cytochrome P450 oxidoreductase (P450R), an often rate-limiting component in P450-dependent reactions. In this study, we determined whether exposure to structurally diverse PPs alters the expression of P450R mRNA and protein. Increases in P450R mRNA levels were observed in male and female F-344 rat livers and in male rat kidneys after chronic exposure of the animals to PPs. Paradoxically, under the same treatment conditions in male rats, liver P450R protein levels decreased after exposure to the PPs Wy-14,643 ([4-chloro-6-(2,3-xylidino)pyrimidynylthio]acetic acid) (WY) or gemfibrozil (GEM). The down-regulation of the P450R protein was sex- and tissue-specific in that exposure to PPs led to increases in P450R protein in female rat livers [di-n-butyl phthalate (DBP) only] and male rat kidneys (WY, GEM, DBP). In male wild-type SV129 mice, P450R mRNA levels increased in livers after exposure to WY and diethylhexyl phthalate (DEHP) and in male kidneys after exposure to DEHP. Induction of mRNA by PPs was not observed in the liver or kidneys of mice, which lack a functional peroxisome proliferator-activated receptor alpha (PPAR alpha), the central mediator of the effects of PPs in the rodent liver. In wild-type male mice, P450R protein was decreased in liver after WY and DEHP treatment and in kidneys after WY treatment. The down-regulation of the P450R protein was not observed in PPAR alpha-null mice. These studies demonstrate the complex regulation of P450R expression by PPs at two different levels, both of which are dependent upon PPAR alpha: up-regulation of transcript levels in liver and kidneys and down-regulation of protein levels in male rat and mouse liver by a novel posttranscriptional mechanism. PMID:12623126

  13. 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 ...

  14. 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...

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

    PubMed Central

    Dejong, Chris A.; Wilson, Joanna Y.

    2014-01-01

    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

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

    PubMed

    Wang, Jinling; de Montellano, Paul R Ortiz

    2003-05-30

    Human heme oxygenase-1 (hHO-1) catalyzes the NADPH-cytochrome P450 reductase-dependent oxidation of heme to biliverdin, CO, and free iron. The biliverdin is subsequently reduced to bilirubin by biliverdin reductase. Earlier kinetic studies suggested that biliverdin reductase facilitates the release of biliverdin from hHO-1 (Liu, Y., and Ortiz de Montellano, P. R. (2000) J. Biol. Chem. 275, 5297-5307). We have investigated the binding of P450 reductase and biliverdin reductase to truncated, soluble hHO-1 by fluorescence resonance energy transfer and site-specific mutagenesis. P450 reductase and biliverdin reductase bind to truncated hHO-1 with Kd = 0.4 +/- 0.1 and 0.2 +/- 0.1 microm, respectively. FRET experiments indicate that biliverdin reductase and P450 reductase compete for binding to truncated hHO-1. Mutation of surface ionic residues shows that hHO-1 residues Lys18, Lys22, Lys179, Arg183, Arg198, Glu19, Glu127, and Glu190 contribute to the binding of cytochrome P450 reductase. The mutagenesis results and a computational analysis of the protein surfaces partially define the binding site for P450 reductase. An overlapping binding site including Lys18, Lys22, Lys179, Arg183, and Arg185 is similarly defined for biliverdin reductase. These results confirm the binding of biliverdin reductase to hHO-1 and define binding sites of the two reductases. PMID:12626517

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

    SciTech Connect

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

    1986-05-01

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

  18. Metabolism of halogenated alkanes by cytochrome P450 enzymes. Aerobic oxidation versus anaerobic reduction.

    PubMed

    Ji, Li; Zhang, Jing; Liu, Weiping; de Visser, Sam P

    2014-04-01

    The cytochromes P450 are a large class of heme-containing enzymes that catalyze a broad range of chemical reactions in biosystems, mainly through oxygen-atom transfer to substrates. A relatively unknown reaction catalyzed by the P450s, but very important for human health, is the activation of halogenated substrates, which may lead to toxicity problems. However, its catalytic mechanism is currently unknown and, therefore, we performed a detailed computational study. To gain insight into the metabolism of halogenated compounds by P450 enzymes, we have investigated the oxidative and reductive P450-mediated activation of tetra- and trichloromethane as halogenated models with density functional theory (DFT) methods. We propose an oxidative halosylation mechanism for CCl4 under aerobic conditions by Compound?I of P450, which follows the typical Groves-type rebound mechanism. By contrast, the metabolism of CHCl3 occurs preferentially via an initial hydrogen-atom abstraction rather than halosylation. Kinetic isotope effect studies should, therefore, be able to distinguish the mechanistic pathways of CCl4 versus CHCl3 . We find a novel mechanism that is different from the well accepted P450 substrate activation mechanisms reported previously. Moreover, the studies highlight the substrate specific activation pathways by P450 enzymes leading to different products. These reactivity differences are rationalized using Marcus theory equations, which reproduce experimental product distributions. PMID:24501011

  19. Climbazole is a new potent inducer of rat hepatic cytochrome P450.

    PubMed

    Kobayashi, Y; Suzuki, M; Ohshiro, N; Sunagawa, T; Sasaki, T; Tokuyama, S; Yamamoto, T; Yoshida, T

    2001-08-01

    We examined the effect of climbazole on the induction of rat hepatic microsomal cytochrome P450 (P450), and compared the induction potency with other N-substituted azole drugs such as clorimazole. We found that climbazole is found to be a potent inducer of rat hepatic microsomal P450 as clorimazole. Induced level of P450 by climbazole was almost similar in extent to clorimazole when compared with other imidazole drugs in a dose- and time-dependent manner. Parallel to the increase in P450, climbazole increased aminopyrine and erythromycin N-demethylase, ethoxycoumarin O-deethylase, and androstenedione 16 beta- and 15 alpha/6 beta hydroxylase activities; however, clorimazole did not induce aminopyrine N-demethylase activity irrespective of its marked increase in P450 content. Immunoblot analyses revealed that climbazole induced CYP2B1, 3A2 and 4A1. The present findings indicate that climbazole is a new potent inducer of hepatic microsomal P450 and drug-metabolizing enzymes like clorimazole, but it may have some differential mechanism(s) for these enzymes' induction in rat liver. PMID:11552297

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

    PubMed Central

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

    2001-01-01

    Plants use a diverse array of cytochrome P450 monooxygenases in their biosynthetic and detoxification pathways. To determine the extent to which various maize P450s are induced in response to chemical inducers, such as naphthalic anhydride (NA), triasulfuron (T), phenobarbital, and bacterial pathogens (Erwinia stuartii, Acidovorax avenae), we have analyzed the response patterns of seven P450 transcripts after treatment of seedlings with these inducers. Each of these P450 transcripts has distinct developmental, tissue-specific, and chemical cues regulating their expression even when they encode P450s within the same biosynthetic pathway. Most notably, the CYP71C1 and CYP71C3 transcripts, encoding P450s in the DIMBOA biosynthetic pathway, are induced to the same level in response to wounding and NA treatment of younger seedlings and differentially in response to NA/T treatment of younger seedlings and NA and NA/T treatment of older seedlings. NA and T induce expression of both CYP92A1 and CYP72A5 transcripts in older seedling shoots, whereas phenobarbital induces CYP92A1 expression in older seedling shoots and highly induces CYP72A5 expression in young and older seedling roots. Expressed sequence tag (EST) 6c06b11 transcripts, encoding an undefined P450 activity, are highly induced in seedling shoots infected with bacterial pathogens. PMID:11161067

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

    PubMed

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

    2009-08-15

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

  2. Cytochrome P450 in Cancer Susceptibility and Treatment.

    PubMed

    Mittal, Balraj; Tulsyan, Sonam; Kumar, Surendra; Mittal, Rama Devi; Agarwal, Gaurav

    2015-01-01

    Cytochrome 450 (CYP450) designates a group of enzymes abundant in smooth endoplasmic reticulum of hepatocytes and epithelial cells of small intestines. The main function of CYP450 is oxidative catalysis of various endogenous and exogenous substances. CYP450 are implicated in phase I metabolism of 80% of drugs currently in use, including anticancer drugs. They are also involved in synthesis of various hormones and influence hormone-related cancers. CYP450 genes are highly polymorphic and their variants play an important role in cancer risk and treatment. Association studies and meta-analyses have been performed to decipher the role of CYP450 polymorphisms in cancer susceptibility. Cancer treatment involves multimodal therapies and evaluation of CYP450 polymorphisms is necessary for pharmacogenetic assessment of anticancer therapy outcomes. In addition, CYP450 inhibitors are being evaluated for improved pharmacokinetics and oral formulation of several anticancer drugs. PMID:26411412

  3. Isopropanol enhancement of cytochrome P-450-dependent monooxygenase activities and its effects on carbon tetrachloride intoxication.

    PubMed

    Ueng, T H; Moore, L; Elves, R G; Alvares, A P

    1983-11-01

    Acute or chronic treatment of rats with isopropanol caused a significant increase in hepatic cytochrome P-450 content and a two- to threefold increase in aniline hydroxylase and 7-ethoxycoumarin O-deethylase activities, but no significant change in ethylmorphine N-demethylase or benzo(a)pyrene hydroxylase activity. In rats treated with isopropanol and challenged with CCl4, liver toxicity of CCl4 was characteristically potentiated, as assessed by elevation of serum glutamic-pyruvic transaminase (SGPT) levels. Isopropanol pretreatment also potentiated CCl4-induced damage to the hepatic monooxygenase system. In addition to a decrease in cytochrome P-450, rats treated with isopropanol and challenged with CCl4 showed a nonspecific decrease not only in aniline hydroxylase and 7-ethoxycoumarin O-deethylase activities, but also in ethylmorphine N-demethylase, benzo(a)pyrene hydroxylase, and NADPH-cytochrome c reductase activities. These results were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of solubilized microsomes. The electrophoretic results showed that isopropanol pretreatment markedly potentiated the CCl4-caused destruction of cytochrome P-450 hemeproteins. The data strongly suggest that isopropanol increases one or more forms of cytochrome P-450 which selectively enhance the metabolism of CCl4 to an active metabolite. This active metabolite then causes a nonselective damage to the microsomal mixed-function oxidase system. PMID:6636185

  4. Cytochrome P450 Monooxygenases for Fatty Acids and Xenobiotics in Marine Macroalgae1

    PubMed Central

    Pflugmacher, Stephan; Sandermann, Heinrich

    1998-01-01

    The metabolism of xenobiotics has mainly been investigated in higher plant species. We studied them in various marine macroalgae of the phyla Chlorophyta, Chromophyta, and Rhodophyta. Microsomes contained high oxidative activities for known cytochrome (Cyt) P450 substrates (fatty acids, cinnamic acid, 3- and 4-chlorobiphenyl, 2,3-dichlorobiphenyl, and isoproturon; up to 54 pkat/mg protein). The presence of Cyt P450 (approximately 50 pmol/mg protein) in microsomes of the three algal families was demonstrated by CO-difference absorption spectra. Intact algal tissue converted 3-chlorobiphenyl to the same monohydroxy-metabolite formed in vitro. This conversion was 5-fold stimulated upon addition of phenobarbital, and was abolished by the known P450 inhibitor, 1-aminobenzotriazole. It is concluded that marine macroalgae contain active species of Cyt P450 and could act as a metabolic sink for marine pollutants. PMID:9576781

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

    PubMed

    Renault, Hugues; Bassard, Jean-Etienne; Hamberger, Bjrn; Werck-Reichhart, Danile

    2014-06-01

    Cytochromes P450 catalyze a broad range of regiospecific, stereospecific and irreversible steps in the biosynthetic routes of plant natural metabolites with important applications in pharmaceutical, cosmetic, fragrance and flavour, or polymer industries. They are consequently essential drivers for the engineered bioproduction of such compounds. Two ground-breaking developments of commercial products driven by the engineering of P450s are the antimalarial drug precursor artemisinic acid and blue roses or carnations. Tedious optimizations were required to generate marketable products. Hurdles encountered in P450 engineering and their potential solutions are summarized here. Together with recent technical developments and novel approaches to metabolic engineering, the lessons from this pioneering work should considerably boost exploitation of the amazing P450 toolkit emerging from accelerated sequencing of plant genomes. PMID:24709279

  6. Induction of liver microsomal cytochrome P450 in cynomolgus monkeys.

    PubMed

    Bullock, P; Pearce, R; Draper, A; Podval, J; Bracken, W; Veltman, J; Thomas, P; Parkinson, A

    1995-07-01

    The aim of this study was to determine whether treatment of cynomolgus monkeys (Macaca fasicularis) with phenobarbital, beta-naphthoflavone, or dexamethasone causes an induction of microsomal crytochrome P450 (CYP) enzymes that are structurally and functionally related to rat enzymes belonging to the CYP1A, CYP2B, and CYP3A gene families. Oral treatment of male and female monkeys with phenobarbital resulted in a marked induction of a protein recognized by antibody against rat CYP2B1, as determined by Western immunoblotting. This protein, presumably a CYP2B enzyme, was not detectable in untreated monkeys, and was modestly inducible by dexamethasone but not beta-naphthoflavone. Induction of this CYP2B enzyme by phenobarbital was associated with a relatively large increase (up to 5-fold) in the rate of testosterone 16 beta-hydroxylation. Antibody, against rat CYP2B1 markedly inhibited this reaction in liver microsomes from phenobarbital-treated monkeys, but not from control monkeys. Consequently, the antibody-inhibitable rate of testosterone 16 beta-hydroxylation increased 17-fold after treatment of monkeys with phenobarbital, which is comparable with the situation in rats. In contrast to the rat CYP2B enzymes, the monkey CYP2B enzyme had little or no capacity to convert testosterone to 16 alpha-hydroxytestosterone or androstenedione, and had negligible capacity to O-dealkylate 7-pentoxyresorufin and 7-benzyloxyresorufin. Oral treatment of male and female monkeys with beta-naphthoflavone resulted in a marked induction of a protein recognized by polyclonal and monoclonal antibodies against rat CYP1A1 or against both CYP1A1 and CYP1A2. This protein was apparently a mixture of CYP1A1 and CYP1A2, neither of which was readily detectable in liver microsomes from control monkeys or monkeys treated with phenobarbital or dexamethasone. Induction of monkey CYP1A1/2 was associated with a marked increase in the O-dealkylation of 7-methoxyresorufin (up to 65-fold), the O-dealkylation of 7-ethoxyresorufin (up to 30-fold), and the N3-demethylation of caffeine (up to 17-fold), but only a 2-fold increase in benzo[a]pyrene 3-hydroxylation. Polyclonal antibodies against CYP1A1 markedly inhibited the N3-demethylation of caffeine and the O-dealkylation of 7-methoxy- and 7-ethoxyresorufin by liver microsomes from beta-naphthoflavone-treated monkeys, and partially inhibited the 3-hydroxylation of benzo[a]pyrene, indicating that monkey CYP1A1 and/or CYP1A2, like the corresponding rat enzymes, can catalyze all four reactions. Treatment of monkeys with phenobarbital resulted in a 2- to 3-fold induction of a protein recognized by antibody against rat CYP3A1. This protein (CYP3A8 or an immunochemically related enzyme) was constitutively expressed in untreated monkeys of both sexes.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:7587963

  7. Effects of avermectin on microsomal cytochrome P450 enzymes in the liver and kidneys of pigeons.

    PubMed

    Zhu, Wen-Jun; Zhang, Zi-Wei; Wang, Xian-Song; Xu, Shi-Wen; Li, Ming; Li, Shu

    2014-09-01

    Residues of avermectin (AVM) drugs have toxic effects on non-target organisms. Analyses of cytochrome P450 enzymes are among the most frequently employed indicators in pharmacology and toxicology studies. In this study, the responses of cytochrome P450 enzymes and pathological changes in the liver and kidney tissues of King pigeons (Columba livia) following subchronic exposure to avermectin for 30, 60 and 90d were investigated. Dose- and time-dependent decreases in the activities of P450 enzymes (i.e., aminopyrine-N-demethylase, erythromycin N-demethylase, aniline 4-hydroxylase and NADPH-cytochrome C reductase) and down-regulation of the P450 and b5 contents were observed. The microscopic structures were clearly altered, the severity of these alterations increased with the concentration of AVM and the exposure time. These results imply that AVM can inhibit the P450 enzyme systems in the liver and kidney tissues of pigeons. This research provides insight into the safe use of AVM and a comprehensive evaluation of the toxicological effects of AVM in birds. PMID:25194326

  8. Task-related differential expression of four cytochrome P450 genes in honeybee appendages.

    PubMed

    Mao, W; Schuler, M A; Berenbaum, M R

    2015-10-01

    In insects, cytochrome P450 monooxygenases (P450s) contribute to phytochemical and pheromone clearance in chemoreception and xenobiotic detoxification in food processing. In eusocial species, P450 expression varies with anatomy and age-related behaviour. Adult honeybees (Apis mellifera) possess appendages differentially equipped for chemoreception; antennae and prothoracic and mesothoracic legs assess food and pheromone signals whereas metathoracic legs transport pollen over long distances. Newly eclosed bees and nurses remain in the hive and neither gather nor process food, whereas foragers collect pollen and nectar, thereby encountering phytochemicals. To understand the functions of cytochrome P450, family 4, subfamily G, polypeptide 11 (CYP4G11) in the honeybee genome, we compared its expression relative to worker age and task to expression of cytochrome P450, family 9, subfamily Q, polypeptides (CYP9Qs) known to metabolize xenobiotics. That CYP4G11 is highly expressed in forager antennae and legs, with highest expression in prothoracic and mesothoracic legs, is consistent with chemosensory perception, whereas weak expression of CYP4G11 in nurses suggests that it may process primarily exogenous rather than endogenous chemical signals. By contrast, and consistent with xenobiotic detoxification, the three CYP9Q transcripts were almost undetectable in newly eclosed workers and highest in foragers, with maximal expression in the metathoracic legs that closely contact pollen phytochemicals. These CYP4G11 expression patterns suggest a role in processing environmental signals, particularly those associated with food. PMID:26190094

  9. Kinetic Analysis of Lauric Acid Hydroxylation by Human Cytochrome P450 4A11

    PubMed Central

    2015-01-01

    Cytochrome P450 (P450) 4A11 is the only functionally active subfamily 4A P450 in humans. P450 4A11 catalyzes mainly ω-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 ω-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 12-2H-substituted lauric acid. However, considerable “metabolic switching” to 11-hydroxylation was observed with [12-2H3]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 [12-3H]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 ω-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 ω-oxidation. PMID:25203493

  10. Down-regulation of hepatic cytochrome P450 in chronic renal failure: role of uremic mediators

    PubMed Central

    Guvin, Carl; Michaud, Jose; Naud, Judith; Leblond, Francois A; Pichette, Vincent

    2002-01-01

    Chronic renal failure (CRF) is associated with a decrease in liver cytochrome P450 (P450). The mechanism remains poorly understood. The present study aimed to investigate the effects of the serum of rats with CRF on liver P450. Normal rat hepatocytes were incubated for 24 h with serum (concentration of 10%) from rats with CRF and from control animals in order to measure (1) total P450 level, (2) protein expression and mRNA levels of major P450 isoforms, and (3) some of their specific metabolic activities (N-demethylation of erythromycin). Time-course experiments (incubation time from 12 to 48 h) and dose-response curves (concentration of serum ranging from 1 to 30%) have been conducted. In normal hepatocytes incubated for 24 h with serum (concentration of 10%) from rats with CRF, total P450 level, protein expression and mRNA levels of several P450 isoforms (CYP2C6, 2C11, 3A1 and 3A2) were decreased by more than 35% (P<0.001) compared to serum from control animals. The protein expression as well as the mRNA levels of CYP2D were similar in hepatocytes incubated with serum from either control or CRF rats. The N-demethylation of erythromycin was decreased by more than 35% (P<0.001) in hepatocytes incubated with serum from rats with CRF. The inhibitory effect of serum from rats with CRF tended to peak at 48 h of incubation and was maximum at a concentration of 20%. In conclusion, uremic serum contains mediator(s) that down-regulate the cytochrome P450 of normal hepatocytes secondary to reduced gene expression. PMID:12429576

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

    PubMed

    Yang, Xin; Atkinson, Karen; Di, Li

    2016-03-01

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

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

    PubMed

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

    2012-12-01

    In order to develop in vitro methods as an alternative to P450 animal testing in the drug discovery process, two main requisites are necessary: 1) gathering of data on animal homologues of the human P450 enzymes, currently very limited, and 2) bypassing the requirement for both the P450 reductase and the expensive cofactor NADPH. In this work, P450 2C20 from Macaca fascicularis, homologue of the human P450 2C8 has been taken as a model system to develop such an alternative in vitro method by two different approaches. In the first approach called "molecular Lego", a soluble self-sufficient chimera was generated by fusing the P450 2C20 domain with the reductase domain of cytochrome P450 BM3 from Bacillus megaterium (P450 2C20/BMR). In the second approach, the need for the redox partner and also NADPH were both obviated by the direct immobilization of the P450 2C20 on glassy carbon and gold electrodes. Both systems were then compared to those obtained from the reconstituted P450 2C20 monooxygenase in presence of the human P450 reductase and NADPH using paclitaxel and amodiaquine, two typical drug substrates of the human P450 2C8. The K(M) values calculated for the 2C20 and 2C20/BMR in solution and for 2C20 immobilized on electrodes modified with gold nanoparticles were 1.9 0.2, 5.9 2.3, 3.0 0.5 ?M for paclitaxel and 1.2 0.2, 1.60.2 and 1.4 0.2 ?M for amodiaquine, respectively. The data obtained not only show that the engineering of M. fascicularis did not affect its catalytic properties but also are consistent with K(M) values measured for the microsomal human P450 2C8 and therefore show the feasibility of developing alternative in vitro animal tests. PMID:22819650

  13. Position specificity in n-hexane hydroxylation by two forms of cytochrome P-450 in rat liver microsomes.

    PubMed

    Morohashi, K; Sadano, H; Okada, Y; Omura, T

    1983-02-01

    The hydroxylation of n-hexane by rat liver microsomes was studied and the contribution of different molecular species of cytochrome P-450 to the hydroxylation reaction was examined. In the case of untreated rats, the products of NADPH-dependent n-hexane hydroxylation were 1-, 2-, and 3-hexanols, and the major one was 2-hexanol. Phenobarbital (PB) treatment of animals resulted in a significant increase of the hydroxylation activity. The formation of 2- and 3-hexanols was much more significantly increased than that of 1-hexanol. On the other hand, 3-methylcholanthrene (MC) treatment stimulated the formation of 3-hexanol and the formation of the other two isomeric alcohols was rather decreased. These observations suggested the position specificities of the PB-inducible form (P-450(PB)) and MC-inducible form (P-450(MC)) of cytochrome P-450 in the hydroxylation of n-hexane. Inhibition experiments using antibodies specific to P-450(PB) and P-450(MC) also indicated that P-450(PB) was more active in the hydroxylation at the 2-position whereas P-450(MC) was more specific for the 3-position. NADPH-dependent n-hexane hydroxylation systems were reconstituted by the use of purified NADPH-cytochrome P-450 reductase and P-450(PB) or P-450(MC), and the activities of the reconstituted systems supported the proposed position specificities of these two forms of cytochrome P-450 in n-hexane hydroxylation. PMID:6404899

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

    PubMed Central

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

    2011-01-01

    Lapatinib, an oral breast cancer drug, has recently been reported to be a mechanism-based inactivator of cytochrome P450 (P450) 3A4 and also an idiosyncratic hepatotoxicant. It was suggested that formation of a reactive quinoneimine metabolite was involved in mechanism-based inactivation (MBI) and/or hepatotoxicity. We investigated the mechanism of MBI of P450 3A4 by lapatinib. Liquid chromatography-mass spectrometry analysis of P450 3A4 after incubation with lapatinib did not show any peak corresponding to irreversible modifications. The enzymatic activity inactivated by lapatinib was completely restored by the addition of potassium ferricyanide. These results indicate that the mechanism of MBI by lapatinib is quasi-irreversible and mediated via metabolic intermediate complex (MI complex) formation. This finding was verified by the increase in a signature Soret absorbance at approximately 455 nm. Two amine oxidation products of the metabolism of lapatinib by P450 3A4 were characterized: N-hydroxy lapatinib (M3) and the oxime form of N-dealkylated lapatinib (M2), suggesting that a nitroso or another related intermediate generated from M3 is involved in MI complex formation. In contrast, P450 3A5 was much less susceptible to MBI by lapatinib via MI complex formation than P450 3A4. In addition, P450 3A5 had a significantly lower ability than 3A4 to generate M3, consistent with N-hydroxylation as the initial step in the pathway to MI complex formation. In conclusion, our results demonstrate that the primary mechanism for MBI of P450 3A4 by lapatinib is not irreversible modification by the quinoneimine metabolite, but quasi-irreversible MI complex formation mediated via oxidation of the secondary amine group of lapatinib. PMID:21363997

  15. Orphans in the human cytochrome P450 superfamily: approaches to discovering functions and relevance in pharmacology.

    PubMed

    Guengerich, F Peter; Cheng, Qian

    2011-09-01

    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

  16. A Multiscale Approach to Modelling Drug Metabolism by Membrane-Bound Cytochrome P450 Enzymes

    PubMed Central

    Sansom, Mark S. P.; Mulholland, Adrian J.

    2014-01-01

    Cytochrome P450 enzymes are found in all life forms. P450s play an important role in drug metabolism, and have potential uses as biocatalysts. Human P450s are membrane-bound proteins. However, the interactions between P450s and their membrane environment are not well-understood. To date, all P450 crystal structures have been obtained from engineered proteins, from which the transmembrane helix was absent. A significant number of computational studies have been performed on P450s, but the majority of these have been performed on the solubilised forms of P450s. Here we present a multiscale approach for modelling P450s, spanning from coarse-grained and atomistic molecular dynamics simulations to reaction modelling using hybrid quantum mechanics/molecular mechanics (QM/MM) methods. To our knowledge, this is the first application of such an integrated multiscale approach to modelling of a membrane-bound enzyme. We have applied this protocol to a key human P450 involved in drug metabolism: CYP3A4. A biologically realistic model of CYP3A4, complete with its transmembrane helix and a membrane, has been constructed and characterised. The dynamics of this complex have been studied, and the oxidation of the anticoagulant R-warfarin has been modelled in the active site. Calculations have also been performed on the soluble form of the enzyme in aqueous solution. Important differences are observed between the membrane and solution systems, most notably for the gating residues and channels that control access to the active site. The protocol that we describe here is applicable to other membrane-bound enzymes. PMID:25033460

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

    PubMed Central

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

    2014-01-01

    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

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

    PubMed Central

    Cheng, Qian

    2011-01-01

    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

  19. mRNA Distribution and Heterologous Expression of Orphan Cytochrome P450 20A1

    PubMed Central

    Stark, Katarina; Wu, Zhong-Liu; Bartleson, Cheryl J.; Guengerich, F. Peter

    2015-01-01

    Cytochrome P450 (P450) 20A1 is one of the so-called orphan P450s without assigned biological function. mRNA expression was detected in human liver and extrahepatic expression was noted in several human brain regions, including substantia nigra, hippocampus, and amygdala, using conventional polymerase chain reaction and RNA dot blot analysis. Adult human liver contained 3-fold higher overall mRNA levels than whole brain, although specific regions (i.e., hippocampus and substantia nigra) exhibited higher mRNA expression levels than liver. Orthologous full-length and truncated transcripts of P450 20A1 were transcribed and sequenced from rat liver, heart, and brain. In rat, the concentrations of full-length transcripts were 34 fold higher in brain and heart than liver. In situ hybridization of rat whole brain sections showed a similar mRNA expression pattern as observed for human P450 20A1, indicating expression in substantia nigra, hippocampus, and amygdala. A number of N-terminal modifications of the codon-optimized human P450 20A1 sequence were prepared and expressed in Escherichia coli, and two of the truncated derivatives showed characteristic P450 spectra (200280 nmol P450/l). Although the recombinant enzyme system oxidized NADPH, no catalytic activity was observed with the heterologously expressed protein when a number of potential steroids and biogenic amines were surveyed as potential substrates. The function of P450 20A1 remains unknown; however, the sites of mRNA expression in human brain and the conservation among species may suggest possible neurophysiological function. PMID:18541694

  20. Adrenodoxin supports reactions catalyzed by microsomal steroidogenic cytochrome P450s

    SciTech Connect

    Pechurskaya, Tatiana A. . E-mail: usanov@iboch.bas-net.by

    2007-02-16

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

  1. Alternative Sampling Strategies for Cytochrome P450 Phenotyping.

    PubMed

    De Kesel, Pieter M M; Lambert, Willy E; Stove, Christophe P

    2016-02-01

    Interindividual variability in the expression and function of drug metabolizing cytochrome P (CYP) 450 enzymes, determined by a combination of genetic, non-genetic and environmental parameters, is a major source of variable drug response. Phenotyping by administration of a selective enzyme substrate, followed by the determination of a specific phenotyping metric, is an appropriate approach to assess the in vivo activity of CYP450 enzymes as it takes into account all influencing factors. A phenotyping protocol should be as simple and convenient as possible. Typically, phenotyping metrics are determined in traditional matrices, such as blood, plasma or urine. Several sampling strategies have been proposed as an alternative for these traditional sampling techniques. In this review, we provide a comprehensive overview of available methods using dried blood spots (DBS), hair, oral fluid, exhaled breath and sweat for in vivo CYP450 phenotyping. We discuss the relation between phenotyping metrics measured in these samples and those in conventional matrices, along with the advantages and limitations of the alternative sampling techniques. Reliable phenotyping procedures for several clinically relevant CYP450 enzymes, including CYP1A2, CYP2C19 and CYP2D6, are currently available for oral fluid, breath or DBS, while additional studies are needed for other CYP450 isoforms, such as CYP3A4. The role of hair analysis for this purpose remains to be established. Being non- or minimally invasive, these sampling strategies provide convenient and patient-friendly alternatives for classical phenotyping procedures, which may contribute to the implementation of CYP450 phenotyping in clinical practice. PMID:26239501

  2. Deletion of 30 murine cytochrome p450 genes results in viable mice with compromised drug metabolism.

    PubMed

    Scheer, Nico; McLaughlin, Lesley A; Rode, Anja; Macleod, A Kenneth; Henderson, Colin J; Wolf, C Roland

    2014-06-01

    In humans, 75% of all drugs are metabolized by the cytochrome P450-dependent monooxygenase system. Enzymes encoded by the CYP2C, CYP2D, and CYP3A gene clusters account for ?80% of this activity. There are profound species differences in the multiplicity of cytochrome P450 enzymes, and the use of mouse models to predict pathways of drug metabolism is further complicated by overlapping substrate specificity between enzymes from different gene families. To establish the role of the hepatic and extrahepatic P450 system in drug and foreign chemical disposition, drug efficacy, and toxicity, we created a unique mouse model in which 30 cytochrome P450 genes from the Cyp2c, Cyp2d, and Cyp3a gene clusters have been deleted. Remarkably, despite a wide range of putative important endogenous functions, Cyp2c/2d/3a KO mice were viable and fertile, demonstrating that these genes have evolved primarily as detoxification enzymes. Although there was no overt phenotype, detailed examination showed Cyp2c/2d/3a KO mice had a smaller body size (15%) and larger livers (20%). Changes in hepatic morphology and a decreased blood glucose (30%) were also noted. A five-drug cocktail of cytochrome P450 isozyme probe substrates were used to evaluate changes in drug pharmacokinetics; marked changes were observed in either the pharmacokinetics or metabolites formed from Cyp2c, Cyp2d, and Cyp3a substrates, whereas the metabolism of the Cyp1a substrate caffeine was unchanged. Thus, Cyp2c/2d/3a KO mice provide a powerful model to study the in vivo role of the P450 system in drug metabolism and efficacy, as well as in chemical toxicity. PMID:24671958

  3. 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...

  4. 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...

  5. 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 ...

  6. METABOLISM OF MYCLOBUTANIL AND TRIADIMEFON BY HUMAN AND RAT CYTOCHROME P450 ENZYMES AND LIVER MICROSOMES.

    EPA Science Inventory

    Metabolism of two triazole-containing antifungal azoles was studied using expressed human and rat cytochrome P450s (CYP) and liver microsomes. Substrate depletion methods were used due to the complex array of metabolites produced from myclobutanil and triadimefon. Myclobutanil wa...

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

    PubMed Central

    Zimmerlin, Alfred; Durst, Francis

    1992-01-01

    Wheat (Triticum aestivum L. cv Etoile de Choisy) microsomes catalyzed the cytochrome P-450-dependent oxidation of the herbicide diclofop to three hydroxy-diclofop isomers. Hydroxylation was predominant at carbon 4, with migration of chlorine to carbon 5 (67%) and carbon 3 (25%). The 2,4-dichloro-5-hydroxy isomer was identified as a minor reaction product (8%). Substrate-specificity studies showed that the activity was not inhibited or was weakly inhibited by a range of xenobiotic or physiological cytochrome P-450 substrates, with the exception of lauric acid. Wheat microsomes also catalyze the metabolism of the herbicides chlorsulfuron, chlortoluron, and 2,4-dichlorophenoxyacetic acid and of the model substrate ethoxycoumarin, as well as the hydroxylation of the endogenous substrates cinnamic and lauric acids. Treatments of wheat seedlings with phenobarbital or the safener naphthalic acid anhydride enhanced the cytochrome P-450 content of the microsomes and all related activities except that of cinnamic acid 4-hydroxylase, which was reduced. The stimulation patterns of diclofop aryl hydroxylase and lauric acid hydroxylase were similar, in contrast with the other activities tested. Lauric acid inhibited competitively (Ki = 9 ?m) the oxidation of diclofop and reciprocally. The similarity of diclofop aryl hydroxylase and lauric acid hydroxylase was further investigated by alternative substrate kinetics, autocatalytic inactivation, and computer-aided molecular modelisation studies, and the results suggest that both reactions are catalyzed by the same cytochrome P-450 isozyme. PMID:16653070

  8. 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...

  9. 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...

  10. 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...

  11. 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...

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

    ERIC Educational Resources Information Center

    Groves, John T.

    1985-01-01

    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)

  13. Sex differences in the diabetes-induced modulation of rat hepatic cytochrome P450 proteins.

    PubMed

    Barnett, C R; Rudd, S; Flatt, P R; Ioannides, C

    1993-01-26

    Sex differences in the diabetes-induced changes in hepatic cytochrome P450 proteins were investigated in rats treated with streptozotocin. Changes in specific cytochrome P450 proteins were monitored using diagnostic substrates and immunologically utilizing specific polyclonal antibodies. When expressed in terms of nmoles of total cytochrome P450, ethoxyresorufin O-deethylase activity was increased by treatment with streptozotocin, the extent of induction being the same in the two sexes. In contrast, lauric acid hydroxylase and ethylmorphine N-demethylase activities were induced only in the male rat. Finally, p-nitrophenol hydroxylase and pentoxyresorufin O-dealkylase were enhanced by the same treatment in both sexes, the effect being more pronounced in the male. These findings indicate that sex-specific changes in certain cytochrome P450 proteins exist in response to insulin-dependent diabetes but these cannot, however, be ascribed to sex differences in the severity of diabetes induced by streptozotocin since the degrees of hyperketonaemia and hyperglycaemia were the same in the two sexes. These are likely to reflect sex-specific differences in growth hormone and triglyceride levels in the diabetic animals. PMID:8435090

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

    EPA Science Inventory

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

  15. 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...

  16. 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...

  17. Influence of age on in vitro effect of cadmium on rat liver cytochrome P-450 concentration and monooxygenases activity.

    PubMed

    Jahn, F; Klinger, W

    1982-02-01

    Cadmium++ added in vitro destroys rat liver cytochrome P-450 (cyt. P-450) with increasing age by 25-50%. Ethylmorphine N-demethylation is inhibited only in rats 30-days old and thereafter. Ethoxycoumarin 0-deethylation is inhibited even in newborn rats, and the maximal inhibition appears to increase with age. It is concluded that in all age groups cadmium resistant cyt. P-450 subspecies are present. Ethoxycoumarin 0-deethylase activity possibly indicates the cadmium sensitive P-450 fraction. PMID:6978593

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

    SciTech Connect

    Arnold, Frances H.

    2012-02-27

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

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

    SciTech Connect

    Stiborova, Marie Arlt, Volker M.; Henderson, Colin J.; Wolf, C. Roland; Kotrbova, Vera; Moserova, Michaela; Hudecek, Jiri; Phillips, David H.; Frei, Eva

    2008-02-01

    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.

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

    PubMed Central

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

    2013-01-01

    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 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

  1. Direct Observation of an Oxepin from a Bacterial Cytochrome P450-Catalyzed Oxidation.

    PubMed

    Stok, Jeanette E; Chow, Sharon; Krenske, Elizabeth H; Farfan Soto, Clementina; Matyas, Csongor; Poirier, Raymond A; Williams, Craig M; De Voss, James J

    2016-03-18

    The cytochromes P450 are hemoproteins that catalyze a range of oxidative C-H functionalization reactions, including aliphatic and aromatic hydroxylation. These transformations are important in a range of biological contexts, including biosynthesis and xenobiotic biodegradation. Much work has been carried out on the mechanism of aliphatic hydroxylation, implicating hydrogen atom abstraction, but aromatic hydroxylation is postulated to proceed differently. One mechanism invokes as the key intermediate an arene oxide (and/or its oxepin tautomer). Conclusive isolation of this intermediate has remained elusive and, currently, direct formation of phenols from a Meisenheimer intermediate is believed to be favored. We report here the identification of a P450 [P450cam (CYP101A1) and P450cin (CYP176A1)]-generated arene oxide as a product of in vitro oxidation of tert-butylbenzene. Computations (CBS-QB3) predict that the arene oxide and oxepin have similar stabilities to other arene oxides/oxepins implicated (but not detected) in P450-mediated transformations, suggesting that arene oxides can be unstable terminal products of P450-catalyzed aromatic oxidation that can explain the origin of some observed metabolites. PMID:26811874

  2. Conformational Plasticity and Structure/Function Relationships in Cytochromes P450

    PubMed Central

    Kazanis, Sophia; Dang, Marina

    2010-01-01

    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, 12731296. PMID:20446763

  3. Induction and inhibition of cytochrome P450 and drug-metabolizing enzymes by climbazole.

    PubMed

    Kobayashi, Yasuna; Suzuki, Michiya; Ohshiro, Naomi; Sunagawa, Takashi; Sasaki, Tadanori; Oguro, Takiko; Tokuyama, Shogo; Yamamoto, Toshinori; Yoshida, Takemi

    2002-01-01

    To determine the effect of climbazole on hepatic microsomal cytochrome P450 (P450) and drug-metabolizing enzymes, four different P450 isoforms (CYP2B1, 3A2, 2E1, and 2C12) were examined in female Long-Evans rats. Treatment of rats with climbazole resulted in the induction of P450 content. Climbazole both induced and inhibited aminopyrine N-demethylase activity, but not erythromycin N-demethylase activity. Uridine 5'-phosphate (UDP)-glucuronosyl transferase and glutathione S-transferase activities were also increased with climbazole treatment. Immunoblot analyses revealed that climbazole induces CYP2B1 and CYP3A2 at the lower dose examined, but it failed to increase CYP2B1 at the higher dose. Northern blot analysis revealed that climbazole markedly increases P450 2B1 mRNA. These results indicate that climbazole induces and inhibits P450-dependent drug-metabolizing enzymes in vivo and may have the dose-differential effect on CYP2B1 in rat liver. PMID:11824557

  4. Cloning of wound-induced cytochrome P450 monooxygenases expressed in pea.

    PubMed Central

    Frank, M R; Deyneka, J M; Schuler, M A

    1996-01-01

    Cytochrome P450 monooxygenases (P450s) mediate a wide range of oxidative reactions involved in the biosynthesis of plant secondary metabolites including phenylpropanoids and phytoalexins. To investigate the regulation of these P450s in the phenylpropanoid biosynthetic pathway of pea (Pisum sativum), partial cDNAs representing four distinct P450s expressed in pea seedlings were cloned using a reverse transcription-polymerase chain reaction strategy. One of the corresponding full-length cDNA clones, designated CYP73A9, encodes pea trans-cinnamic acid 4-hydroxylase, which catalyzes the second core reaction in the phenylpropanoid pathway. As expected from its central role in the production of lignin precursors and defense compounds, northern analysis of poly(A)+ mRNA demonstrates that transcripts encoding CYP73A9 are induced appreciably within 3 h after wounding. A second cDNA clone, designated CYP82, encodes a novel P450, whose transcripts are also induced in response to wounding at approximately the same time as CYP73A9 transcripts. Despite the multitude of environmental stimuli known to induce expression of phenylpropanoid pathway enzymes, genomic DNA Southern analysis indicates that each of these P450s is encoded by a low copy number (possibly a single copy) gene family. PMID:8819874

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

    SciTech Connect

    Otto, S.A.; Marcus, C.M.; Jefcoate, C.R. )

    1990-02-26

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

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

    SciTech Connect

    Letcher, R.J.; Norstrom, R.J.

    1994-12-31

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

  7. Damage to the Brain Serotonergic System Increases the Expression of Liver Cytochrome P450.

    PubMed

    Rysz, Marta; Bromek, Ewa; Haduch, Anna; Sadakierska-Chudy, Anna; Daniel, W?adys?awa A

    2015-09-01

    Genes coding for cytochrome P450 are regulated by endogenous hormones such as the growth hormone, corticosteroids, thyroid, and sex hormones. Secretion of these hormones is regulated by the respective hypothalamus-pituitary-secretory organ axes. Since the brain sends its serotonergic projections from the raphe nuclei to the hypothalamus, we have assumed that damage to these nuclei may affect the neuroendocrine regulation of cytochrome P450 expression in the liver. Thereby, 5,7-dihydroxytryptamine (5,7-DHT), a serotonergic neurotoxin, was injected into the dorsal and median raphe nuclei of male Wistar rats. Ten days after the neurotoxin injections, the brain concentrations of neurotransmitters, serum hormone, and cytokine levels, as well as the expression of cytochrome P450 in the liver were measured. Injection of 5,7-DHT decreased serotonin concentration in the brain followed by a significant rise in the levels of the growth hormone, corticosterone, and testosterone, and a drop in triiodothyronine concentration in the serum. No changes in interleukin (IL) levels (IL-2 and IL-6) were observed. Simultaneously, the activity and protein level of liver CYP1A, CYP3A1, and CYP2C11 rose (the activity of CYP2A/2B/2C6/2D was not significantly changed). Similarly, the mRNA levels of CYP1A1, CYP1A2, CYP2C11, and CYP3A1 were elevated. This is the first report demonstrating the effect of intracerebral administration of serotonergic neurotoxin on liver cytochrome P450. The obtained results indicate involvement of the brain serotonergic system in the neuroendocrine regulation of liver cytochrome P450 expression. The physiologic and pharmacological significance of the findings is discussed. PMID:26059263

  8. The evolutionary history of Cytochrome P450 genes in four filamentous Ascomycetes

    PubMed Central

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

    2007-01-01

    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

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

    SciTech Connect

    Estabrook, Ronald W. . E-mail: Ronald.estabrook@utsouthwestern.edu

    2005-12-09

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

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

    SciTech Connect

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

    2013-02-15

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

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

    PubMed Central

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

    2008-01-01

    Arachidonic acid is an essential constituent of cell membranes that is esterified to the sn-2-position of glycerophospholipids and is released from selected lipid pools by phospholipase cleavage. The released arachidonic acid can be metabolized by three enzymatic pathways: the cyclooxygenase pathway forming prostaglandins and thromboxanes, the lipoxygenase pathway generating leukotrienes and lipoxins, and the cytochrome P450 (cP450) pathway producing epoxyeicosatrienoic acids and hydroxyeicosatetraenoic acids. The present study describes a novel group of cP450 epoxygenase-dependent metabolites of arachidonic acid, termed 2-epoxyeicosatrienoylglycerols (2-EG), including two regioisomers, 2-(11,12-epoxyeicosatrienoyl)glycerol (2-11,12-EG) and 2-(14,15-epoxyeicosatrienoyl)glycerol (2-14,15-EG), which are both produced in the kidney and spleen, whereas 2-11,12-EG is also detected in the brain. Both 2-11,12-EG and 2-14,15-EG activated the two cannabinoid (CB) receptor subtypes, CB1 and CB2, with high affinity and elicited biological responses in cultured cells expressing CB receptors and in intact animals. In contrast, the parental arachidonic acid and epoxyeicosatrienoic acids failed to activate CB1 or CB2 receptors. Thus, these cP450 epoxygenase-dependent metabolites are a novel class of endogenously produced, biologically active lipid mediators with the characteristics of endocannabinoids. This is the first evidence of a cytochrome P450-dependent arachidonate metabolite that can activate G-protein-coupled cell membrane receptors and suggests a functional link between the cytochrome P450 enzyme system and the endocannabinoid system. PMID:18606824

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

    PubMed Central

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

    2015-01-01

    Cytochrome P450 function is dependent on the ability of these enzymes to successfully interact with their redox partners, NADPH-cytochrome P450 reductase (CPR) and cytochrome b5, in the endoplasmic reticulum (ER). Because the ER is heterogeneous in lipid composition, membrane microdomains with different characteristics are formed. Ordered microdomains are more tightly packed, and enriched in saturated fatty acids, sphingomyelin and cholesterol, whereas disordered regions contain higher levels of unsaturated fatty acids. The goal of this study was to determine if the P450 system proteins localize to different regions of the ER. The localization of CYP1A2, CYP2B4, and CYP2E1 within the ER was determined by partial membrane solubilization with Brij 98, centrifugation on a discontinuous sucrose gradient, and immune blotting of the gradient fractions to identify ordered and disordered microdomains. CYP1A2 resided almost entirely in the ordered regions of the ER with CPR also localized predominantly to this region. CYP2B4 was equally distributed between the ordered and disordered domains. In contrast, CYP2E1 localized to the disordered membrane regions. Removal of cholesterol (an important constituent of ordered domains) led to the relocation of CYP1A2, CYP2B4 and CPR to the disordered regions. Interestingly, CYP1A1 and CYP1A2 localized to different membrane microdomains, despite their high degree of sequence similarity. These data demonstrate that P450 system enzymes are organized in specific membrane regions, and their localization can be affected by depletion of membrane cholesterol. The differential localization of different P450s in specific membrane regions may provide a novel mechanism for modulating P450 function. PMID:25236845

  13. Ab Initio Electronic Structure Calculations of Cytochrome P450 -- Ligand Interactions

    NASA Astrophysics Data System (ADS)

    Segall, M. D.; Payne, M. C.; Ellis, S. W.; Tucker, G. T.

    1997-03-01

    The Cytochrome P450 superfamily of enzymes are of great interest in pharmacology as they participate in an enormous range of physiological processes including drug deactivation and xenobiotic detoxification. We apply ab initio electronic structure calculations to model the interactions of the haem molecule at the P450 active site with substrate and inhibitor ligands. These calculations, based on density function theory, were performed with the CETEP code which uses a plane wave basis set and pseudopotentials to perform efficient LDA, GGA and spin dependent calculations. A change in the spin state of the haem iron atom is observed on binding of a substrate molecule, consistent with the accepted reaction mechanism.

  14. Mechanistic aspects of CYP74 allene oxide synthases and related cytochrome P450 enzymes

    PubMed Central

    Brash, Alan R.

    2009-01-01

    The existence of CYP5, CYP8A, and the CYP74 enzymes specialized for reaction with fatty acid peroxide substrates presents opportunities for a “different look” at the catalytic cycle of the cytochrome P450s. This review considers how the properties of the peroxide-metabolizing enzymes are distinctive, and how they tie in with those of the conventional monooxygenase enzymes. Some unusual reactions of each class have parallels in the other. As new enzyme reactions and new P450 structures emerge there will be possibilities for finding their special properties and edging this knowledge into the big picture. PMID:19747698

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

    SciTech Connect

    Karaszkiewicz, J.W.

    1989-01-01

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

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

    PubMed Central

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

    1994-01-01

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

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

    PubMed

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

    2015-05-01

    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

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

    SciTech Connect

    Iyanagi, Takashi . E-mail: iyanagi@spring8.or.jp

    2005-12-09

    NADPH-cytochrome P450 reductase (CPR) and the nitric oxide synthase (NOS) reductase domains are members of the FAD-FMN family of proteins. The FAD accepts two reducing equivalents from NADPH (dehydrogenase flavin) and FMN acts as a one-electron carrier (flavodoxin-type flavin) for the transfer from NADPH to the heme protein, in which the FMNH {sup {center_dot}}/FMNH{sub 2} couple donates electrons to cytochrome P450 at constant oxidation-reduction potential. Although the interflavin electron transfer between FAD and FMN is not strictly regulated in CPR, electron transfer is activated in neuronal NOS reductase domain upon binding calmodulin (CaM), in which the CaM-bound activated form can function by a similar mechanism to that of CPR. The oxygenated form and spin state of substrate-bound cytochrome P450 in perfused rat liver are also discussed in terms of stepwise one-electron transfer from CPR. This review provides a historical perspective of the microsomal mixed-function oxidases including CPR and P450. In addition, a new model for the redox-linked conformational changes during the catalytic cycle for both CPR and NOS reductase domain is also discussed.

  19. 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...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    EPA Science Inventory

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

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

    EPA Science Inventory

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

  3. THE DIFFERENTIAL HEPATOTOXICITY AND CYTOCHROME P450 RESPONSE OF F344 RATS TO THE THREE ISOMERS OF DICHLOROBENZENE

    EPA Science Inventory

    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 toxic thresholds and to further e1ucidate the role of cytochrome P450 in the metabolism and toxici...

  4. Sex- and tissue-specific expression of P450 aromatase (cyp19a1a) in the yellowtail clownfish, Amphiprion clarkii.

    PubMed

    Kobayashi, Yasuhisa; Horiguchi, Ryo; Miura, Saori; Nakamura, Masaru

    2010-02-01

    To investigate the role of estrogen in the gonad of yellowtail clownfish Amphiprion clarkii, we isolated cDNA encoding cytochrome P450 aromatase (Cyp19a1a) from the adult ovary. The full-length cDNA of clownfish cyp19a1a is 1928-bp long and encodes 520 amino acids. Real-time quantitative RT-PCR analysis showed that cyp19a1a was expressed mainly in the ovary of female-phase fish. In situ hybridization and immunohistochemical observations showed that positive signals were restricted to the ovarian follicle of the female-phase fish. In contrast, Cyp19a1a signal was not detected in the ambisexual gonad of the male-phase fish. These findings suggest that Cyp19a1a is involved in oogenesis in the female-phase fish, but not in the ambisexual gonad of male-phase fish. PMID:19913632

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

    NASA Astrophysics Data System (ADS)

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

    2009-03-01

    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.

  6. Combined effect of nitrogen dioxide and cold stress on the activity of the hepatic cytochrome P-450 system in rats.

    PubMed

    Takano, T; Miyazaki, Y

    1984-12-01

    The combined effects of nitrogen dioxide (NO2) and cold stress were assessed on the cytochrome P-450 system by measuring microsomal protein content, cytochrome P-450 content, aminopyrine N-demethylase activity, and aniline hydroxylase activity in the liver of male Wistar rats exposed to 4 ppm NO2 and/or cold environment (4 degrees C) for 24 h, 14 days, and 30 days. Exposure to cold alone changed the activity of the cytochrome P-450 system during the exposure up to 30 days, and exposure to NO2 alone also influenced it after 14 days and 30 days of exposure. Interactions were observed in the effect on the cytochrome P-450 system when rats were exposed to NO2 and cold simultaneously. There was a tendency that NO2 suppressed the increases in activities of the cytochrome P-450 system caused by cold of 24-h and 30-day exposure. PMID:6515658

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

    USGS Publications Warehouse

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

    1996-01-01

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

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

    PubMed

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

    2015-06-01

    Experimental studies indicate that short-term fasting alters drug metabolism. However, the effects of short-term fasting on drug metabolism in humans need further investigation. Therefore, the aim of this study was to evaluate the effects of short-term fasting (36 h) on P450-mediated drug metabolism. In a randomized crossover study design, nine healthy subjects ingested a cocktail consisting of five P450-specific probe drugs [caffeine (CYP1A2), S-warfarin (CYP2C9), omeprazole (CYP2C19), metoprolol (CYP2D6), and midazolam (CYP3A4)] on two occasions (control study after an overnight fast and after 36 h of fasting). Blood samples were drawn for pharmacokinetic analysis using nonlinear mixed effects modeling. In addition, we studied in Wistar rats the effects of short-term fasting on hepatic mRNA expression of P450 isoforms corresponding with the five studied P450 enzymes in humans. In the healthy subjects, short-term fasting increased oral caffeine clearance by 20% (P = 0.03) and decreased oral S-warfarin clearance by 25% (P < 0.001). In rats, short-term fasting increased mRNA expression of the orthologs of human CYP1A2, CYP2C19, CYP2D6, and CYP3A4 (P < 0.05), and decreased the mRNA expression of the ortholog of CYP2C9 (P < 0.001) compared with the postabsorptive state. These results demonstrate that short-term fasting alters cytochrome P450-mediated drug metabolism in a nonuniform pattern. Therefore, short-term fasting is another factor affecting cytochrome P450-mediated drug metabolism in humans. PMID:25795462

  9. Human cytochrome P-450 PB-1: A multigene family involved in mephenytoin and steroid oxidations that maps to chromosome 10

    PubMed Central

    Meehan, R. R.; Gosden, J. R.; Rout, D.; Hastie, N. D.; Friedberg, T.; Adesnik, M.; Buckland, R.; van Heyningen, V.; Fletcher, J.; Spurr, N. K.; Sweeney, J.; Wolf, C. R.

    1988-01-01

    The cytochrome P-450 monooxygenase system possesses catalytic activity toward many exogenous compounds (e.g., drugs, insecticides, and polycyclic aromatic hydrocarbons) and endogenous compounds (e.g., steroids, fatty acids, and prostaglandins). Multiple forms of cytochrome P-450 with different substrate specificities have been isolated. In the present paper we report the isolation and sequence of a cDNA clone for the human hepatic cytochrome P-450 responsible for mephenytoin (an anticonvulsant) oxidation. The mephenytoin cytochrome P-450 is analogous to the rat cytochrome P-450 form termed PB-1 (family P450C2C). We also report that human PB-1 is encoded by one of a small family of related genes all of which map to human chromosome 10q24.1-10q24.3. The endogenous role of this enzyme appears to be in steroid oxidations. This cytochrome P-450 family does not correspond to any of the hepatic cytochrome P-450 gene families previously mapped in humans. ImagesFigure 1Figure 4Figure 5Figure 6 PMID:2827463

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

    PubMed

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

    2011-04-01

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

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

    SciTech Connect

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

    1987-05-01

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

  12. Significance of Neuronal Cytochrome P450 Activity in Opioid-Mediated Stress-Induced Analgesia

    PubMed Central

    Hough, Lindsay B.; Nalwalk, Julia W.; Yang, Weizhu; Ding, Xinxin

    2014-01-01

    Stressful environmental changes can suppress nociceptive transmission, a phenomenon known as “stress-induced analgesia”. Depending on the stressor and the subject, opioid or non-opioid mechanisms are activated. Brain μ opioid receptors mediate analgesia evoked either by exogenous agents (e.g. morphine), or by the release of endogenous opioids following stressful procedures. Recent work with morphine and neuronal cytochrome P450 (P450)-deficient mice proposed a signal transduction role for P450 enzymes in μ analgesia. Since μ opioid receptors also mediate some forms of stress-induced analgesia, the present studies assessed the significance of brain P450 activity in opioid-mediated stress-induced analgesia. Two widely-used models of opioid stress-induced analgesia (restraint and warm water swim) were studied in both sexes of wild-type control and P450-deficient (Null) mice. In control mice, both stressors evoked moderate analgesic responses which were blocked by pretreatment with the opioid antagonist naltrexone, confirming the opioid nature of these responses. Consistent with literature, sex differences (control female > control male) were seen in swim-induced, but not restraint-induced, analgesia. Null mice showed differential responses to the two stress paradigms. As compared with control subjects, Null mice showed highly attenuated restraint-induced analgesia, showing a critical role for neuronal P450s in this response. However, warm water swim-induced analgesia was unchanged in Null vs. control mice. Additional control experiments confirmed the absence of morphine analgesia in Null mice. These results are the first to show that some forms of opioid-mediated stress-induced analgesia require brain neuronal P450 activity. PMID:25020125

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

    PubMed

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

    2004-08-01

    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

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

    PubMed

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

    2014-01-01

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

  15. Mediation of pyrethroid insecticide toxicity to honey bees (Hymenoptera: Apidae) by cytochrome P450 monooxygenases.

    PubMed

    Johnson, Reed M; Wen, Zhimou; Schuler, Mary A; Berenbaum, May R

    2006-08-01

    Honey bees, Apis mellifera L., often thought to be extremely susceptible to insecticides in general, exhibit considerable variation in tolerance to pyrethroid insecticides. Although some pyrethroids, such as cyfluthrin and lambda-cyhalothrin, are highly toxic to honey bees, the toxicity of tau-fluvalinate is low enough to warrant its use to control parasitic mites inside honey bee colonies. Metabolic insecticide resistance in other insects is mediated by three major groups of detoxifying enzymes: the cytochrome P450 monooxygenases (P450s), the carboxylesterases (COEs), and the glutathione S-transferases (GSTs). To test the role of metabolic detoxification in mediating the relatively low toxicity of tau-fluvalinate compared with more toxic pyrethroid insecticides, we examined the effects of piperonyl butoxide (PBO), S,S,S-tributylphosphorotrithioate (DEF), and diethyl maleate (DEM) on the toxicity of these pyrethroids. The toxicity of the three pyrethroids to bees was greatly synergized by the P450 inhibitor PBO and synergized at low levels by the carboxylesterase inhibitor DEF. Little synergism was observed with DEM. These results suggest that metabolic detoxification, especially that mediated by P450s, contributes significantly to honey bee tolerance of pyrethroid insecticides. The potent synergism between tau-fluvalinate and PBO suggests that P450s are especially important in the detoxification of this pyrethroid and explains the ability of honey bees to tolerate its presence. PMID:16937654

  16. Novel approaches to the use of cytochrome P450 activities in wildlife toxicity studies

    SciTech Connect

    VandenBerg, M.; Bosveld, A.T.C.

    1995-12-31

    Many wildlife toxicity studies, e.g. with avian species, use cytochrome P450 activities as markers for biological activities of environmental contaminants. It has been established that induction of CYP1A1 correlates with Ah-receptor mediated toxicity of dioxin-like compounds in many species. In addition, CYP1A1 plays a significant role in bioactivation of polycyclic aromatics. So far very few studies focused on the natural function of P450 isoenzymes in wildlife species. Besides classical hepatic CYP1A(1) associated activities, like EROD and AHH, several new techniques are available to study the activities of various CYP isoenzymes. Caffeine N-demethylation, testosterone and 17ss-estradiol hydroxylation patterns can provide new insights in the physiological function of P450 isoenzymes and the induction of the basal activities by chemicals. So far little interest was given to processes which occur after the DNA-receptor binding, e.g. changes in steroid hormone metabolism and pathways in environmental toxicology. This in spite of the fact that very subtle changes in steroid hormone levels may have significant physiological implications. This presentation will focus on some P450 activities, besides CYP1A(1), which might be important for development and reproduction. Some experimental approaches, limitations and techniques will be discussed which could lead to elucidation of the possible endocrine function of P450s.

  17. Deficits in Neuronal Cytochrome P450 Activity Attenuate Opioid Analgesia but not Opioid Side Effects

    PubMed Central

    Hough, Lindsay B.; Nalwalk, Julia W.; Cleary, Rachel A.; Phillips, James G.; Fang, Cheng; Yang, Weizhu; Ding, Xinxin

    2014-01-01

    Morphine-like analgesics act on ? opioid receptors in the CNS to produce highly effective pain relief, but the same class of receptors also mediates non-therapeutic side effects. The analgesic properties of morphine were recently shown to require the activity of a brain neuronal cytochrome P450 epoxygenase, but the significance of this pathway for opioid side effects is unknown. Here we show that brain P450 activity is not required for three of morphines major side effects (respiratory depression, constipation, and locomotor stimulation). Following systemic or intracerebroventricular administration of morphine, transgenic mice with brain neuronspecific reductions in P450 activity showed highly attenuated analgesic responses as compared with wild-type (control) mice. However, brain P450-deficient mice showed normal morphine-induced side effects (respiratory depression, locomotor stimulation, and inhibition of intestinal motility). Pretreatment of control mice with the P450 inhibitor CC12 similarly reduced the analgesia, but not these side effects of morphine. Because activation of brain ? opioid receptors produces both opioid analgesia and opioid side effects, dissociation of the mechanisms for the therapeutic and therapy-limiting effects of opioids has important consequences for the development of analgesics with reduced side effects and/or limited addiction liability. PMID:25062792

  18. Pungent ginger components modulates human cytochrome P450 enzymes in vitro

    PubMed Central

    Li, Mian; Chen, Pei-zhan; Yue, Qing-xi; Li, Jing-quan; Chu, Rui-ai; Zhang, Wei; Wang, Hui

    2013-01-01

    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 μmol/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

  19. Cytochrome P450 purification and immunological detection in an insecticide resistant strain of German cockroach (Blattella germanica, L.).

    PubMed

    Scharf, M E; Neal, J J; Marcus, C B; Bennett, G W

    1998-01-01

    A German cockroach strain, Munsyana (MA) had 80-fold resistance to the pyrethroid insecticide cypermethrin, 4.5-fold greater total cytochrome P450 content and 2.5-fold greater cytochrome P450-mediated N-demethylation of 4-chloro-N-methylaniline compared to the susceptible Johnson Wax (JWax) strain. Immobilized artificial membrane high performance liquid chromatography (IAM-HPLC) of microsomal proteins from the MA strain enriched cytochrome P450 greater than 70-fold. Following purification, a single protein band of M(r) = 49,000 (P450 MA), was detected by silver-staining SDS PAGE gels. Antiserum to the purified protein from the MA strain (anti-P450 MA) was produced in mice. Anti-P450 MA inhibited cytochrome P450-mediated N-demethylation by 4-fold in both MA and JWax strains. In Western blots of microsomal proteins, anti-P450 MA differentiated single MA and JWax individuals by recognizing and M(r) 49,000 protein band in only the MA strain. In JWax cockroaches, the M(r) 49,000 band was only detectable in Western analysis following induction with pentamethylbenzene (PMB). PMB induction also increases N-demethylation 2.6 and 8.0-fold in the MA and JWax strains, respectively. These results are consistent with the hypothesis that insecticide resistance in the MA strain is due to over-expression of a cytochrome P450. PMID:9612934

  20. Silencing NADPH-cytochrome P450 reductase results in reduced acaricide resistance in Tetranychus cinnabarinus (Boisduval)

    PubMed Central

    Shi, Li; Zhang, Jiao; Shen, Guangmao; Xu, Zhifeng; Wei, Peng; Zhang, Yichao; Xu, Qiang; He, Lin

    2015-01-01

    Cytochrome P450 monooxygenases (P450s) are involved in metabolic resistance to insecticides and require NADPH cytochrome P450 reductase (CPR) to transfer electrons when they catalyze oxidation reactions. The carmine spider mite, Tetranychus cinnabarinus is an important pest mite of crop and vegetable plants worldwide, and its resistance to acaricides has quickly developed. However, the role of CPR on the formation of acaricide-resistance in T. cinnabarinus is still unclear. In this study, a full-length cDNA encoding CPR was cloned and characterized from T. cinnabarinus (designated TcCPR). TcCPR expression was detectable in all developmental stages of T. cinnabarinus, but it’s much lower in eggs. TcCPR was up-regulated and more inducible with fenpropathrin treatment in the fenpropathrin-resistant (FeR) strain compared with the susceptible SS strain. Feeding of double-strand RNA was effective in silencing the transcription of TcCPR in T. cinnabarinus, which resulted in decreasing the activity of P450s and increasing the susceptibility to fenpropathrin in the FeR strain but not in the susceptible strain. The current results provide first evidence that the down-regulation of TcCPR contributed to an increase of the susceptibility to fenpropathrin in resistant mites. TcCPR could be considered as a novel target for the development of new pesticides. PMID:26493678

  1. Silencing NADPH-cytochrome P450 reductase results in reduced acaricide resistance in Tetranychus cinnabarinus (Boisduval).

    PubMed

    Shi, Li; Zhang, Jiao; Shen, Guangmao; Xu, Zhifeng; Wei, Peng; Zhang, Yichao; Xu, Qiang; He, Lin

    2015-01-01

    Cytochrome P450 monooxygenases (P450s) are involved in metabolic resistance to insecticides and require NADPH cytochrome P450 reductase (CPR) to transfer electrons when they catalyze oxidation reactions. The carmine spider mite, Tetranychus cinnabarinus is an important pest mite of crop and vegetable plants worldwide, and its resistance to acaricides has quickly developed. However, the role of CPR on the formation of acaricide-resistance in T. cinnabarinus is still unclear. In this study, a full-length cDNA encoding CPR was cloned and characterized from T. cinnabarinus (designated TcCPR). TcCPR expression was detectable in all developmental stages of T. cinnabarinus, but it's much lower in eggs. TcCPR was up-regulated and more inducible with fenpropathrin treatment in the fenpropathrin-resistant (FeR) strain compared with the susceptible SS strain. Feeding of double-strand RNA was effective in silencing the transcription of TcCPR in T. cinnabarinus, which resulted in decreasing the activity of P450s and increasing the susceptibility to fenpropathrin in the FeR strain but not in the susceptible strain. The current results provide first evidence that the down-regulation of TcCPR contributed to an increase of the susceptibility to fenpropathrin in resistant mites. TcCPR could be considered as a novel target for the development of new pesticides. PMID:26493678

  2. Structural and kinetic basis of steroid 17?,20-lyase activity in teleost fish cytochrome P450 17A1 and its absence in cytochrome P450 17A2.

    PubMed

    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

    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

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

    PubMed Central

    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

    2015-01-01

    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 17α-hydroxylation and a subsequent 17α,20-lyase reaction with both Prog and Preg. Teleost fish contain two 17A P450s; zebrafish P450 17A1 catalyzes both 17α-hydroxylation and lyase reactions with Prog and Preg, and P450 17A2 is more efficient in pregnenolone 17α-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. 17α-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

  4. The role of cytochrome P450s in polycyclic aromatic hydrocarbon carcinogenesis

    SciTech Connect

    Polzer, R.J.

    1993-01-01

    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.

  5. Antagonistic effects of hydrostatic pressure and osmotic pressure on cytochrome P-450cam spin transition.

    PubMed Central

    Di Primo, C; Deprez, E; Hoa, G H; Douzou, P

    1995-01-01

    The combined effects of hydrostatic pressure and osmotic pressure, generated by polyols, on the spin equilibrium of fenchone-bound cytochrome P-450cam were investigated. Hydrostatic pressure indices a high spin to low spin transition, whereas polyols induce the reversed reaction. Of the four solutes used, glycerol, glucose, stachyose, and sucrose, only the last two would act on the spin transition by osmotic stress. The spin volume changes measured by both techniques are different, 29 and -350 ml/mol for hydrostatic pressure and osmotic pressure, respectively. It suggests that even if the two are perturbing water molecules, different properties are probed. From the volume change induced by osmotic stress, 19 water molecules are deduced that would be implicated in the spin transition of the fenchone-bound protein. This result suggests that water molecules other than the well defined ones located in the active site play a key role in modulating the spin equilibrium of cytochrome P-450cam. PMID:7612848

  6. Role of cytochrome P-450 and related enzymes in the pulmonary metabolism of xenobiotics.

    PubMed Central

    Philpot, R M; Smith, B R

    1984-01-01

    The lung metabolizes a wide variety of xenobiotics and, in the process, forms products that may be more or less toxic than the parent compound. The consequence of metabolism, activation or detoxication, is a function of the nature of the substrate and of the characteristics and concentrations of the enzymes involved. As a result, the biotransformation of xenobiotics can lead to their excretion or to the formation of reactive products that produce deleterious effects by binding covalently to tissue macromolecules. Among the enzymes that metabolize xenobiotics, those associated with the cytochrome P-450-dependent monooxygenase system are probably the most important. The route by which a given substrate is metabolized in a tissue or cell is, to a great extent, determined by the types and concentrations of cytochrome P-450 isozymes present. We are just beginning to understand the distribution of these enzymes in lung and to appreciate the species and cellular differences that exist. PMID:6376107

  7. Antagonistic effects of hydrostatic pressure and osmotic pressure on cytochrome P-450cam spin transition.

    PubMed

    Di Primo, C; Deprez, E; Hoa, G H; Douzou, P

    1995-05-01

    The combined effects of hydrostatic pressure and osmotic pressure, generated by polyols, on the spin equilibrium of fenchone-bound cytochrome P-450cam were investigated. Hydrostatic pressure indices a high spin to low spin transition, whereas polyols induce the reversed reaction. Of the four solutes used, glycerol, glucose, stachyose, and sucrose, only the last two would act on the spin transition by osmotic stress. The spin volume changes measured by both techniques are different, 29 and -350 ml/mol for hydrostatic pressure and osmotic pressure, respectively. It suggests that even if the two are perturbing water molecules, different properties are probed. From the volume change induced by osmotic stress, 19 water molecules are deduced that would be implicated in the spin transition of the fenchone-bound protein. This result suggests that water molecules other than the well defined ones located in the active site play a key role in modulating the spin equilibrium of cytochrome P-450cam. PMID:7612848

  8. Isolation and characterization of a cDNA clone from Catharanthus roseus encoding NADPH:cytochrome P-450 reductase, an enzyme essential for reactions catalysed by cytochrome P-450 mono-oxygenases in plants.

    PubMed

    Meijer, A H; Lopes Cardoso, M I; Voskuilen, J T; de Waal, A; Verpoorte, R; Hoge, J H

    1993-07-01

    The membrane-bound flavoprotein NADPH:cytochrome P-450 (cytochrome c) reductase, that functions in electron transfer to cytochrome P-450 monooxygenases, was purified from a cell suspension culture of the higher plant Catharanthus roseus. Anti-serum raised against the purified protein was found to inhibit NADPH:cytochrome c reductase activity as well as the activities of the cytochrome P-450 enzymes geraniol 10-hydroxylase and trans-cinnamate 4-hydroxylase, which are involved in alkaloid biosynthesis and phenylpropanoid biosynthesis, respectively. Immunoscreening of a C. roseus cDNA expression library resulted in the isolation of a partial NADPH: cytochrome P-450 reductase cDNA clone, which was identified on the basis of sequence homology with NADPH:cytochrome P-450 reductases from yeast and animal species. The identify of the cDNA was confirmed by expression in Escherichia coli as a functional protein capable of NADPH-dependent reduction of cytochrome c and neotetrazolium, two in vitro substrates for the reductase. The N-terminal sequence of the reductase, which was not present in the cDNA clone, was determined from a genomic NADPH: cytochrome P-450 reductase clone. It was demonstrated that the reductase probably is encoded by a single copy gene. A sequence comparison of this plant NADPH:cytochrome P-450 reductase with the corresponding enzymes from yeast and animals species showed that functional domains involved in binding of the cofactors FMN, FAD and NADPH are highly conserved between all kingdoms. In C. roseus cell cultures a rapid increase of the reductase steady state mRNA level was observed after the addition of fungal elicitor preparations that are known to induce cytochrome P-450-dependent biosynthetic pathways. PMID:8220474

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

    SciTech Connect

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

    2013-01-25

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

  10. Transformation of Fatty Acids Catalyzed by Cytochrome P450 Monooxygenase Enzymes of Candida tropicalis

    PubMed Central

    Eschenfeldt, William H.; Zhang, Yeyan; Samaha, Hend; Stols, Lucy; Eirich, L. Dudley; Wilson, C. Ronald; Donnelly, Mark I.

    2003-01-01

    Candida tropicalis ATCC 20336 can grow on fatty acids or alkanes as its sole source of carbon and energy, but strains blocked in β-oxidation convert these substrates to long-chain α,ω-dicarboxylic acids (diacids), compounds of potential commercial value (Picataggio et al., Biotechnology 10:894-898, 1992). The initial step in the formation of these diacids, which is thought to be rate limiting, is ω-hydroxylation by a cytochrome P450 (CYP) monooxygenase. C. tropicalis ATCC 20336 contains a family of CYP genes, and when ATCC 20336 or its derivatives are exposed to oleic acid (C18:1), two cytochrome P450s, CYP52A13 and CYP52A17, are consistently strongly induced (Craft et al., this issue). To determine the relative activity of each of these enzymes and their contribution to diacid formation, both cytochrome P450s were expressed separately in insect cells in conjunction with the C. tropicalis cytochrome P450 reductase (NCP). Microsomes prepared from these cells were analyzed for their ability to oxidize fatty acids. CYP52A13 preferentially oxidized oleic acid and other unsaturated acids to ω-hydroxy acids. CYP52A17 also oxidized oleic acid efficiently but converted shorter, saturated fatty acids such as myristic acid (C14:0) much more effectively. Both enzymes, in particular CYP52A17, also oxidized ω-hydroxy fatty acids, ultimately generating the α,ω-diacid. Consideration of these different specificities and selectivities will help determine which enzymes to amplify in strains blocked for β-oxidation to enhance the production of dicarboxylic acids. The activity spectrum also identified other potential oxidation targets for commercial development. PMID:14532054

  11. Two cytochromes P450 catalyze S-heterocyclizations in cabbage phytoalexin biosynthesis.

    PubMed

    Klein, Andrew P; Sattely, Elizabeth S

    2015-11-01

    Phytoalexins are abundant in edible crucifers and have important biological activities, yet no dedicated gene for their biosynthesis is known. Here, we report two new cytochromes P450 from Brassica rapa (Chinese cabbage) that catalyze unprecedented S-heterocyclizations in cyclobrassinin and spirobrassinin biosynthesis. Our results provide genetic and biochemical insights into the biosynthesis of a prominent pair of dietary metabolites and have implications for pathway discovery across >20 recently sequenced crucifers. PMID:26389737

  12. Cytochrome P-450-CO and substrates: lessons from ligand binding under high pressure.

    PubMed

    Jung, Christiane

    2002-03-25

    An overview of the application of high-pressure studies on the carbon monoxide complex of cytochrome P-450 is given. Different approaches to characterize ligand binding steps, the conformational states and substates and the compressibility of the ligand-bound complex are reviewed. A particular focus is the effect of substrates on these properties. It is shown that substrate mobility, compressibility and water accessibility are interrelated and may have functional meaning. PMID:11983405

  13. Plant Omics: Isolation, Identification, and Expression Analysis of Cytochrome P450 Gene Sequences from Coleus forskohlii.

    PubMed

    Awasthi, Praveen; Mahajan, Vidushi; Rather, Irshad Ahmad; Gupta, Ajai Prakash; Rasool, Shafaq; Bedi, Yashbir S; Vishwakarma, Ram A; Gandhi, Sumit G

    2015-12-01

    The omics analyses of plants and the agrigenomics field offer the opportunity to better characterize our ecosystems. In this context, characterization of cytochrome P450 genes (CYP450s), which constitute one of the largest gene families in plants, is important. They play vital roles in biosynthesis of secondary metabolites, phytohormones as well as in detoxification of harmful chemicals. Tuberous roots of Coleus forskohlii accumulate forskolin, a potent and reversible activator of adenylate cyclase, as well as other related diterpenoids. Coleus forskohlii is also known to produce rosmarinic acid, genkwanin (7-O-methylapigenin), and guaiacol glycerin. We report here the isolation of CYP450s from C. forskohlii, expression profiling of CYP450s in different tissues, and how different elicitors/stresses regulate the expression of different CYP450 sequences. Degenerate primers, designed from the conserved regions of CYP450s, were used to amplify fragments from cDNA of C. forskohlii and a library was prepared. Sequences homologous to CYP450s were assembled into seven distinct gene fragments (CfP450C1-C7), belonging to seven CYP450 families. Expression profiling of CYP450s showed that the transcripts of CfP450C1, CfP450C4, CfP450C5, CfP450C6, and CfP450C7 were prominent in aerial tissues (flower, young leaf, and mature leaf), whereas expression of CfP450C3 was dominant in root and root tip. CfP450C2 showed higher expression in flowers and roots as compared to other tissues. Expression profiles of CYP450s, in response to different stresses (abscisic acid, methyl jasmonate, salicylic acid, 2, 4-dichloro-phenoxyacetic acid, UVA, and wounding) were also studied. This study has isolated CYP450s from C. forskohlii, and will help to understand their regulation as well as their functions. This is the first report on the isolation and expression analysis of CYP450s from this herb. PMID:26669713

  14. Transplantation of fetal liver tissue suspension into the spleens of adult syngenic rats: effects of various mitogens and cytotoxins on cytochrome P450 (P450) isoforms expression and on P450 mediated monooxygenase functions.

    PubMed

    Lupp, A; Tralls, M; Fuchs, U; Lucas, N; Danz, M; Klinger, W

    1999-07-01

    Syngenic fetal liver tissue suspensions were transplanted into the spleens of adult male Fisher 344 inbred rats. Four months after surgery, transplant recipients and age matched control rats were treated with various mitogens (fluorene [FEN], fluorenone [FON] and 2-acetylaminofluorene [AAF]) or cytotoxins (allyl alcohol [AAL], bromobenzene [BBZ] and carbon tetrachloride [CCl4]) or the respective solvents 24 or 48 hours before sacrifice. The expression of three cytochrome P450 (P450) isoforms, 1A1, 2B1 and 3A2, within spleens and livers was assessed by immunohistochemistry and P450 mediated monooxygenase functions in spleen and liver 9000 g supernatants by the model reactions ethoxyresorufin O-deethylation (EROD), ethoxycoumarin O-deethylation (ECOD), and ethylmorphine N-demethylation (EMND). The orthotopic livers of both solvent treated transplant recipients and control rats displayed only in few lobules a slight P450 1A1, but in all lobules a moderate P450 2B1 and 3A2 expression, all mainly located in the hepatocytes around the central veins. Correspondingly, regular EROD, ECOD and EMND activities were observed. Each of the three mitogens increased the P450 1A1 expression in the hepatocytes of the perivenous zones of the liver lobules. FON administration caused an additional P450 1A1 immunostaining in the periportal areas, and AAF treatment a P450 1A1 expression in bile duct epithelia. Also the staining for P450 2B1 and 3A2 in the hepatocytes of the perivenous and intermediate zones of the liver lobules was intensified after treatment with any of the mitogens. The three model reactions were significantly increased within the livers after FEN and FON administration, whereas after AAF treatment only ECOD was enhanced, EROD remained unaffected and EMND was decreased. The cytotoxin AAL caused small lesions and fatty degeneration of hepatocytes only in some periportal areas. BBZ only produced a perivenous necrosis of single cells, whereas CCl4 caused complete necrosis of the centrilobular parenchyma. Immunohistochemically, AAL administration led to an increase in the P450 2B1 expression in the perivenous hepatocytes, whereas the staining for P450 1A1 was not affected and that for P450 3A2 was even decreased in the periportal areas. Due to AAL treatment EROD and EMND activities were not affected and ECOD activity was increased. BBZ administration caused a P450 1A1 expression in the periportal hepatocytes but a decrease in this staining of the perivenous cells. The number of hepatocytes positively stained for P450 2B1 and 3A2 in the perivenous and intermediate zones was diminished in comparison to the livers of solvent treated rats. After BBZ treatment, EROD and EMND activities were decreased, ECOD activity was not affected. CCl4 administration caused a strong reduction in the expression of all three P450 isoforms and in the activity of all three model reactions. Spleens of control rats displayed almost no P450 isoforms expression and P450 mediated monooxygenase functions, without as well as after treatment with the mitogens or cytotoxins. Similar to adult liver, the hepatocytes in the transplant containing spleens showed nearly no P450 1A1, but a noticeable P450 2B1 and 3A2 expression. No staining was observed within the bile duct cells of the intrasplenic transplants. PMID:10445401

  15. [Effect of bemethyl on cytochrome P-450-dependent monoxygenases in the human liver and lymphocytes].

    PubMed

    Sorokina, E A; Sibiriak, S V; Sergeeva, S A

    2002-01-01

    Effects of the actoprotector bemithyl (50 mg/kg, p.o.) upon a single or five-fold administration on the cytochrome P-450 and b5 content and the isoform-specific and nonspecific monooxygenase activity [aminopyrine-N-demethylase, aniline-p-hydroxylase, 4-nitroanisole-o-demethylase,2,5-diphenyloxazole-p-hydroxylase, 7-ethoxyresorufin-o-deethylase (EROD), benzyloxyresorufin-o-debenzylase (BROD)] in rat liver were evaluated. In addition, the influence of bemithyl (0.(1)-100 microM) on the development of EROD and BROD activity was studied on the mitogen-stimulated human lymphocytes in vitro. Administered in rats, bemithyl exhibited the properties of a cytochrome P-450 inductor of the mixed type, which was manifested by an increase in the total cytochrome P-450 content in liver microsomes and in the monooxygenase activity related to both Ah-receptor-dependent and -independent isoforms (except for the aniline-p-hydroxylase activity). The induction of the monooxygenase activity realized by Ah-receptor-dependent isoforms (4-nitroanisole-o-demethylase, 2,5-diphenyloxazole-p-hydroxylase, and EROD activity) was more pronounced, reaching maximum upon a single drug administration. Acting upon the human lymphocytes in vitro, high concentrations of bemithyl increased expression of the EROD activity, while low drug concentrations stimulated the BROD activity. PMID:12227093

  16. Cytochrome P450 Inhibitors Reduce Creeping Bentgrass (Agrostis stolonifera) Tolerance to Topramezone.

    PubMed

    Elmore, Matthew T; Brosnan, James T; Armel, Gregory R; Kopsell, Dean A; Best, Michael D; Mueller, Thomas C; Sorochan, John C

    2015-01-01

    Creeping bentgrass (Agrostis stolonifera L.) is moderately tolerant to the p-hydroxyphenylpyruvate dioxygenase-inhibiting herbicide topramezone. However, the contribution of plant metabolism of topramezone to this tolerance is unknown. Experiments were conducted to determine if known cytochrome P450 monooxygenase inhibitors 1-aminobenzotriazole (ABT) and malathion alone or in combination with the herbicide safener cloquintocet-mexyl influence creeping bentgrass tolerance to topramezone. Creeping bentgrass in hydroponic culture was treated with ABT (70 ?M), malathion (70 ?m and 1000 g ha(-1)), or cloquintocet-mexyl (70 ?M and 1000 g ha(-1)) prior to topramezone (8 g ha(-1)) application. Topramezone-induced injury to creeping bentgrass increased from 22% when applied alone to 79 and 41% when applied with malathion or ABT, respectively. Cloquintocet-mexyl (70 ?M and 1000 g ha(-1)) reduced topramezone injury to 1% and increased creeping bentgrass biomass and PSII quantum yield. Cloquintocet-mexyl mitigated the synergistic effects of ABT more than those of malathion. The effects of malathion on topramezone injury were supported by creeping bentgrass biomass responses. Responses to ABT and malathion suggest that creeping bentgrass tolerance to topramezone is influenced by cytochrome P450-catalyzed metabolism. Future research should elucidate primary topramezone metabolites and determine the contribution of cytochrome P450 monooxygenases and glutathione S-transferases to metabolite formation in safened and non-safened creeping bentgrass. PMID:26186714

  17. Cytochrome P450 Inhibitors Reduce Creeping Bentgrass (Agrostis stolonifera) Tolerance to Topramezone

    PubMed Central

    Elmore, Matthew T.; Brosnan, James T.; Armel, Gregory R.; Kopsell, Dean A.; Best, Michael D.; Mueller, Thomas C.; Sorochan, John C.

    2015-01-01

    Creeping bentgrass (Agrostis stolonifera L.) is moderately tolerant to the p-hydroxyphenylpyruvate dioxygenase-inhibiting herbicide topramezone. However, the contribution of plant metabolism of topramezone to this tolerance is unknown. Experiments were conducted to determine if known cytochrome P450 monooxygenase inhibitors 1-aminobenzotriazole (ABT) and malathion alone or in combination with the herbicide safener cloquintocet-mexyl influence creeping bentgrass tolerance to topramezone. Creeping bentgrass in hydroponic culture was treated with ABT (70 μM), malathion (70 μm and 1000 g ha-1), or cloquintocet-mexyl (70 μM and 1000 g ha-1) prior to topramezone (8 g ha-1) application. Topramezone-induced injury to creeping bentgrass increased from 22% when applied alone to 79 and 41% when applied with malathion or ABT, respectively. Cloquintocet-mexyl (70 μM and 1000 g ha-1) reduced topramezone injury to 1% and increased creeping bentgrass biomass and PSII quantum yield. Cloquintocet-mexyl mitigated the synergistic effects of ABT more than those of malathion. The effects of malathion on topramezone injury were supported by creeping bentgrass biomass responses. Responses to ABT and malathion suggest that creeping bentgrass tolerance to topramezone is influenced by cytochrome P450-catalyzed metabolism. Future research should elucidate primary topramezone metabolites and determine the contribution of cytochrome P450 monooxygenases and glutathione S-transferases to metabolite formation in safened and non-safened creeping bentgrass. PMID:26186714

  18. Computational finding of potential inhibitor for Cytochrome P450 Mono-oxygenases Enzyme of Mycobacterium tuberculosis

    PubMed Central

    Sharma, Ashwani; Subbias, Krishna K; Robine, Ophlie; Chaturvedi, Indu; Nigam, Anshul; Sharma, Nishant; Chaudhary, Prem Prashant

    2012-01-01

    Cytochrome P450 mono-oxygenases (2UUQ) enzyme from Mycobacterium tuberculosis catalyzes oxidation of organic compounds such as lipids and steroidal hormones therefore remain as potential drug target. Currently available first line anti-tuberculosis drugs have been caused several side effects in the body as well as resistance development by mycobacterium against these drugs, necessitates the considerable need for finding new drugs. Therefore, we propose a structure based computational method to find a new potential inhibitor for cytochrome P450 mono-oxygenases enzyme. Compounds from several ligand databases were docked against the functional sites of 2UUQ (A) through the standard GEMDOCK v2.0 and AUTODOCK4.0 molecular docking tools. Commercially available chemical compound ZINC00004165 (5-[3-(2-nitroimidazol-1-yl) propyl] phenanthridine) has produced top rank with lowest interaction energy of -113.2 (via GEMDOCK) and lowest docking energy of -9.80 kcal/mol (via AUTODOCK) as compared to first line anti TB compounds. Z score and normal distribution analysis verified that the ZINC00004165 compound has more affinity towards 2UUQ in comparison to large number of random population of compounds. ZINC00004165 is also in agreement with the drug likeness properties of Lipinski rule of five without any violation. Therefore, our finding concludes that the commercial compound ZINC00004165 can act as a potential inhibitor against cytochrome P450 mono-oxygenases enzyme of Mycobacterium tuberculosis. PMID:23144553

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

    USGS Publications Warehouse

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

    1999-01-01

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

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

    SciTech Connect

    Mandai, Takao; Fujiwara, Shinsuke; Imaoka, Susumu

    2009-06-19

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

  1. Studies on the substrate specificity and inducibility of cytochrome P-450meg.

    PubMed Central

    Berg, A; Rafter, J J

    1981-01-01

    The cytochrome P-450-dependent steroid 15 beta-hydroxylase system in Bacillus megaterium A.T.C.C. 13368 was investigated with regard to its appearance in the cell with respect to the growth curve of the organism, with regard to its inducibility by a number of agents (among them some of the classical inducers of the mammalian liver microsomal cytochrome P-450 system) and with regard to its capacity to convert non-steroidal substances into oxygenated compounds. The enzyme was found to reach a maximum concentration in the cell during the stationary phase of the growth curve. Of all the agents tested as inducers, none showed any capacity to induce cytochrome P-450meg. Finally, of the substances tested as substrates only aniline (p-hydroxylation) was metabolized by the microbial enzyme system. This conversion might be related to the general oxygenase activity of haemoproteins. It is concluded that the substrate specificity of the B. megaterium hydroxylase system is narrow. PMID:6797409

  2. 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 ...

  3. Drug interactions between nine antifungal agents and drugs metabolized by human cytochromes P450.

    PubMed

    Niwa, Toshiro; Imagawa, Yurie; Yamazaki, Hiroshi

    2014-01-01

    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 14α-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

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

    PubMed

    Zhang, Jing; Ji, Li; Liu, Weiping

    2015-08-17

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

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

    PubMed Central

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

    2013-01-01

    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

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

    PubMed

    Vang, O; Wallin, H; Doehmer, J; Autrup, H

    1993-11-01

    The role of metabolism of tumour promoters on the inhibition of intercellular communication was investigated in a modified V79 metabolic cooperation system. V79 cells, which stably express different rat cytochrome P450 enzymes (CYP1A1, CYP1A2 or CYP2B1), were used in the metabolic cooperation assay. The inhibitory effect on intercellular communication of four compounds was changed in cells expressing cytochrome P450 enzymes, compared to cells without. The phorbol ester TPA and di(2-ethylhexyl)phthalate blocked intercellular communication in all the cell lines tested, but expression of CYP1A1 enzyme reduced the inhibitory activity in these cells. Diethylstilbestrol caused inhibition only with cells containing cytochrome P450 enzymes. In contrast, the benzene metabolite hydroquinone inhibited metabolic cooperation preferentially in cells without cytochrome P450 enzymes. The inhibition of metabolic cooperation by another benzene metabolite, phenol, was not affected by the cytochrome P450 enzymes. The inhibitory activity of several chemicals that have not been tested previously was analysed in the new metabolic cooperation assay. The inhibitory activity of none of these chemicals was affected by cytochrome P450-associated metabolism. 7-Octylindolactam V was as potent as TPA, whereas the related indolactam V was 100-fold less active. The carcinogenic aromatic amine 4-aminobiphenyl, but not its primary metabolite 4-hydroxyaminobiphenyl, inhibited metabolic cooperation. Other known carcinogens, ochratoxin A, aflatoxin B1 and 4-nitrobiphenyl, did not inhibit metabolic cooperation in either V79 cells expressing or cells not expressing cytochrome P450. We conclude that cytochrome P450-associated metabolism plays an important role in the inhibition of gap junctional intercellular communication of some tumour promoters. The modified metabolic cooperation assay presented here is valuable for detecting some inhibitory chemicals which have been 'false negative' in previous assays for gap junctional intercellular communication. The assay also discloses that cytochrome P450 metabolism alters intercellular communication by a mechanism other than metabolism of the exogenous inhibitor. PMID:8242868

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

    PubMed Central

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

    2014-01-01

    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 knockout and wild-type mice were treated with styrene (6.0 mmol/kg, ip), and cell counts and LDH activity in bronchoalveolar lavage fluids were monitored to evaluate the pulmonary toxicity induced by styrene. Cyp2e1-null mice displayed 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

  8. Solubilization and reconstitution of pisatin demethylase, a cytochrome P-450 from the pathogenic fungus Nectria haematococca

    SciTech Connect

    Desjardins, A.E.; Matthews, D.E.; VanEtten, H.D.

    1984-07-01

    Some isolates of the fungus Nectria haematococca Berk. and Br. can demethylate pisatin, a phytoalexin from pea (Pisum sativum L.). Pisatin demethylation appears to be necessary for tolerance to pisatin and virulence on pea, and is catalyzed by a microsomal cytochrome P-450. We now report solubilization of this enzyme from N. haematococca microsomes. Pisatin demethylase activity was obtained in the high speed supernatant of detergent treated microsomes, if detergent was removed before assay. The CO-binding spectrum of the soluble enzyme preparation indicated the presence of cytochrome P-450. Cholic acids were the most effective of the detergents tested for solubilizing enzyme activity. Loss of enzyme activity during solubilization was reduced by certain protease inhibitors, but not by substrate, reducing agents, antioxidants, or phospholipids. The most effective solubilization medium tests was 1% sodium cholate, 100 millimolar potassium phosphate, 500 millimolar sucrose, 1 millimolar phenylmethylsulfonyl fluoride, pH 7.5, which yielded approximately 30% of the pisatin demethylase and over 95% of the NADPH-cytochrome c reductase in the soluble fraction. Demethylase activity was lost when the reductase was removed by adsorption on 2',5'-ADP-agarose. The demethylase activity of reductase-free fractions could be restored by adding a reductase preparation purified approximately 100-fold from microsomes of N. haematococca isolate 74-8-1, which does not demethylate pisatin. We conclude that pisatin demethylase requires NADPH-cytochrome c reductase for activity. The inability of some isolates to demethylate pisatin appears to be due to the absence of a suitable cytochrome P-450, rather than to a lack of functional reductase. 24 references, 4 figures, 4 tables.

  9. Molecular mechanisms of cytochrome p450 induction: potential for drug-drug interactions.

    TOXLINE Toxicology Bibliographic Information

    Jana S; Paliwal J

    2007-12-01

    The human liver cytochromes P450 (CYP P450s) are superfamily of hemoproteins responsible for catalyzing the oxidative metabolism of drugs and xenobiotics entering human body. Drug-drug/xenobiotic interactions are a major cause of therapeutic failures and adverse events. The concomitant administration of inducers with other drugs that are metabolized by CYP450 can result in their altered metabolism in the gastrointestinal tract and/ or liver. The clinical importance of such interactions includes auto induction leading to suboptimal or failed treatment. It is a major concern for the drug companies while developing new drugs. The present understanding of the mechanisms of induction of CYP P450s enzymes and their regulation has made considerable progress during last few years. However there are still gaps in our understanding on molecular aspects of CYP enzymes. Therefore, it remains the subject of intense scientific research to ascertain their in vivo function, and also better understand how the expression of CYP enzymes is regulated at the molecular level. This review analyzes and presents recent findings and concepts on xenosensors and their target genes. Emphasis is given to the molecular mechanisms and signaling pathways of CYP P450 mediated induction by xenobiotics and their potential for drug-drug interactions.

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

    SciTech Connect

    Jung, Christiane . E-mail: christiane_jung@hotmail.com; Schuenemann, Volker; Lendzian, Friedhelm

    2005-12-09

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

  11. Molecular cloning of a divinyl ether synthase. Identification as a CYP74 cytochrome P-450.

    PubMed

    Itoh, A; Howe, G A

    2001-02-01

    Lipoxygenase-derived fatty acid hydroperoxides are metabolized by CYP74 cytochrome P-450s to various oxylipins that play important roles in plant growth and development. Here, we report the characterization of a Lycopersicon esculentum (tomato) cDNA whose predicted amino acid sequence defines a previously unidentified P-450 subfamily (CYP74D). The recombinant protein, expressed in Escherichia coli, displayed spectral properties of a P-450. The enzyme efficiently metabolized 9-hydroperoxy linoleic acid and 9-hydroperoxy linolenic acid but was poorly active against the corresponding 13-hydroperoxides. Incubation of recombinant CYP74D with 9-hydroperoxy linoleic acid and 9-hydroperoxy linolenic acid yielded divinyl ether fatty acids (colneleic acid and colnelenic acid, respectively), which have been implicated as plant anti-fungal toxins. This represents the first identification of a cDNA encoding a divinyl ether synthase and establishment of the enzyme as a CYP74 P-450. Genomic DNA blot analysis revealed the existence of a single divinyl ether synthase gene located on chromosome one of tomato. In tomato seedlings, root tissue was the major site of both divinyl ether synthase mRNA accumulation and enzyme activity. These results indicate that developmental expression of the divinyl ether synthase gene is an important determinant of the tissue specific synthesis of divinyl ether oxylipins. PMID:11060314

  12. Differential induction of cytochrome P450-mediated triasulfuron metabolism by naphthalic anhydride and triasulfuron.

    PubMed Central

    Persans, M W; Schuler, M A

    1995-01-01

    Cytochrome P450 monooxygenases play paramount roles in the detoxification of herbicides as well as in the synthesis of lignins, flavonoids, and phenolic acids. Biochemical analysis of triasulfuron metabolism in maize (Zea mays) seedlings has demonstrated that the P450(s) responsible for detoxification of this herbicide is induced by naphthalic anhydride (NA), a plant safener, and by triasulfuron, the herbicide itself. Induction studies conducted with seedlings of different ages suggest that two separate response pathways modulate this P-450 activity. Induction by NA is independent of the developmental age of the seedlings up to 6.5 d; induction by triasulfuron is tightly modulated with respect to developmental age in that triasulfuron metabolism can be induced by triasulfuron in young (2.5 d) but not older (6.5 d) seedlings. Induction by NA administered in combination with triasulfuron synergistically enhances triasulfuron metabolism in younger seedlings to levels substantially above that obtained with either herbicide or safener treatment alone. In older seedlings, NA plus triasulfuron treatment induces triasulfuron metabolism to only the level of NA treatment alone, indicating again that the induction cascade responding to triasulfuron is nonfunctional in later development. MnCl2 studies indicate that the triasulfuron insensitivity of older seedlings does not result from a general limitation in the inducibility of this P-450 detoxification system but rather from specific limitations in the triasulfuron-response pathway. PMID:8539299

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

    PubMed

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

    2005-05-01

    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

  14. CYP261 enzymes from deep sea bacteria: a clue to conformational heterogeneity in cytochromes P450.

    PubMed

    Davydov, Dmitri R; Sineva, Elena V; Davydova, Nadezhda Y; Bartlett, Douglas H; Halpert, James R

    2013-01-01

    We have explored the adaptation of the cytochromes P450 (P450) of deep-sea bacteria to high hydrostatic pressures. Strict conservation of the protein fold and functional importance of protein-bound water make P450 a unique subject for the studies of high-pressure adaptation. Earlier, we expressed and purified a fatty-acid binding P450 from the deep-sea bacteria Photobacterium profundum SS9 (CYP261C1). Here, we report purification and initial characterization of its mesophilic ortholog from the shallow-water P. profundum 3TCK (CYP261C2), as well as another piezophilic enzyme, CYP261D1, from deep-sea Moritella sp. PE36. Comparison of the three enzymes revealed a striking peculiarity of the piezophilic enzymes. Both CYP261C1 and CYP261D1 possess an apparent pressure-induced conformational toggle actuated at the pressures commensurate with the physiological pressure of habitation of the host bacteria. Furthermore, in contrast to CYP261C2, the piezophilic CYP261 enzymes may be chromatographically separated into two fractions with different properties, and different thermodynamic parameters of spin equilibrium in particular. According to our concept, the changes in the energy landscape that evolved in pressure-tolerant enzymes must stabilize the less-hydrated, closed conformers, which may be transient in the catalytic mechanisms of nonpiezophilic enzymes. The studies of enzymes of piezophiles should help unravel the mechanisms that control water access during the catalytic cycle. PMID:23586990

  15. The role of tryptophan 97 of cytochrome P450 BM3 from Bacillus megaterium in catalytic function. Evidence against the 'covalent switching' hypothesis of P-450 electron transfer.

    PubMed Central

    Munro, A W; Malarkey, K; McKnight, J; Thomson, A J; Kelly, S M; Price, N C; Lindsay, J G; Coggins, J R; Miles, J S

    1994-01-01

    The 'Covalent Switching' hypothesis suggests that a strongly conserved tryptophan residue acts as a mediator of electron-transfer flow between redox partners in cytochrome P-450 systems [Baldwin, Morris and Richards (1991) Proc. R. Soc. London B 245, 43-51]. We have investigated the effect of alteration of the conserved tryptophan (Trp-97) in cytochrome P-450 BM3 (P-450 102) from Bacillus megaterium. Replacement of Trp-97 with Ala, Phe or Tyr results in a decrease in the natural haem content and alters the resting spin state of the remaining haem in the purified mutant enzymes. However, kinetic analyses indicate that the mutant enzymes retain high levels of catalytic activity. C.d. and e.p.r. spectroscopy also reveal little alteration in secondary structure or change in the pattern of haem ligation. These findings cast doubt on the covalent switching mechanism of intermolecular electron flow in the P-450s, but indicate that this residue plays a role in the association of the haem prosthetic group. PMID:7980400

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

    PubMed

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

    2014-01-01

    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

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

    PubMed Central

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

    2014-01-01

    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 Darwins 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

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

    PubMed Central

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

    2015-01-01

    Fungi are an exceptional source of diverse and novel cytochrome P450 monooxygenases (P450s), heme-thiolate proteins, with catalytic versatility. Agaricomycotina saprophytes have yielded most of the available information on basidiomycete P450s. This resulted in observing similar P450 family types in basidiomycetes with few differences in P450 families among Agaricomycotina saprophytes. The present study demonstrated the presence of unique P450 family patterns in basidiomycete biotrophic plant pathogens that could possibly have originated from the adaptation of these species to different ecological niches (host influence). Systematic analysis of P450s in basidiomycete biotrophic plant pathogens belonging to three different orders, Agaricomycotina (Armillaria mellea), Pucciniomycotina (Melampsora laricis-populina, M. lini, Mixia osmundae and Puccinia graminis) and Ustilaginomycotina (Ustilago maydis, Sporisorium reilianum and Tilletiaria anomala), revealed the presence of numerous putative P450s ranging from 267 (A. mellea) to 14 (M. osmundae). Analysis of P450 families revealed the presence of 41 new P450 families and 27 new P450 subfamilies in these biotrophic plant pathogens. Order-level comparison of P450 families between biotrophic plant pathogens revealed the presence of unique P450 family patterns in these organisms, possibly reflecting the characteristics of their order. Further comparison of P450 families with basidiomycete non-pathogens confirmed that biotrophic plant pathogens harbour the unique P450 families in their genomes. The CYP63, CYP5037, CYP5136, CYP5137 and CYP5341 P450 families were expanded in A. mellea when compared to other Agaricomycotina saprophytes and the CYP5221 and CYP5233 P450 families in P. graminis and M. laricis-populina. The present study revealed that expansion of these P450 families is due to paralogous evolution of member P450s. The presence of unique P450 families in these organisms serves as evidence of how a host/ecological niche can influence shaping the P450 content of an organism. The present study initiates our understanding of P450 family patterns in basidiomycete biotrophic plant pathogens. PMID:26536121

  19. Modulation of the Cytochrome P450 Reductase Redox Potential by the Phospholipid Bilayer

    PubMed Central

    Das, Aditi; Sligar, Stephen G.

    2009-01-01

    Cytochrome P450 reductase (CPR) is a tethered membrane protein which transfers electrons from NADPH to microsomal P450s. We show that the lipid bilayer has a role in defining the redox potential of the CPR flavin domains. In order to quantitate the electrochemical behavior of this central redox protein, full length CPR was incorporated into soluble nanometer scale discoidal membrane bilayers (Nanodiscs) and potentials measured using spectro-potentiometry. The redox potentials of both FMN and FAD were found to shift to more positive values when in a membrane bilayer as compared to a solubilized version of the reductase. The potentials of the semiquinone/hydroquinone couple of both FMN and FAD are altered to a larger extent than the oxidized/semiquinone couple which is understood by a simple electrostatic model. When anionic lipids were used to change the membrane composition of the CPR-Nanodisc, the redox potential of both flavins became more negative, favoring electron transfer from CPR to cytochrome P450. PMID:19908820

  20. Cloning and characterization of the cytochrome P450 oxidoreductase gene from the zygomycete fungus Cunninghamella.

    PubMed

    Yadav, J S; Loper, J C

    2000-02-16

    The filamentous fungus Cunninghamella utilizes cytochrome P450 system(s) in the metabolism of a broad range of polyaromatic and aliphatic pollutants and a variety of drugs, but prior attempts at isolation of P450 system components of this fungus have been generally unsuccessful. We report upon the cytochrome P450 oxidoreductase (CPR) gene from two widely studied species, C. elegans and C. echinulata. The C. elegans CPR gene was obtained by screening a genomic library using as probe a PCR amplicon obtained with degenerate primers based on known CPRs. The 2420 bp coding region contained two apparent introns (149 bp and 138 bp). Northern blot analysis showed that the CPR gene is transcriptionally expressed in C. elegans and appears to be inducible by an alkane substrate, n-tetradecane. Phylogenetic comparison of the deduced C. elegans CPR (710 aa) suggested that it is more closely related to animal CPRs (41-42%) than to yeast (38-41%) and plant (35-36%) forms. A 2074 bp sequence containing most of the CPR gene homolog from C. echinulata was also isolated. PMID:10679206

  1. Catalytic and immunochemical properties of NADPH-cytochrome P450 reductase from fungus Rhizopus nigricans.

    PubMed

    Makovec, Tomaz; Breskvar, Katja

    2002-09-01

    Flavoprotein NADPH-cytochrome P450 reductase (CPR, EC 1.6.2.4) from filamentous fungus Rhizopus nigricans is a membrane bound enzyme which is involved in the reduction of cytochrome P450 during the hydroxylation of progesterone at 11alpha position. After purification of the enzyme from induced mycelia three forms of fungal CPR were detected on SDS-PAGE: a predominant form with an apparent molecular mass of 78kDa and two truncated forms. N-terminal sequences of all three forms were determined as well as some internal sequences of 78kDa form. Dose-dependent immunoinhibition of NADPH-cytochrome c reductase and progesterone 11alpha-hydroxylase activities was observed with mouse anti-CPR antisera. No cross-reactions were obtained on Western blots between mouse anti-CPR antisera and protein preparations from noninduced mycelia and microsomal fraction from fungus Pleurotus osteatus, plant Ginkgo biloba or chicken liver. The kinetic mechanism of CPR was proposed on the basis of model reaction with cytochrome c(3+). Results obtained at high ionic strength suggest a nonclassical two-site ping pong mechanism and at low ionic strength a sequential mechanism of bisubstrate reaction. PMID:12429143

  2. Oxidation of pyrene, 1-hydroxypyrene, 1-nitropyrene and 1-acetylpyrene by human cytochrome P450 2A13.

    PubMed

    Shimada, Tsutomu; Takenaka, Shigeo; Murayama, Norie; Kramlinger, Valerie M; Kim, Joo-Hwan; Kim, Donghak; Liu, Jiawang; Foroozesh, Maryam K; Yamazaki, Hiroshi; Guengerich, F Peter; Komori, Masayuki

    2016-03-01

    1.?The polycyclic hydrocarbons (PAHs), pyrene, 1-hydroxypyrene, 1-nitropyrene and 1-acetylpyrene, were found to induce Type I binding spectra with human cytochrome P450 (P450) 2A13 and were converted to various mono- and di-oxygenated products by this enzyme. 2.?Pyrene was first oxidized by P450 2A13 to 1-hydroxypyrene which was further oxidized to di-oxygenated products, i.e. 1,8- and 1,6-dihydroxypyrene. Of five other human P450s examined, P450 1B1 catalyzed pyrene oxidation to 1-hydroxypyrene at a similar rate to P450 2A13 but was less efficient in forming dihydroxypyrenes. P450 2A6, a related human P450 enzyme, which did not show any spectral changes with these four PAHs, showed lower activities in oxidation of these compounds than P450 2A13. 3.?1-Nitropyrene and 1-acetylpyrene were also found to be efficiently oxidized by P450 2A13 to several oxygenated products, based on mass spectrometry analysis. 4.?Molecular docking analysis supported preferred orientations of pyrene and its derivatives in the active site of P450 2A13, with lower interaction energies (U values) than observed for P450 2A6 and that several amino acid residues (including Ala-301, Asn-297 and Ala-117) play important roles in directing the orientation of these PAHs in the P450 2A13 active site. In addition, Phe-231 and Gly-329 were found to interact with pyrene to orient this compound in the active site of P450 1B1. 5.?These results suggest that P450 2A13 is one of the important enzymes that oxidizes these PAH compounds and may determine how these chemicals are detoxicated and bioactivated in humans. PMID:26247835

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

    PubMed

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

    2014-02-01

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

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

    PubMed Central

    Bezirtzoglou, Eugenia Elefterios Venizelos

    2012-01-01

    Cytochromes P450 (CYPs) enzymes metabolize a large variety of xenobiotic substances. In this vein, a plethora of studies were conducted to investigate their role, as cytochromes are located in both liver and intestinal tissues. The P450 profile of the human intestine has not been fully characterized. Human intestine serves primarily as an absorptive organ for nutrients, although it has also the ability to metabolize drugs. CYPs are responsible for the majority of phase I drug metabolism reactions. CYP3A represents the major intestinal CYP (80%) followed by CYP2C9. CYP1A is expressed at high level in the duodenum, together with less abundant levels of CYP2C8-10 and CYP2D6. Cytochromes present a genetic polymorphism intra- or interindividual and intra- or interethnic. Changes in the pharmacokinetic profile of the drug are associated with increased toxicity due to reduced metabolism, altered efficacy of the drug, increased production of toxic metabolites, and adverse drug interaction. The high metabolic capacity of the intestinal flora is due to its enormous pool of enzymes, which catalyzes reactions in phase I and phase II drug metabolism. Compromised intestinal barrier conditions, when rupture of the intestinal integrity occurs, could increase passive paracellular absorption. It is clear that high microbial intestinal charge following intestinal disturbances, ageing, environment, or food-associated ailments leads to the microbial metabolism of a drug before absorption. The effect of certain bacteria having a benefic action on the intestinal ecosystem has been largely discussed during the past few years by many authors. The aim of the probiotic approach is to repair the deficiencies in the gut flora and establish a protective effect. There is a tentative multifactorial association of the CYP (P450) cytochrome role in the different diseases states, environmental toxic effects or chemical exposures and nutritional status. PMID:23990816

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

    SciTech Connect

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

    2011-08-05

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

  6. Ocular cytochrome P450s and transporters: roles in disease and endobiotic and xenobiotic disposition

    PubMed Central

    Nakano, Mariko; Lockhart, Catherine M.; Kelly, Edward J.; Rettie, Allan E.

    2015-01-01

    Drug metabolism and transport processes in the liver, intestine and kidney that affect the pharmacokinetics and pharmacodynamics of therapeutic agents have been studied extensively. In contrast, comparatively little research has been conducted on these topics as they pertain to the eye. Recently, however, catalytic functions of ocular cytochrome P450 enzymes have gained increasing attention, in large part due to the roles of CYP1B1 and CYP4V2 variants in primary congenital glaucoma and Biettis corneoretinal crystalline dystrophy, respectively. In this review, we discuss challenges to ophthalmic drug delivery, including Phase I drug metabolism and transport in the eye, and the role of three specific P450s, CYP4B1, CYP1B1 and CYP4V2 in ocular inflammation and genetically determined ocular disease. PMID:24856391

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

    DOEpatents

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

    1992-01-01

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

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

    DOEpatents

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

    1993-01-01

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

  9. Comparison of two monooxygenase systems with cytochrome P450 in filamentous fungus Rhizopus nigricans.

    PubMed

    Kunic, B; Makovec, T; Breskvar, K

    2000-01-01

    Besides the progesterone inducible steroid hydroxylase which catalyses the transformation of progesterone to 11alpha-hydroxyprogesterone the presence of another monooxygenase system in filamentous fungus Rhizopus nigricans (R. nigricans)--lanosterol demethylase was confirmed. Lanosterol demethylase was not inducible with progesterone. After subcellular fractionation both monooxygenase systems were found in microsomal fraction of fungus R. nigricans. To find out how to differentiate between the hydroxylase and demethylase systems the influence of inhibitors ketoconazole and metyrapone on both monooxygenase systems was studied. Both substances efficiently inhibited fungal steroid hydroxylase. Spectral studies used to characterize the interaction of inhibitors with cytochromes P450 showed that both inhibitors bind to induced fungal preparations whereas in noninduced fungal preparations interaction with P450 was found only with ketoconazole. This indicated the difference in interaction of the two inhibitors on both monooxygenase systems present in fungus R. nigricans. PMID:10653158

  10. Light-induced cytochrome P450-dependent enzyme in indole alkaloid biosynthesis: tabersonine 16-hydroxylase.

    PubMed

    Schröder, G; Unterbusch, E; Kaltenbach, M; Schmidt, J; Strack, D; De Luca, V; Schröder, J

    1999-09-17

    Vinblastine and vincristine are two medically important bisindole alkaloids from Catharanthus roseus (Madagascar periwinkle). Attempts at production in cell cultures failed because a part of the complex pathway was not active, i.e. from tabersonine to vindoline. It starts with tabersonine 16-hydroxylase (T16H), a cytochrome P450-dependent enzyme. We now show that T16H is induced in the suspension culture by light and we report the cloning of the cDNA. The enzyme was expressed in Escherichia coli as translational fusion with the P450 reductase from C. roseus, and the reaction product was identified by mass spectrometry. The protein (CYP71D12) shares 47-52% identity with other members of the CYP71D subfamily with unknown function. The induction by light was strongly enhanced by a nutritional downshift (transfer into 8% aqueous sucrose). We discuss the possibility that the entire pathway to bisindoles can be expressed in suspension cultures. PMID:10481044

  11. Immobilization and activity assay of cytochrome P450 on patterned lipid membranes

    SciTech Connect

    Ueda, Yoshihiro; Morigaki, Kenichi . E-mail: morigaki-kenichi@aist.go.jp; Tatsu, Yoshiro; Yumoto, Noboru; Imaishi, Hiromasa . E-mail: himaish@kobe-u.ac.jp

    2007-04-20

    We report on a methodology for immobilizing cytochrome P450 on the surface of micropatterned lipid bilayer membranes and measuring the enzymatic activity. The patterned bilayer comprised a matrix of polymeric lipid bilayers and embedded fluid lipid bilayers. The polymeric lipid bilayer domains act as a barrier to confine fluid lipid bilayers in defined areas and as a framework to stabilize embedded membranes. The fluid bilayer domains, on the other hand, can contain lipid compositions that facilitate the fusion between lipid membranes, and are intended to be used as the binding agent of microsomes containing rat CYP1A1. By optimizing the membrane compositions of the fluid bilayers, we could selectively immobilize microsomal membranes on these domains. The enzymatic activity was significantly higher on lipid bilayer substrates compared with direct adsorption on glass. Furthermore, competitive assay experiment between two fluorogenic substrates demonstrated the feasibility of bioassays based on immobilized P450s.

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

    PubMed

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

    1990-05-01

    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

  13. Effects of cadmium and environmental pollution on metallothionein and cytochrome P450 in Tilapia

    SciTech Connect

    Ueng, Y.F.; Meng, L.M.; Hung, Y.Y.; Ueng, T.H.; Liu, C.; Lai, C.F.

    1996-07-01

    Tilapia are widely distributed freshwater fish frequently used for environmental toxicology, comparative biochemistry and physiology studies. Tilapia can persist in a highly polluted habitat and have the potential for the development as a biological monitor of environmental pollution. Metallothioneins (MTs) are a group of small-molecular-weight cytoplasmic proteins induced in many animals including fish, following exposure to metals such as cadmium, copper, zinc, and mercury. An increasing number of reports have indicated that fish MT induction is a sensitive measure of metal contamination in the environment. Fish cytochrome (P450)-dependent monooxygenases are inducible by many environmental pollutants including polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs). Extensive studies have suggested that fish monooxygenase can serve as a biochemical marker for exposure to PAH- and PCB-types of pollutants. Tilapia P450 is highly responsive to the inductive effects of PAH and PCBs. Tilapia collected from a polluted section of a river showed higher levels of P450 and dependent monooxygenase activities than tilapia collected from an unpolluted section. Previous studies showed that pretreatment with Cd decreased microsomal monooxygenase activities in fish such as plaice, bass, and trout. However, direct information regarding the effects of heavy metals on tilapia P450 are not available. Reports concerning the effect of heavy metal on tilapia MT are scarce. The purpose of the present study was to determine the ability of cadmium to modulate P450 and MT in tilapia liver and gill. In addition, we have extended our study to feral tilapia collected from Er-Jen Stream, a polluted river in Taiwan. 16 refs., 1 fig., 2 tabs.

  14. Alterations in cytochrome P-450 levels in adult rats following neonatal exposure to xenobiotics

    SciTech Connect

    Zangar, R.C. Pacific Northwest Laboratories, Richland, WA ); Springer, D.L. ); Buhler, D.R. )

    1993-01-01

    Neonatal exposure to certain xenobiotics has been shown to alter hepatic metabolism in adult rats in a manner that indicates long-term changes in enzyme regulation. Previously, the authors have observed changes in adult testosterone metabolism and in cytochrome P-450 (P-450) mRNA levels in animals neonatally exposed to phenobarbital (PB) or diethylstilbestrol (DES). In order to test for other enzyme alterations, they used Western blot procedures for specific P-450s to analyze hepatic microsomes from adult rats (24 wk old) that had been exposed neonatally to DES, PB, 7,12-dimethylbenz[a]anthracene (DMBA), or pregnenolone 16[alpha]-carbonitrile (PCN). The most striking effects were observed in the DES-treated males: P-4502C6 and an immunologically similar protein were increased 60 and 90%, respectively, relative to control values, but P-4503A2 was decreased by 44%. No changes were observed in the DES-treated males in levels of P-4502E1, P-4502B, or the male-specific P-4502C13. Adult males neonatally treated with PB had 150% increase in levels of anti-P4502B-reactive protein without significant changes in the other enzymes. The DES- and DMBA-treated females had increased levels of the female-specific P-4502C12 of 38 and 48%, respectively, but no other observed alterations. The results confirm that neonatal exposure to DES or PB can cause alterations in adult hepatic cytochrome P-450 levels but show that these chemicals act on different enzymes. Neonatal DMBA resulted in changes in adult females similar to those produced by the synthetic estrogen DES, but did so at about two-thirds lower dose. 37 refs., 5 figs.

  15. Potential Biological Functions of Cytochrome P450 Reductase-dependent Enzymes in Small Intestine

    PubMed Central

    D'Agostino, Jaime; Ding, Xinxin; Zhang, Peng; Jia, Kunzhi; Fang, Cheng; Zhu, Yi; Spink, David C.; Zhang, Qing-Yu

    2012-01-01

    NADPH-cytochrome P450 reductase (POR) is essential for the functioning of microsomal cytochrome P450 (P450) monooxygenases and heme oxygenases. The biological roles of the POR-dependent enzymes in the intestine have not been defined, despite the wealth of knowledge on the biochemical properties of the various oxygenases. In this study, cDNA microarray analysis revealed significant changes in gene expression in enterocytes isolated from the small intestine of intestinal epithelium-specific Por knock-out (named IE-Cpr-null) mice compared with that observed in wild-type (WT) littermates. Gene ontology analyses revealed significant changes in terms related to P450s, transporters, cholesterol biosynthesis, and, unexpectedly, antigen presentation/processing. The genomic changes were confirmed at either mRNA or protein level for selected genes, including those of the major histocompatibility complex class II (MHC II). Cholesterol biosynthetic activity was greatly reduced in the enterocytes of the IE-Cpr-null mice, as evidenced by the accumulation of the lanosterol metabolite, 24-dihydrolanosterol. However, no differences in either circulating or enterocyte cholesterol levels were observed between IE-Cpr-null and WT mice. Interestingly, the levels of the cholesterol precursor farnesyl pyrophosphate and its derivative geranylgeranyl pyrophosphate were also increased in the enterocytes of the IE-Cpr-null mice. Furthermore, the expression of STAT1 (signal transducer and activator of transcription 1), a downstream target of geranylgeranyl pyrophosphate signaling, was enhanced. STAT1 is an activator of CIITA, the class II transactivator for MHC II expression; CIITA expression was concomitantly increased in IE-Cpr-null mice. Overall, these findings provide a novel and mechanistic link between POR-dependent enzymes and the expression of MHC II genes in the small intestine. PMID:22453923

  16. Cytochrome P450 1A2 Detoxicates Aristolochic Acid in the Mouse

    PubMed Central

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

    2010-01-01

    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

  17. Ecologically Appropriate Xenobiotics Induce Cytochrome P450s in Apis mellifera

    PubMed Central

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

    2012-01-01

    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

  18. Coal fly ash induces hepatic and pulmonary cytochrome P-450 and sigma-aminolevulinic acid synthetase in rats

    SciTech Connect

    Srivastava, P.K.; Misra, U.K.

    1987-01-01

    The effect of intratracheal administration of coal fly ash, its benzene-soluble and benzene-insoluble fractions has been studied on the levels of hepatic and pulmonary cytochrome P-450, cytochrome b5, and the activities of sigma-aminolevulinic acid synthetase and heme oxygenase. Fly ash and both its fractions significantly increased the levels of hepatic and pulmonary cytochrome P-450. Benzene-soluble and benzene-insoluble fractions of coal fly ash significantly increased the levels of cytochrome b5 also in both lung and liver. Fly ash and both its fractions increased the activity of sigma-aminolevulinic acid synthetase and reduced the activity of heme oxygenase in lung and liver. Glass bead particles of similar size did not show any effect on hepatic and pulmonary cytochrome P-450 and cytochrome b5.

  19. Identification of a microsomal retinoic acid synthase as a microsomal cytochrome P-450-linked monooxygenase system.

    PubMed

    Tomita, S; Tsujita, M; Matsuo, Y; Yubisui, T; Ichikawa, Y

    1993-12-01

    1. To characterize an enzyme which metabolizes retinal in liver microsomes, several properties of the enzymatic reaction from retinal to retinoic acid were investigated using rabbit liver microsomes. 2. The maximum pH of the reaction in the liver microsomes was 7.6. 3. The Km and Vmax values for all-trans, 9-cis and 13-cis-retinals were determined. 4. The reaction proceeded in the presence of NADPH and molecular oxygen. 5. The incorporation of one atom of molecular oxygen into retinal was confirmed by using oxygen-18, showing that the reaction comprised monooxygenation, not dehydrogenation. 6. The monooxygenase activity was inhibited by carbon monoxide, phenylisocyanide and anti-NADPH-cytochrome P-450 reductase IgG, but not by anti-cytochrome b5 IgG. 7. The enzymatic activity inhibited by carbon monoxide was photoreversibly restored by light of a wavelength of around 450 nm. 8. The retinal-induced spectra of liver microsomes with three isomeric retinals were type I spectra. 9. The microsomal monooxygenase activity induced by phenobarbital or ethanol were more effective than that by 3-methylcholanthrene, clotrimazole or beta-naphthoflavone. 10. These results showed that the monooxygenase reaction from retinal to retinoic acid in liver microsomes is catalyzed by a cytochrome P-450-linked monooxygenase system. PMID:8138015

  20. Regulation of Gap Junction Function and Connexin 43 Expression by Cytochrome P450 Oxidoreductase (CYPOR)

    PubMed Central

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

    2011-01-01

    Cytochrome P450 oxidoreductase (CYPOR) is a microsomal electron-transferring enzyme containing both FAD and FMN as co-factors, which provides the reducing equivalents to various redox partners, such as cytochromes P450 (CYPs), heme oxygenase (HO), cytochrome b5 and squalene monooxygenase. Human patients with severe forms of CYPOR mutation show bone defects such as cranio- and humeroradial synostoses and long bone fractures, known as Antley-Bixler-like Syndrome (ABS). To elucidate the role of CYPOR in bone, we knocked-down CYPOR in multiple osteoblast cell lines using RNAi technology. In this study, knock-down of CYPOR decreased the expression of Connexin43 (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 calvaria 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. PMID:21726529

  1. The role of brain noradrenergic system in the regulation of liver cytochrome P450 expression.

    PubMed

    Sadakierska-Chudy, Anna; Haduch, Anna; Rysz, Marta; Gołembiowska, Krystyna; Daniel, Władysława A

    2013-09-15

    The aim of the present study was to examine the effect of the brain noradrenergic system on the expression of cytochrome P450 in the liver. The experiment was carried out on male Wistar rats. Intracerebroventricular injection of the noradrenergic neurotoxin DSP-4 diminished noradrenaline level in the brain. Simultaneously, significant decreases in the serum concentration of the growth hormone, testosterone and the thyroid hormone thyroxine, as well as an increase in corticosterone level were observed. The concentrations of triiodothyronine and the cytokines interleukine 2 (IL-2) and 6 (IL-6) were not changed by DSP-4. The neurotoxin produced complex changes in the functioning of cytochrome P450. Significant decreases in the activity of liver CYP2C11 (measured as a rate of the 2α- and 16α-hydroxylation of testosterone) and CYP3A (measured as a rate of the 2β- and 6β-hydroxylation of testosterone) were found. In contrast, the activity of CYP1A (measured as a rate of caffeine metabolism) rose, while that of CYP2A (measured as a rate of the 7α-hydroxylation of testosterone), CYP2C6 (measured as a rate of the 7-hydroxylation of warfarin) and CYP2D (the 1'-hydroxylation of bufuralol) remained unchanged. The changes in the activity of CYP1A, CYP2C11 and CYP3A correlated positively with those in CYP protein levels and with the CYP mRNA levels of CYP1A1, CYP2C11 and CYP3A1/2 genes, respectively. The obtained results indicate an important role of the brain noradrenergic system in the neuroendocrine regulation of liver cytochrome P450 expression, which may be of significance in pathological states involving this system, or during pharmacotherapy with drugs affecting noradrenergic transmission. PMID:23924605

  2. Control of heme and cytochrome P-450 metabolism by inorganic metals, organometals and synthetic metalloporphyrins

    SciTech Connect

    Kappas, A.; Drummond, G.S.

    1984-08-01

    The heme-cytochrome P-450 complexes represent sensitive metabolic systems for examining the biological impact of metals on important cellular functions. Many metals, both in the inorganic form and bound to organic moieties, potently induce heme oxygenase, the rate limiting enzyme of heme degradation. The resulting increase in the rate of heme breakdown is reflected in a marked depression of cellular cytochrome P-450 content and impairment of the oxidative metabolism of natural and foreign chemicals dependent on this hemoprotein. Organometal complexes do not mimic in all their aspects the actions of the inorganic elements which they contain. For example, organotins, in contrast to inorganic tin, produce a prolonged induction response of heme oxygenase in the liver but not in the kidney. Co-protoporphyrin is a much more potent inducer of heme oxygenase in liver than is inorganic cobalt; and Sn-protoporphyrin inhibits heme oxygenase activity nearly completely, whereas inorganic tin is a powerful inducer of the renal enzyme. Contrasting effects on heme metabolism exist as well within the metalloporphyrin species as demonstrated by the effects in vivo of Co-protoporphyrin and Sn-protoporphyrin on heme oxygenase activity; the former induces the enzyme whereas the latter potently inhibits it. In vitro, however, both compounds competitively inhibit heme oxidation activity. These differences, among others which characterize metal actions in vivo and in vitro attest to the importance of pharmacokinetic, adaptive and other host factors in defining the responses of the heme-cytochrome P-450 systems to the impact of metals in the whole animal. 21 references, 8 figures, 1 table.

  3. Control of heme and cytochrome P-450 metabolism by inorganic metals, organometals and synthetic metalloporphyrins.

    PubMed Central

    Kappas, A; Drummond, G S

    1984-01-01

    The heme-cytochrome P-450 complexes represent sensitive metabolic systems for examining the biological impact of metals on important cellular functions. Many metals, both in the inorganic form and bound to organic moieties, potently induce heme oxygenase, the rate limiting enzyme of heme degradation. The resulting increase in the rate of heme breakdown is reflected in a marked depression of cellular cytochrome P-450 content and impairment of the oxidative metabolism of natural and foreign chemicals dependent on this hemeprotein. Organometal complexes do not mimic in all their aspects the actions of the inorganic elements which they contain. For example, organotins, in contrast to inorganic tin, produce a prolonged induction response of heme oxygenase in the liver but not in the kidney. Co-protoporphyrin is a much more potent inducer of heme oxygenase in liver than is inorganic cobalt; and Sn-protoporphyrin inhibits heme oxygenase activity nearly completely, whereas inorganic tin is a powerful inducer of the renal enzyme. Contrasting effects on heme metabolism exist as well within the metalloporphyrin species as demonstrated by the effects in vivo of Co-protoporphyrin and Sn-protoporphyrin on heme oxygenase activity; the former induces the enzyme whereas the latter potently inhibits it. In vitro, however, both compounds competitively inhibit heme oxidation activity. These differences, among others which characterize metal actions in vivo and in vitro attest to the importance of pharmacokinetic, adaptive and other host factors in defining the responses of the heme-cytochrome P-450 systems to the impact of metals in the whole animal. Images FIGURE 5. PMID:6548701

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

    PubMed

    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

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

  5. Environmentally Persistent Free Radicals Inhibit Cytochromes P450 Activity in Rat Liver Microsomes

    PubMed Central

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

    2014-01-01

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

  6. Mammalian PC-12 cell genetically engineered for human cytochrome P450 2E1 expression.

    PubMed

    Mapoles, J; Berthou, F; Alexander, A; Simon, F; Ménez, J F

    1993-06-15

    The stable expression of the human cytochrome CYP2E1 (P450 alcohol) was performed in the mammalian cell line PC-12. This cell line expressed cytochrome b5 (58 +/- 12 pmol/mg microsomal protein vs 528 +/- 80 pmol/mg in microsomal human liver) and a high level of NADPH: cytochrome P450 reductase (140 +/- 20 nmol.min-1.mg microsomal protein-1 vs 68 +/- 48 nmol.min-1.mg-1 in microsomal human liver). An expression plasmid was constructed using the cDNA for the human CYP2E1 mRNA and the Rous sarcoma virus (RSV) promoter. This plasmid was co-transfected with the plasmid RSVneo into PC-12 cells. Clones were selected for resistance to the neomycin analog, G418, and then screened for expression of the CYP2E1 isozyme by testing for 6-hydroxylation of chlorzoxazone, a specific substrate for CYP2E1. Expression of CYP2E1 was confirmed in one clone, DB-7, by Western blot analysis and by measurement of monooxygenase activities which were not detectable in PC-12 cells. Chlorzoxazone 6-hydroxylation, n-butanol oxidation and dimethylnitrosamine N-demethylation were localized in microsomes (62, 60 and 63 pmol.min-1.mg microsomal protein-1, respectively) and were inhibited by carbon monoxide and diethyldithiocarbamate, both inhibitors of P450 enzymes. Although the level of the enzyme activities was about a tenth of that measured in human liver microsomes, CYP2E1 expressed in DB-7 cells has catalytic competence similar to human liver CYP2E1. DB-7 cells metabolized acetaminophen and this metabolic activation was shown to be toxic to these cells by release of lactate dehydrogenase. Construction of recombinant cell lines expressing CYP2E1 provides a useful tool for studying the catalytic properties of this enzyme and the consequent cytotoxic effects of substrates metabolized by this enzyme. PMID:8391436

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

    SciTech Connect

    Froehlich, Eleonore; Kueznik, Tatjana; Samberger, Claudia; Roblegg, Eva; Wrighton, Christopher

    2010-02-01

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

  8. Molecular characterization and functional analysis of three pathogenesis-related cytochrome P450 genes from Bursaphelenchus xylophilus (Tylenchida: Aphelenchoidoidea).

    PubMed

    Xu, Xiao-Lu; Wu, Xiao-Qin; Ye, Jian-Ren; Huang, Lin

    2015-01-01

    Bursaphelenchus xylophilus, the causal agent of pine wilt disease, causes huge economic losses in pine forests. The high expression of cytochrome P450 genes in B. xylophilus during infection in P. thunbergii indicated that these genes had a certain relationship with the pathogenic process of B. xylophilus. Thus, we attempted to identify the molecular characterization and functions of cytochrome P450 genes in B. xylophilus. In this study, full-length cDNA of three cytochrome P450 genes, BxCYP33C9, BxCYP33C4 and BxCYP33D3 were first cloned from B. xylophilus using 3' and 5' RACE PCR amplification. Sequence analysis showed that all of them contained a highly-conserved cytochrome P450 domain. The characteristics of the three putative proteins were analyzed with bioinformatic methods. RNA interference (RNAi) was used to assess the functions of BxCYP33C9, BxCYP33C4 and BxCYP33D3. The results revealed that these cytochrome P450 genes were likely to be associated with the vitality, dispersal ability, reproduction, pathogenicity and pesticide metabolism of B. xylophilus. This discovery confirmed the molecular characterization and functions of three cytochrome P450 genes from B. xylophilus and provided fundamental information in elucidating the molecular interaction mechanism between B. xylophilus and its host plant. PMID:25756378

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

    PubMed Central

    Xu, Xiao-Lu; Wu, Xiao-Qin; Ye, Jian-Ren; Huang, Lin

    2015-01-01

    Bursaphelenchus xylophilus, the causal agent of pine wilt disease, causes huge economic losses in pine forests. The high expression of cytochrome P450 genes in B. xylophilus during infection in P. thunbergii indicated that these genes had a certain relationship with the pathogenic process of B. xylophilus. Thus, we attempted to identify the molecular characterization and functions of cytochrome P450 genes in B. xylophilus. In this study, full-length cDNA of three cytochrome P450 genes, BxCYP33C9, BxCYP33C4 and BxCYP33D3 were first cloned from B. xylophilus using 3' and 5' RACE PCR amplification. Sequence analysis showed that all of them contained a highly-conserved cytochrome P450 domain. The characteristics of the three putative proteins were analyzed with bioinformatic methods. RNA interference (RNAi) was used to assess the functions of BxCYP33C9, BxCYP33C4 and BxCYP33D3. The results revealed that these cytochrome P450 genes were likely to be associated with the vitality, dispersal ability, reproduction, pathogenicity and pesticide metabolism of B. xylophilus. This discovery confirmed the molecular characterization and functions of three cytochrome P450 genes from B. xylophilus and provided fundamental information in elucidating the molecular interaction mechanism between B. xylophilus and its host plant. PMID:25756378

  10. Time Course of Induction of Cytochrome P-450, NADPH-Cytochrome c Reductase, and Cinnamic Acid Hydroxylase by Phenobarbital, Ethanol, Herbicides, and Manganese in Higher Plant Microsomes 1

    PubMed Central

    Reichhart, Danile; Salan, Jean-Pierre; Benveniste, Irne; Durst, Francis

    1980-01-01

    The mixed function oxidase trans-cinnamic acid 4-hydroxylase, cytochrome P-450, cytochrome b5, and NADPH-cytochrome c (P-450) reductase were measured in microsomes from aging artichoke tuber slices exposed to manganese, ethanol, phenobarbital, and the herbicides Chloro-IPC, Dichlobenil, and Monuron. Although the microsomal hydroxylating complex is already induced by the slicing and aging process, 25 millimolar MnCl2, 4 millimolar phenobarbital, and 300 millimolar ethanol caused a marked increase of hydroxylase activity and cytochrome P-450 content and shifted their time course. The herbicides, 200 micromolar Dichlobenil and 200 micromolar Monuron, were less effective. Chloro-IPC was slightly inhibitory. NADPH cytochrome c reductase was significantly increased only in phenobarbital-treated slices. Cytochrome b5 was generally the least affected among the parameters being measured. The mechanisms by which these compounds increase cytochrome P-450 content and hydroxylase activity are not yet defined. PMID:16661486

  11. Expression of P450 Aromatase in Granulosa Cell Tumors and Sertoli-Stromal Cell Tumors of the Ovary: Which Cells Are Responsible for Estrogenesis?

    PubMed Central

    Uchigasaki, Shinya; Fukase, Masayuki; Kurose, Akira

    2016-01-01

    Granulosa cell tumors are representative of estrogenic ovarian tumors, and some Sertoli-stromal cell tumors are also estrogenic. The exact cells that are responsible for estrogenesis, however, have yet to be identified. In the present study, 25 sex cord-stromal tumors (20 granulosa cell tumors, 4 Sertoli-Leydig cell tumors, and a Sertoli cell tumor) were immunohistochemically examined for expression of P450 aromatase, which is critical for estrogenesis. All of the tumors had been evaluated for estrogenic function, including contemporaneous endometrial hyperplasia and/or elevation of serum estradiol. Eleven of 14 estrogenic granulosa cell tumors showed sparse or aggregated immunoreactivity for aromatase, whereas 5 of 6 nonestrogenic tumors did not. Aromatase was selectively expressed by plump granulosa cells with eosinophilic or vacuolated cytoplasm, resembling luteinized granulosa cells. Such a localization of aromatase is analogous to that in normal ovaries. Aromatase expression in primary tumors was recapitulated by recurrent tumors. In Sertoli-stromal cell tumors, either undifferentiated plump cells or well-differentiated Sertoli cells expressed aromatase. In conclusion, the expression of P450 aromatase corresponds to specific cell morphology in sex cord-stromal tumors, including recurrent tumors. Aromatase status in granulosa cell tumors provides helpful information on whether serum estradiol could be a marker for recurrence. PMID:26166720

  12. Epoxidation of olefins by cytochrome P-450 model compounds: mechanism of oxygen atom transfer.

    PubMed Central

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

    1984-01-01

    The mechanism of the Mn(III) porphyrin-catalyzed epoxidation of olefins by lithium hypochlorite is examined. The active oxidant is thought to be a high-valent manganese-oxo complex. It is shown that a relatively stable intermediate is reversibly formed upon interaction of the olefin and the oxo complex. The decomposition of this intermediate to Mn(III) porphyrin and epoxide is the rate-determining step of the catalytic cycle. Some analogies to the biochemical epoxidation of olefins catalyzed by cytochrome P-450 are discussed. PMID:6587349

  13. Cytochrome P450-derived versus mitochondrial oxidant stress in acetaminophen hepatotoxicity.

    PubMed

    Jaeschke, Hartmut; McGill, Mitchell R

    2015-06-15

    In evaluating the mechanisms of acetaminophen hepatotoxicity in experimental systems, it is critical to keep in mind the relevance of the model system for humans. Important aspects of the human toxicity include formation of a reactive metabolite by the cytochrome P450 system and protein adduct formation, which is thought to trigger mitochondrial dysfunction and oxidant stress ultimately causing necrotic cell death. If models that miss critical parts of this well-established mechanism are used, the relevance of the new information for the human toxicity has to be questioned. Therefore, we feel it is necessary to express our concern regarding the recent publication by Jiang et al. (2015). PMID:25858113

  14. Induction of cytochrome p-450-ia1 in juvenile fish by creosote-contaminated sediment

    SciTech Connect

    Schoor, W.P.; Williams, D.E.; Takahashi, N.

    1991-01-01

    Intact sediment cores, including their surface layers, were used in simulated field exposure tests of juvenile guppies (Poecilia reticulata) to creosote-contaminated sediments. Mixed-function oxygenase activity was induced in the fish after 43 days of exposure to environmentally realistic, sublethal concentrations of creosote-related compounds. An average 50-fold induction in the cytochrome P-450-IA1 was found in the liver in the absence of any histopathological lesions. The possibility that a threshold level for proliferative liver changes was not reached is discussed in the light of the observed biochemical activation.

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

    SciTech Connect

    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

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

  16. Cytochrome P450-catalysed L-tryptophan nitration in thaxtomin phytotoxin biosynthesis

    PubMed Central

    Barry, Sarah M.; Kers, Johan A.; Johnson, Evan G.; Song, Lijiang; Aston, Philip R.; Patel, Bhumit; Krasnoff, Stuart B.; Crane, Brian R.; Gibson, Donna M.; Loria, Rosemary; Challis, Gregory L.

    2012-01-01

    Thaxtomin phytotoxins produced by plant-pathogenic Streptomyces species contain a nitro group that is essential for phytotoxicity. The N,N-dimethyldiketopiperazine core of thaxtomins is assembled from L-phenylalanine and L-4-nitrotryptophan by a nonribosomal peptide synthetase and nitric oxide synthase-generated NO is incorporated into the nitro group, but the biosynthesis of the non-proteinogenic amino acid L-4-nitrotryptophan is unclear. Here we report that TxtE, a unique cytochrome P450, catalyzes L-tryptophan nitration using NO and O2. PMID:22941045

  17. Cytochrome P450catalyzed L-tryptophan nitration in thaxtomin phytotoxin biosynthesis.

    PubMed

    Barry, Sarah M; Kers, Johan A; Johnson, Evan G; Song, Lijiang; Aston, Philip R; Patel, Bhumit; Krasnoff, Stuart B; Crane, Brian R; Gibson, Donna M; Loria, Rosemary; Challis, Gregory L

    2012-10-01

    Thaxtomin phytotoxins produced by plant-pathogenic Streptomyces species contain a nitro group that is essential for phytotoxicity. The N,N'-dimethyldiketopiperazine core of thaxtomins is assembled from L-phenylalanine and L-4-nitrotryptophan by a nonribosomal peptide synthetase, and nitric oxide synthase-generated NO is incorporated into the nitro group, but the biosynthesis of the nonproteinogenic amino acid L-4-nitrotryptophan is unclear. Here we report that TxtE, a unique cytochrome P450, catalyzes L-tryptophan nitration using NO and O(2). PMID:22941045

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

    PubMed Central

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

    1994-01-01

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

  19. Molecular characterization of aromatase.

    PubMed

    Hong, Yanyan; Li, Hongzhi; Yuan, Yate-Ching; Chen, Shiuan

    2009-02-01

    Aromatase is an estrogen synthetase. Estrogens are female sex hormones involved in the development and growth of breast tumors. Elucidation of the structure-function relationship of aromatase has been of significant interest since its inhibitors have shown great promise in fighting breast cancer. Aromatase belongs to the cytochrome P450 family and forms an electron-transfer complex with its partner, NADPH-cytochrome P450 reductase. Because of the membrane-bound character and heme-binding instability, no crystal structure of aromatase has been reported so far. Much remains to be investigated, including the 3-dimensional structure of aromatase, interaction between aromatase and reductase, catalytic mechanism of estrogen synthesis by aromatase, and the binding mechanism of aromatase inhibitors. This review presents current knowledge about structural and functional characteristics of aromatase to address unsolved mysteries about this enzyme. PMID:19250198

  20. Fungal unspecific peroxygenases: heme-thiolate proteins that combine peroxidase and cytochrome p450 properties.

    PubMed

    Hofrichter, Martin; Kellner, Harald; Pecyna, Marek J; Ullrich, Ren

    2015-01-01

    Eleven years ago, a secreted heme-thiolate peroxidase with promiscuity for oxygen transfer reactions was discovered in the basidiomycetous fungus, Agrocybe aegerita. The enzyme turned out to be a functional mono-peroxygenase that transferred an oxygen atom from hydrogen peroxide to diverse organic substrates (aromatics, heterocycles, linear and cyclic alkanes/alkenes, fatty acids, etc.). Later similar enzymes were found in other mushroom genera such as Coprinellus and Marasmius. Approximately one thousand putative peroxygenase sequences that form two large clusters can be found in genetic databases and fungal genomes, indicating the widespread occurrence of such enzymes in the whole fungal kingdom including all phyla of true fungi (Eumycota) and certain fungus-like heterokonts (Oomycota). This new enzyme type was classified as unspecific peroxygenase (UPO, EC 1.11.2.1) and placed in a separate peroxidase subclass. Furthermore, UPOs and related heme-thiolate peroxidases such as well-studied chloroperoxidase (CPO) represent a separate superfamily of heme proteins on the phylogenetic level. The reactions catalyzed by UPOs include hydroxylation, epoxidation, O- and N-dealkylation, aromatization, sulfoxidation, N-oxygenation, dechlorination and halide oxidation. In many cases, the product patterns of UPOs resemble those of human cytochrome P450 (P450) monooxygenases and, in fact, combine the catalytic cycle of heme peroxidases with the "peroxide shunt" of P450s. Here, an overview on UPOs is provided with focus on their molecular and catalytic properties. PMID:26002742

  1. Structural Adaptability Facilitates Histidine Heme Ligation in a Cytochrome P450

    PubMed Central

    2015-01-01

    Almost all known members of the cytochrome P450 (CYP) superfamily conserve a key cysteine residue that coordinates the heme iron. Although mutation of this residue abolishes monooxygenase activity, recent work has shown that mutation to either serine or histidine unlocks non-natural carbene- and nitrene-transfer activities. Here we present the first crystal structure of a histidine-ligated P450. The T213A/C317H variant of the thermostable CYP119 from Sulfolobus acidocaldarius maintains heme iron coordination through the introduced ligand, an interaction that is accompanied by large changes in the overall protein structure. We also find that the axial cysteine C317 may be substituted with any other amino acid without abrogating folding and heme cofactor incorporation. Several of the axial mutants display unusual spectral features, suggesting that they have active sites with unique steric and electronic properties. These novel, highly stable enzyme active sites will be fruitful starting points for investigations of non-natural P450 catalysis and mechanisms. PMID:26299431

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

    SciTech Connect

    Miao, Yinglong; Baudry, Jerome Y

    2011-01-01

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

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

    NASA Astrophysics Data System (ADS)

    Rosenkrans, Charles; Ezell, Nicholas

    2015-03-01

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

  4. Relationships among ergot alkaloids, cytochrome P450 activity, and beef steer growth.

    PubMed

    Rosenkrans, Charles F; Ezell, Nicholas S

    2015-01-01

    Determining a grazing animal's susceptibility to ergot alkaloids has been a research topic for decades. Our objective was to determine if the Promega P450-Glo assay could be used to indirectly detect ergot alkaloids or their metabolites in urine of steers. The first experiment validated the effects of ergot alkaloids [0, 20, and 40 ?M of ergotamine (ET), dihydroergotamine (DHET), and ergonovine (EN)] on human CYP3A4 using the P450-Glo assay (Promega V9800). With this assay, luminescence is directly proportional to CYP450 activity. Relative inhibition of in vitro cytochrome P450 activity was affected (P < 0.001) by an interaction between alkaloids and concentration. That interaction resulted in no concentration effect of EN, but within ET and DHET 20 and 40 ?M concentrations inhibited CYP450 activity when compared with controls. In experiment 2, urine was collected from Angus-sired crossbred steers (n = 39; 216 2.6 days of age; 203 1.7 kg) after grazing tall fescue pastures for 105 days. Non-diluted urine was added to the Promega P450-Glo assay, and observed inhibition (3.7 % 2.7 of control). Urine content of total ergot alkaloids (331.1 ng/mg of creatinine 325.7) was determined using enzyme linked immunosorbent assay. Urine inhibition of CYP450 activity and total alkaloids were correlated (r = -0.31; P < 0.05). Steers were genotyped at CYP450 single nucleotide polymorphism, C994G. Steer genotype affected (P < 0.03) inhibition of CYP450 activity by urine; heterozygous steers had the least amount of CYP450 inhibition suggesting that genotyping cattle may be a method of identifying animals that are susceptible to ergot alkaloids. Although, additional research is needed, we demonstrate that the Promega P450-Glo assay is sensitive to ergot alkaloids and urine from steers grazing tall fescue. With some refinement the P450-Glo assay has potential as a tool for screening cattle for their exposure to fescue toxins. PMID:25815288

  5. Microbial cytochromes P450: biodiversity and biotechnology. Where do cytochromes P450 come from, what do they do and what can they do for us?

    PubMed Central

    Kelly, Steven L.; Kelly, Diane E.

    2013-01-01

    The first eukaryote genome revealed three yeast cytochromes P450 (CYPs), hence the subsequent realization that some microbial fungal genomes encode these proteins in 1 per cent or more of all genes (greater than 100) has been surprising. They are unique biocatalysts undertaking a wide array of stereo- and regio-specific reactions and so hold promise in many applications. Based on ancestral activities that included 14α-demethylation during sterol biosynthesis, it is now seen that CYPs are part of the genes and metabolism of most eukaryotes. In contrast, Archaea and Eubacteria often do not contain CYPs, while those that do are frequently interesting as producers of natural products undertaking their oxidative tailoring. Apart from roles in primary and secondary metabolism, microbial CYPs are actual/potential targets of drugs/agrochemicals and CYP51 in sterol biosynthesis is exhibiting evolution to resistance in the clinic and the field. Other CYP applications include the first industrial biotransformation for corticosteroid production in the 1950s, the diversion into penicillin synthesis in early mutations in fungal strain improvement and bioremediation using bacteria and fungi. The vast untapped resource of orphan CYPs in numerous genomes is being probed and new methods for discovering function and for discovering desired activities are being investigated. PMID:23297358

  6. A Cytochrome P450 Involved in the Metabolism of Abietane Diterpenoids by Pseudomonas abietaniphila BKME-9

    PubMed Central

    Smith, Daryl J.; Martin, Vincent J. J.; Mohn, William W.

    2004-01-01

    Diterpenoids are naturally occurring plant compounds which have pharmaceutical properties. We have sequenced a 10.4-kbp extension of the dit cluster in Pseudomonas abietaniphila BKME-9, containing genes involved in abietane diterpenoid biodegradation. The ditQ gene was found to encode a cytochrome P450 monooxygenase, P450dit, and to be homologous to the tdtD gene of Pseudomonas diterpeniphila A19-6a. Knocking out ditQ had little effect on growth of BKME-9 on abietic acid but severely impaired growth on dehydroabietic acid (DhA) and palustric acid (PaA), increasing doubling times from 3.8 to 15 h on DhA and from 5.6 to 18.5 h on PaA. A xylE transcriptional fusion showed that transcription of ditQ was induced by a range of diterpenoids. Substrate binding assays of P450dit expressed in Escherichia coli revealed that DhA binds to the enzyme and yields a type I binding spectrum with a Kd of 0.4 ?M. These results indicate that P450dit is involved in the metabolism of DhA and PaA and are consistent with its putative role in converting DhA to 7-hydroxy-DhA. Finally, an amino acid sequence identity of greater than 72% and conserved gene arrangement support the hypothesis that the dit gene cluster of P. abietaniphila BKME-9 and the tdt cluster of P. diterpeniphila A19-6a contain functional homologues. PMID:15150251

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

    PubMed Central

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

    2013-01-01

    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

  8. Exposure to various benzene derivatives differently induces cytochromes P450 2B1 and P450 2E1 in rat liver.

    PubMed

    Gut, I; Terelius, Y; Frantk, E; Linhart, I; Soucek, P; Filipcov, B; Kluckov, H

    1993-01-01

    Benzene (B), toluene (T), ethylbenzene (EB), styrene (S) and xylene isomers (oX, mX, pX) are important environmental pollutants and B is a proved human carcinogen. Their inhalation by male Wistar rats (4 mg/l, 20 h/day, 4 days) caused cytochrome P450 (P450) induction. The degree of P450 2B1 induction increased and that of 2E1 decreased in the series B, T, EB, S, oX, mX and pX, as estimated by Western blots, while neither solvent was as effective for 2B1 induction as phenobarbital and B was more effective for 2E1 than ethanol. The levels of several other P450s decreased after exposure to these solvents, B being most effective. Exposure to these solvents increased in vitro hepatic microsomal oxidation of B and aniline (AN) (2E1 substrates) 3 to 6-fold, indicating induction of this P450. T oxidation was increased 2 to 4-fold and chlorobenzene (ClB) oxidation 3-fold. Sodium phenobarbital (PB, 80 mg/kg/day, 4 days, i.p.) did not increase ethylmorphine (EM) and benzphetamine (BZP) demethylation (2B1 substrates), neither of the B derivatives did so, and oX decreased it; however, pentoxyresorufin O-dealkylation was well related to the immunochemically detected 2B1 levels in control, PB and B microsomes. PB did not increase B, but increased T and ClB oxidation 2-4 and 3-fold, respectively, indicating possible 2B1 role in their oxidation. B oxidation after various inducers was related to immunochemical 2E1 levels, T and ClB oxidation to both 2B1 and 2E1 and AN oxidation to 2E1 and 1A2 levels.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8517779

  9. Discrete species of activated oxygen yield different cytochrome P450 heme adducts from aldehydes.

    PubMed

    Kuo, C L; Raner, G M; Vaz, A D; Coon, M J

    1999-08-10

    Aldehydes are known to inactivate cytochrome P450 in the reconstituted enzyme system containing NADPH and NADPH-cytochrome P450 reductase under aerobic conditions in a mechanism-based reaction involving heme adduct formation [Raner, G. M., Chiang, E. W. , Vaz, A. D. N., and Coon, M. J. (1997) Biochemistry 36, 4895-4902]. In the study presented here, artificial oxidants were used to examine the mechanism of aldehyde activation by purified P450 2B4 in the absence of the usual O(2)-reducing system, and the adducts that were formed were isolated and characterized. With hydrogen peroxide as the oxidant, 3-phenylpropionaldehyde gives an adduct with a mass corresponding to that of native heme modified by a phenylethyl group, presumably arising from the reaction of a peroxy-iron species with the aldehyde to give a peroxyhemiacetal, which upon deformylation yields the alkyl radical. NMR analysis indicated that the substitution is specifically at the gamma-meso position. In contrast, with m-chloroperbenzoic acid as the oxidant, an adduct is formed from 3-phenylpropionaldehyde with a mass that is consistent with the addition of a phenylpropionyl group, apparently arising by hydrogen abstraction from the aldehyde to give the carbonyl carbon radical. m-Chloroperbenzoic acid by itself forms a heme adduct with a mass corresponding to the addition of a chlorobenzoyloxy group apparently derived from homolytic oxygen-oxygen bond cleavage. These and other results with nonanal and 2-trans-nonenal support the concept that this versatile enzyme utilizes discrete oxidizing species in heme adduct formation from aldehydes. PMID:10441147

  10. CYTOCHROME P450 17A1 STRUCTURES WITH PROSTATE CANCER DRUGS ABIRATERONE AND TOK-001

    PubMed Central

    DeVore, Natasha M.; Scott, Emily E.

    2011-01-01

    Cytochrome P450 17A1 (P450c17) catalyzes the biosynthesis of androgens in humans1. Since prostate cancer cells proliferate in response to androgen steroids2,3, CYP17A1 inhibition is a new strategy to prevent androgen synthesis and treat lethal metastatic castration-resistant prostate cancer4, but drug development has been hampered by the lack of a CYP17A1 structure. Here we report the only known structures of CYP17A1, which contain either abiraterone, a first-in-class steroidal inhibitor recently approved by the FDA for late-stage prostate cancer5, or TOK-001, another inhibitor in clinical trials4,6. Both bind the heme iron forming a 60 angle above the heme plane, packing against the central I helix with the 3?-OH interacting with N202 in the F helix. Importantly, this binding mode differs substantially from those predicted by homology models or from steroids in other cytochrome P450 enzymes with known structures, with some features more similar to steroid receptors. While the overall CYP17A1 structure provides a rationale for understanding many mutations found in patients with steroidogenic diseases, the active site reveals multiple steric and hydrogen bonding features that will facilitate better understanding of the enzymes dual hydroxylase and lyase catalytic capabilities and assist in rational drug design. Specifically, structure-based design is expected to aid development of inhibitors that bind only CYP17A1 and solely inhibit its androgen-generating lyase activity to improve treatment of prostate and other hormone-responsive cancers. PMID:22266943

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

    PubMed Central

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

    2013-01-01

    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 Å structure of human CYP1A1 with the inhibitor α-naphthoflavone. α-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

  12. Prediction of drug-like molecular properties: modeling cytochrome p450 interactions.

    PubMed

    Jalaie, Mehran; Arimoto, Rieko; Gifford, Eric; Schefzick, Sabine; Waller, Chris L

    2004-01-01

    Preventing drug-drug interactions and reducing drug-related mortalities dictate cleaner and costlier medicines. The cost to bring a new drug to market has increased dramatically over the last 10 years, with post-discovery activities (preclinical and clinical) costs representing the majority of the spend. With the ever-increasing scrutiny that new drug candidates undergo in the post-discovery assessment phases, there is increasing pressure on discovery to deliver higher-quality drug candidates. Given that compound attrition in the early clinical stages can often be attributed to metabolic liabilities, it has been of great interest lately to implement predictive measures of metabolic stability/ liability in the drug design stage of discovery. The solution to this issue is wrapped in understanding the basic of the cytochrome P450 (CYP) enzymes functions and structures. Recently, experimental information on the structure of a variety of cytochrome P450 enzymes, major contributors to phase I metabolism, has become readily available. This, coupled with the availability of experimental information on substrate specificities, has lead to the development of numerous computational models (macromolecular, pharmacophore, and structure-activity) for the rationalization and prediction of CYP liabilities. A comprehensive review of these models is presented in this chapter. PMID:15141126

  13. PksS from Bacillus subtilis is a cytochrome P450 involved in bacillaene metabolism

    SciTech Connect

    Reddick, Jason J. . E-mail: jjreddic@uncg.edu; Antolak, Stephanie A.; Raner, Gregory M.

    2007-06-22

    As part of the pksX gene cluster of Bacillus subtilis strain 168, pksS has been preliminarily annotated as a cytochrome P450 homolog that hydroxylates the polyketide product of this cluster, which was recently shown to be involved in the biosynthesis of bacillaene and dihydrobacillaene. Here we report that there is a frame-shift error in the reported sequence for pksS, and that we have successfully cloned, overexpressed, and purified the protein encoded by the corrected sequence. By utilizing electronic absorption spectrophotometry, we have observed that the ferrous CO complex of PksS absorbs maximally near 450 nm, which confirms the annotation that this protein is a cytochrome P450. We have also established a cell-free system derived from crude cytosolic B. subtilis protein extracts which provides reductase activity essential to sustaining the putative catalytic cycle of PksS. Using LC-MS analysis we have collected data which suggests that the substrate for PksS is dihydrobacillaene.

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

    PubMed

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

    2012-07-01

    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

  15. Dynamics of cytochrome P450 inducers in polluted sites of So Paulo city reservoirs.

    PubMed

    Rocha-e-Silva, Thomaz A A; Rantin, Francisco T; Matsumura-Tundisi, Jose E; Matsumura-Tundisi, Takako; Tundisi, Jose G; Degterev, Igor A

    2004-09-01

    The first analysis of water pollutants using biomarkers at the Guarapiranga Reservoir, which supplies water for one-third of the population of the So Paulo megalopolis (Brazil), is reported. Studies were performed before and after the start of water pumping to the Guarapiranga from the highly polluted Billings reservoir. Billings's water was purified by passing through the natural wetland located near Guarapiranga. Liver enzymes of Oreochromis niloticus (tilopias) obtained from both reservoirs served as biomarkers of pollution in a comparison with animals obtained from a reference site. Enhanced levels of total cytochromes P450 (3.4 times) and b5 (2.7 times) and activity of cytochrome c (P450) reductase (2.2 times) were observed in specimens collected near the water influx from the Billings before the pumping started. However, these parameters were significantly decreased 3 months later. This effect is probably due to dilution of pollutants because of the increased level of water in the Guarapiranga. PMID:15261731

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

    PubMed Central

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

    2015-01-01

    The exposure of insects to xenobiotics, such as insecticides, triggers a complex defence response necessary for survival. This response includes the induction of genes that encode key Cytochrome P450 monooxygenase detoxification enzymes. Drosophila melanogaster Malpighian (renal) tubules are critical organs in the detoxification and elimination of these foreign compounds, so the tubule response induced by dietary exposure to the insecticide permethrin was examined. We found that expression of the gene encoding Cytochrome P450-4e3 (Cyp4e3) is significantly up-regulated by Drosophila fed on permethrin and that manipulation of Cyp4e3 levels, specifically in the principal cells of the Malpighian tubules, impacts significantly on the survival of permethrin-fed flies. Both dietary exposure to permethrin and Cyp4e3 knockdown cause a significant elevation of oxidative stress-associated markers in the tubules, including H2O2 and lipid peroxidation byproduct, HNE (4-hydroxynonenal). Thus, Cyp4e3 may play an important role in regulating H2O2 levels in the endoplasmic reticulum (ER) where it resides, and its absence triggers a JAK/STAT and NF-κB-mediated stress response, similar to that observed in cells under ER stress. This work increases our understanding of the molecular mechanisms of insecticide detoxification and provides further evidence of the oxidative stress responses induced by permethrin metabolism. PMID:26073628

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

    PubMed

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

    2015-12-01

    The exposure of insects to xenobiotics, such as insecticides, triggers a complex defence response necessary for survival. This response includes the induction of genes that encode key Cytochrome P450 monooxygenase detoxification enzymes. Drosophila melanogaster Malpighian (renal) tubules are critical organs in the detoxification and elimination of these foreign compounds, so the tubule response induced by dietary exposure to the insecticide permethrin was examined. We found that expression of the gene encoding Cytochrome P450-4e3 (Cyp4e3) is significantly up-regulated by Drosophila fed on permethrin and that manipulation of Cyp4e3 levels, specifically in the principal cells of the Malpighian tubules, impacts significantly on the survival of permethrin-fed flies. Both dietary exposure to permethrin and Cyp4e3 knockdown cause a significant elevation of oxidative stress-associated markers in the tubules, including H2O2 and lipid peroxidation byproduct, HNE (4-hydroxynonenal). Thus, Cyp4e3 may play an important role in regulating H2O2 levels in the endoplasmic reticulum (ER) where it resides, and its absence triggers a JAK/STAT and NF-κB-mediated stress response, similar to that observed in cells under ER stress. This work increases our understanding of the molecular mechanisms of insecticide detoxification and provides further evidence of the oxidative stress responses induced by permethrin metabolism. PMID:26073628

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

    SciTech Connect

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

    1995-12-31

    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 aryl hydrocarbon hydroxylase (AHH) and benzyl-oxyresorufin-O-dealkylase (BROD) were modestly elevated ({<=} three-fold) in nestlings from polluted sites. Concentrations of p,p{prime}DDE, other organochlorine pesticides and total PCBs in nestlings were greatest at contaminated sites, although much lower than found in concurrently collected eggs and pipping embryos, At these low pollutant concentrations there was little correlation between monooxygenase activity and contaminant levels in nestlings. These observations markedly contrast the pronounced monooxygenase induction (up to 85-fold) and its significant correlation with total PCBS, aryl hydrocarbon receptor-active PCB congeners and toxic equivalents in concurrently collected night-heron embryos that were often siblings of the nestlings. The present findings suggest that cytochrome P450-associated monooxygenase activity of heron nestlings may have only limited value as a biomarker of exposure at this rapid-growth life stage.

  19. Inhibition of rat liver microsomal NADPH cytochrome P450 reductase by glutathione and glutathione disulfide.

    PubMed

    Scholz, R W; Reddy, P V; Liken, A D; Reddy, C C

    1996-09-13

    Previously we have demonstrated inhibition of lipid peroxidation by reduced glutathione (GSH) in rat liver microsomes that is dependent upon the presence of alpha-tocopheral (alpha-TH) in the membranes. Glutathione disulfide (GSSG) potentiated the inhibitory effect of GSH in an enzymatic (NADPH-dependent) lipid peroxidation system in rat liver microsomes; however, inhibition by GSH + GSSG is independent of alpha-TH. When we repeated these experiments with a non-enzymatic system (ascorbate/ADP) to stimulate lipid peroxidation, GSSG did not potentiate the inhibitory effect of GSH. To delineate the mechanism of inhibition of microsomal lipid peroxidation by GSH + GSSG, we examined the effects of these compounds on cytochrome P-450 reductase (EC 1.6.2.4), an important component of the NADPH-dependent enzymatic lipid peroxidation system. It was observed that GSH alone caused about 25% and 21% inhibition of reductase activity in crude microsomes and partially purified enzyme preparations, respectively. There was no inhibition of reductase activity by GSSG alone in either crude microsomes or partially purified enzyme preparations. However, when added together, GSH and GSSG enhanced the inhibition of reductase activity in crude microsomes and partially purified enzyme by 39% and 56%, respectively. We speculate that one possible mechanism for the inhibition of NADPH-dependent lipid peroxidation by GSH + GSSG is, in part, due to inhibition of NADPH cytochrome P-450 reductase, thus affecting the initiation phase of lipid peroxidation. PMID:8806659

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

    USGS Publications Warehouse

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

    1995-01-01

    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.

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

    PubMed

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

    2015-03-01

    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

  2. Effects of cytochrome p450 inhibitors on itraconazole and fluconazole induced cytotoxicity in hepatocytes.

    PubMed

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

    2009-01-01

    Itraconazole and fluconazole have been reported to induce hepatotoxicity in patients. The present study was designed to investigate the role of cytochrome P450 inhibitors, SKF 525A, and curcumin pretreatment on the cytotoxicity of antifungal drugs fluconazole and itraconazole. For 3 consecutive days, female rats were administered daily SKF 525A or curcumin (5 and 25 mg/kg). Control rats received an equivalent amount of dosed vehicle. The animals were 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

  3. Cytochrome P450 induction and metabolism of alkoxyresorufins, ethylmorphine and testosterone in cultured hepatocytes from goats, sheep and cattle.

    PubMed

    van 't Klooster, G A; Blaauboer, B J; Noordhoek, J; van Miert, A S

    1993-11-17

    Very little is known of cytochrome P450 (P450) patterns and enzyme characteristics in food-producing animal species. Oxidative metabolism of alkoxyresorufins, ethylmorphine (EtM) and testosterone (TST) was used to monitor the effects of the P450 inducers phenobarbital (PB), beta-naphthoflavone (BNF), dexamethasone (DEX) and triacetyloleandomycin (TAO) in primary cultured hepatocytes from goats, sheep and cattle. BNF effectively and specifically induced ethoxyresorufin deethylase (> 20-fold), indicating the presence of an inducible P450 1A form, and down-regulated EtM demethylation and most selected TST hydroxylations. In non-induced hepatocyte cultures, TST was metabolized to 6 beta-, 2 beta-, 12 beta-, and 11 alpha-hydroxy-TST (OHT). PB and, to a lesser extent, DEX non-specifically induced all OHT formations, and EtM demethylation. TAO almost completely inhibited OHT formation and EtM demethylation. These results indicate the involvement of principally one P450 form, or a restricted number of related P450 forms, presumably belonging to the P450 3A subfamily. In western blot analysis, cross reactivity was found with rat anti-P450 3A1 and anti-sheep P450 3A. A more specific PB effect was observed for 16 alpha-OHT, which may be formed though a ruminant P450 2B form. None of the inducers influenced pentoxyresorufin depentylase (PROD) or EtM O-deethylation. Metabolite patterns and inducibility of selected activities in ruminant hepatocytes are in accordance with previous findings in goats in vivo. Cytochrome P450 characteristics in ruminants appear to differ from those in rats whereas similarities to the situation in humans appear to exist. PMID:8250964

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

    SciTech Connect

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

    1992-01-01

    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.

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

    USGS Publications Warehouse

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

    1992-01-01

    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.

  6. Non-specific inhibition of cytochrome P450 activities by chlorophyllin in human and rat liver microsomes.

    PubMed

    Yun, C H; Jeong, H G; Jhoun, J W; Guengerich, F P

    1995-06-01

    Chlorophyllin, a copper/sodium salt of chlorophyll used in the treatment of geriatric patients, inhibits the mutagenicity of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ), 3-amino-1-methyl-5H- pyrido[4,3-b]indole (Trp-P-2), aflatoxin B1 and benzo[a]pyrene (B[a]P). Recent in vitro and in vivo studies have shown that a molecular complex is formed between IQ and chlorophyllin, suggesting that this complex formation might be responsible for the antigenotoxic effect of chlorophyllin observed. Cytochrome P450 (P450) enzymes appear to be the major catalysts in the bioactivation of these carcinogens. We have investigated the in vitro effects of chlorophyllin on several P450 activities including ethoxyresorufin O-deethylation, benzyloxyresorufin O-debenzylation, coumarin 7-hydroxylation, 7-ethoxycourmarin O-deethylation, B[a]P 3-hydroxylation, and chlorzoxazone 6-hydroxylation. Chlorophyllin non-specifically inhibited all of P450 activities observed. Spectrally detectable P450 was also destroyed in microsomes and purified P450 in a reconstituted system in the presence of chlorophyllin and an NADPH-generating system. These results suggest that the antigenotoxic effect of chlorophyllin might be due to inhibition of P450 enzymes involving bioactivation of carcinogens in addition to molecular complex formation between carcinogens and chlorophyllin. Comparison of the apparent Ki for P450 inactivation with previously estimated constants for chlorophyllin-IQ complexation suggest that P450 inhibition should be the dominant mechanism of inhibition. PMID:7788866

  7. Dynamic Interaction Between Membrane-Bound Full-Length Cytochrome P450 and Cytochrome b5 Observed by Solid-State NMR Spectroscopy

    PubMed Central

    Yamamoto, Kazutoshi; Dürr, Ulrich H. N.; Xu, Jiadi; Im, Sang-Choul; Waskell, Lucy; Ramamoorthy, Ayyalusamy

    2013-01-01

    Microsomal monoxygenase enzymes of the cytochrome-P450 family are found in all biological kingdoms, and play a central role in the breakdown of metabolic as well as xenobiotic, toxic and 70% of the drugs in clinical use. Full-length cytochrome-b5 has been shown to be important for the catalytic activity of cytochrome-P450. Despite the significance in understanding the interactions between these two membrane-associated proteins, only limited high-resolution structural information on the full-length cytochrome-P450 and the cytochromes-b5-P450 complex is available. Here, we report a structural study on a functional ~72-kDa cytochromes-b5-P450 complex embedded in magnetically-aligned bicelles without having to freeze the sample. Functional and solid-state NMR (Nuclear Magnetic Resonance) data reveal interactions between the proteins in fluid lamellar phase bilayers. In addition, our data infer that the backbone structure and geometry of the transmembrane domain of cytochrome-b5 is not significantly altered due to its interaction with cytochrome-P450, whereas the mobility of cytochrome-b5 is considerably reduced. PMID:23985776

  8. 1-Ethynylpyrene, a suicide inhibitor of cytochrome P-450 dependent benzo(a)pyrene hydroxylase activity in liver microsomes

    SciTech Connect

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

    1984-08-14

    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.

  9. Human Recombinant Cytochrome P450 Enzymes Display Distinct Hydrogen Peroxide Generating Activities During Substrate Independent NADPH Oxidase Reactions

    PubMed Central

    Mishin, Vladimir; Heck, Diane E.; Laskin, Debra L.; Laskin, Jeffrey D.

    2014-01-01

    Microsomal enzymes generate H2O2 in the presence of NADPH. In this reaction, referred to as “oxidase” activity, H2O2 is generated directly or indirectly via the formation of superoxide anion. In the presence of redox active transition metals, H2O2 can form highly toxic hydroxyl radicals and, depending on the “oxidase” activity of individual cytochrome P450 isoenzymes, this can compromise cellular functioning and contribute to tissue injury. In the present studies, we compared the initial rates of H2O2 generating activity of microsomal preparations containing various human recombinant cytochromes P450s. In the absence of cytochrome P450s the human recombinant NADPH cytochrome P450 reductase (CPR) generated low, but detectable amounts of H2O2 (∼0.04 nmol H2O2/min/100 units of reductase). Significantly greater activity was detected in preparations containing individual cytochrome P450s coexpressed with CPR (from 6.0 nmol H2O2/min/nmol P450 to 0.2 nmol/min/nmol P450); CYP1A1 was the most active, followed by CYP2D6, CYP3A4, CYP2E1, CYP4A11, CYP1A2, and CYP2C subfamily enzymes. H2O2 generating activity of the cytochrome P450s was independent of the ratio of CYP/CPR. Thus, similar H2O2 generating activity was noted with the same cytochrome P450s (CYP3A4, CYP2E1, and CYP2C9) expressed at or near the ratio of CYP/CPR in human liver microsomes (5–7), and when CPR was present in excess (CYP/CPR = 0.2–0.3). Because CYP3A4/5/7 represent up to 40% of total cytochrome P450 in the liver, these data indicate that these enzymes are the major source of H2O2 in human liver microsomes. PMID:25061110

  10. Systematic and searchable classification of cytochrome P450 proteins encoded by fungal and oomycete genomes

    PubMed Central

    2012-01-01

    Background Cytochrome P450 proteins (CYPs) play diverse and pivotal roles in fungal metabolism and adaptation to specific ecological niches. Fungal genomes encode extremely variable CYPomes ranging from one to more than 300 CYPs. Despite the rapid growth of sequenced fungal and oomycete genomes and the resulting influx of predicted CYPs, the vast majority of CYPs remain functionally uncharacterized. To facilitate the curation and functional and evolutionary studies of CYPs, we previously developed Fungal Cytochrome P450 Database (FCPD), which included CYPs from 70 fungal and oomycete species. Here we present a new version of FCPD (1.2) with more data and an improved classification scheme. Results The new database contains 22,940 CYPs from 213 species divided into 2,579 clusters and 115 clans. By optimizing the clustering pipeline, we were able to uncover 36 novel clans and to assign 153 orphan CYP families to specific clans. To augment their functional annotation, CYP clusters were mapped to David Nelsons P450 databases, which archive a total of 12,500 manually curated CYPs. Additionally, over 150 clusters were functionally classified based on sequence similarity to experimentally characterized CYPs. Comparative analysis of fungal and oomycete CYPomes revealed cases of both extreme expansion and contraction. The most dramatic expansions in fungi were observed in clans CYP58 and CYP68 (Pezizomycotina), clans CYP5150 and CYP63 (Agaricomycotina), and family CYP509 (Mucoromycotina). Although much of the extraordinary diversity of the pan-fungal CYPome can be attributed to gene duplication and adaptive divergence, our analysis also suggests a few potential horizontal gene transfer events. Updated families and clans can be accessed through the new version of the FCPD database. Conclusions FCPD version 1.2 provides a systematic and searchable catalogue of 9,550 fungal CYP sequences (292 families) encoded by 108 fungal species and 147 CYP sequences (9 families) encoded by five oomycete species. In comparison to the first version, it offers a more comprehensive clan classification, is fully compatible with Nelsons P450 databases, and has expanded functional categorization. These features will facilitate functional annotation and classification of CYPs encoded by newly sequenced fungal and oomycete genomes. Additionally, the classification system will aid in studying the roles of CYPs in the evolution of fungal adaptation to specific ecological niches. PMID:23033934

  11. Mechanistic studies of 9-ethynylphenanthrene-inactivated cytochrome P450 2B1.

    PubMed

    Roberts, E S; Ballou, D P; Hopkins, N E; Alworth, W L; Hollenberg, P F

    1995-11-10

    The mechanism of inactivation of the major phenobarbital-inducible cytochrome P450 of rat liver, P450 2B1, by 9-ethynylphenanthrene (9EPh) has been investigated. Matrix-assisted laser desorption ionization-mass spectrometry analysis of the cyanogen bromide-generated peptides from 9EPh-inactivated P450 2B1 confirmed the addition of a phenanthrylacetyl group to the peptide corresponding to residues 290 to 314. When this peptide was further digested with pepsin, the site of attachment could be assigned to one of the amino acids in the peptide Phe297 to Leu307 [Roberts, E. S., Hopkins, N. E., Zaluzec, E. J., Gage, D. A., Alworth, W. L., and Hollenberg, P. F. (1995) Arch. Biochem. Biophys. 323, 000-000]. The inactivation by 9EPh resulted in a 90-95% loss in the NADPH-supported deethylation of 7-ethoxy-4-trifluoromethylcoumarin (EFC), but had no effect on the iodosobenzene- or cumene hydroperoxide-supported metabolism of EFC. The loss of NADPH-supported activity was not affected by the addition of cytochrome b5 or the presence of excess levels of reductase. The magnitude of the Type 1 spectral change upon the addition of benzphetamine was decreased with the 9EPh-modified protein. There was no decrease in the ability of modified 2B1 to form the steady-state level of the CO-reduced complex either enzymatically with NADPH and reductase or chemically with sodium dithionite, but the rate of reduction by reductase under anaerobic conditions was 57% that of native protein in the absence of substrate and 35% that of native protein in the presence of substrate. The 9EPh-modified 2B1 had an overall slower rate of NADPH oxidation, H2O2 formation, and formaldehyde formation during metabolism of benzphetamine compared to native 2B1. The ratio of H2O2 to HCHO was 1.0:1.0 for the native and 1.6:1.0 for the modified protein. The ability of the modified protein to form the steady-state level of the oxygen-iron complex in the presence of cyclohexane was decreased. These results are consistent with the idea that the covalent modification of one of the residues in the peptide Phe297 to Leu307 by the phenanthrylacetyl group impairs the reduction of P450 2B1 by reductase and also causes the uncoupling of NADPH utilization and oxygen consumption from product formation. PMID:7487092

  12. 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

    PubMed Central

    Gray, Joshua P.; Mishin, Vladimir; Heck, Diane E.; Laskin, Debra L.; Laskin, Jeffrey D.

    2015-01-01

    Inhalation of vesicants including sulfur mustard can cause significant damage to the upper airways. This is the result of vesicant-induced modifications of proteins important in maintaining the integrity of the lung. Cytochrome 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 ?-naphthoflavone treated rats, and cytochrome P450 reductase from type II lung epithelial cells. Using rat liver microsomes from ?-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. PMID:20561902

  13. 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

    Gray, Joshua P.; Mishin, Vladimir; Heck, Diane E.; Laskin, Debra L.; Laskin, Jeffrey D.

    2010-09-01

    Inhalation of vesicants including sulfur mustard can cause significant damage to the upper airways. This is the result of vesicant-induced modifications of proteins important in maintaining the integrity of the lung. Cytochrome 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.

  14. 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.

    PubMed

    Gray, Joshua P; Mishin, Vladimir; Heck, Diane E; Laskin, Debra L; Laskin, Jeffrey D

    2010-09-01

    Inhalation of vesicants including sulfur mustard can cause significant damage to the upper airways. This is the result of vesicant-induced modifications of proteins important in maintaining the integrity of the lung. Cytochrome 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. PMID:20561902

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

    PubMed Central

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

    2011-01-01

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

  16. Immunohistochemical analysis of cytochrome P-450 17 alpha-hydroxylase in pig adrenal cortex, testis and ovary.

    PubMed

    Sasano, H; Mason, J I; Sasano, N

    1989-04-01

    Cytochrome P-450 specific for steroid 17 alpha-hydroxylation (P-450(17 alpha] was immunohistochemically observed in pig adrenal cortex, testis and ovary by the biotin-streptavidin method using a specific antibody against P-450(17 alpha) purified from neonatal pig testis. In the adrenal cortex, P-450(17 alpha) was present in the zona fasciculata and reticularis while no immunoreactivity was observed in the zona glomerulosa, confirming the absence of 17-hydroxylated steroid synthesis in the zona glomerulosa. In the testis, P-450(17 alpha) was present exclusively in Leydig cells and immunoreactivity was absent in seminiferous tubules. In the ovary, immunoreactivity was observed only in the theca interna but not in the membrana granulosa of follicles. Among the tissues examined, the relative intensity of immunoreactivity was greatest in the Leydig cells, and progressively less in theca interna cells, outer fasciculata cells and inner fasciculata and reticularis cells. PMID:2663569

  17. Conformational Changes of NADPH-Cytochrome P450 Oxidoreductase Are Essential for Catalysis and Cofactor Binding*

    PubMed Central

    Xia, Chuanwu; Hamdane, Djemel; Shen, Anna L.; Choi, Vivian; Kasper, Charles B.; Pearl, Naw May; Zhang, Haoming; Im, Sang-Choul; Waskell, Lucy; Kim, Jung-Ja P.

    2011-01-01

    The crystal structure of NADPH-cytochrome P450 reductase (CYPOR) implies that a large domain movement is essential for electron transfer from NADPH via FAD and FMN to its redox partners. To test this hypothesis, a disulfide bond was engineered between residues Asp147 and Arg514 in the FMN and FAD domains, respectively. The cross-linked form of this mutant protein, designated 147CC514, exhibited a significant decrease in the rate of interflavin electron transfer and large (?90%) decreases in rates of electron transfer to its redox partners, cytochrome c and cytochrome P450 2B4. Reduction of the disulfide bond restored the ability of the mutant to reduce its redox partners, demonstrating that a conformational change is essential for CYPOR function. The crystal structures of the mutant without and with NADP+ revealed that the two flavin domains are joined by a disulfide linkage and that the relative orientations of the two flavin rings are twisted ?20 compared with the wild type, decreasing the surface contact area between the two flavin rings. Comparison of the structures without and with NADP+ shows movement of the Gly631Asn635 loop. In the NADP+-free structure, the loop adopts a conformation that sterically hinders NADP(H) binding. The structure with NADP+ shows movement of the Gly631Asn635 loop to a position that permits NADP(H) binding. Furthermore, comparison of these mutant and wild type structures strongly suggests that the Gly631Asn635 loop movement controls NADPH binding and NADP+ release; this loop movement in turn facilitates the flavin domain movement, allowing electron transfer from FMN to the CYPOR redox partners. PMID:21345800

  18. Potent Mechanism-Based Inactivation Of Cytochrome P450 2B4 By 9-Ethynylphenanthrene: Implications For Allosteric Modulation Of Cytochrome P450 Catalysis1

    PubMed Central

    Zhang, Haoming; Gay, Sean C.; Shah, Manish; Foroozesh, Maryam; Liu, Jiawang; Osawa, Yoichi; Zhang, Qinghai; Stout, C. David; Halpert, James R.; Hollenberg, Paul F.

    2013-01-01

    The mechanism-based inactivation of cytochrome P450 2B4 (CYP2B4) by 9-ethynylphenanthrene (9EP) has been investigated. The partition ratio and kinact are 0.2 and 0.25 min−1, respectively. Intriguingly, the inactivation exhibits sigmoidal kinetics with a Hill coefficient of 2.5 and S50 of 4.5 μM indicative of homotropic cooperativity. Enzyme inactivation led to an increase in mass of the apo-CYP2B4 by 218 Da as determined by ESI-LC/MS, consistent with covalent protein modification. The modified CYP2B4 was purified to homogeneity and its structure determined by X-ray crystallography. The structure showed that 9EP is covalently attached to the Oγ of Thr 302 via an ester bond, which is consistent with the increased mass of the protein. The presence of the bulky phenanthrenyl ring resulted in inward rotations of Phe 297 and Phe 206 leading to a compact active site. Thus, binding of another molecule of 9EP in the active site is prohibited. However, results from the quenching of 9EP fluorescence by unmodified or 9EP-modified CYP2B4 revealed at least two binding sites with distinct affinities, with the low affinity site being the catalytic site and the high affinity site on the protein periphery. Computer-aided docking and MD simulations with one or two ligands bound revealed that the high affinity site is situated at the entrance of a substrate access channel surrounded by the F’ helix, β1/β2 loop and β4 loop and functions as an allosteric site to enhance the efficiency of activation of the acetylenic group of 9EP and subsequent covalent modification of Thr 302. PMID:23276288

  19. Phthalate esters: effects of orally administered dibutylphthalate on cytochrome P-450 mediated metabolism in rat liver and lung.

    PubMed

    Walseth, F; Nilsen, O G

    1986-10-01

    Dibutylphthalate (DBP) was administered to male Sprague Dawley rats in oral doses of 0.01, 0.1 and 1.0 mmol/kg for five days. The lower dose was considered relevant to human intake. Additional groups were given a recovery period of four weeks without DBP. DBP significantly increased the liver microsomal concentration of cytochrome P-450 (48%) at the lowest dose and NADPH-cytochrome-c-reductase activity (28-29%), at the two lower doses. The liver microsomal metabolism of n-hexane increased to about the same extent at all dosage levels. The main increase was found in the formation of the preneurotoxic metabolite 2-hexanol. The induction of cytochrome P-450 and NADPH-cytochrome-c-reductase in liver microsomes did not return to normal after the period of recovery, whereas the metabolism of n-hexane normalized during the same period of time, indicating that the majority of the induced forms of cytochrome P-450 were not related to n-hexane metabolism. No major changes were observed in the liver microsomal metabolism of B(a)P. The only effect found in cytochrome P-450 related metabolism in lung microsomes was a decrease of the B(a)P metabolism, especially in the formation of the 9,10- and 4,5-diol metabolites at lower dosage levels. It is suggested that DBP and its hydrolyzed products formed in the intestine after oral administration exert the same effect on some specific forms of cytochrome P-450 in liver and lung. It is concluded that DBP is a moderate to weak inducer of several minor forms of liver microsomal cytochrome P-450 enzymes in doses which may be relevant to human oral intake. PMID:3099543

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

    SciTech Connect

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

    2009-05-14

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

  1. The effects of milk thistle (Silybum marianum) on human cytochrome P450 activity.

    PubMed

    Kawaguchi-Suzuki, Marina; Frye, Reginald F; Zhu, Hao-Jie; Brinda, Bryan J; Chavin, Kenneth D; Bernstein, Hilary J; Markowitz, John S

    2014-10-01

    Milk thistle (Silybum marianum) extracts are widely used as a complementary and alternative treatment of various hepatic conditions and a host of other diseases/disorders. The active constituents of milk thistle supplements are believed to be the flavonolignans contained within the extracts. In vitro studies have suggested that some milk thistle components may significantly inhibit specific cytochrome P450 (P450) enzymes. However, determining the potential for clinically significant drug interactions with milk thistle products has been complicated by inconsistencies between in vitro and in vivo study results. The aim of the present study was to determine the effect of a standardized milk thistle supplement on major P450 drug-metabolizing enzymes after a 14-day exposure period. CYP1A2, CYP2C9, CYP2D6, and CYP3A4/5 activities were measured by simultaneously administering the four probe drugs, caffeine, tolbutamide, dextromethorphan, and midazolam, to nine healthy volunteers before and after exposure to a standardized milk thistle extract given thrice daily for 14 days. The three most abundant falvonolignans found in plasma, following exposure to milk thistle extracts, were silybin A, silybin B, and isosilybin B. The concentrations of these three major constituents were individually measured in study subjects as potential perpetrators. The peak concentrations and areas under the time-concentration curves of the four probe drugs were determined with the milk thistle administration. Exposure to milk thistle extract produced no significant influence on CYP1A2, CYP2C9, CYP2D6, or CYP3A4/5 activities. PMID:25028567

  2. Evaluation of Inhibition Selectivity for Human Cytochrome P450 2A Enzymes

    PubMed Central

    Stephens, Eva S.; Walsh, Agnes A.

    2012-01-01

    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. Ki values were determined for inhibition of p-nitrophenol 2-hydroxylation, a reaction accomplished by CYP2A13 and CYP2A6 with more similar catalytic efficiencies (kcat/Km 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 Ki 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

  3. Evidence that adrenal hexose-6-phosphate dehydrogenase can effect microsomal P450 cytochrome steroidogenic enzymes

    PubMed Central

    Foster, Christy A.; Mick, Gail J.; Wang, Xudong; McCormick, Kenneth

    2014-01-01

    The role of adrenal hexose-6-phosphate dehydrogenase in providing reducing equivalents to P450 cytochrome steroidogenic enzymes in the endoplasmic reticulum is uncertain. Hexose-6-phosphate dehydrogenase resides in the endoplasmic reticulum lumen and co-localizes with the bidirectional enzyme 11?-hydroxysteroid dehydrogenase 1. Hexose-6-phosphate dehydrogenase likely provides 11?-hydroxysteroid dehydrogenase 1 with NADPH electrons via channeling. Intracellularly, two compartmentalized reactions generate NADPH upon oxidation of glucose-6-phosphate: cytosolic glucose-6-phosphate dehydrogenase and microsomal hexose-6-phosphate dehydrogenase. Because some endoplasmic reticulum enzymes require an electron donor (NADPH), it is conceivable that hexose-6-phosphate dehydrogenase serves in this capacity for these pathways. Besides 11?-hydroxysteroid dehydrogenase 1, we examined whether hexose-6-phosphate dehydrogenase generates reduced pyridine nucleotide for pivotal adrenal microsomal P450 enzymes. 21-hydroxylase activity was increased with glucose-6-phosphate and, also, glucose and glucosamine-6-phosphate. The latter two substrates are only metabolized by hexose-6-phosphate dehydrogenase, indicating that requisite NADPH for 21-hydroxylase activity was not via glucose-6-phosphate dehydrogenase. Moreover, dihydroepiandrostenedione, a non-competitive inhibitor of glucose-6-phosphate dehydrogenase, but not hexose-6-phosphate dehydrogenase, did not curtail activation by glucose-6-phosphate. Finally, the most compelling observation was that the microsomal glucose-6-phosphate transport inhibitor, chlorogenic acid, blunted the activation by glucose-6-phosphate of both 21-hydroxylase and 17-hydroxylase indicating that luminal hexose-6-phosphate dehydrogenase can supply NADPH for these enzymes. Analogous kinetic observations were found with microsomal 17-hydroxylase. These findings indicate that hexose-6-phosphate dehydrogenase can be a source, but not exclusively so, of NADPH for several adrenal P450 enzymes in the steroid pathway. Although the reduced pyridine nucleotides are produced intra-luminally, these compounds may also slowly transverse the endoplasmic reticulum membrane by unknown mechanisms. PMID:23665046

  4. The Effects of Milk Thistle (Silybum marianum) on Human Cytochrome P450 Activity

    PubMed Central

    Kawaguchi-Suzuki, Marina; Frye, Reginald F.; Zhu, Hao-Jie; Brinda, Bryan J.; Chavin, Kenneth D.; Bernstein, Hilary J.

    2014-01-01

    Milk thistle (Silybum marianum) extracts are widely used as a complementary and alternative treatment of various hepatic conditions and a host of other diseases/disorders. The active constituents of milk thistle supplements are believed to be the flavonolignans contained within the extracts. In vitro studies have suggested that some milk thistle components may significantly inhibit specific cytochrome P450 (P450) enzymes. However, determining the potential for clinically significant drug interactions with milk thistle products has been complicated by inconsistencies between in vitro and in vivo study results. The aim of the present study was to determine the effect of a standardized milk thistle supplement on major P450 drug-metabolizing enzymes after a 14-day exposure period. CYP1A2, CYP2C9, CYP2D6, and CYP3A4/5 activities were measured by simultaneously administering the four probe drugs, caffeine, tolbutamide, dextromethorphan, and midazolam, to nine healthy volunteers before and after exposure to a standardized milk thistle extract given thrice daily for 14 days. The three most abundant falvonolignans found in plasma, following exposure to milk thistle extracts, were silybin A, silybin B, and isosilybin B. The concentrations of these three major constituents were individually measured in study subjects as potential perpetrators. The peak concentrations and areas under the time-concentration curves of the four probe drugs were determined with the milk thistle administration. Exposure to milk thistle extract produced no significant influence on CYP1A2, CYP2C9, CYP2D6, or CYP3A4/5 activities. PMID:25028567

  5. Regio-selectivity of purified forms of rabbit liver microsomal cytochrome P-450 in the metabolism of benzo(a)pyrene, n-hexane and 7-ethoxyresorufin.

    PubMed

    Nilsen, O G; Toftgrd, R; Eng, L; Gustafsson, J A

    1981-05-01

    The specificity of electrophoretically homogeneous preparations of rabbit liver microsomal cytochrome P-450LM2-4 towards oxygenation of n-hexane, 7-ethoxyresorufin and benzo(a)pyrene was examined using a reconstituted system consisting of cytochrome P-450, NADPH-cytochrome P-450 reductase and dilauroylphosphatidylcholine. Epoxide hydrase was included when benzo(a)pyrene was used as substrate. Cytochrome P-450LM2 was most active in n-hexane and benzo(a)pyrene oxygenation especially with regard to the formation of 2-hexanol, B(a)P-4,5-dihydrodiol and B(a)P-phenol metabolites. 7-Ethoxyresorufin was, however, a very poor substrate for cytochrome P-450LM2. Cytochrome P-450LM3 had less activity towards the investigated substrates while cytochrome P-450LM4 preferentially formed 2- and 3-hexanol, resorufin and B(a)P-9,10-dihydrodiol. Cytochrome P-450LM4 isolated after pretreatment with 3-methylcholanthrene or phenobarbital showed roughly the same characteristics except in the formation of 1-hexanol where cytochrome P-450LM4 isolated after phenobarbital treatment was the most effective. The formation of B(a)P-4,5- and -9,10-dihydrodiols was greatly increased by incorporation of epoxide hydrase. Our results indicate a certain specificity of the different forms of cytochrome P-450 in the liver microsomes although some overlap in activities was observed. PMID:7336952

  6. Catalytic and immunochemical characterization of hepatic microsomal cytochromes P450 in beluga whale (Delphinapterus leucas).

    PubMed

    White, R D; Hahn, M E; Lockhart, W L; Stegeman, J J

    1994-05-01

    Understanding the effects of environmental contaminants on cetaceans and other marine mammals will require information on the biochemistry of xenobiotic metabolism in these species. We characterized the hepatic microsomal cytochrome P450 system in beluga whales (Delphinapterus leucas) from the Canadian Arctic. The content of native P450 averaged 0.203 and 0.319 nmol/mg microsomal protein, cytochrome b5 content averaged 0.199 and 0.236 nmol/mg, and rates of NADPH-cytochrome c reductase were 79 and 76 nmol/min/mg, for females and males respectively. Ethoxyresorufin O-deethylase (EROD), pentoxyresorufin O-depentylase (PROD), and benzo[a]pyrene (BP) hydroxylase (AHH) activities were significantly greater in males than in females, and were highly correlated with one another (r2 between 0.853 and 0.912). HPLC analysis of in vitro BP metabolites revealed benzo-ring (7,8- and 9,10-) dihydrodiols, consistent with activation of this compound, as well as 4,5-dihydrodiol,3-OH-, 7-OH-, and 9-OH-BP and 1,6- and 3,6-quinones. Estradiol 2-hydroxylase activity did not differ between sexes, and rates did not correlate with those of the other activities. Antibodies against scup P450B (an apparent teleost CYP2B) and rat CYP2B1 did not recognize proteins in beluga liver microsomes, but there was a protein detected by antibodies to PB-inducible rabbit CYP2B4. Antibodies to ethanol and ketone-inducible rat CYP2E1 reacted with two proteins in beluga liver microsomes. Antibodies specific to hydrocarbon-inducible CYP1A1 and/or CYP1A2 forms showed a single protein band, apparently more closely related to CYP1A1. The content of CYP1A was fivefold greater in male than in female beluga. CYP1A content was highly correlated with EROD, PROD, and AHH activities, suggesting that this P450 form is a primary catalyst for these reactions in beluga. CYP1A content and activity were highly correlated with the concentrations in blubber of non-ortho and mono-ortho PCB congeners, compounds that induce CYP1A in other mammals. These results indicate that a CYP1A is a catalyst for the metabolism of aromatic hydrocarbon pollutants in the beluga whale, and strongly suggest that this protein is induced in these organisms by environmental contaminants, including PCBs. The results support the measurement of CYP1A expression as a biomarker of exposure to inducers in marine mammals. The full functional and evolutionary relationships of beluga CYP1A and of beluga proteins immunologically related to other P450 forms are uncertain. PMID:8184432

  7. Expression and inducibility of cytochrome P450 isoforms in 1-year-old intrasplenic liver cell transplants in rats.

    PubMed

    Lupp, Amelie; Danz, Manfred; Mller, Dieter; Klinger, Wolfgang

    2002-03-01

    Syngenic fetal liver tissue suspensions were transplanted into the spleens of 60- to 90-day-old male Fischer 344 inbred rats. Transplant recipients were compared with age-matched control rats. One year after surgery, the animals were treated orally with beta-naphthoflavone (BNF), phenobarbital (PB), dexamethasone (DEX) or the respective solvents 24 or 48 h before being killed. Expression of cytochrome P450 (P450) isoforms in spleens and orthotopic livers was assessed by immunohistochemistry and P450-dependent monooxygenase functions by the model reactions ethoxyresorufin O-deethylation (EROD), ethoxycoumarin O-deethylation (ECOD), pentoxyresorufin O-depentylation (PROD) and ethylmorphine N-demethylation (EMND). Spleens of control animals displayed almost no expression of P450 isoforms and P450-mediated monooxygenase functions. Similar to liver, in the transplanted hepatocytes no P450 1A1 but distinct P450 2B1 and 3A2 expression was observed. Furthermore, the transplant-containing spleens displayed significant EROD, ECOD, PROD and EMND activities. Similar to normal liver, BNF treatment enhanced P450 1A1 and 2B1, PB induced P450 2B1 and 3A2, and DEX induced P450 3A2 expression in the transplanted hepatocytes. Correspondingly, in the transplant-containing spleens EROD, ECOD and PROD activities were significantly enhanced following BNF treatment, EROD, ECOD, PROD and EMND activities after PB administration, and EMND activity by DEX treatment. These results demonstrate that hepatocytes originating from fetal liver tissue suspensions can survive in the spleen at least for 1 year. They have differentiated into adult hepatocytes and even 1 year after transplantation express different P450 isoforms which are inducible by BNF, PB and DEX, corresponding to normal adult liver. PMID:11935166

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

    PubMed

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

    2015-09-01

    Cytochrome P450 (P450) and chloroperoxidase (CPO) are thiolate-ligated haem proteins that catalyse the activation of carbon hydrogen bonds. The principal intermediate in these reactions is a ferryl radical species called compound I. P450 compound I (P450-I) is significantly more reactive than CPO-I, which only cleaves activated C-H bonds. To provide insight into the differing reactivities of these intermediates, we examined CPO-I and P450-I using variable-temperature Mssbauer and X-ray absorption spectroscopies. These measurements indicate that the Fe-S bond is significantly shorter in P450-I than in CPO-I. This difference in Fe-S bond lengths can be understood in terms of variations in the hydrogen-bonding patterns within the 'cys-pocket' (a portion of the proximal helix that encircles the thiolate ligand). Weaker hydrogen bonding in P450-I results in a shorter Fe-S bond, which enables greater electron donation from the axial thiolate ligand. This observation may in part explain P450's greater propensity for C-H bond activation. PMID:26291940

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

    PubMed Central

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

    2015-01-01

    Cytochrome P450 (P450) and chloroperoxidase (CPO) are thiolate ligated heme proteins that catalyze the activation of carbon hydrogen bonds. The principal intermediate in these reactions is a ferryl radical species called compound I. P450 compound I (P450-I) is significantly more reactive than CPO-I, which only cleaves activated C-H bonds. To provide insight into the differing reactivities of these intermediates, we examined CPO-I and P450-I with variable temperature Mössbauer and X-ray absorption spectroscopies. These measurements indicate that the Fe-S bond is significantly shorter in P450-I than in CPO-I. This difference in Fe-S bond lengths can be understood in terms of variations in hydrogen bonding patterns within the “cys-pocket” (a portion of the proximal helix that encircles the thiolate ligand). Weaker hydrogen bonding in P450-I results in a shorter Fe-S bond, which enables greater electron donation from the axial-thiolate ligand. This observation may in part explain P450's greater propensity for C-H bond activation. PMID:26291940

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

    NASA Astrophysics Data System (ADS)

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

    2015-09-01

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

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

    PubMed

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

    1991-08-01

    Plasma growth hormone (GH) profiles are sexually differentiated in many species and regulate the sex-dependence of peripubescent growth rates and liver function, including steroid hydroxylase cytochrome P450 expression, by mechanisms that are poorly understood. By use of an external pump to deliver to hypophysectomized rats pulses of rat GH of varying frequency and amplitude, a critical element for liver discrimination between male and female GH patterns was identified. Liver expression of the male-specific steroid 2 alpha (or 16 alpha)-hydroxylase P450, designated CYP2C11, was stimulated by GH at both physiological and nonphysiological pulse amplitudes, durations, and frequencies, provided that an interpulse interval of no detectable GH was maintained for at least 2.5 hr. This finding suggests that hepatocytes undergo an obligatory recovery period after stimulation by a GH pulse. This period may be required to reset a GH-activated intracellular signaling pathway or may relate to the short-term absence of GH receptors at the hepatocyte surface after a cycle of GH binding and receptor internalization. These requirements were distinguished from those necessary for the stimulation by GH of normal male growth rates in hypophysectomized rats, indicating that different GH responses and, perhaps, different GH-responsive tissues recognize distinct signaling elements in the sexually dimorphic patterns of circulating GH. PMID:1862110

  12. Does Compound I Vary Significantly between Isoforms of Cytochrome P450?

    PubMed Central

    2011-01-01

    The cytochrome P450 (CYP) enzymes are important in many areas, including pharmaceutical development. Subtle changes in the electronic structure of the active species, Compound I, have been postulated previously to account partly for the experimentally observed differences in reactivity between isoforms. Current predictive models of CYP metabolism typically assume an identical Compound I in all isoforms. Here we present a method to calculate the electronic structure and to estimate the FeO bond enthalpy of Compound I, and apply it to several human and bacterial CYP isoforms. Conformational flexibility is accounted for by sampling large numbers of structures from molecular dynamics simulations, which are subsequently optimized with density functional theory (B3LYP) based quantum mechanics/molecular mechanics. The observed differences in Compound I between human isoforms are small: They are generally smaller than the spread of values obtained for the same isoform starting from different initial structures. Hence, it is unlikely that the variation in activity between human isoforms is due to differences in the electronic structure of Compound I. A larger difference in electronic structure is observed between the human isoforms and P450cam and may be explained by the slightly different hydrogen-bonding environment surrounding the cysteinyl sulfur. The presence of substrate in the active site of all isoforms studied appears to cause a slight decrease in the FeO bond enthalpy, apparently due to displacement of water out of the active site, suggesting that Compound I is less stable in the presence of substrate. PMID:21863858

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

    PubMed Central

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

    2013-01-01

    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

  14. Water complexes of cytochrome P450: insights from energy decomposition analysis.

    PubMed

    Thellamurege, Nandun; Hirao, Hajime

    2013-01-01

    Water is a small molecule that nevertheless perturbs, sometimes significantly, the electronic properties of an enzyme's active site. In this study, interactions of a water molecule with the ferric heme and the compound I (Cpd I) intermediate of cytochrome P450 are studied. Energy decomposition analysis (EDA) schemes are used to investigate the physical origins of these interactions. Localized molecular orbital EDA (LMOEDA) implemented in the quantum chemistry software GAMESS and the EDA method implemented in the ADF quantum chemistry program are used. EDA reveals that the electrostatic and polarization effects act as the major driving force in both of these interactions. The hydrogen bonding in the Cpd IH?O complex is similar to that in the water dimer; however, the relative importance of the electrostatic effect is somewhat larger in the water dimer. PMID:23752465

  15. Evaluation on activity of cytochrome p450 enzymes in turbot via a probe drug cocktail.

    PubMed

    Chang, Zhi-Qiang; Li, Jian; Zhai, Qian-Qian

    2014-12-01

    Cytochrome P450s (CYPs) are the main catalytic enzymes for metabolism by a variety of endogenous and exogenous substrates in mammals, fish, insects, etc. We evaluated the application of a multidrug cocktail on changes in CYP1, CYP2, and CYP3 activity in Turbot Scophthalmus maximus. The probe drugs were a combination of caffeine (5 mg/kg body weight), dapsone (5 mg/kg), and chlorzoxazone (10 mg/kg). After a single intraperitoneal injection of the cocktail, the concentration of all three probe drugs in the plasma increased quickly to a peak and then decreased gradually over 24 h. Pharmacokinetic profiles of the three probe drugs were determined using a noncompartmental analysis, and the typical parameters were calculated. In the assay for CYP induction, pretreatment with rifampicin significantly reduced the typical pharmacokinetic metrics for caffeine and chlorzoxazone, but not dapsone, indicating that the activity of CYP1 and CYP2 in turbot were induced by rifampicin. PMID:25369285

  16. Expression of a mammalian PCB-metabolizing cytochrome P-450 in Nicotiana tabacum

    SciTech Connect

    Wall, V.D.; Galbraith, D.W.; Halpert, J.R.; Bourque, D.P. )

    1991-05-01

    Polychlorinated biphenyls (PCBs) are resistant to metabolism in most animal species. The dog possesses the unique ability to metabolize and eliminate certain PCB congeners, as a result of the activity of the cytochrome P-450 isozyme PBD-2. An expressible cDNA coding for PBD-2 has been introduced into the genome of tobacco plants. PBD-2 cDNA and a screenable marker gene coding for neomycin phosphotransferase were introduced into tobacco leaf disks using a binary Agrobacterium tumefaciens vector system. Southern and Western blot analyses have confirmed chromosomal integration of the cDNA and expression of the PBD-2 polypeptide. Differential centrifugation and Western blot analyses have shown the PBD-2 protein to be associated with a membrane fraction in transgenic tobacco leaf homogenates. The authors goal is to develop transgenic plants in which the PBD-2 protein metabolizes PCBs, thus providing a novel method for bioremediation of PCB-contaminated soils.

  17. Interactions between the cytochrome P450 system and the second-generation antipsychotics

    PubMed Central

    Prior, Trevor I.; Baker, Glen B.

    2003-01-01

    Awareness of the metabolism of second-generation antipsychotics by the cytochrome P450 (CYP) system can inform the clinician about how to avoid and manage drug–drug interactions involving these enzymes. Clozapine is metabolized primarily by CYP1A2, with additional contributions by CYP2C19, CYP2D6 and CYP3A4. Risperidone is metabolized primarily by CYP2D6 and to a lesser extent by CYP3A4. Olanzapine is metabolized primarily by CYP1A2 and to a lesser extent by CYP2D6. Quetiapine and ziprasidone are metabolized by CYP3A4. At the usual clinical doses, these drugs appear not to significantly affect the metabolism of other medications. There is, however, a lack of in vivo metabolic data, especially for the 3 newest second-generation antipsychotics: olanzapine, quetiapine and ziprasidone. PMID:12670127

  18. Delicate conformational balance of the redox enzyme cytochrome P450cam

    PubMed Central

    Skinner, Simon P.; Liu, Wei-Min; Hiruma, Yoshitaka; Timmer, Monika; Blok, Anneloes; Hass, Mathias A. S.; Ubbink, Marcellus

    2015-01-01

    The energy landscapes of proteins are highly complex and can be influenced by changes in physical and chemical conditions under which the protein is studied. The redox enzyme cytochrome P450cam undergoes a multistep catalytic cycle wherein two electrons are transferred to the heme group and the enzyme visits several conformational states. Using paramagnetic NMR spectroscopy with a lanthanoid tag, we show that the enzyme bound to its redox partner, putidaredoxin, is in a closed state at ambient temperature in solution. This result contrasts with recent crystal structures of the complex, which suggest that the enzyme opens up when bound to its partner. The closed state supports a model of catalysis in which the substrate is locked in the active site pocket and the enzyme acts as an insulator for the reactive intermediates of the reaction. PMID:26130807

  19. Use of P450 cytochrome inhibitors in studies of enokipodin biosynthesis

    PubMed Central

    Ishikawa, Noemia Kazue; Tahara, Satoshi; Namatame, Tomohiro; Farooq, Afgan; Fukushi, Yukiharu

    2013-01-01

    Enokipodins A, B, C, and D are antimicrobial sesquiterpenes isolated from the mycelial culture medium of Flammulina velutipes, an edible mushroom. The presence of a quaternary carbon stereocenter on the cyclopentane ring makes enokipodins A-D attractive synthetic targets. In this study, nine different cytochrome P450 inhibitors were used to trap the biosynthetic intermediates of highly oxygenated cuparene-type sesquiterpenes of F. velutipes. Of these, 1-aminobenzotriazole produced three less-highly oxygenated biosynthetic intermediates of enokipodins A-D; these were identified as (S)-(?)-cuparene-1,4-quinone and epimers at C-3 of 6-hydroxy-6-methyl-3-(1,2,2-trimethylcyclopentyl)-2-cyclohexen-1-one. One of the epimers was found to be a new compound. PMID:24688524

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

    PubMed

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

    2011-05-11

    The potential for 15 different ales (6), ciders (2 apple and 1 pear), and porters (6) and 2 non-alcoholic products to affect cytochrome P450 (CYP)-mediated biotransformation and P-glycoprotein-mediated efflux of rhodamine was examined. As in our previous study, a wide range of recovered nonvolatile suspended solids dry weights were noted. Aliquots were also found to have varying effects on biotransformation and efflux. Distinct differences in product ability to affect the safety and efficacy of therapeutic products confirmed our initial findings that some porters (stouts) have a potential to affect the safety and efficacy of health products metabolized by CYP2D6 and CYP3A4 isozymes. Most products, except 2 of the ciders and the 2 non-alcoholic products, also have the potential to affect the safety of CYP2C9 metabolized medications and supplements. Further studies are required to determine the clinical significance of these findings. PMID:21476568

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

    PubMed Central

    Miura, Mai; Ito, Kensuke; Hayashi, Maiko; Nakajima, Motowo; Tanaka, Tohru; Ogura, Shun-ichiro

    2015-01-01

    Of late, numerous prodrugs are widely used for therapy. The hemeprotein cytochrome P450 (CYP) catalyzes the activation of prodrugs to form active metabolites. Therefore, the activation of CYP function might allow the use of lower doses of prodrugs and decrease toxicity. We hypothesized that the addition of 5-aminolevulinic acid (ALA), a precursor in the porphyrin biosynthetic pathway, enhances the synthesis of heme, leading to the up-regulation of CYP activity. To test this hypothesis, we treated a human gastric cancer cell line with ALA and determined the effect on CYP-dependent prodrug activation. For this purpose, we focused on the anticancer prodrug tegafur, which is converted to its active metabolite 5-fluorouracil (5-FU) mainly by CYP2A6. We show here that ALA increased CYP2A6-dependent tegafur activation, suggesting that ALA elevated CYP activity and potentiated the activation of the prodrug. PMID:26181717

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

    PubMed

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

    2012-04-01

    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

  3. Pharmacokinetic Interactions of Herbs with Cytochrome P450 and P-Glycoprotein

    PubMed Central

    Cho, Hyun-Jong

    2015-01-01

    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

  4. Discovery of potent and novel S-nitrosoglutathione reductase inhibitors devoid of cytochrome P450 activities.

    PubMed

    Sun, Xicheng; Qiu, Jian; Strong, Sarah A; Green, Louis S; Wasley, Jan W F; Blonder, Joan P; Colagiovanni, Dorothy B; Mutka, Sarah C; Stout, Adam M; Richards, Jane P; Rosenthal, Gary J

    2011-10-01

    The pyrrole based N6022 was recently identified as a potent, selective, reversible, and efficacious S-nitrosoglutathione reductase (GSNOR) inhibitor and is currently undergoing clinical development for the treatment of acute asthma. GSNOR is a member of the alcohol dehydrogenase family (ADH) and regulates the levels of S-nitrosothiols (SNOs) through catabolism of S-nitrosoglutathione (GSNO). Reduced levels of GSNO, as well as other nitrosothiols (SNOs), have been implicated in the pathogenesis of many diseases including those of the respiratory, cardiovascular, and gastrointestinal systems. Preservation of endogenous SNOs through GSNOR inhibition presents a novel therapeutic approach with broad applicability. We describe here the synthesis and structure-activity relationships (SAR) of novel pyrrole based analogues of N6022 focusing on removal of cytochrome P450 inhibition activities. We identified potent and novel GSNOR inhibitors having reduced CYP inhibition activities and demonstrated efficacy in a mouse ovalbumin (OVA) model of asthma. PMID:21855338

  5. X-ray absorption studies of metalloprotein structure: cytochrome P-450, horseradish peroxidase, plastocyanin and laccase

    SciTech Connect

    Penner-Hahn, J.E.

    1984-03-01

    Extended x-ray absorption fine structure (EXAFS) has been developed to determine the structure of metalloproteins. EXAFS data have been collected and analysed for four states in the catalytic cycle of bacterial cytochrome P-450/sub CAM/. This data demonstrates that sulfur is retained as an axial ligand in the reduced forms of the enzyme. EXAFS and edge data have been analysed for the high-valent states of horseradish peroxidase (HRP), and for high-valent iron-porphyrin model compounds. These data provide the first direct confirmation of the presence of a ferryl Fe=O coordination in HRP and in some of the model compounds. The polarized single-crystal EXAFS spectra of plastocyanin have been measured as a function of both orientation and temperature. These data demonstrate that at room temperature the relative motions of the Cu and the S(Met) are essentially uncorrelated.

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

    PubMed

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

    2016-02-01

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

  7. In vivo cytochrome P450 drug metabolizing enzyme characterization using surface-enhanced Raman spectroscopy

    NASA Astrophysics Data System (ADS)

    Li, Yanfang; Bachmann, Kenneth A.; Cameron, Brent D.

    2003-07-01

    The development of a rapid, inexpensive, and accurate in vivo phenotyping methodology for characterizing drug-metabolizing phenotypes with reference to the cytochrome P450 (CYP450) enzymes would be very beneficial. In terms of application, in the wake of the human genome project, considerable interest is focused on the development of new drugs whose uses will be tailored to specific genetic polymorphisms, and on the individualization of dosing regimens that are also tailored to meet individual patient needs depending upon genotype. In this investigation, chemical probes for CYP450 enzymes were characterized and identified with Raman spectroscopy. Furthermore, gold-based metal colloid clusters were utilized to generate surface enhanced Raman spectra for each of the chemical probes. Results will be presented demonstrating the ability of SERS to identify minute quantities of these probes on the order needed for in vivo application.

  8. Mechanism of the N-hydroxylation of primary and secondary amines by cytochrome P450.

    PubMed

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

    2015-04-20

    Cytochrome P450 enzymes (CYPs) metabolize alkyl- and arylamines, generating several different products. For the primary and secondary amines, some of these reactions result in hydroxylated amines, which may be toxic. Thus, when designing new drugs containing amine groups, it is important to be able to predict if a given compound will be a substrate for CYPs, in order to avoid toxic metabolites, and hence to understand the mechanism that is utilized by CYPs. Two possible mechanisms, for the N-hydroxylation of primary and secondary amines mediated by CYPs, are studied by density functional theory (DFT) for four different amines (aniline, N-methylaniline, propan-2-amine, and dimethylamine). The hydrogen abstraction and rebound mechanism is found to be preferred over a direct oxygen transfer mechanism for all four amines. However, in contrast to the same mechanism for the hydroxylation of aliphatic carbon atoms, the rebound step is shown to be rate-limiting in most cases. PMID:25651340

  9. Cryoradiolysis and cryospectroscopy for studies of heme-oxygen intermediates in cytochromes P450

    PubMed Central

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

    2014-01-01

    Cryogenic radiolytic reduction is one of the most simple 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 peroxoferric 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

  10. Gravity persistent signal 1 reveals a novel cytochrome P450 involved in gravitropic signal transduction

    NASA Astrophysics Data System (ADS)

    Wyatt, Sarah

    Understanding gene expression that occurs during gravitopism is important