Sample records for aromatase cytochrome p450

  1. The planetary biology of cytochrome P450 aromatases

    PubMed Central

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

    2004-01-01

    Background Joining a model for the molecular evolution of a protein family to the paleontological and geological records (geobiology), and then to the chemical structures of substrates, products, and protein folds, is emerging as a broad strategy for generating hypotheses concerning function in a post-genomic world. This strategy expands systems biology to a planetary context, necessary for a notion of fitness to underlie (as it must) any discussion of function within a biomolecular system. Results Here, we report an example of such an expansion, where tools from planetary biology were used to analyze three genes from the pig Sus scrofa that encode cytochrome P450 aromatases–enzymes that convert androgens into estrogens. The evolutionary history of the vertebrate aromatase gene family was reconstructed. Transition redundant exchange silent substitution metrics were used to interpolate dates for the divergence of family members, the paleontological record was consulted to identify changes in physiology that correlated in time with the change in molecular behavior, and new aromatase sequences from peccary were obtained. Metrics that detect changing function in proteins were then applied, including KA/KS values and those that exploit structural biology. These identified specific amino acid replacements that were associated with changing substrate and product specificity during the time of presumed adaptive change. The combined analysis suggests that aromatase paralogs arose in pigs as a result of selection for Suoidea with larger litters than their ancestors, and permitted the Suoidea to survive the global climatic trauma that began in the Eocene. Conclusions This combination of bioinformatics analysis, molecular evolution, paleontology, cladistics, global climatology, structural biology, and organic chemistry serves as a paradigm in planetary biology. As the geological, paleontological, and genomic records improve, this approach should become widely useful to make systems biology statements about high-level function for biomolecular systems. PMID:15315709

  2. 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...

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

    Microsoft Academic Search

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

    1997-01-01

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

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

    SciTech Connect

    Yoshida, Nobutaka; Osawa, Yoshio (Medical Foundation of Buffalo Research Institute, NY (USA))

    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.

  5. Regulation of cytochrome P450 aromatase gene expression in adult rat Leydig cells: comparison with estradiol production

    Microsoft Academic Search

    C Genissel; J Levallet; S Carreau

    2001-01-01

    Regulation of aromatase gene expression in purified rat Leydig cells has not yet been investigated. Therefore, using a highly specific quantitative RT-PCR method, we have measured the amount of cytochrome P450 aromatase (P450arom) mRNA and aromatase activity in mature rat Leydig cells submitted to various treatments during 24 h. Estradiol production was enhanced in a dose-related manner in the presence

  6. Purification and characterization of equine testicular cytochrome P-450 aromatase: comparison with the human enzyme.

    PubMed

    Moslemi, S; Vibet, A; Papadopoulos, V; Camoin, L; Silberzahn, P; Gaillard, J L

    1997-09-01

    Cytochrome P-450 aromatase was purified by five chromatographic steps from adult stallion testis. It was first separated from NADPH-cytochrome P-450 reductase (reductase) on omega-aminohexyl-Sepharose 4B then purified to homogeneity on concanavalin A-Sepharose 4B, hydroxyapatite-Sepharose 4B, DEAE-Sepharose CL-6B and on a second hydroxyapatite-Sepharose 4B. On the other hand, purifications of the equine testicular and rat liver reductases, which allowed the reconstitution of aromatase activity in vitro, were achieved for each species in one chromatographic step on an adenosine 2',5'-diphosphate-agarose affinity column. Analysis on SDS/PAGE indicated single bands with apparent molecular masses of 53, 82, and 80 kDa for purified equine testicular cytochrome P-450 aromatase (eAROM), equine testicular reductase and rat liver reductase respectively. eAROM shows a time- and concentration-dependent activity that was stable for at least 2 months when stored at -78 degrees C. It is a highly hydrophobic protein composed from 505 residues and direct sequencing of its N-terminal part showed good homology when compared with human aromatase. When deglycosylated by N-glycosidase-F the apparent molecular mass of eAROM was decreased from 53 to 51 kDa as revealed by electrophoresis, its activity, however, was not impaired. eAROM exhibits much higher affinity for androgens than for 19-norandrogens, Km values were approximately 3, 16 and 170 nM for androstenedione (A), testosterone (T) and 19-nortestosterone (19-NT) respectively. However, it aromatizes 19-norandrostenedione (19-NA) slightly more efficiently than A, the estrone (E1) formed was 4.27 vs 3.54 pmol min-1 micrograms-1 respectively (P < 0.01). After incubation of eAROM with radiolabelled A and separation of steroids on HPLC, E1, 19-hydroxyandrostenedione (19-OHA) and 19-oxoandrostenedione (19-oxoA) were accumulated in the incubation medium in a time-dependent manner. The presence of 4-hydroxyandrostenedione (4-OHA), a suicide inhibitor of aromatase, cause a time-dependent inactivation of the enzyme. Whereas the activity of eAROM was unchanged in the presence of K+ (up to 250 mM), it was increased in the presence of EDTA (up to 50 mM) and decreased in the presence of DTT or Mg2+ (from 25 mM). We conclude that: (a) eAROM is a glycoprotein, however, deglycosylation by N-glycosidase-F does not appear to impair its activity, (b) eAROM aromatizes really both androgens and 19-norandrogens having a higher affinity for androgens, (c) the intermediary compounds of aromatization 19-OHA and 19-oxoA appear to be synthesized by the same active site that synthesizes E1 as the final product, (d) the inhibition of eAROM by increasing concentrations of Mg2+ and the stimulation of its activity by EDTA, taken together, indicate the importance of negatively charged residues in the polypeptide chain of equine aromatase, which play a role in enzymatic activity. PMID:9418012

  7. Immunohistochemical studies of steroidogenic enzymes (aromatase, 17 alpha-hydroxylase and cholesterol side-chain cleavage cytochromes P-450) in sex cord-stromal tumors of the ovary.

    PubMed

    Sasano, H; Okamoto, M; Mason, J I; Simpson, E R; Mendelson, C R; Sasano, N; Silverberg, S G

    1989-05-01

    Aromatase, 17 alpha-hydroxylase, and cholesterol side-chain cleavage P-450 cytochromes (P-450AROM, P-450(17 alpha,) and P-450SCC, respectively) were immunohistochemically localized in nine granulosa cell tumors, 15 thecomas, ten Sertoli-Leydig cell tumors, two steroid cell tumors, five fibromas, and five sclerosing stromal tumors. In the thecomas, P-450SCC and P-450(17 alpha) were positive in luteinized theca cells and in cells with vacuolated cytoplasm, while P-450AROM was not observed. In the steroid cell tumors, all the P-450 cytochromes were intensely stained. P-450SCC and P-450(17 alpha) were present in cells with vacuolated cytoplasm in two cases of sclerosing stromal tumor. P-450AROM was weakly demonstrated in one of the granulosa cell tumors. P-450(17 alpha,) P-450SCC, and P-450AROM were all faintly stained in the Sertoli-Leydig cell tumors. No P-450 cytochrome immunoreactivity was observed in any fibroma. PMID:2540088

  8. 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...

  9. Characterization of aromatase cytochrome P-450 mRNA in rat perinatal brain, ovary and a Leydig tumor cell line: evidence for the existence of brain specific aromatase transcripts

    Microsoft Academic Search

    Edwin D. Lephart; Michele A. Herbst; Michael J. McPhaul

    1995-01-01

    The conversion of androgens to estrogens is catalyzed by the aromatase cytochrome P-450 (P-450AROM) in a veriety of tissues and cell types in vertebrates. The manner in which aromatase activity is regulated appears to be\\u000a quite different, even between different tissues of a single species. In the current study, we have determined the sequence\\u000a of the 5? end of the

  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. Immunoexpression of aromatase cytochrome P450 and 17?-hydroxysteroid dehydrogenase in women’s ovaries after menopause

    PubMed Central

    2014-01-01

    Background Menopause results in a lack of regular menstrual cycles, leading to the reduction of estrogen production. On the other hand, ovarian androgen synthesis is still present at reduced levels and requires expression of several steroidogenic enzymes. Methods This study was performed on 104 postmenopausal women hospitalized due to uterine leiomyomas, endometriosis, and/or a prolapsed uterus. Patients were divided into three groups depending on the time from menopause. Group A patients experienced menopause 1–5 years before enrollment in the study (42 women). Group B included women who had their last menstruation 5–10 years before the study (40 women). Group C consisted of 22 women who were more than 10 years past menopause. Hysterectomy or removal of the uterine corpus with adnexa was performed during laparotomy. We evaluated the expression of aromatase cytochrome P450 (CYP 19) and 17?-hydroxysteroid dehydrogenase (17? HSD) by employing immunohistochemistry. Results Activity of 17?-HSD and CYP19 was demonstrated in the cytoplasm of stromal cells of postmenopausal ovaries, epithelium cells coating the ovaries, vascular endothelial cells, and epithelial inclusion cysts. However, overall expression of both 17?-HSD and CYP 19 decreased with time after menopause. Conclusion Demonstration of the activity of the key enzymes of ovarian steroidogenesis, CYP 19 and 17?-HSD, confirms steroidogenic activity in the ovaries of postmenopausal women. Nevertheless, ovarian steroidogenic activity decreases with time, and its significant decrease occurs 10 years after menopause. PMID:24855493

  12. Ontogenic expression patterns of several nuclear receptors and cytochrome P450 aromatases in brain and gonads of the Nile tilapia Oreochromis niloticus suggests their involvement in sex differentiation

    Microsoft Academic Search

    C. C. Sudhakumari; B. Senthilkumaran; T. Kobayashi; H. Kajiura-Kobayashi; D. S. Wang; M. Yoshikuni; Y. Nagahama

    2005-01-01

    Using semi-quantitative reverse transcriptase polymerase chain reaction we analyzed the ontogenic expression patterns of several\\u000a nuclear receptors (estrogen receptors [ER? and ?], androgen receptors [AR? and ?], Ad4BP\\/SF-1 and Dax-1) and cytochrome P450 aromatases (brain and ovarian types) in whole brain and gonads of the Nile tilapia.\\u000a ER? and ? transcripts were evident in both sexes with a high expression

  13. Porcine hypothalamic aromatase cytochrome P450: isoform characterization, sex-dependent activity, regional expression, and regulation by enzyme inhibition in neonatal boars.

    PubMed

    Corbin, C J; Berger, T; Ford, J J; Roselli, C E; Sienkiewicz, W; Trainor, B C; Roser, J F; Vidal, J D; Harada, N; Conley, A J

    2009-08-01

    Domestic pigs have three CYP19 genes encoding functional paralogues of the enzyme aromatase cytochrome P450 (P450arom) that are expressed in the gonads, placenta, and preimplantation blastocyst. All catalyze estrogen synthesis, but the gonadal-type enzyme is unique in also synthesizing a nonaromatizable biopotent testosterone metabolite, 1OH-testosterone (1OH-T). P450arom is expressed in the vertebrate brain, is higher in males than females, but has not been investigated in pigs, to our knowledge. Therefore, these studies defined which of the porcine CYP19 genes was expressed, and at what level, in adult male and female hypothalamus. Regional expression was examined in mature boars, and regulation of P450arom expression in neonatal boars was investigated by inhibition of P450arom with letrozole, which is known to reprogram testicular expression. Pig hypothalami expressed the gonadal form of P450arom (redesignated the "gonadal/hypothalamic" porcine CYP19 gene and paralogue) based on functional analysis confirmed by cloning and sequencing transcripts. Hypothalamic tissue synthesized 1OH-T and was sensitive to the selective P450arom inhibitor etomidate. Levels were 4-fold higher in male than female hypothalami, with expression in the medial preoptic area and lateral borders of the ventromedial hypothalamus of boars. In vivo, letrozole-treated neonates had increased aromatase activity in hypothalami but decreased activity in testes. Therefore, although the same CYP19 gene is expressed in both tissues, expression is regulated differently in the hypothalamus than testis. These investigations, the first such studies in pig brain to our knowledge, demonstrate unusual aspects of P450arom expression and regulation in the hypothalamus, offering promise of gaining better insight into roles of P450arom in reproductive function. PMID:19403926

  14. Immunolocalization of androgen receptor, aromatase cytochrome P450, estrogen receptor alpha and estrogen receptor beta proteins during the breeding season in scent glands of muskrats (Ondatra zibethicus).

    PubMed

    Lu, Lu; Zhang, Haolin; Lv, Na; Ma, Xiaoting; Tian, Long; Hu, Xiao; Liu, Shuqiang; Xu, Meiyu; Weng, Qiang; Watanabe, Gen; Taya, Kazuyoshi

    2011-10-01

    Aromatase cytochrome P450 (P450arom) is an enzyme that catalyzes the conversion of androgen to estrogen. Expression of P450arom in extra-gonadal sites and locally-synthesized estrogen play an important role in physiological conditions. The purpose of this study was to investigate the cellular immunolocalization of androgen receptor (AR), P450arom, estrogen receptor alpha (ERa) and estrogen receptor beta (ER?) in muskrat scent glands during the breeding season. Histological observation and immunohistochemistry of AR, P450arom, ERa and ER? were performed in the muskrat scent glands. In addition, total proteins were extracted from scent glandular tissues in the breeding season and were used for Western blotting analysis for AR, P450arom, ER? and ER?. Histologically, glandular cells, interstitial cells, epithelial cells of the excretory duct and the excretory tubules were identified in the muskrat scent glands during the breeding season. AR was only observed in glandular cells of scent glands; P450arom was expressed in glandular cells and epithelial cells of the excretory duct; ER? was found in glandular cells, interstitial cells and epithelial cells of the excretory duct, whereas ER? was present in glandular cells and epithelial cells of the excretory duct. Also, the positive signals of AR, P450arom, ER? and ER? by Western blotting were all observed in scent glandular tissues. These results suggested that the scent gland is the target organ of androgens and estrogens, and that estrogens may play an important autocrine or paracrine role in glandular function of the muskrats. PMID:21967219

  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. 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.

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

    PubMed

    Kazeto, Y; Trant, J M

    2005-12-01

    Cytochrome P450 aromatase (CYP19) converts androgens to estrogens. Unlike mammals, teleosts have two CYP19 genes, expressed differentially in ovary (CYP19A1) and neuronal tissues (CYP19A2). The primary purpose of this study was to demonstrate the potential involvement of CYP19A2 in the reproductive endocrinology of teleosts. Channel catfish CYP19A2 (ccCYP19A2) cDNAs were isolated from the brain using a PCR-based strategy. The ccCYP19A2 cDNA putatively encodes 500 amino acids which conferred aromatase activity in transfected COS-7 cells. Additionally, an alternatively spliced transcript was isolated which lacks the first 122 amino acids and is catalytically inactive. The brain and the pituitary were predominant sources of ccCYP19A2 transcript and the abundance in both tissues acutely increased prior to spawning. This preovulatory induction of ccCYP19A2 gene in the pituitary is remarkably similar to the pattern of gene expression for luteinizing hormone-beta (LHbeta). Estradiol-17beta (E(2)) and testosterone enhanced the transcript abundance of ccCYP19A2 and LHbeta in catfish pituitary cells cultured in vitro but the stimulatory effects of testosterone were abolished by an aromatase inhibitor, indicating an important role of E(2), the product of CYP19A2 activity, in the regulation of CYP19A2 and LHbeta. Structural and functional analysis of the 5'-flanking region of the gene suggested that the sequence from -1076 to - 435 bp is critical for the basal promoter activity in the pituitary. This report demonstrates that CYP19A2 functions as an important factor in the reproductive endocrinology of teleosts through the brain-pituitary-gonadal axis. PMID:16326841

  18. Post-translational dual regulation of cytochrome P450 aromatase at the catalytic and protein levels by phosphorylation/dephosphorylation.

    PubMed

    Hayashi, Takanori; Harada, Nobuhiro

    2014-11-01

    The post-translational regulation of aromatase has not been well characterized as compared with transcriptional regulation. Several studies of post-translational regulation have focused on decreases in catalytic activity following phosphorylation. We report here dual post-translational regulation of aromatase, at the catalytic activity and protein levels. Microsomal aromatase prepared from JEG-3 cells was rapidly inactivated and subsequently degraded in the presence of a cytosolic fraction with calcium, magnesium, and ATP. In a reconstituted system consisting of microsomal and cytosolic fractions, aromatase was protected from protein degradation by treatment with alkaline phosphatase, whereas degradation was enhanced by treatment with calcineurin inhibitors (FK506 and cyclosporin A). Furthermore, aromatase was protected from degradation by treatment with kinase inhibitors, especially the calcium/calmodulin kinase inhibitors KN62 and KN93. Similarly to the reconstituted system, aromatase in cultured JEG-3 cells was protected from degradation by KN93, whereas FK503 increased degradation in the presence of cycloheximide, although cellular aromatase mRNA levels were unchanged by these reagents. Knockdown of calcineurin and calcium/calmodulin kinase II (CaMKII) with small interfering RNAs resulted in a dose-dependent increase in aromatase degradation and protection from degradation, respectively. The cytosol fraction-dependent phosphorylation of microsomal aromatase was inhibited by calcineurin, KN62, and KN93, and promoted by CaMKII and FK506. These results indicate that aromatase is regulated acutely at the catalytic activity level and subsequently at the enzyme content level by CaMKII/calcineurin-dependent phosphorylation/dephosphorylation. PMID:25158681

  19. The immunoexpression of androgen receptor, estrogen receptors alpha and beta, vanilloid type 1 receptor and cytochrome p450 aromatase in rats testis chronically treated with letrozole, an aromatase inhibitor.

    PubMed

    Pilutin, Anna; Misiakiewicz-Has, Kamila; Kolasa, Agnieszka; Baranowska-Bosiacka, Irena; Marchlewicz, Mariola; Wiszniewska, Barbara

    2014-01-01

    The function of testis is under hormonal control and any disturbance of hormonal homeostasis can lead to morphological and physiological changes. Therefore the aim of the study was to investigate the expression of androgen and estrogen receptors (AR, ERs), vanilloid receptor (TRPV1), cytochrome P450 aromatase (P450arom), as well as apoptosis of cells in testis of adult rats chronically treated with letrozole (LT), a non-steroidal aromatase inhibitor, for 6 months. The testicular tissues were fixed in Bouin's fixative and embedded in paraffin. Immunohistochemistry with monoclonal antibodies (abs) against AR, ERa, P450arom, and polyclonalabs against ER?, TRPV1, caspase-3 was applied. Long-lasting estradiol deficiency, as an effect of LT treatment, produced changes in the morphology of testis and altered the expression of the studied receptors in cells of the seminiferous tubules and rate of cell apoptosis. The immunostaining for AR was found in the nuclei of Sertoli cells and the cytoplasm of spermatogonia and spermatocytes in III-IV stages of the seminiferous epithelium cycle. The intensity of staining for P450arom was lower in the testis of LT-treated rats as compared to control animals. The immunofluorescence of ER? and ER? was observed exclusively in the nuclei of Leydig cells of LT-treated rats. There were no changes in localization of TRPV1, however, the intensity of reaction was stronger in germ cells of the seminiferous epithelium after LT treatment. The apoptosis in both groups of animals was observed within the population of spermatocytes and spermatids in II and III stages of the seminiferous epithelium cycle. In testis of LT-treated rats the immunoexpression of caspase-3 was additionally found in the germ cells in I and IV stages, and Sertoli, myoid and Leydig cells. In conclusion, our results underline the important role of letrozole treatment in the proper function of male reproductive system, and additionally demonstrate that hormonal imbalance can produce the morphological abnormalities in testis. PMID:25308736

  20. Apoptosis in atretic ovarian follicles is associated with selective decreases in messenger ribonucleic acid transcripts for gonadotropin receptors and cytochrome P450 aromatase.

    PubMed

    Tilly, J L; Kowalski, K I; Schomberg, D W; Hsueh, A J

    1992-10-01

    Although atresia of ovarian follicles is of critical importance during preovulatory follicle selection as well as during normal and premature menopause, the mechanisms underlying atresia remain poorly understood. To study molecular events associated with atresia, we evaluated changes in mRNA levels for cytochrome P450 aromatase, FSH receptor, LH receptor, and a structural protein, beta-actin, during atresia in small (3-mm diameter) and large (6-mm diameter) porcine follicles. In addition, internucleosomal fragmentation of DNA characteristic of apoptosis ("programmed cell death") was assessed in individual healthy and atretic follicles using a sensitive autoradiographic method. Follicles were classified as morphologically healthy or atretic based on the absence or presence of follicular haemorrhagia and the degree of follicular clarity. Morphological signs of atresia in individual follicles were correlated with the occurrence of internucleosomal DNA fragmentation in granulosa cells as well as in thecal cells during advanced stages of atresia. The presence of apoptosis in atretic follicles was also associated with significant decreases in follicular fluid estrogen concentrations compared to those in healthy follicles of the same size. The decline in estrogen synthesis in degenerating follicles was further correlated with decreased levels of a predominant 2.6-kilobase aromatase mRNA. Moreover, substantial declines in both FSH receptor and LH receptor mRNAs were found in atretic follicles, consistent with previous reports of their decreased responsiveness to gonadotropins. The observed decreases in mRNAs for aromatase and gonadotropin receptors could not be attributed to a generalized degradation of cellular RNA during atresia, as evidenced by the presence of intact 18S and 28S ribosomal RNA as well as constitutive expression of beta-actin mRNA in atretic follicles. These data indicate that apoptotic cell death is initiated in both granulosa and thecal cells of porcine follicles during atresia. Associated with internucleosomal DNA fragmentation, decreased transcription of specific ovarian genes or destabilization of their transcripts leads to selective decreases in aromatase and gonadotropin receptor mRNAs. The atresia of ovarian follicles provides an interesting model to further study the molecular events associated with DNA fragmentation and selective mRNA down-regulation during apoptosis. PMID:1396312

  1. Luminogenic cytochrome P450 assays.

    PubMed

    Cali, James J; Ma, Dongping; Sobol, Mary; Simpson, Daniel J; Frackman, Susan; Good, Troy D; Daily, William J; Liu, David

    2006-08-01

    Luminogenic cytochrome P450 (CYP) assays couple CYP enzyme activity to firefly luciferase luminescence in a technology called P450-Glo(TM) (Promega). Luminogenic substrates are used in assays of human CYP1A1, -1A2, -1B1, -2C8, -2C9, -2C19, -2D6, -2J2, -3A4, -3A7, -4A11, -4F3B, -4F12 and -19. The assays detect dose-dependent CYP inhibition by test compounds against recombinant CYP enzymes or liver microsomes. Induction or inhibition of CYP activities in cultured hepatocytes is measured in a nonlytic approach that leaves cells intact for additional analysis. Luminogenic CYP assays offer advantages of speed and safety over HPLC and radiochemical-based methods. Compared with fluorogenic methods the approach offers advantages of improved sensitivity and decreased interference between optical properties of test compound and CYP substrate. These homogenous assays are sensitive and robust tools for high-throughput CYP screening in early drug discovery. PMID:16859410

  2. Reactive Intermediates in Cytochrome P450 Catalysis*

    PubMed Central

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

    2013-01-01

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

  3. Cooperative properties of cytochromes P450

    PubMed Central

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

    2009-01-01

    Cytochromes P450 form a large and important class of heme monooxygenases with a broad spectrum of substrates and corresponding functions, from steroid hormone biosynthesis to the metabolism of xenobiotics. Despite decades of study, the molecular mechanisms responsible for the complex non-Michaelis behavior observed with many members of this super-family during metabolism, often termed ‘cooperativity,’ remain to be fully elucidated. Although there is evidence that oligomerization may play an important role in defining the observed cooperativity, some monomeric cytochromes P450, particularly those involved in xenobiotic metabolism, also display this behavior due to their ability to simultaneously bind several substrate molecules. As a result, formation of distinct enzyme-substrate complexes with different stoichiometry and functional properties can give rise to homotropic and heterotropic cooperative behavior. This review aims to summarize the current understanding of cooperativity in cytochromes P450, with a focus on the nature of cooperative effects in monomeric enzymes. PMID:19555717

  4. 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

  5. Aldehyde Reduction by Cytochrome P450

    PubMed Central

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

    2011-01-01

    This protocol describes the procedure for measuring the relative rates of metabolism of the ?,?-unsaturated aldehydes, 9-anthracene aldehyde (9-AA) and 4-hydroxy-trans-2-nonenal (4-HNE); specifically the aldehyde reduction reactions of cytochrome P450s (CYPs). These assays can be performed using either liver microsomal or other tissue fractions, spherosome preparations of recombinant CYPs, or recombinant CYPs from other sources. The method used here to study the reduction of a model ?,?-unsaturated aldehyde, 9-AA, by CYPs was adapted from the assay used to investigate 9-anthracene oxidation as reported by Marini et al. (Marini et al., 2003). For experiments measuring reduction of the endogenous aldehyde, 4-HNE, the substrate was incubated with CYP in the presence of oxygen and NADPH and the metabolites were separated by High Pressure Liquid Chromatograpy (HPLC), using an adaptation of the method of Srivastava et al. (Srivastava et al., 2010). For study of 9-AA and 4-HNE reduction, the first step involves incubation of the substrate with the CYP in appropriate media, followed by quantification of metabolites through either spectrofluorimetry or analysis by HPLC coupled with a radiometric assay, respectively. Metabolite identification can be achieved by HPLC GC-mass spectrometric analysis. Inhibitors of cytochrome P450 function can be utilized to show the role of the hemoprotein or other enzymes in these reduction reactions. The reduction reactions for CYP’s were not inhibited by either anaerobiosis or inclusion of CO in the gaseous phase of the reaction mixture. These character of these reactions are similar to those reported for some cytochrome P450-catalyzed azo reduction reactions. PMID:21553396

  6. Novel SNPs in cytochrome P450 oxidoreductase.

    PubMed

    Hart, Steven N; Li, Ye; Nakamoto, Kaori; Wesselman, Chris; Zhong, Xiao-bo

    2007-08-01

    Cytochrome P450 oxidoreductase (POR) is the single flavoprotein which donates electrons to the microsomal cytochrome P450 enzymes for oxidation of their substrates. In this study, we sequenced all 15 exons and the surrounding intronic sequences of POR in 100 human liver samples to identify novel and confirm known genetic polymorphisms in POR. Thirty-four single nucleotide polymorphisms (SNPs) were identified including 9 in the coding exons (5 synonymous and 4 nonsynonymous), 20 in the intronic regions, and 5 in the 3'-UTR. Of these, 9 were novel SNPs, including three nonsynonymous SNPs, SNH313003 (817733G>C; K49N), SNH313020 (848661C>A; L420M), and SNH313029 (849577T>C; L577P) with minor allele frequencies of 0.005, 0.045, and 0.020, respectively. We also confirmed a previously reported non-synonymous SNP rs1057868 (A503V) as well as five synonymous SNPs (G5G, T29T, P129P, S485S, and S572S) all with allele frequencies similar to those previously reported. Structurally, these polymorphisms occur in different regions: SNH313003 (K49N) in the amino-terminal tail, SNH313020 (L420M) in the connecting domain, SNH313029 (L577P) in the NADPH-binding domain, and rs1057868 (A503V) in the FAD binding domain. PMID:17827787

  7. Cytochrome P450cam-monoterpene interactions.

    PubMed

    Van Roon, A; Parsons, J R; Govers, H A J

    2005-08-01

    The use of the molecular mechanics AMBER force field (FF) to predict product profiles for the hydroxylation of the monoterpenes 1R-camphor, 1S-camphor, 1R-norcamphor, 1S-norcamphor and camphane by the enzyme cytochrome P450cam from the soil bacterium Pseudomonas putida was investigated. Predictions were carried out by applying multiple substrate (starting) orientations in the enzyme pocket in two procedures: a procedure based on molecular dynamics (MD) and a procedure based on short MD simulations followed by geometry optimisations. The latter (GO) procedure is faster and enabled the use of more monoterpene starting orientations. Monoterpene orientations were transformed into product profiles by applying both energetic and geometrical criteria appropriate for the (monoterpene) hydrogen abstraction reaction. Good predictions compared to experimental data were obtained for most compounds in both the MD and GO procedures. Prior to the product profile calculations, the FF was calibrated by reproducing the experimental data for the binding energy of 1R-camphor and 1S-camphor to P450cam and the energy of vaporisation of water. Focus of the calibration was on the value for the scaling factor for the electrostatic interactions. PMID:16234177

  8. Structural diversity of eukaryotic membrane cytochrome p450s.

    PubMed

    Johnson, Eric F; Stout, C David

    2013-06-14

    X-ray crystal structures are available for 29 eukaryotic microsomal, chloroplast, or mitochondrial cytochrome P450s, including two non-monooxygenase P450s. These structures provide a basis for understanding structure-function relations that underlie their distinct catalytic activities. Moreover, structural plasticity has been characterized for individual P450s that aids in understanding substrate binding in P450s that mediate drug clearance. PMID:23632020

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

    PubMed

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

    2009-11-13

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

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

    PubMed

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

    2012-10-15

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

  11. Rearrangement Reactions Catalyzed by Cytochrome P450s

    PubMed Central

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

    2010-01-01

    Cytochrome P450s promote a variety of rearrangement reactions both as a consequence of the nature of the radical and other intermediates generated during catalysis, and of the neighboring structures in the substrate that can interact either with the initial radical intermediates or with further downstream products of the reactions. This article will review several kinds of previously published cytochrome P450-catalyzed rearrangement reactions, including changes in stereochemistry, radical clock reactions, allylic rearrangements, “NIH” and related shifts, ring contractions and expansions, and cyclizations that result from neighboring group interactions. Although most of these reactions can be carried out by many members of the cytochrome P450 superfamily, some have only been observed with select P450s, including some reactions that are catalyzed by specific endoperoxidases and cytochrome P450s found in plants. PMID:20971058

  12. 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

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

    ClinicalTrials.gov

    2015-01-06

    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

  14. Canine cytochrome P450 (CYP) pharmacogenetics

    PubMed Central

    Court, Michael H.

    2013-01-01

    Synopsis The cytochrome P450 (CYP) drug metabolizing enzymes are essential for the efficient elimination of many clinically used drugs. These enzymes typically display high interindividual variability in expression and function resulting from enzyme induction, inhibition, and genetic polymorphism thereby predisposing patients to adverse drug reactions or therapeutic failure. There are also substantial species differences in CYP substrate specificity and expression that complicate direct extrapolation of information from humans to veterinary species. This article reviews the available published data regarding the presence and impact of genetic polymorphisms on CYP-dependent drug metabolism in dogs in the context of known human-dog CYP differences. Canine CYP1A2, which metabolizes phenacetin, caffeine, and theophylline, is the most widely studied polymorphic canine CYP. A single nucleotide polymorphism resulting in a CYP1A2 premature stop codon (c.1117C>T; R383X) with a complete lack of enzyme is highly prevalent in certain dog breeds including Beagle and Irish wolfhound. This polymorphism was shown to substantially affect the pharmacokinetics of several experimental compounds in Beagles during preclinical drug development. However, the impact on the pharmacokinetics of phenacetin (a substrate specific for human CYP1A2) was quite modest probably because other canine CYPs are capable of metabolizing phenacetin. Other canine CYPs with known genetic polymorphisms include CYP2C41 (gene deletion), as well as CYP2D15, CYP2E1, and CYP3A12 (coding SNPs). However the impact of these variants on drug metabolism in vitro or on drug pharmacokinetics is unknown. Future systematic investigations are needed to comprehensively identify CYP genetic polymorphisms that are predictive of drug effects in canine patients. PMID:23890236

  15. Cytochrome P450 1 family and cancers.

    PubMed

    Go, Ryeo-Eun; Hwang, Kyung-A; Choi, Kyung-Chul

    2015-03-01

    The aryl hydrocarbon receptor (AhR) is a ligand-activated transcriptional factor that dimerizes with aryl hydrocarbon receptor nuclear translocator (ARNT). This complex binds to xenobiotics response element (XREs), and then starts the expressions of downstream genes including cytochrome P450 (CYP) 1 family members: CYP1A1, CYP1A2 and CYP1B1. Role of CYP1 family is involved in the metabolism of endogenous hormones, xenobiotics and drug. The expression of CYP1 family is regulated by estradiol (E2) or xenobiotics in diverse cancers. In breast cancers expressing estrogen receptors (ERs), level of CYP1B1 is increased by E2 and reversed by an estrogen receptor antagonist, ICI 182,780 or 4-hydrotamoxifen, which indicates that the expression of CYP1 family in downstream region of AhR is regulated by an activation of ER?. In metabolic pathways, E2 is converted into 4-hydroxyestradiol by CYP1B1, which can be converted into mainly estradiol-3,4-quinone, a potential carcinogen, by peroxidase. Increased expression of CYP1 family indicates the possibility of carcinogenesis by exposure of xenobiotics in endometrial and ovarian cancers. Apart from roles of CYP1 family in relation with ER pathway, CYP1 family is over-expressed in ER independent cancers. CYP1A1 exhibits hydroxylase activity in oxidation of arachidonic acid, which has been transformed to 12(R)-hydrxyeicosatetraenoic (HETEs), a potent activator of AhR activity. On the basis of results, phytoestrogens and dexamethasone are provided as cancer therapy regulating the expression of CYP1 family. Thus, this review focuses on the role(s) of CYP1 family in ER-dependent or ER-independent cancers and the potential for cancer therapy to target CYP1 family in these cancers. PMID:25448748

  16. Cytochrome P450 pharmacogenetics in African populations.

    PubMed

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

    2013-05-01

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

  17. Expression levels of mRNA for neurosteroidogenic enzymes 17?-HSD, 5?-reductase, 3?-HSD and cytochrome P450 aromatase in the fetal wild type and SF-1 knockout mouse brain.

    PubMed

    Spanic, Tanja; Fabjan, Teja; Majdic, Gregor

    2015-01-01

    The presence of steroidogenic enzymes in the brain suggests de novo synthesis of steroid hormones in the brain. The current study was designed to determine the developmental profiles of cytochrome p450 aromatase (cyp19), 17?-hydroxysteroid dehydrogenase (17?-HSD), 5?-reductase type I and 3?-hydroxysteroid dehydrogenase (3?-HSD) mRNA expression levels in the fetal mouse brain and potential influence of peripheral steroids, and the steroidogenic factor 1 (SF-1) gene on their expression. Brains were collected from WT and SF-1 knockout male and female fetuses at embryonic (E) days E12, E14, E16, and E18. Quantitative PCR analyses revealed age related increases in the expression levels of 17?-HSD and 5?-reductase. Differences between genotypes in the expression levels of 17?-HSD and 5?-reductase were detected on E14, with reduced levels of expression in SF-1 KO males and females for 17?-HSD and only between females for 5?-reductase. Expression of 3?-HSD mRNA did not differ significantly between sexes, age groups or genotypes with the exception of SF-1 KO males, which had an unexplained increase in mRNA for this enzyme on day E18. Expression of cyp19 was at the limit of detection and could not be analyzed effectively. There were no sex differences and, with the exception of small difference on E14 for 17?-HSD and 5?-reductase, no differences between genotypes. The results suggest that gonadal steroids do not influence the production of neurosteroids in the fetal brain, nor does SF-1 play a major role in the regulation of steroidogenic enzyme expression in the brain. PMID:25111584

  18. Genetics Home Reference: Cytochrome P450 oxidoreductase deficiency

    MedlinePLUS

    ... hormones, which are needed for normal development and reproduction. The hormonal changes associated with cytochrome P450 oxidoreductase ... which are essential for normal sexual development and reproduction; corticosteroids, which are involved in the body's response ...

  19. Cytochromes P450 catalyze the reduction of ?,?-unsaturated aldehydes.

    PubMed

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

    2011-08-15

    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, O(2), 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 and 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 a 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 the reduction of ?,?-unsaturated aldehydes in the liver. PMID:21766881

  20. 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

  1. Thiomers: Inhibition of cytochrome P450 activity.

    PubMed

    Iqbal, Javed; Sakloetsakun, Duangkamon; Bernkop-Schnürch, Andreas

    2011-08-01

    The aim of the present study was to investigate the potential of different thiolated polymers (thiomers) on the catalytic activity of CYP450s on one hand and to explore new inhibitors for CYP activity on the other hand. Several thiolated polymers including poly(acrylic acid)-cysteine (PAA-cysteine), chitosan-thioglycolic acid (chitosan-TGA), and thiolated PEG-g-PEI copolymer along with brij 35, myrj 52 and the well-established CYPP450 inhibitor verapamil were screened for their CYP3A4 and CYP2A6 inhibitory activity, and their IC(50) values were determined. Both enzyme inhibition assays were performed in 96-well microtiter plates. 7-Benzyloxy-4-(trifluoromethyl)-coumarin (BFC) and 7-hydroxycoumarin (7-HC) were used as fluorescent substrates in order to determine CYP3A4 and CYP2A6 catalytic activity, respectively. All investigated compounds inhibited CYP3A4 as well as CYP2A6 activity. All tested (thiolated) polymers were found to be more potent inhibitors of CYP3A4 than of CYP2A6 catalytic activity. Apart from verapamil that is a known CYP3A4 inhibitor, brij 35 and myrj 52 were explored as potent inhibitors of CYP3A4 and CYP2A6 catalytic activity. Among the tested polymers, the rank order for CYP3A4 inhibition was PAA-cysteine (100 kDa)>brij 35>thiolated PEG-g-PEI copolymer (16 kDa)>myrj 52>PAA (100 kDa)>PAA-cysteine (450 kDa)>verapamil>PAA (450 kDa)>chitosan-TGA (150 kDa)>chitosan (150 kDa). On the other hand, the rank order of CYP2A6 inhibition was brij 35>PAA-cysteine (100kDa)>chitosan-TGA (150 kDa)>PAA (100 kDa)>thiolated PEG-g-PEI copolymer (16 kDa)>PAA-cysteine (450 kDa)>chitosan (150 kDa)>verapamil>PAA (450 kDa)>myrj 52. Thus, this study suggests that (thiolated) polymers display a promising potential to inhibit cytochrome P450s activity and might turn out to be potentially valuable tools for improving the oral bioavailability of actively secreted compounds by avoiding intestinal metabolism. PMID:21362475

  2. Characterization of Drosophila melanogaster cytochrome P450 genes

    PubMed Central

    Chung, Henry; Sztal, Tamar; Pasricha, Shivani; Sridhar, Mohan; Batterham, Philip; Daborn, Phillip J.

    2009-01-01

    Cytochrome P450s form a large and diverse family of heme-containing proteins capable of carrying out many different enzymatic reactions. In both mammals and plants, some P450s are known to carry out reactions essential for processes such as hormone synthesis, while other P450s are involved in the detoxification of environmental compounds. In general, functions of insect P450s are less well understood. We characterized Drosophila melanogaster P450 expression patterns in embryos and 2 stages of third instar larvae. We identified numerous P450s expressed in the fat body, Malpighian (renal) tubules, and in distinct regions of the midgut, consistent with hypothesized roles in detoxification processes, and other P450s expressed in organs such as the gonads, corpora allata, oenocytes, hindgut, and brain. Combining expression pattern data with an RNA interference lethality screen of individual P450s, we identify candidate P450s essential for developmental processes and distinguish them from P450s with potential functions in detoxification. PMID:19289821

  3. Structural Characterization of Human Cytochrome P450 2C19

    PubMed Central

    Reynald, R. Leila; Sansen, Stefaan; Stout, C. David; Johnson, Eric F.

    2012-01-01

    To identify the structural features underlying the distinct substrate and inhibitor profiles of P450 2C19 relative to the closely related human enzymes, P450s 2C8 and 2C9, the atomic structure (Protein Data Bank code 4GQS) of cytochrome P450 2C19 complexed with the inhibitor (2-methyl-1-benzofuran-3-yl)-(4-hydroxy-3,5-dimethylphenyl)methanone (Protein Data Bank chemical component 0XV) was determined to 2.87 Å resolution by x-ray crystallography. The conformation of the peptide backbone of P450 2C19 is most similar to that of P450 2C8, but the substrate-binding cavity of P450 2C8 is much larger than that of P450 2C19 due to differences in the amino acid residues that form the substrate-binding cavities of the two enzymes. In contrast, the substrate-binding cavity of P450 2C19 is much more similar in size to that of the structure of the P450 2C9 flurbiprofen complex than to that of a modified P450 2C9 or that of P450 2C8. The cavities of the P450 2C19 0XV complex and the P450 2C9 flurbiprofen complex differ, however, because the helix B-C loops of the two enzymes are dissimilar. These conformational differences reflect the effects of adjacent structural elements that interact with the B-C loops and that differ between the two enzymes. The availability of a structure for 2C19 will facilitate computational approaches for predictions of substrate and inhibitor binding to this enzyme. PMID:23118231

  4. Cytochrome P450 monooxygenases and insecticide resistance in insects.

    PubMed Central

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

    1998-01-01

    Cytochrome P450 monooxygenases are involved in many cases of resistance of insects to insecticides. Resistance has long been associated with an increase in monooxygenase activities and with an increase in cytochrome P450 content. However, this increase does not always account for all of the resistance. In Drosophila melanogaster, we have shown that the overproduction of cytochrome P450 can be lost by the fly without a corresponding complete loss of resistance. These results prompted the sequencing of a cytochrome P450 candidate for resistance in resistant and susceptible flies. Several mutations leading to amino-acid substitutions have been detected in the P450 gene CYP6A2 of a resistant strain. The location of these mutations in a model of the 3D structure of the CYP6A2 protein suggested that some of them may be important for enzyme activity of this molecule. This has been verified by heterologous expression of wild-type and mutated cDNA in Escherichia coli. When other resistance mechanisms are considered, relatively few genetic mutations are involved in insecticide resistance, and this has led to an optimistic view of the management of resistance. Our observations compel us to survey in more detail the genetic diversity of cytochrome P450 genes and alleles involved in resistance. PMID:10021770

  5. 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

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

    Microsoft Academic Search

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

    1996-01-01

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

  7. Reconstitution Premixes for Assays Using Purified Recombinant Human Cytochrome P450, NADPH-Cytochrome P450 Reductase, and Cytochrome b 5

    Microsoft Academic Search

    Peter M. Shaw; Natilie A. Hosea; David V. Thompson; Janean M. Lenius; F. Peter Guengerich

    1997-01-01

    The development of enzyme and buffer premixes forin vitrobiotransformation assays is described. The protein premixes contain a mixture of three recombinant human proteins, cytochrome P450 (P450) 3A4, NADPH-P450 reductase, cytochromeb5, and liposomes. The buffer premix contains reagents which, when diluted, provide for optimal metabolic activity with selected P450 3A4 substrates. P450 3A4 premixes were competent in the oxidation of known

  8. Homotropic cooperativity of monomeric cytochrome P450 3A4

    SciTech Connect

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

    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.

  9. Role of cytochrome P450 in drug interactions

    Microsoft Academic Search

    Zakia Bibi

    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

  10. Comparison of Cytochrome P450 Genes from Six Plant Genomes

    Microsoft Academic Search

    David R. Nelson; Ray Ming; Maqsudul Alam; Mary A. Schuler

    2008-01-01

    Plants depend on cytochrome P450 (CYP) enzymes for nearly every aspect of their biology. In several sequenced angiosperms,\\u000a CYP genes constitute up to 1% of the protein coding genes. The angiosperm sequence diversity is encapsulated by 59 CYP families,\\u000a of which 52 families form a widely distributed core set. In the 20 years since the first plant P450 was sequenced, 3,387

  11. 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...

  12. 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

  13. Cytochrome p450 inhibitory properties of common efflux transporter inhibitors.

    PubMed

    Englund, Gunilla; Lundquist, Patrik; Skogastierna, Cristine; Johansson, Jenny; Hoogstraate, Janet; Afzelius, Lovisa; Andersson, Tommy B; Projean, Denis

    2014-03-01

    Drug transporter inhibitors are important tools to elucidate the contribution of transporters to drug disposition both in vitro and in vivo. These inhibitors are often unselective and affect several transporters as well as drug metabolizing enzymes, which can make experimental results difficult to interpret with confidence. We therefore tested 14 commonly used P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and multidrug-resistance associated protein (MRP) inhibitors as inhibitors of cytochrome P450 (P450) enzyme activities using recombinant enzymes. A subset of P-gp and/or CYP3A inhibitors were selected (cyclosporin A, elacridar, ketoconazole, quinidine, reserpine, and tacrolimus) for a comparison of P450 inhibition in human microsomes and hepatocytes. Most P-gp inhibitors showed CYP3A4 inhibition, with potencies often in a similar range as their P-gp inhibition, as well as less potent CYP2C19 inhibition. Other P450 enzymes were not strongly inhibited except a few cases of CYP2D6 inhibition. MRP and BCRP inhibitors showed limited P450 inhibition. Some inhibitors showed less P450 inhibition in human hepatocytes than human liver microsomes, for example, elacridar, probably due to differences in binding, permeability limitations, or active, P-gp mediated efflux of the inhibitor from the hepatocytes. Quinidine was a potent P450 inhibitor in hepatocytes but only showed weak inhibition in microsomes. Quinidine shows an extensive cellular uptake, which may potentiate intracellular P450 inhibition. Elacridar, described as a potent and selective P-gp inhibitor, displayed modest P450 inhibition in this study and is thus a useful model inhibitor to define the role of P-gp in drug disposition without interference with other processes. PMID:24396142

  14. The expression of cytochrome P-450 and cytochrome P-450 reductase genes in the simultaneous transformation of corticosteroids and phenanthrene by Cunninghamella elegans.

    PubMed

    Lisowska, Katarzyna; Szemraj, Janusz; Rózalska, Sylwia; D?ugo?ski, Jerzy

    2006-08-01

    The expression of cytochrome P-450 and cytochrome P-450 reductase (CPR) genes in the conterminous biotransformation of corticosteroids and PAHs was studied in Cunninghamella elegans 1785/21Gp. We had previously used this strain as a microbial eucaryotic model for studying the relationship between mammalian steroid hydroxylation and the metabolization of PAHs. We reported that cytochrome P-450 reductase is involved in the biotransformaton of cortexolone and phenanthrene. RT-PCR and Northern blotting analyses indicated that the cytochrome P-450 and CPR genes appear to be inducible by both steroids and PAHs. The expression of the cytochrome P-450 gene was increased ninefold and the expression of the CPR gene increased 6.4-fold in cultures with cortexolone and/or phenanthrene in comparison with controls. We conclude that the increase in cytochrome P-450 gene expression was accompanied by an increase in cytochrome P-450 enzymatic activity levels. PMID:16907717

  15. Cytochrome P450 arachidonic acid metabolism in bovine corneal epithelium

    SciTech Connect

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

    1986-03-01

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

  16. Effects of Aromatase Inhibitor on Sex Differentiation and Levels of P450 17?and P450 aromMessenger Ribonucleic Acid of Gonads in Chicken Embryos

    Microsoft Academic Search

    K Abinawanto; Kiyoshi Shimada; Kumiko Yoshida; Noboru Saito

    1996-01-01

    On Day 5 of incubation fertilized eggs of single-comb White Leghorn hens were injected with an aromatase inhibitor (AI) and the sex reversal effect and levels of mRNA of P45017?-hydroxylase(P45017?) and P450aromatase(P450arom) were evaluated by observation of gonadal phenotype and by Northern and slot blot analysis. Individual genetic sex was evaluated by Southern blot analysis of red blood cells using

  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. Aromatase P450 expression in human pituitary adenomas.

    PubMed

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

    2015-02-01

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

  19. 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 ...

  20. Aromatase (P450arom) and 11?-hydroxylase (P45011?) genes are differentially expressed during the sex change process of the protogynous rice field eel, monopterus albus

    Microsoft Academic Search

    Ji-Fang Liu; Yann Guiguen; Shao-Jun Liu

    2009-01-01

    Steroids are known to play a crucial role in gonadal sex differentiation in many non-mammalian vertebrates, but also in the\\u000a gonadal sex change of hermaphroditic teleosts. We investigated the expression of two genes encoding key steroidogenic enzymes,\\u000a i.e., cytochrome P450 aromatase (P450arom) and cytochrome P45011?-hydroxylase (P45011?), during the sex change of the protogynous\\u000a rice field eel, Monopterus albus. Using RT-PCR

  1. 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...

  2. 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...

  3. Cytochrome P450 in fluke Opisthorchis felineus: identification and characterization.

    PubMed

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

    2012-02-01

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

  4. Substrates of human hepatic cytochrome P450 3A4

    Microsoft Academic Search

    Albert P. Li; Donald L. Kaminski; Asenath Rasmussen

    1995-01-01

    Cytochrome P450 isozyme 3A4 (CYP3A4) is a major isozyme in the human liver and is known to metabolize a large variety of xenobiotics and endogenous biochemicals. The identities of CYP3A4 substrates are summarized here. A total of 32 chemicals belonging to different structural classes have been evaluated and found to be substrates for CYP3A4. The metabolic pathways for these substrates

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

    Microsoft Academic Search

    Robert P. Hanzlik; Kahhiing John Ling

    1990-01-01

    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

  6. Modulation by phytochemicals of cytochrome P450-linked enzyme activity

    Microsoft Academic Search

    Robert W. Teel; Huong Huynh

    1998-01-01

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

  7. Involvement of cytochrome P450 1A in sanguinarine detoxication

    Microsoft Academic Search

    Ji??? Vrba; Pavel Kosina; Jitka Ulrichová; Martin Modrianský

    2004-01-01

    Sanguinarine (SA), a member of the benzo[c]phenanthridine alkaloids, is a potent anti-microbial agent with anti-inflammatory and anti-neoplastic properties. However, toxicity of the alkaloid severely limits its medical applications. Recent report by Williams et al. [Vet. Hum. Toxicol. 42 (2000) 196] implicated rat hepatic cytochrome P450 (CYP) 1A2 as a likely modulator of SA toxicity. Indeed, the in vitro toxicity of

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

    PubMed Central

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

    2012-01-01

    Dihydrotestosterone is a more potent androgen than testosterone and plays an important role in endocrine function. We demonstrated that, like testosterone, dihydrotestosterone can be oxidized by human cytochrome P450 (P450) 19A1, the steroid aromatase. The products identified include the 19-hydroxy- and 19-oxo derivatives and the resulting ?1,10-, ?5,10-, and ?9,10-dehydro 19-norsteroid products (loss of 19-methyl group). The overall catalytic efficiency of oxidation was ?10-fold higher than reported for 3?-reduction by 3?-hydroxysteroid dehydrogenase, the major enzyme known to deactivate dihydrotestosterone. These and other studies demonstrate the flexibility of P450 19A1 in removing the 1- and 2-hydrogens from 19-norsteroids, the 2-hydrogen from estrone, and (in this case) the 1-, 5?-, and 9?-hydrogens of dihydrotestosterone. Incubation of dihydrotestosterone with human liver microsomes and NADPH yielded the 18- and 19-hydroxy products plus the ?1,10-dehydro 19-nor product identified in the P450 19A1 reaction. The 18- and 19-hydroxylation reactions were attributed to P450 3A4, and 18- and 19-hydroxydihydrotestosterone were identified in human plasma and urine samples. The change in the pucker of the A ring caused by reduction of the ?4,5 bond is remarkable in shifting the course of hydroxylation from the 6?-, 2?-, 1?-, and 15?-methylene carbons (testosterone) to the axial methyl groups (18, 19) in dihydrotestosterone and demonstrates the sensitivity of P450 3A4, even with its large active site, to small changes in substrate structure. PMID:22773874

  9. Epoxidation activities of human cytochromes P450c17 and P450c21.

    PubMed

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

    2014-12-01

    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 16?-hydroxylates progesterone, might catalyze the formation of the 16?,17-epoxide from 16,17-dehydroprogesterone (pregna-4,16-diene-3,20-dione). CYP17A1 catalyzed the novel 16?,17-epoxidation and the ordinarily minor 21-hydroxylation of 16,17-dehydroprogesterone in a 1:1 ratio. CYP17A1 mutation A105L, which has reduced progesterone 16?-hydroxylase activity, gave a 1:5 ratio of epoxide:21-hydroxylated products. In contrast, human P450c21 (steroid 21-hydroxylase, CYP21A2) converted 16,17-dehydroprogesterone to the 21-hydroxylated product and only a trace of epoxide. CYP21A2 mutation V359A, which has significant 16?-hydroxylase activity, likewise afforded the 21-hydroxylated product and slightly more epoxide. CYP17A1 wild-type and mutation A105L do not 21- or 16?-hydroxylate pregnenolone, but the enzymes 21-hydroxylated and 16?,17-epoxidized 16,17-dehydropregnenolone (pregna-5,16-diene-3?-ol-20-one) in 4:1 or 12:1 ratios, respectively. Catalase and superoxide dismutase did not prevent epoxide formation. The progesterone epoxide was not a time-dependent, irreversible CYP17A1 inhibitor. Our substrate modification studies have revealed occult epoxidase and 21-hydroxylase activities of CYP17A1, and the fraction of epoxide formed correlated with the 16?-hydroxylase activity of the enzymes. PMID:25386927

  10. 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

  11. Cytochrome P450 polymorphism--molecular, metabolic and pharmacogenetic aspects. I. Mechanisms of activity of cytochrome P450 monooxygenases.

    PubMed

    Pachecka, Jan; Tomaszewski, Piotr; Kubiak-Tomaszewska, Grazyna

    2008-01-01

    Cytochrome P450, initially perceived as a type of cell pigment, was soon identified as a hemoprotein with an enzymatic activity characteristic for monooxygenases with an affinity for differentiated endo- or exogenous substrates, including drugs. So far in the human organism 58 CYP isoenzymes belonging to 18 families have been described. Most from the CYP monooxygenases superfamily turned out to be integral elements of hepatocytic reticular monooxygenase complexes which also contain NADPH-dependent cytochrome P450 reductase (CPR). Later investigations indicated the possibility of the participation in electron transport for reticular CYP isoenzymes, alternative NADH-dependent reticular system composed of cytochrome b5 reductase (CBR) and cytochrome b5. The demonstration of the activity of some CYP superfamily isoenzymes not only in hepatocytes but also in many other cells of the human organism, numerous plant and animal tissues and even in cells of fungi, protists and prokaryotes has contributed to the significantly increased understanding of the role of CYP in biological systems. In addition, some CYP isoenzymes were found to be characteristic for the inner mitochondrial membrane monooxygenase complexes which contain NADPH-dependent adrenodoxin reductase (AR) and adrenodoxin (Ad), which is identical with ferredoxin-1 (Fd-1) and hepatoredoxin (Hd). PMID:18646549

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

    PubMed Central

    Honkakoski, P; Negishi, M

    2000-01-01

    Members of the nuclear-receptor superfamily mediate crucial physiological functions by regulating the synthesis of their target genes. Nuclear receptors are usually activated by ligand binding. Cytochrome P450 (CYP) isoforms often catalyse both formation and degradation of these ligands. CYPs also metabolize many exogenous compounds, some of which may act as activators of nuclear receptors and disruptors of endocrine and cellular homoeostasis. This review summarizes recent findings that indicate that major classes of CYP genes are selectively regulated by certain ligand-activated nuclear receptors, thus creating tightly controlled networks. PMID:10749660

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

    PubMed

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

    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

  14. 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

  15. Resveratrol Is a Selective Human Cytochrome P450 1A1 Inhibitor

    Microsoft Academic Search

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

    1999-01-01

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

  16. Isolation and characterization of Streptomyces griseolus deletion mutants affected in cytochrome P-450-mediated herbicide metabolism

    Microsoft Academic Search

    Patricia A. Harder; Daniel P. O'Keefe; James A. Romesser; Kenneth J. Leto; Charles A. Omer

    1991-01-01

    Metabolism of sulfonylurea herbicides by Streptomyces griseolus ATCC 11796 is carried out via two cytochromes P-450, P-450SU1 and P-450SU2. Mutants of S. griseolus, selected by their reduced ability to metabolize a fluorescent sulfonylurea, do not synthesize cytochrome P-450SU1 when grown in the presence of sulfonylureas. Genetic evidence indicated that this phenotype was the result of a deletion of > 15

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

    PubMed Central

    2012-01-01

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

  18. Cytochrome P450-mediated metabolism of xanthotoxin by Papilio multicaudatus.

    PubMed

    Mao, Wenfu; Berhow, Mark A; Zangerl, Arthur R; McGovern, Jennifer; Berenbaum, May R

    2006-03-01

    Within the genus Papilio, the P. glaucus group contains the most polyphagous Papilio species within the Papilionidae. The majority of Papilio species are associated with hostplants in the families Rutaceae and Apiaceae, and characterizing most are secondary metabolites called furanocoumarins. Recent phylogenetic studies suggest that furanocoumarin metabolism is an ancestral trait, with the glaucus group derived from ancestors associated with furanocoumarin-containing Rutaceae. In this study, we examined this relationship by conducting a gravimetric analysis of growth that used various concentrations of the furanocoumarin xanthotoxin. Papilio multicaudatus, the putative ancestor of the glaucus group, includes at least one furanocoumarin-containing rutaceous species among its hostplants; this species can consume leaf tissue containing up to 0.3% xanthotoxin with no detectable effect on relative growth rate, relative consumption rate, or efficiency of conversion of ingested food. As is the case for other Papilio species, xanthotoxin metabolism is mediated by cytochrome P450 monooxygenases (P450s). Ingestion of xanthotoxin by ultimate instar P. multicaudatus increases activity up to 30-fold in a dose-dependent fashion. Midguts of induced larvae can also effectively metabolize six other furanocoumarins, including both linear (bergapten, isopimpinellin, imperatorin) and angular (angelicin, sphondin) forms. A metabolite of xanthotoxin in the frass from xanthotoxin-treated larvae, identified as 6-(7-hydroxy-8-methoxycoumaryl)-acetic acid by MS-MS and NMR analyses, is identical to one from the frass of P. polyxenes. The occurrence of this metabolite in two swallowtails and the presence of a second metabolite of xanthotoxin, 6-(7-hydroxy-8-methoxycoumaryl)-hydroxyethanol in the frass of both P. polyxenes and Depressaria pastinacella are consistent with the suggestion that lepidopterans share as the first step of xanthotoxin metabolism the P450-mediated epoxidation of the furan ring 2'-3' double bond. PMID:16572296

  19. Determinants of Cytochrome P450 2C8 Substrate Binding

    PubMed Central

    Schoch, Guillaume A.; Yano, Jason K.; Sansen, Stefaan; Dansette, Patrick M.; Stout, C. David; Johnson, Eric F.

    2008-01-01

    Although a crystal structure and a pharmacophore model are available for cytochrome P450 2C8, the role of protein flexibility and specific ligand-protein interactions that govern substrate binding are poorly understood. X-ray crystal structures of P450 2C8 complexed with montelukast (2.8 Å), troglitazone (2.7 Å), felodipine (2.3 Å), and 9-cis-retinoic acid (2.6 Å) were determined to examine ligand-protein interactions for these chemically diverse compounds. Montelukast is a relatively large anionic inhibitor that exhibits a tripartite structure and complements the size and shape of the active-site cavity. The inhibitor troglitazone occupies the upper portion of the active-site cavity, leaving a substantial part of the cavity unoccupied. The smaller neutral felodipine molecule is sequestered with its dichlorophenyl group positioned close to the heme iron, and water molecules fill the distal portion of the cavity. The structure of the 9-cis-retinoic acid complex reveals that two substrate molecules bind simultaneously in the active site of P450 2C8. A second molecule of 9-cis-retinoic acid is located above the proximal molecule and can restrain the position of the latter for more efficient oxygenation. Solution binding studies do not discriminate between cooperative and noncooperative models for multiple substrate binding. The complexes with structurally distinct ligands further demonstrate the conformational adaptability of active site-constituting residues, especially Arg-241, that can reorient in the active-site cavity to stabilize a negatively charged functional group and define two spatially distinct binding sites for anionic moieties of substrates. PMID:18413310

  20. High-Level Expression in Escherichia coli of Enzymatically Active Fusion Proteins Containing the Domains of Mammalian Cytochromes P450 and NADPH P450 Reductase Flavoprotein

    Microsoft Academic Search

    Charles W. Fisher; Manjunath S. Shet; Deborah L. Caudle; Cheryl A. Martin-Wixtrom; Ronald W. Estabrook

    1992-01-01

    This report describes the properties of two mammalian cytochromes P450 that have been expressed at high levels in Escherichia coli as enzymatically active fusion proteins containing the flavoprotein domain of rat NADPH-cytochrome P450 reductase (EC 1.6.2.4). Fusion proteins were prepared by engineering the cDNAs for the steroid-metabolizing bovine adrenal P450 17A with the cDNA for rat liver NADPH-P450 reductase with

  1. 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.

  2. Cytochrome P450 eicosanoids in hypertension and renal disease

    PubMed Central

    Fan, Fan; Muroya, Yoshikazu; Roman, Richard J.

    2015-01-01

    Purpose of review Cytochrome (CYP) P450 metabolites of arachidonic acid, 20-hydroxyeicosatetraenoic acid (20-HETE) and epoxyeicosatrienoic acids (EETs) contribute to the regulation of renal tubular and vascular function. This review highlights the results of the recent genetic studies in humans and rodent models, indicating that these eicosanoids participate in the control of blood pressure (BP), chronic kidney disease (CKD), renal ischemia–reperfusion injury (IRI) and polycystic kidney disease (PKD). Recent findings Endogenous 20-HETE has been reported to play an essential role in the myogenic and tubuloglomerular feedback responses in the afferent arteriole, and a deficiency of 20-HETE contributes to the development of hypertension and renal injury in Dahl S rats. Mutations in CYP4A11 and CYP4F2 have been linked to elevated BP in humans. EETs have been shown to regulate epithelial sodium channel in the collecting duct, lower BP and have renoprotective properties. 20-HETE also opposes the development of CKD and IRI, and may play a role in PKD. Summary These studies indicate that CYP P450 metabolites of arachidonic acid play an important role in the control of BP, CKD, AKI and PKD. Drugs targeting these pathways could be useful in the treatment of IRI and CKD. PMID:25427230

  3. Ab initio dynamics of the cytochrome P450 hydroxylation reaction

    NASA Astrophysics Data System (ADS)

    Elenewski, Justin E.; Hackett, John C.

    2015-02-01

    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.

  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. 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.

  6. 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

  7. Sterol 14?-Demethylase Cytochrome P450 (CYP51), a P450 in all Biological Kingdoms

    PubMed Central

    Lepesheva, Galina I.; Waterman, Michael R.

    2007-01-01

    Summary The CYP51 family is an intriguing subject for fundamental P450 structure/function studies and is also an important clinical drug target. This review updates information on the variety of the CYP51 family members, including their physiological roles, natural substrates and substrate preferences, and catalytic properties in vitro. We present experimental support for the notion that specific conserved regions in the P450 sequences represent a CYP51 signature. Two possible roles of CYP51 in P450 evolution are discussed and the major approaches for CYP51 inhibition are summarized. PMID:16963187

  8. Validated assays for human cytochrome P450 activities.

    PubMed

    Walsky, Robert L; Obach, R Scott

    2004-06-01

    The measurement of the effect of new chemical entities on human cytochrome P450 marker activities using in vitro experimentation represents an important experimental approach in drug development. In vitro drug interaction data can be used in guiding the design of clinical drug interaction studies, or, when no effect is observed in vitro, the data can be used in place of an in vivo study to claim that no interaction will occur in vivo. To make such a claim, it must be assured that the in vitro experiments are performed with absolute confidence in the methods used and data obtained. To meet this need, 12 semiautomated assays for human P450 marker substrate activities have been developed and validated using approaches described in the GLP (good laboratory practices) as per the code of U.S. Federal Regulations. The assays that were validated are: phenacetin O-deethylase (CYP1A2), coumarin 7-hydroxylase (CYP2A6), bupropion hydroxylase (CYP2B6), amodiaquine N-deethylase (CYP2C8), diclofenac 4'-hydroxylase and tolbutamide methylhydroxylase (CYP2C9), (S)-mephenytoin 4'-hydroxylase (CYP2C19), dextromethorphan O-demethylase (CYP2D6), chlorzoxazone 6-hydroxylase (CYP2E1), felodipine dehydrogenase, testosterone 6 beta-hydroxylase, and midazolam 1'-hydroxylase (CYP3A4 and CYP3A5). High-pressure liquid chromatography-tandem mass spectrometry, using stable isotope-labeled internal standards, has been applied as the analytical method. This analytical approach, through its high sensitivity and selectivity, has permitted the use of very low incubation concentrations of microsomal protein (0.01-0.2 mg/ml). Analytical assay accuracy and precision values were excellent. Enzyme kinetic and inhibition parameters obtained using these methods demonstrated high precision and were within the range of values previously reported in the scientific literature. These methods should prove useful in the routine assessments of the potential for new drug candidates to elicit pharmacokinetic drug interactions via inhibition of cytochrome P450 activities. PMID:15155557

  9. P450-aromatase activity and expression in human testicular tissues with severe spermatogenic failure.

    PubMed

    Lardone, M C; Castillo, P; Valdevenito, R; Ebensperger, M; Ronco, A M; Pommer, R; Piottante, A; Castro, A

    2010-08-01

    There is evidence that impaired spermatogenesis is associated with an imbalance in the oestradiol/testosterone ratio and with Leydig cell (LC) dysfunction. In testis, P450-aromatase, encoded by CYP19, is responsible for the conversion of testosterone to oestradiol. The aims of this study were to quantify CYP19 mRNA expression, aromatase activity and protein localization, and to measure the oestradiol to testosterone ratio in testicular tissues of men with spermatogenic impairment. Twenty-four men with complete Sertoli cell-only syndrome (SCOS), 14 with focal SCOS, 14 with maturation arrest (MA), 8 with mixed atrophy and 30 controls with normal spermatogenesis were subjected to testicular biopsy. All subjects underwent a physical examination, cytogenetic and serum hormonal studies. Testicular CYP19 mRNA was quantified using real time RT-PCR. Testicular aromatase activity was measured using the (3)H(2)0 assay and protein expression was evaluated using immunohistochemistry. In cases, serum testosterone and oestradiol were normal, but the testosterone/LH ratio was lower compared with controls (p < 0.05). Aromatase was localized in the Leydig, Sertoli and germ cells of all tissues, although stronger intensity was observed in LC. Aromatase mRNA and activity were not altered in cases and correlated positively with LC number (r = 0.516 and r = 0.369; p < 0.008). The intratesticular oestradiol/testosterone ratio was elevated (p = 0.005) in complete SCOS patients compared with controls. In conclusion, testicular aromatase seems to be normal in most subjects with impaired spermatogenesis. However, an altered intratesticular oestradiol/testosterone ratio in some patients with complete SCOS suggests that aromatase is increased, which might contribute to Leydig cell dysfunction. PMID:19906189

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

    Microsoft Academic Search

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

    2000-01-01

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

  11. Analysis of cytochrome P-450 side-chain cleavage gene promoter activation during trophoblast cell differentiation

    Microsoft Academic Search

    Toshiya Yamamoto; Belinda M. Chapman; Jeffrey W. Clemens; JoAnne S. Richards; Michael J. Soares

    1995-01-01

    Trophoblast giant cell differentiation is accompanied by transcriptional activation of the cytochrome P-450 side-chain cleavage (P450scc) gene. The Rcho-1 trophoblast cell line has the capacity to differentiate along the trophoblast giant cell lineage and has been used to study trophoblast-specific P450scc gene expression. In this report, P450scc gene promoter activities in trophoblast cells have been mapped and the involvement of

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

    Microsoft Academic Search

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

    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

  13. Comparative structural and immunochemical characterization of recombinant and natural cytochrome p450scc (CYPXIAI).

    PubMed

    Lepesheva, G I; Usanov, S A

    1998-02-01

    Optimization of the conditions for heterologous expression of recombinant cytochrome P450scc in E. coli provided an expression level of about 420 nmoles of cytochrome P450scc per liter of bacterial culture. A new procedure for purification of recombinant protein in substrate-bound high-spin and substrate-free low-spin form is described. Highly purified electrophoretically homogeneous recombinant cytochrome P450scc contains 12.3 and 16.7 nmoles heme per mg protein for substrate-free and substrate-bound forms, respectively. The recombinant and natural cytochrome P450scc from bovine adrenocortical mitochondria were compared functionally and immunochemically. The dissociation constants for the complexes of cytochrome P450scc with cholesterol and adrenodoxin, the efficiency of enzymatic reduction in the reconstituted system (NADPH--adrenodoxin reductase--adrenodoxin), and cholesterol side-chain cleavage activity were determined. It was found that limited proteolysis of the recombinant cytochrome P450scc with trypsin forms two main fragments which are electrophoretically and immunochemically identical with the fragments F1 (29.8 kD) and F2 (26.6 kD) formed during proteolysis of bovine adrenocortical cytochrome P450scc. The quantitative values of the studied parameters are practically identical in natural and substrate-bound recombinant cytochrome P450scc, while there were great differences between substrate-bound and substrate-free forms of recombinant cytochrome P450scc both of functional (decrease of cholesterol side-chain cleavage activity, efficiency of enzymatic reduction in the reconstituted system, and affinity to adrenodoxin for substrate-free cytochrome P450scc) as well as structural (increase in accessibility to exogenous and endogenous proteolysis) character. The identity of the folding process for recombinant and natural proteins as well as the nature of a stabilizing and activating effect of cholesterol on cytochrome P450scc is discussed. PMID:9526119

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

    PubMed Central

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

    2009-01-01

    In a previous study from our lab (Drug Metab. Disp. (2006) 34, 660–666), we found several limitations with published methods (cholate gel filtration and cholate dialysis) for the incorporation of cytochromes P450 and P450 reductase into phospholipid vesicles. We found that a significant proportion of reductase was not incorporated in the vesicles when the amount of reductase was equal to or greater than that of CYP2B4 in the systems reconstituted with phosphatidylcholine. Furthermore, implementation of these methods compromised the ability of the CYP2B4 to form a ferrous carbon monoxy complex. In the current study, a comparison of results using the detergent-dialysis method with five similar detergents having the “bile salt” ring structure finds that glycocholate results in the greatest incorporation of reductase and the least loss in carbon monoxy ferrous CYP2B4 complex. The method is further improved by using Biobeads SM-2 to remove detergent instead of the lengthy dialysis procedure or the size exclusion chromatography which significantly dilutes the protein and lipid concentrations of the preparation. The method is shown to be applicable over a range of lipid to CYP2B4 ratios; and by employing assay methods for total lipid, reductase, and CYP2B4, this improved reconstitution method resulted in increased incorporation efficiencies while minimizing the protein degradation inherent with these procedures. PMID:18048487

  15. Expression, Purification, and Biochemical Characterization of A Human Cytochrome P450 CYP2D6NADPH Cytochrome P450 Reductase Fusion Protein

    Microsoft Academic Search

    Yusuf Y. Deeni; Mark J. I. Paine; Andrew D. Ayrton; Stephen E. Clarke; Richard Chenery; C. Roland Wolf

    2001-01-01

    Cytochrome P450 CYP2D6 metabolizes a wide range of pharmaceutical compounds. A CYP2D6 fusion enzyme (CYP2D6F), containing an amino-terminal human CYP2D6 sequence and a carboxyterminal human NADPH-cytochrome P450 oxidoreductase (CPR) moiety, was constructed. High levels of expression were achieved in Escherichia coli (60–100 nmol\\/liter) and the enzyme was catalytically active with optimal activities achieved in the presence of the antioxidant, GSH.

  16. Taurine Modulates Catalytic Activity of Cytochrome P450 3A4.

    PubMed

    Shumyantseva, V V; Makhova, A A; Bulko, T V; Bernhardt, R; Kuzikov, A V; Shich, E V; Kukes, V G; Archakov, A I

    2015-03-01

    The influence of the biologically active compound taurine on the stability and catalytic properties of the hemoprotein cytochrome P450 3A4 has been investigated. The catalytic properties were analyzed by electrochemical methods (cyclic and square-wave voltammetry) using cytochrome P450 3A4 immobilized on the electrode. Taurine at concentrations in the range 10-70 µM stimulated the electrochemical reduction of cytochrome P450 3A4, and the reduction was the highest (115 ± 3%) in the presence of 50 µM taurine. Taurine pronouncedly attenuated the itraconazol-caused inhibition of the P450 isoenzyme P450 3A4. Taurine protected cytochrome P450 3A4 due to stabilizing it during electrolysis at controlled voltage in the presence of erythromycin as a substrate. This protection was manifested by an increase in the amount of the "residual" reduced form of the hemoprotein (52 ± 5 and 71 ± 8%, respectively). PMID:25761690

  17. An inducible NADPH-cytochrome P450 reductase from Picrorhiza kurrooa - an imperative redox partner of cytochrome P450 enzymes.

    PubMed

    Bhat, Wajid Waheed; Rana, Satiander; Dhar, Niha; Razdan, Sumeer; Pandith, Shahzad A; Vishwakarma, Ram; Lattoo, Surrinder K

    2014-06-01

    Picrorhiza kurrooa synthesizes a large array of pharmacologically important monoterpenoid iridoid glycosides called picrosides. Although chemical profile and pharmacological activities of P. kurrooa have been extensively studied, limited attempts have been made to decipher the biosynthetic route and to identify the key regulatory genes involved in picroside biosynthesis. In the present study, NADPH-cytochrome P450 reductase, a key enzyme involved in electron transfer to cytochrome P450s was identified from P. kurrooa. The full length cDNA (2679 bp) contained an open reading frame of 2133 bp, corresponding to 710 amino acids. PkCPR was heterologously expressed in Escherichia coli and the kinetic parameters of the recombinant enzyme were determined. Specific activity, V max and K m of PkCPR were found to be 5.8?±?0.05 ?mol min(-1) mg(-1), 8.1?±?0.12 ?mol min(-1) mg(-1) and 7.8 ?M, respectively. PkCPR was found to be spatially regulated at transcript level, being maximally expressed in leaf tissues. Altitude was found to have a positive effect on the picroside concentration and the picroside content positively correlated with the PkCPR transcript levels in samples collected at varied altitudes. Further, transcript profiling under methyl jasmonate, salicylic acid, 2,4-dicholorophenoxy acetic acid and UV-B elicitations displayed differential transcriptional regulation of PkCPR that fully corroborated with the identified cis-elements within the PkCPR promoter. Expression of PkCPR was inducible by UV-B and phytohormone elicitation, indicating that the PkCPR is possibly related to defence reactions, including biosynthesis of secondary metabolites. Present study is so far the only report of identification and functional characterization of CPR ortholog from P. kurrooa. PMID:24522789

  18. 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...

  19. Bacterial Cytochrome P450 System Catabolizing the Fusarium Toxin Deoxynivalenol

    PubMed Central

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

    2013-01-01

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

  20. Ethynyl and Propynylpyrene Inhibitors of Cytochrome P450.

    PubMed

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

    2010-04-01

    The single-crystal X-ray structures and in vivo activities of three aryl acetylenic inhibitors of cytochromes P450 1A1, 1A2, 2A6, and 2B1 have been determined and are reported herein. These are 1-ethynylpyrene, 1-propy-nylpyrene, and 4-propynylpyrene. To investigate electronic influences on the mechanism of enzyme inhibition, the experimental electron density distribution of 1-ethynylpy-rene has been determined using low-temperature X-ray diffraction measurements, and the resulting net atomic charges compared with various theoretical calculations. A total of 82,390 reflections were measured with Mo K? radiation to a (sin?/?)(max) = 0.985 Å(-1). Averaging symmetry equivalent reflections yielded 8,889 unique reflections. A least squares refinement procedure was used in which multipole parameters were added to describe the distortions of the atomic electron distributions from spherical symmetry. A map of the model electron density distribution of 1-ethynylpyrene was obtained. Net atomic charges calculated from refined monopole population parameters yielded charges that showed that the terminal acetylenic carbon atom (C18) is more negative than the internal carbon (C17). Net atomic charges calculated by ab initio, density functional theory, and semi-empirical methods are consistent with this trend suggesting that the terminal acetylenic carbon atom is more likely to be the site of oxidation. This is consistent with the inhibition mechanism pathway that results in the formation of a reactive ketene intermediate. This is also consistent with assay results that determined that 1-ethynylpyrene acts as a mechanism-based inhibitor of P450s 1A1 and 1A2 and as a reversible inhibitor of P450 2B1. Crystallographic data: 1-ethynylpyrene, C(18)H(10), P2(1)/c, a = 14.571(2) Å, b = 3.9094(5) Å, c = 20.242(3) Å, ? = 105.042(2)°, V = 1,113.5(2) Å(3); 1-propynylpyrene, C(19)H(12), P2(1)/n, a = 8.970(2) Å, b = 10.136(1) Å, c = 14.080(3) Å, ? = 99.77(2)°, V = 1,261.5(4) Å(3); 4-propynylpyrene, C(19)H(12), Pbca, a = 9.904(1) Å, b = 13.174(2) Å, c = 19.401(1) Å, V = 2,531.4(5) Å(3). PMID:20473363

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

    SciTech Connect

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

    2011-09-09

    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.

  2. Transfer learning for cytochrome P450 isozyme selectivity prediction.

    PubMed

    Teramoto, Reiji; Kato, Tsuyoshi

    2011-08-01

    In the drug discovery process, the metabolic fate of drugs is crucially important to prevent drug-drug interactions. Therefore, P450 isozyme selectivity prediction is an important task for screening drugs of appropriate metabolism profiles. Recently, large-scale activity data of five P450 isozymes (CYP1A2 CYP2C9, CYP3A4, CYP2D6, and CYP2C19) have been obtained using quantitative high-throughput screening with a bioluminescence assay. Although some isozymes share similar selectivities, conventional supervised learning algorithms independently learn a prediction model from each P450 isozyme. They are unable to exploit the other P450 isozyme activity data to improve the predictive performance of each P450 isozyme's selectivity. To address this issue, we apply transfer learning that uses activity data of the other isozymes to learn a prediction model from multiple P450 isozymes. After using the large-scale P450 isozyme selectivity dataset for five P450 isozymes, we evaluate the model's predictive performance. Experimental results show that, overall, our algorithm outperforms conventional supervised learning algorithms such as support vector machine (SVM), Weighted k-nearest neighbor classifier, Bagging, Adaboost, and latent semantic indexing (LSI). Moreover, our results show that the predictive performance of our algorithm is improved by exploiting the multiple P450 isozyme activity data in the learning process. Our algorithm can be an effective tool for P450 selectivity prediction for new chemical entities using multiple P450 isozyme activity data. PMID:21776607

  3. Presence of functional cytochrome P-450 on isolated rat hepatocyte plasma membrane.

    PubMed

    Loeper, J; Descatoire, V; Maurice, M; Beaune, P; Feldmann, G; Larrey, D; Pessayre, D

    1990-05-01

    Antibodies against cytochrome P-450 are found in some children with autoimmune hepatitis (antiliver/kidney microsome 1) and in patients with ticrynafen hepatitis (antiliver/kidney microsome 2). For an immune reaction against cytochrome P-450 to possibly destroy the hepatocytes, one must assume that cytochrome P-450 is present on the plasma membrane surface of hepatocytes. In a first series of experiments, plasma membranes were prepared with a technique based on the electrostatic attachment of isolated hepatocytes to polyethyleneimine-coated beads. After vortexing, beads were coated with a very pure plasma membrane fraction. Microsomal contamination, judged from the specific activities of glucose-6-phosphatase or NADH-cytochrome c reductase, was less than 1%. Nevertheless, the specific content (per milligram of protein) of CO-binding cytochrome P-450 was 20% of that in microsomes; the specific benzo(a)pyrene hydroxylase activity was 25%, and ethoxycoumarin deethylase 11%. Immunoblots showed the presence of cytochromes P-450 UT-A, UT-H, PB-B, ISF-G and PCN-E, the last three isoenzymes being inducible by, respectively, phenobarbital, 3-methylcholanthrene and dexamethasone. In a second series of experiments, nonpermeabilized isolated hepatocytes from untreated rats were incubated with anticytochrome P-450 antibodies. Immunofluorescence and immunoperoxidase staining confirmed the presence of cytochromes P-450 UT-A, PB-B and ISF-G on the membrane. In a last series of experiments, human antiliver-kidney microsomal 1 antibodies were found to react specifically with rat liver plasma membrane cytochrome P-450 UT-H (IID subfamily). We conclude that several cytochrome P-450 isoenzymes are present, active and inducible on the plasma membrane surface of hepatocytes. It is therefore conceivable that immunization against plasma membrane cytochrome P-450 might lead to the immunological destruction of hepatocytes in some patients. PMID:2112112

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

    PubMed

    Rendic, Slobodan Petar; Guengerich, F Peter

    2014-12-01

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

  5. Redox-dependent dynamics in cytochrome P450cama

    PubMed Central

    Pochapsky, Susan Sondej; Dang, Marina; OuYang, Bo; Simorellis, Alana K.; Pochapsky, Thomas C.

    2009-01-01

    Local protein backbone dynamics of the camphor hydroxylase cytochrome P450cam (CYP101) depend upon the oxidation and ligation state of the heme iron. 1H,15N correlation nuclear magnetic resonance experiments were used to compare backbone dynamics of oxidized and reduced forms of this 414-residue metalloenzyme via hydrogen-deuterium exchange kinetics (H/D exchange) and 15N relaxation measurements, and these results are compared with previously published results obtained by H/D exchange mass spectrometry. In general, the reduced enzyme shows lower amplitude motions of secondary structural features than the oxidized enzyme on all of the time scales accessible to these experiments, and these differences are more pronounced in regions of the enzyme involved in substrate access to the active site (B? helix, ?3 and ?5 sheets) and binding of putidaredoxin (C and L helices) the iron-sulfur protein that acts as effector and reductant of CYP101 in vivo. These results are interpreted in terms of local structural effects of changes in the heme oxidation state, and relevance of the observed effects to the enzyme mechanism is discussed. PMID:19366254

  6. Effect of natamycin on cytochrome P450 enzymes in rats.

    PubMed

    Martínez, María Aránzazu; Martínez-Larrañaga, María Rosa; Castellano, Victor; Martínez, Marta; Ares, Irma; Romero, Alejandro; Anadón, Arturo

    2013-12-01

    Natamycin is a polyene macrolide antibiotic widely used in the food industry as a feed additive to prevent mold contamination of foods. There are many contradictory results on the genotoxic effects of macrolides which could suggest a potential risk for humans. In the present study, the effects of natamycin on the activities of some drug metabolizing enzymes in rat liver microsomes were determined in vivo. Rats were treated orally with natamycin at doses of 0.3, 1, 3 and 10 mg/kg body weight (bw)/day for 6 days. Determinations of cytochrome P450 (CYP) enzyme activities were carried out in hepatic microsomes isolated from rats treated. The activities of CYP2E1, CYP1A1/2 CYP2B1/2 and CYP4A1/2 enzymes significantly decreased after treatment with 1, 3 and 10 mg/kg bw/day, in a dose-dependent manner as compared to control. This effect was not observed after natamycin treatment at dose of 0.3 mg/kg bw/day. Our results suggest that natamycin may not potentiate the toxicity of many xenobiotics via metabolic activation and/or accumulation of reactive metabolites but also might affect the clearance of other xenobiotics detoxified by the studied CYP enzymes. PMID:24001439

  7. The cytochrome P450 complement (CYPome) of Mycosphaerella graminicola.

    PubMed

    Newsome, Alun W; Nelson, David; Corran, Andrew; Kelly, Steven L; Kelly, Diane E

    2013-01-01

    Mycosphaerella graminicola is a key fungal pathogen of wheat and a major target for azole fungicides, many of whose central mode of action is through inhibition of cytochrome P450 51 (lanosterol 14?-demethylase) in the ergosterol biosynthetic pathway. The range of activities of other fungal CYPs is thought to be a reflection of the differences between different organisms and their range of secondary metabolic pathways as a response to their niche environments, for example, in the production of mycotoxins. The present study collates information from a range of databases, to classify the CYPs found in M. graminicola and assign them an internationally recognized nomenclature, which, when referenced to the recent publication of the JGI version 2.0 genome model, creates a current, robust model for the CYP complement (CYPome) of M. graminicola. These CYPome data, which examined 82 CYPs and one pseudo-gene, may be utilized not only to further characterize and describe the physiology of the organism but also to enhance our understanding of CYP function and diversity. PMID:23586992

  8. Immunotoxicology and expression of human cytochrome P450 in microorganisms.

    PubMed

    Beaune, P; Bourdi, M; Belloc, C; Gautier, J C; Guengerich, F P; Valadon, P

    1993-10-01

    Drug-induced hepatitis can be caused by an abnormal immunological response. In the case of tienilic acid- and dihydralazine-induced hepatitis, we postulated a scheme in which a P450 produced a reactive metabolite (step 1); this reactive metabolite bound to the P450 producing it (step 2) leading to a neoantigen triggering the immune response (step 3); the autoantibodies produced during the immune response recognized the P450 producing the reactive metabolite (step 4). The use of microorganisms (yeast or bacteria) expressing cloned human P450 helped in proving some steps of this postulated scheme, particularly steps 1 and 4. PMID:8236281

  9. Homology modeling of plant cytochrome P450s

    Microsoft Academic Search

    Sanjeewa Rupasinghe; Mary A. Schuler

    2006-01-01

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

  10. 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

  11. Human Cytochrome P450 2E1: Functional Comparison to Cytochrome 2A13 and 2A6

    E-print Network

    Blevins, Melanie

    2008-05-05

    ) The structre of human cytochrome P450 29 complexd with flurbiprofen at 2.0-A resolution. J Biol Che 279, 35630- 35637 39 Wiliams, P. A., Cosme, J., Ward, A., ngove, H. C., Matk Vinkovic, D. and Jhoti, H. (203) rystal structre of human cytochrome P450 2C9...

  12. Expression and Characterization of Truncated Recombinant Human Cytochrome P450 2J2

    PubMed Central

    Park, Hyoung-Goo; Lim, Young-Ran; Han, Songhee

    2014-01-01

    The human cytochrome P450 2J2 catalyzes an epoxygenase reaction to oxidize various fatty acids including arachidonic acid. In this study, three recombinant enzyme constructs of P450 2J2 were heterologously expressed in Escherichia coli and their P450 proteins were successfully purified using a Ni2+-NTA affinity column. Deletion of 34 amino acid residues in N-terminus of P450 2J2 enzyme (2J2-D) produced the soluble enzyme located in the cytosolic fraction. The enzymatic analysis of this truncated protein indicated the typical spectral characteristics and functional properties of P450 2J2 enzyme. P450 2J2-D enzymes from soluble fraction catalyzed the oxidation reaction of terfenadine to the hydroxylated product. However, P450 2J2-D enzymes from membrane fraction did not support the P450 oxidation reaction although it displayed the characteristic CO-binding spectrum of P450. Our finding of these features in the N-terminal modified P450 2J2 enzyme could help understand the biological functions and the metabolic roles of P450 2J2 enzyme and make the crystallographic analysis of the P450 2J2 structure feasible for future studies. PMID:24795797

  13. Mechanisms that Regulate Production of Reactive Oxygen Species by Cytochrome P450

    SciTech Connect

    Zangar, Richard C.; Davydov, Dmitri R.; Verma, Seema

    2004-09-15

    Mammalian cytochromes P450 (P450) are a family of heme-thiolate enzymes involved in the oxidative metabolism of a variety of endogenous and exogenous lipophilic compounds. Poor coupling of the P450 catalytic cycle results in continuous production of reactive oxygen species (ROS), which affect signaling pathways and other cellular functions. P450 generation of ROS is tightly controlled by regulation of gene transcription, as well as by modulation of interactions between protein constituents of the monooxygenase that affects its activity, coupling and stability. Malfunction of these mechanisms may result in a burst of ROS production, which can cause lipid peroxidation and oxidative stress. In turn, oxidative stress downregulates P450 levels by a variety of feedback mechanisms. This review provides an overview of recent advances in our understanding of these feedback mechanisms that serve to limit P450 production of ROS. Some of the more likely physiological and cellular effects of P450 generation of ROS are also discussed.

  14. Cytochrome P450 and Steroid Hydroxylase Activity in Mouse Olfactory and Vomeronasal Mucosa

    Microsoft Academic Search

    Jun Gu; Carol Dudley; Ting Su; David C. Spink; Qing-Yu Zhang; Robert L. Moss; Xinxin Ding

    1999-01-01

    The aims of this study are to identify the sex steroid-metabolizing cytochrome P450 enzymes of the vomeronasal organ (VNO) and to determine the activities of VNO microsomes to metabolize estradiol, progesterone, and testosterone. Several P450 isoforms, including CYP1A2, CYP2A, CYP2B, CYP2C, CYP2G1, and CYP3A, NADPH P450-reductase, and microsomal epoxide hydrolase were detected in mouse VNO, although their expression levels were

  15. Cloning and characterisation of cytochrome P450 genes from barley (Hordeum vulgare)

    Microsoft Academic Search

    Linh Nguyen; Angela Delves; Peter C Bundock; Timothy A Holton

    2001-01-01

    Plant P450s belong to the cytochrome group that are membrane-bound enzymes, usually found in plant endoplasmic reticulum. This gene family is diverse in structure and function, which enables these enzymes to participate in numerous biosynthetic and degradative pathways. In plants, P450s are known to play important roles in production of hormones, pigments, oils, and defensive compounds. P450s are also involved

  16. Formation of nitric oxide by cytochrome P450-catalyzed oxidation of aromatic amidoximes.

    PubMed

    Andronik-Lion, V; Boucher, J L; Delaforge, M; Henry, Y; Mansuy, D

    1992-05-29

    Rat liver microsomes catalyze the oxidation of para-hexyloxy-benzamidoxime 1 to the corresponding arylamide 2 and NO2-, by NADPH and O2. Involvement of cytochromes P450 as catalysts of this reaction was shown by the strong inhibitory effects of CO and miconazole and the spectacular increase of the activity upon treatment of rats with dexamethasone, a specific inducer of cytochromes P450 of the 3A subfamily. Formation of NO during oxidation of 1 was shown by detection of the formation of cytochrome P450- and cytochrome P420-Fe(II)-NO complexes by visible and EPR spectroscopy. The formation of these complexes should be responsible, at least in part, for the fast decrease of the rate of microsomal oxidation of 1 with time. These results suggest that exogenous compounds containing amidine or amidoxime functions could act as precursors of NO in vivo after in situ oxidation by cytochromes P450. PMID:1599484

  17. Thiophene derivatives as new mechanism-based inhibitors of cytochromes P-450: inactivation of yeast-expressed human liver cytochrome P-450 2C9 by tienilic acid.

    PubMed

    López-Garcia, M P; Dansette, P M; Mansuy, D

    1994-01-11

    Oxidation of tienilic acid (TA) by microsomes of yeast expressing two closely related human liver cytochrome P-450s (P450), P450 2C9 and 2C10, led to catalysis-dependent loss of activity of these P450s. Under identical conditions, oxidation of a tienilic acid isomer (TAI) failed to give any P450 inactivation. The loss of P450 activity during TA oxidation was concomitant with product (5-hydroxytienilic acid, 5-OHTA) formation, showed pseudo-first-order and saturation kinetics, and was inhibited by an alternative substrate, tolbutamide. Covalent binding of TA metabolites to microsomal proteins occurred in parallel with enzyme inactivation and was partially inhibited by the presence of glutathione in the reaction medium. However, glutathione did not protect P450 enzyme from inactivation. Thus, TA exhibited all of the characteristics of a mechanism-based inactivator for P450 2C9 and 2C10 enzymes. The following kinetic parameters were determined in the case of P450 2C10: t1/2,max = 3.4 min, k(inact) = 3.6 10(-3) s-1, KI = 4.3 microM, k(inact)/KI = 813 L mol-1 s-1, and partition ratio = 11.6. Moreover, a specific covalent binding of 0.9 mol of TA metabolite per mole of P450 2C10 was found to occur before the complete loss of enzyme activity (in incubations performed in the presence of glutathione). A plausible mechanism for P450 2C10 (2C9) inactivation during TA oxidation is proposed. It involves the intermediate formation of an electrophilic thiophene sulfoxide, which may react at position 5 of its thiophene ring either with H2O to give 5-OHTA or with a nucleophilic group of an amino acid residue of the P450 active site, which results in its covalent binding to P450 protein. This alkylation and inactivation of P450 2C9 (2C10) by TA could be a starting point for the appearance of anti-P450 2C antibodies detected in patients treated with TA and suffering from immunoallergic hepatitis. PMID:8286335

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

    PubMed

    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

  19. 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

  20. Cytochrome P450IA mRNA expression in feral Hudson River tomcod.

    PubMed

    Kreamer, G L; Squibb, K; Gioeli, D; Garte, S J; Wirgin, I

    1991-06-01

    We sought to determine if levels of cytochrome P450IA gene expression are environmentally induced in feral populations of Hudson River tomcod, a cancer prone fish, and whether laboratory exposure of tomcod to artificially spiked and naturally contaminated Hudson sediments can elicit a significant response. Using Northern blot analysis, we found levels of P450IA mRNA in tomcod collected from two Hudson River sites higher than those in tomcod from a river in Maine. Depuration of environmentally induced Hudson tomcod P450IA mRNA was rapid, with an initial detectable decline in P450 gene expression by 8 hr and basal levels reached by 5 days. Intraperitoneal injection of beta-napthoflavone in depurated Hudson tomcod resulted in a 15-fold induction of P450 gene expression within 26 hr. Exposure of depurated Hudson tomcod to natural sediment spiked with two PAHs resulted in a 7-fold induction of P450 gene expression. Exposure of depurated tomcod to sediment from a contaminated Hudson site also resulted in a 7- to 15-fold induction of P450IA mRNA expression. Northern blot analysis revealed a second polymorphic cytochrome P450IA mRNA band in some tomcod which was also detected by Southern blot analysis. Induction of cytochrome P450IA mRNA in Atlantic tomcod may provide a sensitive biomarker of environmentally relevant concentrations of some pollutants in the Hudson and other northeastern tidal rivers. PMID:1855491

  1. Global Incorporation of Norleucine in Place of Methionine in Cytochrome P450

    E-print Network

    Arnold, Frances H.

    Global Incorporation of Norleucine in Place of Methionine in Cytochrome P450 BM-3 Heme Domain, and also an efficient way to test whether inactivation during peroxide-driven P450 catalysis involves- portant in drug metabolism and drug design, and their cata- lytic capabilities are increasingly valued

  2. 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...

  3. 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...

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

    EPA Science Inventory

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

  5. Cytochrome P450: A Target for Drug Development for Skin Diseases

    Microsoft Academic Search

    Nihal Ahmad; Hasan Mukhtar

    2004-01-01

    Enzymes of the cytochrome P450 (P450 or CYP) super family are the most versatile and important class of drug-metabolizing enzymes that are induced in mammalian skin in response to xenobiotic exposure. At the same time, CYP have numerous important roles in endogenous and exogenous substrate metabolism in the skin. For example, they participate in the metabolism of therapeutic drugs, fatty

  6. Directed Evolution of a Cytochrome P450 Monooxygenase for Alkane Oxidation

    E-print Network

    Arnold, Frances H.

    Directed Evolution of a Cytochrome P450 Monooxygenase for Alkane Oxidation Edgardo T. Farinas alcohols and amides. How- ever, it is not known to oxidize alkanes. Here we re- port that P450 BM-3 = slight affinity) accurately reflects the nature of these com- pounds: alkanes are notoriously inert

  7. The Interplay between Tubulins and P450 Cytochromes during Plasmodium berghei Invasion of Anopheles gambiae Midgut

    PubMed Central

    Félix, Rute C.; Silveira, Henrique

    2011-01-01

    Background Plasmodium infection increases the oxidative stress inside the mosquito, leading to a significant alteration on transcription of Anopheles gambiae detoxification genes. Among these detoxification genes several P450 cytochromes and tubulins were differently expressed, suggesting their involvement in the mosquito's response to parasite invasion. P450 cytochromes are usually involved in the metabolism and detoxification of several compounds, but are also regulated by several pathogens, including malaria parasite. Tubulins are extremely important as components of the cytoskeleton, which rearrangement functions as a response to malaria parasite invasion. Methodology/Principal Findings Gene silencing methods were used to uncover the effects of cytochrome P450 reductase, tubulinA and tubulinB silencing on the A. gambiae response to Plasmodium berghei invasion. The role of tubulins in counter infection processes was also investigated by inhibiting their effect. Colchicine, vinblastine and paclitaxel, three different tubulin inhibitors were injected into A. gambiae mosquitoes. Twenty-four hours post injection these mosquitoes were infected with P. berghei through a blood meal from infected CD1 mice. Cytochrome P450 gene expression was measured using RT-qPCR to detect differences in cytochrome expression between silenced, inhibited and control mosquitoes. Results showed that cytochrome P450 reductase silencing, as well as tubulin (A and B) silencing and inhibition affected the efficiency of Plasmodium infection. Silencing and inhibition also affected the expression levels of cytochromes P450. Conclusions Our results suggest the existence of a relationship between tubulins and P450 cytochromes during A. gambiae immune response to P. berghei invasion. One of the P450 cytochromes in this study, CYP6Z2, stands out as the potential link in this association. Further work is needed to fully understand the role of tubulin genes in the response to Plasmodium infection. PMID:21912622

  8. Immunoquantitation of cytochrome. beta. /sub 5/ and methylcholanthrene-induced cytochromes P-450

    SciTech Connect

    Shires, T.K.; Krieter, P.A.; Shawver, L.K.; Seidel, S.L.

    1987-06-01

    The enzyme-linked immunosorbent assay (ELISA) has been investigated for its ability to quantitate hydrophobic proteins like cytochromes ..beta../sub 5/ and P-450 at the subnanogram level. Issues encountered that have broad significance not only for ELISA, but for other qualitative and quantitative immunoassays as well, include the effects of detergent, the discriminatory capacity of ELISA, and the method for determining an assay's selectivity.

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

    PubMed Central

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

    1988-01-01

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

  10. 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

  11. New Trends in Cytochrome P450 Research at the Half-Century Mark*

    PubMed Central

    Guengerich, F. Peter

    2013-01-01

    Cytochrome P450 enzymes have major roles in the metabolism of steroids, drugs, carcinogens, eicosanoids, and numerous other chemicals. The P450s are collectively considered the most diverse catalysts known in biochemistry, although they operate from a basic structural fold and catalytic mechanism. The four minireviews in this thematic series deal with the unusual aspects of catalytic reactions and electron transfer pathway organization, the structural diversity of P450s, and the expanding roles of P450s in disease and medicine. PMID:23632015

  12. Approaches to Deorphanization of Human and Microbial Cytochrome P450 Enzymes

    PubMed Central

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

    2010-01-01

    One of the general problems in biology today is that we are characterizing genomic sequences much faster than identifying the functions of the gene products, and the same problem exists with cytochromes P450 (P450). One-fourth of the human P450s are not well-characterized and therefore considered “orphans.” A number of approaches to deorphanization are discussed generally. Several liquid chromatography-mass spectrometry approaches have been applied to some of the human and Streptomyces coelicolor P450s. One current limitation is that too many fatty acid oxidations have been identified and we are probably missing more relevant substrates, possibly due to limits of sensitivity. PMID:20493973

  13. Homotropic cooperativity of monomeric cytochrome P450 3A4 in a nanoscale native bilayer environment

    Microsoft Academic Search

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

    2004-01-01

    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

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

    NASA Astrophysics Data System (ADS)

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

    2003-04-01

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

  15. Induction of cytochrome P450-mediated detoxification of xanthotoxin in the black swallowtail.

    PubMed

    Cohen, M B; Berenbaum, M R; Schuler, M A

    1989-09-01

    Xanthotoxin is a phototoxic allomone found in many of the host plants of the black swallowtail,Papilio polyxenes (Lepidoptera: Papilionidae). When added to the diet of final instar larvae, xanthotoxin can induce the cytochrome P450 monooxygenase (P450) activity in midgut microsomes by which it is detoxified. Induction is dose-dependent, increasing sevenfold when larvae feed on parsley treated topically with xanthotoxin at 0.5 or 1.0% fresh weight. Although xanthotoxin exerts much of its toxic effects when photoactivated by ultraviolet light, induction of P450 activity did not differ in the presence or absence of ultraviolet light. Despite a 4.7-fold induction of xanthotoxin-metabolizing P450 activity, total P450 content measured in the same microsomal samples did not increase significantly. These data indicate that multiple forms of P450 exist in the black swallowtail midgut and that they are differentially induced by xanthotoxin. PMID:24272422

  16. Influence of nutrients and other dietary materials on cytochrome P-450 enzymes.

    PubMed

    Guengerich, F P

    1995-03-01

    The cytochrome P-450 (P-450) enzymes are collectively responsible for the bulk of oxidation of xenobiotic chemicals, including drugs, pesticides, and carcinogens. This biotransformation can result in either increased or decreased toxicity, depending on the situation. The regulation of individual P-450 enzymes is a complex subject, with examples of induction and direct inhibition and stimulation. Nutrients and food additives can modify P-450 activities and consequently influence toxicity. P-450s also influence the toxicity of potentially harmful materials found in foods, as well as some vitamins and natural products. Some of the foodstuffs and conditions that influence P-450 in experimental animals and in humans are protein, carbohydrate, lipid, obesity and fasting, water- and fat-soluble vitamins, minerals, sulfides, isothiocyanates, indoles, ellagic acid, capsaicin, terpenes, flavones, butylated hydroxytoluene and hydroxyanisole, charbroiled foods, ethanol, and (monosodium) glutamate and aspartate. Consideration is given, when possible, to differences in responses between animal models and humans. PMID:7879733

  17. Involvement of D-Asp in P450 aromatase activity and estrogen receptors in boar testis.

    PubMed

    Lamanna, C; Assisi, L; Botte, V; Di Fiore, M M

    2007-01-01

    Mammalian testis contains D-aspartic acid (D-Asp), which enhances testosterone production. D-Asp, on other hand, also stimulates 17beta-estradiol synthesis in the ovary of some lower vertebrates. We studied boar testis in order to determine if D-Asp intervenes in 17beta-estradiol synthesis in the testis of those mammals which produce significant amounts of estrogens as well as testosterone. The boar testis contains D-Asp (40 +/- 3.6 nmol/g tissue) which, according to immunohistological techniques, is localized mainly in Leydig cells, and, to a lesser extent, in sustentacular (Sertoli), peritubular and some germ cells. The enzyme P450aromatase is present in Leydig cells and few germ cells. In vitro experiments showed that the addition of D-Asp to testicular tissue extracts induced a significant increase of aromatase activity, as evaluated by testosterone conversion into 17beta-estradiol. The enzyme's K(m) was not affected by D-Asp (about 25 nM in both control and D-Asp added tests). On the basis of these results we suggest that, as in the ovary, D-Asp is involved in the local control of aromatase activity of boar testis and, therefore, it intervenes in the 17beta-estradiol production. In the testis, the D-Asp targets are presumably the Leydig cells, which having also a nuclear estrogen receptor are, in turn, one of the putative targets of the 17beta-estradiol that they produce (autocrine effect). PMID:17469225

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

    PubMed

    Clejan, L A; Cederbaum, A I

    1993-11-01

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

  19. 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

  20. Electrospray ionization mass spectrometric analysis of intact cytochrome P450: identification of tienilic acid adducts to P450 2C9.

    PubMed

    Koenigs, L L; Peter, R M; Hunter, A P; Haining, R L; Rettie, A E; Friedberg, T; Pritchard, M P; Shou, M; Rushmore, T H; Trager, W F

    1999-02-23

    A general scheme for the purification of baculovirus-expressed cytochrome P450s (P450s) from the crude insect cell pastes has been designed which renders the P450s suitable for analysis by high-performance liquid chromatography (HPLC) electrospray ionization mass spectrometry (ESI-MS). An HPLC/ESI-MS procedure has been developed to analyze small amounts of intact purified P450 (P450s cam-HT, 1A1, 1A2, 2A6, 2B1, 2C9, 2C9 C175R, 3A4, 3A4-HT) and rat NADPH cytochrome P450 reductase (P450 reductase). The experimentally determined and predicted (based on the amino acid sequences) molecular masses (MMs) of the various proteins had identical rank orders. For each individual protein, the difference between the experimentally determined (+/-SD, based on experiments performed on at least 3 different days) and predicted MMs ranged from 0.002 to 0.035%. Each experimentally determined MM had a standard deviation of less than 0.09% (based on the charge state distribution). Application of this HPLC/ESI-MS technique made the detection of the covalent modification to P450 2C9 following mechanism-based inactivation by tienilic acid possible. In the absence of glutathione, three P450 2C9 species were detected that produced ESI mass spectra corresponding to native P450 2C9 and both a monoadduct and a diadduct of tienilic acid to P450 2C9. In the presence of glutathione, only native P450 2C9 and the monoadduct were detected. Based on the observed mass shifts for the P450 2C9/tienilic acid adducts, a mechanism for the inactivation of P450 2C9 by tienilic acid is proposed. PMID:10029524

  1. 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…

  2. Genomic and Bioinformatic Analysis of NADPH-Cytochrome P450 Reductase in Anopheles stephensi (Diptera: Culicidae).

    PubMed

    Suwanchaichinda, C; Brattsten, L B

    2014-01-01

    The cytochrome P450 monooxygenase (P450) enzyme system is a major mechanism of xenobiotic biotransformation. The nicotinamide adenine dinucleotide phosphate (NADPH)-cytochrome P450 reductase (CPR) is required for transfer of electrons from NADPH to P450. One CPR gene was identified in the genome of the malaria-transmitting mosquito Anopheles stephensi Liston (Diptera: Culicidae). The gene encodes a polypeptide containing highly conserved flavin mononucleotide-, flavin adenine dinucleotide-, and NADPH-binding domains, a unique characteristic of the reductase. Phylogenetic analysis revealed that the A. stephensi and other known mosquito CPRs belong to a monophyletic group distinctly separated from other insects in the same order, Diptera. Amino acid residues of CPRs involved in binding of P450 and cytochrome c are conserved between A. stephensi and the Norway rat Rattus norvegicus Berkenhout (Rodentia: Muridae). However, gene structure particularly within the coding region is evidently different between the two organisms. Such difference might arise during the evolution process as also seen in the difference of P450 families and isoforms found in these organisms. CPR in the mosquito A. stephensi is expected to be active and serve as an essential component of the P450 system. PMID:25368081

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

    NASA Astrophysics Data System (ADS)

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

    2006-03-01

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

  4. Cofactor-free light-driven whole-cell cytochrome P450 catalysis.

    PubMed

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

    2015-01-12

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

  5. 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.

  6. 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.

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

    PubMed

    Baliga, R; Zhang, Z; Baliga, M; Shah, S V

    1996-02-01

    Iron has been implicated to play an important role in several models of tissue injury, including myoglobinuric acute renal failure. In this model, myoglobin released from the injured muscle is generally accepted as a source of iron. In the present study we measured the bleomycin-detectable iron (iron capable of catalyzing free radical reactions) in the kidneys and examined the role of cytochrome P-450 as a source of catalytic iron in glycerol-induced model of myoglobinuric acute renal failure. Rats were injected with 50% glycerol (8 ml/kg) i.m. after overnight water deprivation and sacrificed 24 hours later. There was a marked and a specific increase in the bleomycin-detectable iron content accompanied by a marked decrease in the cytochrome P-450 content in the kidneys of glycerol treated rats. We then examined the effects of two different cytochrome P-450 inhibitors, cimetidine (with ranitidine as a control) and piperonyl butoxide. Cimetidine, but not ranitidine, significantly prevented the increase of bleomycin-detectable iron in the kidneys of glycerol-treated rats. The loss of cytochrome P-450 content was substantially blocked by both inhibitors, cimetidine and piperonyl butoxide, but not by ranitidine. Both the inhibitors of cytochrome P-450 provided functional (as measured by BUN and creatinine) and histological protection against glycerol-induced acute renal failure. Our data thus demonstrate a marked increase in bleomycin-detectable iron in the kidneys of glycerol-treated rats. Our data also indicate that inhibitors of cytochrome P-450 provide protection against glycerol-induced acute renal failure and that cytochrome P-450 may be a significant source of this iron in this model of acute renal failure. PMID:8821818

  8. Production of hydroxy-fatty acid derivatives from waste oil by Escherichia coli cells producing fungal cytochrome P450foxy

    Microsoft Academic Search

    Tatsuya Kitazume; Yuya Yamazaki; Shigeru Matsuyama; Hirofumi Shoun; Naoki Takaya

    2008-01-01

    Cytochrome P450foxy (P450foxy) is a fatty acid (FA) monooxygenase that is characterized by self-sufficient catalysis and high\\u000a turnover numbers due to the fused structure of cytochrome P450 and its reductase. Here we found that resting recombinant Escherichia coli cells producing P450foxy converted saturated FA with a chain length of 7–16 carbon atoms to their ? ? 1 to ? ?

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

    SciTech Connect

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

    2007-03-30

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

  10. Cytochrome P-450 mediates tissue-damaging hydroxyl radical formation during reoxygenation of the kidney.

    PubMed

    Paller, M S; Jacob, H S

    1994-07-19

    Renal reperfusion injury results from oxygen radical generation. During reoxygenation of hypoxic kidney cells, xanthine oxidase produces superoxide radical, which eventuates in hydroxyl radical formation by the Fenton reaction. This reaction, catalyzed by transition metals such as iron, is particularly important because hydroxyl radical is highly reactive with a wide variety of biomolecules. We tested the hypothesis that this catalytic function is fostered by iron released from the heme moiety of cytochrome P-450. Primary cultures of rat proximal tubule epithelial cells studied in a subconfluent stage were subjected to 60 min of hypoxia and 30 min of reoxygenation. When cells were pretreated with one of three cytochrome P-450 inhibitors (piperonyl butoxide, cimetidine, or ketoconazole), lethal cell injury was attenuated. There was the expected increase in O2-. production during hypoxia/reoxygenation that cytochrome P-450 inhibitors did not prevent; on the other hand, inhibitors did prevent reoxygenation-induced hydroxyl radical formation. Analogously, the increase in catalytic iron (bleomycin-detectable iron) that accompanies hypoxia/reoxygenation did not occur in the presence of cytochrome P-450 inhibitors. In vivo studies confirmed a protective effect of cytochrome P-450 inhibition because glomerular filtration rate was better preserved in rats pretreated with cimetidine and then subjected to renal artery occlusion. In summary, several chemically distinct cytochrome P-450 inhibitors reduced iron release, and thereby, hydroxyl radical formation and reoxygenation-induced lethal cell injury, without inhibiting superoxide radical formation. We conclude that highly labile P-450 may act as an Fe-donating catalyst for Fenton reaction production of HO.-mediated reperfusion injury. PMID:8041736

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

    PubMed

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

    1992-12-01

    The steroid 11ß-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 11ß-hydroxyprogesterone/h·mg protein in the 100,000 g pellet fraction. The 11ß-hydroxylation was dependent on NADPH. The formation of 11ß-hydroxyprogesterone correlated linearly with the cytochrome P450 concentration. The fungal 11ß-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 P45011ß 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 11ß-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

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

    SciTech Connect

    Kaspera, Ruediger; Sahele, Tariku; Lakatos, Kyle [Department of Medicinal Chemistry, University of Washington, Box 357610, Seattle, WA 98195-7610 (United States)] [Department of Medicinal Chemistry, University of Washington, Box 357610, Seattle, WA 98195-7610 (United States); Totah, Rheem A., E-mail: rtotah@u.washington.edu [Department of Medicinal Chemistry, University of Washington, Box 357610, Seattle, WA 98195-7610 (United States)

    2012-02-17

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

  13. 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

  14. Structural characterization of human cytochrome P450 2C19: active site differences between P450s 2C8, 2C9, and 2C19.

    PubMed

    Reynald, R Leila; Sansen, Stefaan; Stout, C David; Johnson, Eric F

    2012-12-28

    To identify the structural features underlying the distinct substrate and inhibitor profiles of P450 2C19 relative to the closely related human enzymes, P450s 2C8 and 2C9, the atomic structure (Protein Data Bank code 4GQS) of cytochrome P450 2C19 complexed with the inhibitor (2-methyl-1-benzofuran-3-yl)-(4-hydroxy-3,5-dimethylphenyl)methanone (Protein Data Bank chemical component 0XV) was determined to 2.87 Å resolution by x-ray crystallography. The conformation of the peptide backbone of P450 2C19 is most similar to that of P450 2C8, but the substrate-binding cavity of P450 2C8 is much larger than that of P450 2C19 due to differences in the amino acid residues that form the substrate-binding cavities of the two enzymes. In contrast, the substrate-binding cavity of P450 2C19 is much more similar in size to that of the structure of the P450 2C9 flurbiprofen complex than to that of a modified P450 2C9 or that of P450 2C8. The cavities of the P450 2C19 0XV complex and the P450 2C9 flurbiprofen complex differ, however, because the helix B-C loops of the two enzymes are dissimilar. These conformational differences reflect the effects of adjacent structural elements that interact with the B-C loops and that differ between the two enzymes. The availability of a structure for 2C19 will facilitate computational approaches for predictions of substrate and inhibitor binding to this enzyme. PMID:23118231

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

    Microsoft Academic Search

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

    1986-01-01

    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

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

    Microsoft Academic Search

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

    1996-01-01

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

  17. Interaction of methadone with substrates of human hepatic cytochrome P450 3A4

    Microsoft Academic Search

    C. Iribarne; Y. Dréano; L. G. Bardou; J. F. Ménez; F. Berthou

    1997-01-01

    Methadone, a synthetic drug, is one of the most widely used drugs for opiate dependency treatment. This drug has been demonstrated to be extensively metabolized by cytochrome P450 3A4 in human liver microsomes. Thus, the aim of this in vitro study was to determine if methadone is an inhibitor of other P450s characterized by their specific catalytic activities. Enzymatic activities

  18. Time course for induction of cytochrome P450 - Dependent activities by ethylbenzene

    Microsoft Academic Search

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

    1992-01-01

    The goal of this study was to examine the time course for ethylbenzene-mediated induction of cytochrome P450-dependent activities. Male Holtzman rats were treated with a single i.p. injection of ethylbenzene (EB) suspended in corn oil. In this study, the rats were injected at different times so all animals were killed at the same time P450 levels were transiently elevated at

  19. Cytochrome P450 genes from Helicoverpa armigera: Expression in a pyrethroid-susceptible and -resistant strain

    Microsoft Academic Search

    Barry Pittendrigh; Kate Aronstein; Eva Zinkovsky; Olga Andreev; Bronwyn Campbell; Joanne Daly; Stephen Trowell; Richard H. Ffrench-Constant

    1997-01-01

    The molecular basis of metabolic resistance to pyrethroids in Helicoverpa armigera is currently under debate. Substantial indirect evidence supports a role for both esterase- and cytochromeP450-mediated metabolism. However, the relative roles played by these two mechanisms in field-based resistance is uncertain. Our understanding of the importance of P450-mediated metabolism is hindered by the paucity of cloned genes from this species,

  20. Induction of cytochrome P450-mediated detoxification of xanthotoxin in the black swallowtail

    Microsoft Academic Search

    Michael B. Cohen; May R. Berenbaum; Mary A. Schuler

    1989-01-01

    Xanthotoxin is a phototoxic allomone found in many of the host plants of the black swallowtail,Papilio polyxenes (Lepidoptera: Papilionidae). When added to the diet of final instar larvae, xanthotoxin can induce the cytochrome P450 monooxygenase (P450) activity in midgut microsomes by which it is detoxified. Induction is dose-dependent, increasing sevenfold when larvae feed on parsley treated topically with xanthotoxin at

  1. Characterization and Functional Analysis of Two Common Human Cytochrome P450 1B1 Variants

    Microsoft Academic Search

    Roman A. McLellan; Mikael Oscarson; Mats Hidestrand; Brith Leidvik; Eva Jonsson; Charlotta Otter; Magnus Ingelman-Sundberg

    2000-01-01

    Cytochrome P450 1B1 (CYP1B1) is a human extrahepatic P450 that activates procarinogens, metabolizes 17?-estradiol, and may well have a role in the pathogenesis of some forms of cancer. Besides rare deleterious mutations reported for the CYP1B1 gene, six single-nucleotide polymorphisms have been reported, of which four cause amino acid exchanges. We have expressed two of the common CYP1B1 alleles in

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

    Microsoft Academic Search

    Kenji Matsui; Mizuyoshi Shibutani; Toshiharu Hase; Tadahiko Kajiwara

    1996-01-01

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

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

    Microsoft Academic Search

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

    2003-01-01

    Cytochromes P450 catalyse extremely diverse and often complex regiospecific and\\/or stereospecific reactions in the biosynthesis or catabolism of plant bioactive molecules. Engineered P450 expression is needed for low-cost production of antineoplastic drugs such as taxol or indole alkaloids and offers the possibility to increase the content of nutraceuticals such as phytoestrogens and antioxidants in plants. Natural products may serve important

  4. 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

  5. Enolization as an alternative proton delivery pathway in human aromatase (P450 19A1).

    PubMed

    Krámos, Balázs; Oláh, Julianna

    2014-01-16

    Human aromatase catalyzes the last step of estrogen biosynthesis, the aromatization of ring A of androstenedione (ASD) and testosterone leading to estrone and estradiol. The enolization of the substrate molecule has been suggested to play an essential role in this process. In this work using quantum mechanical and hybrid QM/MM calculations, the reaction mechanism of enolization was investigated. It is shown that the energetically unfavorable enolization of andostenedione occurs in a coupled process with the energetically favorable protonation of the ferrous superoxo complex (traditionally called ferric peroxo complex) via a low barrier of about 5 kcal/mol. This mechanism implies an alternative way for protonation of the ferrous superoxo complex to form compound 0, which occurs via the Asp309-water-ASD proton delivery pathway instead of the Asp-water-Thr pathway suggested for other P450 enzymes. It is also shown that Thr310, which is known experimentally to be important for catalysis, plays a key role in the conversion of compound 0 to compound I. PMID:24369956

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed Central

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

    2014-01-01

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

  8. Substrate-, hormone-, and cAMP-regulated cytochrome P450 degradation.

    PubMed Central

    Eliasson, E; Johansson, I; Ingelman-Sundberg, M

    1990-01-01

    The hepatic cytochrome P450 system, with numerous different P450 enzymes, is characterized by its inducibility by a variety of endogenous and exogenous compounds. Specific forms of P450, exhibiting distinct but partially overlapping substrate specificities, are increased in response to a given chemical. Consequently, the rate of elimination of the inducing compound is often enhanced and the system is in this respect adaptive to changes in the environment. Transcriptional activation mechanisms for the endo- or xenobiotically controlled P450 synthesis are well documented. Here we describe a mechanism for posttranslational ligand-dependent stabilization of ethanol-inducible P450IIE1 in hepatocyte cultures. Glucagon or 8-bromoadenosine 3',5'-cyclic monophosphate causes an enhanced rate of P450IIE1 degradation in the hepatocytes as well as phosphorylation on Ser-129, a reaction which denatures the protein under in vitro conditions. Substrates for the enzyme, such as ethanol and imidazole, protect the enzyme from phosphorylation and degradation in hepatocytes but do not influence phosphorylation or degradation of phenobarbital-inducible P450IIB1. Our proposed mechanism, which remains to be shown under in vivo conditions, describes the P450 molecules as receptors for the compounds in question and might provide a way by which endo- and xenobiotics regulate their own rate of metabolism. Images PMID:2326279

  9. Computational biotransformation profile of paracetamol catalyzed by cytochrome p450.

    PubMed

    Ji, Li; Schüürmann, Gerrit

    2015-04-20

    The P450-catalyzed biotransformation of the analgesic drug paracetamol (PAR) is a long-debated topic, involving different mechanistic hypotheses as well as experimental evidence for the metabolites N-acetyl-p-benzoquinone imine (NAPQI), p-benzoquinone, acetamide, and 3-hydroxy-PAR. During the catalytic cycle of P450, a high-valent iron(IV)-oxo species known as Compound I (Cpd I) is formed as the ultimate oxidant, featuring two energetically close-lying ground states in the doublet (low-spin) and quartet (high-spin) spin states, respectively. In order to clarify the catalytic mechanism, a computational chemistry analysis has been undertaken for both the high- and low-spin routes, employing density functional theory (DFT) including PCM (polarized continuum-solvation model) that yields an approximate simulation of the bulk polarization exerted through the protein. The results demonstrate that hydrogen abstraction transfer (HAT) by the P450 oxidant Cpd I (FeO) is kinetically strongly preferred over the alternative pathways of an oxygen addition reaction (OAR) or two consecutive single-electron transfers (SET). Moreover, only the respective high-spin route yields N-acetyl-p-semiquinone imine (NAPSQI) as an intermediate that is converted to the electrophile N-acetyl-p-benzoquinone imine (NAPQI). By contrast, 3-hydroxy-PAR, acetamide, and p-benzoquinone as electrophilic and redox-active agent are formed predominantly in the low-spin state through reactions that do not involve NAPSQI. Thus, all experimentally observed PAR metabolites are in accord with an initial HAT from the phenolic oxygen, and NAPSQI should indeed be the precursor of NAPQI, both of which are generated only via the high-spin pathway. PMID:25548954

  10. 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

  11. Molecular and Cellular Endocrinology 231 (2005) 7585 Steroid and xenobiotic receptor (SXR), cytochrome P450 3A4 and

    E-print Network

    Blumberg, Bruce

    2005-01-01

    ), cytochrome P450 3A4 and multidrug resistance gene 1 in human adult and fetal tissues Yasuhiro Mikia,, Takashi an important role in the regulation of the cytochrome P450 3A4 gene (CYP3A4) and multidrug resistance gene 1PAR; Bertilsson et al., 1998). SXR positively regulated transcription of cytochrome P450 3A4 (CYP3A4; Bertilsson

  12. Cytochrome p450 purification and immunological detection in an insecticide resistant strain of german cockroach ( Blattella germanica, l.)

    Microsoft Academic Search

    MICHAEL E. SCHARF; JONATHAN J. NEAL; CRAIG B. MARCUS; GARY W. BENNETT

    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

  13. Nitric oxide formation during microsomal hepatic denitration of glyceryl trinitrate: involvement of cytochrome P-450.

    PubMed

    Servent, D; Delaforge, M; Ducrocq, C; Mansuy, D; Lenfant, M

    1989-09-29

    Glyceryl trinitrate was denitrated by rat liver microsomes in the presence of NADPH with formation of a mixture of glyceryl dinitrates and glyceryl mononitrates. The highest activity was obtained under anaerobic conditions and the reaction was inhibited by O2 indicating that it is a reductive denitration. It was also inhibited by CO, metyrapone and miconazole showing that it was catalyzed by cytochrome P-450. Finally the formation of the cytochrome P-450-Fe(II)-NO complex during this reaction was shown by visible spectroscopy. These data demonstrate that microsomal reductive denitration of glyceryl trinitrate is catalyzed by cytochrome P-450 and can be involved in the formation of the endothelium-derived relaxing factor (EDRF = nitric oxide). PMID:2506859

  14. Potent cytochrome P450 2C19 genotype–related interaction between voriconazole and the cytochrome P450 3A4 inhibitor ritonavir

    Microsoft Academic Search

    Gerd Mikus; Verena Schöwel; Magdalena Drzewinska; Jens Rengelshausen; Reinhard Ding; Klaus-Dieter Riedel; Jürgen Burhenne; Johanna Weiss; Torben Thomsen; Walter E. Haefeli

    2006-01-01

    Objectives: Cytochrome P450 (CYP) 2C19 and CYP3A4 are the major enzymes responsible for voriconazole elimination. Because the activity of CYP2C19 is under genetic control, the extent of inhibition with a CYP3A4 inhibitor was expected to be modulated by the CYP2C19 metabolizer status. This study thus assessed the effect of the potent CYP3A4 inhibitor ritonavir after short-term administration on voriconazole pharmacokinetics

  15. Probing the cytochrome P-450 2B1 active site with diamantoid compounds.

    PubMed

    Hodek, P; Burkhard, J; Jank?, J

    1995-06-01

    Hydrocarbone diamantane has been shown to be a specific substrate with a high affinity for the binding site of PB-inducible cytochrome P-450 2B1 (Hodek et al. 1988). Using a difference spectroscopy approach, a battery of diamantane analogues and diamantane oxygen containing derivatives were examined for their interaction with P-450 2B1 active site. Of the compounds (diamantane and its analogues, adamantane and triamantane) tested, diamantane had the lowest value of a spectral dissociation constant Ks = 0.5 mumol/l, indicating that diamantane was accommodated well to the cytochrome P-450 2B1, hence values of 0.46 nm and 0.66 nm for the width and length of the diamantane molecule, respectively, were used to describe of the dimensions the cytochrome P-450 binding site. Adamantane (Ks = 1.3 mumol/l) is relatively small and thus it binds loosely whereas triamantane (Ks = 4.3 mumol/l) is bulky enough to fit the binding site. This conclusion has been confirmed by spectral competition experiments as well as metabolic studies. Of all oxygen containing derivatives diamantane 1,6-dicarboxylic acid dimethylester only exhibited a pronounced ligand interaction with cytochrome P-450. Using molecular dimensions of this derivative the distance of 0.56 nm from the heme iron to the center of the substrate binding site was estimated. PMID:8586256

  16. Oxidative cleavage of esters and amides to carbonyl products by cytochrome P450.

    PubMed

    Peng, H M; Raner, G M; Vaz, A D; Coon, M J

    1995-04-20

    A series of esters and several amides were shown to undergo oxidative cleavage with the formation of carbonyl products in the presence of purified isoforms of liver microsomal cytochrome P450 (P450) in a reconstituted enzyme system. The reaction also requires NADPH and NADPH-cytochrome P450 reductase and is stimulated by phosphatidylcholine. Kinetic constants were determined in experiments in which the predicted aldehyde product was identified and quantitated by gas chromatography. A relationship was seen with P450 2E1 between the structures of the esters and the Vmax values, with the rates decreasing in the series of methyl formate to methyl valerate, and similarly in the series of methyl, ethyl, propyl, butyl, and amyl acetates. Furthermore, a clear correlation exists between the Km values of the ethyl esters examined and the log of the octanol/water partition coefficients of these substrates. With P450 2E1, the Km decreases significantly between one and four carbon atoms in the chain length of the acyl component of the ester but is unaffected by a further increase in length. However, no correlation was found between the Km value and the chain length of the alcohol moiety of the esters. Similarly, with P450 2B4 a large decrease in Km occurs between one and five carbons in the acyl component of the ethyl esters but is unaffected by a further increase in chain length. The observed correlation is presumed to arise from hydrophobic interactions between the access channel to the active site of P450 and the acyl side chain of the esters. P450 1A2 is also active in ester cleavage, and the three cytochromes examined with esters are active in the conversion of N-alkyl amides to aldehydes, as are P450s 2C3, 1A1, and 3A6. Studies on 2-butyl acetate oxidation by P450 2B4 in the presence of 18O2 showed 88% 18O incorporation into the product, 2-butanone. This is consistent with a mechanism that involves hydroxylation at the alpha-carbon of the alcohol component of the ester to yield an unstable geminal hydroxy ester, as proposed earlier by F. P. Guengerich et al. (1988, J. Biol. Chem. 263, 8176-8183) for several dihydropyridine carboxylic esters. Our results further indicate that such an intermediate decomposes by a nonhydrolytic mechanism and also rule out the possibility of transient ester hydrolysis with subsequent oxidation of the alcohol formed. In addition, they establish that oxidative cleavage is a widespread reaction among P450 cytochromes and commonly used esters and amides. PMID:7733661

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

    PubMed Central

    Halkier, Barbara Ann; Møller, Birger Lindberg

    1991-01-01

    The biosynthesis of the tyrosine-derived cyanogenic glucoside dhurrin involves N-hydroxytyrosine, (E)- and (Z)-p-hydroxyphenylacetaldehyde oxime, p-hydroxyphenylacetonitrile, and p-hydroxymandelonitrile as intermediates and has been studied in vitro using a microsomal enzyme system obtained from etiolated sorghum (Sorghum bicolor [L.] Moench) seedlings. The biosynthesis is inhibited by carbon monoxide and the inhibition is reversed by 450 nm light demonstrating the involvement of cytochrome P-450. The combined use of two differently prepared microsomal enzyme systems and of tyrosine, p-hydroxyphenylacetaldehyde oxime, and p-hydroxyphenylacetonitrile as substrates identify two cytochrome P-450-dependent monooxygenases: the N-hydroxylase which converts tyrosine into N-hydroxytyrosine and the C-hydroxylase converting p-hydroxyphenylacetonitrile into p-hydroxymandelonitrile. The inhibitory effect of a number of putative cytochrome P-450 inhibitors confirms the involvement of cytochrome P-450. Monospecific polyclonal antibodies raised toward NADPH-cytochrome P-450-reductase isolated from sorghum inhibits the same metabolic conversions as carbon monoxide. No cytochrome P-450-dependent monooxygenase catalyzing an N-hydroxylation reaction has previously been reported in plants. The metabolism of p-hydroxyphenylacetaldehyde oxime is completely dependent on the presence of NADPH and oxygen and results in the production of p-hydroxymandelonitrile with no accumulation of the intermediate p-hydroxyphenylacetonitrile in the reaction mixture. The apparent NADPH and oxygen requirements of the oxime-metabolizing enzyme are identical to those of the succeeding C-hydroxylase converting p-hydroxyphenylacetonitrile to p-hydroxymandelonitrile. Due to the complex kinetics of the microsomal enzyme system, these requirements may not appertain to the oxime-metabolizing enzyme, which may convert p-hydroxyphenylacetaldehyde oxime to p-hydroxyacetonitrile by a simple dehydration. ImagesFigure 3Figure 4 PMID:16668136

  18. 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

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

    SciTech Connect

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

    2006-07-15

    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.

  20. Involvement of human cytochrome P450 3A4 in reduced haloperidol oxidation

    Microsoft Academic Search

    S. Kudo; M. Odomi

    1998-01-01

    Objective: The present study was conducted to identify in vitro the cytochrome P450(CYP) isoform involved in the metabolic conversion\\u000a of reduced haloperidol to haloperidol using microsomes derived from human AHH-1 TK +\\/? cells expressing human cytochrome P450s.\\u000a The inhibitory and\\/or stimulatory effects of reduced haloperidol or haloperidol on CYP2D6-catalyzed carteolol 8-hydroxylase\\u000a activity were also investigated.\\u000a \\u000a \\u000a \\u000a Results: The CYP isoform involved

  1. Styrene metabolism in Exophiala jeanselmei and involvement of a cytochrome P-450-dependent styrene monooxygenase.

    PubMed Central

    Cox, H H; Faber, B W; Van Heiningen, W N; Radhoe, H; Doddema, H J; Harder, W

    1996-01-01

    The yeast-like fungus Exophiala jeanselmei degrades styrene via initial oxidation of the vinyl side chain to phenylacetic acid, which is subsequently hydroxylated to homogentisic acid. The initial reactions are catalyzed by a NADPH- and flavin adenine dinucleotide-dependent styrene monooxygenase, a styrene oxide isomerase, and a NAD(+)-dependent phenylacetaldehyde dehydrogenase. The reduced CO-difference spectrum of microsomal preparations of styrene-grown cells shows a characteristic absorption maximum at 450 nm, which strongly suggests the involvement of a cytochrome P-450-dependent styrene monooxygenase. Inhibition of styrene monooxygenase activity in cell extracts by cytochrome P-450 inhibitors SKF-525-A, metyrapone, and CO confirms this assumption. PMID:8919815

  2. Expression of Cytochrome P450 3A7 in Escherichia coli:Effects of 5? Modification and Catalytic Characterization of Recombinant Enzyme Expressed in Bicistronic Format with NADPH-Cytochrome P450 Reductase

    Microsoft Academic Search

    Elizabeth M. J. Gillam; Rebecca M. Wunsch; Yune-Fang Ueng; Tsutomu Shimada; Paul E. B. Reilly; Tetsuya Kamataki; F. Peter Guengerich

    1997-01-01

    Cytochrome P450 3A7 is the major P450 form present in fetal liver tissue and may be responsible for the detoxification of many drugs that reach the fetal circulation. We report the development of bacterial expression systems for P450 3A7. Maximal yields (up to 50 nmol P450\\/liter culture) were obtained with a construct in which the 5?-terminus of the 3A7 cDNA

  3. 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 ...

  4. 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.

  5. 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

  6. 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

  7. Extending the diversity of cytochrome P450 enzymes by DNA family shuffling.

    PubMed

    Rosic, Nedeljka N; Huang, Weiliang; Johnston, Wayne A; DeVoss, James J; Gillam, Elizabeth M J

    2007-06-15

    The cytochrome P450 enzymes involved in xenobiotic metabolism are an excellent starting point for the directed evolution of novel biocatalysts due to their wide substrate specificity. A shuffled library of three highly homologous mammalian genes (for P450 2C9, P450 2C11 and P450 2C19) was constructed by applying a modified DNA family shuffling procedure. The modifications made to the traditional DNA shuffling protocols involved non-random digestion via the use of different combinations of restriction enzymes (REs) followed by isolation of fragments under 300 bp by size-selective filtration. Shuffled cytochrome P450 mutants were co-expressed in Escherichia coli with their redox partner, NADPH-cytochrome P450 reductase (NPR). We report here how non-random fragmentation may help in chimeragenesis within the areas of low sequence similarity such as substrate recognition sites (SRSs) that are generally underrepresented in recombination using the random fragmentation process. Size-selective filtration was used to limit recovery of incompletely digested fragments and consequently minimize the chances for contamination of the shuffled library with parental forms. No parental forms could be detected in the shuffled library using restriction fragment length polymorphism (RFLP) analysis, suggesting the library was free of parental contamination. Sequencing of randomly selected mutants demonstrated a high level of chimeragenesis with on average of 8.0+/-2.2 crossovers and a low level of mutagenesis with 5.2+/-2.8 spontaneous mutations per approximately 1.5 kbp of the full-length P450 sequence. The proportion of properly folded protein as indicated by the observation of characteristic Fe(II).CO vs. Fe(II) difference spectra was 15% (4/27) of analysed mutants. Screening of the shuffled library for indole oxidation revealed four clones with similar or higher levels of indigo pigment production to those of the parental P450s and two clones with elevated P450 expression. In this paper we present a method for the effective family shuffling of cytochrome P450 enzymes, applicable to the creation of mutant libraries with expanded metabolic diversity and with a significant proportion of functional clones. PMID:17400405

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

    PubMed

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

    2014-10-01

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

  9. CYTOCHROME P450 INHIBITION USING RECOMBINANT PROTEINS AND MASS SPECTROMETRY\\/MULTIPLE REACTION MONITORING TECHNOLOGY IN A CASSETTE INCUBATION

    Microsoft Academic Search

    RICHARD WEAVER; KEN S. GRAHAM; IAIN G. BEATTIE; ROB J. RILEY

    2003-01-01

    Detailed cytochrome P450 (P450) inhibition profiles are now re- quired for the registration of novel molecular entities. This method uses combined substrates (phenacetin, diclofenac, S-mepheny- toin, bufuralol, and midazolam) with combined recombinant P450 enzymes (CYP1A2, 2C9, 2C19, 2D6, and 3A4) in an attempt to limit interactions with other more minor P450s and associated reduc- tases. Kinetic analysis of single substrate

  10. A Host-Inducible Cytochrome P-450 from a Host-Specific Caterpillar: Molecular Cloning and Evolution

    Microsoft Academic Search

    Michael B. Cohen; Mary A. Schuler; May R. Berenbaum

    1992-01-01

    Cytochrome P-450 monooxygenases (P-450s) play a critical role in the detoxification of natural and synthetic toxins in a wide range of organisms. We have isolated and sequenced cDNA clones encoding a P-450, CYP6B1, from larvae of Papilio polyxenes (Lepidoptera: Papilionidae), the black swallowtail butterfly. This P-450, cloned from a herbivorous insect, is highly inducible by xanthotoxin, a secondary metabolite abundant

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

    PubMed

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

    2012-12-01

    In order to develop in vitro methods as an alternative to P450 animal testing in the drug discovery process, two main requisites are necessary: 1) gathering of data on animal homologues of the human P450 enzymes, currently very limited, and 2) bypassing the requirement for both the P450 reductase and the expensive cofactor NADPH. In this work, P450 2C20 from Macaca fascicularis, homologue of the human P450 2C8 has been taken as a model system to develop such an alternative in vitro method by two different approaches. In the first approach called "molecular Lego", a soluble self-sufficient chimera was generated by fusing the P450 2C20 domain with the reductase domain of cytochrome P450 BM3 from Bacillus megaterium (P450 2C20/BMR). In the second approach, the need for the redox partner and also NADPH were both obviated by the direct immobilization of the P450 2C20 on glassy carbon and gold electrodes. Both systems were then compared to those obtained from the reconstituted P450 2C20 monooxygenase in presence of the human P450 reductase and NADPH using paclitaxel and amodiaquine, two typical drug substrates of the human P450 2C8. The K(M) values calculated for the 2C20 and 2C20/BMR in solution and for 2C20 immobilized on electrodes modified with gold nanoparticles were 1.9 ± 0.2, 5.9 ± 2.3, 3.0 ± 0.5 ?M for paclitaxel and 1.2 ± 0.2, 1.6±0.2 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

  12. The cytochrome P450scc system opens an alternate pathway of vitamin D3 metabolism

    PubMed Central

    Slominski, Andrzej; Semak, Igor; Zjawiony, Jordan; Wortsman, Jacobo; Li, Wei; Szczesniewski, Andre; Tuckey, Robert C.

    2008-01-01

    We show that cytochrome P450scc (CYP11A1) in either a reconstituted system or in isolated adrenal mitochondria can metabolize vitamin D3. The major products of the reaction with reconstituted enzyme were 20-hydroxycholecalciferol and 20,22-dihydroxycholecalciferol, with yields of 16 and 4%, respectively, of the original vitamin D3 substrate. Trihydroxycholecalciferol was a minor product, likely arising from further metabolism of dihydroxycholecalciferol. Based on NMR analysis and known properties of P450scc we propose that hydroxylation of vitamin D3 by P450scc occurs sequentially and stereospecifically with initial formation of 20(S)-hydroxyvitamin D3. P450scc did not metabolize 25-hydroxyvitamin D3, indicating that modification of C25 protected it against P450scc action. Adrenal mitochondria also metabolized vitamin D3 yielding 10 hydroxyderivatives, with UV spectra typical of vitamin D triene chromophores. Aminogluthimide inhibition showed that the three major metabolites, but not the others, resulted from P450scc action. It therefore appears that non-P450scc enzymes present in the adrenal cortex to some extent contribute to metabolism of vitamin D3. We conclude that purified P450scc in a reconstituted system or P450scc in adrenal mitochondria can add one hydroxyl group to vitamin D3 with subsequent hydroxylation being observed for reconstituted enzyme but not for adrenal mitochondria. Additional vitamin D3 metabolites arise from the action of other enzymes in adrenal mitochondria. These findings appear to define novel metabolic pathways involving vitamin D3 that remain to be characterized. PMID:16098191

  13. mRNA distribution and heterologous expression of orphan cytochrome P450 20A1.

    PubMed

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

    2008-09-01

    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 3- to 4-fold higher in brain and heart than in liver. In situ hybridization of rat whole brain sections showed an mRNA expression pattern similar to that 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 (200-280 nmol of 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

  14. 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

  15. 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

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

    PubMed Central

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

    2013-01-01

    Updated models of the Rat Cytochrome P450 2D enzymes are produced based on the recent x-ray structures of the Human P450 2D6 enzyme both with and without a ligand bound. The differences in species selectivity between the epimers quinine and quinidine are rationalised using these models and the results are discussed with regard to previous studies. A close approach to the heme is not observed in this study. The x-ray structure of the enzyme with a ligand bound is shown to be a better model for explaining the observed experimental binding of quinine and quinidine. Hence models with larger closed binding sites are recommended for comparative docking studies. This is consistent with molecular recognition in Cytochrome P450 enzymes being the result of a number of non-specific interactions in a large binding site. PMID:23691026

  17. VECTOR CONTROL, PEST MANAGEMENT, RESISTANCE, REPELLENTS A Rapid Luminescent Assay for Measuring Cytochrome P450 Activity

    E-print Network

    Hammock, Bruce D.

    Cytochrome P450 Activity in Individual Larval Culex pipiens Complex Mosquitoes (Diptera: Culicidae) A. B using the pyrethroid resistant Marin strain of Culex pipiens pipiens form molestus easily be adapted to be used for other arthropod species. KEY WORDS mosquito, Culex pipiens complex

  18. Cytochrome P450 enzymes: Central players in cardiovascular health and disease

    Microsoft Academic Search

    Reem H. Elbekai; Ayman O. S. El-Kadi

    2006-01-01

    Cardiovascular disease (CVD) is a human health crisis that remains the leading cause of death worldwide. The cytochrome P450 (CYP) class of enzymes are key metabolizers of both xenobiotics and endobiotics. Many CYP enzyme families have been identified in the heart, endothelium and smooth muscle of blood vessels. Furthermore, mounting evidence points to the role of endogenous CYP metabolites, such

  19. Enalapril hepatotoxicity in the rat. Effects of modulators of cytochrome P450 and glutathione.

    PubMed

    Jurima-Romet, M; Huang, H S

    1992-11-01

    The effects of modulators of cytochrome P450 and reduced glutathione (GSH) on the hepatotoxicity of enalapril maleate (EN) were investigated in Fischer 344 rats. Twenty-four hours following the administration of EN (1.5 to 1.8 g/kg), increased serum transaminases (ALT and AST) and hepatic necrosis were observed. Pretreatment of the animals with pregnenolone-16 alpha-carbonitrile, a selective inducer of the cytochrome P450IIIA gene subfamily, enhanced EN-induced hepatotoxicity, whereas pretreatment with the cytochrome P450 inhibitor, cobalt protoporphyrin, reduced the liver injury. Depletion of hepatic non-protein sulfhydryls (NPSHs), an indicator of GSH, by combined treatment with buthionine sulfoximine (BSO) and diethyl maleate (DEM) produced marked elevations in serum transaminases by 6 hr after EN treatment. Administered on its own, EN decreased hepatic NPSH content and when combined with the BSO/DEM pretreatment, the liver was nearly completely devoid of NPSHs. Protection from EN-induced hepatotoxicity was observed in animals administered L-2-oxothiazolidine-4-carboxylic acid, a cysteine precursor. Together, these observations suggest the involvement of cytochrome P450 in EN bioactivation and GSH in detoxification. The results corroborate previous in vitro observations pertaining to the mechanism of EN-induced cytotoxicity towards primary cultures of rat hepatocytes. Although the doses of EN used in this study were far in excess of therapeutic doses, under certain circumstances, this metabolism-mediated toxicologic mechanism could form the basis for idiosyncratic liver injury in patients receiving EN therapy. PMID:1449535

  20. Unusual regioselectivity and active site topology of human cytochrome P450 2J2

    E-print Network

    Boyer, Edmond

    .S.A. daniel.mansuy@univ-paris5.fr RECEIVED DATE TITLE RUNNING HEAD: CYP2J2 regioselectivity and active site1 Unusual regioselectivity and active site topology of human cytochrome P450 2J2 . : This work Sciences (USA). Pierre Lafite , François André§ , Darryl C. Zeldin , Patrick M. Dansette , Daniel Mansuy

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

    PubMed Central

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

    2013-01-01

    Summary NADPH-cytochrome P450 reductase is a key component of the P450 mono-oxygenase drug-metabolizing system. There is evidence for a conformational equilibrium involving large-scale domain motions in this enzyme. We now show, using small-angle X-ray scattering (SAXS) and small-angle neutron scattering, that delivery of two electrons to cytochrome P450 reductase leads to a shift in this equilibrium from a compact form, similar to the crystal structure, toward an extended form, while coenzyme binding favors the compact form. We present a model for the extended form of the enzyme based on nuclear magnetic resonance and SAXS data. Using the effects of changes in solution conditions and of site-directed mutagenesis, we demonstrate that the conversion to the extended form leads to an enhanced ability to transfer electrons to cytochrome c. This structural evidence shows that domain motion is linked closely to the individual steps of the catalytic cycle of cytochrome P450 reductase, and we propose a mechanism for this. PMID:23911089

  2. Polymorphisms in cytochrome P450 17A1 and risk of non-Hodgkin lymphoma

    E-print Network

    California at Berkeley, University of

    Polymorphisms in cytochrome P450 17A1 and risk of non-Hodgkin lymphoma Immune function, believed of lymphoproliferative diseases such as non- Hodgkin lymphoma (NHL) may be influenced by hormonal modulation. Few studies )34T>C and CYP17A1 IVS2 105A>C polymorphisms were analyzed in a non-Hodgkin lymphoma (NHL) population

  3. 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...

  4. 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...

  5. 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)

  6. 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...

  7. Cytochrome P450 enzyme functionalized-quantum dots as photocatalysts for drug metabolism.

    PubMed

    Xu, Xuan; Qian, Jing; Yu, Jiachao; Zhang, Yuanjian; Liu, Songqin

    2014-07-21

    On the basis of the photo-induced electron transfer (PET) from CdTe quantum dots (QDs) to cytochrome P450 2C9 (CYP2C9), a light-controlled drug metabolism system was successfully designed by using CYP2C9 functionalized-CdTe QDs as photocatalysts. PMID:24821498

  8. 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 ...

  9. 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...

  10. Differential induction of cytochrome P450 transcripts in Papilio polyxenes by linear and angular furanocoumarins

    Microsoft Academic Search

    Chien-Fu Hung; Hataichanoke Prapaipong; May R. Berenbaum; Mary A. Schuler

    1995-01-01

    Cytochrome P450 monooxygenases play critical roles in the detoxification of linear and angular furanocoumarins present in the host plants of Papilio polyxenes (black swallowtail) larvae. To determine the spectrum of linear and angular furanocoumarins that induce isozymes responsible for these detoxifications, the metabolic activities of individual P. polyxenes larvae were monitored in response to individual furanocoumarins. Two linear furanocoumarins, xanthotoxin

  11. 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...

  12. 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...

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

    Microsoft Academic Search

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

    1997-01-01

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

  14. A diverse family of thermostable cytochrome P450s created by recombination of stabilizing fragments

    E-print Network

    Snow, Christopher

    A diverse family of thermostable cytochrome P450s created by recombination of stabilizing fragments­9. In an effort to create new biocatalysts for these applications, we used structure-guided SCHEMA recombination of the activities of 14 chimeras demonstrated that the sequence diversity created by SCHEMA recombination also

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

    EPA Science Inventory

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

  16. 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...

  17. Cloning, Functional Expression, and Subcellular Localization of Multiple NADPH-Cytochrome P450 Reductases from Hybrid Poplar

    Microsoft Academic Search

    Dae-Kyun Ro; Jurgen Ehlting; Carl J. Douglas

    2002-01-01

    NADPH:cytochrome P450 reductase (CPR) provides reducing equivalents to diverse cytochrome P450 monooxygenases. We isolated cDNAs for three CPR genes (CPR1, CPR2, and CPR3) from hybrid poplar (Populus trichocarpa Populus deltoides). Deduced CPR2 and CPR3 amino acid sequences were 91% identical, but encoded isoforms divergent from CPR1 (72% identity). CPR1 and CPR2 were co-expressed together with the P450 enzyme cinnamate-4-hydroxylase (C4H)

  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. 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. 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...

  1. 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...

  2. 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...

  3. 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...

  4. 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...

  5. 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 ...

  6. Isoform-Specific Regulation of Cytochromes P450 Expression by Estradiol and Progesterone

    PubMed Central

    Choi, Su-Young; Koh, Kwi Hye

    2013-01-01

    Results from clinical studies suggest that pregnancy alters hepatic drug metabolism in a cytochrome P450 (P450) isoform-specific manner, and rising concentrations of female hormones are potentially responsible for the changes. The objective of this study was to comprehensively characterize the effects of estrogen and progesterone on the expression and activity of major drug-metabolizing P450s. To this end, primary human hepatocytes were treated with estradiol and progesterone, and mRNA expression and activity levels of 10 different P450 isoforms were determined. The results showed that estradiol enhances CYP2A6, CYP2B6, and CYP3A4 expression, whereas progesterone induces CYP2A6, CYP2B6, CYP2C8, CYP3A4, and CYP3A5 expression. The induction was mainly observed when the average hormone concentrations were at the levels reached during pregnancy, suggesting that these effects are likely pregnancy-specific. Estradiol also increased enzyme activities of CYP2C9 and CYP2E1 without affecting the mRNA expression levels by unknown mechanisms. Taken together, our results show differential effects of estrogen and progesterone on P450 expression, suggesting involvement of different regulatory mechanisms in female hormone-mediated P450 regulation. Our findings potentially provide a basis in mechanistic understanding for altered drug metabolism during pregnancy. PMID:22837389

  7. 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 Å resolution crystal structure of this enzyme ligand complex reveals that the highly plastic 2B6 utilizes previously unrecognized rearrangements of protein motifs. The results indicate that the specific components of enthalpic contribution to ligand binding are closely tied to the degree of enzyme flexibility. PMID:23786449

  8. 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, 1273–1296. PMID:20446763

  9. 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

  10. The effects of the phosphorothioate insecticide fenitrothion on mammalian cytochrome P450-dependent metabolism of estradiol.

    PubMed

    Berger, C W; Sultatos, L G

    1997-06-01

    Phosphorothioate insecticides, such as fenitrothion, are suicide substrates of cytochromes P450 (P450). These compounds undergo oxidative desulfuration by P450 resulting in the release and subsequent binding of atomic sulfur to the enzyme. Consequently, the P450-dependent metabolism of certain endogenous substrates could be inhibited by exposure to these insecticides. Formation of 2-hydroxyestradiol (2-OHE2), 4-hydroxyestradiol (4-OHE2), 16 alpha-hydroxyestrone (16 alpha-OHE1), and estriol in mammals occurs by P450-dependent hydroxylation of estradiol at various positions on the steroid nucleus. In the present study, pretreatment of male Swiss Webster mice with increasing doses of fenitrothion resulted in dose-dependent biphasic decreases in 2-OHE2 and 4-OHE2 production in mouse hepatic microsomes compared to control, with substantial decreases even at a dosage as low as 7 mg/kg. Fenitrothion pretreatment also resulted in dose-dependent biphasic increases in 16 alpha-OHE1 and estriol production, along with substantial increases in estrone formation, probably as a result of shunting from the inhibition of 2- and 4-hydroxylation. These data suggest that exposure to fenitrothion might alter estradiol metabolism by inhibition of certain P450 isozymes. PMID:9242588

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

    SciTech Connect

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

    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.

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

    SciTech Connect

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

    1994-12-31

    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.

  13. 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

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

    PubMed

    Liu, Huijun

    2010-01-01

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

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

    2008-02-01

    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.

  16. 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.

  17. Dynamic Mobility of Genetically Expressed Fusion Protein between Cytochrome P4501A1 and NADPH-Cytochrome P450 Reductase in Yeast Microsomes

    E-print Network

    Kawato, Suguru

    Ved March 19, 1999 ABSTRACT: A fusion protein of rat liver CYP1A1 with NADPH-cytochrome P450 reductase reductase in microsomal membranes. Cytochrome P450 is the terminal enzyme of the hepatic microsomal substrates (1-3). The monooxygenase systems consist of several membrane proteins such as NADPH- cytochrome P

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

    SciTech Connect

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

    2013-02-15

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

  19. Cytochromes P-450 catalyze the formation of marchantins A and CinMarchantia polymorpha

    E-print Network

    Susanne Friederich A; Martina Rue€er A; Yoshinori Asakawa B; Meinhart H. Zenk A

    1999-01-01

    Two speci®c cytochrome P-450 enzymes were detected in cell suspension cultures of Marchantia polymorpha; the ®rst catalyzes the coupling of two molecules of lunularic acid to form marchantin C and CO2 and the second hydroxylates marchantin C to marchantin A. Cell free experiments using 3 H = 14 C doubly-labeled substrates demonstrated that lunularic acid, and neither lunularine nor prelunularic acid, is the sole substrate for the coupling reaction. Both enzymes are dependent on the presence of oxygen and NADPH. Both reactions were inhibited in the presence of CO in the dark; this inhibition was partially reversed by white light. In addition, both reactions were inhibited by typical inhibitors of cytochrome P-450 enzymes such as cytochrome c

  20. Inhibitory and non-inhibitory monoclonal antibodies to human cytochrome P450 3A3\\/4

    Microsoft Academic Search

    Harry V. Gelboin; Kristopher W. Krausz; Inna Goldfarb; Jeroen T. M. Buters; Shen K. Yang; Frank J. Gonzalez; Kenneth R. Korzekwa; Magang Shou

    1995-01-01

    Cytochromes P450 3A3\\/4 are inordinately important P450 enzymes catalyzing the metabolism of a large variety of clinically useful drugs, steroids, and carcinogens. Two monoclonal antibodies, MAb 3-29-9 and MAb 275-1-2, were prepared to human P450 3A4 from mice immunized with baculovirus-expressed human P450 3A4. MAb 3-29-9 was a powerful inhibitor of the enzymatic activity of P450 3A3\\/4\\/5. MAb 3-29-9 inhibited

  1. 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

  2. UNDERSTANDING THE MECHANISM OF CYTOCHROME P450 3A4: RECENT ADVANCES AND REMAINING PROBLEMS

    PubMed Central

    Sevrioukova, Irina F.; Poulos, Thomas L.

    2013-01-01

    Cytochromes P450 (CYPs) represent a diverse group of heme-thiolate proteins found in almost all organisms. CYPs share a common protein fold but differ in substrate selectivity and catalyze a wide variety of monooxygenation reactions via activation of molecular oxygen. Among 57 human P450s, the 3A4 isoform (CYP3A4) is the most abundant and the most important because it metabolizes the majority of the administered drugs. A remarkable feature of CYP3A4 is its extreme promiscuity in substrate specificity and cooperative substrate binding, which often leads to undesirable drug-drug interactions and toxic side effects. Owing to its importance in drug development and therapy, CYP3A4 has been the most extensively studied mammalian P450. In this review we provide an overview on recent progress and remaining problems in the CYP3A4 research. PMID:23018626

  3. 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.

  4. Expression, purification, and biochemical characterization of a human cytochrome P450 CYP2D6-NADPH cytochrome P450 reductase fusion protein.

    PubMed

    Deeni, Y Y; Paine, M J; Ayrton, A D; Clarke, S E; Chenery, R; Wolf, C R

    2001-12-01

    Cytochrome P450 CYP2D6 metabolizes a wide range of pharmaceutical compounds. A CYP2D6 fusion enzyme (CYP2D6F), containing an amino-terminal human CYP2D6 sequence and a carboxyterminal human NADPH-cytochrome P450 oxidoreductase (CPR) moiety, was constructed. High levels of expression were achieved in Escherichia coli (60-100 nmol/liter) and the enzyme was catalytically active with optimal activities achieved in the presence of the antioxidant, GSH. Turnover values for bufuralol 1'-hydroxylation, metoprolol alpha-hydroxylation, O-desmethylation, and dextromethorphan O-demethylation, using membranes expressing the fusion enzyme, were 5.6, 0.4, 0.72, and 6.19 min(-1), respectively. These values were similar to E. coli membranes which coexpressed human CYP2D6 and CPR (CYP2D6/R). The K(m) and k(cat) values for bufuralol metabolism were estimated to be 10.2 microM and 4.1 min(-1), respectively. The enzyme was purified using ion-exchange chromatography, affinity chromatography (2'-5' ADP-Sepharose), and gel filtration. Estimated turnover rates for bufuralol 1'-hydroxylation, metoprolol alpha-hydroxylation, O-desmethylation, and dextromethorphan O-demethylation were 1.2, 0.52, 0.79, and 0.76 min(-1), respectively. Bufuralol 1'-hydroxylase activity by purified CYP2D6F was enhanced by phospholipids and added CPR. The CYP2D6F enzyme was able to stimulate CYP3A4 testosterone 6beta-hydroxylase activity in a reconstitution system indicating that electron transfer may be largely intermolecular. The catalytically self-sufficient CYP2D6F enzyme will facilitate investigations of P450-CPR interactions and the development of new biocatalysts. PMID:11716457

  5. Cytochrome P450 genes from Helicoverpa armigera: expression in a pyrethroid-susceptible and -resistant strain.

    PubMed

    Pittendrigh, B; Aronstein, K; Zinkovsky, E; Andreev, O; Campbell, B; Daly, J; Trowell, S; Ffrench-Constant, R H

    1997-06-01

    The molecular basis of metabolic resistance to pyrethroids in Helicoverpa armigera is currently under debate. Substantial indirect evidence supports a role for both esterase- and cytochrome-P450-mediated metabolism. However, the relative roles played by these two mechanisms in field-based resistance is uncertain. Our understanding of the importance of P450-mediated metabolism is hindered by the paucity of cloned genes from this species, and the corresponding absence of data on rates of insecticide metabolism by functionally expressed P450s. To facilitate P450 gene isolation from H. armigera we used degenerate primers in the reverse transcriptase-polymerase chain reaction (RT-PCR) to clone P450 gene fragments from the RNA of a pyrethroid-resistant strain. Here we report the isolation of eight new P450 genes: seven from the CYP4 family and one CYP9. One of these genes, CYP4G8, is two-fold over-expressed in the resistant strain, whereas the other CYP4s showed either similar or undetectable levels of expression. CYP9A3 appears to be a homolog of the putatively resistance-associated CYP9A1 of Heliothis virescens. However, no difference in expression between the H. armigera strains was detected. CYP6B2, a gene previously reported to be over-expressed in a different pyrethroid-resistant strain of H. armigera, also revealed non-detectable levels of expression in both strains. These observations suggest that different P450s may be over-expressed in different resistant strains, and emphasize that recombinant expression will be necessary in order to define precisely their individual substrate specificities and ability to metabolize pyrethroids. The gene fragments described here represent an important first step in this direction. PMID:9304792

  6. Comparison of cytochrome P450 3A enzymes in cynomolgus monkeys and humans.

    PubMed

    Iwasaki, Kazuhide; Murayama, Norie; Koizumi, Ryo; Uno, Yasuhiro; Yamazaki, Hiroshi

    2010-01-01

    Drug metabolizing activities of cytochromes P450 (P450s, or CYPs) 3A4 and 3A5 in liver microsomes from the cynomolgus monkey [Macaca fascicularis (mf)] were investigated and compared with those of human P450 3A enzymes. Low activities for dealkylation of ethoxyresorufin and pentoxyresorufin were seen in recombinant monkey mfCYP3A4 and mfCYP3A5 and in recombinant human CYP3A4 and CYP3A5 expressed in bacterial membranes. Hydroxylation activities of mfCYP3A4 and mfCYP3A5 toward coumarin, paclitaxel, diclofenac, flurbiprofen, and S-mephenytoin were below detectable levels, as was also true for CYP3A4 and CYP3A5. Monkey mfCYP3A5 and mfCYP3A4 were highly active in bufuralol 1'-hydroxylation. mfCYP3A5 was efficient at dextromethorphan O-demethylation, although human CYP3A5 was unable to catalyze this reaction. Apparent bufuralol 1'-hydroxylation and dextromethorphan O-demethylation activities of monkey liver microsomes were higher than those of human liver microsomes, possibly because of contributions of mfCYP3A5 to these P450 2D-dependent drug oxidations. mfCYP3A5 and CYP3A5 catalyzed midazolam 1'-hydroxylation at a low substrate concentration more efficiently than the corresponding CYP3A4. mfCYP3A5 had higher testosterone 6beta-hydroxylase activity than mfCYP3A4, but the reverse relationship was observed in oxidation of nifedipine and hydroxylation of dexamethasone. These results demonstrate that monkey P450 3A enzymes have similar substrate selectivity to that of human P450 3A enzymes, but exhibit wider substrate selectivity toward P450 2D substrates. PMID:20814160

  7. Signals for the incorporation and orientation of cytochrome P450 in the endoplasmic reticulum membrane

    PubMed Central

    1988-01-01

    Cytochrome P450b is an integral membrane protein of the rat hepatocyte endoplasmic reticulum (ER) which is cotranslationally inserted into the membrane but remains largely exposed on its cytoplasmic surface. The extreme hydrophobicity of the amino-terminal portion of P450b suggests that it not only serves to initiate the cotranslational insertion of the nascent polypeptide but that it also halts translocation of downstream portions into the lumen of the ER and anchors the mature protein in the membrane. In an in vitro system, we studied the cotranslational insertion into ER membranes of the normal P450b polypeptide and of various deletion variants and chimeric proteins that contain portion of P450b linked to segments of pregrowth hormone or bovine opsin. The results directly established that the amino-terminal 20 residues of P450b function as a combined insertion-halt-transfer signal. Evidence was also obtained that suggests that during the early stages of insertion, this signal enters the membrane in a loop configuration since, when the amino-terminal hydrophobic segment was placed immediately before a signal peptide cleavage site, cleavage by the luminally located signal peptidase took place. After entering the membrane, the P450b signal, however, appeared to be capable of reorienting within the membrane since a bovine opsin peptide segment linked to the amino terminus of the signal became translocated into the microsomal lumen. It was also found that, in addition to the amino- terminal combined insertion-halt-transfer signal, only one other segment within the P450b polypeptide, located between residues 167 and 185, could serve as a halt-transfer signal and membrane-anchoring domain. This segment was shown to prevent translocation of downstream sequences when the amino-terminal combined signal was replaced by the conventional cleavable insertion signal of a secretory protein. PMID:3047140

  8. 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

  9. 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.

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

    SciTech Connect

    VandenBerg, M. [Utrecht Univ. (Netherlands). Research Inst. of Toxicology; 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.

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

    PubMed

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

    2015-02-01

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

  12. Black swallowtail (Papilio polyxenes) alleles encode cytochrome P450s that selectively metabolize linear furanocoumarins.

    PubMed

    Ma, R; Cohen, M B; Berenbaum, M R; Schuler, M A

    1994-05-01

    Cytochrome P450 monooxygenases are involved in metabolism of hostplant allelochemicals by larval Lepidoptera. Biochemical purification of the P450 polypeptide induced in Papilio polyxenes (black swallowtail) larvae in response to xanthotoxin, a linear furanocoumarin, has allowed us to clone cDNAs encoding two allelic variants of the CYP6B1 locus. Expression of these alleles in lepidopteran cell lines using baculovirus expression vectors has demonstrated that both P450 isoforms metabolize substantial amounts of linear furanocoumarins, such as xanthotoxin and bergapten, but not angular furanocoumarins, such as angelicin and sphondin. These linear furanocoumarins are ubiquitous constituents of the hostplants of P. polyxenes. The efficiency of linear furanocoumarin metabolism is strongly affected by the nature of the substituents on the benzene ring; methoxy-derivatives are metabolized more efficiently than are derivatives with smaller (hydroxy-) or larger (8-O isopentenyl) groups. Metabolism of either bergapten or xanthotoxin is inhibited in the presence of the other. In addition, metabolism of linear furanocoumarins is inhibited by the presence of nonmetabolizable angular furanocoumarins, indicating that the active site of CYP6B1 binds angular furanocoumarins. The reactivities described here indicate that P. polyxenes larvae express at least two selective furanocoumarin-metabolic P450s: CYP6B1, which metabolizes a discrete set of linear furanocoumarins, and another P450, as yet unidentified, which metabolizes angular furanocoumarins more efficiently than does CYP6B1. PMID:8179316

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

    SciTech Connect

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

    2013-01-25

    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.

  14. Effects of polyoxyethylene (40) stearate on the activity of P-glycoprotein and cytochrome P450

    Microsoft Academic Search

    Saijie Zhu; Rongqin Huang; Minghuang Hong; Yanyan Jiang; Zhuohan Hu; Chen Liu; Yuanying Pei

    2009-01-01

    The present study was aimed to investigate the effects of polyoxyethylene (40) stearate (PS), a non-ionic surfactant, on the activity of P-glycoprotein (P-gp) and six major cytochrome P450 (CYP) isoforms. An in vitro diffusion chamber system was utilized to estimate the effects of PS concentration on the transport characteristics of Rhodamine 123 (R123) and Rhodamine 110 (R110), a standard P-gp

  15. Colorectal cancer-specific cytochrome P450 2W1: intracellular localization, glycosylation, and catalytic activity.

    PubMed

    Gomez, Alvin; Nekvindova, Jana; Travica, Sandra; Lee, Mi-Young; Johansson, Inger; Edler, David; Mkrtchian, Souren; Ingelman-Sundberg, Magnus

    2010-12-01

    Cytochrome P450 2W1 (CYP2W1) is expressed at high levels in colorectal cancer cells. Moreover, we have shown previously that a higher tumor expression is associated with less survival. In this study, we characterize post-translational modification, inverted endoplasmic reticulum (ER) topology, and catalytic activity of CYP2W1. The analysis of colorectal normal and cancer tissues and CYP2W1 overexpressing human embryonic kidney (HEK) 293 cells showed that a fraction of CYP2W1 is modified by N-glycosylation. Bioinformatic analysis identified Asn177 as the only possible glycosylation site of CYP2W1, which was supported by the inability of an N177A mutant to be glycosylated in HEK 293 cells. Analysis of the membrane topology indicated that unlike other cytochromes P450, CYP2W1 in HEK 293-transfected cells and in nontransfected Caco2TC7 and HepG2 cells is oriented toward the lumen of the ER, a topology making CYP2W1 available to the ER glycosylation machinery. Immunofluorescence microscopy and cell surface biotinylation experiments revealed approximately 8% of the CYP2W1 on the cell surface. Despite the reverse orientation of CYP2W1 in the ER membrane, apparently making functional interactions with NADPH-cytochrome P450 reductase impossible, CYP2W1 in HEK 293 cells was active in the metabolism of indoline substrates and was able to activate aflatoxin B1 into cytotoxic products. The study identifies for the first time a cytochrome P450 enzyme with a luminal ER orientation and still retaining catalytic activity. Together, these results suggest the possibility of using CYP2W1 as a drug target in the treatment of colon cancer using antibodies and/or specific CYP2W1 activated prodrugs. PMID:20805301

  16. In vivo effects of interleukin-10 on human cytochrome P450 activity

    Microsoft Academic Search

    J. Christopher Gorski; Stephen D. Hall; Paula Becker; Melton B. Affrime; David L. Cutler; Barbara Haehner-Daniels

    2000-01-01

    Background: Injection of lipopolysaccharide into human volunteers leads to an increase in serum interleukin-1?, interleukin-6, and tumor necrosis factor-? and a significant decrease in cytochrome P450 (CYP)–mediated drug metabolism. The in vivo effects of the noninflammatory cytokine interleukin-10 (IL-10) on CYP–mediated drug metabolism was examined.Methods: IL-10 (8 ?g\\/kg) and placebo were administered for 6 days to 12 healthy volunteers in

  17. The Effect of Echinacea (Echinacea purpurea Root) on Cytochrome P450 Activity in Vivo

    Microsoft Academic Search

    J. Christopher Gorski; Shiew-Mei Huang; Amar Pinto; Mitchell A. Hamman; Janna K. Hilligoss; Narjis A. Zaheer; Mehul Desai; Margaret Miller; Stephen D. Hall

    2004-01-01

    Background: Echinacea is a widely available over-the-counter herbal remedy. Tinctures of echinacea have been shown to inhibit cytochrome P450 (CYP) in vitro. The effect of echinacea (Echinacea purpurea root) on CYP activity in vivo was assessed by use of the CYP probe drugs caffeine (CYP1A2), tolbutamide (CYP2C9), dextromethorphan (CYP2D6), and midazolam (hepatic and intestinal CYP3A).Methods: Twelve healthy subjects (6 men)

  18. Artemisinin antimalarials moderately affect cytochrome P450 enzyme activity in healthy subjects

    Microsoft Academic Search

    Sara Asimus; Doaa Elsherbiny; Trinh N. Hai; Britt Jansson; Nguyen V. Huong; Max G. Petzold; Ulrika S. H. Simonsson; Michael Ashton

    2007-01-01

    The aim of this study was to investigate which principal human cytochrome P450 (CYP450) enzymes are affected by artemisinin and to what degree the artemisinin derivatives differ with respect to their respective induction and inhibition capacity. Seventy-five healthy adults were randomized to receive therapeutic oral doses of artemisinin, dihydroartemisinin, arteether, artemether or artesunate for 5 days (days 1-5). A six-drug

  19. Metabolism of eicosapentaenoic and docosahexaenoic acids by recombinant human cytochromes P450

    Microsoft Academic Search

    Maude Fer; Yvonne Dréano; Danièle Lucas; Laurent Corcos; Jean-Pierre Salaün; François Berthou; Yolande Amet

    2008-01-01

    Epoxidation and hydroxylation of arachidonic acid (AA) are both catalyzed by cytochromes P450s (CYPs). The oxidized metabolites are known to be involved in the regulation of vascular tone and renal function. By using a panel of 15 human recombinant CYPs, this study demonstrates that other polyunsaturated long-chain fatty acids (PUFA-LC), especially the ?3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic

  20. Effects of aprepitant on cytochrome P450 3A4 activity using midazolam as a probe

    Microsoft Academic Search

    Anup K. Majumdar; Jacqueline B. McCrea; Deborah L. Panebianco; Michael Hesney; James Dru; Marvin Constanzer; Michael R. Goldberg; Gail Murphy; Keith M. Gottesdiener; Christopher R. Lines; Kevin J. Petty; Robert A. Blum

    2003-01-01

    Background: Aprepitant is a neurokinin1 receptor antagonist that enhances prevention of chemotherapy-induced nausea and vomiting when added to conventional therapy with a corticosteroid and a 5-hydroxytryptamine3 (5-HT3) antagonist. Because aprepitant may be used with a variety of chemotherapeutic agents and ancillary support drugs, which may be substrates of cytochrome P450 (CYP) 3A4, assessment of the potential of this drug to

  1. Concomitant use of cytochrome P450 3A4 inhibitors and simvastatin

    Microsoft Academic Search

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

    1999-01-01

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

  2. Effect of Cytochrome P450 3A4 Inhibition on the Pharmacokinetics of Docetaxel

    Microsoft Academic Search

    Frederike K. Engels; Albert J. ten Tije; Sharyn D. Baker; Carlton K. K. Lee; Walter J. Loos; Arnold G. Vulto; Jaap Verweij; Alex Sparreboom

    2004-01-01

    Objective: In vitro studies indicate that the anticancer drug docetaxel is primarily eliminated by cytochrome P450 (CYP) 3A4–mediated metabolism. Coadministration of drugs that modulate the activity of CYP3A4 is, therefore, likely to have undesirable clinical consequences. We investigated the effects of the potent CYP3A4 inhibitor ketoconazole on the pharmacokinetics of docetaxel in patients with cancer.Methods: Seven patients were treated in

  3. From the Cover: Structural basis for ligand promiscuity in cytochrome P450 3A4

    Microsoft Academic Search

    Marika Ekroos; Tove Sjögren

    2006-01-01

    Cytochrome P450 (CYP) 3A4 is the most promiscuous of the human CYP enzymes and contributes to the metabolism of 50% of marketed drugs. It is also the isoform most often involved in unwanted drug-drug interactions. A better understanding of the molecular mechanisms governing CYP3A4-ligand interaction therefore would be of great importance to any drug discovery effort. Here, we present crystal

  4. Monocrotophos Induced Apoptosis in PC12 Cells: Role of Xenobiotic Metabolizing Cytochrome P450s

    Microsoft Academic Search

    Mahendra Pratap Kashyap; Abhishek Kumar Singh; Vivek Kumar; Vinay Kumar Tripathi; Ritesh Kumar Srivastava; Megha Agrawal; Vinay Kumar Khanna; Sanjay Yadav; Swatantra Kumar Jain; Aditya Bhushan Pant; Neeraj Vij

    2011-01-01

    Monocrotophos (MCP) is a widely used organophosphate (OP) pesticide. We studied apoptotic changes and their correlation with expression of selected cytochrome P450s (CYPs) in PC12 cells exposed to MCP. A significant induction in reactive oxygen species (ROS) and decrease in glutathione (GSH) levels were observed in cells exposed to MCP. Following the exposure of PC12 cells to MCP (10?5 M),

  5. Fatal intoxication cases: cytochrome P 450 2D6 and 2C19 genotype distributions

    Microsoft Academic Search

    A. L. Zackrisson; P. Holmgren; A. B. Gladh; J. Ahlner; B. Lindblom

    2004-01-01

    Objective Many commonly used pharmaceuticals, such as antidepressants and neuroleptics as well as some illegal drugs, are metabolised by the cytochrome P 450 enzyme debrisoquine 4-hydroxylase (CYP2D6). Of Caucasians, 7–10% lack this enzyme, which can, upon administration of drugs in normal therapeutic doses, lead to adverse reactions and unexpected intoxication, leading in turn even to a fatal outcome in some

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

    PubMed

    Nielsen, J S; Moller, B L

    1999-08-01

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

  7. Characterization and tissue distribution of a novel human cytochrome P450—CYP2U1

    Microsoft Academic Search

    Maria Karlgren; Maria Backlund; Inger Johansson; Mikael Oscarson; Magnus Ingelman-Sundberg

    2004-01-01

    A novel human cytochrome P450 cDNA designated CYP2U1 was identified using homology searches, and the corresponding gene is located on chromosome 4. The deduced 544 amino acid sequence displays up to 39% identity to other CYP2 family members, with closest resemblance to CYP2R1 and is highly conserved between species. CYP2U1 shows some structural differences compared to other CYP2 family members.

  8. CYPalleles: a web page for nomenclature of human cytochrome P450 alleles.

    PubMed

    Oscarson, Mikael; Ingelman-Sundberg, Magnus

    2002-01-01

    Genetic variations in the genes encoding the cytochrome P450s (CYPs) are important determinants for interindividual differences in sensitivity to drugs and environmental chemicals as well as for the pathogenesis of several human diseases. In order to standardise the nomenclature of the rapidly increasing number of alleles described, a web page was established a few years ago. Here, we describe the present status of the web page and summarise the principles used for CYP allele nomenclature. PMID:15618703

  9. Cytochromes P-450 catalyze the formation of marchantins A and C in Marchantia polymorpha

    Microsoft Academic Search

    Susanne Friederich; Martina Rueffer; Yoshinori Asakawa; Meinhart H. Zenk

    1999-01-01

    Two specific cytochrome P-450 enzymes were detected in cell suspension cultures of Marchantia polymorpha; the first catalyzes the coupling of two molecules of lunularic acid to form marchantin C and CO2 and the second hydroxylates marchantin C to marchantin A. Cell free experiments using 3H\\/14C doubly-labeled substrates demonstrated that lunularic acid, and neither lunularine nor prelunularic acid, is the sole

  10. Amount and type of dietary lipid modulate rat hepatic cytochrome P?450 activity

    Microsoft Academic Search

    1997-01-01

    The influence of the amount and type of dietary lipid on rat hepatic cytochrome P?450 activities in the presence and absence of inducer administration was investigated. Weanling male Sprague?Dawley rats were fed fat?free or 20% beef tallow, olive oil, corn oil, linseed oil, or menhaden oil diets in combination with one of the following three treatments: no inducer, intraperitoneal injection

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

    Microsoft Academic Search

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

    2002-01-01

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

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

    Microsoft Academic Search

    Vasilis P Androutsopoulos; Aristidis M Tsatsakis; Demetrios A Spandidos

    2009-01-01

    CYP1A1 is one of the main cytochrome P450 enzymes, examined extensively for its capacity to activate compounds with carcinogenic properties. Continuous exposure to inhalation chemicals and environmental carcinogens is thought to increase the level of CYP1A1 expression in extrahepatic tissues, through the aryl hydrocarbon receptor (AhR). Although the latter has long been recognized as a ligand-induced transcription factor, which is

  13. The human genome project and novel aspects of cytochrome P450 research

    Microsoft Academic Search

    Magnus Ingelman-Sundberg

    2005-01-01

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

  14. Bovine Bladder Mucosa Microsomal Cytochrome P-450 and 4Aminobiphenyl \\/V-Hydroxylase Activity1

    Microsoft Academic Search

    Jay M. Poupko; Jack L. Radomski; W. Lee Hearn

    The potential for metabolic activation of bladder carcinogens by the bladder mucosa was examined by determining if bladder mucosa microsomes contain cytochrome P-450, exhibit typical microsomal-substrate interactions, and are capable of mediat ing the N-hydroxylation of the bladder carcinogen, 4-aminobi- phenyl (4-ABP). The carbon monoxide difference spectrum of reduced bovine bladder microsomes exhibited an absorption maximum at 450 nm and

  15. Genetic and molecular analysis of a cytochrome P450 based pyrethroid resistance in the red flour beetle, Tribolium castaneum (Herbst)

    Microsoft Academic Search

    Suparna Roy Ray

    1998-01-01

    Detoxification by cytochrome P450 monooxygenases (P450s) is a major mechanism by which insect pests become resistant to insecticides. The purpose of the present study was to investigate the P450 basis of a high level pyrethroid resistance in the field derived Tribolium castaneum strain QTC279. QTC279 was shown to carry a major gene for pyrethroid resistance, PyrR, on linkage group 9

  16. 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.

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

    SciTech Connect

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

    2009-06-19

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

  18. Water oxidation by a cytochrome p450: mechanism and function of the reaction.

    PubMed

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

    2013-01-01

    P450(cam) (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 P450(cam) catalysis is controlled by oxygen levels: at high O2 concentration, P450(cam) catalyzes the known oxidation reaction, whereas at low O2 concentration the enzyme catalyzes the reduction of camphor to borneol. We confirmed, using (17)O and (2)H 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 P450(cam) , 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 P450(cam) 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

  19. Induction of renal cytochrome P450 arachidonic acid epoxygenase activity by dietary gamma-linolenic acid.

    PubMed

    Yu, Zhigang; Ng, Valerie Y; Su, Ping; Engler, Marguerite M; Engler, Mary B; Huang, Yong; Lin, Emil; Kroetz, Deanna L

    2006-05-01

    Dietary gamma-linolenic acid (GLA), a omega-6 polyunsaturated fatty acid found in borage oil (BOR), lowers systolic blood pressure in spontaneously hypertensive rats (SHRs). GLA is converted into arachidonic acid (AA) by elongation and desaturation steps. Epoxyeicosatrienoic acids (EETs) and 20-hydroxyeicosatetraenoic acid (20-HETE) are cytochrome P450 (P450)-derived AA eicosanoids with important roles in regulating blood pressure. This study tested the hypothesis that the blood pressure-lowering effect of a GLA-enriched diet involves alteration of P450-catalyzed AA metabolism. Microsomes and RNA were isolated from the renal cortex of male SHRs fed a basal fat-free diet for 5 weeks to which 11% by weight of sesame oil (SES) or BOR was added. There was a 2.6- to 3.5-fold increase in P450 epoxygenase activity in renal microsomes isolated from the BOR-fed SHRs compared with the SES-fed rats. Epoxygenase activity accounted for 58% of the total AA metabolism in the BOR-treated kidney microsomes compared with 33% in the SES-treated rats. More importantly, renal 14,15- and 8,9-EET levels increased 1.6- to 2.5-fold after dietary BOR treatment. The increase in EET formation is consistent with increases in CYP2C23, CYP2C11, and CYP2J protein levels. There were no differences in the level of renal P450 epoxygenase mRNA between the SES- and BOR-treated rats. Enhanced synthesis of the vasodilatory EETs and decreased formation of the vasoconstrictive 20-HETE suggests that changes in P450-mediated AA metabolism may contribute, at least in part, to the blood pressure-lowering effect of a BOR-enriched diet. PMID:16421287

  20. 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

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed Central

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

    2014-01-01

    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

  3. Constitutive overexpression of multiple cytochrome P450 genes associated with pyrethroid resistance in Helicoverpa armigera.

    PubMed

    Yang, Yihua; Chen, Song; Wu, Shuwen; Yue, Lina; Wu, Yidong

    2006-10-01

    Our previous studies showed that enhanced cytochrome P450-mediated detoxification is a major mechanism responsible for pyrethroid resistance in a laboratory-selected strain (YGF) of Helicoverpa armigera (Hübner). Two new cytochrome P450 genes, CYP9A12 and CYP9A14 (encoding 531 and 530 amino acid residues, respectively) were isolated from the fat body of the YGF strain of H. armigera. The mRNA expression levels of these two CYP9 P450 genes, together with CYP6B7 and CYP4G8 (previously reported to be overexpressed in pyrethroid-resistant strains), were assessed by real-time quantitative reverse transcription-polymerase chain reaction in the final instars of a field-derived YG strain (with seven-fold resistance to the pyrethroid fenvalerate) and the YGF strain (selected from the YG strain with fenvalerate in the laboratory, with 1,690-fold resistance to fenvalerate). The mRNA expression levels of CYP9A12, CYP9A14, and CYP6B7 in the fat body of the YGF strain increased to 433-, 59-, and 9.3-fold, respectively, compared with the YG strain, whereas no overexpression was revealed for CYP4G8. CYP9A12 and CYP9A14 had 19- and 4.3-fold overexpression in the midgut of the YGF strain compared with the YG strain, respectively, but CYP6B7 and CYP4G8 were not overexpressed. The current study provided evidence that constitutive overexpression of multiple cytochrome P450 genes (CYP9A12, CYP9A14, and CYP6B7) is associated with pyrethroid resistance in H. armigera. PMID:17066813

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

    PubMed Central

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

    2014-01-01

    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-see 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 non-piezophilic enzymes. The studies of enzymes of piezophiles should help unravel the mechanisms that control water access during the catalytic cycle. PMID:23586990

  5. Oxidation of 7-dehydrocholesterol and desmosterol by human cytochrome P450 46A1.

    PubMed

    Goyal, Sandeep; Xiao, Yi; Porter, Ned A; Xu, Libin; Guengerich, F Peter

    2014-09-01

    Cytochrome P450 (P450 or CYP) 46A1 is expressed in brain and has been characterized by its ability to oxidize cholesterol to 24S-hydroxycholesterol. In addition, the same enzyme is known to further oxidize 24S-hydroxycholesterol to the 24,25- and 24,27-dihydroxy products, as well as to catalyze side-chain oxidations of 7?-hydroxycholesterol and cholestanol. As precursors in the biosynthesis of cholesterol, 7-dehydrocholesterol has not been found to be a substrate of P450 46A1 and desmosterol has not been previously tested. However, 24-hydroxy-7-dehydrocholesterol was recently identified in brain tissues, which prompted us to reexamine this enzyme and its potential substrates. Here we report that P450 46A1 oxidizes 7-dehydrocholesterol to 24-hydroxy-7-dehydrocholesterol and 25-hydroxy-7-dehydrocholesterol, as confirmed by LC-MS and GC-MS. Overall, the catalytic rates of formation increased in the order of 24-hydroxy-7-dehydrocholesterol < 24-hydroxycholesterol < 25-hydroxy-7-dehydrocholesterol from their respective precursors, with a ratio of 1:2.5:5. In the case of desmosterol, epoxidation to 24S,25-epoxycholesterol and 27-hydroxylation was observed, at roughly equal rates. The formation of these oxysterols in the brain may be of relevance in Smith-Lemli-Opitz syndrome, desmosterolosis, and other relevant diseases, as well as in signal transduction by lipids. PMID:25017465

  6. 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

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

    PubMed

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

    2012-09-01

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

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

    EPA Science Inventory

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

  9. Isolation and effects of citrus limonoids on cytochrome p450 inhibition, apoptotic induction and cytotoxicity on human cancer cells.

    E-print Network

    Poulose, Shibu M.

    2007-04-25

    This dissertation illustrates an efficient purification method for citrus limonoids and flavonoids, while examining their effects on cytochrome P450 inhibition and apoptotic induction on human neuroblastoma (SH-SY5Y) and colonic adenocarcinoma (Caco...

  10. Resveratrol, a red wine constituent, is a mechanism-based inactivator of cytochrome P450 3A4

    Microsoft Academic Search

    William K. Chan; Anthony B. Delucchi

    2000-01-01

    Resveratrol, a phytoalexin found in red wine, has been shown to possess antioxidant and antimutagenic properties. Incubation of resveratrol with Sf 9 insect microsomes containing baculovirus-derived human cytochrome P450 3A4 (CYP3A4) and NADPH-cytochrome P450 reductase showed that resveratrol inactivated CYP3A4 in a time- and NADPH-dependent manner. Resveratrol, erythromycin and troleandomycin inactivated CYP3A4 at a similar rate (as reflected by kinact)

  11. Assessing the Geometric Diversity of Cytochrome P450 Ligand Conformers by Hierarchical Clustering with a Stop Criterion

    E-print Network

    Paris-Sud XI, Université de

    is valid for all small molecules. 1. INTRODUCTION The cytochrome CYP 3A4 is a member of the P450 isoforms of the P450s family,3,4 we need to consider each ligand of the CYP 3A4 as being the union applied on a database containing 70 ligands of the cytochrome CYP 3A4, showing that the structural

  12. Role of the pepper cytochrome P450 gene CaCYP450A in defense responses against microbial pathogens

    Microsoft Academic Search

    In Sun Hwang; Byung Kook Hwang

    2010-01-01

    Plant cytochrome P450 enzymes are involved in a wide range of biosynthetic reactions, leading to various fatty acid conjugates,\\u000a plant hormones, or defensive compounds. Herein, we have identified the pepper cytochrome P450 gene CaCYP450A, which is differentially induced during Xanthomonas campestris pv. vesicatoria (Xcv) infection. CaCYP450A contains a heme-binding motif, PXFXXGXRXCXG, located in the C-terminal region and a hydrophobic membrane

  13. Crystal structure of the FMN-binding domain of human cytochrome P450 reductase at 1.93 A resolution.

    PubMed Central

    Zhao, Q.; Modi, S.; Smith, G.; Paine, M.; McDonagh, P. D.; Wolf, C. R.; Tew, D.; Lian, L. Y.; Roberts, G. C.; Driessen, H. P.

    1999-01-01

    The crystal structure of the FMN-binding domain of human NADPH-cytochrome P450 reductase (P450R-FMN), a key component in the cytochrome P450 monooxygenase system, has been determined to 1.93 A resolution and shown to be very similar both to the global fold in solution (Barsukov I et al., 1997, J Biomol NMR 10:63-75) and to the corresponding domain in the 2.6 A crystal structure of intact rat P450R (Wang M et al., 1997, Proc Nat Acad Sci USA 94:8411-8416). The crystal structure of P450R-FMN reported here confirms the overall similarity of its alpha-beta-alpha architecture to that of the bacterial flavodoxins, but reveals differences in the position, number, and length of the helices relative to the central beta-sheet. The marked similarity between P450R-FMN and flavodoxins in the interactions between the FMN and the protein, indicate a striking evolutionary conservation of the FMN binding site. The P450R-FMN molecule has an unusual surface charge distribution, leading to a very strong dipole, which may be involved in docking cytochrome P450 into place for electron transfer near the FMN. Several acidic residues near the FMN are identified by mutagenesis experiments to be important for electron transfer to P4502D6 and to cytochrome c, a clear indication of the part of the molecular surface that is likely to be involved in substrate binding. Somewhat different parts are found to be involved in binding cytochrome P450 and cytochrome c. PMID:10048323

  14. Purification of Cytochrome P450 and Ferredoxin, Involved in Bisphenol A Degradation, from Sphingomonas sp. Strain AO1

    PubMed Central

    Sasaki, Miho; Akahira, Ayako; Oshiman, Ko-ichi; Tsuchido, Tetsuaki; Matsumura, Yoshinobu

    2005-01-01

    In a previous study (M. Sasaki, J. Maki, K. Oshiman, Y. Matsumura, and T. Tsuchido, Biodegradation 16:449-459, 2005), the cytochrome P450 monooxygenase system was shown to be involved in bisphenol A (BPA) degradation by Sphingomonas sp. strain AO1. In the present investigation, we purified the components of this monooxygenase, cytochrome P450 (P450bisd), ferredoxin (Fdbisd), and ferredoxin reductase (Redbisd). We demonstrated that P450bisd and Fdbisd are homodimeric proteins with molecular masses of 102.3 and 19.1 kDa, respectively, by gel filtration chromatography analysis. Spectroscopic analysis of Fdbisd revealed the presence of a putidaredoxin-type [2Fe-2S] cluster. P450bisd, in the presence of Fdbisd, Redbisd, and NADH, was able to convert BPA. The Km and kcat values for BPA degradation were 85 ± 4.7 ?M and 3.9 ± 0.04 min?1, respectively. NADPH, spinach ferredoxin, and spinach ferredoxin reductase resulted in weak monooxygenase activity. These results indicated that the electron transport system of P450bisd might exhibit strict specificity. Two BPA degradation products of the P450bisd system were detected by high-performance liquid chromatography analysis and were thought to be 1,2-bis(4-hydroxyphenyl)-2-propanol and 2,2-bis(4-hydroxyphenyl)-1-propanol based on mass spectrometry-mass spectrometry analysis. This is the first report demonstrating that the cytochrome P450 monooxygenase system in bacteria is involved in BPA degradation. PMID:16332782

  15. Expression of human cytochrome P450 enzymes in yeast and bacteria and relevance to studies on catalytic specificity.

    PubMed

    Guengerich, F P; Gillam, E M; Ohmori, S; Sandhu, P; Brian, W R; Sari, M A; Iwasaki, M

    1993-10-01

    Heterologous expression systems can be utilized to great advantage in the study of cytochrome P450 (P450) and other enzymes involved in the biotransformation of drugs and other xenobiotics. The list of studies made possible with the technology includes discernment of catalytic specificity, elucidation of structure-activity relationships, and various biophysical measurements. There are advantages and disadvantages to each of the vector systems and choices must be made on the basis of needs. Yeast expression systems were used to establish that different P450 2C enzymes are involved in the hydroxylations of tolbutamide and (S)-mephenytion. P450 3A4 was also expressed in yeast and its very broad catalytic specificity was confirmed. Recently, it has been possible to express P450 3A4 as well as other human and animal P450s in bacteria after slight modification of their 5'-coding sequences. PMID:8236276

  16. 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

  17. 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 [The University of Texas Health Science Center at San Antonio, Department of Biochemistry, San Antonio, TX 78229 (United States)] [The University of Texas Health Science Center at San Antonio, Department of Biochemistry, San Antonio, TX 78229 (United States); Panda, Satya P., E-mail: panda@uthscsa.edu [The University of Texas Health Science Center at San Antonio, Department of Biochemistry, San Antonio, TX 78229 (United States)

    2011-08-05

    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.

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

    DOEpatents

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

    1993-01-01

    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.

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

    DOEpatents

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

    1992-01-01

    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.

  20. Decarboxylation of Fatty acids to terminal alkenes by cytochrome p450 compound I.

    PubMed

    Grant, Job L; Hsieh, Chun H; Makris, Thomas M

    2015-04-22

    OleTJE, a cytochrome P450, catalyzes the conversion of fatty acids to terminal alkenes using hydrogen peroxide as a cosubstrate. Analytical studies with an eicosanoic acid substrate show that the enzyme predominantly generates nonadecene and that carbon dioxide is the one carbon coproduct of the reaction. The addition of hydrogen peroxide to a deuterated substrate-enzyme (E-S) complex results in the transient formation of an iron(IV) oxo ? cation radical (Compound I) intermediate which is spectroscopically indistinguishable from those that perform oxygen insertion chemistries. A kinetic isotope effect for Compound I decay suggests that it abstracts a substrate hydrogen atom to initiate fatty acid decarboxylation. Together, these results indicate that the initial mechanism for alkene formation, which does not result from oxygen rebound, is similar to that widely suggested for P450 monooxygenation reactions. PMID:25843451

  1. Cytochrome P450scc-dependent metabolism of 7-dehydrocholesterol in placenta and epidermal keratinocytes.

    PubMed

    Slominski, Andrzej T; Kim, Tae-Kang; Chen, Jianjun; Nguyen, Minh N; Li, Wei; Yates, Charles R; Sweatman, Trevor; Janjetovic, Zorica; Tuckey, Robert C

    2012-11-01

    The discovery that 7-dehydrocholesterol (7DHC) is an excellent substrate for cytochrome P450scc (CYP11A1) opens up new possibilities in biochemistry. To elucidate its biological significance we tested ex vivo P450scc-dependent metabolism of 7DHC by tissues expressing high and low levels of P450scc activity, placenta and epidermal keratinocytes, respectively. Incubation of human placenta fragments with 7DHC led to its conversion to 7-dehydropregnenolone (7DHP), which was inhibited by dl-aminoglutethimide, and stimulated by forskolin. Final proof for P450scc involvement was provided in isolated placental mitochondria where production of 7DHP was almost completely inhibited by 22R-hydroxycholesterol. 7DHC was metabolized by placental mitochondria at a faster rate than exogenous cholesterol, under both limiting and saturating conditions of substrate transport, consistent with higher catalytic efficiency (k(cat)/K(m)) with 7DHC as substrate than with cholesterol. Ex vivo experiments showed five 5,7-dienal intermediates with MS spectra of dihydroxy and mono-hydroxy-7DHC and retention time corresponding to 20,22(OH)(2)7DHC and 22(OH)7DHC. The chemical structure of 20,22(OH)(2)7DHC was defined by NMR. 7DHP was further metabolized by either placental fragments or placental microsomes to 7-dehydroprogesterone as defined by UV, MS and NMR, and to an additional product with a 5,7-dienal structure and MS corresponding to hydroxy-7DHP. Furthermore, epidermal keratinocytes transformed either exogenous or endogenous 7DHC to 7DHP. 7DHP inhibited keratinocytes proliferation, while the product of its pholytic transformation, pregcalciferol, lost this capability. In conclusion, tissues expressing P450scc can metabolize 7DHC to biologically active 7DHP with 22(OH)7DHC and 20,22(OH)(2)7DHC serving as intermediates, and with further metabolism to 7-dehydroprogesterone and (OH)7DHP. PMID:22877869

  2. Involvement of a Cytochrome P450 Monooxygenase in Thaxtomin A Biosynthesis by Streptomyces acidiscabies

    PubMed Central

    Healy, F. G.; Krasnoff, S. B.; Wach, M.; Gibson, D. M.; Loria, R.

    2002-01-01

    The biosynthesis of the thaxtomin cyclic dipeptide phytotoxins proceeds nonribosomally via the thiotemplate mechanism. Acyladenylation, thioesterification, N-methylation, and cyclization of two amino acid substrates are catalyzed by the txtAB-encoded thaxtomin synthetase. Nucleotide sequence analysis of the region 3? of txtAB in Streptomyces acidiscabies 84.104 identified an open reading frame (ORF) encoding a homolog of the P450 monooxygenase gene family. It was proposed that thaxtomin A phenylalanyl hydroxylation was catalyzed by the monooxygenase homolog. The ORF was mutated in S. acidiscabies 84.104 by using an integrative gene disruption construct, and culture filtrate extracts of the mutant were assayed for the presence of dehydroxy derivatives of thaxtomin A. Reversed-phase high-performance liquid chromatography (HPLC) and HPLC-mass spectrometry indicated that the major component in culture filtrate extracts of the mutant was less polar and smaller than thaxtomin A. Comparisons of electrospray mass spectra as well as 1H- and 13C-nuclear magnetic resonance spectra of the purified compound with those previously reported for thaxtomins confirmed the structure of the compound as 12,15-N-dimethylcyclo-(l-4-nitrotryptophyl-l-phenylalanyl), the didehydroxy analog of thaxtomin A. The ORF, designated txtC, was cloned and the recombinant six-His-tagged fusion protein produced in Escherichia coli and purified from cell extracts. TxtC produced in E. coli exhibited spectral properties similar to those of cytochrome P450-type hemoproteins that have undergone conversion to the catalytically inactive P420 form. Based on these properties and the high similarity of TxtC to other well-characterized P450 enzymes, we conclude that txtC encodes a cytochrome P450-type monooxygenase required for postcyclization hydroxylation of the cyclic dipeptide. PMID:11889110

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

    PubMed

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

    2012-12-15

    Cynomolgus monkeys are widely used as primate models in preclinical studies. However, some differences are occasionally seen between monkeys and humans in the activities of cytochrome P450 enzymes. R- and S-warfarin are model substrates for stereoselective oxidation in humans. In this current research, the activities of monkey liver microsomes and 14 recombinantly expressed monkey cytochrome P450 enzymes were analyzed with respect to R- and S-warfarin 6- and 7-hydroxylation. Monkey liver microsomes efficiently mediated both R- and S-warfarin 7-hydroxylation, in contrast to human liver microsomes, which preferentially catalyzed S-warfarin 7-hydroxylation. R-Warfarin 7-hydroxylation activities in monkey liver microsomes were not inhibited by ?-naphthoflavone or ketoconazole, and were roughly correlated with P450 2C19 levels and flurbiprofen 4-hydroxylation activities in microsomes from 20 monkey livers. In contrast, S-warfarin 7-hydroxylation activities were not correlated with the four marker drug oxidation activities used. Among the 14 recombinantly expressed monkey P450 enzymes tested, P450 2C19 had the highest activities for R- and S-warfarin 7-hydroxylations. Monkey P450 3A4 and 3A5 slowly mediated R- and S-warfarin 6-hydroxylations. Kinetic analysis revealed that monkey P450 2C19 had high V(max) and low K(m) values for R-warfarin 7-hydroxylation, comparable to those for monkey liver microsomes. Monkey P450 2C19 also mediated S-warfarin 7-hydroxylation with V(max) and V(max)/K(m) values comparable to those for recombinant human P450 2C9. R-warfarin could dock favorably into monkey P450 2C19 modeled. These results collectively suggest high activities for monkey liver P450 2C19 toward R- and S-warfarin 6- and 7-hydroxylation in contrast to the saturation kinetics of human P450 2C9-mediated S-warfarin 7-hydroxylation. PMID:23041648

  4. Evaluation of time-dependent cytochrome P450 inhibition using cultured human hepatocytes.

    PubMed

    McGinnity, Dermot F; Berry, Amanda J; Kenny, Jane R; Grime, Ken; Riley, Robert J

    2006-08-01

    Primary human hepatocytes in culture are commonly used to evaluate cytochrome P450 (P450) induction via an enzyme activity endpoint. However, other processes can confound data interpretation. To this end, the impact of time-dependent P450 inhibition in this system was evaluated. Using a substrate-cassette approach, P450 activities were determined after incubation with the prototypic inhibitors tienilic acid (CYP2C9), erythromycin, troleandomycin, and fluoxetine (CYP3A4). Kinetic analysis of enzyme inactivation in hepatocytes was used to describe the effect of these time-dependent inhibitors and derive the inhibition parameters kinact and KI) which generally were in good agreement with the values derived using recombinant P450s and human liver microsomes (HLMs). Tienilic acid selectively inhibited CYP2C9-dependent diclofenac 4'-hydroxylation activity, and erythromycin, troleandomycin, and fluoxetine inhibited CYP3A4-dependent midazolam 1'-hydroxylation in a time- and concentration-dependent manner. Fluoxetine also inhibited CYP2C19-dependent S-mephenytoin 4'-hydroxylation in a time- and concentration-dependent manner in hepatocytes, HLMs, and recombinant CYP2C19 (KI 0.4 microM and kinact 0.5 min(-1)). As expected, the effect of fluoxetine on CYP2D6 in hepatocytes was consistent with potent yet reversible inhibition. A very weak time-dependent CYP2C9 inhibitor (AZ1, a proprietary AstraZeneca compound; KI 30 microM and kinact 0.02 min(-1)) effectively abolished CYP2C9 activity over 24 h at low (micromolar) concentrations in primary cultured human hepatocytes. This work demonstrates that caution is warranted in the interpretation of enzyme induction studies with metabolically stable, weak time-dependent inhibitors, which may have dramatic inhibitory effects on P450 activity in this system. Therefore, in addition to enzyme activity, mRNA and/or protein levels should be measured to fully evaluate the P450 induction potential of a drug candidate. PMID:16679385

  5. 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

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

    PubMed

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

    2014-07-25

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

  7. 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 ?m in diameter) has been shown to negatively influence pulmonary and cardiovascular functions in living organisms. The goal of this study was to determine if these EPFRs have a direct effect on cytochrome P450 function. This was accomplished by direct addition of the EPFRs to rat liver microsomal preparations and measurement of several P450 activities using form-selective substrates. The EPFRs used in this study were formed by heating vapors from an organic compound (either monochlorophenol (MCP230) or 1,2-dichlorobenzene (DCB230)) and 5% copper oxide supported on silica (approximately 0.2 ?m in diameter) to 230°C under vacuum. Both types of EPFRs (but not silica, physisorbed silica, or silica impregnated with copper oxide) dramatically inhibited the activities of CYP1A, CYP2B, CYP2E1, CYP2D2 and CYP3A when incubated at concentrations less than 0.1 mg/ml with microsomes and NADPH. Interestingly, at the same concentrations, the EPFRs did not inhibit HO-1 activity or the reduction of cytochrome c by NADPH-cytochrome P450 reductase. CYP2D2-selective metabolism by rat liver microsomes was examined in more detail. The inhibition of CYP2D2-selective metabolism by both DCB230- and MCP230-EPFRs appeared to be largely noncompetitive and was attenuated in the presence of catalase suggesting that reactive oxygen species may be involved in the mechanism of inhibition. PMID:24713513

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

    SciTech Connect

    Froehlich, Eleonore, E-mail: eleonore.froehlich@klinikum-graz.a [Center for Medical Research, Medical University of Graz (Austria); Department of Internal Medicine, Division of Endocrinology and Nuclear Medicine, Medical University of Graz (Austria); Kueznik, Tatjana [Center for Medical Research, Medical University of Graz (Austria); Samberger, Claudia [Center for Medical Research, Medical University of Graz (Austria); Department of Internal Medicine, Division of Endocrinology and Nuclear Medicine, Medical University of Graz (Austria); Roblegg, Eva [Institute of Pharmaceutical Sciences, Department of Pharmaceutical Technology, Karl-Franzens-University of Graz (Austria); Wrighton, Christopher [Institute of Medical Technologies and Health Management, Joanneum Research, Auenbruggerplatz 20/3, A-8036 Graz (Austria); Pieber, Thomas R. [Department of Internal Medicine, Division of Endocrinology and Nuclear Medicine, Medical University of Graz (Austria)

    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.

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

    2010-09-01

    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.

  10. Generation of hepatocytes expressing functional cytochromes P450 from a pancreatic progenitor cell line in vitro.

    PubMed Central

    Marek, Carylyn J; Cameron, Gary A; Elrick, Lucy J; Hawksworth, Gabrielle M; Wright, Matthew C

    2003-01-01

    The proliferating AR42J-B13 pancreatic cell line is known to respond to glucocorticoid treatment by producing foci of cells that express the liver-specific albumin gene. We demonstrate that this cell line also expresses liver-specific or liver-enriched functional cytochrome P450 proteins when stimulated to trans-differentiate into hepatocytes by glucocorticoid. These data suggest that this cell line has an unusual ability to trans-differentiate into functional hepatocytes and that it could be possible to generate a limitless supply of functional hepatocyte-like cells in vitro. PMID:12542397

  11. Prediction of irinotecan pharmacokinetics by use of cytochrome P450 3A4 phenotyping probes

    Microsoft Academic Search

    Ron H. J. Mathijssen; Jong de F. A; Schaik van R. H. N; E. R. Lepper; L. E. Friberg; T. Rietveld; Bruijn de P; W. J. Graveland; W. D. Figg; J. Verweij; A. Sparreboom

    2004-01-01

    BACKGROUND: Irinotecan is a topoisomerase I inhibitor that has been\\u000a approved for use as a first- and second-line treatment for colorectal\\u000a cancer. The response to irinotecan is variable, possibly because of\\u000a interindividual variation in the expression of the enzymes that metabolize\\u000a irinotecan, including cytochrome P450 3A4 (CYP3A4) and uridine diphosphate\\u000a glucuronosyltransferase 1A1 (UGT1A1). We prospectively explored the\\u000a relationships between CYP3A

  12. Valence bond all the way: from the degenerate H-exchange to cytochrome P450.

    PubMed

    Shaik, Sason

    2010-08-21

    This is a personalized Perspective on the development of a valence bond (VB) view of chemical reactivity (J. Am. Chem. Soc., 1981, 103, 3692) as a LEGO process whereby one constructs "reactivity objects", such as barriers, transition states, and reaction intermediates from VB building blocks, and thereby understands and predicts chemical reactivity in a unified manner. In so doing, I have tried to give the reader a panoramic set of applications, from the simple H-exchange reaction all the way to alkane hydroxylation by cytochrome P450 (Prog. Phys. Org. Chem. 1985, 15, 197; Angew. Chem., Int. Ed., 1999, 38, 586). PMID:20574583

  13. Cloning and expression of cytochrome P450 genes controlling flower colour.

    PubMed

    Holton, T A; Brugliera, F; Lester, D R; Tanaka, Y; Hyland, C D; Menting, J G; Lu, C Y; Farcy, E; Stevenson, T W; Cornish, E C

    1993-11-18

    Blue and violet flowers generally contain derivatives of delphinidin; red and pink flowers generally contain derivatives of cyanidin or pelargonidin. Differences in hydroxylation patterns of these three major classes of anthocyanidins are controlled by the cytochrome P450 enzymes flavonoid 3'-hydroxylase and flavonoid 3',5'-hydroxylase. Here we report on the isolation of complementary DNA clones of two different flavonoid 3',5'-hydroxylase genes that are expressed in petunia flowers. Restriction-fragment length polymorphism mapping and complementation of mutant petunia lines showed that the flavonoid 3',5'-hydroxylase genes correspond to the genetic loci Hf1 and Hf2. PMID:8232589

  14. Differential inhibition of rat and human hepatic cytochrome P450 by Andrographis paniculata extract and andrographolide

    Microsoft Academic Search

    D. Pekthong; H. Martin; C. Abadie; A. Bonet; B. Heyd; G. Mantion; L. Richert

    2008-01-01

    The inhibitory effect of Andrographis paniculata extract (APE) and andrographolide (AND), the most medicinally active phytochemical in the extract, on hepatic cytochrome P450s (CYPs) activities was examined using rat and human liver microsomes. For this purpose, CYP1A2-dependent ethoxyresorufin-O-deethylation, CYP2B1-dependent benzyloxyresorufin-O-dealkylation, CYP2B6-dependent bupropion hydroxylation, CYP2C-dependent tolbutamide hydroxylation, CYP2E1-dependent p-nitrophenol hydroxylation and CYP3A-dependent testosterone 6?-hydroxylation activities, were determined in the presence and

  15. Cytochrome P450 3A4 inhibitory constituents of the wood of Taxus yunnanensis.

    PubMed

    Tezuka, Yasuhiro; Morikawa, Kouhei; Li, Feng; Auw, Lidyawati; Awale, Suresh; Nobukawa, Takahiro; Kadota, Shigetoshi

    2011-01-28

    From the aqueous extract of the wood of Taxus yunnanensis, which showed cytochrome P450 3A4 (CYP3A4) inhibition, a new isoflavan [(3S,4R)-4'-hydroxy-6,3'-dimethoxyisoflavan-4-ol (1)], a new degraded lignan [2,3-bis(hydroxymethyl)-7-hydroxy-6-methoxy-1-tetralone (2)], and a new lignan [(7R)-7-hydroxytaxiresinol (3)] were isolated, together with nine known lignans. Among the isolates obtained, ?-conidendrin (12) showed strong CYP3A4 inhibition with an IC(50) value of 0.2 ?M. PMID:21138310

  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. Bioactivation of aflatoxin B1 by turkey liver microsomes: responsible cytochrome P450 enzymes.

    PubMed

    Diaz, G J; Murcia, H W; Cepeda, S M

    2010-12-01

    1. A study was conducted to determine the cytochrome P450 enzymes responsible for the bioactivation of aflatoxin B1 into its epoxide form (AFBO) in turkey liver microsomes. 2. The strategies used included the measurement of prototype substrate activity for specific human P450s, use of selective inhibitors, determination of correlation between aflatoxin bioactivation and enzymatic activity of prototype substrates and the determination of immunoreactive proteins using antibodies against human P450s. 3. Enzymatic activity and immunoreactive proteins corresponding to the turkey orthologs CYP1A1, CYP1A2, CYP2A6 and CYP3A4 were detected, but not for the CYP2D6 ortholog. 4. The results of the inhibition and correlation studies strongly suggest that the turkey CYP2A6 ortholog and, to a lesser extent, the CYP1A1 ortholog, are involved in the bioactivation of aflatoxin B1 in turkey liver microsomes. 5. This is the first study reporting the role of CYP2A6 in the bioactivation of AFB1 in an avian species and the role of CYP1A1 in any species. PMID:21161791

  18. Microfluidic electrochemical array for detection of reactive metabolites formed by cytochrome P450 enzymes.

    PubMed

    Wasalathanthri, Dhanuka P; Mani, Vigneshwaran; Tang, Chi K; Rusling, James F

    2011-12-15

    A novel, simple, rapid microfluidic array using bioelectronically driven cytochrome P450 enzyme catalysis for reactive metabolite screening is reported for the first time. The device incorporates an eight-electrode screen-printed carbon array coated with thin films of DNA, [Ru(bpy)(2)(PVP)(10)](ClO(4)) {RuPVP}, and rat liver microsomes (RLM) as enzyme sources. Catalysis features electron donation to cyt P450 reductase in the RLMs and subsequent cyt P450 reduction while flowing an oxygenated substrate solution past sensor electrodes. Metabolites react with DNA in the film if they are able, and damaged DNA is detected by catalytic square wave voltammetry (SWV) utilizing the RuPVP polymer. The microfluidic device was tested for a set of common pollutants known to form DNA-reactive metabolites. Logarithmic turnover rates based on SWV responses gave excellent correlation with the rodent liver TD(50) toxicity metric, supporting the utility of the device for toxicity screening. The microfluidic array gave much better S/N and reproducibility than single-electrode sensors based on similar principles. PMID:22040095

  19. CYP6B3: a second furanocoumarin-inducible cytochrome P450 expressed in Papilio polyxenes.

    PubMed

    Hung, C F; Harrison, T L; Berenbaum, M R; Schuler, M A

    1995-08-01

    Cytochrome P450 monooxygenases in the larvae of Papilio polyxenes (black swallowtail) (Lepidoptera: Papilionidae) are capable of detoxifying linear and angular furanocoumarins found in their host plants. The CYP6B1 locus, which is transcriptionally induced in these larvae in response to xanthotoxin, encodes a P450 that principally metabolizes linear furanocoumarins such as xanthotoxin and bergapten. We have now cloned CYP6B3 cDNA derived from a second locus that is evolutionarily related to the CYP6B1 locus. Reverse transcription-PCR Southern analyses have demonstrated that CYP6B3 transcripts are expressed in response to a wider range of linear and angular furanocoumarins but at lower abundance than CYP6B1 transcripts. Whereas CYP6B1 transcripts are expressed at a low detectable level in uninduced control larvae and at high levels in xanthotoxin-induced larvae, CYP6B3 transcripts are nearly undetectable in control larvae and are highly induced by xanthotoxin and bergapten (linear furanocoumarins) as well as by angelicin and sphondin (angular furanocoumarins). The fact that these two CYP6B loci are differentially regulated by these four furanocoumarins indicates that P. polyxenes has adapted to the presence of the wide range of furanocoumarins in its host plants by diversifying its P450 isozyme structure and its furanocoumarin-responsive regulatory cascades. PMID:8589841

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

    SciTech Connect

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

    2011-01-01

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

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

    PubMed

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

    2005-01-01

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

  2. Structural basis for the 4'-hydroxylation of diclofenac by a microbial cytochrome P450 monooxygenase.

    PubMed

    Xu, Lian-Hua; Ikeda, Haruo; Liu, Ling; Arakawa, Takatoshi; Wakagi, Takayoshi; Shoun, Hirofumi; Fushinobu, Shinya

    2015-04-01

    Diclofenac is a nonsteroidal anti-inflammatory drug. It undergoes hydroxylation by mammalian cytochrome P450 enzymes at 4'- and/or 5'-positions. A bacterial P450 enzyme, CYP105D7 from Streptomyces avermitilis, has been shown to catalyze hydroxylation of 1-deoxypentalenic acid and an isoflavone daidzein. Here, we demonstrated that CYP105D7 also catalyzes hydroxylation of diclofenac at the C4'-position. A spectroscopic analysis showed that CYP105D7 binds diclofenac in a slightly cooperative manner with an affinity of 65 ?M and a Hill coefficient of 1.16. The crystal structure of CYP105D7 in complex with diclofenac was determined at 2.2 Å resolution. The distal pocket of CYP105D7 contains two diclofenac molecules, illustrating drug recognition with a double-ligand-binding mode. The C3' and C4' atoms of the dichlorophenyl ring of one diclofenac molecule are positioned near the heme iron, suggesting that it is positioned appropriately for aromatic hydroxylation to yield the 4'-hydroxylated product. However, recognition of diclofenac by CYP105D7 was completely different from that of rabbit CYP2C5, which binds one diclofenac molecule with a cluster of water molecules. The distal pocket of CYP105D7 contains four arginine residues, forming a wall of the substrate-binding pocket, and the arginine residues are conserved in bacterial P450s in the CYP105 family. PMID:25341403

  3. 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.

  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. Expression of Cytochrome-P450-3A5 in Escherichia Coli: Effects of 5? Modification, Purification, Spectral Characterization, Reconstitution Conditions, and Catalytic Activities

    Microsoft Academic Search

    E. M. J. Gillam; Z. Y. Guo; Y. F. Ueng; H. Yamazaki; I. Cock; P. E. B. Reilly; W. D. Hooper; F. P. Guengerich

    1995-01-01

    Cytochrome P450 (P450) 3A5 is a human enzyme with 85% amino acid sequence identity to the more predominantly expressed P450 3A4 and has been reported to have overlapping catalytic specificity. The 5?-terminus of a P450 3A5 cDNA was modified for optimal expression in Escherichia coli using the vector pCW, by aligning the MALLLAVFL N-terminal sequence of recombinant bovine P450 17A

  6. Metabolism of linear and angular furanocoumarins by Papilio polyxenes CYP6B1 co-expressed with NADPH cytochrome P450 reductase

    Microsoft Academic Search

    Zhimou Wen; Liping Pan; May R Berenbaum; Mary A Schuler

    2003-01-01

    One challenge in the heterologous expression of microsomal cytochrome P450 monooxygenases (P450s) is fulfilling their obligatory requirement for electrons transferred from NADPH P450 reductase. We have established co-expression parameters for Papilio polyxenes CYP6B1 and house fly P450 reductase in baculovirus-infected Sf9 cells that allow for efficient expression of both components and significantly enhance metabolic turnover of this insect P450’s substrates.

  7. 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 ?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 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.

  8. Relationships among ergot alkaloids, cytochrome P450 activity, and beef steer growth

    PubMed Central

    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

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed Central

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

    2005-01-01

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

  11. Do multiple cytochrome P450 isoforms contribute to parathion metabolism in man?

    PubMed

    Mutch, Elaine; Daly, Ann K; Leathart, Julian B S; Blain, Peter G; Williams, Faith M

    2003-06-01

    Phosphorothioate compounds are widely used in agriculture and public health for the control of unwanted pests. The phosphorothioate parathion was metabolised to the toxic metabolite paraoxon (0.038-0.683 nmol/min per mg protein) and p-nitrophenol (0.023-2.10 nmol/min per mg protein) by human liver microsomes ( n=27) in an NADPH-dependent reaction. There was a significant correlation ( P<0.02) between nifedipine oxidation and paraoxon formation from parathion (200 micro M) by human liver microsomes and with cytochrome P450 (CYP) 3A4/5 expression ( P<0.05), although not with midazolam 1'-hydroxylation or testosterone 6beta-hydroxylation. Paclitaxel 6'-hydroxylation and CYP2C8 expression correlated with paraoxon formation ( P<0.01), indicating CYP2C8 involvement. Of nine recombinant P450 isoforms, CYPs 3A4, 3A5, 1A2 and 2D6 activated parathion to paraoxon at the highest rates (6.5, 8.5, 5.7 and 6.2 pmol/pmol P450 per h) with K(m) values of 12.6, 2.7, 1.5 and 9.2 micro M, respectively. Similar K(m) values were seen with the human liver microsomes. These data indicate that CYP3A4/5 and CYP2C8, which constitute up to 40% of human liver P450s, are the most significant participants in the metabolism of parathion. However, several other isoforms could play an important role when CYP3A and CYP2C8 are poorly expressed due to environmental factors or the presence of a genetic polymorphism. PMID:12669189

  12. Induction of "male-specific" cytochrome P450 isozymes in female rats by oxandrolone.

    PubMed

    Waskiewicz, M J; Choudhuri, S; Vanderbeck, S M; Zhang, X J; Thomas, P E

    1995-11-01

    Oxandrolone (OXA) (5 alpha-androstan-2-oxa-17 alpha-methyl-17 beta-ol-3-one) is a clinically useful, synthetic, anabolic androgen steroid hormone. OXA was administered to rats orally twice daily for 3 days at 75 mg/kg to study the effect on hepatic cytochrome P450 (P450) isozymes. Western blots were performed on the hepatic microsomal fraction and probed with isozyme-specific monoclonal antibodies. Microsomes were also tested for catalytic activity in a testosterone metabolism assay. Data from Western blots revealed that, in female rats, there were increased levels of two male-specific isozymes, P4502C11 and P4503A2, as well as P4503A1. In contrast, male rats showed little or no change in expression of these P450 isozymes after OXA treatment. The 6 beta-hydroxylation of testosterone, which is catalyzed predominantly by P4503A1 and P4503A2, increased approximately 10-fold in female rats after treatment with OXA (from 0.05 +/- 0.01 to 0.52 +/- 0.05 nmol/min/mg protein), but only relatively small changes were seen in the male rats (from 1.02 +/- 0.05 to 1.38 +/- 0.07 nmol/min/mg protein). To investigate if the changes seen in P4503A1 and P4503A2 protein and activity were caused, at least in part, by an increase in mRNA levels, Northern blot analysis was performed. P4503A2 mRNA was increased dramatically in the female rat liver after OXA treatment, but only small increases in P4503A1 mRNA were seen. This data indicate that OXA induces P450 isozymes in the female but not in the male rat liver, probably through transcriptional activation, and some of these induced isozymes are male-specific. PMID:8591733

  13. 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.

  14. O-demethylation of epipodophyllotoxins is catalyzed by human cytochrome P450 3A4.

    PubMed

    Relling, M V; Nemec, J; Schuetz, E G; Schuetz, J D; Gonzalez, F J; Korzekwa, K R

    1994-02-01

    We previously demonstrated that O-demethylation of the pendant dimethoxyphenol ring of epipodophyllotoxins to produce their respective catechol metabolites is catalyzed by cytochrome(s) P450 in human liver microsomes. Our objective was to identify the specific human cytochrome(s) P450 responsible for catechol formation. Using a panel of prototypical substrates and inhibitors for specific cytochromes P450, we identified substrates for CYP3A4 (midazolam, erythromycin, cyclosporin, and dexamethasone) as inhibitors of catechol formation from both etoposide and teniposide. Dexamethasone inhibition was competitive, with Ki values of 60 and 45 microM for etoposide and teniposide, respectively. In 58 human livers, the correlation coefficients for teniposide catechol formation versus 1'- and 4-hydroxymidazolam formation were 80% and 85%, respectively; for etoposide catechol formation versus 1'- and 4-hydroxymidazolam formation r2 was 83% and 79%, respectively. Teniposide and etoposide catechol formation rates were also significantly correlated with immunodetectable CYP3A (r2 = 49% and 51%, respectively) and not with immunodetectable CYP1A2, 2E1, or 2C8. Finally, cDNAs for human CYP3A4, 3A5, 2A6, 2B6, 2C8, and 2C9 were functionally expressed in HepG2 cells, using a vaccinia viral vector. Teniposide and etoposide catechol formation was catalyzed primarily by 3A4 (15.4 and 40.9 pmol/pmol/hr, respectively) and to a lesser degree by 3A5 (1.94 and 11.3 pmol/pmol/hr, respectively), whereas there was no detectable O-demethylation of epipodophyllotoxins by 2A6, 2B6, 2C8, 2C9, or the control virus alone. Moreover, the relative activities of midazolam hydroxylation, compared with O-demethylation of epipodophyllotoxins, were similar for heterologously expressed 3A4 and for human liver microsomes. We conclude that catechol formation from teniposide and etoposide is primarily mediated by human CYP3A4, making these reactions susceptible to inhibition by prototypical 3A substrates and inhibitors. PMID:8114683

  15. Identification and analysis of NADPH-cytochrome P450 reductase in Aedes sollicitans (Diptera: Culicidae).

    PubMed

    Suwanchaichinda, C; Sun, D; Brattsten, L B

    2014-09-01

    An NADPH-cytochrome P450 reductase (CPR) gene was identified in Aedes sollicitans Walker (Diptera: Culicidae). The open reading frame is 2,040 bp long, encoding a 679-residue polypeptide. Amino acid sequence analysis indicates that the Ae. sollicitans CPR carries conserved ligand-binding domains and belongs to the same phylogenetic group as CPRs in other mosquitoes. The cDNA of the CPR gene was cloned, and the recombinant protein was expressed in Escherichia coli. Cytochrome c reductase activity was detected in the bacterial cytosolic and membrane fractions and in larval microsomes from two New Jersey Ae. sollicitans populations, indicating that the Ae. sollicitans CPR is enzymatically functional and closely related to that in other dipterans. PMID:25276923

  16. 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 enzyme’s 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

  17. Modulation of rat cytochrome P-450 by an investigational HIV protease inhibitor.

    PubMed

    Nishime, J A; Wang, R W; Lin, J H; Chiba, M

    1999-09-01

    Previous studies in vitro have revealed that L-754,394, an HIV protease inhibitor, is a potent suicide inhibitor of cytochrome P-450 enzymes. The present report examines the effect of chronic treatment of L-754,394 on hepatic cytochrome P-450s in adult male rats. L-754,394 was administered orally once a day for 7 days and resulted in significant changes in marker activities. An unusual parabolic (ascending, then descending) profile was observed for testosterone 2beta-/6beta-(CYP 3A1/2-catalyzed) hydroxylase activities during the 7-day treatment with 20 mg/kg L-754,394. These activities, which were elevated 2-fold on day 2, returned to basal levels by day 8. In contrast, testosterone 2alpha-/16alpha-(CYP2C11-catalyzed) hydroxylase activities showed an opposite parabolic (descending, then ascending) profile during the same period, reducing to 40% of control activities on day 4, followed by a rebounding trend. Immunoquantitation of CYP 3A1/2 and 2C11 showed that the expressed protein levels were in parallel with the associated activities. Furthermore, mRNA levels of CYP 3A2 and CYP2C11 showed the same trends as the protein expression of the respective isoforms. These observations show that L-754,394 perturbs the relative abundance of P-450 isoforms in rat liver by affecting the regulation at a pretranslational step. This may further involve a disturbance of hormonal homeostasis. Although serum levels of testosterone did not show a marked change during treatment, thyroxine and triiodothyronine markedly decreased on days 2 and 4, and subsequently increased to basal levels. PMID:10460793

  18. 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

  19. Activities of cytochrome P450 1A2, N-acetyltransferase 2, xanthine oxidase, and cytochrome P450 2D6 are unaltered in children with cystic fibrosis.

    PubMed

    Kennedy, Mary Jayne; Scripture, Charity D; Kashuba, Angela D M; Scott, Christy S; Gaedigk, Andrea; Kearns, Gregory L

    2004-03-01

    The activities of hepatic cytochrome P450 (CYP) 1A2, N-acetyltransferase 2 (NAT-2), xanthine oxidase (XO), and CYP2D6 were evaluated in 12 young children (aged 3-8 years) with mild cystic fibrosis (CF) and 12 age-matched healthy control subjects by use of standard caffeine and dextromethorphan phenotyping methods. Subjects were given 4 oz of Coca-Cola (approximately 35 mg caffeine) (The Coca-Cola Company, Atlanta, Ga) and a single 0.5-mg/kg dose of dextromethorphan. Urine was collected for 8 hours after biomarker administration, and enzyme activity was assessed by use of previously validated caffeine and dextromethorphan molar ratios. CYP2D6 genotyping was also performed in 10 of 12 subjects with CF and 11 of 12 control subjects. There were no significant differences in the urinary molar ratios for any of the enzyme systems evaluated. These data suggest that CF does not alter the activities of CYP1A2, NAT-2, XO, and CYP2D6. Altered biotransformation of drugs in this patient population is likely enzyme- and isoform-specific and thus is apparent for only selected compounds that are substrates for enzymes other than CYP1A2, NAT-2, XO, and CYP2D6. PMID:15001967

  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. Effect of xenoestrogen exposure on the expression of cytochrome P450 isoforms in rainbow trout liver.

    PubMed

    Katchamart, Sirinmas; Miranda, Cristobal L; Henderson, Marilyn C; Pereira, Clifford B; Buhler, Donald R

    2002-11-01

    We studied the estrogenic effects of model chemicals in one-year-old juvenile rainbow trout. Methoxychlor (20 mg/kg), diethylstilbestrol (15 mg/kg), 4-tert-octylphenol (25 and 50 mg/kg), and biochanin A (25 and 50 mg/kg) were injected intraperitoneally on days 1, 4, and 7. Fish were sacrificed on day 9 and examined for multiple biomarkers. All of the test chemicals caused increases in plasma vitellogenin levels, a biomarker of estrogenicity. Treatment with the xenoestrogens decreased hepatic lauric acid hydroxylase activity and, as shown by Western blots, also generally reduced expression of hepatic cytochrome P450s 2K1 (CYP2K1), 2M1 (CYP2M1), and 3A27 (CYP3A27) at the protein level. Both doses of biochanin A also significantly induced P4501A (CYPIA) and greatly increased hepatic 7-ethoxyresorufin-O-deethylase (EROD) activity. These findings suggest that methoxychlor, diethylstilbestrol, 4-tert-octylphenol, and biochanin A were all estrogenic and mimicked 17beta-estradiol (E2) in repressing the expression of cytochrome P450 isoforms (CYP2KI, CYP2M1, and CYP3A27) in the rainbow trout liver. Additionally, biochanin A was found to induce CYPIA in this fish species. PMID:12389925

  2. Anti-cytochrome P450 autoantibodies in drug-induced disease.

    PubMed

    Beaune, P H; Lecoeur, S; Bourdi, M; Gauffre, A; Belloc, C; Dansette, P; Mansuy, D

    1996-01-01

    Drugs may induce hepatitis through immune mechanisms. In this review we have used the examples of 2 drugs to elucidate the first steps leading to the triggering of such disease, namely tienilic acid (TA) and dihydralazine (DH). These drugs are transformed into reactive metabolite(s) by cytochrome P450 (2C9 for TA and 1A2 for DH) (step 1). The reactive metabolites produced are very short-lived and bind directly to the enzymes which generated them (step 2). A neoantigen is thus formed which triggers an immune response (step 3), characterized by the presence of autoantibodies in the patient's serum (step 4). The autoantibodies are directed against the cytochrome P450 which generated the metabolite(s). Although the process by which TA and DH induce-hepatitis has been elucidated, further studies are necessary to generalize this mechanism. In addition, an animal model will also be useful to fully understand the immune mechanism of this type of disease. PMID:8987248

  3. 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

  4. Functional expression and characterization of cytochrome P450 52A21 from Candida albicans* S

    PubMed Central

    Kim, Donghak; Cryle, Max J.; De Voss, James J.; Ortiz de Montellano, Paul R.

    2007-01-01

    Candida albicans contains ten putative cytochrome P450 (CYP) genes coding for enzymes that appear to play important roles in fungal survival and virulence. Here, we report the characterization of CYP52A21, a putative alkane/fatty acid hydroxylase. The recombinant CYP52A21 protein containing a 6 × (His)-tag was expressed in Escherichia coli and was purified. The purified protein, reconstituted with rat NADPH-cytochrome P450 reductase, ?-hydroxylated dodecanoic acid to give 12-hydroxydodecanoic acid, but to a lesser extent also catalyzed (?-1)-hydroxylation to give 11-hydroxydodecanoic acid. When 12,12,12-d3-dodecanoic acid was used as the substrate, there was a major shift in the oxidation from the ?- to the (?-1)-hydroxylated product. The regioselectivity of fatty acid hydroxylation was examined with the 12-iodo-, 12-bromo-, and 12-chlorododecanoic acids. Although all three 12-halododecanoic acids bound to CYP52A21 with similar affinities, the production of 12-oxododecanoic acid decreased as the size of the terminal halide increased. The regioselectivity of CYP52A21 fatty acid oxidation is thus consistent with presentation of the terminal end of the fatty acid chain for oxidation via a narrow channel that limits access to other atoms of the fatty acid chain. This constricted access, in contrast to that proposed for the CYP4A family of enzymes, does not involve covalent binding of the heme to the protein. PMID:17400174

  5. Human NADPH-P450 oxidoreductase modulates the level of cytochrome P450 CYP2D6 holoprotein via haem oxygenase-dependent and -independent pathways.

    PubMed Central

    Ding, S; Yao, D; Deeni, Y Y; Burchell, B; Wolf, C R; Friedberg, T

    2001-01-01

    NADPH-P450 oxidoreductase (CPR) is essential for the activity of cytochrome P450 (P450). Previous studies demonstrated that CPR regulates the levels of various P450 isoforms in vitro. We investigated the mechanistic basis for this regulation. By transfection of Chinese hamster ovary DUKXB11 cells we obtained the cell line DUKX/2D6, which expressed human CYP2D6, a P450 isoform. Subsequently, DUKX/2D6 cells were transfected with human CPR cDNA to generate the cell line DUKX/2D6/CPR-3. Expression of recombinant CPR decreased the level of spectrally detectable CYP2D6 holoprotein in DUKX/2D6/CPR-3 cells by 70%, whereas the level of immunodetectable apoprotein remained unchanged. Addition of the radical scavenger DMSO increased levels of CYP2D6 holoenzyme in DUKX/2D6/CPR-3 cells but not in DUKX/2D6 cells. A similar effect was noted when cells were grown in the presence of hemin. Importantly, combined treatment with DMSO and hemin increased levels of CYP2D6 holoenzyme in DUKX/2D6/CPR-3 but not in DUKX/2D6 cells even further than either treatment alone. None of these treatments affected the level of immunodetectable CYP2D6. This demonstrates that expression of CPR increases production of damaging radicals but also that CPR may alter haem homoeostasis. In agreement with this, the activity of haem oxygenase, a rate-limiting enzyme in haem metabolism, was compared with that in DUKX/DHFR control cells (expressing dihydrofolate reductase), and was 3-fold higher in DUKX/2D6/CPR-3 but similar in DUKX/2D6 cells. Furthermore, treatment of cells with sodium arsenite increased levels of haem oxygenase concomitant with a marked decrease of spectrally detectable CYP2D6 and a rise in levels of ferritin, which sequesters free iron released from the destruction of haem. These data demonstrate that CPR regulates P450 activity by supplying electrons and also by altering P450 levels via radical-and haem oxygenase-mediated pathways. PMID:11368792

  6. Human NADPH-P450 oxidoreductase modulates the level of cytochrome P450 CYP2D6 holoprotein via haem oxygenase-dependent and -independent pathways.

    PubMed

    Ding, S; Yao, D; Deeni, Y Y; Burchell, B; Wolf, C R; Friedberg, T

    2001-06-01

    NADPH-P450 oxidoreductase (CPR) is essential for the activity of cytochrome P450 (P450). Previous studies demonstrated that CPR regulates the levels of various P450 isoforms in vitro. We investigated the mechanistic basis for this regulation. By transfection of Chinese hamster ovary DUKXB11 cells we obtained the cell line DUKX/2D6, which expressed human CYP2D6, a P450 isoform. Subsequently, DUKX/2D6 cells were transfected with human CPR cDNA to generate the cell line DUKX/2D6/CPR-3. Expression of recombinant CPR decreased the level of spectrally detectable CYP2D6 holoprotein in DUKX/2D6/CPR-3 cells by 70%, whereas the level of immunodetectable apoprotein remained unchanged. Addition of the radical scavenger DMSO increased levels of CYP2D6 holoenzyme in DUKX/2D6/CPR-3 cells but not in DUKX/2D6 cells. A similar effect was noted when cells were grown in the presence of hemin. Importantly, combined treatment with DMSO and hemin increased levels of CYP2D6 holoenzyme in DUKX/2D6/CPR-3 but not in DUKX/2D6 cells even further than either treatment alone. None of these treatments affected the level of immunodetectable CYP2D6. This demonstrates that expression of CPR increases production of damaging radicals but also that CPR may alter haem homoeostasis. In agreement with this, the activity of haem oxygenase, a rate-limiting enzyme in haem metabolism, was compared with that in DUKX/DHFR control cells (expressing dihydrofolate reductase), and was 3-fold higher in DUKX/2D6/CPR-3 but similar in DUKX/2D6 cells. Furthermore, treatment of cells with sodium arsenite increased levels of haem oxygenase concomitant with a marked decrease of spectrally detectable CYP2D6 and a rise in levels of ferritin, which sequesters free iron released from the destruction of haem. These data demonstrate that CPR regulates P450 activity by supplying electrons and also by altering P450 levels via radical-and haem oxygenase-mediated pathways. PMID:11368792

  7. Soft-hydrothermal processing of red cedar bedding reduces its induction of cytochrome P450 in mouse liver.

    PubMed

    Li, Z; Okano, S; Yoshinari, K; Miyamoto, T; Yamazoe, Y; Shinya, K; Ioku, K; Kasai, N

    2009-04-01

    Red cedar-derived bedding materials cause changes in cytochrome P450-dependent microsomal enzyme systems in laboratory animals. We examined the effect of essential oil of red cedar (EORC), as well as the effect of bedding from which it had been removed, on the hepatic expression cytochrome P450s in mice. EORC was obtained from liquid extracts of red cedar bedding by a soft-hydrothermal process and was administered orally to mice. Between days 1 and 2 after administration, hepatic P450s were significantly induced as follows: CYP3As, 7.1x; CYP1As, 1.6x; CYP2E1, 1.5x; CYP2Cs, 1.6x. A housing study of mice indicated that red cedar bedding increased the levels of these P450s in mouse liver, whereas mice housed in cedar bedding from which EORC had been removed (ST-cedar bedding) showed significantly lower levels of P450s, especially CYP3As, CYP1As and CYP2E1. Soft-hydrothermal processing partially removed many components of EORC. In particular, several volatile sesquiterpenes, naphthalene-derived aromatics and 4,4-dimethyl-13alpha-androst-5-ene were decreased in the ST-cedar bedding, suggesting that these may be responsible for P450 induction. This study demonstrated that the removal of these volatile compounds by soft-hydrothermal processing can decrease the hepatic P450-inducing effect of red cedar bedding. PMID:19116287

  8. Altered Expression of Hepatic Cytochromes P-450 in Mice Deficient in One or More mdr1 Genes

    Microsoft Academic Search

    ERIN G. SCHUETZ; DIANE R. UMBENHAUER; KAZUTO YASUDA; CYNTHIA BRIMER; LAN NGUYEN; MARY V. RELLING; JOHN D. SCHUETZ; ALFRED H. SCHINKEL

    We hypothesized that the drug efflux protein P-glycoprotein (Pgp), the product of the multidrug resistance gene MDR1, might influence hepatic expression of CYP3A or other cyto- chromes P-450 (P-450s) because Pgp can transport endoge- nous regulators of these cytochromes. We began with variants of a CF-1 mouse strain containing a defective mdr1a gene that is inherited in a Mendelian fashion.

  9. Involvement of cytochrome P450 3A4 in N-dealkylation of buprenorphine in human liver microsomes

    Microsoft Academic Search

    Christelle Iribarne; Daniel Picart; Yvonne Dréano; Jean-Pierre Bail; François Berthou

    1997-01-01

    Buprenorphine is a long acting analgesic of the opiate family. Recently, it has been proposed for the opioid dependency treatment at a large scale. The drug is extensively metabolized by the hepatic cytochrome P450 in man, yielding a N-dealkylated metabolite, norbuprenorphine. The specific forms of P450 involved in this oxidative N-demethylation were examined in a panel of 18 human liver

  10. Regulation of an insect cytochrome P450 monooxygenase gene ( CYP6B1) by aryl hydrocarbon and xanthotoxin response cascades

    Microsoft Academic Search

    Rebecca Petersen Brown; Cynthia M. McDonnell; May R. Berenbaum; Mary A. Schuler

    2005-01-01

    Many organisms respond to toxic compounds in their environment by inducing regulatory networks controlling the expression and activity of cytochrome P450 monooxygenase (P450s) detoxificative enzymes. In particular, black swallowtail (Papilio polyxenes) caterpillars respond to xanthotoxin, a toxic phytochemical in their hostplants, by activating transcription of the CYP6B1 promoter via several regions located within 150 nt of the transcription initiation site.

  11. Structure, Genetic Mapping, and Function of the Cytochrome P450 3A37 Gene in the Turkey (Meleagris gallopavo)

    Microsoft Academic Search

    S. Rawal; K. M. Mendoza; K. M. Reed

    2009-01-01

    Cytochromes P450 (P450 for protein; CYP for gene) are a superfamily of membrane-bound hemoproteins that oxidize a large number of endogenous and exogenous compounds. Through oxidation reactions, these enzymes are often responsible for the toxic and carcinogenic effects of natural food-borne toxicants, such as the mycotoxin aflatoxin B1 (AFB1). Previous studies in our laboratory have shown that the extreme sensitivity

  12. Isozyme-specific monoclonal antibody-directed assessment of induction of hepatic cytochrome p-450 by clotrimazole.

    PubMed

    Khan, W A; Kuhn, C; Merk, H F; Park, S S; Gelboin, H V; Bickers, D R; Mukhtar, H

    1989-01-01

    Clotrimazole, an N-substituted imidazole widely used as an antifungal agent, has been shown to both inhibit and induce hepatic cytochrome P-450 and related monooxygenase activities. In this study the profile of hepatic cytochrome P-450 isozyme(s) induced by clotrimazole treatment of male Sprague-Dawley rats was investigated. Clotrimazole administration (100 mg/kg, daily for 4 days, ig) resulted in 86% induction of spectrally detectable cytochrome P-450 in hepatic microsomes. In these microsomes 7-ethoxycoumarin O-deethylase (126%), aminopyrine N-demethylase (176%), benzphetamine N-demethylase (117%), p-nitrophenol hydroxylase (89%), and 7-ethoxyresorufin O-deethylase (62%) activities were significantly induced, whereas aryl hydrocarbon hydroxylase activity remained unchanged. Characterization of cytochrome P-450 isozyme(s) in hepatic microsomes prepared from clotrimazole-treated animals was based on the immunoreactivity of these microsomes with highly specific monoclonal antibodies (MAbs) raised against 3-methylcholanthrene-specific P-450 (MAb 1-7-1), phenobarbital-specific P-450 (MAb 2-66-3), pregnenolone-16 alpha-carbonitrile-specific P-450 (MAb C2), and ethanol-inducible P-450 (MAb 1-98-1). Western blot analysis of hepatic microsomes prepared from clotrimazole-treated animals with MAb 2-66-3, MAb 1-98-1, and MAb C2 revealed strong immunoreactive bands, whereas moderate reactivity was observed with MAb 1-7-1. MAb 2-66-3 significantly inhibited 7-ethoxycoumarin O-deethylase activity 45%), whereas MAb 1-7-1 moderately inhibited 7-ethoxyresorufin O-deethylase activity (-30%) in clotrimazole-treated animals.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2571472

  13. Selective Filling of Nanowells in Nanowell Arrays Fabricated Using Polystyrene Nanosphere Lithography with Cytochrome P450 Enzymes

    PubMed Central

    Wollenberg, Lance A.; Jett, John E.; Wu, Yueting; Flora, Darcy R.; Wu, Nianqiang; Tracy, Timothy S.; Gannett, Peter M.

    2012-01-01

    This work describes an original and simple technique for protein immobilization into nanowells, fabricated using nanopatterned-array fabrication methods, while ensuring the protein retains the normal biological activity. Nanosphere-lithography was used to fabricate a nanowell array with nanowells that were 100 nm in diameter and a periodicity of 500 nm. The base of the nanowells was gold and the surrounding material was silicon dioxide. The different surface chemistries of these materials were used to attach two different self-assembled monolayers (SAM) with different affinities for the protein used here, cytochrome P450 (P450). The nanowell SAM, a methyl terminated thiol, had high affinity for the P450. The surrounding SAM, a polyethylene glycol silane, displayed very little affinity toward the P450 isozyme CYP2C9, as demonstrated by x-ray photoelectron spectroscopy and surface plasmon resonance. The regularity of the nanopatterned array was examined by scanning electron microscopy and atomic force microscopy. P450-mediated metabolism experiments of known substrates demonstrated that the nanowell bound P450 enzyme exceeded its normal activity, as compared to P450 solutions, when bound to the methyl terminated self-assembled monolayer. The nanopatterned array chips bearing P450 display long term stability and give reproducible results making them potentially useful for high throughput screening assays or as nanoelectrode arrays. PMID:22947619

  14. Selective filling of nanowells in nanowell arrays fabricated using polystyrene nanosphere lithography with cytochrome P450 enzymes

    NASA Astrophysics Data System (ADS)

    Wollenberg, Lance A.; Jett, John E.; Wu, Yueting; Flora, Darcy R.; Wu, Nianqiang; Tracy, Timothy S.; Gannett, Peter M.

    2012-09-01

    This work describes an original and simple technique for protein immobilization into nanowells, fabricated using nanopatterned array fabrication methods, while ensuring the protein retains normal biological activity. Nanosphere lithography was used to fabricate a nanowell array with nanowells 100 nm in diameter with a periodicity of 500 nm. The base of the nanowells was gold and the surrounding material was silicon dioxide. The different surface chemistries of these materials were used to attach two different self-assembled monolayers (SAM) with different affinities for the protein used here, cytochrome P450 (P450). The nanowell SAM, a methyl terminated thiol, had high affinity for the P450. The surrounding SAM, a polyethylene glycol silane, displayed very little affinity toward the P450 isozyme CYP2C9, as demonstrated by x-ray photoelectron spectroscopy and surface plasmon resonance. The regularity of the nanopatterned array was examined by scanning electron microscopy and atomic force microscopy. P450-mediated metabolism experiments of known substrates demonstrated that the nanowell bound P450 enzyme exceeded its normal activity, as compared to P450 solutions, when bound to the methyl terminated self-assembled monolayer. The nanopatterned array chips bearing P450 display long term stability and give reproducible results making them potentially useful for high-throughput screening assays or as nanoelectrode arrays.

  15. 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 Nelson’s 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 Nelson’s 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

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

    PubMed Central

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

    2008-01-01

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

  17. Camphor hydroxylase of Pseudomonas putida: vestiges of sequence homology in cytochrome P-450CAM, putidaredoxin, and related proteins.

    PubMed Central

    Dus, K M

    1984-01-01

    The amino acid sequences of cytochrome P-450CAM and putidaredoxin of the camphor hydroxylase [camphor, reduced-putida-ferredoxin:oxygen oxidoreductase (5-hydroxylating), EC 1.14.15.1] of Pseudomonas putida are compared to each other and then to the sequences of bovine adrenodoxin and cytochrome b5. The comparisons reveal areas of homology indicating that these four proteins may share a common evolutionary origin. Moreover, homologous segments can be recognized by proper alignment of the sequence of cytochrome P-450CAM to recently determined sequences of other P-450 hemeproteins. Further scrutiny indicates vestiges of internal sequence duplications that have been conserved in limited segments by the constraints of selection over a long time period, while other segments of the sequence diverged. It is predicted that the corresponding reductases will show a similar sequence pattern. The conserved regions of internal sequence repetition may serve a special purpose in protein-protein interactions within the multienzyme systems. PMID:6584899

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

    SciTech Connect

    Lee, Richard F.; Anderson, Jack W.

    2005-07-01

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

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

    PubMed

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

    2012-09-01

    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. K(i) values were determined for inhibition of p-nitrophenol 2-hydroxylation, a reaction accomplished by CYP2A13 and CYP2A6 with more similar catalytic efficiencies (k(cat)/K(m) 0.19 and 0.12 ?M?¹ · min?¹, respectively) than hydroxylation of the classic substrate coumarin (0.11 and 0.53 ?M?¹ · min?¹, respectively). Of the nine compounds assayed, only tranylcypromine and (R)-(+)-menthofuran had a greater than 10-fold preference for CYP2A6 inhibition versus CYP2A13 inhibition. Most compounds evaluated [tryptamine, 4-dimethylaminobenzaldehyde, phenethyl isothiocyanate, ?-nicotyrine, (S)-nicotine, and pilocarpine] demonstrated only moderate or no preference for inhibition of one CYP2A enzyme over the other. However, 8-methoxypsoralen has a 6-fold lower K(i) for CYP2A13 than for CYP2A6. This information is useful to inform reinterpretation of previous data with these inhibitors and to guide future studies seeking to determine which human CYP2A enzyme is responsible for the in vivo metabolism of compounds in human tissues expressing both enzymes. PMID:22696418

  20. 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

  1. Oxidative dehalogenation of perhalogenated benzenes by cytochrome P450 compound I.

    PubMed

    Hackett, John C; Sanan, Toby T; Hadad, Christopher M

    2007-05-22

    Resolution of the identity PBE (RI-PBE) and B3LYP density functional theory calculations are used to understand the cytochrome P450-catalyzed, Compound I-mediated oxidation of perchlorobenzenes, perfluorobenzenes, their phenols, and mixed chlorofluorobenzenes to form benzoquinones. Addition of Compound I to the chlorine-bearing carbon of perchlorobenzenes and perchlorophenols results in an apparently barrierless 1,2-shift of the chlorine atom to form hexachlorocyclohexadienones and hydroxypentachlorocyclohexadienones, respectively. Hexachlorocyclohexadienone has a significant electron affinity, and its radical anion expels chloride in a facile manner to give the pentachlorophenoxyl radical. Deprotonation of hydroxypentachlorocyclohexadienones results in the expulsion of chloride and provides a direct route to the production of tetrachloroquinones. Barrier heights for Compound I addition to fluorine-bearing carbons of hexafluorobenzene and pentafluorophenol are comparable to those computed for oxidation of benzene via an analogous reaction path. In contrast to the chlorinated cases, fluorine migration to cyclohexadienones occurs with a moderate barrier. Additionally, gas-phase elimination of fluoride from the hexafluorocyclohexadienone radical anion and deprotonated hydroxypentafluorocyclohexadienone are not facile. Rather, consideration of implicit and explicit solvent is required to achieve favorable thermochemistry for fluoride elimination and generation of the experimentally observed products. Finally, the theoretical approach described herein is predictive of the experimentally observed preferential elimination of fluorine from chloropentafluorobenzene and 1,3,5-trichloro-2,4,6-trifluorobenzene. These studies illustrate the effectiveness of P450 Compound I as an oxidant of halogenated aromatic hydrocarbons, which are persistent environmental contaminants, and the potential utility of such computational methods for predicting P450 metabolism. PMID:17455915

  2. Roles of Cytochrome b 5in the Oxidation of Testosterone and Nifedipine by Recombinant Cytochrome P450 3A4 and by Human Liver Microsomes

    Microsoft Academic Search

    Hiroshi Yamazaki; Masami Nakano; Yoshio Imai; Yune-Fang Ueng; F. Peter Guengerich; Tsutomu Shimada

    1996-01-01

    NADH-dependent testosterone 6?-hydroxylation and nifedipine oxidation activities could be reconstituted in systems containing cytochrome b5(b5), NADH–b5reductase, and bacterial recombinant cytochrome P450 (P450) 3A4 with a synthetic phospholipid mixture, cholate, MgCl2, and reduced glutathione. Replacement of NADH–b5reductase with NADPH–P450 reductase produced an eightfold increase in testosterone 6?-hydroxylation activity. Further stimulation could be obtained when NADPH was used as an electron donor

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

    PubMed Central

    2012-01-01

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

  4. 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}.

  5. Heme Exporter FLVCR1a Regulates Heme Synthesis and Degradation and Controls Activity of Cytochromes P450

    PubMed Central

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

    2014-01-01

    Background & Aims The liver has one of the highest rates of heme synthesis of any organ. More than 50% of the heme synthesized in the liver is used for synthesis of P450 enzymes, which metabolize exogenous and endogenous compounds that include natural products, hormones, drugs, and carcinogens. Feline leukemia virus subgroup C cellular receptor 1a (FLVCR1a) is plasma membrane heme exporter that is ubiquitously expressed and controls intracellular heme content in hematopoietic lineages. We investigated the role of Flvcr1a in liver function in mice. Methods We created mice with conditional disruption of Mfsd7b, which encodes Flvcr1a, in hepatocytes (Flvcr1afl/fl;alb-cre mice). Mice were analyzed under basal conditions, after phenylhydrazine-induced hemolysis, and after induction of cytochromes P450 synthesis. Livers were collected and analyzed by histologic, quantitative real-time polymerase chain reaction, and immunoblot analyses. Hepatic P450 enzymatic activities were measured. Results Flvcr1afl/fl;alb-cre mice accumulated heme and iron in liver despite up-regulation of heme oxygenase 1, ferroportin, and ferritins. Hepatic heme export activity of Flvcr1a was closely associated with heme biosynthesis, which is required to sustain cytochrome induction. Upon cytochromes P450 stimulation, Flvcr1afl/fl;alb-cre mice had reduced cytochrome activity, associated with accumulation of heme in hepatocytes. The expansion of the cytosolic heme pool in these mice was likely responsible for the early inhibition of heme synthesis and increased degradation of heme, which reduced expression and activity of cytochromes P450. Conclusions In livers of mice, Flvcr1a maintains a free heme pool that regulates heme synthesis and degradation as well as cytochromes P450 expression and activity. These findings have important implications for drug metabolism. PMID:24486949

  6. Cloning and expression of cDNA encoding a bovine adrenal cytochrome P-450 specific for steroid 21-hydroxylation.

    PubMed Central

    White, P C; New, M I; Dupont, B

    1984-01-01

    We isolated a cDNA clone encoding a bovine adrenal cytochrome P-450 specific for steroid 21-hydroxylation (P-450C21). Serum from rabbits immunized with purified P-450C21 precipitated a single protein from the products of an in vitro translation reaction using bovine adrenal mRNA. This protein migrated with P-450C21 on NaDodSO4/polyacrylamide gel electrophoresis. After sucrose gradient sedimentation, mRNA encoding P-450C21 was found in the 19S fraction. This fraction was reverse transcribed into double-stranded cDNA and inserted into the Pst I site of pBR322 by the dC X dG tailing procedure. Escherichia coli cells transformed with recombinant plasmids were screened with an in situ immunoassay using anti-P-450C21 serum and 125I-labeled staphylococcal protein A. Two colonies consistently bound anti-P-450C21 serum. They were identified as carrying the same plasmid by restriction mapping. This plasmid, pC21a, contains an insert of 520 base pairs. It hybridizes with mRNA encoding P-450C21. The peptide encoded by the insert in pC21a is highly homologous to two peptides isolated from porcine P-450C21 and shows limited homology to the P-450 induced by phenobarbital in rat liver. This clone may be useful in studying the molecular genetics of human congenital adrenal hyperplasia due to 21-hydroxylase deficiency. Images PMID:6609358

  7. Inhibition Selectivity of Grapefruit Juice Components on Human Cytochromes P450

    Microsoft Academic Search

    Wichittra Tassaneeyakul; Lian-Qing Guo; Katsuyuki Fukuda; Tomihisa Ohta; Yasushi Yamazoe

    2000-01-01

    Five compounds including furanocoumarin monomers (bergamottin, 6?,7?-dihydroxybergamottin (DHB)), furanocoumarin dimers (4-[[6-hydroxy-7[[1-[(1-hydroxy-1-methyl)ethyl]-4-methyl-6-(7-oxo-7H-furo[3,2-g][1]benzopyran-4-yl)-4-hexenyl]oxy]-3,7-dimethyl- 2-octenyl]oxy]-7H-furo[3,2-g][1]benzopyran-7-one (GF-I-1) and 4-[[6-hydroxy-7[[4-methyl-1-(1-methylethenyl)-6-(7-oxo-7H-furo[3,2-g][1]benzopyran-4-yl)-4-hexenyl]oxy]-3,7-dimethyl-2-octenyl]oxy]-7H-furo[3,2-g][1]benzopyran-7-one (GF-I-4)), and a sesquiterpene nootkatone have been isolated from grapefruit juice and screened for their inhibitory effects toward human cytochrome P450 (P450) forms using selective substrate probes. Addition of ethyl acetate extract of grapefruit juice into an incubation mixture resulted in decreased activities of CYP3A4,

  8. Molecular cloning and characterization of a cytochrome P450 in sanguinarine biosynthesis from Eschscholzia californica cells.

    PubMed

    Takemura, Tomoya; Ikezawa, Nobuhiro; Iwasa, Kinuko; Sato, Fumihiko

    2013-07-01

    Benzophenanthridine alkaloids, such as sanguinarine, are produced from reticuline, a common intermediate in benzylisoquinoline alkaloid biosynthesis, via protopine. Four cytochrome P450s are involved in the biosynthesis of sanguinarine from reticuline; i.e. cheilanthifoline synthase (CYP719A5; EC 1.14.21.2.), stylopine synthase (CYP719A2/A3; EC 1.14.21.1.), N-methylstylopine hydroxylase (MSH) and protopine 6-hydroxylase (P6H; EC 1.14.13.55.). In this study, a cDNA of P6H was isolated from cultured Eschscholzia californica cells, based on an integrated analysis of metabolites and transcript expression profiles of transgenic cells with Coptis japonica scoulerine-9-O-methyltransferase. Using the full-length candidate cDNA for P6H (CYP82N2v2), recombinant protein was produced in Saccharomyces cerevisiae for characterization. The microsomal fraction containing recombinant CYP82N2v2 showed typical reduced CO-difference spectra of P450, and production of dihydrosanguinarine and dihydrochelerythrine from protopine and allocryptopine, respectively. Further characterization of the substrate-specificity of CYP82N2v2 indicated that 6-hydroxylation played a role in the reaction. PMID:22421633

  9. Production of a highly active, soluble form of the cytochrome P450 reductase (CPR A) from Candida tropicalis

    DOEpatents

    Donnelly, Mark

    2006-08-01

    The present invention provides soluble cytochrome p450 reductase (CPR) proteins from Candida sp. having an altered N-terminal region which results in reduced hydrophobicity of the N-terminal region. Also provided are host cells comprising the subject soluble CPR proteins. In addition, the present invention provides nucleotide and corresponding amino acid sequences for soluble CPR proteins and vectors comprising the nucleotide sequences. Methods for producing a soluble CPR, for increasing production of a dicarboxylic acid, and for detecting a cytochrome P450 are also provided.

  10. 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. (Univ. of Arizona, Tucson (United States))

    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.

  11. 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

  12. Repression of hepatic cytochrome P450 2D expression in mice during Babesia microti infection.

    PubMed

    Shimamoto, Yoshinori; Watanabe, Kensuke; Ikadai, Hiromi; Okamura, Masashi; Ishizuka, Mayumi

    2014-08-01

    To examine the effect of Babesia infection on the level of the drug-metabolizing enzyme hepatic cytochrome P450 (CYP) 2D, we intraperitoneally inoculated Babesia microti into male ICR mice. CYP2D protein and CYP2D9 mRNA were significantly decreased at 12 days after infection with B. microti. The activity of bunitrolol 4-hydroxylase, which is catalyzed by CYP2D, was also significantly decreased. The mRNA levels of transcriptional regulators of CYP2D9, hepatocyte nuclear factor 4? and signal transducer and activator of transcription 5b, were markedly suppressed. These results suggest that Babesia infection represses CYP2D expression in the mouse liver. The decline in CYP2D-dependent drug metabolism might be involved in the incidence of adverse drug reactions in patients with babesiosis. PMID:24784440

  13. Repression of Hepatic Cytochrome P450 2D Expression in Mice during Babesia microti Infection

    PubMed Central

    SHIMAMOTO, Yoshinori; WATANABE, Kensuke; IKADAI, Hiromi; OKAMURA, Masashi; ISHIZUKA, Mayumi

    2014-01-01

    ABSTRACT To examine the effect of Babesia infection on the level of the drug-metabolizing enzyme hepatic cytochrome P450 (CYP) 2D, we intraperitoneally inoculated Babesia microti into male ICR mice. CYP2D protein and CYP2D9 mRNA were significantly decreased at 12 days after infection with B. microti. The activity of bunitrolol 4-hydroxylase, which is catalyzed by CYP2D, was also significantly decreased. The mRNA levels of transcriptional regulators of CYP2D9, hepatocyte nuclear factor 4? and signal transducer and activator of transcription 5b, were markedly suppressed. These results suggest that Babesia infection represses CYP2D expression in the mouse liver. The decline in CYP2D-dependent drug metabolism might be involved in the incidence of adverse drug reactions in patients with babesiosis. PMID:24784440

  14. 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

  15. 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

  16. Role of water in molecular docking simulations of cytochrome P450 2D6.

    PubMed

    Santos, Rita; Hritz, Jozef; Oostenbrink, Chris

    2010-01-01

    Active-site water molecules form an important component in biological systems, facilitating promiscuous binding or an increase in specificity and affinity. Taking water molecules into account in computational approaches to drug design or site-of-metabolism predictions is currently far from straightforward. In this study, the effects of including water molecules in molecular docking simulations of the important metabolic enzyme cytochrome P450 2D6 are investigated. The structure and dynamics of water molecules that are present in the active site simultaneously with a selected substrate are described, and based on this description, water molecules are selected to be included in docking experiments into multiple protein conformations. Apart from the parent substrate, 11 similar and 53 dissimilar substrates are included to investigate the transferability of active-site hydration sites between substrates. The role of water molecules appears to be highly dependent on the protein conformation and the substrate. PMID:19899781

  17. 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

  18. Using Fluorescence Spectroscopy to Evaluate Hill Parameters and Heterogeneity of Ligand Binding to Cytochromes P450

    NASA Astrophysics Data System (ADS)

    Marsch, Glenn A.; Carlson, Benjamin; Hansen, Jennifer; Mihelc, Elaine; Martin, Martha V.; Guengerich, F. Peter

    2009-03-01

    The cytochromes P450 (CYPs) are hemoproteins that oxidize many drugs and carcinogens. Binding interactions of two CYPs with Nile Red, pyrene, and alpha-naphthoflavone were studied using fluorescence quenching. Upon interaction with CYPs, fluorescence from pyrene excited-state dimers was quenched more efficiently than fluorescence from pyrene monomers. Quenching data was fit to the Hill equation to determine binding affinities and the Hill parameter n for the interaction of substrates with CYPs. All ligands showed strong binding to the CYPs, especially alpha-naphthoflavone, but exhibited little or no cooperativity in the interaction. Modified Stern-Volmer plots were used to confirm binding affinities, and suggested heterogeneous populations of amino acid fluorophores. Fluorescence anisotropy experiments suggest that CYP molecules tumble more rapidly when alpha-naphthoflavone is added.

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

    PubMed Central

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

    2014-01-01

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

  20. Maintenance of in vivo induced cytochrome P-450s in hepatocyte monolayers at non freezing temperatures.

    PubMed

    Evans, Peter J

    2015-04-01

    Cytochrome P450s (CYPs) induced in rats by 3-methylcholanthrene (3-MC), phenobarbital (PB) and dexamethasone (Dex) were investigated. The inducers had no effect on hepatocyte yield, viability, attachment or spreading on collagen. 3-MC induced ethoxyresorufin deethylase (EROD). Under normothermic conditions the activity fell in culture. However, it was maintained when cells were preserved at 10°C under a gelatin gel. Upon reactivation the activity mirrored that of freshly isolated cells at 37°C. Induced levels were stable for at least 6h, the time to form a confluent monolayer. The investigation was extended to other CYPs by looking at patterns of testosterone metabolism. Phenobarbital had the greatest influence in terms of the quantity and number of metabolites. Culture at 37°C decreased the peaks dramatically within 24h. All 7 peaks were maintained in the preservation system. PMID:25666303

  1. 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

  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. 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.

  4. 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 for studying the processes that link the perception of gravity to the growth response. Arabidopsis plants with a mutation in the GRAVITY PERSISTENT SIGNAL (GPS)1 locus show a "no response" phenotype during gravistimulation experiments. Basepital auxin transport in gps1 mutant was unaffected by the mutation, but auxin was not laterally redistributed after gravistimulation. GPS1 encodes CYP705A22, a cytochrome P450 protein (P450) of unknown function. The wild type CYP705A22 gene was transformed into the gps1 mutant background and successfully rescued the mutant phenotype. Data mining of microarray data collected from gravistimulated root tips of Arabidopsis indicated that although CYP705A22 was not expressed in roots, a family member CYP705A5 was up-regulated within 3 minutes after gravistimulation. Expression profiling of CYP705A5, using real-time quantitative PCR, showed that CYP705A5 was up-regulated nearly five fold within minutes of gravity stimulation. And reporter gene fusions that link the CYP705A5 gene to the green fluorescent protein showed that CYP705A5 was expressed in the root zones of elongation and maturation. Computer modeling of the catalytic domain of CYP705A22 and CYP705A5 and in silico substrate docking simulations generated a list of 130 compounds that are potential substrates of the P450s. Many of the compounds are phenylpropanoid derivatives. Heterologous expression of CYP705A5 in baculovirus and Type 1 binding studies indicate the substrate of the P450 may be quercitin or myricetin. A mutation affecting CYP705A5 expression resulted in a delayed gravity response in roots. The mutant phenotype could be chemically complemented, and DPBA staining in the CYP705A5 mutant indicated a 1.5 fold accumulation of quercetin in mutant roots as compared to WT. These data, taken together, may indicate that we have identified a flavonoid pathway that regulates auxin distribution and thus is involved in gravitropic signal transduction. (Partially support by NSF: 0618506 to SEW)

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

    PubMed

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

    2014-01-01

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

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

    PubMed

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

    2014-09-01

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

  7. A host-inducible cytochrome P-450 from a host-specific caterpillar: molecular cloning and evolution.

    PubMed Central

    Cohen, M B; Schuler, M A; Berenbaum, M R

    1992-01-01

    Cytochrome P-450 monooxygenases (P-450s) play a critical role in the detoxification of natural and synthetic toxins in a wide range of organisms. We have isolated and sequenced cDNA clones encoding a P-450, CYP6B1, from larvae of Papilio polyxenes (Lepidoptera: Papilionidae), the black swallowtail butterfly. This P-450, cloned from a herbivorous insect, is highly inducible by xanthotoxin, a secondary metabolite abundant in the host plants of this specialized herbivore. On Northern blots, mRNAs crossreactive with CYP6B1 were detected in three Papilio species that, like the black swallowtail, have high levels of xanthotoxin-metabolic P-450 activity and encounter xanthotoxin or related compounds in their host plants; in contrast, no crossreactive mRNAs were detectable in three papilinid species that never encounter xanthotoxin in their host plants and lack detectable xanthotoxin-metabolic activity. These results provide evidence that new P-450s can arise as herbivores colonize different host plants and support the hypothesis that interactions between herbivores and their toxin-producing host plants have contributed to the diversification of the P-450 superfamily. Images PMID:1279697

  8. A host-inducible cytochrome P-450 from a host-specific caterpillar: molecular cloning and evolution.

    PubMed

    Cohen, M B; Schuler, M A; Berenbaum, M R

    1992-11-15

    Cytochrome P-450 monooxygenases (P-450s) play a critical role in the detoxification of natural and synthetic toxins in a wide range of organisms. We have isolated and sequenced cDNA clones encoding a P-450, CYP6B1, from larvae of Papilio polyxenes (Lepidoptera: Papilionidae), the black swallowtail butterfly. This P-450, cloned from a herbivorous insect, is highly inducible by xanthotoxin, a secondary metabolite abundant in the host plants of this specialized herbivore. On Northern blots, mRNAs crossreactive with CYP6B1 were detected in three Papilio species that, like the black swallowtail, have high levels of xanthotoxin-metabolic P-450 activity and encounter xanthotoxin or related compounds in their host plants; in contrast, no crossreactive mRNAs were detectable in three papilinid species that never encounter xanthotoxin in their host plants and lack detectable xanthotoxin-metabolic activity. These results provide evidence that new P-450s can arise as herbivores colonize different host plants and support the hypothesis that interactions between herbivores and their toxin-producing host plants have contributed to the diversification of the P-450 superfamily. PMID:1279697

  9. A Fluorescence Spectroscopic Analysis of the Binding of Pyrene to Cytochromes P450 1A2 and 3A4.

    NASA Astrophysics Data System (ADS)

    Henry, Judah; Guengerich, F. Peter; Marsch, Glenn

    2007-03-01

    Fluorescence spectroscopy was used to study cytochromes P450 1A2 and 3A4. Spectra of P450s were acquired in the presence and absence of acrylamide quencher. In both P450s, quenching revealed three distinguishable species of amino acid fluorescence, with maxima at 297, 323, and 345 nm. The 345 nm tryptophan fluorescence was quenched by low levels of acrylamide; the 297 nm tyrosine fluorescence was resistant to quenching. The 323 nm fluorescence was observed at intermediate concentrations of quencher. Stern-Volmer plots of P450 quenching were non-linear, but were well-fitted to a superposition of linear plots for each fluorophore species. The effect of the P450's binding on pyrene fluorescence was also examined. Upon binding to P450 1A2, the intensity of the 383 nm pyrene vibronic band was decreased relative to the intensities of the 372 and 393 nm bands. Both P450's showed binding of the pyrene, but 1A2 demonstrated significantly more excimer emission than did the 3A4, which suggests that more than one pyrene molecule binds to 1A2's active site. The results of these analyses will be used in further characterization of these enzymes.

  10. Effect of crude extract of Eugenia jambolana Lam. on human cytochrome P450 enzymes.

    PubMed

    Chinni, Santhivardhan; Dubala, Anil; Kosaraju, Jayasankar; Khatwal, Rizwan Basha; Satish Kumar, M N; Kannan, Elango

    2014-11-01

    The fruit of Eugenia jambolana Lam. is very popular for its anti-diabetic property. Previous studies on the crude extract of E.?jambolana (EJE) have successfully explored the scientific basis for some of its traditional medicinal uses. Considering its wide use and consumption as a seasonal fruit, the present study investigates the ability of E.?jambolana to interact with cytochrome P450 enzymes. The standardized EJE was incubated with pooled human liver microsomes to assess the CYP2C9-, CYP2D6-, and CYP3A4-mediated metabolism of diclofenac, dextromethorphan, and testosterone, respectively. The metabolites formed after the enzymatic reactions were quantified by high performance liquid chromatography. EJE showed differential effect on cytochrome P450 activities with an order of inhibitory potential as CYP2C9?>?CYP3A4?>?CYP2D6 having IC50 of 76.69, 359.02, and 493.05?µg/mL, respectively. The selectivity of EJE for CYP2C9 rather than CYP3A4 and CYP2D6 led to perform the enzyme kinetics to explicate the mechanism underlying the inhibition of CYP2C9-mediated diclofenac 4'-hydroxylation. EJE was notably potent in inhibiting the reaction in a non-competitive manner with Ki of 84.85?±?5.27?µg/mL. The results revealed the CYP2C9 inhibitory potential of EJE with lower Ki value suggesting that EJE should be examined for its potential pharmacokinetic and pharmacodynamic interactions when concomitantly administered with other drugs. PMID:24590863

  11. Inhibition of human cytochrome P450 enzymes by hops (Humulus lupulus) and hop prenylphenols

    PubMed Central

    Nikoli?, Dejan; Chen, Shao-Nong; Huang, Ke; Li, Guannan; Pauli, Guido F.; van Breemen, Richard B.

    2014-01-01

    As hops (Humulus lupulus L.) are used in the brewing of beer and by menopausal women as estrogenic dietary supplements, the potential for hop extracts and hop constituents to cause drug-botanical interactions by inhibiting human cytochrome P450 enzymes was investigated. Inhibition of major human cytochrome P450 enzymes by a standardized hop extract and isolated hop prenylated phenols was evaluated using a fast and efficient assay based on ultrahigh pressure liquid chromatography-tandem mass spectrometry. The hop extract at 5 ?g/mL inhibited CYP2C8 (93%), CYP2C9 (88%), CYP2C19 (70%), and CYP1A2 (27%) with IC50 values of 0.8, 0.9, 3.3, and 9.4 ?g/mL, respectively, but time-dependent inactivation was observed only for CYP1A2. Isoxanthohumol from hops was the most potent inhibitor of CYP2C8 with an IC50 of 0.2 ?M, whereas 8-prenylnaringenin was the most potent inhibitor of CYP1A2, CYP2C9 and CYP2C19 with IC50 values of 1.1 ?M, 1.1 ?M and 0.4 ?M, respectively. Extracts of hops contain prenylated compounds such as the flavanones isoxanthohumol and 8-prenylnaringenin and the chalcone xanthohumol that can inhibit CYP450s, especially the CYP2C family, which may affect the efficacy and safety of some CYP2C substrate drugs when co-administered. PMID:24342125

  12. Cytochrome P450 Reductase: A Harbinger of Diffusible Reduced Oxygen Species

    PubMed Central

    Manoj, Kelath Murali; Gade, Sudeep Kumar; Mathew, Lazar

    2010-01-01

    The bi-enzymatic system of cytochrome P450 (CYP, a hemoprotein) and cytochrome P450 reductase (CPR, a diflavoenzyme) mediate the redox metabolism of diverse indigenous and xenobiotic molecules in various cellular and organ systems, using oxygen and NADPH. Curiously, when a 1?1 ratio is seen to be optimal for metabolism, the ubiquitous CYP:CPR distribution ratio is 10 to 100?1 or higher. Further, the NADPH equivalents consumed in these in vitro or in situ assemblies usually far exceeded the amount of substrate metabolized. We aimed to find the rationale to explain for these two oddities. We report here that CPR is capable of activating molecular oxygen on its own merit, generating diffusible reduced oxygen species (DROS). Also, in the first instance for a flavoprotein, CPR is shown to deplete peroxide via diffusible radical mediated process, thereby leading to the formation of water (but without significant evolution of oxygen). We also quantitatively demonstrate that the rate of oxygen activation and peroxide depletion by CPR accounts for the major reactivity in the CYP+CPR mixture. We show unambiguously that CPR is able to regulate the concentration of diffusible reduced oxygen species in the reaction milieu. These findings point out that CPR mediated processes are bound to be energetically ‘wasteful’ and potentially ‘hazardous’ owing to the unavoidable nature of the CPR to generate and deplete DROS. Hence, we can understand that CPR is distributed at low densities in cells. Some of the activities that were primarily attributed to the heme-center of CYP are now established to be a facet of the flavins of CPR. The current approach of modeling drugs to minimize “uncoupling” on the basis of erstwhile hypothesis stands questionable, considering the ideas brought forth in this work. PMID:20967245

  13. Inhibition of human cytochrome P450 enzymes by hops (Humulus lupulus) and hop prenylphenols.

    PubMed

    Yuan, Yang; Qiu, Xi; Nikoli?, Dejan; Chen, Shao-Nong; Huang, Ke; Li, Guannan; Pauli, Guido F; van Breemen, Richard B

    2014-03-12

    As hops (Humulus lupulus L.) are used in the brewing of beer and by menopausal women as estrogenic dietary supplements, the potential for hop extracts and hop constituents to cause drug-botanical interactions by inhibiting human cytochrome P450 enzymes was investigated. Inhibition of major human cytochrome P450 enzymes by a standardized hop extract and isolated hop prenylated phenols was evaluated using a fast and efficient assay based on ultrahigh pressure liquid chromatography-tandem mass spectrometry. The hop extract at 5 ?g/mL inhibited CYP2C8 (93%), CYP2C9 (88%), CYP2C19 (70%), and CYP1A2 (27%) with IC50 values of 0.8, 0.9, 3.3, and 9.4 ?g/mL, respectively, but time-dependent inactivation was observed only for CYP1A2. Isoxanthohumol from hops was the most potent inhibitor of CYP2C8 with an IC50 of 0.2 ?M, whereas 8-prenylnaringenin was the most potent inhibitor of CYP1A2, CYP2C9 and CYP2C19 with IC50 values of 1.1 ?M, 1.1 ?M and 0.4 ?M, respectively. Extracts of hops contain prenylated compounds such as the flavanones isoxanthohumol and 8-prenylnaringenin and the chalcone xanthohumol that can inhibit CYP450s, especially the CYP2C family, which may affect the efficacy and safety of some CYP2C substrate drugs when co-administered. PMID:24342125

  14. Inhibitory effect of valproic acid on cytochrome P450 2C9 activity in epilepsy patients.

    PubMed

    Gunes, Arzu; Bilir, Erhan; Zengil, Hakan; Babaoglu, Melih O; Bozkurt, Atila; Yasar, Umit

    2007-06-01

    Drug interactions constitute a major problem in the treatment of epilepsy because drug combinations are so common. Valproic acid is a widely used anticonvulsant drug with a broad therapeutic spectrum. Case reports suggest interaction between valproic acid and other drugs metabolized mainly by cytochrome P450 isoforms. The aim of this study was to evaluate the inhibitory effect of valproic acid on cytochrome P450 2C9 (CYP2C9) activity by using losartan oxidation as a probe in epilepsy patients. Patients were prescribed sodium valproate (mean 200 mg/day for the first week and 400 mg/day in the following period) according to their clinical need. A single oral dose of 25 mg losartan was given to patients before and after the first dose, first week and 4 weeks of valproic acid treatment. Losartan and E3174, the CYP2C9-derived carboxylic acid metabolite of losartan in 8 hr urine were assayed by using high pressure liquid chromatography. Urinary losartan/E3174 ratio did not change significantly on the first day (0.9, 0.3-3.5; median, range), and first week (0.6, 0.2-3.8; median, range), while a significant increase was observed after 4 weeks of valproic acid treatment (1.1, 0.3-5.7; median, range) as compared to that of measured before valproic acid administration (0.6, 0.1-2.1; median, range) (P = 0.039). The degree of inhibition was correlated with the steady-state plasma concentrations of valproic acid (r(2) = 0.70, P = 0.04). The results suggest an inhibitory effect of valproic acid on CYP2C9 enzyme activity in epilepsy patients at steady state. The risk of pharmacokinetic drug-drug interactions should be taken into account during concomitant use of valproic acid and CYP2C9 substrates. PMID:17516991

  15. Effect of age on hepatic cytochrome P450 of Ross 708 broiler chickens.

    PubMed

    Hu, S X

    2013-05-01

    Age has significant impact on hepatic cytochrome P450 (CYP450) systems in animals. Ross 708 broiler chicken is a breed of chicken with fast growth characteristics. Cytochrome P450 in the livers of Ross 708 broiler chicken of different ages has been investigated. The birds were raised under standard husbandry conditions. A certain number of chickens was randomly sampled weekly for liver collection from d 1 to 56 posthatch. The chicken body and liver weights were recorded. The chicken livers were processed for liver microsomes though a multiple-step procedure at low temperature. Total CYP450 content in chicken liver homogenates and liver microsomes was measured using a UV/visible spectroscopic method. The enzymatic activities of CYP450 in the chicken liver microsomes were determined through incubation of CYP450 isoform substrates followed by measurement of formation of their metabolites. The chicken showed an opposite age pattern in hepatic CYP450 content and activities compared with most mammals. The hepatic CYP450 content and activities of chicken at d 1 posthatch were higher than at other ages. The total hepatic CYP450 content in chickens at d 1 posthatch was more than twice the average hepatic value of the chickens at d 7 to 28. This high CYP450 fell quickly in the first week posthatch and slightly rose from d 28 to 56. Hepatic CYP450 activities of CYP1A, 3A, 2C, 2D, and 2H were much higher in the chicken at d 1 posthatch. The differences of these enzymatic activities between d 1 and other ages of chicken were CYP450 isoform dependent. This result suggests that embryonic development of chicken livers has a significant impact on the age profile of hepatic CYP450 content and activities of posthatch chickens. PMID:23571338

  16. Structural basis for human NADPH-cytochrome P450 oxidoreductase deficiency

    SciTech Connect

    Xia, Chuanwu; Panda, Satya P.; Marohnic, Christopher C.; Martásek, Pavel; Masters, Bettie Sue; Kim, Jung-Ja P. (MCW); (Charles U); (UTSMC)

    2012-03-15

    NADPH-cytochrome P450 oxidoreductase (CYPOR) is essential for electron donation to microsomal cytochrome P450-mediated monooxygenation in such diverse physiological processes as drug metabolism (approximately 85-90% of therapeutic drugs), steroid biosynthesis, and bioactive metabolite production (vitamin D and retinoic acid metabolites). Expressed by a single gene, CYPOR's role with these multiple redox partners renders it a model for understanding protein-protein interactions at the structural level. Polymorphisms in human CYPOR have been shown to lead to defects in bone development and steroidogenesis, resulting in sexual dimorphisms, the severity of which differs significantly depending on the degree of CYPOR impairment. The atomic structure of human CYPOR is presented, with structures of two naturally occurring missense mutations, V492E and R457H. The overall structures of these CYPOR variants are similar to wild type. However, in both variants, local disruption of H bonding and salt bridging, involving the FAD pyrophosphate moiety, leads to weaker FAD binding, unstable protein, and loss of catalytic activity, which can be rescued by cofactor addition. The modes of polypeptide unfolding in these two variants differ significantly, as revealed by limited trypsin digestion: V492E is less stable but unfolds locally and gradually, whereas R457H is more stable but unfolds globally. FAD addition to either variant prevents trypsin digestion, supporting the role of the cofactor in conferring stability to CYPOR structure. Thus, CYPOR dysfunction in patients harboring these particular mutations may possibly be prevented by riboflavin therapy in utero, if predicted prenatally, or rescued postnatally in less severe cases.

  17. Role of polymorphic human cytochrome P450 enzymes in estrone oxidation.

    PubMed

    Cribb, Alastair E; Knight, M Joy; Dryer, Dagny; Guernsey, Judy; Hender, Kimberly; Tesch, Marvin; Saleh, Tarek M

    2006-03-01

    Estrogen and its metabolites are believed to play important roles in breast cancer. The influence of genetic polymorphisms in the enzymes responsible for formation and disposition of estrogen on breast cancer risk may shed light on the importance of estrogen metabolites in this disease. However, for such studies to be valid, it is important to correctly identify the enzymes involved in estrogen bioactivation. Therefore, we assessed the human cytochrome P450-dependent oxidation of estrone using substrate concentrations that more closely approximate the maximum expected concentrations in breast tissue. The in vitro metabolism of estrone by recombinant human cytochrome P450 enzymes and human liver microsomes was studied. The formation of estrone metabolites (2-hydroxyestrone, 4-hydroxyestrone, and 16alpha-hydroxyestrone) was monitored by high-performance liquid chromatography. 2-Hydroxyestrone formation was catalyzed predominantly by CYP1A2, CYP1A1, and CYP1B1 enzymes; 4-hydroxyestrone formation was catalyzed predominantly by CYP1B1, CYP1A2, and CYP1A1 enzymes; and 16alpha-hydroxyestrone formation was catalyzed predominantly by CYP2C19, CYP1A1, and CYP3A5. This study confirms the important role of members of the CYP1 family in the 2-hydroxylation and 4-hydroxylation of estrone, but the enzymes identified as responsible for the 16alpha-hydroxylation of estrone are different from those previously identified. The relative importance of these enzymes in vivo would depend on the specific tissue expression of the enzymes. These enzymes are all known to be genetically variant in the human population, and additional studies to assess the role CYP1A2, CYP2C19, and CYP3A5 in breast cancer risk are indicated. PMID:16537715

  18. Transcription profiling of 12 asian gypsy moth (Lymantria dispar) cytochrome P450 genes in response to insecticides.

    PubMed

    Sun, Lili; Wang, Zhiying; Zou, Chuanshan; Cao, Chuanwang

    2014-04-01

    As the main group of detoxification enzymes, cytochrome P450 monoxygenases (P450s) catalyse an extremely diverse range of reactions that play an important role in the detoxification of foreign compounds. Transcription profiling of 12 Lymantria dispar P450 genes from the CYP6 subfamily believed to be involved in insecticide metabolism was performed in this study. Life-stage transcription profiling of CYP6 genes revealed significant variations between eggs, larvae, pupae, and adult males and females. Exposure of larvae to sublethal doses of deltamethrin, omethoate, and carbaryl enhanced the transcription of most of the CYP6 P450 genes, with induction peaking between 24 and 72 h after exposure. Transcription profiles were dependent on the levels of insecticide exposure and the various developmental stages. PMID:24488622

  19. Role of cytochrome P-450 in hydrogen peroxide-induced cytotoxicity to LLC-PK1 cells.

    PubMed

    Baliga, R; Zhang, Z; Shah, S V

    1996-10-01

    The current study was designed to test the role of cytochrome P-450 in hydrogen peroxide-induced cytotoxicity, and to determine whether it may serve as a source of catalytic iron. Hydrogen peroxide led to iron release (as measured by iron/bathophenanthroline complex) from the microsomes prepared from LLC-PK1 cells. Cimetidine, which inhibits cytochrome P-450 by interacting with the heme iron, significantly blocked iron release, whereas ranitidine, which has a similar structure as cimetidine but is a weak inhibitor of P-450, did not have an effect (H2O2, 0.42 +/- 0.04; H2O2 + cimetidine, 0.23 +/- 0.02 nmol/mg protein; N = 4, P < 0.01). Exposure of LLC-PK1 cells to hydrogen peroxide (2.5 mM) resulted in a significant increase in the bleomycin-detectable iron (iron capable of catalyzing free radical reactions) content that was prevented by cimetidine, but not ranitidine. We then examined the effect of the inhibitors of cytochrome P-450 on cell death (as measured by Trypan blue exclusion) after exposure of LLC-PK1 cells to 2.5 mM hydrogen peroxide for 120 minutes. Inhibition of cytochrome P-450 by cimetidine significantly reduced the cell death; the effect was observed with 0.05 mM and was concentration dependent with 1 mM affording almost complete protection (H2O2, 59 +/- 1.3% vs. H2O2 + cimetidine, 11 +/- 0.7%; N = 5, P < 0.01). In contrast, ranitidine did not show any protection. We confirmed that the protective effect of cimetidine was not related to scavenging hydrogen peroxide or hydroxyl radicals or chelating iron. A second inhibitor of cytochrome P-450, piperonyl butoxide, had a similar dose-dependent beneficial effect against hydrogen peroxide-induced cell injury. Our data thus indicate an important role of cytochrome P-450 in hydrogen peroxide-induced cytotoxicity to LLC-PK1 cells and suggest that cytochrome P-450 may serve as a source of catalytic iron. PMID:8887268

  20. Effects of diet and ethanol on the expression and localization of cytochromes P450 2E1 and P450 2C7 in the colon of male rats

    Microsoft Academic Search

    Reza Hakkak; Soheila Korourian; Martin J. J. Ronis; Magnus Ingelman-Sundberg; Thomas M. Badger

    1996-01-01

    Local activation of procarcinogens in target tissues such as the colon by cytochrome P450-dependent microsomal monooxygenases is considered to be an important factor in the etiology of cancer. Diet and alcohol consumption are considered risk factors in colon cancer, and the cytochrome P450 isozymes CYP2E1 and CYP2C7 have been implicated in the biochemical mechanisms underlying colon cancer. The current study

  1. Significantly reduced cytochrome P450 3A4 expression and activity in liver from humans with diabetes mellitus

    PubMed Central

    Dostalek, Miroslav; Court, Michael H; Yan, Bingfang; Akhlaghi, Fatemeh

    2011-01-01

    BACKGROUND AND PURPOSE Patients with diabetes mellitus require pharmacotherapy with numerous medications. However, the effect of diabetes on drug biotransformation is not well understood. Our goal was to investigate the effect of diabetes on liver cytochrome P450 3As, the most abundant phase I drug-metabolizing enzymes in humans. EXPERIMENTAL APPROACH Human liver microsomal fractions (HLMs) were prepared from diabetic (n = 12) and demographically matched nondiabetic (n = 12) donors, genotyped for CYP3A4*1B and CYP3A5*3 polymorphisms. Cytochrome P450 3A4, 3A5 and 2E1 mRNA expression, protein level and enzymatic activity were compared between the two groups. KEY RESULTS Midazolam 1?- or 4-hydroxylation and testosterone 6?-hydroxylation, catalyzed by P450 3A, were markedly reduced in diabetic HLMs, irrespective of genotype. Significantly lower P450 3A4 protein and comparable mRNA levels were observed in diabetic HLMs. In contrast, neither P450 3A5 protein level nor mRNA expression differed significantly between the two groups. Concurrently, we have observed increased P450 2E1 protein level and higher chlorzoxazone 6-hydroxylation activity in diabetic HLMs. CONCLUSIONS AND IMPLICATIONS These studies indicate that diabetes is associated with a significant decrease in hepatic P450 3A4 enzymatic activity and protein level. This finding could be clinically relevant for diabetic patients who have additional comorbidities and are receiving multiple medications. To further characterize the effect of diabetes on P450 3A4 activity, a well-controlled clinical study in diabetic patients is warranted. PMID:21323901

  2. cDNA and deduced protein sequence of Cyp6D1: the putative gene for a cytochrome p450 responsible for pyrethroid resistance in house fly

    Microsoft Academic Search

    Takashi Tomita; Jeffrey G. Scott

    1995-01-01

    A microsomal cytochrome P450 from the house fly (Musca domestica), termed P450lpr, is involved in P450 monooxygenase-mediated pyrethroid resistance and is expressed at 8-fold higher levels in the insecticide resistant LPR strain compared to a susceptible strain. An internal cDNA sequence was amplified by polymerase chain reaction (PCR) using degenerate primers based on known P450lpr, polypeptide sequences, and the remainder

  3. Structure-activity relationships in the induction of hepatic microsomal cytochrome P450 by clotrimazole and its structurally related compounds in rats.

    PubMed

    Matsuura, Y; Kotani, E; Iio, T; Fukuda, T; Tobinaga, S; Yoshida, T; Kuroiwa, Y

    1991-06-15

    We investigated the structure-activity relationship in the induction of hepatic microsomal cytochrome P450 by clotrimazole and its structurally related compounds. For this purpose, we synthesized various compounds structurally analogous to clotrimazole and injected them into rats at a fixed dose of 0.2 mmol/kg. We found that the chlorine atom in clotrimazole was not necessary for the induction of cytochrome P450. The imidazole moiety of clotrimazole, however, was a very important structural component for the induction of cytochrome P450; triazole, but not pyrrole, could be substituted for this moiety. 1-Tritylimidazole, 1-diphenylmethylimidazole and 1-benzylimidazole were also found to be inducers of cytochrome P450, but, to a somewhat lesser extent, with a decreasing number of substituted phenyl groups. Thus, 1-benzylimidazole was a minimum structurally active unit for inducing cytochrome P450. In addition, 4-diphenylmethylpyridine and 4-benzylpyridine also induced cytochrome P450 to extents similar to those induced by the corresponding imidazole derivatives, but 4-benzylpiperidine lacked this effect. When the methylene unit of clotrimazole-related compounds was introduced by a hydroxy or amino group instead of imidazole, there was a less extensive increase in cytochrome P450 content. This inducing effect was lost completely by the lack of an imidazole moiety and imidazole itself. 1-Phenylethylimidazole and 1-benzylimidazole induced cytochrome P450 to a similar extent. All of these findings suggest that 1-substituted heteroaromatic compounds having two or more nitrogen atoms are likely to be required for inducing cytochrome P450. Immunoblot analysis revealed that clotrimazole and other various inducers found in this study increased cytochrome P450b/e content. These results could provide information on the effects of drugs and chemicals on cytochrome P450 induction. PMID:2039545

  4. 7-Ethynylcoumarins: Selective Inhibitors of Human Cytochrome P450s 1A1 and 1A2

    PubMed Central

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

    2012-01-01

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

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

    USGS Publications Warehouse

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

    2009-01-01

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

  6. Induction of cytochrome P450 and\\/or detoxication enzymes by various extracts of rosemary: description of specific patterns

    Microsoft Academic Search

    P Debersac; J.-M Heydel; M.-J Amiot; H Goudonnet; Y Artur; M Suschetet; M.-H Siess

    2001-01-01

    The ability of rosemary to modulate cytochrome P450 (CYP) and detoxication enzymes in rat liver was evaluated by comparing the effects of dried leaves and leaf extracts with different chemical compositions: essential oil (EO) containing monoterpenes, a dichloromethane extract (DCME) containing phenolic diterpenes and a water-soluble extract (WSE) containing phenolic compounds such as rosmarinic acid and flavonoids. Chemical analyses were

  7. Induction of cytochrome p?450 in the norway rat, rattus norvegicus, following exposure to potential environmental contaminants

    Microsoft Academic Search

    Raymond W. Nims; Ronald A. Lubet

    1995-01-01

    Cytochrome P?450 (CYP) induction (consisting of increases in cellular RNA and protein content and associated catalytic activities) occurs predominantly in the liver, but also in small intestine, lung, kidney, and placenta, of Norway rats (Rattus norvegicus.) exposed to certain types of potential environmental contaminants. The specific isoform(s) induced in the rat and the magnitudes of the increases observed depend upon

  8. Cytochrome P450 2E1 inhibition prevents hepatic carcinogenesis induced by diethylnitrosamine in alcohol-fed rats

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Chronic alcohol ingestion increases hepatic cytochrome P450 2E1 (CYP2E1), which is associated with hepatocarcinogenesis. We investigated whether treatment with chlormethiazole (CMZ), a CYP2E1 inhibitor, protects against alcohol-associated hepatic carcinogenesis in rats. Rats were fed either an ethan...

  9. Multiple forms of cytochrome P-450 in phenobarbital- and 3-methylcholanthrene-treated rats. Separation and spectral properties.

    PubMed

    Ryan, D; Lu, A Y; West, S; Levin, W

    1975-03-25

    Multiple forms of liver microsomal cytochrome P-450 isolated from immature male rats pretreated with phenobarbital or 3-methylcholanthrene are described. Afraction of low specific content (Fraction A. 1.7 TO 4.0 nmol of cytochrome P-450 per mg of protein) and a fraction substantially purified (Fraction B, 9.0 TO 11.0 NMOL of cytochrome P-450 per mg of protein) are obtained by DEAE-cellulose chromatography of a partially purified cytochrome P-450 preparation in the presence of Emulgen 911. Shifts in the absorption maxima in the CO-reduced and ethyl isocyanide difference spectra are observed in the fractions derived from 3-methylcholanthrene-treated rats. The fractions derived from phenobarbital-treated rats exhibit different 455:430 ratios and pH intercepts in the ethyl isocyanide difference spectra. The absolute oxidized spectra and n-octylamine binding spectra at room temperature and EPR analysis at the temperature of liquid helium characterize all the fractions, except the Fraction A from 3-methylcholanthrene-treated rats, as low spin ferric hemeproteins. The A hemeprotein fractions from both 3-methylcholanthrene- and phenobarbital-treated rats have poor catalytic activity for the metabolism of benzphentamine and 3,4-benzo-[a]pyrene in comparison to the B hemeprotein fractions which may be due to the presence of a high concentration of Emulgen 911 in the A fractions. However, the presence of Emulgen 911 cannot account for the spectral differences among the fractions. PMID:1117002

  10. An evaluation of the potential for pharmacokinetic interaction between escitalopram and the cytochrome P450 3A4 inhibitor ritonavir

    Microsoft Academic Search

    Marcelo M. Gutierrez; Jeffrey Rosenberg; Wattanaporn Abramowitz

    2003-01-01

    Background: Depression often coexists with a number of disease states, and patients with a diagnosis of depression often receive multiple medications. Thus, it is desirable to avoid coadministration of agents that have a potential for drug interactions in these patients. Although escitalopram and its metabolites are weak to negligible inhibitors of the cytochrome P450 (CYP) 3A4 isozyme and are therefore

  11. Metabolism of levo--Acetylmethadol (LAAM) by Human Liver Cytochrome P450: Involvement of CYP3A4 Characterized by

    E-print Network

    Steinbach, Joe Henry

    Metabolism of levo- -Acetylmethadol (LAAM) by Human Liver Cytochrome P450: Involvement of CYP3A4-LAAM formation, respectively. LAAM and nor-LAAM metabolism was inhibited by the CYP3A4-selective inhibitors and 3A4 had the highest activity toward LAAM and nor-LAAM at both low (2 M) and high (250 M) substrate

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

    E-print Network

    Steinbach, Joe Henry

    ), catalyzed in vitro by intestinal, hepatic, and expressed cytochrome P450 (CYP) 3A4. However, the role of CYP3A4 in human methadone disposition in vivo is unclear. This investigation tested the hypothesis. They received intravenous (IV) midazolam (to assess CYP3A4 activity) and then simultaneous oral deuterium

  13. Genome-wide identification and characterization of cytochrome P450 monooxygenase genes in the ciliate Tetrahymena thermophila

    Microsoft Academic Search

    Chengjie Fu; Jie Xiong; Wei Miao

    2009-01-01

    BACKGROUND: Cytochrome P450 monooxygenases play key roles in the metabolism of a wide variety of substrates and they are closely associated with endocellular physiological processes or detoxification metabolism under environmental exposure. To date, however, none has been systematically characterized in the phylum Ciliophora. T. thermophila possess many advantages as a eukaryotic model organism and it exhibits rapid and sensitive responses

  14. Prediction of Cytochrome P450 Profiles of Environmental Chemicals with QSAR Models Built from Drug-like Molecules

    EPA Science Inventory

    The human cytochrome P450 (CYP450) enzyme family is involved in the biotransformation of many environmental chemicals. As part of the U.S. Tox21 effort, we profiled the CYP450 activity of ~2800 chemicals predominantly of environmental concern against CYP1A2, CYP2C19, CYP2C9, CYP2...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

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

    EPA Science Inventory

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

  17. Interaction of ligands with cytochrome P-450. On the 442 nm spectral species generated during the oxidative metabolism of pyridine.

    PubMed Central

    Hlavica, P; Mietaschk, J; Baden, I

    1982-01-01

    When added to aerobic rabbit liver microsomal fractions fortified with an NADPH-generating system, pyridine initially produces a type II difference spectrum such as is observed with other aromatic amines. There is a time-dependent conversion of this perturbation into a new spectral species characterized by an absorbance maximum at 442 nm and a minor peak at 389 nm. Experiments with inhibitors of the cytochrome P-450-dependent electron-transport chain suggest that these species originate from binding to the haemoprotein of metabolic intermediate(s) derived from the amine substrate. Analysis of the incubation mixtures by t.l.c., high-pressure liquid chromatography, u.v.- and mass-spectrometry reveals the presence of a single metabolite arising from cytochrome P-450-catalysed oxidation of the heteroaromatic tertiary amine, which was identified as pyridine N-oxide, obviously accounting for adduct formation. This view is supported by comparative studies on the spectral changes generated by exogenous amine oxide with NADPH-reduced cytochrome P-450. Moreover, dithiothreitol, a potent N-oxidase inhibitor, strongly suppresses development of the 442 nm and 389 nm complexes. The ability of forming low-spin adducts with ferrous cytochrome P-450 absorbing around 440 nm appears to be an inherent property of different types of N-oxides. Considering the dipole character of the N+-O- function, a co-ordinate iron-oxygen bond is proposed to be formed in these complexes. PMID:7115339

  18. COMPETITIVE AND NON-COMPETITIVE MODE OF INHIBITION BY CITRUS LIMONOIDS ON HUMAN CYTOCHROME P450 ISOENZYMES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cytochrome P450 enzymes are known to play a vital role in the metabolic activation and detoxification of environmental toxic substances and drugs. Activation often results in the formation of reactive oxygen species, which consequently interact with DNA, leading to the etiology of cancer. Limonoids,...

  19. Induction of cytochrome P450 CYP6B7 and cytochrome b5 mRNAs from Helicoverpa armigera (Hubner) by pyrethroid insecticides in organ culture.

    PubMed

    Ranasinghe, C; Hobbs, A A

    1999-11-01

    An organ culture system derived from Helicoverpa armigera has been used to study the expression of cytochrome P450 and cytochrome b5 mRNAs. Northern analysis showed that levels of the mRNAs for cytochrome P450s, CYP6B2, CYP6B6 and CYP6B7, and cytochrome b5 in control tissue were commensurate with those in the tissue of whole larvae. Substantial induction of cytochrome P450, CYP6B7 and cytochrome b5 mRNAs by alpha-pinene, and the pyrethroids, fenvalerate, cypermethrin and permethrin were observed in fat body culture. Neither mRNA was induced, either in midgut or integument organ cultures. In contrast, the relatively water-soluble compound phenobarbital, could induce CYP6B7 mRNA but not cytochrome b5 mRNA in fat body cultures. As for pyrethroids, phenobarbital had no effect on the other tissues in culture. These results confirm a previous conclusion that pyrethroids could induce CYP6B7 mRNA, which was based upon a very slight induction observed in living insects. Because many cytochrome P450 substrates can act as their inducers, these results support a previous conclusion that CYP6B7 could be the enzyme that is involved in pyrethroid resistance in H. armigera. PMID:10620039

  20. Cloning and expression of koala (Phascolarctos cinereus) liver cytochrome P450 reductase.

    PubMed

    Kong, Sandra; Ngo, Suong N T; McKinnon, Ross A; Stupans, Ieva

    2009-07-01

    The cloning, expression and characterization of hepatic NADPH-cytochrome P450 reductase (CPR) from koala (Phascolarctos cinereus) is described. Two 2059 bp koala liver CPR cDNAs, designated CPR1 and CPR2, were cloned by reverse transcription-polymerase chain reaction and rapid amplification of cDNA ends. The koala CPR cDNAs encode proteins of 678 amino acids and share 85% amino acid sequence identity to human CPR. Transfection of the koala CPR cDNAs into Cos-7 cells resulted in the expression of proteins, which were recognized by a goat-antihuman CPR antibody. The koala CPR1 and 2 cDNA-expressed enzymes catalysed cytochrome c reductase at the rates of 4.9 +/- 0.5 and 2.6 +/- 0.4 nmol/min/mg protein (mean +/- SD, n = 3), respectively which were comparable to that of rat CPR cDNA-expressed enzyme. The apparent Km value for CPR activity in koala liver microsomes was 11.61 +/- 6.01 microM, which is consistent with that reported for rat CPR enzyme. Northern analysis detected a CPR mRNA band of approximately 2.6 kb. Southern analysis suggested a single PCR gene across species. The present study provides primary molecular data regarding koala CPR1 and CPR2 genes in this unique marsupial species. PMID:19444989

  1. Role of cytochrome P-450 isozyme 3a in microsomal metabolism of three nasal carcinogens

    SciTech Connect

    Ding, X.; Coon, M.J.

    1986-05-01

    Cytochrome P-450 isozyme 3a, first isolated from rabbit liver microsomes, also occurs in nasal mucosal microsomes. The role of the cytochrome in the metabolism of three nasal carcinogens, hexamethylphosphoramide (HMPA), N-nitrosodiethylamine (NDEA), and phenacetin (PA) was examined in the present study. Microsomal preparations from rabbit nasal ethmoturbinates catalyze the O- or N-deakylation of the above compounds at much greater rates than do liver microsomes. The participation of isozyme 3a was shown by immunochemical experiments involving inhibition by anti-isozyme 3a antibodies. With 1.0 mM substrate present, anti-3a IgG inhibited the activities in nasal membrane microsomes as follows: HMPA N-demethylation, 61%; NDEA N-deethylation, 67%; and PA O-deethylation, 75%. Similar results were obtained in experiments with liver microsomes except for PA O-deethylation, which was only slightly inhibited (18%) by the antibody. The involvement of isozyme 3a in the dealkylation activities was further indicated by a correlation between the increase in anti-3a-inhibited rates and the induction of isozyme 3a in liver microsomes from animals chronically administered ethanol. They conclude that isozyme 3a plays a significant role in the metabolism and, presumably, the known carcinogenicity of HMPA, NDEA, and PA in nasal mucosa.

  2. Low dose trichloroethylene alters cytochrome P450 - 2C subfamily expression in the developing chick heart

    PubMed Central

    Makwana, Om; Ahles, Lauren; Lencinas, Alejandro; Selmin, Ornella I.; Runyan, Raymond B.

    2013-01-01

    Trichloroethylene (TCE) is an organic solvent and common environmental contaminant. TCE exposure is associated with heart defects in humans and animal models. Primary metabolism of TCE in adult rodent models is by specific hepatic cytochrome P450 enzymes (Lash et al., 2000). As association of TCE exposure with cardiac defects is in exposed embryos prior to normal liver development, we investigated metabolism of TCE in the early embryo. Developing chick embryos were dosed in ovo with environmentally relevant doses of TCE (8 ppb and 800 ppb) and RNA was extracted from cardiac and extra-cardiac tissue (whole embryo without heart). Real time PCR showed upregulation of CYP2H1 transcripts in response to TCE exposure in the heart. No detectable cytochrome expression was found in extra-cardiac tissue. As seen previously, the dose response was non-monotonic and 8ppb elicited stronger upregulation than 800 ppb. Immunostaining for CYP2C subfamily expression confirmed protein expression and showed localization in both myocardium and endothelium. TCE exposure increased protein expression in both tissues. These data demonstrate that the earliest embryonic expression of phase I detoxification enzymes is in the developing heart. Expression of these CYPs is likely to be relevant to the susceptibility of the developing heart to environmental teratogens. PMID:22855351

  3. POSSIBLE PATHWAY(S) OF TESTOSTERONE EGRESS FROM THE ACTIVE SITE OF CYTOCHROME P450 2B1: A STEERED MOLECULAR DYNAMICS SIMULATION

    E-print Network

    Gräter, Frauke

    ), and human 2C8 (Schoch et al., 2004), 2C9 (Williams et al., 2003), and 3A4 (Williams et al., 2004; Yano et alPOSSIBLE PATHWAY(S) OF TESTOSTERONE EGRESS FROM THE ACTIVE SITE OF CYTOCHROME P450 2B1: A STEERED(s) in cytochrome P450 (P450) 2B1 and to clarify the role of residues previously identified by site

  4. Cis Acting Sequences from the Rat Cytochrome P450 2B1 Gene Confer Pulmonary and Phenobarbital-Inducible Expression in Transgenic Mice

    Microsoft Academic Search

    Tobias Skarin; Rune Becher; Anders Bucht; Kristina Duvefelt; Staffan Bohm; Toril Ranneberg-Nilsen; Edel Marie Lilleaas; Per Everhard Schwarze; Rune Toftgård

    Specific cytochrome P450 enzymes show tissue-specific induction, and different regulatory units for ex- pression of these enzymes have been identified. The regulation of the phenobarbital (PB)-inducible P450 genes has been relatively well characterized in terms of PB induction, but less so with regard to tissue-spe- cific expression. CYP2B2 is not expressed in the rat lung, whereas cytochrome P450 2B1 (CYP2B1)

  5. Role of cytochrome P450 2C9 and an allelic variant in the 4?-hydroxylation of ( R)- and ( S)-flurbiprofen

    Microsoft Academic Search

    Timothy S. Tracy; Brady W. Rosenbluth; Steven A. Wrighton; Frank J. Gonzalez; Kenneth R. Korzekwa

    1995-01-01

    Flurbiprofen is a chiral non-steroidal anti-inflammatory drug used in the treatment of pain or inflammation. The primary routes of biotransformation for (R)- and (S)-flurbiprofen are oxidation (presumably cytochrome P450) and conjugation. To date, the specific cytochrome P450 (P450) involved in the oxidative metabolism of this compound (specifically 4?-hydroxylation) has not been elucidated. Experiments were conducted to characterize the kinetic parameters

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

    PubMed Central

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

    2014-01-01

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

  7. Drug-induced liver graft toxicity caused by cytochrome P450 poor metabolism

    PubMed Central

    Kóbori, László; Kõhalmy, Krisztina; Porrogi, Pálma; Sárváry, Enikõ; Gerlei, Zsuzsa; Fazakas, János; Nagy, Péter; Járay, Jenõ; Monostory, Katalin

    2008-01-01

    Aims The drug-metabolizing capacity of transplanted liver highly influences drug efficacy or toxicity, particularly in the early postoperative period. The aim of our study was to predict therapeutic failures or severe adverse drug reactions by phenotyping for cytochrome P450 (P450) polymorphism resulting in reduced or no activity of the key drug-metabolizing enzymes. Methods A validated analytical system with metabolomic tools has been developed for estimation of the drug-metabolizing capacity of transplanted liver, which allows the prediction of potential poor metabolizer phenotypes of donors and facilitates improvement of the individual recipient therapy. Results Of the 109 liver donors in Hungary, the frequency of poor metabolizers was found to be 0.92%, 5.5% and 8.3% for CYP2C9, CYP2C19 and CYP2D6, respectively. In the present study, two liver grafts transplanted in paediatric recipients were reported to be poor metabolizer phenotypes. The liver grafts presented normal function in the early postoperative days; 2 weeks after transplantation, however, increasing liver enzymes were detected. Histological investigation of a liver biopsy suggested drug toxicity. The test of drug metabolizing status showed one of the liver grafts to be a CYP2C9 poor metabolizer, and the other was found to be a CYP2C19 poor metabolizer. Rationalization of the medication resulted in the recovery of both the grafts and the recipients within 1 week. Conclusions Prospective investigation of the P450 status may lead to the optimization of drug choice and/or dose for a more effective therapy, avoid serious adverse effects, and decrease medical costs. Phenotyping donor livers and tailored medication can contribute to the improvement of graft and recipient survival. What is already known about this subject The activity of drug-metabolizing enzymes, primarily cytochrome P450 enzymes, can determine a patient's response to a drug.Therapeutic failure or drug toxicity in the postoperative period after liver transplantation is influenced by the drug metabolizing capacity of the graft.Dose adjustment or selection of an alternative drug, which is not a substrate for the polymorphic enzyme may prevent the development of side-effects in recipients of poor metabolizer liver grafts. What this study adds A validated analytical system with metabolomic tools has been developed to estimate the drug-metabolizing capacity of transplanted liver, which allows the prediction of potential poor metabolizer phenotypes of donors and facilitates the improvement of individual recipient therapy.In the test of drug-metabolizing status, one of the liver grafts was found to be a CYP2C9 poor metabolizer, while the other was a CYP2C19 poor metabolizer.Rationalization of the medication resulted in the recovery of both the grafts and the recipients within 1 week. PMID:18070218

  8. Characterization of Human Cytochrome P450s Involved in the Bioactivation of Tri-ortho-cresyl Phosphate (ToCP).

    PubMed

    Reinen, Jelle; Nematollahi, Leyla; Fidder, Alex; Vermeulen, Nico P E; Noort, Daan; Commandeur, Jan N M

    2015-04-20

    Tri-ortho-cresyl phosphate (ToCP) is a multipurpose organophosphorus compound that is neurotoxic and suspected to be involved in aerotoxic syndrome in humans. It has been reported that not ToCP itself but a metabolite of ToCP, namely, 2-(ortho-cresyl)-4H-1,2,3-benzodioxaphosphoran-2-one (CBDP), may be responsible for this effect as it can irreversibly bind to human butyrylcholinesterase (BuChE) and human acetylcholinesterase (AChE). The bioactivation of ToCP into CBDP involves Cytochrome P450s (P450s). However, the individual human P450s responsible for this bioactivation have not been identified yet. In the present study, we aimed to investigate the metabolism of ToCP by different P450s and to determine the inhibitory effect of the in vitro generated ToCP-metabolites on human BuChE and AChE. Human liver microsomes, rat liver microsomes, and recombinant human P450s were used for that purpose. The recombinant P450s 2B6, 2C18, 2D6, 3A4 and 3A5 showed highest activity of ToCP-bioactivation to BuChE-inhibitory metabolites. Inhibition experiments using pooled human liver microsomes indicated that P450 3A4 and 3A5 were mainly involved in human hepatic bioactivation of ToCP. In addition, these experiments indicated a minor role for P450 1A2. Formation of CBDP by in-house expressed recombinant human P450s 1A2 and 3A4 was proven by both LC-MS and GC-MS analysis. When ToCP was incubated with P450 1A2 and 3A4 in the presence of human BuChE, CBDP-BuChE-adducts were detected by LC-MS/MS which were not present in the corresponding control incubations. These results confirmed the role of human P450s 1A2 and 3A4 in ToCP metabolism and demonstrated that CBDP is the metabolite responsible for the BuChE inactivation. Interindividual differences at the level of P450 1A2 and 3A4 might play an important role in the susceptibility of humans in developing neurotoxic effects, such as aerotoxic syndrome, after exposure to ToCP. PMID:25706813

  9. Nature of cytochromes P450 involved in the 2-/4-hydroxylations of estradiol in human liver microsomes.

    PubMed

    Kerlan, V; Dreano, Y; Bercovici, J P; Beaune, P H; Floch, H H; Berthou, F

    1992-11-01

    Kinetics of the 2- and 4-hydroxylations of estradiol (E2) by human liver microsomal samples were studied to determine the major P450 isoform involved in these endogenous reactions. Thirty human liver microsomal samples were analysed. Metabolism of 25 microM [14C]E2 produced 2-hydroxy and 4-hydroxy derivatives with a ratio of 3.2 +/- 1.5 and a great inter-individual variation. Kinetic analysis of the 2- and 4-hydroxylations of E2 exhibited a curvilinear double reciprocal plot with an apparent Km of 15 microM. Further experiments demonstrated that alpha-naphthoflavone, testosterone and progesterone increased the 2-hydroxylation activity, suggesting the involvement of a substrate activation mechanism. These two hydroxylations of E2 were shown to be catalysed by cytochrome P450 with an apparent dissociation constant Ks of 0.8 microM. These 2- and 4-hydroxylations inter-correlated significantly (r = 0.93; N = 30). The 2-hydroxylation of E2 correlated with four monooxygenase activities known to be supported by P450 3A4/3A5, namely nifedipine oxidation (r = 0.78; N = 29); erythromycin N-demethylation (r = 0.69; N = 27), testosterone 6 beta-hydroxylation (r = 0.66; N = 25) and tamoxifen N-demethylation (r = 0.64; N = 29). On the other hand, E2-hydroxylations did not correlate with activities supported by P450 1A2 and P450 2E1. Furthermore, drugs as cyclosporin, diltiazem, triacetyl-oleandomycin and 17 alpha-ethynylestradiol inhibited more than 90% of the E2-hydroxylations at concentrations < 250 microM, while weak inhibition was shown with 500 microM cimetidine and no significant inhibition with caffeine, phenacetin and omeprazole. Finally, 2- and 4-hydroxylations of E2 correlated significantly with the content of P450 3A4/3A5 immunodetected by a monoclonal antibody anti-human P450-nifedipine (r = 0.84; N = 28). E2-hydroxylation activities were inhibited by more than 80% with polyclonal anti-human anti-P450-nifedipine. Preincubation of human liver microsomes with 100 microM gestodene (a suicide substrate of P450 3A4) inactivated this P450 isoform and accordingly allowed evaluation of the contribution of other P450 isoforms to the E2 metabolism to about 21% (+/- 17%, N = 29). All these results taken together suggest that P450 3A4/3A5 are the major forms involved in the formation of catecholestrogens in the human liver microsomes. PMID:1449532

  10. Supramolecular protein assembly supports immobilization of a cytochrome P450 monooxygenase system as water-insoluble gel

    PubMed Central

    Tan, Cheau Yuaan; Hirakawa, Hidehiko; Nagamune, Teruyuki

    2015-01-01

    Diverse applications of the versatile bacterial cytochrome P450 enzymes (P450s) are hampered by their requirement for the auxiliary proteins, ferredoxin reductases and ferredoxins, that transfer electrons to P450s. Notably, this limits the use of P450s as immobilized enzymes for industrial purposes. Herein, we demonstrate the immobilization of a bacterial P450 and its redox protein partners by supramolecular complex formation using a self-assembled heterotrimeric protein. Employment of homodimeric phosphite dehydrogenase (PTDH) for cross-linking “proliferating cell nuclear antigen-utilized protein complex of P450 and its two electron transfer-related proteins” (PUPPET) yielded a gelling PUPPET-PTDH system capable of regenerating NADH for electron supply owing to its phosphite oxidation activity. The protein gel catalyzed monooxygenation in the presence of phosphite and NAD+. The gel was completely water-insoluble and could be reused. This concept of oligomeric protein-insolubilized enzymes can be widely applied to various multienzymatic reactions such as cascade reactions and coupling reactions. PMID:25733255

  11. Supramolecular protein assembly supports immobilization of a cytochrome P450 monooxygenase system as water-insoluble gel.

    PubMed

    Tan, Cheau Yuaan; Hirakawa, Hidehiko; Nagamune, Teruyuki

    2015-01-01

    Diverse applications of the versatile bacterial cytochrome P450 enzymes (P450s) are hampered by their requirement for the auxiliary proteins, ferredoxin reductases and ferredoxins, that transfer electrons to P450s. Notably, this limits the use of P450s as immobilized enzymes for industrial purposes. Herein, we demonstrate the immobilization of a bacterial P450 and its redox protein partners by supramolecular complex formation using a self-assembled heterotrimeric protein. Employment of homodimeric phosphite dehydrogenase (PTDH) for cross-linking "proliferating cell nuclear antigen-utilized protein complex of P450 and its two electron transfer-related proteins" (PUPPET) yielded a gelling PUPPET-PTDH system capable of regenerating NADH for electron supply owing to its phosphite oxidation activity. The protein gel catalyzed monooxygenation in the presence of phosphite and NAD(+). The gel was completely water-insoluble and could be reused. This concept of oligomeric protein-insolubilized enzymes can be widely applied to various multienzymatic reactions such as cascade reactions and coupling reactions. PMID:25733255

  12. Functional Coupling of ATP-binding Cassette Transporter Abcb6 to Cytochrome P450 Expression and Activity in Liver.

    PubMed

    Chavan, Hemantkumar; Li, Feng; Tessman, Robert; Mickey, Kristen; Dorko, Kenneth; Schmitt, Timothy; Kumer, Sean; Gunewardena, Sumedha; Gaikwad, Nilesh; Krishnamurthy, Partha

    2015-03-20

    Although endogenous mechanisms that negatively regulate cytochrome P450 (P450) monooxygenases in response to physiological and pathophysiological signals are not well understood, they are thought to result from alterations in the level of endogenous metabolites, involved in maintaining homeostasis. Here we show that homeostatic changes in hepatic metabolite profile in Abcb6 (mitochondrial ATP-binding cassette transporter B6) deficiency results in suppression of a specific subset of hepatic P450 activity. Abcb6 null mice are more susceptible to pentobarbital-induced sleep and zoxazolamine-induced paralysis, secondary to decreased expression and activity of Cyp3a11 and Cyp2b10. The knock-out mice also show decrease in both basal and xeno-inducible expression and activity of a subset of hepatic P450s that appear to be related to changes in hepatic metabolite profile. These data, together with the observation that liver extracts from Abcb6-deficient mice suppress P450 expression in human primary hepatocytes, suggest that this mouse model may provide an opportunity to understand the physiological signals and the mechanisms involved in negative regulation of P450s. PMID:25623066

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

    PubMed Central

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

    2012-01-01

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

  14. In vitro and in vivo evaluation of the effect of puerarin on hepatic cytochrome p450-mediated drug metabolism.

    PubMed

    Kim, Sang-Bum; Yoon, In-Soo; Kim, Kyu-Sang; Cho, Sung-Jun; Kim, Yeong Shik; Cho, Hyun-Jong; Chung, Suk-Jae; Chong, Saeho; Kim, Dae-Duk

    2014-05-01

    Puerarin (8-?-D-glucopyranosyl-7-hydroxy-3-(4-hydroxyphenyl)-4H-1-benzopyran-4-one) is a major pharmacological component of Puerariae Radix, the root of Pueraria lobata. We investigated the effect of puerarin on hepatic cytochrome P450-mediated drug metabolism in rats and humans. The in vitro cytochrome P450 inhibitory effect of puerarin in human and rat liver microsomes was evaluated using the following model cytochrome P450 substrates: phenacetin for CYP1A, diclofenac for CYP2C, dextromethorphan for CYP2D, and testosterone for CYP3A. The in vivo pharmacokinetics of intravenous and oral buspirone, a probe substrate for CYP3A, was studied with single simultaneous intravenous coadministration of puerarin in rats. In the in vitro cytochrome P450 inhibition study, the rate of disappearance of testosterone was significantly reduced in the presence of 10?µM PU, while that of other cytochrome P450 substrates was not significantly affected in both human and rat liver microsomes, suggesting that puerarin inhibits the in vitro hepatic CYP3A-mediated metabolism in the human and rat systems (IC50?=?15.5?±?3.9?µM). After intravenous administration of buspirone with single simultaneous coadministration of intravenous puerarin at a dose of 10?mg/kg in rats, the total area under the plasma concentration-time curve from time zero to time infinity was increased while time-averaged total body clearance decreased. When buspirone was orally administered in rats with the 10?mg/kg intravenous puerarin coadministration, both total area under the plasma concentration-time curve from time zero to time infinity and the extent of absolute oral bioavailability were significantly increased. Therefore, results of the in vitro microsomal and in vivo pharmacokinetic studies suggest the possible inhibition of hepatic CYP3A-mediated drug metabolism by puerarin administration, potentially leading to metabolism-mediated herb-drug interactions with clinical significance. PMID:24710899

  15. Inhibition of cytochrome P450 2B4 by environmentally persistent free radical-containing particulate matter.

    PubMed

    Reed, James R; Cruz, Albert Leo N Dela; Lomnicki, Slawo M; Backes, Wayne L

    2015-05-15

    Combustion processes generate particulate matter (PM) that can affect human health. The presence of redox-active metals and aromatic hydrocarbons in the post-combustion regions results in the formation of air-stable, environmentally persistent free radicals (EPFRs) on entrained particles. Exposure to EPFRs has been shown to negatively influence pulmonary and cardiovascular functions. Cytochromes P450 (P450/CYP) are endoplasmic reticulum resident proteins that are responsible for the metabolism of foreign compounds. Previously, it was shown that model EPFRs, generated by exposure of silica containing 5% copper oxide (CuO-Si) to either dicholorobenzene (DCB230) or 2-monochlorophenol (MCP230) at ? 230°C, inhibited six forms of P450 in rat liver microsomes (Toxicol. Appl. Pharmacol. (2014) 277:200-209). In this study, the inhibition of P450 by MCP230 was examined in more detail by measuring its effect on the rate of metabolism of 7-ethoxy-4-trifluoromethylcoumarin (7EFC) and 7-benzyloxyresorufin (7BRF) by the purified, reconstituted CYP2B4 system. MCP230 inhibited the CYP2B4-mediated metabolism of 7EFC at least 10-fold more potently than non-EPFR controls (CuO-Si, silica, and silica generated from heating silica and MCP at 50°C, so that EPFRs were not formed (MCP50)). The inhibition by EPFRs was specific for the P450 and did not affect the ability of the redox partner, P450 reductase (CPR) from reducing cytochrome c. All of the PM inhibited CYP2B4-mediated metabolism noncompetitively with respect to substrate. When CYP2B4-mediated metabolism of 7EFC was measured as a function of the CPR concentration, the mechanism of inhibition was competitive. EPFRs likely inhibit CYP2B4-mediated substrate metabolism by physically disrupting the CPR·P450 complex. PMID:25817938

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

    SciTech Connect

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

    1986-05-01

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

  17. Diversification of furanocoumarin-metabolizing cytochrome P450 monooxygenases in two papilionids: Specificity and substrate encounter rate.

    PubMed

    Li, Weimin; Schuler, Mary A; Berenbaum, May R

    2003-11-25

    Diversification of cytochrome P450 monooxygenases (P450s) is thought to result from antagonistic interactions between plants and their herbivorous enemies. However, little direct evidence demonstrates the relationship between selection by plant toxins and adaptive changes in herbivore P450s. Here we show that the furanocoumarin-metabolic activity of CYP6B proteins in two species of swallowtail caterpillars is associated with the probability of encountering host plant furanocoumarins. Catalytic activity was compared in two closely related CYP6B4 and CYP6B17 groups in the polyphagous congeners Papilio glaucus and Papilio canadensis. Generally, P450s from P. glaucus, which feeds occasionally on furanocoumarin-containing host plants, display higher activities against furanocoumarins than those from P. canadensis, which normally does not encounter furanocoumarins. These P450s in turn catalyze a larger range of furanocoumarins at lower efficiency than CYP6B1, a P450 from Papilio polyxenes, which feeds exclusively on furanocoumarin-containing host plants. Reconstruction of the ancestral CYP6B sequences using maximum likelihood predictions and comparisons of the sequence and geometry of their active sites to those of contemporary CYP6B proteins indicate that host plant diversity is directly related to P450 activity and inversely related to substrate specificity. These predictions suggest that, along the lineage leading to Papilio P450s, the ancestral, highly versatile CYP6B protein presumed to exist in a polyphagous species evolved through time into a more efficient and specialized CYP6B1-like protein in Papilio species with continual exposure to furanocoumarins. Further diversification of Papilio CYP6Bs has likely involved interspersed events of positive selection in oligophagous species and relaxation of functional constraints in polyphagous species. PMID:12968082

  18. Diversification of furanocoumarin-metabolizing cytochrome P450 monooxygenases in two papilionids: Specificity and substrate encounter rate

    PubMed Central

    Li, Weimin; Schuler, Mary A.; Berenbaum, May R.

    2003-01-01

    Diversification of cytochrome P450 monooxygenases (P450s) is thought to result from antagonistic interactions between plants and their herbivorous enemies. However, little direct evidence demonstrates the relationship between selection by plant toxins and adaptive changes in herbivore P450s. Here we show that the furanocoumarin-metabolic activity of CYP6B proteins in two species of swallowtail caterpillars is associated with the probability of encountering host plant furanocoumarins. Catalytic activity was compared in two closely related CYP6B4 and CYP6B17 groups in the polyphagous congeners Papilio glaucus and Papilio canadensis. Generally, P450s from P. glaucus, which feeds occasionally on furanocoumarin-containing host plants, display higher activities against furanocoumarins than those from P. canadensis, which normally does not encounter furanocoumarins. These P450s in turn catalyze a larger range of furanocoumarins at lower efficiency than CYP6B1, a P450 from Papilio polyxenes, which feeds exclusively on furanocoumarin-containing host plants. Reconstruction of the ancestral CYP6B sequences using maximum likelihood predictions and comparisons of the sequence and geometry of their active sites to those of contemporary CYP6B proteins indicate that host plant diversity is directly related to P450 activity and inversely related to substrate specificity. These predictions suggest that, along the lineage leading to Papilio P450s, the ancestral, highly versatile CYP6B protein presumed to exist in a polyphagous species evolved through time into a more efficient and specialized CYP6B1-like protein in Papilio species with continual exposure to furanocoumarins. Further diversification of Papilio CYP6Bs has likely involved interspersed events of positive selection in oligophagous species and relaxation of functional constraints in polyphagous species. PMID:12968082

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

    PubMed Central

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

    2013-01-01

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

  20. Cold induces catalytic iron release of cytochrome P-450 origin: a critical step in cold storage-induced renal injury.

    PubMed

    Huang, Hong; Salahudeen, Abdulla K

    2002-08-01

    Earlier experimental studies have suggested a role for iron in cold-storage-induced organ injury. Whether the cytochrome P-450 enzymes, shown to be a source for iron in several injury models, contribute to cold-induced iron release is not known. Storage of human proximal tubular epithelial (RPTE) cells at 4 degrees C in the University of Wisconsin (UW) solution caused a significant and time-dependent increase in bleomycin-detectable iron (BDI). To identify the cellular source of BDI, RPTE cells were subfractionated and stored at 4 degrees C for 4 h. Bleomycin-detectable iron release was highest in the microsomes, next in the cytosol and none in the mitochondria. As microsomes are rich in iron-containing cytochrome P-450 enzymes, microsomes were cold stored with P-450 inhibitors, cimetidine and piperonyl butoxide. P-450 inhibitors significantly reduced cold-induced BDI release. Furthermore, cimetidine and iron chelator deferoxamine (DFO) significantly reduced cold-induced cell injury, suggesting a role for P-450-derived iron in cold-induced cell injury. In rat kidney experiments, BDI and LDH release were significantly higher in cold-stored kidneys than in control kidneys. Inclusion of cimetidine and DFO in the cold-storage solution significantly suppressed the BDI and LDH release, and reduced the ultrastructural changes. Our data demonstrate for the first time that cold-induced catalytic iron release may be at least in part of microsomal cytochrome P-450 origin, and that it participates in cold-storage-induced renal injury. In the clinical setting, sequestering free iron released during cold storage is possible and may prove to be useful in limiting organ injury. PMID:12201364

  1. Vascular characterization of mice with endothelial expression of cytochrome P450 4F2.

    PubMed

    Cheng, Jennifer; Edin, Matthew L; Hoopes, Samantha L; Li, Hong; Bradbury, J Alyce; Graves, Joan P; DeGraff, Laura M; Lih, Fred B; Garcia, Victor; Shaik, Jafar Sadik B; Tomer, Kenneth B; Flake, Gordon P; Falck, John R; Lee, Craig R; Poloyac, Samuel M; Schwartzman, Michal L; Zeldin, Darryl C

    2014-07-01

    Cytochrome P450 (CYP) 4A and 4F enzymes metabolize arachidonic acid to 20-hydroxyeicosatetraenoic acid (20-HETE). Although CYP4A-derived 20-HETE is known to have prohypertensive and proangiogenic properties, the effects of CYP4F-derived metabolites are not well characterized. To investigate the role of CYP4F2 in vascular disease, we generated mice with endothelial expression of human CYP4F2 (Tie2-CYP4F2-Tr). LC/MS/MS analysis revealed 2-foldincreases in 20-HETE levels in tissues and endothelial cells (ECs), relative to wild-type (WT) controls. Tie2-CYP4F2-Tr ECs demonstrated increases in growth (267.1 ± 33.4 vs. 205.0 ± 13% at 48 h) and tube formation (7.7 ± 1.1 vs. 1.6 ± 0.5 tubes/field) that were 20-HETE dependent and associated with up-regulation of prooxidant NADPH oxidase and proangiogenic VEGF. Increases in VEGF and NADPH oxidase levels were abrogated by inhibitors of NADPH oxidase and MAPK, respectively, suggesting the possibility of crosstalk between pathways. Interestingly, IL-6 levels in Tie2-CYP4F2-Tr mice (18.6 ± 2.7 vs. 7.9 ± 2.7 pg/ml) were up-regulated via NADPH oxidase- and 20-HETE-dependent mechanisms. Although Tie2-CYP4F2-Tr aortas displayed increased vasoconstriction, vasorelaxation and blood pressure were unchanged. Our findings indicate that human CYP4F2 significantly increases 20-HETE production, CYP4F2-derived 20-HETE mediates EC proliferation and angiogenesis via VEGF- and NADPH oxidase-dependent manners, and the Tie2-CYP4F2-Tr mouse is a novel model for examining the pathophysiological effects of CYP4F2-derived 20-HETE in the vasculature.-Cheng, J., Edin, M. L., Hoopes, S. L., Li, H., Bradbury, J. A., Graves, J. P., DeGraff, L. M., Lih, F. B., Garcia, V., Shaik, J. S. B., Tomer, K. B., Flake, G. P., Falck, J. R., Lee, C. R., Poloyac, S. M., Schwartzman, M. L., Zeldin, D. C. Vascular characterization of mice with endothelial expression of cytochrome P450 4F2. PMID:24668751

  2. Inhibitory effect of mitragynine on human cytochrome P450 enzyme activities

    PubMed Central

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

    2013-01-01

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

  3. In vitro characterization of clobazam metabolism by recombinant cytochrome P450 enzymes: importance of CYP2C19.

    PubMed

    Giraud, Carole; Tran, Agnès; Rey, Elisabeth; Vincent, Jean; Tréluyer, Jean-Marc; Pons, Gérard

    2004-11-01

    The specific cytochrome P450 (P450) isoforms mediating the biotransformations of clobazam (CLB) and those of its major metabolites, N-desmethylclobazam (NCLB) and 4'-hydroxyclobazam were identified using cDNA-expressed P450 and P450-specific chemical inhibitors. Among the 13 cDNA-expressed P450 isoforms tested, CLB was mainly demethylated by CYP3A4, CYP2C19, and CYP2B6 and 4'-hydroxylated by CYP2C19 and CYP2C18. CYP2C19 and CYP2C18 catalyzed the 4'-hydroxylation of NCLB. The kinetics of the major biotransformations were studied: CYP3A4, CYP2C19, and CYP2B6 mediated the formation of NCLB with Km = 29.0, 31.9, and 289 microM, Vmax = 6.20, 1.15, and 5.70 nmol/min/nmol P450, and intrinsic clearance (CLint) = 214, 36.1, and 19.7 microl/min/nmol P450, respectively. NCLB was hydroxylated to 4'-hydroxydesmethylclobazam by CYP2C19 with Km = 5.74 microM, Vmax = 0.219 nmol/min/nmol P450, and CLint = 38.2 microl/min/nmol P450 (Hill coefficient = 1.54). These findings were supported by chemical inhibition studies in human liver microsomes. Indeed, ketoconazole (1 microM) inhibited the demethylation of CLB by 70% and omeprazole (10 microM) by 19%; omeprazole inhibited the hydroxylation of NCLB by 26%. Twenty-two epileptic patients treated with CLB were genotyped for CYP2C19. The NCLB/CLB plasma metabolic ratio was significantly higher in the subjects carrying one CYP2C19*2 mutated allele than in those carrying the wild-type genotype. CYP3A4 and CYP2C19 are the main P450s involved in clobazam metabolism. Interactions with other drugs metabolized by these P450s can occur; moreover, the CYP2C19 genetic polymorphism could be responsible for interindividual variations of plasma concentrations of N-desmethylclobazam and thus for occurrence of adverse events. PMID:15483195

  4. The regulation of cytochrome P450 2E1 during LPS-induced inflammation in the rat

    SciTech Connect

    Abdulla, Dalya [Department of Pharmacology, Sir Charles Tupper Medical Bldg., Dalhousie University, Halifax, Nova Scotia, B3H 4H7 (Canada); Goralski, Kerry B. [Department of Pharmacology, Sir Charles Tupper Medical Bldg., Dalhousie University, Halifax, Nova Scotia, B3H 4H7 (Canada); College of Pharmacy, Burbidge Building, Dalhousie University, Halifax, Nova Scotia, B3H 3J5 (Canada); Renton, Kenneth W. [Department of Pharmacology, Sir Charles Tupper Medical Bldg., Dalhousie University, Halifax, Nova Scotia, B3H 4H7 (Canada)]. E-mail: Ken.Renton@dal.ca

    2006-10-01

    It is well known that inflammatory and infectious conditions differentially regulate cytochrome P450 (P450)-mediated drug metabolism in the liver. We have previously outlined a potential pathway for the downregulation in hepatic cytochrome P450 following LPS-mediated inflammation in the CNS (Abdulla, D., Goralski, K.B., Garcia Del Busto Cano, E., Renton, K.W., 2005. The signal transduction pathways involved in hepatic cytochrome P450 regulation in the rat during an LPS-induced model of CNS inflammation. Drug Metab. Dispos). The purpose of this study was to outline the effects of LPS-induced peripheral and central nervous system inflammation on hepatic cytochrome P450 2E1 (CYP2E1) in vivo, an enzyme that plays an important role in various physiological and pathological states. We report an increase in hepatic mRNA expression of CYP2E1 that occurred as early as 2-3 h following either the intraperitoneal (i.p.) injection of 5 mg/kg LPS or i.c.v. administration of 25 {mu}g of LPS. This increase in CYP2E1 mRNA expression was sustained for 24 h. In sharp contrast to the increase in hepatic CYP2E1 mRNA, we observed a significant reduction in the catalytic activity of this enzyme 24 h following either the i.c.v. or i.p. administration of LPS. Cycloheximide or actinomycin-D did not change the LPS-mediated downregulation in hepatic CYP2E1 catalytic activity. Our results support the idea that LPS acts at two different levels to regulate hepatic CYP2E1: a transcriptional level to increase CYP2E1 mRNA expression and a post-transcriptional level to regulate CYP2E1 protein and activity.

  5. Development of a plant viral-vector-based gene expression assay for the screening of yeast cytochrome p450 monooxygenases.

    PubMed

    Hanley, Kathleen; Nguyen, Long V; Khan, Faizah; Pogue, Gregory P; Vojdani, Fakhrieh; Panda, Sanjay; Pinot, Franck; Oriedo, Vincent B; Rasochova, Lada; Subramanian, Mani; Miller, Barbara; White, Earl L

    2003-02-01

    Development of a gene discovery tool for heterologously expressed cytochrome P450 monooxygenases has been inherently difficult. The activity assays are labor-intensive and not amenable to parallel screening. Additionally, biochemical confirmation requires coexpression of a homologous P450 reductase or complementary heterologous activity. Plant virus gene expression systems have been utilized for a diverse group of organisms. In this study we describe a method using an RNA vector expression system to phenotypically screen for cytochrome P450-dependent fatty acid omega-hydroxylase activity. Yarrowia lipolytica CYP52 gene family members involved in n-alkane assimilation were amplified from genomic DNA, cloned into a plant virus gene expression vector, and used as a model system for determining heterologous expression. Plants infected with virus vectors expressing the yeast CYP52 genes (YlALK1-YlALK7) showed a distinct necrotic lesion phenotype on inoculated plant leaves. No phenotype was detected on negative control constructs. YlALK3-, YlALK5-, and YlALK7-inoculated plants all catalyzed the terminal hydroxylation of lauric acid as confirmed using thin-layer and gas chromatography/mass spectrometry methods. The plant-based cytochrome P450 phenotypic screen was tested on an n-alkane-induced Yarrowia lipolytica plant virus expression library. A subset of 1,025 random library clones, including YlALK1-YlALK7 constructs, were tested on plants. All YlALK gene constructs scored positive in the randomized screen. Following nucleotide sequencing of the clones that scored positive using a phenotypic screen, approximately 5% were deemed appropriate for further biochemical analysis. This report illustrates the utility of a plant-based system for expression of heterologous cytochrome P450 monooxygenases and for the assignment of gene function. PMID:15090141

  6. Human-liver cytochromes P-450 expressed in yeast as tools for reactive-metabolite formation studies. Oxidative activation of tienilic acid by cytochromes P-450 2C9 and 2C10.

    PubMed

    Lopez Garcia, M P; Dansette, P M; Valadon, P; Amar, C; Beaune, P H; Guengerich, F P; Mansuy, D

    1993-04-01

    Human liver cytochromes P-450 (P450) 2C9 and 2C10 expressed in yeast reproduce all the metabolic features of the oxidation of tienilic acid (2-aryloxo-thiophene) and its isomer (3-aroylthiophene) by human liver microsomes. Microsomes of yeast expressing either P450 2C9 or P450 2C10 catalyze (a) the 5-hydroxylation of tienilic acid by NADPH and O2 (Km = 6 microM, Vmax = 2.5 turnover/min), (b) the activation of tienilic acid and its isomer into electrophilic metabolites which covalently bind to proteins, and (c) the formation of a mercaptoethanol adduct which results from the trapping of the tienilic acid isomer sulfoxide by this thiol. Microsomes of yeast expressing human liver P450 3A4, 1A1 and 1A2 are unable to catalyze these reactions. There is a striking similarity between the quantitative characteristics of the oxidation of tienilic acid (and its isomer) by yeast-expressed P450 2C9 (or 2C10) and by human liver microsomes: (a) analogous Km values (around 10 microM) for tienilic acid 5-hydroxylation, (b) a strong inhibition of tienilic acid oxidation by human sera containing anti-(liver kidney microsomes type 2) (anti-LKM2) antibodies, and (c) almost identical relative ratios of tienilic acid metabolic activation/5-hydroxylation and of tienilic acid activation/the activation of its isomer with both systems. Rates of oxidation of tienilic acid (and its isomer) by yeast microsomes are 6-8 fold higher than those found in human liver microsomes, which would be in agreement with the previously reported amount of P450 2C9 in human liver. These results not only suggest the important role of P450 2C9 in the oxidative metabolism of tienilic acid in human liver, but also indicate that the 5-hydroxylation reaction could be a useful marker for P450 2C9 activity and underline the interest of human liver P450s expressed in yeast as tools for studying the formation of reactive metabolites. PMID:8477697

  7. Insights into the Role of Substrates on the Interaction between Cytochrome b5 and Cytochrome P450 2B4 by NMR

    PubMed Central

    Zhang, Meng; Le Clair, Stéphanie V.; Huang, Rui; Ahuja, Shivani; Im, Sang-Choul; Waskell, Lucy; Ramamoorthy, Ayyalusamy

    2015-01-01

    Mammalian cytochrome b5 (cyt b5) is a membrane-bound protein capable of donating an electron to cytochrome P450 (P450) in the P450 catalytic cycle. The interaction between cyt b5 and P450 has been reported to be affected by the substrates of P450; however, the mechanism of substrate modulation on the cyt b5-P450 complex formation is still unknown. In this study, the complexes between full-length rabbit cyt b5 and full-length substrate-free/substrate-bound cytochrome P450 2B4 (CYP2B4) are investigated using NMR techniques. Our findings reveal that the population of complexes is ionic strength dependent, implying the importance of electrostatic interactions in the complex formation process. The observation that the cyt b5-substrate-bound CYP2B4 complex shows a weaker dependence on ionic strength than the cyt b5-substrate-free CYP2B4 complex suggests the presence of a larger fraction of steoreospecific complexes when CYP2B4 is substrate-bound. These results suggest that a CYP2B4 substrate likely promotes specific interactions between cyt b5 and CYP2B4. Residues D65, V66, T70, D71 and A72 are found to be involved in specific interactions between the two proteins due to their weak response to ionic strength change. These findings provide insights into the mechanism underlying substrate modulation on the cyt b5-P450 complexation process. PMID:25687717

  8. Insights into the Role of Substrates on the Interaction between Cytochrome b5 and Cytochrome P450 2B4 by NMR

    NASA Astrophysics Data System (ADS)

    Zhang, Meng; Le Clair, Stéphanie V.; Huang, Rui; Ahuja, Shivani; Im, Sang-Choul; Waskell, Lucy; Ramamoorthy, Ayyalusamy

    2015-02-01

    Mammalian cytochrome b5 (cyt b5) is a membrane-bound protein capable of donating an electron to cytochrome P450 (P450) in the P450 catalytic cycle. The interaction between cyt b5 and P450 has been reported to be affected by the substrates of P450; however, the mechanism of substrate modulation on the cyt b5-P450 complex formation is still unknown. In this study, the complexes between full-length rabbit cyt b5 and full-length substrate-free/substrate-bound cytochrome P450 2B4 (CYP2B4) are investigated using NMR techniques. Our findings reveal that the population of complexes is ionic strength dependent, implying the importance of electrostatic interactions in the complex formation process. The observation that the cyt b5-substrate-bound CYP2B4 complex shows a weaker dependence on ionic strength than the cyt b5-substrate-free CYP2B4 complex suggests the presence of a larger fraction of steoreospecific complexes when CYP2B4 is substrate-bound. These results suggest that a CYP2B4 substrate likely promotes specific interactions between cyt b5 and CYP2B4. Residues D65, V66, T70, D71 and A72 are found to be involved in specific interactions between the two proteins due to their weak response to ionic strength change. These findings provide insights into the mechanism underlying substrate modulation on the cyt b5-P450 complexation process.

  9. Photosystem I from plants as a bacterial cytochrome P450 surrogate electron donor: terminal hydroxylation of branched hydrocarbon chains

    PubMed Central

    Jensen, Kenneth; Johnston, Jonathan B.; Ortiz de Montellano, Paul R.

    2013-01-01

    The ability of cytochrome P450 enzymes to catalyze highly regio- and stereospecific hydroxylations makes them attractive alternatives to approaches based on chemical synthesis but they require expensive cofactors, e.g. NAD(P)H, which limits their commercial potential. Ferredoxin (Fdx) is a multifunctional electron carrier that in plants accepts electrons from photosystem I (PSI) and facilitates photoreduction of NADP+ to NADPH mediated by ferredoxin-NAD(P)H oxidoreductase (FdR). In bacteria, the electron flow is reversed and Fdx accepts electrons from NADPH via FdR and serves as the direct electron donor to bacterial P450s. By combining the two systems, we demonstrate that irradiation of PSI can drive the activity of a bacterial P450, CYP124 from Mycobacterium tuberculosis. The substitution of the costly cofactor NADPH with sunlight illustrates the potential of the light-driven hydroxylation system for biotechnology applications. PMID:21983973

  10. The Cytochrome P450 Epoxygenase Pathway Regulates the Hepatic Inflammatory Response in Fatty Liver Disease

    PubMed Central

    Schuck, Robert N.; Zha, Weibin; Edin, Matthew L.; Gruzdev, Artiom; Vendrov, Kimberly C.; Miller, Tricia M.; Xu, Zhenghong; Lih, Fred B.; DeGraff, Laura M.; Tomer, Kenneth B.; Jones, H. Michael; Makowski, Liza; Huang, Leaf; Poloyac, Samuel M.; Zeldin, Darryl C.; Lee, Craig R.

    2014-01-01

    Fatty liver disease is an emerging public health problem without effective therapies, and chronic hepatic inflammation is a key pathologic mediator in its progression. Cytochrome P450 (CYP) epoxygenases metabolize arachidonic acid to biologically active epoxyeicosatrienoic acids (EETs), which have potent anti-inflammatory effects. Although promoting the effects of EETs elicits anti-inflammatory and protective effects in the cardiovascular system, the contribution of CYP-derived EETs to the regulation of fatty liver disease-associated inflammation and injury is unknown. Using the atherogenic diet model of non-alcoholic fatty liver disease/non-alcoholic steatohepatitis (NAFLD/NASH), our studies demonstrated that induction of fatty liver disease significantly and preferentially suppresses hepatic CYP epoxygenase expression and activity, and both hepatic and circulating levels of EETs in mice. Furthermore, mice with targeted disruption of Ephx2 (the gene encoding soluble epoxide hydrolase) exhibited restored hepatic and circulating EET levels and a significantly attenuated induction of hepatic inflammation and injury. Collectively, these data suggest that suppression of hepatic CYP-mediated EET biosynthesis is an important pathological consequence of fatty liver disease-associated inflammation, and that the CYP epoxygenase pathway is a central regulator of the hepatic inflammatory response in NAFLD/NASH. Future studies investigating the utility of therapeutic strategies that promote the effects of CYP-derived EETs in NAFLD/NASH are warranted. PMID:25310404

  11. Impact of age on hepatic cytochrome P450 of domestic male Camborough-29 pigs.

    PubMed

    Hu, S X

    2015-04-01

    Swine is not only an important species in veterinary medicine research but also a popular animal model for human drug discovery. It is valuable to understand the impact of pig age on abundance and activity of porcine hepatic cytochrome P450 (CYP450). Liver microsomes were prepared from Camborough-29 intact male pigs at the age of 1 day and 2 weeks and the castrated male pigs at the age of 5, 10, and 20 weeks. Hepatic CYP450 content in the liver microsomes was measured using a UV/visible spectroscopic method. The activities of CYP450s were evaluated by metabolism of phenacetin, coumarin, tolbutamide, bufuralol, chlorzoxazone, and midazolam. The porcine hepatic CYP450 content increased with age with a plateau between age 2 and 5 weeks. Activities of all CYP450 enzymes increased with age of pigs too. The bufuralol 1'-hydroxylase showed the highest hepatic activities compared with other CYP enzymes at all ages of pigs. The average activities at the age of 20 weeks were about five times higher than those at the age of 5 weeks for most of the CYP enzymes. With compensation of the ratio of liver to body weights, the overall CYP450 metabolism capability of the pigs may be peaked around ages of 10 to 20 weeks. Those findings suggest that metabolism can be significantly different in growing phase of pigs and that the age may be an important factor in porcine medicine evaluation and pig model development. PMID:25230135

  12. Involvement of cytochrome P450-mediated metabolism in tienilic acid hepatotoxicity in rats.

    PubMed

    Nishiya, Takayoshi; Kato, Michiyuki; Suzuki, Takami; Maru, Chikako; Kataoka, Hiroko; Hattori, Chiharu; Mori, Kazuhiko; Jindo, Toshimasa; Tanaka, Yorihisa; Manabe, Sunao

    2008-12-15

    Tienilic acid is reported to be converted into electrophilic metabolites by cytochrome P450 (CYP) in vitro. In vivo, however, the metabolites have not been detected and their effect on liver function is unknown. We previously demonstrated that tienilic acid decreased the GSH level and upregulated genes responsive to oxidative/electrophilic stresses, such as heme oxygenase-1 (Ho-1), glutamate-cysteine ligase modifier subunit (Gclm) and NAD(P)H dehydrogenase quinone 1 (Nqo1), in rat liver, as well as inducing hepatotoxicity by co-treatment with the glutathione biosynthesis inhibitor l-buthionine-(S,R)-sulfoximine (BSO). In this study, for the first time, we identified a glutathione-tienilic acid adduct, a stable conjugate of putative electrophilic metabolites with glutathione (GSH), in the bile of rats given a single oral dose of tienilic acid (300mg/kg). Furthermore, a tienilic acid-induced decrease in the GSH level and upregulation of Ho-1, Gclm and Nqo1 were completely blocked by pretreatment with the CYP inhibitor 1-aminobenzotriazole (ABT, 66mg/kg, i.p.). The increase in the serum ALT level and hepatocyte necrosis resulting from the combined dosing of BSO and tienilic acid was prevented by ABT, despite a low hepatic GSH level. These findings suggest that the electrophilic metabolites of tienilic acid produced by CYP induce electrophilic/oxidative stresses in the rat liver and this contributes to the hepatotoxicity of tienilic acid under impaired GSH biosynthesis. PMID:18992796

  13. Effect of cyclosporine and tacrolimus on cytochrome p450 activities in human liver microsomes.

    PubMed

    Niwa, Toshiro; Yamamoto, Sachiko; Saito, Miho; Shiraga, Toshifumi; Takagi, Akira

    2007-01-01

    The effects of cyclosporine and tacrolimus on cytochrome P450 (CYP) 1A2-mediated 7-ethoxyresorufin O-deethylation, CYP2C9-mediated tolbutamide hydroxylation, CYP2C19-mediated S-mephenytoin 4'-hydroxylation, CYP2D6-mediated debrisoquine 4-hydroxylation, CYP2E1-mediated chlorzoxazone 6-hydroxylation, CYP3A4-mediated nifedipine oxidation, and CYP3A4-mediated testosterone 6beta-hydroxylation activities in human liver microsomes were compared. Cyclosporine and tacrolimus, at concentrations of 0.2 or 2 muM, neither inhibited nor stimulated any of the metabolic activities except for those of CYP3A4. On the other hand, cyclosporine and tacrolimus competitively inhibited CYP3A4-mediated nifedipine oxidation activity, with inhibition constants (K(i)) of 1.42 and 0.36 muM, respectively. In addition, 20 muM cyclosporine inhibited CYP2C19 and CYP2D6 activities by 29% and 30%, respectively. These results suggest that tacrolimus would not cause clinically significant interactions with other drugs, which are metabolized by CYPs, via the inhibition of hepatic metabolism and that the reason why cyclosporine, but not tacrolimus, has a pharmacokinetic inhibitory effect might be that the dosage and/or the unbound concentrations around its metabolic enzymes are higher than those of tacrolimus, rather than the differences in the inhibition potential. Obvious substrate-dependent effects on CYP3A4-inhibition potential were not observed. PMID:17202802

  14. 2011 CCNP Heinz Lehmann Award paper: Cytochrome P450–mediated drug metabolism in the brain

    PubMed Central

    Miksys, Sharon; Tyndale, Rachel F.

    2013-01-01

    Cytochrome P450 enzymes (CYPs) metabolize many drugs that act on the central nervous system (CNS), such as antidepressants and antipsychotics; drugs of abuse; endogenous neurochemicals, such as serotonin and dopamine; neurotoxins; and carcinogens. This takes place primarily in the liver, but metabolism can also occur in extrahepatic organs, including the brain. This is important for CNS-acting drugs, as variation in brain CYP-mediated metabolism may be a contributing factor when plasma levels do not predict drug response. This review summarizes the characterization of CYPs in the brain, using examples from the CYP2 subfamily, and discusses sources of variation in brain CYP levels and metabolism. Some recent experiments are described that demonstrate how changes in brain CYP metabolism can influence drug response, toxicity and drug-induced behaviours. Advancing knowledge of brain CYP-mediated metabolism may help us understand why patients respond differently to drugs used in psychiatry and predict risk for psychiatric disorders, including neurodegenerative diseases and substance abuse. PMID:23199531

  15. Evaluation of cytochrome P450 2D6 phenotyping in healthy adult Western Indians

    PubMed Central

    Gogtay, Nithya J.; Mali, Nitin B.; Iyer, Krishna; Kadam, Prashant P.; Sridharan, Kannan; Shrimal, Divya; Thatte, Urmila M.

    2014-01-01

    Background: Cytochrome P450 2D6 (CYP2D6) metabolizes around 25% of the drugs used in therapeutics and different polymorphisms have been identified in various populations. This study aimed at finding the prevalence of CYP2D6 polymorphisms using dextromethorphan as a probe drug. Materials and Methods: Healthy participants were administered 60 mg dextromethorphan after an overnight fast and 5 ml of blood was collected 3 h postdose. A validated laboratory method was used to measure both dextromethorphan and its active metabolite, dextrorphan from plasma. Metabolic ratio (MR) of dextromethorphan to dextrorphan was calculated for each of the participants. Probit analysis was done and antimode was defined. Individuals with log MR equal to or higher than the antimode were classified as poor metabolizers (PMs) and those with values less than antimode were categorized as extensive metabolizers (EMs). Results: Data from a total of 149 participants were evaluated and the median (range) of MR was 0.25 (0.03-3.01). The polynomial equation obtained in probit analysis gave an antimode for MR of 1.39. Five (3.36%) participants were PMs and 144 (96.64%) were found to be EMs. One participant had reported mild drowsiness 2 h postdose that subsided spontaneously without any intervention. Conclusion: The prevalence of CYP2D6 polymorphism in Western Indian population is low (3.36%) and is similar to other populations. PMID:24987171

  16. Effects of repeated allopurinol administration on rat cytochrome P450 activity.

    PubMed

    Xu, Ren-Ai; Xu, Zhi-Sheng; Hu, Lu-Feng; Zhang, Chun-Hong; Pan, Xiao-Feng; Shi, Da-Wei; Ye, Xiao-Lan; Llu, Yun-Jie; Zhang, Xiu-Hua

    2013-05-01

    Allopurinol is a popular and widely-prescribed anti-hyperuricemic agent that has been implicated in drug interactions with substrates of several cytochrome P450 (CYP) enzymes. The effect of repeated allopurinol administration (20 mg/kg, once daily for 14 days) on metabolic activity of CYP was assessed in rats. This was a randomized, double-blind, two-way crossover study with a 4-week washout period between phases. The substrates used in this study were phenacetin (CYP1A2), tolbutamide (CYP2C9), omeprazole (CYP2C19) and dextromethorphan (CYP2D6). Validated HPLC-MS/MS was used to quantify all compounds. Our study showed that allopurinol administration inhibited CYP1A2 activity, causing a significant increase in AUC (0-infinity) (P < 0.01) and t1/2 (P < 0.05) of phenacetin, and a distinct decline in CL (P < 0.01). However, there were no significant differences of another three probe drugs in plasma concentrations and the corresponding pharmacokinetic parameters between the allopurinol-treated and normal saline-treated rats. The findings in this study suggested that allopurinol could inhibit CYP1A2 but did not influence CYP2C9, CYP2C19 and CYP2D6 enzymes. PMID:23802435

  17. Effect of Cholesterol on the Structure of Membrane-Attached Cytochrome P450 3A4.

    PubMed

    Navrátilová, Veronika; Paloncýová, Markéta; Kajšová, Michaela; Berka, Karel; Otyepka, Michal

    2015-03-23

    Cholesterol is a widely researched component of biological membranes that significantly influences membrane properties. Human cytochrome P450 3A4 (CYP3A4) is an important drug-metabolizing enzyme, wherein the catalytic domain is attached to a membrane by an N-terminal ?-helical transmembrane anchor. We analyzed the behavior of CYP3A4 immersed in a 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) membrane with various amounts of cholesterol. The presence of cholesterol caused ordering and thickening of the membrane and led to greater immersion and inclination of CYP3A4 toward the membrane. Cholesterol also lowered the flexibility of and tended to concentrate around membrane-immersed parts of CYP3A4. Further, the pattern of the CYP3A4 active-site access channels was altered in the presence of cholesterol. In summary, cholesterol in the membrane affected the positioning and structural features of CYP3A4, which in turn may have implications for the activity of this enzyme in various membranes and membrane parts with different cholesterol content. PMID:25654496

  18. Monocrotophos induced apoptosis in PC12 cells: role of xenobiotic metabolizing cytochrome P450s.

    PubMed

    Kashyap, Mahendra Pratap; Singh, Abhishek Kumar; Kumar, Vivek; Tripathi, Vinay Kumar; Srivastava, Ritesh Kumar; Agrawal, Megha; Khanna, Vinay Kumar; Yadav, Sanjay; Jain, Swatantra Kumar; Pant, Aditya Bhushan

    2011-01-01

    Monocrotophos (MCP) is a widely used organophosphate (OP) pesticide. We studied apoptotic changes and their correlation with expression of selected cytochrome P450s (CYPs) in PC12 cells exposed to MCP. A significant induction in reactive oxygen species (ROS) and decrease in glutathione (GSH) levels were observed in cells exposed to MCP. Following the exposure of PC12 cells to MCP (10(-5) M), the levels of protein and mRNA expressions of caspase-3/9, Bax, Bcl(2), P(53), P(21), GSTP1-1 were significantly upregulated, whereas the levels of Bclw, Mcl1 were downregulated. A significant induction in the expression of CYP1A1/1A2, 2B1/2B2, 2E1 was also observed in PC12 cells exposed to MCP (10(-5) M), whereas induction of CYPs was insignificant in cells exposed to 10(-6) M concentration of MCP. We believe that this is the first report showing altered expressions of selected CYPs in MCP-induced apoptosis in PC12 cells. These apoptotic changes were mitochondria mediated and regulated by caspase cascade. Our data confirm the involvement of specific CYPs in MCP-induced apoptosis in PC12 cells and also identifies possible cellular and molecular mechanisms of organophosphate pesticide-induced apoptosis in neuronal cells. PMID:21445290

  19. Monocrotophos Induced Apoptosis in PC12 Cells: Role of Xenobiotic Metabolizing Cytochrome P450s

    PubMed Central

    Kashyap, Mahendra Pratap; Singh, Abhishek Kumar; Kumar, Vivek; Tripathi, Vinay Kumar; Srivastava, Ritesh Kumar; Agrawal, Megha; Khanna, Vinay Kumar; Yadav, Sanjay; Jain, Swatantra Kumar; Pant, Aditya Bhushan

    2011-01-01

    Monocrotophos (MCP) is a widely used organophosphate (OP) pesticide. We studied apoptotic changes and their correlation with expression of selected cytochrome P450s (CYPs) in PC12 cells exposed to MCP. A significant induction in reactive oxygen species (ROS) and decrease in glutathione (GSH) levels were observed in cells exposed to MCP. Following the exposure of PC12 cells to MCP (10?5 M), the levels of protein and mRNA expressions of caspase-3/9, Bax, Bcl2, P53, P21, GSTP1-1 were significantly upregulated, whereas the levels of Bclw, Mcl1 were downregulated. A significant induction in the expression of CYP1A1/1A2, 2B1/2B2, 2E1 was also observed in PC12 cells exposed to MCP (10?5 M), whereas induction of CYPs was insignificant in cells exposed to 10?6 M concentration of MCP. We believe that this is the first report showing altered expressions of selected CYPs in MCP-induced apoptosis in PC12 cells. These apoptotic changes were mitochondria mediated and regulated by caspase cascade. Our data confirm the involvement of specific CYPs in MCP-induced apoptosis in PC12 cells and also identifies possible cellular and molecular mechanisms of organophosphate pesticide-induced apoptosis in neuronal cells. PMID:21445290

  20. Effect of buagafuran on liver microsomal cytochrome P450 in rats.

    PubMed

    Li, En; Hu, Jin-Ping; Wang, Bao-Lian; Li, Yan

    2010-05-01

    Buagafuran (BF), derived from alpha-agarofuran, is a promising anti-anxiety drug in phase I clinical trials. The present study was undertaken to examine the regulation of BF on liver cytochrome P450 (CYP) isoforms in rats. After being administered (4, 16, and 64 mg/kg) by gavage for 7 continuous days, the activities of CYP isoforms were measured by the qualification of six metabolites from CYP probe substrates using LC-MS/MS analysis. The mRNA and protein levels of CYPs were detected by reverse transcription polymerase chain reaction and Western blotting assay, respectively. Using phenacetin and chlorzoxazone as probe drugs, the activities of CYP1A2 and CYP2E1 were monitored in vivo. The result indicated that BF significantly increased the activity and protein levels of CYP1A2 and CYP2E1, while the mRNA levels were elevated to a certain extent. CYP2C6 and CYP2C11 were also slightly induced by BF, but no effect on liver CYP3A was detected in rats. Treatment of BF orally resulted in the decreasing of AUC, MRT and increasing of CL/F of phenacetin as well as production of acetaminophen in rats. The similar pharmacokinetic changes were also observed when using chlorzoxazone as a probe drug. Collectively, BF has inducing potential of liver CYP1A2 and CYP2E1 and may influence the corresponding pharmacokinetics of other drugs. PMID:20496194

  1. Concurrent Assessment of Hepatic and Intestinal Cytochrome P450 3A Activities Using Deuterated Alfentanil

    PubMed Central

    Kharasch, Evan D.; Vangveravong, Suwanna; Buck, Nikki; London, Amy; Kim, Thomas; Blood, Jane; Mach, Robert H

    2013-01-01

    Alfentanil is a validated probe for hepatic, first-pass, and intestinal cytochrome P450 (CYP) 3A activity, using plasma clearances, single-point concentrations and noninvasive pupil diameter change (miosis). Assessing intravenous and oral drug disposition typically requires separate dosing. This investigation evaluated concurrent administration of oral deuterated and intravenous unlabeled alfentanil, to assess both intestinal and hepatic CYP3A, and compare sequential and simultaneous dosing. Alfentanil disposition was evaluated after strong hepatic and/or intestinal CYP3A induction and inhibition by rifampin, ketoconazole, and grapefruit juice. Using plasma alfentanil concentrations and area under the curve, clearance, or single-point concentrations, both simultaneous and sequential dosing provided equivalent results and detected hepatic and intestinal CYP3A induction and inhibition. Miosis better detected CYP3A modulation with sequential vs simultaneous dosing. These results show that concurrent oral deuterated and intravenous alfentanil, administered either sequentially or simultaneously, is an efficient and effective approach to assessing hepatic and intestinal CYP3A activity. PMID:21346758

  2. Strength of Axial Water Ligation in Substrate-Free Cytochrome P450s Is Isoform Dependent

    PubMed Central

    2015-01-01

    The heme-containing cytochrome P450s exhibit isoform-dependent ferric spin equilibria in the resting state and differential substrate-dependent spin equilibria. The basis for these differences is not well understood. Here, magnetic circular dichroism (MCD) reveals significant differences in the resting low spin ligand field of CYPs 3A4, 2E1, 2C9, 125A1, and 51B1, which indicates differences in the strength of axial water ligation to the heme. The near-infrared bands that specifically correspond to charge-transfer porphyrin-to-metal transitions span a range of energies of nearly 2 kcal/mol. In addition, the experimentally determined MCD bands are not entirely in agreement with the expected MCD energies calculated from electron paramagnetic resonance parameters, thus emphasizing the need for the experimental data. MCD marker bands of the high spin heme between 500 and 680 nm were also measured and suggest only a narrow range of energies for this ensemble of high spin Cys(S–) ? Fe3+ transitions among these isoforms. The differences in axial ligand energies between CYP isoforms of the low spin states likely contribute to the energetics of substrate-dependent spin state perturbation. However, these ligand field energies do not correlate with the fraction of high spin vs low spin in the resting state enzyme, suggestive of differences in water access to the heme or isoform-dependent differences in the substrate-free high spin states as well. PMID:24576089

  3. Insights on Cytochrome P450 Enzymes and Inhibitors Obtained Through QSAR Studies

    PubMed Central

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

    2013-01-01

    The cytochrome P450 (CYP) superfamily of heme enzymes play an important role in the metabolism of a large number of endogenous and exogenous compounds, including most of the drugs currently on the market. Inhibitors of CYP enzymes have important roles in the treatment of several disease conditions such as numerous cancers and fungal infections in addition to their critical role in drug-drug interactions. Structure activity relationships (SAR), and three-dimensional quantitative structure activity relationships (3D-QSAR) represent important tools in understanding the interactions of the inhibitors with the active sites of the CYP enzymes. A comprehensive account of the QSAR studies on the major human CYPs 1A1, 1A2, 1B1, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4 and a few other CYPs are detailed in this review which will provide us with an insight into the individual/common characteristics of the active sites of these enzymes and the enzyme-inhibitor interactions. PMID:22864238

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

    SciTech Connect

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

    2006-01-15

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

  5. Identification of Cytochrome P450 ( CYP) genes in Zhikong scallop ( Chlamys farreri)

    NASA Astrophysics Data System (ADS)

    Guo, Huihui; Bao, Zhenmin; Du, Huixia; Zhang, Lingling; Wang, Shi; Sun, Luyang; Mou, Xiaoyu; Hu, Xiaoli

    2013-03-01

    Cytochrome P450 ( CYP) superfamily is one of the membership largest and function most diverse protein superfamily recogniozed among living beings. Members of this superfamily were further assigned to different families and subfamilies based on their amino acid similarities. According to their phylogenetic relationships, the CYP genes which likely diverged from common ancestor gene and may share common functions were grouped into one clan. Widely distributing scallops are a group of the most conspicuous bivalve; however the studies on their CYP is acarce. In this study, we searched the genome and expressed sequence tags of Zhikong scallop ( Chlamys farreri) for CYP genes. In total, 88 non-redundant CYP were identified, which were homed in 13 CYPs gene families. Phylogenetic analysis divided these genes into 4 CYP clans. As in deuterostomes, Clan 2 was the largest, which contained 33 genes belonging to CYP1, CYP2, CYP17 and CYP356 families. Clan 3 contgained 19 genes belonging to CYP3, CYP5 and CYP30 families. Clan 4 contained 23 genes, all belonging to CYP4 family. The mitochondrial CYP clan contained 9 genes belonging to CYP10 and CYP24 families. In comparison, protostomes ( C. farreri, D. pluex, D. melanogaster) contained more CYP genes than deuterostomes ( S. purpuratus and vertebrates) in Clan 2 but less genes in Clan 3 and Clan 4. Our findings will aid to deciphering CYP function and evolution in scallops and bivalves.

  6. In vivo inhibitory effects of puerarin on selected rat cytochrome P450 isoenzymes.

    PubMed

    Guo, Yu-Jin; Liang, Dong-Lou; Xu, Zhi-Sheng; Ye, Qiang

    2014-05-01

    Puerarin, the major bioactive constituent in kudzu root, is used widely in China for the treatment of cardiovascular diseases and diabetes. The purpose of this study was to find out whether puerarin influences the effect on rat cytochrome P450 (CYP) enzymes (CYP2B6, CYP2C9 and CYP3A4) by using cocktail probe drugs in vivo. A cocktail solution at a dose of 5 mL/kg, which contained bupropion (20 mg/kg), tolbutamide (5 mg/kg) and midazolam (20 mg/kg), was given as oral administration to rats treated with 10 days oral administration of puerarin. Blood samples were collected at a series of time-points and the concentrations of probe drugs in plasma were determined by HPLC-MS/MS. The results showed that treatment with multiple doses of puerarin had inhibitory effects on rat CYP2B6, CYP2C9 and CYP3A4 enzyme activities. Therefore, caution is needed when puerarin is co-administered with CYP substrates, in view of herb-drug interactions. PMID:24855829

  7. Spectroscopic and molecular docking studies of the in vitro interaction between puerarin and cytochrome P450.

    PubMed

    Shao, Jiangjuan; Chen, Jianwei; Li, Tingting; Zhao, Xiaoli

    2014-01-01

    Puerarin, an isoflavone glycoside extracted from Pueraria plants, has various medical functions. Cytochrome P450s (CYPs) are crucial phase I metabolizing enzymes, which have been spotlighted for their effects on drug metabolism. The interaction between puerarin and CYPs (CYP1A2 and CYP2D6) was investigated by fluorescence, UV-Vis and circular dichroism spectroscopies, as well as molecular docking, to explore the underlying mechanism under simulated physiological conditions. The molecular docking results indicated that puerarin interacted with CYPs mainly by hydrophobic force and hydrogen bonding. The fluorescences of CYPs were quenched statically. Binding constants (Ka) and number of binding sites (n) at different temperatures were calculated, with the results being consistent with those of molecular docking. At the same temperature, puerarin bound to CYP1A2 more weakly than it did to CYP2D6. UV-Vis and circular dichroism spectroscopies confirmed the micro-environmental and conformational changes of CYP1A2 and CYP2D6. The findings provide reliable evidence for clarifying the structures and functions of CYPs. PMID:24743933

  8. Monitoring compound integrity with cytochrome P450 assays and qHTS

    PubMed Central

    MacArthur, Ryan; Leister, William; Veith, Henrike; Shinn, Paul; Southall, Noel; Austin, Christopher P.; Inglese, James; Auld, Douglas S.

    2012-01-01

    We describe how room temperature storage of a 1,120 member compound library prepared in either DMSO or in a hydrated DMSO/water (67/33) mixture affects the reproducibility of potency values as monitored using cytochrome P450 1A2 and 2D6 isozyme assays. The bioluminescent assays showed Z?-factors of 0.71 and 0.62, with 18% and 32% of the library found as active against the CYP 1A2 and 2D6 isozymes respectively. We tested the library using quantitative high-throughput screening to generate potency values for every library member which was measured at seven time intervals spanning 37 weeks. We calculated the minimum significant ratio (MSR) from these potency values at each time interval and we found that for the library stored in DMSO, the CYP 1A2 and 2D6 assay MSRs progressed from approximately 2.0 to 5.0. The hydrated conditions showed similar performance in both MSR progression and analytical QC results. Based on this study we recommend that DMSO samples be stored in 1,536-well plates for < 4 months at room temperature. Further, the study shows the magnitude of potency changes that can occur in a robust bioassay due to compound sample storage. PMID:19483146

  9. Improved feature-based prediction of SNPs in human cytochrome P450 enzymes.

    PubMed

    Li, Li; Xiong, Yi; Zhang, Zhuo-Yu; Guo, Quan; Xu, Qin; Liow, Hien-Haw; Zhang, Yong-Hong; Wei, Dong-Qing

    2015-03-01

    Single nucleotide polymorphisms (SNPs) make up the most common form of mutations in human cytochrome P450 enzymes family, and have the potential to bring with different drug responses or specific diseases in individual patients. Here, based on machine learning technology, we aim to explore an effective set of sequence-based features for improving prediction of SNPs by using support vector machine algorithms. The features are derived from the target residues and flanking protein sequences, such as amino acid types, sequences composition, physicochemical properties, position-specific scoring matrix, phylogenetic entropy and the number of possible codons of target residues. In order to deal with the imbalance data with a majority of non-SNPs and a minority of SNPs, a preprocessing strategy based on fuzzy set theory was applied to the datasets. Our final model achieves the performance of 93.8% in sensitivity, 88.8% in specif