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Sample records for cytochrome p450 activities

  1. Cytochrome P450-activated prodrugs

    PubMed Central

    Ortiz de Montellano, Paul R

    2013-01-01

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

  2. 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.

  3. Epoxidation Activities of Human Cytochromes P450c17 and P450c21

    PubMed Central

    2015-01-01

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

  4. 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

  5. First principles calculation of the activity of cytochrome P450

    NASA Astrophysics Data System (ADS)

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

    1998-04-01

    The cytochrome P450 superfamily of enzymes is of enormous interest in the biological sciences due to the wide range of endogenous and xenobiotic compounds which it metabolises, including many drugs. We describe the use of first principles quantum mechanical modeling techniques, based on density functional theory, to determine the outcome of interactions between an enzyme and a number of compounds. Specifically, we calculate the spin state of an Fe3+ ion present in a haem moiety at the active site of these enzymes. The spin state of this ion indicates if the catalytic reaction will proceed. The computational results obtained compare favorably with experimental data. Only the principle components of the active site of the enzyme are included in the computational models, demonstrating that only a small fragment of the protein needs to be included in the models in order to accurately reproduce this aspect of the enzymes' function. These results open the way for further investigation of this superfamily of enzymes using the methods detailed in this paper.

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

    PubMed Central

    Wright, Aaron T.; Cravatt, Benjamin F.

    2007-01-01

    The cytochrome P450 (P450) superfamily metabolizes many endogenous signaling molecules and drugs. P450 enzymes are regulated by post-translational mechanisms in vivo, which hinders their functional characterization by conventional genomic or proteomic methods. Here, we describe a chemical proteomic strategy to profile P450 activities directly in living systems. Derivatization of a mechanism-based inhibitor with a “clickable” handle provided an activity-based probe that labels multiple P450s both in proteomic extracts and in vivo. This probe was used to record alterations in liver P450 activities triggered by chemical agents, including inducers of P450 expression and direct P450 inhibitors. The chemical proteomic strategy described herein thus offers a versatile method to monitor P450 activities and small molecule interactions in any biological system and, through doing so, should facilitate the functional characterization of this large and diverse enzyme class. PMID:17884636

  7. The active site of cytochrome P-450 nifedipine oxidase: a model-building study.

    PubMed

    Ferenczy, G G; Morris, G M

    1989-12-01

    A model of the active site of cytochrome P-450 nifedipine oxidase is built on the basis of sequence homology with cytochrome P-450CAM. Substrates are docked into the binding pocket, and molecular mechanical energy minimization is performed to analyze the forces between the substrates and the enzyme.

  8. Metabolic conditions determining the composition and catalytic activity of cytochrome P-450 monooxygenases in Candida tropicalis.

    PubMed Central

    Sanglard, D; Käppeli, O; Fiechter, A

    1984-01-01

    In the microsomal fraction of Candida tropicalis cells, two distinct monooxygenases were detected, depending on the growth conditions. The distinction of the two monooxygenases was evident from: (i) the absorption maxima in the reduced CO difference spectra of the terminal oxidases (cytochromes P-450 and P-448); (ii) the contents of the monooxygenase components (cytochromes P-450/P-448, NADPH-cytochrome c (P-450) reductase, and cytochrome b5) and (iii) the catalytic activity of the complete system (aliphatic hydroxylation and N-demethylation activity). The occurrence of the respective monooxygenases could be related to the carbon source (n-alkanes or glucose). Oxygen limitation led to a significant increase of cytochrome P-450/P-448 content, independent of the carbon source utilized by the cells. An improved method for the isolation of microsomes enabled us to demonstrate the presence of cytochrome P-448 in glucose-grown cells. PMID:6690424

  9. Hepatic Cytochrome P450 Activity, Abundance, and Expression Throughout Human Development

    PubMed Central

    Sadler, Natalie C.; Nandhikonda, Premchendar; Webb-Robertson, Bobbie-Jo; Ansong, Charles; Anderson, Lindsey N.; Smith, Jordan N.; Corley, Richard A.

    2016-01-01

    Cytochrome P450s are oxidative metabolic enzymes that play critical roles in the biotransformation of endogenous compounds and xenobiotics. The expression and activity of P450 enzymes varies considerably throughout human development; the deficit in our understanding of these dynamics limits our ability to predict environmental and pharmaceutical exposure effects. In an effort to develop a more comprehensive understanding of the ontogeny of P450 enzymes, we employed a multi-omic characterization of P450 transcript expression, protein abundance, and functional activity. Modified mechanism-based inhibitors of P450s were used as chemical probes for isolating active P450 proteoforms in human hepatic microsomes with developmental stages ranging from early gestation to late adult. High-resolution liquid chromatography–mass spectrometry was used to identify and quantify probe-labeled P450s, allowing for a functional profile of P450 ontogeny. Total protein abundance profiles and P450 rRNA was also measured, and our results reveal life-stage–dependent variability in P450 expression, abundance, and activity throughout human development and frequent discordant relationships between expression and activity. We have significantly expanded the knowledge of P450 ontogeny, particularly at the level of individual P450 activity. We anticipate that these results will be useful for enabling predictive therapeutic dosing, and for avoiding potentially adverse and harmful reactions during maturation from both therapeutic drugs and environmental xenobiotics. PMID:27084891

  10. Cytochromes P450 in Nanodiscs

    PubMed Central

    Denisov, Ilia G.; Sligar, Stephen G.

    2010-01-01

    Nanodiscs have proven to be a versatile tool for the study all types of membrane proteins, including receptors, transporters, enzymes and viral antigens. The self-assembled Nanodisc system provides a robust and common means for rendering these targets soluble in aqueous media while providing a native like bilayer environment that maintains functional activity. This system has thus provided a means for studying the extensive collection of membrane bound cytochromes P450 with the same biochemical and biophysical tools that have been previously limited to use with the soluble P450s. These include a plethora of spectroscopic, kinetic and surface based methods. Significant improvements in homogeneity and stability of these preparations open new possibilities for detailed analysis of equilibrium and steady-state kinetic characteristics of catalytic mechanisms of human cytochromes P450 involved in xenobiotic metabolism and in steroid biosynthesis. The experimental methods developed for physico-chemical and functional studies of membrane cytochromes P450 incorporated in Nanodiscs allow for more detailed understanding of the scientific questions along the lines pioneered by Professor Klaus Ruckpaul and his array of colleagues and collaborators. PMID:20685623

  11. Hepatic cytochrome P450 activity, abundance, and expression throughout human development

    SciTech Connect

    Sadler, Natalie C.; Nandhikonda, Premchendar; Webb-Robertson, Bobbie-Jo M.; Ansong, Charles; Anderson, Lindsey N.; Smith, Jordan N.; Corley, Richard A.; Wright, Aaron T.

    2016-07-01

    Cytochrome P450s are Phase I metabolic enzymes that play critical roles in the biotransformation of endogenous compounds and xenobiotics. The expression and activity of P450 enzymes can vary considerably throughout human development, especially when comparing fetal development to neonates, children, and adults. In an effort to develop a more comprehensive understanding of the ontogeny of P450 expression and activity we employed a multi-omic characterization of P450 transcript expression, protein abundance, and functional activity. To quantify the functional activity of individual P450s we employ activity-based protein profiling, which uses modified mechanism-based inhibitors of P450s as chemical probes, in tandem with proteomic analyses to quantify activity. Our results reveal life-stage-dependent variability in P450 expression, abundance, and activity throughout human development and frequent discordant relationships between expression and activity. The results were used to distribute P450s into three general classes based upon developmental stage of expression and activity. We have significantly expanded the knowledge of P450 ontogeny, particularly at the level of individual P450 activity. We anticipate that our ontogeny results will be useful for enabling predictive therapeutic dosing, and for avoiding potentially adverse and harmful reactions during maturation from both therapeutic drugs and environmental xenobiotics.

  12. The cytochrome p450 homepage.

    PubMed

    Nelson, David R

    2009-10-01

    The Cytochrome P450 Homepage is a universal resource for nomenclature and sequence information on cytochrome P450 ( CYP ) genes. The site has been in continuous operation since February 1995. Currently, naming information for 11,512 CYPs are available on the web pages. The P450 sequences are manually curated by David Nelson, and the nomenclature system conforms to an evolutionary scheme such that members of CYP families and subfamilies share common ancestors. The organisation and content of the Homepage are described.

  13. 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…

  14. 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

  15. Cancer Activation and Polymorphisms of Human Cytochrome P450 1B1

    PubMed Central

    Chun, Young-Jin; Kim, Donghak

    2016-01-01

    Human cytochrome P450 enzymes (P450s, CYPs) are major oxidative catalysts that metabolize various xenobiotic and endogenous compounds. Many carcinogens induce cancer only after metabolic activation and P450 enzymes play an important role in this phenomenon. P450 1B1 mediates bioactivation of many procarcinogenic chemicals and carcinogenic estrogen. It catalyzes the oxidation reaction of polycyclic aromatic carbons, heterocyclic and aromatic amines, and the 4-hydroxylation reaction of 17β-estradiol. Enhanced expression of P450 1B1 promotes cancer cell proliferation and metastasis. There are at least 25 polymorphic variants of P450 1B1 and some of these have been reported to be associated with eye diseases. In addition, P450 1B1 polymorphisms can greatly affect the metabolic activation of many procarcinogenic compounds. It is necessary to understand the relationship between metabolic activation of such substances and P450 1B1 polymorphisms in order to develop rational strategies for the prevention of its toxic effect on human health. PMID:27123158

  16. The Cytochrome P450 Homepage

    PubMed Central

    2009-01-01

    The Cytochrome P450 Homepage is a universal resource for nomenclature and sequence information on cytochrome P450 (CYP) genes. The site has been in continuous operation since February 1995. Currently, naming information for 11,512 CYPs are available on the web pages. The P450 sequences are manually curated by David Nelson, and the nomenclature system conforms to an evolutionary scheme such that members of CYP families and subfamilies share common ancestors. The organisation and content of the Homepage are described. PMID:19951895

  17. Cytochrome P450 CYP1B1 activity in renal cell carcinoma.

    PubMed

    McFadyen, M C E; Melvin, W T; Murray, G I

    2004-08-31

    Renal cell carcinoma (RCC) is the most common malignancy of the kidney and has a poor prognosis due to its late presentation and resistance to current anticancer drugs. One mechanism of drug resistance, which is potentially amenable to therapeutic intervention, is based on studies in our laboratory. CYP1B1 is a cytochrome P450 enzyme overexpressed in a variety of malignant tumours. Our studies are now elucidating a functional role for CYP1B1 in drug resistance. Cytochrome P450 reductase (P450R) is required for optimal metabolic activity of CYP1B1. Both CYP1B1 and P450R can catalyse the biotransformation of anticancer drugs at the site of the tumour. In this investigation, we determined the expression of CYP1B1 and P450R in samples of normal kidney and RCC (11 paired normal and tumour and a further 15 tumour samples). The O-deethylation of ethoxyresorufin to resorufin was used to measure CYP1B1 activity in RCC. Cytochrome P450 reductase activity was determined by following the reduction of cytochrome c at 550 nm. The key finding of this study was the presence of active CYP1B1 in 70% of RCC. Coincubation with the CYP1B1 inhibitor alpha-naphthoflavone (10 nM) inhibited this activity. No corresponding CYP1B1 activity was detected in any of the normal tissue examined (n=11). Measurable levels of active P450R were determined in all normal (n=11) and tumour samples (n=26). The presence of detectable CYP1B1, which is capable of metabolising anticancer drugs in tumour cells, highlights a novel target for therapeutic intervention.

  18. Metazoan cytochrome P450 evolution.

    PubMed

    Nelson, D R

    1998-11-01

    There are 37 cytochrome P450 families currently identified in animals. The concept of higher order groupings of P450 families called P450 CLANS is introduced. The mammalian CYP3 and CYP5 families belong to the same clan as insect CYP6 and CYP9. All mitochondrial P450s seem to belong to the same clan. Lack of mitochondrial P450s in C. elegans suggests that mitochondrial P450s probably arose from the mistargeting of a microsomal P450 after the coelomates diverged from acoelomates and pseudocoelomates. Different taxonomic groups appear to have recruited different ancestral P450s for expansion as they evolved, since each major taxon seems to have one large cluster of P450s. In insects, this cluster derives from the ancestor to the CYP4 family. Vertebrates and C. elegans may have used the same ancestor independently to generate the CYP1, 2, 17, and 21 families in vertebrates and a large distinctive clan with 45 genes in C. elegans.

  19. Cytochromes P450 and species differences in xenobiotic metabolism and activation of carcinogen.

    PubMed Central

    Lewis, D F; Ioannides, C; Parke, D V

    1998-01-01

    The importance of cytochrome P450 isoforms to species differences in the metabolism of foreign compounds and activation of procarcinogens has been identified. The possible range of P450 isozymes in significant variations in toxicity exhibited by experimental rodent species may have a relevance to chemical risk assessment, especially as human P450s are likely to show changes in the way they metabolize xenobiotics. Consequently, in the safety evaluation of chemicals, we should be cautious in extrapolating results from experimental animal models to humans. This paper focuses on examples in which species differences in P450s lead to significant alterations in carcinogenic response, and includes a discussion of the current procedures for toxicity screening, with an emphasis on short-term tests. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:9755138

  20. 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...

  1. Expression and enzymatic activity of recombinant cytochrome P450 17 alpha-hydroxylase in Escherichia coli.

    PubMed Central

    Barnes, H J; Arlotto, M P; Waterman, M R

    1991-01-01

    When the cDNA encoding bovine microsomal 17 alpha-hydroxylase cytochrome P450 (P45017 alpha) containing modifications within the first seven codons which favor expression in Escherichia coli is placed in a highly regulated tac promoter expression plasmid, as much as 16 mg of spectrally detectable P45017 alpha per liter of culture can be synthesized and integrated into E. coli membranes. The known enzymatic activities of bovine P45017 alpha can be reconstituted by addition of purified rat liver NADPH-cytochrome P450 reductase to isolated E. coli membrane fractions containing the recombinant P45017 alpha enzyme. Surprisingly, it is found that E. coli contain an electron-transport system that can substitute for the mammalian microsomal NADPH-cytochrome P450 reductase in supporting both the 17 alpha-hydroxylase and 17,20-lyase activities of P45017 alpha. Thus, not only can E. coli express this eukaryotic membrane protein at relatively high levels, but as evidenced by metabolism of steroids added directly to the cells, the enzyme is catalytically active in vivo. These studies establish E. coli as an efficacious heterologous expression system for structure-function analysis of the cytochrome P450 system. Images PMID:1829523

  2. Photoaffinity ligands in the study of cytochrome p450 active site structure.

    PubMed

    Gartner, Carlos Augusto

    2003-04-01

    While photoaffinity ligands have been widely used to probe the structures of many receptors and nucleic acid binding proteins, their effective use in the study of cytochrome p450 structure is less established. Nevertheless, significant advances in this field have been made since the technique was first applied to p450cam in 1979. In several cases, especially studies involving p450s of the 1A and 2B families, peptides covalently modified with photoaffinity ligands have been isolated and characterized. Some of these peptides were predicted by molecular modeling to line substrate binding regions of the enzymes. Other data obtained from such studies were more difficult to reconcile with theory. This review addresses the status of photoaffinity labeling as a tool for studying cytochrome p450 structure. In addition, potential future directions in this field are discussed, including the development of heme-directed agents and validation of their effectiveness as photoaffinity ligands using sperm whale myoglobin as a test protein. The potential for hydroxyaromatic compounds to serve as photoactivated probes of active site nucleophiles is also discussed. This class of compounds and its derivatives has long been known in the fields of photochemistry and photophysics to be precursors of reactive radicals and quinone methides that are likely to serve as effective active site probes of the p450s.

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

    SciTech Connect

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

    1994-12-31

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

  4. [Activity of cytochromes P-450p and P-450h in liver microsomes and blood corticosteroid levels in experimental animals under the action of physical factors].

    PubMed

    Zolotareva, T A; Gorchakova, G A; Konovalenko, V L; Konovalenko, L N; Grishanova, A Iu; Guliaeva, L F; Liakhovich, V V

    1992-05-01

    In experiments on male Wistar rats it has been found that physical factors applied in medicine (laser radiation of low intensity with wave length 0.89 microns, microwaves of centimeter range of 2450 MHz, and ultrasound of low intensity 880 KHz) changed catalytic activity of liver microsomal and rostenedione 16 alpha- and 6 beta-hydroxylating cytochromes P-450h and P-450p and blood corticosteroids level. Activities of these two steroid-metabolizing cytochromes decreased under ultrasonic skin application on liver region and increased under microwave and laser action. Contents of physiologically inactive form of corticosterone were not changed by the physical factors action while level of active hormone was increased under ultrasonic and microwave action. These findings suggest association of the activity of liver steroid-metabolizing cytochromes P-450 and level of physiologically active form of corticosterone in blood under physical factors skin application on liver region.

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

    SciTech Connect

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

    1995-12-31

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

  6. Enzymatic metabolism of ergosterol by cytochrome p450scc to biologically active 17alpha,24-dihydroxyergosterol.

    PubMed

    Slominski, Andrzej; Semak, Igor; Zjawiony, Jordan; Wortsman, Jacobo; Gandy, Michael N; Li, Jinghu; Zbytek, Blazej; Li, Wei; Tuckey, Robert C

    2005-08-01

    We demonstrate the metabolism of ergosterol by cytochrome P450scc in either a reconstituted system or isolated adrenal mitochondria. The major reaction product was identified as 17alpha,24-dihydroxyergosterol. Purified P450scc also generated hydroxyergosterol as a minor product, which is probably an intermediate in the synthesis of 17alpha,24-dihydroxyergosterol. In contrast to cholesterol and 7-dehydrocholesterol, cleavage of the ergosterol side chain was not observed. NMR analysis clearly located one hydroxyl group to C24, with evidence that the second hydroxyl group is at C17. 17alpha,24-Dihydroxyergosterol inhibited cell proliferation of HaCaT keratinocytes and melanoma cells. Thus, in comparison with cholesterol and 7-dehydrocholesterol, the 24-methyl group and the C22-C23 double bond of ergosterol prevent side chain cleavage by P450scc and change the enzyme's hydroxylase activity from C22 and C20, to C24 and C17, generating bioactive product.

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

    PubMed Central

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

    2014-01-01

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

  8. Catalytic activities of human liver cytochrome P-450 IIIA4 expressed in Saccharomyces cerevisiae.

    PubMed

    Brian, W R; Sari, M A; Iwasaki, M; Shimada, T; Kaminsky, L S; Guengerich, F P

    1990-12-25

    A human liver cytochrome P-450 (P-450) IIIA4 cDNA clone was inserted behind an alcohol dehydrogenase promoter in the plasmid vector pAAH5 and expressed in Saccharomyces cerevisiae (D12 and AH22 strains). A cytochrome P-450 with typical spectral properties was expressed at a level of approximately 8 x 10(5) molecules/cell in either strain of yeast. The expressed P-450 IIIA4 had the same apparent monomeric Mr as the corresponding protein in human liver microsomes (P-450NF) and could be isolated from yeast microsomes. Catalytic activity of the yeast microsomes toward putative P-450 IIIA4 substrates was seen in the reactions supported by cumene hydroperoxide but was often lower and variable when supported by the physiological donor NADPH. The catalytic activity of purified P-450 IIIA4 was also poor in some systems reconstituted with rabbit liver NADPH-P-450 reductase and best when both the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate and a lipid extract (from liver or yeast microsomes) or L-alpha-1,2-dilauroyl-sn-glycero-3-phosphocholine were present. Under these conditions the expressed P-450 IIIA4 was an efficient catalyst for nifedipine oxidation, 6 beta-hydroxylation of testosterone and cortisol, 2-hydroxylation of 17 beta-estradiol and 17 alpha-ethynylestradiol, N-oxygenation and 3-hydroxylation of quinidine, 16 alpha-hydroxylation of dehydroepiandrosterone 3-sulfate, erythromycin N-demethylation, the 10-hydroxylation of (R)-warfarin, the formation of 9,10-dehydrowarfarin from (S)-warfarin, and the activation of aflatoxins B1 and G1, sterigmatocystin, 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (both + and - diastereomers), 3,4-dihydroxy-3,4-dihydrobenz[a]anthracene, 3,4-dihydroxy-3,4-dihydro-7, 12-dimethylbenz[a]anthracene, 9,10-dihydroxy-9,10-dihydrobenzo[b]fluoranthene, 6-aminochrysene, and tris(2,3-dibromopropyl) phosphate to products genotoxic in a Salmonella typhimurium TA1535/pSK1002 system where a chimeric umuC' 'lacZ plasmid is

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

    PubMed

    Chiapella, C; Radovan, R D; Moreno, J A; Casares, L; Barbé, J; Llagostera, M

    2000-10-31

    To know the mechanisms involved in the activation of promutagenic aromatic amines mediated by plants, we used Persea americana S117 system (S117) for the activation of 2-aminofluorene (2-AF) and m-phenylenediamine (m-PDA) in Ames assays. In these assays, the effect of the diphenylene iodonium (DPI), an inhibitor of flavin-containing monooxygenases (FMOs), of the 1-aminobenzotriazole (1-ABT), an inhibitor of cytochromes P450 (cyt-P450s) and of the methimazole, a high-affinity substrate for FMOs, was studied. The efficacy of both inhibitors and of the methimazole was verified to find that they did partially inhibit the mutagenesis of both aromatic amines, activated with rat liver S9. Similarly, both inhibitors and methimazole did produce a significant decrease in 2-AF and m-PDA mutagenesis, when the activation system was S117, indicating that, similar to what occurs in mammalian systems, plant FMOs and cyt-P450s can metabolize aromatic amines to mutagenic product(s). However, the affinity of both FMOs and cyt-P450s of plant for 2-AF and m-PDA was different. Data obtained indicate that the activities of plant FMOs must be the main enzymatic system of m-PDA activation while, in 2-AF activation, plant cyt-P450s have the most relevant activities. In addition, peroxidases of the S117 system must contribute to 2-AF activation and some isoforms of FMOs and/or cyt-P450s of the S117 system, uninhibited by the inhibitors used, must be the responsible for a partial activation of m-PDA.

  10. Peroxidase activity of bacterial cytochrome P450 enzymes: modulation by fatty acids and organic solvents.

    PubMed

    Rabe, Kersten S; Erkelenz, Michael; Kiko, Kathrin; Niemeyer, Christof M

    2010-08-01

    The modulation of peroxidase activity by fatty acid additives and organic cosolvents was determined and compared for four bacterial cytochrome P450 enzymes, thermostable P450 CYP119A1, the P450 domain of CYP102A1 (BMP), CYP152A1 (P450(bsbeta)), and CYP101A1 (P450(cam)). Utilizing a high-throughput microplate assay, we were able to readily screen more than 100 combinations of enzymes, additives and cosolvents in a convenient and highly reproducible assay format. We found that, in general, CYP119A1 and BMP showed an increase in peroxidative activity in the presence of fatty acids, whereas CYP152A1 revealed a decrease in activity and CYP101A1 was only slightly affected. In particular, we observed that the conversion of the fluorogenic peroxidase substrate Amplex Red by CYP119A1 and BMP was increased by a factor of 38 or 11, respectively, when isopropanol and lauric acid were present in the reaction mixture. The activity of CYP119A1 could thus be modulated to reach more than 90% of the activity of CYP152A1 without effectors, which is the system with the highest peroxidative activity. For all P450s investigated we found distinctive reactivity patterns, which suggest similarities in the binding site of CYP119A1 and BMP in contrast with the other two proteins studied. Therefore, this study points towards a role of fatty acids as activators for CYP enzymes in addition to being mere substrates. In general, our detailed description of fatty acid- and organic solvent-effects is of practical interest because it illustrates that optimization of modulators and cosolvents can lead to significantly increased yields in biocatalysis.

  11. [Heterologous expression of functionally active human cytochrome P-450s. Cytochrome P-450IIIA4 catalyzes the biotransformation of the anabolic steroid hormone methandrostenolone].

    PubMed

    Krynetskiĭ, E Iu; Kovaleva, I E; Luzikov, V N

    1994-02-01

    The expression of the cytochrome P450IIIA4 gene in the Saccharomyces cerevisiae yeast using the shuttle vector pYeDP1-8/2 has been carried out. The microsomal fraction isolated from the transformed yeast cells was used for biotransformation of the anabolic steroid hormone-methandrostenolone (MA). The microsomal oxidation products were analyzed by HPLC and two-dimensional TLC. It was shown that microsomes of the yeasts expressing human cytochrome P450IIIA4 catalyze the MA conversion into its 6 beta-hydroxy derivative. An identical product is formed via a reaction catalyzed by human liver microsomes. The use of the heterological system of cytochrome P450IIIA4 expression has made it possible to establish its role in MA metabolism. The experimental system simulates the first phase of the drug biotransformation in liver cells.

  12. 13C-Methyl isocyanide as an NMR probe for cytochrome P450 active sites

    PubMed Central

    McCullough, Christopher R.; Pullela, Phani Kumar; Im, Sang-Choul; Waskell, Lucy

    2012-01-01

    The cytochromes P450 (CYPs) play a central role in many biologically important oxidation reactions, including the metabolism of drugs and other xenobiotic compounds. Because they are often assayed as both drug targets and anti-targets, any tools that provide: (a) confirmation of active site binding and (b) structural data, would be of great utility, especially if data could be obtained in reasonably high throughput. To this end, we have developed an analog of the promiscuous heme ligand, cyanide, with a 13CH3-reporter attached. This 13C-methyl isocyanide ligand binds to bacterial (P450cam) and membrane-bound mammalian (CYP2B4) CYPs. It can be used in a rapid 1D experiment to identify binders, and provides a qualitative measure of structural changes in the active site. PMID:19199046

  13. Cytochrome P450s and molecular epidemiology

    NASA Astrophysics Data System (ADS)

    Gonzalez, Frank J.; Gelboin, Harry V.

    1993-03-01

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

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

    PubMed Central

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

    2015-01-01

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

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

    SciTech Connect

    Miao, Yinglong; Baudry, Jerome Y

    2011-01-01

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

  16. Nerval influences on liver cytochrome P450.

    PubMed

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

    1995-09-01

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

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

    SciTech Connect

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

    2014-06-01

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

  18. Relationships among Ergot Alkaloids, Cytochrome P450 Activity, and Beef Steer Growth

    NASA Astrophysics Data System (ADS)

    Rosenkrans, Charles; Ezell, Nicholas

    2015-03-01

    Determining a grazing animal’s susceptibility to ergot alkaloids has been a research topic for decades. Our objective was to determine if the Promega™ P450-Glo assay could be used to indirectly detect ergot alkaloids or their metabolites in urine of steers. The first experiment validated the effects of ergot alkaloids [0, 20, and 40 μM of ergotamine (ET), dihydroergotamine (DHET), and ergonovine (EN)] on human CYP3A4 using the P450-Glo assay (Promega™ V9800). With this assay, luminescence is directly proportional to CYP450 activity. Relative inhibition of in vitro cytochrome P450 activity was affected (P < 0.001) by an interaction between alkaloids and concentration. That interaction resulted in no concentration effect of EN, but within ET and DHET 20 and 40 µM concentrations inhibited CYP450 activity when compared with controls. In experiment 2, urine was collected from Angus-sired crossbred steers (n = 39; 216 ± 2.6 d of age; 203 ± 1.7 kg) after grazing tall fescue pastures for 105 d. Non-diluted urine was added to the Promega™ P450-Glo assay, and observed inhibition (3.7 % ± 2.7 of control). Urine content of total ergot alkaloids (331.1 ng/mg of creatinine ± 325.7) was determined using enzyme linked immunosorbent assay. Urine inhibition of CYP450 activity and total alkaloids were correlated (r = -0.31; P < 0.05). Steers were genotyped at CYP450 single nucleotide polymorphism, C994G. Steer genotype affected (P < 0.03) inhibition of CYP450 activity by urine; heterozygous steers had the least amount of CYP450 inhibition suggesting that genotyping cattle may be a method of identifying animals that are susceptible to ergot alkaloids. Although, additional research is needed, we demonstrate that the Promega™ P450-Glo assay is sensitive to ergot alkaloids and urine from steers grazing tall fescue. With some refinement the P450-Glo assay has potential as a tool for screening cattle for their exposure to fescue toxins.

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

    SciTech Connect

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

    2010-02-01

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

  20. Inhibition of cytochrome P450 activity enhances the systemic availability of triclabendazole metabolites in sheep.

    PubMed

    Virkel, G; Lifschitz, A; Sallovitz, J; Ballent, M; Scarcella, S; Lanusse, C

    2009-02-01

    Understanding the disposition kinetics and the pattern of metabolism is critical to optimise the flukicidal activity of triclabendazole (TCBZ) in ruminants. TCBZ is metabolised by both flavin-monooxygenase (FMO) and cytochrome P450 (P450) in the liver. Interference with these metabolic pathways may be useful to increase the systemic availabilities of TCBZ metabolites, which may improve the efficacy against Fasciola hepatica. The plasma disposition of TCBZ metabolites was evaluated following TCBZ co-administration with FMO [methimazole (MTZ)] and P450 [piperonyl butoxyde (PB) and ketoconazole (KTZ)] inhibitors in sheep. Twenty (20) healthy Corriedale x Merino weaned female lambs were randomly allocated into four experimental groups. Animals of each group were treated as follow: Group A, TCBZ alone (5 mg/kg, IV route); Group B, TCBZ (5 mg/kg, IV) + MTZ (3 mg/kg, IV); Group C, TCBZ (5 mg/kg, IV) + PB (30 mg/kg, IV) and Group D, TCBZ (5 mg/kg, IV) + KTZ (10 mg/kg, orally). Blood samples were taken over 240 h post-treatment and analysed by HPLC. TCBZ sulphoxide and sulphone were the main metabolites recovered in plasma. MTZ did not affect TCBZ disposition kinetics. TCBZ sulphoxide Cmax values were significantly increased (P < 0.05) after the TCBZ + PB (62%) and TCBZ + KTZ (37%) treatments compared to those measured in the TCBZ alone treatment. TCBZ sulphoxide plasma AUCs were higher (P < 0.05) in the presence of both PB (99%) and KTZ (41%). Inhibition of TCBZ P450-mediated oxidation in the liver accounted for the increased systemic availability of its active metabolite TCBZ sulphoxide. This work contributes to the search of different strategies to improve the use of this flukicidal drug in ruminants.

  1. 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 μM. 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

  2. Cytochrome P450 (CYP450) Tests

    MedlinePlus

    ... By Mayo Clinic Staff Your doctor may use cytochrome P450 (CYP450) tests to help determine how your body processes (metabolizes) a drug. The human body contains P450 enzymes to process medications. Because of inherited (genetic) traits ...

  3. Pyrethroid Activity-Based Probes for Profiling Cytochrome P450 Activities Associated with Insecticide Interactions

    SciTech Connect

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

    2014-01-18

    Pyrethroid insecticides are used to control a diverse spectrum of diseases spread by arthropods. We have developed a suite of pyrethroid mimetic activity based probes (PyABPs) to selectively label and identify P450s associated with pyrethroid metabolism. The probes were screened against pyrethroid metabolizing and non-metabolizing mosquito P450s, as well as rodent microsomes to measure labeling specificity, plus CPR and b5 knockout mouse livers to validate P450 activation and establish the role for b5 in probe activation. Using a deltamethrin mimetic PyABP we were able to profile active enzymes in rat liver microsomes and identify pyrethroid metabolizing enzymes in the target tissue. The most reactive enzyme was a P450, CYP2C11, which is known to metabolize deltamethrin. Furthermore, several other pyrethroid metabolizers were identified (CYPs 2C6, 3A4, 2C13 and 2D1) along with related detoxification enzymes, notably UDP-g’s 2B1 - 5, suggesting a network of associated pyrethroid metabolizing enzymes, or ‘pyrethrome’. Considering the central role that P450s play in metabolizing insecticides, we anticipate that PyABPs will aid the identification and profiling of P450s associated with insecticide pharmacology in a wide range of species, improving understanding of P450-insecticide interactions and aiding the development of new tools for disease control.

  4. The cytochrome P-450 active site. Regiospecificity of prosthetic heme alkylation by olefins and acetylenes.

    PubMed

    Kunze, K L; Mangold, B L; Wheeler, C; Beilan, H S; Ortiz de Montellano, P R

    1983-04-10

    Hepatic microsomal cytochrome P-450 from phenobarbital-pretreated rats is inactivated during the metabolism of linear olefins (ethylene, propene, and octene) and acetylenes (acetylene, propyne, and octyne). As expected from previous work, the inactivation is due to N-alkylation of the prosthetic heme group by the substrate. The N-alkyl group in each adduct is formally obtained by addition of a porphyrin nitrogen to the terminal carbon and of an oxygen atom (as a hydroxyl function) to the internal carbon of the pi-bond. The oxygen is shown here by 18O studies to be catalytically introduced by the enzyme. The olefins exclusively alkylate the nitrogen of pyrrole ring D, but the acetylenes alkylate that of pyrrole ring A. Acetylene is an exception in that it reacts with more than one nitrogen. Circular dichroism studies of the ethylene adduct and of the ring D regioisomer of N-ethylprotoporphyrin IX obtained by alkylation of the prosthetic heme of hemoglobin have been used to determine which face of cytochrome P-450 heme is alkylated by the unsaturated substrates. These results implicate an active site that is sterically encumbered in the region over pyrrole ring B and has a lipophilic binding site that accommodates chains of at least six carbon atoms over pyrrole ring C.

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

    PubMed Central

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

    1985-01-01

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

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

    PubMed

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

    2014-01-01

    The electrochemical analysis of cytochrome Р450 3А4 catalytic activity has shown that vitamins C, A and Е influence on electron transfer and Fe3+/Fe2+ reduction process of cytochrome Р450 3А4. These data allow to assume possibility of cross effects and interference of vitamins-antioxidants with drugs metabolised by cytochrome Р450 3А4, at carrying out of complex therapy. This class of vitamins shows antioxidant properties that lead to increase of the cathodic current corresponding to heme reduction of this functionally significant haemoprotein. Ascorbic acid of 0.028-0.56 mM concentration stimulates cathodic peak (an electrochemical signal) of cytochrome Р450 3А4. At the presence of diclofenac (Voltaren) - a typical substrate of cytochrome Р450 3А4 - the increase growth of a catalytic current testifying to an electrocatalysis and stimulating action of ascorbic acid is observed. In the presence of vitamins A and Е also is registered dose-dependent (in a range of 10-100 M) increase in a catalytic current of cytochrome Р450 3А4: the maximum increase corresponds to 229 ± 20% for 100 M of vitamin A, and 162±10% for 100 M of vitamin E. Vitamin E in the presence of P450's inhibitor itraconazole doesn't give essential increase in a reductive current, unlike retinol (vitamin A). This effect can manifest substrate properties of tocopherol (vitamin E). The electrochemical approach for the analysis of catalytic activity of cytochrome Р450 3А4 and studies of influence of biologically active compounds on an electrocatalysis is the sensitive and effective sensor approach, allowing to use low concentration of protein on an electrode (till 10-15 mol/electrode), to carry out the analysis without participation of protein redox partners, and to reveal drug-drug or drug-vitamins interaction in pre-clinical experiments.

  7. 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.

  8. Indole hydroxylation by bacterial cytochrome P450 BM-3 and modulation of activity by cumene hydroperoxide.

    PubMed

    Li, Qing-Shan; Ogawa, Jun; Schmid, Rolf D; Shimizu, Sakayu

    2005-02-01

    Cytochrome P450 BM-3 from Bacillus megaterium catalyzed NADPH-supported indole hydroxylation under alkaline conditions with homotropic cooperativity toward indole. The activity was also found with the support of H2O2, tert-butyl hydroperoxide (tBuOOH), or cumene hydroperoxide (CuOOH). Enhanced activity and heterotropic cooperativity were observed in CuOOH-supported hydroxylation, and both the Hill coefficient and substrate concentration required for half-maximal activity in the CuOOH-supported reaction were much lower than those in the H2O2-, tBuOOH-, or NADPH-supported reactions. CuOOH greatly enhanced NADPH consumption and indole hydroxylation in the NADPH-supported reaction. However, when CuOOH was replaced by tBuOOH or H2O2, heterotropic cooperativity was not observed. Spectral studies also confirmed that CuOOH stimulated indole binding to P450 BM-3. Interestingly, a mutant enzyme with enhanced indole-hydroxylation activity, F87V (Phe87 was replaced by Val), lost homotropic cooperativity towards indole and heterotropic cooperativity towards CuOOH, indicating that the active-site structure affects the cooperativities.

  9. Human Recombinant Cytochrome P450 Enzymes Display Distinct Hydrogen Peroxide Generating Activities During Substrate Independent NADPH Oxidase Reactions

    PubMed Central

    Mishin, Vladimir; Heck, Diane E.; Laskin, Debra L.; Laskin, Jeffrey D.

    2014-01-01

    Microsomal enzymes generate H2O2 in the presence of NADPH. In this reaction, referred to as “oxidase” activity, H2O2 is generated directly or indirectly via the formation of superoxide anion. In the presence of redox active transition metals, H2O2 can form highly toxic hydroxyl radicals and, depending on the “oxidase” activity of individual cytochrome P450 isoenzymes, this can compromise cellular functioning and contribute to tissue injury. In the present studies, we compared the initial rates of H2O2 generating activity of microsomal preparations containing various human recombinant cytochromes P450s. In the absence of cytochrome P450s the human recombinant NADPH cytochrome P450 reductase (CPR) generated low, but detectable amounts of H2O2 (∼0.04 nmol H2O2/min/100 units of reductase). Significantly greater activity was detected in preparations containing individual cytochrome P450s coexpressed with CPR (from 6.0 nmol H2O2/min/nmol P450 to 0.2 nmol/min/nmol P450); CYP1A1 was the most active, followed by CYP2D6, CYP3A4, CYP2E1, CYP4A11, CYP1A2, and CYP2C subfamily enzymes. H2O2 generating activity of the cytochrome P450s was independent of the ratio of CYP/CPR. Thus, similar H2O2 generating activity was noted with the same cytochrome P450s (CYP3A4, CYP2E1, and CYP2C9) expressed at or near the ratio of CYP/CPR in human liver microsomes (5–7), and when CPR was present in excess (CYP/CPR = 0.2–0.3). Because CYP3A4/5/7 represent up to 40% of total cytochrome P450 in the liver, these data indicate that these enzymes are the major source of H2O2 in human liver microsomes. PMID:25061110

  10. The Effects of Milk Thistle (Silybum marianum) on Human Cytochrome P450 Activity

    PubMed Central

    Kawaguchi-Suzuki, Marina; Frye, Reginald F.; Zhu, Hao-Jie; Brinda, Bryan J.; Chavin, Kenneth D.; Bernstein, Hilary J.

    2014-01-01

    Milk thistle (Silybum marianum) extracts are widely used as a complementary and alternative treatment of various hepatic conditions and a host of other diseases/disorders. The active constituents of milk thistle supplements are believed to be the flavonolignans contained within the extracts. In vitro studies have suggested that some milk thistle components may significantly inhibit specific cytochrome P450 (P450) enzymes. However, determining the potential for clinically significant drug interactions with milk thistle products has been complicated by inconsistencies between in vitro and in vivo study results. The aim of the present study was to determine the effect of a standardized milk thistle supplement on major P450 drug-metabolizing enzymes after a 14-day exposure period. CYP1A2, CYP2C9, CYP2D6, and CYP3A4/5 activities were measured by simultaneously administering the four probe drugs, caffeine, tolbutamide, dextromethorphan, and midazolam, to nine healthy volunteers before and after exposure to a standardized milk thistle extract given thrice daily for 14 days. The three most abundant falvonolignans found in plasma, following exposure to milk thistle extracts, were silybin A, silybin B, and isosilybin B. The concentrations of these three major constituents were individually measured in study subjects as potential perpetrators. The peak concentrations and areas under the time-concentration curves of the four probe drugs were determined with the milk thistle administration. Exposure to milk thistle extract produced no significant influence on CYP1A2, CYP2C9, CYP2D6, or CYP3A4/5 activities. PMID:25028567

  11. Discovery of potent and novel S-nitrosoglutathione reductase inhibitors devoid of cytochrome P450 activities.

    PubMed

    Sun, Xicheng; Qiu, Jian; Strong, Sarah A; Green, Louis S; Wasley, Jan W F; Blonder, Joan P; Colagiovanni, Dorothy B; Mutka, Sarah C; Stout, Adam M; Richards, Jane P; Rosenthal, Gary J

    2011-10-01

    The pyrrole based N6022 was recently identified as a potent, selective, reversible, and efficacious S-nitrosoglutathione reductase (GSNOR) inhibitor and is currently undergoing clinical development for the treatment of acute asthma. GSNOR is a member of the alcohol dehydrogenase family (ADH) and regulates the levels of S-nitrosothiols (SNOs) through catabolism of S-nitrosoglutathione (GSNO). Reduced levels of GSNO, as well as other nitrosothiols (SNOs), have been implicated in the pathogenesis of many diseases including those of the respiratory, cardiovascular, and gastrointestinal systems. Preservation of endogenous SNOs through GSNOR inhibition presents a novel therapeutic approach with broad applicability. We describe here the synthesis and structure-activity relationships (SAR) of novel pyrrole based analogues of N6022 focusing on removal of cytochrome P450 inhibition activities. We identified potent and novel GSNOR inhibitors having reduced CYP inhibition activities and demonstrated efficacy in a mouse ovalbumin (OVA) model of asthma.

  12. 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

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

    EPA Science Inventory

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

  14. Reduced Duodenal Cytochrome P450 3A Protein Expression and Catalytic Activity in Patients with Cirrhosis

    PubMed Central

    McConn, Donavon J.; Lin, Yvonne S.; Mathisen, Terri L.; Blough, David K.; Xu, Yang; Hashizume, Takanori; Taylor, Shari L.; Thummel, Kenneth E.; Shuhart, Margaret C.

    2009-01-01

    The small intestine and liver express high levels of cytochrome P450 3A (CYP3A), an enzyme subfamily contributing significantly to drug metabolism. In patients with cirrhosis, reduced metabolism of drugs is typically attributed to decreased liver function, but it is unclear whether intestinal drug metabolism is also compromised. In this study, we compared CYP3A protein expression and in vitro midazolam hydroxylation in duodenal mucosal biopsies from subjects with normal liver function (controls; n=20) and subjects with varying severity of cirrhosis (n=23). Compared to samples from controls, duodenal CYP3A expression and total midazolam hydroxylation was reduced by 47% and 34%, respectively in samples from subjects with cirrhosis. Greater decreases in CYP3A expression were seen in subjects with increasing severity of cirrhosis. Thus, patients with advanced cirrhosis may have increased drug exposure following oral dosing as a result of both impaired liver function and decreased intestinal CYP3A expression and activity. PMID:19212316

  15. The role of highly purified forms of rat liver cytochrome P-450 in the dimethylation of dimethylnitrosamine and its activation to mutagens.

    PubMed

    Masson, H A; Ioannides, C; Gibson, G G

    1983-06-01

    Highly purified NADPH-cytochrome P-450 reductase and the major phenobarbital (PB) and beta-naphthoflavone (beta NF) forms of cytochrome P-450 were used in reconstituted systems to study the demethylation and subsequent activation of dimethylnitrosamine (DMN) to mutagenic intermediates. Both forms of cytochrome P-450 were active in the demethylation of DMN, cytochrome P-450 from PB-treated animals being more efficient, generating nearly twice as much formaldehyde per nmol of haemoprotein. Neither form of the cytochrome could activate DMN to mutagens in the Ames test. These findings indicate that DMN demethylation does not lead to its activation to mutagenic products.

  16. In vivo indirect measurement of cytochrome P450-associated activities in freshwater gastropod molluscs.

    PubMed

    Gagnaire, Beatrice; Geffard, Olivier; Noury, Patrice; Garric, Jeanne

    2010-12-01

    The cytochrome P450 (CYP) system is widely distributed across phyla and plays a key role in the metabolism of xenobiotic compounds. However, most studies on CYP system were developed on vertebrates and among invertebrates, gastropod molluscs are rarely used. In this context, ethoxycoumarin-O-deethylase (ECOD), ethoxyresorufin-O-deethylase (EROD), and pentoxyresorufin-O-dealkylase (PROD) activities, which are indirect measurements of CYP system, were characterized in two freshwater gastropod molluscs, Potamopyrgus antipodarum, and Valvata piscinalis, to ascertain their potential interest as biomarkers of exposure to chemicals. Activities were measured using an in vivo non lethal method based on the measurement of formed product (resorufin or hydroxycoumarin). This in vivo assay allowed to measure the three activities in P. antipodarum and two of them (ECOD and PROD) in V. piscinalis. The detection of activities and the optimization of experimental design were carried out first and allowed to measure the selected activities for one individual. The modulation of the detected activities was secondly assessed using a polycyclic aromatic hydrocarbon (Benzo(a)pyrene). Based on this non destructive measurement, effect of BaP exposure could be detected on ECOD and EROD activity in P. antipodarum, as well on PROD activity of V. piscinalis after 96 h of exposure. Such an in vivo assay must be further developed to be valuably used to screen the exposure of gastropod species to CYP inducer chemicals and its consequences in terms of fitness of the organisms and of the population.

  17. Effects of phenol on metabolic activities and transcription profiles of cytochrome P450 enzymes in Chironomus kiinensis larvae.

    PubMed

    Cao, C W; Sun, L L; Niu, F; Liu, P; Chu, D; Wang, Z Y

    2016-02-01

    Phenol, also known as carbolic acid or phenic acid, is a priority pollutant in aquatic ecosystems. The present study has investigated metabolic activities and transcription profiles of cytochrome P450 enzymes in Chironomus kiinensis under phenol stress. Exposure of C. kiinensis larvae to three sublethal doses of phenol (1, 10 and 100 µM) inhibited cytochrome P450 enzyme activity during the 96 h exposure period. The P450 activity measured after the 24 h exposure to phenol stress could be used to assess the level (low or high) of phenol contamination in the environment. To investigate the potential of cytochrome P450 genes as molecular biomarkers to monitor phenol contamination, the cDNA of ten CYP6 genes from the transcriptome of C. kiinensis were identified and sequenced. The open reading frames of the CYP6 genes ranged from 1266 to 1587 bp, encoding deduced polypeptides composed of between 421 and 528 amino acids, with predicted molecular masses from 49.01 to 61.94 kDa and isoelectric points (PI) from 6.01 to 8.89. Among the CYP6 genes, the mRNA expression levels of the CYP6EW3, CYP6EV9, CYP6FV1 and CYP6FV2 genes significantly altered in response to phenol exposure; therefore, these genes could potentially serve as biomarkers in the environment. This study shows that P450 activity combined with one or multiple CYP6 genes could be used to monitor phenol pollution.

  18. Potent inhibition by star fruit of human cytochrome P450 3A (CYP3A) activity.

    PubMed

    Hidaka, Muneaki; Fujita, Ken-ichi; Ogikubo, Tetsuya; Yamasaki, Keishi; Iwakiri, Tomomi; Okumura, Manabu; Kodama, Hirofumi; Arimori, Kazuhiko

    2004-06-01

    There has been very limited information on the capacities of tropical fruits to inhibit human cytochrome P450 3A (CYP3A) activity. Thus, the inhibitory effects of tropical fruits on midazolam 1'-hydroxylase activity of CYP3A in human liver microsomes were evaluated. Eight tropical fruits such as common papaw, dragon fruit, kiwi fruit, mango, passion fruit, pomegranate, rambutan, and star fruit were tested. We also examined the inhibition of CYP3A activity by grapefruit (white) and Valencia orange as controls. The juice of star fruit showed the most potent inhibition of CYP3A. The addition of a star fruit juice (5.0%, v/v) resulted in the almost complete inhibition of midazolam 1'-hydroxylase activity (residual activity of 0.1%). In the case of grape-fruit, the residual activity was 14.7%. The inhibition depended on the amount of fruit juice added to the incubation mixture (0.2-6.0%, v/v). The elongation of the preincubation period of a juice from star fruit (1.25 or 2.5%, v/v) with the microsomal fraction did not alter the CYP3A inhibition, suggesting that the star fruit did not contain a mechanism-based inhibitor. Thus, we discovered filtered extracts of star fruit juice to be inhibitors of human CYP3A activity in vitro.

  19. Unusual Cytochrome P450 Enzymes and Reactions*

    PubMed Central

    Guengerich, F. Peter; Munro, Andrew W.

    2013-01-01

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

  20. Cytochrome P450-inhibitory activity of parabens and phthalates used in consumer products.

    PubMed

    Ozaki, Hitomi; Sugihara, Kazumi; Watanabe, Yoko; Ohta, Shigeru; Kitamura, Shigeyuki

    2016-01-01

    The in vitro cytochrome P450 (CYP)-inhibitory effects of 11 parabens and 7 phthalates used in consumer products, as well as their hydrolytic metabolites, were investigated, using rat liver microsomes as an enzyme source. The effects on individual CYP isozymes were evaluated by assaying inhibition of activities towards specific substrates, i.e., ethoxyresorufin O-dealkylase (EROD), methoxyresorufin O-dealkylase (MROD), pentoxyresorufin O-dealkylase (PROD), 7-benzyloxy-4-trifluoromethylcoumarin dealkylase (BFCD), 7-methoxy-4-trifluoromethylcoumarin dealkylase (MFCD) and 7-ethoxy-4-trifluoromethylcoumarin dealkylase (EFCD) activities. These activities were dose-dependently inhibited, most potently by medium-side-chain parabens (C6-9) and phthalates (C4-6), and less potently by shorter- and longer-side-chain esters. The hydrolytic product of parabens, 4-hydroxybenzoic acid, was not inhibitory, while those of phthalates, phthalic acid monoesters, showed lower inhibitory activity than the parent phthalates. Parabens showed relatively potent inhibition of MFCD activity, considered to be mainly due to CYP2C, and phthalates showed relatively potent inhibition of PROD activity, considered to be mainly due to CYP2B.

  1. Involvement of cytochrome P-450 enzyme activity in the selectivity and safening action of pyrazosulfuron-ethyl.

    PubMed

    Yun, M S; Shim, I S; Usui, K

    2001-03-01

    To investigate the selectivity and safening action of the sulfonylurea herbicide pyrazosulfuron-ethyl (PSE), pyrazosulfuron-ethyl O-demethylase (PSEOD) activity involving oxidative metabolism by cytochrome P-450 was studied in rice (Oryza sativa L cv Nipponbare) and Cyperus serotinus Rottb. Cytochrome P-450-dependent activity was demonstrated by the use of the inducers 1,8-naphthalic anhydride and ethanol, the herbicides PSE, bensulfuron-methyl, dimepiperate and dymron, or the inhibitor piperonyl butoxide (PBO). Growth inhibition in C serotinus seedlings was more severe than that in rice seedlings. O-Dealkylation activities of PSE were induced differently in rice and in C serotinus, with distinctly higher activity in rice seedlings. The induced PSEOD activities were slightly inhibited by PBO in rice seedlings, whereas they were strongly inhibited in C serotinus seedlings. Dimepiperate and dymron were effective safeners of rice against PSE treatment. Treatments with herbicide alone resulted in less induction of PSEOD activity compared with combined treatments of the herbicide and safener. PSEOD activity in rice seedlings induced with herbicide alone was strongly inhibited by PBO, whereas it was weakly inhibited in rice seedlings induced with combinations of PSE and two safeners. These results suggest that O-demethylation by cytochrome P-450 enzymes may be involved in the metabolism of PSE and may contribute to its selectivity and safening action. Furthermore, these results suggest the existence of a multiple form of cytochrome P-450 in plants.

  2. Posttranslational modification by an isolevuglandin diminishes activity of the mitochondrial cytochrome P450 27A1.

    PubMed

    Charvet, Casey D; Laird, James; Xu, Yunfeng; Salomon, Robert G; Pikuleva, Irina A

    2013-05-01

    Posttranslational modification by isolevuglandins (isoLGs), arachidonate oxidation products, is an important yet understudied process associated with altered protein properties. This type of modification is detected in cytochrome P450 27A1 (CYP27A1), a multifunction enzyme expressed in almost every cell and involved in the metabolism of cholesterol and other sterols. Previously, the CYP27A1 Lys(358)-isoLG adduct was found in human retina afflicted with age-related macular degeneration. Yet, the effect of Lys(358) modification on enzyme activity was not investigated. Herein, we characterized catalytic properties of Lys(358) as well as Lys(476) CYP27A1 mutants before and after isoLG treatment and quantified the extent of modification by multiple reaction monitoring. The K358R mutant was less susceptible to isoLG-induced loss of catalytic activity than the wild type (WT), whereas the K476R mutant was nearly as vulnerable as the WT. Both mutants showed less isoLG modification than WT. Thus, modification of Lys(358), a residue involved in redox partner interactions, is the major contributor to isoLG-associated loss of CYP27A1 activity. Our data show the specificity of isoLG modification, provide direct evidence that isoLG adduction impairs enzyme activity, and support our hypothesis that isoLG modification in the retina is detrimental to CYP27A1 enzyme activity, potentially disrupting cholesterol homeostasis.

  3. Five of 12 forms of vaccinia virus-expressed human hepatic cytochrome P450 metabolically activate aflatoxin B sub 1

    SciTech Connect

    Aoyama, Toshifumi; Yamano, Shigeru; Gelboin, H.V.; Gonzalez, F.J. ); Guzelian, P.S. )

    1990-06-01

    Twelve forms of human hepatic cytochrome P450 were expressed in hepatoma cells by means of recombinant vaccinia viruses. The expressed P450s were analyzed for their abilities to activate the potent hepatocarcinogen aflatoxin B{sub 1} to metabolites having mutagenic or DNA-binding properties. Five forms, P450s IA2, IIA3, IIB7, IIIA3, and IIIA4, activated aflatoxin B{sub 1} to mutagenic metabolites as assessed by the production of His revertants of Salmonella typhimurium in the Ames test. The same P450s catalyzed conversion of aflatoxin B{sub 1} to DNA-bound derivatives as judged by an in situ assay in which the radiolabeled carcinogen was incubated with cells expressing the individual P450 forms. Seven other human P450s, IIC8, IIC9, IID6, IIE1, IIF1, and IIIA5, and IVB1, did not significantly activate aflatoxin B{sub 1} as measured by both the Ames test and the DNA-binding assay. Moreover, polyclonal anti-rat liver P450 antibodies that crossreact with individual human P450s IA2, IIA3, IIIA3, and IIIA4 each inhibited aflatoxin B{sub 1} activation catalyzed by human liver S-9 extracts. Inhibition ranged from as low as 10% with antibody against IIA3 to as high as 65% with antibody against IIIA3 and IIIA4. These results establish that metabolic activation of aflatoxin B{sub 1} in human liver involves the contribution of multiple forms of P450.

  4. Novel extrahepatic cytochrome P450s

    SciTech Connect

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

    2005-09-01

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

  5. Effects of cytochrome P450 inhibitors on potassium currents and mechanical activity in rat portal vein.

    PubMed Central

    Edwards, G.; Zygmunt, P. M.; Högestätt, E. D.; Weston, A. H.

    1996-01-01

    1. The effects of the cytochrome P450 inhibitors, proadifen, clotrimazole and 17-octadecynoic acid (17-ODYA) on K-currents in freshly-isolated single cells derived from rat portal vein and on mechanical activity in whole veins were studied. 2. When cells were stepped from -90 mV to a series of test potentials (from -80 to +50 mV), a delayed rectifier current (IK(V)) and an A-type current (IK(A)) could be identified. Proadifen (10 microM), clotrimazole (30 microM) and 17-ODYA (5 microM) each inhibited IK(V) but had little effect on IK(A). 3. When cells were held at -10 mV to inactivate the time-dependent K-currents, IK(V) and IK(A), levcromakalim (3 microM) induced a time-independent outward K-current (IK(ATP)) which was totally inhibited by clotrimazole (30 microM) and almost fully inhibited by proadifen (10 microM). 17-ODYA (5 microM) had no effect on IK(ATP) and exerted only a minor inhibitory action on this current at 20 microM. 4. 17-ODYA (5 microM) potentiated current flow through the large conductance, Ca-sensitive K-channel (BKCa). In contrast, proadifen (10 microM) had no effect on IBK(Ca) whereas clotrimazole (30 microM) exerted a small but significant inhibitory action. 5. Proadifen (10 microM) and clotrimazole (30 microM) each inhibited the magnitude but increased the frequency of spontaneous contractions in whole portal veins. 17-ODYA (5 microM) had no effect on spontaneous contractions but these were inhibited when the concentration of 17-ODYA was increased to 50 microM. 6. The spasmolytic effect of levcromakalim on spontaneous contractions was antagonized by proadifen (10-30 microM) in a concentration-dependent manner but 17-ODYA (up to 50 microM) was without effect. 7. These results in portal vein show that cytochrome P450 inhibitors exert profound effects on a variety of K-channel subtypes. This suggests that enzymes dependent on this cofactor may be important regulators of K-channel activity in smooth muscle. The relevance of these findings for the

  6. Effects of interferon-tau and steroids on cytochrome P450 activity in bovine endometrial epithelial cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective of the current study was to examine cyclooxygenase (COX), cytochrome P450 1A (CYP1A) and 2C (CYP2C) activity in bovine endometrial cell cultures following exposure to oxytocin (OT), interferon-t (IFN), estradiol (E2) and/or progesterone (P4). Bovine endometrial epithelial cells were tr...

  7. Measuring cytochrome P450 activity in aquatic invertebrates: a critical evaluation of in vitro and in vivo methods.

    PubMed

    Gottardi, Michele; Kretschmann, Andreas; Cedergreen, Nina

    2016-03-01

    The first step in xenobiotic detoxification in aquatic invertebrates is mainly governed by the cytochrome P450 mixed function oxidase system. The ability to measure cytochrome P450 activity provides an important tool to understand macroinvertebrates' responses to chemical stressors. However, measurements of P450 activity in small aquatic invertebrates have had variable success and a well characterized assay is not yet available. The general lack of success has been scarcely investigated and it is therefore the focus of the present work. In particular, the suitability of the substrate selected for the assay, the sensitivity of the assay and the possible inhibition/attenuation of enzymatic activity caused by endogenous substances were investigated. 7-ethoxycoumarin-O-dealkylation activity of Daphnia magna, Chironomus riparius larvae and Hyalella azteca was assessed in vivo and in vitro and possible inhibition of enzymatic activity by macroinvertebrates homogenate was investigated. Activities of D. magna and C. riparius larvae measured in vivo were 1.37 ± 0.08 and 2.2 ± 0.2 pmol h(-1) organism(-1), respectively, while activity of H. azteca could not be detected. In vitro activity could be measured in C. riparius larvae only (500-1000 pmol h(-1) mg microsomal protein(-1)). The optimization of the in vitro assay has been especially long and resource consuming and particularly for D. magna, substances that inhibited cytochrome P450 activity seemed to be released during tissue homogenization preventing activity measurements in vitro. We therefore recommend testing the P450 inhibition potential of homogenate preparations prior to any investigation of P450 activity in vitro in macroinvertebrates.

  8. Multiple, Ligand-Dependent Routes from the Active Site of Cytochrome P450 2C9

    SciTech Connect

    Cojocaru, Vlad; Winn, Peter J.; Wade, Rebecca C.

    2012-02-13

    The active site of liver-specific, drug-metabolizing cytochrome P450 (CYP) monooxygenases is deeply buried in the protein and is connected to the protein surface through multiple tunnels, many of which were found open in different CYP crystal structures. It has been shown that different tunnels could serve as ligand passage routes in different CYPs. However, it is not understood whether one CYP uses multiple routes for substrate access and product release and whether these routes depend on ligand properties. From 300 ns of molecular dynamics simulations of CYP2C9, the second most abundant CYP in the human liver we found four main ligand exit routes, the occurrence of each depending on the ligand type and the conformation of the F-G loop, which is likely to be affected by the CYP-membrane interaction. A non-helical F-G loop favored exit towards the putative membrane-embedded region. Important protein features that direct ligand exit include aromatic residues that divide the active site and whose motions control access to two pathways. The ligands interacted with positively charged residues on the protein surface through hydrogen bonds that appear to select for acidic substrates. The observation of multiple, ligand-dependent routes in a CYP aids understanding of how CYP mutations affect drug metabolism and provides new possibilities for CYP inhibition.

  9. The effects of acute hydrogen sulfide poisoning on cytochrome P450 isoforms activity in rats.

    PubMed

    Wang, Xianqin; Chen, Mengchun; Chen, Xinxin; Ma, Jianshe; Wen, Congcong; Pan, Jianchun; Hu, Lufeng; Lin, Guanyang

    2014-01-01

    Hydrogen sulfide (H2S) is the second leading cause of toxin related death (after carbon monoxide) in the workplace. H2S is absorbed by the upper respiratory tract mucosa, and it causes histotoxic hypoxemia and respiratory depression. Cocktail method was used to evaluate the influences of acute H2S poisoning on the activities of cytochrome P450 isoforms CYP2B6, CYP2D6, CYP3A4, CYP1A2, CYP2C19, and CYP2C9, which were reflected by the changes of pharmacokinetic parameters of six specific probe drugs, bupropion, metoprolol, midazolam, phenacetin, omeprazole, and tolbutamide, respectively. The experimental rats were randomly divided into two groups, control group and acute H2S poisoning group (inhaling 300 ppm for 2 h). The mixture of six probes was given to rats by oral administration and the blood samples were obtained at a series of time points through the caudal vein. The concentrations of probe drugs in rat plasma were measured by LC-MS. The results for acute H2S poisoning and control groups were as follows: there was a statistically significant difference in the AUC and C max for bupropion, metoprolol, phenacetin, and tolbutamide, while there was no statistical pharmacokinetic difference for midazolam and omeprazole. Acute H2S poisoning could inhibit the activity of CYP2B6, CYP2D6, CYP1A2, and CYP2C9 in rats.

  10. Assessment of regional cytochrome P450 activities in rat liver slices using resorufin substrates and fluorescence confocal laser cytometry.

    PubMed Central

    Heinonen, J T; Sidhu, J S; Reilly, M T; Farin, F M; Omiecinski, C J; Eaton, D L; Kavanagh, T J

    1996-01-01

    Characterizing constitutive activities and inducibility of various cytochrome P450 isozymes is important for elucidating species and individual differences in susceptibility to many toxicants. Although expression of certain P450s has been studied in homogenized tissues, the ability to assess functional enzyme activity without tissue disruption would further our understanding of interactive factors that modulate P450 activities. We used precision-cut, viable rat liver slices and confocal laser cytometry to determine the regional enzyme activities of P450 isozymes in situ. Livers from control and beta-naphthoflavone (beta NF)-treated rats were sectioned with a Krumdieck tissue slicer into 250-microns thick sections. A slice perfusion chamber that mounts on the cytometer stage was developed to allow for successive measurement of region-specific P450-dependent O-dealkylation of 7-ethoxy-, 7-pentoxy-, and 7-benzyloxyresorufin (EROD, PROD, and BROD activity, respectively) in the same liver slice. Images of the accumulated fluorescent resorufin product within the tissue were acquired using a confocal laser cytometer in confocal mode. As expected, slices isolated from beta NF-treated rats showed high levels of centrilobular EROD activity compared to slices from control rats, whereas PROD and BROD activities remained at control levels. These techniques should allow for the accurate quantification of regional and cell-specific P450 enzyme activity and, with subsequent analysis of the same slice, the ability to correlate specific P450 mRNAs or other factors with enzymatic activity. Moreover, these techniques should be amenable to examination of similar phenomena in other tissues such as lung and kidney, where marked heterogeneity in cellular P450 expression patterns is also known to occur. Images Figure 1. Figure 2. Figure 3. Figure 3. Figure 4. Figure 4. Figure 5. Figure 6. PMID:8743442

  11. Activation of the anticancer drugs cyclophosphamide and ifosfamide by cytochrome P450 BM3 mutants.

    PubMed

    Vredenburg, Galvin; den Braver-Sewradj, Shalenie; van Vugt-Lussenburg, Barbara M A; Vermeulen, Nico P E; Commandeur, Jan N M; Vos, J Chris

    2015-01-05

    Cyclophosphamide (CPA) and ifosfamide (IFA) are widely used anticancer agents that require metabolic activation by cytochrome P450 (CYP) enzymes. While 4-hydroxylation yields DNA-alkylating and cytotoxic metabolites, N-dechloroethylation results in the generation of neuro- and nephrotoxic byproducts. Gene-directed enzyme prodrug therapies (GDEPT) have been suggested to facilitate local CPA and IFA bioactivation by expressing CYP enzymes within the tumor cells, thereby increasing efficacy. We screened bacterial CYP BM3 mutants, previously engineered to metabolize drug-like compounds, for their ability to catalyze 4-hydroxylation of CPA and IFA. Two CYP BM3 mutants showed very rapid initial bioactivation of CPA and IFA, followed by a slower phase of product formation. N-dechloroethylation by these mutants was very low (IFA) to undetectable (CPA). Using purified CYP BM3 as an extracellular bioactivation tool, cytotoxicity of CPA and IFA metabolism was confirmed in U2OS cells. This novel application of CYP BM3 possibly provides a clean and catalytically efficient alternative to liver microsomes or S9 for the study of CYP-mediated drug toxicity. To our knowledge, the observed rate of CPA and IFA 4-hydroxylation by these CYP BM3 mutants is the fastest reported to date, and might be of potential interest for CPA and IFA GDEPT.

  12. Rapid estimation of activation enthalpies for cytochrome-P450-mediated hydroxylations.

    PubMed

    Mayeno, Arthur N; Robinson, Jonathan L; Reisfeld, Brad

    2011-03-01

    Cytochrome P450 (CYP) enzymes play a critical role in detoxication and bioactivation of xenobiotics; thus, the ability to predict the biotransformation rates and regioselectivity of CYP enzymes toward substrates is an important goal in toxicology and pharmacology. Here, we present the use of the semiempirical quantum chemistry method SAM1 to rapidly estimate relative activation enthalpies (ΔH(‡)) for the hydroxylation of aliphatic carbon centers of various substrates. The ΔH(‡) were determined via a reaction path calculation, in the reverse direction (RRP), using the iron-hydroxo-porphine intermediate and the substrate radical. The SAM1 ΔH(‡) were calculated via unrestricted Hartree-Fock (UHF) and configuration interaction (CI) formalisms for both the doublet and quartet spin states. The SAM1 RRP ΔH(‡), after subtracting a correction factor, were compared with density functional theory (DFT) B3LYP activation energies for two sets of substrates and showed R(2) ranging from 0.69 to 0.89, and mean absolute differences ranging from 1.2 ± 1.0 to 1.7 ± 1.5 kcal/mol. SAM1 UHF and CI RRP calculation times were, on average, more than 200 times faster than those for the corresponding forward reaction path DFT calculations. Certain key transition-state (TS) geometry measurements, such as the forming O···H bond length, showed good correlation with the DFT values. These results suggest that the SAM1 RRP approach can be used to rapidly estimate the DFT activation energy and some key TS geometry measurements and can potentially be applied to estimate substrate hydroxylation rates and regioselectivity by CYP enzymes.

  13. Benzene metabolism by human liver microsomes in relation to cytochrome P450 2E1 activity.

    PubMed

    Seaton, M J; Schlosser, P M; Bond, J A; Medinsky, M A

    1994-09-01

    Low levels of benzene from sources including cigarette smoke and automobile emissions are ubiquitous in the environment. Since the toxicity of benzene probably results from oxidative metabolites, an understanding of the profile of biotransformation of low levels of benzene is critical in making a valid risk assessment. To that end, we have investigated metabolism of a low concentration of [14C]benzene (3.4 microM) by microsomes from human, mouse and rat liver. The extent of phase I benzene metabolism by microsomal preparations from 10 human liver samples and single microsomal preparations from both mice and rats was then related to measured activities of cytochrome P450 (CYP) 2E1. Measured CYP 2E1 activities, as determined by hydroxylation of p-nitrophenol, varied 13-fold (0.253-3.266 nmol/min/mg) for human samples. The fraction of benzene metabolized in 16 min ranged from 10% to 59%. Also at 16 min, significant amounts of oxidative metabolites were formed. Phenol was the main metabolite formed by all but two human microsomal preparations. In those samples, both of which had high CYP 2E1 activity, hydroquinone was the major metabolite formed. Both hydroquinone and catechol formation showed a direct correlation with CYP 2E1 activity over the range of activities present. A simulation model was developed based on a mechanism of competitive inhibition between benzene and its oxidized metabolites, and was fit to time-course data for three human liver preparations. Model calculations for initial rates of benzene metabolism ranging from 0.344 to 4.442 nmol/mg/min are directly proportional to measured CYP 2E1 activities. The model predicted the dependence of benzene metabolism on the measured CYP 2E1 activity in human liver samples, as well as in mouse and rat liver samples. These results suggest that differences in measured hepatic CYP 2E1 activity may be a major factor contributing to both interindividual and interspecies variations in hepatic metabolism of benzene

  14. Impact of visceral leishmaniasis and curative chemotherapy on cytochrome P450 activity in Brazilian patients

    PubMed Central

    Lanchote, Vera Lucia; Almeida, Roque; Barral, Aldina; Barral-Netto, Manoel; Marques, Maria Paula; Moraes, Natália V; da Silva, Angela M; Souza, Tania M V; Suarez-Kurtz, Guilherme

    2015-01-01

    Aims The aim of the present study was to investigate the impact of human visceral leishmaniasis (VL) and curative chemotherapy on the activity of cytochrome P450 (CYP) 3A, CYP2C9 and CYP2C19 in patients from an endemic region in Brazil. Methods Adult patients with parasitologically confirmed VL were given a CYP phenotyping cocktail, comprising midazolam, omeprazole and losartan, immediately before (Study phase 1), 2–3 days (phase 2) and 3–6 months (phase 3) after curative VL chemotherapy. CYP activity was assessed by the apparent clearance of midazolam (CYP3A), omeprazole/5-hydroxyomeprazol ratio in plasma (CYP2C19) and losartan/E3174 ratio in urine (CYP2C9). Results Mean values (95% confidence interval) in phases 1, 2 and 3 were, respectively: log apparent midazolam clearance, 1.21 (1.10–1.31), 1.45 (1.32–1.57) and 1.35 (1.26–1.44) ml min–1 kg–1; omeprazole/5-hydroxyomeprazole ratio, 0.78 (0.61–0.94), 0.45 (0.27–0.63) and 0.37 (0.20-0.55); losartan/E3174 ratio, 0.66 (0.39–0.92), 0.35 (0.20–0.50) and 0.35 (0.16–0.53). Analysis of variance revealed significant differences in CYP3A (P = 0.018) and CYP2C19 (P = 0.008), but not CYP2C9 (P = 0.11) phenotypic activity, across the three study phases. Conclusion The phenotypic activities of CYP3A4 and CYP2C19 were significantly reduced during acute VL compared with post-chemotherapy. We propose that increased plasma concentrations of proinflammatory cytokines during active disease account for the suppression of CYP activity. The failure to detect significant changes in CYP2C9 activity in the overall cohort may reflect differential effects of the inflammatory process on the expression of CYP isoforms, although the possibility of insufficient statistical power cannot be dismissed. PMID:25940755

  15. Cytochrome P450 3A, NADPH cytochrome P450 reductase and cytochrome b5 in the upper airways in horse.

    PubMed

    Tydén, E; Olsén, L; Tallkvist, J; Tjälve, H; Larsson, P

    2008-08-01

    Gene and protein expression as well as catalytic activity of cytochrome P450 (CYP) 3A were studied in the nasal olfactory and respiratory mucosa and the tracheal mucosa of the horse. We also examined the activity of NADPH cytochrome P450 reductase (NADPH P450 reductase), the amount of cytochrome b(5) and the total CYP content in these tissues. Comparative values for the above were obtained using liver as a control. The CYP3A related catalytic activity in the tissues of the upper airways was considerably higher than in the liver. The CYP3A gene and protein expression, on the other hand, was higher in the liver than in the upper airway tissues. Thus, the pattern of CYP3A metabolic activity does not correlate with the CYP3A gene and protein expression. Our results showed that the activity of NADPH P450 reductase and the level of cytochrome b(5) in the relation to the gene and protein expression of CYP3A were higher in the tissues of the upper airways than in the liver. It is concluded that CYP3A related metabolism in horse is not solely dependent on the expression of the enzyme but also on adequate levels of NADPH P450 reductase and cytochrome b(5).

  16. Comparative modelling of cytochromes P450.

    PubMed

    Kirton, Stewart B; Baxter, Carol A; Sutcliffe, Michael J

    2002-03-31

    The superfamily of enzymes known as the cytochromes P450 (P450s) comprises a wide-ranging class of proteins with diverse functions. They are known, amongst other things, to be involved in the hormonal regulation of metabolism and reproduction, as well as having a major clinical significance through their association with diseases such as cancer, diabetes and hepatitis. Knowledge of the three-dimensional (3D) structure of a protein gives insight into its function. The 3D structures of P450s are therefore of considerable scientific interest. A number of high-resolution structures of P450s have been determined by X-ray crystallography and studies of these structures have provided valuable insights into the mechanism of these enzymes. Only one of these structures is mammalian and as yet there is no structural information on human P450s in the public domain. Until such a structure is solved it is necessary to employ alternative methods to gain structural insight into how human P450s perform their biological function. Here we report on the use of comparative modelling to predict the structure of human P450s based on knowledge of their amino acid sequences plus the 3D structures of other (not human) P450s. As an illustrative example of these techniques we have modelled the structure of P450 2C5 using five bacterial P450 structures as templates. We examine the importance of selecting suitable templates, obtaining a good amino acid sequence alignment, and evaluating the models generated. To improve the quality of the models an iterative cycle of sequence alignment, model building, and model evaluation is employed. The result is a model with excellent stereochemistry, good amino acid side chain environment properties, and a Calpha trace similar to the crystal structure.

  17. Induction of hepatic cytochrome P-450 activity in wild cotton rats (Sigmodon hispidus) by phenobarbital and 3-methylcholanthrene

    SciTech Connect

    Elangbam, C.S.; Qualls, C.W.,Jr.; Bauduy, M. )

    1989-05-01

    Wild cotton rats (Sigmodon hispidus) are ubiquitous throughout the Southeast quadrant of the United States, easy to capture, have a generation interval of less than one year and a limited range of movement (less than one hectare). This species may prove to be an excellent model for monitoring environmental contamination. Traditionally, cytochrome P-450 inducing agents are grouped into two classes. One, represented by phenobarbital, induces P-450b and P-450e; the other, represented by 3-methylcholanthrene, induces P-450c and P-450d isoenzymes. The types and amounts of cytochrome P-450 vary among species, organs, health status, sex, and stress of the animal. If the levels of cytochrome P-450 of wild cotton rats are to be used in monitoring environmental pollution, it is necessary to characterize the inducibility and concentration of cytochrome P-450 in this species. This study was designed to determine the concentration and inducibility of cytochrome P-450 in the livers of cotton rats after intraperitoneal (ip) administration of phenobarbital and 3-methylcholanthrene.

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

    PubMed Central

    Im, Sang-Choul; Waskell, Lucy

    2010-01-01

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

  19. Molecular modeling of cytochrome P450 3A4

    NASA Astrophysics Data System (ADS)

    Szklarz, Grazyna D.; Halpert, James R.

    1997-05-01

    The three-dimensional structure of human cytochrome P450 3A4 was modeled based on crystallographic coordinates of four bacterial P450s: P450 BM-3, P450cam, P450terp, and P450eryF. The P450 3A4 sequence was aligned to those of the known proteins using a structure-based alignment of P450 BM-3, P450cam, P450terp, and P450eryF. The coordinates of the model were then calculated using a consensus strategy, and the final structure was optimized in the presence of water. The P450 3A4 model resembles P450 BM-3 the most, but the B' helix is similar to that of P450eryF, which leads to an enlarged active site when compared with P450 BM-3, P450cam, and P450terp. The 3A4 residues equivalent to known substrate contact residues of the bacterial proteins and key residues of rat P450 2B1 are located in the active site or the substrate access channel. Docking of progesterone into the P450 3A4 model demonstrated that the substrate bound in a 6β-orientation can interact with a number of active site residues, such as 114, 119, 301, 304, 305, 309, 370, 373, and 479, through hydrophobic interactions. The active site of the enzyme can also accommodate erythromycin, which, in addition to the residues listed for progesterone, also contacts residues 101, 104, 105, 214, 215, 217, 218, 374, and 478. The majority of 3A4 residues which interact with progesterone and/or erythromycin possess their equivalents in key residues of P450 2B enzymes, except for residues 297, 480 and 482, which do not contact either substrate in P450 3A4. The results from docking of progesterone and erythromycin into the enzyme model make it possible to pinpoint residues which may be important for 3A4 function and to target them for site-directed mutagenesis.

  20. Species difference among experimental rodents in the activity and induction of cytochrome P-450 isozymes for mutagenic activation of carcinogenic aromatic amines.

    PubMed

    Degawa, M; Agatsuma, T; Hashimoto, Y

    1990-12-01

    The expressions of hepatic microsomal cytochrome P-450 isozymes in male rats, mice, hamsters and guinea pigs were studied comparatively with or without an ip injection of a cytochrome P-450 inducer. The activity and quantity of microsomal cytochrome P-450 isozymes were determined respectively by a bacterial mutation assay with Salmonella typhimurium TA98 and immunochemical assays using monoclonal antibodies against rat cytochrome P-450 isozymes. 3-Methoxy-4-aminoazobenzene (3-MeO-AAB), 2-amino-3-methyl-9H-pyrido[2,3-b]indole acetate (MeA alpha C) and 3-methylcholanthrene were used as cytochrome P-450 inducers, and 7 carcinogenic aromatic amines including 3-MeO-AAB and MeA alpha C were used as substrates for the mutation assay. By means of these assays, we examined the species differences among rodents in the activity and induction rate of hepatic cytochrome P-450 isozymes responsible for the mutagenic activation of carcinogenic aromatic amines.

  1. Bio-activation of 4-alkyl analogs of 1,4-dihydropyridine mediated by cytochrome P450 enzymes.

    PubMed

    Li, Xiao-Xi; Zhang, Xiaoqian; Zheng, Qing-Chuan; Wang, Yong

    2015-06-01

    4-Alkyl-substituted 1,4-dihydropyridines (DHP) exhibit inhibitory activity toward certain cytochrome P450 enzymes (P450) during their biotransformation by these enzymes, which is called mechanism-based inactivation. Though much experimental evidence had proved the essentiality of alkyl radical for P450 inactivation, the underlying mechanism of such radical formation remains elusive. In the present study, density functional calculations were employed to investigate the dealkylation mechanism of 4-alkyl-substituted DHPs mediated by P450. Interestingly, our results indicate that the initial N-H activation proceeds via a proton-coupled electron transfer process, not via the long presumed hydrogen atom transfer mechanism or the stepwise electron transfer/proton transfer one, to form the amino radical and Cpd II complex. Subsequently, homolytic C-C bond cleavage at the 4-position occurs to afford the product complex involving an alkyl radical, an aromatic pyridine derivative. This C-C cleavage step is rate determining for the whole metabolic reaction, with an energy barrier of 7.9/7.9 kcal/mol on the quartet/doublet state, to which aromatization contributes as an essential intrinsic driving force. The 4-substituent groups induce differences in activation energy barriers and in the transition state structures of hydrogen abstraction process. The substrate reactivity correlates well with the stability of the generated alkyl radical as well as the C-C bond dissociation energy. Understanding the metabolic mechanism of DHP analogs is indeed essential for the related design of safer and more efficient drugs. Furthermore, our findings also enrich the mechanistic picture of amine oxidation catalyzed by P450.

  2. Interaction of isoflavonoids with human liver microsomal cytochromes P450: inhibition of CYP enzyme activities.

    PubMed

    Kopečná-Zapletalová, Michaela; Krasulová, Kristýna; Anzenbacher, Pavel; Hodek, Petr; Anzenbacherová, Eva

    2017-04-01

    1. The possibility of interaction of isoflavonoids with concomitantly taken drugs to determined isoflavonoids safety was studied. Inhibition of nine forms of cytochrome P450 (CYP3A4, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C19, CYP2C9, CYP2D6 and CYP2E1) by 12 isoflavonoids (daidzein, genistein, biochanin A, formononetin, glycitein, equol and six glucosides, daidzin, puerarin, genistin, sissotrin, ononin and glycitin) was studied systematically. 2. The most potent inhibitors were genistein and daidzein inhibiting noncompetitively the CYP2C9 with Ki of 35.95 ± 6.96 and 60.56 ± 3.53 μmol/l and CYP3A4 (inhibited by genistein with Ki of 23.25 ± 5.85 μmol/l also by a noncompetitive mechanism). Potent inhibition of CYP3A4 was observed also with biochanin A (Ki of 57.69 ± 2.36 μmol/l) and equol (Ki of 38.47 ± 2.32 μmol/l). 3. Genistein and daidzein inhibit noncompetitively CYP3A4 and CYP2C9. With plasma levels in micromolar range, a clinically important interaction with concomitantly taken drugs does not seem to be probable.

  3. Relation among cytochrome P450, Ah-active PCB congeners and dioxin equivalents in pipping black-crowned night-heron embryos

    SciTech Connect

    Rattner, B.A.; Hatfield, J.S.; Melancon, M.J.; Custer, T.W. . Patuxent Environmental Science Center); Tillitt, D.E. . Midwest Science Center)

    1994-11-01

    Pipping black-crowned night-heron (Nycticorax nycticorax) embryos were collected from a relatively uncontaminated site (next to 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). Hepatic cytochrome P450-associated mono-oxygenates and cytochrome P450 proteins, induced up to 85-fold relative to the reference site, were associated with concentrations of total polychlorinated biphenyls (PCBs) and 11 PCB congeners that are presumed to express toxicity through the arylhydrocarbon (Ah) receptor. Multiple regression revealed that up to 86% of the variation of cytochrome P450 measurements was accounted for by variation in the concentration of these PCB congeners. Toxic equivalents (TEQs) of sample extracts, predicted mathematically (summed product of PCB congener concentrations and toxic equivalency factors), and dioxin equivalents (TCDD-EQs), derived by bioassay (ethoxyresorufin-O-dealkylase activity of treated H4IIE rat hepatoma cells), were greatest in Cat Island samples. Cytochrome P450-associated monooxygenases and cytochrome P450 proteins were related to TEQs and TCDD-EQs; adjusted r[sup 2] often exceeded 0.5 for the relation among mathematically predicted TEQs and cytochrome P450 measurements. These data extend previous observations in heron embryos of an association between P450 and total PCB burdens to include Ah-active PCB congeners, and presumably other compounds, which interact similarly with the AH receptor. Benzyloxyresorufin O-dealkylase, ethoxyresorufin O-dealkylase, and cytochrome P450 1A appear to be the most reliable measures of exposure to Ah-active PCB congeners in black-crowned night-heron embryos. These findings provide further evidence that cytochrome P450-associated parameters have considerable value as a biomarker for assessing environmental contamination of wetlands.

  4. Relation among cytochrome P450, Ah-active PCB congeners and dioxin equivalents in pipping black- crowned night-heron embryos

    USGS Publications Warehouse

    Rattner, B.A.; Hatfield, J.S.; Melancon, M.J.; Custer, T.W.; Tillitt, D.E.

    1994-01-01

    Pipping black-crowned night-heron (Nycticorax nycticorax) embryos were collected from a relatively uncontaminated site (next to 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). Hepatic cytochrome P450-associated monooxygenases and cytochrome P450 proteins, induced up to 85- fold relative to the reference site, were associated with concentrations of total polychlorinated biphenyls (PCBs) and 11 PCB congeners that are presumed to express toxicity through the arylhydrocarbon (Ah) receptor. Multiple regression revealed that up to 86% of the variation of cytochrome P450 measurements was accounted for by variation in the concentration of these PCB congeners. Toxic equivalents (TEQs) of sample extracts, predicted mathematically (summed product of PCB congener concentrations and toxic equivalency factors), and dioxin equivalents (TCDD-EQs), derived by bioassay (ethoxyresorufin-O-dealkylase activity of treated H4IIE rat hepatoma cells), were greatest in Cat Island samples. Cytochrome P450-associated monooxygenases and cytochrome P450 proteins were related to TEQs and TCDD-EQs; adjusted r super(2) often exceeded 0.5 for the relation among mathematically predicted TEQs and cytochrome P450 measurements. These data extend previous observations in heron embryos of an association between P450 and total PCB burdens to include Ah- active PCB congeners, and presumably other compounds, which interact similarly with the Ah receptor. Benzyloxyresorufin O-dealkylase, ethoxyresorufin O-dealkylase, and cytochrome P450 1A appear to be the most reliable measures of exposure to Ah-active PCB congeners in black-crowned night-heron embryos. These findings provide further evidence that cytochrome P450-associated parameters have considerable value as a biomarker for assessing environmental contamination of wetlands.

  5. Relation among cytochrome P450, AH-active PCB congeners and dioxin equivalents in pipping black-crowned night-heron embryos

    USGS Publications Warehouse

    Rattner, B.A.; Hatfield, J.S.; Melancon, M.J.; Custer, T.W.; Tillitt, D.E.

    1994-01-01

    Pipping black-crowned night-heron (Nycticorax nycticorax) embryos were collected from a relatively uncontaminated site (next to 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). Hepatic cytochrome P-450-associated monooxygenases and cytochrome P-450 proteins, induced up to 85-fold relative to the reference site, were associated with concentrations of total polychlorinated biphenyls (PCBs) and 11 PCB congeners that are presumed to express toxicity through the arylhydrocarbon (Ah) receptor. Multiple regression revealed that up to 86% of the variation of cytochrome P450 measurements was accounted for by variation in the concentration of these PCB congeners. Toxic equivalents (TEQs) of sample extracts, predicted mathematically (summed product of PCB congener concentrations and toxic equivalency factors), and dioxin equivalents (TCDD-EQs), derived by bioassay (ethoxyresorufin-O-dealkylase activity of treated H4IIE rat hepatoma cells), were greatest in Cat Island samples. Cytochrome P450-associated monooxygenases and cytochrome P450 proteins were related to TEQs and TCDD-EQs; adjusted r-2 often exceeded 0.5 for the relation among mathematically predicted TEQs and cytochrome P450 measurements. These data extend previous observations in heron embryos of an association between P450 and total PCB burdens to include Ah-active PCB congeners, and presumably other compounds, which interact similarly with the Ah receptor. Benzyloxyresorufin O-dealkylase, ethoxyresorufin O-dealkylase, and cytochrome P450 1A appear to be the most reliable measures of exposure to Ah-active PCB congeners in black-crowned night-heron embryos. These findings provide further evidence that cytochrome P450-associated parameters have considerable value as a biomarker for assessing environmental contamination of wetlands.

  6. Detection of human lung cytochromes P450 that are immunochemically related to cytochrome P450IIE1 and cytochrome P450IIIA.

    PubMed

    Wheeler, C W; Wrighton, S A; Guenthner, T M

    1992-07-07

    We have used monoclonal antibodies that were prepared against and specifically recognize human hepatic cytochromes P450 as probes for solid phase radioimmunoassay and Western immunoblotting to directly demonstrate the presence in human lung microsomes of cytochromes P450 immunochemically related to human liver cytochromes P450IIE1 (CYP2E1) and P450IIIA (CYP3A). The detected levels of these cytochromes are much lower than levels in human liver microsomes, but similar to the levels seen in microsomes from untreated baboon lung. Proteins immunochemically related to two other constitutive hepatic cytochromes P450, cytochrome P450IIC8 (CYP2C8) and cytochrome P450IIC9 (CYP2C9), were not detectable in lung microsomes.

  7. Stable expression of rat cytochrome P-450IIB1 cDNA in Chinese hamster cells (V79) and metabolic activation of aflatoxin B sub 1

    SciTech Connect

    Doehmer, J.; Dogra, S.; Friedberg, T.; Monier, S.; Adesnik, M.; Glatt, H.; Oesch, F. )

    1988-08-01

    V79 Chinese hamster fibroblasts are widely used for mutagenicity testing but have the serious limitation that they do not express cytochromes P-450, which are needed for the activation of many promutagens to mutagenic metabolites. A full-length cDNA clone encoding the monooxygenase cytochrome P-450IIB1 under control of the simian virus 40 early promoter was constructed and cointroduced with the selection marker neomycin phosphotransferase (conferring resistance to G418) into V79 Chinese hamster cells. G418-resistant cells were selected, established as cell lines, and tested for cytochrome P-450IIB1 expression and enzymatic activity. Two cell lines (SD1 and SD3) were found that stably produce cytochrome P-450IIB1. Although purified cytochromes P-450 possess monooxygenase activity only after reconstitution with cytochrome P-450 reductase and phospholipid, the gene product of the construct exhibited this activity. This implies that the gene product is intracellularly localized in a way that allows access to the required components. If compared with V79 cells, the mutation rate for the hypoxanthine phosphoribosyltranferase (HPRT) locus in SD1 cells is markedly increased when exposed to aflatoxin B{sub 1}, which is activated by this enzyme.

  8. Predictive three-dimensional quantitative structure-activity relationship of cytochrome P450 1A2 inhibitors.

    PubMed

    Korhonen, Laura E; Rahnasto, Minna; Mähönen, Niina J; Wittekindt, Carsten; Poso, Antti; Juvonen, Risto O; Raunio, Hannu

    2005-06-02

    The purpose of this study was to determine the cytochrome P450 1A2 (CYP1A2) inhibition potencies of structurally diverse compounds to create a comprehensive three-dimensional quantitative structure-activity relationship (3D-QSAR) model of CYP1A2 inhibitors and to use this model to predict the inhibition potencies of an external set of compounds. Fifty-two compounds including naphthalene, lactone and quinoline derivatives were assayed in a 96-well plate format for CYP1A2 inhibition activity using 7-ethoxyresorufin O-dealkylation as the probe reaction. The IC50 values of the tested compounds varied from 2.3 microM to over 40,000 microM. On the basis of this data set, a comparative molecular field analysis (CoMFA) and GRID/GOLPE models were created that yielded novel structural information about the interaction between inhibitory molecules and the CYP1A2 active site. The created CoMFA model was able to accurately predict inhibitory potencies of several structurally unrelated compounds, including selective inhibitors of other cytochrome P450 forms.

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

    PubMed

    Almadhidi, J; Moslemi, S; Drosdowsky, M A; Séralini, G E

    1996-09-01

    Aromatase (estrogen synthetase) is a steroidogenic enzyme complex which catalyzes the conversion of androgens to estrogens (termed aromatization). This enzyme was purified from adult equine testis to homogeneity by five chromatographic steps. The ability of purified and reconstituted equine aromatase to exhibit an estrogen 2-hydroxylase activity was tested and compared to testosterone aromatization. Enzymatic activities were assessed by tritiated water release from labelled estradiol and testosterone. Kinetic analysis of estradiol 2-hydroxylation showed an apparent K(m) of 23 microM and a V(max) of 18 nmol/min/mg, whereas the values for testosterone aromatization were a K(m) of 15.7 nM and a V(max) of 34.6 pmol/min/mg. A specific antiserum raised against purified testicular equine P450arom and known to inhibit aromatase activity [1] was also found to inhibit the estrogen hydroxylase activity of equine placental microsomes in a dose-dependent manner with an IC50 value of 15 microl serum: 0.5 ml incubate. The estrogen hydroxylase activity was inhibited in a dose-dependent manner by two classes of aromatase inhibitors, i.e. steroidal-- (4-hydroxyandrostenedione and 7alpha-([4-aminophenyl]thio)-androst-4-ene-3, 17-dione)--and non-steroidal--(fadrozole and miconazole). The IC50 values were approximately 300 and 890 nM for 4-hydroxyandrostenedione and 7alpha-([4-aminophenyl]thio)-androst-4-ene-3, 17-dione, and 92 and 285 nM, for fadrozole and miconazole, respectively. Furthermore, 4-hydroxyandrostenedione caused a time-dependent inactivation of estrogen hydroxylase activity. We conclude that equine aromatase is able to use estradiol as a substrate, and converts it to catechol estradiol in vitro, possibly using the active site of aromatization. This is the first demonstration that equine aromatase functions as an estrogen 2-hydroxylase, in addition to transforming androgens into estrogen.

  10. Formation of the active species of cytochrome p450 by using iodosylbenzene: a case for spin-selective reactivity.

    PubMed

    Cho, Kyung-Bin; Moreau, Yohann; Kumar, Devesh; Rock, Dan A; Jones, Jeffrey P; Shaik, Sason

    2007-01-01

    The generation of the active species for the enzyme cytochrome P450 by using the highly versatile oxygen surrogate iodosylbenzene (PhIO) often produces different results compared with the native route, in which the active species is generated through O(2) uptake and reduction by NADPH. One of these differences that is addressed here is the deuterium kinetic isotope effect (KIE) jump observed during N-dealkylation of N,N-dimethylaniline (DMA) by P450, when the reaction conditions change from the native to the PhIO route. The paper presents a theoretical analysis targeted to elucidate the mechanism of the reaction of PhIO with heme, to form the high-valent iron-oxo species Compound I (Cpd I), and define the origins of the KIE jump in the reaction of Cpd I with DMA. It is concluded that the likely origin of the KIE jump is the spin-selective chemistry of the enzyme cytochrome P450 under different preparation procedures. In the native route, the reaction proceeds via the doublet spin state of Cpd I and leads to a low KIE value. PhIO, however, diverts the reaction to the quartet spin state of Cpd I, which leads to the observed high KIE values. The KIE jump is reproduced here experimentally for the dealkylation of N,N-dimethyl-4-(methylthio)aniline, by using intra-molecular KIE measurements that avoid kinetic complexities. The effect of PhIO is compared with N,N-dimethylaniline-N-oxide (DMAO), which acts both as the oxygen donor and the substrate and leads to the same KIE values as the native route.

  11. Cytochrome P450-mediated metabolism of vitamin D

    PubMed Central

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

    2014-01-01

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

  12. Enhanced expression of cytochrome P450 in stomach cancer.

    PubMed Central

    Murray, G. I.; Taylor, M. C.; Burke, M. D.; Melvin, W. T.

    1998-01-01

    The cytochromes P450 have a central role in the oxidative activation and detoxification of a wide range of xenobiotics, including many carcinogens and several anti-cancer drugs. Thus the cytochrome P450 enzyme system has important roles in both tumour development and influencing the response of tumours to chemotherapy. Stomach cancer is one of the commonest tumours of the alimentary tract and environmental factors, including dietary factors, have been implicated in the development of this tumour. This type of tumour has a poor prognosis and responds poorly to current therapies. In this study, the presence and cellular localization of several major forms of P450, CYP1A, CYP2E1 and CYP3A have been investigated in stomach cancer and compared with their expression in normal stomach. There was enhanced expression of CYP1A and CYP3A in stomach cancer with CYP1A present in 51% and CYP3A present in 28% of cases. In contrast, no P450 was identified in normal stomach. The presence of CYP1A and CYP3A in stomach cancer provides further evidence for the enhanced expression of specific forms of cytochrome P450 in tumours and may be important therapeutically for the development of anti-cancer drugs that are activated by these forms of P450. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:9569036

  13. 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 ERα. 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.

  14. 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.

  15. Inhibition of cytochrome P450 3A4 activity by schisandrol A and gomisin A isolated from Fructus Schisandrae chinensis.

    PubMed

    Wan, C-K; Tse, A K; Yu, Z-L; Zhu, G-Y; Wang, H; Fong, D W F

    2010-07-01

    We studied the effects of schisandrol A (SCH) and gomisin A (GOM), two of the main bioactive components of Fructus Schisandrae chinensis, on cytochrome P450-3A4 (CYP3A4) activity and cellular glutathione (GSH) level. In a cell-free system both SCH and GOM inhibited CYP3A4 activity with IC(50) values of 32.02 microM and 1.39 microM, respectively. SCH or GOM at concentrations up to 100 microM did not alter cellular GSH level in regular HepG2 cells and P-glycoprotein overexpressing HepG2-DR cells. Since SCH and GOM may reverse multidrug resistance (MDR) by impeding the activity of P-glycoprotein, a membrane xenobiotic exporter, SCH or GOM could affect cellular drug metabolism in addition to drug uptake.

  16. Cytochrome P450 gene polymorphism and cancer.

    PubMed

    Agundez, Jose A G

    2004-06-01

    Human cytochrome P450 (CYP) enzymes play a key role in the metabolism of drugs and environmental chemicals. Several CYP enzymes metabolically activate procarcinogens to genotoxic intermediates. Phenotyping analyses revealed an association between CYP enzyme activity and the risk to develop several forms of cancer. Research carried out in the last decade demonstrated that several CYP enzymes are polymorphic due to single nucleotide polymorphisms, gene duplications and deletions. As genotyping procedures became available for most human CYP, an impressive number of association studies on CYP polymorphisms and cancer risk were conducted. Here we review the findings obtained in these studies regarding CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1, CYP3A4, CYP3A5, CYP3A7, CYP8A1 and CYP21 gene polymorphisms. Consistent evidences for association between CYP polymorphisms and lung, head and neck, and liver cancer were reported. Controversial findings suggest that colorectal and prostate cancers may be associated to CYP polymorphisms, whereas no evidences for a relevant association with breast or bladder cancers were reported. We summarize the available information related to the association of CYP polymorphisms with leukaemia, lymphomas and diverse types of cancer that were investigated only for some CYP genes, including brain, esophagus, stomach, pancreas, pituitary, cervical epithelium, melanoma, ovarian, kidney, anal and vulvar cancers. This review discusses on causes of heterogeneity in the proposed associations, controversial findings on cancer risk, and identifies topics that require further investigation. In addition, some recommendations on study design, in order to obtain more conclusive findings in further studies, are provided.

  17. Flower colour and cytochromes P450.

    PubMed

    Tanaka, Yoshikazu; Brugliera, Filippa

    2013-02-19

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

  18. Purification of the pyrazole-inducible cytochrome P-450 isozyme

    SciTech Connect

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

    1987-05-01

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

  19. Conformational selectivity in cytochrome P450 redox partner interactions

    PubMed Central

    Hollingsworth, Scott A.; Batabyal, Dipanwita; Nguyen, Brian D.; Poulos, Thomas L.

    2016-01-01

    The heme iron of cytochromes P450 must be reduced to bind and activate molecular oxygen for substrate oxidation. Reducing equivalents are derived from a redox partner, which requires the formation of a protein–protein complex. A subject of increasing discussion is the role that redox partner binding plays, if any, in favoring significant structural changes in the P450s that are required for activity. Many P450s now have been shown to experience large open and closed motions. Several structural and spectral studies indicate that the well-studied P450cam adopts the open conformation when its redox partner, putidaredoxin (Pdx), binds, whereas recent NMR studies indicate that this view is incorrect. Given the relevance of this discrepancy to P450 chemistry, it is important to determine whether Pdx favors the open or closed form of P450cam. Here, we have used both computational and experimental isothermal titration calorimetry studies that unequivocally show Pdx favors binding to the open form of P450cam. Analyses of molecular-dynamic trajectories also provide insights into intermediate conformational states that could be relevant to catalysis. PMID:27439869

  20. Cytochrome b5 is a major determinant of human cytochrome P450 CYP2D6 and CYP3A4 activity in vivo.

    PubMed

    Henderson, Colin J; McLaughlin, Lesley A; Scheer, Nico; Stanley, Lesley A; Wolf, C Roland

    2015-04-01

    The cytochrome P450-dependent mono-oxygenase system is responsible for the metabolism and disposition of chemopreventive agents, chemical toxins and carcinogens, and >80% of therapeutic drugs. Cytochrome P450 (P450) activity is regulated transcriptionally and by the rate of electron transfer from P450 reductase. In vitro studies have demonstrated that cytochrome b5 (Cyb5) also modulates P450 function. We recently showed that hepatic deletion of Cyb5 in the mouse (HBN) markedly alters in vivo drug pharmacokinetics; a key outstanding question is whether Cyb5 modulates the activity of the major human P450s in drug disposition in vivo. To address this, we crossed mice humanized for CYP2D6 or CYP3A4 with mice carrying a hepatic Cyb5 deletion. In vitro triazolam 4-hydroxylation (probe reaction for CYP3A4) was reduced by >50% in hepatic microsomes from CYP3A4-HBN mice compared with controls. Similar reductions in debrisoquine 4-hydroxylation and metoprolol α-hydroxylation were observed using CYP2D6-HBN microsomes, indicating a significant role for Cyb5 in the activity of both enzymes. This effect was confirmed by the concentration-dependent restoration of CYP3A4-mediated triazolam turnover and CYP2D6-mediated bufuralol and debrisoquine turnover on addition of Escherichia coli membranes containing recombinant Cyb5. In vivo, the peak plasma concentration and area under the concentration time curve from 0 to 8 hours (AUC0-8 h) of triazolam were increased 4- and 5.7-fold, respectively, in CYP3A4-HBN mice. Similarly, the pharmacokinetics of bufuralol and debrisoquine were significantly altered in CYP2D6-HBN mice, the AUC0-8 h being increased ∼1.5-fold and clearance decreased by 40-60%. These data demonstrate that Cyb5 can be a major determinant of CYP3A4 and CYP2D6 activity in vivo, with a potential impact on the metabolism, efficacy, and side effects of numerous therapeutic drugs.

  1. 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...

  2. Interactions among Cytochromes P450 in Microsomal Membranes

    PubMed Central

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

    2015-01-01

    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 α-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

  3. Diversity and evolution of cytochrome P450 monooxygenases in Oomycetes.

    PubMed

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

    2015-07-01

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

  4. Diversity and evolution of cytochrome P450 monooxygenases in Oomycetes

    PubMed Central

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

    2015-01-01

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

  5. Suppression of human cytochrome P450 aromatase activity by monoclonal and recombinant antibody fragments and identification of a stable antigenic complex.

    PubMed

    Lala, Puloma; Higashiyama, Tadayoshi; Erman, Mary; Griswold, Jennifer; Wagner, Traci; Osawa, Yoshio; Ghosh, Debashis

    2004-03-01

    Human cytochrome P450 aromatase (P450arom) is responsible for biosynthesis of estrogens from androgens. Monoclonal antibody MAb3-2C2 to P450arom specifically binds to a conformational epitope and suppresses the enzyme activity in a dose-dependent manner. The crystal structure of the Fab fragment of MAb3-2C2 has been used to engineer a recombinant single chain antibody fragment (scFv) and a homodimeric variable domain of the light chain (VL(2)). These recombinant antibody fragments have been expressed in Escherichia coli and purified. Here, we show that the recombinant scFv suppresses P450arom activity with an IC(50) value similar to that of natural MAb3-2C2 F(ab')(2). The recombinant VL(2) also exhibits dose-dependent suppression of the P450arom activity, but at a reduced level, demonstrating that the homodimer is unable to fully mimic the complementarity determining region (CDR) of a variable heavy chain (VH)-VL heterodimer. We prepare and purify a stable complex of P450arom with MAb3-2C2 F(ab')(2) and show that the complex migrates and precipitates as a single molecular assembly. Efforts to crystallize P450arom for structure-function studies have yielded small single crystals. Our results suggest that formation of stable complexes with fragments of the monoclonal antibody could provide an alternative method for crystallization of P450arom.

  6. Biological activity of phenolic compounds. Hepatic cytochrome P-450, cytochrome b/sub 5/ and NADPH cytochrome c reductase in chicks and rats fed phenolic monomers, polymers, and glycosides

    SciTech Connect

    Klasing, S.A.; Mora, M.I.; Wilson, W.C.; Fahey, G.C. Jr.; Garst, J.E.

    1985-09-01

    Experiments were conducted to determine effects of a phenolic polymer (Kraft wood lignin, Indulin), phenolic glycosides (cane molasses and wood molasses), and phenolic monomers (vanillin, vanillic acid, ferulic acid, and p-coumaric acid) on liver cytochromes P-450, cytochrome b/sub 5/, and NADPH cytochrome c reductase in chicks and rats. Chicks fed 6.0% lignin had a higher cytochromes P-450 content than did chicks fed 0% fiber, 6.0% wood cellulose, or 6.0% arenaceous flour. Chicks fed 12.0% wood molasses had a higher cytochromes P-450 level than did chicks fed 0% fiber or 6.0% wood molasses. Cane molasses incorporated at both 6.0 and 12.0% of the diet induced cytochromes P-450 content over those of control-fed birds. Chicks fed 6.0% lignin, with or without antibiotic, had a higher cytochromes P-450 level than did chicks fed control diets, with or without antibiotic. Additionally, chicks fed 6.0% lignin had lower intestinal diaminopimelic acid (DAP) levels than did chicks fed 0% fiber. Rats fed 0% fiber, 6.0% wood cellulose, 6.0% arenaceous flour, or 6.0% lignin exhibited no difference in cytochrome level or activity among treatments. Chicks fed 0.5% vanillin, 0.5% vanillic acid, 0.5% ferulic acid, or 0.5% p-coumaric acid had comparable cytochromes level and activity compared with chicks fed no phenolics. Chicks fed 0.5% p-coumaric acid had lower rates of gain than did chicks fed control or other phenolic-containing diets. Rats fed these phenolics had similar cytochromes P-450 content among treatments.

  7. 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.

  8. Cytochrome P450 expression in oesophageal cancer.

    PubMed Central

    Murray, G I; Shaw, D; Weaver, R J; McKay, J A; Ewen, S W; Melvin, W T; Burke, M D

    1994-01-01

    The cytochrome P450 superfamily of enzymes play a central part in the metabolism of carcinogens and anti-cancer drugs. The expression, cellular localisation, and distribution of different forms of P450 and the functionally associated enzymes epoxide hydrolase and glutathione S-transferases have been investigated in oesophageal cancer and non-neoplastic oesophageal tissue using immunohistochemistry. Expression of the different enzymes was confined to epithelial cells in both non-neoplastic samples and tumour samples except the CYP3A was also identified in mast cells and glutathione S-transferase pi was present in chronic inflammatory cells. CYP1A was present in a small percentage of non-neoplastic samples but both CYP2C and CYP3A were absent. Epoxide hydrolase was present in half of the non-neoplastic samples and the different classes of glutathione S-transferase were present in a low number of samples. In carcinomas CYP1A, CYP3A, epoxide hydrolase, and glutathione S-transferase pi were expressed in at least 60% of samples. The expression of glutathione S-transferases alpha and mu were significantly less in adenocarcinoma compared with squamous carcinoma. Images Figure 1 Figure 2 Figure 3 PMID:8200549

  9. Flower colour and cytochromes P450

    PubMed Central

    Tanaka, Yoshikazu; Brugliera, Filippa

    2013-01-01

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

  10. Bioactivation of dibrominated biphenyls by cytochrome P450 activity to metabolites with estrogenic activity and estrogen sulfotransferase inhibition capacity.

    PubMed

    van Lipzig, Marola M H; Commandeur, Jan N; de Kanter, Frans J J; Damsten, Micaela C; Vermeulen, Nico P E; Maat, Evelina; Groot, Ed J; Brouwer, Abraham; Kester, Monique H A; Visser, Theo J; Meerman, John H N

    2005-11-01

    Exposure of humans and wildlife to xenobiotics, such as halogenated biphenyls, that interfere with the endogenous estrogen balance may lead to endocrine disruption. Such compounds may either mimic or block estradiol's action by agonistic or antagonistic action, respectively. They may also affect endogenous estradiol concentrations by induction or inhibition of enzymes that metabolize estradiol. In the present study, we demonstrate that estrogenic metabolites of two brominated biphenyls, 2,2'-dibromobiphenyl (2,2'-DBB) and 4,4'-dibromobiphenyl (4,4'-DBB), are formed by rat liver microsomal cytochrome P450 (CYP) activity. Bioactivation of 2,2'-DBB and 4,4'-DBB yielded various mono- and dihydroxylated bromobiphenyl metabolites, which were collected by preparative HPLC and analyzed by LC/MS. Several of the metabolites bound to the estrogen receptor (ER) activated the ER and inhibited human estrogen sulfotransferase (hEST). Seven monohydroxylated metabolites were positively identified using synthetic monohydroxylated reference compounds. These synthetic monohydroxylated bromobiphenyls also bound to and activated the ER and inhibited hEST. The highest ER affinity was observed for 4-OH-2,2'-DBB, with an EC50 of 6.6 nM. The highest ER activation was observed for 4-OH-3,4'-DBB (EC50 of 74 nM) while 4-OH-4'-MBB and 4-OH-2,2'-DBB induced a supramaximal (as compared to estradiol) ER activation. The strongest hEST inhibition was found with 4-OH-3,4'-DBB (EC50 = 40 nM). In conclusion, we show that two dibrominated biphenyls are bioactivated by CYP activity into very potent estrogenic metabolites and inhibitors of hEST. These findings are of vital importance for accurate risk assessment of exposure to environmental contaminants, such as halogenated biphenyls. Neglecting bioactivation through biotransformation will lead to underestimation of health risks of this class of xenobiotics.

  11. Cytochromes P450 in the bioactivation of chemicals.

    PubMed

    Ioannides, Costas; Lewis, David F V

    2004-01-01

    The initial view that the cytochrome P450 enzyme system functions simply in the deactivation of xenobiotics is anachronistic on the face of mounting evidence that this system can also transform many innocuous chemicals to toxic products. However, not all xenobiotic-metabolising cytochrome P450 subfamilies show the same propensity in the bioactivation of chemicals. For example, the CYP2C, 2B and 2D subfamilies play virtually no role in the bioactivation of toxic and carcinogenic chemicals, whereas the CYP1A, 1B and 2E subfamilies are responsible for the bioactivation of the majority of xenobiotics. Electronic and molecular structural features of organic chemicals appear to predispose them to either bioactivation by one cytochrome P450 enzyme or deactivation by another. Consequently, the fate of a chemical in the body is largely dependent on the cytochrome P450 profile at the time of exposure. Any factor that modulates the enzymes involved in the metabolism of a certain chemical will also influence its toxicity and carcinogenicity. For example, many chemical carcinogens bioactivated by CYP1, on repeated administration, selectively induce this family, thus exacerbating their carcinogenicity. CYP1 induction potency by chemicals appears to be determined by a combination of their molecular shape and electron activation. The function of cytochromes P450 in the bioactivation of chemicals is currently being exploited to design systems that can be used clinically to facilitate the metabolic conversion of prodrugs to their biologically-active metabolites in cells that poorly express them, such as tumour cells, in the so-called gene-directed prodrug therapy.

  12. Hepatocarcinogenic heterocyclic aromatic amines that induce cytochrome P-448 isozymes, mainly cytochrome P-448H (P-450IA2), responsible for mutagenic activation of the carcinogens in rat liver.

    PubMed

    Degawa, M; Tanimura, S; Agatsuma, T; Hashimoto, Y

    1989-06-01

    Male F344 rats were treated with hepatocarcinogenic heterocyclic aromatic amines such as amino acid- and protein-pyrolysate components (Trp P-1, Trp P-2, Glu P-1, Glu P-2, A alpha C, MeA alpha C, IQ and MeIQx) and changes in microsomal cytochrome P-450 isozymes in the livers were examined by means of immuno-Western blotting using anti-rat cytochrome P-450 monoclonal antibodies. The results suggested that all chemicals tested induce cytochrome P-448 isozymes, particularly cytochrome P-448H (P-450IA2), which efficiently mediate mutagenic activation of the carcinogens. This was substantiated by the enzymatic analyses with the substrates showing different characters to rat cytochrome P-450 isozyme-mediated mutagenesis.

  13. Bosentan and Rifampin Interactions Modulate Influx Transporter and Cytochrome P450 Expression and Activities in Primary Human Hepatocytes.

    PubMed

    Han, Kyoung-Moon; Ahn, Sun-Young; Seo, Hyewon; Yun, Jaesuk; Cha, Hye Jin; Shin, Ji-Soon; Kim, Young-Hoon; Kim, Hyungsoo; Park, Hye-Kyung; Lee, Yong-Moon

    2017-02-06

    The incidence of polypharmacy-which can result in drug-drug interactions-has increased in recent years. Drug-metabolizing enzymes and drug transporters are important polypharmacy modulators. In this study, the effects of bosentan and rifampin on the expression and activities of organic anion-transporting peptide (OATP) and cytochrome P450 (CYP450) 2C9 and CYP3A4 were investigated in vitro. HEK293 cells and primary human hepatocytes overexpressing the target genes were treated with bosentan and various concentrations of rifampin, which decreased the uptake activities of OATP transporters in a dose-dependent manner. In primary human hepatocytes, CYP2C9 and CYP3A4 gene expression and activities decreased upon treatment with 20 μM bosentan+200 μM rifampin. Rifampin also reduced gene expression of OATP1B1, OATP1B3, and OATP2B1 transporter, and inhibited bosentan influx in human hepatocytes at increasing concentrations. These results confirm rifampin- and bosentan-induced interactions between OATP transporters and CYP450.

  14. Two azole fungicides (carcinogenic triadimefon and non-carcinogenic myclobutanil) exhibit different hepatic cytochrome P450 activities in medaka fish.

    PubMed

    Lin, Chun-Hung; Chou, Pei-Hsin; Chen, Pei-Jen

    2014-07-30

    Conazoles are a class of imidazole- or triazole-containing drugs commonly used as fungicides in agriculture and medicine. The broad application of azole drugs has led to the contamination of surface aquifers receiving the effluent of municipal or hospital wastewater or agricultural runoff. Several triazoles are rodent carcinogens; azole pollution is a concern to environmental safety and human health. However, the carcinogenic mechanisms associated with cytochrome P450 enzymes (CYPs) of conazoles remain unclear. We exposed adult medaka fish (Oryzias latipes) to continuous aqueous solutions of carcinogenic triadimefon and non-carcinogenic myclobutanil for 7 to 20 days at sub-lethal or environmentally relevant concentrations and assessed hepatic CYP activity and gene expression associated with CYP-mediated toxicity. Both triadimefon and myclobutanil induced hepatic CYP3A activity, but only triadimefon enhanced CYP1A activity. The gene expression of cyp3a38, cyp3a40, pregnane x receptor (pxr), cyp26b, retinoid acid receptor γ1 (rarγ1) and p53 was higher with triadimefon than myclobutanil. As well, yeast-based reporter gene assay revealed that 4 tested conazoles were weak agonists of aryl hydrocarbon receptor (AhR). We reveal differential CYP gene expression with carcinogenic and non-carcinogenic conazoles in a lower vertebrate, medaka fish. Liver CYP-enzyme induction may be a key event in conazole-induced tumorigenesis. This information is essential to evaluate the potential threat of conazoles to human health and fish populations in the aquatic environment.

  15. Metabolic activation of the antibacterial agent triclocarban by cytochrome P450 1A1 yielding glutathione adducts.

    PubMed

    Schebb, Nils Helge; Muvvala, Jaya B; Morin, Dexter; Buckpitt, Alan R; Hammock, Bruce D; Rice, Robert H

    2014-07-01

    Triclocarban (3,4,4'-trichlorocarbanilide; TCC) is an antibacterial agent used in personal care products such as bar soaps. Small amounts of chemical are absorbed through the epidermis. Recent studies show that residues of reactive TCC metabolites are bound covalently to proteins in incubations with keratinocytes, raising concerns about the potential toxicity of this antimicrobial agent. To obtain additional information on metabolic activation of TCC, this study characterized the reactive metabolites trapped as glutathione conjugates. Incubations were carried out with (14)C-labeled TCC, recombinant CYP1A1 or CYP1B1, coexpressed with cytochrome P450 reductase, glutathione-S-transferases (GSH), and an NADPH-generating system. Incubations containing CYP1A1, but not 1B1, led to formation of a single TCC-GSH adduct with a conversion rate of 1% of parent compound in 2 hours. Using high-resolution mass spectrometry and diagnostic fragmentation, the adduct was tentatively identified as 3,4-dichloro-3'-glutathionyl-4'-hydroxycarbanilide. These findings support the hypothesis that TCC is activated by oxidative dehalogenation and oxidation to a quinone imine. Incubations of TCDD-induced keratinocytes with (14)C-TCC yielded a minor radioactive peak coeluting with TCC-GSH. Thus, we conclude that covalent protein modification by TCC in TCDD-induced human keratinocyte incubations is mainly caused by activation of TCC by CYP1A1 via a dehalogenated TCC derivative as reactive species.

  16. Inhibitory effects of polyphenols on human cytochrome P450 3A4 and 2C9 activity.

    PubMed

    Kimura, Yuka; Ito, Hideyuki; Ohnishi, Ryoko; Hatano, Tsutomu

    2010-01-01

    Polyphenols present in foods and supplements may contribute to human health by preventing cardiovascular diseases and cancer. Drug-food or drug-herb interactions have recently come into focus but, except for some phytochemicals, few components of food or herbs participate in such interactions. In this study, we systematically evaluated the inhibitory effects of 60 polyphenols and related compounds on human cytochrome P450 (CYP) 3A4 and CYP2C9 activity by in vitro assay to investigate whether some polyphenols induce drug interactions. In addition, the kinetics of potent CYP inhibitors was investigated by Lineweaver-Burk plot analysis. Three coumarins and 12 flavonoids significantly suppressed CYP3A4 or CYP2C9 activities. Lineweaver-Burk plot analysis indicated that apigenin and its dimer amentoflavone and imperatorin displayed a mixed type of inhibition on CYP3A4 or CYP2C9. Among the inhibitors, amentoflavone was the most potent inhibitor of CYP3A4 and CYP2C9 activities with IC(50) values of 0.07 and 0.03 microM, respectively. The K(i) value of amentoflavone was significantly lower than that of the CYP2C9 inhibition positive control sulfaphenazole. These findings suggest that some dietary polyphenols may have the potential to inhibit the metabolism of clinical drugs.

  17. Influence of different proton pump inhibitors on activity of cytochrome P450 assessed by [(13)C]-aminopyrine breath test.

    PubMed

    Kodaira, Chise; Uchida, Shinya; Yamade, Mihoko; Nishino, Masafumi; Ikuma, Mutsuhiro; Namiki, Noriyuki; Sugimoto, Mitsushige; Watanabe, Hiroshi; Hishida, Akira; Furuta, Takahisa

    2012-03-01

    Aminopyrine is metabolized by cytochrome P450 (CYP) in the liver. The investigators evaluated influences of different PPIs on CYP activity as assessed by the [(13)C]-aminopyrine breath test ([(13)C]-ABT). Subjects were 15 healthy volunteers with different CYP2C19 status (5 rapid metabolizers [RMs], 5 intermediate metabolizers [IMs], and 5 poor metabolizers [PMs]). Breath samples were collected before and every 15 to 30 minutes for 3 hours after oral ingestion of [(13)C]-aminopyrine 100 mg on day 8 of each of the following regimens: control; omeprazole 20 mg and 80 mg, lansoprazole 30 mg, and rabeprazole 20 mg. Changes in carbon isotope ratios in carbon dioxide ((13)CO(2)/(12)CO(2)) in breath samples were measured by infrared spectrometry and expressed as delta-over-baseline (DOB) ratios (‰). Mean areas under the curve of DOB from 0 to 3 h (AUC(0-3h) of DOB) were significantly decreased by omeprazole 20 mg and lansoprazole 30 mg but not by rabeprazole 20 mg. Conversely, higher PPI dose (ie, omeprazole 80 mg) seemed to further decrease AUC(0-3h) of DOB in RMs but increased it in PMs. Omeprazole and lansoprazole at the standard doses inhibit CYP activity but rabeprazole does not, whereas high-dose omeprazole seems to induce CYPs.

  18. [Effects of posttranslational modification on the activity of cytochrome P450: current progress].

    PubMed

    Li, Yu-hua; Bi, Hui-chang; Huang, Min

    2011-05-01

    Regulation of the activity of CYP450 has always been research focus of drug metabolism. The effect of compounds on the mRNA and protein expression level of CYP450 is the main purpose of most of the existing reports. In recent years, the protein modification in the posttranslation level has been found to participate in maintaining the proper function of CYP450, thus effect of posttranslational modification on the enzyme activity has been paid more and more attention. Posttranslational modifications including phosphorylation, nitration, and ubiquitination have been described to regulate the activity of CYP450. In this paper, recent developments in the effects of posttranslational modifications on the activity of CYP450 have been reviewed.

  19. Cytochrome P450-mediated activation of the fragrance compound geraniol forms potent contact allergens

    SciTech Connect

    Hagvall, Lina; Baron, Jens Malte; Boerje, Anna; Weidolf, Lars; Merk, Hans; Karlberg, Ann-Therese

    2008-12-01

    Contact sensitization is caused by low molecular weight compounds which penetrate the skin and bind to protein. In many cases, these compounds are activated to reactive species, either by autoxidation on exposure to air or by metabolic activation in the skin. Geraniol, a widely used fragrance chemical, is considered to be a weak allergen, although its chemical structure does not indicate it to be a contact sensitizer. We have shown that geraniol autoxidizes and forms allergenic oxidation products. In the literature, it is suggested but not shown that geraniol could be metabolically activated to geranial. Previously, a skin-like CYP cocktail consisting of cutaneous CYP isoenzymes, was developed as a model system to study cutaneous metabolism. In the present study, we used this system to investigate CYP-mediated activation of geraniol. In incubations with the skin-like CYP cocktail, geranial, neral, 2,3-epoxygeraniol, 6,7-epoxygeraniol and 6,7-epoxygeranial were identified. Geranial was the main metabolite formed followed by 6,7-epoxygeraniol. The allergenic activities of the identified metabolites were determined in the murine local lymph node assay (LLNA). Geranial, neral and 6,7-epoxygeraniol were shown to be moderate sensitizers, and 6,7-epoxygeranial a strong sensitizer. Of the isoenzymes studied, CYP2B6, CYP1A1 and CYP3A5 showed high activities. It is likely that CYP1A1 and CYP3A5 are mainly responsible for the metabolic activation of geraniol in the skin, as they are expressed constitutively at significantly higher levels than CYP2B6. Thus, geraniol is activated through both autoxidation and metabolism. The allergens geranial and neral are formed via both oxidation mechanisms, thereby playing a large role in the sensitization to geraniol.

  20. Effect of vanillin and ethyl vanillin on cytochrome P450 activity in vitro and in vivo.

    PubMed

    Chen, Xiao-min; Wei, Min; Zhang, Hai-mou; Luo, Cheng-hao; Chen, Yi-kun; Chen, Yong

    2012-06-01

    Food safety is of extreme importance to human health. Vanillin and ethyl vanillin are the widely used food additives and spices in foods, beverages, cosmetics and drugs. The objective of the present work was to evaluate the impact of vanillin and ethyl vanillin on the activities of CYP2C9, CYP2E1, CYP3A4, CYP2B6 and CYP1A2 in human liver microsomes (HLM) in vitro, and impact on the activities of CYP1A2, CYP2C, CYP3A and CYP2E1 in rat liver microsomes (RLM) in vivo. The in vitro results demonstrated that vanillin and ethyl vanillin had no significant effect on the activity of five human CYP450 enzymes with concentration ranged from 8 to 128 μM. However, after rats were orally administered vanillin or ethyl vanillin once a day for seven consecutive days, CYP2E1 activity was increased and CYP1A2 activity was decreased in RLM. The in vivo results revealed that drug interaction between vanillin/ethyl vanillin and the CYP2E1/CYP1A2-metabolizing drugs might be possible, and also suggested that the application of the above additives in foods and drugs should not be unlimited so as to avoid the adverse interaction.

  1. Cytochrome P450 2D6 Activity Predicts Discontinuation of Tamoxifen Therapy in Breast Cancer Patients

    PubMed Central

    Rae, James M.; Sikora, Matthew J.; Henry, N. Lynn; Li, Lang; Kim, Seongho; Oesterreich, Steffi; Skaar, Todd; Nguyen, Anne T.; Desta, Zeruesenay; Storniolo, Anna Maria; Flockhart, David A.; Hayes, Daniel F.; Stearns, Vered

    2009-01-01

    The selective estrogen receptor modulator tamoxifen is routinely used for treatment and prevention of estrogen receptor positive breast cancer. Studies of tamoxifen adherence suggest that over half of patients discontinue treatment before the recommended 5 years. We hypothesized that polymorphisms in CYP2D6, the enzyme responsible for tamoxifen activation, predict for tamoxifen discontinuation. Tamoxifen-treated women (n = 297) were genotyped for CYP2D6 variants and assigned a “score” based on predicted allele activities from 0 (no activity) to 2 (high activity). Correlation between CYP2D6 score and discontinuation rates at 4 months were tested. We observed a strong non-linear correlation between higher CYP2D6 score and increased rates of discontinuation (r2 = 0.935, p = 0.018). These data suggest that presence of active CYP2D6 alleles may predict for higher likelihood of tamoxifen discontinuation. Therefore, patients who may be most likely to benefit from tamoxifen may paradoxically be most likely to discontinue treatment prematurely. PMID:19421167

  2. Chemotherapy pro-drug activation by biocatalytic virus-like nanoparticles containing cytochrome P450.

    PubMed

    Sánchez-Sánchez, Lorena; Cadena-Nava, Rubén D; Palomares, Laura A; Ruiz-Garcia, Jaime; Koay, Melissa S T; Cornelissen, Jeroen J M T; Vazquez-Duhalt, Rafael

    2014-06-10

    This work shows, for the first time, the encapsulation of a highly relevant protein in the biomedical field into virus-like particles (VLPs). A bacterial CYP variant was effectively encapsulated in VLPs constituted of coat protein from cowpea chlorotic mottle virus (CCMV). The catalytic VLPs are able to transform the chemotherapeutic pro-drug, tamoxifen, and the emerging pro-drug resveratrol. The chemical nature of the products was identified, confirming similar active products than those obtained with human CYP. The enzymatic VLPs remain stable after the catalytic reaction. The potential use of these biocatalytic nanoparticles as targeted CYP carriers for the activation of chemotherapy drugs is discussed.

  3. Constitutive expression and activity of cytochrome P450 in conventional pigs.

    PubMed

    Nielsen, Søren Drud; Bauhaus, Yvonne; Zamaratskaia, Galia; Junqueira, Matheus Antunes; Blaabjerg, Karoline; Petrat-Melin, Bjørn; Young, Jette Feveile; Rasmussen, Martin Krøyer

    2017-04-01

    Pigs have often been suggested to be a useful model for humans, when investigating CYP dependent events, like drug metabolism. However, comprehensive knowledge about the constitutive expression of the major CYP and corresponding transcription factors is limited. We compared the constitutive mRNA expression of aryl hydrocarbon receptor, constitutive androstane receptor and pregnane X receptor and CYP1A1, CYP1A2, CYP2A, CYP2E1 and CYP3A in liver, adipose tissue, muscle and small intestine in pigs, as well as the expression along the length of the small intestine and colon. Tissue samples were taken from female pigs, and analyzed for gene expression, as well as CYP dependent activity using qPCR and specific probe substrates, respectively. For all investigated transcription factors and CYPs the mRNA expression and activity was highest in the liver. CYP1A1 and CYP3A mRNA expression and activity was shown in all investigated tissues. Along the small intestine and colon the mRNA expression and activity of CYP1A1 and CYP3A was gradually decreased. The results demonstrated, similarity to that reported for humans, and hence adds to the use of pigs as a model for humans.

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

    PubMed

    Keller, Jennifer M; McClellan-Green, Patricia

    2004-01-01

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

  5. Effects of mace and nutmeg on human cytochrome P450 3A4 and 2C9 activity.

    PubMed

    Kimura, Yuka; Ito, Hideyuki; Hatano, Tsutomu

    2010-01-01

    Pharmacokinetic or pharmacodynamic interactions between herbal medicines or food constituents and drugs have been studied as crucial factors determining therapeutic efficacy and outcome. Most of these interactions are attributed to inhibition or induction of activity of cytochrome P450 (CYP) metabolic enzymes. Inhibition or induction of CYP enzymes by beverages, including grapefruit, pomegranate, or cranberry juice, has been well documented. Because spices are a common daily dietary component, other studies have reported inhibition of CYP activity by spices or their constituents/derivatives. However, a systematic evaluation of various spices has not been performed. In this study, we investigated effects of 55 spices on CYP3A4 and CYP2C9 activity. Cinnamon, black or white pepper, ginger, mace, and nutmeg significantly inhibited CYP3A4 or CYP2C9 activity. Furthermore, bioassay-guided fractionation of mace (Myristica fragrans) led to isolation and structural characterization of a new furan derivative (1) along with other 16 known compounds, including an acylphenol, neolignans, and phenylpropanoids. Among these isolates, (1S,2R)-1-acetoxy-2-(4-allyl-2,6-dimethoxyphenoxy)-1-(3,4-dimethoxyphenyl)propane (9) exhibited the most potent CYP2C9 inhibitory activity with an IC₅₀ value comparable to that of sulfaphenazole, a CYP2C9 inhibitor. Compound 9 competitively inhibited CYP2C9-mediated 4'-hydroxylation of diclofenac. The inhibitory constant (K(i)) of 9 was determined to be 0.037 µM. Compound 9 was found to be 14-fold more potent than was sulfaphenazole.

  6. Menadione Suppresses Benzo(α)pyrene-Induced Activation of Cytochromes P450 1A: Insights into a Possible Molecular Mechanism

    PubMed Central

    Pivovarova, Elena N.; Markel, Arkady L.; Lyakhovich, Vyacheslav V.; Grishanova, Alevtina Y.

    2016-01-01

    Oxidative reactions that are catalyzed by cytochromes P450 1A (CYP1A) lead to formation of carcinogenic derivatives of arylamines and polycyclic aromatic hydrocarbons (PAHs), such as the widespread environmental pollutant benzo(α)pyrene (BP). These compounds upregulate CYP1A at the transcriptional level via an arylhydrocarbon receptor (AhR)-dependent signaling pathway. Because of the involvement of AhR-dependent genes in chemically induced carcinogenesis, suppression of this signaling pathway could prevent tumor formation and/or progression. Here we show that menadione (a water-soluble analog of vitamin K3) inhibits BP-induced expression and enzymatic activity of both CYP1A1 and CYP1A2 in vivo (in the rat liver) and BP-induced activity of CYP1A1 in vitro. Coadministration of BP and menadione reduced DNA-binding activity of AhR and increased DNA-binding activity of transcription factors Oct-1 and CCAAT/enhancer binding protein (C/EBP), which are known to be involved in negative regulation of AhR-dependent genes, in vivo. Expression of another factor involved in downregulation of CYP1A—pAhR repressor (AhRR)—was lower in the liver of the rats treated with BP and menadione, indicating that the inhibitory effect of menadione on CYP1A is not mediated by this protein. Furthermore, menadione was well tolerated by the animals: no signs of acute toxicity were detected by visual examination or by assessment of weight gain dynamics or liver function. Taken together, our results suggest that menadione can be used in further studies on animal models of chemically induced carcinogenesis because menadione may suppress tumor formation and possibly progression. PMID:27167070

  7. Peroxisome proliferator-activated receptor alpha, PPARα, directly regulates transcription of cytochrome P450 CYP2C8

    PubMed Central

    Thomas, Maria; Winter, Stefan; Klumpp, Britta; Turpeinen, Miia; Klein, Kathrin; Schwab, Matthias; Zanger, Ulrich M.

    2015-01-01

    The cytochrome P450, CYP2C8, metabolizes more than 60 clinically used drugs as well as endogenous substances including retinoic acid and arachidonic acid. However, predictive factors for interindividual variability in the efficacy and toxicity of CYP2C8 drug substrates are essentially lacking. Recently we demonstrated that peroxisome proliferator-activated receptor alpha (PPARα), a nuclear receptor primarily involved in control of lipid and energy homeostasis directly regulates the transcription of CYP3A4. Here we investigated the potential regulation of CYP2C8 by PPARα. Two linked intronic SNPs in PPARα (rs4253728, rs4823613) previously associated with hepatic CYP3A4 status showed significant association with CYP2C8 protein level in human liver samples (N = 150). Furthermore, siRNA-mediated knock-down of PPARα in HepaRG human hepatocyte cells resulted in up to ∼60 and ∼50% downregulation of CYP2C8 mRNA and activity, while treatment with the PPARα agonist WY14,643 lead to an induction by >150 and >100%, respectively. Using chromatin immunoprecipitation scanning assay we identified a specific upstream gene region that is occupied in vivo by PPARα. Electromobility shift assay demonstrated direct binding of PPARα to a DR-1 motif located at positions –2762/–2775 bp upstream of the CYP2C8 transcription start site. We further validated the functional activity of this element using luciferase reporter gene assays in HuH7 cells. Moreover, based on our previous studies we demonstrated that WNT/β-catenin acts as a functional inhibitor of PPARα-mediated inducibility of CYP2C8 expression. In conclusion, our data suggest direct involvement of PPARα in both constitutive and inducible regulation of CYP2C8 expression in human liver, which is further modulated by WNT/β-catenin pathway. PPARA gene polymorphism could have a modest influence on CYP2C8 phenotype. PMID:26582990

  8. AM-2201 Inhibits Multiple Cytochrome P450 and Uridine 5'-Diphospho-Glucuronosyltransferase Enzyme Activities in Human Liver Microsomes.

    PubMed

    Kim, Ju-Hyun; Kwon, Soon-Sang; Kong, Tae Yeon; Cheong, Jae Chul; Kim, Hee Seung; In, Moon Kyo; Lee, Hye Suk

    2017-03-10

    AM-2201 is a synthetic cannabinoid that acts as a potent agonist at cannabinoid receptors and its abuse has increased. However, there are no reports of the inhibitory effect of AM-2201 on human cytochrome P450 (CYP) or uridine 5'-diphospho-glucuronosyltransferase (UGT) enzymes. We evaluated the inhibitory effect of AM-2201 on the activities of eight major human CYPs (1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4) and six major human UGTs (1A1, 1A3, 1A4, 1A6, 1A9, and 2B7) enzymes in pooled human liver microsomes using liquid chromatography-tandem mass spectrometry to investigate drug interaction potentials of AM-2201. AM-2201 potently inhibited CYP2C9-catalyzed diclofenac 4'-hydroxylation, CYP3A4-catalyzed midazolam 1'-hydroxylation, UGT1A3-catalyzed chenodeoxycholic acid 24-acyl-glucuronidation, and UGT2B7-catalyzed naloxone 3-glucuronidation with IC50 values of 3.9, 4.0, 4.3, and 10.0 μM, respectively, and showed mechanism-based inhibition of CYP2C8-catalyzed amodiaquine N-deethylation with a Ki value of 2.1 μM. It negligibly inhibited CYP1A2, CYP2A6, CYP2B6, CYP2C19, CYP2D6, UGT1A1, UGT1A4, UGT1A6, and UGT1A9 activities at 50 μM in human liver microsomes. These in vitro results indicate that AM-2201 needs to be examined for potential pharmacokinetic drug interactions in vivo due to its potent inhibition of CYP2C8, CYP2C9, CYP3A4, UGT1A3, and UGT2B7 enzyme activities.

  9. Inhibitory effects of seven components of danshen extract on catalytic activity of cytochrome P450 enzyme in human liver microsomes.

    PubMed

    Qiu, Furong; Zhang, Rong; Sun, Jianguo; Jiye, A; Hao, Haiping; Peng, Ying; Ai, Hua; Wang, Guangji

    2008-07-01

    The potential for herb-drug interactions has recently received greater attention worldwide, considering the fact that the use of herbal products becomes more and more widespread. The goal of this work was to examine the potential for the metabolism-based drug interaction arising from seven active components (danshensu, protocatechuic aldehyde, protocatechuic acid, salvianolic acid B, tanshinone I, tanshinone IIA, and cryptotanshinone) of danshen extract. Probe substrates of cytochrome P450 enzymes were incubated in human liver microsomes (HLMs) with or without each component of danshen extract. IC(50) and K(i) values were estimated, and the types of inhibition were determined. Among the seven components of danshen extract, tanshinone I, tanshinone IIA, and cryptotanshinone were potent competitive inhibitors of CYP1A2 (K(i) = 0.48, 1.0, and 0.45 microM, respectively); danshensu was a competitive inhibitor of CYP2C9 (K(i) = 35 microM), and cryptotanshinone was a moderate mixed-type inhibitor of CYP2C9 (K(i) = 8 microM); cryptotanshinone inhibited weakly and in mixed mode against CYP2D6 activity (K(i) = 68 microM), and tanshinone I was a weak inhibitor of CYP2D6 (IC(50) = 120 microM); and protocatechuic aldehyde was a weak inhibitor of CYP3A4 (IC(50) = 130 and 160 microM for midazolam and testosterone, respectively). These findings provided some useful information for safe and effective use of danshen preparations in clinical practice. Our data indicated that it was necessary to study the in vivo interactions between drugs and pharmaceuticals with danshen extract.

  10. 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

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

    PubMed Central

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

    2015-01-01

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

  12. Phenotyping studies to assess the effects of phytopharmaceuticals on in vivo activity of main human cytochrome p450 enzymes.

    PubMed

    Zadoyan, Gregor; Fuhr, Uwe

    2012-09-01

    The extensive use of herbal drugs and their multiple components and modes of action suggests that they may also cause drug interactions by changing the activity of human cytochrome P450 enzymes. The purpose of the present review is to present the available data for the top 14 herbal drug sales in the U. S. Studies describing the effects of herbal drugs on phenotyping substrates for individual CYPs were identified by a comprehensive MEDLINE search. Drugs included Allium sativum (Liliaceae), Echinacea purpurea (Asteraceae), Serenoa repens (Arecaceae), Ginkgo biloba (Ginkgoaceae), Vaccinium macrocarpon (Ericaceae), Glycine max (Fabaceae), Panax ginseng (Araliaceae), Actea racemosa (Ranunculaceae), Hypericum perforatum (Hypericaceae), Silybum marianum (Asteraceae), Camellia sinensis (Theaceae), Valeriana officinalis (Valerianaceae), Piper methysticum (Piperaceae), and Hydrastis canadensis (Ranunculaceae) preparations. We identified 70 clinical studies in 69 publications. The majority of the herbal drugs appeared to have no clear effects on most of the CYPs examined. If there was an effect, there was mild inhibition in almost all cases, as seen with garlic or kava effects on CYP2E1 and with soybean components on CYP1A2. The most pronounced effects were induction of CYP3A and other CYPs by St. John's wort and the inhibitory effect of goldenseal on CYP3A and CYP2D6, both being borderline between mild and moderate in magnitude. With the exceptions of St.John's wort and goldenseal, the information currently available suggests that concomitant intake of the herbal drugs addressed here is not a major risk for drugs that are metabolized by CYPs.

  13. In vitro inhibitory activities of the extract of Hibiscus sabdariffa L. (family Malvaceae) on selected cytochrome P450 isoforms.

    PubMed

    Johnson, Showande Segun; Oyelola, Fakeye Titilayo; Ari, Tolonen; Juho, Hokkanen

    2013-01-01

    Literature is scanty on the interaction potential of Hibiscus sabdariffa L., plant extract with other drugs and the affected targets. This study was conducted to investigate the cytochrome P450 (CYP) isoforms that are inhibited by the extract of Hibiscus sabdariffa L. in vitro. The inhibition towards the major drug metabolizing CYP isoforms by the plant extract were estimated in human liver microsomal incubations, by monitoring the CYP-specific model reactions through previously validated N-in-one assay method. The ethanolic extract of Hibiscus sabdariffa showed inhibitory activities against nine selected CYP isoforms: CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4. The concentrations of the extract which produced 50% inhibition of the CYP isoforms ranged from 306 µg/ml to 1660 µg/ml, and the degree of inhibition based on the IC50 values for each CYP isoform was in the following order: CYP1A2 > CYP2C8 > CYP2D6 > CYP2B6 > CYP2E1 > CYP2C19 > CYP3A4 > CYP2C9 > CYP2A6. Ethanolic extract of Hibiscus sabdariffa caused inhibition of CYP isoforms in vitro. These observed inhibitions may not cause clinically significant herb-drug interactions; however, caution may need to be taken in co-administering the water extract of Hibiscus sabdariffa with other drugs until clinical studies are available to further clarify these findings.

  14. Camptothecin Attenuates Cytochrome P450 3A4 Induction by Blocking the Activation of Human Pregnane X ReceptorS⃞

    PubMed Central

    Chen, Yakun; Tang, Yong; Robbins, Gregory T.

    2010-01-01

    Differential regulation of drug-metabolizing enzymes (DMEs) is a common cause of adverse drug effects in cancer therapy. Due to the extremely important role of cytochrome P450 3A4 (CYP3A4) in drug metabolism and the dominant regulation of human pregnane X receptor (hPXR) on CYP3A4, finding inhibitors for hPXR could provide a unique tool to control drug efficacies in cancer therapy. Camptothecin (CPT) was demonstrated as a novel and potent inhibitor (IC50 = 0.58 μM) of an hPXR-mediated transcriptional regulation on CYP3A4 in this study. In contrast, one of its analogs, irinotecan (CPT-11), was found to be an hPXR agonist in the same tests. CPT disrupted the interaction of hPXR with steroid receptor coactivator-1 but had effects on neither the competition of ligand binding nor the formation of the hPXR and retinoid X receptor α heterodimer, nor the interaction between the regulatory complex and DNA-responsive elements. CPT treatment resulted in delayed metabolism of nifedipine in human hepatocytes treated with rifampicin, suggesting a potential prevention of drug-drug interactions between CYP3A4 inducers and CYP3A4-metabolized drugs. Because CPT is the leading compound of topoisomerase I inhibitors, which comprise a quickly developing class of anticancer agents, the findings indicate the potential of a new class of compounds to modify hPXR activity as agonists/inhibitors and are important in the development of CPT analogs. PMID:20504912

  15. Applications of microbial cytochrome P450 enzymes in biotechnology and synthetic biology.

    PubMed

    Girvan, Hazel M; Munro, Andrew W

    2016-04-01

    Cytochrome P450 enzymes (P450s) are a superfamily of monooxygenase enzymes with enormous potential for synthetic biology applications. Across Nature, their substrate range is vast and exceeds that of other enzymes. The range of different chemical transformations performed by P450s is also substantial, and continues to expand through interrogation of the properties of novel P450s and by protein engineering studies. The ability of P450s to introduce oxygen atoms at specific positions on complex molecules makes these enzymes particularly valuable for applications in synthetic biology. This review focuses on the enzymatic properties and reaction mechanisms of P450 enzymes, and on recent studies that highlight their broad applications in the production of oxychemicals. For selected soluble bacterial P450s (notably the high-activity P450-cytochrome P450 reductase enzyme P450 BM3), variants with a multitude of diverse substrate selectivities have been generated both rationally and by random mutagenesis/directed evolution approaches. This highlights the robustness and malleability of the P450 fold, and the capacity of these biocatalysts to oxidise a wide range of chemical scaffolds. This article reviews recent research on the application of wild-type and engineered P450s in the production of important chemicals, including pharmaceuticals and drug metabolites, steroids and antibiotics. In addition, the properties of unusual members of the P450 superfamily that do not follow the canonical P450 catalytic pathway are described.

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

    PubMed Central

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

    2010-01-01

    Inhaled glucocorticoid (GC) therapy is a vital part of the management of chronic asthma. GCs are metabolized by members of the cytochrome P450 3A family in both liver and lung, but the enzymes are differentially expressed. Selective inhibition of one or more P450 3A enzymes could substantially modify target and systemic concentrations of GCs. In this study, we have evaluated the mechanism-based inactivation of P450 3A4, 3A5 and 3A7 enzymes by GCs. Among the five major inhaled GCs approved for clinical use in the United States, fluticasone propionate (FLT) was the most potent mechanism-based inactivator of P450 3A5, the predominant P450 enzyme in the lung. FLT inactivated P450 3A5 in a time- and concentration-dependent manner with KI, kinact and partition ratio of 16 μM, 0.027 min-1 and 3, respectively. In contrast, FLT minimally inactivated P450 3A4 and did not inactivate 3A7, even with a concentration of 100 μM. The inactivation of P450 3A5 by FLT was irreversible because dialysis did not restore enzyme activity. In addition, the exogenous nucleophilic scavenger GSH did not attenuate inactivation. The prosthetic heme of P450 3A5 was not modified by FLT. The loss of P450 3A5 activity in lung cells could substantially decrease the metabolism of FLT, which would increase the effective FLT concentration at its target site, the respiratory epithelium. Also, inactivation of lung P450 3A5 could increase the absorption of inhaled FLT, which could lead to high systemic concentrations and adverse effects, such as life-threatening adrenal crises or cataracts that have been documented in children receiving high doses of inhaled GCs. PMID:20707410

  17. Cytochrome p450nor, a novel class of mitochondrial cytochrome P450 involved in nitrate respiration in the fungus Fusarium oxysporum.

    PubMed

    Takaya, N; Suzuki, S; Kuwazaki, S; Shoun, H; Maruo, F; Yamaguchi, M; Takeo, K

    1999-12-15

    Fusarium oxysporum, an imperfect filamentous fungus performs nitrate respiration under limited oxygen. In the respiratory system, Cytochrome P450nor (P450nor) is thought to catalyze the last step; reduction of nitric oxide to nitrous oxide. We examined its intracellular localization using enzymatic, spectroscopic, and immunological analyses to show that P450nor is found in both the mitochondria and the cytosol. Translational fusions between the putative mitochondrial targeting signal on the amino terminus of P450nor and Escherichia coli beta-galactosidase resulted in significant beta-galactosidase activity in the mitochondrial fraction of nitrate-respiring cells, suggesting that one of the isoforms of P450nor (P450norA) is in anaerobic mitochondrion of F. oxysporum and acts as nitric oxide reductase. Furthermore, these findings suggest the involvement of P450nor in nitrate respiration in mitochondria.

  18. Vasoactive intestinal peptide-induced expression of cytochrome P450 cholesterol side-chain cleavage and 17 alpha-hydroxylase enzyme activity in hen granulosa cells.

    PubMed

    Johnson, A L; Li, Z; Gibney, J A; Malamed, S

    1994-08-01

    Experiments were conducted to determine whether vasoactive intestinal peptide (VIP) can regulate expression of cytochrome P450 side-chain cleavage (P450scc) and P450 17 alpha-hydroxylase (P450 17 alpha-OH) mRNA levels and enzyme activity in granulosa cells from nonhierarchal (6-8-mm) follicles. Initial studies demonstrated that immunoreactive VIP is localized within the theca (but not granulosa) layer of both resting (< 0.5-mm follicles) and 6-8-mm follicles, thus providing a potential paracrine mechanism of action for VIP. While short-term (3 h) incubation of granulosa cells with VIP (0.001-1.0 microM) failed to stimulate progesterone production from 6-8-mm follicle granulosa cells, a 4-h culture period in the presence of VIP resulted in increased cyclic AMP (cAMP) accumulation, and a 24-h culture period resulted in progesterone synthesis and increased P450scc mRNA levels; control levels of each endpoint measurement were not altered within the period observed. By contrast, culture with the growth factor transforming growth factor alpha (TGF alpha) in the presence of VIP (1 microM) prevented increases in P450scc mRNA levels and progesterone production. Similar effects of VIP and TGF alpha in the presence of VIP were demonstrated for P450 17 alpha-OH mRNA levels and enzyme activity. Finally, there was an additive effect of VIP (0.1 microM) plus recombinant human (rh) FSH (100 mIU) on the initiation of progesterone production in cultured 6-8-mm follicle granulosa cells compared to the addition of VIP or rhFSH alone.(ABSTRACT TRUNCATED AT 250 WORDS)

  19. Suppression of Peroxisomal Enzyme Activities and Cytochrome P450 4A Isozyme Expression by Congeneric Polybrominated and Polychlorinated Biphenyls

    PubMed Central

    Robertson, Larry W.; Berberian, Isabelle; Borges, Tim; Chen, Li-Chuan; Chow, Ching K.; Glauert, Howard P.; Filser, Johannes G.; Thomas, Helmut

    2007-01-01

    The purpose of this study was to determine the effects of PCBs and PBBs on peroxisome proliferator-activated receptor-α-(PPARα-) associated enzyme activities or protein levels. Male Sprague-Dawley rats were administered a single IP injection (150 μ mol/kg) of either 3,3′,4,4′-tetrabromobiphenyl, 3,3′,4,4′-tetrachlorobiphenyl, 3,3′,5,5′-tetrabromobiphenyl, 2′,3,3′,4,5-pentachlorobiphenyl, 3,3′,4,4′,5-pentachlorobiphenyl, 2,2′,3,3′,5,5′-hexachlorobiphenyl, or 3,3′,4,4′,5,5′-hexabromobiphenyl in corn oil (10 ml/kg). One week later, the activities of catalase, peroxisomal fatty acyl-CoA oxidase, and peroxisomal beta-oxidation as well as cytochrome P450 4A (CYP4A) protein content were determined in subcellular liver fractions. None of the peroxisomal enzyme activities were significantly increased by any of the halogenated biphenyl congeners tested. Except for minor (approx. 25%) increases in the total CYP4A content following treatment with 2,2′,3,3′,5,5′-hexachlorobiphenyl and 3,3′,5,5′-tetrabromobiphenyl, CYP4A protein contents were not increased by any treatment. The two Ah receptor agonists, 3,3′,4,4′-tetrabromobiphenyl and 3,3′,4,4′,5-pentachlorobiphenyl, significantly diminished the liver content of CYP4A proteins and activities of the peroxisomal enzymes studied. Since a range of congeners with different biologic and toxicologic activities were selected for this study, it may be concluded that the polyhalogenated biphenyls do not induce peroxisome proliferation in the male rat, but rather certain members of this class of compounds down regulate peroxisome-associated enzymes. Since PCBs and PBBs do not increase enzyme activities and expression of proteins associated with PPARα, these agents are therefore exerting their carcinogenic and promoting activities by some other mechanism. PMID:18274624

  20. Overexpression of cerebral and hepatic cytochrome P450s alters behavioral activity of rat offspring following prenatal exposure to lindane

    SciTech Connect

    Johri, Ashu; Yadav, Sanjay; Dhawan, Alok; Parmar, Devendra

    2007-12-15

    Oral administration of different doses (0.0625, 0.125 or 0.25 mg/kg corresponding to 1/1400th, 1/700th or 1/350th of LD{sub 50}) of lindane to the pregnant Wistar rats from gestation days 5 to 21 were found to produce a dose-dependent increase in the activity of cytochrome P450 (CYP)-dependent 7-ethoxyresorufin-O-deethylase (EROD), 7-pentoxyresorufin-O-dealkylase (PROD) and N-nitrosodimethylamine demethylase (NDMA-d) in brain and liver of offspring postnatally at 3 weeks. The increase in the activity of CYP monooxygenases was found to be associated with the increase in the mRNA and protein expression of xenobiotic metabolizing CYP1A, 2B and 2E1 isoenzymes in the brain and liver of offspring. Dose-dependent alterations in the parameters of spontaneous locomotor activity in the offspring postnatally at 3 weeks have suggested that increase in CYP activity may possibly lead to the formation of metabolites to the levels that may be sufficient to alter the behavioral activity of the offspring. Interestingly, the inductive effect on cerebral and hepatic CYPs was found to persist postnatally up to 6 weeks in the offspring at the relatively higher doses (0.125 and 0.25 mg/kg) of lindane and up to 9 weeks at the highest dose (0.25 mg/kg), though the magnitude of induction was less than that observed at 3 weeks. Alterations in the parameters of spontaneous locomotor activity in the offspring postnatally at 6 and 9 weeks, though significant only in the offspring at 3 and 6-week of age, have further indicated that due to the reduced activity of the CYPs during the ontogeny, lindane and its metabolites may not be effectively cleared from the brain. The data suggest that low dose prenatal exposure to the pesticide has the potential to produce overexpression of xenobiotic metabolizing CYPs in brain and liver of the offspring which may account for the behavioral changes observed in the offspring.

  1. Characterization of cytochrome P-450 2D1 activity in rat brain: high-affinity kinetics for dextromethorphan.

    PubMed

    Tyndale, R F; Li, Y; Li, N Y; Messina, E; Miksys, S; Sellers, E M

    1999-08-01

    We investigated the enzymatic function, stability, and regional distribution of rat brain cytochrome P-450 (CYP) 2D1 activity. CYP2D1 is the homolog of human CYP2D6, a genetically variable enzyme that activates or inactivates many clinical drugs acting on the central nervous system (e.g., antidepressants, monoamine oxidase inhibitors, serotonin uptake inhibitors, and neuroleptics), drugs of abuse (e.g., amphetamine and codeine), neurotoxins (e.g., 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, 1,2,3, 4-tetrahydroquinoline), and endogenous neurochemicals (e.g., tryptamine). The CYP2D family has been identified in rodent, canine, and primate brain. Conversion of dextromethorphan to dextrorphan by rat brain membranes was assayed by HPLC and was dependent on NADPH, protein concentration, and incubation time. Significant loss of activity was observed in some homogenizing buffers and after freezing of whole tissues or membrane preparations. Dextromethorphan (0.5-640 microM) metabolism was mediated by high- and low-affinity enzyme systems; K(m1) was 2.7 +/- 2.6 and K(m2) was 757 +/- 156 microM (n = 3 rats, mean +/- S.E.). The enzyme activity was significantly (p <.01) and stereoselectively inhibited by CYP2D1 inhibitors quinine and quinidine (not by CYP2C or CYP3A inhibitors), and by anti-CYP2D6 peptide antiserum (not by anti-CYP2C, -CYP2B, or -CYP3A antibodies). The enzymatic activity demonstrated significant brain regional variation (n = 10 regions, p <.001). These data characterize CYP2D1-mediated dextromethorphan metabolism in rat brain and suggest that localized metabolism of other CYP2D1 substrates (drugs, neurotoxins, and possibly endogenous compounds) within the brain will occur. In humans, CYP2D6 is genetically polymorphic; the variable expression of brain CYP2D6 may result in interindividual differences in central drug and neurotoxin metabolism, possibly contributing to interindividual differences in drug effects and neurotoxicity.

  2. 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 ...

  3. Cytochrome P450 expression and activities in human tongue cells and their modulation by green tea extract

    SciTech Connect

    Yang, S.-P.; Raner, Gregory M. . E-mail: gmraner@uncg.edu

    2005-01-15

    The expression, inducibility, and activities of several cytochrome P450 (CYP) enzymes were investigated in a human tongue carcinoma cell model, CAL 27, and compared with the human liver model HepG2 cells. The modulation effects of green tea on various CYP isoforms in both cell lines were also examined. RT-PCR analysis of CAL 27 cells demonstrated constitutive expression of mRNA for CYPs 1A1, 1A2, 2C, 2E1, 2D6, and 4F3. The results were negative for CYP2A6, 2B6/7, 3A3/4, and 3A7. Both cell lines displayed identical expression and induction profiles for all of the isoforms examined in this study except 3A7 and 2B6/7, which were produced constitutively in HepG2 but not Cal-27 cells. CYP1A1 and 1A2 were both induced by treatment with {beta}-napthoflavone as indicated by RT-PCR and Western blotting, while CYP2C mRNA was upregulated by all-trans retinoic acid and farnesol. RT-PCR and Western blot analysis showed that the expressions of CYP1A1 and 1A2 were induced by green tea extract (GTE), which also caused an increase in mRNA for CYP2E1, CYP2D6, and CYP2C isoforms. The four tea catechins, EGC, EC, EGCG and ECG, applied to either HepG2 or Cal-27 cells at the concentration found in GTE failed to induce CYP1A1 or CYP1A2, as determined by RT-PCR. Of the isoforms that were apparently induced by GTE, only 7-ethoxycoumarin deethylase (ECOD) activity could be detected in CAL 27 or HepG2 cells. Interestingly, mRNA and protein for CYP1A1 and CYP1A2 were detected in both cell lines, and although protein and mRNA levels of CYP1A1 and CYP1A2 were increased by GTE, the observed ECOD activity in both cell lines was decreased.

  4. 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

  5. Insecticide resistance and cytochrome-P450 activation in unfed and blood-fed laboratory and field populations of Culex pipiens pallens.

    PubMed

    Chang, Kyu-Sik; Kim, Heung-Chul; Klein, Terry A; Ju, Young Ran

    2017-01-01

    Understanding the mechanisms of insecticide resistance to vector mosquitoes is critical for the implementation of effective control measures. A nulliparous susceptible Culex pipiens pallens (KSCP) laboratory colony and two field strains from Paju (PAJ) and Jeonju (JEO) Korea were evaluated for susceptibility to five pesticides by microapplication techniques. Unfed PAJ and JEO females demonstrated increased resistance compared to unfed KSCP females, respectively. While blood-fed KSCP females demonstrated <10-fold decreased susceptibility to pesticides compared to unfed KSCP females, blood-fed PAJ and JEO females demonstrated 25.0-50.0- and 16.0-38.6-fold increased resistance compared to unfed PAJ and JEO females, respectively. Unfed and blood-fed groups were assayed for α- and β-esterase, glutathione S-transferases, and cytochrome P-450 (P450) enzyme activity assays. P450 activity was 58.8- and 72.8-fold higher for unfed PAJ and JEO females, respectively, than unfed KSCP females. P450 enzyme activity of KSCP females assayed 1 and 7 days after a blood meal increased by 14.5- and 11.8-fold, respectively, compared to unfed KSCP females, while PAJ and JEO females demonstrated 164.9- and 148.5- and 170.7- and 160.4-fold increased activity, respectively, compared to unfed females of each population. However, other three resistance-related metabolic enzymes showed low activation at <10-fold after a blood meal. The data demonstrate that P450 acts on elevated insecticide resistance after blood meals in resistant field populations. Our findings might reveal that suppressing of the P450 protein by artificial gene mutation increases insecticidal susceptibility of Cx. pipiens and will promise effective vector mosquito control.

  6. Monoclonal antibody-directed analysis of cytochrome P-450-dependent monooxygenases and mutagen activation in the livers of DBA/2 and C57BL/6 mice.

    PubMed

    Hietanen, E; Malaveille, C; Friedman, F K; Park, S S; Béréziat, J C; Brun, G; Bartsch, H; Gelboin, H V

    1986-02-01

    Monoclonal antibodies (MAb 1-7-1 and Mab 2-66-3) specific for cytochrome P-450 (cyt. P-450) isozymes inhibited the metabolism of carcinogens, other xenobiotics, and endogenous compounds in two strains of mice. Postmitochondrial liver supernatant (S9) was prepared from untreated, 3-methylcholanthrene-treated, phenobarbital-treated, and pregnenolone 16 alpha-carbonitrile-treated C57BL/6 (B6) and DBA/2 (D2) mice. The modifying effect of two types of MAb to a 3-methylcholanthrene-induced cyt. P-450 and a phenobarbital-induced cyt. P-450 was investigated for: (a) S9-mediated mutagenicity of aflatoxin B1, benzo(a)pyrene 7,8-dihydrodiol, 2-acetylaminofluorene, and N-nitrosomorpholine in Salmonella typhimurium strains; and (b) the activity of aryl hydrocarbon hydroxylase, ethoxycoumarin O-deethylase, ethoxyresorufin O-deethylase, aminopyrine N-demethylase, and testosterone 6 beta-, 7 alpha-, and 16 beta-hydroxylases. With certain S9s, MAb-1-7-1 inhibited only those cytochrome P-450 isozymes involved predominantly in activity of aryl hydrocarbon hydroxylase, ethoxyresorufin O-deethylase, and ethoxycoumarin O-deethylase and mutagenicity of 2-acetylaminofluorene and benzo(a)pyrene 7,8-dihydrodiol; MAb 2-66-3 inhibited only those involved in aminopyrine N-demethylase and testosterone 6 beta-, 7 alpha, and 16 beta-hydroxylase activity and aflatoxin B1 mutagenicity. Both Mab 1-7-1 and MAb 2-66-3 inhibited cytochrome P-450 isozyme(s) implicated predominantly in testosterone 7 alpha-hydroxylation in S9 from pregnenolone 16 alpha-carbonitrile-treated B6 mice. MAb 1-7-1 did not inhibit N-nitrosomorpholine mutagenicity and MAb 2-66-3 increased it by 2- to 6-fold depending on the source of S9. Using these MAbs, it is thus possible to identify the contribution of the epitope-defined single or class of cyt. P-450 to specific metabolic reactions in S9 from untreated and inducer-treated mice.

  7. 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

  8. Microsomal cytochrome P450 as a target for drug discovery and repurposing.

    PubMed

    El-Sherbeni, Ahmed A; El-Kadi, Ayman O S

    2017-02-01

    Cytochrome P450 (P450) enzymes are ancient electron-transfer-chain system of remarkable biological importance. Microsomal P450 enzymes are the P450 attached to endoplasmic reticulum, which, in humans, are critical for body's defenses against xenobiotics by mediating their metabolism, and cell signaling by mediating arachidonic acid (AA) transformation to several potent bioactive molecules. Only recently, modulating P450-mediated AA metabolism has risen as a promising new drug target. This review presents the therapeutic potential of finding effective, selective and safe treatments targeting P450-mediated AA metabolism, and the several approaches that have been used to find these treatments; among which, our focus was on modulators of P450 activities. We detailed the efforts done to develop new molecular entities designed to modulate P450, and the more recent efforts tried to employ our previous knowledge on drug metabolism to repurpose old drugs with the capacity to alter P450-mediated drug metabolism to target AA metabolism. Because of the long recognition of P450 role in xenobiotic metabolism, several clinically approved agents were identified to alter P450 activity. Repurposing old drugs as P450 modulators can facilitate bringing treatments targeting P450-mediated AA metabolism to clinical trials. However, the capacity of the modulation of P450-derived AA metabolites of clinically approved drugs has to be systematically investigated and validated for their new use in humans.

  9. Impact of six fruits--banana, guava, mangosteen, pineapple, ripe mango and ripe papaya--on murine hepatic cytochrome P450 activities.

    PubMed

    Chatuphonprasert, Waranya; Jarukamjorn, Kanokwan

    2012-12-01

    The effects of six Thai fruits, namely banana, guava, mangosteen, pineapple, ripe mango and ripe papaya, on cytochrome P450 (P450) activities were investigated. The median inhibitory concentrations (IC(50) ) of each of the fruit juices on CYP1A1, CYP1A2, CYP2E1 and CYP3A11 activities were determined. Pineapple juice showed the strongest inhibitory effect against all the evaluated P450 isozyme activities in mouse hepatic microsomes, followed by mangosteen, guava, ripe mango, ripe papaya and banana. The study was further performed in male ICR mice given pineapple juice intragastrically at doses of 10, 20 and 40 mg kg(-1) per day for 7 or 28 days. In a concentration-dependent fashion, the pineapple juice raised ethoxyresorufin O-deethylase, aniline hydroxylase and erythromycin N-demethylase activities, which are marker enzymatic reactions responsible for CYP1A1, CYP2E1 and CYP3A11, respectively. The effect of pineapple juice on the expression of CYP1A1, CYP2E1 and CYP3A11 mRNAs corresponded to their enzymatic activities. However, the pineapple juice significantly decreased methoxyresorufin O-demethylase activity. These observations supported that the six Thai fruits were a feasible cause of food-drug interaction or adverse drug effects owing to their potential to modify several essential P450 activities. Individuals consuming large quantities of pineapple for long periods of time should be cautioned of these potential adverse effects.

  10. Identification of camphor oxidation and reduction products in Pseudomonas putida: new activity of the cytochrome P450cam system.

    PubMed

    Prasad, Brinda; Rojubally, Adina; Plettner, Erika

    2011-06-01

    P450 enzymes are known for catalyzing hydroxylation reactions of non-activated C-H bonds. For example, P450(cam) from Pseudomonas putida oxidizes (1R)-(+)-camphor to 5-exo-hydroxy camphor and further to 5-ketocamphor. This hydroxylation reaction proceeds via a catalytic cycle in which the reduction of dioxygen (O(2)) is coupled to the oxidation of the substrate. We have observed that under conditions of low oxygen, P. putida and isolated P450(cam) reduce camphor to borneol. We characterized the formation of borneol under conditions of low oxygen or when the catalytic cycle is shunted by artificial oxidants like m-chloro perbenzoic acid, cumene hydroperoxide, etc. We also tested the toxicity of camphor and borneol with P. putida and Escherichia coli. We have found that in P. putida borneol is less toxic than camphor, whereas in E. coli borneol is more toxic than camphor. We discuss a potental ecological advantage of the camphor reduction reaction for P. putida.

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

    ClinicalTrials.gov

    2016-10-24

    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

  12. Xenobiotic biotransformation in unicellular green algae. Involvement of cytochrome P450 in the activation and selectivity of the pyridazinone pro-herbicide metflurazon.

    PubMed Central

    Thies, F; Backhaus, T; Bossmann, B; Grimme, L H

    1996-01-01

    The N-demethylation of the pyridazinone pro-herbicide metflurazon into norflurazon implies a toxification in photosynthetic organisms. This is confirmed by quantitative structure activity relationships determined for two unicellular green algae, Chlorella sorokiniana and Chlorella fusca; however, the latter is 25 to 80 times more sensitive to metflurazon. This sensitivity is linked to differences in the N-demethylase activity of both algae, as determined by an optimized in vivo biotransformation assay. Apparent K(m) values of the metflurazon-N-demethylase indicate a 10-fold higher affinity for this xenobiotic substrate for Chlorella fusca. Furthermore, algal metflurazon-N-demethylation is characterized by distinct variations in activity, depending on the stage of cell development within the cell cycle. Several well-established inhibitors of cytochrome P450-mediated reactions, including piperonylbutoxide, 1-aminobenzotriazole, 1-phenoxy-3-(1H-1,2,4-triol-1yl)-4-hydroxy-5,5-dimethylhexane++ +, and tetcyclacis, as well as cinnamic acid, a potential endogenous substrate, inhibited the N-demethylation of metflurazon. The results suggest that the N-demethylation of metflurazon by both algae is mediated by a cytochrome P450 monooxygenase. The determination of antigenic cross-reactivity of algal proteins with heterologous polyclonal antibodies originally raised against plant P450s, anti-cinnamic acid 4-hydroxylase (CYP73A1), anti-ethoxycoumarin-O-dealkylase, anti-tulip allene oxidase (CYP74), and an avocado P450 (CYP71A1) or those of bacterial origin, CYP105A1 and CYP105B1, suggests the presence of distinct P450 isoforms in both algae. PMID:8819332

  13. Inactivation of cytochrome P450 (P450) 3A4 but not P450 3A5 by OSI-930, a thiophene-containing anticancer drug.

    PubMed

    Lin, Hsia-lien; Zhang, Haoming; Medower, Christine; Hollenberg, Paul F; Johnson, William W

    2011-02-01

    An investigational anticancer agent that contains a thiophene moiety, 3-[(quinolin-4-ylmethyl)-amino]-N-[4-trifluoromethox)phenyl] thiophene-2-carboxamide (OSI-930), was tested to investigate its ability to modulate the activities of several cytochrome P450 enzymes. Results showed that OSI-930 inactivated purified, recombinant cytochrome P450 (P450) 3A4 in the reconstituted system in a mechanism-based manner. The inactivation was dependent on cytochrome b(5) and required NADPH. Catalase did not protect against the inactivation. No inactivation was observed in studies with human 2B6, 2D6, or 3A5 either in the presence or in the absence of b(5). The inactivation of 3A4 by OSI-930 was time- and concentration-dependent. The inactivation of the 7-benzyloxy-4-(trifluoromethyl)coumarin catalytic activity of 3A4 was characterized by a K(I) of 24 μM and a k(inact) of 0.04 min(-1). This K(I) is significantly greater than the clinical OSI-930 C(max) of 1.7 μM at the maximum tolerated dose, indicating that clinical drug interactions of OSI-930 via this pathway are not likely. Spectral analysis of the inactivated protein indicated that the decrease in the reduced CO spectrum at 450 nm was comparable to the amount of inactivation, thereby suggesting that the inactivation was primarily due to modification of the heme. High-pressure liquid chromatography (HPLC) analysis with detection at 400 nm showed a loss of heme comparable to the activity loss, but a modified heme was not detected. This result suggests either that the heme must have been modified enough so as not to be observed in a HPLC chromatograph or, possibly, that it was destroyed. The partition ratio for the inactivation of P450 3A4 was approximately 23, suggesting that this P450 3A4-mediated pathway occurs with approximately 4% frequency during the metabolism of OSI-930. Modeling studies on the binding of OSI-930 to the active site of the P450 3A4 indicated that OSI-930 would be oriented properly in the active site

  14. Mechanism-based inactivation of cytochrome P-450 dependent benzo(a)pyrene hydroxylase activity by acetylenic and olefinic polycyclic arylhydrocarbons

    SciTech Connect

    Gan, L.S.

    1986-01-01

    A series of aryl acetylenes and aryl olefins have been examined as substrates and inhibitors of cytochrome P-450 dependent monooxygenases in liver microsomes from 5,6-benzoflavone or phenobarbital pretreated rats. 1-Ethynylpyrene (EP), 3-ethynylperylene (EPL), cis- and trans-1-(2-bromo-vinyl)pyrene (c-BVP and t-BVP), and 1-allylpyrene (AP) serve as mechanism-based irreversible inactivators (suicide inhibitors) of benzo(a)pyrene (BP) hydroxylase, while 1-vinyl-pyrene (VP) and phenyl 1-pyrenyl acetylene (PPA) do not cause a detectable suicide inhibition of the BP hydroxylase. The mechanism-based loss of BP hydroxylase activity caused by the aryl acetylenes is not accompanied by a corresponding loss of the P-450 content of the microsomes. In the presence of NADPH, /sup 3/H-labeled EP covalently attached to P-450 isozymes with a measured stoichiometry of one mole of EP per mole of the P-450 heme. The results of the effects of these aryl derivatives in the mammalian cell-mediated mutagenesis assay and toxicity assay show that none of the compounds examined nor any of the their metabolites produced in the incubation system are cytotoxic to V79 cells.

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

    PubMed Central

    Oinonen, T; Lindros, K O

    1998-01-01

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

  16. 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...

  17. ROS-sensitive cytochrome P450 activity maintains endothelial dilatation in ageing but is transitory in dyslipidaemic mice

    PubMed Central

    Krummen, Stéphane; Drouin, Annick; Gendron, Marie-Ève; Falck, John R; Thorin, Eric

    2006-01-01

    Risk factors for cardiovascular diseases (CVD) have been proposed to accelerate the vascular endothelial dysfunction that develops during the normal ageing process. The objective of this work was to study the impact of dyslipidaemia (DL) on the dilatory efficacy of the non-NO/non-PGI2 endothelium-derived hyperpolarising factor (EDHF) through maturation and ageing. We isolated and pressurised (80 mmHg) gracilis arterial segments from 3, 12 and 20-month-old (m/o) DL mice expressing the human apolipoprotein B-100 and wild-type (WT) C57BL/6 mice. EDHF-dependent dilatations to acetylcholine (ACh) were measured in the presence of L-NNA (100 μM, NOS inhibitor) and indomethacin (INDO; 10 μM, COX inhibitor). Data are expressed as mean±s.e.m. EDHF-mediated maximal dilatation of arteries isolated from WT mice declined by 44% with ageing, from 86±3% at 3 months to 66±8% at 12 and 48±4% at 20 months of age (P<0.05). This decline was magnified by DL to 73%, characterised by an early increased efficacy at 3 m/o (95±2%, P<0.05) and a worsening of the dysfunction at 20 m/o (26±2%, P<0.05). 17-Octadecynoic acid (17-ODYA), a cytochrome P450/epoxygenase inhibitor, reduced by 56% (P<0.05) ACh-induced EDHF-dependent dilatation of arteries isolated from 3 m/o DL – but not WT – mice, an effect of 17-ODYA disappearing in older DL mice. 17-ODYA, however, reduced (P<0.05) ACh-induced EDHF-dependent dilatation in arteries isolated from 12 m/o WT mice by 35% and from 20 m/o WT mice by 31% (P<0.05). Reactive oxygen species production was increased in arteries isolated from 12 m/o DL mice. The antioxidant N-acetyl-L-cystein (NAC) restored the 17-ODYA-sensitive responses in arteries isolated from 12 – but not 20 – m/o DL mice (84±3% from an Emax of 57±8%; P<0.05). NAC did not affect the dilatation of arteries isolated from WT mice. Our data suggest that the decline in EDHF-dependent dilatation is hastened by DL despite the early expression of a 17-ODYA-sensitive pathway

  18. Homotropic cooperativity of monomeric cytochrome P450 3A4

    SciTech Connect

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

    2010-11-16

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

  19. Special issue: Cytochrome P450 structure and function: introduction.

    PubMed

    Munro, Andrew W; Leys, David

    2012-05-01

    The 17th International Conference on Cytochrome P450 Biochemistry, Biophysics and Structure was held in Manchester, UK from 26-30 June 2011. This issue of FEBS J. contains review and primary research articles reflecting the breadth of science covered at this conference, and reflecting the impact of P450-related research in fields as diverse as steroid metabolism, plant biochemistry, structural biology and biotechnology.

  20. Cytochrome P450 structure, function and clinical significance: A review.

    PubMed

    Palrasu, Manikandan; Nagini, Siddavaram

    2017-01-25

    The cytochrome P450 (CYP) enzymes are membrane-bound hemoproteins that play a pivotal role in the detoxification of xenobiotics, cellular metabolism and homeostasis. Induction or inhibition of CYP enzymes is a major mechanism that underlies drug-drug interactions. CYP enzymes can be transcriptionally activated by various xenobiotics and endogenous substrates through receptor-dependent mechanisms. CYP enzyme inhibition is a principal mechanism for metabolism-based drug-drug interactions. Many chemotherapeutic drugs can cause drug interactions due to their ability to either inhibit or induce the CYP enzyme system. Predictions based on in silico analyses followed by validation have identified several microRNAs that regulate CYPs. Genetic polymorphisms and epigenetic changes in CYP genes may be responsible for inter-individual and inter-ethnic variations in disease susceptibility and the therapeutic efficacy of drugs. Knowledge about the substrates, inducers, inhibitors of CYP isoforms, and the polymorphisms of CYP enzymes may be used as an aid by clinicians to determine therapeutic strategy, and treatment doses for drugs that are metabolized by CYP gene products. The present review is a comprehensive compilation of cytochrome P450 structure, function, pharmacogenetics, and pharmacoepigenetics and clinical significance.

  1. Induction of rat hepatic cytochromes P450 by toxic ingredients in plants: lack of correlation between toxicity and inductive activity.

    PubMed

    Yamada, H; Nakamura, T; Oguri, K

    1998-12-01

    "Animal-Plant Warfare" is one of the hypotheses for the evolution of drug-metabolizing P450s. To address the validity of this hypothesis, we examined the induction of xenobiotic-metabolizing P450s by 12 plant toxins in rats, using hepatic activity for testosterone metabolism as the index. The compounds tested were aconitine, morphine, tubocurarine, physostigmine, pilocarpine, muscarine, cocaine, atropine, amygdalin, digitonin, nicotine and solanine. Drinking water containing a test compound was given to rats for 4 days, and the hepatic activity of testosterone metabolism was determined together with monitoring body weight gain and liver weight as the indices of toxicity. The results showed that while cocaine and nicotine have a minor ability to increase testosterone 16 beta-hydroxylase activity, a marker activity for the CYP2B1 and 2, all other compounds did not have any such effect. No correlation was observed between a change in 16 beta-hydroxylase and toxicity caused by toxins. Therefore, these results did not support the idea that the inducibility of the CYP2B subfamily in animals is acquired through "Animal-Plant Warfare". Several compounds examined here increased or decreased hepatic activities of testosterone 2 alpha-, 6 beta-, 7 alpha- and 16 alpha-hydroxylation and 17-oxidation, indicating a possible effect on the CYP2A, 2C and 3A subfamily. Of these effects, a moderate correlation (r < 0.49) was observed in the changes in the activities of 2 alpha-/16 alpha-hydroxylation and 17-oxidation vs. that in toxicity. It is therefore suggested that inhibition or suppression of the expression of CYP2C11 is one of the mechanisms in the toxicity of plant toxins for rats, although it comes from an examination using limited numbers of compounds.

  2. Hyper- and Hypo- Induction of Cytochrome P450 activities with Aroclor 1254 and 3-Methylcholanthrene in Cyp1a2(−/−) mice

    PubMed Central

    Barker, Melissa L.; Hathaway, Laura B.; Arch, Dorinda D.; Westbroek, Mark L.; Kushner, James P.; Phillips, John D.; Franklin, Michael R.

    2009-01-01

    The response of hepatic mono-oxygenase activities to Aroclor 1254 or 3-methylcholanthrene was investigated in wild-type and Cyp1a2(−/−) mice. Cytochrome P450 concentrations were similar in naïve Cyp1a2(−/−) and wild-type mice. There was no difference between naïve wild-type and Cyp1a2(−/−) animals in 7-ethoxyresorufin and 7-ethoxy-4-trifluoromethylcoumarin dealkylase activities, nor was the induction response after 3-methylcholanthrene any different between the two genotypes. However, both activities were induced to a higher extent in Cyp1a2(−/−) mice after Aroclor 1254. In contrast, 7-pentoxyresorufin dealkylation activity was lower in Cyp1a2(−/−) mice and this differential was maintained during induction by both agents. 7-Methoxy- and 7-benzoxyresorufin dealkylation activities were also lower than wild-type in naïve Cyp1a2(−/−) animals and during 3-methylcholanthrene induction, but showed accelerated induction in Cyp1a2(−/−) mice with Aroclor 1254. Bufuralol 1′- and testosterone 6β-hydroxylation activities, and P450 characteristics were evaluated 48 hours after inducer administration. Bufuralol 1′-hydroxylation, a sexual dimorphic activity (female > male) showed no genotype differences in naïve animals. Activity changes varied across gender and genotype, with 3-methylcholanthrene and Aroclor 1254 inducing in male Cyp1a2(−/−), and Aroclor 1254 inducing in female wild-type. Testosterone 6β-hydroxylation activity was 16% higher in Cyp1a2(−/−) mice and neither 3-methylcholanthrene nor Aroclor 1254 elicited induction. After Aroclor 1254, a 24% increase in P450 concentration with a hypsochromic shift in the ferrous-CO maximum characteristic of CYP1A enzymes occurred in wild-type, compared to no change in either parameter in Cyp1a2(−/−) mice. Induction changes with 3-methylcholanthrene were greater in wild-type mice, a 60% increase in concentration and ~2 nm hypsochromic shift versus a 10% increase and ~1 nm hypsochromic

  3. 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...

  4. Examination of microsomal cytochrome P450-catalyzed in vitro activation of o-phenylphenol to DNA binding metabolite(s) by 32P-postlabeling technique.

    PubMed

    Pathak, D N; Roy, D

    1992-09-01

    It has been previously reported that the reactive metabolites phenylsemiquinone and phenylbenzoquinone are generated during microsomal cytochrome P450-catalyzed redox cycling of o-phenylphenol (OPP). However, covalent modification of DNA by OPP-reactive metabolites has yet not been demonstrated. In the present study we have investigated the covalent binding in DNA by OPP-reactive metabolites using 32P-postlabeling. Analysis of adducts by 32P-postlabeling in products of chemical reaction of DNA with phenylbenzoquinone revealed four major and several minor adducts. The chemical reaction of deoxyguanosine 3'-phosphate with phenylbenzoquinone also showed four major adducts. The chromatographic mobility of major adducts of deoxyguanosine 3'-phosphate-phenylbenzoquinone was identical to that of major adducts of DNA-phenylbenzoquinone. The major adducts are demonstrated to be stable. The total covalent binding in deoxyguanosine 3'-phosphate by phenylbenzoquinone (686,000-687,000 amol/nmol nucleotide) was higher than that observed in DNA (26,500-28,000 amol/nmol nucleotides). Reaction of DNA with OPP or a hydroxylated metabolite of OPP, phenylhydroquinone, in the presence of microsomes and NADPH or cumene hydroperoxide showed four major adducts. Adduct formation in DNA by OPP or phenylhydroquinone in the presence of the microsomal activation system was drastically decreased by known inhibitors of cytochrome P450. The chromatographic mobility of major adducts in DNA by OPP or phenylhydroquinone in the presence of microsomal activation system matched with those major adducts observed in deoxyguanosine 3'-phosphate or DNA reacted with pure phenylbenzoquinone. These data demonstrate that OPP or phenylhydroquinone, a hydroxylated metabolite of OPP, is able to bind covalently to DNA in the presence of a microsomal cytochrome P450 activation system. Phenylbenzoquinone is one of the DNA-binding metabolite(s) of OPP. It is concluded that OPP is genotoxic in an in vitro system and

  5. Cytochrome P450 1B1 gene polymorphisms as predictors of anticancer drug activity: studies with in vitro models.

    PubMed

    Laroche-Clary, Audrey; Le Morvan, Valérie; Yamori, Takao; Robert, Jacques

    2010-12-01

    Cytochrome P450 1B1 (CYP1B1) is found in tumor tissue and is suspected to play a role in oncogenesis and drug resistance. CYP1B1 gene polymorphisms have been associated with the risk of developing lung and other cancers. They may be associated with tumor response to anticancer drugs. We have determined 4 frequent nonsynonymous gene polymorphisms of CYP1B1 in the human tumor cell lines panels of the National Cancer Institute (NCI) and the Japanese Foundation for Cancer Research (JFCR): rs10012 (R48G), rs1056827 (A119S), rs1056836 (L432V), and rs1800440 (N453S). Numerous anticancer drugs have been tested against these panels that offer the opportunity to detect associations between gene polymorphisms and drug sensitivity. CYP1B1 single nucleotide polymorphisms were in marked linkage disequilibrium. The L432V allelic variants were significantly associated with reduced sensitivity to DNA-interacting anticancer agents, alkylators, camptothecins, topoisomerase II inhibitors, and some antimetabolites. For instance, in the NCI panel, cell lines homozygous for the V432 allele were globally 2-fold resistant to alkylating agents (P = 5 × 10(-10)) and 4.5-fold to camptothecins (P = 6.6 × 10(-9)) than cell lines homozygous for the L432 allele. Similar features were exhibited by the JFCR panel. Cell lines homozygous for the V432 allele were globally less sensitive to DNA-interfering drugs than cell lines having at least 1 common allele. There was no significant association between mRNA expression of CYP1B1 and CYP1B1 genotype, and no significant association between CYP1B1 mRNA expression and drug cytotoxicity. These observations open the way to clinical studies exploring the role of CYP1B1 gene polymorphisms for predicting tumor sensitivity to chemotherapy.

  6. Spectroscopic quantitation of cytochrome P-450 in human lung microsomes.

    PubMed

    Wheeler, C W; Guenthner, T M

    1990-01-01

    The cytochrome P-450 content of human lung microsomes was measured by difference spectroscopy of the carbon monoxide-complexed hemoprotein. These measurements were only possible after the microsome preparation had been subjected to centrifugation over a discontinuous sucrose gradient, to remove an opaque black contaminant. The specific concentration of total cytochrome P-450 in human lung microsomes is essentially identical to that of microsomes prepared under identical conditions from untreated baboon lungs, but is only 0.7% of the specific content found in lung microsomes from untreated rabbits. These measurements correspond well to the observed metabolic capacities of the various microsome samples.

  7. Amino-steroids as inhibitors and probes of the active site of cytochrome P-450scc. Effects on the enzyme from different sources.

    PubMed

    Kellis, J T; Sheets, J J; Vickery, L E

    1984-02-01

    A series of analogues of cholesterol, each having a primary amine attached to a shortened side chain, were tested for their effects on cytochrome P-450scc from several different sources. Reconstituted enzyme systems using disrupted mitochondria from bovine adrenal and placenta, adult human adrenal and placenta, neonatal human adrenal, and rat adrenal and testis were used to assay for inhibitory effects on the side chain cleavage of cholesterol to pregnenolone. Two of the derivatives tested, 22-amino-23,24-bisnor-5-cholen-3 beta-ol and 23-amino-24-nor-5-cholen-3 beta-ol, were found to be potent inhibitors of this reaction; the derivatives in which the amine was attached closer to or further from the steroid ring, (20 R and S)-20-amino-5-pregnen-3 beta-ol and 24-amino-5-cholen-3 beta-ol, were much weaker inhibitors. In addition, spectral studies with rat adrenal mitochondria and a soluble preparation of human placental cytochrome P-450scc showed that binding of the 22-amine derivative to the enzyme produces difference spectra characteristic of nitrogen bonding to the heme; this indicates that the heme is positioned close to C-22 in the steroid-enzyme complex. These findings on the relative effectiveness of the amino-steroid inhibitors and the type of complex formed are similar to results obtained with purified bovine adrenocortical cytochrome P-450scc. This establishes that the proximity of the substrate binding site and the heme-iron catalytic site is a feature common to the enzyme from several sources and is therefore likely to be a necessary property of the active site structure.

  8. Enzyme kinetics of oxidative metabolism: cytochromes P450.

    PubMed

    Korzekwa, Ken

    2014-01-01

    The cytochrome P450 enzymes (CYPs) are the most important enzymes in the oxidative metabolism of hydrophobic drugs and other foreign compounds (xenobiotics). The versatility of these enzymes results in some unusual kinetic properties, stemming from the simultaneous interaction of multiple substrates with the CYP active site. Often, the CYPs display kinetics that deviate from standard hyperbolic saturation or inhibition kinetics. Non-Michaelis-Menten or "atypical" saturation kinetics include sigmoidal, biphasic, and substrate inhibition kinetics (see Chapter 3 ). Interactions between substrates include competitive inhibition, noncompetitive inhibition, mixed inhibition, partial inhibition, activation, and activation followed by inhibition (see Chapter 4 ). Models and equations that can result in these kinetic profiles will be presented and discussed.

  9. Monooxygenation of small hydrocarbons catalyzed by bacterial cytochrome p450s.

    PubMed

    Shoji, Osami; Watanabe, Yoshihito

    2015-01-01

    Cytochrome P450s (P450s) catalyze the NAD(P)H/O2-dependent monooxygenation of less reactive organic molecules under mild conditions. The catalytic activity of bacterial P450s is very high compared with P450s isolated from animals and plants, and the substrate specificity of bacterial P450s is also very high. Accordingly, their catalytic activities toward nonnative substrates are generally low especially toward small hydrocarbons. However, mutagenesis approaches have been very successful for engineering bacterial P450s for the hydroxylation of small hydrocarbons. On the other hand, "decoy" molecules, whose structures are very similar to natural substrates, can be used to trick the substrate recognition of bacterial P450s, allowing the P450s to catalyze oxidation reactions of nonnative substrates without any substitution of amino acid residues in the presence of decoy molecules. Thus, the hydroxylation of small hydrocarbons such as ethane, propane, butane and benzene can be catalyzed by P450BM3, a long-alkyl-chain hydroxylase, using substrate misrecognition of P450s induced by decoy molecules. Furthermore, a number of H2O2-dependent bacterial P450s can catalyze the peroxygenation of a variety of nonnative substrates through a simple substrate-misrecognition trick, in which catalytic activities and enantioselectivity are dependent on the structure of decoy molecules.

  10. Prevention of LDL-suppression of HMG-CoA reductase (HMGR) activity by progesterone (PG): evidence for cytochrome P-450 involvement

    SciTech Connect

    Sexton, R.C.; Gupta, A.; Panini, S.R.; Rudney, H.

    1987-05-01

    Incubation of rat intestinal epithelial cells (IEC-6) with PG has been reported by us to prevent the suppression of HMGR activity by LDL. In the present study, addition of LDL and PG to IEC-6 cells resulted in a 2 fold increase in cellular free cholesterol (CH) in 24 h, while HMGR activity remained elevated. PG did not affect the internalization and degradation of (/sup 125/I) LDL nor the accumulation of free (/sup 3/H) CH in cells incubated with (/sup 3/H-cholesteryl linoleate)-LDL. Also, PG did not affect the intracellular transport of LDL-derived (/sup 3/H) CH to the plasma membrane nor the efflux of the (/sup 3/H) CH into medium containing human high density lipoprotein. Addition of LDL to cells, in which the cellular CH was radiolabeled from (/sup 3/H) acetate, resulted in an increased formation of radiolabeled oxysterols, detected by HPLC, and a corresponding decrease in HMGR activity. PG attenuated both the LDL-induced formation of oxysterols and suppression of HMGR activity. PG inhibited cytochrome P-450 dependent oxidation of benzphetamine, aminopyrine and aniline by liver microsomes from phenobarbitol treated rats. These results suggest PG may prevent LDL suppression of HMGR activity in IEC-6 cells by inhibiting cytochrome P-450 dependent formation of regulatory oxysterols.

  11. The role of active-site Phe87 in modulating the organic co-solvent tolerance of cytochrome P450 BM3 monooxygenase

    PubMed Central

    Kuper, Jochen; Tee, Kang Lan; Wilmanns, Matthias; Roccatano, Danilo; Schwaneberg, Ulrich; Wong, Tuck Seng

    2012-01-01

    Understanding the effects of organic co-solvents on protein structure and function is pivotal to engineering enzymes for biotransformation in non-aqueous solvents. The effects of DMSO on the catalytic activity of cytochrome P450 BM3 have previously been investigated and the importance of Phe87 in its organic co-solvent tolerance was identified. To probe the DMSO inactivation mechanism and the functional role of Phe87 in modulating the organic co-solvent tolerance of P450 BM3, the haem domain (Thr1–Leu455) of the F87A variant was cocrystallized in the presence of 14%(v/v) and 28%(v/v) DMSO. At both DMSO concentrations the protein retained the canonical structure of the P450 haem domain without any sign of partial or global unfolding. Interestingly, a DMSO molecule was found in the active site of both structures, with its O atom pointing towards the haem iron. The orientation of the DMSO molecule indicated a dynamic coordination process that was in competition with the active-site water molecule. The ability of the DMSO molecule to coordinate the haem iron is plausibly the main reason why P450 BM3 is inactivated at elevated DMSO concentrations. The data allowed an interesting comparison with the wild-type structures reported previously. A DMSO molecule was found when the wild-type protein was placed in 28%(v/v) DMSO, in which the DMSO molecule coordinated the haem iron directly via its S atom. Intriguingly, no DMSO molecule was observed at 14%(v/v) DMSO for the wild-type structure. These results suggested that the bulky phenyl side chain of Phe87 protects the haem from being accessed by the DMSO molecule and explains the higher tolerance of the wild-type enzyme towards organic co-solvents compared with its F87A variant. PMID:22949185

  12. Defective Cytochrome P450-Catalysed Drug Metabolism in Niemann-Pick Type C Disease

    PubMed Central

    Wassif, Christopher A.; Gray, James; Burkert, Kathryn R.; Smith, David A.; Morris, Lauren; Cologna, Stephanie M.; Peer, Cody J.; Sissung, Tristan M.; Uscatu, Constantin-Daniel; Figg, William D.; Pavan, William J.; Vite, Charles H.; Porter, Forbes D.; Platt, Frances M.

    2016-01-01

    Niemann-Pick type C (NPC) disease is a neurodegenerative lysosomal storage disease caused by mutations in either the NPC1 or NPC2 gene. NPC is characterised by storage of multiple lipids in the late endosomal/lysosomal compartment, resulting in cellular and organ system dysfunction. The underlying molecular mechanisms that lead to the range of clinical presentations in NPC are not fully understood. While evaluating potential small molecule therapies in Npc1-/- mice, we observed a consistent pattern of toxicity associated with drugs metabolised by the cytochrome P450 system, suggesting a potential drug metabolism defect in NPC1 disease. Investigation of the P450 system in the context of NPC1 dysfunction revealed significant changes in the gene expression of many P450 associated genes across the full lifespan of Npc1-/- mice, decreased activity of cytochrome P450 reductase, and a global decrease of multiple cytochrome P450 catalysed dealkylation reactions. In vivo drug metabolism studies using a prototypic P450 metabolised drug, midazolam, confirmed dysfunction in drug clearance in the Npc1-/- mouse. Expression of the Phase II enzyme uridinediphosphate-glucuronosyltransferase (UGT) was also significantly reduced in Npc1-/- mice. Interestingly, reduced activity within the P450 system was also observed in heterozygous Npc1+/- mice. The reduced activity of P450 enzymes may be the result of bile acid deficiency/imbalance in Npc1-/- mice, as bile acid treatment significantly rescued P450 enzyme activity in Npc1-/- mice and has the potential to be an adjunctive therapy for NPC disease patients. The dysfunction in the cytochrome P450 system were recapitulated in the NPC1 feline model. Additionally, we present the first evidence that there are alterations in the P450 system in NPC1 patients. PMID:27019000

  13. Cholesterol Ester Oxidation by Mycobacterial Cytochrome P450*

    PubMed Central

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

    2014-01-01

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

  14. Application of nanodisc technology for direct electrochemical investigation of plant cytochrome P450s and their NADPH P450 oxidoreductase

    PubMed Central

    Bavishi, Krutika; Laursen, Tomas; Martinez, Karen L.; Møller, Birger Lindberg; Della Pia, Eduardo Antonio

    2016-01-01

    Direct electrochemistry of cytochrome P450 containing systems has primarily focused on investigating enzymes from microbes and animals for bio-sensing applications. Plant P450s receive electrons from NADPH P450 oxidoreductase (POR) to orchestrate the bio-synthesis of a plethora of commercially valuable compounds. In this report, full length CYP79A1, CYP71E1 and POR of the dhurrin pathway in Sorghum bicolor were reconstituted individually in nanoscale lipid patches, “nanodiscs”, and directly immobilized on unmodified gold electrodes. Cyclic voltammograms of CYP79A1 and CYP71E1 revealed reversible redox peaks with average midpoint potentials of 80 ± 5 mV and 72 ± 5 mV vs. Ag/AgCl, respectively. POR yielded two pairs of redox peaks with midpoint potentials of 90 ± 5 mV and −300 ± 10 mV, respectively. The average heterogeneous electron transfer rate constant was calculated to be ~1.5 s−1. POR was electro-catalytically active while the P450s generated hydrogen peroxide (H2O2). These nanodisc-based investigations lay the prospects and guidelines for construction of a simplified platform to perform mediator-free, direct electrochemistry of non-engineered cytochromes P450 under native-like conditions. It is also a prelude for driving plant P450 systems electronically for simplified and cost-effective screening of potential substrates/inhibitors and fabrication of nano-bioreactors for synthesis of high value natural products. PMID:27386958

  15. tert-Butylphenylacetylene Is a Potent Mechanism-Based Inactivator of Cytochrome P450 2B4: Inhibition of Cytochrome P450 Catalysis by Steric Hindrance

    PubMed Central

    Zhang, Haoming; Lin, Hsia-lien; Walker, Vyvyca J.; Hamdane, Djemel

    2009-01-01

    We have demonstrated that 4-(tert-butyl)-phenylacetylene (tBPA) is a potent mechanism-based inactivator for cytochrome P450 2B4 (P450 2B4) in the reconstituted system. It inactivates P450 2B4 in a NADPH- and time-dependent manner with a KI of 0.44 μM and kinact of 0.12 min−1. The partition ratio was approximately zero, indicating that inactivation occurs without the reactive intermediate leaving the active site. Liquid chromatography-mass spectrometry analyses revealed that tBPA forms a protein adduct with a 1:1 stoichiometry. Peptide mapping of the tBPA-modified protein provides evidence that tBPA is covalently bound to Thr302. This is consistent with results of molecular modeling that show the terminal carbon of the acetylenic group is only 3.65 Å away from Thr302. To characterize the effect of covalent modification of Thr302, tBPA-modified P450 2B4 was purified to homogeneity from the reconstituted system. The Soret band of tBPA-modified protein is red-shifted by 5 to 422 nm compared with unmodified protein. Benzphetamine binding to the modified P450 2B4 causes no spin shift, indicating that substrate binding and/or the heme environment has been altered by covalently bound tBPA. Cytochrome P450 reductase reduces the unmodified and tBPA-modified P450s at approximately the same rate. However, addition of benzphetamine stimulates the rate of reduction of unmodified P450 2B4 by ∼20-fold but only marginally stimulates reduction of the tBPA-modified protein. This large discrepancy in the stimulation of the first electron transfer by benzphetamine strongly suggests that the impairment of P450 catalysis is due to inhibition of benzphetamine binding to the tBPA-modified P450 2B4. PMID:19720728

  16. Inhibitory effects of curcumin on activity of cytochrome P450 2C9 enzyme in human and 2C11 in rat liver microsomes.

    PubMed

    Wang, Zhe; Sun, Wei; Huang, Cheng-Ke; Wang, Li; Xia, Meng-Ming; Cui, Xiao; Hu, Guo-Xin; Wang, Zeng-Shou

    2015-04-01

    Cytochrome P450 2C9 (CYP2C9), one of the most important phase I drug metabolizing enzymes, could catalyze the reactions that convert diclofenanc into diclofenac 4'-hydroxylation. Evaluation of the inhibitory effects of compounds on CYP2C9 is clinically important because inhibition of CYP2C9 could result in serious drug-drug interactions. The objective of this work was to investigate the effects of curcumin on CYP2C9 in human and cytochrome P450 2C11 (CYP2C11) in rat liver microsomes. The results showed that curcumin inhibited CYP2C9 activity (10 µmol L(-1) diclofenac) with half-maximal inhibition or a half-maximal inhibitory concentration (IC50) of 15.25 µmol L(-1) and Ki = 4.473 µmol L(-1) in human liver microsomes. Curcumin's mode of action on CYP2C9 activity was noncompetitive for the substrate diclofenanc and uncompetitive for the cofactor NADPH. In contrast to its potent inhibition of CYP2C9 in human, diclofenanc had lesser effects on CYP2C11 in rat, with an IC50 ≥100 µmol L(-1). The observations imply that curcumin has the inhibitory effects on CYP2C9 activity in human. These in vitro findings suggest that more attention should be paid to special clinical caution when intake of curcumin combined with other drugs in treatment.

  17. Expression of cytochrome P450 genes in CD34+ hematopoietic stem and progenitor cells.

    PubMed

    Soucek, Pavel; Anzenbacher, Pavel; Skoumalová, Ivana; Dvorák, Michal

    2005-10-01

    Expression of major cytochrome P450 forms (P450) was followed in preparation of purified hematopoietic CD34+ stem and progenitor cells. Levels of transcripts as well as mature proteins were traced by quantitative real-time polymerase chain reaction and by Northern and Western blotting. P450 1B1 and P450 2E1 proteins and respective mRNAs were found in all cases. On the other hand, no expression of P450 3A4, P450 3A7, and P450 2C9 was found. The results showed that expression of various P450 enzymes starts at different stages of cell differentiation. Both P450 forms found are known to be connected with cancer cells and with activation of procarcinogens (P450 1B1, polycyclic aromatic hydrocarbons; P450 2E1, nitrosamines, and solvents). Hence, cells at the early stage of differentiation already may be influenced by interaction with xenobiotics. This fact should also be taken into consideration when hematopoietic cell transplant therapy is applied.

  18. Molecular evolutionary dynamics of cytochrome P450 monooxygenases across kingdoms: Special focus on mycobacterial P450s

    PubMed Central

    Parvez, Mohammad; Qhanya, Lehlohonolo Benedict; Mthakathi, Ntsane Trevor; Kgosiemang, Ipeleng Kopano Rosinah; Bamal, Hans Denis; Pagadala, Nataraj Sekhar; Xie, Ting; Yang, Haoran; Chen, Hengye; Theron, Chrispian William; Monyaki, Richie; Raselemane, Seiso Caiphus; Salewe, Vuyani; Mongale, Bogadi Lorato; Matowane, Retshedisitswe Godfrey; Abdalla, Sara Mohamed Hasaan; Booi, Wool Isaac; van Wyk, Mari; Olivier, Dedré; Boucher, Charlotte E.; Nelson, David R.; Tuszynski, Jack A.; Blackburn, Jonathan Michael; Yu, Jae-Hyuk; Mashele, Samson Sitheni; Chen, Wanping; Syed, Khajamohiddin

    2016-01-01

    Since the initial identification of cytochrome P450 monooxygenases (CYPs/P450s), great progress has been made in understanding their structure-function relationship, diversity and application in producing compounds beneficial to humans. However, the molecular evolution of P450s in terms of their dynamics both at protein and DNA levels and functional conservation across kingdoms still needs investigation. In this study, we analyzed 17 598 P450s belonging to 113 P450 families (bacteria −42; fungi −19; plant −28; animal −22; plant and animal −1 and common P450 family −1) and found highly conserved and rapidly evolving P450 families. Results suggested that bacterial P450s, particularly P450s belonging to mycobacteria, are highly conserved both at protein and DNA levels. Mycobacteria possess the highest P450 diversity percentage compared to other microbes and have a high coverage of P450s (≥1%) in their genomes, as found in fungi and plants. Phylogenetic and functional analyses revealed the functional conservation of P450s despite belonging to different biological kingdoms, suggesting the adherence of P450s to their innate function such as their involvement in either generation or oxidation of steroids and structurally related molecules, fatty acids and terpenoids. This study’s results offer new understanding of the dynamic structural nature of P450s. PMID:27616185

  19. Ab initio dynamics of the cytochrome P450 hydroxylation reaction

    PubMed Central

    Elenewski, Justin E.; Hackett, John C

    2015-01-01

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

  20. Ab initio dynamics of the cytochrome P450 hydroxylation reaction

    SciTech Connect

    Elenewski, Justin E.; Hackett, John C

    2015-02-14

    The iron(IV)-oxo porphyrin π-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.

  1. Oxidations of p-alkoxyacylanilides catalyzed by human cytochrome P450 1A2: structure-activity relationships and simulation of rate constants of individual steps in catalysis.

    PubMed

    Yun, C H; Miller, G P; Guengerich, F P

    2001-04-10

    Human cytochrome P450 (P450) 1A2 is involved in the oxidation of many important drugs and carcinogens. The prototype substrate phenacetin is oxidized to an acetol as well as the O-dealkylation product [Yun, C.-H., Miller, G. P., and Guengerich, F. P. (2000) Biochemistry 39, 11319-11329]. In an effort to improve rates of catalysis of P450 1A2 enzymes, we considered a set of p-alkoxyacylanilide analogues of phenacetin and found that variations in the O-alkyl and N-acyl substituents altered the rates of the two oxidation reactions and the ratio of acetol/phenol products. Moving one methylene group of phenacetin from the O-alkyl group to the N-acyl moiety increased rates of both oxidations approximately 5-fold and improved the coupling efficiency (oxidation products formed/NADPH consumed) from 6% to 38%. Noncompetitive kinetic deuterium isotope effects of 2-3 were measured for all O-dealkylation reactions examined with wild-type P450 1A2 and the E225I mutant, which has 6-fold higher activity. A trend of decreasing kinetic deuterium isotope effect for E225I > wild-type > mutant D320A was observed for O-demethylation of p-methoxyacetanilide, which follows the trend for k(cat). The set of O-dealkylation and acetol formation results for wild-type P450 1A2 and the E225I mutant with several of the protiated and deuterated substrates were fit to a model developed for the basic catalytic cycle and a set of microscopic rate constants in which the only variable was the rate of product formation (substrate oxygenation, including hydrogen abstraction). In this model, k(cat) is considerably less than any of the microscopic rate constants and is affected by several individual rate constants, including the rate of formation of the oxygenating species, the rate of substrate oxidation by the oxygenating species, and the rates of generation of reduced oxygen species (H(2)O(2), H(2)O). This analysis of the effects of the individual rate constants provides a framework for consideration of

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

    SciTech Connect

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

    1993-09-01

    Cytochrome P450-associated monooxygenase activities and cytochrome P450 proteins were measured in pipping black-crowned night heron (Nycticorax nycticorax) embryos collected from a reference site (next to the Chincoteague National Wildlife Refuge, VA) and three polluted sites (Cat Island, Green Bay, Lake Michigan, WI; Bair Island, San Francisco Bay, CA; West Marin Island, San Francisco Bay, CA). In a laboratory study, artificially incubated night heron embryos from the reference site were treated with 3-methylcholanthrene or phenobarbital. Compared to controls, 3-methylcholanthrene induced a greater than fivefold increase in activities of several monooxygenases and a greater than 100-fold increase in the concentration of immunodetected cytochrome P450 1A (CYP1A). Phenobarbital treatment resulted in only a slight increase in BROD activity but induced proteins recognized by antibodies to cytochrome P450 2B (CYP2B) by 2,000-fold. In a field study, activities of AHH, BROD, EROD, and ethoxycoumarin-O-dealkylase (ECOD) were up to 85-fold higher in pipping black-crowned night herons collected from Cat Island compared to other sites. Hepatic CYP1A and CYP2B cross-reactive proteins were detected in significantly more individuals from Cat Island than from the reference site. Greatest burdens of total PCBs and p,p[prime]-DDE were detected in embryos from Cat Island. Cytochrome P450-associated monooxygenase activities and cytochrome P450 proteins (AHH, BROD, EROD, ECOD, CYP1A, CYP1B) were significantly associated with total PCB burdens.

  3. Cytochrome P450 Enzyme Metabolites in Lead Discovery and Development

    PubMed Central

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

    2015-01-01

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

  4. Personalized Cancer Therapy Considering Cytochrome P450 Variability.

    PubMed

    Preissner, Saskia; Simmaco, Maurizio; Gentile, Giovanna; Preissner, Robert

    2015-01-01

    The individual variability of pharmacokinetics is underestimated and few systematic studies exist in this field. In most cases, this leads to unwanted side effects or toxicity. In polychemotherapy, prodrugs (like ifosfamide), which have to be activated by cytochrome P450 enzymes (CYPs), play an important role. If patients are poor metabolizers for these drugs, the therapy will be ineffective. Furthermore, CYPs and transporters can be (over)expressed in target tissues, which is also not examined and considered in clinical routine. Here, we present a body map showing relevant enzymes in some organs and tissues. Finally, a typical case of a Caucasian chemotherapy patient with breast cancer is presented and discussed regarding a personalized cancer therapy considering the single nucleotide polymorphisms found via genotyping.

  5. Immunohistochemical localization of cytochrome P450 aromatase in equine gonads.

    PubMed

    Almadhidi, J; Seralini, G E; Fresnel, J; Silberzahn, P; Gaillard, J L

    1995-06-01

    Estrogens are the major steroids produced by equine gonads. To identify the cells responsible for estrogen synthesis, an antiserum against purified equine testicular cytochrome P450 aromatase was produced in rabbits. The reactivity and specificity of the antiserum were assessed by ELISA, immunoblot analysis, and immunoneutralization studies. Immunofluorescence microscopy demonstrated that in the male gonad, cytochrome P450 aromatase (P450arom) was localized in the interstitial tissue, whereas, under the experimental conditions used, the Sertoli and germ cells did not show any specific staining. In the ovary, the granulosa cells of small follicles exhibited faint immunofluorescent staining for P450arom and the granulosa cells of large, viable more follicles showed a high degree of immunoreactivity. In the corpus luteum, all the luteinized cells showed immunoreactivity. No immunoreactivity was detected in other cells of small and large viable follicles. Immunolocalization of P450arom in the equine testicular Leydig cells and in ovarian granulosa and luteinized cells indicates that these cells have the ability to metabolize androgens to estrogens and possibly to catechol estrogens.

  6. Selective expression and induction of cytochrome P450PB and P450MC during the development of hereditary hepatitis and hepatoma of LEC rats.

    PubMed

    Sugiyama, T; Suzuki, K; Ookawara, T; Kurosawa, T; Taniguchi, N

    1989-11-01

    The Long-Evans rat with a cinnamon-like coat color (LEC rat) is a mutant strain displaying hereditary hepatitis with severe jaundice. The age related difference in microsomal dealkylation of pentoxyresorufin and ethoxyresorufin was examined. The enzyme activity levels of pentoxyresorufin O-depentylase in LEC rats were decreased to 25% of the levels in control [Long-Evans rats with an agouti coat color (LEA rats)]. In contrast, ethoxyresorufin O-deethylase exhibited a much less marked difference between the strains. In parallel with these strain differences in enzyme activities, a decrease in phenobarbital (PB) inducible P450 isozymes, mainly P450b and P450e, was observed by Western blot analysis. The level of P450PB in LEC rats was more markedly depressed than in the LEA strain. On the other hand, microsomes from uninduced LEC rat liver had more 3-methylcholanthrene (MC) inducible P450MC, mainly P450c and P450d, than microsomes from LEA rat liver and these isozymes in the LEC were markedly induced by 3-methylcholanthrene treatment. The great difference in cytochrome P450PB content of the liver microsomes between LEC and LEA rats and the maintained constitutive levels of hepatic cytochrome P450MC in the LEC rats suggest a possible role of these cytochrome isozymes in the onset of spontaneous hepatitis and hepatoma.

  7. A three-dimensional model of aromatase cytochrome P450.

    PubMed Central

    Graham-Lorence, S.; Amarneh, B.; White, R. E.; Peterson, J. A.; Simpson, E. R.

    1995-01-01

    P450 hemeproteins comprise a large gene superfamily that catalyzes monooxygenase reactions in the presence of a redox partner. Because the mammalian members are, without exception, membrane-bound proteins, they have resisted structure-function analysis by means of X-ray crystallographic methods. Among P450-catalyzed reactions, the aromatase reaction that catalyzes the conversion of C19 steroids to estrogens is one of the most complex and least understood. Thus, to better understand the reaction mechanism, we have constructed a three-dimensional model of P450arom not only to examine the active site and those residues potentially involved in catalysis, but to study other important structural features such as substrate recognition and redox-partner binding, which require examination of the entire molecule (excepting the putative membrane-spanning region). This model of P450arom was built based on a "core structure" identified from the structures of the soluble, bacterial P450s (P450cam, P450terp, and P450BM-P) rather than by molecular replacement, after which the less conserved elements and loops were added in a rational fashion. Minimization and dynamic simulations were used to optimize the model and the reasonableness of the structure was evaluated. From this model we have postulated a membrane-associated hydrophobic region of aliphatic and aromatic residues involved in substrate recognition, a redox-partner binding region that may be unique compared to other P450s, as well as residues involved in active site orientation of substrates and an inhibitor of P450arom, namely vorozole. We also have proposed a scheme for the reaction mechanism in which a "threonine switch" determines whether oxygen insertion into the substrate molecule involves an oxygen radical or a peroxide intermediate. PMID:7549871

  8. 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

  9. 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.

  10. 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.

  11. Catechins in tea suppress the activity of cytochrome P450 1A1 through the aryl hydrocarbon receptor activation pathway in rat livers.

    PubMed

    Fukuda, Itsuko; Nishiumi, Shin; Mukai, Rie; Yoshida, Ken-ichi; Ashida, Hitoshi

    2015-05-01

    Polycyclic aromatic hydrocarbons (PAHs) and halogenated aromatic hydrocarbons (HAHs) develop various adverse effects through activation of an aryl hydrocarbon receptor (AhR). The suppressive effects of brewed green tea and black tea on 3-methylcholanthrene (MC)-induced AhR activation and its downstream events were examined in the liver of rats. Ad-libitum drinking of green tea and black tea suppressed MC-induced AhR activation and elevation of ethoxyresorufin O-deethylase activity in the liver, whereas the teas themselves did not induce them. Tea showed a suppressive fashion on the expression of cytochrome P450 1A1 (CYP1A1). Tea suppressed the AhR activation induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) ex vivo. A part of catechins and theaflavins was present in plasma and liver as conjugated and intact forms. The results of this study suggested that active component(s) of tea are incorporated in the liver and suppress the activity of CYP1As through the AhR activation pathway.

  12. Biocatalytic Conversion of Avermectin to 4"-Oxo-Avermectin: Characterization of Biocatalytically Active Bacterial Strains and of Cytochrome P450 Monooxygenase Enzymes and Their Genes

    PubMed Central

    Jungmann, Volker; Molnár, István; Hammer, Philip E.; Hill, D. Steven; Zirkle, Ross; Buckel, Thomas G.; Buckel, Dagmar; Ligon, James M.; Pachlatko, J. Paul

    2005-01-01

    4"-Oxo-avermectin is a key intermediate in the manufacture of the agriculturally important insecticide emamectin benzoate from the natural product avermectin. Seventeen biocatalytically active Streptomyces strains with the ability to oxidize avermectin to 4"-oxo-avermectin in a regioselective manner have been discovered in a screen of 3,334 microorganisms. The enzymes responsible for this oxidation reaction in these biocatalytically active strains were found to be cytochrome P450 monooxygenases (CYPs) and were termed Ema1 to Ema17. The genes for Ema1 to Ema17 have been cloned, sequenced, and compared to reveal a new subfamily of CYPs. Ema1 to Ema16 have been overexpressed in Escherichia coli and purified as His-tagged recombinant proteins, and their basic enzyme kinetic parameters have been determined. PMID:16269732

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

    EPA Science Inventory

    The gene for the alkane-inducible cytochrome P450, P450alk, has been isolated from the yeast Candida tropicalis by immunoscreening a λgt11 library. Isolation of the gene has been identified on the basis of its inducibility and partial DNA sequence. Transcripts of this gene were i...

  14. A predicted three-dimensional structure of human cytochrome P450: implications for substrate specificity.

    PubMed

    Zvelebil, M J; Wolf, C R; Sternberg, M J

    1991-02-01

    A three-dimensional structure for human cytochrome P450IA1 was predicted based on the crystal coordinates of cytochrome P450cam from Pseudomonas putida. As there was only 15% residue identity between the two enzymes, additional information was used to establish an accurate sequence alignment that is a prerequisite for model building. Twelve representative eukaryotic sequences were aligned and a net prediction of secondary structure was matched against the known alpha-helices and beta-sheets of P450cam. The cam secondary structure provided a fixed main-chain framework onto which loops of appropriate length from the human P450IA1 structure were added. The model-built structure of the human cytochrome conformed to the requirements for the segregation of polar and nonpolar residues between the core and the surface. The first 44 residues of human cytochrome P450 could not be built into the model and sequence analysis suggested that residues 1-26 formed a single membrane-spanning segment. Examination of the sequences of cytochrome P450s from distinct gene families suggested specific residues that could account for the differences in substrate specificity. A major substrate for P450IA1, 3-methyl-cholanthrene, was fitted into the proposed active site and this planar aromatic molecule could be accommodated into the available cavity. Residues that are likely to interact with the haem were identified. The sequence similarity between 59 eukaryotic enzymes was represented as a dendrogram that in general clustered according to gene family. Until a crystallographic structure is available, this model-building study identifies potential residues in cytochrome P450s important in the function of these enzymes and these residues are candidates for site-directed mutagenesis.

  15. 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.

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

    PubMed Central

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

    1995-01-01

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

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

    SciTech Connect

    Torres, Eduardo; Hayen, Heiko; Niemeyer, Christof M.; 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.

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

    PubMed Central

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

    1990-01-01

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

  19. Reasons for reduced activities of 17 alpha-hydroxylase and C17-C20 lyase in spite of increased contents of cytochrome P-450 in mature rat testis fetally irradiated with 60Co.

    PubMed

    Inano, H; Ishii-Ohba, H; Suzuki, K; Ikeda, K

    1990-05-01

    Pregnant rats received whole body irradiation with 2.6 Gy gamma-ray from a 60Co source at Day 20 of gestation. When pups were 4 months old, activities of electron transport system and steroid monooxygenase in tests were assayed. The content of total cytochrome P-450 in the irradiated testes had increased to 170% of that in non-irradiated rats, but NADPH-cytochrome P-450 reductase activity had reduced to 36% of the control. Also, amounts of cytochrome b5 in testicular microsomal fraction were decreased markedly after irradiation, but no significant change of NADH-cytochrome b5 reductase activity was observed in the treated pups. Because both 17 alpha-hydroxylase and C17-C20 lyase activities tended to be decreased by fetal irradiation, testosterone production from progesterone and 17 alpha-hydroxyprogesterone was reduced to about 30% of the control. From these results, it has been suggested that the testicular cytochrome P-450 is radioresistant but steroid monooxygenase activities are reduced after the fetal irradiation. We propose that the discrepancy arises from the marked decrement of NADPH-cytochrome P-450 reductase activity.

  20. Cytochrome P450IA1 induction and localization in endothelium of vertebrate (teleost) heart.

    PubMed

    Stegeman, J J; Miller, M R; Hinton, D E

    1989-11-01

    Previous studies have shown that high levels of cytochrome P450 can occur in cardiac microsomes of vertebrates [Mol. Pharmacol. 21:517-526, (1982)]. Here we identify the dominant cardiac P450 in the marine fish scup as P450E, a teleost representative of P450IA1, and we describe its restricted cellular localization in the heart. Treatment of scup with beta-naphthoflavone produced an unusually strong (10-fold) induction of spectrally measured P450 in cardiac microsomes, with specific content reaching levels (0.5 nmol/mg) similar to those induced in scup liver. Microsomal ethoxyresorufin O-deethylase and aryl hydrocarbon hydroxylase activities, catalytic functions of scup P450E, were induced in parallel with P450 content. Similar induction was seen in both atrium and ventricle. Immunoblot analysis with monoclonal antibody 1-12-3, specific to scup P450E and other vertebrate P450IA1 proteins, showed that this hydrocarbon-inducible P450 is the dominant and possibly sole P450 form in heart microsomes of experimentally induced animals. Immunohistochemical analysis of scup heart sections (2-4-microns) with monoclonal antibody 1-12-3 revealed that P450E was detectable only in endothelial cells of the endocardium and of the coronary vasculature. A similar endothelial cell localization of the monoclonal antibody 1-12-3 epitope was observed in heart of rainbow trout, induced with beta-naphthoflavone, indicating a general nature for the endothelial localization of induced cardiac P450. Morphometric analysis showed that endothelium could constitute 8-9% of the volume of teleost heart, from which we calculate that P450IA1 could account for as much as 25% of the endothelial cell microsomal protein. Heart microsomes of untreated animals from contaminated environments also contained high levels of P450E, indicating that induction like that caused by beta-naphthoflavone could occur with chemicals in the environment. Strongly induced P450E (P450IA1) in endothelium could play a critical

  1. Cytochrome P450-derived eicosanoids: the neglected pathway in cancer

    PubMed Central

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

    2010-01-01

    Endogenously produced lipid autacoids are locally acting small molecule mediators that play a central role in the regulation of inflammation and tissue homeostasis. A well-studied group of autacoids are the products of arachidonic acid metabolism, among which the prostaglandins and leukotrienes are the best known. They are generated by two pathways controlled by the enzyme systems cyclooxygenase and lipoxygenase, respectively. However, arachidonic acid is also substrate for a third enzymatic pathway, the cytochrome P450 (CYP) system. This third eicosanoid pathway consists of two main branches: ω-hydroxylases convert arachidonic acid to hydroxyeicosatetraenoic acids (HETEs) and epoxygenases convert it to epoxyeicosatrienoic acids (EETs). This third CYP pathway was originally studied in conjunction with inflammatory and cardiovascular disease. Arachidonic acid and its metabolites have recently stimulated great interest in cancer biology; but, unlike prostaglandins and leukotrienes the link between cytochome P450 metabolites and cancer has received little attention. In this review, the emerging role in cancer of cytochrome P450 metabolites, notably 20-HETE and EETs, are discussed. PMID:20941528

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

    PubMed Central

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

    2013-01-01

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

  3. Occurrence of a barbiturate-inducible catalytically self-sufficient 119,000 dalton cytochrome P-450 monooxygenase in bacilli.

    PubMed

    Fulco, A J; Ruettinger, R T

    1987-05-04

    In a recent publication (Narhi, L.O. and Fulco, A.J.[1986] J. Biol. Chem. 261, 7160-7169) we described the characterization of a catalytically self-sufficient 119,000 Dalton cytochrome P-450 fatty acid monooxygenase (P-450BM-3) induced by barbiturates in Bacillus megaterium ATCC 14581. We have now examined cell-free preparations from 12 distinct strains of B. megaterium and from one or two strains each of B. alvei, B. brevis, B. cereus, B. licheniformis, B. macerans, B. pumilis and B. subtilis for the presence of this inducible enzyme. Using Western blot analyses in combination with assays for fatty acid hydroxylase activity and cytochrome P-450, we were able to show that 11 of the 12 B. megaterium strains contained not only a strongly pentobarbital-inducible fatty acid monooxygenase identical to or polymorphic with P-450BM-3 but also significant levels of two smaller P-450 cytochromes that were the same as or similar to cytochromes P-450BM-1 and P-450BM-2 originally found in ATCC 14581. Unlike the 119,000 Dalton P-450, however, the two smaller P-450s were generally easily detectable in cultures grown to stationary phase in the absence of barbiturates and, with some exceptions, were not strongly induced by pentobarbital. None of the non-megaterium species of Bacillus tested exhibited significant levels of either fatty acid monooxygenase activity or cytochrome P-450. The one strain of B. megaterium that lacked inducible P-450BM-3 was also negative for BM-1 and BM-2. However, this strain (ATCC 13368) did contain a small but significant level of another P-450 cytochrome that others have identified as the oxygenase component of a steroid 15-beta-hydroxylase system. Our evidence suggests that the BM series of P-450 cytochromes is encoded by chromosomal (rather than by plasmid) DNA.

  4. 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

  5. Fusion of Ferredoxin and Cytochrome P450 Enables Direct Light-Driven Biosynthesis

    PubMed Central

    2016-01-01

    Cytochrome P450s (P450s) are key enzymes in the synthesis of bioactive natural products in plants. Efforts to harness these enzymes for in vitro and whole-cell production of natural products have been hampered by difficulties in expressing them heterologously in their active form, and their requirement for NADPH as a source of reducing power. We recently demonstrated targeting and insertion of plant P450s into the photosynthetic membrane and photosynthesis-driven, NADPH-independent P450 catalytic activity mediated by the electron carrier protein ferredoxin. Here, we report the fusion of ferredoxin with P450 CYP79A1 from the model plant Sorghum bicolor, which catalyzes the initial step in the pathway leading to biosynthesis of the cyanogenic glucoside dhurrin. Fusion with ferredoxin allows CYP79A1 to obtain electrons for catalysis by interacting directly with photosystem I. Furthermore, electrons captured by the fused ferredoxin moiety are directed more effectively toward P450 catalytic activity, making the fusion better able to compete with endogenous electron sinks coupled to metabolic pathways. The P450-ferredoxin fusion enzyme obtains reducing power solely from its fused ferredoxin and outperforms unfused CYP79A1 in vivo. This demonstrates greatly enhanced electron transfer from photosystem I to CYP79A1 as a consequence of the fusion. The fusion strategy reported here therefore forms the basis for enhanced partitioning of photosynthetic reducing power toward P450-dependent biosynthesis of important natural products. PMID:27119279

  6. Cytochrome P450 of wood-rotting basidiomycetes and biotechnological applications.

    PubMed

    Ichinose, Hirofumi

    2013-01-01

    Wood-rotting basidiomycetes possess superior metabolic functions to degrade woody biomass, and these activities are indispensable for the carbon cycle of the biosphere. As well as basic studies of the biochemistry of basidiomycetes, many researchers have been focusing on utilizing basidiomycetes and/or their enzymes in the biotechnology sector; therefore, the unique activities of their extracellular and intracellular enzymes have been widely demonstrated. A rich history of applied study has established that basidiomycetes are capable of metabolizing a series of endogeneous and exogeneous compounds using cytochrome P450s (P450s). Recently, whole genome sequence analyses have revealed large-scale divergences in basidiomycetous P450s. The tremendous variation in P450s implies that basidiomycetes have vigorously diversified monooxygenase functions to acquire metabolic adaptations such as lignin degradation, secondary metabolite production, and xenobiotics detoxification. However, fungal P450s discovered from genome projects are often categorized into novel families and subfamilies, making it difficult to predict catalytic functions by sequence comparison. Experimental screening therefore remains essential to elucidate the catalytic potential of individual P450s, even in this postgenomic era. This paper archives the known metabolic capabilities of basidiomycetes, focusing on their P450s, outlines the molecular diversity of basidiomycetous P450s, and introduces new functions revealed by functionomic studies using a recently developed, rapid, functional screening system.

  7. Toxaphene detoxification and acclimation in Daphnia magna: do cytochrome P-450 enzymes play a role?

    PubMed

    Kashian, Donna R

    2004-01-01

    Toxaphene is a persistent environmental contaminant that has been shown to alter male production in Daphnia magna and to induce P-450 activity in mammals. Cytochrome P-450-mediated metabolism may lead to xenobiotic detoxification resulting in acclimation. To determine if D. magna acclimate to toxaphene via P-450 pathways, chronic and acute toxicity tests were conducted with D. magna exposed to toxaphene in the presence and absence of piperonyl butoxide (PBO), an inhibitor of cytochrome P-450 enzymes. Toxaphene exposure increased male production in acute but not chronic assays, indicating that D. magna may acclimate to chronic toxaphene exposure. Upon co-administration of toxaphene and PBO in chronic tests, D. magna exhibited a decline in growth rate, fecundity and survival. The observed toxaphene acclimation in chronic tests, along with its increased toxicity in the presence of a P-450 suppressor, suggests that P-450 enzymes may contribute to detoxification and subsequent acclimation of D. magna to chronic toxaphene exposure. Additional chronic toxicity tests indicated that toxaphene acclimation occurs between 7 and 12 days following initial exposure, at which time sex determination is no longer affected. Thus, sublethal toxaphene toxicity effects such as reproductive impairments may be detectable with acute but not chronic tests, potentially due to the upregulation of P-450 isozymes.

  8. Formation of estrogenic metabolites of benzo[a]pyrene and chrysene by cytochrome P450 activity and their combined and supra-maximal estrogenic activity.

    PubMed

    van Lipzig, Marola M H; Vermeulen, Nico P E; Gusinu, Renato; Legler, Juliette; Frank, Heinz; Seidel, Albrecht; Meerman, John H N

    2005-01-01

    Metabolism of polycyclic aromatic hydrocarbons (PAHs) has been studied intensively, and potential metabolites with estrogenic activity have been identified previously. However, little attention has been paid to the metabolic pathways in mammalians and to the combined effect of individual metabolites. Several hydroxylated metabolites of benzo[a]pyrene (BaP) and chrysene (CHN) were formed by rat liver microsomal cytochrome P450 (CYP) activity, some of which possess estrogenic activity. All mono- and several dihydroxylated metabolites of BaP and CHN were tested for ER affinity and estrogenic activity in a proliferation assay (E-screen) and in a reporter-gene assay (ER-CALUX). Twelve estrogenic metabolites were identified with EC50 values ranging from 40nM to 0.15mM. The combined effect of a mixture of seven PAH-metabolites was also studied in the ER binding assay. At concentrations that show little activity themselves, their joint action clearly exhibited significant estrogenic activity. BaP itself exhibited estrogenicity in the ER-CALUX assay due to bio-activation into estrogenic metabolites, probably via aryl hydrocarbon receptor (AhR) induced CYP activity. Furthermore, 2-hydroxy-CHN (2-OHCHN) induced supra-maximal (400%) estrogenic effects in the ER-CALUX assay. This effect was entirely ER-mediated, since the response was completely blocked with the ER-antagonist ICI182,780. We showed that 2-OHCHN increased ER-concentration, using ELISA techniques, which may explain the observed supra-maximal effects. Co-treatment with the AhR-antagonist 3',4'-dimethoxyflavone (DMF) enhanced ER-signaling, possibly via blockage of AhR-ER inhibitory cross-talk.

  9. Semi-rational engineering of cytochrome P450sca-2 in a hybrid system for enhanced catalytic activity: insights into the important role of electron transfer.

    PubMed

    Ba, Lina; Li, Pan; Zhang, Hui; Duan, Yan; Lin, Zhanglin

    2013-11-01

    Hybrid P450 systems in which P450 monooxygenases are reconstituted with non-native or surrogate redox partners have become important for the engineering of this class of versatile enzymes. P450sca-2 from Streptomyces carbophilus stereoselectively hydroxylates mevastatin to yield pravastatin, a cholesterol-lowering drug. While S. carbophilus has been successfully applied in the industrial biotransformation process for pravastatin, the molecular study and engineering of P450sca-2 has been very limited. We have previously established a functional P450sca-2/Pdx/Pdr hybrid system. In this study, on the basis of a more active P450sca-2 mutant (R8-5C), five sites located in the substrate binding pocket, substrate access entrance, and presumed Pdx interaction interface were rationally chosen, and systematically subjected to site-directed saturation mutagenesis (SDSM), and three rounds of iterative saturation mutagenesis (ISM). A best mutant (Variant III) was obtained, which showed a whole cell biotransformation activity (377.5 mg/L) and an overall apparent k(cat) (6.37 min⁻¹) that was 7.1- and 10.0-fold that of the starting template R8-5C, respectively. Kinetic characterization revealed that most of the improvements seen for the SDSM and ISM mutants came from enhanced overall electron transfer, with the two sites at the interface between P450sca-2 and Pdx (T119 and N363) being most critical. Our study underscores the important role of electron transfer in a hybrid P450 system, and also demonstrates the utility of ISM in optimizing the redox partner interface. This should facilitate engineering of this and other important hybrid P450 systems.

  10. A molecular model for the interaction between vorozole and other non-steroidal inhibitors and human cytochrome P450 19 (P450 aromatase).

    PubMed

    Koymans, L M; Moereels, H; Vanden Bossche, H

    1995-06-01

    In a previous study (Vanden Bossche et al., Breast Cancer Res. Treat. 30 (1994) 43) the interaction between (+)-S-vorozole and the I-helix of cytochrome P450 19 (P450 aromatase) has been reported. In the present study we extended the "I-helix model" by incorporating the C-terminus of P450 aromatase. The crystal structures of P450 101 (P450 cam), 102 (P450 BM-3) and 108 (P450 terp) reveal that the C-terminus is structurally conserved and forms part of their respective substrate binding pocket. Furthermore, the present study is extended to the interaction between P450 aromatase and its natural substrate androstenedione and the non-steroidal inhibitors (-)-R-vorozole, (-)-S-fadrozole, R-liarozole and (-)-R-aminoglutethimide. It is found that (+)-S-vorozole, (-)-S-fadrozole and R-liarozole bind in a comparable way to P450 aromatase and interact with both the I-helix (Glu302 and Asp309) and C-terminus (Ser478 and His480). The weak activity of (-)-R-aminoglutethimide might be attributed to a lack of interaction with the C-terminus.

  11. Influence of Various Polymorphic Variants of Cytochrome P450 Oxidoreductase (POR) on Drug Metabolic Activity of CYP3A4 and CYP2B6

    PubMed Central

    Naranmandura, Hua; Zeng, Su; Chen, Shu Qing

    2012-01-01

    Cytochrome P450 oxidoreductase (POR) is known as the sole electron donor in the metabolism of drugs by cytochrome P450 (CYP) enzymes in human. However, little is known about the effect of polymorphic variants of POR on drug metabolic activities of CYP3A4 and CYP2B6. In order to better understand the mechanism of the activity of CYPs affected by polymorphic variants of POR, six full-length mutants of POR (e.g., Y181D, A287P, K49N, A115V, S244C and G413S) were designed and then co-expressed with CYP3A4 and CYP2B6 in the baculovirus-Sf9 insect cells to determine their kinetic parameters. Surprisingly, both mutants, Y181D and A287P in POR completely inhibited the CYP3A4 activity with testosterone, while the catalytic activity of CYP2B6 with bupropion was reduced to approximately ∼70% of wild-type activity by Y181D and A287P mutations. In addition, the mutant K49N of POR increased the CLint (Vmax/Km) of CYP3A4 up to more than 31% of wild-type, while it reduced the catalytic efficiency of CYP2B6 to 74% of wild-type. Moreover, CLint values of CYP3A4-POR (A115V, G413S) were increased up to 36% and 65% of wild-type respectively. However, there were no appreciable effects observed by the remaining two mutants of POR (i.e., A115V and G413S) on activities of CYP2B6. In conclusion, the extent to which the catalytic activities of CYP were altered did not only depend on the specific POR mutations but also on the isoforms of different CYP redox partners. Thereby, we proposed that the POR-mutant patients should be carefully monitored for the activity of CYP3A4 and CYP2B6 on the prescribed medication. PMID:22719896

  12. A QM/MM study of the active species of the human cytochrome P450 3A4, and the influence thereof of the multiple substrate binding

    PubMed Central

    Fishelovitch, Dan; Hazan, Carina; Hirao, Hajime; Wolfson, Haim J.; Nussinov, Ruth; Shaik, Sason

    2008-01-01

    Cytochrome P450 3A4 is involved in the metabolism of 50% of all swallowed drugs. The enzyme functions by means of a high-valent iron-oxo species, called Compound I (Cpd I), which is formed after entrance of the substrate to the active site. We explored the features of Cpd I using hybrid quantum mechanical/molecular mechanical calculations on various models that are either substrate-free or containing one and two molecules of diazepam as a substrate. Mössbauer parameters of Cpd I were computed. Our major finding shows that without the substrate, Cpd I tends to elongate its Fe-S bond, localize the radical on the sulfur, and form hydrogen bonds with A305 and T309, which may hypothetically lead to Cpd I consumption by H-abstraction. However, the positioning of diazepam close to Cpd I, as enforced by the effector molecule, was found to strengthen the NH---S interactions of the conserved I443 and G444 residues with the proximal cysteinate ligand. These interactions are known to stabilize the Fe-S bond, and as such, the presence of the substrate leads to a shorter Fe-S bond and it prevents the localization of the radical on the sulfur. This diazepam-Cpd I stabilization was manifested in the 1W0E conformer. The effector substrate did not influence Cpd I directly but rather by positioning the active substrate close to Cpd I, thus displacing the hydrogen bonds with A305 and T309, and thereby giving preference to substrate oxidation. It is hypothesized that these effects on Cpd I, promoted by the restrained substrate, may be behind the special metabolic behavior observed in cases of multiple substrate binding (called also cooperative binding). This restraint constitutes a mechanism whereby substrates stabilize Cpd I sufficiently long to affect monooxygenation by P450s at the expense of Cpd I destruction by the protein residues. PMID:18020326

  13. Inducible cytochrome P-450 from rat liver mitochondria

    SciTech Connect

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

    1987-05-01

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

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

    PubMed

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

    2015-10-01

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

  15. Cumene hydroperoxide effected hydroperoxidation by cytochrome P-450.

    PubMed

    Chen, C; Gurka, D P

    1985-04-01

    9-Methylfluorene was found to be oxygenated to 9-hydroperoxy-9-methylfluorene and 9-hydroxy-9-methylfluorene by cytochrome P-450 in the presence of cumene hydroperoxide. Molecular oxygen is required and carbon monoxide is inhibitory. The reaction is inhibited by SKF-525A and metyrapone. Metyrapone and cumene hydroperoxide also retard the conversion of 9-hydroperoxy-9-methylfluorene to 9-hydroxy-9-methylfluorene. The reaction is different from hydroperoxide-supported oxygenation, since the cumene hydroperoxide appears to act as an effector of the enzyme rather than oxygen donor. It is suggested that substrates with stable radicals can be dioxygenated in this manner.

  16. Cytochromes P450 for terpene functionalisation and metabolic engineering.

    PubMed

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

    2015-01-01

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

  17. 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

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

    PubMed Central

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

    2009-01-01

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

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

    PubMed

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

    2012-01-01

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

  20. Human Hepatic Cytochrome P450-Specific Metabolism of the Organophosphorus Pesticides Methyl Parathion and Diazinon

    PubMed Central

    Tian, Yuan; Knaak, James B.; Kostyniak, Paul J.; Olson, James R.

    2012-01-01

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

  1. Analysis of mammalian cytochrome P450 structure and function by site-directed mutagenesis.

    PubMed

    Domanski, T L; Halpert, J R

    2001-06-01

    Over the past decade, site-directed mutagenesis has become an essential tool in the study of mammalian cytochrome P450 structure-function relationships. Residues affecting substrate specificity, cooperativity, membrane localization, and interactions with redox partners have been identified using a combination of amino-acid sequence alignments, homology modeling, chimeragenesis, and site-directed mutagenesis. As homology modeling and substrate docking technology continue to improve, the ability to predict more precise functions for specific residues will also advance, making it possible to utilize site-directed mutagenesis to test these predictions. Future studies will employ site-directed mutagenesis to learn more about cytochrome P450 substrate access channels, to define the role of residues that do not lie within substrate recognition sites, to engineer additional soluble forms of microsomal cytochromes P450 for x-ray crystallography, and to engineer more efficient enzymes for drug activation and/or bioremediation.

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

    PubMed

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

    2010-12-01

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

  3. Function and membrane topology of wild-type and mutated cytochrome P-450c21.

    PubMed Central

    Hu, M C; Hsu, L C; Hsu, N C; Chung, B C

    1996-01-01

    We have studied membrane topology of cytochrome P-450c21 (P450c21) using the approaches of mutagenesis and protease digestion. P450c21 is located at the cytoplasm with an N-terminal hydrophobic domain integrated into microsomal membranes. When this hydrophobic domain was replaced by a secretory signal peptide, P450c21 was translocated into the lumen and lost enzymic activity. No other topogenic sequence was detected in the bulk of the P450c21 peptide. A mutant protein with Pro-30 replaced by Leu (L30) corresponding to the mutation found in the diseased state was created. L30 protein lost 90% of enzymic activity, while a double mutant (L30R32) with an additional Leu-32 to Arg mutation had slightly higher residual enzymic activity. Apart from lower activity, L30 was also present in the cell at a lower level than wild-type P450c21. This lower level is probably due to increased degradation, as L30 is synthesized at a normal rate. Both L30 and L30R32 proteins, however, were integrated into membranes normally. Therefore the Pro-30 --> Leu mutation did not affect membrane integration, but affected the abundance and enzymic activity of P450c21. PMID:8645225

  4. Effects of Cu(2+) and Pb(2+) on different fish species: liver cytochrome P450-dependent monooxygenase activities and FTIR spectra.

    PubMed

    Henczová, Mária; Deér, Aranka Kiss; Filla, Adrienn; Komlósi, Viktória; Mink, János

    2008-07-01

    The effects of Cu(2+)-sulfate and Pb(2+)-acetate on carp (Cyprinus carpio L.), silver carp (Hypopthalmichtys molitrix V.) and wels (Silurus glanis L.) were studied. The liver microsomal Cyt P450 content, the EROD, ECOD and APND monooxygenase activities were measured. In vivo treatment with 1 mg L(-1) Cu(2+) significantly elevated the activities of these enzymes and Cyt P450 content in silver carp livers. The high-dose Cu(2+) treatment (10 mg L(-1)) on silver carp caused two-fold higher induction in the P450 dependent monooxygenase isoensymes than in wels. Although the 2 mg kg(-1) treatment with Pb(2+) in carp elevated significantly the P450 content, the EROD isoenzyme activities were significantly decreased after 1 day, showing the destructive effect of metal ion on the enzyme system. In vitro, Cu(2+) and Pb(2+) decreased the Cyt P450 content in the carp liver microsomes and the absorption peak shifted to higher wavelength. Fourier Transform Infrared (FTIR) spectroscopy was used to detect the damaging effects of the heavy metals. According to the inhibitory potency to Cu(2+), the most sensitive isoenzyme was the EROD in wels, the least was the silver carp's isoenzyme. The investigated fish P450 isoenzymes showed, that the Cu(2+) was a stronger inhibitor than Pb(2+).

  5. [Effects of repeated skin applications of heavy pyrolysis resin on cytochrome P-450 level and glutathione transferase activity in liver microsomes and cytosol in rats in relation to the level of toxic effects of pyrolysis resin on internal organs].

    PubMed

    Kravchenko, M N; Loginov, A S; Petrova, L P; Ausheva, L Kh; Bendikov, E A

    1990-10-01

    Rats received 20 skin applications of heavy pyrolysis resin, containing about 30% of polycyclic aromatic hydrocarbons. The every exposure duration was 4 hours a day. Applications have been carried out daily 5 days a week in the course of 4 weeks. Induction of cytochrome P-450 (P-450) by 79%, significant induction of microsomal (GTm) and cytosol (GTc) glutathione-S-transferase activity (by 46 and 85%, respectively) and small increase of reduced glutathione level (by 9%) also were registered as a result of these exposures. Lipid peroxidation level determined by TBK-reactive products quantity didn't alter significantly. Close correlation between ratio values of P-450/GTm levels and P-450/GTc levels and toxic effect indices of heavy pyrolysis resin on rat immune and endocrine systems.

  6. Novel CAR-mediated mechanism for synergistic activation of two distinct elements within the human cytochrome P450 2B6 gene in HepG2 cells.

    PubMed

    Swales, Karen; Kakizaki, Satoru; Yamamoto, Yukio; Inoue, Kaoru; Kobayashi, Kaoru; Negishi, Masahiko

    2005-02-04

    The constitutive active receptor (CAR) regulates the induction of the cytochrome P450 2B6 (CYP2B6) gene by phenobarbital-type inducers, such as 1,4 bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP) via the distal phenobarbital-responsive enhancer module (PBREM, at -1732/-1685 bp). Activation of the PBREM by TCPOBOP generated a 10-fold induction of CYP2B6 mRNA in HepG2 cells stably expressing mouse CAR (Ym17). Co-treatment with the protein phosphatase inhibitor okadaic acid (OA) synergistically increased this induction over 100-fold without directly activating CAR or the PBREM. Although OA synergy required the presence of PBREM, deletion assays delineated the OA-responsive activity to a proximal 24-bp (-256/-233) sequence (OARE) in the CYP2B6 promoter. CAR did not directly bind to the OARE in electrophoretic mobility shift assays. However, both DNA affinity and chromatin immunoprecipitation assays showed a significant increase in CAR association with the OARE after co-treatment with TCPOBOP and OA, indicating the indirect binding of CAR to the OARE. The two cis-acting elements, the distal PBREM and the proximal OARE, within the chromatin structure are both regulated by CAR in response to TCPOBOP and OA, respectively, to maximally induce the CYP2B6 promoter. This functional interaction between the two sites expands the current understanding of the mechanism of CAR-mediated inducible transcription.

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

    SciTech Connect

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

    1984-10-01

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

  8. A preliminary 3D model for cytochrome P450 2D6 constructed by homology model building.

    PubMed

    Koymans, L M; Vermeulen, N P; Baarslag, A; Donné-Op den Kelder, G M

    1993-06-01

    A homology model building study of cytochrome P450 2D6 has been carried out based on the crystal structure of cytochrome P450 101. The primary sequences of P450 101 and P450 2D6 were aligned by making use of an automated alignment procedure. This alignment was adjusted manually by matching alpha-helices (C, D, G, I, J, K and L) and beta-sheets (beta 3/beta 4) of P450 101 that are proposed to be conserved in membrane-bound P450s (Ouzounis and Melvin [Eur. J. Biochem., 198 (1991) 307]) to the corresponding regions in the primary amino acid sequence of P450 2D6. Furthermore, alpha-helices B, B' and F were found to be conserved in P450 2D6. No significant homology between the remaining regions of P450 101 and P450 2D6 could be found and these regions were therefore deleted. A 3D model of P450 2D6 was constructed by copying the coordinates of the residues from the crystal structure of P450 101 to the corresponding residues in P450 2D6. The regions without a significant homology with P450 101 were not incorporated into the model. After energy-minimization of the resulting 3D model of P450 2D6, possible active site residues were identified by fitting the substrates debrisoquine and dextrometorphan into the proposed active site. Both substrates could be positioned into a planar pocket near the heme region formed by residues Val370, Pro371, Leu372, Trp316, and part of the oxygen binding site of P450 2D6. Furthermore, the carboxylate group of either Asp100 or Asp301 was identified as a possible candidate for the proposed interaction with basic nitrogen atom(s) of the substrates.(ABSTRACT TRUNCATED AT 250 WORDS)

  9. In Vitro Cytochrome P450 Formation of a Mono-Hydroxylated Metabolite of Zearalenone Exhibiting Estrogenic Activities: Possible Occurrence of This Metabolite in Vivo

    PubMed Central

    Bravin, Frederique; Duca, Radu C.; Balaguer, Patrick; Delaforge, Marcel

    2009-01-01

    The mycoestrogen zearalenone (ZEN), as well as its reduced metabolites, which belong to the endocrine disruptor bio-molecule family, are substrates for various enzymes involved in steroid metabolism. In addition to its reduction by the steroid dehydrogenase pathway, ZEN also interacts with hepatic detoxification enzymes, which convert it into hydroxylated metabolites (OH-ZEN). Due to their structures to that of estradiol, ZEN and its derived metabolites bind to the estrogen receptors and are involved in endocrinal perturbations and are possibly associated with estrogen-dependent cancers. The primary aim of this present study was to identify the enzymatic cytochrome P450 isoforms responsible for the formation of the most abundant OH-ZEN. We thus studied its in vitro formation using hepatic microsomes in a range of animal model systems including man. OH-ZEN was also recovered in liver and urine of rats treated orally with ZEN. Finally we compared the activity of ZEN and its active metabolites (α-ZAL and OH-ZEN) on estrogen receptors using HeLa ER-α and ER-β reporter cell lines as reporters. OH-ZEN estrogenic activities were revealed to be limited and not as significant as those of ZEN or α-ZAL. PMID:19468341

  10. Activating nuclear xenobiotic receptors and triggering ER stress and hepatic cytochromes P450 systems in quails (Coturnix C. coturnix) during atrazine exposure.

    PubMed

    Du, Zheng-Hai; Qin, Lei; Lin, Jia; Sun, Yan-Chun; Xia, Jun; Zhang, Cong; Li, Xue-Nan; Li, Jin-Long

    2017-02-10

    Atrazine (ATR) is one of the most widely detected contaminant in the ecosystem. Nuclear xenobiotic receptors are activated by herbicides and induce the transcription of CYP450 isoforms involved in xenobiotic metabolism and transport. However, little is known about hepatic nuclear xenobiotic receptors in birds are responsible for ATR-induced hepatotoxicity via regulating the cytochrome P450 enzyme systems (CYP450s). The objective of this study was to investigate the mechanism of ATR hepatotoxicity in quails. For this purpose, male quails were dosed by oral gavage from sexual immaturity to maturity with 0, 50, 250, and 500 mg/kg/day ATR for 45 days. The results showed that ATR exposure caused the hepatotoxicity damage and endoplasmic reticulum (ER) degeneration. It suggested that ER is a target organelle of ATR toxicity in hepatocytes. ATR exposure disrupted the hepatic CYP450s homeostasis. This study also demonstrated that ATR triggered the CYP450 isoforms transcription via activating the hepatic CAR/PXR pathway. The present study provides new insights regarding the mechanism of the ATR-induced hepatotoxicity through activating nuclear xenobiotic receptors and triggering ER stress and hepatic CYP450s in quails.

  11. Species differences in hepatic and intestinal metabolic activities for 43 human cytochrome P450 substrates between humans and rats or dogs.

    PubMed

    Nishimuta, Haruka; Nakagawa, Tetsuya; Nomura, Naruaki; Yabuki, Masashi

    2013-11-01

    1. Prediction of human pharmacokinetics might be made more precise by using species with similar metabolic activities to humans. We had previously reported the species differences in intestinal and hepatic metabolic activities of 43 cytochrome P450 (CYP) substrates between cynomolgus monkeys and humans. However, the species differences between humans and rats or dogs had not yet been determined using comparable data sets with sufficient number of compounds. 2. Here, we investigated metabolic stabilities in intestinal and liver microsomes obtained from rats, dogs and humans using 43 substrates of human CYP1A2, CYP2J2, CYP2C, CYP2D6 and CYP3A. 3. Hepatic intrinsic clearance (CLint) values for most compounds in dogs were comparable to those in humans (within 10-fold), whereas in rats, those for the human CYP2D6 substrates were much higher and showed low correlation with humans. In dog intestine, as with human intestine, CLint values for almost all human CYP1A2, CYP2C, CYP2D6 substrates were not determined because they were very low. Intestinal CLint values for human CYP3A substrates in rats and dogs appeared to be lower for most of the compounds and showed moderate correlation with those in humans. 4. In conclusion, dogs showed the most similar metabolic activity to humans.

  12. Membrane properties induced by anionic phospholipids and phosphatidylethanolamine are critical for the membrane binding and catalytic activity of human cytochrome P450 3A4.

    PubMed

    Kim, Keon-Hee; Ahn, Taeho; Yun, Chul-Ho

    2003-12-30

    Human cytochrome P450 (CYP) 3A4, a membrane anchoring protein, is the major CYP enzyme present in both liver and small intestine. The enzyme plays a major role in the metabolism of many drugs and procarcinogens. The roles of individual phospholipids and membrane properties in the catalytic activity, membrane binding, and insertion into the membrane of CYP3A4 are poorly understood. Here we report that the catalytic activity of testosterone 6beta-hydroxylation, membrane binding, and membrane insertion of CYP3A4 increase as a function of anionic phospholipid concentration in the order phosphatidic acid (PA) > phosphatidylserine (PS) in a binary system of phosphatidylcholine (PC)/anionic phospholipid and as a function of phosphatidylethanolamine (PE) content in ternary systems of PC/PE/PA or PC/PE/PS having a fixed concentration of anionic phospholipids. These results suggest that PA and PE might help the binding of CYP3A4 to the membrane and the interaction with NPR. Cytochrome b(5) (b(5)) and apolipoprotein b(5) further enhanced the testosterone 6beta-hydroxylation activities of CYP3A4 in all tested phospholipids vesicles with various compositions. Phospholipid-dependent changes of the CYP3A4 conformation were also revealed by altered Trp fluorescence and CD spectra. We also found that PE induced the formation of anionic phospholipid-enriched domains in ternary systems using extrinsic fluorescent probes incorporated into lipid bilayers. Taken together, it can be suggested that the chemical and physical properties of membranes induced by anionic phospholipids and PE are critical for the membrane binding and catalytic activity of CYP3A4.

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

    PubMed

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

    2007-07-01

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

  14. Stimulatory effect of sesamin on hepatic cytochrome P450 activities in Atlantic salmon (Salmo salar L.) is not directly associated with expression of genes related to xenobiotic metabolism.

    PubMed

    Zlabek, Vladimir; Vestergren, AnnaLotta Schiller; Trattner, Sofia; Wagner, Liane; Pickova, Jana; Zamaratskaia, Galia

    2015-01-01

    1. This study examined hepatic cytochrome P450 (CYP450) response to dietary sesamin in combination with different n-6/n-3 fatty acid ratios in fish diet. Over a period of 4 months, fish were fed seven different experimental diets an n-6/n-3 FA ratio of either 0.5 or 1.0 in combination with two sesamin levels: low sesamin = 1.16 g/kg feed and high sesamin = 5.8 g/kg feed. Control diets did not contain sesamin. 2. The CYP450-associated activities of ethoxyresorufin O-deethylase (EROD), 7-benzyloxy-4-trifluoromethylcoumarin O-debenzylation (BFCOD), pentoxyresorufin O-depentylase (PROD), coumarin hydroxylase (COH), methoxyresorufin O-deethylase (MROD) and p-nitrophenol hydroxylase (PNPH) were significantly induced by dietary sesamin in a dose-related manner. 3. Expressions of the genes CYP1A1, CYP1A3, CYP3A, AhR1α, AhR2β, AhR2δ and PXR involved in the regulation of CYP450 activities, was not the primary source of this induction.

  15. Eicosapentaenoic acid increases cytochrome P-450 2J2 gene expression and epoxyeicosatrienoic acid production via peroxisome proliferator-activated receptor γ in endothelial cells.

    PubMed

    Wang, Dahai; Hirase, Tetsuaki; Nitto, Takeaki; Soma, Masaaki; Node, Koichi

    2009-12-01

    ω-3 fatty acids, such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), have beneficial effects on cardiovascular diseases. Cytochrome P-450 (CYP) 2J2 that is expressed in endothelial cells metabolizes arachidonic acids to biologically active epoxyeicosatrienoic acids (EETs) that possess anti-inflammatory and anti-thrombotic effects. We studied the effects of EPA and DHA on the expression of CYP 2J2 mRNA by reverse transcription-polymerase chain reaction in cultured human umbilical vein endothelial cells and found that EPA, but not DHA, increased the expression of CYP 2J2 mRNA in a dose-dependent and a time-dependent manner. EPA-induced CYP 2J2 expression was significantly inhibited by pretreatment with a peroxisome proliferator-activated receptor (PPAR) γ antagonist, GW9662. EPA, but not DHA, caused a significant increase in cellular levels of 11,12-dihydroxyeicosatrienoic acid that is a stable metabolite of 11,12-EET, which was blocked by pretreatment with GW9662. These data demonstrate that EPA increases CYP 2J2 mRNA expression and 11,12-EET production via PPARγ in endothelial cells and indicate a novel protective role of EPA and PPARγ against vascular inflammation.

  16. In vitro identification of human cytochrome P450 isoforms involved in the metabolism of Geissoschizine methyl ether, an active component of the traditional Japanese medicine Yokukansan.

    PubMed

    Matsumoto, Takashi; Kushida, Hirotaka; Maruyama, Takeshi; Nishimura, Hiroaki; Watanabe, Junko; Maemura, Kazuya; Kase, Yoshio

    2016-01-01

    1. Yokukansan (YKS) is a traditional Japanese medicine also called kampo, which has been used to treat neurosis, insomnia, and night crying and peevishness in children. Geissoschizine methyl ether (GM), a major indole alkaloid found in Uncaria hook, has been identified as a major active component of YKS with psychotropic effects. Recently, GM was reported to have a partial agonistic effect on serotonin 5-HT1A receptors. However, there is little published information on GM metabolism in humans, although several studies reported the blood kinetics of GM in rats and humans. In this study, we investigated the GM metabolic pathways and metabolizing enzymes in humans. 2. Using recombinant human cytochrome P450 (CYP) isoforms and polyclonal antibodies to CYP isoforms, we found that GM was metabolized into hydroxylated, dehydrogenated, hydroxylated+dehydrogenated, demethylated and water adduct forms by some CYP isoforms. 3. The relative activity factors in human liver microsomes were calculated to determine the relative contributions of individual CYP isoforms to GM metabolism in human liver microsomes (HLMs). We identified CYP3A4 as the CYP isoform primarily responsible for GM metabolism in human liver microsomes. 4. These findings provide an important basis for understanding the pharmacokinetics and pharmacodynamics of GM and YKS.

  17. In vitro chemopreventive potential of fucophlorethols from the brown alga Fucus vesiculosus L. by anti-oxidant activity and inhibition of selected cytochrome P450 enzymes.

    PubMed

    Parys, Sabine; Kehraus, Stefan; Krick, Anja; Glombitza, Karl-Werner; Carmeli, Shmuel; Klimo, Karin; Gerhäuser, Clarissa; König, Gabriele M

    2010-02-01

    Within a project focusing on the chemopreventive potential of algal phenols, two phloroglucinol derivatives, belonging to the class of fucophlorethols, and the known fucotriphlorethol A were obtained from the ethanolic extract of the brown alga Fucus vesiculosus L. The compounds trifucodiphlorethol A and trifucotriphlorethol A are composed of six and seven units of phloroglucinol, respectively. The compounds were examined for their cancer chemopreventive potential, in comparison with the monomer phloroglucinol. Trifucodiphlorethol A, trifucotriphlorethol A as well as fucotriphlorethol A were identified as strong radical scavengers, with IC(50) values for scavenging of 1,1-diphenyl-2 picrylhydrazyl radicals (DPPH) in the range of 10.0-14.4 microg/ml. All three compounds potently scavenged peroxyl radicals in the oxygen radical absorbance capacity (ORAC) assay. In addition, the compounds were shown to inhibit cytochrome P450 1A activity with IC(50) values in the range of 17.9-33 microg/ml, and aromatase (Cyp19) activity with IC(50) values in the range of 1.2-5.6 microg/ml.

  18. Effects of protein flexibility and active site water molecules on the prediction of sites of metabolism for cytochrome P450 2C19 substrates.

    PubMed

    Li, Junhao; Cai, Jinya; Su, Haixia; Du, Hanwen; Zhang, Juan; Ding, Shihui; Liu, Guixia; Tang, Yun; Li, Weihua

    2016-03-01

    Structure-based prediction of sites of metabolism (SOMs) mediated by cytochrome P450s (CYPs) is of great interest in drug discovery and development. However, protein flexibility and active site water molecules remain a challenge for accurate SOM prediction. CYP2C19 is one of the major drug-metabolizing enzymes and has attracted considerable attention because of its polymorphism and capability of metabolizing ∼7% clinically used drugs. In this study, we systematically evaluated the effects of protein flexibility and active site water molecules on SOM prediction for CYP2C19 substrates. Multiple conformational sampling techniques including GOLD flexible residues sampling, molecular dynamics (MD) and tCONCOORD side-chain sampling were adopted for assessing the influence of protein flexibility on SOM prediction. The prediction accuracy could be significantly improved when protein flexibility was considered using the tCONCOORD sampling method, which indicated that the side-chain conformation was important for accurate prediction. However, the inclusion of the crystallographic or MD-derived water molecule(s) does not necessarily improve the prediction accuracy. Finally, a combination of docking results with SMARTCyp was found to be able to increase the SOM prediction accuracy.

  19. Characterization of maize cytochrome P450 monooxygenases induced in response to safeners and bacterial pathogens.

    PubMed

    Persans, M W; Wang, J; Schuler, M A

    2001-02-01

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

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

    PubMed Central

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

    2001-01-01

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

  1. 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.

  2. cDNA and derived amino acid sequence of ethanol-inducible rabbit liver cytochrome P-450 isozyme 3a (P-450ALC).

    PubMed Central

    Khani, S C; Zaphiropoulos, P G; Fujita, V S; Porter, T D; Koop, D R; Coon, M J

    1987-01-01

    Administration of ethanol to rabbits is known to induce a unique liver microsomal cytochrome P-450, termed isozyme 3a or P-450ALC, which is responsible for the increased oxidation of ethanol and other alcohols and the activation of toxic or carcinogenic compounds such as acetaminophen and N-nitrosodimethylamine. To further characterize this cytochrome P-450 we have identified cDNA clones to isozyme 3a by immunoscreening, DNA hybridization, and hybridization-selection. The cDNA sequence determined from two overlapping clones contains an open reading frame of 1416 nucleotides, and the first 25 amino acids of this reading frame correspond to residues 21-45 of cytochrome P-450 3a. The complete polypeptide, including residues 1 to 20, contains 492 amino acids and has a molecular weight of 56,820. Cytochrome P-450 3a is approximately 55% identical in sequence to P-450 isozymes 1 and 3b and 48% identical to isozyme 2. Hybridization of clone p3a-2 to electrophoretically fractionated rabbit liver poly(A)+ RNA revealed multiple bands, but, with a probe derived from the 3' nontranslated portion of this cDNA, only a 1.9-kilobase band was observed. Treatment of rabbits with imidazole, which increases the content of isozyme 3a, resulted in a transient increase in form 3a mRNA, but this was judged to be insufficient to account for the known 4.5-fold increase in form 3a protein. Genomic DNA analysis indicated that the cytochrome P-450 3a gene does not belong to a large subfamily. Images PMID:3027695

  3. Adrenodoxin supports reactions catalyzed by microsomal steroidogenic cytochrome P450s

    SciTech Connect

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

    2007-02-16

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

  4. A Comparison of the In Vitro Inhibitory Effects of Thelephoric Acid and SKF-525A on Human Cytochrome P450 Activity.

    PubMed

    Song, Min; Do, Hyunhee; Kwon, Oh Kwang; Yang, Eun-Ju; Bae, Jong-Sup; Jeong, Tae Cheon; Song, Kyung-Sik; Lee, Sangkyu

    2014-02-01

    Thelephoric acid is an antioxidant produced by the hydrolysis of polyozellin, which is isolated from Polyozellus multiplex. In the present study, the inhibitory effects of polyozellin and thelephoric acid on 9 cytochrome P450 (CYP) family members (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4) were examined in pooled human liver microsomes (HLMs) using a cocktail probe assay. Polyozellin exhibited weak inhibitory effects on the activities of all 9 CYPs examined, whereas thelephoric acid exhibited dose- and time-dependent inhibition of all 9 CYP isoforms (IC50 values, 3.2-33.7 μM). Dixon plots of CYP inhibition indicated that thelephoric acid was a competitive inhibitor of CYP1A2 and CYP3A4. In contrast, thelephoric acid was a noncompetitive inhibitor of CYP2D6. Our findings indicate that thelephoric acid may be a novel, non-specific CYP inhibitor, suggesting that it could replace SKF-525A in inhibitory studies designed to investigate the effects of CYP enzymes on the metabolism of given compounds.

  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. Effects of Eleutheroside B and Eleutheroside E on activity of cytochrome P450 in rat liver microsomes

    PubMed Central

    2014-01-01

    Background Chemicals of herbal products may cause unexpected toxicity or adverse effect by the potential for alteration of the activity of CYP450 when co-administered with other drugs. Eleutherococcus senticosus (ES), has been widely used as a traditional herbal medicine and popular herbal dietary supplements, and often co-administered with many other drugs. The main bioactive constituents of ES were considered to be eleutherosides including eleutheroside B (EB) and eleutheroside E (EE). This study was to investigate the effects of EB and EE on CYP2C9, CYP2D6, CYP2E1 and CYP3A4 in rat liver microsomes in vitro. Method Probe drugs of tolbutamide (TB), dextromethorphan (DM), chlorzoxazone (CLZ) and testosterone (TS) as well as eleutherosides of different concentrations were added to incubation systems of rat liver microsomes in vitro. After incubation, validated HPLC methods were used to quantify relevant metabolites. Results The results suggested that EB and EE exhibited weak inhibition against the activity of CYP2C9 and CYP2E1, but no effects on CYP2D6 and CYP3A4 activity. The IC50 values for EB and EE were calculated to be 193.20 μM and 188.36 μM for CYP2E1, 595.66 μM and 261.82 μM for CYP2C9, respectively. Kinetic analysis showed that inhibitions of CYP2E1 by EB and EE were best fit to mixed-type with Ki value of 183.95 μM and 171.63 μM, respectively. Conclusions These results indicate that EB and EE may inhibit the metabolism of drugs metabolized via CYP2C9 and CYP2E1, and have the potential to increase the toxicity of the drugs. PMID:24383621

  7. Minor activities and transition state properties of the human steroid hydroxylases cytochromes P450c17 and P450c21, from reactions observed with deuterium-labeled substrates

    PubMed Central

    Yoshimoto, Francis K.; Zhou, Yishan; Peng, Hwei-Ming; Stidd, David; Yoshimoto, Jennifer A.; Sharma, Kamalesh K.; Matthew, Susan; Auchus, Richard J.

    2012-01-01

    The steroid hydroxylases CYP17A1 (P450c17, 17-hydroxylase/17,20-lyase) and CYP21A2 (P450c21, 21-hydroxylase) catalyze progesterone hydroxylation at one or more sites within a 2 Å radius. We probed their hydrogen atom abstraction mechanisms and regiochemical plasticity with deuterium-labeled substrates: 17-[2H]-pregnenolone; 17-[2H]-, 16α-[2H]-, 21,21,21-[2H3]-, and 21-[2H]-progesterone; and 21,21,21-[2H3]-17-hydroxyprogesterone. Product distribution and formation rates with recombinant human P450-oxidoreductase and wild-type human CYP17A1 or mutation A105L (reduced progesterone 16α-hydroxylation) and wild-type human CYP21A2 or mutation V359A (substantial progesterone 16α-hydroxylation) were used to calculate intramolecular and intermolecular kinetic isotope effects (KIEs). The intramolecular KIEs for CYP17A1 and mutation A105L were 4.1 and 3.8, respectively, at H-17 and 2.9 and 5.1, respectively, at H-16α. Mutation A105L 21-hydroxylates progesterone (5% of products), and wild-type CYP17A1 also catalyzes a trace of 21-hydroxylation, which increases with 16α-[2H]- and 17-[2H]-progesterone. The intramolecular KIEs with CYP21A2 mutation V359A and progesterone were 6.2 and 3.8 at H-21 and H-16α, respectively. Wild-type CYP21A2 also forms a trace of 16α-hydroxyprogesterone, which increased with 21,21,21-[2H3]-progesterone substrate. Competitive intermolecular KIEs paralleled the intramolecular KIE values, with DV values of 1.4–5.1 and DV/K values of 1.8–5.1 for these reactions. CYP17A1 and CYP21A2 mutation V359A both 16α-hydroxylate 16α-[2H]-progesterone with 33–44% deuterium retention, indicating stereochemical inversion. We conclude that human CYP17A1 has progesterone 21-hydroxylase activity and human CYP21A2 has progesterone 16α-hydroxylase activity, both of which are enhanced with deuterated substrates. The transition states for C-H bond cleavage in these hydroxylation reactions are either significantly non-linear and/or asymmetric, and C-H bond

  8. Expression of cytochromes P-450 in rat hepatoma cells. Analysis by monoclonal antibodies specific for cytochromes P-450 from rat liver induced by 3-methylcholanthrene or phenobarbital.

    PubMed

    Wiebel, F J; Park, S S; Kiefer, F; Gelboin, H V

    1984-12-17

    We have studied the expression of aldrin eposidase (AE), 7-ethoxycoumarin-O-deethylase (ECDE), and aryl hydrocarbon (benzo[a]pyrene) hydroxylase (AHH) in nine differentiated or dedifferentiated cell lines derived from H4IIEC3 rat hepatoma cells. The nature of the cytochromes P-450 mediating AE, ECDE and AHH activities was analysed using monoclonal antibodies (MAb) made to the major 3-methylcholanthrene-induced cytochrome P-450 (MAb-MC) or phenobarbital-induced cytochrome P-450 (MAb-PB) from rat liver. The cells were treated with 5 microM dexamethasone for 30 h to increase the levels of the monoxygenase activities. (a) The six differentiated cell lines examined (Faza967, Fao, HF1-4, 2sFou, C2Rev7, and H4IIEC3/G-) contained MAb-PB-sensitive AE comprising 30-75% of the total AE activity. In most of these cell lines MAb-PB also markedly inhibited ECDE; however, the antibody had a considerably weaker effect on AHH. (b) MAb-PB-sensitive AHH, ECDE and AE activities were also observed in untreated and phenobarbital-treated cells. (c) MAb-MC inhibited AHH and ECDE in the two dedifferentiated lines HF1 and H5 by 50-80%. The antibody also inhibited AHH activities in the poorly differentiated line H4IIEC3/T and in the majority of the differentiated lines by 40-65%. MAb-MC-sensitive AHH was found in Fao cells after treatment with benz[a]anthracene but induced AHH in H4IIEC3/T, H4IIEC3/G-, and 2sFou cells 20-30-fold and in Faza967 and Fao cells 3-5-fold. Benz[a]anthracene remained without effect on AHH activity in C2Rev7 cells. The results show that the hepatoma cells examined express to various degrees phenobarbital-inducible cytochrome P-450 and/or 3-methylcholanthrene-inducible cytochrome P-450. These cell lines are versatile tools for studying the regulation of monooxygenase activities and analysing their role in the activation and inactivation of xenobiotics such as carcinogens, drugs and pesticides.

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

    PubMed Central

    2015-01-01

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

  10. 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

    2014-10-07

    Cytochrome P450 (P450) 4A11 is the only functionally active subfamily 4A P450 in humans. P450 4A11 catalyzes mainly ω-hydroxylation of fatty acids in liver and kidney; this process is not a major degradative pathway, but at least one product, 20-hydroxyeicosatetraenoic acid, has important signaling properties. We studied catalysis by P450 4A11 and the issue of rate-limiting steps using lauric acid ω-hydroxylation, a prototypic substrate for this enzyme. Some individual reaction steps were studied using pre-steady-state kinetic approaches. Substrate and product binding and release were much faster than overall rates of catalysis. Reduction of ferric P450 4A11 (to ferrous) was rapid and not rate-limiting. Deuterium kinetic isotope effect (KIE) experiments yielded low but reproducible values (1.2-2) for 12-hydroxylation with 12-(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 ω-hydroxylation ∼2-fold; the apoprotein was ineffective, indicating that electron transfer is involved in the b5 enhancement. The rate of b5 reoxidation was increased in the presence of ferrous P450 mixed with O2. Collectively, the results indicate that both the transfer of an electron to the ferrous·O2 complex and C-H bond-breaking limit the rate of P450 4A11 ω-oxidation.

  11. Inhibition and induction of cytochrome P450 2B1 in rat liver by promazine and chlorpromazine.

    PubMed

    Murray, M

    1992-09-25

    Phenothiazine tranquilizers have been associated with pharmacokinetic drug interactions in man. In this study the in vivo and in vitro effects of the clinically important phenothiazines promazine (PZ) and chlorpromazine (CPZ) on drug oxidations catalysed by specific cytochrome P450 (P450) enzymes were investigated in the rat. In vitro, the two drugs were relatively ineffective inhibitors of constitutive P450 activities, but were inhibitory toward the principal phenobarbital-inducible P450 2B1 and, to a lesser extent, P450 1A1. Administration of PZ and CPZ to male rats did not markedly influence the total microsomal P450 content of the liver. However, the quantitatively important male-specific P450 2C11 was down-regulated by CPZ and concomitant induction of P450 2B1 and associated 7-pentylresorufin O-depentylase activity were noted. A small increase in the activity of microsomal 7-ethylresorufin O-deethylase was also observed following administration of both drugs to rats, suggesting induction of P450 1A1/2. Considered together, it is apparent that the two phenothiazines are preferential inhibitors and inducers of P450 2B1 in rat liver. Drug interactions in humans involving phenothiazines may reflect a combined effect of induction and inhibition processes as well as down-regulation of other P450s, such as that produced by CPZ on P450 2C11.

  12. 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

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

    PubMed Central

    Zimmerlin, Alfred; Durst, Francis

    1992-01-01

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

  14. Spaceflight Effects on Cytochrome P450 Content in Mouse Liver

    PubMed Central

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

    2015-01-01

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

  15. Strain differences in cytochrome P450 mRNA and protein expression, and enzymatic activity among Sprague Dawley, Wistar, Brown Norway and Dark Agouti rats.

    PubMed

    Nishiyama, Yoshihiro; Nakayama, Shouta M M; Watanabe, Kensuke P; Kawai, Yusuke K; Ohno, Marumi; Ikenaka, Yoshinori; Ishizuka, Mayumi

    2016-05-03

    Rat cytochrome P450 (CYP) exhibits inter-strain differences, but their analysis has been scattered across studies under different conditions. To identify these strain differences in CYP more comprehensively, mRNA expression, protein expression and metabolic activity among Wistar (WI), Sprague Dawley (SD), Dark Agouti (DA) and Brown Norway (BN) rats were compared. The mRNA level and enzymatic activity of CYP1A1 were highest in SD rats. The rank order of Cyp3a2 mRNA expression mirrored its protein expression, i.e., DA>BN>SD>WI, and was similar to the CYP3A2-dependent warfarin metabolic activity, i.e., DA>SD>BN>WI. These results suggest that the strain differences in CYP3A2 enzymatic activity are caused by differences in mRNA expression. Cyp2b1 mRNA levels, which were higher in DA rats, did not correlate with its protein expression or enzymatic activity. This suggests that the strain differences in enzymatic activity are not related to Cyp2b1 mRNA expression. In conclusion, WI rats tended to have the lowest CYP1A1, 2B1 and 3A2 mRNA expression, protein expression and enzymatic activity among the strains. In addition, SD rats had the highest CYP1A1 mRNA expression and activity, while DA rats had higher CYP2B1 and CYP3A2 mRNA and protein expression. These inter-strain differences in CYP could influence pharmacokinetic considerations in preclinical toxicological studies.

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

    PubMed Central

    Yoshimoto, Francis K.; Auchus, Richard J.

    2014-01-01

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

  17. Structures of Cytochrome P450 2B6 Bound to 4-Benzylpyridine and 4-(4-Nitrobenzyl)pyridine: Insight into Inhibitor Binding and Rearrangement of Active Site Side Chains

    PubMed Central

    Pascual, Jaime; Zhang, Qinghai; Stout, C. David; Halpert, James R.

    2011-01-01

    The biochemical, biophysical, and structural analysis of the cytochrome P450 2B subfamily of enzymes has provided a wealth of information regarding conformational plasticity and substrate recognition. The recent X-ray crystal structure of the drug-metabolizing P450 2B6 in complex with 4-(4-chlorophenyl)imidazole (4-CPI) yielded the first atomic view of this human enzyme. However, knowledge of the structural basis of P450 2B6 specificity and inhibition has remained limited. In this study, structures of P450 2B6 were determined in complex with the potent inhibitors 4-benzylpyridine (4-BP) and 4-(4-nitrobenzyl)pyridine (4-NBP). Comparison of the present structures with the previous P450 2B6-4-CPI complex showed that reorientation of side chains of the active site residue Phe206 on the F-helix and Phe297 on the I-helix was necessary to accommodate the inhibitors. However, P450 2B6 does not require any major side chain rearrangement to bind 4-NBP compared with 4-BP, and the enzyme provides no hydrogen-bonding partners for the polar nitro group of 4-NBP within the hydrophobic active site. In addition, on the basis of these new structures, substitution of residue 172 with histidine as observed in the single nucleotide polymorphism Q172H and in P450 2B4 may contribute to a hydrogen bonding network connecting the E- and I-helices, thereby stabilizing active site residues on the I-helix. These results provide insight into the role of active site side chains upon inhibitor binding and indicate that the recognition of the benzylpyridines in the closed conformation structure of P450 2B6 is based solely on hydrophobicity, size, and shape. PMID:21875942

  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

  19. Cytochrome P450 1A2 (CYP1A2) activity and risk factors for breast cancer: a cross-sectional study

    PubMed Central

    Hong, Chi-Chen; Tang, Bing-Kou; Hammond, Geoffrey L; Tritchler, David; Yaffe, Martin; Boyd, Norman F

    2004-01-01

    Introduction Breast cancer risk may be determined by various genetic, metabolic, and lifestyle factors that alter sex hormone metabolism. Cytochrome P450 1A2 (CYP1A2) is responsible for the metabolism of estrogens and many exogenous compounds, including caffeine. Methods In a cross-sectional study of 146 premenopausal and 149 postmenopausal women, we examined the relationships between CYP1A2 activity and known or suspected risk factors for breast cancer. Blood levels of sex hormones, lipids, and growth factors were measured. In vivo CYP1A2 activity was assessed by measuring caffeine metabolites in urine. Stepwise and maximum R regression analyses were used to identify covariates related to CYP1A2 activity after adjustment for ethnicity. Results In both menopausal groups CYP1A2 activity was positively related to smoking and levels of sex hormone binding globulin. In premenopausal women, CYP1A2 activity was also positively related to insulin levels, caffeine intake, age, and plasma triglyceride levels, and negatively related with total cholesterol levels and body mass index. In postmenopausal women CYP1A2 activity was positively associated with insulin-like growth factor-1, and negatively associated with plasma triglyceride, high-density lipoprotein cholesterol, and age at menarche. Conclusion These results suggest that CYP1A2 activity is correlated with hormones, blood lipids, and lifestyle factors associated with breast cancer risk, although some of the observed associations were contrary to hypothesized directions and suggest that increased CYP1A2 function may be associated with increased risk for breast cancer. PMID:15217502

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

    SciTech Connect

    Wang, Yue-Ming; Lin, Wenwei; Chai, Sergio C.; Wu, Jing; Ong, Su Sien; Schuetz, Erin G.; Chen, Taosheng

    2013-10-01

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

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

    PubMed Central

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

    1993-01-01

    Cytochrome P-450lin catalyzes the 8-methyl hydroxylation of linalool as the first committed step of its utilization by Pseudomonas incognita as the sole carbon source. By using a polymerase chain reaction-based cloning strategy, a 2.1-kb DNA fragment containing the cytochrome P-450lin gene (linC) was isolated. An open reading frame of 406 amino acids has been identified as that of P-450lin on the basis of amino acid sequence data from peptides of the native protein. Heterologous expression of functional holoprotein is exhibited by Escherichia coli transformed with pUC18 containing the subcloned linC gene under constitutive transcriptional control of the lac promoter. The G+C content of linC was found to be 55% overall and 58% in the third codon position. An optimized amino acid sequence alignment of P-450lin with cytochrome P-450cam shows that the two enzymes have only 25% identity. P-450lin was found to exhibit the expected conservation in the axial cysteine heme ligand-containing peptide and the threonine region postulated to form an O2-binding pocket (T. L. Poulos, B. C. Finzel, and A. J. Howard, J. Mol. Biol. 195:687-700, 1987). The low amino acid sequence identity between P-450lin and all other P-450 sequences has shown that P-450lin is the first member of the CYP111 P-450 gene family. PMID:8376348

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

    PubMed Central

    Wanwimolruk, Sompon; Phopin, Kamonrat; Prachayasittikul, Virapong

    2014-01-01

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

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

    PubMed Central

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

    2015-01-01

    Cytochrome P450 (P450) 20A1 is one of the so-called “orphan” P450s without assigned biological function. mRNA expression was detected in human liver and extrahepatic expression was noted in several human brain regions, including substantia nigra, hippocampus, and amygdala, using conventional polymerase chain reaction and RNA dot blot analysis. Adult human liver contained 3-fold higher overall mRNA levels than whole brain, although specific regions (i.e., hippocampus and substantia nigra) exhibited higher mRNA expression levels than liver. Orthologous full-length and truncated transcripts of P450 20A1 were transcribed and sequenced from rat liver, heart, and brain. In rat, the concentrations of full-length transcripts were 3–4 fold higher in brain and heart than liver. In situ hybridization of rat whole brain sections showed a similar mRNA expression pattern as observed for human P450 20A1, indicating expression in substantia nigra, hippocampus, and amygdala. A number of N-terminal modifications of the codon-optimized human P450 20A1 sequence were prepared and expressed in Escherichia coli, and two of the truncated derivatives showed characteristic P450 spectra (200–280 nmol P450/l). Although the recombinant enzyme system oxidized NADPH, no catalytic activity was observed with the heterologously expressed protein when a number of potential steroids and biogenic amines were surveyed as potential substrates. The function of P450 20A1 remains unknown; however, the sites of mRNA expression in human brain and the conservation among species may suggest possible neurophysiological function. PMID:18541694

  4. Systemic exposure of topical erythromycin in comparison to oral administration and the effect on cytochrome P450 3A4 activity

    PubMed Central

    Carls, Alexandra; Jedamzik, Julia; Witt, Lukas; Hohmann, Nicolas; Burhenne, Juergen; Mikus, Gerd

    2014-01-01

    Aims Erythromycin is a macrolide antibiotic, which is frequently used as a topical formulation for the treatment of acne. It is also known as an inhibitor of the cytochrome P450 (CYP) isoenzyme 3A4. In this study, the systemic availability of topical erythromycin, hence the influence on the activity of CYP3A, is evaluated in comparison to orally administered erythromycin. Methods Sixteen healthy volunteers received consecutively topical (two applications of 800 mg) and oral erythromycin (two dose groups, 250 and 1000 mg, with n = 8) to assess erythromycin pharmacokinetics. A microdose of midazolam (3 μg orally) was used to determine the effect on CYP3A activity. Results After topical administration, erythromycin was detected in the plasma of every participant without causing a statistically significant alteration of CYP3A activity. After oral administration, the dose-normalized erythromycin exposure (AUC∞) was 1335 h ng ml−1 after 250 mg and 3-fold higher after the 1000 mg dose (4051 h ng ml−1; P < 0.01), suggesting nonlinear pharmacokinetics of erythromycin. Both oral doses inhibited CYP3A activity; midazolam clearance was decreased to 61% (250 mg) and 21% (1000 mg). The relationship between erythromycin exposure and CYP3A activity (Hill equation) revealed a 50% reduction of CYP3A activity by an erythromycin AUC∞ of 2106 h ng ml−1. Conclusions Topical erythromycin did not cause clinically relevant CYP3A alterations, although low systemic availability of erythromycin was observed. This supports the assumption that treatment with topical erythromycin is not critical in terms of CYP3A inhibition. Furthermore, substantial nonlinearity of erythromycin pharmacokinetics after two different oral doses was observed, possibly due to autoinhibition. PMID:25139487

  5. Multiple doses of saw palmetto (Serenoa repens) did not alter cytochrome P450 2D6 and 3A4 activity in normal volunteers.

    PubMed

    Markowitz, John S; Donovan, Jennifer L; Devane, C Lindsay; Taylor, Robin M; Ruan, Ying; Wang, Jun-Sheng; Chavin, Kenneth D

    2003-12-01

    Saw palmetto (Serenoa repens) is the most commonly used herbal preparation in the treatment of benign prostatic hyperplasia. The objective of this study was to determine whether a characterized saw palmetto product affects the activity of cytochrome P450 (CYP) 2D6 or 3A4 in healthy volunteers (6 men and 6 women). The probe substrates dextromethorphan (CYP2D6 activity) and alprazolam (CYP3A4 activity) were administered orally at baseline and again after exposure to saw palmetto (320-mg capsule once daily) for 14 days. Dextromethorphan metabolic ratios and alprazolam pharmacokinetics were determined at baseline and after saw palmetto treatment. The mean ratio of dextromethorphan to its metabolite was 0.038 +/- 0.044 at baseline and 0.048 +/- 0.080 after 14 days of saw palmetto administration (P =.704, not significant [NS]), indicating a lack of effect on CYP2D6 activity. The area under the plasma alprazolam concentration versus time curve was 476 +/- 178 h. ng. mL(-1) at baseline and 479 +/- 125 h. ng. mL(-1) after saw palmetto treatment (P =.923, NS), indicating a lack of effect on CYP3A4 activity. The elimination half-life of alprazolam was 11.4 +/- 3.1 hours at baseline and 11.6 +/- 2.7 hours after saw palmetto treatment (P =.770, NS), also indicating a lack of effect on CYP3A4 activity. Our results indicate that extracts of saw palmetto at generally recommended doses are unlikely to alter the disposition of coadministered medications primarily dependent on the CYP2D6 or CYP3A4 pathways for elimination. These conclusions must be weighed in the context of the study's limited assessments and regarded as only the initial investigation into the drug interaction potential of saw palmetto.

  6. Reverse type I binding spectra of human cytochrome P450 1B1 induced by flavonoid, stilbene, pyrene, naphthalene, phenanthrene, and biphenyl derivatives that inhibit catalytic activity: a structure-function relationship study.

    PubMed

    Shimada, Tsutomu; Tanaka, Katsuhiro; Takenaka, Shigeo; Foroozesh, Maryam K; Murayama, Norie; Yamazaki, Hiroshi; Guengerich, F Peter; Komori, Masayuki

    2009-07-01

    Fifty-one chemicals including derivatives of 16 flavonoids, three stilbenes, six pyrenes, seven naphthalenes, seven phenanthrenes, 10 biphenyls, 17beta-estradiol, and estrone were examined for their abilities to induce reverse type I binding spectra with human cytochrome P450 (P450) 1B1 and to inhibit 7-ethoxyresorufin O-deethylation (EROD) activities catalyzed by P450 1B1. Forty-nine chemicals showed reverse type I spectra with P450 1B1, and we found that 3,5,7-trihydroxyflavone, 3',4'-dimethoxy-5,7-dihydroxyflavone, 4'-methoxy-5,7-dihydroxyflavone, alpha- and beta-naphthoflavones, 2,4,3',5'-tetramethoxystilbene, pyrene, and several acetylenic pyrenes and phenanthrenes were strong inducers of the spectra and also potent inhibitors of EROD activities catalyzed by P450 1B1. The spectral dissociation constant (K(s)) and the magnitude of the binding (DeltaA(max)/K(s)) of 49 chemicals were correlated with the inhibition potencies of EROD activities by these chemicals [correlation coefficients (r) of 0.72 and 0.74, respectively]. The K(s) and DeltaA(max)/K(s) values were more correlated with IC(50) values when compared in a group of derivatives of flavonoids, stilbenes, and estrogens (r = 0.81 and 0.88, respectively) or a group of derivatives of pyrenes, naphthalenes, phenanthrenes, and biphenyls (r = 0.88 and 0.91, respectively). Among 14 flavonoids examined, 3,5,7-trihydroxyflavone and 4'-methoxy- and 3',4'-dimethoxy-5,7-dihydroxyflavone were more active than flavone in interacting with P450 1B1, but the respective 7,8-dihydroxyflavones were less active. Pyrene itself was highly active in interacting with P450 1B1, but its binding was slightly decreased when substituted with acetylenic groups. In contrast, substitution of naphthalene with methyl and ethyl propargyl ethers led to more interaction with P450 1B1 than with naphthalene itself. Similarly, substitution on phenanthrene or biphenyl with acetylenic groups and propargyl ethers increased affinities to P450 1B1

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

    SciTech Connect

    Gray, Joshua P.; Mishin, Vladimir; Heck, Diane E.; Laskin, Debra L.; Laskin, Jeffrey D.

    2010-09-01

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

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

    PubMed Central

    Wanwimolruk, Sompon; Prachayasittikul, Virapong

    2014-01-01

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

  9. Interindividual Variability in Cytochrome P450–Mediated Drug Metabolism

    PubMed Central

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

    2016-01-01

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

  10. Valence tautomerism in synthetic models of cytochrome P450

    PubMed Central

    Das, Pradip Kumar; Samanta, Subhra; McQuarters, Ashley B.; Lehnert, Nicolai

    2016-01-01

    CytP450s have a cysteine-bound heme cofactor that, in its as-isolated resting (oxidized) form, can be conclusively described as a ferric thiolate species. Unlike the native enzyme, most synthetic thiolate-bound ferric porphyrins are unstable in air unless the axial thiolate ligand is sterically protected. Spectroscopic investigations on a series of synthetic mimics of cytP450 indicate that a thiolate-bound ferric porphyrin coexists in organic solutions at room temperature (RT) with a thiyl-radical bound ferrous porphyrin, i.e., its valence tautomer. The ferric thiolate state is favored by greater enthalpy and is air stable. The ferrous thiyl state is favored by entropy, populates at RT, and degrades in air. These ground states can be reversibly interchanged at RT by the addition or removal of water to the apolar medium. It is concluded that hydrogen bonding and local electrostatics protect the resting oxidized cytP450 active site from degradation in air by stabilizing the ferric thiolate ground state in contrast to its synthetic analogs. PMID:27302948

  11. Limitations of in silico predictability of specificity of co-immobilised cytochromes P450 and mimics in food-bioprocessing.

    PubMed

    Wiseman, Alan

    2003-04-01

    Cytochromes P450 (EC 1.14.14.1) are mixed function oxidases (oxygenases) that can catalyse redox bioconversions of food components. Also, efficacious removal of undesirable components can be achieved using solid-support immobilised enzyme (IME) of a selection from 2700 isoforms of cytochromes P450 (CYP). Cytochromes P450 co-immobilised with other enzymes, or protein receptors, may be used to confer a secondary order of regio- or stereo-specificity of chiral bioconversion: these can be predictable in silico by utilisation of QSARs (quantitative structure/activity relationships).

  12. The aflatoxin B1 -fumonisin B1 toxicity in BRL-3A hepatocytes is associated to induction of cytochrome P450 activity and arachidonic acid metabolism.

    PubMed

    Mary, Verónica S; Arias, Silvina L; Otaiza, Santiago N; Velez, Pilar A; Rubinstein, Héctor R; Theumer, Martín G

    2017-02-09

    Human oral exposure to aflatoxin B1 (AFB1 ) and fumonisin B1 (FB1 ) is associated with increased hepatocellular carcinoma. Although evidence suggested interactive AFB1 -FB1 hepatotoxicity, the underlying mechanisms remain mostly unidentified. This work was aimed at evaluating the possible AFB1 -FB1 interplay to induce genetic and cell cycle toxicities in BRL-3A rat hepatocytes, reactive oxygen species (ROS) involvement, and the AFB1 metabolizing pathways cytochrome P450 (CYP) and arachidonic acid (ArAc) metabolism as ROS contributors. Flow cytometry of stained BRL-3A hepatocytes was used to study the cell cycle (propidium iodide), ROS intracellular production (DCFH-DA, HE, DAF-2 DA), and phospholipase A activity (staining with bis-BODIPY FL C11-PC). The CYP1A activity was assessed by the 7-ethoxyresorufin-O-deethylase (EROD) assay. Despite a 48-h exposure to FB1 (30 μM) not being genotoxic, the AFB1 (20 μM)-induced micronucleus frequency was overcome by the AFB1 -FB1 mixture (MIX), presumably showing toxin interaction. The mycotoxins blocked G1/S-phase, but only MIX caused cell death. Overall, the oxidative stress led these alterations as the pretreatment with N-acetyl-l-cysteine reduced such toxic effects. While AFB1 had a major input to the MIX pro-oxidant activity, with CYP and ArAc metabolism being ROS contributors, these pathways were not involved in the FB1 -elicited weak oxidative stress. The MIX-induced micronucleus frequency in N-acetyl-l-cysteine pretreated cells was greater than that caused by AFB1 without antioxidants, suggesting enhanced AFB1 direct genotoxicity probably owing to the higher CYP activity and ArAc metabolism found in MIX. The metabolic pathways modulation by AFB1 -FB1 mixtures could raise its hepatocarcinogenic properties.

  13. Isolation and characterization of a complementary DNA specific for human aromatase-system cytochrome P-450 mRNA.

    PubMed Central

    Evans, C T; Ledesma, D B; Schulz, T Z; Simpson, E R; Mendelson, C R

    1986-01-01

    A cloned complementary DNA sequence has been isolated from a human placental cDNA library in the bacteriophage expression vector lambda gt11 after screening with polyclonal antibodies against human placental aromatase-system cytochrome P-450 (P-450Arom). A single recombinant clone, lambda hAROM1, was characterized by its ability to generate a beta-galactosidase fusion protein that reacted independently with polyclonal antibodies raised against beta-galactosidase and cytochrome P-450Arom and with monoclonal antibodies specific for cytochrome P-450Arom. The cDNA insert, which was found to be 1.8 kilobases in length, was radiolabeled and used to analyze poly(A)+ RNA isolated from human placenta and total RNA isolated from human adipose stromal cells cultured in the absence or presence of regulatory factors. The radiolabeled cDNA hybridized to several size species of mRNA in both placental and adipose stromal cell RNA fractions. Changes in the levels of adipose stromal cell RNA that hybridized to the cDNA insert were associated with comparable changes in the levels of translatable cytochrome P-450Arom mRNA and aromatase system activity. These findings are indicative that lambda hAROM1 contains DNA sequences complementary to human cytochrome P-450Arom mRNA and are suggestive that regulatory factors affect aromatase activity by altering the transcriptional activity of the cytochrome P-450Arom gene. Images PMID:3018730

  14. 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.

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

    SciTech Connect

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

    1984-09-28

    Electrophoresis of Drosophila microsomes resolves two major hemecontaining protein bands with apparent molecular weights of 59,290 (band a) and 55,750 (band b). The hemoproteins in these two bands can account for most of the cytochrome P-450 in the organism. Band a is present in all strains examined: band b is not. Dimethylnitrosamine demethylase, a P-450 enzyme, is a component of band b. Numerous studies have shown that P-450-attributed activities of Drosophila are genotype dependent. Drosophila, therefore, represents a unique system for studying the genetics of, and the molecular mechanisms that regulate, the expression of constitutive levels of P-450 isozymes. Here we explore the molecular basis for the large differences in P-450 expression between strains. Microsomal proteins from several wild-type strains were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Significant differences in the protein bands that contained P-450 were observed between strains with high or low mixed-function oxidase activity. 22 references, 2 figures, 1 table.

  16. Electron transfer properties and catalytic competence of cytochrome b5 in the fusion protein Hmwb5-EGFP in reactions catalyzed by cytochrome P450 3A4.

    PubMed

    Yantsevich, A V; Gilep, A A; Usanov, S A

    2009-08-01

    In the present paper we describe studies on molecular mechanisms of protein-protein interactions between cytochrome P450 3A4 (CYP3A4) and cytochrome b(5), the latter being incorporated into the artificial recombinant protein Hmwb(5)-EGFP containing full-length cytochrome b(5) (functional module) and a mutant form of the green fluorescent protein EGFP (signal module) fused into a single polypeptide chain. It is shown that cytochrome b(5) within the fusion protein Hmwb(5)-EGFP can be reduced by NADPH-cytochrome P450 reductase in the presence of NADPH, the rate of reduction being dependent on solution ionic strength, indicating that the signal module does not prevent the interaction of the flavo- and hemeproteins. Interaction of cytochrome P450 3A4 and Hmwb(5)-EGFP was estimated based on spin equilibrium shift of cytochrome P450 3A4 to high-spin state in the presence of Hmwb(5)-EGFP, as well as based on steady-state fluorescence anisotropy of the EGFP component of the fusion protein in the presence of CYP3A4. The engineering of chimeric protein Hmwb(5)-EGFP gives an independent method to determine dissociation constant for the complex of cytochrome P450 and cytochrome b(5) that is less sensitive to environmental factors compared to spectrophotometric titration used before. Reconstitution of catalytic activity of cytochrome P450 3A4 in the reaction of testosterone 6beta-hydroxylation in the presence of Hmwb(5)-EGFP indicates that cytochrome b(5) in the fusion protein is able to stimulate the hydroxylation reaction. Using other fusion proteins containing either cytochrome b(5) or its hydrophilic domain to reconstitute catalytic activity of cytochrome P450 3A4 showed that the hydrophobic domain of cytochrome b(5) participates not only in hemeprotein interaction, but also in electron transfer from cytochrome b(5) to cytochrome P450.

  17. Screening of bacterial cytochrome P450s responsible for regiospecific hydroxylation of (iso)flavonoids.

    PubMed

    Pandey, Bishnu Prasad; Lee, Nahum; Choi, Kwon-Young; Jung, Eunok; Jeong, Da-Hye; Kim, Byung-Gee

    2011-04-07

    Screening of cytochrome P450 monoxygenases responsible for the regiospecific hydroxylation of flavones, isoflavones and chalcones was attempted using a P450 library constructed from Streptomyces avermitilis MA4680, Bacillus and Nocardia farcinica IFM10152 strains. As electron transfer redox partners with the P450s in Escherichia coli system, putidaredoxin reductase (PdR) and putidaredoxin (Pdx) from Pseudomonas putida were used. Among the 50 soluble P450s in the library screened, three cytochrome P450s, i.e. CYP107Y1, CYP125A2 and CYP107P2 from S. avermitilis MA4680 showed good hydroxylation activities towards flavones and isoflavones. However, low product yields prevented us from identifying complete structure of the products. By using S. avermitilis MA4680 as their expression host, further analysis identified that CYP107Y1(SAV2377), CYP125A2(SAV5841) and CYP107P2(SAV4539) showed good regiospecific hydroxylation activities towards genistein (4',5,7-trihydroxyisoflavone), chrysin (5,7-dihydroxyisoflavone) and apigenin (4',5,7-dihydroxyisoflavone) to produce 3',4',5,7,-tetrahydroxyisoflavone, B-ring hydroxylated 5,7-dihydroxyflavone and 3',4',5,7,-tetrahydroxyflavone, respectively. Analyses of the reaction products were performed using HPLC, ESI-MS-MS and GC-MS and 1H NMR.

  18. Inhibition effects of Vernonia cinerea active compounds against cytochrome P450 2A6 and human monoamine oxidases, possible targets for reduction of tobacco dependence.

    PubMed

    Prasopthum, Aruna; Pouyfung, Phisit; Sarapusit, Songklod; Srisook, Ekaruth; Rongnoparut, Pornpimol

    2015-04-01

    The human cytochrome P450 2A6 (CYP2A6) and monoamine oxidases (MAO-A and MAO-B), catalyzing nicotine and dopamine metabolisms, respectively, are two therapeutic targets of nicotine dependence. Vernonia cinerea, a medicinal plant commonly used for treatment of diseases such as asthma and bronchitis, has been shown reducing tobacco dependence effect among tobacco users. In the present study, we found eight active compounds isolated from V. cinerea that comprise inhibitory activity toward CYP2A6 and MAO-A and MAO-B enzymes using activity-guided assays, with coumarin as substrate of CYP2A6 and kynuramine of MAOs. These compounds were three flavones (apigenin, chrysoeriol, luteolin), one flavonol (quercetin), and four hirsutinolide-type sesquiterpene lactones (8α-(2-methylacryloyloxy)-hirsutinolide-13-O-acetate, 8α-(4-hydroxymethacryloyloxy)-hirsutinolide-13-O-acetate, 8α-tigloyloxyhirsutinolide-13-O-acetate, and 8α-(4-hydroxytigloyloxy)-hirsutinolide-13-O-acetate). Modes and kinetics of inhibition against the three enzymes were determined. Flavonoids possessed strong inhibitory effect on CYP2A6 in reversible mode, while inhibition by hirsutinolides was mechanism-based (NADPH-, concentration-, and time-dependence) and irreversible. Inhibition by hirsutinolides could not be reversed by dialysis and by addition of trapping agents or potassium ferricyanide. Flavonoids inhibited MAOs with variable degrees and were more prominent in inhibition toward MAO-A than hirsutinolides, while two of hirsutinolides inhibited MAO-B approximately comparable to two flavonoids. These results could have implications in combination of drug therapy for smoking cessation.

  19. Structural features of cytochrome P450 1A associated with the absence of EROD activity in liver of the of the loricariid catfish Pterygoplichthys sp

    PubMed Central

    Parente, T.E.M.; Rebelo, M.F.; da-Silva, M.L.; Woodin, B.R.; Goldstone, J. V.; Bisch, P.M.; Paumgartten, F.J.R.; Stegeman, J.J.

    2011-01-01

    The Amazon catfish genus Pterygoplichthys (Loricariidae, Siluriformes) is closely related to the loricariid genus Hypostomus, in which at least two species lack detectable ethoxyresorufin-O-deethylase (EROD) activity, typically catalyzed by cytochrome P450 1 (CYP1) enzymes. Pterygoplichthys sp. liver microsomes also lacked EROD, as well as activity with other substituted resorufins, but aryl hydrocarbon receptor agonists induced hepatic CYP1A mRNA and protein suggesting structural/functional differences in Pterygoplichthys CYP1s from those in other vertebrates. Comparing the sequences of CYP1As of Pterygoplichthys sp. and of two phylogenetically-related siluriform species that do catalyze EROD (Ancistrus sp., Loricariidae and Corydoras sp., Callichthyidae) showed that these three proteins share amino acids at 17 positions that are not shared by any fish in a set of 24 other species. Pterygoplichthys and Ancistrus (the loricariids) have an additional 22 amino acid substitutions in common that are not shared by Corydoras or by other fish species. Pterygoplichthys has six exclusive amino acid substitutions. Molecular docking and dynamics simulations indicate that Pterygoplichthys CYP1A has a weak affinity for ER, which binds infrequently in a productive orientation, and in a less stable conformation than in CYP1As of species that catalyze EROD. ER also binds with the carbonyl moiety proximal to the heme iron. Pterygoplichthys CYP1A has amino acids substitutions that reduce the frequency of correctly oriented ER in the AS preventing the detection of EROD activity. The results indicate that loricariid CYP1As may have a peculiar substrate selectivity that differs from CYP1As of most vertebrates. PMID:21840383

  20. Engineering of a hybrid biotransformation system for cytochrome P450sca-2 in Escherichia coli.

    PubMed

    Ba, Lina; Li, Pan; Zhang, Hui; Duan, Yan; Lin, Zhanglin

    2013-07-01

    P450sca-2 is an industrially important enzyme that stereoselectively converts mevastatin into pravastatin. However, little information or engineering efforts have been reported for this enzyme or its redox partner. In this study, we successfully reconstituted the P450sca-2 activity in Escherichia coli by co-expression with putidaredoxin reductase (Pdr) and putidaredoxin (Pdx) from the Pseudomonas putida cytochrome P450cam system. With an HPLC-based screening assay, random mutagenesis was applied to yield a mutant (R8-5C) with a pravastatin yield of the whole-cell biotransformation 4.1-fold that of the wild type. P450sca-2 wild-type and R8-5C were characterized in terms of mevastatin binding and hydroxylation, electron transfer, and circular dichroism spectroscopy. R8-5C showed an active P450 expression level that was 3.8-fold that of the wild type, with relatively smaller changes in the apparent k(cat)/K(M) with respect to the substrate mevastatin (1.3-fold) or Pdx (1.5-fold) compared with the wild type. Thus, the increase in the pravastatin yield of the whole-cell biotransformation primarily came from the improved active P450 expression, which has resulted largely from better heme incorporation, although none of the six mutations of R8-5C are located near the heme active site. These results will facilitate further engineering of this P450sca-2 system and provide useful clues for improving other hybrid P450 systems.

  1. 7,12-Dimethylbenzanthracene induces apoptosis in RL95-2 human endometrial cancer cells: Ligand-selective activation of cytochrome P450 1B1

    SciTech Connect

    Kim, Ji Young; Lee, Seung Gee; Chung, Jin-Yong; Kim, Yoon-Jae; Park, Ji-Eun; Oh, Seunghoon; Lee, Se Yong; Choi, Hong Jo; Yoo, Young Hyun; and others

    2012-04-15

    7,12-Dimethylbenzanthracene (DMBA), a polycyclic aromatic hydrocarbon, exhibits mutagenic, carcinogenic, immunosuppressive, and apoptogenic properties in various cell types. To achieve these functions effectively, DMBA is modified to its active form by cytochrome P450 1 (CYP1). Exposure to DMBA causes cytotoxicity-mediated apoptosis in bone marrow B cells and ovarian cells. Although uterine endometrium constitutively expresses CYP1A1 and CYP1B1, their apoptotic role after exposure to DMBA remains to be elucidated. Therefore, we chose RL95-2 endometrial cancer cells as a model system for studying DMBA-induced cytotoxicity and cell death and hypothesized that exposure to DMBA causes apoptosis in this cell type following CYP1A1 and/or CYP1B1 activation. We showed that DMBA-induced apoptosis in RL95-2 cells is associated with activation of caspases. In addition, mitochondrial changes, including decrease in mitochondrial potential and release of mitochondrial cytochrome c into the cytosol, support the hypothesis that a mitochondrial pathway is involved in DMBA-induced apoptosis. Exposure to DMBA upregulated the expression of AhR, Arnt, CYP1A1, and CYP1B1 significantly; this may be necessary for the conversion of DMBA to DMBA-3,4-diol-1,2-epoxide (DMBA-DE). Although both CYP1A1 and CYP1B1 were significantly upregulated by DMBA, only CYP1B1 exhibited activity. Moreover, knockdown of CYP1B1 abolished DMBA-induced apoptosis in RL95-2 cells. Our data show that RL95-2 cells are susceptible to apoptosis by exposure to DMBA and that CYP1B1 plays a pivotal role in DMBA-induced apoptosis in this system. -- Highlights: ► Cytotoxicity-mediated apoptogenic action of DMBA in human endometrial cancer cells. ► Mitochondrial pathway in DMBA-induced apoptosis of RL95-2 endometrial cancer cells. ► Requirement of ligand-selective activation of CYP1B1 in DMBA-induced apoptosis.

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

    SciTech Connect

    Verschoyle, R.D.; Dinsdale, D. )

    1990-04-01

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

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

    SciTech Connect

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

    2005-12-09

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

  4. Monitoring of cytochrome P-450 1A activity by determination of the urinary pattern of caffeine metabolites in Wistar and hyperbilirubinemic Gunn rats.

    PubMed

    Jorritsma, U; Schrader, E; Klaunick, G; Kapitulnik, J; Hirsch-Ernst, K I; Kahl, G F; Foth, H

    2000-04-03

    Various studies suggest that induction of cytochrome P-450 1A (CYP1A) might be a valuable therapeutic modality for reducing the hyperbilirubinemia of infants with Crigler-Najjar syndrome type I (CNS-I), a severe form of congenital jaundice. To evaluate inducers of CYP1A as possible tools in the treatment of hyperbilirubinemia, a novel assay was established, based on the analysis of the urinary pattern of caffeine metabolites in rats. Wistar rats received [1-Me-(14)C]-caffeine (10 mg/kg i.p.), before and 48h after administration of the potent CYP1A inducer 5,6-benzoflavone (BNF) (80 mg/kg, i.p.). A substantial increase in the fractions of the terminal caffeine metabolites 1-methyluric acid (1-U), 1-methylxanthine (1-X), and a concomitant decrease in the caffeine demethylation product 1,7-dimethylxanthine (1,7-X) was observed after application of BNF. The ratio of the caffeine metabolites (1-U+1-X)/1,7-X may serve as an index of CYP1A activity in rats in vivo. Hyperbilirubinemic, homozygous (jj) Gunn rats are an accepted model for human CNS-I. In male jj Gunn rats treated with BNF or with indole-3-carbinol (I3C, 80 mg/kg, oral gavage), the inducing effect of BNF and 13C on CYP1A activity was confirmed by the urinary pattern of caffeine metabolites, and was parallelled by a decrease in plasma bilirubin levels. These data demonstrate the usefulness of the established caffeine assay for the evaluation of inducers of CYP1A as tools for reducing hyperbilirubinemia and further confirm the potential value of I3C in the treatment of CNS-I.

  5. The Arabidopsis LUT1 locus encodes a member of the cytochrome P450 family that is required for carotenoid ε-ring hydroxylation activity

    PubMed Central

    Tian, Li; Musetti, Valeria; Kim, Joonyul; Magallanes-Lundback, Maria; DellaPenna, Dean

    2004-01-01

    Lutein, a dihydroxy xanthophyll, is the most abundant carotenoid in plant photosynthetic tissues and plays crucial structural and functional roles in the light-harvesting complexes. Carotenoid β-and ε-hydroxylases catalyze the formation of lutein from α-carotene (β,ε-carotene). In contrast to the well studied β-hydroxylases that have been cloned and characterized from many organisms, the ε-hydroxylase has only been genetically defined by the lut1 mutation in Arabidopsis. We have isolated the LUT1 gene by positional cloning and found that, in contrast to all known carotenoid hydroxylases, which are the nonheme diiron monooxygenases, LUT1 encodes a cytochrome P450-type monooxygenase, CYP97C1. Introduction of a null mutant allele of LUT1, lut1-3, into the β-hydroxylase 1/β-hydroxylase 2 (b1 b2) double-mutant background, in which both Arabidopsis β-hydroxylases are disrupted, yielded a genotype (lut1-3 b1 b2) in which all three known carotenoid hydroxylase activities are eliminated. Surprisingly, hydroxylated β-rings were still produced in lut1-3 b1 b2, suggesting that a fourth unknown carotenoid β-hydroxylase exists in vivo that is structurally unrelated to β-hydroxylase 1 or 2. A second chloroplast-targeted member of the CYP97 family, CYP97A3, is 49% identical to LUT1 and hypothesized as a likely candidate for this additional β-ring hydroxylation activity. Overall, LUT1 defines a class of carotenoid hydroxylases that has evolved independently from and uses a different mechanism than nonheme diiron β-hydroxylases. PMID:14709673

  6. Role of cytochrome P450 2E1 in the metabolism of acrylamide and acrylonitrile in mice.

    PubMed

    Sumner, S C; Fennell, T R; Moore, T A; Chanas, B; Gonzalez, F; Ghanayem, B I

    1999-11-01

    Acrylonitrile (AN) and acrylamide (AM) are commonly used in the synthesis of plastics and polymers. In rodents, AM and AN are metabolized to the epoxides glycidamide and cyanoethylene oxide, respectively. The aim of this study was to determine the role of cytochrome P450 in the metabolism of AM and AN in vivo. Wild-type (WT) mice, WT mice pretreated with aminobenzotriazole (ABT, 50 mg/kg ip, 2 h pre-exposure), and mice devoid of cytochrome P450 2E1 (P450 2E1-null) were treated with 50 mg/kg [(13)C]AM po. WT mice and P450 2E1-null mice were treated with 2.5 or 10 mg/kg [(13)C]AN po. Urine was collected for 24 h, and metabolites were characterized using (13)C NMR. WT mice excreted metabolites derived from the epoxides and from direct GSH conjugation with AM or AN. Only metabolites derived from direct GSH conjugation with AM or AN were observed in the urine from ABT-pretreated WT mice and P450 2E1-null mice. On the basis of evaluation of urinary metabolites at these doses, these data suggest that P450 2E1 is possibly the only cytochrome P450 enzyme involved in the metabolism of AM and AN in mice, that inhibiting total P450 activity does not result in new pathways of non-P450 metabolism of AM, and that mice devoid of P450 2E1 do not excrete metabolites of AM or AN that would be produced by oxidation by other cytochrome P450s. P450 2E1-null mice may be an appropriate model for the investigation of the role of oxidative metabolism in the toxicity or carcinogenicity of these compounds.

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

    PubMed Central

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

    2008-01-01

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

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

    SciTech Connect

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

    1990-02-26

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

  10. Crystal Structure of Human Cytochrome P450 2D6 with Prinomastat Bound*

    PubMed Central

    Wang, An; Savas, Uzen; Hsu, Mei-Hui; Stout, C. David; Johnson, Eric F.

    2012-01-01

    Human cytochrome P450 2D6 contributes to the metabolism of >15% of drugs used in clinical practice. This study determined the structure of P450 2D6 complexed with a substrate and potent inhibitor, prinomastat, to 2.85 Å resolution by x-ray crystallography. Prinomastat binding is well defined by electron density maps with its pyridyl nitrogen bound to the heme iron. The structure of ligand-bound P450 2D6 differs significantly from the ligand-free structure reported for the P450 2D6 Met-374 variant (Protein Data Bank code 2F9Q). Superposition of the structures reveals significant differences for β sheet 1, helices A, F, F′, G″, G, and H as well as the helix B-C loop. The structure of the ligand complex exhibits a closed active site cavity that conforms closely to the shape of prinomastat. The closure of the open cavity seen for the 2F9Q structure reflects a change in the direction and pitch of helix F and introduction of a turn at Gly-218, which is followed by a well defined helix F′ that was not observed in the 2F9Q structure. These differences reflect considerable structural flexibility that is likely to contribute to the catalytic versatility of P450 2D6, and this new structure provides an alternative model for in silico studies of substrate interactions with P450 2D6. PMID:22308038

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

    PubMed Central

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

    2017-01-01

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

  12. Selective Targeting of Heme Protein in Cytochrome P450 and Nitric Oxide Synthase by Diphenyleneiodonium.

    PubMed

    Szilagyi, John T; Mishin, Vladimir; Heck, Diane E; Jan, Yi-Hua; Aleksunes, Lauren M; Richardson, Jason R; Heindel, Ned D; Laskin, Debra L; Laskin, Jeffrey D

    2016-05-01

    Cytochrome P450 (CYP) enzymes mediate mixed-function oxidation reactions important in drug metabolism. The aromatic heterocyclic cation, diphenyleneiodonium (DPI), binds flavin in cytochrome P450 reductase and inhibits CYP-mediated activity. DPI also inhibits CYP by directly interacting with heme. Herein, we report that DPI effectively inhibits a number of CYP-related monooxygenase reactions including NADPH oxidase, a microsomal enzyme activity that generates hydrogen peroxide in the absence of metabolizing substrates. Inhibition of monooxygenase by DPI was time and concentration dependent with IC50's ranging from 0.06 to 1.9 μM. Higher (4.6-23.9 μM), but not lower (0.06-1.9 μM), concentrations of DPI inhibited electron flow via cytochrome P450 reductase, as measured by its ability to reduce cytochrome c and mediate quinone redox cycling. Similar results were observed with inducible nitric oxide synthase (iNOS), an enzyme containing a C-terminal reductase domain homologous to cytochrome P450 reductase that mediates reduction of cytochrome c, and an N-terminal heme-thiolate oxygenase domain mediating nitric oxide production. Significantly greater concentrations of DPI were required to inhibit cytochrome c reduction by iNOS (IC50 = 3.5 µM) than nitric oxide production (IC50 = 0.16 µM). Difference spectra of liver microsomes, recombinant CYPs, and iNOS demonstrated that DPI altered heme-carbon monoxide interactions. In the presence of NADPH, DPI treatment of microsomes and iNOS yielded a type II spectral shift. These data indicate that DPI interacts with both flavin and heme in CYPs and iNOS. Increased sensitivity for inhibition of CYP-mediated metabolism and nitric oxide production by iNOS indicates that DPI targets heme moieties within the enzymes.

  13. NADPH:cytochrome c (P450) reductase activates tirapazamine (SR4233) to restore hypoxic and oxic cytotoxicity in an aerobic resistant derivative of the A549 lung cancer cell line

    PubMed Central

    Saunders, M P; Patterson, A V; Chinje, E C; Harris, A L; Stratford, I J

    2000-01-01

    Tirapazamine (TPZ, SR4233, WIN 59075) is a bioreductive drug that is activated in regions of low oxygen tension to a cytotoxic radical intermediate. This labile metabolite shows high selective toxicity towards hypoxic cells, such as those found in solid tumours. Under aerobic conditions, redox cycling occurs with subsequent generation of superoxide radicals, which are also cytotoxic. NADPH:cytochrome c (P450) reductase (P450R) is a one-electron reducing enzyme that efficiently activates TPZ. Recently a derivative of the A549 non-small cell lung cancer cell line (A549c50) was generated that showed substantially reduced P450R activity compared to its parental line (Elwell et al (1997) Biochem Pharmacol54: 249–257). Here, it is demonstrated that the A549c50 cells are markedly more resistant to TPZ under both aerobic and hypoxic conditions. In addition, these cells have a dramatically impaired ability to metabolize TPZ to its two-electron reduction product, SR4317, under hypoxic conditions when compared to wild-type cells. P450R activity in the A549c50 cells was reintroduced to similar levels as that seen in the parental A549 cells by transfection of the full-length cDNA for human P450R. These P450R over-expressing cells exhibit restored sensitivity to TPZ under both aerobic and hypoxic conditions, comparable to that found in the original parental A549 cells. Further, the ability of the transfected cells to metabolize TPZ to SR4317 under hypoxic conditions is also shown to be restored. This provides further evidence that P450R can play an important role in the activation, metabolism and toxicity of this lead bioreductive drug. © 2000 Cancer Research Campaign PMID:10682679

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

    NASA Astrophysics Data System (ADS)

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

    1997-03-01

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

  15. UPLC-MS-MS method for simultaneous determination of caffeine, tolbutamide, metoprolol, and dapsone in rat plasma and its application to cytochrome P450 activity study in rats.

    PubMed

    Liu, Yan; Li, Xiang; Yang, Chunjuan; Tai, Sheng; Zhang, Xiangning; Liu, Gaofeng

    2013-01-01

    A specific ultra-performance liquid chromatography tandem mass spectrometry method has been described for the simultaneous determination of caffeine, tolbutamide, metoprolol and dapsone in rat plasma, which are the four probe drugs of the four cytochrome P450 (CYP450) isoforms CYP1A2, CYP2C9, CYP2D6 and CYP3A4. The chromatographic separation was achieved using a Waters Acquity UPLC BEH HILIC C(18) column (2.1 × 50 mm, 1.7 µm). The mobile phase consisted of acetonitrile and water (containing 0.1% formic acid) (15:85, v/v). The triple quadrupole mass spectrometric detection was operated by positive electrospray ionization. Phenacetin was chosen as internal standard. Plasma samples were extracted with dichloromethane-butanol (10:1, v/v). The recoveries ranged from 67.5% to 98.5%. The calibration curves in plasma were linear in the range of 2.5-1,000 ng/mL for caffeine and dapsone, 5-5,000 ng/mL for tolbutamide and 2.5-250 ng/mLfor metoprolol, with correlation coefficient (r(2)) of 0.9936, 0.9966, 0.9990 and 0.9998, respectively. The method was successfully applied to pharmacokinetic studies of the four probe drugs of the four CYP450 isoforms and used to evaluate the effects of breviscapine on the activities of CYP1A2, CYP2C9, CYP2D6 and CYP3A4 in rats.

  16. Oxidized linoleic acid metabolite-cytochrome P450 system (OLAM-CYP) is active in biopsy samples from patients with inflammatory dental pain.

    PubMed

    Ruparel, Shivani; Hargreaves, Kenneth M; Eskander, Michael; Rowan, Spencer; de Almeida, Jose F A; Roman, Linda; Henry, Michael A

    2013-11-01

    Endogenous TRPV1 agonists such as oxidized linoleic acid metabolites (OLAMs) and the enzymes releasing them [eg, cytochrome P450 (CYP)] are up-regulated after inflammation in the rat. However, it is not known whether such agonists are elevated in human inflammatory pain conditions. Because TRPV1 is expressed in human dental pulp nociceptors, we hypothesized that OLAM-CYP machinery is active in this tissue type and is increased under painful inflammatory conditions such as irreversible pulpitis (IP). The aim of this study was to compare CYP expression and linoleic acid (LA) metabolism in normal vs inflamed human dental pulp. Our data showed that exogenous LA metabolism was significantly increased in IP tissues compared to normal tissues and that pretreatment with a CYP inhibitor, ketoconazole, significantly inhibited LA metabolism. Additionally, extracts obtained from LA-treated inflamed tissues evoked significant inward currents in trigeminal ganglia neurons and were blocked by pretreatment with the TRPV1 antagonist IRTX. Moreover, extracts obtained from ketoconazole-pretreated inflamed tissues significantly reduced inward currents in trigeminal ganglia neurons. These data suggest that LA metabolites produced in human inflamed tissues act as TRPV1 agonists and that the metabolite production can be targeted by CYP inhibition. In addition, immunohistochemical analysis of 2 CYP isoforms, CYP2J and CYP3A1, were shown to be predominately expressed in immune cells infiltrating the inflamed dental pulp, emphasizing the paracrine role of CYP enzymes in OLAM regulation. Collectively, our data indicate that the machinery responsible for OLAM production is up-regulated during inflammation and can be targeted to develop potential analgesics for inflammatory-induced dental pain.

  17. Effects of contaminated sediment from Cork Harbour, Ireland on the cytochrome P450 system of turbot.

    PubMed

    Kilemade, M; Hartl, M G J; O'Halloran, J; O'Brien, N M; Sheehan, D; Mothersill, C; van Pelt, F N A M

    2009-03-01

    Hatchery-reared juvenile turbot (Scophthalmus maximus L.) were exposed for 3 weeks, under laboratory conditions, to inter-tidal sediments collected from polluted sites in Cork Harbour (Whitegate and Agahda) and a reference site at Ballymacoda Co., Cork, Ireland. The potential of the sediment exposure to induce cytochrome P450 activities and CYP1A1 in the fish was assessed. Chemical analysis revealed that the sediments originating from the reference and harbour sites were contaminated principally with PAHs-the harbour sites having double the levels of those at the reference site. Following 3 weeks exposure to the sediments western blotting demonstrated a strong immunogenic response for CYP1A1 in the liver, but not for gill or intestine. P450 activities were generally significantly higher than those exposed to reference site sediment. Liver was the most responsive tissue with significantly greater P450 activities compared with gill and intestinal tissues.

  18. Interaction of epicatechins derived from green tea with rat hepatic cytochrome P-450.

    PubMed

    Wang, Z Y; Das, M; Bickers, D R; Mukhtar, H

    1988-01-01

    Green tea has been used for generations in China and Asia as an antipyretic and diuretic. Prior studies have shown that extracts of green tea inhibit the mutagenicity of polycyclic aromatic hydrocarbons and aflatoxin B1. In this study, we investigated the interaction of certain flavonoid components of green tea epicatechin derivatives including (-)-epicatechin (EC), (-)-epigallocatechin (EGC), (-)-epicatechin-3-gallate (ECG), and (-)-epigallocatechin-3-gallate (EGCG) with rat hepatic microsomal cytochrome P-450 (P-450). The addition of EC, EGC, ECG, and EGCG to hepatic microsomes prepared from phenobarbital (PB)-treated rats resulted in spectral changes characterized by absorbance maxima at 420 nm and minima at 380 nm, typical of modified Type II (reverse Type I) binding. Of the epicatechin derivatives, EGCG and ECG showed greater spectral change with oxidized P-450 and time- and concentration-dependent inhibition of the binding of carbon monoxide to dithionite-reduced cytochrome P-450. The addition of EC, EGC, ECG, and EGCG to microsomes prepared from control, PB- or 3-methylcholanthrene-treated rats resulted in a dose-dependent inhibition of cytochrome P-450-dependent aryl hydrocarbon hydroxylase, 7-ethoxycoumarin O-deethylase, and 7-ethoxyresorufin O-deethylase activities. EGCG was the most potent in this regard. Green tea polyphenols and epicatechin derivatives also significantly inhibited NADPH-cytochrome c reductase activity. An examination of the structure activity relationship of epicatechin derivatives suggests that the inhibitory effect on the microsomal enzyme system may be due to the galloyl groups or hydroxyl groups on the molecule. Our data indicate that these extracts of green tea may have potential as anticarcinogens.

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

    PubMed

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

    2014-02-01

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

  20. 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 α-naphthoflavone or ketoconazole, and were roughly correlated with P450 2C19 levels and flurbiprofen 4-hydroxylation activities in microsomes from 20 monkey livers. In contrast, S-warfarin 7-hydroxylation activities were not correlated with the four marker drug oxidation activities used. Among the 14 recombinantly expressed monkey P450 enzymes tested, P450 2C19 had the highest activities for R- and S-warfarin 7-hydroxylations. Monkey P450 3A4 and 3A5 slowly mediated R- and S-warfarin 6-hydroxylations. Kinetic analysis revealed that monkey P450 2C19 had high V(max) and low K(m) values for R-warfarin 7-hydroxylation, comparable to those for monkey liver microsomes. Monkey P450 2C19 also mediated S-warfarin 7-hydroxylation with V(max) and V(max)/K(m) values comparable to those for recombinant human P450 2C9. R-warfarin could dock favorably into monkey P450 2C19 modeled. These results collectively suggest high activities for monkey liver P450 2C19 toward R- and S-warfarin 6- and 7-hydroxylation in contrast to the saturation kinetics of human P450 2C9-mediated S-warfarin 7-hydroxylation.

  1. 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.

  2. The gap junction inhibitor 2-aminoethoxy-diphenyl-borate protects against acetaminophen hepatotoxicity by inhibiting cytochrome P450 enzymes and c-jun N-terminal kinase activation

    SciTech Connect

    Du, Kuo; Williams, C. David; McGill, Mitchell R.; Xie, Yuchao; Farhood, Anwar; Vinken, Mathieu; Jaeschke, Hartmut

    2013-12-15

    Acetaminophen (APAP) hepatotoxicity is the leading cause of acute liver failure in the US. Although many aspects of the mechanism are known, recent publications suggest that gap junctions composed of connexin32 function as critical intercellular communication channels which transfer cytotoxic mediators into neighboring hepatocytes and aggravate liver injury. However, these studies did not consider off-target effects of reagents used in these experiments, especially the gap junction inhibitor 2-aminoethoxy-diphenyl-borate (2-APB). In order to assess the mechanisms of protection of 2-APB in vivo, male C56Bl/6 mice were treated with 400 mg/kg APAP to cause extensive liver injury. This injury was prevented when animals were co-treated with 20 mg/kg 2-APB and was attenuated when 2-APB was administered 1.5 h after APAP. However, the protection was completely lost when 2-APB was given 4–6 h after APAP. Measurement of protein adducts and c-jun-N-terminal kinase (JNK) activation indicated that 2-APB reduced both protein binding and JNK activation, which correlated with hepatoprotection. Although some of the protection was due to the solvent dimethyl sulfoxide (DMSO), in vitro experiments clearly demonstrated that 2-APB directly inhibits cytochrome P450 activities. In addition, JNK activation induced by phorone and tert-butylhydroperoxide in vivo was inhibited by 2-APB. The effects against APAP toxicity in vivo were reproduced in primary cultured hepatocytes without use of DMSO and in the absence of functional gap junctions. We conclude that the protective effect of 2-APB was caused by inhibition of metabolic activation of APAP and inhibition of the JNK signaling pathway and not by blocking connexin32-based gap junctions. - Highlights: • 2-APB protected against APAP-induced liver injury in mice in vivo and in vitro • 2-APB protected by inhibiting APAP metabolic activation and JNK signaling pathway • DMSO inhibited APAP metabolic activation as the solvent of 2-APB

  3. The involvement of cytochrome P450 peroxidase in the metabolic bioactivation of cumene hydroperoxide by isolated rat hepatocytes.

    PubMed

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

    1996-09-01

    Organic hydroperoxides are believed to be primarily detoxified in cells by the GSH peroxidase/GSSG reductase system and activated to cytotoxic radical species by non-heme iron. However, organic hydroperoxides seem to be bioactivated by cytochrome P450 (P450) in isolated hepatocytes as various P450 (particularly P450 2E1) inhibitors inhibited cumene hydroperoxide (CumOOH) metabolism and attenuated subsequent cytotoxic effects including antimycin A-resistant respiration, lipid peroxidation, iron mobilization, ATP depletion, and cell membrane disruption. CumOOH metabolism was also faster in P450 1A-induced hepatocytes and was inhibited by the P450 1A inhibitor alpha-naphthoflavone. The ferric chelator deferoxamine also prevented cytotoxicity even after CumOOH had been metabolized but had no effect on CumOOH metabolism. This emphasizes the toxicological significance of the iron released following hydroperoxide metabolic activation by cytochrome P450. The radical trap, 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO), had no effect on CumOOH metabolism but prevented CumOOH-induced antimycin A-resistant respiration, lipid peroxidation, iron mobilization, and loss of membrane integrity. These results suggest that CumOOH is metabolically activated by some P450 enzymes (e.g., P450 2E1) in hepatocytes to form reactive radical metabolites or oxidants that cause lipid peroxidation and cytotoxicity.

  4. Repurposing Resveratrol and Fluconazole To Modulate Human Cytochrome P450-Mediated Arachidonic Acid Metabolism.

    PubMed

    El-Sherbeni, Ahmed A; El-Kadi, Ayman O S

    2016-04-04

    Cytochrome P450 (P450) enzymes metabolize arachidonic acid (AA) to several biologically active epoxyeicosatrienoic acids (EETs) and hydroxyeicosatetraenoic acids (HETEs). Repurposing clinically-approved drugs could provide safe and readily available means to control EETs and HETEs levels in humans. Our aim was to determine how to significantly and selectively modulate P450-AA metabolism in humans by clinically-approved drugs. Liquid chromatography-mass spectrometry was used to determine the formation of 15 AA metabolites by human recombinant P450 enzymes, as well as human liver and kidney microsomes. CYP2C19 showed the highest EET-forming activity, while CYP1B1 and CYP2C8 showed the highest midchain HETE-forming activities. CYP1A1 and CYP4 showed the highest subterminal- and 20-HETE-forming activity, respectively. Resveratrol and fluconazole produced the most selective and significant modulation of hepatic P450-AA metabolism, comparable to investigational agents. Monte Carlo simulations showed that 90% of human population would experience a decrease by 6-22%, 16-39%, and 16-35% in 16-, 18-, and 20-HETE formation, respectively, after 2.5 g daily of resveratrol, and by 22-31% and 14-23% in 8,9- and 14,15-EET formation after 50 mg of fluconazole. In conclusion, clinically-approved drugs can provide selective and effective means to modulate P450-AA metabolism, comparable to investigational drugs. Resveratrol and fluconazole are good candidates to be repurposed as new P450-based treatments.

  5. Cytochrome P450 monooxygenases involved in anthracene metabolism by the white-rot basidiomycete Phanerochaete chrysosporium.

    PubMed

    Chigu, Nomathemba Loice; Hirosue, Sinji; Nakamura, Chie; Teramoto, Hiroshi; Ichinose, Hirofumi; Wariishi, Hiroyuki

    2010-08-01

    Cytochrome P450 monooxygenases (P450s) involved in anthracene metabolism by the white-rot basidiomycete Phanerochaete chrysosporium were identified by comprehensive screening of both catalytic potentials and transcriptomic profiling. Functional screening of P. chrysosporium P450s (PcCYPs) revealed that 14 PcCYP species catalyze stepwise conversion of anthracene to anthraquinone via intermediate formation of anthrone. Moreover, transcriptomic profiling explored using a complementary DNA microarray system demonstrated that 12 PcCYPs are up-regulated in response to exogenous addition of anthracene. Among the up-regulated PcCYPs, five species showed catalytic activity against anthracene. Based upon both catalytic and transcriptional properties, these five species are most likely to play major roles in anthracene metabolic processes in vivo. Thus, the combination of functional screening and a microarray system may provide a novel strategy for obtaining a thorough understanding of the catalytic functions and biological impacts of PcCYPs.

  6. Key Mutations Alter the Cytochrome P450 BM3 Conformational Landscape and Remove Inherent Substrate Bias*

    PubMed Central

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

    2013-01-01

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

  7. High-throughput mass spectrometric cytochrome P450 inhibition screening.

    PubMed

    Lim, Kheng B; Ozbal, Can C; Kassel, Daniel B

    2013-01-01

    We describe here a high-throughput assay to support rapid evaluation of drug discovery compounds for possible drug-drug interaction (DDI). Each compound is evaluated for its DDI potential by incubating over a range of eight concentrations and against a panel of six cytochrome P450 (CYP) enzymes: 1A2, 2C8, 2C9, 2C19, 2D6, and 3A4. The method utilizes automated liquid handling for sample preparation, and online solid-phase extraction/tandem mass spectrometry (SPE/MS/MS) for sample analyses. The system is capable of generating two 96-well assay plates in 30 min, and completes the data acquisition and analysis of both plates in about 30 min. Many laboratories that perform the CYP inhibition screening automate only part of the processes leaving a throughput bottleneck within the workflow. The protocols described in this chapter are aimed to streamline the entire process from assay to data acquisition and processing by incorporating automation and utilizing high-precision instrument to maximize throughput and minimize bottleneck.

  8. Cytochrome P450 ω-Hydroxylases in Inflammation and Cancer

    PubMed Central

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

    2015-01-01

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

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

    PubMed

    Zhu, Yi; Zhang, Qing-Yu

    2012-11-01

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

  10. In vitro characterization of the cytochrome P450 isoforms involved in the metabolism of 6-methoxy-2-napthylacetic acid, an active metabolite of the prodrug nabumetone.

    PubMed

    Matsumoto, Kaori; Nemoto, Eiichi; Hasegawa, Tetsuya; Akimoto, Masayuki; Sugibayashi, Kenji

    2011-01-01

    The cytochrome P450 (CYP) isoforms that catalyze the oxidation metabolism of 6-methoxy-2-napthylacetic acid (6-MNA), an active metabolite of nabumetone, were studied in rats and humans. Using an extractive reversed-phase HPLC assay with fluorescence detection, monophasic Michaelis-Menten kinetics was obtained for the formation of 6-hydroxy-2-naphthylacetic acid (6-HNA) in liver microsomes of rats and humans, and kinetic analysis showed that the K(m) and V(max) values for the formation of 6-HNA in humans and rats were 640.0 ± 30.9 and 722.9 ± 111.7 µM, and 1167.5 ± 33.0 and 1312.7 ± 73.8 pmol min⁻¹ mg protein⁻¹, respectively. The CYPs responsible for metabolism of 6-MNA in liver microsomes of rats and humans were identified using correlation study, recombinant CYP supersomes, and specific CYP inhibitors and antibodies. Recombinant human CYP2C9 exhibited appreciable catalytic activity with respect to 6-HNA formation from 6-MNA. Among 14 recombinant rat CYPs examined, CYP2C6, CYP2C11 and CYP1A2 were involved in the metabolism of 6-MNA. Sulfaphenazole (a selective inhibitor of CYP2C9) inhibited the formation of 6-HNA in pooled human microsomes by 89%, but failed to inhibit this reaction in rat liver microsomes. The treatment of pooled human liver microsomes with an antibody against CYP2C9 inhibited the formation of 6-HNA by about 80%. The antibody against CYP2C11 suppressed the activity by 20 to 30% in rat microsomes, whereas that of CYP1A2 microsomes did not show drastic inhibition. These findings suggest that CYP2C9 has the highest catalytic activity of 6-MNA metabolism in humans. In contrast, metabolism of 6-MNA is suggested to be mediated mainly by CYP2C6 and CYP2C11 in rats.

  11. Traditional Herbal Formulas to as Treatments for Musculoskeletal Disorders: Their Inhibitory Effects on the Activities of Human Microsomal Cytochrome P450s and UDP-glucuronosyltransferases

    PubMed Central

    Jin, Seong Eun; Seo, Chang-Seob; Shin, Hyeun-Kyoo; Ha, Hyekyung

    2016-01-01

    Objective: The aim of this study was to assess the influence of traditional herbal formulas, including Bangpungtongseong-san (BPTSS; Fangfengtongsheng-san, Bofu-tsusho-san), Ojeok-san (OJS; Wuji-san, Goshaku-san), and Oyaksungi-san (OYSGS; Wuyaoshungi-san, Uyakujyunki-san), on the activities of the human cytochrome P450s (CYP450s) and UDP-glucuronosyltransferases (UGTs), which are drug-metabolizing enzymes. Materials and Methods: The activities of the major human CYP450 isozymes (CYP1A2, CYP3A4, CYP2B6, CYP2C9, CYP2C19, CYP2D6, and CYP2E1) and UGTs (UGT1A1, UGT1A4, and UGT2B7) were investigated using in vitro fluorescence-based and luminescence-based enzyme assays, respectively. The inhibitory effects of the herbal formulas were characterized, and their IC50 values were determined. Results: BPTSS inhibited the activities of CYP1A2, CYP2C19, CYP2E1, and UGT1A1 while it exerted relatively weak inhibition on CYP2B6, CYP2C9, CYP2D6, and CYP3A4. BPTSS also negligibly inhibited the activities of UGT1A4 and UGT2B7, with IC50 values in the excess of 1000 μg/mL. OJS and OYSGS inhibited the activity of CYP2D6, whereas they exhibited no inhibition of the UGT1A4 activity at doses <1000 μg/mL. In addition, OJS inhibited the CYP1A2 activity but exerted a relatively weak inhibition on the activities of CYP2C9, CYP2C19, CYP2E1, and CYP3A4. Conversely, OJS negligibly inhibited the activities of CYP2B6, UGT1A1, and UGT2B7 with IC50 values in excess of 1000 μg/mL. OYSGS weakly inhibited the activities of CYP1A2, CYP2C19, CYP2E1, CYP3A4, and UGT1A1, with a negligible inhibition on the activities of CYP2B6, CYP2C9, and UGT2B7, with IC50 values in excess of 1000 μg/mL. Conclusions: These results provide information regarding the safety and effectiveness of BPTSS, OJS, and OYSGS when combined with conventional drugs. SUMMARY Bangpungtongseong-san inhibited the activities of human microsomal CYP1A2, CYP2C19, CYP2E1, and UGT1A1, with a negligibly inhibition on the activities of CYP2B6

  12. Active site-directed inhibitors of cytochrome P-450scc. Structural and mechanistic implications of a side chain-substituted series of amino-steroids.

    PubMed

    Sheets, J J; Vickery, L E

    1983-10-10

    A series of analogues of cholesterol, each having a shortened side chain and a primary amine group, were prepared and tested for their effects on bovine adrenocortical cholesterol side chain cleavage cytochrome P-450 (P-450scc). A previous study had shown that one derivative, 22-amino-23,24-bisnor-5-cholen-3 beta-ol, is a potent competitive inhibitor of the enzyme and forms a complex in which the steroid ring binds to the cholesterol site and the side chain amine forms a bond with the heme iron (Sheets, J. J., and Vickery, L. E. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 5773-5777). In the studies reported here, the 23-amine derivative, 23-amino-24-nor-5-cholen-3 beta-ol, was found to be an equally potent inhibitor and to be competitive with respect to cholesterol (Ki = 38 nM). Binding of the 23-amine to P-450scc also caused formation of a low spin complex with an absorption maximum at 422 nm, indicative of a nitrogen-donor ligand. Other derivatives in which the side chain amine was linked closer to the steroid, 17 beta-amino-5-androsten-3 beta-ol and (20 R + S)-20-amino-5-pregnen-3 beta-ol, were found to be only very weak inhibitors (I50 greater than 100 microM) and did not produce the 422 nm spectral form when bound. Derivatives in which the amine was attached a greater distance from the steroid ring, 24-amino-5-cholen-3 beta-ol and 25-amino-26,27-bisnor-5-cholesten-3 beta-ol, caused a progressive decrease in inhibitory potency and a failure to produce the 422 nm form on binding. The dependence of the type of interaction of these amino-steroids with P-450scc upon the amine position establishes that the steroid binding site and the heme catalytic site of the enzyme are fixed within a specific distance of one another. The heme appears to be located sufficiently close to the position that the side chain of cholesterol would occupy to allow for direct attack of an iron-bound oxidant to occur during hydroxylation and side chain cleavage.

  13. Fusion to Hydrophobin HFBI Improves the Catalytic Performance of a Cytochrome P450 System

    PubMed Central

    Schulz, Sebastian; Schumacher, Dominik; Raszkowski, Daniel; Girhard, Marco; Urlacher, Vlada B.

    2016-01-01

    Cytochrome P450 monooxygenases (P450) are heme-containing enzymes that oxidize a broad range of substrates in the presence of molecular oxygen and NAD(P)H. For their activity, most P450s rely on one or two redox proteins responsible for the transfer of electrons from the cofactor NAD(P)H to the heme. One of the challenges when using P450s in vitro, especially when non-physiological redox proteins are applied, is the inefficient transfer of electrons between the individual proteins resulting in non-productive consumption of NAD(P)H – referred to as uncoupling. Herein, we describe the improvement of the coupling efficiency between a P450 and its redox partner – diflavin reductase – by fusing both enzymes individually to the hydrophobin HFBI – a small self-assembling protein of the fungus Trichoderma reesei. The separated monooxygenase (BMO) and reductase (BMR) domains of P450 BM3 from Bacillus megaterium were chosen as a P450-reductase model system and individually fused to HFBI. The fusion proteins could be expressed in soluble form in Escherichia coli. When HFBI-fused BMO and BMR were mixed in vitro, substantially higher coupling efficiencies were measured as compared with the respective non-fused enzymes. Consequently, myristic acid conversion increased up to 20-fold (after 6 h) and 5-fold (after 24 h). Size exclusion chromatography demonstrated that in vitro the hydrophobin-fused enzymes build multimeric protein assemblies. Thus, the higher activity is hypothesized to be due to HFBI-mediated self-assembly arranging BMO and BMR in close spatial proximity in aqueous solution. PMID:27458582

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

    PubMed Central

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

    2013-01-01

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

  15. Purification and properties of a shortened form of cytochrome P-450 2E1: deletion of the NH2-terminal membrane-insertion signal peptide does not alter the catalytic activities.

    PubMed Central

    Larson, J R; Coon, M J; Porter, T D

    1991-01-01

    As reported previously, alcohol-inducible cytochrome P-450 2E1 lacking the hydrophobic NH2-terminal segment is located primarily in the inner cell membrane when expressed in Escherichia coli and is active with a typical substrate. To study the catalytic properties in detail, we have purified the truncated P-450 lacking residues 3-29 to electrophoretic homogeneity from the solubilized bacterial membrane fraction in the presence of 4-methylpyrazole as a stabilizing agent. The resulting heme protein with a specific content of 15.8 nmol of P-450 per mg of protein has a reduced CO difference spectrum identical to that of the full-length enzyme, with a Soret maximum at 452 nm. The rates of catalysis of four reactions in the reconstituted enzyme system, including the oxygenation of ethanol to give acetaldehyde, the oxidative dealkylation of N-nitrosodiethylamine to give ethylene and acetaldehyde, and the ring hydroxylation of aniline and p-nitrophenol, are the same with the shortened and full-length enzymes. The apparent Km of p-nitrophenol is also the same, as is that for NADPH-cytochrome P-450 reductase and for cytochrome b5, which stimulates p-nitrocatechol formation about 3-fold. Moreover, the requirement for phosphatidylcholine for full catalytic activity is unchanged despite the absence of the NH2-terminal segment. Although this highly hydrophobic segment is believed to play a role in the intact cell as a membrane-insertion signal sequence, we conclude that it has no function in the catalytic activity of the cytochrome as an oxygenase, including interactions with the other components of the enzyme system. Images PMID:1656462

  16. Human Cytochrome P450 21A2, the Major Steroid 21-Hydroxylase

    PubMed Central

    Pallan, Pradeep S.; Wang, Chunxue; Lei, Li; Yoshimoto, Francis K.; Auchus, Richard J.; Waterman, Michael R.; Guengerich, F. Peter; Egli, Martin

    2015-01-01

    Cytochrome P450 (P450) 21A2 is the major steroid 21-hydroxylase, and deficiency of this enzyme is involved in ∼95% of cases of human congenital adrenal hyperplasia, a disorder of adrenal steroidogenesis. A structure of the bovine enzyme that we published previously (Zhao, B., Lei, L., Kagawa, N., Sundaramoorthy, M., Banerjee, S., Nagy, L. D., Guengerich, F. P., and Waterman, M. R. (2012) Three-dimensional structure of steroid 21-hydroxylase (cytochrome P450 21A2) with two substrates reveals locations of disease-associated variants. J. Biol. Chem. 287, 10613–10622), containing two molecules of the substrate 17α-hydroxyprogesterone, has been used as a template for understanding genetic deficiencies. We have now obtained a crystal structure of human P450 21A2 in complex with progesterone, a substrate in adrenal 21-hydroxylation. Substrate binding and release were fast for human P450 21A2 with both substrates, and pre-steady-state kinetics showed a partial burst but only with progesterone as substrate and not 17α-hydroxyprogesterone. High intermolecular non-competitive kinetic deuterium isotope effects on both kcat and kcat/Km, from 5 to 11, were observed with both substrates, indicative of rate-limiting C–H bond cleavage and suggesting that the juxtaposition of the C21 carbon in the active site is critical for efficient oxidation. The estimated rate of binding of the substrate progesterone (kon 2.4 × 107 m−1 s−1) is only ∼2-fold greater than the catalytic efficiency (kcat/Km = 1.3 × 107 m−1 s−1) with this substrate, suggesting that the rate of substrate binding may also be partially rate-limiting. The structure of the human P450 21A2-substrate complex provides direct insight into mechanistic effects of genetic variants. PMID:25855791

  17. Role of zebrafish cytochrome P450 CYP1C genes in the reduced mesencephalic vein blood flow caused by activation of AHR2

    SciTech Connect

    Kubota, Akira; Stegeman, John J.; Woodin, Bruce R.; Iwanaga, Toshihiko; Harano, Ryo; Peterson, Richard E.; Hiraga, Takeo; Teraoka, Hiroki

    2011-06-15

    2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) causes various signs of toxicity in early life stages of vertebrates through activation of the aryl hydrocarbon receptor (AHR). We previously reported a sensitive and useful endpoint of TCDD developmental toxicity in zebrafish, namely a decrease in blood flow in the dorsal midbrain, but downstream genes involved in the effect are not known. The present study addressed the role of zebrafish cytochrome P450 1C (CYP1C) genes in association with a decrease in mesencephalic vein (MsV) blood flow. The CYP1C subfamily was recently discovered in fish and includes the paralogues CYP1C1 and CYP1C2, both of which are induced via AHR2 in zebrafish embryos. We used morpholino antisense oligonucleotides (MO or morpholino) to block initiation of translation of the target genes. TCDD-induced mRNA expression of CYP1Cs and a decrease in MsV blood flow were both blocked by gene knockdown of AHR2. Gene knockdown of CYP1C1 by two different morpholinos and CYP1C2 by two different morpholinos, but not by their 5 nucleotide-mismatch controls, was effective in blocking reduced MsV blood flow caused by TCDD. The same CYP1C-MOs prevented reduction of blood flow in the MsV caused by {beta}-naphthoflavone (BNF), representing another class of AHR agonists. Whole-mount in situ hybridization revealed that mRNA expression of CYP1C1 and CYP1C2 was induced by TCDD most strongly in branchiogenic primordia and pectoral fin buds. In situ hybridization using head transverse sections showed that TCDD increased the expression of both CYP1Cs in endothelial cells of blood vessels, including the MsV. These results indicate a potential role of CYP1C1 and CYP1C2 in the local circulation failure induced by AHR2 activation in the dorsal midbrain of the zebrafish embryo. - Research Highlights: > We examine the roles of zebrafish CYP1C1 and CYP1C2 in TCDD developmental toxicity. > TCDD induces mRNA expression of both CYP1Cs in the mesencephalic vein. > Knockdown of each

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

    SciTech Connect

    Polzer, R.J.

    1993-01-01

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

  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. 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...

  1. Effect of fermented red ginseng on cytochrome P450 and P‐glycoprotein activity in healthy subjects, as evaluated using the cocktail approach

    PubMed Central

    Kim, Min‐Gul; Kim, Yunjeong; Jeon, Ji‐Young

    2016-01-01

    Aims We assessed the drug interaction profile of fermented red ginseng with respect to the activity of major cytochrome (CYP) P450 enzymes and of a drug transporter protein, P‐glycoprotein (P‐gp), in healthy volunteers. Methods This study was an open‐label crossover study. The CYP probe cocktail drugs caffeine, losartan, dextromethorphan, omeprazole, midazolam and fexofenadine were administered before and after 2 weeks of fermented red ginseng administration. Plasma samples were collected, and tolerability was assessed. Pharmacokinetic parameters were calculated, and the 90% confidence intervals (CIs) of the geometric mean ratios of the parameters were determined from logarithmically transformed data. Values were compared between before and after fermented red ginseng administration using analysis of variance (anova). Results Fifteen healthy male subjects were evaluated, none of whom were genetically defined as a poor CYP2C9, CYP2C19 or CYP2D6 metabolizer based on genotyping. Before and after fermented red ginseng administration, the geometric least‐square mean metabolic ratio (90% CI) was 0.901 (0.830–0.979) for caffeine (CYP1A2) to paraxanthine, 0.774 (0.720–0.831) for losartan (CYP2C9) to EXP3174, 1.052 (0.925–1.197) for omeprazole (CYP2C19) to 5‐hydroxyomeprazole, 1.150 (0.860–1.538) for dextromethorphan (CYP2D6) to dextrorphan, and 0.816 (0.673–0.990) for midazolam (CYP3A4) to 1‐hydroxymidazolam. The geometric mean ratio of the area under the curve of the last sampling time (AUClast) for fexofenadine (P‐gp) was 1.322 (1.112–1.571). Conclusion No significantly different drug interactions were observed between fermented red ginseng and the CYP probe substrates following the two‐week administration of concentrated fermented red ginseng. However, the inhibition of P‐gp was significantly different between fermented red ginseng and the CYP probe substrates. The use of fermented red ginseng requires close attention due to the potential

  2. Size- and time-dependent alteration in metabolic activities of human hepatic cytochrome P450 isozymes by gold nanoparticles via microsomal coincubations

    NASA Astrophysics Data System (ADS)

    Ye, Meiling; Tang, Ling; Luo, Mengjun; Zhou, Jing; Guo, Bin; Liu, Yangyuan; Chen, Bo

    2014-11-01

    Nano-sized particles are known to interfere with drug-metabolizing cytochrome P450 (CYP) enzymes, which can be anticipated to be a potential source of unintended adverse reactions, but the mechanisms underlying the inhibition are still not well understood. Herein we report a systematic investigation of the impacts of gold nanoparticles (AuNPs) on five major CYP isozymes under in vitro incubations of human liver microsomes (HLMs) with tannic acid (TA)-stabilized AuNPs in the size range of 5 to 100 nm. It is found that smaller AuNPs show more pronounced inhibitory effects on CYP2C9, CYP2C19, CYP2D6, and CYP3A4 in a dose-dependent manner, while 1A2 is the least susceptible to the AuNP inhibition. The size- and dose-dependent CYP-specific inhibition and the nonspecific drug-nanogold binding in the coincubation media can be significantly reduced by increasing the concentration ratio of microsomal proteins to AuNPs, probably via a noncompetitive mode. Remarkably, AuNPs are also found to exhibit a slow time-dependent inactivation of 2D6 and 3A4 in a β-nicotinamide adenine dinucleotide 2'-phosphate reduced tetrasodium salt hydrate (NADPH)-independent manner. During microsomal incubations, UV-vis spectroscopy, dynamic light scattering, and zeta-potential measurements were used to monitor the changes in particle properties under the miscellaneous AuNP/HLM/CYP dispersion system. An improved stability of AuNPs by mixing HLM with the gold nanocolloid reveals that the stabilization via AuNP-HLM interactions may occur on a faster time scale than the salt-induced nanoaggregation by incubation in phosphate buffer. The results suggest that the AuNP induced CYP inhibition can be partially attributed to its adhesion onto the enzymes to alter their structural conformations or onto the HLM membrane therefore impairing the integral membrane proteins. Additionally, AuNPs likely block the substrate pocket on the CYP surface, depending on both the particle characteristics and the

  3. Inhibition of cytochrome p450 enzymes by enrofloxacin in the sea bass (Dicentrarchus labrax).

    PubMed

    Vaccaro, E; Giorgi, M; Longo, V; Mengozzi, G; Gervasi, P G

    2003-01-10

    Currently, there are no reports on the effects of enrofloxacin (EF), a fluoroquinolone antibiotic, on the cytochrome p450 enzymes in fish, although its use as antimicrobial agent in aquaculture has been put forward. Therefore, the in vivo and in vitro effects of EF on hepatic p450 enzymes of sea bass, a widespread food-producing fish, have been evaluated. Sea bass pretreated with a single dose of EF (3 mg/kg i.p.) or with three daily doses of EF (1 mg/kg i.p.) markedly depressed the microsomal N-demethylation of aminopyrine, erythromycin, the O-deethylation of 7-ethoxycoumarin, ethoxyresorufin and the 6beta-testosterone hydroxylase. In vitro experiments showed that EF at 10 microM inhibited the above-mentioned activities and, in particular, the erythromycin N-demethylase (ERND) and 6beta-testosterone-hydroxylase, likely dependant on a p450 3A isoform. When the nature of ERND inhibition by EF was specifically studied with sea bass liver microsomes, it was found that EF is a potent mechanism-based inhibitor, with K(i) of 3.7 microM and a K(inact) of 0.045 min(-1). An immunoblot analysis with anti p450 3A27 of trout showed that the p450 3A isoform, constitutively expressed in sea bass, is particularly susceptible to inactivation by EF. In vitro experiments with sea bass microsomes have also demonstrated that EF is oxidative deethylated by the p450 system to ciprofloxacin (CF) and that this compound maintains the ability to inactivate the p450 enzymes. The mechanism by which EF or CF inactivate the p450 enzymes has not been studied but an attack of p450 on the cyclopropan ring, present, both in EF and CF structure, with the formation of electrophilic intermediates (i.e. radicals) has been postulated. In conclusion, the EF seems to be a powerful inhibitor of p450s in the sea bass. Therefore, the clinical use of this antibiotic in aquaculture has to be considered with caution.

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

    PubMed Central

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

    2015-01-01

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

  5. Structural features of cytochromes P450 and ligands that affect drug metabolism as revealed by X-ray crystallography and NMR.

    PubMed

    Gay, Sean C; Roberts, Arthur G; Halpert, James R

    2010-09-01

    Cytochromes P450 (P450s) play a major role in the clearance of drugs, toxins, and environmental pollutants. Additionally, metabolism by P450s can result in toxic or carcinogenic products. The metabolism of pharmaceuticals by P450s is a major concern during the design of new drug candidates. Determining the interactions between P450s and compounds of very diverse structures is complicated by the variability in P450-ligand interactions. Understanding the protein structural elements and the chemical attributes of ligands that dictate their orientation in the P450 active site will aid in the development of effective and safe therapeutic agents. The goal of this review is to describe P450-ligand interactions from two perspectives. The first is the various structural elements that microsomal P450s have at their disposal to assume the different conformations observed in X-ray crystal structures. The second is P450-ligand dynamics analyzed by NMR relaxation studies.

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

    PubMed Central

    Klein, Andrew P; Sattely, Elizabeth S

    2015-01-01

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

  7. Differential cumene hydroperoxide sensitivity of cytochrome P-450 enzymes IA1 and IIB1 determined by their way of membrane incorporation.

    PubMed

    Balvers, W G; Boersma, M G; Veeger, C; Rietjens, I M

    1992-09-15

    The cytochrome P-450-dependent O-dealkylation of alkoxyresorufins was used to study the effect of cumene hydroperoxide on cytochrome P-450 IIB1 and IA1 in microsomal and reconstituted systems. In liver microsomal systems from respectively phenobarbital and 3-methylcholanthrene pretreated male Wistar rats, cytochrome P-450 IIB1-dependent pentoxyresorufin-O-dealkylation appeared to be more sensitive to cumene hydroperoxide treatment than cytochrome P-450 IA1-dependent ethoxyresorufin-O-dealkylation. This phenomenon was also observed when the cumene hydroperoxide sensitivity of P-450 IIB1 and IA1 was studied in an isosafrole pretreated rat liver microsomal system. The decrease in alkoxy-O-dealkylating activities appeared to proceed by destruction of the cytochrome P-450 component of the enzyme system. Purification and reconstitution of the enzyme system components in a system in which the isolated proteins were not incorporated into a membrane resulted in the disappearance of the difference in sensitivity between the two P-450 enzymes. However, in a reconstituted system with membrane incorporated proteins, again cytochrome P-450 IIB1 expressed a higher sensitivity towards cumene hydroperoxide than cytochrome P-450 IA1. From this it was concluded that the differential cumene hydroperoxide sensitivity of cytochrome P-450 IIB1 and IA1 is not caused by an intrinsic difference in their sensitivity but by a differential effect of membrane incorporation on their cumene hydroperoxide sensitivity.

  8. A thiolate-ligated nonheme oxoiron(IV) complex relevant to cytochrome P450.

    PubMed

    Bukowski, Michael R; Koehntop, Kevin D; Stubna, Audria; Bominaar, Emile L; Halfen, Jason A; Münck, Eckard; Nam, Wonwoo; Que, Lawrence

    2005-11-11

    Thiolate-ligated oxoiron(IV) centers are postulated to be the key oxidants in the catalytic cycles of oxygen-activating cytochrome P450 and related enzymes. Despite considerable synthetic efforts, chemists have not succeeded in preparing an appropriate model complex. Here we report the synthesis and spectroscopic characterization of [FeIV(O)(TMCS)]+ where TMCS is a pentadentate ligand that provides a square pyramidal N4(SR)apical, where SR is thiolate, ligand environment about the iron center, which is similar to that of cytochrome P450. The rigidity of the ligand framework stabilizes the thiolate in an oxidizing environment. Reactivity studies suggest that thiolate coordination favors hydrogen-atom abstraction chemistry over oxygen-atom transfer pathways in the presence of reducing substrates.

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

    PubMed Central

    Philpot, R M; Smith, B R

    1984-01-01

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

  10. 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.

  11. Synergistic Effects of Mutations in Cytochrome P450cam Designed to Mimic CYP101D1

    PubMed Central

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

    2013-01-01

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

  12. A possible role of cAMP dependent phosphorylation of hepatic microsomal cytochrome P450: a mechanism to increase lipid peroxidation in response to hormone.

    PubMed

    Mkrtchian, S L; Andersson, K K

    1990-01-30

    Enzymatic lipid peroxidation in hepatocytes is believed to involve cytochrome P450. cAMP dependent phosphorylation of cytochrome P450 was found to increase the NADPH dependent production of malondialdehyde (lipid peroxidation) by about 30%. The cytochrome P450 inhibitor cyanide abolished this activity. The presence of spermine decreased the cytochrome P450 dependent lipid peroxidation in non-phosphorylated microsomes, phosphorylation partially reversed this effect. Thus, phosphorylation of cytochrome P450 and the associated increased lipid peroxidation may be a hormone dependent response to pathological conditions e.g. stress Phosphorylation was observed to subtly alter other properties of cytochrome P450. The rate of 7-ethoxycoumarin deethylase activity was reduced and the microwave power required to saturate the EPR spectrum of the low spin cytochrome P450 was decreased. It is hypothesized that phosphorylation of cytochrome P450 alters the interaction between the components of the cytochrome P450 system, which may enhance production of free radical species, initiating lipid peroxidation.

  13. Metabolism of N-nitroso-2,6-dimethylmorpholine by isozymes of rabbit liver microsomal cytochrome P-450.

    PubMed

    Kokkinakis, D M; Koop, D R; Scarpelli, D G; Coon, M J; Hollenberg, P F

    1985-02-01

    The cis isomer of N-nitroso-2,6-dimethylmorpholine (NNDM), a pancreatic carcinogen for the Syrian golden hamster, is metabolized by hamster liver microsomes to yield N-nitroso(2-hydroxypropyl)(2-oxopropyl)amine (HPOP) as the major product. Rabbit liver microsomes catalyze the metabolism of cis-NNDM to HPOP at a rate slower than that observed with hamster microsomes, but significantly faster than that obtained with rat microsomes. Pretreatment of rabbits with phenobarbital results in a 6-fold increase in the cis-NNDM hydroxylase activity of the rabbit microsomes to levels equal to that observed with the hamster; pretreatment of rabbits with other xenobiotics had no effect on the hydroxylation of cis-NNDM. The role of rabbit liver microsomal cytochrome P-450 in the metabolism of the cis isomer of NNDM was studied in the reconstituted system consisting of NADPH:cytochrome P-450 reductase, phospholipid, and cytochrome P-450. Cytochrome P-450LM2, which is induced by pretreatment with phenobarbital, exhibited the highest activity for the metabolism of cis-NNDM. The Vmax for the formation of HPOP was 1.78 nmol/min/nmol cytochrome P-450LM2, and the apparent Km was 360 microM. Cytochrome P-450LM3a also catalyzed the metabolism of NNDM to HPOP at a significant rate (0.25 nmol/min/nmol cytochrome P-450). Of the four other isozymes of cytochrome P-450 (forms 3b, 3c, 4, and 6) tested in the reconstituted system, only forms 3b and 3c exhibited measurable activities (approximately 0.04 nmol of HPOP formed/min/nmol cytochrome P-450). The addition of antibodies to isozyme 2 to microsomes from phenobarbital-treated rabbits resulted in approximately 95% inhibition of the metabolism of NNDM, while the addition of antibodies to LM3a inhibited NNDM metabolism by only 7%. In microsomes from untreated rabbits, inhibition by anti-LM2 and anti-LM3a antibodies was 50 and 64%, respectively. The addition of antibodies to isozyme 3a to microsomes isolated from ethanol-treated rabbits caused

  14. 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.

  15. Concurrent Cooperativity and Substrate Inhibition in the Epoxidation of Carbamazepine by Cytochrome P450 3A4 Active Site Mutants Inspired by Molecular Dynamics Simulations

    PubMed Central

    2015-01-01

    Cytochrome P450 3A4 (CYP3A4) is the major human P450 responsible for the metabolism of carbamazepine (CBZ). To explore the mechanisms of interactions of CYP3A4 with this anticonvulsive drug, we carried out multiple molecular dynamics (MD) simulations, starting with the complex of CYP3A4 manually docked with CBZ. On the basis of these simulations, we engineered CYP3A4 mutants I369F, I369L, A370V, and A370L, in which the productive binding orientation was expected to be stabilized, thus leading to increased turnover of CBZ to the 10,11-epoxide product. In addition, we generated CYP3A4 mutant S119A as a control construct with putative destabilization of the productive binding pose. Evaluation of the kinetics profiles of CBZ epoxidation demonstrate that CYP3A4-containing bacterial membranes (bactosomes) as well as purified CYP3A4 (wild-type and mutants I369L/F) exhibit substrate inhibition in reconstituted systems. In contrast, mutants S119A and A370V/L exhibit S-shaped profiles that are indicative of homotropic cooperativity. MD simulations with two to four CBZ molecules provide evidence that the substrate-binding pocket of CYP3A4 can accommodate more than one molecule of CBZ. Analysis of the kinetics profiles of CBZ metabolism with a model that combines the formalism of the Hill equation with an allowance for substrate inhibition demonstrates that the mechanism of interactions of CBZ with CYP3A4 involves multiple substrate-binding events (most likely three). Despite the retention of the multisite binding mechanism in the mutants, functional manifestations reveal an exquisite sensitivity to even minor structural changes in the binding pocket that are introduced by conservative substitutions such as I369F, I369L, and A370V. PMID:25545162

  16. Concurrent cooperativity and substrate inhibition in the epoxidation of carbamazepine by cytochrome P450 3A4 active site mutants inspired by molecular dynamics simulations.

    PubMed

    Müller, Christian S; Knehans, Tim; Davydov, Dmitri R; Bounds, Patricia L; von Mandach, Ursula; Halpert, James R; Caflisch, Amedeo; Koppenol, Willem H

    2015-01-27

    Cytochrome P450 3A4 (CYP3A4) is the major human P450 responsible for the metabolism of carbamazepine (CBZ). To explore the mechanisms of interactions of CYP3A4 with this anticonvulsive drug, we carried out multiple molecular dynamics (MD) simulations, starting with the complex of CYP3A4 manually docked with CBZ. On the basis of these simulations, we engineered CYP3A4 mutants I369F, I369L, A370V, and A370L, in which the productive binding orientation was expected to be stabilized, thus leading to increased turnover of CBZ to the 10,11-epoxide product. In addition, we generated CYP3A4 mutant S119A as a control construct with putative destabilization of the productive binding pose. Evaluation of the kinetics profiles of CBZ epoxidation demonstrate that CYP3A4-containing bacterial membranes (bactosomes) as well as purified CYP3A4 (wild-type and mutants I369L/F) exhibit substrate inhibition in reconstituted systems. In contrast, mutants S119A and A370V/L exhibit S-shaped profiles that are indicative of homotropic cooperativity. MD simulations with two to four CBZ molecules provide evidence that the substrate-binding pocket of CYP3A4 can accommodate more than one molecule of CBZ. Analysis of the kinetics profiles of CBZ metabolism with a model that combines the formalism of the Hill equation with an allowance for substrate inhibition demonstrates that the mechanism of interactions of CBZ with CYP3A4 involves multiple substrate-binding events (most likely three). Despite the retention of the multisite binding mechanism in the mutants, functional manifestations reveal an exquisite sensitivity to even minor structural changes in the binding pocket that are introduced by conservative substitutions such as I369F, I369L, and A370V.

  17. 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.

  18. Construction of a 3D model of cytochrome P450 2B4.

    PubMed

    Chang, Y T; Stiffelman, O B; Vakser, I A; Loew, G H; Bridges, A; Waskell, L

    1997-02-01

    A three-dimensional structural model of rabbit phenobarbital-inducible cytochrome P450 2B4 (LM2) was constructed by homology modeling techniques previously developed for building and evaluating a 3D model of the cytochrome P450choP isozyme. Four templates with known crystal structures including cytochrome P450cam, terp, BM-3 and eryF were used in multiple sequence alignments and construction of the cytochrome P450 2B4 coordinates. The model was evaluated for its overall quality using available protein analysis programs and found to be satisfactory. The model structure was stable at room temperature during a 140 ps unconstrained full protein molecular dynamics simulation. A putative substrate access channel and binding site were identified. Two different substrates, benzphetamine and androstenedione, that are metabolized by cytochrome P450 2B4 with pronounced product specificity were docked into the putative binding site. Two orientations were found for each substrate that could lead to the observed preferred products. Using a geometric fit method three regions on the surface of the model cytochrome P450 structure were identified as possible sites for interaction with cytochrome b5, a redox partner of P450 2B4. Residues that may interact with the substrates and with cytochrome b5 have been identified and mutagenesis studies are currently in progress.

  19. Tumor-specific expression of cytochrome P450 CYP1B1.

    PubMed

    Murray, G I; Taylor, M C; McFadyen, M C; McKay, J A; Greenlee, W F; Burke, M D; Melvin, W T

    1997-07-15

    Cytochrome P450 CYP1B1 is a recently cloned dioxin-inducible form of the cytochrome P450 family of xenobiotic metabolizing enzymes. An antibody raised against a peptide specific for CYP1B1 was found to recognize CYP1B1 expressed in human lymphoblastoid cells but not to recognize other forms of cytochrome P450, particularly CYP1A1 and CYP1A2. Using this antibody, the cellular distribution and localization of CYP1B1 were investigated by immunohistochemistry in a range of malignant tumors and corresponding normal tissues. CYP1B1 was found to be expressed at a high frequency in a wide range of human cancers of different histogenetic types, including cancers of the breast, colon, lung, esophagus, skin, lymph node, brain, and testis. There was no detectable immunostaining for CYP1B1 in normal tissues. These results provide the basis for the development of novel methods of cancer diagnosis based on the identification of CYP1B1 in tumor cells and the development of anticancer drugs that are selectively activated in tumors by CYP1B1.

  20. Cytochrome P450 CYP1B1 over-expression in primary and metastatic ovarian cancer

    PubMed Central

    McFadyen, M C E; Cruickshank, M E; Miller, I D; McLeod, H L; Melvin, W T; Haites, N E; Parkin, D; Murray, G I

    2001-01-01

    Ovarian cancer is the most frequent cause of death from gynaecological malignancies world wide. Little improvement has been made in the long-term outcome of this disease, with the 5-year survival of patients only 30%. This poor prognosis is due to the late presentation of the disease and to the unpredictable response of ovarian cancer to chemotherapy. The cytochrome P450 enzymes are a superfamily of haemoproteins, known to be involved in the metabolic activation and/or detoxification of a number of anti-cancer drugs. CYP1B1 is a tumour-related form of cytochrome P450 which is over expressed in a wide variety of primary tumours of different histological type. The presence of CYP1B1 may be of importance in the modulation of these tumours to anti-cancer drugs. We have conducted a comprehensive immunohistochemical investigation, into the presence of cytochrome P450 CYP1B1 in primary and metastatic ovarian cancer. The key findings of this study are the increased expression of CYP1B1 in the majority of ovarian cancers investigated (92%), with a strong correlation demonstrated between CYP1B1 expression in both primary and metastatic ovarian cancer (P= 0.005 Spearman's rank correlation test). In contrast no detectable CYP1B1 was found in normal ovary. © 2001 Cancer Research Campaign http://www.bjcancer.com PMID:11461084

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

    SciTech Connect

    Mandai, Takao; Fujiwara, Shinsuke; Imaoka, Susumu

    2009-06-19

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

  2. Cytochrome P450 CYP1B1 over-expression in primary and metastatic ovarian cancer.

    PubMed

    McFadyen, M C; Cruickshank, M E; Miller, I D; McLeod, H L; Melvin, W T; Haites, N E; Parkin, D; Murray, G I

    2001-07-20

    Ovarian cancer is the most frequent cause of death from gynaecological malignancies world wide. Little improvement has been made in the long-term outcome of this disease, with the 5-year survival of patients only 30%. This poor prognosis is due to the late presentation of the disease and to the unpredictable response of ovarian cancer to chemotherapy. The cytochrome P450 enzymes are a superfamily of haemoproteins, known to be involved in the metabolic activation and/or detoxification of a number of anti-cancer drugs. CYP1B1 is a tumour-related form of cytochrome P450 which is over expressed in a wide variety of primary tumours of different histological type. The presence of CYP1B1 may be of importance in the modulation of these tumours to anti-cancer drugs. We have conducted a comprehensive immunohistochemical investigation, into the presence of cytochrome P450 CYP1B1 in primary and metastatic ovarian cancer. The key findings of this study are the increased expression of CYP1B1 in the majority of ovarian cancers investigated (92%), with a strong correlation demonstrated between CYP1B1 expression in both primary and metastatic ovarian cancer (P = 0.005 Spearman's rank correlation test). In contrast no detectable CYP1B1 was found in normal ovary.

  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. 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.

  5. Interaction of sanguinarine alkaloid, isolated from argemone oil, with hepatic cytochrome p450 in rats.

    PubMed

    Reddy, Naveen P; Das, Mukul

    2008-01-01

    ABSTRACT Prior studies have shown that argemone oil (AO), responsible for 'Epidemic dropsy', causes inhibition of catalytic activities of Cytochrome P450 (P450). In this study interaction of sanguinarine (SAN) alkaloid, isolated from AO, with rat hepatic P450 was investigated. Hepatic microsomes prepared from 3-methylcholantherene (3MC) treated rats when incubated with SAN (1-3 muM) resulted in a spectral peak at 385 nm and a trough at 415 nm, indicative of Type I binding. Incubation of SAN (50-200 muM) with hepatic microsomes prepared from phenobarbitone (PB) treated rats also showed a Type I spectra with a peak at 395 nm and a trough at 420 nm. Relative binding efficiency (DeltaA(max)/K(s)(app) factor) of SAN with P450 was found to be 1540 and 1030 absorbance units/nmol CYP/M for 3MC and PB induced microsomes, respectively. In a P450 spectral inhibition study SAN showed higher affinity towards 3MC eliciting inhibition at much lesser concentrations (0.25-5 muM) as compared to PB (100-300 muM). The IC50s of SAN with different catalytic markers of P450 isoforms, i.e. ethoxyresorufin-O-deethylase (EROD) for CYP1A1, was 2.8 muM and for methoxyresorufin-O-deethylase (MROD) for CYP1A2 was 2.2 muM in 3MC induced microsomes, while benzoyloxyresorufin-O-deethylase (BROD) for CYP 2B1/1A1 showed an IC50 of 50 muM but pentoxyresorufin-O-deethylase (PROD) for CYP2B1 showed no inhibition even at higher concentrations of SAN (> 60 muM) in PB-induced microsomes. These results indicate that higher affinity of SAN binding towards the CYP1A family may have a role in SAN toxicity.

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

    PubMed

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

    2005-03-01

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

  7. Molecular genetic analysis of the cytochrome P450-debrisoquine hydroxylase locus and association with cancer susceptibility.

    PubMed Central

    Smith, C A; Moss, J E; Gough, A C; Spurr, N K; Wolf, C R

    1992-01-01

    The cytochrome P450-dependent monooxygenases play a central role in the metabolism of chemical carcinogens. The action of these enzymes can lead to either carcinogen detoxication or activation. Differences in P450 expression in animal models give rise to large differences in susceptibility to chemical carcinogens, so genetic polymorphisms in P450 expression may be expected to be an important factor in individual human susceptibility to cancer. Of particular interest is the genetic polymorphism at the cytochrome P450-debrisoquine/sparteine hydroxylase locus (CYP2D6). Although this is a minor liver P450, its polymorphic expression is associated with the abnormal metabolism of at least 30 therapeutic drugs, including beta-blockers and tricyclic antidepressants. Conflicting reports have been made on the association of this polymorphism with cancer susceptibility. This disagreement may be attributable to limitations of the phenotyping assay used to identify affected individuals (poor metabolizers, PMs). In order to clarify these anomalies, we have developed a simple DNA-based assay with which we can identify the majority of PMs. The assay is centered around the primary gene defect responsible for the polymorphism, a G to A transition at the junction of intron 3/exon 4 which results in a frame-shift in the resultant mRNA. The frequency of this mutation is 70-80% in PMs. We have studied the frequency of mutated alleles in a control population and in a wide range of cancer patients. No association between this polymorphism and lung cancer susceptibility was observed; however, in other populations of cancer patients some very interesting shifts were found in the proportion of PMs and heterozygotes from that in the normal population. PMID:1486838

  8. Hepatic microsomal cytochrome P450 enzyme activity in relation to in vitro metabolism/inhibition of polychlorinated biphenyls and testosterone in Baltic grey seal (Halichoerus grypus).

    PubMed

    Li, Hongxia; Boon, Jan P; Lewis, Wilma E; van den Berg, Martin; Nyman, Madeleine; Letcher, Robert J

    2003-03-01

    Among other factors, cytochrome P450 (CYP) enzyme activity determines polychlorinated biphenyl (PCB) bioaccumulation, biotransformation, and toxicity in exposed species. We measured the oxidative metabolism in vitro of 12 PCB congeners, representing structural groups based on the number and position of the chlorine atoms, by the hepatic microsomes of one Baltic grey seal (Halichoerus grypus). Microsomal metabolism was observed for several PCBs with vicinal H atoms exclusively in the ortho and meta positions and without any ortho-Cl substituents (CB-15 [4,4'-Cl2] and CB-77 [3,3',4,4'-Cl4]), vicinal meta and para-H atoms (CB-52 [2,2',5,5'-Cl4], and -101 [2,2',4,5,5'-Cl5]) or with both characteristics in combination with either only one ortho-Cl (CB-26 [2,3',5-Cl3], CB-31 [2,4',5-Cl3]) or two ortho-Cl substituents (CB-44 [2,2',3,5'-Cl4]). To allocate PCB biotransformation to specific CYPs, the inhibitive effect of compounds with known CYP-specific inhibition properties was assessed on in vitro PCB metabolism and on regio- and stereospecific testosterone hydroxylase activities. Metabolic inhibition was considered relevant at concentrations < or = 1.0 microM because these inhibitors became decreasingly selective at higher concentrations. At < 1.0 microM, ellipticine (CYPIAI/2 inhibitor) selectively inhibited CB-15, -26, -31, and -77 metabolism, with no significant inhibition of CB-44, -52, and -101 metabolism. Inhibition of CB-52 and -101 metabolism by chloramphenicol (CYP2B inhibitor) started at 1.0 microM and maximized at about 100% at 10 microM. Ketoconazole (CYP3A inhibitor) appeared to selectively inhibit CB-26, -31, and -44 metabolism relative to CB-15, -77, and -52 at concentrations < or = 1.0 microM. Major testosterone metabolites formed in vitro were 2beta-(CYP3A), 6beta- (CYP3A, CYPIA), and 16beta- (CYP2B) hydroxytestosterone and androstenedione (CYP2B, CYP2C11). The CYP forms indicated are associated with the specific metabolism of testosterone in laboratory

  9. Specific and non-specific effects of potassium cations on substrate-protein interactions in cytochromes P450cam and P450lin.

    PubMed

    Deprez, Eric; Gill, Edward; Helms, Volkhard; Wade, Rebecca C; Hui Bon Hoa, Gaston

    2002-09-20

    Substrate binding to cytochrome P450cam is generally considered to be a two-step process. The first step corresponds to the entrance of the substrate, camphor, into the heme pocket. The second step corresponds to a spin transition (low spin-->high spin) of the iron in the protein-substrate complex. This spin transition is related to the mobility of the substrate inside the active site [Biochim Biophys Acta 1338 (1997) 77]. Potassium cations (K(+)) have a specific effect on the spin equilibrium. This is generally attributed to the K(+) ion-induced conformational change of tyrosine 96, the hydroxyl group of which is hydrogen bonded to the keto group of camphor and results in optimum substrate orientation and reduced mobility of this substrate in the active site. In the present paper, we show that K(+) not only affects the substrate-Tyr 96 couple, but acts more globally since K(+) effects are also observed in the Tyr96Phe mutant as well as in complexes with camphor-analogues. Large compounds, that fit well in the heme pocket and bind with higher affinity than camphor, display high spin contents that are less dependent on the presence of K(+). In contrast, K(+) has a significant effect on the high spin content of substrate-cytochrome P450cam complexes with looser interactions. We conclude that large compounds with higher affinities than camphor have more van der Waals contacts with the active site residues. Their mobilities are then reduced and less dependent on the presence of K(+). In this study, we also explored, for comparison, the K(+) effect on the spin transition state of another member of the P450 superfamily, cytochrome P450lin. This effect is not as strong as those observed for cytochrome P450cam. Even though the spin equilibrium does not change dramatically in the presence of K(+) or Na(+), the value of the dissociation constant (K(d)) for linalool binding is significantly affected by ionic strength. Analysis of the thermodynamic parameters for the linalool

  10. A cytochrome P450 terpenoid hydroxylase linked to the suppression of insect juvenile hormone synthesis.

    PubMed

    Sutherland, T D; Unnithan, G C; Andersen, J F; Evans, P H; Murataliev, M B; Szabo, L Z; Mash, E A; Bowers, W S; Feyereisen, R

    1998-10-27

    A cDNA encoding a cytochrome P450 enzyme was isolated from a cDNA library of the corpora allata (CA) from reproductively active Diploptera punctata cockroaches. This P450 from the endocrine glands that produce the insect juvenile hormone (JH) is most closely related to P450 proteins of family 4 and was named CYP4C7. The CYP4C7 gene is expressed selectively in the CA; its message could not be detected in the fat body, corpora cardiaca, or brain, but trace levels of expression were found in the midgut and caeca. The levels of CYP4C7 mRNA in the CA, measured by ribonuclease protection assays, were linked to the activity cycle of the glands. In adult females, CYP4C7 expression increased immediately after the peak of JH synthesis, reaching a maximum on day 7, just before oviposition. mRNA levels then declined after oviposition and during pregnancy. The CYP4C7 protein was produced in Escherichia coli as a C-terminal His-tagged recombinant protein. In a reconstituted system with insect NADPH cytochrome P450 reductase, cytochrome b5, and NADPH, the purified CYP4C7 metabolized (2E,6E)-farnesol to a more polar product that was identified by GC-MS and by NMR as (10E)-12-hydroxyfarnesol. CYP4C7 converted JH III to 12-trans-hydroxy JH III and metabolized other JH-like sesquiterpenoids as well. This omega-hydroxylation of sesquiterpenoids appears to be a metabolic pathway in the corpora allata that may play a role in the suppression of JH biosynthesis at the end of the gonotrophic cycle.

  11. Identification of a new plasmid-encoded cytochrome P450 CYP107DY1 from Bacillus megaterium with a catalytic activity towards mevastatin.

    PubMed

    Milhim, Mohammed; Putkaradze, Natalia; Abdulmughni, Ammar; Kern, Fredy; Hartz, Philip; Bernhardt, Rita

    2016-12-20

    In the current work, we describe the identification and characterization of the first plasmid-encoded P450 (CYP107DY1) from a Bacillus species. The recombinant CYP107DY1 exhibits characteristic P450 absolute and reduced CO-bound difference spectra. Reconstitution with different redox systems revealed the autologous one, consisting of BmCPR and Fdx2, as the most effective one. Screening of a library of 18 pharmaceutically relevant compounds displayed activity towards mevastatin to produce pravastatin. Pravastatin is an important therapeutic drug to treat hypercholesterolemia, which was described to be produced by oxyfunctionlization of mevastatin (compactin) by members of CYP105 family. The hydroxylation at C6 of mevastatin was also suggested by docking this compound into a computer model created for CYP107DY1. Moreover, in view of the biotechnological application, CYP107DY1 as well as its redox partners (BmCPR and Fdx2) were successfully utilized to establish an E. coli based whole-cell system for an efficient biotransformation of mevastatin. The in vitro and in vivo application of the CYP07DY1 also offers the possibility for the screening of more substrates, which could open up further biotechnological usage of this enzyme.

  12. BAP31 is involved in the retention of cytochrome P450 2C2 in the endoplasmic reticulum.

    PubMed

    Szczesna-Skorupa, Elzbieta; Kemper, Byron

    2006-02-17

    Microsomal cytochrome P450 2C2 is an integral endoplasmic reticulum (ER) membrane protein that is directly retained in the ER and excluded from transport vesicles. We have used bimolecular fluorescence complementation and co-immunoprecipitation to show that a ubiquitous ER membrane protein (BAP31) interacts with P450 2C2 in transfected COS-1 cells. A chimera containing only the N-terminal signal anchor of P450 2C1 (P450 2C1-(1-29)) also interacted with BAP31, which is consistent with interaction of the two proteins via their transmembrane domains. Down-regulation of BAP31 expression with small interfering RNA resulted in redistribution of green fluorescent protein-tagged P450 2C2 or P450 2C1-(1-29) from the ER into the nuclear membrane and compact perinuclear compartment structures as well as the cell surface in a small fraction of the cells. In Bap31-null embryonic stem cells, a significant fraction of P450 2C2 or P450 2C1-(1-29) was detected at the cell surface and nuclear envelope, but was redistributed to the ER by expression of BAP31. The expression level of P450 2C2 was significantly increased in COS-1 cells with repressed levels of BAP31. Formation of the pro-apoptotic p20 fragment of BAP31 was detected in transfected COS-1 cells expressing P450 2C2, and annexin V staining was consistent with the activation of an apoptotic pathway in these cells. Down-regulation of BAP31 with small interfering RNA partially reversed the apoptosis. These results suggest that interaction of P450 2C2 with BAP31 is important for its ER retention and expression level and that BAP31 may be involved in the regulation of apoptosis induced by the ER overload response to increased expression of P450.

  13. Characterization of benidipine and its enantiomers' metabolism by human liver cytochrome P450 enzymes.

    PubMed

    Yoon, Yune-Jung; Kim, Kwon-Bok; Kim, Hyunmi; Seo, Kyung-Ah; Kim, Ho-Sook; Cha, In-June; Kim, Eun-Young; Liu, Kwang-Hyeon; Shin, Jae-Gook

    2007-09-01

    Benidipine is a dihydropyridine calcium antagonist that has been used clinically as an antihypertensive and antianginal agent. It is used clinically as a racemate, containing the (-)-alpha and (+)-alpha isomers of benidipine. This study was performed to elucidate the metabolism of benidipine and its enantiomers in human liver microsomes (HLMs) and to characterize the cytochrome P450 (P450) enzymes that are involved in the metabolism of benidipine. Human liver microsomal incubation of benidipine in the presence of NADPH resulted in the formation of two metabolites, N-desbenzylbenidipine and dehydrobenidipine. The intrinsic clearance (CL(int)) of the formation of N-desbenzylbenidipine and dehydrobenidipine metabolites from (-)-alpha isomer was similar to those from the (+)-alpha isomer (1.9 +/- 0.1 versus 2.3 +/- 2.3 microl/min/pmol P450 and 0.5 +/- 0.2 versus 0.6 +/- 0.6 microl/min/pmol P450, respectively). Correlation analysis between the known P450 enzyme activities and the rate of the formation of benidipine metabolites in the 15 HLMs showed that benidipine metabolism is correlated with CYP3A activity. The P450 isoform-selective inhibition study in liver microsomes and the incubation study of cDNA-expressed enzymes also showed that theN-debenzylation and dehydrogenation of benidipine are mainly mediated by CYP3A4 and CYP3A5. The total CL(int) values of CYP3A4-mediated metabolite formation from (-)-alpha isomer were similar to those from (+)-alpha isomer (17.7 versus 14.4 microl/min/pmol P450, respectively). The total CL(int) values of CYP3A5-mediated metabolite formation from (-)-alpha isomer were also similar to those from (+)-alpha isomer (8.3 versus 11.0 microl/min/pmol P450, respectively). These findings suggest that CYP3A4 and CYP3A5 isoforms are major enzymes contributing to the disposition of benidipine, but stereoselective disposition of benidipine in vivo may be influenced not by stereoselective metabolism but by other factors.

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

    PubMed Central

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

    1990-01-01

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

  15. Cytochrome P450IA mRNA expression in feral Hudson River tomcod

    SciTech Connect

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

    1991-06-01

    The authors 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, they 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.

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

    PubMed

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

    2013-12-01

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

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

    DOEpatents

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

    1993-01-01

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

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

    DOEpatents

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

    1992-01-01

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

  19. 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...

  20. Significantly shorter Fe–S bond in cytochrome P450-I is consistent with greater reactivity relative to chloroperoxidase

    DOE PAGES

    Krest, Courtney M.; Silakov, Alexey; Rittle, Jonathan; ...

    2015-08-03

    Cytochrome P450 (P450) and chloroperoxidase (CPO) are thiolate-ligated haem proteins that catalyse the activation of carbon hydrogen bonds. The principal intermediate in these reactions is a ferryl radical species called compound I. P450 compound I (P450-I) is significantly more reactive than CPO-I, which only cleaves activated C–H bonds. In this paper, to provide insight into the differing reactivities of these intermediates, we examined CPO-I and P450-I using variable-temperature Mössbauer and X-ray absorption spectroscopies. These measurements indicate that the Fe–S bond is significantly shorter in P450-I than in CPO-I. This difference in Fe–S bond lengths can be understood in terms ofmore » variations in the hydrogen-bonding patterns within the ‘cys-pocket’ (a portion of the proximal helix that encircles the thiolate ligand). Weaker hydrogen bonding in P450-I results in a shorter Fe–S bond, which enables greater electron donation from the axial thiolate ligand. Finally, this observation may in part explain P450's greater propensity for C–H bond activation.« less

  1. Significantly shorter Fe–S bond in cytochrome P450-I is consistent with greater reactivity relative to chloroperoxidase

    SciTech Connect

    Krest, Courtney M.; Silakov, Alexey; Rittle, Jonathan; Yosca, Timothy H.; Onderko, Elizabeth L.; Calixto, Julio C.; Green, Michael T.

    2015-08-03

    Cytochrome P450 (P450) and chloroperoxidase (CPO) are thiolate-ligated haem proteins that catalyse the activation of carbon hydrogen bonds. The principal intermediate in these reactions is a ferryl radical species called compound I. P450 compound I (P450-I) is significantly more reactive than CPO-I, which only cleaves activated C–H bonds. In this paper, to provide insight into the differing reactivities of these intermediates, we examined CPO-I and P450-I using variable-temperature Mössbauer and X-ray absorption spectroscopies. These measurements indicate that the Fe–S bond is significantly shorter in P450-I than in CPO-I. This difference in Fe–S bond lengths can be understood in terms of variations in the hydrogen-bonding patterns within the ‘cys-pocket’ (a portion of the proximal helix that encircles the thiolate ligand). Weaker hydrogen bonding in P450-I results in a shorter Fe–S bond, which enables greater electron donation from the axial thiolate ligand. Finally, this observation may in part explain P450's greater propensity for C–H bond activation.

  2. Significantly shorter Fe-S bond in cytochrome P450-I is consistent with greater reactivity relative to chloroperoxidase

    PubMed Central

    Krest, Courtney M.; Silakov, Alexey; Rittle, Jonathan; Yosca, Timothy H.; Onderko, Elizabeth L.; Calixto, Julio C.; Green, Michael T.

    2015-01-01

    Cytochrome P450 (P450) and chloroperoxidase (CPO) are thiolate ligated heme proteins that catalyze the activation of carbon hydrogen bonds. The principal intermediate in these reactions is a ferryl radical species called compound I. P450 compound I (P450-I) is significantly more reactive than CPO-I, which only cleaves activated C-H bonds. To provide insight into the differing reactivities of these intermediates, we examined CPO-I and P450-I with variable temperature Mössbauer and X-ray absorption spectroscopies. These measurements indicate that the Fe-S bond is significantly shorter in P450-I than in CPO-I. This difference in Fe-S bond lengths can be understood in terms of variations in hydrogen bonding patterns within the “cys-pocket” (a portion of the proximal helix that encircles the thiolate ligand). Weaker hydrogen bonding in P450-I results in a shorter Fe-S bond, which enables greater electron donation from the axial-thiolate ligand. This observation may in part explain P450's greater propensity for C-H bond activation. PMID:26291940

  3. Relevance of cytochrome P450s in plants: also one of Ron Estabrook's research interests.

    PubMed

    Shet, Manjunath S

    2007-01-01

    I worked with Dr. Ronald Estabrook for nearly 10 years at The University of Texas Southwestern Medical Center in Dallas, Texas. In Ron's lab, when I joined I was initially involved in the isolation, purification, and characterization of cytochrome P450s and NADPH-P450(c) reductase(s) from plants, which was his new exploratory project at the time. We developed methods for the isolation, solubilization, and purification of P450s and NADPH-P450(c) reductase from plant tissue microsomes. We carried out number of in vitro experiments to study the involvement P450s and NADPH-P450(c) reductase in the biosynthesis of number of phytoalexins. We successfully isolated, purified, and cloned NADPH-P450(c) reductase from etiolated mung bean (Vigna radiate) seedlings. In addition, a series of studies were undertaken to show that purified mung bean NADPH-P450(c) reductase was able to catalyze P450-supported reactions for mammalian and bacterial P450s. My stay in Ron's lab was very educational and productive. He provided the necessary support and led the way through the maze in different research projects in the lab, which allowed me to understand the roles of P450s in humans, animals, plants, and microorganisms. He liked to teach and discover new things everyday in the lab. He is a great scientist, as well as loving and caring mentor.

  4. Genomewide annotation and comparative genomics of cytochrome P450 monooxygenases (P450s) in the polypore species Bjerkandera adusta, Ganoderma sp. and Phlebia brevispora.

    PubMed

    Syed, Khajamohiddin; Nelson, David R; Riley, Robert; Yadav, Jagjit S

    2013-01-01

    Genomewide annotation of cytochrome P450 monooxygenases (P450s) in three white-rot species of the fungal order Polyporales, namely Bjerkandera adusta, Ganoderma sp. and Phlebia brevispora, revealed a large contingent of P450 genes (P450ome) in their genomes. A total of 199 P450 genes in B. adusta and 209 P450 genes each in Ganoderma sp. and P. brevispora were identified. These P450omes were classified into families and subfamilies as follows: B. adusta (39 families, 86 subfamilies), Ganoderma sp. (41 families, 105 subfamilies) and P. brevispora (42 families, 111 subfamilies). Of note, the B. adusta genome lacked the CYP505 family (P450foxy), a group of P450-CPR fusion proteins. The three polypore species revealed differential enrichment of individual P450 families in their genomes. The largest CYP families in the three genomes were CYP5144 (67 P450s), CYP5359 (46 P450s) and CYP5344 (43 P450s) in B. adusta, Ganoderma sp. and P. brevispora, respectively. Our analyses showed that tandem gene duplications led to expansions in certain P450 families. An estimated 33% (72 P450s), 28% (55 P450s) and 23% (49 P450s) of P450ome genes were duplicated in P. brevispora, B. adusta and Ganoderma sp., respectively. Family-wise comparative analysis revealed that 22 CYP families are common across the three Polypore species. Comparative P450ome analysis with Ganoderma lucidum revealed the presence of 143 orthologs and 56 paralogs in Ganoderma sp. Multiple P450s were found near the characteristic biosynthetic genes for secondary metabolites, namely polyketide synthase (PKS), non-ribosomal peptide synthetase (NRPS), terpene cyclase and terpene synthase in the three genomes, suggesting a likely role of these P450s in secondary metabolism in these Polyporales. Overall, the three species had a richer P450 diversity both in terms of the P450 genes and P450 subfamilies as compared to the model white-rot and brown-rot polypore species Phanerochaete chrysosporium and Postia placenta.

  5. The cytochrome P450 superfamily: biochemistry, evolution and drug metabolism in humans.

    PubMed

    Danielson, P B

    2002-12-01

    Cytochrome p450s comprise a superfamily of heme-thiolate proteins named for the spectral absorbance peak of their carbon-monoxide-bound species at 450 nm. Having been found in every class of organism, including Archaea, the p450 superfamily is believed to have originated from an ancestral gene that existed over 3 billion years ago. Repeated gene duplications have subsequently given rise to one of the largest of multigene families. These enzymes are notable both for the diversity of reactions that they catalyze and the range of chemically dissimilar substrates upon which they act. Cytochrome p450s support the oxidative, peroxidative and reductive metabolism of such endogenous and xenobiotic substrates as environmental pollutants, agrochemicals, plant allelochemicals, steroids, prostaglandins and fatty acids. In humans, cytochrome p450s are best know for their central role in phase I drug metabolism where they are of critical importance to two of the most significant problems in clinical pharmacology: drug interactions and interindividual variability in drug metabolism. Recent advances in our understanding of cytochrome p450-mediated drug metabolism have been accelerated as a result of an increasing emphasis on functional genomic approaches to p450 research. While human cytochrome p450 databases have swelled with a flood of new human sequence variants, however, the functional characterization of the corresponding gene products has not kept pace. In response researchers have begun to apply the tools of proteomics as well as homology-based and ab initio modeling to salient questions of cytochrome p450 structure/function. This review examines the latest advances in our understanding of human cytochrome p450s.

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

    SciTech Connect

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

    2011-08-05

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

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

    PubMed Central

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

    2016-01-01

    Summary Cytochrome P450 (P450) 4X1 is one of the so-called “orphan” P450s without assigned biological function. Codon-optimized P450 4X1 and a number of N-terminal modified sequences were expressed in Escherichia coli. Native P450 4X1 showed a characteristic P450 spectrum but low expression in E. coli DH5α cells (<100 nmol P450/L). The highest level of expression (300-450 nmol P450/L culture) was achieved with a bicistronic P450 4X1 construct (N-terminal MAKKTSSKGKL, change of E2A, amino acids 3-44 truncated). Anandamide (arachidonoyl ethanolamide) has emerged as an important signaling molecule in the neurovascular cascade. Recombinant P450 4X1 protein, co-expressed with human NADPH-P450 reductase in E. coli, was found to convert the natural endocannabinoid anandamide to a single monooxygenated product, 14,15-epoxyeicosatrienoic (EET) ethanolamide. A stable anandamide analog (CD-25) was also converted to a monooxygenated product. Arachidonic acid was oxidized more slowly to 14,15- and 8,9-EETs but only in the presence of cytochrome b5. Other fatty acids were investigated as putative substrates but showed only little or minor oxidation. Real-time PCR analysis demonstrated extrahepatic mRNA expression, including several human brain structures (cerebellum, amygdala, and basal ganglia), in addition to expression in human heart, liver, prostate, and breast. The highest mRNA expression levels were detected in amygdala and skin. The ability of P450 4X1 to generate anandamide derivatives and the mRNA distribution pattern suggest a potential role for P450 4X1 in anandamide signaling in the brain. PMID:18549450

  8. 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.

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

    PubMed Central

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

    1991-01-01

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

  10. Pharmacokinetics of Lipophilic Agents Following Preexposure: Non-Cytochrome P-450 Mediated Mechanisms

    DTIC Science & Technology

    1990-05-30

    FOLLOWING PREEXPOSUR G - AFOSR-87- 185 U’) NON -CYTOCHROME P-450 MEDIATED MECHAN’ICS PE - 61102F (NJ . AUTHOKS) PR - 2312 N TA - A5 Dr Lawrence R Curtis, Dr...Z39- I*. PHARMACOKINETICS OF LIPOPHILIC AGENTS FOLLOWING PREEXPOSURE: NON -CYTOCHROME P-450 MEDIATED MECHANISMS Air Force Grant No. 87-0185 Hillary M...amounts located in non -hepatic tissues. These studies showed that, despite the differences in PDR, the systems involved respond in a dose related manner

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

    PubMed

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

    2015-01-01

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

  12. 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.

  13. Exploiting the versatility of human cytochrome P450 enzymes: the promise of blue roses from biotechnology.

    PubMed

    Gillam, E M; Guengerich, F P

    2001-12-01

    The cytochrome P450 (P450) enzymes involved in drug metabolism are among the most versatile biological catalysts known. A small number of discrete forms of human P450 are capable of catalyzing the monooxygenation of a practically unlimited variety of xenobiotic substrates, with each enzyme showing a more or less wide and overlapping substrate range. This versatility makes P450s ideally suited as starting materials for engineering designer catalysts for industrial applications. In the course of heterologous expression of P450s in bacteria, we observed the unexpected formation of blue pigments. Although this was initially assumed to be an artifact, subsequent work led to the discovery of a new function of P450s in intermediary metabolism and toxicology, new screens for protein engineering, and potential applications in the dye and horticulture industries.

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

    PubMed Central

    Kullman, S W; Matsumura, F

    1997-01-01

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

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

    SciTech Connect

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

    1991-01-01

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

  16. Relationship between hepatic phenotype and changes in gene expression in cytochrome P450 reductase (POR) null mice

    PubMed Central

    Wang, Xiu Jun; Chamberlain, Mark; Vassieva, Olga; Henderson, Colin J.; Wolf, C. Roland

    2005-01-01

    Cytochrome P450 reductase is the unique electron donor for microsomal cytochrome P450s; these enzymes play a major role in the metabolism of endogenous and xenobiotic compounds. In mice with a liver-specific deletion of cytochrome P450 reductase, hepatic cytochrome P450 activity is ablated, with consequent changes in bile acid and lipid homoeostasis. In order to gain insights into the metabolic changes resulting from this phenotype, we have analysed changes in hepatic mRNA expression using microarray analysis and real-time PCR. In parallel with the perturbations in bile acid levels, changes in the expression of key enzymes involved in cholesterol and lipid homoeostasis were observed in hepatic cytochrome P450 reductase null mice. This was characterized by a reduced expression of Cyp7b1, and elevation of Cyp7a1 and Cyp8b1 expression. The levels of mRNAs for other cytochrome P450 genes, including Cyp2b10, Cyp2c29, Cyp3a11 and Cyp3a16, were increased, demonstrating that endogenous factors play a role in regulating the expression of these proteins and that the increases are due, at least in part, to altered levels of transcripts. In addition, levels of mRNAs encoding genes involved in glycolysis and lipid transport were also increased; the latter may provide an explanation for the increased hepatic lipid content observed in the hepatic null mice. Serum testosterone and oestradiol levels were lowered, accompanied by significantly decreased expression of Hsd3b2 (3β-hydroxy-Δ5-steroid dehydrogenase-2), Hsd3b5 (3β-hydroxy-Δ5-steroid dehydrogenase-5) and Hsd11b1 (11β-hydroxysteroid dehydrogenase type 1), key enzymes in steroid hormone metabolism. These microarray data provide important insights into the control of metabolic pathways by the cytochrome system. PMID:15717863

  17. 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

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

    PubMed

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

    2013-09-15

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

  19. Phylogenetic analysis of Bacillus P450 monooxygenases and evaluation of their activity towards steroids.

    PubMed

    Furuya, Toshiki; Shibata, Daisuke; Kino, Kuniki

    2009-11-01

    Cytochrome P450 (P450) open reading frames (ORFs) identified in genome sequences of Bacillus species are potential resources for new oxidation biocatalysts. Phylogenetic analysis of 29 Bacillus P450 ORFs revealed that the P450s consist of a limited number of P450 families, CYP102, CYP106, CYP107, CYP109, CYP134, CYP152, and CYP197. Previously, we identified the catalytic activities of three P450s of Bacillus subtilis towards steroids by rapid substrate screening using Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR/MS). Here, we further applied this method to evaluate the activity of Bacillus cereus P450s towards steroids. Five P450 genes were cloned from B. cereus ATCC 10987 based on its genomic sequence and were expressed in Escherichia coli. These P450s were reacted with a mixture of 30 compounds that mainly included steroids, and the reaction mixtures were analyzed using FT-ICR/MS. We found that BCE_2659 (CYP106) catalyzed the monooxygenation of methyltestosterone, progesterone, 11-ketoprogesterone, medroxyprogesterone acetate, and chlormadinone acetate. BCE_2654 (CYP107) monooxygenated testosterone enanthate, and BCE_3250 (CYP109) monooxygenated testosterone and compactin. Based on the phylogenetic relationship and the known substrate specificities including ones identified in this study, we discuss the catalytic potential of Bacillus P450s towards steroids.

  20. Structures of cytochrome P450 17A1 with prostate cancer drugs abiraterone and TOK-001.

    PubMed

    DeVore, Natasha M; Scott, Emily E

    2012-01-22

    Cytochrome P450 17A1 (also known as CYP17A1 and cytochrome P450c17) catalyses the biosynthesis of androgens in humans. As prostate cancer cells proliferate in response to androgen steroids, CYP17A1 inhibition is a new strategy to prevent androgen synthesis and treat lethal metastatic castration-resistant prostate cancer, but drug development has been hampered by lack of information regarding the structure of CYP17A1. Here we report X-ray crystal structures of CYP17A1, which were obtained in the presence of either abiraterone, a first-in-class steroidal inhibitor recently approved by the US Food and Drug Administration for late-stage prostate cancer, or TOK-001, an inhibitor that is currently undergoing clinical trials. Both of these inhibitors bind the haem iron, forming a 60° angle above the haem plane and packing against the central I helix with the 3β-OH interacting with aspargine 202 in the F helix. Notably, this binding mode differs substantially from those that are predicted by homology models and from steroids in other cytochrome P450 enzymes with known structures, and some features of this binding mode are more similar to steroid receptors. Whereas the overall structure of CYP17A1 provides a rationale for understanding many mutations that are found in patients with steroidogenic diseases, the active site reveals multiple steric and hydrogen bonding features that will facilitate a 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.

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

    PubMed Central

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

    1984-01-01

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

  2. 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

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

    PubMed

    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

  4. Interaction of Angeli's salt with cytochrome P450 1A2 distal mutants: an optical absorption spectral study.

    PubMed

    Shibata, Y; Sato, H; Sagami, I; Shimizu, T

    1997-11-14

    Angeli's salt, Na2N2O3 or O-N=N+-(OH)(O-) in aqueous solution, is known to release NO- or NO., which relaxes vascular tissue and lowers blood pressure. In the liver, the most abundant heme enzyme is cytochrome P450. In the present study, we studied the effect of rat liver cytochrome P450 1A2 (P450 1A2) in regard to its catalysis of the N=N bond scission of Angeli's salt with optical absorption spectra. Also, we examined the contribution of putative distal amino acids of P450 1A2 to the reaction with the salt. We found that wild-type Fe3+ P450 1A2 markedly enhances the N=N scission of the salt up to 100 fold in terms of absorption spectroscopy. A Fe3+ P450 1A2-NO complex with an absorption peak at 435 nm was formed when the salt was added and the complex was then changed to a 6-coordinated Fe2+-NO complex having a 440-nm peak. Glu318Asp, Glu318Ala and Thr319Ala mutants at the putative distal site of P450 1A2 formed a 5-coordinated Fe2+-NO complex having a 400-nm absorption, that was not formed with the wild type. The Glu318Ala mutant, in particular, did not form the Fe3+-NO complex with the addition of Angeli's salt. The presence of L-Cys, reduced glutathione, catalase or superoxide dismutase markedly stabilized the Fe3+ wild type-NO complex. Thus, our data suggests that the N=N bond of Angeli's salt is cleaved with the P450 1A2 active site and NO- or NO. is released. We discuss mechanisms of redox and ligand changes of the P450 heme.

  5. Cytochrome P450 Bioconjugate as a Nanovehicle for Improved Chemotherapy Treatment.

    PubMed

    Quester, Katrin; Juarez-Moreno, Karla; Secundino, Isamel; Roseinstein, Yvonne; Alejo, Karla P; Huerta-Saquero, Alejandro; Vazquez-Duhalt, Rafael

    2016-11-28

    Cancer is still a growing public health problem, especially breast cancer that is one of the most important cancers in women. Chemotherapy, even though a successful treatment, is accompanied by severe side effects. Moreover, most of the drugs used for chemotherapy are administered as prodrugs and need to be transformed to the active form by cytochromes P450 (CYPs). In addition, increasing numbers of cancer tissues show lower CYP activity than the surrounding healthy tissues in which prodrugs are preferentially activated causing cytotoxicity. Here, the design of a functionalized cytochrome P450 bioconjugate is reported as nanovehicle for the enzyme direct delivery to the tumor tissue in order to improve the local drug activation. MCF-7 breast cancer cells are treated with CYP-polyethylene glycol bioconjugate functionalized folic acid, where it activates the prodrug tamoxifen and significantly reduces the dose of tamoxifen needed to kill the tumor cells. The CYP bioconjugate covered with polyethylene glycol shows no immunogenic activity. The advantages of increasing the site-specific CYP activity in tumor tissues are discussed.

  6. Cumene hydroperoxide supported demethylation of N,N-dimethylaniline by cytochrome P-450 from adrenal cortex mitochondria.

    PubMed

    Akhrem, A A; Khatyleva SYu; Shkumatov, V M; Chashchin, V L; Kiselev, P A

    1982-01-01

    The interaction of highly purified cytochrome P-450 from bovine adrenal cortex mitochondria (cytochrome P-450scc) with N,N-dimethylaniline (DMA), aniline, N-dimethylcyclohexylamine and cumene hydroperoxide (CHP) has been investigated. The formation of complexes between cytochrome P-450scc and the above listed compounds could be demonstrated. The reaction of oxidative demethylation of DMA by cumene hydroperoxide involving cytochrome P-450scc has been carried out at 37 degrees C; the mechanism of this process is discussed. Incubation of cytochrome P-450scc with negatively charged phospholipids, phosphatidylglycerol (PG), and phosphatidylinosite (PI) exerts an inhibiting effect on the reaction of oxidative demethylation. The interaction of cytochrome P-450scc with CHP is accompanied by hemoprotein destruction in a complex biphasic way. The process of oxidative demethylation of DMA in the system of cytochrome P-450scc-CHP has been concluded to have a predominantly radical character.

  7. In vitro identification of the cytochrome P450 isozymes involved in the N-demethylation of the active opioid metabolite nortilidine to bisnortilidine.

    PubMed

    Wustrow, Isabel; Riedel, Klaus-Dieter; Mikus, Gerd; Weiss, Johanna

    2012-06-01

    Tilidine exhibits the highest consumption of opioids in Germany. The prodrug is hepatically metabolised in a sequential N-demethylation reaction. Its primary metabolite nortilidine is a selective μ-opioid receptor agonist which can penetrate the blood-brain barrier. Cytochrome P450 isozymes (CYP) 3A4 and CYP2C19 were previously identified as isozymes mediating the formation of nortilidine. This study was set up to identify the enzymes and kinetics of the subsequent N-demethylation to bisnortilidine, thus being able to understand clinical interactions. Human liver microsomes and recombinant CYPs were used to investigate the metabolism of nortilidine to bisnortilidine. Nortilidine and bisnortilidine were quantified using liquid chromatography tandem mass spectrometry. Inhibitor screening kits were used to quantify the inhibition of CYP3A4, CYP2C19, CYP2B6 and CYP2D6 by bisnortilidine. Nortilidine metabolism to bisnortilidine followed the Michaelis-Menten kinetics with K (m) = 141.6 ± 15 μM and V (max) = 46.2 ± 3 nmol/mg/h. Inhibitors of CYP3A4, CYP2C19 and CYP2B6 inhibited this reaction. Assays with recombinant CYPs verified that the N-demethylation is catalysed by CYP3A4, CYP2C19 and CYP2B6. Our results also demonstrated that the metabolism from tilidine to nortilidine is not only mediated by CYP3A4 and CYP2C19, but also by CYP2B6. Moreover, bisnortilidine is a weak inhibitor of CYP3A4 and CYP2B6, a strong inhibitor of CYP2D6, but not an inhibitor of CYP2C19. Our study demonstrated that nortilidine is metabolised via the same CYP isozymes as the prodrug tilidine, whereas the formation of bisnortilidine appears to be the rate-limiting step in the metabolism of tilidine. Pharmacokinetic interactions can be expected with inhibitors or inducers of CYP3A4, CYP2C19 or CYP2B6.

  8. In vitro inhibition and induction of human liver cytochrome p450 enzymes by milnacipran.

    PubMed

    Paris, Brandy L; Ogilvie, Brian W; Scheinkoenig, Julie A; Ndikum-Moffor, Florence; Gibson, Remi; Parkinson, Andrew

    2009-10-01

    Milnacipran (Savella) inhibits both norepinephrine and serotonin reuptake and is distinguished by a nearly 3-fold greater potency in inhibiting norepinephrine reuptake in vitro compared with serotonin. We evaluated the ability of milnacipran to inhibit and induce human cytochrome P450 enzymes in vitro. In human liver microsomes, milnacipran did not inhibit CYP1A2, 2B6, 2C8, 2C9, 2C19, or 2D6 (IC(50) >or= 100 microM); whereas, a comparator with dual reuptake properties [duloxetine (Cymbalta)] inhibited CYP2D6 (IC(50) = 7 microM) and CYP2B6 (IC(50) = 15 microM) with a relatively high potency. Milnacipran inhibited CYP3A4/5 in a substrate-dependent manner (i.e., midazolam 1'-hydroxylation IC(50) approximately 30 microM; testosterone 6beta-hydroxylation IC(50) approximately 100 microM); whereas, duloxetine inhibited both CYP3A4/5 activities with equal potency (IC(50) = 37 and 38 microM, respectively). Milnacipran produced no time-dependent inhibition (<10%) of P450 activity, whereas duloxetine produced time-dependent inhibition of CYP1A2, 2B6, 2C19, and 3A4/5. To evaluate P450 induction, freshly isolated human hepatocytes (n = 3) were cultured and treated once daily for 3 days with milnacipran (3, 10, and 30 microM), after which microsomal P450 activities were measured. Whereas positive controls (omeprazole, phenobarbital, and rifampin) caused anticipated P450 induction, milnacipran had minimal effect on CYP1A2, 2C8, 2C9, or 2C19 activity. The highest concentration of milnacipran (30 microM; >10 times plasma C(max)) produced 2.6- and 2.2-fold increases in CYP2B6 and CYP3A4/5 activity (making it 26 and 34% as effective as phenobarbital and rifampin, respectively). Given these results, milnacipran is not expected to cause clinically significant P450 inhibition or induction.

  9. Mechanism-based inactivators as probes of cytochrome P450 structure and function.

    PubMed

    Kent, U M; Juschyshyn, M I; Hollenberg, P F

    2001-09-01

    The cytochromes P450 superfamily of enzymes is a group of hemeproteins that catalyze the metabolism of an extensive series of compounds including drugs, chemical carcinogens, fatty acids, and steroids. They oxidize substrates ranging in size from ethylene to cyclosporin. Although significant efforts have been made to obtain structural information on the active sites of the microbial P450s, relatively little is currently known regarding the identities of the critical amino acid residues in the P450 active sites that are involved in substrate binding and catalysis. Since information on the crystal structures of the eukaryotic P450s has been relatively limited, investigators have used a variety of other techniques in attempts to elucide the structural features that play a role in the catalytic properties and substrate specificity at the enzyme active site. These include site-directed mutagenesis, natural mutations, homology modeling, mapping with aryl-iron complexes, affinity and photoaffinity labeling, and mechanism-based inactivators. A variety of different mechanism-based inactivators have proven to be useful in identifiying active site amino acid residues involved in substrate binding and catalysis. In this review we present a sampling of the types of studies that can be conducted using mechanism-based inactivators and highlight studies with several classes of compounds including acetylenes, isothiocyanates, xanthates, aminobenzotriazoles, phencyclidine, and furanocoumarins. Labeled peptides isolated from the inactivated proteins have been analyzed by N-terminal amino acid sequencing in conjunction with mass spectrometry to determine the sites of covalent modification. Mechanistic studies aimed at identifying the basis for the inactivation following adduct formation are also presented.

  10. Induction and characterization of a cytochrome P-450-dependent camphor hydroxylase in tissue cultures of common sage (Salvia officinalis)

    SciTech Connect

    Funk, C.; Croteau, R. )

    1993-04-01

    (+)-Camphor, a major monoterpene of the essential oil of common sage (Salvia officinalis), is catabolized in senescent tissue, and the pathway for the breakdown of this bicyclic ketone has been previously elucidated in sage cell-suspension cultures. In the initial step of catabolism, camphor is oxidized to 6-exo-hydroxycamphor, and the corresponding NADPH- and O[sub 2]-dependent hydroxylase activity was demonstrated in microsomal preparations of sage cells. Several well-established inhibitors of cytochrome P-450-dependent reactions, including cytochrome c, clotrimazole, and CO, inhibited the hydroxylation of camphor, and CO-dependent inhibition was partially reversed by blue light. Upon treatment of sage suspension cultures with 30 mM MnCl[sub 2], camphor-6-hydroxylase activity was induced up to 7-fold. A polypeptide with estimated molecular mass of 58 kD from sage microsomal membranes exhibited antigenic cross-reactivity in western blot experiments with two heterologous polyclonal antibodies raised against cytochrome P-450 camphor-5-exo-hydroxylase from Pseudomonas putida and cytochrome P-450 limonene-6S-hydroxylase from spearmint (Mentha spicata). Dot blotting indicated that the concentration of this polypeptide increased with camphor hydroxylase activity in microsomes of Mn[sup 2+]-induced sage cells. These results suggest that camphor-6-exo-hydroxylase from sage is a microsomal cytochrome P-450 monooxygenase that may share common properties and epitopes with bacterial and other plant monoterpene hydroxylases. 44 refs., 6 figs., 2 tabs.

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

    PubMed Central

    Funk, C.; Croteau, R.

    1993-01-01

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

  12. 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 (activity and contaminant levels in nestlings. These observations markedly contrast the pronounced monooxygenase induction (up to 85-fold) and its significant correlation with total PCBs, arylhydrocarbon receptor-active PCB congeners and toxic equivalents in concurrently collected night-heron embryos that were often siblings of the nestlings. The present findings suggest that cytochrome P450-associated monooxygenase activity of heron nestlings may have only limited value as a biomarker of exposure at this rapid-growth life stage.

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

    SciTech Connect

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

    1995-12-31

    Hepatic cytochrome P450-associated monooxygenase activities were measured in 11-day-old nestling black-crowned night-herons (Nycticorax nycticorax) collected from a reference site (next to the Chincoteague National Wildlife Refuge, Virginia) and three polluted sites (Cat Island, Green Bay, Lake Michigan, Wisconsin; Bair Island, San Francisco Bay, California; West Marin Island, San Francisco Bay, California). Activities of aryl hydrocarbon hydroxylase (AHH) and benzyl-oxyresorufin-O-dealkylase (BROD) were modestly elevated ({<=} three-fold) in nestlings from polluted sites. Concentrations of p,p{prime}DDE, other organochlorine pesticides and total PCBs in nestlings were greatest at contaminated sites, although much lower than found in concurrently collected eggs and pipping embryos, At these low pollutant concentrations there was little correlation between monooxygenase activity and contaminant levels in nestlings. These observations markedly contrast the pronounced monooxygenase induction (up to 85-fold) and its significant correlation with total PCBS, aryl hydrocarbon receptor-active PCB congeners and toxic equivalents in concurrently collected night-heron embryos that were often siblings of the nestlings. The present findings suggest that cytochrome P450-associated monooxygenase activity of heron nestlings may have only limited value as a biomarker of exposure at this rapid-growth life stage.

  14. Genetic polymorphism of human cytochrome P-450 (S)-mephenytoin 4-hydroxylase. Studies with human autoantibodies suggest a functionally altered cytochrome P-450 isozyme as cause of the genetic deficiency

    SciTech Connect

    Meier, U.T.; Meyer, U.A.

    1987-12-15

    The metabolism of the anticonvulsant mephenytoin is subject to a genetic polymorphism. In 2-5% of Caucasians and 18-23% of Japanese subjects a specific cytochrome P-450 isozyme, P-450 meph, is functionally deficient or missing. The authors have accumulated evidence that autoimmune antibodies observed in sera of patients with tienilic acid induced hepatitis (anti-liver kidney microsome 2 or anti-LKM2 antibodies) specifically recognize the cytochrome P-450 involved in the mephrenytoin hydroxylation polymorphism. This is demonstrated by immunoinhibition and immunoprecipitation of microsomal (S)-mephenytoin 4-hydroxylation activity and by the recognition by anti-LKM2 antibodies of a single (/sup 125/I)-protein band on immunoblots of human liver microsomes after sodium dodecyl sulfate-polyacrylamide gel electrophoresis or isoelectric focusing. The cytochrome P-450 recognized by anti-LKM2 antibodies was immunopurified from microsomes derived from livers of extensive (EM) or poor metabolizers (PM) of (S)-mephenytoin. Comparison of the EM-type cytochrome P-450 to that isolated from PM livers revealed no difference in regard to immuno-cross-reactivity, molecular weight, isoelectric point, relative content in microsomes, two-dimensional tryptic peptide maps, one-dimensional peptide maps with three proteases, amino acid composition, and amino-terminal protein sequence. Finally, the same protein was precipitated from microsomes prepared from the liver biopsy of a subject phenotyped in vivo as a poor metabolizer of mephenytoin. These data strongly suggest that the mephenytoin hydroxylation deficiency is caused by a minor structural change leading to a functionally altered cytochrome P-450 isozyme.

  15. Mutation effects of a conserved threonine (Thr243) of cytochrome P450nor on its structure and function.

    PubMed

    Obayashi, E; Shimizu, H; Park, S Y; Shoun, H; Shiro, Y

    2000-11-01

    Threonine 243 of cytochrome P450nor (fungal nitric oxide reductase) corresponds to the 'conserved' Thr in the long I helix of monooxygenase cytochrome P450s. In P450nor, the replacement of Thr243 with Asn, Ala or Val makes the enzymatic activity dramatically reduce. In order to understand the roles of Thr243 in the reduction reaction of NO by P450nor, the crystal structures of three Thr243 mutants (Thr243-->Asn, Thr243-->Val, Thr243-->Ala) of P450nor were determined at a 1.4-A resolution and at cryogenic temperature. However, the hydrogen-bonding pattern in the heme pocket of these mutants is essentially similar for that of the WT enzyme. This suggests that the determination of the structure of the NADH complex of P450nor is required, in order to evaluate the role of Thr243 in its enzymatic reaction. We attempted to crystallize the NADH complex under several conditions, but have not yet been successful.

  16. Diversity in mechanisms of substrate oxidation by cytochrome P450 2D6. Lack of an allosteric role of NADPH-cytochrome P450 reductase in catalytic regioselectivity.

    PubMed

    Hanna, I H; Krauser, J A; Cai, H; Kim, M S; Guengerich, F P

    2001-10-26

    Cytochrome P450 (P450) 2D6 was first identified as the polymorphic human debrisoquine hydroxylase and subsequently shown to catalyze the oxidation of a variety of drugs containing a basic nitrogen. Differences in the regioselectivity of oxidation products formed in systems containing NADPH-P450 reductase/NADPH and the model oxidant cumene hydroperoxide have been proposed by others to be due to an allosteric influence of the reductase on P450 2D6 (Modi, S., Gilham, D. E., Sutcliffe, M. J., Lian, L.-Y., Primrose, W. U., Wolf, C. R., and Roberts, G. C. K. (1997) Biochemistry 36, 4461-4470). We examined the differences in the formation of oxidation products of N-methyl-4-phenyl-1,2,5,6-tetrahydropyridine, metoprolol, and bufuralol between reductase-, cumene hydroperoxide-, and iodosylbenzene-supported systems. Catalytic regioselectivity was not influenced by the presence of the reductase in any of the systems supported by model oxidants, ruling out allosteric influences. The presence of the reductase had little effect on the affinity of P450 2D6 for any of these three substrates. The addition of the reaction remnants of the model oxidants (cumyl alcohol and iodobenzene) to the reductase-supported system did not affect reaction patterns, arguing against steric influences of these products on catalytic regioselectivity. Label from H(2)18O was quantitatively incorporated into 1'-hydroxybufuralol in the iodosylbenzene- but not in the reductase- or cumene hydroperoxide-supported reactions. We conclude that the P450 systems utilizing NADPH-P450 reductase, cumene hydroperoxide, and iodosylbenzene use similar but distinct chemical mechanisms. These differences are the basis for the variable product distributions, not an allosteric influence of the reductase.

  17. Pi-pi Stacking Mediated Cooperative Mechanism for Human Cytochrome P450 3A4.

    PubMed

    Fa, Botao; Cong, Shan; Wang, Jingfang

    2015-04-24

    Human Cytochrome P450 3A4 (CYP3A4) is an important member of the cytochrome P450 superfamily with responsibility for metabolizing ~50% of clinical drugs. Experimental evidence showed that CYP3A4 can adopt multiple substrates in its active site to form a cooperative binding model, accelerating substrate metabolism efficiency. In the current study, we constructed both normal and cooperative binding models of human CYP3A4 with antifungal drug ketoconazoles (KLN). Molecular dynamics simulation and free energy calculation were then carried out to study the cooperative binding mechanism. Our simulation showed that the second KLN in the cooperative binding model had a positive impact on the first one binding in the active site by two significant pi-pi stacking interactions. The first one was formed by Phe215, functioning to position the first KLN in a favorable orientation in the active site for further metabolism reactions. The second one was contributed by Phe304. This pi-pi stacking was enhanced in the cooperative binding model by the parallel conformation between the aromatic rings in Phe304 and the dioxolan moiety of the first KLN. These findings can provide an atomic insight into the cooperative binding in CYP3A4, revealing a novel pi-pi stacking mechanism for drug-drug interactions.

  18. Resveratrol stimulates cortisol biosynthesis by activating SIRT-dependent deacetylation of P450scc.<