Sample records for nadph-cytochrome p-450 reductase

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Iyanagi, Takashi

    2005-12-09

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

  2. Fluorescence labelling of NADPH-cytochrome P-450 reductase with the monobromomethyl derivative of syn-9,10-dioxabimane.

    PubMed Central

    Vogel, F; Lumper, L

    1983-01-01

    The kinetics of thiol-group alkylation in NADPH-cytochrome P-450 reductase during its inactivation by monobromobimane has been studied using the fluorimetric determination of S-bimane-L-cysteine by high-performance liquid chromatography. Loss of activity during the reaction of NADPH-cytochrome P-450 reductase with monobromobimane is caused by the alkylation of one single critical cysteine residue, which can be protected against thiol-specific reagents by NADP(H). The chemical stability of the bimane group allows the digestion of bimane-labelled NADPH-cytochrome P-450 reductase by CNBr. The critical cysteine residue could be located in a CNBr-cleaved peptide purified to homogeneity with Mr 10 500 +/- 1 000 and valine as N-terminus. Images Fig. 2. PMID:6414464

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

    EPA Science Inventory

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

  4. Influence of acute and chronic administration of methadone hydrochloride on NADPH-cytochrome c reductase and cytochrome P-450 of mouse liver microsomes.

    PubMed

    Datta, R K; Johnson, E A; Bhattacharjee, G; Stenger, R J

    1976-03-01

    Administration of a single acute dose (20 mg/kg body weight) of methadone hydrochloride to both male and female mice increased the specific activity of NADPH-cytochrome c reductase and did not change much the content of cytochrome P-450 of their liver microsomes. Administration of multiple acute doses of methadone in male mice increased the specific activity of cytochrome c reductase and the content of cytochrome P-450 of their liver microsomes. Chronic administration of progressively increasing doses of methadone (up to 40 mg/kg body weight) to male mice increased the specific activity of c reductase. Similar chronic administration of methadone up to 28 mg/kg body weight also increased the microsomal content of P-450, but with higher doses of methadone, the content of P-450 declined and finally dropped slightly below control levels. The levels of c reductase activity and P-450 content returned to normal about two weeks after discontinuation of methadone administration.

  5. Functional expression and characterization of recombinant NADPH-P450 reductase from Malassezia globosa.

    PubMed

    Lee, Hwayoun; Park, Hyoung-Goo; Lim, Young-Ran; Lee, Im-Soon; Kim, Beom Joon; Seong, Cheul-Hun; Chun, Young-Jin; Kim, Donghak

    2012-01-01

    Malassezia globosa is a common pathogenic fungus that causes skin diseases including dandruff and seborrheic dermatitis in humans. Analysis of its genome identified a gene (MGL_1677) coding for a putative NADPH-P450 reductase (NPR) to support the fungal cytochrome P450 enzymes. The heterologously expressed recombinant M. globosa NPR protein was purified, and its functional features were characterized. The purified protein generated a single band on SDS-PAGE at 80.74 kDa and had an absorption maximum at 452 nm, indicating its possible function as an oxidized flavin cofactor. It evidenced NADPH-dependent reducing activity for cytochrome c or nitroblue tetrazolium. Human P450 1A2 and 2A6 were able to successfully catalyze the O-deethylation of 7- ethoxyresorufin and the 7-hydroxylation of coumarin, respectively, with the support of the purified NPR. These results demonstrate that purified NPR is an orthologous reductase protein that supports cytochrome P450 enzymes in M. globosa.

  6. Enhanced Purification of Recombinant Rat NADPH-P450 Reductase by Using a Hexahistidine-Tag.

    PubMed

    Park, Hyoung-Goo; Lim, Young-Ran; Han, Songhee; Jeong, Dabin; Kim, Donghak

    2017-05-28

    NADPH-P450 reductase (NPR) transfers electrons from NADPH to cytochrome P450 and heme oxygenase enzymes to support their catalytic activities. This protein is localized within the endoplasmic reticulum membrane and utilizes FMN, FAD, and NADPH as cofactors. Although NPR is essential toward enabling the biochemical and pharmacological analyses of P450 enzymes, its production as a recombinant purified protein requires a series of tedious efforts and a high cost due to the use of NADP + in the affinity chromatography process. In the present study, the rat NPR clone containing a 6× Histidine-tag (NPR-His) was constructed and heterologously expressed. The NPR-His protein was purified using Ni 2+ -affinity chromatography, and its functional features were characterized. A single band at 78 kDa was observed from SDS-PAGE and the purified protein displayed a maximum absorbance at 455 nm, indicating the presence of an oxidized flavin cofactor. Cytochrome c and nitroblue tetrazolium were reduced by purified NPR-His in an NADPH-dependent manner. The purified NPR-His successfully supported the catalytic activities of human P450 1A2 and 2A6 and fungal CYP52A21, yielding results similar to those obtained using conventional purified rat reductase. This study will facilitate the use of recombinant NPR-His protein in the various fields of P450 research.

  7. Molecular cloning and functional characterization of multiple NADPH-cytochrome P450 reductases from Andrographis paniculata.

    PubMed

    Lin, Huixin; Wang, Jian; Qi, Mengdie; Guo, Juan; Rong, Qixian; Tang, Jinfu; Wu, Yisheng; Ma, Xiaojing; Huang, Luqi

    2017-09-01

    Andrographis paniculata (Burm.f.) Wall. ex Nees is widely used as medicinal herb in Southern and Southeastern Asia and andrographolide is its main medicinal constituent. Based on the structure of andrographolide, it has been proposed that cytochrome P450 enzymes play vital roles on its biosynthesis. NADPH:cytochrome P450 reductase (CPR) is the most important redox partner of multiple P450s. In this study, three CPRs were identified in the genomic data of A. paniculata (namely ApCPR1, ApCPR2, and ApCPR3), and their coding regions were cloned. They varied from 62% to 70% identities to each other at the amino acid sequence level. ApCPR1 belongs to Class I of dicotyledonous CPR while both ApCPR2 and ApCPR3 are grouped to Class II. The recombinant enzymes ApCPR1 and ApCPR2 reduced cytochrome c and ferricyanide in an NADPH-dependent manner. In yeast, they supported the activity of CYP76AH1, a ferruginol-forming enzyme. However, ApCPR3 did not show any enzymatic activities either in vitro or in vivo. Quantitative real-time PCR analysis showed that both ApCPR1 and ApCPR2 expressed in all tissues examined, but ApCPR2 showed higher expression in leaves. Expression of ApCPR2 was inducible by MeJA and its pattern matched with andrographolide accumulation. Present investigation suggested ApCPR2 involves in the biosynthesis of secondary metabolites including andrographolide. Copyright © 2017. Published by Elsevier B.V.

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

    PubMed

    Wang, Jinling; de Montellano, Paul R Ortiz

    2003-05-30

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

  9. Functional characterization of NADPH-cytochrome P450 reductase from Bactrocera dorsalis: Possible involvement in susceptibility to malathion

    PubMed Central

    Huang, Yong; Lu, Xue-Ping; Wang, Luo-Luo; Wei, Dong; Feng, Zi-Jiao; Zhang, Qi; Xiao, Lin-Fan; Dou, Wei; Wang, Jin-Jun

    2015-01-01

    NADPH cytochrome P450 reductase (CPR) is essential for cytochrome P450 catalysis, which is important in the detoxification and activation of xenobiotics. In this study, two transcripts of Bactrocera dorsalis CPR (BdCPR) were cloned, and the deduced amino-acid sequence had an N-terminus membrane anchor for BdCPR-X1 and three conserved binding domains (FMN, FAD, and NADP), as well as an FAD binding motif and catalytic residues for both BdCPR-X1 and BdCPR-X2. BdCPR-X1 was detected to have the high expression levels in adults and in Malpighian tubules, fat bodies, and midguts of adults, but BdCPR-X2 expressed lowly in B. dorsalis. The levels of BdCPRs were similar in malathion-resistant strain compared to susceptible strain. However, injecting adults with double-stranded RNA against BdCPR significantly reduced the transcript levels of the mRNA, and knockdown of BdCPR increased adult susceptibility to malathion. Expressing complete BdCPR-X1 cDNA in Sf9 cells resulted in high activity determined by cytochrome c reduction and these cells had higher viability after exposure to malathion than control. The results suggest that BdCPR could affect the susceptibility of B. dorsalis to malathion and eukaryotic expression of BdCPR would lay a solid foundation for further investigation of P450 in B. dorsalis. PMID:26681597

  10. Molecular cloning and functional characterization of NADPH-dependent cytochrome P450 reductase from the green microalga Botryococcus braunii, B race.

    PubMed

    Tsou, Chung-Yau; Matsunaga, Shigeki; Okada, Shigeru

    2018-01-01

    The green microalga Botryococcus braunii of the B race accumulates various lipophilic compounds containing a 10,11-oxidosqualene epoxide moiety in addition to large amounts of triterpene hydrocarbons. While 2,3-squalene epoxidases have already been isolated and characterized from the alga, the enzyme that catalyzes the 10,11-epoxidation of squalene has remained elusive. In order to obtain a molecular tool to explore a 10,11-squalene epoxidase, cDNA cloning of an NADPH-dependent cytochrome P450 reductase (CPR) that is required by both squalene epoxidases and cytochrome P450 enzymes was carried out. The isolated cDNA contained an open reading frame (1998 bp) that encoded for a protein with 665 amino acid residues with a predicted molecular weight of 71.46 kDa and a theoretical pI of 5.49. Analysis of the deduced amino acid sequence revealed the presence of conserved motifs, including FMN, FAD, and NADPH binding domains, which are typical of other CPRs and necessary for enzyme activity. By truncation of the N-terminal transmembrane anchor and addition of a 6× His-tag, BbCPR was heterologously produced in Escherichia coli and purified by Ni-NTA affinity chromatography. The purified recombinant enzyme showed optimal reducing activity of cytochrome c at around a neutral pH at a temperature range of 30-37°C. For steady state kinetic parameters, the recombinant enzyme had a k m for cytochrome c and NADPH of 11.7±1.6 and 9.4±1.4 μM, and a k cat for cytochrome c and NADPH of 2.78±0.09 and 3.66±0.11 μmol/min/mg protein, respectively. This is the first study to perform the functional characterization of a CPR from eukaryotic microalgae. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  11. Three-dimensional structure of NADPH–cytochrome P450 reductase: Prototype for FMN- and FAD-containing enzymes

    PubMed Central

    Wang, Ming; Roberts, David L.; Paschke, Rosemary; Shea, Thomas M.; Masters, Bettie Sue Siler; Kim, Jung-Ja P.

    1997-01-01

    Microsomal NADPH–cytochrome P450 reductase (CPR) is one of only two mammalian enzymes known to contain both FAD and FMN, the other being nitric-oxide synthase. CPR is a membrane-bound protein and catalyzes electron transfer from NADPH to all known microsomal cytochromes P450. The structure of rat liver CPR, expressed in Escherichia coli and solubilized by limited trypsinolysis, has been determined by x-ray crystallography at 2.6 Å resolution. The molecule is composed of four structural domains: (from the N- to C- termini) the FMN-binding domain, the connecting domain, and the FAD- and NADPH-binding domains. The FMN-binding domain is similar to the structure of flavodoxin, whereas the two C-terminal dinucleotide-binding domains are similar to those of ferredoxin–NADP+ reductase (FNR). The connecting domain, situated between the FMN-binding and FNR-like domains, is responsible for the relative orientation of the other domains, ensuring the proper alignment of the two flavins necessary for efficient electron transfer. The two flavin isoalloxazine rings are juxtaposed, with the closest distance between them being about 4 Å. The bowl-shaped surface near the FMN-binding site is likely the docking site of cytochrome c and the physiological redox partners, including cytochromes P450 and b5 and heme oxygenase. PMID:9237990

  12. The Hinge Segment of Human NADPH-Cytochrome P450 Reductase in Conformational Switching: The Critical Role of Ionic Strength

    PubMed Central

    Campelo, Diana; Lautier, Thomas; Urban, Philippe; Esteves, Francisco; Bozonnet, Sophie; Truan, Gilles; Kranendonk, Michel

    2017-01-01

    NADPH-cytochrome P450 reductase (CPR) is a redox partner of microsomal cytochromes P450 and is a prototype of the diflavin reductase family. CPR contains 3 distinct functional domains: a FMN-binding domain (acceptor reduction), a linker (hinge), and a connecting/FAD domain (NADPH oxidation). It has been demonstrated that the mechanism of CPR exhibits an important step in which it switches from a compact, closed conformation (locked state) to an ensemble of open conformations (unlocked state), the latter enabling electron transfer to redox partners. The conformational equilibrium between the locked and unlocked states has been shown to be highly dependent on ionic strength, reinforcing the hypothesis of the presence of critical salt interactions at the interface between the FMN and connecting FAD domains. Here we show that specific residues of the hinge segment are important in the control of the conformational equilibrium of CPR. We constructed six single mutants and two double mutants of the human CPR, targeting residues G240, S243, I245 and R246 of the hinge segment, with the aim of modifying the flexibility or the potential ionic interactions of the hinge segment. We measured the reduction of cytochrome c at various salt concentrations of these 8 mutants, either in the soluble or membrane-bound form of human CPR. All mutants were found capable of reducing cytochrome c yet with different efficiency and their maximal rates of cytochrome c reduction were shifted to lower salt concentration. In particular, residue R246 seems to play a key role in a salt bridge network present at the interface of the hinge and the connecting domain. Interestingly, the effects of mutations, although similar, demonstrated specific differences when present in the soluble or membrane-bound context. Our results demonstrate that the electrostatic and flexibility properties of the hinge segment are critical for electron transfer from CPR to its redox partners. PMID:29163152

  13. Cloning, functional characterization, and expression profiles of NADPH-cytochrome P450 reductase gene from the Asiatic rice striped stem borer, Chilo suppressalis (Lepidoptera: Pyralidae).

    PubMed

    Liu, Su; Liang, Qing-Mei; Huang, Yuan-Jie; Yuan, Xin; Zhou, Wen-Wu; Qiao, Fei; Cheng, Jiaan; Gurr, Geoff M; Zhu, Zeng-Rong

    2013-01-01

    NADPH-cytochrome P450 reductase (CPR) is one of the most important components of the cytochrome P450 enzyme system. It catalyzes electron transfer from NADPH to all known P450s, thus plays central roles not only in the metabolism of exogenous xenobiotics but also in the regulation of endogenous hormones in insects. In this study, a full-length cDNA encoding of a CPR (named CsCPR) was isolated from the Asiatic rice striped stem borer, Chilo suppressalis, by using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) methods. The cDNA contains a 2061 bp open reading frame, which encodes an enzyme of 686 amino acid residues, with a calculated molecular mass of 77.6 kDa. The deduced peptide has hallmarks of typical CPR, including an N-terminal membrane anchor and the FMN, FAD and NADPH binding domains. The N-terminal-truncated protein fused with a 6 × His·tag was heterologously expressed in Escherichia coli Rosetta (DE3) cells and purified, specific activity and the Km values of the recombinant enzyme were determined. Tissue- and developmental stage-dependent expression of CsCPR mRNA was investigated by real-time quantitative PCR. The CsCPR mRNA was noticeably expressed in the digestive, metabolic, and olfactory organs of the larvae and adults of C. suppressalis. Our initial results would provide valuable information for further study on the interactions between CPR and cytochrome P450 enzyme systems. © 2013.

  14. NADPH-cytochrome P450 reductase-mediated denitration reaction of 2,4,6-trinitrotoluene to yield nitrite in mammals.

    PubMed

    Shinkai, Yasuhiro; Nishihara, Yuya; Amamiya, Masahiro; Wakayama, Toshihiko; Li, Song; Kikuchi, Tomohiro; Nakai, Yumi; Shimojo, Nobuhiro; Kumagai, Yoshito

    2016-02-01

    While the biodegradation of 2,4,6-trinitrotoluene (TNT) via the release of nitrite is well established, mechanistic details of the reaction in mammals are unknown. To address this issue, we attempted to identify the enzyme from rat liver responsible for the production of nitrite from TNT. A NADPH-cytochrome P450 reductase (P450R) was isolated and identified from rat liver microsomes as the enzyme responsible for not only the release of nitrite from TNT but also formation of superoxide and 4-hydroxyamino-2,6-dinitrotoluene (4-HADNT) under aerobic conditions. In this context, reactive oxygen species generated during P450R-catalyzed TNT reduction were found to be, at least in part, a mediator for the production of 4-HADNT from TNT via formation of 4-nitroso-2,6-dinitrotoluene. P450R did not catalyze the formation of the hydride-Meisenheimer complex (H(-)-TNT) that is thought to be an intermediate for nitrite release from TNT. Furthermore, in a time-course experiment, 4-HADNT formation reached a plateau level and then declined during the reaction between TNT and P450R with NADPH, while the release of nitrite was subjected to a lag period. Notably, the produced 4-HADNT can react with the parent compound TNT to produce nitrite and dimerized products via formation of a Janovsky complex. Our results demonstrate for the first time that P450R-mediated release of nitrite from TNT results from the process of chemical interaction of TNT and its 4-electron reduction metabolite 4-HADNT. Copyright © 2015 Elsevier Inc. All rights reserved.

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

    PubMed

    Ishihara, Yasuhiro; Shiba, Dai; Shimamoto, Norio

    2006-07-15

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

  16. The endogenous adrenodoxin reductase-like flavoprotein arh1 supports heterologous cytochrome P450-dependent substrate conversions in Schizosaccharomyces pombe.

    PubMed

    Ewen, Kerstin M; Schiffler, Burkhard; Uhlmann-Schiffler, Heike; Bernhardt, Rita; Hannemann, Frank

    2008-05-01

    Mitochondrial cytochromes P450 are essential for biosynthesis of steroid hormones, vitamin D and bile acids. In mammals, the electrons needed for these reactions are provided via adrenodoxin and adrenodoxin reductase (AdR). Recently, Schizosaccharomyces pombe was introduced as a new host for the functional expression of human mitochondrial steroid hydroxylases without the coexpression of their natural redox partners. This fact qualifies S. pombe for the biotechnological production of steroids and for application as inhibitor test organism of heterologously expressed cytochromes P450. In this paper, we present evidence that the S. pombe ferredoxin reductase, arh1, and ferredoxin, etp1fd provide mammalian class I cytochromes P450 with reduction equivalents. The recombinant reductase showed an unusual weak binding of flavin adenine dinucleotide (FAD), which was mastered by modifying the FAD-binding region by site-directed mutagenesis yielding a stable holoprotein. The modified reductase arh1_A18G displayed spectroscopic characteristics similar to AdR and was shown to be capable of accepting electrons with no evident preference for NADH or NADPH, respectively. Arh1_A18G can substitute for AdR by interacting not only with its natural redox partner etp1fd but also with the mammalian homolog adrenodoxin. Cytochrome P450-dependent substrate conversion with all combinations of the mammalian and yeast redox proteins was evaluated in a reconstituted system.

  17. RNA Interference of NADPH-Cytochrome P450 Reductase Results in Reduced Insecticide Resistance in the Bed Bug, Cimex lectularius

    PubMed Central

    Zhu, Fang; Sams, Sarah; Moural, Tim; Haynes, Kenneth F.; Potter, Michael F.; Palli, Subba R.

    2012-01-01

    Background NADPH-cytochrome P450 reductase (CPR) plays a central role in cytochrome P450 action. The genes coding for P450s are not yet fully identified in the bed bug, Cimex lectularius. Hence, we decided to clone cDNA and knockdown the expression of the gene coding for CPR which is suggested to be required for the function of all P450s to determine whether or not P450s are involved in resistance of bed bugs to insecticides. Methodology/Principal Findings The full length Cimex lectularius CPR (ClCPR) cDNA was isolated from a deltamethrin resistant bed bug population (CIN-1) using a combined PCR strategy. Bioinformatics and in silico modeling were employed to identify three conserved binding domains (FMN, FAD, NADP), a FAD binding motif, and the catalytic residues. The critical amino acids involved in FMN, FAD, NADP binding and their putative functions were also analyzed. No signal peptide but a membrane anchor domain with 21 amino acids which facilitates the localization of ClCPR on the endoplasmic reticulum was identified in ClCPR protein. Phylogenetic analysis showed that ClCPR is closer to the CPR from the body louse, Pediculus humanus corporis than to the CPRs from the other insect species studied. The ClCPR gene was ubiquitously expressed in all tissues tested but showed an increase in expression as immature stages develop into adults. We exploited the traumatic insemination mechanism of bed bugs to inject dsRNA and successfully knockdown the expression of the gene coding for ClCPR. Suppression of the ClCPR expression increased susceptibility to deltamethrin in resistant populations but not in the susceptible population of bed bugs. Conclusions/Significance These data suggest that P450-mediated metabolic detoxification may serve as one of the resistance mechanisms in bed bugs. PMID:22347424

  18. RNA interference of NADPH-cytochrome P450 reductase results in reduced insecticide resistance in the bed bug, Cimex lectularius.

    PubMed

    Zhu, Fang; Sams, Sarah; Moural, Tim; Haynes, Kenneth F; Potter, Michael F; Palli, Subba R

    2012-01-01

    NADPH-cytochrome P450 reductase (CPR) plays a central role in cytochrome P450 action. The genes coding for P450s are not yet fully identified in the bed bug, Cimex lectularius. Hence, we decided to clone cDNA and knockdown the expression of the gene coding for CPR which is suggested to be required for the function of all P450s to determine whether or not P450s are involved in resistance of bed bugs to insecticides. The full length Cimex lectularius CPR (ClCPR) cDNA was isolated from a deltamethrin resistant bed bug population (CIN-1) using a combined PCR strategy. Bioinformatics and in silico modeling were employed to identify three conserved binding domains (FMN, FAD, NADP), a FAD binding motif, and the catalytic residues. The critical amino acids involved in FMN, FAD, NADP binding and their putative functions were also analyzed. No signal peptide but a membrane anchor domain with 21 amino acids which facilitates the localization of ClCPR on the endoplasmic reticulum was identified in ClCPR protein. Phylogenetic analysis showed that ClCPR is closer to the CPR from the body louse, Pediculus humanus corporis than to the CPRs from the other insect species studied. The ClCPR gene was ubiquitously expressed in all tissues tested but showed an increase in expression as immature stages develop into adults. We exploited the traumatic insemination mechanism of bed bugs to inject dsRNA and successfully knockdown the expression of the gene coding for ClCPR. Suppression of the ClCPR expression increased susceptibility to deltamethrin in resistant populations but not in the susceptible population of bed bugs. These data suggest that P450-mediated metabolic detoxification may serve as one of the resistance mechanisms in bed bugs.

  19. Inactivation of the hepatic cytochrome P450 system by conditional deletion of hepatic cytochrome P450 reductase.

    PubMed

    Henderson, Colin J; Otto, Diana M E; Carrie, Dianne; Magnuson, Mark A; McLaren, Aileen W; Rosewell, Ian; Wolf, C Roland

    2003-04-11

    Cytochrome P450 (CYP) monooxygenases catalyze the oxidation of a large number of endogenous compounds and the majority of ingested environmental chemicals, leading to their elimination and often to their metabolic activation to toxic products. This enzyme system therefore provides our primary defense against xenobiotics and is a major determinant in the therapeutic efficacy of pharmacological agents. To evaluate the importance of hepatic P450s in normal homeostasis, drug pharmacology, and chemical toxicity, we have conditionally deleted the essential electron transfer protein, NADH:ferrihemoprotein reductase (EC, cytochrome P450 reductase, CPR) in the liver, resulting in essentially complete ablation of hepatic microsomal P450 activity. Hepatic CPR-null mice could no longer break down cholesterol because of their inability to produce bile acids, and whereas hepatic lipid levels were significantly increased, circulating levels of cholesterol and triglycerides were severely reduced. Loss of hepatic P450 activity resulted in a 5-fold increase in P450 protein, indicating the existence of a negative feedback pathway regulating P450 expression. Profound changes in the in vivo metabolism of pentobarbital and acetaminophen indicated that extrahepatic metabolism does not play a major role in the disposition of these compounds. Hepatic CPR-null mice developed normally and were able to breed, indicating that hepatic microsomal P450-mediated steroid hormone metabolism is not essential for fertility, demonstrating that a major evolutionary role for hepatic P450s is to protect mammals from their environment.

  20. RNA interference of NADPH-cytochrome P450 reductase of the rice brown planthopper, Nilaparvata lugens, increases susceptibility to insecticides.

    PubMed

    Liu, Su; Liang, Qing-Mei; Zhou, Wen-Wu; Jiang, Yan-Dong; Zhu, Qing-Zi; Yu, Hang; Zhang, Chuan-Xi; Gurr, Geoff M; Zhu, Zeng-Rong

    2015-01-01

    NADPH-cytochrome P450 reductase (CPR) is essential for numerous biological reactions catalysed by microsomal cytochrome P450 monooxygenases (P450s). Knockdown of CPR in several insects leads to developmental defects and increased susceptibility to insecticides. However, information about the role of CPR in the brown planthopper, Nilaparvata lugens, is still unavailable. A full-length cDNA encoding CPR was cloned from N. lugens (NlCPR). The deduced amino acid sequence showed marked features of classical CPRs, such as an N-terminus membrane anchor, conserved domains for flavin mononucleotide, flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide phosphate binding, as well as an FAD-binding motif and catalytic residues. Phylogenetic analysis revealed that NlCPR was located in a branch along with bed bug and pea aphid hemipteran insects. NlCPR mRNA was detectable in all tissues and developmental stages of N. lugens, as determined by real-time quantitative PCR. NlCPR transcripts were most abundant in the abdomen in adults, and in first-instar nymphs. Injection of N. lugens with double-strand RNA (dsRNA) against NlCPR significantly reduced the transcription level of the mRNA, and silencing of NlCPR resulted in increased susceptibility in N. lugens to beta-cypermethrin and imidacloprid. The results provide first evidence that NlCPR contributes to the susceptibility to beta-cypermethrin and imidacloprid in N. lugens. © 2014 Society of Chemical Industry.

  1. Identification and characterization of NADPH-dependent cytochrome P450 reductase gene and cytochrome b₅ gene from Plutella xylostella: possible involvement in resistance to beta-cypermethrin.

    PubMed

    Chen, Xi'en; Zhang, Yalin

    2015-03-10

    NADPH-cytochrome P450 reductase (CPR) and cytochrome b5 (b5) are essential for cytochrome P450 mediated biological reactions. CPR and b5 in several insects have been found to be associated with insecticide resistance. However, CPR and b5 in the diamondback moth (DBM), Plutella xylostella, are not characterized and their roles remain undefined. A full-length cDNA of CPR encoding 678 amino acids and a full-length cDNA of b5 encoding 127 amino acids were cloned from DBM. Their deduced amino acid sequences shared high identities with those of other insects and showed characteristics of classical CPRs and b5s, respectively. The mRNAs of both genes were detectable in all developmental stages with the highest expression levels occurring in the 4th instar larvae. Tissue-specific expression analysis showed that their transcripts were most abundant in gut. Transcripts of CPR and b5 in the beta-cypermethrin resistant DBM strain were 13.2- and 2.84-fold higher than those in the beta-cypermethrin susceptible strain, respectively. The expression levels of CPR and b5 were enhanced by beta-cypermethrin at the concentration of 12 mg L(-1) (~LC10). The results indicate that CPR and b5 may play essential roles in the P450 mediated resistance of DBM to beta-cypermethrin or even other insecticides. Copyright © 2015 Elsevier B.V. All rights reserved.

  2. Conformational Changes of NADPH-Cytochrome P450 Oxidoreductase Are Essential for Catalysis and Cofactor Binding*

    PubMed Central

    Xia, Chuanwu; Hamdane, Djemel; Shen, Anna L.; Choi, Vivian; Kasper, Charles B.; Pearl, Naw May; Zhang, Haoming; Im, Sang-Choul; Waskell, Lucy; Kim, Jung-Ja P.

    2011-01-01

    The crystal structure of NADPH-cytochrome P450 reductase (CYPOR) implies that a large domain movement is essential for electron transfer from NADPH via FAD and FMN to its redox partners. To test this hypothesis, a disulfide bond was engineered between residues Asp147 and Arg514 in the FMN and FAD domains, respectively. The cross-linked form of this mutant protein, designated 147CC514, exhibited a significant decrease in the rate of interflavin electron transfer and large (≥90%) decreases in rates of electron transfer to its redox partners, cytochrome c and cytochrome P450 2B4. Reduction of the disulfide bond restored the ability of the mutant to reduce its redox partners, demonstrating that a conformational change is essential for CYPOR function. The crystal structures of the mutant without and with NADP+ revealed that the two flavin domains are joined by a disulfide linkage and that the relative orientations of the two flavin rings are twisted ∼20° compared with the wild type, decreasing the surface contact area between the two flavin rings. Comparison of the structures without and with NADP+ shows movement of the Gly631–Asn635 loop. In the NADP+-free structure, the loop adopts a conformation that sterically hinders NADP(H) binding. The structure with NADP+ shows movement of the Gly631–Asn635 loop to a position that permits NADP(H) binding. Furthermore, comparison of these mutant and wild type structures strongly suggests that the Gly631–Asn635 loop movement controls NADPH binding and NADP+ release; this loop movement in turn facilitates the flavin domain movement, allowing electron transfer from FMN to the CYPOR redox partners. PMID:21345800

  3. Thyroid hormone stimulation of NADPH P450 reductase expression in liver and extrahepatic tissues. Regulation by multiple mechanisms.

    PubMed

    Ram, P A; Waxman, D J

    1992-02-15

    The role of thyroid hormone in regulating the expression of the flavoprotein NADPH cytochrome P450 reductase was studied in adult rats. Depletion of circulating thyroid hormone by hypophysectomy, or more selectively, by treatment with the anti-thyroid drug methimazole led to a 75-85% depletion of hepatic microsomal P450 reductase activity and protein in both male and female rats. Thyroxine substantially restored P450 reductase activity at a dose that rendered the thyroid-depleted rats euthyroid. Microsomal P450 reductase activity in several extrahepatic tissues was also dependent on thyroid hormone, but to a lesser extent than in liver (30-50% decrease in kidney, adrenal, lung, and heart but not in testis from hypothyroid rats). Hepatic P450 reductase mRNA levels were also decreased in the hypothyroid state, indicating that the loss of P450 reductase activity is not a consequence of the associated decreased availability of the FMN and FAD cofactors of P450 reductase. Parallel analysis of S14 mRNA, which has been studied extensively as a model thyroid-regulated liver gene product, indicated that P450 reductase and S14 mRNA respond similarly to these changes in thyroid state. In contrast, while the expression of S14 and several other thyroid hormone-dependent hepatic mRNAs is stimulated by feeding a high carbohydrate, fat-free diet, hepatic P450 reductase expression was not increased by this lipogenic diet. Injection of hypothyroid rats with T3 at a supraphysiologic, receptor-saturating dose stimulated a major induction of hepatic P450 reductase mRNA that was detectable 4 h after the T3 injection, and peaked at approximately 650% of euthyroid levels by 12 h. However, this same treatment stimulated a biphasic increase in P450 reductase protein and activity that required 3 days to reach normal euthyroid levels. T3 treatment of euthyroid rats also stimulated a major induction of P450 reductase mRNA that was maximal (12-fold increase) by 12 h, but in this case no major

  4. Drug-enhanced carbon monoxide production from heme by cytochrome P450 reductase.

    PubMed

    Vukomanovic, Dragic; Rahman, Mona N; Jia, Zongchao; Nakatsu, Kanji

    2017-01-01

    Carbon monoxide (CO) formed endogenously is considered to be cytoprotective, and the vast majority of CO formation is attributed to the degradation of heme by heme oxygenases-1 and -2 (HO-1, HO-2). Previously, we observed that brain microsomes containing HO-2 produced many-fold more CO in the presence of menadione and its congeners; herein we explored these observations further. We determined the effects of various drugs on CO production of rat brain microsomes and recombinant human cytochrome P450 reductase (CPR); CO was measured by gas chromatography with reductive detection. Brain microsomes of Sprague-Dawley rats or recombinant human cytochrome P450 reductase (CPR) were incubated with NADPH and various drugs in closed vials in phosphate buffer at pH 7.4 and 37°C. After 15 minutes, the reaction was stopped by cooling in dry ice, and the headspace gas was analyzed for CO production using gas chromatography with reductive (mercuric oxide) detection. We observed drug-enhanced CO production in the presence of both microsomes and recombinant CPR alone; the presence of HO was not required. A range of structurally diverse drugs were capable of amplifying this CO formation; these molecules had structures consistent with redox cycling capability. The addition of catalase to a reaction mixture, that contained activating drugs, inhibited the production of CO. Drug-enhanced CO formation can be catalyzed by CPR. The mechanism of CPR activation was not through classical drug-receptor mediation. Redox cycling may be involved in the drug-induced amplification of CO production by CPR through the production of reactive oxygen species.

  5. Engineering of a functional human NADH-dependent cytochrome P450 system

    PubMed Central

    Döhr, Olaf; Paine, Mark J. I.; Friedberg, Thomas; Roberts, Gordon C. K.; Wolf, C. Roland

    2001-01-01

    A functional human NADH-dependent cytochrome P450 system has been developed by altering the cofactor preference of human NADPH cytochrome P450 reductase (CPR), the redox partner for P450s. This has been achieved by a single amino acid change of the conserved aromatic amino acid Trp-676, which covers the re-side of the FAD isoalloxazine ring in the nicotinamide-binding site. Of the mutations made, the substitution of Trp-676 with alanine (W676A) resulted in a functional NADH-dependent enzyme, which catalyzed the reduction of cytochrome c and ferricyanide as well as facilitated the metabolism of 7-ethoxyresorufin by CYP1A2. Kinetic analysis measuring cytochrome c activity revealed that the NADH-dependent kcat of W676A is equivalent (90%) to the NADPH-dependent kcat of the wild-type enzyme, with W676A having an approximately 1,000-fold higher specificity for NADH. The apparent KMNADPH and KMNADH values of W676A are 80- and 150-fold decreased, respectively. In accordance with structural data, which show a bipartite binding mode of NADPH, substitution of Trp-676 does not affect 2′-AMP binding as seen by the inhibition of both wild-type CPR and the W676A mutant. Furthermore, NADPH was a potent inhibitor of the W676A NADH-dependent cytochrome c reduction and CYP1A2 activity. Overall, the results show that Trp-676 of human CPR plays a major role in cofactor discrimination, and substitution of this conserved aromatic residue with alanine results in an efficient NADH-dependent cytochrome P450 system. PMID:11136248

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Jan, Yi-Hua; Richardson, Jason R., E-mail: jricha3@eohsi.rutgers.edu; Baker, Angela A.

    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,more » 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 (40 mg/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. - Highlights: • Menadione redox cycles with cytochrome P450 reductase and generates reactive oxygen species. • Redox cycling inhibits cytochrome P450-mediated parathion metabolism. • Short term administration of menadione inhibits parathion toxicity by inhibiting paraoxon formation.« less

  7. Knockdown of NADPH-cytochrome P450 reductase results in reduced resistance to buprofezin in the small brown planthopper, Laodelphax striatellus (fallén).

    PubMed

    Zhang, Yueliang; Wang, Yaming; Wang, Lihua; Yao, Jing; Guo, Huifang; Fang, Jichao

    2016-02-01

    NADPH-cytochrome P450 reductase (CPR) plays an important role in cytochrome P450 function, and CPR knockdown in several insects leads to increased susceptibility to insecticides. However, a putative CPR gene has not yet been fully characterized in the small brown planthopper Laodelphax striatellus, a notorious agricultural pest in rice that causes serious damage by transmitting rice stripe and rice black-streaked dwarf viruses. The objective of this study was to clone the cDNA and to knock down the expression of the gene that encodes L. striatellus CPR (LsCPR) to further determine whether P450s are involved in the resistance of L. striatellus to buprofezin. First, the full-length cDNA of LsCPR was cloned and found to contain an open reading frame (ORF) encoding a polypeptide of 679 amino acids with a calculated molecular mass and isoelectric point of 76.92kDa and 5.37, respectively. The deduced amino acid sequence shares high identity with the CPRs of other insects (98%, 97%, 75% and 68% for Sogatella furcifera, Nilaparvata lugens, Cimex lectularius and Anopheles gambiae, respectively) and possesses the characteristic features of classical CPRs, such as an N-terminal membrane anchor and conserved domains for flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide phosphate (NADPH) binding. Phylogenetic analysis revealed that LsCPR is located in a branch along with the CPRs of other hemipteran insects. LsCPR mRNA was detectable in all examined body parts and developmental stages of L. striatellus, as determined by real-time quantitative PCR (qPCR), and transcripts were most abundant in the adult abdomen and in first-instar nymphs and adults. Ingestion of 200μg/mL of LsCPR double-stranded RNA (dsLsCPR) by the planthopper for 5days significantly reduced the transcription level of LsCPR. Moreover, silencing of LsCPR caused increased susceptibility to buprofezin in a buprofezin-resistant (YN-BPF) strain but not in a

  8. Differentially regulated NADPH:cytochrome P450 oxidoreductases in parsley

    PubMed Central

    Koopmann, Edda; Hahlbrock, Klaus

    1997-01-01

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

  9. Effects of 3G cell phone exposure on the structure and function of the human cytochrome P450 reductase.

    PubMed

    Tanvir, Shazia; Thuróczy, György; Selmaoui, Brahim; Silva Pires Antonietti, Viviane; Sonnet, Pascal; Arnaud-Cormos, Delia; Lévêque, Philippe; Pulvin, Sylviane; de Seze, René

    2016-10-01

    Cell phones increase exposure to radiofrequency (RF) electromagnetic fields (EMFs). Whether EMFs exert specific effects on biological systems remains debatable. This study investigated the effect of cell phone exposure on the structure and function of human NADPH-cytochrome P450 reductase (CPR). CPR plays a key role in the electron transfer to cytochrome P450, which takes part in a wide range of oxidative metabolic reactions in various organisms from microbes to humans. Human CPR was exposed for 60min to 1966-MHz RF inside a transverse electromagnetic cell (TEM-cell) placed in an incubator. The specific absorption rate (SAR) was 5W·kg(-1). Conformation changes have been detected through fluorescent spectroscopy of flavin and tryptophan residues, and investigated through circular dichroism, dynamic light scattering and microelectrophoresis. These showed that CPR was narrowed. By using cytochrome C reductase activity to assess the electron flux through the CPR, the Michaelis Menten constant (Km) and the maximum initial velocity (Vmax) decreased by 22% as compared with controls. This change was due to small changes in the tertiary and secondary structures of the protein at 37°C. The relevance of these findings to an actual RF exposure scenario demands further biochemical and in-vivo confirmation. Copyright © 2016 Elsevier B.V. All rights reserved.

  10. Expression and characterization of truncated human heme oxygenase (hHO-1) and a fusion protein of hHO-1 with human cytochrome P450 reductase.

    PubMed

    Wilks, A; Black, S M; Miller, W L; Ortiz de Montellano, P R

    1995-04-04

    A human heme oxygenase (hHO-1) gene without the sequence coding for the last 23 amino acids has been expressed in Escherichia coli behind the pho A promoter. The truncated enzyme is obtained in high yields as a soluble, catalytically-active protein, making it available for the first time for detailed mechanistic studies. The purified, truncated hHO-1/heme complex is spectroscopically indistinguishable from that of the rat enzyme and converts heme to biliverdin when reconstituted with rat liver cytochrome P450 reductase. A self-sufficient heme oxygenase system has been obtained by fusing the truncated hHO-1 gene to the gene for human cytochrome P450 reductase without the sequence coding for the 20 amino acid membrane binding domain. Expression of the fusion protein in pCWori+ yields a protein that only requires NADPH for catalytic turnover. The failure of exogenous cytochrome P450 reductase to stimulate turnover and the insensitivity of the catalytic rate toward changes in ionic strength establish that electrons are transferred intramolecularly between the reductase and heme oxygenase domains of the fusion protein. The Vmax for the fusion protein is 2.5 times higher than that for the reconstituted system. Therefore, either the covalent tether does not interfere with normal docking and electron transfer between the flavin and heme domains or alternative but equally efficient electron transfer pathways are available that do not require specific docking.

  11. Radiometric assay for cytochrome P-450-catalyzed progesterone 16 alpha-hydroxylation and determination of an apparent isotope effect

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Osawa, Y.; Coon, M.J.

    1987-08-01

    In the course of studies on the oxygenation of steroids by purified P-450 cytochromes, particularly rabbit liver microsomal cytochrome P-450 form 3b, a rapid and reliable radiometric assay has been devised for progesterone 16 alpha-hydroxylation. In view of the lack of a commercially available, suitably tritiated substrate, (1,2,6,7,16,17-3H)progesterone was treated with alkali to remove the label from potential hydroxylation sites other than the 16 alpha position. The resulting (1,7,16-3H)progesterone was added to a reconstituted enzyme system containing cytochrome P-450 form 3b, NADPH-cytochrome P-450 reductase, and NADPH, and the rate of 16 alpha-hydroxylation was measured by the formation of /sup 3/H/submore » 2/O. This reaction was shown to be linear with respect to time and to the cytochrome P-450 concentration. An apparent tritium isotope effect of 2.1 was observed by comparison of the rates of formation of tritium oxide and 16 alpha-hydroxyprogesterone, and the magnitude of the isotope effect was confirmed by an isotope competition assay in which a mixture of (1,7,16-/sup 3/H)progesterone and (4-14C)progesterone was employed.« less

  12. NADPH-Cytochrome P450 Reductase: Molecular Cloning and Functional Characterization of Two Paralogs from Withania somnifera (L.) Dunal

    PubMed Central

    Rana, Satiander; Lattoo, Surrinder K.; Dhar, Niha; Razdan, Sumeer; Bhat, Wajid Waheed; Dhar, Rekha S.; Vishwakarma, Ram

    2013-01-01

    Withania somnifera (L.) Dunal, a highly reputed medicinal plant, synthesizes a large array of steroidal lactone triterpenoids called withanolides. Although its chemical profile and pharmacological activities have been studied extensively during the last two decades, limited attempts have been made to decipher the biosynthetic route and identification of key regulatory genes involved in withanolide biosynthesis. Cytochrome P450 reductase is the most imperative redox partner of multiple P450s involved in primary and secondary metabolite biosynthesis. We describe here the cloning and characterization of two paralogs of cytochrome P450 reductase from W. somnifera. The full length paralogs of WsCPR1 and WsCPR2 have open reading frames of 2058 and 2142 bp encoding 685 and 713 amino acid residues, respectively. Phylogenetic analysis demonstrated that grouping of dual CPRs was in accordance with class I and class II of eudicotyledon CPRs. The corresponding coding sequences were expressed in Escherichia coli as glutathione-S-transferase fusion proteins, purified and characterized. Recombinant proteins of both the paralogs were purified with their intact membrane anchor regions and it is hitherto unreported for other CPRs which have been purified from microsomal fraction. Southern blot analysis suggested that two divergent isoforms of CPR exist independently in Withania genome. Quantitative real-time PCR analysis indicated that both genes were widely expressed in leaves, stalks, roots, flowers and berries with higher expression level of WsCPR2 in comparison to WsCPR1. Similar to CPRs of other plant species, WsCPR1 was un-inducible while WsCPR2 transcript level increased in a time-dependent manner after elicitor treatments. High performance liquid chromatography of withanolides extracted from elicitor-treated samples showed a significant increase in two of the key withanolides, withanolide A and withaferin A, possibly indicating the role of WsCPR2 in withanolide biosynthesis

  13. Adrenodoxin supports reactions catalyzed by microsomal steroidogenic cytochrome P450s

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Pechurskaya, Tatiana A.; Harnastai, Ivan N.; Grabovec, Irina P.

    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 rolemore » 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.« less

  14. Mutation of the Inducible ARABIDOPSIS THALIANA CYTOCHROME P450 REDUCTASE2 Alters Lignin Composition and Improves Saccharification1[W][OPEN

    PubMed Central

    Sundin, Lisa; Vanholme, Ruben; Geerinck, Jan; Goeminne, Geert; Höfer, René; Kim, Hoon; Ralph, John; Boerjan, Wout

    2014-01-01

    ARABIDOPSIS THALIANA CYTOCHROME P450 REDUCTASE1 (ATR1) and ATR2 provide electrons from NADPH to a large number of CYTOCHROME P450 (CYP450) enzymes in Arabidopsis (Arabidopsis thaliana). Whereas ATR1 is constitutively expressed, the expression of ATR2 appears to be induced during lignin biosynthesis and upon stresses. Therefore, ATR2 was hypothesized to be preferentially involved in providing electrons to the three CYP450s involved in lignin biosynthesis: CINNAMATE 4-HYDROXYLASE (C4H), p-COUMARATE 3-HYDROXYLASE1 (C3H1), and FERULATE 5-HYDROXYLASE1 (F5H1). Here, we show that the atr2 mutation resulted in a 6% reduction in total lignin amount in the main inflorescence stem and a compositional shift of the remaining lignin to a 10-fold higher fraction of p-hydroxyphenyl units at the expense of syringyl units. Phenolic profiling revealed shifts in lignin-related phenolic metabolites, in particular with the substrates of C4H, C3H1 and F5H1 accumulating in atr2 mutants. Glucosinolate and flavonol glycoside biosynthesis, both of which also rely on CYP450 activities, appeared less affected. The cellulose in the atr2 inflorescence stems was more susceptible to enzymatic hydrolysis after alkaline pretreatment, making ATR2 a potential target for engineering plant cell walls for biofuel production. PMID:25315601

  15. Altered heme catabolism by heme oxygenase-1 caused by mutations in human NADPH cytochrome P450 reductase

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Pandey, Amit V., E-mail: amit@pandeylab.org; Flueck, Christa E.; Mullis, Primus E.

    2010-09-24

    Research highlights: {yields} Mutations in POR identified from patients lead to reduced HO-1 activities. {yields} POR mutation Y181D affecting FMN binding results in total loss of HO-1 activity. {yields} POR mutations A287P, C569Y and V608F, lost 50-70% activity. {yields} Mutations in FAD binding domain, R457H, Y459H and V492E lost all HO-1 activity. {yields} POR polymorphisms P228L, R316W, G413S, A503V and G504R have normal activity. -- Abstract: Human heme oxygenase-1 (HO-1) carries out heme catabolism supported by electrons supplied from the NADPH through NADPH P450 reductase (POR, CPR). Previously we have shown that mutations in human POR cause a rare formmore » of congenital adrenal hyperplasia. In this study, we have evaluated the effects of mutations in POR on HO-1 activity. We used purified preparations of wild type and mutant human POR and in vitro reconstitution with purified HO-1 to measure heme degradation in a coupled assay using biliverdin reductase. Here we show that mutations in POR found in patients may reduce HO-1 activity, potentially influencing heme catabolism in individuals carrying mutant POR alleles. POR mutants Y181D, A457H, Y459H, V492E and R616X had total loss of HO-1 activity, while POR mutations A287P, C569Y and V608F lost 50-70% activity. The POR variants P228L, R316W and G413S, A503V and G504R identified as polymorphs had close to WT activity. Loss of HO-1 activity may result in increased oxidative neurotoxicity, anemia, growth retardation and iron deposition. Further examination of patients affected with POR deficiency will be required to assess the metabolic effects of reduced HO-1 activity in affected individuals.« less

  16. Dependence of microsomal methoxyflurane O-demethylation on cytochrome P-450 reductase and the stoichiometry of fluoride ion and formaldehyde release.

    PubMed

    Waskell, L; Gonzales, J

    1982-07-01

    In order to characterize further the in vitro liver microsomal O-demethylation and defluorination of the volatile anesthetic methoxyflurane, and obtain additional information regarding the participation of cytochrome P-450 in the oxidation, the stoichiometry of the reaction was determined and the effect of antibody to cytochrome P-450 reductase on this unique biotransformation was examined. Liver microsomes were isolated from rabbits and rats in which enzyme induction had previously been produced by phenobarbital. The O-demethylation of methoxyflurane by phenobarbital-induced microsomes results in the production of 1 mol of formaldehyde for every 2 mol of fluoride ion produced. Dichloroacetic acid is also a product of methoxyflurane O-demethylation. Antibody to cytochrome P-450 reductase inhibits by 85% the amount of fluoride ion produced by the microsomal metabolism of methoxyflurane. Thus critical indirect supportive data are contributed to the hypothesis that at least one, but perhaps more, cytochrome P-450 is indeed responsible for methoxyflurane O-demethylation and defluorination.

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

    PubMed

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

    2012-12-01

    In order to develop in vitro methods as an alternative to P450 animal testing in the drug discovery process, two main requisites are necessary: 1) gathering of data on animal homologues of the human P450 enzymes, currently very limited, and 2) bypassing the requirement for both the P450 reductase and the expensive cofactor NADPH. In this work, P450 2C20 from Macaca fascicularis, homologue of the human P450 2C8 has been taken as a model system to develop such an alternative in vitro method by two different approaches. In the first approach called "molecular Lego", a soluble self-sufficient chimera was generated by fusing the P450 2C20 domain with the reductase domain of cytochrome P450 BM3 from Bacillus megaterium (P450 2C20/BMR). In the second approach, the need for the redox partner and also NADPH were both obviated by the direct immobilization of the P450 2C20 on glassy carbon and gold electrodes. Both systems were then compared to those obtained from the reconstituted P450 2C20 monooxygenase in presence of the human P450 reductase and NADPH using paclitaxel and amodiaquine, two typical drug substrates of the human P450 2C8. The K(M) values calculated for the 2C20 and 2C20/BMR in solution and for 2C20 immobilized on electrodes modified with gold nanoparticles were 1.9 ± 0.2, 5.9 ± 2.3, 3.0 ± 0.5 μM for paclitaxel and 1.2 ± 0.2, 1.6±0.2 and 1.4 ± 0.2 μM for amodiaquine, respectively. The data obtained not only show that the engineering of M. fascicularis did not affect its catalytic properties but also are consistent with K(M) values measured for the microsomal human P450 2C8 and therefore show the feasibility of developing alternative in vitro animal tests. Copyright © 2012 Elsevier Inc. All rights reserved.

  18. Exposure to benzo[a]pyrene of Hepatic Cytochrome P450 Reductase Null (HRN) and P450 Reductase Conditional Null (RCN) mice: Detection of benzo[a]pyrene diol epoxide-DNA adducts by immunohistochemistry and 32P-postlabelling.

    PubMed

    Arlt, Volker M; Poirier, Miriam C; Sykes, Sarah E; John, Kaarthik; Moserova, Michaela; Stiborova, Marie; Wolf, C Roland; Henderson, Colin J; Phillips, David H

    2012-09-03

    Benzo[a]pyrene (BaP) is a widespread environmental carcinogen activated by cytochrome P450 (P450) enzymes. In Hepatic P450 Reductase Null (HRN) and Reductase Conditional Null (RCN) mice, P450 oxidoreductase (Por) is deleted specifically in hepatocytes, resulting in the loss of essentially all hepatic P450 function. Treatment of HRN mice with a single i.p. or oral dose of BaP (12.5 or 125mg/kg body weight) resulted in higher DNA adduct levels in liver (up to 10-fold) than in wild-type (WT) mice, indicating that hepatic P450s appear to be more important for BaP detoxification in vivo. Similar results were obtained in RCN mice. We tested whether differences between hepatocytes and non-hepatocytes in P450 activity may underlie the increased liver BaP-DNA binding in HRN mice. Cellular localisation by immunohistochemistry of BaP-DNA adducts showed that HRN mice have ample capacity for formation of BaP-DNA adducts in liver, indicating that the metabolic process does not result in the generation of a reactive species different from that formed in WT mice. However, increased protein expression of cytochrome b(5) in hepatic microsomes of HRN relative to WT mice suggests that cytochrome b(5) may modulate the P450-mediated bioactivation of BaP in HRN mice, partially substituting the function of Por. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

  19. 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. Copyright © 2015 Elsevier Inc. All rights reserved.

  20. Comparison of in Vitro Bioactivation of Flutamide and Its Cyano Analogue: Evidence for Reductive Activation by Human NADPH:Cytochrome P450 Reductase

    PubMed Central

    Wen, Bo; Coe, Kevin J.; Rademacher, Peter; Fitch, William L.; Monshouwer, Mario; Nelson, Sidney D.

    2009-01-01

    Flutamide (FLU), a nonsteroidal antiandrogen drug widely used in the treatment of prostate cancer, has been associated with idiosyncratic hepatotoxicity in patients. It is proposed that bioactivation of FLU and subsequent binding of reactive metabolite(s) to cellular proteins play a causative role. A toxicogenomic study comparing FLU and its nitro to cyano analogue (CYA) showed that the nitroaromatic group of FLU enhanced cytotoxicity to hepatocytes, indicating that reduction of the nitroaromatic group may represent a potential route of FLU-induced hepatotoxicity [Coe et al. (2007) Chem. Res. Toxicol. 20, 1277–1290]. In the current study, we compared in vitro bioactivation of FLU and CYA in human liver microsomes and cryopreserved human hepatocytes. A nitroreduction metabolite FLU-6 was formed in liver microsomal incubations of FLU under atmospheric oxygen levels and, to a greater extent, under anaerobic conditions. Seven glutathione (GSH) adducts of FLU, FLU-G1–7, were tentatively identified in human liver microsomal incubations using liquid chromatography–tandem mass spectrometry (LC/MS/MS), while CYA formed only four corresponding GSH adducts, CYA-G1–4, under the same conditions. Of particular interest was the formation of FLU-G5–7 from FLU, where the nitroaromatic group of FLU was reduced to an amino group. A tentative pathway is that upon nitroreduction, the para-diamines undergo cytochrome P450 (P450)-catalyzed two-electron oxidations to form corresponding para-diimine intermediates that react with GSH to form GSH adducts FLU-G5–7, respectively. The identities of FLU-G5–7 were further confirmed by LC/MS/MS analyses of microsomal incubations of a synthesized standard FLU-6. In an attempt to identify enzymes involved in the nitroreduction of FLU, NADPH:cytochrome P450 reductase (CPR) was shown to reduce FLU to FLU-6 under both aerobic and anaerobic conditions. Furthermore, the formation of FLU-G5–7 was completely blocked by the addition of a

  1. Comparison of in vitro bioactivation of flutamide and its cyano analogue: evidence for reductive activation by human NADPH:cytochrome P450 reductase.

    PubMed

    Wen, Bo; Coe, Kevin J; Rademacher, Peter; Fitch, William L; Monshouwer, Mario; Nelson, Sidney D

    2008-12-01

    Flutamide (FLU), a nonsteroidal antiandrogen drug widely used in the treatment of prostate cancer, has been associated with idiosyncratic hepatotoxicity in patients. It is proposed that bioactivation of FLU and subsequent binding of reactive metabolite(s) to cellular proteins play a causative role. A toxicogenomic study comparing FLU and its nitro to cyano analogue (CYA) showed that the nitroaromatic group of FLU enhanced cytotoxicity to hepatocytes, indicating that reduction of the nitroaromatic group may represent a potential route of FLU-induced hepatotoxicity [Coe et al. (2007) Chem. Res. Toxicol. 20, 1277-1290]. In the current study, we compared in vitro bioactivation of FLU and CYA in human liver microsomes and cryopreserved human hepatocytes. A nitroreduction metabolite FLU-6 was formed in liver microsomal incubations of FLU under atmospheric oxygen levels and, to a greater extent, under anaerobic conditions. Seven glutathione (GSH) adducts of FLU, FLU-G1-7, were tentatively identified in human liver microsomal incubations using liquid chromatography-tandem mass spectrometry (LC/ MS/MS), while CYA formed only four corresponding GSH adducts, CYA-G1-4, under the same conditions. Of particular interest was the formation of FLU-G5-7 from FLU, where the nitroaromatic group of FLU was reduced to an amino group. A tentative pathway is that upon nitroreduction, the para-diamines undergo cytochrome P450 (P450)-catalyzed two-electron oxidations to form corresponding para-diimine intermediates that react with GSH to form GSH adducts FLU-G5-7, respectively. The identities of FLU-G5-7 were further confirmed by LC/MS/MS analyses of microsomal incubations of a synthesized standard FLU-6. In an attempt to identify enzymes involved in the nitroreduction of FLU, NADPH:cytochrome P450 reductase (CPR) was shown to reduce FLU to FLU-6 under both aerobic and anaerobic conditions. Furthermore, the formation of FLU-G5-7 was completely blocked by the addition of a reversible CPR

  2. Effects of atrazine on cytochrome P450 enzymes of zebrafish (Danio rerio).

    PubMed

    Dong, Xiaoli; Zhu, Lusheng; Wang, Jinhua; Wang, Jun; Xie, Hui; Hou, Xinxin; Jia, Wentao

    2009-10-01

    In this study, the effects of atrazine (2-chloro-4-ethylamino-6-isopropylamino-1,3,5-triazine) in males and females of adult zebrafish (Danio rerio) were studied. The liver microsomal cytochrome P450 content, NADPH-P450 reductase, aminopyrine N-demethylase (APND), and erythromycin N-demethylase (ERND) activity were measured. Zebrafish were exposed to control and 3 treatments (0.01, 0.1, and 1 mg L(-1)) of atrazine for 5, 10, 15, 20, and 25 days. The results indicated that, within the range of test atrazine concentrations, either P450 content or P450 isozyme activities could be induced by atrazine. Compared to controls, P450 content was significantly increased at all atrazine concentrations at days 10, 15, and 20; thereafter, at day 25, all concentrations decreased to approximately the control levels, both in males and females. In addition, the strongest induction of P450 content was observed on day 15 in males and day 10 in females at treatment concentrations of 1 mg L(-1). NADPH-P450 reductase activities showed mild increase in males; however, the females exhibited significant induction on days 15, 20, and 25; especially, at concentrations of 0.01 mg L(-1), the induction level was consistently increased during the experiment. The inducements of APND and ERND in males were mainly observed on the days 5, 10, and 15, which showed less distinct induction, while significant induction was observed in cases of treatments during all days in females. In conclusion, atrazine induces P450 enzymes in zebrafish, and the effects may function as significant toxicity mechanisms in zebrafish. Additionally, it also confirms the importance of using a combined multi-time and multi-index diagnostic method to enhance the sensitivity and effectiveness of the indices adopted.

  3. A possible role of NADPH-dependent cytochrome P450nor isozyme in glycolysis under denitrifying conditions.

    PubMed

    Watsuji, Tomo-o; Takaya, Naoki; Nakamura, Akira; Shoun, Hirofumi

    2003-05-01

    The denitrifying fungus Cylindrocarpon tonkinense contains two isozymes of cytochrome P450nor. One isozyme, P450nor1, uses NADH specifically as its electron donor whereas the other isozyme P450nor2 prefers NADPH to NADH. Here we show that P450nor1 is localized in both cytosol and mitochondria, like P450nor of Fusarium oxysporum, while P450nor2 is exclusively in cytosol. We also found that the addition of glucose as a carbon source to the culture media leads to the production of much more P450nor2 in the fungal cells than a non-fermentable substrate (glycerol or acetate) does. These results suggest that the NADP-dependent pentose phosphate cycle acts predominantly in C. tonkinense as the glycolysis pathway under the denitrifying conditions, which was confirmed by the observation that glucose induced enzyme activities involved in the cycle. These results showed that P450nor2 should act as the electron sink under anaerobic, denitrifying conditions to regenerate NADP+ for the pentose phosphate cycle.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Xia, Chuanwu; Panda, Satya P.; Marohnic, Christopher C.

    2012-03-15

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

  5. Cytochrome P450 Organization and Function Are Modulated by Endoplasmic Reticulum Phospholipid Heterogeneity.

    PubMed

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

    2016-12-01

    Cytochrome P450s (P450s) comprise a superfamily of proteins that catalyze numerous monooxygenase reactions in animals, plants, and bacteria. In eukaryotic organisms, these proteins not only carry out reactions necessary for the metabolism of endogenous compounds, but they are also important in the oxidation of exogenous drugs and other foreign compounds. Eukaryotic P450 system proteins generally reside in membranes, primarily the endoplasmic reticulum or the mitochondrial membrane. These membranes provide a scaffold for the P450 system proteins that facilitate interactions with their redox partners as well as other P450s. This review focuses on the ability of specific lipid components to influence P450 activities, as well as the role of the membrane in P450 function. These studies have shown that P450s and NADPH-cytochrome P450 reductase appear to selectively associate with specific phospholipids and that these lipid-protein interactions influence P450 activities. Finally, because of the heterogeneous nature of the endoplasmic reticulum as well as other biologic membranes, the phospholipids are not arranged randomly but associate to generate lipid microdomains. Together, these characteristics can affect P450 function by 1) altering the conformation of the proteins, 2) influencing the P450 interactions with their redox partners, and 3) affecting the localization of the proteins into specific membrane microdomains. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    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.

  7. Functional expression of a putative geraniol 8-hydroxylase by reconstitution of bacterially expressed plant CYP76F45 and NADPH-cytochrome P450 reductase CPR I from Croton stellatopilosus Ohba.

    PubMed

    Sintupachee, Siriluk; Promden, Worrawat; Ngamrojanavanich, Nattaya; Sitthithaworn, Worapan; De-Eknamkul, Wanchai

    2015-10-01

    While attempting to isolate the enzyme geranylgeraniol 18-hydroxylase, which is involved in plaunotol biosynthesis in Croton stellatopilosus (Cs), the cDNAs for a cytochrome P450 monooxygenase(designated as CYP76F45) and an NADPH-cytochrome P450 reductase (designated as CPR I based on its classification) were isolated from the leaf. The CYP76F45 and CsCPR I genes have open reading frames (ORFs) encoding 507- and 711-amino acid proteins with predicted relative molecular weights of 56.7 and 79.0 kDa,respectively. Amino acid sequence comparison showed that both CYP76F45 (63–73%) and CsCPR I (79–83%) share relatively high sequence identities with homologous proteins in other plant species.Phylogenetic tree analysis confirmed that CYP76F45 belongs to the CYP76 family and that CsCPR I belongs to Class I of dicotyledonous CPRs, with both being closely related to Ricinus communis genes. Functional characterization of both enzymes, each expressed separately in Escherichia coli as recombinant proteins,showed that only simultaneous incubation of the membrane bound proteins with the substrate geraniol (GOH) and the coenzyme NADPH could form 8-hydroxygeraniol. The enzyme mixture could also utilize acyclic sesquiterpene farnesol (FOH) with a comparable substrate preference ratio (GOH:FOH) of 54:46. The levelsof the CYP76F45 and CsCPR I transcripts in the shoots, leaves and twigs of C. stellatopilosus were correlated with the levels of a major monoterpenoid indole alkaloid, identified tentatively as 19-Evallesamine,that accumulated in these plant parts. These results suggested that CYP76F45 and CPR I function as the enzyme geraniol-8-hydroxylase (G8H), which is likely to be involved in the biosynthesis of the indole alkaloid in C. stellatopilosus [corrected]. Copyright © 2015 Elsevier Ltd. All rights reserved.

  8. Co-expression of human cytochrome P4501A1 (CYP1A1) variants and human NADPH-cytochrome P450 reductase in the baculovirus/insect cell system.

    PubMed

    Schwarz, D; Kisselev, P; Honeck, H; Cascorbi, I; Schunck, W H; Roots, I

    2001-06-01

    1. Three human cytochrome P4501A1 (CYP1A1) variants, wild-type (CYP1A1.1), CYP1A1.2 (1462V) and CYP1A1.4 (T461N), were co-expressed with human NADPH-P450 reductase (OR) in Spodoptera frugiperda (Sf9) insect cells by baculovirus co-infection to elaborate a suitable system for studying the role of CYPA1 polymorphism in the metabolism of exogenous and endogenous substrates. 2. A wide range of conditions was examined to optimize co-expression with regard to such parameters as relative multiplicity of infection (MOI), time of harvest, haem precursor supplementation and post-translational stabilization. tinder optimized conditions, almost identical expression levels and molar OR/CYP1A1 ratios (20:1) were attained for all CYP1A1 variants. 3. Microsomes isolated from co-infected cells demonstrated ethoxyresorufin deethlylase activities (nmol/min(-1) nmol(-1) CYP1A1) of 16.0 (CYP1A1.1), 20.5 (CYP1A1.2) and 22.5 (CYP1A1.4). Pentoxyresorufin was dealkylated approximately 10-20 times slower with all enzyme variants. 4. All three CYP1A1 variants were active in metabolizing the precarcinogen benzo[a]pyrene (B[a]P), with wild-type enzyme showing the highest activity, followed by CYP1A1.4 (60%) and CYP1A1.2 (40%). Each variant produced all major metabolites including B[a]P-7,8-dihydrodiol, the precursor of the ultimate carcinogenic species. 5. These studies demonstrate that the baculovirus-mediated co-expression-by-co-infection approach all CYP1A1 variants yields functionally active enzyme systems with similar molar OR/CYP1A1 ratios, thus providing suitable preconditions to examine the metabolism of and environmental chemicals by the different CY1A1 variants.

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

    PubMed

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

    2011-04-01

    Tractable plasmids (pAC-Mv-based plasmids) for Escherichia coli were constructed, which carried a mevalonate-utilizing gene cluster, towards an efficient functional analysis of cytochromes P450 involved in sesquiterpene biosynthesis. They included genes coding for a series of redox partners that transfer the electrons from NAD(P)H to a P450 protein. The redox partners used were ferredoxin reductases (CamA and NsRED) and ferredoxins (CamB and NsFER), which are derived from Pseudomonas putida and cyanobacterium Nostoc sp. strain PCC 7120, respectively, as well as three higher-plant NADPH-P450 reductases, the Arabidopsis thaliana ATR2 and two corresponding enzymes derived from ginger (Zingiber officinale), named ZoRED1 and ZoRED2. We also constructed plasmids for functional analysis of two P450s, α-humulene-8-hydroxylase (CYP71BA1) from shampoo ginger (Zingiber zerumbet) and germacrene A hydroxylase (P450NS; CYP110C1) from Nostoc sp. PCC 7120, and co-transformed E. coli with each of the pAC-Mv-based plasmids. Production levels of 8-hydroxy-α-humulene with recombinant E. coli cells (for CYP71BA1) were 1.5- to 2.3-fold higher than that of a control strain without the mevalonate-pathway genes. Level of the P450NS product with the combination of NsRED and NsFER was 2.9-fold higher than that of the CamA and CamB. The predominant product of P450NS was identified as 1,2,3,5,6,7,8,8a-octahydro-6-isopropenyl-4,8a-dimethylnaphth-1-ol with NMR analyses. © Springer-Verlag 2011

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

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

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

  11. Gene engineering in yeast for biodegradation: Immunological cross-reactivity among cytochrome p-450 system proteins of saccharomyces cerevisiae and candida tropicalis

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Loper, J.C.; Chen, C.; Dey, C.R.

    1993-01-01

    Yeasts are eukaryotic microorganisms whose cytochrome P-450 monooxygenase systems may be amenable to genetic engineering for the hydroxylation and detoxication of polychlorinated aromatic hydrocarbons. The molecular genetic properties of strains of bakers yeast, Saccharomyces cerevisiae, and an n-alkane utilizing yeast, Candida tropicalis ATCC750 are examined. Standard methods were used to purify cytochrome P-450 and NADPH-cytochrome c (P-450) reductase proteins from cells cultured by semi-anaerobic glucose fermentation (S. cerevisiae, C. tropicalis) and by growth on tetradecane (C. tropicalis). Polyvalent antisera prepared in rabbits to some of these proteins were used in tests of immunological relatedness among the purified proteins using sodiummore » dodecyl sulfate-polyacrylamide gel electrophoresis and nitrocellulose filter immunoblots. The results provide evidence for gene relationships which should prove useful in gene isolation and subsequent engineering of P-450 enzyme systems in yeast.« less

  12. Purification and characterization of NADPH--cytochrome c reductase from the midgut of the southern armyworm (Spodoptera eridania).

    PubMed Central

    Crankshaw, D L; Hetnarski, K; Wilkinson, C F

    1979-01-01

    1. NADPH-cytochrome c reductase was solubilized with bromelain and purified about 400-fold from sucrose/pyrophosphate-washed microsomal fractions from southern armyworm (Spodoptera eridania) larval midguts. 2. The enzyme has a mol.wt. of 70 035 +/- 1300 and contained 2 mol of flavin/mol of enzyme consisting of almost equimolar amounts of FMN and FAD. 3. Aerobic titration of the enzyme with NADPH caused the formation of a stable half-reduced state at 0.5 mol of NADPH/mol of flavin. 4. Kinetic analysis showed that the reduction of cytochrome c proceeded by a Bi Bi Ping Pong mechanism. 5. Apparent Km values for NADPH and cytochrome c and Ki values for NADP+ and 2'-AMP were considerably higher for the insect reductase than for the mammalian liver enzyme. 6. These are discussed in relation to possible differences in the active sites of the enzymes. Images Fig. 3. PMID:117798

  13. Purification and characterization of NADPH--cytochrome c reductase from the midgut of the southern armyworm (Spodoptera eridania).

    PubMed

    Crankshaw, D L; Hetnarski, K; Wilkinson, C F

    1979-09-01

    1. NADPH-cytochrome c reductase was solubilized with bromelain and purified about 400-fold from sucrose/pyrophosphate-washed microsomal fractions from southern armyworm (Spodoptera eridania) larval midguts. 2. The enzyme has a mol.wt. of 70 035 +/- 1300 and contained 2 mol of flavin/mol of enzyme consisting of almost equimolar amounts of FMN and FAD. 3. Aerobic titration of the enzyme with NADPH caused the formation of a stable half-reduced state at 0.5 mol of NADPH/mol of flavin. 4. Kinetic analysis showed that the reduction of cytochrome c proceeded by a Bi Bi Ping Pong mechanism. 5. Apparent Km values for NADPH and cytochrome c and Ki values for NADP+ and 2'-AMP were considerably higher for the insect reductase than for the mammalian liver enzyme. 6. These are discussed in relation to possible differences in the active sites of the enzymes.

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

    PubMed Central

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

    2013-01-01

    Malaria is one of the most dangerous mosquito-borne diseases in many tropical countries, including Thailand. Studies in a deltamethrin resistant strain of Anopheles minimus mosquito, suggest cytochrome P450 enzymes contribute to the detoxification of pyrethroid insecticides. Purified A. minimus CYPOR enzyme (AnCYPOR), which is the redox partner of cytochrome P450s, loses flavin-adenosine di-nucleotide (FAD) and FLAVIN mono-nucleotide (FMN) cofactors that affect its enzyme activity. Replacement of leucine residues at positions 86 and 219 with phenylalanines in FMN binding domain increases FMN binding, enzyme stability, and cytochrome c reduction activity. Membrane-Bound L86F/L219F-AnCYPOR increases A. minimus P450-mediated pyrethroid metabolism in vitro. In this study, we constructed a comparative model structure of AnCYPOR using a rat CYPOR structure as a template. Overall model structure is similar to rat CYPOR, with some prominent differences. Based on primary sequence and structural analysis of rat and A. minimus CYPOR, C427R, W678A, and W678H mutations were generated together with L86F/L219F resulting in three soluble Δ55 triple mutants. The C427R triple AnCYPOR mutant retained a higher amount of FAD binding and increased cytochrome c reduction activity compared to wild-type and L86F/L219F-Δ55AnCYPOR double mutant. However W678A and W678H mutations did not increase FAD and NAD(P)H bindings. The L86F/L219F double and C427R triple membrane-bound AnCYPOR mutants supported benzyloxyresorufin O-deakylation (BROD) mediated by mosquito CYP6AA3 with a two-to three-fold increase in efficiency over wild-type AnCYPOR. The use of rat CYPOR in place of AnCYPOR most efficiently supported CYP6AA3-mediated BROD compared to all AnCYPORs. PMID:23325047

  15. Analysis of the complex formation, interaction and electron transfer pathway between the "open" conformation of NADPH-cytochrome P450 reductase and aromatase.

    PubMed

    Dai, Yuejie; Zhen, Jing; Zhang, Xiuli; Zhong, Yonghui; Liu, Shaodan; Sun, Ziyue; Guo, Yue; Wu, Qingli

    2015-09-01

    The complex structure of human aromatase (CYP19) and the open form of ΔTGEE mutant NADPH-cytochrome P450 reductase (mCPR) was constructed using template-based protein alignment method. Dynamic simulation of formed complex was performed on NAMD 2.9, in which CHARMm all 27_prot_lipid_na force field and an explicit TIP3P water solvent model were applied. The result showed mCPR in its open conformation could steadily combine with aromatase from the proximal face. Data analysis indicates hydrogen bonds and four salt bridges on the binding surface enhance the interaction between the two protein molecules. Amino acid, Lys108 plays a key role in aromatase activity through the formation of a salt bridge with Asp147 and two hydrogen bonds with Asp147 and Gln150 in mCPR. The optimal pathway for the first electron transfer from CPR to aromatase was revealed and calculated using HARLEM software. The rates for solvent mediated and non-solvent mediated electron transfer from FMNH2 to heme were determined as 1.04×10(6)s(-)(1) and 4.86×10(5)s(-)(1) respectively, which indicates the solvent water can facilitate the electron transfer from FMNH2 to heme. This study presents a novel strategy for the study of the protein-protein interactions based on the template-based protein alignment, which may help new aromtase development targeting the electron transfer between mCPR and aromatase. Copyright © 2015 Elsevier Inc. All rights reserved.

  16. A collection of cytochrome P450 monooxygenase genes involved in modification and detoxification of herbicide atrazine in rice (Oryza sativa) plants.

    PubMed

    Rong Tan, Li; Chen Lu, Yi; Jing Zhang, Jing; Luo, Fang; Yang, Hong

    2015-09-01

    Plant cytochrome P450 monooxygenases constitute one of the largest families of protein genes involved in plant growth, development and acclimation to biotic and abiotic stresses. However, whether these genes respond to organic toxic compounds and their biological functions for detoxifying toxic compounds such as herbicides in rice are poorly understood. The present study identified 201 genes encoding cytochrome P450s from an atrazine-exposed rice transcriptome through high-throughput sequencing. Of these, 69 cytochrome P450 genes were validated by microarray and some of them were confirmed by real time PCR. Activities of NADPH-cytochrome P450 reductase (CPR) and p-nitroanisole O-demethylase (PNOD) related to toxicity were determined and significantly induced by atrazine exposure. To dissect the mechanism underlying atrazine modification and detoxification by P450, metabolites (or derivatives) of atrazine in plants were analyzed by ultra performance liquid chromatography mass spectrometry (UPLC/MS). Major metabolites comprised desmethylatrazine (DMA), desethylatrazine (DEA), desisopropylatrazine (DIA), hydroxyatrazine (HA), hydroxyethylatrazine (HEA) and hydroxyisopropylatrazine (HIA). All of them were chemically modified by P450s. Furthermore, two specific inhibitors of piperonyl butoxide (PBO) and malathion (MAL) were used to assess the correlation between the P450s activity and rice responses including accumulation of atrazine in tissues, shoot and root growth and detoxification. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. Cytochrome P450 monooxygenases: perspectives for synthetic application.

    PubMed

    Urlacher, Vlada B; Eiben, Sabine

    2006-07-01

    Cytochrome P450 monooxygenases are versatile biocatalysts that introduce oxygen into a vast range of molecules. These enzymes catalyze diverse reactions in a regio- and stereoselective manner, and their properties have been used for drug development, bioremediation and the synthesis of fine chemicals and other useful compounds. However, the potential of P450 monooxygenases has not been fully exploited; there are some drawbacks limiting the broader implementation of these catalysts for commercial needs. Protein engineering has produced P450 enzymes with widely altered substrate specificities, substantially increased activity and higher stability. Furthermore, electrochemical and enzymatic approaches for the replacement or regeneration of NAD(P)H have been developed, enabling the more cost-effective use of P450 enzymes. In this review, we focus on the aspects relevant to the synthetic applications of P450 enzymes and their optimization for commercial needs.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Reed, James R., E-mail: rreed@lsuhsc.edu; The Stanley S. Scott Cancer Center, Louisiana State University Health Science Center, 533 Bolivar St., New Orleans, LA 70112; Cawley, George F.

    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 severalmore » 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

  19. Cytochrome P450 (CYP450) Tests

    MedlinePlus

    ... P450 (CYP450) tests Overview 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 ...

  20. The central role of mosquito cytochrome P450 CYP6Zs in insecticide detoxification revealed by functional expression and structural modelling.

    PubMed

    Chandor-Proust, Alexia; Bibby, Jaclyn; Régent-Kloeckner, Myriam; Roux, Jessica; Guittard-Crilat, Emilie; Poupardin, Rodolphe; Riaz, Muhammad Asam; Paine, Mark; Dauphin-Villemant, Chantal; Reynaud, Stéphane; David, Jean-Philippe

    2013-10-01

    The resistance of mosquitoes to chemical insecticides is threatening vector control programmes worldwide. Cytochrome P450 monooxygenases (CYPs) are known to play a major role in insecticide resistance, allowing resistant insects to metabolize insecticides at a higher rate. Among them, members of the mosquito CYP6Z subfamily, like Aedes aegypti CYP6Z8 and its Anopheles gambiae orthologue CYP6Z2, have been frequently associated with pyrethroid resistance. However, their role in the pyrethroid degradation pathway remains unclear. In the present study, we created a genetically modified yeast strain overexpressing Ae. aegypti cytochrome P450 reductase and CYP6Z8, thereby producing the first mosquito P450-CPR (NADPH-cytochrome P450-reductase) complex in a yeast recombinant system. The results of the present study show that: (i) CYP6Z8 metabolizes PBAlc (3-phenoxybenzoic alcohol) and PBAld (3-phenoxybenzaldehyde), common pyrethroid metabolites produced by carboxylesterases, producing PBA (3-phenoxybenzoic acid); (ii) CYP6Z8 transcription is induced by PBAlc, PBAld and PBA; (iii) An. gambiae CYP6Z2 metabolizes PBAlc and PBAld in the same way; (iv) PBA is the major metabolite produced in vivo and is excreted without further modification; and (v) in silico modelling of substrate-enzyme interactions supports a similar role of other mosquito CYP6Zs in pyrethroid degradation. By playing a pivotal role in the degradation of pyrethroid insecticides, mosquito CYP6Zs thus represent good targets for mosquito-resistance management strategies.

  1. In vitro modulation of cytochrome P450 reductase supported indoleamine 2,3-dioxygenase activity by allosteric effectors cytochrome b(5) and methylene blue.

    PubMed

    Pearson, Josh T; Siu, Sophia; Meininger, David P; Wienkers, Larry C; Rock, Dan A

    2010-03-30

    Indoleamine 2,3-dioxygenase (IDO) is a heme-containing dioxygenase involved in the degradation of several indoleamine derivatives and has been indicated as an immunosuppressive. IDO is an attractive target for therapeutic intervention in diseases which are known to capitalize on immune suppression, including cancer, HIV, and inflammatory diseases. Conventionally, IDO activity is measured through chemical reduction by the addition of ascorbate and methylene blue. Identification of potential coenzymes involved in the reduction of IDO in vivo should improve in vitro reconstitution systems used to identify potential IDO inhibitors. In this study we show that NADPH-cytochrome P450 reductase (CPR) is capable of supporting IDO activity in vitro and that oxidation of l-Trp follows substrate inhibition kinetics (k(cat) = 0.89 +/- 0.04 s(-1), K(m) = 0.72 +/- 0.15 microM, and K(i) = 9.4 +/- 2.0 microM). Addition of cytochrome b(5) to CPR-supported l-Trp incubations results in modulation from substrate inhibition to sigmoidal kinetics (k(cat) = 1.7 +/- 0.3 s(-1), K(m) = 1.5 +/- 0.9 microM, and K(i) = 1.9 +/- 0.3). CPR-supported d-Trp oxidations (+/-cytochrome b(5)) exhibit Michaelis-Menten kinetics. Addition of methylene blue (minus ascorbate) to CPR-supported reactions resulted in inhibition of d-Trp turnover and modulation of l-Trp kinetics from allosteric to Michaelis-Menten with a concurrent decrease in substrate affinity for IDO. Our data indicate that CPR is capable of supporting IDO activity in vitro and oxidation of tryptophan by IDO displays substrate stereochemistry dependent atypical kinetics which can be modulated by the addition of cytochrome b(5).

  2. Studies on the interactions between drugs and estrogen: analytical method for prediction system of gynecomastia induced by drugs on the inhibitory metabolism of estradiol using Escherichia coli coexpressing human CYP3A4 with human NADPH-cytochrome P450 reductase.

    PubMed

    Satoh, T; Fujita, K I; Munakata, H; Itoh, S; Nakamura, K; Kamataki, T; Itoh, S; Yoshizawa, I

    2000-11-15

    To establish a prediction system for drug-induced gynecomastia in clinical fields, a model reaction system was developed to explain numerically this side effect. The principle is based on the assumption that 50% inhibition concentration (IC(50)) of drugs on the in vitro metabolism of estradiol (E2) to its major product 2-hydroxyestradiol (2-OH-E2) can be regarded as the index for achieving this purpose. By using human cytochrome P450s coexpressed with human NADPH-cytochrome P450 reductase in Escherichia coli as the enzyme, the reaction was examined. Among the nine enzymes (CYP1A1, 1A2, 2A6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4) tested, CYP3A4 having a V(max)/K(m) (ml/min/nmol P450) value of 0.32 for production of 2-OH-E2 was shown to be the most suitable enzyme as the reagent. The inhibitory effects of ketoconazole, cyclosporin A, and cimetidine toward the 2-hydroxylation of E2 catalyzed by CYP3A4 were obtained, and their IC(50) values were 7 nM, 64 nM, and 290 microM, respectively. The present results suggest that IC(50) values thus obtained can be substituted as the prediction index for gynecomastia induced by drugs, considering the patients' individual information. Copyright 2000 Academic Press.

  3. Insights into electron leakage in the reaction cycle of cytochrome P450 BM3 revealed by kinetic modeling and mutagenesis

    PubMed Central

    Lim, Joseph B; Barker, Kimberly A; Eller, Kristen A; Jiang, Linda; Molina, Veronica; Saifee, Jessica F; Sikes, Hadley D

    2015-01-01

    As a single polypeptide, cytochrome P450 BM3 fuses oxidase and reductase domains and couples each domain's function to perform catalysis with exceptional activity upon binding of substrate for hydroxylation. Mutations introduced into the enzyme to change its substrate specificity often decrease coupling efficiency between the two domains, resulting in unproductive consumption of cofactors and formation of water and/or reactive species. This phenomenon can correlate with leakage, in which P450 BM3 uses electrons from NADPH to reduce oxygen to water and/or reactive species even without bound substrate. The physical basis for leakage is not yet well understood in this particular member of the cytochrome P450 family. To clarify the relationship between leakage and coupling, we used simulations to illustrate how different combinations of kinetic parameters related to substrate-free consumption of NADPH and substrate hydroxylation can lead to either minimal effects on coupling or a dramatic decrease in coupling as a result of leakage. We explored leakage in P450 BM3 by introducing leakage-enhancing mutations and combining these mutations to assess whether doing so increases leakage further. The variants in this study provide evidence that while a transition to high spin may be vital for coupled hydroxylation, it is not required for enhanced leakage; substrate binding and the consequent shift in spin state are not necessary as a redox switch for catalytic oxidation of NADPH. Additionally, the variants in this study suggest a tradeoff between leakage and stability and thus evolvability, as the mutations we investigated were far more deleterious than other mutations that have been used to change substrate specificity. PMID:26311413

  4. Phytomonitoring and phytoremediation of agrochemicals and related compounds based on recombinant cytochrome P450s and aryl hydrocarbon receptors (AhRs).

    PubMed

    Shimazu, Sayuri; Inui, Hideyuki; Ohkawa, Hideo

    2011-04-13

    Molecular mechanisms of metabolism and modes of actions of agrochemicals and related compounds are important for understanding selective toxicity, biodegradability, and monitoring of biological effects on nontarget organisms. It is well-known that in mammals, cytochrome P450 (P450 or CYP) monooxygenases metabolize lipophilic foreign compounds. These P450 species are inducible, and both CYP1A1 and CYP1A2 are induced by aryl hydrocarbon receptor (AhR) combined with a ligand. Gene engineering of P450 and NADPH cytochrome P450 oxidoreductase (P450 reductase) was established for bioconversion. Also, gene modification of AhRs was developed for recombinant AhR-mediated β-glucronidase (GUS) reporter assay of AhR ligands. Recombinant P450 genes were transformed into plants for phytoremediation, and recombinant AhR-mediated GUS reporter gene expression systems were each transformed into plants for phytomonitoring. Transgenic rice plants carrying CYP2B6 metabolized the herbicide metolachlor and remarkably reduced the residues in the plants and soils under paddy field conditions. Transgenic Arabidopsis plants carrying recombinant guinea pig (g) AhR-mediated GUS reporter genes detected PCB126 at the level of 10 ng/g soils in the presence of biosurfactants MEL-B. Both phytomonitoring and phytoremediation plants were each evaluated from the standpoint of practical uses.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kaspera, Ruediger; Sahele, Tariku; Lakatos, Kyle

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

  6. The central role of mosquito cytochrome P450 CYP6Zs in insecticide detoxification revealed by functional expression and structural modelling

    PubMed Central

    Chandor-Proust, Alexia; Bibby, Jaclyn; Régent-Kloeckner, Myriam; Roux, Jessica; Guittard-Crilat, Emilie; Poupardin, Rodolphe; Riaz, Muhammad Asam; Paine, Mark; Dauphin-Villemant, Chantal; Reynaud, Stéphane; David, Jean-Philippe

    2013-01-01

    The resistance of mosquitoes to chemical insecticides is threatening vector control programmes worldwide. Cytochrome P450 monooxygenases (CYPs) are known to play a major role in insecticide resistance, allowing resistant insects to metabolize insecticides at a higher rate. Among them, members of the mosquito CYP6Z subfamily, like Aedes aegypti CYP6Z8 and its Anopheles gambiae orthologue CYP6Z2, have been frequently associated with pyrethroid resistance. However, their role in the pyrethroid degradation pathway remains unclear. In the present study, we created a genetically modified yeast strain overexpressing Ae. aegypti cytochrome P450 reductase and CYP6Z8, thereby producing the first mosquito P450–CPR (NADPH-cytochrome P450-reductase) complex in a yeast recombinant system. The results of the present study show that: (i) CYP6Z8 metabolizes PBAlc (3-phenoxybenzoic alcohol) and PBAld (3-phenoxybenzaldehyde), common pyrethroid metabolites produced by carboxylesterases, producing PBA (3-phenoxybenzoic acid); (ii) CYP6Z8 transcription is induced by PBAlc, PBAld and PBA; (iii) An. gambiae CYP6Z2 metabolizes PBAlc and PBAld in the same way; (iv) PBA is the major metabolite produced in vivo and is excreted without further modification; and (v) in silico modelling of substrate–enzyme interactions supports a similar role of other mosquito CYP6Zs in pyrethroid degradation. By playing a pivotal role in the degradation of pyrethroid insecticides, mosquito CYP6Zs thus represent good targets for mosquito-resistance management strategies. PMID:23844938

  7. Rat oesophageal cytochrome P450 (CYP) monooxygenase system: comparison to the liver and relevance in N-nitrosodiethylamine carcinogenesis.

    PubMed

    Pinto, L F; Moraes, E; Albano, R M; Silva, M C; Godoy, W; Glisovic, T; Lang, M A

    2001-11-01

    N-nitrosodiethylamine (NDEA) is able to induce tumours in the rat oesophagus. It has been suggested that this could be due to tissue specific expression of NDEA activating cytochrome P450 enzymes. We investigated this by characterizing the oesophageal monooxygenase complex of male Wistar rats and comparing it with that of the liver. Total amount of cytochrome P450, NADPH P450 reductase, cytochrome b5 and cytochrome b5 reductase of the oesophageal mucosa was approximately 7% of what was found in the liver. In addition, major differences were found in the cytochrome P450 isoenzyme composition between these organs: CYP 2B1/2B2 and CYP3A were found only in the liver, whereas CYP1A1 was constitutively expressed only in the oesophagus. Of the two well-known nitrosamine metabolizing enzymes, CYP2A3 was found only in the oesophagus whereas CYP2E1 was exclusively expressed in the liver. Catalytic studies, western blotting and RT-PCR analyses confirmed the expression of CYP2A3 in the oesophagus. CYP2A enzymes are known to be good catalysts of NDEA metabolism. Oesophageal microsomes had a K(m) for NDEA metabolism, which was about one-third of that of hepatic microsomes, but they showed similar activities when compared per nmol of total P450. NDEA activity in the oesophagus was significantly increased by coumarin (CO), which also induced oesophageal CYP2A3. Immunoinhibition of the microsomal NDEA activity showed that up to 70% of this reaction is catalysed by CYP2A3 in the oesophagus, whereas no inhibition of the hepatic NDEA activity could be achieved by the anti-CYP2A5 antibody. NDEA, but not N-nitrosodimethylamine (NDMA) inhibited the oesophageal metabolism of CO. The results of the present investigation show major differences in the enzyme composition of the oesophageal and hepatic monooxygenase complexes, and are in accordance with the hypothesis that the NDEA organotropism could, to a large extent, be due to the tissue specific expression of the activating enzymes.

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

    PubMed Central

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

    2015-01-01

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

  9. Molecular dynamics simulations give insight into the conformational change, complex formation, and electron transfer pathway for cytochrome P450 reductase

    PubMed Central

    Sündermann, Axel; Oostenbrink, Chris

    2013-01-01

    Cytochrome P450 reductase (CYPOR) undergoes a large conformational change to allow for an electron transfer to a redox partner to take place. After an internal electron transfer over its cofactors, it opens up to facilitate the interaction and electron transfer with a cytochrome P450. The open conformation appears difficult to crystallize. Therefore, a model of a human CYPOR in the open conformation was constructed to be able to investigate the stability and conformational change of this protein by means of molecular dynamics simulations. Since the role of the protein is to provide electrons to a redox partner, the interactions with cytochrome P450 2D6 (2D6) were investigated and a possible complex structure is suggested. Additionally, electron pathway calculations with a newly written program were performed to investigate which amino acids relay the electrons from the FMN cofactor of CYPOR to the HEME of 2D6. Several possible interacting amino acids in the complex, as well as a possible electron transfer pathway were identified and open the way for further investigation by site directed mutagenesis studies. PMID:23832577

  10. Expression of Xanthophyllomyces dendrorhous cytochrome-P450 hydroxylase and reductase in Mucor circinelloides.

    PubMed

    Csernetics, Árpád; Tóth, Eszter; Farkas, Anita; Nagy, Gábor; Bencsik, Ottó; Vágvölgyi, Csaba; Papp, Tamás

    2015-02-01

    Carotenoids are natural pigments that act as powerful antioxidants and have various beneficial effects on human and animal health. Mucor circinelloides (Mucoromycotina) is a carotenoid producing zygomycetes fungus, which accumulates β-carotene as the main carotenoid but also able to produce the hydroxylated derivatives of β-carotene (i.e. zeaxanthin and β-cryptoxanthin) in low amount. These xanthophylls, together with the ketolated derivatives of β-carotene (such as canthaxanthin, echinenone and astaxanthin) have better antioxidant activity than β-carotene. In this study our aim was to modify and enhance the xanthophyll production of the M. circinelloides by expression of heterologous genes responsible for the astaxanthin biosynthesis. The crtS and crtR genes, encoding the cytochrome-P450 hydroxylase and reductase, respectively, of wild-type and astaxanthin overproducing mutant Xanthophyllomyces dendrorhous strains were amplified from cDNA and the nucleotide and the deduced amino acid sequences were compared to each other. Introduction of the crtS on autonomously replicating plasmid in the wild-type M. circinelloides resulted enhanced zeaxanthin and β-cryptoxanthin accumulation and the presence of canthaxanthin, echinenone and astaxanthin in low amount; the β-carotene hydroxylase and ketolase activity of the X. dendrorhous cytochrome-P450 hydroxylase in M. circinelloides was verified. Increased canthaxanthin and echinenone production was observed by expression of the gene in a canthaxanthin producing mutant M. circinelloides. Co-expression of the crtR and crtS genes led to increase in the total carotenoid and slight change in xanthophyll accumulation in comparison with transformants harbouring the single crtS gene.

  11. A cellular stress response (CSR) that interacts with NADPH-P450 reductase (NPR) is a new regulator of hypoxic response.

    PubMed

    Oguro, Ami; Koyama, Chika; Xu, Jing; Imaoka, Susumu

    2014-02-28

    NADPH-P450 reductase (NPR) was previously found to contribute to the hypoxic response of cells, but the mechanism was not clarified. In this study, we identified a cellular stress response (CSR) as a new factor interacting with NPR by a yeast two-hybrid system. Overexpression of CSR enhanced the induction of erythropoietin and hypoxia response element (HRE) activity under hypoxia in human hepatocarcinoma cell lines (Hep3B), while knockdown of CSR suppressed them. This new finding regarding the interaction of NPR with CSR provides insight into the function of NPR in hypoxic response. Copyright © 2014 Elsevier Inc. All rights reserved.

  12. Methoxyflurane acts at the substrate binding site of cytochrome P450 LM2 to induce a dependence on cytochrome b5.

    PubMed

    Lipka, J J; Waskell, L A

    1989-01-01

    Rabbit cytochrome P450 isozyme 2 requires cytochrome b5 to metabolize the volatile anesthetic methoxyflurane but not the substrate benzphetamine [E. Canova-Davis and L. Waskell (1984) J. Biol. Chem. 259, 2541-2546]. To determine whether the requirement for cytochrome b5 for methoxyflurane oxidation is mediated by an allosteric effect on cytochrome P450 LM2 or cytochrome P450 reductase, we have investigated whether this anesthetic can induce a role for cytochrome b5 in benzphetamine metabolism. Using rabbit liver microsomes and antibodies raised in guinea pigs against rabbit cytochrome b5, we found that methoxyflurane did not create a cytochrome b5 requirement for benzphetamine metabolism. Methoxyflurane also failed to induce a role for cytochrome b5 in benzphetamine metabolism in the purified, reconstituted mixed function oxidase system. Studies of the reaction kinetics established that in the absence of cytochrome b5, methoxyflurane and benzphetamine are competitive inhibitors, and that in the presence of cytochrome b5, benzphetamine and methoxyflurane are two alternate substrates in competition for a single site on the same enzyme. These results all indicate that the methoxyflurane-induced cytochrome b5 dependence of the mixed function oxidase cytochrome P450 LM2 system is a direct result of the interaction between methoxyflurane and the substrate binding site of cytochrome P450 LM2 and suggest the focus of future studies of this question.

  13. 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.

  14. Comparison of the metabolism of parathion by a rat liver reconstituted mixed-function oxidase enzyme system and by a system containing cumene hydroperoxide and purified rat liver cytochrome P-450.

    PubMed

    Yoshihara, S; Neal, R A

    1977-01-01

    The metabolism of parathion by a reconstituted mixed-function oxidase enzyme system (rat liver cytochrome P-450, NADPH-cytochrome c reductase, dilauroyl phosphatidylcholine, deoxycholate, and NADPH) or a cumene hydroperoxide system (cytochrome P-450, dilauroyl phosphatidylcholine, and cumene hydroperoxide) have been compared. The products formed on incubation of parathion with both systems were paraoxon, diethyl phosphorothioic acid, diethyl phosphoric acid, p-nitrophenol, and atomic sulfur. The apparent KM values for parathion for formation of paraoxon and diethyl phosphorothioic acid with the cumene hydroperoxide system were 55 and 39 X 10(-6) M, respectively. These KM values are not significantly different. When the reconstituted system was used, apparent KM values of 2.8 x 10(-6) M for formation of paraoxon and 3.9 x 10(-6) M for The formation of diethyl phosphorothioic acid and diethyl phosphoric acid were determined. These KM values are also not significantly different. covalent binding of the sulfur atom, released in the metabolism of parathion to paraoxon, to the proteins of the reconstituted system and to cytochrome P-450 of the cumene hydroperoxide system was also examined. With both the reconstituted system and the cumene hydroperoxide system approximately 65% of the sulfur released became bound to the proteins of these enzyme systems. The binding of the sulfur atome resulted in a progressive inhibition of the metabolism of parathion by these two systems.

  15. 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

  16. Positive correlation between decreased cellular uptake, NADPH-glutathione reductase activity and adriamycin resistance in Ehrlich ascites tumor lines.

    PubMed

    Scheulen, M E; Hoensch, H; Kappus, H; Seeber, S; Schmidt, C G

    1987-01-01

    From a wild type strain of Ehrlich ascites tumor (EATWT) sublines resistant to daunorubicin (EATDNM), etoposide (EATETO), and cisplatinum (EATCIS) have been developed in vivo. Increase in survival and cure rate caused by adriamycin (doxorubicin) have been determined in female NMRI mice which were inoculated i.p. with EAT cells. Adriamycin concentrations causing 50% inhibition of 3H-thymidine (ICT) and 3H-uridine incorporation (ICU) and intracellular adriamycin steady-state concentrations (SSC) were measured in vitro. Adriamycin resistance increased and SSC decreased in the following sequence: EATWT - EATCIS - EATDNM - EATETO. When ICT and ICU were corrected for intracellular adriamycin concentrations in consideration of the different SSC (ICTc, ICUc), ICTc and ICUc still varied up to the 3.2 fold in EATCIS, EATDNM and EATETO in comparison to EATWT. Thus, in addition to different SSC other factors must be responsible for adriamycin resistance. Therefore, enzymes which may play a role in the cytotoxicity related to adriamycin metabolism (NADPH-cytochrome P-450 reductase, NADPH-glutathione reductase, NADP-glucose-6-phosphate dehydrogenase, NADP-isocitrate dehydrogenase) were measured. In contrast to the other parameters determined, NADPH-glutathione reductase was significantly (p less than 0.01) increased up to the 3.2 fold parallel to adriamycin resistance as determined by increase in life span, cure rate, ICTc, and ICUc, respectively. It is concluded that high activities of NADPH-glutathione reductase may contribute to an increase in adriamycin resistance of malignant tumors.

  17. Novel approaches to mitigating parathion toxicity: targeting cytochrome P450-mediated metabolism with menadione.

    PubMed

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

    2016-08-01

    Accidental or intentional exposures to parathion, an organophosphorus (OP) pesticide, can cause severe poisoning in humans. Parathion toxicity is dependent on its metabolism by the cytochrome P450 (CYP) system to paraoxon (diethyl 4-nitrophenyl phosphate), a highly poisonous nerve agent and potent inhibitor of acetylcholinesterase. We have been investigating inhibitors of CYP-mediated bioactivation of OPs as a method of preventing or reversing progressive parathion toxicity. It is well recognized that NADPH-cytochrome P450 reductase, an enzyme required for the transfer of electrons to CYPs, mediates chemical redox cycling. In this process, the enzyme diverts electrons from CYPs to support chemical redox cycling, which results in inhibition of CYP-mediated biotransformation. Using menadione as the redox-cycling chemical, we discovered that this enzymatic reaction blocks metabolic activation of parathion in rat and human liver microsomes and in recombinant CYPs important to parathion metabolism, including CYP1A2, CYP2B6, and CYP3A4. Administration of menadione to rats reduces metabolism of parathion, as well as parathion-induced inhibition of brain cholinesterase activity. This resulted in inhibition of parathion neurotoxicity. Menadione has relatively low toxicity and is approved by the Food and Drug Administration for other indications. Its ability to block parathion metabolism makes it an attractive therapeutic candidate to mitigate parathion-induced neurotoxicity. © 2016 New York Academy of Sciences.

  18. The Role of Protein-Protein and Protein-Membrane Interactions on P450 Function

    PubMed Central

    Scott, Emily E.; Wolf, C. Roland; Otyepka, Michal; Humphreys, Sara C.; Reed, James R.; Henderson, Colin J.; McLaughlin, Lesley A.; Paloncýová, Markéta; Navrátilová, Veronika; Berka, Karel; Anzenbacher, Pavel; Dahal, Upendra P.; Barnaba, Carlo; Brozik, James A.; Jones, Jeffrey P.; Estrada, D. Fernando; Laurence, Jennifer S.; Park, Ji Won

    2016-01-01

    This symposium summary, sponsored by the ASPET, was held at Experimental Biology 2015 on March 29, 2015, in Boston, Massachusetts. The symposium focused on: 1) the interactions of cytochrome P450s (P450s) with their redox partners; and 2) the role of the lipid membrane in their orientation and stabilization. Two presentations discussed the interactions of P450s with NADPH-P450 reductase (CPR) and cytochrome b5. First, solution nuclear magnetic resonance was used to compare the protein interactions that facilitated either the hydroxylase or lyase activities of CYP17A1. The lyase interaction was stimulated by the presence of b5 and 17α-hydroxypregnenolone, whereas the hydroxylase reaction was predominant in the absence of b5. The role of b5 was also shown in vivo by selective hepatic knockout of b5 from mice expressing CYP3A4 and CYP2D6; the lack of b5 caused a decrease in the clearance of several substrates. The role of the membrane on P450 orientation was examined using computational methods, showing that the proximal region of the P450 molecule faced the aqueous phase. The distal region, containing the substrate-access channel, was associated with the membrane. The interaction of NADPH-P450 reductase (CPR) with the membrane was also described, showing the ability of CPR to “helicopter” above the membrane. Finally, the endoplasmic reticulum (ER) was shown to be heterogeneous, having ordered membrane regions containing cholesterol and more disordered regions. Interestingly, two closely related P450s, CYP1A1 and CYP1A2, resided in different regions of the ER. The structural characteristics of their localization were examined. These studies emphasize the importance of P450 protein organization to their function. PMID:26851242

  19. Construction and application of a functional library of cytochrome P450 monooxygenases from the filamentous fungus Aspergillus oryzae.

    PubMed

    Nazir, K H M Nazmul Hussain; Ichinose, Hirofumi; Wariishi, Hiroyuki

    2011-05-01

    A functional library of cytochrome P450 monooxygenases from Aspergillus oryzae (AoCYPs) was constructed in which 121 isoforms were coexpressed with yeast NADPH-cytochrome P450 oxidoreductase in Saccharomyces cerevisiae. Using this functional library, novel catalytic functions of AoCYPs, such as catalytic potentials of CYP57B3 against genistein, were elucidated for the first time. Comprehensive functional screening promises rapid characterization of catalytic potentials and utility of AoCYPs.

  20. 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

  1. Photosystem I from plants as a bacterial cytochrome P450 surrogate electron donor: terminal hydroxylation of branched hydrocarbon chains.

    PubMed

    Jensen, Kenneth; Johnston, Jonathan B; de Montellano, Paul R Ortiz; Møller, Birger Lindberg

    2012-02-01

    The ability of cytochrome P450 enzymes to catalyze highly regio- and stereospecific hydroxylations makes them attractive alternatives to approaches based on chemical synthesis but they require expensive cofactors, e.g. NAD(P)H, which limits their commercial potential. Ferredoxin (Fdx) is a multifunctional electron carrier that in plants accepts electrons from photosystem I (PSI) and facilitates photoreduction of NADP(+) to NADPH mediated by ferredoxin-NAD(P)H oxidoreductase (FdR). In bacteria, the electron flow is reversed and Fdx accepts electrons from NADPH via FdR and serves as the direct electron donor to bacterial P450s. By combining the two systems, we demonstrate that irradiation of PSI can drive the activity of a bacterial P450, CYP124 from Mycobacterium tuberculosis. The substitution of the costly cofactor NADPH with sunlight illustrates the potential of the light-driven hydroxylation system for biotechnology applications.

  2. Leukotriene B4 omega-hydroxylase in human polymorphonuclear leukocytes. Partial purification and identification as a cytochrome P-450.

    PubMed

    Shak, S; Goldstein, I M

    1985-09-01

    Human polymorphonuclear leukocytes (PMN) not only synthesize and respond to leukotriene B4 (LTB4), but also catabolize this mediator of inflammation rapidly and specifically by omega-oxidation. To characterize the enzyme(s) responsible for omega-oxidation of LTB4, human PMN were disrupted by sonication and subjected to differential centrifugation to yield membrane, granule, and cytosol fractions (identified by biochemical markers). LTB4 omega-hydroxylase activity was concentrated (together with NADPH cytochrome c reductase activity) only in the membrane fraction (specific activity increased 10-fold as compared to whole sonicates, 41% recovery). Negligible activity was detected in granule or cytosol fractions. LTB4 omega-hydroxylase activity in isolated PMN membranes was linear with respect to duration of incubation and protein concentration, was maximal at pH 7.4, had a Km for LTB4 of 0.6 microM, and was dependent on oxygen and on reduced pyridine nucleotides (apparent Km for NADPH = 0.5 microM; apparent Km for NADH = 223 microM). The LTB4 omega-hydroxylase was inhibited significantly by carbon monoxide, ferricytochrome c, SKF-525A, and Triton X-100, but was not affected by alpha-naphthoflavone, azide, cyanide, catalase, and superoxide dismutase. Finally, isolated PMN membranes exhibited a carbon monoxide difference spectrum with a peak at 452 nm. Thus, we have partially purified the LTB4 omega-hydroxylase in human PMN and identified the enzyme as a membrane-associated, NADPH-dependent cytochrome P-450.

  3. 1-Ethynylpyrene, a suicide inhibitor of cytochrome P-450 dependent benzo(a)pyrene hydroxylase activity in liver microsomes

    DOE Office of Scientific and Technical Information (OSTI.GOV)

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

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

  4. High throughput assay for cytochrome P450 BM3 for screening libraries of substrates and combinatorial mutants.

    PubMed

    Tsotsou, Georgia Eleni; Cass, Anthony Edward George; Gilardi, Gianfranco

    2002-01-01

    A rapid method for identifying compounds that are potential substrates for the drug metabolising enzyme cytochrome P450 is described. The strategy is based on the detection of a degradation product of NAD(P)H oxidation during substrate turnover by the enzyme expressed in Escherichia coli cells spontaneously lysed under the experimental conditions. The performance of the method has been tested on two known substrates of the wild-type cytochrome P450 BM3, arachidonic (AA) and lauric (LA) acids, and two substrates with environmental significance, the anionic surfactant sodium dodecyl sulfate (SDS), and the solvent 1,1,2,2-tetrachloroethane (TCE). The minimal background signal given from cells expressing cytochrome P450 BM3 in the absence of added substrate is only 3% of the signal in the presence of saturating substrate. Control experiments have proven that this method is specifically detecting NADPH oxidation by catalytic turnover of P450 BM3. The assay has been adapted to a microtitre plate format and used to screen a series of furazan derivatives as potential substrates. Three derivatives were identified as substrates. The method gave a significant different signal for two isomeric furazan derivatives. All results found on the cell lysate were verified and confirmed with the purified enzyme. This strategy opens the way to automated high throughput screening of NAD(P)H-linked enzymatic activity of molecules of pharmacological and biotechnological interest and libraries of random mutants of NAD(P)H-dependent biocatalysts.

  5. Engineering human cytochrome P450 enzymes into catalytically self-sufficient chimeras using molecular Lego.

    PubMed

    Dodhia, Vikash Rajnikant; Fantuzzi, Andrea; Gilardi, Gianfranco

    2006-10-01

    The membrane-bound human cytochrome P450s have essential roles in the metabolism of endogenous compounds and drugs. Presented here are the results on the construction and characterization of three fusion proteins containing the N-terminally modified human cytochrome P450s CYP2C9, CY2C19 and CYP3A4 fused to the soluble NADPH-dependent oxidoreductase domain of CYP102A1 from Bacillus megaterium. The constructs, CYP2C9/BMR, CYP2C19/BMR and CYP3A4/BMR are well expressed in Escherichia coli as holo proteins. The chimeras can be purified in the absence of detergent and the purified enzymes are both active and correctly folded in the absence of detergent, as demonstrated by circular dichroism and functional studies. Additionally, in comparison with the parent P450 enzyme, these chimeras have greatly improved solubility properties. The chimeras are catalytically self-sufficient and present turnover rates similar to those reported for the native enzymes in reconstituted systems, unlike previously reported mammalian cytochrome P450 fusion proteins. Furthermore the specific activities of these chimeras are not dependent on the enzyme concentration present in the reaction buffer and they do not require the addition of accessory proteins, detergents or phospholipids to be fully active. The solubility, catalytic self-sufficiency and wild-type like activities of these chimeras would greatly simplify the studies of cytochrome P450 mediated drug metabolism in solution.

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

    PubMed Central

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

    2013-01-01

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

  7. NAD(P)H-dependent quinone oxidoreductase 1 (NQO1) and cytochrome P450 oxidoreductase (CYP450OR) differentially regulate menadione-mediated alterations in redox status, survival and metabolism in pancreatic β-cells.

    PubMed

    Gray, Joshua P; Karandrea, Shpetim; Burgos, Delaine Zayasbazan; Jaiswal, Anil A; Heart, Emma A

    2016-11-16

    NQO1 (NAD(P)H-quinone oxidoreductase 1) reduces quinones and xenobiotics to less-reactive compounds via 2-electron reduction, one feature responsible for the role of NQO1 in antioxidant defense in several tissues. In contrast, NADPH cytochrome P450 oxidoreductase (CYP450OR), catalyzes the 1-electron reduction of quinones and xenobiotics, resulting in enhanced superoxide formation. However, to date, the roles of NQO1 and CYP450OR in pancreatic β-cell metabolism under basal conditions and oxidant challenge have not been characterized. Using NQO1 inhibition, over-expression and knock out, we have demonstrated that, in addition to protection of β-cells from toxic concentrations of the redox cycling quinone menadione, NQO1 also regulates the basal level of reduced-to-oxidized nucleotides, suggesting other role(s) beside that of an antioxidant enzyme. In contrast, over-expression of NADPH cytochrome P450 oxidoreductase (CYP450OR) resulted in enhanced redox cycling activity and decreased cellular viability, consistent with the enhanced generation of superoxide and H 2 O 2 . Basal expression of NQO1 and CYP450OR was comparable in isolated islets and liver. However, NQO1, but not CYP450OR, was strongly induced in β-cells exposed to menadione. NQO1 and CYP450OR exhibited a reciprocal preference for reducing equivalents in β-cells: while CYP450OR preferentially utilized NADPH, NQO1 primarily utilized NADH. Together, these results demonstrate that NQO1 and CYP450OR reciprocally regulate oxidant metabolism in pancreatic β-cells. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  8. NAD(P)H-dependent Quinone Oxidoreductase 1 (NQO1) and Cytochrome P450 Oxidoreductase (CYP450OR) differentially regulate menadione-mediated alterations in redox status, survival and metabolism in pancreatic β-cells

    PubMed Central

    Gray, Joshua P.; Karandrea, Shpetim; Burgos, Delaine Zayasbazan; Jaiswal, Anil A; Heart, Emma A.

    2017-01-01

    NQO1 (NAD(P)H-quinone oxidoreductase 1) reduces quinones and xenobiotics to less-reactive compounds via 2-electron reduction, one feature responsible for the role of NQO1 in antioxidant defense in several tissues. In contrast, NADPH cytochrome P450 oxidoreductase (CYP450OR), catalyzes the 1-electron reduction of quinones and xenobiotics, resulting in enhanced superoxide formation. However, to date, the roles of NQO1 and CYP450OR in pancreatic β-cell metabolism under basal conditions and oxidant challenge have not been characterized. Using NQO1 inhibition, over-expression and knock out, we have demonstrated that, in addition to protection of β-cells from toxic concentrations of the redox cycling quinone menadione, NQO1 also regulates the basal level of reduced-to-oxidized nucleotides, suggesting other role(s) beside that of an antioxidant enzyme. In contrast, over-expression of NADPH cytochrome P450 oxidoreductase (CYP450OR) resulted in enhanced redox cycling activity and decreased cellular viability, consistent with the enhanced generation of superoxide and H2O2. Basal expression of NQO1 and CYP450OR was comparable in isolated islets and liver. However, NQO1, but not CYP450OR, was strongly induced in β-cells exposed to menadione. NQO1 and CYP450OR exhibited a reciprocal preference for reducing equivalents in β-cells: while CYP450OR preferentially utilized NADPH, NQO1 primarily utilized NADH. Together, these results demonstrate that NQO1 and CYP450OR reciprocally regulate oxidant metabolism in pancreatic β-cells. PMID:27558805

  9. A world of cytochrome P450s

    PubMed Central

    Nelson, David R.

    2013-01-01

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

  10. Cytochromes P450

    PubMed Central

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

    2011-01-01

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

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Mandai, Takao; Fujiwara, Shinsuke; Imaoka, Susumu, E-mail: imaoka@kwansei.ac.jp

    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.more » 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.« less

  12. Biosynthesis of Costunolide, Dihydrocostunolide, and Leucodin. Demonstration of Cytochrome P450-Catalyzed Formation of the Lactone Ring Present in Sesquiterpene Lactones of Chicory

    PubMed Central

    de Kraker, Jan-Willem; Franssen, Maurice C.R.; Joerink, Maaike; de Groot, Aede; Bouwmeester, Harro J.

    2002-01-01

    Chicory (Cichorium intybus) is known to contain guaianolides, eudesmanolides, and germacranolides. These sesquiterpene lactones are postulated to originate from a common germacranolide, namely (+)-costunolide. Whereas a pathway for the formation of germacra-1(10),4,11(13)-trien-12-oic acid from farnesyl diphosphate had previously been established, we now report the isolation of an enzyme activity from chicory roots that converts the germacrene acid into (+)-costunolide. This (+)-costunolide synthase catalyzes the last step in the formation of the lactone ring present in sesquiterpene lactones and is dependent on NADPH and molecular oxygen. Incubation of the germacrene acid in the presence of 18O2 resulted in the incorporation of one atom of 18O into (+)-costunolide. The label was situated at the ring oxygen atom. Hence, formation of the lactone ring most likely occurs via C6-hydroxylation of the germacrene acid and subsequent attack of this hydroxyl group at the C12-atom of the carboxyl group. Blue light-reversible CO inhibition and experiments with cytochrome P450 inhibitors demonstrated that the (+)-costunolide synthase is a cytochrome P450 enzyme. In addition, enzymatic conversion of (+)-costunolide into 11(S),13-dihydrocostunolide and leucodin, a guaianolide, was detected. The first-mentioned reaction involves an enoate reductase, whereas the formation of leucodin from (+)-costunolide probably involves more than one enzyme, including a cytochrome P450 enzyme. PMID:12011356

  13. Biosynthesis of costunolide, dihydrocostunolide, and leucodin. Demonstration of cytochrome p450-catalyzed formation of the lactone ring present in sesquiterpene lactones of chicory.

    PubMed

    de Kraker, Jan-Willem; Franssen, Maurice C R; Joerink, Maaike; de Groot, Aede; Bouwmeester, Harro J

    2002-05-01

    Chicory (Cichorium intybus) is known to contain guaianolides, eudesmanolides, and germacranolides. These sesquiterpene lactones are postulated to originate from a common germacranolide, namely (+)-costunolide. Whereas a pathway for the formation of germacra-1(10),4,11(13)-trien-12-oic acid from farnesyl diphosphate had previously been established, we now report the isolation of an enzyme activity from chicory roots that converts the germacrene acid into (+)-costunolide. This (+)-costunolide synthase catalyzes the last step in the formation of the lactone ring present in sesquiterpene lactones and is dependent on NADPH and molecular oxygen. Incubation of the germacrene acid in the presence of 18O2 resulted in the incorporation of one atom of 18O into (+)-costunolide. The label was situated at the ring oxygen atom. Hence, formation of the lactone ring most likely occurs via C6-hydroxylation of the germacrene acid and subsequent attack of this hydroxyl group at the C12-atom of the carboxyl group. Blue light-reversible CO inhibition and experiments with cytochrome P450 inhibitors demonstrated that the (+)-costunolide synthase is a cytochrome P450 enzyme. In addition, enzymatic conversion of (+)-costunolide into 11(S),13-dihydrocostunolide and leucodin, a guaianolide, was detected. The first-mentioned reaction involves an enoate reductase, whereas the formation of leucodin from (+)-costunolide probably involves more than one enzyme, including a cytochrome P450 enzyme.

  14. Defining the in Vivo Role for cytochrome b5 in cytochrome P450 function through the conditional hepatic deletion of microsomal cytochrome b5.

    PubMed

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

    2008-11-14

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

  15. 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

  16. Conformational change of cytochrome P450 1A2 induced by phospholipids and detergents.

    PubMed

    Yun, C H; Song, M; Kim, H

    1997-08-08

    . It was found that the activity increase of rabbit P450 1A2 coincides with the conformational change including raised alpha-helix content. It is proposed that the interaction with the phospholipid molecules surrounding P450 1A2 in the endoplasmic reticulum is important for a functional conformation of P450 1A2 in a monooxygenase system including NADPH-P450 reductase.

  17. Molecular LEGO by domain-imprinting of cytochrome P450 BM3.

    PubMed

    Jetzschmann, K J; Yarman, A; Rustam, L; Kielb, P; Urlacher, V B; Fischer, A; Weidinger, I M; Wollenberger, U; Scheller, F W

    2018-04-01

    Electrosynthesis of the MIP nano-film after binding of the separated domains or holo-cytochrome BM3 via an engineered anchor should result in domain-specific cavities in the polymer layer. Both the two domains and the holo P450 BM3 have been bound prior polymer deposition via a N-terminal engineered his6-anchor to the electrode surface. Each step of MIP preparation was characterized by cyclic voltammetry of the redox-marker ferricyanide. Rebinding after template removal was evaluated by quantifying the suppression of the diffusive permeability of the signal for ferricyanide and by the NADH-dependent reduction of cytochrome c by the reductase domain (BMR). The working hypothesis is verified by the discrimination of the two domains by the respective MIPs: The holoenzyme P450 BM3 was ca. 5.5 times more effectively recognized by the film imprinted with the oxidase domain (BMO) as compared to the BMR-MIP or the non-imprinted polymer (NIP). Obviously, a cavity is formed during the imprinting process around the his 6 -tag-anchored BMR which cannot accommodate the broader BMO or the P450 BM3. The affinity of the MIP towards P450 BM3 is comparable with that to the monomer in solution. The his 6 -tagged P450 BM3 binds (30 percent) stronger which shows the additive effect of the interaction with the MIP and the binding to the electrode. Copyright © 2018. Published by Elsevier B.V.

  18. 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.

  19. Purification, Reconstitution, and Inhibition of Cytochrome P-450 Sterol Δ22-Desaturase from the Pathogenic Fungus Candida glabrata

    PubMed Central

    Lamb, David C.; Maspahy, Segula; Kelly, Diane E.; Manning, Nigel J.; Geber, Antonia; Bennett, John E.; Kelly, Steven L.

    1999-01-01

    Sterol Δ22-desaturase has been purified from a strain of Candida glabrata with a disruption in the gene encoding sterol 14α-demethylase (cytochrome P-45051; CYP51). The purified cytochrome P-450 exhibited sterol Δ22-desaturase activity in a reconstituted system with NADPH–cytochrome P-450 reductase in dilaurylphosphatidylcholine, with the enzyme kinetic studies revealing a Km for ergosta-5,7-dienol of 12.5 μM and a Vmax of 0.59 nmol of this substrate metabolized/min/nmol of P-450. This enzyme is encoded by CYP61 (ERG5) in Saccharomyces cerevisiae, and homologues have been shown in the Candida albicans and Schizosaccharomyces pombe genome projects. Ketoconazole, itraconazole, and fluconazole formed low-spin complexes with the ferric cytochrome and exhibited type II spectra, which are indicative of an interaction between the azole moiety and the cytochrome heme. The azole antifungal compounds inhibited reconstituted sterol Δ22-desaturase activity by binding to the cytochrome with a one-to-one stoichiometry, with total inhibition of enzyme activity occurring when equimolar amounts of azole and cytochrome P-450 were added. These results reveal the potential for sterol Δ22-desaturase to be an antifungal target and to contribute to the binding of drugs within the fungal cell. PMID:10390230

  20. Isolation and Expression Analysis of CYP9A11 and Cytochrome P450 Reductase Gene in the Beet Armyworm (Lepidoptera: Noctuidae)

    PubMed Central

    Zhao, Chunqing; Feng, Xiaoyun; Tang, Tao; Qiu, Lihong

    2015-01-01

    Cytochrome P450 monooxygenases (CYPs), as an enzyme superfamily, is widely distributed in organisms and plays a vital function in the metabolism of exogenous and endogenous compounds by interacting with its obligatory redox partner, CYP reductase (CPR). A novel CYP gene (CYP9A11) and CPR gene from the agricultural pest insect Spodoptera exigua were cloned and characterized. The complete cDNA sequences of SeCYP9A11 and SeCPR are 1,931 and 3,919 bp in length, respectively, and contain open reading frames of 1,593 and 2,070 nucleotides, respectively. Analysis of the putative protein sequences indicated that SeCYP9A11 contains a heme-binding domain and the unique characteristic sequence (SRFALCE) of the CYP9 family, in addition to a signal peptide and transmembrane segment at the N-terminal. Alignment analysis revealed that SeCYP9A11 shares the highest sequence similarity with CYP9A13 from Mamestra brassicae, which is 66.54%. The putative protein sequence of SeCPR has all of the classical CPR features, such as an N-terminal membrane anchor; three conserved domain flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), and nicotinamide adenine dinucleotide phosphate (NADPH) domain; and characteristic binding motifs. Phylogenetic analysis revealed that SeCPR shares the highest identity with HaCPR, which is 95.21%. The SeCYP9A11 and SeCPR genes were detected in the midgut, fat body, and cuticle tissues, and throughout all of the developmental stages of S. exigua. The mRNA levels of SeCYP9A11 and SeCPR decreased remarkably after exposure to plant secondary metabolites quercetin and tannin. The results regarding SeCYP9A11 and SeCPR genes in the current study provide foundation for the further study of S. exigua P450 system. PMID:26320261

  1. Suicide inactivation of cytochrome P-450 by methoxsalen. Evidence for the covalent binding of a reactive intermediate to the protein moiety

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Labbe, G.; Descatoire, V.; Beaune, P.

    Incubation of rat liver microsomes with (3H)methoxsalen and NADPH resulted in the covalent binding of a methoxsalen intermediate to proteins comigrating with cytochromes P-450 UT-A, PB-B/D, ISF-G and PCN-E. Binding was increased by pretreatments with phenobarbital, beta-naphthoflavone (beta NF) and dexamethasone. Such pretreatments also increased the loss of CO-binding capacity either after administration of methoxsalen, or after incubation of hepatic microsomes with methoxsalen and NADPH. Immunoprecipitation of the methoxsalen metabolite-protein adducts in phenobarbital-induced microsomes was moderate with anti-UT-A antibodies, but marked with anti-PB-B/D and anti-PCN-E antibodies. Immunoprecipitation was observed also with anti-ISF-G (anti-beta NF-B) antibodies in beta NF-induced microsomes. Methoxsalenmore » (0.25 mM) inhibited markedly the benzphetamine demethylase activity of phenobarbital-induced microsomes and the erythromycin demethylase activity of dexamethasone-induced microsomes. Whereas methoxsalen itself did not produce any binding spectrum, in contrast either in vivo administration of methoxsalen or incubation in vitro with methoxsalen and NADPH resulted in a low-to-high spin conversion of cytochrome P-450 as suggested by the appearance of a spectrum analogous to a type I binding spectrum. This low-to-high spin conversion was apparently due to a methoxsalen intermediate (probably, covalently bound to the protein and preventing partial sixth ligation of the iron). We conclude that suicide inactivation of cytochrome P-450 by methoxsalen is related to the covalent binding of a methoxsalen intermediate to the protein moiety of several cytochrome P-450 isoenzymes (including UT-A, PB-B/D, PCN-E as well as ISF-G and/or beta NF-B).« less

  2. Hydroxylation of salicylate by microsomal fractions and cytochrome P-450. Lack of production of 2,3-dihydroxybenzoate unless hydroxyl radical formation is permitted.

    PubMed Central

    Ingelman-Sundberg, M; Kaur, H; Terelius, Y; Persson, J O; Halliwell, B

    1991-01-01

    Attack by hydroxyl radicals (.OH) upon salicylate (2-hydroxybenzoate) leads to formation of both 2,3-dihydroxybenzoate (2,3-DHB) and 2,5-dihydroxybenzoate (gentisate, 2,5-DHB). It has been suggested that formation of 2,3-DHB from salicylate is a means of monitoring .OH formation. Production of 2,3-DHB and 2,5-DHB by liver microsomal fractions and isoforms of cytochrome P-450 was investigated. Liver microsomes prepared from variously treated rats and rabbits catalysed the formation of 2,5-DHB but not 2,3-DHB. Formation of 2,5-DHB was inhibited by CO, metyrapone and SKF-525A, but not by the .OH scavengers mannitol and formate or by the iron chelator desferrioxamine. Purified P-450s IIE1, IIB4 or IA2 from rabbit liver microsomes, reconstituted together with NADPH-cytochrome P-450 reductase, led to formation of equal amounts of 2,3-DHB and 2,5-DHB in reactions that were almost completely inhibited by mannitol or formate. Addition of Fe3+/EDTA either to microsomes or to membranes containing reconstituted P-450 caused formation of approximately equal amounts of 2,3-DHB and 2,5-DHB, consistent with an .OH-dependent attack on salicylate. The data indicate that the microsomal P-450 system catalyses hydroxylation of salicylate to 2,5-DHB, but not formation of 2,3-DHB. Hence measurement of 2,3-DHB might provide a means of monitoring .OH formation. Care must be taken in studies of substrate hydroxylation by microsomes or reconstituted P-450 systems to avoid artefacts resulting from .OH generation. PMID:2064611

  3. Inactivation of Cytochrome P450 (P450) 3A4 but not P450 3A5 by OSI-930, a Thiophene-Containing Anticancer DrugS⃞

    PubMed Central

    Lin, Hsia-lien; Zhang, Haoming; Medower, Christine; Johnson, William W.

    2011-01-01

    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 b5 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 b5. 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 KI of 24 μM and a kinact of 0.04 min−1. This KI is significantly greater than the clinical OSI-930 Cmax 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 for oxidation

  4. Cytochromes P450 Catalyze the Reduction of α,β-Unsaturated Aldehydes

    PubMed Central

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

    2011-01-01

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

  5. EXPRESSION AND CHARACTERIZATION OF FULL-LENGTH HUMAN HEME OXYGENASE-1: PRESENCE OF INTACT MEMBRANE-BINDING REGION LEADS TO INCREASED BINDING AFFINITY FOR NADPH-CYTOCHROME P450 REDUCTASE

    PubMed Central

    Huber, Warren J.; Backes, Wayne L.

    2009-01-01

    Heme oxygenase (HO) is the chief regulatory enzyme in the oxidative degradation of heme to biliverdin. In the process of heme degradation, this NADPH and cytochrome P450 reductase (CPR)-dependent oxidation of heme also releases free iron and carbon monoxide. Much of the recent research involving heme oxygenase is done using a 30-kDa soluble form of the enzyme, which lacks the membrane binding region (C-terminal 23 amino acids). The goal of this study was to express and purify a full-length human HO-1 (hHO-1) protein; however, due to the lability of the full-length form, a rapid purification procedure was required. This was accomplished by use of a GST-tagged hHO-1 construct. Although the procedure permitted the generation of a full-length HO-1, this form was contaminated with a 30-kDa degradation product that could not be eliminated. Therefore, we attempted to remove a putative secondary thrombin cleavage site by a conservative mutation of amino acid 254, which replaces lysine with arginine. This mutation allowed the expression and purification of a full length hHO-1 protein. Unlike wild-type HO-1, the K254R mutant could be purified to a single 32-kDa protein capable of degrading heme at the same rate as the wild-type enzyme. The K254R full-length form had a specific activity of ~200–225 nmol bilirubin hr−1nmol−1 HO-1 as compared to ~140–150 nmol bilirubin hr−1nmol−1 for the WT form, which contains the 30-kDa contaminant. This is a 2–3-fold increase from the previously reported soluble 30-kDa HO-1, suggesting that the C-terminal 23 amino acids are essential for maximal catalytic activity. Because the membrane spanning domain is present, the full-length hHO-1 has the potential to incorporate into phospholipid membranes, which can be reconstituted at known concentrations, in combination with other ER-resident enzymes. PMID:17915953

  6. Single-molecule height measurements on microsomal cytochrome P450 in nanometer-scale phospholipid bilayer disks

    NASA Astrophysics Data System (ADS)

    Bayburt, Timothy H.; Sligar, Stephen G.

    2002-05-01

    The architecture of membrane proteins in their native environment of the phospholipid bilayer is critical for understanding physiological function, but has been difficult to realize experimentally. In this communication we describe the incorporation of a membrane-anchored protein into a supported phospholipid bilayer. Cytochrome P450 2B4 solubilized and purified from the hepatic endoplasmic reticulum was incorporated into phospholipid bilayer nanostructures and oriented on a surface for visualization by atomic force microscopy. Individual P450 molecules were observed protruding from the bilayer surface. Problems associated with deformation of the protein by the atomic force microscopy probe were avoided by analyzing force-dependent height measurements to quantitate the height of the protein above the bilayer surface. Measurements of the atomic force microscopy cantilever deflection as a function of probe-sample separation reveal that the top of the P450 opposite the N-terminal membrane anchor region sits 3.5 nanometers above the phospholipid-water boundary. Models of the orientation of the enzyme are presented and discussed in relation to membrane interactions and interaction with cytochrome P450 reductase.

  7. Nitrate transport is independent of NADH and NAD(P)H nitrate reductases in barley seedlings

    NASA Technical Reports Server (NTRS)

    Warner, R. L.; Huffaker, R. C.

    1989-01-01

    Barley (Hordeum vulgare L.) has NADH-specific and NAD(P)H-bispecific nitrate reductase isozymes. Four isogenic lines with different nitrate reductase isozyme combinations were used to determine the role of NADH and NAD(P)H nitrate reductases on nitrate transport and assimilation in barley seedlings. Both nitrate reductase isozymes were induced by nitrate and were required for maximum nitrate assimilation in barley seedlings. Genotypes lacking the NADH isozyme (Az12) or the NAD(P)H isozyme (Az70) assimilated 65 or 85%, respectively, as much nitrate as the wild type. Nitrate assimilation by genotype (Az12;Az70) which is deficient in both nitrate reductases, was only 13% of the wild type indicating that the NADH and NAD(P)H nitrate reductase isozymes are responsible for most of the nitrate reduction in barley seedlings. For all genotypes, nitrate assimilation rates in the dark were about 55% of the rates in light. Hypotheses that nitrate reductase has direct or indirect roles in nitrate uptake were not supported by this study. Induction of nitrate transporters and the kinetics of net nitrate uptake were the same for all four genotypes indicating that neither nitrate reductase isozyme has a direct role in nitrate uptake in barley seedlings.

  8. Contrasting Influence of NADPH and a NADPH-Regenerating System on the Metabolism of Carbonyl-Containing Compounds in Hepatic Microsomes

    EPA Science Inventory

    Carbonyl containing xenobiotics may be susceptible to NADPH-dependent cytochrome P450 (P450) and carbonyl-reduction reactions. In vitro hepatic microsome assays are routinely supplied NADPH either by direct addition of NADPH or via an NADPH-regenerating system (NRS). In contrast ...

  9. Mobility of cytochrome P450 in the endoplasmic reticulum membrane.

    PubMed

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

    1998-12-08

    Cytochrome P450 2C2 is a resident endoplasmic reticulum (ER) membrane protein that is excluded from the recycling pathway and contains redundant retention functions in its N-terminal transmembrane signal/anchor sequence and its large, cytoplasmic domain. Unlike some ER resident proteins, cytochrome P450 2C2 does not contain any known retention/retrieval signals. One hypothesis to explain exclusion of resident ER proteins from the transport pathway is the formation of networks by interaction with other proteins that immobilize the proteins and are incompatible with packaging into the transport vesicles. To determine the mobility of cytochrome P450 in the ER membrane, chimeric proteins of either cytochrome P450 2C2, its catalytic domain, or the cytochrome P450 2C1 N-terminal signal/anchor sequence fused to green fluorescent protein (GFP) were expressed in transiently transfected COS1 cells. The laurate hydroxylase activities of cytochrome P450 2C2 or the catalytic domain with GFP fused to the C terminus were similar to the native enzyme. The mobilities of the proteins in the membrane were determined by recovery of fluorescence after photobleaching. Diffusion coefficients for all P450 chimeras were similar, ranging from 2.6 to 6.2 x 10(-10) cm2/s. A coefficient only slightly larger (7.1 x 10(-10) cm2/s) was determined for a GFP chimera that contained a C-terminal dilysine ER retention signal and entered the recycling pathway. These data indicate that exclusion of cytochrome P450 from the recycling pathway is not mediated by immobilization in large protein complexes.

  10. Nitrous oxide-forming codenitrification catalyzed by cytochrome P450nor.

    PubMed

    Su, Fei; Takaya, Naoki; Shoun, Hirofumi

    2004-02-01

    Intact cells of the denitrifying fungus Fusarium oxysporum were previously shown to catalyze codenitrification to form a hybrid nitrous oxide (N2O) species from nitrite and other nitrogen compounds such as azide and ammonia. Here we show that cytochrome P450nor can catalyze the codenitrification reaction to form N2O from nitric oxide (NO) but not nitrite, and azide or ammonia. The results show that the direct substrate of the codenitrification by intact cells should not be nitrite but NO, which is formed from nitrite by the reaction of a dissimilatory nitrite reductase.

  11. Albendazole sulfonation by rat liver cytochrome P-450c.

    PubMed

    Souhaili-El Amri, H; Mothe, O; Totis, M; Masson, C; Batt, A M; Delatour, P; Siest, G

    1988-08-01

    The metabolism of albendazole (ABZ) was studied in perfused livers from control and ABZ-treated rats (10.6 mg/kg, per os, each day for 10 days). In the perfusion fluid, the concentration of ABZ-sulfoxide (SO-ABZ) remained unchanged in treated, as compared to control animals, whereas ABZ-sulfone (SO2-ABZ) was increased in treated animals. In bile, only SO-ABZ was present. The transformation kinetics of SO-ABZ to SO2-ABZ in microsomes from rats treated with ABZ, 3-methylcholanthrene, Aroclor and isosafrole were biphasic. This suggests that enzyme activity was a consequence of two enzyme systems, one characterized by low affinity and high capacity, the other by high affinity and low capacity, the latter could be induced by 3-methylcholanthrene, ABZ, Aroclor and isosafrole. Cytochrome P-450c was induced potently in vivo by ABZ as proven by increased monooxygenase (7-ethoxyresorufin and 7-ethoxycoumarin-O-deethylase) activities and by Elisa test (a 5-fold increase in hemoprotein concentration was observed). Purified and reconstituted cytochrome P-450c from 3-methylcholanthrene or ABZ-treated rat liver were able to produce SO2-ABZ (2.01 and 1.70 nmol/mg/15 min, respectively, whereas cytochrome P-450b produced 10 times less SO2-ABZ). Immunological assays, as well as activity measurements showed a relationship between cytochrome P-450c-3-methylcholanthrene and cytochrome P-450c-ABZ. We conclude that induction of cytochrome P-450c by ABZ is the probable explanation for the enhanced formation of SO2-ABZ in vivo.

  12. DOE Office of Scientific and Technical Information (OSTI.GOV)

    Gray, Joshua P.; Department of Pharmacology and Toxicology, Rutgers University, Piscataway, NJ; Mishin, Vladimir

    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 cytochromemore » 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.« less

  13. 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

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

    ClinicalTrials.gov

    2017-08-15

    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

  15. Reduction in hepatic drug metabolizing CYP3A4 activities caused by P450 oxidoreductase mutations identified in patients with disordered steroid metabolism

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Flueck, Christa E.; Mullis, Primus E.; Pandey, Amit V., E-mail: amit@pandeylab.org

    2010-10-08

    Research highlights: {yields} Cytochrome P450 3A4 (CYP3A4), metabolizes 50% of drugs in clinical use and requires NADPH-P450 reductase (POR). {yields} Mutations in human POR cause congenital adrenal hyperplasia from diminished activities of steroid metabolizing P450s. {yields} We are reporting that mutations in POR may reduce CYP3A4 activity. {yields} POR mutants Y181D, A457H, Y459H, V492E and R616X lost 99%, while A287P, C569Y and V608F lost 60-85% CYP3A4 activity. {yields} Reduction of CYP3A4 activity may cause increased risk of drug toxicities/adverse drug reactions in patients with POR mutations. -- Abstract: Cytochrome P450 3A4 (CYP3A4), the major P450 present in human liver metabolizesmore » approximately half the drugs in clinical use and requires electrons supplied from NADPH through NADPH-P450 reductase (POR, CPR). Mutations in human POR cause a rare form of congenital adrenal hyperplasia from diminished activities of steroid metabolizing P450s. In this study we examined the effect of mutations in POR on CYP3A4 activity. We used purified preparations of wild type and mutant human POR and in vitro reconstitution with purified CYP3A4 to perform kinetic studies. We are reporting that mutations in POR identified in patients with disordered steroidogenesis/Antley-Bixler syndrome (ABS) may reduce CYP3A4 activity, potentially affecting drug metabolism in individuals carrying mutant POR alleles. POR mutants Y181D, A457H, Y459H, V492E and R616X had more than 99% loss of CYP3A4 activity, while POR mutations A287P, C569Y and V608F lost 60-85% activity. Loss of CYP3A4 activity may result in increased risk of drug toxicities and adverse drug reactions in patients with POR mutations.« less

  16. 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

  17. Nitrate Transport Is Independent of NADH and NAD(P)H Nitrate Reductases in Barley Seedlings 1

    PubMed Central

    Warner, Robert L.; Huffaker, Ray C.

    1989-01-01

    Barley (Hordeum vulgare L.) has NADH-specific and NAD(P)H-bispecific nitrate reductase isozymes. Four isogenic lines with different nitrate reductase isozyme combinations were used to determine the role of NADH and NAD(P)H nitrate reductases on nitrate transport and assimilation in barley seedlings. Both nitrate reductase isozymes were induced by nitrate and were required for maximum nitrate assimilation in barley seedlings. Genotypes lacking the NADH isozyme (Az12) or the NAD(P)H isozyme (Az70) assimilated 65 or 85%, respectively, as much nitrate as the wild type. Nitrate assimilation by genotype (Az12;Az70) which is deficient in both nitrate reductases, was only 13% of the wild type indicating that the NADH and NAD(P)H nitrate reductase isozymes are responsible for most of the nitrate reduction in barley seedlings. For all genotypes, nitrate assimilation rates in the dark were about 55% of the rates in light. Hypotheses that nitrate reductase has direct or indirect roles in nitrate uptake were not supported by this study. Induction of nitrate transporters and the kinetics of net nitrate uptake were the same for all four genotypes indicating that neither nitrate reductase isozyme has a direct role in nitrate uptake in barley seedlings. PMID:11537465

  18. The effects of endosulfan on cytochrome P450 enzymes and glutathione S-transferases in zebrafish (Danio rerio) livers.

    PubMed

    Dong, Miao; Zhu, Lusheng; Shao, Bo; Zhu, Shaoyuan; Wang, Jun; Xie, Hui; Wang, Jinhua; Wang, Fenghua

    2013-06-01

    Endosulfan, an organochlorine pesticide, has been used worldwide in the past decades. The present study was performed to investigate the effect of endosulfan on liver microsomal cytochrome P450 (CYP) enzymes and glutathione S-transferases (GST) in zebrafish. Male and female zebrafish were separated and exposed to a control and four concentrations of endosulfan (0.01, 0.1, 1, and 10μgL(-1)) and were sampled on days 7, 14, 21, and 28. After exposure to endosulfan, the content of CYP increased and later gradually fell back to control level in most sampling time intervals. A similar tendency was also found in the activities of NADPH-P450 reductase (NCR), aminopyrine N-demethylase (APND) and erythromycin N-demethylase (ERND). GST activities were generally higher in treatment groups than control groups. Regarding sex-based differences, the induction degree of the activity of NCR was generally higher in males than females. Similar differences were also found on the 28th day in the activities of APND and ERND, as well as GST activity on the 7th day. Overall, the present results demonstrate the toxicity at low doses of endosulfan and indicated marked induction of CYP and GST enzymes in zebrafish liver. Copyright © 2012 Elsevier Inc. All rights reserved.

  19. Optimization of the Bacterial Cytochrome P450 BM3 System for the Production of Human Drug Metabolites

    PubMed Central

    Di Nardo, Giovanna; Gilardi, Gianfranco

    2012-01-01

    Drug metabolism in human liver is a process involving many different enzymes. Among them, a number of cytochromes P450 isoforms catalyze the oxidation of most of the drugs commercially available. Each P450 isoform acts on more than one drug, and one drug may be oxidized by more than one enzyme. As a result, multiple products may be obtained from the same drug, and as the metabolites can be biologically active and may cause adverse drug reactions (ADRs), the metabolic profile of a new drug has to be known before this can be commercialized. Therefore, the metabolites of a certain drug must be identified, synthesized and tested for toxicity. Their synthesis must be in sufficient quantities to be used for metabolic tests. This review focuses on the progresses done in the field of the optimization of a bacterial self-sufficient and efficient cytochrome P450, P450 BM3 from Bacillus megaterium, used for the production of metabolites of human enzymes. The progress made in the improvement of its catalytic performance towards drugs, the substitution of the costly NADPH cofactor and its immobilization and scale-up of the process for industrial application are reported. PMID:23443101

  20. Electrochemistry of cytochrome P450 17α-hydroxylase/17,20-lyase (P450c17).

    PubMed

    Martin, Lisandra L; Kubeil, Clemens; Simonov, Alexandr N; Kuznetsov, Vladimir L; Corbin, C Jo; Auchus, Richard J; Conley, Alan J; Bond, Alan M; Rodgers, Raymond J

    2017-02-05

    Within the superfamily of cytochrome P450 enzymes (P450s), there is a small class which is functionally employed for steroid biosynthesis. The enzymes in this class appear to have a small active site to accommodate the steroid substrates specifically and snuggly, prior to the redox transformation or hydroxylation to form a product. Cytochrome P450c17 is one of these and is also a multi-functional P450, with two activities, the first 17α-hydroxylation of pregnenolone is followed by a subsequent 17,20-lyase transformation to dehydroepiandrosterone (DHEA) as the dominant pathways to cortisol precursors or androgens in humans, respectively. How P450c17 regulates these two redox reactions is of special interest. There is a paucity of direct electrochemical studies on steroidogenic P450s, and in this mini-review we provide an overview of these studies with P450c17. Historical consideration as to the difficulties in obtaining reliable electrochemistry due to issues of handling proteins on an electrode, together with advances in the electrochemical techniques are addressed. Recent work using Fourier transformed alternating current voltammetry is highlighted as this technique can provide both catalytic information simultaneously with the underlying redox transfer with the P450 haem. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  1. Improved NADPH Regeneration for Fungal Cytochrome P450 Monooxygenase by Co-Expressing Bacterial Glucose Dehydrogenase in Resting-Cell Biotransformation of Recombinant Yeast.

    PubMed

    Jeon, Hyunwoo; Durairaj, Pradeepraj; Lee, Dowoo; Ahsan, Md Murshidul; Yun, Hyungdon

    2016-12-28

    Fungal cytochrome P450 (CYP) enzymes catalyze versatile monooxygenase reactions and play a major role in fungal adaptations owing to their essential roles in the production avoid metabolites critical for pathogenesis, detoxification of xenobiotics, and exploitation avoid substrates. Although fungal CYP-dependent biotransformation for the selective oxidation avoid organic compounds in yeast system is advantageous, it often suffers from a shortage avoid intracellular NADPH. In this study, we aimed to investigate the use of bacterial glucose dehydrogenase (GDH) for the intracellular electron regeneration of fungal CYP monooxygenase in a yeast reconstituted system. The benzoate hydroxylase FoCYP53A19 and its homologous redox partner FoCPR from Fusarium oxysporum were co-expressed with the BsGDH from Bacillus subtilis in Saccharomyces cerevisiae for heterologous expression and biotransformations. We attempted to optimize several bottlenecks concerning the efficiency of fungal CYP-mediated whole-cell-biotransformation to enhance the conversion. The catalytic performance of the intracellular NADPH regeneration system facilitated the hydroxylation of benzoic acid to 4-hydroxybenzoic acid with high conversion in the resting-cell reaction. The FoCYP53A19 +FoCPR+BsGDH reconstituted system produced 0.47 mM 4-hydroxybenzoic acid (94% conversion) in the resting-cell biotransformations performed in 50 mM phosphate buffer (pH 6.0) containing 0.5 mM benzoic acid and 0.25% glucose for 24 h at 30°C. The "coupled-enzyme" system can certainly improve the overall performance of NADPH-dependent whole-cell biotransformations in a yeast system.

  2. Stable expression of rat cytochrome P-450IIB1 cDNA in Chinese hamster cells (V79) and metabolic activation of aflatoxin B1.

    PubMed Central

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

    1988-01-01

    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 phosphoribosyltransferase (HPRT) locus in SD1 cells is markedly increased when exposed to aflatoxin B1, which is activated by this enzyme. Images PMID:3137560

  3. Identification and heterologous expression of the cytochrome P450 oxidoreductase from the white-rot basidiomycete Coriolus versicolor.

    PubMed

    Ichinose, H; Wariishi, H; Tanaka, H

    2002-09-01

    A cDNA encoding cytochrome P450 oxidoreductase (CPR) from the lignin-degrading basidiomycete Coriolus versicolor was identified using RT-PCR. The full-length cDNA consisted of 2,484 nucleotides with a poly(A) tail, and contained an open reading frame. The G+C content of the cDNA isolated was 60%. A deduced protein contained 730 amino acid residues with a calculated molecular weight of 80.7 kDa. The conserved amino acid residues involved in functional domains such as FAD-, FMN-, and NADPH-binding domains, were all found in the deduced protein. A phylogenetic analysis demonstrated that C. versicolor CPR is significantly similar to CPR of the basidiomycete Phanerochaete chrysosporium and that they share the same major branch in the fungal cluster. A recombinant CPR protein was expressed using a pET/ Escherichia coli system. The recombinant CPR protein migrated at 81 kDa on SDS polyacrylamide gel electrophoresis. It exhibited an NADPH-dependent cytochrome c reducing activity.

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

    PubMed Central

    2015-01-01

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

  5. The role of renal proximal tubule P450 enzymes in chloroform-induced nephrotoxicity: Utility of renal specific P450 reductase knockout mouse models

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Liu, Senyan; Wadsworth Center, New York State Department of Health, and School of Public Health, State University of New York, Albany, NY 12201; Yao, Yunyi

    The kidney is a primary target for numerous toxic compounds. Cytochrome P450 enzymes (P450) are responsible for the metabolic activation of various chemical compounds, and in the kidney are predominantly expressed in proximal tubules. The aim of this study was to test the hypothesis that renal proximal tubular P450s are critical for nephrotoxicity caused by chemicals such as chloroform. We developed two new mouse models, one having proximal tubule-specific deletion of the cytochrome P450 reductase (Cpr) gene (the enzyme required for all microsomal P450 activities), designated proximal tubule-Cpr-null (PTCN), and the other having proximal tubule-specific rescue of CPR activity withmore » the global suppression of CPR activity in all extra-proximal tubular tissues, designated extra-proximal tubule-Cpr-low (XPT-CL). The PTCN, XPT-CL, Cpr-low (CL), and wild-type (WT) mice were treated with a single oral dose of chloroform at 200 mg/kg. Blood, liver and kidney samples were obtained at 24 h after the treatment. Renal toxicity was assessed by measuring BUN and creatinine levels, and by pathological examination. The blood and tissue levels of chloroform were determined. The severity of toxicity was less in PTCN and CL mice, compared with that of WT and XPT-CL mice. There were no significant differences in chloroform levels in the blood, liver, or kidney, between PTCN and WT mice, or between XPT-CL and CL mice. These findings indicate that local P450-dependent activities play an important role in the nephrotoxicity induced by chloroform. Our results also demonstrate the usefulness of these novel mouse models for studies of chemical-induced kidney toxicity. - Highlights: • New mouse models were developed with varying P450 activities in the proximal tubule. • These mouse models were treated with chloroform, a nephrotoxicant. • Studies showed the importance of local P450s in chloroform-induced nephrotoxicity.« less

  6. Expression and characterization of full-length human heme oxygenase-1: the presence of intact membrane-binding region leads to increased binding affinity for NADPH cytochrome P450 reductase.

    PubMed

    Huber, Warren J; Backes, Wayne L

    2007-10-30

    Heme oxygenase-1 (HO-1) is the chief regulatory enzyme in the oxidative degradation of heme to biliverdin. In the process of heme degradation, HO-1 receives the electrons necessary for catalysis from the flavoprotein NADPH cytochrome P450 reductase (CPR), releasing free iron and carbon monoxide. Much of the recent research involving heme oxygenase has been done using a 30 kDa soluble form of the enzyme, which lacks the membrane binding region (C-terminal 23 amino acids). The goal of this study was to express and purify a full-length human HO-1 (hHO-1) protein; however, due to the lability of the full-length form, a rapid purification procedure was required. This was accomplished by use of a glutathione-s-transferase (GST)-tagged hHO-1 construct. Although the procedure permitted the generation of a full-length HO-1, this form was contaminated with a 30 kDa degradation product that could not be eliminated. Therefore, attempts were made to remove a putative secondary thrombin cleavage site by a conservative mutation of amino acid 254, which replaces arginine with lysine. This mutation allowed the expression and purification of a full-length hHO-1 protein. Unlike wild type (WT) HO-1, the R254K mutant could be purified to a single 32 kDa protein capable of degrading heme at the same rate as the WT enzyme. The R254K full-length form had a specific activity of approximately 200-225 nmol of bilirubin h-1 nmol-1 HO-1 as compared to approximately 140-150 nmol of bilirubin h-1 nmol-1 for the WT form, which contains the 30 kDa contaminant. This is a 2-3-fold increase from the previously reported soluble 30 kDa HO-1, suggesting that the C-terminal 23 amino acids are essential for maximal catalytic activity. Because the membrane-spanning domain is present, the full-length hHO-1 has the potential to incorporate into phospholipid membranes, which can be reconstituted at known concentrations, in combination with other endoplasmic reticulum resident enzymes.

  7. Cytochrome P450 humanised mice

    PubMed Central

    2004-01-01

    Humans are exposed to countless foreign compounds, typically referred to as xenobiotics. These can include clinically used drugs, environmental pollutants, food additives, pesticides, herbicides and even natural plant compounds. Xenobiotics are metabolised primarily in the liver, but also in the gut and other organs, to derivatives that are more easily eliminated from the body. In some cases, however, a compound is converted to an electrophile that can cause cell toxicity and transformation leading to cancer. Among the most important xenobiotic-metabolising enzymes are the cytochromes P450 (P450s). These enzymes represent a superfamily of multiple forms that exhibit marked species differences in their expression and catalytic activities. To predict how humans will metabolise xenobiotics, including drugs, human liver extracts and recombinant P450s have been used. New humanised mouse models are being developed which will be of great value in the study of drug metabolism, pharmacokinetics and pharmacodynamics in vivo, and in carrying out human risk assessment of xenobiotics. Humanised mice expressing CYP2D6 and CYP3A4, two major drug-metabolising P450s, have revealed the feasibility of this approach. PMID:15588489

  8. Identification of human cytochrome P450s as autoantigens.

    PubMed

    Manns, M P; Johnson, E F

    1991-01-01

    Antimicrosomal antibodies in inflammatory liver diseases all seem to be directed against members of the cytochrome P450 family of proteins. These autoantigens seem to be genetically polymorphic, the autoantibodies are inhibitory, and the autoepitopes are generally conserved among species. Anti-P450 autoantibodies share these characteristics with other autoantibodies, for example, antinuclear antibodies in systemic lupus erythematosus. The identification of P450s as human autoantigens is clinically important. Diagnostic tests will be developed on the basis of cloned antigen, facilitating a better diagnosis of drug-induced and idiopathic autoimmune hepatitis. It is unknown what triggers autoantibody production against cytochrome P450 proteins. Furthermore, their pathogenetic role and thus their involvement in tissue destruction is unclear. In this context LKM1 autoantibodies may serve as a model. Although LKM1 antibodies are inhibitory, all LKM1 antibody-positive patients tested so far are extensive metabolizers for drug metabolism mediated by P450IID6 and express this protein in their livers. Thus, the inhibitory LKM1 autoantibody does not sufficiently penetrate through the intact liver cell membrane to inhibit enzyme function in vivo. Presumably, tissue destruction in autoimmune hepatitis is mediated by liver-infiltrating T lymphocytes. T lymphocytes have been cloned from liver tissue that specifically proliferate in the presence of recombinant cytochrome P450IID6. The construction of overlapping cDNA subclones is also valuable to identify immunodominant B cell as well as relevant T cell epitopes.

  9. Cytochrome P450 systems--biological variations of electron transport chains.

    PubMed

    Hannemann, Frank; Bichet, Andreas; Ewen, Kerstin M; Bernhardt, Rita

    2007-03-01

    Cytochromes P450 (P450) are hemoproteins encoded by a superfamily of genes nearly ubiquitously distributed in different organisms from all biological kingdoms. The reactions carried out by P450s are extremely diverse and contribute to the biotransformation of drugs, the bioconversion of xenobiotics, the bioactivation of chemical carcinogens, the biosynthesis of physiologically important compounds such as steroids, fatty acids, eicosanoids, fat-soluble vitamins and bile acids, the conversion of alkanes, terpenes and aromatic compounds as well as the degradation of herbicides and insecticides. Cytochromes P450 belong to the group of external monooxygenases and thus receive the necessary electrons for oxygen cleavage and substrate hydroxylation from different redox partners. The classical as well as the recently discovered P450 redox systems are compiled in this paper and classified according to their composition.

  10. NADPH–Cytochrome P450 Oxidoreductase: Roles in Physiology, Pharmacology, and Toxicology

    PubMed Central

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

    2013-01-01

    This is a report on a symposium sponsored by the American Society for Pharmacology and Experimental Therapeutics and held at the Experimental Biology 2012 meeting in San Diego, California, on April 25, 2012. The symposium speakers summarized and critically evaluated our current understanding of the physiologic, pharmacological, and toxicological roles of NADPH–cytochrome P450 oxidoreductase (POR), a flavoprotein involved in electron transfer to microsomal cytochromes P450 (P450), cytochrome b5, squalene mono-oxygenase, and heme oxygenase. Considerable insight has been derived from the development and characterization of mouse models with conditional Por deletion in particular tissues or partial suppression of POR expression in all tissues. Additional mouse models with global or conditional hepatic deletion of cytochrome b5 are helping to clarify the P450 isoform- and substrate-specific influences of cytochrome b5 on P450 electron transfer and catalytic function. This symposium also considered studies using siRNA to suppress POR expression in a hepatoma cell–culture model to explore the basis of the hepatic lipidosis phenotype observed in mice with conditional deletion of Por in liver. The symposium concluded with a strong translational perspective, relating the basic science of human POR structure and function to the impacts of POR genetic variation on human drug and steroid metabolism. PMID:23086197

  11. Effect of calcium(ion) uptake by rat adrenal mitochondria on pregnenolone formation and spectral properties of cytochrome P-450.

    PubMed

    Simpson, E R; Williams-Smith, D L

    1975-10-09

    The effect of calcium on pregnenolone formation from endogenous precursors has been studied in mitochondria from rat decapsulated and capsular adrenal glands. In the presence of succinate, addition of calcium chloride in the concentration range 20-150 muM caused an inhibition of pregnenolone formation in both decapsulated and capsular adrenal mitochondria. 11beta-hydroxylation of added deoxycosticosterone in decapsulated adrenal mitochondria was also inhibited. Under these conditions, calcium inhibited the reduction of adrenodoxin, a component of the cytochrome P-450 reductase system, presumably because uptake of calcium by the mitochondria competes with energy-linked transhydrogenase for high-energy intermediates. For this reason, incubations were carried out in the presence of succinate plus isocitrate plus NADP+. Under these conditions, calcium chloride in the concentration range 120-875 muM caused a 2-4-fold stimulation of pregnenolone formation, but had no effect on corticosterone formation from added deoxycorticosterone. The effect of calcium on the optical spectra of cytochrome P-450 has also been examined in mitochondria from decapsulated and capsular rat adrenals. In the presence of succinate, calcium induced a spectral change resembling a type I difference spectrum of cytochrome P-450. Thus it appears that uptake of calcium by adrenal mitochondria can stimulate pregnenolone formation by increasing the interaction of mitochondrial cytochrome P-450 with endogenous substrate.

  12. Generation of a mouse model with a reversible hypomorphic cytochrome P450 reductase gene: utility for tissue-specific rescue of the reductase expression, and insights from a resultant mouse model with global suppression of P450 reductase expression in extrahepatic tissues.

    PubMed

    Wei, Yuan; Zhou, Xin; Fang, Cheng; Li, Lei; Kluetzman, Kerri; Yang, Weizhu; Zhang, Qing-Yu; Ding, Xinxin

    2010-07-01

    A mouse model termed Cpr-low (CL) was recently generated, in which the expression of the cytochrome P450 reductase (Cpr) gene was globally down-regulated. The decreased CPR expression was accompanied by phenotypical changes, including reduced embryonic survival, decreases in circulating cholesterol, increases in hepatic P450 expression, and female infertility (accompanied by elevated serum testosterone and progesterone levels). In the present study, a complementary mouse model [named reversible-CL (r-CL)] was generated, in which the reduced CPR expression can be reversed in an organ-specific fashion. The neo cassette, which was inserted into the last Cpr intron in r-CL mice, can be deleted by Cre recombinase, thus returning the structure of the Cpr gene (and hence CPR expression) to normal in Cre-expressing cells. All previously identified phenotypes of the CL mice were preserved in the r-CL mice. As a first application of the r-CL model, we have generated an extrahepatic-CL (xh-CL) mouse for testing of the functions of CPR-dependent enzymes in all extrahepatic tissues. The xh-CL mice, generated by mating of r-CL mice with albumin-Cre mice, had normal CPR expression in hepatocytes but down-regulated CPR expression elsewhere. They were indistinguishable from wild-type mice in body and liver weights, circulating cholesterol levels, and hepatic microsomal P450 expression and activities; however, they still showed elevated serum testosterone and progesterone levels and sterility in females. Embryonic lethality was prevented in males, but apparently not in females, indicating a critical role for fetal hepatic CPR-dependent enzymes in embryonic development, at least in males.

  13. Enhanced hepatic and kidney cytochrome p-450 activities in nandrolone decanoate treated albino mice.

    PubMed

    Acharjee, B K; Mahanta, R

    2009-04-01

    Anabolic androgenic steroids are the xenobiotic substrates that are metabolized in the body by the protective enzyme systems. Mixed function oxygenase enzymes include a group of enzymes which play an essential role in the metabolism of a broad range of xenobiotics including endogenous and exogenous substrates. Cytochrome P-450, a member of mixed function oxygenase enzymes, plays an important role in oxidative metabolism of drugs and xenobiotics entering human body. Various anabolic steroids are found either to increase or decrease the activity of cytochrome P-450. However, effect of nandrolone decanoate, most commonly abused anabolic steroid, on cytochrome P-450 activity is still fragmentary. In the present study, albino mice were administered intramuscular 2.5 mg of nandrolone decanoate injection at 15 days interval. Cytochrome P-450 activity is determined by following the method of Omura and Sato (1964) in liver and kidney tissues of both normal and experimental groups upto 90 days. Investigation shows a significant (p <0.01) increase of cytochrome P-450 (nmol/mg) activity in liver tissue as compared to that of kidney tissues. A tissue specific and dose specific increase of cytochrome P-450 activity is observed. Mean cytochrome P-450 is found highest in liver tissue on 45(th) day whereas the activity in kidney tissue is noticed on 90(th) day of treatment. From the above observation, nandrolone decanoate can be suggested as a potent inducer of cytochrome P-450 activity like other anabolic steroids.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Flueck, Christa E., E-mail: christa.flueck@dkf.unibe.ch; Mallet, Delphine; Hofer, Gaby

    2011-09-09

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

  15. Cytochrome P450 diversity in the tree of life.

    PubMed

    Nelson, David R

    2018-01-01

    Sequencing in all areas of the tree of life has produced >300,000 cytochrome P450 (CYP) sequences that have been mined and collected. Nomenclature has been assigned to >41,000 CYP sequences and the majority of the remainder has been sorted by BLAST searches into clans, families and subfamilies in preparation for naming. The P450 sequence space is being systematically explored and filled in. Well-studied groups like vertebrates are covered in greater depth while new insights are being added into uncharted territories like horseshoe crab (Limulus polyphemus), tardigrades (Hypsibius dujardini), velvet worm (Euperipatoides_rowelli), and basal land plants like hornworts, liverworts and mosses. CYPs from the fungi, one of the most diverse groups, are being explored and organized as nearly 800 fungal species are now sequenced. The CYP clan structure in fungi is emerging with 805 CYP families sorting into 32 CYP clans. >3000 bacterial sequences are named, mostly from terrestrial or freshwater sources. Of 18,379 bacterial sequences downloaded from the CYPED database, all are >43% identical to named CYPs. Therefore, they fit in the 602 named P450 prokaryotic families. Diversity in this group is becoming saturated, however 25% of 3305 seawater bacterial P450s did not match known P450 families, indicating marine bacterial CYPs are not as well sampled as land/freshwater based bacterial CYPs. Future sequencing plans of the Genome 10K project, i5k and GIGA (Global Invertebrate Genomics Alliance) are expected to produce more than one million cytochrome P450 sequences by 2020. This article is part of a Special Issue entitled: Cytochrome P450 biodiversity and biotechnology, edited by Erika Plettner, Gianfranco Gilardi, Luet Wong, Vlada Urlacher, Jared Goldstone. Copyright © 2017 Elsevier B.V. All rights reserved.

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

    PubMed Central

    2010-01-01

    Background Cytochrome P450 monooxygenases (P450s) catalyze oxidation of various substrates using oxygen and NAD(P)H. Plant P450s are involved in the biosynthesis of primary and secondary metabolites performing diverse biological functions. The recent availability of the soybean genome sequence allows us to identify and analyze soybean putative P450s at a genome scale. Co-expression analysis using an available soybean microarray and Illumina sequencing data provides clues for functional annotation of these enzymes. This approach is based on the assumption that genes that have similar expression patterns across a set of conditions may have a functional relationship. Results We have identified a total number of 332 full-length P450 genes and 378 pseudogenes from the soybean genome. From the full-length sequences, 195 genes belong to A-type, which could be further divided into 20 families. The remaining 137 genes belong to non-A type P450s and are classified into 28 families. A total of 178 probe sets were found to correspond to P450 genes on the Affymetrix soybean array. Out of these probe sets, 108 represented single genes. Using the 28 publicly available microarray libraries that contain organ-specific information, some tissue-specific P450s were identified. Similarly, stress responsive soybean P450s were retrieved from 99 microarray soybean libraries. We also utilized Illumina transcriptome sequencing technology to analyze the expressions of all 332 soybean P450 genes. This dataset contains total RNAs isolated from nodules, roots, root tips, leaves, flowers, green pods, apical meristem, mock-inoculated and Bradyrhizobium japonicum-infected root hair cells. The tissue-specific expression patterns of these P450 genes were analyzed and the expression of a representative set of genes were confirmed by qRT-PCR. We performed the co-expression analysis on many of the 108 P450 genes on the Affymetrix arrays. First we confirmed that CYP93C5 (an isoflavone synthase gene) is

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

    PubMed Central

    2012-01-01

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

  18. Suppression of Cytochrome P450 Reductase (POR) Expression in Hepatoma Cells Replicates the Hepatic Lipidosis Observed in Hepatic POR-Null Mice

    PubMed Central

    Banerjee, Subhashis; Stolarczyk, Elzbieta I.; Zou, Ling

    2011-01-01

    Cytochrome P450 reductase (POR) is a microsomal electron transport protein essential to cytochrome P450-mediated drug metabolism and sterol and bile acid synthesis. The conditional deletion of hepatic POR gene expression in mice results in a marked decrease in plasma cholesterol levels counterbalanced by the accumulation of triglycerides in lipid droplets in hepatocytes. To evaluate the role of cholesterol and bile acid synthesis in this hepatic lipidosis, as well as the possible role of lipid transport from peripheral tissues, we developed a stable, small interfering RNA (siRNA)-mediated cell culture model for the suppression of POR. POR mRNA and protein expression were decreased by greater than 50% in McArdle-RH7777 rat hepatoma cells 10 days after transfection with a POR-siRNA expression plasmid, and POR expression was nearly completely extinguished by day 20. Immunofluorescent analysis revealed a marked accumulation of lipid droplets in cells by day 15, accompanied by a nearly 2-fold increase in cellular triglyceride content, replicating the lipidosis seen in hepatic POR-null mouse liver. In contrast, suppression of CYP51A1 (lanosterol demethylase) did not result in lipid accumulation, indicating that loss of cholesterol synthesis is not the basis for this lipidosis. Indeed, addition of cholesterol to the medium appeared to augment the lipidosis in POR-suppressed cells, whereas removal of lipids from the medium reversed the lipidosis. Oxysterols did not accumulate in POR-suppressed cells, discounting a role for liver X receptor in stimulating triglyceride synthesis, but addition of chenodeoxycholate significantly repressed lipid accumulation, suggesting that the absence of bile acids and loss of farnesoid X receptor stimulation lead to excessive triglyceride synthesis. PMID:21368239

  19. Suppression of cytochrome P450 reductase (POR) expression in hepatoma cells replicates the hepatic lipidosis observed in hepatic POR-null mice.

    PubMed

    Porter, Todd D; Banerjee, Subhashis; Stolarczyk, Elzbieta I; Zou, Ling

    2011-06-01

    Cytochrome P450 reductase (POR) is a microsomal electron transport protein essential to cytochrome P450-mediated drug metabolism and sterol and bile acid synthesis. The conditional deletion of hepatic POR gene expression in mice results in a marked decrease in plasma cholesterol levels counterbalanced by the accumulation of triglycerides in lipid droplets in hepatocytes. To evaluate the role of cholesterol and bile acid synthesis in this hepatic lipidosis, as well as the possible role of lipid transport from peripheral tissues, we developed a stable, small interfering RNA (siRNA)-mediated cell culture model for the suppression of POR. POR mRNA and protein expression were decreased by greater than 50% in McArdle-RH7777 rat hepatoma cells 10 days after transfection with a POR-siRNA expression plasmid, and POR expression was nearly completely extinguished by day 20. Immunofluorescent analysis revealed a marked accumulation of lipid droplets in cells by day 15, accompanied by a nearly 2-fold increase in cellular triglyceride content, replicating the lipidosis seen in hepatic POR-null mouse liver. In contrast, suppression of CYP51A1 (lanosterol demethylase) did not result in lipid accumulation, indicating that loss of cholesterol synthesis is not the basis for this lipidosis. Indeed, addition of cholesterol to the medium appeared to augment the lipidosis in POR-suppressed cells, whereas removal of lipids from the medium reversed the lipidosis. Oxysterols did not accumulate in POR-suppressed cells, discounting a role for liver X receptor in stimulating triglyceride synthesis, but addition of chenodeoxycholate significantly repressed lipid accumulation, suggesting that the absence of bile acids and loss of farnesoid X receptor stimulation lead to excessive triglyceride synthesis.

  20. An Isotopic Labelling Strategy to Study Cytochrome P450 Oxidations of Terpenes.

    PubMed

    Rinkel, Jan; Litzenburger, Martin; Bernhardt, Rita; Dickschat, Jeroen Sidney

    2018-04-26

    The cytochrome P450 monooxygenase CYP267B1 from Sorangium cellulosum was applied for enzymatic oxidation of the sesquiterpene alcohols T-muurolol and isodauc-8-en-11-ol. Various isotopically labelled geranyl and farnesyl diphosphates were used for product identification from micro-scale reactions, for determination of the absolute configurations of unknown compounds, to follow the stereochemical course of a cytochrome P450-catalysed hydroxylation step, and to investigate kinetic isotope effects. Overall, this study demonstrates that isotopically labelled terpene precursors are highly useful to follow cytochrome P450 dependent oxidations of terpenes. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Isolation and reconstitution of cytochrome P450ox and in vitro reconstitution of the entire biosynthetic pathway of the cyanogenic glucoside dhurrin from sorghum.

    PubMed Central

    Kahn, R A; Bak, S; Svendsen, I; Halkier, B A; Møller, B L

    1997-01-01

    A cytochrome P450, designated P450ox, that catalyzes the conversion of (Z)-p-hydroxyphenylacetaldoxime (oxime) to p-hydroxymandelonitrile in the biosynthesis of the cyanogenic glucoside beta-D-glucopyranosyloxy-(S)-p-hydroxymandelonitrile (dhurrin), has been isolated from microsomes prepared from etiolated seedlings of sorghum (Sorghum bicolor L. Moench). P450ox was solubilized using nonionic detergents, and isolated by ion-exchange chromatography, Triton X-114 phase partitioning, and dye-column chromatography. P450ox has an apparent molecular mass of 55 kD, its N-terminal amino acid sequence is -ATTATPQLLGGSVP, and it contains the internal sequence MDRLVADLDRAAA. Reconstitution of P450ox with NADPH-P450 oxidoreductase in micelles of L-alpha-dilauroyl phosphatidylcholine identified P450ox as a multifunctional P450 catalyzing dehydration of (Z)-oxime to p-hydroxyphenylaceto-nitrile (nitrile) and C-hydroxylation of p-hydroxyphenylacetonitrile to nitrile. P450ox is extremely labile compared with the P450s previously isolated from sorghum. When P450ox is reconstituted in the presence of a soluble uridine diphosphate glucose glucosyltransferase, oxime is converted to dhurrin. In vitro reconstitution of the entire dhurrin biosynthetic pathway from tyrosine was accomplished by the insertion of CYP79 (tyrosine N-hydroxylase), P450ox, and NADPH-P450 oxidoreductase in lipid micelles in the presence of uridine diphosphate glucose glucosyltransferase. The catalysis of the conversion of Tyr into nitrile by two multifunctional P450s explains why all intermediates in this pathway except (Z)-oxime are channeled. PMID:9414567

  2. Olfactory cytochrome P-450. Studies with suicide substrates of the haemoprotein.

    PubMed Central

    Reed, C J; Lock, E A; De Matteis, F

    1988-01-01

    1. The olfactory epithelium of male hamsters has been found to be extremely active in the cumene hydroperoxide-supported oxidation of tetramethylphenylenediamine, and this peroxidase activity has been shown to be cytochrome P-450-dependent. 2. The interaction of a series of suicide substrates of cytochrome P-450 with the hepatic and olfactory mono-oxygenase systems has been assessed by determination of peroxidase, 7-ethoxycoumarin O-de-ethylase (ECOD) and 7-ethoxyresorufin O-de-ethylase (EROD) activities after treatment in vivo with these compounds. Chloramphenicol, OOS-trimethylphosphorothiolate and two dihydropyridines [DDC (3,5-diethoxycarbonyl-1,4-dihydrocollidine) and 4-ethyl DDC (3,5-diethoxycarbonyl-4-ethyl-1,4-dihydro-2,6-dimethylpyridine)] all caused similar percentage inhibitions of hepatic and olfactory activities, but the absolute amounts of enzymic activity lost were considerably greater in the latter tissue. In contrast, halothane had little effect upon hepatic cytochrome P-450-dependent reactions, whereas it severely inhibited those of the olfactory epithelium. 3. The time course of loss and recovery of hepatic and olfactory peroxidase, ECOD and EROD activities after a single dose of 4-ethyl DDC was studied. The rates of loss of activity observed were very similar, irrespective of tissue or reaction examined. In the olfactory epithelium, all three activities recovered concurrently and at a rate similar to that of the hepatic peroxidase activity. In contrast, the hepatic de-ethylation of 7-ethoxycoumarin and 7-ethoxy-resorufin recovered significantly more rapidly. 4. It is suggested that this behaviour is due to 4-ethyl DDC acting not only as a suicidal inhibitor but also as an inducer of certain forms of cytochrome P-450 in the liver; in the olfactory epithelium, however, inactivation, but not induction, occurs. Classical inducing agents were reported to have no effect upon olfactory cytochrome P-450, and in the present study neither phenobarbitone

  3. Evidence for complexation of P-450 IIC6 by an orphenadrine metabolite.

    PubMed

    Reidy, G F; Murray, M

    1990-01-30

    Removal of the orphenadrine metabolite from its complex with rat liver P-450 IIB1 is associated with a discrepancy in the reactivation of IIB1 activity. Two possible explanations are that either (1) NADPH-P-450-reductase is inaccessible to the restored IIB1, or (2) complexation of other P-450s may occur. Exogenous P-450 reductase increased all pathways of steroid hydroxylation (1.9 to 3.6-fold) but did not enhance reactivation of IIB1-dependent steroid 16 beta-hydroxylation. Instead, P-450 IIC6-dependent progesterone 21-hydroxylase activity was increased after dissociation to 122% of control. IIC6 activity was also inhibited in vitro in microsomes from phenobarbital-induced rats (ki = 151 microM). Thus, orphenadrine appears to complex P-450 IIC6 as well as IIB1 in rat liver.

  4. Differential effects of traumatic brain injury on the cytochrome p450 system: a perspective into hepatic and renal drug metabolism.

    PubMed

    Kalsotra, Auinash; Turman, Cheri M; Dash, Pramod K; Strobel, Henry W

    2003-12-01

    Traumatic brain injury is known to cause several secondary effects, one of which is altered drug clearance. Given the fact that patients who sustain TBI are subsequently treated with a variety of pharmacological agents for the purpose of either neuroprotection or physiological support, it is imperative to clarify changes in expression and/or activities of enzymes involved in clearing drugs. The mixed function oxidase system, which consists of cytochrome P450 and cytochrome P450 reductase, plays a vital role in phase I drug metabolism. This paper addresses the issue as to what extent TBI affects the levels and activity of various rat CYP450 subfamilies. Our results show that TBI induces tissue-specific and time-dependent alterations. Total hepatic CYP450 content showed a biphasic response with a decrease seen at 24 h followed by an increase at 2 weeks. CYP450 reductase, in contrast, showed an opposite temporal profile. Immunoblot analyses and marker substrate metabolism demonstrated a clear decrease in hepatic CYP1A levels while a significant increase in kidney was seen at both 24 h and 2 weeks. A dramatic induction of CYP3A was evident at 2 weeks in liver, while no changes were noticed in CYP2B or CYP2D subfamilies. CYP4F subfamily showed induction in kidney only. Collectively, the data reveal the differential effects of TBI on hepatic and renal drug metabolism.

  5. Effect of carbon source on the accumulation of cytochrome P-450 in the yeast Saccharomyces cerevisiae.

    PubMed

    Kärenlampi, S O; Marin, E; Hänninen, O O

    1981-02-15

    The appearance of cytochrome P-450 in the yeast Saccharomyces cerevisiae depended on the substrate supporting growth. Cytochrome P-450 was apparent in yeast cells grown on a strongly fermentable sugar such as D-glucose, D-fructose or sucrose. When yeast was grown on D-galactose, D-mannose or maltose, where fermentation and respiration occurred concomitantly, cytochrome P-450 was also formed. The cytochrome P-450 concentration was maximal at the beginning of the stationary phase of the culture. Thereafter the concentration decreased, reaching zero at a late-stationary phase. When the yeast was grown on a medium that contained lactose or pentoses (L-arabinose, L-rhamnose, D-ribose and D-xylose), cytochrome P-450 did not occur. When a non-fermentable energy source (glycerol, lactate or ethanol) was used, no cytochrome P-450 was detectable. Transfer of cells from D-glucose medium to ethanol medium caused a slow disappearance of cytochrome P-450, although the amount of the haemoprotein still continued to increase in the control cultures. Cytochrome P-450 appeared thus to accumulate in conditions where the rate of growth was fast and fermentation occurred. Occurrence of this haemoprotein is not necessarily linked, however, with the repression of mitochondrial haemoprotein synthesis.

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

    PubMed Central

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

    1988-01-01

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

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

    PubMed

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

    1988-11-01

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

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

    EPA Science Inventory

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

  9. 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.

  10. Inhibition of cytochrome P450 2B4 by environmentally persistent free radical-containing particulate matter

    PubMed Central

    Reed, James R.; dela Cruz, Albert Leo N.; Lomnicki, Slawo M.; Backes, Wayne L.

    2015-01-01

    Combustion processes generate particulate matter (PM) that can affect human health. The presence of redox-active metals and aromatic hydrocarbons in the post-combustion regions results in the formation of air-stable, environmentally persistent free radicals (EPFRs) on entrained particles. Exposure to EPFRs has been shown to negatively influence pulmonary and cardiovascular functions. Cytochromes P450 (P450/CYP) are endoplasmic reticulum resident proteins that are responsible for the metabolism of foreign compounds. Previously, it was shown that model EPFRs, generated by exposure of silica containing 5% copper oxide (CuO-Si) to either dicholorobenzene (DCB230) or 2-monochlorophenol (MCP230) at ≥ 230°C, inhibited six forms of P450 in rat liver microsomes (Toxicol. Appl. Pharmacol. (2014) 277:200-209). In this study, the inhibition of P450 by MCP230 was examined in more detail by measuring its effect on the rate of metabolism of 7-ethoxy-4-trifluoromethylcoumarin (7EFC) and 7-benzyloxyresorufin (7BRF) by the purified, reconstituted CYP2B4 system. MCP230 inhibited the CYP2B4-mediated metabolism of 7EFC at least 10-fold more potently than non-EPFR controls (CuO-Si, silica, and silica generated from heating silica and MCP at 50°C, so that EPFRs were not formed (MCP50)). The inhibition by EPFRs was specific for the P450 and did not affect the ability of the redox partner, P450 reductase (CPR) from reducing cytochrome c. All of the PM inhibited CYP2B4-mediated metabolism noncompetitively with respect to substrate. When CYP2B4-mediated metabolism of 7EFC was measured as a function of the CPR concentration, the mechanism of inhibition was competitive. EPFRs likely inhibit CYP2B4-mediated substrate metabolism by physically disrupting the CPR•P450 complex. PMID:25817938

  11. Phenobarbital induction of cytochromes P-450. High-level long-term responsiveness of primary rat hepatocyte cultures to drug induction, and glucocorticoid dependence of the phenobarbital response.

    PubMed Central

    Waxman, D J; Morrissey, J J; Naik, S; Jauregui, H O

    1990-01-01

    The induction of hepatic cytochromes P-450 by phenobarbital (PB) was studied in rat hepatocytes cultured for up to 5 weeks on Vitrogen-coated plates in serum-free modified Chee's medium then exposed to PB (0.75 mM) for an additional 4 days. Immunoblotting analysis indicated that P-450 forms PB4 (IIB1) and PB5 (IIB2) were induced dramatically (greater than 50-fold increase), up to levels nearly as high as those achieved in PB-induced rat liver in vivo. The newly synthesized cytochrome P-450 was enzymically active, as shown by the major induction of the P-450 PB4-dependent steroid 16 beta-hydroxylase and pentoxyresorufin O-dealkylase activities in the PB-induced hepatocyte microsomes (up to 90-fold increase). PB induction of these P-450s was markedly enhanced by the presence of dexamethasone (50 nM-1 microM), which alone was not an affective inducing agent, and was inhibited by greater than 90% by 10% fetal bovine serum. The PB response was also inhibited (greater than 85%) by growth hormone (250 ng/ml), indicating that this hormone probably acts directly on the hepatocyte when it antagonizes the induction of P-450 PB4 in intact rats. In untreated hepatocytes, P-450 RLM2 (IIA2), P-450 3 (IIA1) and NADPH P-450 reductase levels were substantially maintained in the cultures for 10-20 days. The latter two enzymes were also inducible by PB to an extent (3-4 fold elevation) that is comparable with that observed in the liver in vivo. Moreover, P-450c (IA1) and P-450 3 (IIA1) were highly inducible by 3-methylcholanthrene (5 microM; 48 h exposure) even after 3 weeks in culture. In contrast, the male-specific pituitary-regulated P-450 form 2c (IIC11) was rapidly lost upon culturing the hepatocytes, suggesting that supplementation of appropriate hormonal factors may be necessary for its expression. The present hepatocyte culture system exhibits a responsiveness to drug inducers that is qualitatively and quantitatively comparable with that observed in vivo, and should prove

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

    PubMed

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

    2012-09-01

    Six proton pump inhibitors (PPIs), omeprazole, lansoprazole, esomeprazole, dexlansoprazole, pantoprazole, and rabeprazole, were shown to be weak inhibitors of cytochromes P450 (CYP3A4, -2B6, -2D6, -2C9, -2C8, and -1A2) in human liver microsomes. In most cases, IC₅₀ values were greater than 40 μM, except for dexlansoprazole and lansoprazole with CYP1A2 (IC₅₀ = ∼8 μM) and esomeprazole with CYP2C8 (IC₅₀ = 31 μM). With the exception of CYP2C19 inhibition by omeprazole and esomeprazole (IC₅₀ ratio, 2.5 to 5.9), there was no evidence for a marked time-dependent shift in IC₅₀ (IC₅₀ ratio, ≤ 2) after a 30-min preincubation with NADPH. In the absence of preincubation, lansoprazole (IC₅₀ = 0.73 μM) and esomeprazole (IC₅₀ = 3.7 μM) were the most potent CYP2C19 inhibitors, followed by dexlansoprazole and omeprazole (IC₅₀ = ∼7.0 μM). Rabeprazole and pantoprazole (IC₅₀ = ≥ 25 μM) were the weakest. A similar ranking was obtained with recombinant CYP2C19. Despite the IC₅₀ ranking, after consideration of plasma levels (static and dynamic), protein binding, and metabolism-dependent inhibition, it is concluded that omeprazole and esomeprazole are the most potent CYP2C19 inhibitors. This was confirmed after the incubation of the individual PPIs with human primary hepatocytes (in the presence of human serum) and by monitoring their impact on diazepam N-demethylase activity at a low concentration of diazepam (2 μM). Data described herein are consistent with reports that PPIs are mostly weak inhibitors of cytochromes P450 in vivo. However, two members of the PPI class (esomeprazole and omeprazole) are more likely to serve as clinically relevant inhibitors of CYP2C19.

  13. Cytochrome P450IA mRNA expression in feral Hudson River tomcod

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kreamer, G.L.; Squibb, K.; Gioeli, D.

    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 bymore » 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.« less

  14. 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 ...

  15. Metabolic capabilities of cytochrome P450 enzymes in Chinese liver microsomes compared with those in Caucasian liver microsomes

    PubMed Central

    Yang, Junling; He, Minxia M; Niu, Wei; Wrighton, Steven A; Li, Li; Liu, Yang; Li, Chuan

    2012-01-01

    AIM The most common causes of variability in drug response include differences in drug metabolism, especially when the hepatic cytochrome P450 (CYP) enzymes are involved. The current study was conducted to assess the differences in CYP activities in human liver microsomes (HLM) of Chinese or Caucasian origin. METHODS The metabolic capabilities of CYP enzymes in 30 Chinese liver microsomal samples were compared with those of 30 Caucasian samples utilizing enzyme kinetics. Phenacetin O-deethylation, coumarin 7-hydroxylation, bupropion hydroxylation, amodiaquine N-desethylation, diclofenac 4′-hydroxylation (S)-mephenytoin 4′-hydroxylation, dextromethorphan O-demethylation, chlorzoxazone 6-hydroxylation and midazolam 1′-hydroxylation/testosterone 6β-hydroxylation were used as probes for activities of CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A, respectively. Mann-Whitney U test was used to assess the differences. RESULTS The samples of the two ethnic groups were not significantly different in cytochrome-b5 concentrations but were significantly different in total CYP concentrations and NADPH-P450 reductase activity (P < 0.05). Significant ethnic differences in intrinsic clearance were observed for CYP1A2, CYP2C9, CYP2C19 and CYP2E1; the median values of the Chinese group were 54, 58, 26, and 35% of the corresponding values of the Caucasian group, respectively. These differences were associated with differences in Michaelis constant or maximum velocity. Despite negligible difference in intrinsic clearance, the Michaelis constant of CYP2B6 appeared to have a significant ethnic difference. No ethnic difference was observed for CYP2A6, CYP2C8, CYP2D6 and CYP3A. CONCLUSIONS These data extend our knowledge on the ethnic differences in CYP enzymes and will have implications for drug discovery and drug therapy for patients from different ethnic origins. PMID:21815912

  16. Cloning, expression and characterisation of P450-Hal1 (CYP116B62) from Halomonas sp. NCIMB 172: A self-sufficient P450 with high expression and diverse substrate scope.

    PubMed

    Porter, Joanne L; Sabatini, Selina; Manning, Jack; Tavanti, Michele; Galman, James L; Turner, Nicholas J; Flitsch, Sabine L

    2018-06-01

    Cytochrome P450 monooxygenases are able to catalyse a range of synthetically challenging reactions ranging from hydroxylation and demethylation to sulfoxidation and epoxidation. As such they have great potential for biocatalytic applications but are underutilised due to often-poor expression, stability and solubility in recombinant bacterial hosts. The use of self-sufficient P450 s with fused haem and reductase domains has already contributed heavily to improving catalytic efficiency and simplifying an otherwise more complex multi-component system of P450 and redox partners. Herein, we present a new addition to the class VII family with the cloning, sequencing and characterisation of the self-sufficient CYP116B62 Hal1 from Halomonas sp. NCIMB 172, the genome of which has not yet been sequenced. Hal1 exhibits high levels of expression in a recombinant E. coli host and can be utilised from cell lysate or used in purified form. Hal1 favours NADPH as electron donor and displays a diverse range of activities including hydroxylation, demethylation and sulfoxidation. These properties make Hal1 suitable for future biocatalytic applications or as a template for optimisation through engineering. Copyright © 2018 Elsevier Inc. All rights reserved.

  17. 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...

  18. Regiospecificity determinants of human heme oxygenase: differential NADPH- and ascorbate-dependent heme cleavage by the R183E mutant.

    PubMed

    Wang, Jinling; Lad, Latesh; Poulos, Thomas L; Ortiz de Montellano, Paul R

    2005-01-28

    The ability of the human heme oxygenase-1 (hHO-1) R183E mutant to oxidize heme in reactions supported by either NADPH-cytochrome P450 reductase or ascorbic acid has been compared. The NADPH-dependent reaction, like that of wild-type hHO-1, yields exclusively biliverdin IXalpha. In contrast, the R183E mutant with ascorbic acid as the reductant produces biliverdin IXalpha (79 +/- 4%), IXdelta (19 +/- 3%), and a trace of IXbeta. In the presence of superoxide dismutase and catalase, the yield of biliverdin IXdelta is decreased to 8 +/- 1% with a corresponding increase in biliverdin IXalpha. Spectroscopic analysis of the NADPH-dependent reaction shows that the R183E ferric biliverdin complex accumulates, because reduction of the iron, which is required for sequential iron and biliverdin release, is impaired. Reversal of the charge at position 183 makes reduction of the iron more difficult. The crystal structure of the R183E mutant, determined in the ferric and ferrous-NO bound forms, shows that the heme primarily adopts the same orientation as in wild-type hHO-1. The structure of the Fe(II).NO complex suggests that an altered active site hydrogen bonding network supports catalysis in the R183E mutant. Furthermore, Arg-183 contributes to the regiospecificity of the wild-type enzyme, but its contribution is not critical. The results indicate that the ascorbate-dependent reaction is subject to a lower degree of regiochemical control than the NADPH-dependent reaction. Ascorbate may be able to reduce the R183E ferric and ferrous dioxygen complexes in active site conformations that cannot be reduced by NADPH-cytochrome P450 reductase.

  19. The stoichiometry of the cytochrome P-450-catalyzed metabolism of methoxyflurane and benzphetamine in the presence and absence of cytochrome b5.

    PubMed

    Gruenke, L D; Konopka, K; Cadieu, M; Waskell, L

    1995-10-20

    The complete stoichiometry of the metabolism of the cytochrome b5 (cyt b5)-requiring substrate, methoxyflurane, by purified cytochrome P-450 2B4 was compared to that of another substrate, benzphetamine, which does not require cyt b5 for its metabolism. Cyt b5 invariably improved the efficiency of product formation. That is, in the presence of cyt b5 a greater percentage of the reducing equivalents from NADPH were utilized to generate substrate metabolites, primarily at the expense of the side product, superoxide. With methoxyflurane, cyt b5 addition always resulted in an increased rate of product formation, while with benzphetamine the rate of product formation remained unchanged, increased or decreased. The apparently contradictory observations of increased reaction efficiency but decrease in total product formation for benzphetamine can be explained by a second effect of cyt b5. Under some experimental conditions cyt b5 inhibits total NADPH consumption. Whether stimulation, inhibition, or no change in product formation is observed in the presence of cyt b5 depends on the net effect of the stimulatory and inhibitory effects of cyt b5. When total NADPH consumption is inhibited by cyt b5, the rapidly metabolized, highly coupled (approximately equal to 50%) substrate, benzphetamine, undergoes a net decrease in metabolism not counterbalanced by the increase in the efficiency (2-20%) of the reaction. In contrast, in the presence of the slowly metabolized, poorly coupled (approximately equal to 0.5-3%) substrate, methoxyflurane, inhibition of total NADPH consumption by cyt b5 was never sufficient to overcome the stimulation of product formation due to an increase in efficiency of the reaction.

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

    EPA Science Inventory

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

  1. Luminogenic cytochrome P450 assays.

    PubMed

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

    2006-08-01

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

  2. Cytochrome P450 isozyme protein verified in the skin of southern hemisphere humpback whales (Megaptera novaeangliae): implications for biochemical biomarker assessment.

    PubMed

    Waugh, Courtney A; Huston, Wilhelmina M; Noad, Michael J; Bengtson Nash, Susan

    2011-04-01

    Large mysticete whales represent a unique challenge for chemical risk assessment. Few epidemiological investigations are possible due to the low incidence of adult stranding events. Similarly their often extreme life-history adaptations of prolonged migration and fasting challenge exposure assumptions. Molecular biomarkers offer the potential to complement information yielded through tissue chemical analysis, as well as providing evidence of a molecular response to chemical exposure. In this study we confirm the presence of cytochrome P450 reductase (CPR) and cytochrome P450 isoenzyme 1A1 (CYP1A1) in epidermal tissue of southern hemisphere humpback whales (Megaptera novaeangliae). The detection of CYP1A1 in the integument of the humpback whale affords the opportunity for further quantitative non-destructive investigations of enzyme activity as a function of chemical stress. Copyright © 2011 Elsevier Ltd. All rights reserved.

  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. [Immunomodulators with an 8-azasteroid structure as inducers of liver cytochrome P-450].

    PubMed

    Kuz'mitskiĭ, B B; Dad'kov, I G; Mashkovich, A E; Stoma, O V; Slepneva, L M

    1990-01-01

    Two structural analogues of D-homo-8-azasteroids, both an immunostimulant and an immunodepressant, are inductors of the liver cytochrome P-450 in animals. This capability was shown by means of both a decrease of the hexenal sleep duration in the pharmacological test and an increase of the quantity of cytochrome P-450 and the rate of N-demethylation of aminopyrine in the biochemical assays.

  5. FTIR studies of the redox partner interaction in cytochrome P450: the Pdx-P450cam couple.

    PubMed

    Karyakin, Andrey; Motiejunas, Domantas; Wade, Rebecca C; Jung, Christiane

    2007-03-01

    Recently we have developed a new approach to study protein-protein interactions using Fourier transform infrared spectroscopy in combination with titration experiments and principal component analysis (FTIR-TPCA). In the present paper we review the FTIR-TPCA results obtained for the interaction between cytochrome P450 and the redox partner protein in two P450 systems, the Pseudomonas putida P450cam (CYP101) with putidaredoxin (P450cam-Pdx), and the Bacillus megaterium P450BM-3 (CYP102) heme domain with the FMN domain (P450BMP-FMND). Both P450 systems reveal similarities in the structural changes that occur upon redox partner complex formation. These involve an increase in beta-sheets and alpha-helix content, a decrease in the population of random coil/3(10)-helix structure, a redistribution of turn structures within the interacting proteins and changes in the protonation states or hydrogen-bonding of amino acid carboxylic side chains. We discuss in detail the P450cam-Pdx interaction in comparison with literature data and conclusions drawn from experiments obtained by other spectroscopic techniques. The results are also interpreted in the context of a 3D structural model of the Pdx-P450cam complex.

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

    PubMed

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

    2005-10-01

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

  7. 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.

  8. 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.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Gan, L.S.

    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 themore » 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.« less

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

    PubMed Central

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

    2013-01-01

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

  12. 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

  13. Contrasting influence of NADPH and a NADPH-regenerating system on the metabolism of carbonyl-containing compounds in hepatic microsomes.

    PubMed

    Mazur, Christopher S; Kenneke, John F; Goldsmith, Michael-Rock; Brown, Cather

    2009-09-01

    Carbonyl containing xenobiotics may be susceptible to NADPH-dependent cytochrome P450 (P450) and carbonyl-reduction reactions. In vitro hepatic microsome assays are routinely supplied NADPH either by direct addition of NADPH or via an NADPH-regenerating system (NRS). In contrast to oxidative P450 transformations, which occur on the periphery of a microsome vesicle, intraluminal carbonyl reduction depends on transport of cofactors across the endoplasmic reticulum (ER) membrane into the lumen. Glucose 6-phosphate, a natural cofactor and component of the NRS matrix, is readily transported across the ER membrane and facilitates intraluminal NADPH production, whereas direct addition of NADPH has limited access to the lumen. In this study, we compared the effects of direct addition of NADPH and use of an NRS on the P450-mediated transformation of propiconazole and 11 beta-hydroxysteroid dehydrogenase type 1 (HSD1) carbonyl reduction of cortisone and the xenobiotic triadimefon in hepatic microsomes. Our results demonstrate that the use of NADPH rather than NRS can underestimate the kinetic rates of intraluminal carbonyl reduction, whereas P450-mediated transformations were unaffected. Therefore, in vitro depletion rates measured for a carbonyl-containing xenobiotic susceptible to both intraluminal carbonyl reduction and P450 processes may not be properly assessed with direct addition of NADPH. In addition, we used in silico predictions as follows: 1) to show that 11 beta-HSD1 carbonyl reduction was energetically more favorable than oxidative P450 transformation; and 2) to calculate chemical binding score and the distance between the carbonyl group and the hydride to be transferred by NADPH to identify other 11 beta-HSD1 substrates for which reaction kinetics may be underestimated by direct addition of NADPH.

  14. The use of electrochemistry for the synthesis of 17 alpha-hydroxyprogesterone by a fusion protein containing P450c17.

    PubMed

    Estabrook, R W; Shet, M S; Faulkner, K; Fisher, C W

    1996-11-01

    A method has been developed for the commercial application of the unique oxygen chemistry catalyzed by various cytochrome P450s. This is illustrated here for the synthesis of hydroxylated steroids. This method requires the preparation of large amounts of enzymatically functional P450 proteins that can serve as catalysts and a technique for providing electrons at an economically acceptable cost. To generate large amounts of enzymatically active recombinant P450s we have engineered the cDNAs for various P450s, including bovine adrenal P450c17, by linking them to a modified cDNA for rat NADPH-P450 reductase and placing them in the plasmid pCWori+. Transformation of E. coli results in the high level expression of an enzymatically active protein that can be easily purified by affinity chromatography. Incubation of the purified enzyme with steroid in a reaction vessel containing a platinum electrode and a Ag/AgCl electrode couple poised at -650 mV, together with the electromotively active redox mediator, cobalt sepulchrate, results in the 17 alpha-hydroxylation of progesterone at rates as high as 25 nmoles of progesterone hydroxylated/min/nmole of P450. Thus, high concentrations of hydroxylated steroids can be produced with incubation conditions of hours duration without the use of costly NADPH. Similar experiments have been carried out for the generation of the 6 beta-hydroxylation product of testosterone (using a fusion protein containing human P450 3A4). It is apparent that this method is applicable to many other P450 catalyzed reactions for the synthesis of large amounts of hydroxylated steroid metabolites. The electrochemical system is also applicable to drug discovery studies for the characterization of drug metabolites.

  15. 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

  16. Cytochrome P450 3A4 activity after surgical stress.

    PubMed

    Haas, Curtis E; Kaufman, David C; Jones, Carolyn E; Burstein, Aaron H; Reiss, William

    2003-05-01

    To evaluate the relationship between the acute inflammatory response after surgical trauma and changes in hepatic cytochrome P450 3A4 activity, compare changes in cytochrome P450 3A4 activity after procedures with varying degrees of surgical stress, and to explore the time course of any potential drug-cytokine interaction after surgery. Prospective, open-label study with each patient serving as his or her own control. University-affiliated, acute care, general hospital. A total of 16 patients scheduled for elective repair of an abdominal aortic aneurysm (n = 5), complete or partial colectomy (n = 6), or peripheral vascular surgery with graft (n = 5). Cytochrome P450 3A4 activity was estimated using the carbon-14 [14C]erythromycin breath test (ERMBT) before surgery and 24, 48, and 72 hrs after surgery. Abdominal aortic aneurysm and colectomy patients also had an ERMBT performed at discharge. Blood samples were obtained before surgery, immediately after surgery, and 6, 24, 32, 48, and 72 hrs after surgery for determination of plasma concentrations of interleukin-6, interleukin-1beta, and tumor necrosis factor-alpha. Clinical markers of surgical stress that were collected included duration of surgery, estimated blood loss, and volume of fluids administered in the operating room. ERMBT results significantly declined in all three surgical groups, with the lowest value at the time of the 72-hr study in all three groups. There was a trend toward differences in ERMBT results among groups that did not reach statistical significance (p =.06). The nadir ERMBT result was significantly and negatively correlated with both peak interleukin-6 concentration (r(s) = -.541, p =.03) and log interleukin-6 area under the curve from 0 to 72 hrs (r(s) = -.597, p =.014). Subjects with a peak interleukin-6 of >100 pg/mL had a significantly lower nadir ERMBT compared with subjects with a peak interleukin-6 of <100 pg/mL (35.5% +/- 5.2% vs. 74.7% +/- 5.1%, p <.001). Acute inflammation after

  17. Degradation of Morpholine by an Environmental Mycobacterium Strain Involves a Cytochrome P-450

    PubMed Central

    Poupin, P.; Truffaut, N.; Combourieu, B.; Besse, P.; Sancelme, M.; Veschambre, H.; Delort, A. M.

    1998-01-01

    A Mycobacterium strain (RP1) was isolated from a contaminated activated sludge collected in a wastewater treatment unit of a chemical plant. It was capable of utilizing morpholine and other heterocyclic compounds, such as pyrrolidine and piperidine, as the sole source of carbon, nitrogen, and energy. The use of in situ 1H nuclear magnetic resonance (1H NMR) spectroscopy allowed the determination of two intermediates in the biodegradative pathway, 2-(2-aminoethoxy)acetate and glycolate. The inhibitory effects of metyrapone on the degradative abilities of strain RP1 indicated the involvement of a cytochrome P-450 in the biodegradation of morpholine. This observation was confirmed by spectrophotometric analysis and 1H NMR. Reduced cell extracts from morpholine-grown cultures, but not succinate-grown cultures, gave rise to a carbon monoxide difference spectrum with a peak near 450 nm, which indicated the presence of a soluble cytochrome P-450. 1H NMR allowed the direct analysis of the incubation medium containing metyrapone, a specific inhibitor of cytochrome P-450. The inhibition of morpholine degradation was dependent on the morpholine/metyrapone ratio. The heme-containing monooxygenase was also detected in pyrrolidine- and piperidine-grown cultures. The abilities of different compounds to support strain growth or the induction of a soluble cytochrome P-450 were assayed. The results suggest that this enzyme catalyzes the cleavage of the C—N bond of the morpholine ring. PMID:9435074

  18. Novel approaches to mitigating parathion toxicity: targeting cytochrome P450–mediated metabolism with menadione

    PubMed Central

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

    2016-01-01

    Accidental or intentional exposures to parathion, an organophosphorus (OP) pesticide, can cause severe poisoning in humans. Parathion toxicity is dependent on its metabolism by the cytochrome P450 (CYP) system to paraoxon (diethyl 4-nitrophenyl phosphate), a highly poisonous nerve agent and potent inhibitor of acetylcholinesterase (AChE). We have been investigating inhibitors of CYP-mediated bioactivation of OPs as a method of preventing or reversing progressive parathion toxicity. It is well recognized that NADPH–cytochrome P450 reductase, an enzyme required for the transfer of electrons to CYPs, mediates chemical redox cycling. In this process, the enzyme diverts electrons from CYPs to support chemical redox cycling, which results in inhibition of CYP-mediated biotransformation. Using menadione as the redox-cycling chemical, we discovered that this enzymatic reaction blocks metabolic activation of parathion in rat and human liver microsomes and in recombinant CYPs important to parathion metabolism, including CYP1A2, CYP2B6, and CYP3A4. Administration of menadione to rats reduces metabolism of parathion, as well as parathion-induced inhibition of brain cholinesterase activity. This resulted in inhibition of parathion neurotoxicity. Menadione has relatively low toxicity and is approved by the FDA for other indications. Its ability to block parathion metabolism makes it an attractive therapeutic candidate to mitigate parathion-induced neurotoxicity. PMID:27441453

  19. Thermodynamics of camphor migration in cytochrome P450cam by atomistic simulations.

    PubMed

    Rydzewski, J; Nowak, W

    2017-08-10

    Understanding the mechanisms of ligand binding to enzymes is of paramount importance for the design of new drugs. Here, we report on the use of a novel biased molecular dynamics (MD) methodology to study the mechanism of camphor binding to cytochrome P450cam. Microsecond-long MD simulations allowed us to observe reaction coordinates characterizing ligand diffusion from the active site of cytochrome P450cam to solvent via three egress routes. These atomistic simulations were used to estimate thermodynamic quantities along the reaction coordinates and indicate diverse binding configurations. The results suggest that the diffusion of camphor along the pathway near the substrate recognition site (SRS) is thermodynamically preferred. In addition, we show that the diffusion near the SRS is triggered by a transition from a heterogeneous collection of closed ligand-bound conformers to the basin comprising the open conformations of cytochrome P450cam. The conformational change accompanying this switch is characterized by the retraction of the F and G helices and the disorder of the B' helix. These results are corroborated by experimental studies and provide detailed insight into ligand binding and conformational behavior of the cytochrome family. The presented methodology is general and can be applied to other ligand-protein systems.

  20. Interactions between Cytochromes P450 2B4 (CYP2B4) and 1A2 (CYP1A2) Lead to Alterations in Toluene Disposition and P450 Uncoupling

    PubMed Central

    Reed, James R.; Cawley, George F.; Backes, Wayne L.

    2013-01-01

    The goal of this study was to characterize the effects of CYP1A2•CYP2B4 complex formation on the rates and efficiency of toluene metabolism by comparing the results from simple reconstituted systems containing P450 reductase (CPR) and a single P450 to those using a mixed system containing CPR and both P450s. In the mixed system, the rates of formation of CYP2B4-specific benzyl alcohol and p-cresol were inhibited, whereas that of CYP1A2-specific o-cresol was increased, results consistent with the formation of a CYP1A2•CYP2B4 complex where the CYP1A2 moiety has higher affinity for CPR binding. Comparison of the rates of NADPH oxidation and production of hydrogen peroxide and excess water by the simple and mixed systems indicated that excess water formed at a much lower rate in the mixed system. The commensurate increase in the rate of CYP1A2-specific product formation suggested the P450P450 interaction increased the putative rate-limiting step of CYP1A2 catalysis, abstraction of a hydrogen radical from the substrate. Cumene hydroperoxide-supported metabolism was measured to determine whether the effects of the P450P450 interaction required the presence of CPR. Peroxidative metabolism was not affected by the interaction of the two P450s, even with CPR present. However, CPR did stimulate peroxidative metabolism by the simple system containing CYP1A2. These results suggest the major functional effects of the P450P450 interaction are mediated by changes in the relative abilities of the P450s to receive electrons from CPR. Furthermore, CPR may play an effector role by causing a conformation change in CYP1A2 that makes its metabolism more efficient. PMID:23675771

  1. Aromatic Hydroxylation of Salicylic Acid and Aspirin by Human Cytochromes P450

    PubMed Central

    Bojić, Mirza; Sedgeman, Carl A.; Nagy, Leslie D.; Guengerich, F. Peter

    2015-01-01

    Aspirin (acetylsalicylic acid) is a well-known and widely-used analgesic. It is rapidly deacetylated to salicylic acid, which forms two hippuric acids—salicyluric acid and gentisuric acid—and two glucuronides. The oxidation of aspirin and salicylic acid has been reported with human liver microsomes, but data on individual cytochromes P450 involved in oxidation is lacking. In this study we monitored oxidation of these compounds by human liver microsomes and cytochrome P450 (P450) using UPLC with fluorescence detection. Microsomal oxidation of salicylic acid was much faster than aspirin. The two oxidation products were 2,5-dihydroxybenzoic acid (gentisic acid, documented by its UV and mass spectrum) and 2,3-dihydroxybenzoic acid. Formation of neither product was inhibited by desferrioxamine, suggesting a lack of contribution of oxygen radicals under these conditions. Although more liphophilic, aspirin was oxidized less efficiently, primarily to the 2,5-dihydroxy product. Recombinant human P450s 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4 all catalyzed the 5-hydroxylation of salicylic acid. Inhibitor studies with human liver microsomes indicated that all six of the previously mentioned P450s could contribute to both the 5- and 3-hydroxylation of salicylic acid and that P450s 2A6 and 2B6 have contributions to 5-hydroxylation. Inhibitor studies indicated that the major human P450 involved in both 3- and 5-hydroxylation of salicylic acid is P450 2E1. PMID:25840124

  2. Formation of cytochrome P-450 containing haem or cobalt-protoporphyrin in liver homogenates of rats treated with phenobarbital and allylisopropylacetamide.

    PubMed Central

    Bonkovsky, H L; Sinclair, J F; Healey, J F; Sinclair, P R; Smith, E L

    1984-01-01

    The potent porphyrogen allylisopropylacetamide and related compounds decrease hepatic concentrations of cytochrome P-450. This decrease occurs particularly in phenobarbital-induced cytochrome P-450 and is caused by suicidal breakdown of the haem of cytochrome P-450. Quantitative rocket immunoelectrophoresis showed that the protein moiety of the major phenobarbital-inducible form of hepatic cytochrome P-450 was not diminished up to 1 h, but was markedly decreased (to 43% of that of the phenobarbital-treated control) at 20 h after allylisopropylacetamide treatment. In contrast, the concentration of total cytochrome P-450, measured spectrophotometrically, decreased to 30-40% of the control at both 1 and 20 h after allylisopropylacetamide. Cytochrome P-450-dependent demethylations of ethylmorphine and benzphetamine decreased to a similar extent. When liver homogenates from rats treated with allylisopropylacetamide 1 h before being killed were incubated with haem, functional holocytochrome P-450 could be reconstituted from the apoprotein. Incubation with haem increased spectrophotometrically measurable cytochrome P-450 to 69%, ethylmorphine demethylase to 64% and benzphetamine demethylase to 93% of the activities in rats treated with phenobarbital alone. At 20 h after allylisopropylacetamide treatment, however, little or no reconstitution of cytochrome P-450 occurred after incubation with haem. When liver homogenates were incubated with cobalt and protoporphyrin, and microsomal proteins were then subjected to polyacrylamide-gel electrophoresis, cobalt-protoporphyrin was found specifically associated with proteins of Mr 50 000-53 000. When homogenates from rats given allylisopropylacetamide for 1 h or 20 h were compared, it was found that the extent of this association was higher in livers from the rats containing more apocytochrome P-450, suggesting that cobalt-protoporphyrin had associated with the apocytochrome. The data provide insight into the association of haem

  3. Cytochrome P450 2D6 polymorphism and character traits.

    PubMed

    Suzuki, Eiji; Kitao, Yoshie; Ono, Yutaka; Iijima, Yoshimi; Inada, Toshiya

    2003-06-01

    It has been suggested that cytochrome P450 2D6 (CYP2D6) is involved in dopamine metabolism within the brain. The dopamine system is suggested to play a role in determining normal character. The purpose of this study was to examine whether character traits are dependent on cytochrome P450 2D6 activity. We investigated the association between temperament and CYP2D6 gene polymorphism. The subjects were all Japanese and the polymorphism genotyped in the present study was CYP2D6*10. Character traits were assessed using the Temperament and Character Inventory. There was no overall or specific association between personality traits and the CYP2D6*10 allele and genotype frequencies. The present results do not support the hypothesis that CYP2D6 activity affects temperament and character.

  4. A human cytochrome P-450 is recognized by anti-liver/kidney microsome antibodies in autoimmune chronic hepatitis.

    PubMed

    Kiffel, L; Loeper, J; Homberg, J C; Leroux, J P

    1989-02-28

    1- Anti-liver/kidney microsome autoantibodies type 1 (anti-LKM1), observed in some children with chronic active hepatitis, were used to isolate their antigen in human liver microsomes. A protein, called P-LKM1 was thus purified. This protein was recognized by a rabbit antiserum directed against the related human cytochromes P-450 bufI and P-450 bufII. 2- A human liver microsomal protein immunoprecipitated with anti-LKM1 sera was also recognized by anti cytochromes P-450 bufI/II antibodies. 3- Anti-LKM1 antibodies potently inhibited microsomal bufuralol 1'-hydroxylation. These results displayed the possible identity between cytochrome P-450 bufI/II and LKM1 antigen.

  5. 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

  6. 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.

  7. 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…

  8. Inhibition of oxidative drug metabolism by orphenadrine: in vitro and in vivo evidence for isozyme-specific complexation of cytochrome P-450 and inhibition kinetics.

    PubMed

    Reidy, G F; Mehta, I; Murray, M

    1989-05-01

    The anti-parkinsonian agent orphenadrine has been shown to form an in vitro metabolic intermediate (MI) complex in hepatic microsomes isolated from phenobarbital (PB)-treated rats. The present study was undertaken to assess the cytochrome P-450 isozyme specificity of inhibition and MI complexation. Spectral studies with untreated and PB-induced rat hepatic microsomes confirmed earlier reports on the selectivity of P-450 complexation by orphenadrine; MI complex formation was only observed with PB-induced microsomes. Inhibition studies with the P-450 substrates androst-4-ene-3,17-dione (androstenedione) and 7-pentoxyresorufin revealed selective inhibition of P-450 PB-B/D-associated monooxygenase activity. Thus, in microsomes from untreated male rats, orphenadrine failed to significantly inhibit (less than 50% inhibition up to a concentration of 300 microM) any of the major pathways of P-450-associated androstenedione metabolism. Preincubation of these microsomal fractions with orphenadrine and NADPH was not associated with increased inhibition of androstenedione metabolism. However, in PB-induced microsomes, P-450 PB-B/D-specific androstenedione 16 beta-hydroxylase activity was significantly and selectively inhibited (IC50 = 90 microM). Preincubation of orphenadrine with NADPH-supplemented PB-induced microsomes for 2, 4, or 8 min before androstenedione addition resulted in increased inhibition toward 16 beta-hydroxylase activity, lowering the observed IC50 to 6.6, 0.47, and 0.06 microM), respectively. Preincubation did not affect the selectivity of inhibition. In the absence of preincubation, orphenadrine appeared to be a potent mixed (competitive/noncompetitive)-type inhibitor of P-450 PB-B/D-associated pentoxyresorufin O-depentylation (Ki = 3.8 microM). Preincubation of orphenadrine with NADPH-supplemented microsomal fractions for 4 min resulted in a 30-fold lowering of the apparent inhibitor constant (Ki = 0.13 microM) and a change in the apparent inhibition

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

    PubMed

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

    2015-03-06

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

  10. An indole-deficient Escherichia coli strain improves screening of cytochromes P450 for biotechnological applications.

    PubMed

    Brixius-Anderko, Simone; Hannemann, Frank; Ringle, Michael; Khatri, Yogan; Bernhardt, Rita

    2017-05-01

    Escherichia coli has developed into an attractive organism for heterologous cytochrome P450 production, but, in some cases, was restricted as a host in view of a screening of orphan cytochromes P450 or mutant libraries in the context of molecular evolution due to the formation of the cytochrome P450 inhibitor indole by the enzyme tryptophanase (TnaA). To overcome this effect, we disrupted the tnaA gene locus of E. coli C43(DE3) and evaluated the new strain for whole-cell substrate conversions with three indole-sensitive cytochromes P450, myxobacterial CYP264A1, and CYP109D1 as well as bovine steroidogenic CYP21A2. For purified CYP264A1 and CYP21A2, the half maximal inhibitory indole concentration was determined to be 140 and 500 μM, which is within the physiological concentration range occurring during cultivation of E. coli in complex medium. Biotransformations with C43(DE3)_∆tnaA achieved a 30% higher product formation in the case of CYP21A2 and an even fourfold increase with CYP264A1 compared with C43(DE3) cells. In whole-cell conversion based on CYP109D1, which converts indole to indigo, we could successfully avoid this reaction. Results in microplate format indicate that our newly designed strain is a suitable host for a fast and efficient screening of indole-influenced cytochromes P450 in complex medium. © 2016 International Union of Biochemistry and Molecular Biology, Inc.

  11. Cytochrome P450 3A expression and activity in the rabbit lacrimal gland: glucocorticoid modulation and the impact on androgen metabolism.

    PubMed

    Attar, Mayssa; Ling, Kah-Hiing John; Tang-Liu, Diane D-S; Neamati, Nouri; Lee, Vincent H L

    2005-12-01

    Cytochrome P450 3A (CYP3A) is an enzyme of paramount importance to drug metabolism. The expression and activity of CYP3A, an enzyme responsible for active androgen clearance, was investigated in the rabbit lacrimal gland. Analysis of CYP3A expression and activity was performed on lacrimal gland tissues obtained from naïve untreated and treated New Zealand White rabbits. For 5 days, treated rabbits received daily administration of vehicle or 0.1% or 1.0% dexamethasone, in the lower cul-de-sac of each eye. Changes in mRNA expression were monitored by real-time RT-PCR. Protein expression was confirmed by Western blot. Functional activity was measured by monitoring the metabolism of CYP3A probe substrates-namely, 7-benzyloxyquinoline (BQ) and [3H]testosterone. Cytochrome P450 heme protein was detected at a concentration of 44.6 picomoles/mg protein, along with its redox partner NADPH reductase and specifically CYP3A6 in the naïve rabbit lacrimal gland. Genes encoding CYP3A6, in addition to the pregnane-X-receptor (PXR) and P-glycoprotein (P-gp) were expressed in the untreated tissue. BQ dealkylation was measured in the naïve rabbit lacrimal gland at a rate of 14 +/- 7 picomoles/mg protein per minute. Changes in CYP3A6, P-gp, and androgen receptor mRNA expression levels were detected after dexamethasone treatment. In addition, dexamethasone treatment resulted in significant increases in BQ dealkylation and CYP3A6-mediated [3H]testosterone metabolism. Concomitant increases in CYP3A6-mediated hydroxylated testosterone metabolites were observed in the treated rabbits. Furthermore, ketoconazole, all-trans retinoic acid, and cyclosporine inhibited CYP3A6 mediated [3H]testosterone 6beta hydroxylation in a concentration-dependent manner, with IC50 ranging from 3.73 to 435 microM. The results demonstrate, for the first time, the expression and activity of CYP3A6 in the rabbit lacrimal gland. In addition, this pathway was shown to be subject to modulation by a commonly

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

    PubMed

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

    2015-03-10

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

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

    PubMed Central

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

    2015-01-01

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

  14. Stereoselective metabolism of endosulfan by human liver microsomes and human cytochrome P450 isoforms.

    PubMed

    Lee, Hwa-Kyung; Moon, Joon-Kwan; Chang, Chul-Hee; Choi, Hoon; Park, Hee-Won; Park, Byeoung-Soo; Lee, Hye-Suk; Hwang, Eul-Chul; Lee, Young-Deuk; Liu, Kwang-Hyeon; Kim, Jeong-Han

    2006-07-01

    Endosulfan (6,7,8,9,10,10-hexachloro-1,5,5a,6,9,9a-hexahydro-6,9-methano-2,3,4-benzo(e)dioxathiepin-3-oxide) is a broad-spectrum chlorinated cyclodiene insecticide. This study was performed to elucidate the stereoselective metabolism of endosulfan in human liver microsomes and to characterize the cytochrome P450 (P450) enzymes that are involved in the metabolism of endosulfan. Human liver microsomal incubation of endosulfan in the presence of NADPH resulted in the formation of the toxic metabolite, endosulfan sulfate. The intrinsic clearances (CL(int)) of endosulfan sulfate from beta-endosulfan were 3.5-fold higher than those from alpha-endosulfan, suggesting that beta-endosulfan would be cleared more rapidly than alpha-endosulfan. Correlation analysis between the known P450 enzyme activities and the rate of the formation of endosulfan sulfate in the 14 human liver microsomes showed that alpha-endosulfan metabolism is significantly correlated with CYP2B6-mediated bupropion hydroxylation and CYP3A-mediated midazolam hydroxylation, and that beta-endosulfan metabolism is correlated with CYP3A activity. The P450 isoform-selective inhibition study in human liver microsomes and the incubation study of cDNA-expressed enzymes also demonstrated that the stereoselective sulfonation of alpha-endosulfan is mediated by CYP2B6, CYP3A4, and CYP3A5, and that that of beta-endosulfan is transformed by CYP3A4 and CYP3A5. The total CL(int) values of endosulfan sulfate formation catalyzed by CYP3A4 and CYP3A5 were consistently higher for beta-endosulfan than for the alpha-form (CL(int) of 0.67 versus 10.46 microl/min/pmol P450, respectively). CYP2B6 enantioselectively metabolizes alpha-endosulfan, but not beta-endosulfan. These findings suggest that the CYP2B6 and CYP3A enzymes are major enzymes contributing to the stereoselective disposition of endosulfan.

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

    PubMed Central

    Brault, D

    1985-01-01

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

  16. Buprofezin Is Metabolized by CYP353D1v2, a Cytochrome P450 Associated with Imidacloprid Resistance in Laodelphax striatellus.

    PubMed

    Elzaki, Mohammed Esmail Abdalla; Miah, Mohammad Asaduzzaman; Han, Zhaojun

    2017-11-29

    CYP353D1v2 is a cytochrome P450 related to imidacloprid resistance in Laodelphax striatellus . This work was conducted to examine the ability of CYP353D1v2 to metabolize other insecticides. Carbon monoxide difference spectra analysis indicates that CYP353D1v2 was successfully expressed in insect cell Sf9. The catalytic activity of CYP353D1v2 relating to degrading buprofezin, chlorpyrifos, and deltamethrin was tested by measuring substrate depletion and analyzing the formation of metabolites. The results showed the nicotinamide-adenine dinucleotide phosphate (NADPH)-dependent depletion of buprofezin (eluting at 8.7 min) and parallel formation of an unknown metabolite (eluting 9.5 min). However, CYP353D1v2 is unable to metabolize deltamethrin and chlorpyrifos. The recombinant CYP353D1v2 protein efficiently catalyzed the model substrate p -nitroanisole with a maximum velocity of 9.24 nmol/min/mg of protein and a Michaelis constant of Km = 6.21 µM. In addition, imidacloprid was metabolized in vitro by the recombinant CYP353D1v2 microsomes (catalytic constant Kcat) 0.064 pmol/min/pmol P450, Km = 6.41 µM. The mass spectrum of UPLC-MS analysis shows that the metabolite was a product of buprofezin, which was buprofezin sulfone. This result provided direct evidence that L. striatellus cytochrome P450 CYP353D1v2 is capable of metabolizing imidacloprid and buprofezin.

  17. Buprofezin Is Metabolized by CYP353D1v2, a Cytochrome P450 Associated with Imidacloprid Resistance in Laodelphax striatellus

    PubMed Central

    Elzaki, Mohammed Esmail Abdalla; Miah, Mohammad Asaduzzaman; Han, Zhaojun

    2017-01-01

    CYP353D1v2 is a cytochrome P450 related to imidacloprid resistance in Laodelphax striatellus. This work was conducted to examine the ability of CYP353D1v2 to metabolize other insecticides. Carbon monoxide difference spectra analysis indicates that CYP353D1v2 was successfully expressed in insect cell Sf9. The catalytic activity of CYP353D1v2 relating to degrading buprofezin, chlorpyrifos, and deltamethrin was tested by measuring substrate depletion and analyzing the formation of metabolites. The results showed the nicotinamide–adenine dinucleotide phosphate (NADPH)-dependent depletion of buprofezin (eluting at 8.7 min) and parallel formation of an unknown metabolite (eluting 9.5 min). However, CYP353D1v2 is unable to metabolize deltamethrin and chlorpyrifos. The recombinant CYP353D1v2 protein efficiently catalyzed the model substrate p-nitroanisole with a maximum velocity of 9.24 nmol/min/mg of protein and a Michaelis constant of Km = 6.21 µM. In addition, imidacloprid was metabolized in vitro by the recombinant CYP353D1v2 microsomes (catalytic constant Kcat) 0.064 pmol/min/pmol P450, Km = 6.41 µM. The mass spectrum of UPLC-MS analysis shows that the metabolite was a product of buprofezin, which was buprofezin sulfone. This result provided direct evidence that L. striatellus cytochrome P450 CYP353D1v2 is capable of metabolizing imidacloprid and buprofezin. PMID:29186030

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Sadler, Natalie C.; Nandhikonda, Premchendar; Webb-Robertson, Bobbie-Jo M.

    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 proteomicmore » 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.« less

  19. Identification of novel cytochrome P450s in the Acari

    USDA-ARS?s Scientific Manuscript database

    Cytochrome P450s are the major phase I drug metabolising enzymes found in most organisms, including arthropods. Much of the work within the area of xenobiotic metabolism in this group of animals has centered around mosquito species, e.g. Anopheles gambiae and Culex quinquefasciatus, due to their rol...

  20. 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

  1. Structure and expression of the rat CYP3A1 gene: isolation of the gene (P450/6betaB) and characterization of the recombinant protein.

    PubMed

    Nagata, K; Ogino, M; Shimada, M; Miyata, M; Gonzalez, F J; Yamazoe, Y

    1999-02-15

    A P450 gene (P450/6betaB) of the CYP3A subfamily was isolated from a rat genomic library. Nucleotide sequencing of the exons revealed a high similarity with P450PCN1 cDNA (Gonzalez et al. (1985), J. Biol. Chem. 260, 7345-7441), but differed in 41 nucleotides, resulting in 11 changes and 2 deletions of amino acid residues. The P450/6betaB spanned about 30 kbp and consisted of 13 exons, and was in exon number and size identical with CYP3A2 gene except in the 6th exon, which was shorter than that of CYP3A2. 6beta-B mRNA, which may be transcribed from P450/6betaB, was detected on Northern blotting and by reverse transcription-polymerase chain reaction (RT-PCR). Profiles of the developmental change and induction by a treatment with several chemicals were very similar to those of P450PCN1 mRNA reported previously. P450PCN1 mRNA and gene, however, were not detected by PCR in rats. To determine whether P450/6betaB encodes an active protein, a cDNA was isolated and expressed. Expression of 6beta-B cDNA in COS-1 cells was carried out and revealed that the recombinant protein comigrated with purified P4506beta-4 previously identified as CYP3A1. The recombinant 6beta-B protein showed similar turnover rate and regioselectivity for testosterone with purified P4506beta-4 by the simultaneous addition of NADPH-cytochrome P450 reductase and cytochrome b5. These data suggest that P450/6betaB encodes an active P450 form corresponding to CYP3A1 and P450PCN1 reported previously does not exist in rats. Copyright 1999 Academic Press.

  2. 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).

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

    PubMed

    Renault, Hugues; Bassard, Jean-Etienne; Hamberger, Björn; Werck-Reichhart, Danièle

    2014-06-01

    Cytochromes P450 catalyze a broad range of regiospecific, stereospecific and irreversible steps in the biosynthetic routes of plant natural metabolites with important applications in pharmaceutical, cosmetic, fragrance and flavour, or polymer industries. They are consequently essential drivers for the engineered bioproduction of such compounds. Two ground-breaking developments of commercial products driven by the engineering of P450s are the antimalarial drug precursor artemisinic acid and blue roses or carnations. Tedious optimizations were required to generate marketable products. Hurdles encountered in P450 engineering and their potential solutions are summarized here. Together with recent technical developments and novel approaches to metabolic engineering, the lessons from this pioneering work should considerably boost exploitation of the amazing P450 toolkit emerging from accelerated sequencing of plant genomes. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

  4. Inhibitors of steroidal cytochrome p450 enzymes as targets for drug development.

    PubMed

    Baston, Eckhard; Leroux, Frédéric R

    2007-01-01

    Cytochrome P450's are enzymes which catalyze a large number of biological reactions, for example hydroxylation, N-, O-, S- dealkylation, epoxidation or desamination. Their substrates include fatty acids, steroids or prostaglandins. In addition, a high number of various xenobiotics are metabolized by these enzymes. The enzyme 17alpha-hydroxylase-C17,20-lyase (P450(17), CYP 17, androgen synthase), a cytochrome P450 monooxygenase, is the key enzyme for androgen biosynthesis. It catalyzes the last step of the androgen biosynthesis in the testes and adrenal glands and produces androstenedione and dehydroepiandrosterone from progesterone and pregnenolone. The microsomal enzyme aromatase (CYP19) transforms these androgens to estrone and estradiol. Estrogens stimulate tumor growth in hormone dependent breast cancer. In addition, about 80 percent of prostate cancers are androgen dependent. Selective inhibitors of these enzymes are thus important alternatives to treatment options like antiandrogens or antiestrogens. The present article deals with recent patents (focus on publications from 2000 - 2006) concerning P450 inhibitor design where steroidal substrates are involved. In this context a special focus is provided for CYP17 and CYP19. Mechanisms of action will also be discussed. Inhibitors of CYP11B2 (aldosterone synthase) will also be dealt with.

  5. Evidence that ligand formation is a mechanism underlying the maintenance of cytochrome P-450 in rat liver cell culture. Potent maintenance by metyrapone.

    PubMed Central

    Paine, A J; Villa, P; Hockin, L J

    1980-01-01

    The loss of cytochrome P-450 in cultured rat hepatocytes can be prevented by substituted pyridines, especially isonicotinamide, 3-hydroxypyridine and metyrapone. The effect of these compounds is independent of protein synthesis, suggesting that they maintain pre-existing cytochrome P-450. The efficiency of pyridines at maintaining cytochrome P-450 in hepatocyte culture is highly correlated with their ability to bind to this cytochrome, suggesting that ligand formation with cytochrome P-450 prevents its accelerated turnover in liver cell culture. PMID:7470047

  6. Cytochrome P450 drug interactions with statin therapy.

    PubMed

    Goh, Ivanna Xin Wei; How, Choon How; Tavintharan, Subramaniam

    2013-03-01

    Statins are commonly used in the treatment of hyperlipidaemia. Although the benefits of statins are well-documented, they have the potential to cause myopathy and rhabdomyolysis due to the complex interactions of drugs, comorbidities and genetics. The cytochrome P450 family consists of major enzymes involved in drug metabolism and bioactivation. This article aims to highlight drug interactions involving statins, as well as provide updated recommendations and approaches regarding the safe and appropriate use of statins in the primary care setting.

  7. 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

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

    PubMed

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

    2015-06-01

    Experimental studies indicate that short-term fasting alters drug metabolism. However, the effects of short-term fasting on drug metabolism in humans need further investigation. Therefore, the aim of this study was to evaluate the effects of short-term fasting (36 h) on P450-mediated drug metabolism. In a randomized crossover study design, nine healthy subjects ingested a cocktail consisting of five P450-specific probe drugs [caffeine (CYP1A2), S-warfarin (CYP2C9), omeprazole (CYP2C19), metoprolol (CYP2D6), and midazolam (CYP3A4)] on two occasions (control study after an overnight fast and after 36 h of fasting). Blood samples were drawn for pharmacokinetic analysis using nonlinear mixed effects modeling. In addition, we studied in Wistar rats the effects of short-term fasting on hepatic mRNA expression of P450 isoforms corresponding with the five studied P450 enzymes in humans. In the healthy subjects, short-term fasting increased oral caffeine clearance by 20% (P = 0.03) and decreased oral S-warfarin clearance by 25% (P < 0.001). In rats, short-term fasting increased mRNA expression of the orthologs of human CYP1A2, CYP2C19, CYP2D6, and CYP3A4 (P < 0.05), and decreased the mRNA expression of the ortholog of CYP2C9 (P < 0.001) compared with the postabsorptive state. These results demonstrate that short-term fasting alters cytochrome P450-mediated drug metabolism in a nonuniform pattern. Therefore, short-term fasting is another factor affecting cytochrome P450-mediated drug metabolism in humans. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

  9. Cytochrome P450 1B1 contributes to angiotensin II-induced hypertension and associated pathophysiology.

    PubMed

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

    2010-10-01

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

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

    PubMed Central

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

    2010-01-01

    Hypertension is the leading cause of cardiovascular diseases, and angiotensin II is one of the major components of the mechanisms that contribute to the development of hypertension. However, the precise mechanisms for the development of hypertension are unknown. Our recent study that angiotensin II-induced vascular smooth muscle cell growth is dependent on cytochrome P450 1B1 led us to investigate its contribution to hypertension caused by this peptide. Angiotensin II was infused via miniosmotic pump into rats (150 ng/kg/min) or mice (1000 μg/kg/day) for 13 days resulting in increased blood pressure, increased cardiac and vascular hypertrophy, increased vascular reactivity to vasoconstrictor agents, increased reactive oxygen species production, and endothelial dysfunction in both species. The increase in blood pressure and associated pathophysiological changes were minimized by the cytochrome P450 1B1 inhibitor, 2,3′,4,5′-tetramethoxystilbene in both species and was markedly reduced in Cyp1b1-/- mice. These data suggest that cytochrome P450 1B1 contributes to angiotensin II-induced hypertension and associated pathophysiological changes. Moreover, 2,3′,4,5′-tetramethoxystilbene which prevents both cytochrome P450 1B1-dependent and independent components of angiotensin II-induced hypertension and inhibits associated pathophysiological changes could be clinically useful in the treatment of hypertension and associated cardiovascular and inflammatory diseases. PMID:20805442

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    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 inmore » 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

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

    EPA Science Inventory

    The acute hepatotoxicity and response of hepatic cytochrome P450 to treatment with the three isomers of dichlorobenzene (DCB) have been investigated. The objectives were to estimate toxic thresholds and to further e1ucidate the role of cytochrome P450 in the metabolism and toxici...

  13. Effects of thalidomide, cytochrome P-450 and TNF-alpha on angiogenesis in a three-dimensional collagen gel-culture.

    PubMed

    Fujita, Keiko; Asami, Yoshiko; Murata, Eiko; Akita, Masumi; Kaneko, Katsuji

    2002-10-01

    The anti-angiogenic effects of thalidomide were examined in mouse aortae grown in a three-dimensional collagen gel-culture. In our in vitro model, (+/-)-thalidomide and (-)-thalidomide exhibited no anti-angiogenic effects. On the other hand, when the culture was treated with thalidomide plus cytochrome P-450, both types of thalidomides significantly inhibited angiogenesis. Co-administration of 100 microg/ml thalidomide plus 200 microg/ml cytochrome P-450 inhibited angiogenesis more strongly than thalidomide plus cytochrome P-450 at other concentrations (10 microg/ml + 200 microg/ml and 100 microg/ml + 20 microg/ml). To study the relation between the anti-angiogenic effect and TNF-alpha, we also evaluated the concentration of TNF-alpha in the culture medium. We found that the concentration of TNF-alpha was correlated to the strength of the anti-angiogenic effect. The inhibition of angiogenesis by thalidomide and cytochrome P-450 takes place through a suppression of TNF-alpha and involves the metabolism of the thalidomide.

  14. New reactions and products resulting from alternative interactions between the P450 enzyme and redox partners.

    PubMed

    Zhang, Wei; Liu, Yi; Yan, Jinyong; Cao, Shaona; Bai, Fali; Yang, Ying; Huang, Shaohua; Yao, Lishan; Anzai, Yojiro; Kato, Fumio; Podust, Larissa M; Sherman, David H; Li, Shengying

    2014-03-05

    Cytochrome P450 enzymes are capable of catalyzing a great variety of synthetically useful reactions such as selective C-H functionalization. Surrogate redox partners are widely used for reconstitution of P450 activity based on the assumption that the choice of these auxiliary proteins or their mode of action does not affect the type and selectivity of reactions catalyzed by P450s. Herein, we present an exceptional example to challenge this postulate. MycG, a multifunctional biosynthetic P450 monooxygenase responsible for hydroxylation and epoxidation of 16-membered ring macrolide mycinamicins, is shown to catalyze the unnatural N-demethylation(s) of a range of mycinamicin substrates when partnered with the free Rhodococcus reductase domain RhFRED or the engineered Rhodococcus-spinach hybrid reductase RhFRED-Fdx. By contrast, MycG fused with the RhFRED or RhFRED-Fdx reductase domain mediates only physiological oxidations. This finding highlights the larger potential role of variant redox partner protein-protein interactions in modulating the catalytic activity of P450 enzymes.

  15. Recollection of the early years of the research on cytochrome P450

    PubMed Central

    OMURA, Tsuneo

    2011-01-01

    Since the publication of the first paper on “cytochrome P450” in 1962, the biochemical research on this novel hemoprotein expanded rapidly in the 1960s and the 1970s as its principal roles in various important metabolic processes including steroid hormone biosynthesis in the steroidogenic organs and drug metabolism in the liver were elucidated. Establishment of the purification procedures of microsomal and mitochondrial P450s in the middle of the 1970s together with the introduction of molecular biological techniques accelerated the remarkable expansion of the research on P450 in the following years. This review paper summarizes the important developments in the research on P450 in the early years, for about two decades from the beginning, together with my personal recollections. PMID:22156409

  16. 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

  17. EVIDENCE FOR BROMODICHLOROMETHANE METABOLISM BY CYTOCHROME P-450 1A2

    EPA Science Inventory

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

    Bromodichlorometh...

  18. Microsomal P-450 induction by some secondary products from thermal oxidation of dietary lipids: epidermal hyperplasia, mutagenicity and cytochrome P-450 activities.

    PubMed

    Crawford, L; Wheeler, E L

    1983-12-01

    Distillable secondary products from roasted fowl were found to be cytotoxic but not mutagenic when assayed with Salmonella typhimurium strains TA98, TA100 and TA1537. A crudely separated fraction of the volatiles produced focal hyperplasia and damage to the epidermis of the backs of mice. The volatiles also caused an apparent synthesis of non-constitutive forms of rat hepatic cytochromes P-450 which metabolize benzo[a]pyrene B [a]P differently from the constitutive P-450.

  19. 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

  20. [Cytochrome P-450 and the response to antimalarial drugs].

    PubMed

    Guzmán, Valentina; Carmona-Fonseca, Jaime

    2006-01-01

    To assess the relationship between the genetic and phenotypic factors linked to the cytochrome P-450 enzyme system and the response to the antimalarial drugs chloroquine, amodiaquine, mefloquine, and proguanil, as well as to determine how certain biological and social factors of the host influence the behavior of this enzymatic complex. We performed a systematic review of the medical bibliographic databases PubMed, Excerpta Medica, LILACS, and SciELO by using the following Spanish and English descriptors: "CYP-450" and "citocromo P-450" in combination with "proguanil" (and with "mefloquina," "cloroquina," and "amodiaquina"), "farmacocinética de proguanil" (and the same using "mefloquina," "cloroquina," and "amodiaquina"), "resistencia a proguanil" (and the same using "mefloquina," "cloroquina," and "amodiaquina"), "metabolismo," "farmacogenética," "enfermedad," "inflamación," "infección," "enfermedad hepática," "malaria," "nutrición," and "desnutrición." The same terms were used in English. The search included only articles published in Spanish, English, and Portuguese on or before 30 June 2005 that dealt with only four antimalarial drugs: amodiaquine, chloroquine, mefloquine, and proguanil. Some genetic factors linked to human cytochrome P-450 (mainly its polymorphism), as well as other biological and social factors (the presence of disease itself, or of inflammation and infection, the use of antimalarials in their various combinations, and the patient's nutritional status) influence the behavior of this complex enzymatic system. It has only been in the last decade that the genetics of the cytochromes has been explored and that the mechanisms underlying some therapeutic interactions and aspects of drug metabolism have been uncovered, making it possible to characterize the biotransformation pathway of amodiaquine and chloroquine. Hopefully new research will help answer the questions that still remain, some of which pertain to the metabolism of other

  1. 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.

  2. Anti-liver-kidney microsome antibody type 1 recognizes human cytochrome P450 db1.

    PubMed

    Gueguen, M; Yamamoto, A M; Bernard, O; Alvarez, F

    1989-03-15

    Anti-liver-kidney microsome antibody type 1 (LKM1), present in the sera of a group of children with autoimmune hepatitis, was recently shown to recognize a 50 kDa protein identified as rat liver cytochromes P450 db1 and db2. High homology between these two members of the rat P450 IID subfamily and human P450 db1 suggested that anti-LKM1 antibody is directed against this human protein. To test this hypothesis, a human liver cDNA expression library in phage lambda GT-11 was screened using rat P450 db1 cDNA as a probe. Two human cDNA clones were found to be identical to human P450 db1 by restriction mapping. Immunoblot analysis using as antigen, the purified fusion protein from one of the human cDNA clones showed that only anti-LKM1 with anti-50 kDa reactivity recognized the fusion protein. This fusion protein was further used to develop an ELISA test that was shown to be specific for sera of children with this disease. These results: 1) identify the human liver antigen recognized by anti-LKM1 auto-antibodies as cytochrome P450 db1, 2) allow to speculate that mutation on the human P450 db1 gene could alter its expression in the hepatocyte and make it auto-antigenic, 3) provide a simple and specific diagnostic test for this disease.

  3. Immunohistochemical study of temporal variations in cytochrome P-450 isozymes in rat testis and their modifications by the inductive effects of cadinenes

    NASA Astrophysics Data System (ADS)

    Kobayashi, Yasuhito; Motohashi, Yutaka; Miyazaki, Yoshifumi; Yatagai, Mitsuyoshi; Takano, Takehito

    1991-12-01

    Temporal variations in cytochrome P-450 isozymes of rat testis, PB-P-450 (forms of cytochrome P-450 strongly induced by phenobarbital) and MC-P-448 (forms of cytochrome P-450 strongly induced by 3-methylcholanthrene), were investigated immunohistochemically by the avidin-biotin-complex method using specific antibodies against PB-P-450 and MC-P-448 isozymes. Immunoreactivity to both PB-P-450 and MC-P-448 isozymes was observed in Leydig cells. The number of PB-P-450 positive Leydig cells was found to undergo significant time-of-day variation with a peak time of 0000 hours (light phase from 0800 to 2000 hours). Injection of cadinenes (300 mg/kg per day intraperitoneally at 48 and 96 h before sacrifice) induced PB-P-450 isozyme but did not induce MC-P-448 isozyme. The induction of PB-P-450 isozyme by cadinenes was time dependent, and the early dark phase (2000 and 0000 hours) was most sensitive. These results suggest that temporal variation of cytochrome P-450 isozymes is one of the important physiological variations in detoxification and activation of various xenobiotics and chemicals in the testis.

  4. Cytochrome P450 Activity in Ex Vivo Cornea Models and a Human Cornea Construct.

    PubMed

    Kölln, Christian; Reichl, Stephan

    2016-07-01

    The pharmacokinetic behaviors of novel ophthalmic drugs are often preliminarily investigated in preclinical studies using ex vivo animal cornea or corneal cell culture models. During transcorneal passage, topically applied drugs may be affected by drug metabolizing enzymes. The knowledge regarding the functional expression of metabolic enzymes in corneal tissue is marginal; thus, the aim of this study was to investigate cytochrome P450 activity in an organotypic three-dimensional human cornea construct and to compare it with porcine and rabbit corneas, which are commonly used ex vivo cornea models. The total cytochrome P450 activity was determined by measuring the transformation of 7-ethoxycoumarin. Furthermore, the expression of the cytochrome P450 enzyme 2D6 (CYP2D6) was investigated at the protein level using immunohistochemistry and western blotting. CYP2D6 activity measurements were performed using a d-luciferin-based assay. In summary, similar levels of the total cytochrome P450 activity were identified in all 3 cornea models. The protein expression of CYP2D6 was confirmed in the human cornea construct and porcine cornea, whereas the signals in the rabbit cornea were weak. The analysis of the CYP2D6 activity indicated similar values for the human cornea construct and porcine cornea; however, a distinctly lower activity was observed in the rabbit cornea. Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

  5. Complexation of cytochrome P-450 isozymes in hepatic microsomes from SKF 525-A-induced rats.

    PubMed

    Murray, M

    1988-05-01

    Potassium ferricyanide-elicited reactivation of steroid hydroxylase activities, in hepatic microsomes from SKF 525-A-induced male rats, was used as an indicator of complex formation between individual cytochrome P-450 isozymes and the SKF 525-A metabolite. Induction of male rats with SKF 525-A (50 mg/kg for three days) led to apparent increases in androst-4-ene-3,17-dione 16 beta- and 6 beta-hydroxylation to 6.7- and 3-fold of control activities. Steroid 7 alpha-hydroxylase activity was decreased to 0.8-fold of control and 16 alpha-hydroxylation was unchanged. Ferricyanide-elicited dissociation of the SKF 525-A metabolite-P-450 complex revealed an even greater induction of 16 beta- and 6 beta-hydroxylase activities (to 1.8- and 1.6-fold of activities in the absence of ferricyanide). Androst-4-ene-3,17-dione 16 alpha-hydroxylase activity increased 2-fold after ferricyanide but 7 alpha-hydroxylase activity was unaltered. An antibody directed against the male-specific cytochrome P-450 UT-A decreased androst-4-ene-3,17-dione 16 alpha-hydroxylase activity to 13% of control in hepatic microsomes from untreated rats. In contrast, 16 alpha-hydroxylase activity in microsomes from SKF 525-A-induced rats, before and after dissociation with ferricyanide, was reduced by anti UT-A IgG to 32 and 19% of the respective uninhibited controls. Considered together, these observations strongly suggest that the phenobarbital-inducible cytochrome P-450 isozymes PB-B and PCN-E are present in an inactive complexed state in microsomes from SKF 525-A-induced rat liver. Further, the increased susceptibility of androst-4-ene-3,17-dione 16 alpha-hydroxylase activity to inhibition by an antibody to cytochrome P-450 UT-A, following ferricyanide treatment of microsomes, suggests that this male sexually differentiated enzyme is also complexed after in vivo SKF 525-A dosage. In contrast, the constitutive isozyme cytochrome P-450 UT-F, which is active in steroid 7 alpha-hydroxylation, does not appear

  6. Structure and Dynamics of the Membrane-Bound Cytochrome P450 2C9

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Cojocaru, Vlad; Balali-Mood, Kia; Sansom, Mark S.

    The microsomal, membrane-bound, human cytochrome P450 (CYP) 2C9 is a liver-specific monooxygenase essential for drug metabolism. CYPs require electron transfer from the membrane-bound CYP reductase (CPR) for catalysis. The structural details and functional relevance of the CYP-membrane interaction are not understood. From multiple coarse grained molecular simulations started with arbitrary configurations of protein-membrane complexes, we found two predominant orientations of CYP2C9 in the membrane, both consistent with experiments and conserved in atomic-resolution simulations. The dynamics of membrane-bound and soluble CYP2C9 revealed correlations between opening and closing of different tunnels from the enzyme’s buried active site. The membrane facilitated the openingmore » of a tunnel leading into it by stabilizing the open state of an internal aromatic gate. Other tunnels opened selectively in the simulations of product-bound CYP2C9. We propose that the membrane promotes binding of liposoluble substrates by stabilizing protein conformations with an open access tunnel and provide evidence for selective substrate access and product release routes in mammalian CYPs. The models derived here are suitable for extension to incorporate other CYPs for oligomerization studies or the CYP reductase for studies of the electron transfer mechanism, whereas the modeling procedure is generally applicable to study proteins anchored in the bilayer by a single transmembrane helix.« less

  7. An artificial self-sufficient cytochrome P450 directly nitrates fluorinated tryptophan analogs with a different regio-selectivity.

    PubMed

    Zuo, Ran; Zhang, Yi; Huguet-Tapia, Jose C; Mehta, Mishal; Dedic, Evelina; Bruner, Steven D; Loria, Rosemary; Ding, Yousong

    2016-05-01

    Aromatic nitration is an immensely important industrial process to produce chemicals for a variety of applications, but it often suffers from multiple unsolved challenges. Enzymes as biocatalysts have been increasingly used for organic chemistry synthesis due to their high selectivity and environmental friendliness, but nitration has benefited minimally from the development of biocatalysis. In this work, we aimed to develop TxtE as practical biocatalysts for aromatic nitration. TxtE is a unique class I cytochrome P450 enzyme that nitrates the indole of l-tryptophan. To develop cost-efficient nitration processes, we fused TxtE with the reductase domains of CYP102A1 (P450BM3) and of P450RhF to create class III self-sufficient biocatalysts. The best engineered fusion protein was comparable with wild type TxtE in terms of nitration performance and other key biochemical properties. To demonstrate the application potential of the fusion enzyme, we nitrated 4-F-dl-tryptophan and 5-F-l-tryptophan in large scale enzymatic reactions. Tandem MS/MS and NMR analyses of isolated products revealed altered nitration sites. To our knowledge, these studies represent the first practice in developing biological nitration approaches and lay a solid basis to the use of TxtE-based biocatalysts for the production of valuable nitroaromatics. Copyright © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. High-Fat Diets Alter the Modulatory Effects of Xenobiotics on Cytochrome P450 Activities

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Sadler, Natalie C.; Webb-Robertson, Bobbie-Jo M.; Clauss, Therese R.

    Cytochrome P450 monooxygenases (P450) are key to the metabolism of myriad endogenous chemicals and xenobiotics, including the majority of therapeutic drugs. Dysregulated P450 activities can lead to altered drug metabolism and toxicity, oxidative stress, and inflammation; all physiological states frequently charged as the impetus for various chronic pathologies. We characterized the impact of common xenobiotic exposures, specifically high-fat diet and active or passive cigarette smoke, on the functional capacity of hepatic and pulmonary P450s. We employed an activity-based protein profiling approach to characterize the identity and activity level of measured individual P450 isoforms. Our results confirm expectations of significant alterationsmore » in pulmonary P450s due to cigarette smoke, but now reveal the repressive impact of high-fat diet-induced obesity on many hepatic P450s activities, and the dynamic alterations due to concomitant diet and smoke exposures on liver and lung P450 activities impacting drug metabolism and pathways of inflammation.« less

  9. Transfer of the cytochrome P450-dependent dhurrin pathway from Sorghum bicolor into Nicotiana tabacum chloroplasts for light-driven synthesis

    PubMed Central

    Gnanasekaran, Thiyagarajan; Karcher, Daniel; Nielsen, Agnieszka Zygadlo; Martens, Helle Juel; Ruf, Stephanie; Kroop, Xenia; Olsen, Carl Erik; Motawie, Mohammed Saddik; Pribil, Mathias; Møller, Birger Lindberg; Bock, Ralph; Jensen, Poul Erik

    2016-01-01

    Plant chloroplasts are light-driven cell factories that have great potential to act as a chassis for metabolic engineering applications. Using plant chloroplasts, we demonstrate how photosynthetic reducing power can drive a metabolic pathway to synthesise a bio-active natural product. For this purpose, we stably engineered the dhurrin pathway from Sorghum bicolor into the chloroplasts of Nicotiana tabacum (tobacco). Dhurrin is a cyanogenic glucoside and its synthesis from the amino acid tyrosine is catalysed by two membrane-bound cytochrome P450 enzymes (CYP79A1 and CYP71E1) and a soluble glucosyltransferase (UGT85B1), and is dependent on electron transfer from a P450 oxidoreductase. The entire pathway was introduced into the chloroplast by integrating CYP79A1, CYP71E1, and UGT85B1 into a neutral site of the N. tabacum chloroplast genome. The two P450s and the UGT85B1 were functional when expressed in the chloroplasts and converted endogenous tyrosine into dhurrin using electrons derived directly from the photosynthetic electron transport chain, without the need for the presence of an NADPH-dependent P450 oxidoreductase. The dhurrin produced in the engineered plants amounted to 0.1–0.2% of leaf dry weight compared to 6% in sorghum. The results obtained pave the way for plant P450s involved in the synthesis of economically important compounds to be engineered into the thylakoid membrane of chloroplasts, and demonstrate that their full catalytic cycle can be driven directly by photosynthesis-derived electrons. PMID:26969746

  10. Effects of aging and rifampicin pretreatment on the pharmacokinetics of human cytochrome P450 probes caffeine, warfarin, omeprazole, metoprolol and midazolam in common marmosets genotyped for cytochrome P450 2C19.

    PubMed

    Toda, Akiko; Uehara, Shotaro; Inoue, Takashi; Utoh, Masahiro; Kusama, Takashi; Shimizu, Makiko; Uno, Yasuhiro; Mogi, Masayuki; Sasaki, Erika; Yamazaki, Hiroshi

    2018-07-01

    1. The pharmacokinetics were investigated for human cytochrome P450 probes after single intravenous and oral administrations of 0.20 and 1.0 mg/kg, respectively, of caffeine, warfarin, omeprazole, metoprolol and midazolam to aged (10-14 years old, n = 4) or rifampicin-treated/young (3 years old, n = 3) male common marmosets all genotyped as heterozygous for a cytochrome P450 2C19 variant. 2. Slopes of the plasma concentration-time curves after intravenous administration of warfarin and midazolam were slightly, but significantly (two-way analysis of variance), decreased in aged marmosets compared with young marmosets. The mean hepatic clearances determined by in silico fitting for individual pharmacokinetic models of warfarin and midazolam in the aged group were, respectively, 23% and 56% smaller than those for the young group. 3. Significantly enhanced plasma clearances of caffeine, warfarin, omeprazole and midazolam were evident in young marmosets pretreated with rifampicin (25 mg/kg daily for 4 days). Two- to three-fold increases in hepatic intrinsic clearance values were observed in the individual pharmacokinetic models. 4. The in vivo dispositions of multiple simultaneously administered drugs in old, young and P450-enzyme-induced marmosets were elucidated. The results suggest that common marmosets could be experimental models for aged, induced or polymorphic P450 enzymes in P450-dependent drug metabolism studies.

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

    PubMed

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

    2002-01-01

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

  12. An improved microphotometry system for measurement of cytochrome P-450 in hepatocyte cytoplasm.

    PubMed

    Watanabe, J; Kanamura, S

    1991-05-01

    To measure cytochrome P-450 (P-450) content in hepatocyte cytoplasm, we developed a dual monochromator-equipped microphotometry system (KWSP-1). Simultaneous measurements of absorbance at 450 and 490 nm with narrow band width (0.5 nm) and small spot size (2 microns) were accomplished by this system. Corresponding fields in serial sections could be easily and rapidly identified under the Nomarski imaging mode of KWSP-1. Photometric accuracy and repeatability of wavelength setting of KWSP-1 were also satisfactory for measurement of P-450. With this system, it is thus possible to measure the extinction of P-450 from many small measuring areas and to precisely determine P-450 content in the cytoplasm of rat hepatocytes. A microphotometric method was developed using cuvette slides and two serial 10-microns thick sections (mapping method). The intracellular distribution of P-450 in individual hepatocytes could be visualized by the mapping method with KWSP-1. However, this method was not applicable to tissue sections containing hemoglobin larger than 4 microM.

  13. Inkjet-printed selective microfluidic biosensor using CNTs functionalized by cytochrome P450 enzyme

    NASA Astrophysics Data System (ADS)

    Krivec, Matic; Leitner, Raimund; Überall, Florian; Hochleitner, Johannes

    2017-05-01

    An additive manufacturing concept, consisting of 3D photopolymer printing and Ag nanoparticle printing, was investigated for the construction of a microfluidic biosensor based on immobilized cytochrome P450 enzyme. An acylate-type microfluidic chamber composed of two parts, i.e. chamber-housing and chamber-lid was printed with a polyjet 3D printer. A 3-electrode sensor structure was inkjet-printed on the lid using a combination of Ag and graphene printing. The working electrode was covered with carbon nanotubes by drop-casting and immobilized with cytochrome P450 2D6 enzyme. The microfluidic sensor shows a significant response to a test xenobiotic, i.e. dextromethorphan; the cyclic voltammetrical measurements show a corresponding oxidation peak at 0.4 V with around 5 μM detection limit.

  14. The regulation of cytochrome P450 2E1 during LPS-induced inflammation in the rat

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Abdulla, Dalya; Goralski, Kerry B.; College of Pharmacy, Burbidge Building, Dalhousie University, Halifax, Nova Scotia, B3H 3J5

    2006-10-01

    It is well known that inflammatory and infectious conditions differentially regulate cytochrome P450 (P450)-mediated drug metabolism in the liver. We have previously outlined a potential pathway for the downregulation in hepatic cytochrome P450 following LPS-mediated inflammation in the CNS (Abdulla, D., Goralski, K.B., Garcia Del Busto Cano, E., Renton, K.W., 2005. The signal transduction pathways involved in hepatic cytochrome P450 regulation in the rat during an LPS-induced model of CNS inflammation. Drug Metab. Dispos). The purpose of this study was to outline the effects of LPS-induced peripheral and central nervous system inflammation on hepatic cytochrome P450 2E1 (CYP2E1) in vivo,more » an enzyme that plays an important role in various physiological and pathological states. We report an increase in hepatic mRNA expression of CYP2E1 that occurred as early as 2-3 h following either the intraperitoneal (i.p.) injection of 5 mg/kg LPS or i.c.v. administration of 25 {mu}g of LPS. This increase in CYP2E1 mRNA expression was sustained for 24 h. In sharp contrast to the increase in hepatic CYP2E1 mRNA, we observed a significant reduction in the catalytic activity of this enzyme 24 h following either the i.c.v. or i.p. administration of LPS. Cycloheximide or actinomycin-D did not change the LPS-mediated downregulation in hepatic CYP2E1 catalytic activity. Our results support the idea that LPS acts at two different levels to regulate hepatic CYP2E1: a transcriptional level to increase CYP2E1 mRNA expression and a post-transcriptional level to regulate CYP2E1 protein and activity.« less

  15. Hepatotoxicity of Herbal Supplements Mediated by Modulation of Cytochrome P450

    PubMed Central

    Chen, Taosheng

    2017-01-01

    Herbal supplements are a significant source of drug-drug interactions (DDIs), herb-drug interactions, and hepatotoxicity. Cytochrome P450 (CYP450) enzymes metabolize a large number of FDA-approved pharmaceuticals and herbal supplements. This metabolism of pharmaceuticals and supplements can be augmented by concomitant use of either pharmaceuticals or supplements. The xenobiotic receptors constitutive androstane receptor (CAR) and the pregnane X receptor (PXR) can respond to xenobiotics by increasing the expression of a large number of genes that are involved in the metabolism of xenobiotics, including CYP450s. Conversely, but not exclusively, many xenobiotics can inhibit the activity of CYP450s. Induction of the expression or inhibition of the activity of CYP450s can result in DDIs and toxicity. Currently, the United States (US) Food and Drug Administration does not require the investigation of the interactions of herbal supplements and CYP450s. This review provides a summary of herbal supplements that inhibit CYP450s, induce the expression of CYP450s, and/or whose toxicity is mediated by CYP450s. PMID:29117101

  16. Engineering and improvement of the efficiency of a chimeric [P450cam-RhFRed reductase domain] enzyme.

    PubMed

    Robin, Aélig; Roberts, Gareth A; Kisch, Johannes; Sabbadin, Federico; Grogan, Gideon; Bruce, Neil; Turner, Nicholas J; Flitsch, Sabine L

    2009-05-14

    A chimeric oxygenase, in which the P450cam domain was fused to the reductase host domains of a P450RhF from Rhodococcus sp. strain NCIMB 9784 was optimised to allow for a biotransformation at 30 mM substrate in 80% overall yield, with the linker region between P450 and FMN domain proving to be important for the effective biotransformation of (+)-camphor to 5-exo-hydroxycamphor.

  17. Contribution of cytochrome P450 1B1 to hypertension and associated pathophysiology: a novel target for antihypertensive agents.

    PubMed

    Malik, Kafait U; Jennings, Brett L; Yaghini, Fariborz A; Sahan-Firat, Seyhan; Song, Chi Young; Estes, Anne M; Fang, Xiao R

    2012-08-01

    The aim of this review is to discuss the contribution of cytochrome P450 (CYP) 1B1 in vascular smooth muscle cell growth, hypertension, and associated pathophysiology. CYP1B1 is expressed in cardiovascular and renal tissues, and mediates angiotensin II (Ang II)-induced activation of NADPH oxidase and generation of reactive oxygen species (ROS), and vascular smooth muscle cell migration, proliferation, and hypertrophy. Moreover, CYP1B1 contributes to the development and/or maintenance of hypertension produced by Ang II-, deoxycorticosterone (DOCA)-salt-, and N(ω)-nitro-L-arginine methyl ester-induced hypertension and in spontaneously hypertensive rats. The pathophysiological changes, including cardiovascular hypertrophy, increased vascular reactivity, endothelial and renal dysfunction, injury and inflammation associated with Ang II- and/or DOCA-salt induced hypertension in rats, and Ang II-induced hypertension in mice are minimized by inhibition of CYP1B1 activity with 2,4,3',5'-tetramethoxystilbene or by Cyp1b1 gene disruption in mice. These pathophysiological changes appear to be mediated by increased production of ROS via CYP1B1-dependent NADPH oxidase activity and extracellular signal-regulated kinase 1/2, p38 mitogen-activated protein kinase, and c-Src. Copyright © 2011 Elsevier Inc. All rights reserved.

  18. Contribution of Cytochrome P450 1B1 to Hypertension and Associated Pathophysiology: A Novel Target for Antihypertensive Agents

    PubMed Central

    Malik, Kafait U.; Jennings, Brett L.; Yaghini, Fariborz A.; Sahan-Firat, Seyhan; Song, Chi Young; Estes, Anne M.; Fang, Xiao R.

    2012-01-01

    The aim of this review is to discuss the contribution of cytochrome P450 (CYP) 1B1 in vascular smooth muscle cell growth, hypertension, and associated pathophysiology. CYP1B1 is expressed in cardiovascular and renal tissues, and mediates angiotensin II (Ang II)-induced activation of NADPH oxidase and generation of reactive oxygen species (ROS), and vascular smooth muscle cell migration, proliferation, and hypertrophy. Moreover, CYP1B1 contributes to the development and/or maintenance of hypertension produced by Ang II-, deoxycorticosterone Nω-nitro-(DOCA)-salt-, and L-arginine methyl ester-induced hypertension and in spontaneously hypertensive rats. The pathophysiological changes, including cardiovascular hypertrophy, increased vascular reactivity, endothelial and renal dysfunction, injury and inflammation associated with Ang II- and/or DOCA-salt induced hypertension in rats, and Ang II-induced hypertension in mice are minimized by inhibition of CYP1B1 activity with 2,4,3′,5′-tetramethoxystilbene or by Cyp1b1 gene disruption in mice. These pathophysiological changes appear to be mediated by increased production of ROS via CYP1B1-dependent NADPH oxidase activity and extracellular signal-regulated kinase 1/2, p38 mitogen-activated protein kinase, and c-Src. PMID:22210049

  19. Identification of two new cytochrome P450 genes and RNA interference to evaluate their roles in detoxification of commonly used insecticides in Locusta migratoria.

    PubMed

    Guo, Yanqiong; Zhang, Jianzhen; Yu, Rongrong; Zhu, Kun Yan; Guo, Yaping; Ma, Enbo

    2012-05-01

    Cytochrome P450 monooxygenases (cytochrome P450s), found in virtually all living organisms, play an important role in the metabolism of xenobiotics such as drugs, pesticides, and plant toxins. We have previously evaluated the responses of the oriental migratory locust (Locusta migratoria) to the pyrethroid insecticide deltamethrin and revealed that increased cytochrome P450 enzyme activity was due to increased transcription of multiple cytochrome P450 genes. In this study, we identified for the first time two new cytochrome P450 genes, which belong to two novel cytochrome P450 gene families. CYP409A1 belongs to CYP409 family whereas CYP408B1 belongs to CYP408 family. Our molecular analysis indicated that CYP409A1 was mainly expressed in fatbodies, midgut, gastric caecum, foregut and Malpighian tubules of the third- and fourth-instar nymphs, whereas CYP408B1 was mainly expressed in foregut, hindgut and muscle of the insects at all developmental stages examined. The expression of these two cytochrome P450 genes were differentially affected by three representative insecticides, including carbaryl (carbamate), malathion (organophosphate) and deltamethrin (pyrethroid). The exposure of the locust to carbaryl, malathion and deltamethrin resulted in reduced, moderately increased and significantly increased transcript levels, respectively, of the two cytochrome P450 genes. Our further analysis of their detoxification roles by using RNA interference followed by deltamethrin bioassay showed increased nymph mortalities by 21.1% and 16.7%, respectively, after CYP409A1 and CYP408B1 were silenced. These results strongly support our notion that these two new cytochrome P450 genes play an important role in deltamethrin detoxification in the locust. Copyright © 2011 Elsevier Ltd. All rights reserved.

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

    PubMed Central

    Pflugmacher, Stephan; Sandermann, Heinrich

    1998-01-01

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

  1. Transfer of the cytochrome P450-dependent dhurrin pathway from Sorghum bicolor into Nicotiana tabacum chloroplasts for light-driven synthesis.

    PubMed

    Gnanasekaran, Thiyagarajan; Karcher, Daniel; Nielsen, Agnieszka Zygadlo; Martens, Helle Juel; Ruf, Stephanie; Kroop, Xenia; Olsen, Carl Erik; Motawie, Mohammed Saddik; Pribil, Mathias; Møller, Birger Lindberg; Bock, Ralph; Jensen, Poul Erik

    2016-04-01

    Plant chloroplasts are light-driven cell factories that have great potential to act as a chassis for metabolic engineering applications. Using plant chloroplasts, we demonstrate how photosynthetic reducing power can drive a metabolic pathway to synthesise a bio-active natural product. For this purpose, we stably engineered the dhurrin pathway from Sorghum bicolor into the chloroplasts of Nicotiana tabacum (tobacco). Dhurrin is a cyanogenic glucoside and its synthesis from the amino acid tyrosine is catalysed by two membrane-bound cytochrome P450 enzymes (CYP79A1 and CYP71E1) and a soluble glucosyltransferase (UGT85B1), and is dependent on electron transfer from a P450 oxidoreductase. The entire pathway was introduced into the chloroplast by integrating CYP79A1, CYP71E1, and UGT85B1 into a neutral site of the N. tabacum chloroplast genome. The two P450s and the UGT85B1 were functional when expressed in the chloroplasts and converted endogenous tyrosine into dhurrin using electrons derived directly from the photosynthetic electron transport chain, without the need for the presence of an NADPH-dependent P450 oxidoreductase. The dhurrin produced in the engineered plants amounted to 0.1-0.2% of leaf dry weight compared to 6% in sorghum. The results obtained pave the way for plant P450s involved in the synthesis of economically important compounds to be engineered into the thylakoid membrane of chloroplasts, and demonstrate that their full catalytic cycle can be driven directly by photosynthesis-derived electrons. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  2. [Participation of nitric oxide and arachidonic acid metabolites via cytochrome - P450 in the regulation of arterial blood pressure].

    PubMed

    Sánchez-Mendoza, M Alicia; Martínez-Ayala, Sonia O; Hernández-Hernández, José A; Zúñiga-Sosa, Leonor; Pastelín-Hernández, Gustavo; Escalante-Acosta, Bruno A

    2003-01-01

    Nitric oxide and cytochrome P450 arachidonic acid metabolites participate in blood pressure regulation. The synthesis of these autacoids leads to arterial hypertension. However, it is not known whether there is an interaction between them. Therefore, we studied the modulatory effect of nitric oxide and cytochrome P450-arachidonic acid metabolites, their interaction on blood pressure, and the renal content of cytochrome P450. Male Wistar rats were divided: 1) control, 2) L-NAME (100 mg/kg/d p.o.), 3) L-NAME + SnCl2 (10 mg/kg/d i.p.), and 4) L-NAME + dexamethasone (1 mg/kg/d s.c.). We measured blood pressure and collected urine and blood for nitric oxide measurement. NO2 was quantified by HPLC. Blood pressure was: control, 97 +/- 7 mmHg; L-NAME, 151 +/- 4.6 mmHg; L-NAME + SnCl2, 133 +/- 3 mmHg, and L-NAME + dexamethasone 152 +/- 4.5 mmHg. Urine nitrite concentration was: 1) 1.832 +/- 0.32, 2) 1.031 +/- 0.23, 3) 1.616 +/- 0.33, and 4) 1.244 +/- 0.33 mumol/mL, while the concentration in blood was: 1) 0.293 +/- 0.06, 2) 0.150 +/- 0.05, 3) 0.373 +/- 0.13, and 4) 0.373 +/- 0.07 mumol/mL. L-NAME + SnCl2 decreased cytochrome P450 renal content, and L-NAME + dexamethasone showed a similar response. In conclusion, both, nitric oxide and CYP-arachidonic acid metabolites play a role in the regulation of blood pressure. Nitric oxide also partially regulates renal cytochrome P450 content.

  3. Isolation of the heme-thiolate enzyme cytochrome P-450TYR, which catalyzes the committed step in the biosynthesis of the cyanogenic glucoside dhurrin in Sorghum bicolor (L.) Moench.

    PubMed Central

    Sibbesen, O; Koch, B; Halkier, B A; Møller, B L

    1994-01-01

    The cytochrome P-450 enzyme (hemethiolate enzyme) that catalyzes the N-hydroxylation of L-tyrosine to N-hydroxytyrosine, the committed step in the biosynthesis of the cyanogenic glucoside dhurrin, has been isolated from microsomes prepared from etiolated seedlings of Sorghum bicolor (L.) Moench. The cytochrome P-450 enzyme was solubilized with the detergents Renex 690, reduced Triton X-100, and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate and isolated by ion-exchange (DEAE-Sepharose) and dye (Cibacron blue and reactive red 120) column chromatography. To prevent irreversible aggregation of the cytochrome P-450 enzyme, the isolation procedure was designed without any concentration step--i.e., with dilution of the ion-exchange gel with gel filtration material. The isolated enzyme, which we designate the cytochrome P-450TYR enzyme, gives rise to the specific formation of a type I substrate binding spectrum in the presence of L-tyrosine. The microsomal preparation contains 0.2 nmol of total cytochrome P-450/mg of protein. The cytochrome P-450TYR enzyme is estimated to constitute approximately 20% of the total cytochrome P-450 content of the microsomal membranes and about 0.2% of their total protein content. The apparent molecular mass of the cytochrome P-450TYR enzyme is 57 kDa, and the N-terminal amino acid sequence is ATMEVEAAAATVLAAP. A polyclonal antibody raised against the isolated cytochrome P-450TYR enzyme is specific as monitored by Western blot analysis and inhibits the in vitro conversion of L-tyrosine to p-hydroxymandelonitrile catalyzed by the microsomal system. The cytochrome P-450TYR enzyme exhibits high substrate specificity and acts as an N-hydroxylase on a single endogenous substrate. The reported isolation procedure based on dye columns constitutes a gentle isolation method for cytochrome P-450 enzymes and is of general use as indicated by its ability to separate cytochrome P-450TYR from the cytochrome P-450 enzyme catalyzing the C

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

    PubMed Central

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

    1994-01-01

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

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

    USGS Publications Warehouse

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

    1992-01-01

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

  6. 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

    2017-05-01

    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. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Regulation of Porcine Hepatic Cytochrome P450 — Implication for Boar Taint

    PubMed Central

    Rasmussen, Martin Krøyer; Zamaratskaia, Galia

    2014-01-01

    Cytochrome P450 (CYP450) is the major family of enzymes involved in the metabolism of several xenobiotic and endogenous compounds. Among substrates for CYP450 is the tryptophan metabolite skatole (3-methylindole), one of the major contributors to the off-odour associated with boar-tainted meat. The accumulation of skatole in pigs is highly dependent on the hepatic clearance by CYP450s. In recent years, the porcine CYP450 has attracted attention both in relation to meat quality and as a potential model for human CYP450. The molecular regulation of CYP450 mRNA expression is controlled by several nuclear receptors and transcription factors that are targets for numerous endogenously and exogenously produced agonists and antagonists. Moreover, CYP450 expression and activity are affected by factors such as age, gender and feeding. The regulation of porcine CYP450 has been suggested to have more similarities with human CYP450 than other animal models, including rodents. This article reviews the available data on porcine hepatic CYP450s and its implications for boar taint. PMID:25408844

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

    PubMed

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

    2013-12-03

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

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

    PubMed Central

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

    2013-01-01

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

  10. Detection and diversity of fungal nitric oxide reductase genes ( p450nor) in agricultural soils

    DOE PAGES

    Higgins, Steven A.; Welsh, Allana; Orellana, Luis H.; ...

    2016-03-11

    Members of the Fungi convert nitrate (NO 3 -) and nitrite (NO 2 -) to gaseous nitrous oxide (N 2O) (denitrification), but the fungal contributions to N-loss from soil remain uncertain. Cultivation-based methodologies that include antibiotics to selectively assess fungal activities have limitations and complementary molecular approaches to assign denitrification potential to fungi are desirable. Microcosms established with soils from two representative U.S. Midwest agricultural regions produced N 2O from added NO 3 - or NO 2 - in the presence of antibiotics to inhibit bacteria. Cultivation efforts yielded 214 fungal isolates belonging to at least 15 distinct morphological groups,more » of which 151 produced N 2O from NO 2 -. Novel PCR primers targeting the p450nor gene that encodes the nitric oxide (NO) reductase responsible for N 2O production in fungi yielded 26 novel p450nor amplicons from DNA of 37 isolates and 23 amplicons from environmental DNA obtained from two agricultural soils. The sequences shared 54-98% amino acid identity to reference P450nor sequences within the phylum Ascomycota, and expand the known fungal P450nor sequence diversity. p450nor was detected in all fungal isolates that produced N 2O from nitrite, whereas nirK (encoding the NO-forming nitrite reductase) was amplified in only 13-74% of the N 2O-forming isolates using two separate nirK primer sets. Altogether, our findings demonstrate the value of p450nor-targeted PCR to complement existing approaches to assess the fungal contributions to denitrification and N 2O formation.« less

  11. Detection and diversity of fungal nitric oxide reductase genes ( p450nor) in agricultural soils

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Higgins, Steven A.; Welsh, Allana; Orellana, Luis H.

    Members of the Fungi convert nitrate (NO 3 -) and nitrite (NO 2 -) to gaseous nitrous oxide (N 2O) (denitrification), but the fungal contributions to N-loss from soil remain uncertain. Cultivation-based methodologies that include antibiotics to selectively assess fungal activities have limitations and complementary molecular approaches to assign denitrification potential to fungi are desirable. Microcosms established with soils from two representative U.S. Midwest agricultural regions produced N 2O from added NO 3 - or NO 2 - in the presence of antibiotics to inhibit bacteria. Cultivation efforts yielded 214 fungal isolates belonging to at least 15 distinct morphological groups,more » of which 151 produced N 2O from NO 2 -. Novel PCR primers targeting the p450nor gene that encodes the nitric oxide (NO) reductase responsible for N 2O production in fungi yielded 26 novel p450nor amplicons from DNA of 37 isolates and 23 amplicons from environmental DNA obtained from two agricultural soils. The sequences shared 54-98% amino acid identity to reference P450nor sequences within the phylum Ascomycota, and expand the known fungal P450nor sequence diversity. p450nor was detected in all fungal isolates that produced N 2O from nitrite, whereas nirK (encoding the NO-forming nitrite reductase) was amplified in only 13-74% of the N 2O-forming isolates using two separate nirK primer sets. Altogether, our findings demonstrate the value of p450nor-targeted PCR to complement existing approaches to assess the fungal contributions to denitrification and N 2O formation.« less

  12. The interaction of representative members from two classes of antimycotics--the azoles and the allylamines--with cytochromes P-450 in steroidogenic tissues and liver.

    PubMed

    Schuster, I

    1985-06-01

    Spectrophotometric studies with ketoconazole, clotrimazole and miconazole show strong type-II interactions with several cytochromes P-450, particularly (Ks greater than 10(7)M-1; pH7.4; 25 degrees C) with the 11 beta-hydroxylase of adrenal mitochondria, with the 17 alpha/20 lyase of testis microsomes and with some forms of cytochromes P-450 of liver. A tight binding of the azoles also occurs to the reduced cytochromes, giving rise to an impeded CO binding to the haem iron. The binding of the azoles to 11 beta-hydroxylase and 17 alpha/20 lyase is much tighter than the binding of endogenous substrates, and consequently inhibition of steroidogenesis will occur at these sites. The metabolism of xenobiotic substrates by the cytochromes P-450 of liver will also be severely impeded. In contrast, the allylamines naftifine and SF 86-327 are type-I substrates for a small portion of cytochromes P-450 of liver microsomes only and there is no spectral evidence for binding to the cytochromes P-450 involved in steroid biosynthesis.

  13. Identification of three cytochrome P450 genes in the Chagas' disease vector Triatoma infestans: Expression analysis in deltamethrin susceptible and resistant populations.

    PubMed

    Grosso, Carla G; Blariza, María J; Mougabure-Cueto, Gastón; Picollo, María I; García, Beatriz A

    2016-10-01

    Cytochrome P450 monooxygenases play a predominant role in the metabolism of insecticides. Many insect P450 genes have frequently been associated with detoxification processes allowing the insect to become tolerant or resistant to insecticides. The increases of expression of P450 genes at transcriptional level are often consider responsible for increasing the metabolism of insecticides and seems to be a common phenomenon in the evolution of resistance development in insects. As pyrethroid resistance has been detected in Triatoma infestans, it was of interest to analyze genes associated with resistance to insecticides such as those encoding for cytochromes P450. With this purpose, the cDNA sequences of three cytochrome P450 genes (CYP4EM7, CYP3085B1, and CYP3092A6) were identified in this species. Primers and specific Taqman probes were designed from these sequences to determine their expression by quantitative PCR. The mRNA levels of the cytochrome P450 genes identified were determined from total RNA extracted from pools of fat body collected from individuals of different resistant and susceptible strains of T. infestans, and at different interval times after the topical application of the lethal doses 50% (LD50) of deltamethrin on the ventral abdomen of insects belonging to the different populations analyzed. It was detected overexpression of the CYP4EM7 gene in the most resistant strain of T. infestans and the expression of the three cytochrome P450 genes isolated was induced by deltamethrin in the susceptible and resistant populations included in this study. These results suggest that these genes would be involved in the detoxification of deltamethrin and support the hypothesis that considers to the cytochrome P450 genes of importance in the development of pyrethroid resistance. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. Molecular and functional characterization of CYP6BQ23, a cytochrome P450 conferring resistance to pyrethroids in European populations of pollen beetle, Meligethes aeneus.

    PubMed

    Zimmer, Christoph T; Bass, Chris; Williamson, Martin S; Kaussmann, Martin; Wölfel, Katharina; Gutbrod, Oliver; Nauen, Ralf

    2014-02-01

    The pollen beetle (Meligethes aeneus F.) is widespread throughout much of Europe where it is a major coleopteran pest of oilseed rape (Brassica napus). The reliance on synthetic insecticides for control, particularly the pyrethroid class, has led to the development of populations with high levels of resistance. Resistance to pyrethroids is now widespread throughout Europe and is thought to be mediated by enhanced detoxification by cytochrome P450ś and/or mutation of the pyrethroid target-site, the voltage-gated sodium channel. However, in the case of cytochrome P450 mediated detoxification, the specific enzyme(s) involved has (have) not yet been identified. In this study a degenerate PCR approach was used to identify ten partial P450 gene sequences from pollen beetle. Quantitative PCR was then used to examine the level of expression of these genes in a range of pollen beetle populations that showed differing levels of resistance to pyrethroids in bioassays. The study revealed a single P450 gene, CYP6BQ23, which is significantly and highly overexpressed (up to ∼900-fold) in adults and larvae of pyrethroid resistant strains compared to susceptible strains. CYP6BQ23 overexpression is significantly correlated with both the level of resistance and with the rate of deltamethrin metabolism in microsomal preparations of these populations. Functional recombinant expression of full length CYP6BQ23 along with cytochrome P450 reductase in an insect (Sf9) cell line showed that it is able to efficiently metabolise deltamethrin to 4-hydroxy deltamethrin. Furthermore we demonstrated by detection of 4-hydroxy tau-fluvalinate using ESI-TOF MS/MS that functionally expressed CYP6BQ23 also metabolizes tau-fluvalinate. A protein model was generated and subsequent docking simulations revealed the predicted substrate-binding mode of both deltamethrin and tau-fluvalinate to CYP6BQ23. Taken together these results strongly suggest that the overexpression of CYP6BQ23 is the primary

  15. 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.

  16. Evidence for induction of cytochrome P-450I in patients with tropical chronic pancreatitis.

    PubMed

    Chaloner, C; Sandle, L N; Mohan, V; Snehalatha, C; Viswanathan, M; Braganza, J M

    1990-06-01

    Theophylline kinetics, as an in vivo probe for the potentially toxic cytochrome P-450I pathway of drug metabolism, were studied in 11 healthy volunteers and 11 patients with calcific chronic pancreatitis at Madras, South India. Theophylline clearance was faster in the patients than controls [median 69 (range 39-114) vs 45 (33-56) ml h-1 kg-1, p = 0.003]. In keeping with this finding, detailed social histories identified a higher exposure level in the patients to xenobiotics that are inducers of cytochrome P-450I and/or yield reactive metabolites upon processing thereby (score 7, 4-11 vs 3, 2-9, p = 0.002). However, the concentration of D-glucaric acid in urine, as a marker of phase II conjugating pathways of drug metabolism, was similar in patients and controls. This pattern of drug metabolism could predispose to oxidant stress: hence micronutrient antioxidant supplements may have therapeutic (or even prophylactic) value in tropical chronic pancreatitis.

  17. 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)

  18. Molecular cloning of a family of xenobiotic-inducible drosophilid cytochrome P450s: Evidence for involvement in host-plant allelochemical resistance

    PubMed Central

    Danielson, Phillip B.; MacIntyre, Ross J.; Fogleman, James C.

    1997-01-01

    Cytochrome P450s constitute a superfamily of genes encoding mostly microsomal hemoproteins that play a dominant role in the metabolism of a wide variety of both endogenous and foreign compounds. In insects, xenobiotic metabolism (i.e., metabolism of insecticides and toxic natural plant compounds) is known to involve members of the CYP6 family of cytochrome P450s. Use of a 3′ RACE (rapid amplification of cDNA ends) strategy with a degenerate primer based on the conserved cytochrome P450 heme-binding decapeptide loop resulted in the amplification of four cDNA sequences representing another family of cytochrome P450 genes (CYP28) from two species of isoquinoline alkaloid-resistant Drosophila and the cosmopolitan species Drosophila hydei. The CYP28 family forms a monophyletic clade with strong regional homologies to the vertebrate CYP3 family and the insect CYP6 family (both of which are involved in xenobiotic metabolism) and to the insect CYP9 family (of unknown function). Induction of mRNA levels for three of the CYP28 cytochrome P450s by toxic host-plant allelochemicals (up to 11.5-fold) and phenobarbital (up to 49-fold) corroborates previous in vitro metabolism studies and suggests a potentially important role for the CYP28 family in determining patterns of insect–host-plant relationships through xenobiotic detoxification. PMID:9380713

  19. 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.

  20. NADPH-dependent coenzyme Q reductase is the main enzyme responsible for the reduction of non-mitochondrial CoQ in cells.

    PubMed

    Takahashi, Takayuki; Okuno, Masaaki; Okamoto, Tadashi; Kishi, Takeo

    2008-01-01

    We purified an NADPH-dependent coenzyme Q reductase (NADPH-CoQ reductase) in rat liver cytosol and compared its enzymatic properties with those of the other CoQ10 reductases such as NADPH: quinone acceptor oxidoreductase 1 (NQO1), lipoamide dehydrogenase, thioredoxine reductase and glutathione reductase. NADPH-CoQ reductase was the only enzyme that preferred NADPH to NADH as an electron donor and was also different from the other CoQ10 reductases in the sensitivities to its inhibitors and stimulators. Especially, Zn2+ was the most powerful inhibitor for NADPH-CoQ reductase, but CoQ10 reduction by the other CoQ10 reductases could not be inhibited by Zn2+. Furthermore, the reduction of the CoQ9 incorporated into HeLa cells was also inhibited by Zn2+ in the presence of pyrithione, a zinc ionophore. Moreover, NQO1 gene silencing in HeLa cells by transfection of a small interfering RNA resulted in lowering of both the NQO1 protein level and the NQO1 activity by about 75%. However, this transfection did not affect the NADPH-CoQ reductase activity and the reduction of CoQ9 incorporated into the cells. These results suggest that the NADPH-CoQ reductase located in cytosol may be the main enzyme responsible for the reduction of non-mitochondrial CoQ in cells.

  1. Functional analysis of human cytochrome P450 21A2 variants involved in congenital adrenal hyperplasia

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wang, Chunxue; Pallan, Pradeep S.; Zhang, Wei

    Cytochrome P450 (P450, CYP) 21A2 is the major steroid 21-hydroxylase, converting progesterone to 11-deoxycorticosterone and 17α-hydroxyprogesterone (17α-OH-progesterone) to 11-deoxycortisol. More than 100 CYP21A2 variants give rise to congenital adrenal hyperplasia (CAH). We previously reported a structure of WT human P450 21A2 with bound progesterone and now present a structure bound to the other substrate (17α-OH-progesterone). We found that the 17α-OH-progesterone- and progesterone-bound complex structures are highly similar, with only some minor differences in surface loop regions. Twelve P450 21A2 variants associated with either salt-wasting or nonclassical forms of CAH were expressed, purified, and analyzed. The catalytic activities of these 12more » variants ranged from 0.00009% to 30% of WT P450 21A2 and the extent of heme incorporation from 10% to 95% of the WT. Substrate dissociation constants (Ks) for four variants were 37–13,000-fold higher than for WT P450 21A2. Cytochrome b5, which augments several P450 activities, inhibited P450 21A2 activity. Similar to the WT enzyme, high noncompetitive intermolecular kinetic deuterium isotope effects (≥ 5.5) were observed for all six P450 21A2 variants examined for 21-hydroxylation of 21-d3-progesterone, indicating that C–H bond breaking is a rate-limiting step over a 104-fold range of catalytic efficiency. Using UV-visible and CD spectroscopy, we found that P450 21A2 thermal stability assessed in bacterial cells and with purified enzymes differed among salt-wasting- and nonclassical-associated variants, but these differences did not correlate with catalytic activity. Our in-depth investigation of CAH-associated P450 21A2 variants reveals critical insight into the effects of disease-causing mutations on this important enzyme.« less

  2. Electron transfer in the complex of membrane-bound human cytochrome P450 3A4 with the flavin domain of P450BM-3: the effect of oligomerization of the heme protein and intermittent modulation of the spin equilibrium†

    PubMed Central

    Davydov, Dmitri R.; Sineva, Elena V.; Sistla, Srinivas; Davydova, Nadezhda Y.; Frank, Daniel J.; Sligar, Stephen G.; Halpert, James R.

    2009-01-01

    We studied the kinetics of NADPH-dependent reduction of human CYP3A4 incorporated into Nanodiscs (CYP3A4-ND) and proteoliposomes in order to probe the effect of P450 oligomerization on its reduction. The flavin domain of cytochrome P450-BM3 (BMR) was used as a model electron donor partner. Unlike CYP3A4 oligomers, where only 50% of the enzyme was shown to be reducible by BMR, CYP3A4-ND could be reduced almost completely. High reducibility was also observed in proteoliposomes with a high lipid-to-protein ratio (L/P=910), where the oligomerization equilibrium is displaced towards monomers. In contrast, the reducibililty in proteoliposomes with L/P=76 did not exceed 55 ± 6%. The effect of the surface density of CYP3A4 in proteoliposomes on the oligomerization equilibrium was confirmed with a FRET-based assay employing a cysteine-depleted mutant labeled on Cys-468 with BODIPY iodoacetamide. These results confirm a pivotal role of CYP3A4 oligomerization in its functional heterogeneity. Furthermore, the investigation of the initial phase of the kinetics of CYP3A4 reduction showed that the addition of NADPH causes a rapid low-to-high spin transition in the CYP3A4-BMR complex, which is followed by a partial slower reversal. This observation reveals a mechanism whereby the CYP3A4 spin equilibrium is modulated by the redox state of the bound flavoprotein. PMID:20026040

  3. Prediction of Cytochrome P450 Profiles of Environmental Chemicals with QSAR Models Built from Drug-like Molecules

    EPA Science Inventory

    The human cytochrome P450 (CYP450) enzyme family is involved in the biotransformation of many environmental chemicals. As part of the U.S. Tox21 effort, we profiled the CYP450 activity of ~2800 chemicals predominantly of environmental concern against CYP1A2, CYP2C19, CYP2C9, CYP2...

  4. Role of the NAD(P)H quinone oxidoreductase NQR and the cytochrome b AIR12 in controlling superoxide generation at the plasma membrane.

    PubMed

    Biniek, Catherine; Heyno, Eiri; Kruk, Jerzy; Sparla, Francesca; Trost, Paolo; Krieger-Liszkay, Anja

    2017-04-01

    The quinone reductase NQR and the b-type cytochrome AIR12 of the plasma membrane are important for the control of reactive oxygen species in the apoplast. AIR12 and NQR are two proteins attached to the plant plasma membrane which may be important for generating and controlling levels of reactive oxygen species in the apoplast. AIR12 (Auxin Induced in Root culture) is a single gene of Arabidopsis that codes for a mono-heme cytochrome b. The NADPH quinone oxidoreductase NQR is a two-electron-transferring flavoenzyme that contributes to the generation of O 2 •- in isolated plasma membranes. A. thaliana double knockout plants of both NQR and AIR12 generated more O 2 •- and germinated faster than the single mutant affected in AIR12. To test whether NQR and AIR12 are able to interact functionally, recombinant purified proteins were added to plasma membranes isolated from soybean hypocotyls. In vitro NADH-dependent O 2 •- production at the plasma membrane in the presence of NQR was reduced upon addition of AIR12. Electron donation from semi-reduced menadione to AIR12 was shown to take place. Biochemical analysis showed that purified plasma membrane from soybean hypocotyls or roots contained phylloquinone and menaquinone-4 as redox carriers. This is the first report on the occurrence of menaquinone-4 in eukaryotic photosynthetic organisms. We propose that NQR and AIR12 interact via the quinone, allowing an electron transfer from cytosolic NAD(P)H to apoplastic monodehydroascorbate and control thereby the level of reactive oxygen production and the redox state of the apoplast.

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

    EPA Science Inventory

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

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

    PubMed

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

    1995-12-01

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

  7. 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. Copyright © 2012 Elsevier Inc. All rights reserved.

  8. [Induction of NAD(P)H: quinone reductase by anticarcinogenic ingredients of tea].

    PubMed

    Qi, L; Han, C

    1998-09-30

    By assaying the activity of NAD(P)H: quinone reductase (QR) in Hep G2 cells exposed to inducing agents, a variety of ingredients in tea, we compared their abilities on inducing QR and preventing cancer. The results showed that tea polyphenols, tea pigments and mixed tea were all able to induce the activity of QR significantly. The single-component ingredients of tea polyphenols and tea pigments, including thearubigens, EGCG and ECG, also enhanced the activity of QR. But EGC, EC, theaflavins, tea polysaccharide and tea caffeine, showed no apparent induction of QR. We found that among those tea ingredients studied, the multi-component ingredients were more effective than the single-component ones. So we thought that the abilities of antioxidation and cancer prevention of tea depended on the combined effects of several kinds of active ingredients, which mainly include tea polyphenols and tea pigments.

  9. Ecologically Appropriate Xenobiotics Induce Cytochrome P450s in Apis mellifera

    PubMed Central

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

    2012-01-01

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

  10. Status of Resistance of Bemisia tabaci (Hemiptera: Aleyrodidae) to Neonicotinoids in Iran and Detoxification by Cytochrome P450-Dependent Monooxygenases.

    PubMed

    Basij, M; Talebi, K; Ghadamyari, M; Hosseininaveh, V; Salami, S A

    2017-02-01

    Nine Bemisia tabaci (Gennadius) populations were collected from different regions of Iran. In all nine populations, only one biotype (B biotype) was detected. Susceptibilities of these populations to imidacloprid and acetamiprid were assayed. The lethal concentration 50 values (LC 50 ) for different populations showed a significant discrepancy in the susceptibility of B. tabaci to imidacloprid (3.76 to 772.06 mg l -1 ) and acetamiprid (4.96 to 865 mg l -1 ). The resistance ratio of the populations ranged from 9.72 to 205.20 for imidacloprid and 6.38 to 174.57 for acetamiprid. The synergistic effects of piperonylbutoxide (PBO) and S,S,S-tributylphosphorotrithioate (DEF) were evaluated for the susceptible (RF) and resistant (JR) populations for the determination of the involvement of cytochrome P450-dependent monooxygenase and carboxylesterase, respectively, in their resistance mechanisms. The results showed that PBO overcame the resistance of the JR population to both imidacloprid and acetamiprid, with synergistic ratios of 72.7 and 106.9, respectively. Carboxylesterase, glutathione S-transferase and cytochrome P450-dependent monooxygenase were studied biochemically, for the purpose of measuring the activity of the metabolizing enzymes in order to determine which enzymes are directly involved in neonicotinoid resistance. There was an increase in the activity of cytochrome P450-dependent monooxygenase up to 17-fold in the resistant JR population (RR = 205.20). The most plausible activity of cytochrome P450-dependent monooxygenase correlated with the resistances of imidacloprid and acetamiprid, and this suggests that cytochrome P450-dependent monooxygenase is the only enzyme system responsible for neonicotinoid resistance in the nine populations of B. tabaci.

  11. Development of cytochromes P450 in avian species as a biomarker for environmental contaminant exposure and effect: Procedures and baseline values

    USGS Publications Warehouse

    Melancon, M.J.; Bengston, David A.; Henshel, Diane S.

    1996-01-01

    As in mammals and fish, birds respond to many environmental contaminants with induction of hepatic cytochromes P450. In order to monitor cytchromes P450 in specific avian species, for assessing the status of that species or the habitat it utilizes, it is necessary to have background information on the appropriate assay conditions and the responsiveness of cytochrome P450 induction in that species. Assay of four monooxygenases which give resorufin as product using a fluorescence microwell plate scanner has proven to be an effective approach. Information is provided on the incubation conditions and baseline activity for twenty avian species at ages ranging from pipping embryo to adult. Induction responsiveness is presented for sixteen of them. This information can serve as a guide for those who wish to utilize cytochrome P450 as a biomarker for contaminant exposure and effect to aid in selection of appropriate species, age, and monooxygenase assay(s).

  12. Heterologous expression of equine CYP3A94 and investigation of a tunable system to regulate co-expressed NADPH P450 oxidoreductase levels.

    PubMed

    Dettwiler, Ramona; Schmitz, Andrea L; Plattet, Philippe; Zielinski, Jana; Mevissen, Meike

    2014-01-01

    The activity of cytochrome P450 enzymes depends on the enzyme NADPH P450 oxidoreductase (POR). The aim of this study was to investigate the activity of the equine CYP3A94 using a system that allows to regulate the POR protein levels in mammalian cells. CYP3A94 and the equine POR were heterologously expressed in V79 cells. In the system used, the POR protein regulation is based on a destabilizing domain (DD) that transfers its instability to a fused protein. The resulting fusion protein is therefore degraded by the ubiquitin-proteasome system (UPS). Addition of "Shield-1" prevents the DD fusion protein from degradation. The change of POR levels at different Shield-1 concentrations was demonstrated by cytochrome c reduction, Western immunoblot analysis, and immunocytochemistry. The alteration of CYP3A94 activity was investigated using a substrate (BFC) known to detect CYP3A4 activity. Equine CYP3A94 was demonstrated to be metabolically active and its activity could be significantly elevated by co-expression of POR. Cytochrome c reduction was significantly increased in V79-CYP3A94/DD-POR cells compared to V79-CYP3A94 cells. Surprisingly, incubation with different Shield-1 concentrations resulted in a decrease in POR protein shown by Western immunoblot analysis. Cytochrome c reduction did not change significantly, but the CYP3A94 activity decreased more than 4-fold after incubation with 500 nM and 1 µM Shield-1 for 24 hours. No differences were obtained when V79-CYP3A94 POR cells with and without Shield-1 were compared. The basal activity levels of V79-CYP3A94/DD-POR cells were unexpectedly high, indicating that DD/POR is not degraded without Shield-1. Shield-1 decreased POR protein levels and CYP3A94 activity suggesting that Shield-1 might impair POR activity by an unknown mechanism. Although regulation of POR with the pPTuner system could not be obtained, the cell line V79-CYP3A94/DD-POR system can be used for further experiments to characterize the equine CYP3A94

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

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

    2013-02-15

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

  14. Biocatalytic Conversion of Avermectin to 4"-Oxo-Avermectin: Heterologous Expression of the ema1 Cytochrome P450 Monooxygenase

    PubMed Central

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

    2005-01-01

    The cytochrome P450 monooxygenase Ema1 from Streptomyces tubercidicus R-922 and its homologs from closely related Streptomyces strains are able to catalyze the regioselective oxidation of avermectin into 4"-oxo-avermectin, a key intermediate in the manufacture of the agriculturally important insecticide emamectin benzoate (V. Jungmann, I. Molnár, P. E. Hammer, D. S. Hill, R. Zirkle, T. G. Buckel, D. Buckel, J. M. Ligon, and J. P. Pachlatko, Appl. Environ. Microbiol. 71:6968-6976, 2005). The gene for Ema1 has been expressed in Streptomyces lividans, Streptomyces avermitilis, and solvent-tolerant Pseudomonas putida strains using different promoters and vectors to provide biocatalytically competent cells. Replacing the extremely rare TTA codon with the more frequent CTG codon to encode Leu4 in Ema1 increased the biocatalytic activities of S. lividans strains producing this enzyme. Ferredoxins and ferredoxin reductases were also cloned from Streptomyces coelicolor and biocatalytic Streptomyces strains and tested in ema1 coexpression systems to optimize the electron transport towards Ema1. PMID:16269733

  15. Soybean meal fermented by Aspergillus awamori increases the cytochrome P-450 content of the liver microsomes of mice.

    PubMed

    Kishida, T; Ataki, H; Takebe, M; Ebihara, K

    2000-04-01

    The effect of soybean meal fermented by Aspergillus awamori on the acute lethality of acetaldehyde, pentobarbital sleeping time, and cytochrome P-450 content of the hepatic microsomes was studied in mice. Most of the daidzin and genistin in soybean meal (SBM) were converted into the respective aglycones, daidzein and genistein, by fermentation. In experiment 1, mice were fed isonitrogenic test diets with one of the following five protein sources for 28 d: casein, SBM, fermented and hot-air-dried SBM (FSBM-HD), fermented and freeze-dried SBM (FSBM-FD), or methanol-extracted FSBM-FD (FSMB-FD-R). The acute lethality of acetaldehyde in mice fed the FSBM-FD diet was significantly lower than that in mice fed the SBM, FSBM-HD, or FSBM-FD-R diet. In experiments 2 and 3, mice were fed isonitrogenic test diets with one of the following four protein sources for 28 d: casein, SBM, FSBM-FD, and FSBM-FD-R. The pentobarbital sleeping time was significantly shorter and the cytochrome P-450 content was significantly higher in the mice fed the FSBM-FD diet than the respective value in mice fed the other test diets. In experiment 4, mice were fed one of eight diets which contained different levels of aglycone obtained by varying the proportion of FSBM-FD and FSBM-FD-R, for 28 d. The cytochrome P-450 content in hepatic microsomes increased as the dietary level of isoflavonoid aglycones increased, but there was a saturation phenomenon. These results suggest that soy isoflavonoid aglycones are more potent inducers of cytochrome P-450 than isoflavonoid glycosides.

  16. Cytochrome P450, CYP93A1, as a defense marker in soybean

    USDA-ARS?s Scientific Manuscript database

    CYP93A1 is a cytochrome P450 that is involved in the synthesis of the phytoalexin glyceollin in soybean (Glycine max L. Merr). The gene encoding CYP93A1 has been used as a defense marker in soybean cell cultures, however, little is known regarding how this gene is expressed in the intact plant. To f...

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2014-01-21

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

  19. Detection and Diversity of Fungal Nitric Oxide Reductase Genes (p450nor) in Agricultural Soils

    PubMed Central

    Welsh, Allana; Orellana, Luis H.; Konstantinidis, Konstantinos T.; Chee-Sanford, Joanne C.; Sanford, Robert A.; Schadt, Christopher W.

    2016-01-01

    ABSTRACT Members of the Fungi convert nitrate (NO3−) and nitrite (NO2−) to gaseous nitrous oxide (N2O) (denitrification), but the fungal contributions to N loss from soil remain uncertain. Cultivation-based methodologies that include antibiotics to selectively assess fungal activities have limitations, and complementary molecular approaches to assign denitrification potential to fungi are desirable. Microcosms established with soils from two representative U.S. Midwest agricultural regions produced N2O from added NO3− or NO2− in the presence of antibiotics to inhibit bacteria. Cultivation efforts yielded 214 fungal isolates belonging to at least 15 distinct morphological groups, 151 of which produced N2O from NO2−. Novel PCR primers targeting the p450nor gene, which encodes the nitric oxide (NO) reductase responsible for N2O production in fungi, yielded 26 novel p450nor amplicons from DNA of 37 isolates and 23 amplicons from environmental DNA obtained from two agricultural soils. The sequences shared 54 to 98% amino acid identity with reference P450nor sequences within the phylum Ascomycota and expand the known fungal P450nor sequence diversity. p450nor was detected in all fungal isolates that produced N2O from NO2−, whereas nirK (encoding the NO-forming NO2− reductase) was amplified in only 13 to 74% of the N2O-forming isolates using two separate nirK primer sets. Collectively, our findings demonstrate the value of p450nor-targeted PCR to complement existing approaches to assess the fungal contributions to denitrification and N2O formation. IMPORTANCE A comprehensive understanding of the microbiota controlling soil N loss and greenhouse gas (N2O) emissions is crucial for sustainable agricultural practices and addressing climate change concerns. We report the design and application of a novel PCR primer set targeting fungal p450nor, a biomarker for fungal N2O production, and demonstrate the utility of the new approach to assess fungal denitrification

  20. Expression patterns of bark beetle cytochromes P450 during host colonization: Likely physiological functions and potential targets for pest management

    Treesearch

    Dezene P. W. Huber; Melissa Erickson; Christian Leutenegger; Joerg Bohlmann; Steven J. Seybold

    2007-01-01

    Cytochromes P450 family genes (P450s) are found in a diverse array of organisms ranging from bacteria to mammals to plants to arthropods. Although there are exceptions to this rule, organisms generally contain a fairly large number of P450 genes and pseudogenes in their genomes. For instance, among arthropods whose genomes are well characterized, the mosquito,

  1. Deltamethrin is metabolized by CYP6FU1, a cytochrome P450 associated with pyrethroid resistance, in Laodelphax striatellus.

    PubMed

    Elzaki, Mohammed Esmail Abdalla; Miah, Mohammad Asaduzzaman; Peng, Yingchuan; Zhang, Haomiao; Jiang, Ling; Wu, Min; Han, Zhaojun

    2018-06-01

    Cytochrome P450s (CYPs) are known to play a major role in metabolizing a wide range compounds. CYP6FU1 has been found to be over-expressed in a deltamethrin-resistant strain of Laodelphax striatellus. This study was conducted to express CYP6FU1 in Sf9 cells as a recombinant protein, to confirm its ability to degrade deltamethrin, chlorpyrifos, imidacloprid and traditional P450 probing substrates. Carbon monoxide difference spectrum analysis indicated that the intact CYP6FU1 protein was expressed in insect Sf9 cells. Catalytic activity tests with four traditional P450 probing substrates revealed that the expressed CYP6FU1 preferentially metabolized p-nitroanisole and ethoxyresorufin, but not ethoxycoumarin and luciferin-HEGE. The enzyme kinetic parameters were tested using p-nitroanisole. The michaelis constant (K m ) and catalytic constant (K cat ) values were 17.51 ± 4.29 µm and 0.218 ± 0.001 pmol min -1 mg -1 protein, respectively. Furthermore, CYP6FU1 activity for degradation of insecticides was tested by measuring substrate depletion and metabolite formation. The chromatogram analysis showed obvious nicotinamide-adenine dinucleotide phosphate (NADPH)-dependent depletion of deltamethrin, and formation of the unknown metabolite. Mass spectra and the molecular docking model showed that the metabolite was 4-hydroxy-deltamethrin. However, the recombinant CYP6FU1 could not metabolize imidacloprid and chlorpyrifos. These results confirmed that the over-expressed CYP6FU1 contributes to deltamethrin resistance in L. striatellus, and p-nitroanisole might be a potential diagnostic probe for deltamethrin metabolic resistance detection and monitoring. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

  2. Electrochemistry of Canis familiaris cytochrome P450 2D15 with gold nanoparticles: An alternative to animal testing in drug discovery.

    PubMed

    Rua, Francesco; Sadeghi, Sheila J; Castrignanò, Silvia; Valetti, Francesca; Gilardi, Gianfranco

    2015-10-01

    This work reports for the first time the direct electron transfer of the Canis familiaris cytochrome P450 2D15 on glassy carbon electrodes to provide an analytical tool as an alternative to P450 animal testing in the drug discovery process. Cytochrome P450 2D15, that corresponds to the human homologue P450 2D6, was recombinantly expressed in Escherichia coli and entrapped on glassy carbon electrodes (GC) either with the cationic polymer polydiallyldimethylammonium chloride (PDDA) or in the presence of gold nanoparticles (AuNPs). Reversible electrochemical signals of P450 2D15 were observed with calculated midpoint potentials (E1/2) of −191 ± 5 and −233 ± 4 mV vs. Ag/AgCl for GC/PDDA/2D15 and GC/AuNPs/2D15, respectively. These experiments were then followed by the electro-catalytic activity of the immobilized enzyme in the presence of metoprolol. The latter drug is a beta-blocker used for the treatment of hypertension and is a specific marker of the human P450 2D6 activity. Electrocatalysis data showed that only in the presence of AuNps the expected α-hydroxy-metoprolol product was present as shown by HPLC. The successful immobilization of the electroactive C. familiaris cytochrome P450 2D15 on electrode surfaces addresses the ever increasing demand of developing alternative in vitromethods for amore detailed study of animal P450 enzymes' metabolism, reducing the number of animals sacrificed in preclinical tests.

  3. 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.

  4. 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.

  5. Autoantibodies against cytochrome P450s in sera of children treated with immunosuppressive drugs

    PubMed Central

    LYTTON, S D; BERG, U; NEMETH, A; INGELMAN-SUNDBERG, M

    2002-01-01

    Treatment with the immunosuppressive drugs cyclosporin and tacrolimus, the mainstays of anti-graft rejection and autoimmune disease therapy, is limited by their hepato-and nephrotoxicity. The metabolic conversion of these compounds to more easily excretable products is catalysed mainly by hepatic cytochrome P4503A4 (CYP3A4) but also involves extrahepatic CYP3A5 and other P450 forms. We set out to study whether or not exposure to cyclosporin and FK506 in children undergoing organ transplantation leads to formation of autoantibodies against P450s. Immunoblotting analysis revealed anti-CYP reactivity in 16% of children on CyA for anti-graft rejection or treatment of nephrosis (n = 67), 31% of kidney transplant patients switched from CyA to FK506 (n = 16), and 21% of kidney and or liver transplant patients on FK506 (n = 14). In contrast, the frequency of reactive immunoblots was only 8·5% among the normal paediatric controls (n = 25) and 7% among adult kidney transplant patients on CyA or FK506 (n = 30). The CYP2C9+ sera were able to immunoprecipitate in vitro translated CYP2C9 and the immunoblot reactivity showed striking correlation to peaks in the age at onset of drug exposure. Sera were isoform selective as evidenced from Western blotting using human liver microsomes and heterologously expressed human P450s. These findings suggest that anti-cytochrome P450 autoantibodies, identified on the basis of their specific binding in immunoblots, are significantly increased among children on immunosuppressive drugs and in some cases are associated with drug toxicity and organ rejection. PMID:11876753

  6. Autoantibodies against cytochrome P450s in sera of children treated with immunosuppressive drugs.

    PubMed

    Lytton, S D; Berg, U; Nemeth, A; Ingelman-Sundberg, M

    2002-02-01

    Treatment with the immunosuppressive drugs cyclosporin and tacrolimus, the mainstays of anti-graft rejection and autoimmune disease therapy, is limited by their hepato- and nephrotoxicity. The metabolic conversion of these compounds to more easily excretable products is catalysed mainly by hepatic cytochrome P4503A4 (CYP3A4) but also involves extrahepatic CYP3A5 and other P450 forms. We set out to study whether or not exposure to cyclosporin and FK506 in children undergoing organ transplantation leads to formation of autoantibodies against P450s. Immunoblotting analysis revealed anti-CYP reactivity in 16% of children on CyA for anti-graft rejection or treatment of nephrosis (n = 67), 31% of kidney transplant patients switched from CyA to FK506 (n = 16), and 21% of kidney and or liver transplant patients on FK506 (n = 14). In contrast, the frequency of reactive immunoblots was only 8.5% among the normal paediatric controls (n = 25) and 7% among adult kidney transplant patients on CyA or FK506 (n = 30). The CYP2C9+ sera were able to immunoprecipitate in vitro translated CYP2C9 and the immunoblot reactivity showed striking correlation to peaks in the age at onset of drug exposure. Sera were isoform selective as evidenced from Western blotting using human liver microsomes and heterologously expressed human P450s. These findings suggest that anti-cytochrome P450 autoantibodies, identified on the basis of their specific binding in immunoblots, are significantly increased among children on immunosuppressive drugs and in some cases are associated with drug toxicity and organ rejection.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Polusani, Srikanth R.; Kar, Rekha; Riquelme, Manuel A.

    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 themore » 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.« less

  8. Mechanism of Lethal Interaction of Hazardous Chemicals at Subtoxic Doses

    DTIC Science & Technology

    1991-09-20

    Mehendale, H. M. Phenobarbital-induced cytosolic cytoprotective mechanisms that offset increases in NADPH cytochrome P-450 reductase activity in menadione ...9. Utley, W. M. and Mehendale, H. M. Phenobarbital induced cytoprotective mechanisms in menadione metabolism: The role of glutathione reductase and...Mehendale, H. M. The contribution of DT-diaphorase in hepatocytes isolated from naive and phenobarbital preireaieu rats during menadione metabolism. FASEB J

  9. Functional evolution and structural conservation in chimeric cytochromes p450: calibrating a structure-guided approach.

    PubMed

    Otey, Christopher R; Silberg, Jonathan J; Voigt, Christopher A; Endelman, Jeffrey B; Bandara, Geethani; Arnold, Frances H

    2004-03-01

    Recombination generates chimeric proteins whose ability to fold depends on minimizing structural perturbations that result when portions of the sequence are inherited from different parents. These chimeric sequences can display functional properties characteristic of the parents or acquire entirely new functions. Seventeen chimeras were generated from two CYP102 members of the functionally diverse cytochrome p450 family. Chimeras predicted to have limited structural disruption, as defined by the SCHEMA algorithm, displayed CO binding spectra characteristic of folded p450s. Even this small population exhibited significant functional diversity: chimeras displayed altered substrate specificities, a wide range in thermostabilities, up to a 40-fold increase in peroxidase activity, and ability to hydroxylate a substrate toward which neither parent heme domain shows detectable activity. These results suggest that SCHEMA-guided recombination can be used to generate diverse p450s for exploring function evolution within the p450 structural framework.

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

    USGS Publications Warehouse

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

    1996-01-01

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

  11. In vitro activity of commercial valerian root extracts against human cytochrome P450 3A4.

    PubMed

    Lefebvre, Tania; Foster, Brian C; Drouin, Cathy E; Krantis, Anthony; Livesey, John F; Jordan, Scott A

    2004-08-12

    Valerian root ( Valeriana officinalis L.) has been used since antiquity as a medicinal herb. Recent studies have found that certain herbal products used concomitantly with conventional therapeutic products can markedly affect drug disposition. The in vitro effect of aliquots from 14 commercially available single-entity and blended products containing valerian root on cytochrome P450 CYP3A4-mediated metabolism and P-glycoprotein transport has been determined with aqueous, ethanol and acetonitrile extracts. Hydroxyvalerenic acid, acetoxyvalerenic acid and valerenic acid content was analyzed and wide variation was found between samples and compared to the concentrations noted on the product labels. Valerian extracts from the products tested also exhibited a marked capacity to inhibit cytochrome P450 3A4-mediated metabolism and P-glycoprotein transport based upon the ATPase assay. There is wide variation between commercially available samples of valerian root. The findings from this study suggest that valerian root may have an initial inhibitory effect when taken with therapeutic products. Further work is warranted to determine whether valerian root can affect other CYP450 isozymes and how the results of this in vitro investigation can be extrapolated to in vivo situations.

  12. N-Heterocyclic Carbene Capture by Cytochrome P450 3A4

    PubMed Central

    Jennings, Gareth K.; Ritchie, Caroline M.; Shock, Lisa S.; Lyons, Charles E.

    2016-01-01

    Cytochrome P450 3A4 (CYP3A4) is the dominant P450 enzyme involved in human drug metabolism, and its inhibition may result in adverse interactions or, conversely, favorably reduce the systemic elimination rates of poorly bioavailable drugs. Herein we describe a spectroscopic investigation of the interaction of CYP3A4 with N-methylritonavir, an analog of ritonavir, widely used as a pharmacoenhancer. In contrast to ritonavir, the binding affinity of N-methylritonavir for CYP3A4 is pH-dependent. At pH <7.4, the spectra are definitively type I, whereas at pH ≥7.4 the spectra have split Soret bands, including a red-shifted component characteristic of a P450-carbene complex. Variable-pH UV-visible spectroscopy binding studies with molecular fragments narrows the source of this pH dependence to its N-methylthiazolium fragment. The C2 proton of this group is acidic, and variable-pH resonance Raman spectroscopy tentatively assigns it a pKa of 7.4. Hence, this fragment of N-methylritonavir is expected to be readily deprotonated under physiologic conditions to yield a thiazol-2-ylidene, which is an N-heterocyclic carbene that has high-affinity for and is presumed to be subsequently captured by the heme iron. This mechanism is supported by time-dependent density functional theory with an active site model that accurately reproduces distinguishing features of the experimental UV-visible spectra of N-methylritonavir bound to CYP3A4. Finally, density functional theory calculations support that this novel interaction is as strong as the tightest-binding azaheterocycles found in P450 inhibitors and could offer new avenues for inhibitor development. PMID:27126611

  13. [Consumption of vitamin A in intact and castrated rats. Relation to tocopherol and cytochrome P 450].

    PubMed

    Ferrando, R; Truhaut, R; Fourlon, C

    1979-05-07

    There are significant differences between castrated and non castrated male Rats in regard to vitamine A hepatic storage and plasma level. Cytochrom P 450 levels are the same in both groups. Those results are discussed.

  14. Effects of chlorpyrifos on life cycle parameters, cytochrome P450S expression, and antioxidant systems in the monogonont rotifer Brachionus koreanus.

    PubMed

    Kim, Ryeo-Ok; Kim, Bo-Mi; Jeong, Chang-Bum; Lee, Jae-Seong; Rhee, Jae-Sung

    2016-06-01

    Chlorpyrifos is a widely used organophosphorus insecticide for controlling diverse insect pests of crops. In the monogonont rotifer Brachionus koreanus, population growth retardation with the inhibition of lifespan, fecundity, and individual body size of ovigerous females was shown over 10 d in response to chlorpyrifos exposure. At the molecular and biochemical levels, the rotifer B. koreanus defensome, composed of cytochrome P450 complements, heat shock protein 70, and antioxidant enzymatic systems (i.e., glutathione, glutathione peroxidase, glutathione reductase, and glutathione S-transferase), was significantly induced in response to different concentrations of chlorpyrifos. Thus, chlorpyrifos strongly induced a defensome system to mitigate the deleterious effects of chlorpyrifos at in vivo and in vitro levels as a trade-off in fitness costs. Environ Toxicol Chem 2016;35:1449-1457. © 2015 SETAC. © 2015 SETAC.

  15. In vitro metabolism of an estrogen-related receptor γ modulator, GSK5182, by human liver microsomes and recombinant cytochrome P450s.

    PubMed

    Joo, Jeongmin; Wu, Zhexue; Lee, Boram; Shon, Jong Cheol; Lee, Taeho; Lee, In-Kyu; Sim, Taebo; Kim, Kyung-Hee; Kim, Nam Doo; Kim, Seong Heon; Liu, Kwang-Hyeon

    2015-04-01

    GSK5182 (4-[(Z)-1-[4-(2-dimethylaminoethyloxy)phenyl]-hydroxy-2-phenylpent-1-enyl]phenol) is a specific inverse agonist for estrogen-related receptor γ, a member of the orphan nuclear receptor family that has important functions in development and homeostasis. This study was performed to elucidate the metabolites of GSK5182 and to characterize the enzymes involved in its metabolism. Incubation of human liver microsomes with GSK5182 in the presence of NADPH resulted in the formation of three metabolites, M1, M2 and M3. M1 and M3 were identified as N-desmethyl-GSK5182 and GSK5182 N-oxide, respectively, on the basis of liquid chromatography-tandem mass spectrometric (LC-MS/MS) analysis. M2 was suggested to be hydroxy-GSK5182 through interpretation of its MS/MS fragmentation pattern. In addition, the specific cytochrome P450 (P450) and flavin-containing monooxygenase (FMO) isoforms responsible for GSK5182 oxidation to the three metabolites were identified using a combination of correlation analysis, chemical inhibition in human liver microsomes and metabolism by expressed recombinant P450 and FMO isoforms. GSK5182 N-demethylation and hydroxylation is mainly mediated by CYP3A4, whereas FMO1 and FMO3 contribute to the formation of GSK5182 N-oxide from GSK5182. The present data will be useful for understanding the pharmacokinetics and drug interactions of GSK5182 in vivo. Copyright © 2014 John Wiley & Sons, Ltd.

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

    PubMed Central

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

    2014-01-01

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

  17. A multiscale approach to modelling drug metabolism by membrane-bound cytochrome P450 enzymes.

    PubMed

    Lonsdale, Richard; Rouse, Sarah L; Sansom, Mark S P; Mulholland, Adrian J

    2014-07-01

    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.

  18. Evolution of the scientific literature of cytochrome P450 from 1977 to 2008.

    PubMed

    Robert, Claude; Wilson, Concepción S; Guengerich, F Peter; Arreto, Charles-Daniel

    2010-02-01

    This study traces the evolution of the scientific literature on cytochrome P450 (P450) published during the last 30+ years (1977-2008). Using the Web of Science, P450 articles from the Science Citation Index Expanded published from 1977 to 2008 were retrieved and analyzed. The number of P450 papers has increased from 342 articles in 1977-1978 to 2,357 in 2007-2008, and the number of contributing countries has grown from 23 countries for 1977-1978 to 76 for 2007-2008. While the USA and Japan were the most productive countries, along with several industrialized countries (e.g. UK, Germany and Canada), two Asian countries have recently joined the group of leading countries (in 2007-2008 China ranked 4th and South Korea, 7th). During 1977-2008, the number of journals publishing papers in P450 research increased more than seven-fold (7.7): 94 journals in 1977-1978 and 724 in 2007-2008; however, citation by readers (as measured by the journal impact factor) of the top-ten leading journals increased only slightly from 3.25 for 1977-1978 to 3.81 for 2007-2008. While Biochemistry & Molecular Biology and Pharmacology and Pharmacy are the two main targeted subject areas for P450 research during the period considered, there has been a gradual shift from the biophysical and biochemical fields of interest to aspects of genomics and clinical approaches. The rapid evolution of P450 research in the last 30+ years was accompanied by important changes in the landscape of the contributing countries, in the subject domains, and consequently in the scientific journals targeted by researchers.

  19. Seasonal changes in the activity of cytochrome P450(C17) from the testis of Bufo arenarum.

    PubMed

    Solari, J J F; Pozzi, A G; Ceballos, N R

    2002-12-01

    In Bufo arenarum, the biosynthesis of testosterone and 5alpha-dihydrotestosterone takes place through a complete 5-ene pathway, 5-androsten-3beta,17beta-diol being the immediate precursor of testosterone. Besides androgens, testes are able to synthesise 5alpha-pregnan-3,20-dione and several 3alpha and 20alpha reduced derivatives. During the breeding season, steroid biosynthesis turns from androgen to C21-steroid production. As a consequence, the cytochrome P450 17-hydroxylase, C17,20-lyase (CypP450(c17)) could be a key enzyme in that metabolic shift. The present study demonstrates that in testes of B. arenarum, CypP450(c17) co-localises with glucose-6-phosphatase in the microsomal fraction. CypP450(c17) possesses more affinity for pregnenolone than for progesterone in both non-reproductive (Km = 43.76 +/- 4.63 nM and 2,170 +/- 630 nM, respectively) and reproductive (Km = 37.46 +/- 4.19 nM and 3,060 +/- 190 nM, respectively) seasons. These results could explain the predominance of the 5-ene pathway for testosterone biosynthesis. Toad CypP450(c17) activity is higher in the non-reproductive period than the reproductive period, suggesting that this enzyme is an important factor in toad steroidogenic changes. Animals in reproductive conditions showed a significant reduction in circulating androgens. This is in agreement with the decrease in Vmax of cytochrome P450 17-hydroxylase activity, enhancing the physiological relevance of these in vitro results.

  20. Functional characterisation of an engineered multidomain human P450 2E1 by molecular Lego.

    PubMed

    Fairhead, Michael; Giannini, Silva; Gillam, Elizabeth M J; Gilardi, Gianfranco

    2005-12-01

    The human cytochrome P450s constitute an important family of monooxygenase enzymes that carry out essential roles in the metabolism of endogenous compounds and foreign chemicals. We present here results of a fusion between a human P450 enzyme and a bacterial reductase that for the first time is shown does not require the addition of lipids or detergents to achieve wild-type-like activities. The fusion enzyme, P450 2E1-BMR, contains the N-terminally modified residues 22-493 of the human P450 2E1 fused at the C-terminus to residues 473-1049 of the P450 BM3 reductase (BMR). The P450 2E1-BMR enzyme is active, self-sufficient and presents the typical marker activities of the native human P450 2E1: the hydroxylation of p-nitrophenol (KM=1.84+/-0.09 mM and kcat of 2.98+/-0.04 nmol of p-nitrocatechol formed per minute per nanomole of P450) and chlorzoxazone (KM=0.65+/-0.08 mM and kcat of 0.95+/-0.10 nmol of 6-hydroxychlorzoxazone formed per minute per nanomole of P450). A 3D model of human P450 2E1 was generated to rationalise the functional data and to allow an analysis of the surface potentials. The distribution of charges on the model of P450 2E1 compared with that of the FMN domain of BMR provides the ground for the understanding of the interaction between the fused domains. The results point the way to successfully engineer a variety of catalytically self-sufficient human P450 enzymes for drug metabolism studies in solution.

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

    PubMed Central

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

    2013-01-01

    P450cam (CYP101A1) is a bacterial monooxygenase that is known to catalyze the oxidation of camphor, the first committed step in camphor degradation, with simultaneous reduction of oxygen (O2). We report that P450cam catalysis is controlled by oxygen levels: at high O2 concentration, P450cam catalyzes the known oxidation reaction, whereas at low O2 concentration the enzyme catalyzes the reduction of camphor to borneol. We confirmed, using 17O and 2H NMR, that the hydrogen atom added to camphor comes from water, which is oxidized to hydrogen peroxide (H2O2). This is the first time a cytochrome P450 has been observed to catalyze oxidation of water to H2O2, a difficult reaction to catalyze due to its high barrier. The reduction of camphor and simultaneous oxidation of water are likely catalyzed by the iron-oxo intermediate of P450cam, and we present a plausible mechanism that accounts for the 1∶1 borneol:H2O2 stoichiometry we observed. This reaction has an adaptive value to bacteria that express this camphor catabolism pathway, which requires O2, for two reasons: 1) the borneol and H2O2 mixture generated is toxic to other bacteria and 2) borneol down-regulates the expression of P450cam and its electron transfer partners. Since the reaction described here only occurs under low O2 conditions, the down-regulation only occurs when O2 is scarce. PMID:23634216

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

    EPA Science Inventory

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

  3. 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...

  4. 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...

  5. Evidence for involvement of multiple forms of cytochrome P-450 in aflatoxin B sup 1 metabolism in human liver

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Forrester, L.M.; Wolf, C.R.; Neal, G.E.

    Liver cancer is a major cause of premature death in many areas of Africa and Asia and its incidence is strongly correlated with exposure to aflatoxin B{sub 1} (AFB{sub 1}). Because AFB{sub 1} requires metabolic activation to achieve a biological response, there is a need for detailed knowledge of the mechanism of activation to assess individual risk. The authors carried out an extensive study using a total of 19 human liver samples to determine the individual variability in the metabolism of the toxin to mutagenic or detoxification products and to identify the specific cytochrome P-450 forms involved in these processes.more » Metabolism to the toxic 8,9-epoxide or to products mutagenic in the Ames test was found to exhibit very large individual variation. These data demonstrate that, although P450IIIA probably plays an important role in AFB{sub 1} activation, several other cytochrome P-450 forms have the capacity to activate the toxin. Similar considerations apply to detoxifying metabolism to aflatoxin Q{sub 1} and aflatoxin M{sub 1}. The levels of expression of many of the forms of cytochrome P-450 involved in AFB{sub 1} metabolism are known to be highly sensitive to environmental factors. This indicates that such factors will be an important determinant in individual susceptibility to the tumorigenic action of AFB{sub 1}.« less

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

    PubMed Central

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

    2015-01-01

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

  7. Nanoscale Electron Transport Measurements of Immobilized Cytochrome P450 Proteins

    PubMed Central

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

    2015-01-01

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

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

    EPA Science Inventory

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

  9. Bioavailable flavonoids: cytochrome P450-mediated metabolism of methoxyflavones.

    PubMed

    Walle, U Kristina; Walle, Thomas

    2007-11-01

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

  10. Structural organization and classification of cytochrome P450 genes in flax (Linum usitatissimum L.).

    PubMed

    Babu, Peram Ravindra; Rao, Khareedu Venkateswara; Reddy, Vudem Dashavantha

    2013-01-15

    Flax CYPome analysis resulted in the identification of 334 putative cytochrome P450 (CYP450) genes in the cultivated flax genome. Classification of flax CYP450 genes based on the sequence similarity with Arabidopsis orthologs and CYP450 nomenclature, revealed 10 clans representing 44 families and 98 subfamilies. CYP80, CYP83, CYP92, CYP702, CYP705, CYP708, CYP728, CYP729, CYP733 and CYP736 families are absent in the flax genome. The subfamily members exhibited conserved sequences, length of exons and phasing of introns. Similarity search of the genomic resources of wild flax species Linum bienne with CYP450 coding sequences of the cultivated flax, revealed the presence of 127 CYP450 gene orthologs, indicating amplification of novel CYP450 genes in the cultivated flax. Seven families CYP73, 74, 75, 76, 77, 84 and 709, coding for enzymes associated with phenylpropanoid/fatty acid metabolism, showed extensive gene amplification in the flax. About 59% of the flax CYP450 genes were present in the EST libraries. Copyright © 2012 Elsevier B.V. All rights reserved.

  11. Plant Expression of a Bacterial Cytochrome P450 That Catalyzes Activation of a Sulfonylurea Pro-Herbicide.

    PubMed Central

    O'Keefe, D. P.; Tepperman, J. M.; Dean, C.; Leto, K. J.; Erbes, D. L.; Odell, J. T.

    1994-01-01

    The Streptomyces griseolus gene encoding herbicide-metabolizing cytochrome P450SU1 (CYP105A1) was expressed in transgenic tobacco (Nicotiana tabacum). Because this P450 can be reduced by plant chloroplast ferredoxin in vitro, chloroplast-targeted and nontargeted expression were compared. Whereas P450SU1 antigen was found in the transgenic plants regardless of the targeting, only those with chloroplast-directed enzyme performed P450SU1-mediated N-dealkylation of the sulfonylurea 2-methylethyl-2,3-dihydro-N-[(4,6-dimethoxypyrimidin-2-yl)aminocarbonyl]-1, 2-benzoisothiazole- 7-sulfonamide-1,1-dioxide (R7402). Chloroplast targeting appears to be essential for the bacterial P450 to function in the plant. Because the R7402 metabolite has greater phytotoxicity than R7402 itself, plants bearing active P450SU1 are susceptible to injury from R7402 treatment that is harmless to plants without P450SU1. Thus, P450SU1 expression and R7402 treatment can be used as a negative selection system in plants. Furthermore, expression of P450SU1 from a tissue-specific promoter can sequester production of the phytotoxic R7402 metabolite to a single plant tissue. In tobacco expressing P450SU1 from a tapetum-specific promoter, treatment of immature flower buds with R7402 caused dramatically lowered pollen viability. Such treatment could be the basis for a chemical hybridizing agent. PMID:12232216

  12. Heterologous Expression of Equine CYP3A94 and Investigation of a Tunable System to Regulate Co-Expressed NADPH P450 Oxidoreductase Levels

    PubMed Central

    Dettwiler, Ramona; Schmitz, Andrea L.; Plattet, Philippe; Zielinski, Jana; Mevissen, Meike

    2014-01-01

    The activity of cytochrome P450 enzymes depends on the enzyme NADPH P450 oxidoreductase (POR). The aim of this study was to investigate the activity of the equine CYP3A94 using a system that allows to regulate the POR protein levels in mammalian cells. CYP3A94 and the equine POR were heterologously expressed in V79 cells. In the system used, the POR protein regulation is based on a destabilizing domain (DD) that transfers its instability to a fused protein. The resulting fusion protein is therefore degraded by the ubiquitin-proteasome system (UPS). Addition of “Shield-1” prevents the DD fusion protein from degradation. The change of POR levels at different Shield-1 concentrations was demonstrated by cytochrome c reduction, Western immunoblot analysis, and immunocytochemistry. The alteration of CYP3A94 activity was investigated using a substrate (BFC) known to detect CYP3A4 activity. Equine CYP3A94 was demonstrated to be metabolically active and its activity could be significantly elevated by co-expression of POR. Cytochrome c reduction was significantly increased in V79-CYP3A94/DD-POR cells compared to V79-CYP3A94 cells. Surprisingly, incubation with different Shield-1 concentrations resulted in a decrease in POR protein shown by Western immunoblot analysis. Cytochrome c reduction did not change significantly, but the CYP3A94 activity decreased more than 4-fold after incubation with 500 nM and 1 µM Shield-1 for 24 hours. No differences were obtained when V79-CYP3A94 POR cells with and without Shield-1 were compared. The basal activity levels of V79-CYP3A94/DD-POR cells were unexpectedly high, indicating that DD/POR is not degraded without Shield-1. Shield-1 decreased POR protein levels and CYP3A94 activity suggesting that Shield-1 might impair POR activity by an unknown mechanism. Although regulation of POR with the pPTuner system could not be obtained, the cell line V79-CYP3A94/DD-POR system can be used for further experiments to characterize the equine CYP3A

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

    USDA-ARS?s Scientific Manuscript database

    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...

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

    EPA Science Inventory

    Abstract

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

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

    EPA Science Inventory

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

  16. Resonance Raman study on the structure of the active sites of microsomal cytochrome P-450 isozymes LM2 and LM4.

    PubMed

    Hildebrandt, P; Greinert, R; Stier, A; Taniguchi, H

    1989-12-08

    The isozymes 2 and 4 of rabbit microsomal cytochrome P-450 (LM2, LM4) have been studied by resonance Raman spectroscopy. Based on high quality spectra, a vibrational assignment of the porphyrin modes in the frequency range between 100-1700 cm-1 is presented for different ferric states of cytochrome P-450 LM2 and LM4. The resonance Raman spectra are interpreted in terms of the spin and ligation state of the heme iron and of heme-protein interactions. While in cytochrome P-450 LM2 the six-coordinated low-spin configuration is predominantly occupied, in the isozyme LM4 the five-coordinated high-spin form is the most stable state. The different stability of these two spin configurations in LM2 and LM4 can be attributed to the structures of the active sites. In the low-spin form of the isozymes LM4 the protein matrix forces the heme into a more rigid conformation than in LM2. These steric constraints are removed upon dissociation of the sixth ligand leading to a more flexible structure of the active site in the high-spin form of the isozyme LM4. The vibrational modes of the vinyl groups were found to be characteristic markers for the specific structures of the heme pockets in both isozymes. They also respond sensitively to type-I substrate binding. While in cytochrome P-450 LM4 the occupation of the substrate-binding pocket induces conformational changes of the vinyl groups, as reflected by frequency shifts of the vinyl modes, in the LM2 isozyme the ground-state conformation of these substituents remain unaffected, suggesting that the more flexible heme pocket can accommodate substrates without imposing steric constraints on the porphyrin. The resonance Raman technique makes structural changes visible which are induced by substrate binding in addition and independent of the changes associated with the shift of the spin state equilibrium: the high-spin states in the substrate-bound and substrate-free enzyme are structurally different. The formation of the inactive form

  17. 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 (p,p?DDE, other organochlorinepesticides 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, 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.

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

    PubMed

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

    2012-11-01

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

  19. Cytochrome b5 reductase is the component from neuronal synaptic plasma membrane vesicles that generates superoxide anion upon stimulation by cytochrome c.

    PubMed

    Samhan-Arias, Alejandro K; Fortalezas, Sofia; Cordas, Cristina M; Moura, Isabel; Moura, José J G; Gutierrez-Merino, Carlos

    2018-05-01

    In this work, we measured the effect of cytochrome c on the NADH-dependent superoxide anion production by synaptic plasma membrane vesicles from rat brain. In these membranes, the cytochrome c stimulated NADH-dependent superoxide anion production was inhibited by antibodies against cytochrome b 5 reductase linking the production to this enzyme. Measurement of the superoxide anion radical generated by purified recombinant soluble and membrane cytochrome b 5 reductase corroborates the production of the radical by different enzyme isoforms. In the presence of cytochrome c, a burst of superoxide anion as well as the reduction of cytochrome c by cytochrome b 5 reductase was measured. Complex formation between both proteins suggests that cytochrome b 5 reductase is one of the major partners of cytochrome c upon its release from mitochondria to the cytosol during apoptosis. Superoxide anion production and cytochrome c reduction are the consequences of the stimulated NADH consumption by cytochrome b 5 reductase upon complex formation with cytochrome c and suggest a major role of this enzyme as an anti-apoptotic protein during cell death. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  20. Characterization and identification of an indirect cytochrome P-450-initiated denitrosation of 2,6-dichloro-4-nitroaniline in rat hepatic microsomes.

    PubMed

    Myers, L A; Witmer, C M; Gallo, M A

    1988-08-01

    The metabolism of 2,6-dichloro-4-nitroaniline (DCNA) to a unique denitrosated product, 3,5-dichloro-p-aminophenol (DCAP), was investigated in rat hepatic microsomes using an HPLC system containing a reverse-phase column and an electrochemical detector. The parent compound appears to induce its own metabolism. The characterization of this induction was studied by polyacrylamide gel electrophoresis, catalytic enzymatic activity, and immunochemistry. The in vitro microsomal aerobic production of DCAP was increased 4- to 6.5-fold with respect to controls after animals were treated with DCNA. The microsomal production of DCAP can be inhibited by the addition of specific antibodies to cytochrome P-450d, thus indicating that the removal of the nitro group and subsequent replacement with a hydroxyl group was initiated by cytochrome P-450d in the mixed-function oxidase system. Finally, it was demonstrated by the addition of H218O to the assay that this hydroxyl group came from H2O and not molecular oxygen. It is concluded that cytochrome P-450 initiated this novel reaction by the formation of an N-hydroxylamine, followed by a non-P-450-mediated attack of water causing the removal of nitrous acid and the formation of the phenol.

  1. 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.

  2. Pyridine Nucleotide Complexes with Bacillus anthracis Coenzyme A-Disulfide Reductase: A Structural Analysis of Dual NAD(P)H Specificity

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wallen,J.; Paige, C.; Mallett, T.

    2008-01-01

    We have recently reported that CoASH is the major low-molecular weight thiol in Bacillus anthracis, and we have now characterized the kinetic and redox properties of the B. anthracis coenzyme A-disulfide reductase (CoADR, BACoADR) and determined the crystal structure at 2.30 Angstroms resolution. While the Staphylococcus aureus and Borrelia burgdorferi CoADRs exhibit strong preferences for NADPH and NADH, respectively, B. anthracis CoADR can use either pyridine nucleotide equally well. Sequence elements within the respective NAD(P)H-binding motifs correctly reflect the preferences for S. aureus and Bo. burgdorferi CoADRs, but leave questions as to how BACoADR can interact with both pyridine nucleotides.more » The structures of the NADH and NADPH complexes at ca. 2.3 Angstroms resolution reveal that a loop consisting of residues Glu180-Thr187 becomes ordered and changes conformation on NAD(P)H binding. NADH and NADPH interact with nearly identical conformations of this loop; the latter interaction, however, involves a novel binding mode in which the 2'-phosphate of NADPH points out toward solvent. In addition, the NAD(P)H-reduced BACoADR structures provide the first view of the reduced form (Cys42-SH/CoASH) of the Cys42-SSCoA redox center. The Cys42-SH side chain adopts a new conformation in which the conserved Tyr367'-OH and Tyr425'-OH interact with the nascent thiol(ate) on the flavin si-face. Kinetic data with Y367F, Y425F, and Y367, 425F BACoADR mutants indicate that Tyr425' is the primary proton donor in catalysis, with Tyr367' functioning as a cryptic alternate donor in the absence of Tyr425'.« less

  3. A novel cytochrome P450 CYP6AB14 gene in spodoptera litura (Lepidoptera: Noctuidae) and its potential role in plant allelochemical detoxification

    USDA-ARS?s Scientific Manuscript database

    Cytochrome P450 monooxygenases (P450) play a prominent role in the adaptation of insects to host plant chemical defenses. To investigate the potential role of P450s in adaptation of the lepidopteran pest Spodoptera litura to host plant allelochemicals, an expressed sequence data set derived from 6th...

  4. A novel cytochrome P450 CYP6AB14 gene in Spodoptera litura (Lepidoptera: Noctuidae) and its potential role in plant allelochemical detoxification

    USDA-ARS?s Scientific Manuscript database

    Cytochrome P450 monooxygenases (P450) play a prominent role in the adaptation of insects to host plant chemical defenses. To investigate the potential role of P450s in adaptation of the lepidopteran pest Spodoptera litura to host plant allelochemicals, an expressed sequence data set derived from 6th...

  5. Flexible docking-based molecular dynamics/steered molecular dynamics calculations of protein-protein contacts in a complex of cytochrome P450 1A2 with cytochrome b5.

    PubMed

    Jeřábek, Petr; Florián, Jan; Stiborová, Marie; Martínek, Václav

    2014-10-28

    Formation of transient complexes of cytochrome P450 (P450) with another protein of the endoplasmic reticulum membrane, cytochrome b5 (cyt b5), dictates the catalytic activities of several P450s. Therefore, we examined formation and binding modes of the complex of human P450 1A2 with cyt b5. Docking of soluble domains of these proteins was performed using an information-driven flexible docking approach implemented in HADDOCK. Stabilities of the five unique binding modes of the P450 1A2-cyt b5 complex yielded by HADDOCK were evaluated using explicit 10 ns molecular dynamics (MD) simulations in aqueous solution. Further, steered MD was used to compare the stability of the individual P450 1A2-cyt b5 binding modes. The best binding mode was characterized by a T-shaped mutual orientation of the porphyrin rings and a 10.7 Å distance between the two redox centers, thus satisfying the condition for a fast electron transfer. Mutagenesis studies and chemical cross-linking, which, in the absence of crystal structures, were previously used to deduce specific P450-cyt b5 interactions, indicated that the negatively charged convex surface of cyt b5 binds to the positively charged concave surface of P450. Our simulations further elaborate structural details of this interface, including nine ion pairs between R95, R100, R138, R362, K442, K455, and K465 side chains of P450 1A2 and E42, E43, E49, D65, D71, and heme propionates of cyt b5. The universal heme-centric system of internal coordinates was proposed to facilitate consistent classification of the orientation of the two porphyrins in any protein complex.

  6. Role of Ca2+-independent phospholipase A2 and cytochrome P-450 in store-operated calcium entry in 3T6 fibroblasts.

    PubMed

    Martínez, Javier; Moreno, Juan J

    2005-09-01

    Store-operated calcium (SOC) channels and capacitative Ca2+ entry play a key role in cellular functions, but their mechanism of activation remains unclear. Here, we show that thapsigargin induces [3H] arachidonic acid (AA) release, 45Ca2+ influx and a subsequent enhancement of intracellular calcium concentration ([Ca2+]i. Thapsigargin-induced elevation of [Ca2+]i was inhibited by cytochrome P-450 inhibitors and by cytochrome P-450 epoxygenase inhibitor and was reverted by 11,12 EET addition. However, cyclooxygenase and lipoxygenase inhibitors have no effect. Moreover, we observed that four EETs were able to induce 45Ca2+ influx. Finally, we reported that the effect of 11,12 EET on 45Ca2+ influx was sensible to receptor-operated Ca2+ channel blockers (NiCl2, LaCl3) but not to voltage-dependent Ca2+ channel blocker as verapamil. Thus, AA released by Ca2+-independent phospholipase A2 and AA metabolism through cytochrome P-450 pathway may be crucial molecular determinant in thapsigargin activation of SOC channels and store-operated Ca2+ entry pathway in 3T6 fibroblasts. Moreover, EETs, the main cytochrome P-450 epoxygenase metabolites of AA, are involved in thapsigargin-stimulated Ca2+ influx. In summary, our results suggest that EETs are components of calcium influx factor(s).

  7. Functioning of Microsomal Cytochrome P450s: Murburn Concept Explains the Metabolism of Xenobiotics in Hepatocytes.

    PubMed

    Manoj, Kelath Murali; Parashar, Abhinav; Gade, Sudeep K; Venkatachalam, Avanthika

    2016-01-01

    Using oxygen and NADPH, the redox enzymes cytochrome P450 (CYP) and its reductase (CPR) work in tandem to carry out the phase I metabolism of a vast majority of drugs and xenobiotics. As per the erstwhile understanding of the catalytic cycle, binding of the substrate to CYP's heme distal pocket allows CPR to pump electrons through a CPR-CYP complex. In turn, this trigger (a thermodynamic push of electrons) leads to the activation of oxygen at CYP's heme-center, to give Compound I, a two-electron deficient enzyme reactive intermediate. The formation of diffusible radicals and reactive oxygen species (DROS, hitherto considered an undesired facet of the system) was attributed to the heme-center. Recently, we had challenged these perceptions and proposed the murburn ("mured burning" or "mild unrestricted burning") concept to explain heme enzymes' catalytic mechanism, electron-transfer phenomena and the regulation of redox equivalents' consumption. Murburn concept incorporates a one-electron paradigm, advocating obligatory roles for DROS. The new understanding does not call for high-affinity substrate-binding at the heme distal pocket of the CYP (the first and the most crucial step of the erstwhile paradigm) or CYP-CPR protein-protein complexations (the operational backbone of the erstwhile cycle). Herein, the dynamics of reduced nicotinamide nucleotides' consumption, peroxide formation and depletion, product(s) formation, etc. was investigated with various controls, by altering reaction variables, environments and through the incorporation of diverse molecular probes. In several CYP systems, control reactions lacking the specific substrate showed comparable or higher peroxide in milieu, thereby discrediting the foundations of the erstwhile hypothesis. The profiles obtained by altering CYP:CPR ratios and the profound inhibitions observed upon the incorporation of catalytic amounts of horseradish peroxidase confirm the obligatory roles of DROS in milieu, ratifying

  8. Homology modelling of Drosophila cytochrome P450 enzymes associated with insecticide resistance.

    PubMed

    Jones, Robert T; Bakker, Saskia E; Stone, Deborah; Shuttleworth, Sally N; Boundy, Sam; McCart, Caroline; Daborn, Phillip J; ffrench-Constant, Richard H; van den Elsen, Jean M H

    2010-10-01

    Overexpression of the cytochrome P450 gene Cyp6g1 confers resistance against DDT and a broad range of other insecticides in Drosophila melanogaster Meig. In the absence of crystal structures of CYP6G1 or complexes with its substrates, structural studies rely on homology modelling and ligand docking to understand P450-substrate interactions. Homology models are presented for CYP6G1, a P450 associated with resistance to DDT and neonicotinoids, and two other enzymes associated with insecticide resistance in D. melanogaster, CYP12D1 and CYP6A2. The models are based on a template of the X-ray structure of the phylogenetically related human CYP3A4, which is known for its broad substrate specificity. The model of CYP6G1 has a much smaller active site cavity than the template. The cavity is also 'V'-shaped and is lined with hydrophobic residues, showing high shape and chemical complementarity with the molecular characteristics of DDT. Comparison of the DDT-CYP6G1 complex and a non-resistant CYP6A2 homology model implies that tight-fit recognition of this insecticide is important in CYP6G1. The active site can accommodate differently shaped substrates ranging from imidacloprid to malathion but not the pyrethroids permethrin and cyfluthrin. The CYP6G1, CYP12D1 and CYP6A2 homology models can provide a structural insight into insecticide resistance in flies overexpressing P450 enzymes with broad substrate specificities.

  9. Human Cytochrome P450 Enzyme Modulation by Gymnema sylvestre: A Predictive Safety Evaluation by LC-MS/MS

    PubMed Central

    Rammohan, Bera; Samit, Karmakar; Chinmoy, Das; Arup, Saha; Amit, Kundu; Ratul, Sarkar; Sanmoy, Karmakar; Dipan, Adhikari; Tuhinadri, Sen

    2016-01-01

    Background: Traditionally GS is used to treat diabetes mellitus. Drug-herb interaction of GS via cytochrome P450 enzyme system by substrate cocktail method using HLM has not been reported. Objective: To evaluate the in-vitro modulatory effects of GS extracts (aqueous, methanol, ethyl acetate, chloroform and n-hexane) and deacylgymnemic acid (DGA) on human CYP1A2, 2C8, 2C9, 2D6 and 3A4 activities in HLM. Material and Methods: Probe substrate-based LCMS/MS method was established for all CYPs. The metabolite formations were examined after incubation of probe substrates with HLM in the presence or absence of extracts and DGA. The inhibitory effects of GS extracts and DGA were characterized with kinetic parameters IC50 and Ki values. Results: GS extracts showed differential effect on CYP activities in the following order of inhibitory potency: ethyl acetate > Chloroform > methanol > n-hexane > aqueous > DGA. This differential effect was observed against CYP1A2, 2C9 and less on CYP3A4 and 2C8 but all CYPs were unaffected by aqueous extract and DGA. The ethyl acetate and chloroform extract exhibited moderate inhibition towards CYP1A2 and 3A4. The aqueous extract and DGA however showed negligible inhibition towards all five major human CYPs with very high IC50 values (>90μg/ml). Conclusion: The results of our study revealed that phytoconstituents contained in GS, particularly in ethyl acetate and chloroform extracts, were able to inhibit CYP1A2, 3A4 and 2C9. The presence of relatively small, lipophillic yet slightly polar compounds within the GS extracts may be attributed for inhibition activities. These suggest that the herb or its extracts should be examined for potential pharmacokinetic drug interactions in vivo. Abbreviations used: GS: Gymnema sylvestre, GSE: Gymnema sylvestre extract, DGA: deacyl gymnemic acid, CYP: cytochrome P450, DMSO: dimethylsulphoxide, HLM: human liver microsomes, LC-MS/MS: liquid chromatography tandem mass spectroscopy, NADPH: reduced

  10. Human Cytochrome P450 Enzyme Modulation by Gymnema sylvestre: A Predictive Safety Evaluation by LC-MS/MS.

    PubMed

    Rammohan, Bera; Samit, Karmakar; Chinmoy, Das; Arup, Saha; Amit, Kundu; Ratul, Sarkar; Sanmoy, Karmakar; Dipan, Adhikari; Tuhinadri, Sen

    2016-07-01

    Traditionally GS is used to treat diabetes mellitus. Drug-herb interaction of GS via cytochrome P450 enzyme system by substrate cocktail method using HLM has not been reported. To evaluate the in-vitro modulatory effects of GS extracts (aqueous, methanol, ethyl acetate, chloroform and n -hexane) and deacylgymnemic acid (DGA) on human CYP1A2, 2C8, 2C9, 2D6 and 3A4 activities in HLM. Probe substrate-based LCMS/MS method was established for all CYPs. The metabolite formations were examined after incubation of probe substrates with HLM in the presence or absence of extracts and DGA. The inhibitory effects of GS extracts and DGA were characterized with kinetic parameters IC50 and Ki values. GS extracts showed differential effect on CYP activities in the following order of inhibitory potency: ethyl acetate > Chloroform > methanol > n -hexane > aqueous > DGA. This differential effect was observed against CYP1A2, 2C9 and less on CYP3A4 and 2C8 but all CYPs were unaffected by aqueous extract and DGA. The ethyl acetate and chloroform extract exhibited moderate inhibition towards CYP1A2 and 3A4. The aqueous extract and DGA however showed negligible inhibition towards all five major human CYPs with very high IC50 values (>90μg/ml). The results of our study revealed that phytoconstituents contained in GS, particularly in ethyl acetate and chloroform extracts, were able to inhibit CYP1A2, 3A4 and 2C9. The presence of relatively small, lipophillic yet slightly polar compounds within the GS extracts may be attributed for inhibition activities. These suggest that the herb or its extracts should be examined for potential pharmacokinetic drug interactions in vivo . Abbreviations used: GS: Gymnema sylvestre , GSE: Gymnema sylvestre extract, DGA: deacyl gymnemic acid, CYP: cytochrome P450, DMSO: dimethylsulphoxide, HLM: human liver microsomes, LC-MS/MS: liquid chromatography tandem mass spectroscopy, NADPH: reduced nicotinamide adeninedinucleotide phosphate, NRS: nicotinamide

  11. 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

  12. Molecular Interface of S100A8 with Cytochrome b558 and NADPH Oxidase Activation

    PubMed Central

    Berthier, Sylvie; Hograindleur, Marc-André; Paclet, Marie-Hélène; Polack, Benoît; Morel, Françoise

    2012-01-01

    S100A8 and S100A9 are two calcium binding Myeloid Related Proteins, and important mediators of inflammatory diseases. They were recently introduced as partners for phagocyte NADPH oxidase regulation. However, the precise mechanism of their interaction remains elusive. We had for aim (i) to evaluate the impact of S100 proteins on NADPH oxidase activity; (ii) to characterize molecular interaction of either S100A8, S100A9, or S100A8/S100A9 heterocomplex with cytochrome b 558; and (iii) to determine the S100A8 consensus site involved in cytochrome b 558/S100 interface. Recombinant full length or S100A9-A8 truncated chimera proteins and ExoS-S100 fusion proteins were expressed in E. coli and in P. aeruginosa respectively. Our results showed that S100A8 is the functional partner for NADPH oxidase activation contrary to S100A9, however, the loading with calcium and a combination with phosphorylated S100A9 are essential in vivo. Endogenous S100A9 and S100A8 colocalize in differentiated and PMA stimulated PLB985 cells, with Nox2/gp91phox and p22phox. Recombinant S100A8, loaded with calcium and fused with the first 129 or 54 N-terminal amino acid residues of the P. aeruginosa ExoS toxin, induced a similar oxidase activation in vitro, to the one observed with S100A8 in the presence of S100A9 in vivo. This suggests that S100A8 is the essential component of the S100A9/S100A8 heterocomplex for oxidase activation. In this context, recombinant full-length rS100A9-A8 and rS100A9-A8 truncated 90 chimera proteins as opposed to rS100A9-A8 truncated 86 and rS100A9-A8 truncated 57 chimeras, activate the NADPH oxidase function of purified cytochrome b 558 suggesting that the C-terminal region of S100A8 is directly involved in the molecular interface with the hemoprotein. The data point to four strategic 87HEES90 amino acid residues of the S100A8 C-terminal sequence that are involved directly in the molecular interaction with cytochrome b558 and then in the phagocyte NADPH oxidase

  13. Crystal structure of conjugated polyketone reductase (CPR-C1) from Candida parapsilosis IFO 0708 complexed with NADPH.

    PubMed

    Qin, Hui-Min; Yamamura, Akihiro; Miyakawa, Takuya; Kataoka, Michihiko; Maruoka, Shintaro; Ohtsuka, Jun; Nagata, Koji; Shimizu, Sakayu; Tanokura, Masaru

    2013-11-01

    Conjugated polyketone reductase (CPR-C1) from Candida parapsilosis IFO 0708 is a member of the aldo-keto reductase (AKR) superfamily and reduces ketopantoyl lactone to d-pantoyl lactone in a NADPH-dependent and stereospecific manner. We determined the crystal structure of CPR-C1.NADPH complex at 2.20 Å resolution. CPR-C1 adopted a triose-phosphate isomerase (TIM) barrel fold at the core of the structure in which Thr25 and Lys26 of the GXGTX motif bind uniquely to the adenosine 2'-phosphate group of NADPH. This finding provides a novel structural basis for NADPH binding of the AKR superfamily. Copyright © 2013 Wiley Periodicals, Inc.

  14. Induction by carrot allelochemicals of insecticide-metabolising enzymes in the southern armyworm (Spodoptera eridania).

    PubMed

    Brattsten, L B; Evans, C K; Bonetti, S; Zalkow, L H

    1984-01-01

    Carrot foliage monoterpenes induce cytochrome P-450 up to 2.9-fold, NADPH cytochrome c (P-450) reductase up to 1.6-fold, NADPH-oxidation up to 3.8-fold, aldrin epoxidation up to 1.5-fold in southern armyworm larval midgut tissues when incorporated in their diet at 0.2% for 3 days. Stigmasterol and ergosterol did not substantially induce microsomal oxidase activities and significantly inhibited GSH S-aryltransferase activity and sulfotransferase activity. Coumarin did not substantially affect microsomal oxidase and sulfotransferase activity but is the most potent inducer of GSH S-aryltransferase activity, increasing this activity 7-fold. None of the chemicals is acutely toxic to the sixth instar larvae or affect the larval weight gain except coumarin which significantly depressed the maximal body weight attained.

  15. Environmentally persistent free radical-containing particulate matter competitively inhibits metabolism by cytochrome P450 1A2

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Reed, James R., E-mail: rreed@lsuhsc.edu; Cruz, Albert Leo N. dela, E-mail: adelac2@tigers.lsu.edu; Lomnicki, Slawo M., E-mail: slomni1@lsu.edu

    Combustion processes generate different types of particulate matter (PM) that can have deleterious effects on the pulmonary and cardiovascular systems. Environmentally persistent free radicals (EPFRs) represent a type of particulate matter that is generated after combustion of environmental wastes in the presence of redox-active metals and aromatic hydrocarbons. Cytochromes P450 (P450/CYP) are membrane-bound enzymes that are essential for the phase I metabolism of most lipophilic xenobiotics. The EPFR formed by chemisorption of 2-monochlorophenol to silica containing 5% copper oxide (MCP230) has been shown to generally inhibit the activities of different forms of P450s without affecting those of cytochrome P450 reductasemore » and heme oxygenase-1. The mechanism of inhibition of rat liver microsomal CYP2D2 and purified rabbit CYP2B4 by MCP230 has been shown previously to be noncompetitive with respect to substrate. In this study, MCP230 was shown to competitively inhibit metabolism of 7-benzyl-4-trifluoromethylcoumarin and 7-ethoxyresorufin by the purified, reconstituted rabbit CYP1A2. MCP230 is at least 5- and 50-fold more potent as an inhibitor of CYP1A2 than silica containing 5% copper oxide and silica, respectively. Thus, even though PM generally inhibit multiple forms of P450, PM interacts differently with the forms of P450 resulting in different mechanisms of inhibition. P450s function as oligomeric complexes within the membrane. We also determined the mechanism by which PM inhibited metabolism by the mixed CYP1A2–CYP2B4 complex and found that the mechanism was purely competitive suggesting that the CYP2B4 is dramatically inhibited when bound to CYP1A2. - Highlights: • Combustion of organic pollutants generates long-lived particulate radicals (EPFRs). • Particulate matter (PM) competitively inhibited CYP1A2 activity. • EPFRs were much more potent CYP1A2 inhibitors than other types of PM. • PM interacts differently with different forms of P450.

  16. Cyp15F1: A novel cytochrome P450 gene linked to juvenile hormone-dependent caste differention in the termite R. flavipes

    USDA-ARS?s Scientific Manuscript database

    Termites are eusocial insects that perform social interactions that facilitate chemical signaling. Previous research identified two cytochrome P450s that have homology to other insect p450s responsible for the production of juvenile hormone. Juvenile hormone is an important morphogenic hormone tha...

  17. Effects of tributyltin on the MFO system of the clam Ruditapes decussata: a laboratory and field approach.

    PubMed

    Solé, M

    2000-01-01

    The in vivo interaction of tributyltin (TBT) with the microsomal monooxygenase (MFO) system of the clam Ruditapes decussata was studied. For this purpose, two experiments were designed: (1) a laboratory exposure to increasing nominal doses of TBT (90, 454 and 2268 ng l(-1)) for 1 week and (2) a clam transplant from a clean area to an organotin polluted marina for periods of up to 5 weeks. Chemical analysis of organotins in clam tissue was used to relate TBT body burden to the MFO response. Neither the laboratory nor the field transplant experiment showed any significant TBT effect on the clam's digestive gland MFO components (cytochrome P450 and cytochrome b(5)). However, a significant elevation in the NADPH cytochrome (P450) reductases at the low and medium TBT doses in the laboratory and a significant decrease in NADH cytochrome (b(5)) reductases, 1 week after the field transplant, was observed with further recovery to control levels thereafter.

  18. Evolutionary interplay between sister cytochrome P450 genes shapes plasticity in plant metabolism.

    PubMed

    Liu, Zhenhua; Tavares, Raquel; Forsythe, Evan S; André, François; Lugan, Raphaël; Jonasson, Gabriella; Boutet-Mercey, Stéphanie; Tohge, Takayuki; Beilstein, Mark A; Werck-Reichhart, Danièle; Renault, Hugues

    2016-10-07

    Expansion of the cytochrome P450 gene family is often proposed to have a critical role in the evolution of metabolic complexity, in particular in microorganisms, insects and plants. However, the molecular mechanisms underlying the evolution of this complexity are poorly understood. Here we describe the evolutionary history of a plant P450 retrogene, which emerged and underwent fixation in the common ancestor of Brassicales, before undergoing tandem duplication in the ancestor of Brassicaceae. Duplication leads first to gain of dual functions in one of the copies. Both sister genes are retained through subsequent speciation but eventually return to a single copy in two of three diverging lineages. In the lineage in which both copies are maintained, the ancestral functions are split between paralogs and a novel function arises in the copy under relaxed selection. Our work illustrates how retrotransposition and gene duplication can favour the emergence of novel metabolic functions.

  19. 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

  20. Reduction of aromatic and heterocyclic aromatic N-hydroxylamines by human cytochrome P450 2S1.

    PubMed

    Wang, Kai; Guengerich, F Peter

    2013-06-17

    Many aromatic amines and heterocyclic aromatic amines (HAAs) are known carcinogens for animals, and there is also strong evidence of some in human cancer. The activation of these compounds, including some arylamine drugs, involves N-hydroxylation, usually by cytochrome P450 enzymes (P450) in Family 1 (1A2, 1A1, and 1B1). We previously demonstrated that the bioactivation product of the anticancer agent 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203), an N-hydroxylamine, can be reduced by P450 2S1 to its amine precursor under anaerobic conditions and, to a lesser extent, under aerobic conditions [Wang, K., and Guengerich, F. P. (2012) Chem. Res. Toxicol. 25, 1740-1751]. In the study presented here, we tested the hypothesis that P450 2S1 is involved in the reductive biotransformation of known carcinogenic aromatic amines and HAAs. The N-hydroxylamines of 4-aminobiphenyl (4-ABP), 2-naphthylamine (2-NA), and 2-aminofluorene (2-AF) were synthesized and found to be reduced by P450 2S1 under both anaerobic and aerobic conditions. The formation of amines due to P450 2S1 reduction also occurred under aerobic conditions but was less apparent because the competitive disproportionation reactions (of the N-hydroxylamines) also yielded amines. Further, some nitroso and nitro derivatives of the arylamines could also be reduced by P450 2S1. None of the amines tested were oxidized by P450 2S1. These results suggest that P450 2S1 may be involved in the reductive detoxication of several of the activated products of carcinogenic aromatic amines and HAAs.

  1. Reduction of Aromatic and Heterocyclic Aromatic N-Hydroxylamines by Human Cytochrome P450 2S1

    PubMed Central

    Wang, Kai; Guengerich, F. Peter

    2013-01-01

    Many aromatic amines and heterocyclic aromatic amines (HAAs) are known carcinogens for animals and there is also strong evidence for some in human cancer. The activation of these compounds, including some arylamine drugs, involves N-hydroxylation, usually by cytochrome P450 enzymes (P450) in Family 1 (1A2, 1A1, and 1B1). We previously demonstrated that the bioactivation product of the anti-cancer agent 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203), an N-hydroxylamine, can be reduced by P450 2S1 to its amine precursor under anaerobic conditions and, to a lesser extent, under aerobic conditions (Wang, K., and Guengerich, F. P. (2012) Chem. Res. Toxicol. 25, 1740–1751). In the present study, we tested the hypothesis that P450 2S1 is involved in the reductive biotransformation of known carcinogenic aromatic amines and HAAs. The N-hydroxylamines of 4-aminobiphenyl (4-ABP), 2-naphthylamine (2-NA), and 2-aminofluorene (2-AF) were synthesized and found to be reduced by P450 2S1 under both anaerobic and aerobic conditions. The formation of amines due to P450 2S1 reduction also occurred under aerobic conditions but was less apparent because the competitive disproportionation reactions (of the N-hydroxylamines) also yielded amines. Further, some nitroso and nitro derivatives of the arylamines could also be reduced by P450 2S1. None of the amines tested were oxidized by P450 2S1. These results suggest that P450 2S1 may be involved in the reductive detoxication of several of the activated products of carcinogenic aromatic amines and HAAs. PMID:23682735

  2. Human cytochrome P450scc (CYP11A1) catalyzes epoxide formation with ergosterol.

    PubMed

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

    2012-03-01

    Cytochrome P450scc (P450scc) catalyzes the cleavage of the side chain of both cholesterol and the vitamin D(3) precursor, 7-dehydrocholesterol. The aim of this study was to test the ability of human P450scc to metabolize ergosterol, the vitamin D(2) precursor, and define the structure of the major products. P450scc incorporated into the bilayer of phospholipid vesicles converted ergosterol to two major and four minor products with a k(cat) of 53 mol · min(-1) · mol P450scc(-1) and a K(m) of 0.18 mol ergosterol/mol phospholipid, similar to the values observed for cholesterol metabolism. The reaction of ergosterol with P450scc was scaled up to make enough of the two major products for structural analysis. From mass spectrometry, NMR, and comparison of the NMR data to that for similar molecules, we determined the structures of the two major products as 20-hydroxy-22,23-epoxy-22,23-dihydroergosterol and 22-keto-23-hydroxy-22,23-dihydroergosterol. Molecular modeling and nuclear Overhauser effect (or enhancement) spectroscopy spectra analysis helped to establish the configurations at C20, C22, and C23 and determine the final structures of major products as 22R,23S-epoxyergosta-5,7-diene-3β,20α-diol and 3β,23S-dihydroxyergosta-5,7-dien-22-one. It is likely that the formation of the second product is through a 22,23-epoxy (oxirane) intermediate followed by C22 hydroxylation with the formation of strained 22-hydroxy-22,23-epoxide (oxiranol), which is immediately transformed to the more stable α-hydroxyketone. Molecular modeling of ergosterol into the P450scc crystal structure positioned the ergosterol side chain consistent with formation of the above products. Thus, we have shown that P450scc efficiently catalyzes epoxide formation with ergosterol giving rise to novel epoxy, hydroxy, and keto derivatives, without causing cleavage of the side chain.

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

    PubMed

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

    1987-07-14

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

  4. Purification and Characterization of Ferredoxin-Nicotinamide Adenine Dinucleotide Phosphate Reductase from a Nitrogen-Fixing Bacterium

    PubMed Central

    Yoch, Duane C.

    1973-01-01

    Evidence suggesting that Bacillus polymyxa has an active ferredoxin-NADP+ reductase (EC 1.6.99.4) was obtained when NADPH was found to provide reducing power for the nitrogenase of this organism; direct evidence was provided when it was shown that B. polymyxa extracts could substitute for the native ferredoxin-NADP+ reductase in the photochemical reduction of NADP+ by blue-green algal particles. The ferredoxin-NADP+ reductase was purified about 80-fold by a combination of high-speed centrifugation, ammonium sulfate fractionation, and chromatography on Sephadex G-100 and diethylaminoethyl-cellulose. The molecular weight was estimated by gel filtration to be 60,000. A small amount of the enzyme was further purified by polyacrylamide gel electrophoresis and shown to be a flavoprotein. The reductase was specific for NADPH in the ferredoxin-dependent reduction of cytochrome c and methyl viologen diaphorase reactions; furthermore, NADP+ was the acceptor of preference when the electron donor was photoreduced ferredoxin. The reductase also has an irreversible NADPH-NAD+ transhydrogenase (reduced-NADP:NAD oxidoreductase, EC 1.6.1.1) activity, the rate of which was proportional to the concentration of NAD (Km = 5.0 × 10−3M). The reductase catalyzed electron transfer from NADPH not only to B. polymyxa ferredoxin but also to the ferredoxins of Clostridium pasteurianum, Azotobacter vinelandii, and spinach chloroplasts, although less effectively. Rubredoxin from Clostridium acidi-urici and azotoflavin from A. vinelandii also accept electrons from the B. polymyxa reductase. The pH optima for the various reactions catalyzed by the B. polymyxa ferredoxin-NADP reductase are similar to those of the chloroplast reductase. NAD and acetyl-coenzyme A, which obligatorily activate NADPH- and NADH-ferredoxin reductases, respectively, in Clostridium kluyveri, have no effect on B. polymyxa reductase. PMID:4147648

  5. Scutellarin inhibits cytochrome P450 isoenzyme 1A2 (CYP1A2) in rats.

    PubMed

    Jian, Tun-Yu; He, Jian-Chang; He, Gong-Hao; Feng, En-Fu; Li, Hong-Liang; Bai, Min; Xu, Gui-Li

    2012-08-01

    Scutellarin is the most important flavone glycoside in the herbal drug Erigeron breviscapus (Vant.) Hand.-Mazz. It is used frequently in the clinic to treat ischemic vascular diseases in China. However, the direct relationship between scutellarin and cytochrome P450 (CYP450) is unclear. The present study investigated the in vitro and in vivo effects of scutellarin on cytochrome P450 1A2 (CYP 1A2) metabolism. According to in vitro experiments, scutellarin (10-250 µM) decreased the formation of 4-acetamidophenol in a concentration-dependent manner, with an IC₅₀ value of 108.20 ± 0.657 µM. Furthermore, scutellarin exhibited a weak mixed-type inhibition against the activity of CYP1A2 in rat liver microsomes, with a K(i) value of 95.2 µM. Whereas in whole animal studies, scutellarin treatment for 7 days (at 5, 15, 30 mg/kg, i.p.) decreased the clearance (CL), and increased the T(1/2) (at 15, 30 mg/kg, i.p.), it did not affect the V(d) of phenacetin. Scutellarin treatment (at 5, 15, 30 mg/kg, i.p.) increased the AUC(0-∞) by 14.3%, 67.3% and 159.2%, respectively. Scutellarin at 30 mg/kg also weakly inhibited CYP1A2 activity, in accordance with our in vitro study. Thus, the results indicate that CYP1A2 is inhibited directly, but weakly, by scutellarin in vivo, and provide useful information on the safe and effective use of scutellarin in clinical practice. Copyright © 2012 John Wiley & Sons, Ltd.

  6. Disparity in holoprotein/apoprotein ratios of different standards used for immunoquantification of hepatic cytochrome P450 enzymes.

    PubMed

    Perrett, H F; Barter, Z E; Jones, B C; Yamazaki, H; Tucker, G T; Rostami-Hodjegan, A

    2007-10-01

    An analysis of reported hepatic abundances of CYP3A4 and 3A5 indicated that values determined by immunoquantification using commercially available, unpurified recombinant enzymes as standards are significantly lower than those determined using purified enzymes or human liver microsomes characterized with lysosomal peptides (CYP3A4: mean 45 versus 121 pmol/mg protein, p < 0.01; CYP3A5: mean 28 versus 83 pmol/mg protein, p < 0.05). When immunoquantifying cytochromes P450 (P450s), it is assumed that the holoprotein (holo)/apoprotein ratio is the same in the samples and the standard. Estimates of holo/apoprotein ratios from data reported for a range of P450s purified from human liver and non-commercial recombinant systems indicated less than complete and variable heme coupling dependent on enzyme and system.

  7. A role for cytochrome b5 in the in vivo disposition of anti-cancer and cytochrome P450 probe drugs in mice

    PubMed Central

    Henderson, Colin J.; McLaughlin, Lesley A.; Finn, Robert D.; Ronseaux, Sebastien; Kapelyukh, Yury; Wolf, C. Roland

    2014-01-01

    The role of microsomal cytochrome b5 (Cyb5) in defining the rate of drug metabolism and disposition has been intensely debated for several decades. Recently we described mouse models involving the hepatic or global deletion of Cyb5, demonstrating its central role in in vivo drug disposition. We have now used the cytochrome b5 complete null (BCN) model to determine the role of Cyb5 in the metabolism of ten pharmaceuticals metabolised by a range of cytochrome P450s, including five anti-cancer drugs, in vivo and in vitro. The extent to which metabolism was significantly affected by the absence of Cyb5 was substrate-dependent, with AUC increased (75-245%), and clearance decreased (35-72%), for phenacetin, metoprolol and chlorzoxazone. Tolbutamide disposition was not significantly altered by Cyb5 deletion, while for midazolam clearance was decreased by 66%. The absence of Cyb5 had no effect on gefitinib and paclitaxel disposition, while significant changes in the in vivo pharmacokinetics of cyclophosphamide were measured (Cmax and terminal half-life increased 55% and 40%, respectively), tamoxifen (AUClast and Cmax increased 370% and 233%, respectively) and anastrozole (AUC and terminal half-life increased 125% and 62%, respectively; clearance down 80%). These data from provide strong evidence that both hepatic and extra-hepatic Cyb5 levels are an important determinant of in vivo drug disposition catalysed by a range of cytochrome P450s, including currently-prescribed anti-cancer agents, and that individuality in Cyb5 expression could be a significant determinant in rates of drug disposition in man. PMID:24115751

  8. 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.

  9. Expression, function and regulation of mouse cytochrome P450 enzymes: comparison with human P450 enzymes.

    PubMed

    Hrycay, E G; Bandiera, S M

    2009-12-01

    The present review focuses on the expression, function and regulation of mouse cytochrome P450 (Cyp) enzymes. Information compiled for mouse Cyp enzymes is compared with data collected for human CYP enzymes. To date, approximately 40 pairs of orthologous mouse-human CYP genes have been identified that encode enzymes performing similar metabolic functions. Recent knowledge concerning the tissue expression of mouse Cyp enzymes from families 1 to 51 is summarized. The catalytic activities of microsomal, mitochondrial and recombinant mouse Cyp enzymes are discussed and their involvement in the metabolism of exogenous and endogenous compounds is highlighted. The role of nuclear receptors, such as the aryl hydrocarbon receptor, constitutive androstane receptor and pregnane X receptor, in regulating the expression of mouse Cyp enzymes is examined. Targeted disruption of selected Cyp genes has generated numerous Cyp null mouse lines used to decipher the role of Cyp enzymes in metabolic, toxicological and biological processes. In conclusion, the laboratory mouse is an indispensable model for exploring human CYP-mediated activities.

  10. Identification of a novel cytochrome P450 gene, CYP321E1 from the diamondback moth, Plutella xylostella (L.) and RNA interference to evaluate its role in chlorantraniliprole resistance.

    PubMed

    Hu, Z; Lin, Q; Chen, H; Li, Z; Yin, F; Feng, X

    2014-12-01

    Insect cytochrome P450 monooxygenases (P450s) play an important role in catalysis of many reactions leading to insecticides resistance. Our previous studies on transcriptome analysis of chlorantraniliprole-resistant development in the diamondback moth, Plutella xylostella revealed that up-regulation of cytochrome P450s are one of the main factors leading to the development of chlorantraniliprole resistance. Here, we report for the first time a novel cytochrome P450 gene CYP321E1, which belongs to the cytochrome P450 gene family CYP321. Real-time quantitative PCR (RT-qPCR) analyses indicated that CYP321E1 was expressed at all developmental stages of P. xylostella but was highest in the fourth-instar larvae; furthermore, the relatively high expression was observed in the midgut of the fourth-instar larvae, followed by fat bodies and epidermis. The expression of CYP321E1 in P. xylostella was differentially affected by three representative insecticides, including alphamethrin, abamectin and chlorantraniliprole. Among them, the exposure to chlorantraniliprole resulted in the largest transcript level of this cytochrome P450 gene. The findings suggested potential involvement of CYP321E1 in chlorantraniliprole resistance of P. xylostella. To assess the functional link of CYP321E1 to chlorantraniliprole resistance, RNA interference (RNAi)-mediated gene silencing by double stranded RNA (dsRNA) injecting was used. Results revealed that injection delivery of dsRNA can greatly reduce gene expression after 24 h. As a consequence of RNAi, a significant increment in mortality of larvae injected CYP321E1 dsRNA was observed after 24 h of exposure to chlorantraniliprole. These results strongly support our notion that this novel cytochrome P450 gene plays an important role in chlorantraniliprole detoxification in the diamondback moth and is partly responsible for its resistance.

  11. Cytochrome P450 2E1 inhibition prevents hepatic carcinogenesis induced by diethylnitrosamine in alcohol-fed rats

    USDA-ARS?s Scientific Manuscript database

    Chronic alcohol ingestion increases hepatic cytochrome P450 2E1 (CYP2E1), which is associated with hepatocarcinogenesis. We investigated whether treatment with chlormethiazole (CMZ), a CYP2E1 inhibitor, protects against alcohol-associated hepatic carcinogenesis in rats. Rats were fed either an ethan...

  12. Human Liver Cytochrome P450 3A4 Ubiquitination

    PubMed Central

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

    2015-01-01

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

  13. Electrochemistry of Cytochrome P450 BM3 in Sodium Dodecyl Sulfate Films

    PubMed Central

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

    2008-01-01

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

  14. Different structure of the complexes of two cytochrome P-450 isozymes with acetanilide by 1H-NMR relaxation and spectrophotometry.

    PubMed

    Woldman YaYu; Weiner, L M; Lyakhovich, V V

    1993-05-28

    The functional and spectral characteristics of the interaction of acetanilide with phenobarbital- and methylcholanthrene- induced rat liver microsomes, as well as with corresponding major isozymes (cytochromes P-450b and P-450c) have been compared. The magnitude of the reverse 1st type binding spectra proved to be negatively correlated with the acetanilide oxidation on isozymes under study. The data on paramagnetic relaxation of acetanilide protons in the presence of P-450 have shown the structure of the enzyme-substrate complex to be different for two isozymes, acetanilide molecule being closer to Fe ion in the active site in the case of P-450c, which is active towards acetanilide oxidation. For the P-450c-acetanilide complex the group oxidized (phenyl) is the closest to Fe ion.

  15. Ectopic expression of an apple cytochrome P450 gene MdCYPM1 negatively regulates plant photomorphogenesis and stress response in Arabidopsis.

    PubMed

    An, Jian-Ping; Li, Rui; Qu, Feng-Jia; You, Chun-Xiang; Wang, Xiao-Fei; Hao, Yu-Jin

    2017-01-29

    Cytochrome P450s play an important role in plant growth and are involved in multiple stresses response. However, little is known about the functions of cytochrome P450s in apple. Here, a Malus × domestica cytochrome P450 monooxygenase 1 gene, MdCYPM1, was identified and subsequently cloned from apple 'Gala' (Malus × domestica). To verify the functions of MdCYPM1, we generated transgenic Arabidopsis plants expressing the apple MdCYPM1 gene under the control of the Cauliflower mosaic virus 35S promoter. Four transgenic lines (#3, #5, #7 and #8) were selected for further study. The transgenic plants exhibited a series of skotomorphogenesis phenotypes relative to wild-type controls, such as reduction of the chlorophyll, anthocyanins content and hypocotyls elongation. In addition, overexpression of MdCYPM1 influenced auxin transport and flowering time in transgenic Arabidopsis. Furthermore, MdCYPM1 expression was induced by salt and mannitol treatments, and the transgenic plants were negatively regulated by salinity and osmotic stresses during germination. These results suggest that MdCYPM1 plays a vital role in plant growth and development. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Constituents of Indonesian medicinal plant Averrhoa bilimbi and their cytochrome P450 3A4 and 2D6 inhibitory activities.

    PubMed

    Auw, Lidyawati; Subehan; Sukrasno; Kadota, Shigetoshi; Tezuka, Yasuhiro

    2015-01-01

    As constituents of Averrhoa bilimbi leaves we identified three new compounds (1-3) together with 12 known ones (4-15); their inhibitory activities on cytochrome P450 3A4 (CYP3A4) and 2D6 (CYP2D6) were examined. Among the isolated compounds, the mixture of 1 and 2, and compounds 4 and 9 showed strong inhibition on CYP3A4, but mild or no inhibition on CYP2D6. These compounds revealed the characteristics of 1) time- and concentration-dependent inhibition, 2) requirement of NADPH for the inhibition, 3) no protection by nucleophiles, and 4) suppression of the inhibition by competitive inhibitor. Thus, they are suggested to be mechanism-based inactivators of CYP3A4 and CYP2D6. The kinetic parameters for the inactivation (k(inact) and K(I)) were 0.19 min(-1) and 36.7 μM for the mixture of 1 and 2, 0.126 min(-1) and 10.5 μM for 4, and 0.29 min(-1) and 23.4 μM for 9.

  17. 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

  18. The Use of Human Liver Cell Model and Cytochrome P450 Substrate-Inhibitor Panel for Studies of Dasatinib and Warfarin Interactions.

    PubMed

    Zakharyants, A A; Burmistrova, O A; Poloznikov, A A

    2017-02-01

    The possibility of interactions between warfarin and dasatinib and their interactions with other drugs metabolized by cytochrome P450 isoform CYP3A4 was demonstrated using a previously created cytochrome P450 substrate-inhibitor panel for preclinical in vitro studies of drug biotransformation on a 3D histotypical microfluidic cell model of human liver (liver-on-a-chip technology). Dasatinib and warfarin are inhibitors of CYP2C19 isoform and hence, can interfere the drugs metabolized by this isoform. Our findings are in line with the data obtained on primary culture of human hepatocytes and suggest that the model can be used in preclinical in vitro studies of drugs.

  19. Pharmacokinetics and Differential Regulation of Cytochrome P450 Enzymes in Type 1 Allergic Mice.

    PubMed

    Tanino, Tadatoshi; Komada, Akira; Ueda, Koji; Bando, Toru; Nojiri, Yukie; Ueda, Yukari; Sakurai, Eiichi

    2016-12-01

    Type 1 allergic diseases are characterized by elevated production of specific immunoglobulin E (IgE) for each antigen and have become a significant health problem worldwide. This study investigated the effect of IgE-mediated allergy on drug pharmacokinetics. To further understand differential suppression of hepatic cytochrome P450 (P450) activity, we examined the inhibitory effect of nitric oxide (NO), a marker of allergic conditions. Seven days after primary sensitization (PS7) or secondary sensitization (SS7), hepatic CYP1A2, CYP2C, CYP2E1, and CYP3A activities were decreased to 45%-75% of the corresponding control; however, CYP2D activity was not downregulated. PS7 and SS7 did not change the expression levels of five P450 proteins. Disappearance of CYP1A2 and CYP2D substrates from the plasma was not significantly different between allergic mice and control mice. In contrast, the area under the curve of a CYP1A2-mediated metabolite in PS7 and SS7 mice was reduced by 50% of control values. Total clearances of a CYP2E1 substrate in PS7 and SS7 mice were significantly decreased to 70% and 50% respectively, of the control without altering plasma protein binding. Hepatic amounts of CYP1A2 and CYP2E1 substrates were enhanced by allergic induction, being responsible for each downregulated activity. NO scavenger treatment completely improved the downregulated P450 activities. Therefore, our data suggest that the onset of IgE-mediated allergy alters the pharmacokinetics of major P450-metabolic capacity-limited drugs except for CYP2D drugs. NO is highly expected to participate in regulatory mechanisms of the four P450 isoforms. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

  20. Regulation of cytochrome P-450 4A activity by peroxisome proliferator-activated receptors in the rat kidney.

    PubMed

    Ishizuka, Tsuneo; Ito, Osamu; Tan, Liping; Ogawa, Susumu; Kohzuki, Masahiro; Omata, Ken; Takeuchi, Kazuhisa; Ito, Sadayoshi

    2003-11-01

    The localization of cytochrome P-450 4A, peroxisome proliferator-activated receptor (PPAR) alpha, and PPARgamma proteins, and the inducibility of P-450 4A expression and activity by PPAR agonists were determined in the rat kidney. The expressions of these proteins in isolated nephron segments were evaluated by immunoblot analysis, and the production of 20-hydroxyeicosatetraenoic acid (20-HETE) was measured as P-450 4A activity. P-450 4A proteins were expressed predominantly in the proximal tubule (PT), with lower expression in the preglomerular arteriole (Art), glomerulus (Glm), and medullary thick ascending limb (mTAL), but their expression was not detected in the inner medullary collecting duct (IMCD). PPARalpha protein was expressed in the PT and mTAL, and PPARgamma protein was expressed in the IMCD and mTAL. Treatment with clofibrate, the PPARalpha agonist, increased P-450 4A protein levels and the production of 20-HETE in microsomes prepared from the renal cortex, whereas treatment with pioglitazone, the PPARgamma agonist, affected neither of them. These results indicate that PPARalpha and PPARgamma proteins are localized in different nephron segments and the inducibility of P-450 4A expression and activity by the PPAR agonists correlates with the nephron-specific localization of the respective PPAR isoforms.

  1. The effects of iron deficiency on rat liver enzymes.

    PubMed Central

    Bailey-Wood, R.; Blayney, L. M.; Muir, J. R.; Jacobs, A.

    1975-01-01

    The effect of iron deficiency on a number or iron containing enzymes in rat liver has been examined. In addition, 6-phosphogluconate dehydrogenase and glucose 6-phosphate dehydrogenase have been assayed. Of the mitochondrial electron transport reactions only succinate-cytochrome C reductase activity was decreased in iron deficient animals. Microsomal reductase enzymes associated with the NADPH-oxidase system were also markedly decreased although cytochrome P450 concentrations were unaffected. Both 6-phosphogluconate dehydrogenase and glucose 6-phosphate dehydrogenase were reduced in young iron deficient rats but the former had returned to control levels at the age of 14 weeks. PMID:172099

  2. Physical Studies of P450P450 Interactions: Predicting Quaternary Structures of P450 Complexes in Membranes from Their X-ray Crystal Structures

    PubMed Central

    Reed, James R.; Backes, Wayne L.

    2017-01-01

    Cytochrome P450 enzymes, which catalyze oxygenation reactions of both exogenous and endogenous chemicals, are membrane bound proteins that require interaction with their redox partners in order to function. Those responsible for drug and foreign compound metabolism are localized primarily in the endoplasmic reticulum of liver, lung, intestine, and other tissues. More recently, the potential for P450 enzymes to exist as supramolecular complexes has been shown by the demonstration of both homomeric and heteromeric complexes. The P450 units in these complexes are heterogeneous with respect to their distribution and function, and the interaction of different P450s can influence P450-specific metabolism. The goal of this review is to examine the evidence supporting the existence of physical complexes among P450 enzymes. Additionally, the review examines the crystal lattices of different P450 enzymes derived from X-ray diffraction data to make assumptions regarding possible quaternary structures in membranes and in turn, to predict how the quaternary structures could influence metabolism and explain the functional effects of specific P450P450 interactions. PMID:28194112

  3. The identification of the heat-stable microsomal protein required for methoxyflurane metabolism as cytochrome b5.

    PubMed

    Canova-Davis, E; Waskell, L

    1984-02-25

    Methoxyflurane is an anesthetic whose metabolism by cytochrome P-450LM2 has been shown to be dependent upon a heat-stable microsomal protein (Canova-Davis, E., and Waskell, L. A. (1982) Biochem. Biophys. Res. Commun. 108, 1264-1270). Treatment of this protein with diethylpyrocarbonate, which modifies selected amino acids, caused a dose-dependent loss in its ability to effect the metabolism of methoxyflurane by purified cytochrome P-450LM2. This protein factor has been identified as cytochrome b5 by demonstrating that cytochrome b5 and the heat-stable factor coelute during cytochrome b5 purification. Neither ferriheme nor apocytochrome b5 was able to substitute for the activating factor, while cytochrome b5 reconstituted from apocytochrome b5 and heme exhibited an activity similar to that of native b5. Examination of the cytochrome b5 molecule by computer graphics suggested that diethylpyrocarbonate did not inactivate b5 by reacting with the anionic surface of the cytochrome b5 molecule. Maximal rates of methoxyflurane metabolism were obtained at a ratio of 1:1:1 of the three proteins, cytochrome P-450LM2:reductase:cytochrome b5. In summary, it has been demonstrated that the heat-stable protein, cytochrome b5, is obligatory for the metabolism of methoxyflurane by cytochrome P-450LM2. These data also suggest that cytochrome b5 may be acting as an electron donor to P-450LM2 in the O-demethylation of methoxyflurane.

  4. Overexpression of cytochrome P450 CYP6BG1 may contribute to chlorantraniliprole resistance in Plutella xylostella (L.).

    PubMed

    Li, Xiuxia; Li, Ran; Zhu, Bin; Gao, Xiwu; Liang, Pei

    2018-06-01

    The diamondback moth Plutella xylostella (L.) is the most widely distributed pest of cruciferous crops and has developed resistance to most commonly used insecticides, including chlorantraniliprole. Resistance to chlorantraniliprole is likely caused by mutations of the target, the ryanodine receptor, and/or mediated by an increase in detoxification enzyme activities. Although target-site resistance is documented in detail, resistance mediated by increased metabolism has rarely been reported. The activity of cytochrome P450 was significantly higher in two resistant P. xylostella populations than in a susceptible one. Among ten detected cytochrome P450 genes, CYP6BG1 was significantly overexpressed (over 80-fold) in a field-resistant population compared with expression in a susceptible one. Knockdown of CYP6BG1 by RNA interference dramatically reduced the 7-ethoxycoumarin-O-deethylase (7-ECOD) activity of P450 by 45.5% and increased the toxicity of chlorantraniliprole toward P. xylostella by 26.8% at 48 h postinjection of double-stranded RNA. By contrast, overexpression of CYP6BG1 in a transgenic Drosophila melanogaster line significantly decreased the toxicity of the insecticide to the transgenic flies. Overexpression of CYP6BG1 may contribute to chlorantraniliprole resistance in P. xylostella. Our findings will provide new insights into the mechanisms of resistance to diamide insecticides in other insect pests. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

  5. [Character of changes of the level of serotonin-modulating anticonsolidation protein and of cytochrome P-450 in tissues of the eastern alburnoid Alburnoides bipunctatus eichwaldi from rivers of Azerbaijan].

    PubMed

    Mustafayev, M J; Mekhtiev, A A

    2014-01-01

    The paper deals with study by the method of solid-phase indirect immunoenzyme analysis of levels of the novel serotonin-modulated anticonsolidation protein (SMAP) that is directly correlated with serotonin level as well as of biomarker cytochrome P-450 in the liver, gills, and brain of the eastern alburnoid (Alburnoides bipunctatus eichwaldi) caught in the rivers Khudat, Akstafachai, Kura, and Araks flowing at the territory of Azerbaijan. There was revealed a marked downregulation of cytochrome P-450 and SMAP in the liver and gills of the fish caught in the Akstafachai River relatively to values in the fish from the Khudat River not contaminated with pollutants. In the liver and gills in the fish from the Kura and Araks rivers, a significant differently directed changes of the cytochrome P-450 and SMAP levels were observed: downregulations of the cytochrome P-450 versus an upregulation of SMAP. In the brain of the fish from the River Akstafachai there was observed some downregulation of cytochrome P-450, whereas in fish from the Kura and Araks rivers--a significant upregulation of the SMAP level. The obtained results are analyzed from standpoint of processes of adaptation and disadaptation of aquatic organisms to impact of pollutants.

  6. Genomic organization of human fetal specific P-450IIIA7 (cytochrome P-450HFLa)-related gene(s) and interaction of transcriptional regulatory factor with its DNA element in the 5' flanking region.

    PubMed

    Itoh, S; Yanagimoto, T; Tagawa, S; Hashimoto, H; Kitamura, R; Nakajima, Y; Okochi, T; Fujimoto, S; Uchino, J; Kamataki, T

    1992-03-24

    P-450IIIA7 is a form of cytochrome P-450 which was isolated from human fetal livers and termed P-450HFLa. This form has been clarified to be expressed during fetal life specifically (Komori, M., Nishio, K., Kitada, M., Shiramatsu, K., Muroya, K., Soma, M., Nagashima, K. and Kamataki, T. (1990) Biochemistry 29, 4430-4433). In the present study, we isolated five independent clones which probably corresponded to the human P-450IIIA7 gene. These clones were completely sequenced, all exons, exon-intron junctions and the 5' flanking region from the cap site to-869. Although the sequences in the coding region were completely identical to P-450IIIA7, it is possible that genomic fragments sequenced in this study encode portions of other P-450IIIA7-related genes since we could not obtain a complete overlapping set of genomic clones. Within its 5' flanking sequence, the putative binding sites of several transcriptional regulatory factors existed. Among them, it was shown that a basic transcription element binding factor (BTEB) actually interacted with the 5' flanking region of this gene.

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

    USDA-ARS?s Scientific Manuscript database

    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...

  8. Endotoxin administration to humans inhibits hepatic cytochrome P450-mediated drug metabolism.

    PubMed Central

    Shedlofsky, S I; Israel, B C; McClain, C J; Hill, D B; Blouin, R A

    1994-01-01

    In experimental animals, injection of gram-negative endotoxin (LPS) decreases hepatic cytochrome P450-mediated drug metabolism. To evaluate this phenomenon in a human model of gram-negative sepsis, LPS was administered on two consecutive days to healthy male volunteers during which time a cocktail of antipyrine (AP-250 mg), hexobarbital (HB-500 mg), and theophylline (TH-150 mg) was ingested and the apparent oral clearance of each drug determined. Each subject had a control drug clearance study with saline injections. In the first experiment, six subjects received the drug cocktail 0.5 h after the first dose of LPS. In the second experiment, another six subjects received the drug cocktail 0.5 h after the second dose of LPS. In both experiments, LPS caused the expected physiologic responses of inflammation including fever with increases in serum concentrations of TNF alpha, IL-1 beta, IL-6, and acute phase reactants. In the first experiment, only minor decreases in clearances of the probe drugs were observed (7-12%). However in the second experiment, marked decreases in the clearances of AP (35, 95% CI 18-48%), HB (27, 95% CI 14-34%), and TH (22, 95% CI 12-32%) were seen. The decreases in AP clearance correlated with initial peak values of TNF alpha (r = 0.82) and IL-6 (r = 0.86). These data show that in humans the inflammatory response to even a very low dose of LPS significantly decreases hepatic cytochrome P450-mediated drug metabolism and this effect evolves over a 24-h period. It is likely that septic patients with much higher exposures to LPS have more profound inhibition of drug metabolism. PMID:7989576

  9. Food Polyphenol Apigenin Inhibits the Cytochrome P450 Monoxygenase Branch of the Arachidonic Acid Cascade.

    PubMed

    Steuck, Maryvonne; Hellhake, Stefan; Schebb, Nils Helge

    2016-11-30

    The product of cytochrome P450 monooxygenase (P450) ω-hydroxylation of arachidonic acid (AA), 20- hydroxyeicosatetraenoic acid (HETE), is a potent vasoconstrictor. Utilizing microsomes as well as individual CYP4 isoforms we demonstrate here that flavonoids can block 20-HETE formation. Apigenin inhibits CYP4F2 with an IC 50 value of 4.6 μM and 20-HETE formation in human liver and kidney microsomes at 2.4-9.8 μM. Interestingly, the structurally similar naringenin shows no relevant effect on the formation of 20-HETE. Based on these in vitro data, it is impossible to evaluate if a relevant blockade of 20-HETE formation can result in humans from intake of polyphenols with the diet. However, the potency of apigenin is comparable to those of P450 inhibitors such as ketoconazole. Moreover, an IC 50 value in the micromolar range is also described for the inhibition of CYP-mediated drug metabolism leading to food-drug interactions. The modulation of the arachidonic acid cascade by food polyphenols therefore warrants further investigation.

  10. To Analyze the Amelioration of Phenobarbital Induced Oxidative Stress by Erucin, as Indicated by Biochemical and Histological Alterations.

    PubMed

    Arora, Rohit; Bhushan, Sakshi; Kumar, Rakesh; Mannan, Rahul; Kaur, Pardeep; Singh, Bikram; Sharma, Ritika; Vig, Adarsh Pal; Singh, Balbir; Singh, Amrit Pal; Arora, Saroj

    2016-01-01

    Phenobarbital is a commonly employed antidepressant and anti-epileptic drug. The cancer promoting activity of this genotoxic xenobiotic is often ignored. It is responsible for oxidative stress leading to modulation in xenobiotic and antioxidative enzymes. Glucosinolates and more specifically their hydrolytic products are known for their antioxidative and anticancer activities. The present study involves the analysis of hepatoprotective effect of erucin (isolated from Eruca sativa (Mill.) Thell.) against phenobarbital mediated hepatic damage in male wistar rats. The liver homogenate was analyzed for oxidative stress (superoxide dismutase, catalase, guaiacol peroxidase, ascorbate peroxidase, glutathione reductase and lactate dehydrogenase), other oxidative parameters (thiobarbituric acid reactive species, conjugated dienes and lipid hydroperoxide), phase I enzymes (NADPH-cytochrome P450 reductase, NADH-cytochrome b5 reductase, cytochrome P420, cytochrome P450 and cytochrome b5), phase II enzymes (γ-glutamyl transpeptidase, DT-diaphorase and glutathione-S-transferase), serum parameters (alkaline phosphatase, serum glutamic oxaloacetic transaminase, serum glutamic pyruvic transaminase, direct bilirubin and total bilirubin) and certain histological parameters. Erucin accorded protection from phenobarbital induced hepatic damage by normalizing antioxidative enzymes, other oxidative parameters, phase I, II, and serum parameters. Erucin, an analogue of sulforaphane has the potential to act as an anticancer agent by regulating various biochemical parameters.

  11. Molecular Regulation of the Induction of Cytochrome P-450E in the Estuarine Fish Fundulus Heteroclitus.

    DTIC Science & Technology

    1989-02-01

    and reassemble scientific arguments. Drs. Harry V. Gelboin and Sang S. Park (National Cancer Institute, NIH ) produced crucial monoclonal antibodies and...3-methyicholanthrene ALA(S): amninolevulinic acid (synthase) MFO: mixed-function oxidase ApND: aniinopyrine N-demethylase K~: apparent molecular...humans) have historically been the main focus of study. In this overview, based on the primary literature and Cytochrome P-450: Structure , Mechanism

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

    PubMed Central

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

    2014-01-01

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

  13. Use of P450 cytochrome inhibitors in studies of enokipodin biosynthesis

    PubMed Central

    Ishikawa, Noemia Kazue; Tahara, Satoshi; Namatame, Tomohiro; Farooq, Afgan; Fukushi, Yukiharu

    2013-01-01

    Enokipodins A, B, C, and D are antimicrobial sesquiterpenes isolated from the mycelial culture medium of Flammulina velutipes, an edible mushroom. The presence of a quaternary carbon stereocenter on the cyclopentane ring makes enokipodins A-D attractive synthetic targets. In this study, nine different cytochrome P450 inhibitors were used to trap the biosynthetic intermediates of highly oxygenated cuparene-type sesquiterpenes of F. velutipes. Of these, 1-aminobenzotriazole produced three less-highly oxygenated biosynthetic intermediates of enokipodins A-D; these were identified as (S)-(−)-cuparene-1,4-quinone and epimers at C-3 of 6-hydroxy-6-methyl-3-(1,2,2-trimethylcyclopentyl)-2-cyclohexen-1-one. One of the epimers was found to be a new compound. PMID:24688524

  14. Differentiation‐associated urothelial cytochrome P450 oxidoreductase predicates the xenobiotic‐metabolizing activity of “luminal” muscle‐invasive bladder cancers

    PubMed Central

    Arlt, Volker M.; Indra, Radek; Joel, Madeleine; Stiborová, Marie; Eardley, Ian; Ahmad, Niaz; Otto, Wolfgang; Burger, Maximilian; Rubenwolf, Peter; Phillips, David H.; Southgate, Jennifer

    2018-01-01

    Extra‐hepatic metabolism of xenobiotics by epithelial tissues has evolved as a self‐defence mechanism but has potential to contribute to the local activation of carcinogens. Bladder epithelium (urothelium) is bathed in excreted urinary toxicants and pro‐carcinogens. This study reveals how differentiation affects cytochrome P450 (CYP) activity and the role of NADPH:P450 oxidoreductase (POR). CYP1A1 and CYP1B1 transcripts were inducible in normal human urothelial (NHU) cells maintained in both undifferentiated and functional barrier‐forming differentiated states in vitro. However, ethoxyresorufin O‐deethylation (EROD) activity, the generation of reactive BaP metabolites and BaP‐DNA adducts, were predominantly detected in differentiated NHU cell cultures. This gain‐of‐function was attributable to the expression of POR, an essential electron donor for all CYPs, which was significantly upregulated as part of urothelial differentiation. Immunohistology of muscle‐invasive bladder cancer (MIBC) revealed significant overall suppression of POR expression. Stratification of MIBC biopsies into “luminal” and “basal” groups, based on GATA3 and cytokeratin 5/6 labeling, showed POR over‐expression by a subgroup of the differentiated luminal tumors. In bladder cancer cell lines, CYP1‐activity was undetectable/low in basal PORlo T24 and SCaBER cells and higher in the luminal POR over‐expressing RT4 and RT112 cells than in differentiated NHU cells, indicating that CYP‐function is related to differentiation status in bladder cancers. This study establishes POR as a predictive biomarker of metabolic potential. This has implications in bladder carcinogenesis for the hepatic versus local activation of carcinogens and as a functional predictor of the potential for MIBC to respond to prodrug therapies. PMID:29323757

  15. A current review of cytochrome P450 interactions of psychotropic drugs.

    PubMed

    Madhusoodanan, Subramoniam; Velama, Umamaheswararao; Parmar, Jeniel; Goia, Diana; Brenner, Ronald

    2014-05-01

    The number of psychotropic drugs has expanded tremendously over the past few decades with a proportional increase in drug-drug interactions. The majority of psychotropic agents are biotransformed by hepatic enzymes, which can lead to significant drug-drug interactions. Most drug-drug interactions of psychotropics occur at metabolic level involving the hepatic cytochrome P450 enzyme system. We searched the National Library of Medicine, PsycINFO, and Cochrane reviews from 1981 to 2012 for original studies including clinical trials, double-blind, placebo-controlled studies, and randomized controlled trials. In addition, case reports, books, review articles, and hand-selected journals were utilized to supplement this review. Based on the clinical intensity of outcome, cytochrome interactions can be classified as severe, moderate, and mild. Severe interactions include effects that might be acutely life threatening. They are mainly inhibitory interactions with cardiovascular drugs. Moderate interactions include efficacy issues. Mild interactions include nonserious side effects, such as somnolence. Psychotropic drugs may interact with other prescribed medications used to treat concomitant medical illnesses. A thorough understanding of the most prescribed medications and patient education will help reduce the likelihood of potentially fatal drug-drug interactions.

  16. Expression of a ripening-related cytochrome P450 cDNA in Cavendish banana (Musa acuminata cv. Williams).

    PubMed

    Pua, Eng-Chong; Lee, Yi-Chuan

    2003-02-13

    As part of a study to understand the molecular basis of fruit ripening, this study reports the isolation and characterization of a banana cytochrome P450 (P450) cDNA, designated as MAP450-1, which was associated with fruit ripening of banana. MAP450-1 encoded a single polypeptide of 507 amino acid residues that shared an overall identity of 27-45% with that of several plant P450s, among which MAP450-1 was most related phylogenetically to the avocado P450 CYP71A1. The polypeptide that possessed residue domains conserved in all P450s was classified as CYP71N1. Expression of CYP71N1 varied greatly between banana organs. Transcripts were detected only in peel and pulp of the ripening fruit and not in unripe fruit tissues at all developmental stages or other organs (root, leaf, ovary and flower). During ripening, transcripts were barely detectable in pre-climacteric and climacteric fruits but, as ripening progressed, they began to accumulate and reached a maximum in post-climacteric fruits. CYP71N1 expression in pre-climacteric fruit could be upregulated by exogenous application of ethylene (1-5 ppm) and treatment of overripe fruit with exogenous sucrose (50-300 mM) but not glucose downregulated the expression. These results indicate that P450s may not play a role in fruit development and its expression is associated with ripening, which may be regulated, in part, by ethylene and/or sucrose, at the transcript level.

  17. Identification and functional analysis of cytochrome P450 complement in Streptomyces virginiae IBL14

    PubMed Central

    2013-01-01

    Background As well known, both natural and synthetic steroidal compounds are powerful endocrine disrupting compounds (EDCs) which can cause reproductive toxicity and affect cellular development in mammals and thus are generally regarded as serious contributors to water pollution. Streptomyces virginiae IBL14 is an effective degradative strain for many steroidal compounds and can also catalyze the C25 hydroxylation of diosgenin, the first-ever biotransformation found on the F-ring of diosgenin. Results To completely elucidate the hydroxylation function of cytochrome P450 genes (CYPs) found during biotransformation of steroids by S. virginiae IBL14, the whole genome sequencing of this strain was carried out via 454 Sequencing Systems. The analytical results of BLASTP showed that the strain IBL14 contains 33 CYPs, 7 ferredoxins and 3 ferredoxin reductases in its 8.0 Mb linear chromosome. CYPs from S. virginiae IBL14 are phylogenetically closed to those of Streptomyces sp. Mg1 and Streptomyces sp. C. One new subfamily was found as per the fact that the CYP Svu001 in S. virginiae IBL14 shares 66% identity only to that (ZP_05001937, protein identifer) from Streptomyces sp. Mg1. Further analysis showed that among all of the 33 CYPs in S. virginiae IBL14, three CYPs are clustered with ferredoxins, one with ferredoxin and ferredoxin reductase and three CYPs with ATP/GTP binding proteins, four CYPs arranged with transcriptional regulatory genes and one CYP located on the upstream of an ATP-binding protein and transcriptional regulators as well as four CYPs associated with other functional genes involved in secondary metabolism and degradation. Conclusions These characteristics found in CYPs from S. virginiae IBL14 show that the EXXR motif in the K-helix is not absolutely conserved in CYP157 family and I-helix not absolutely essential for the CYP structure, too. Experimental results showed that both CYP Svh01 and CYP Svu022 are two hydroxylases, capable of bioconverting

  18. The effect of varying halogen substituent patterns on the cytochrome P450 catalysed dehalogenation of 4-halogenated anilines to 4-aminophenol metabolites.

    PubMed

    Cnubben, N H; Vervoort, J; Boersma, M G; Rietjens, I M

    1995-05-11

    The cytochrome P450 catalysed biotransformation of 4-halogenated anilines was studied in vitro with special emphasis on the dehalogenation to 4-aminophenol metabolites. The results demonstrated that a fluorine substituent at the C4 position was more easily eliminated from the aromatic ring than a chloro-, bromo- or iodo-substituent. HPLC analysis of in vitro biotransformation patterns revealed that the dehalogenation of the C4-position was accompanied by formation of non-halogenated 4-aminophenol, without formation of NIH-shifted metabolites. Changes in the apparent Vmax for the microsomal oxidative dehalogenation appeared to correlate with the electronegativity of the halogen substituent at C4, the fluorine substituent being the one most easily eliminated. A similar decrease in the rate of dehalogenation from a fluoro- to a chloro- to a bromo- to an iodo-substituent was observed in a system with purified reconstituted cytochrome P450 IIB1, in a tertiair butyl hydroperoxide supported microsomal cytochrome P450 system as well as in a system with microperoxidase 8. This microperoxidase 8 is a haem-based mini-enzyme without a substrate binding site, capable of catalysing cytochrome P450-like reaction chemistry. Together, these results excluded the possibility that the difference in the rate of dehalogenation with a varying C4-halogen substituent arose from a change in the contribution of cytochrome P450 enzymes involved in oxidative dehalogenation with a change in the halogen substituent. Rather, they strongly suggested that the difference was indeed due to an intrinsic electronic parameter of the various C4 halogenated anilines dependent on the type of halogen substituent. Additional in vitro experiments with polyfluorinated anilines demonstrated that elimination of the C4-fluorine substituent became more difficult upon the introduction of additional electron withdrawing fluorine substituents in the aniline-ring. 19F-NMR analysis of the metabolite patterns showed

  19. CYTOCHROME P450 17A1 STRUCTURES WITH PROSTATE CANCER DRUGS ABIRATERONE AND TOK-001

    PubMed Central

    DeVore, Natasha M.; Scott, Emily E.

    2011-01-01

    Cytochrome P450 17A1 (P450c17) catalyzes the biosynthesis of androgens in humans1. Since prostate cancer cells proliferate in response to androgen steroids2,3, CYP17A1 inhibition is a new strategy to prevent androgen synthesis and treat lethal metastatic castration-resistant prostate cancer4, but drug development has been hampered by the lack of a CYP17A1 structure. Here we report the only known structures of CYP17A1, which contain either abiraterone, a first-in-class steroidal inhibitor recently approved by the FDA for late-stage prostate cancer5, or TOK-001, another inhibitor in clinical trials4,6. Both bind the heme iron forming a 60° angle above the heme plane, packing against the central I helix with the 3β-OH interacting with N202 in the F helix. Importantly, this binding mode differs substantially from those predicted by homology models or from steroids in other cytochrome P450 enzymes with known structures, with some features more similar to steroid receptors. While the overall CYP17A1 structure provides a rationale for understanding many mutations found in patients with steroidogenic diseases, the active site reveals multiple steric and hydrogen bonding features that will facilitate better understanding of the enzyme’s dual hydroxylase and lyase catalytic capabilities and assist in rational drug design. Specifically, structure-based design is expected to aid development of inhibitors that bind only CYP17A1 and solely inhibit its androgen-generating lyase activity to improve treatment of prostate and other hormone-responsive cancers. PMID:22266943

  20. The Chemically Inducible Plant Cytochrome P450 CYP76B1 Actively Metabolizes Phenylureas and Other Xenobiotics1

    PubMed Central

    Robineau, Tiburce; Batard, Yannick; Nedelkina, Svetlana; Cabello-Hurtado, Francisco; LeRet, Monique; Sorokine, Odile; Didierjean, Luc; Werck-Reichhart, Danièle

    1998-01-01

    Cytochrome P450s (P450s) constitute one of the major classes of enzymes that are responsible for detoxification of exogenous molecules both in animals and plants. On the basis of its inducibility by exogenous chemicals, we recently isolated a new plant P450, CYP76B1, from Jerusalem artichoke (Helianthus tuberosus) and showed that it was capable of dealkylating a model xenobiotic compound, 7-ethoxycoumarin. In the present paper we show that CYP76B1 is more strongly induced by foreign compounds than other P450s isolated from the same plant, and metabolizes with high efficiency a wide range of xenobiotics, including alkoxycoumarins, alkoxyresorufins, and several herbicides of the class of phenylureas. CYP76B1 catalyzes the double N-dealkylation of phenylureas with turnover rates comparable to those reported for physiological substrates and produces nonphytotoxic compounds. Potential uses for CYP76B1 thus include control of herbicide tolerance and selectivity, as well as soil and groundwater bioremediation. PMID:9808750

  1. Isolation and Characterization of a Soluble NADPH-Dependent Fe(III) Reductase from Geobacter sulfurreducens

    PubMed Central

    Kaufmann, Franz; Lovley, Derek R.

    2001-01-01

    NADPH is an intermediate in the oxidation of organic compounds coupled to Fe(III) reduction in Geobacter species, but Fe(III) reduction with NADPH as the electron donor has not been studied in these organisms. Crude extracts of Geobacter sulfurreducens catalyzed the NADPH-dependent reduction of Fe(III)-nitrilotriacetic acid (NTA). The responsible enzyme, which was recovered in the soluble protein fraction, was purified to apparent homogeneity in a four-step procedure. Its specific activity for Fe(III) reduction was 65 μmol · min−1 · mg−1. The soluble Fe(III) reductase was specific for NADPH and did not utilize NADH as an electron donor. Although the enzyme reduced several forms of Fe(III), Fe(III)-NTA was the preferred electron acceptor. The protein possessed methyl viologen:NADP+ oxidoreductase activity and catalyzed the reduction of NADP+ with reduced methyl viologen as electron donor at a rate of 385 U/mg. The enzyme consisted of two subunits with molecular masses of 87 and 78 kDa and had a native molecular mass of 320 kDa, as determined by gel filtration. The purified enzyme contained 28.9 mol of Fe, 17.4 mol of acid-labile sulfur, and 0.7 mol of flavin adenine dinucleotide per mol of protein. The genes encoding the two subunits were identified in the complete sequence of the G. sulfurreducens genome from the N-terminal amino acid sequences derived from the subunits of the purified protein. The sequences of the two subunits had about 30% amino acid identity to the respective subunits of the formate dehydrogenase from Moorella thermoacetica, but the soluble Fe(III) reductase did not possess formate dehydrogenase activity. This soluble Fe(III) reductase differs significantly from previously characterized dissimilatory and assimilatory Fe(III) reductases in its molecular composition and cofactor content. PMID:11443080

  2. High-Throughput Cytochrome P450 Cocktail Inhibition Assay for Assessing Drug-Drug and Drug-Botanical Interactions

    PubMed Central

    Li, Guannan; Huang, Ke; Nikolic, Dejan

    2015-01-01

    Detection of drug-drug interactions is essential during the early stages of drug discovery and development, and the understanding of drug-botanical interactions is important for the safe use of botanical dietary supplements. Among the different forms of drug interactions that are known, inhibition of cytochrome P450 (P450) enzymes is the most common cause of drug-drug or drug-botanical interactions. Therefore, a rapid and comprehensive mass spectrometry–based in vitro high-throughput P450 cocktail inhibition assay was developed that uses 10 substrates simultaneously against nine CYP isoforms. Including probe substrates for CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and two probes targeting different binding sites of CYP3A4/5, this cocktail simultaneously assesses at least as many P450 enzymes as previous assays while remaining among the fastest due to short incubation times and rapid analysis using ultrahigh pressure liquid chromatography–tandem mass spectrometry. The method was validated using known inhibitors of each P450 enzyme and then shown to be useful not only for single-compound testing but also for the evaluation of potential drug-botanical interactions using the botanical dietary supplement licorice (Glycyrrhiza glabra) as an example. PMID:26285764

  3. INTERINDIVIDUAL VARIANCE OF CYTOCHROME P450 FORMS IN HUMAN HEPATIC MICROSOMES: CORRELATION OF INDIVIDUAL FORMS WITH XENOBIOTIC METABOLISM AND IMPLICATIONS IN RISK ASSESSMENT

    EPA Science Inventory

    Differences in biotransformation activities may alter the bioavailability or efficacy of drugs, provide protection from certain xenobiotic and environmental agents, or increase toxicity of others. Cytochrome P450 (CYP450) enzymes are responsible for the majority of oxidation reac...

  4. Cycle affects imidacloprid efficiency by mediating cytochrome P450 expression in the brown planthopper Nilaparvata lugens.

    PubMed

    Kang, K; Yang, P; Pang, R; Yue, L; Zhang, W

    2017-10-01

    Circadian clocks influence most behaviours and physiological activities in animals, including daily fluctuations in metabolism. However, how the clock gene cycle influences insects' responses to pesticides has rarely been reported. Here, we provide evidence that cycle affects imidacloprid efficacy by mediating the expression of cytochrome P450 genes in the brown planthopper (BPH) Nilaparvata lugens, a serious insect pest of rice. Survival bioassays showed that the susceptibility of BPH adults to imidacloprid differed significantly between the two time points tested [Zeitgeber Time 8 (ZT8) and ZT4]. After cloning the cycle gene in the BPH (Nlcycle), we found that Nlcycle was expressed at higher levels in the fat body and midgut, and its expression was rhythmic with two peaks. Knockdown of Nlcycle affected the expression levels and rhythms of cytochrome P450 genes as well as susceptibility to imidacloprid. The survival rates of BPH adults after treatment with imidacloprid did not significantly differ between ZT4 and ZT8 after double-stranded Nlcycle treatment. These findings can be used to improve pesticide use and increase pesticide efficiency in the field. © 2017 The Royal Entomological Society.

  5. Promising Tools in Prostate Cancer Research: Selective Non-Steroidal Cytochrome P450 17A1 Inhibitors

    PubMed Central

    Bonomo, Silvia; Hansen, Cecilie H.; Petrunak, Elyse M.; Scott, Emily E.; Styrishave, Bjarne; Jørgensen, Flemming Steen; Olsen, Lars

    2016-01-01

    Cytochrome P450 17A1 (CYP17A1) is an important target in the treatment of prostate cancer because it produces androgens required for tumour growth. The FDA has approved only one CYP17A1 inhibitor, abiraterone, which contains a steroidal scaffold similar to the endogenous CYP17A1 substrates. Abiraterone is structurally similar to the substrates of other cytochrome P450 enzymes involved in steroidogenesis, and interference can pose a liability in terms of side effects. Using non-steroidal scaffolds is expected to enable the design of compounds that interact more selectively with CYP17A1. Therefore, we combined a structure-based virtual screening approach with density functional theory (DFT) calculations to suggest non-steroidal compounds selective for CYP17A1. In vitro assays demonstrated that two such compounds selectively inhibited CYP17A1 17α-hydroxylase and 17,20-lyase activities with IC50 values in the nanomolar range, without affinity for the major drug-metabolizing CYP2D6 and CYP3A4 enzymes and CYP21A2, with the latter result confirmed in human H295R cells. PMID:27406023

  6. Electrochemical Detection of Anti-Breast-Cancer Agents in Human Serum by Cytochrome P450-Coated Carbon Nanotubes

    PubMed Central

    Baj-Rossi, Camilla; De Micheli, Giovanni; Carrara, Sandro

    2012-01-01

    We report on the electrochemical detection of anti-cancer drugs in human serum with sensitivity values in the range of 8–925 nA/μM. Multi-walled carbon nanotubes were functionalized with three different cytochrome P450 isoforms (CYP1A2, CYP2B6, and CYP3A4). A model used to effectively describe the cytochrome P450 deposition onto carbon nanotubes was confirmed by Monte Carlo simulations. Voltammetric measurements were performed in phosphate buffer saline (PBS) as well as in human serum, giving well-defined current responses upon addition of increasing concentrations of anti-cancer drugs. The results assert the capability to measure concentration of drugs in the pharmacological ranges in human serum. Another important result is the possibility to detect pairs of drugs present in the same sample, which is highly required in case of therapies with high side-effects risk and in anti-cancer pharmacological treatments based on mixtures of different drugs. Our technology holds potentials for inexpensive multi-panel drug-monitoring in personalized therapy. PMID:22778656

  7. Antiepileptic drugs affect neuronal androgen signaling via a cytochrome P450-dependent pathway.

    PubMed

    Gehlhaus, Marcel; Schmitt, Nina; Volk, Benedikt; Meyer, Ralf P

    2007-08-01

    Recent data imply an important role for brain cytochrome P450 (P450) in endocrine signaling. In epileptic patients, treatment with P450 inducers led to reproductive disorders; in mouse hippocampus, phenytoin treatment caused concomitant up-regulation of CYP3A11 and androgen receptor (AR). In the present study, we established specific in vitro models to examine whether CYP3A isoforms cause enhanced AR expression and activation. Murine Hepa1c1c7 cells and neuronal-type rat PC-12 cells were used to investigate P450 regulation and its effects on AR after phenytoin and phenobarbital administration. In both cell lines, treatment with antiepileptic drugs (AEDs) led to concomitant up-regulation of CYP3A (CYP3A11 in Hepa1c1c7 and CYP3A2 in PC-12) and AR mRNA and protein. Inhibition of CYP3A expression and activity by the CYP3A inhibitor ketoconazole or by CYP3A11-specific short interfering RNA molecules reduced AR expression to basal levels. The initial up-regulation of AR signal transduction, measured by an androgen-responsive element chloramphenicol-acetyltransferase reporter gene assay, was completely reversed after specific inhibition of CYP3A11. Withdrawal of the CYP3A11 substrate testosterone prevented AR activation, whereas AR mRNA expression remained up-regulated. In addition, recombinant CYP3A11 was expressed heterologously in PC-12 cells, thereby eliminating any direct drug influence on the AR. Again, the initial up-regulation of AR mRNA and activity was reduced to basal levels after silencing of CYP3A11. In conclusion, we show here that CYP3A2 and CYP3A11 are crucial mediators of AR expression and signaling after AED application. These findings point to an important and novel function of P450 in regulation of steroid hormones and their receptors in endocrine tissues such as liver and brain.

  8. Exploring the Electrical Conductivity of Cytochrome P450 by Nano-Electrode and Conductive Atomic Force Microscopy

    NASA Astrophysics Data System (ADS)

    Li, Debin; Gu, Jianhua; Chye, Yewhee; Lederman, David; Kabulski, Jarod; Gannett, Peter; Tracy, Timothy

    2006-03-01

    There is a growing interest in measuring the conductivity of electron-transfer proteins. The cytochrome P450 (CP450) enzymes represent an important class of heme-containing enzymes. Immobilizing CP450 enzymes on a surface can be used for studying a single enzyme with respect to electron transfer. The spin state of the heme iron can change upon binding of a substrate. In our experiment, CP450 (diameter ˜ 5 nm) has been bonded to a metal surface. Nano-electrodes (gap < 10 nm) were fabricated by defining a bridge via e-beam lithography and then breaking the junction by electromigration at low temperatures. We have examined the electronic properties of CP450 by itself and after binding CP450 with flurbiprofen. The room temperature I-V conductivity is reminiscent to cyclic voltammetry measurements, indicating the presence of strong ionic transfer. At lower temperatures (100 K) the I-V characteristics indicate electronic transport dominated by tunneling processes. The conductive AFM is an additional method used to examine the enzyme's electronic properties. The results from two methods will be discussed..

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

    PubMed Central

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

    2012-01-01

    The discovery that 7-dehydrocholesterol (7DHC) is an excellent substrate for cytochrome P450scc (CYP11A1) opens up new possibilities in biochemistry. To elucidate its biological significance we tested ex-vivo P450scc-dependent metabolism of 7DHC by tissues expressing high and low levels of P450scc activity, placenta and epidermal keratinocytes, respectively. Incubation of human placenta fragments with 7DHC led to its conversion to 7-dehydropregnenolone (7DHP), which was inhibited by DL-aminoglutethimide, and stimulated by forskolin. Final proof for P450scc involvement was provided in isolated placental mitochondria where production of 7DHP was almost completely inhibited by 22R-hydroxycholesterol. 7DHC was metabolized by placental mitochondria at a faster rate than exogenous cholesterol, under both limiting and saturating conditions of substrate transport, consistent with higher catalytic efficiency (kcat/Km) with 7DHC as substrate than with cholesterol. Ex-vivo experiments showed five 5,7-dienal intermediates with MS spectra of dihydroxy and mono-hydroxy-7DHC and retention time corresponding to 20,22(OH)27DHC and 22(OH)7DHC. The chemical structure of 20,22(OH)27DHC was defined by NMR. 7DHP was further metabolized by either placental fragments or placental microsomes to 7-dehydroprogesterone as defined by UV, MS and NMR, and to an additional product with a 5,7-dienal structure and MS corresponding to hydroxy-7DHP. Furthermore, epidermal keratinocytes transformed either exogenous or endogenous 7DHC to 7DHP. 7DHP inhibited keratinocytes proliferation, while the product of its pholytic transformation, pregcalciferol, lost this capability. In conclusion, tissues expressing P450scc can metabolize 7DHC to biologically active 7DHP with 22(OH)7DHC and 20,22(OH)27DHC serving as intermediates, and with further metabolism to 7-dehydroprogesterone and (OH)7DHP. PMID:22877869

  10. Mechanism of chloroform-induced renal toxicity: Non-involvement of hepatic cytochrome P450-dependent metabolism

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Fang Cheng; Behr, Melissa; Xie Fang

    2008-02-15

    Chloroform causes hepatic and renal toxicity in a number of species. In vitro studies have indicated that chloroform can be metabolized by P450 enzymes in the kidney to nephrotoxic intermediate, although direct in vivo evidence for the role of renal P450 in the nephrotoxicity has not been reported. This study was to determine whether chloroform renal toxicity persists in a mouse model with a liver-specific deletion of the P450 reductase (Cpr) gene (liver-Cpr-null). Chloroform-induced renal toxicity and chloroform tissue levels were compared between the liver-Cpr-null and wild-type mice at 24 h following differing doses of chloroform. At a chloroform dosemore » of 150 mg/kg, the levels of blood urea nitrogen (BUN) were five times higher in the exposed group than in the vehicle-treated one for the liver-Cpr-null mice, but they were only slightly higher in the exposed group than in the vehicle-treated group for the wild-type mice. Severe lesions were found in the kidney of the liver-Cpr-null mice, while only mild lesions were found in the wild-type mice. At a chloroform dose of 300 mg/kg, severe kidney lesions were observed in both strains, yet the BUN levels were still higher in the liver-Cpr-null than in the wild-type mice. Higher chloroform levels were found in the tissues of the liver-Cpr-null mice. These findings indicated that loss of hepatic P450-dependent chloroform metabolism does not protect against chloroform-induced renal toxicity, suggesting that renal P450 enzymes play an essential role in chloroform renal toxicity.« less

  11. Structure of Hordeum vulgare NADPH-dependent thioredoxin reductase 2. Unwinding the reaction mechanism

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kirkensgaard, Kristine G.; Enzyme and Protein Chemistry, Department of Systems BioIogy, Technical University of Denmark; Hägglund, Per

    2009-09-01

    The first crystal structure of a cereal NTR, a protein involved in seed development and germination, has been determined. The structure is in a conformation that excludes NADPH binding and indicates that a domain reorientation facilitated by Trx binding precedes NADPH binding in the reaction mechanism. Thioredoxins (Trxs) are protein disulfide reductases that regulate the intracellular redox environment and are important for seed germination in plants. Trxs are in turn regulated by NADPH-dependent thioredoxin reductases (NTRs), which provide reducing equivalents to Trx using NADPH to recycle Trxs to the active form. Here, the first crystal structure of a cereal NTR,more » HvNTR2 from Hordeum vulgare (barley), is presented, which is also the first structure of a monocot plant NTR. The structure was determined at 2.6 Å resolution and refined to an R{sub cryst} of 19.0% and an R{sub free} of 23.8%. The dimeric protein is structurally similar to the structures of AtNTR-B from Arabidopsis thaliana and other known low-molecular-weight NTRs. However, the relative position of the two NTR cofactor-binding domains, the FAD and the NADPH domains, is not the same. The NADPH domain is rotated by 25° and bent by a 38% closure relative to the FAD domain in comparison with AtNTR-B. The structure may represent an intermediate between the two conformations described previously: the flavin-oxidizing (FO) and the flavin-reducing (FR) conformations. Here, analysis of interdomain contacts as well as phylogenetic studies lead to the proposal of a new reaction scheme in which NTR–Trx interactions mediate the FO to FR transformation.« less

  12. Modifications of hepatic drug metabolizing enzyme activities in rats fed baobab seed oil containing cyclopropenoid fatty acids.

    PubMed

    Andrianaivo-Rafehivola, A A; Siess, M H; Gaydou, E M

    1995-05-01

    The effects on drug metabolizing enzymes of cyclopropenoid fatty acids present in baobab seed oil were evaluated in rats fed either a diet with baobab seed oil (1.27% cyclopropenoid fatty acids in the diet) or a diet with heated baobab seed oil (0.046% cyclopropenoid fatty acids in the diet). Comparison was made with rats fed a mixture of oils that contained no cyclopropenoid fatty acid. Rats fed baobab oil showed retarded growth. In comparison with the other groups, the relative liver weights were markedly increased whereas cytochrome P-450 content and NADPH cytochrome c reductase and NADH cytochrome c reductase activities were decreased. In rats fed the heated baobab oil the relative liver weight was decreased and the cytochrome P-450 level and reductase activities were increased relative to levels in rats fed the unheated oil. Ethoxycoumarin deethylase, ethoxyresorufin deethylase and pentoxyresorufin depentylase activities, expressed on the basis of cytochrome P-450, were greater in the group fed unheated baobab seed oil. Cytosolic glutathione transferase activity was markedly decreased in rats fed fresh baobab seed oil and heating the oil, which reduced the content of cyclopropenoid fatty acids, led to a considerable increase of this activity. UDP-glucuronyl transferase activities were not modified by the type of oil included in the diet. It is possible that the mechanisms of action of cyclopropenoid fatty acids are related to alterations of membrane lipid composition or microsomal proteins.

  13. 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

  14. Multiple Cytochrome P450 genes: their constitutive overexpression and permethrin induction in insecticide resistant mosquitoes, Culex quinquefasciatus.

    PubMed

    Liu, Nannan; Li, Ting; Reid, William R; Yang, Ting; Zhang, Lee

    2011-01-01

    Four cytochrome P450 cDNAs, CYP6AA7, CYP9J40, CYP9J34, and CYP9M10, were isolated from mosquitoes, Culex quinquefasciatus. The P450 gene expression and induction by permethrin were compared for three different mosquito populations bearing different resistance phenotypes, ranging from susceptible (S-Lab), through intermediate (HAmCq(G0), the field parental population) to highly resistant (HAmCq(G8), the 8(th) generation of permethrin selected offspring of HAmCq(G0)). A strong correlation was found for P450 gene expression with the levels of resistance and following permethrin selection at the larval stage of mosquitoes, with the highest expression levels identified in HAmCq(G8), suggesting the importance of CYP6AA7, CYP9J40, CYP9J34, and CYP9M10 in the permethrin resistance of larva mosquitoes. Only CYP6AA7 showed a significant overexpression in HAmCq(G8) adult mosquitoes. Other P450 genes had similar expression levels among the mosquito populations tested, suggesting different P450 genes may be involved in the response to insecticide pressure in different developmental stages. The expression of CYP6AA7, CYP9J34, and CYP9M10 was further induced by permethrin in resistant mosquitoes. Taken together, these results indicate that multiple P450 genes are up-regulated in insecticide resistant mosquitoes through both constitutive overexpression and induction mechanisms, thus increasing the overall expression levels of P450 genes.

  15. Metabolism of agrochemicals and related environmental chemicals based on cytochrome P450s in mammals and plants.

    PubMed

    Ohkawa, Hideo; Inui, Hideyuki

    2015-06-01

    A yeast gene expression system originally established for mammalian cytochrome P450 monooxygenase cDNAs was applied to functional analysis of a number of mammalian and plant P450 species, including 11 human P450 species (CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1 and CYP3A4). The human P450 species CYP1A1, CYP1A2, CYP2B6, CYP2C18 and CYP2C19 were identified as P450 species metabolising various agrochemicals and environmental chemicals. CYP2C9 and CYP2E1 specifically metabolised sulfonylurea herbicides and halogenated hydrocarbons respectively. Plant P450 species metabolising phenylurea and sulfonylurea herbicides were also identified mainly as the CYP71 family, although CYP76B1, CYP81B1 and CYP81B2 metabolised phenylurea herbicides. The transgenic plants expressing these mammalian and plant P450 species were applied to herbicide tolerance as well as phytoremediation of agrochemical and environmental chemical residues. The combined use of CYP1A1, CYP2B6 and CYP2C19 belonging to two families and three subfamilies covered a wide variety of herbicide tolerance and phytoremediation of these residues. The use of 2,4-D-and bromoxynil-induced CYP71AH11 in tobacco seemed to enhance herbicide tolerance and selectivity. © 2014 Society of Chemical Industry.

  16. Engineering Herbicide Metabolism in Tobacco and Arabidopsis with CYP76B1, a Cytochrome P450 Enzyme from Jerusalem Artichoke1

    PubMed Central

    Didierjean, Luc; Gondet, Laurence; Perkins, Roberta; Lau, Sze-Mei Cindy; Schaller, Hubert; O'Keefe, Daniel P.; Werck-Reichhart, Danièle

    2002-01-01

    The Jerusalem artichoke (Helianthus tuberosus) xenobiotic inducible cytochrome P450, CYP76B1, catalyzes rapid oxidative dealkylation of various phenylurea herbicides to yield nonphytotoxic metabolites. We have found that increased herbicide metabolism and tolerance can be achieved by ectopic constitutive expression of CYP76B1 in tobacco (Nicotiana tabacum) and Arabidopsis. Transformation with CYP76B1 conferred on tobacco and Arabidopsis a 20-fold increase in tolerance to linuron, a compound detoxified by a single dealkylation, and a 10-fold increase in tolerance to isoproturon or chlortoluron, which need successive catalytic steps for detoxification. Two constructs for expression of translational fusions of CYP76B1 with P450 reductase were prepared to test if they would yield even greater herbicide tolerance. Plants expressing these constructs had lower herbicide tolerance than CYP76B1 alone, which is apparently a consequence of reduced stability of the fusion proteins. In all cases, increased herbicide tolerance results from more extensive metabolism, as demonstrated with exogenously fed phenylurea. Beside increased herbicide tolerance, expression of CYP76B1 has no other visible phenotype in the transgenic plants. Our data indicate that CYP76B1 can function as a selectable marker for plant transformation, allowing efficient selection in vitro and in soil-grown plants. Plants expressing CYP76B1 may also be a potential tool for phytoremediation of contaminated sites. PMID:12226498

  17. Biallelic mutations in the ferredoxin reductase gene cause novel mitochondriopathy with optic atrophy

    PubMed Central

    Peng, Yanyan; Shinde, Deepali N; Valencia, C Alexander; Mo, Jun-Song; Rosenfeld, Jill; Truitt Cho, Megan; Chamberlin, Adam; Li, Zhuo; Liu, Jie; Gui, Baoheng; Brockhage, Rachel; Basinger, Alice; Alvarez-Leon, Brenda; Heydemann, Peter; Magoulas, Pilar L; Lewis, Andrea M; Scaglia, Fernando; Gril, Solange; Chong, Shuk Ching; Bower, Matthew; Monaghan, Kristin G; Willaert, Rebecca; Plona, Maria-Renee; Dineen, Rich; Milan, Francisca; Hoganson, George; Helbig, Katherine L; Keller-Ramey, Jennifer; Harris, Belinda; Anderson, Laura C; Green, Torrian; Sukoff Rizzo, Stacey J; Kaylor, Julie; Chen, Jiani; Guan, Min-Xin; Sellars, Elizabeth; Sparagana, Steven P; Gibson, James B; Reinholdt, Laura G; Tang, Sha; Huang, Taosheng

    2017-01-01

    Abstract Iron–sulfur (Fe-S) clusters are ubiquitous cofactors essential to various cellular processes, including mitochondrial respiration, DNA repair, and iron homeostasis. A steadily increasing number of disorders are being associated with disrupted biogenesis of Fe–S clusters. Here, we conducted whole-exome sequencing of patients with optic atrophy and other neurological signs of mitochondriopathy and identified 17 individuals from 13 unrelated families with recessive mutations in FDXR, encoding the mitochondrial membrane-associated flavoprotein ferrodoxin reductase required for electron transport from NADPH to cytochrome P450. In vitro enzymatic assays in patient fibroblast cells showed deficient ferredoxin NADP reductase activity and mitochondrial dysfunction evidenced by low oxygen consumption rates (OCRs), complex activities, ATP production and increased reactive oxygen species (ROS). Such defects were rescued by overexpression of wild-type FDXR. Moreover, we found that mice carrying a spontaneous mutation allelic to the most common mutation found in patients displayed progressive gait abnormalities and vision loss, in addition to biochemical defects consistent with the major clinical features of the disease. Taken together, these data provide the first demonstration that germline, hypomorphic mutations in FDXR cause a novel mitochondriopathy and optic atrophy in humans. PMID:29040572

  18. Insights into Hydrocarbon Assimilation by Eurotialean and Hypocrealean Fungi: Roles for CYP52 and CYP53 Clans of Cytochrome P450 Genes.

    PubMed

    Huarte-Bonnet, Carla; Kumar, Suresh; Saparrat, Mario C N; Girotti, Juan R; Santana, Marianela; Hallsworth, John E; Pedrini, Nicolás

    2018-03-01

    Several filamentous fungi are able to concomitantly assimilate both aliphatic and polycyclic aromatic hydrocarbons that are the biogenic by-products of some industrial processes. Cytochrome P450 monooxygenases catalyze the first oxidation reaction for both types of substrate. Among the cytochrome P450 (CYP) genes, the family CYP52 is implicated in the first hydroxylation step in alkane-assimilation processes, while genes belonging to the family CYP53 have been linked with oxidation of aromatic hydrocarbons. Here, we perform a comparative analysis of CYP genes belonging to clans CYP52 and CYP53 in Aspergillus niger, Beauveria bassiana, Metarhizium robertsii (formerly M. anisopliae var. anisopliae), and Penicillium chrysogenum. These species were able to assimilate n-hexadecane, n-octacosane, and phenanthrene, exhibiting a species-dependent modification in pH of the nutrient medium during this process. Modeling of the molecular docking of the hydrocarbons to the cytochrome P450 active site revealed that both phenanthrene and n-octacosane are energetically favored as substrates for the enzymes codified by genes belonging to both CYP52 and CYP53 clans, and thus appear to be involved in this oxidation step. Analyses of gene expression revealed that CYP53 members were significantly induced by phenanthrene in all species studied, but only CYP52X1 and CYP53A11 from B. bassiana were highly induced with n-alkanes. These findings suggest that the set of P450 enzymes involved in hydrocarbon assimilation by fungi is dependent on phylogeny and reveal distinct substrate and expression specificities.

  19. Immobilized Cytochrome P450 2C9 (CYP2C9): Applications for Metabolite Generation, Monitoring Protein-Protein Interactions, and Improving In-vivo Predictions Using Enhanced In-vitro Models

    NASA Astrophysics Data System (ADS)

    Wollenberg, Lance A.

    Cytochrome P450 (P450) enzymes are a family of oxoferroreductase enzymes containing a heme moiety and are well known to be involved in the metabolism of a wide variety of endogenous and xenobiotic materials. It is estimated that roughly 75% of all pharmaceutical compounds are metabolized by these enzymes. Traditional reconstituted in-vitro incubation studies using recombinant P450 enzymes are often used to predict in-vivo kinetic parameters of a drug early in development. However, in many cases, these reconstituted incubations are prone to aggregation which has been shown to affect the catalytic activity of an enzyme. Moreover, the presence of other isoforms of P450 enzymes present in a metabolic incubation, as is the case with microsomal systems, may affect the catalytic activity of an enzyme through isoform-specific protein-protein interactions. Both of these effects may result in inaccurate prediction of in-vivo drug metabolism using in-vitro experiments. Here we described the development of immobilized P450 constructs designed to elucidate the effects of aggregation and protein-protein interactions between P450 isoforms on catalytic activities. The long term objective of this project is to develop a system to control the oligomeric state of Cytochrome P450 enzymes to accurately elucidate discrepancies between in vitro reconstituted systems and actual in vivo drug metabolism for the precise prediction of metabolic activity. This approach will serve as a system to better draw correlations between in-vivo and in-vitro drug metabolism data. The central hypothesis is that Cytochrome P450 enzymes catalytic activity can be altered by protein-protein interactions occurring between Cytochrome P450 enzymes involved in drug metabolism, and is dependent on varying states of protein aggregation. This dissertation explains the details of the construction and characterization of a nanostructure device designed to control the state of aggregation of a P450 enzyme. Moreover

  20. Transposable elements are enriched within or in close proximity to xenobiotic-metabolizing cytochrome P450 genes

    PubMed Central

    Chen, Song; Li, Xianchun

    2007-01-01

    Background Transposons, i.e. transposable elements (TEs), are the major internal spontaneous mutation agents for the variability of eukaryotic genomes. To address the general issue of whether transposons mediate genomic changes in environment-adaptation genes, we scanned two alleles per each of the six xenobiotic-metabolizing Helicoverpa zea cytochrome P450 loci, including CYP6B8, CYP6B27, CYP321A1, CYP321A2, CYP9A12v3 and CYP9A14, for the presence of transposon insertions by genome walking and sequence analysis. We also scanned thirteen Drosophila melanogaster P450s genes for TE insertions by in silico mapping and literature search. Results Twelve novel transposons, including LINEs (long interspersed nuclear elements), SINEs (short interspersed nuclear elements), MITEs (miniature inverted-repeat transposable elements), one full-length transib-like transposon, and one full-length Tcl-like DNA transpson, are identified from the alleles of the six H. zea P450 genes. The twelve transposons are inserted into the 5'flanking region, 3'flanking region, exon, or intron of the six environment-adaptation P450 genes. In D. melanogaster, seven out of the eight Drosophila P450s (CYP4E2, CYP6A2, CYP6A8, CYP6A9, CYP6G1, CYP6W1, CYP12A4, CYP12D1) implicated in insecticide resistance are associated with a variety of transposons. By contrast, all the five Drosophila P450s (CYP302A1, CYP306A1, CYP307A1, CYP314A1 and CYP315A1) involved in ecdysone biosynthesis and developmental regulation are free of TE insertions. Conclusion These results indicate that TEs are selectively retained within or in close proximity to xenobiotic-metabolizing P450 genes. PMID:17381843

  1. Detoxification of ivermectin by ATP binding cassette transporter C4 and cytochrome P450 monooxygenase 6CJ1 in the human body louse, Pediculus humanus humanus.

    PubMed

    Kim, J H; Gellatly, K J; Lueke, B; Kohler, M; Nauen, R; Murenzi, E; Yoon, K S; Clark, J M

    2018-02-01

    We previously observed that ivermectin-induced detoxification genes, including ATP binding cassette transporter C4 (PhABCC4) and cytochrome P450 6CJ1 (CYP6CJ1) were identified from body lice following a brief exposure to a sublethal dose of ivermectin using a non-invasive induction assay. In this current study, the functional properties of PhABCC4 and CYP6CJ1 were investigated after expression in either X. laevis oocytes or using a baculovirus expression system, respectively. Efflux of [ 3 H]-9-(2-phosphonomethoxyethyl) adenine ([ 3 H]-PMEA), a known ABCC4 substrate in humans, was detected from PhABCC4 cRNA-injected oocytes by liquid scintillation spectrophotometric analysis and PhABCC4 expression in oocytes was confirmed using ABC transporter inhibitors. Efflux was also determined to be ATP-dependent. Using a variety of insecticides in a competition assay, only co-injection of ivermectin and dichlorodiphenyltrichloroethane led to decreased efflux of [ 3 H]-PMEA. PhABCC4-expressing oocytes also directly effluxed [ 3 H]-ivermectin, which increased over time. In addition, ivermectin appeared to be oxidatively metabolized and/or sequestered, although at low levels, following functional expression of CYP6CJ1 along with cytochrome P450 reductase in Sf9 cells. Our study suggests that PhABCC4 and perhaps CYP6CJ1 are involved in the Phase III and Phase I xenobiotic metabolism of ivermectin, respectively, and may play an important role in the evolution of ivermectin resistance in lice and other insects as field selection occurs. © 2017 The Royal Entomological Society.

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

    PubMed

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

    2015-02-01

    Spodoptera frugiperda is a polyphagous lepidopteran pest that encounters a wide range of toxic plant metabolites in its diet. The ability of this insect to adapt to its chemical environment might be explained by the action of major detoxification enzymes such as cytochrome P450s (or CYP). Forty-two sequences coding for P450s were identified and most of the transcripts were found to be expressed in the midgut, Malpighian tubules and fat body of S. frugiperda larvae. Relatively few P450s were expressed in the established cell line Sf9. In order to gain information on how these genes respond to different chemical compounds, larvae and Sf9 cells were exposed to plant secondary metabolites (indole, indole-3-carbinol, quercetin, 2-tridecanone and xanthotoxin), insecticides (deltamethrin, fipronil, methoprene, methoxyfenozide) or model inducers (clofibrate and phenobarbital). Several genes were induced by plant chemicals such as P450s from the 6B, 321A and 9A subfamilies. Only a few genes responded to insecticides, belonging principally to the CYP9A family. There was little overlap between the response in vivo measured in the midgut and the response in vitro in Sf9 cells. In addition, regulatory elements were detected in the promoter region of these genes. In conclusion, several P450s were identified that could potentially be involved in the adaptation of S. frugiperda to its chemical environment. © 2014 The Royal Entomological Society.

  3. Structure of conjugated polyketone reductase from Candida parapsilosis IFO 0708 reveals conformational changes for substrate recognition upon NADPH binding.

    PubMed

    Qin, Hui-Min; Yamamura, Akihiro; Miyakawa, Takuya; Kataoka, Michihiko; Nagai, Takahiro; Kitamura, Nahoko; Urano, Nobuyuki; Maruoka, Shintaro; Ohtsuka, Jun; Nagata, Koji; Shimizu, Sakayu; Tanokura, Masaru

    2014-01-01

    Conjugated polyketone reductase C2 (CPR-C2) from Candida parapsilosis IFO 0708, identified as a nicotinamide adenine dinucleotide phosphate (NADPH)-dependent ketopantoyl lactone reductase, belongs to the aldo-keto reductase superfamily. This enzyme reduces ketopantoyl lactone to D-pantoyl lactone in a strictly stereospecific manner. To elucidate the structural basis of the substrate specificity, we determined the crystal structures of the apo CPR-C2 and CPR-C2/NADPH complex at 1.70 and 1.80 Å resolutions, respectively. CPR-C2 adopted a triose-phosphate isomerase barrel fold at the core of the structure. Binding with the cofactor NADPH induced conformational changes in which Thr27 and Lys28 moved 15 and 5.0 Å, respectively, in the close vicinity of the adenosine 2'-phosphate group of NADPH to form hydrogen bonds. Based on the comparison of the CPR-C2/NADPH structure with 3-α-hydroxysteroid dehydrogenase and mutation analyses, we constructed substrate binding models with ketopantoyl lactone, which provided insight into the substrate specificity by the cofactor-induced structure. The results will be useful for the rational design of CPR-C2 mutants targeted for use in the industrial manufacture of ketopantoyl lactone.

  4. Kinetic consequences of introducing a proximal selenocysteine ligand into cytochrome P450cam.

    PubMed

    Vandemeulebroucke, An; Aldag, Caroline; Stiebritz, Martin T; Reiher, Markus; Hilvert, Donald

    2015-11-10

    The structural, electronic, and catalytic properties of cytochrome P450cam are subtly altered when the cysteine that coordinates to the heme iron is replaced with a selenocysteine. To map the effects of the sulfur-to-selenium substitution on the individual steps of the catalytic cycle, we conducted a comparative kinetic analysis of the selenoenzyme and its cysteine counterpart. Our results show that the more electron-donating selenolate ligand has only negligible effects on substrate, product, and oxygen binding, electron transfer, catalytic turnover, and coupling efficiency. Off-pathway reduction of oxygen to give superoxide is the only step significantly affected by the mutation. Incorporation of selenium accelerates this uncoupling reaction approximately 50-fold compared to sulfur, but because the second electron transfer step is much faster, the impact on overall catalytic turnover is minimal. Density functional theory calculations with pure and hybrid functionals suggest that superoxide formation is governed by a delicate interplay of spin distribution, spin state, and structural effects. In light of the remarkably similar electronic structures and energies calculated for the sulfur- and selenium-containing enzymes, the ability of the heavier atom to enhance the rate of spin crossover may account for the experimental observations. Because the selenoenzyme closely mimics wild-type P450cam, even at the level of individual steps in the reaction cycle, selenium represents a unique mechanistic probe for analyzing the role of the proximal ligand and spin crossovers in P450 chemistry.

  5. The crystal structure of NADPH:ferredoxin reductase from Azotobacter vinelandii.

    PubMed Central

    Sridhar Prasad, G.; Kresge, N.; Muhlberg, A. B.; Shaw, A.; Jung, Y. S.; Burgess, B. K.; Stout, C. D.

    1998-01-01

    NADPH:ferredoxin reductase (AvFPR) is involved in the response to oxidative stress in Azotobacter vinelandii. The crystal structure of AvFPR has been determined at 2.0 A resolution. The polypeptide fold is homologous with six other oxidoreductases whose structures have been solved including Escherichia coli flavodoxin reductase (EcFldR) and spinach, and Anabaena ferredoxin:NADP+ reductases (FNR). AvFPR is overall most homologous to EcFldR. The structure is comprised of a N-terminal six-stranded antiparallel beta-barrel domain, which binds FAD, and a C-terminal five-stranded parallel beta-sheet domain, which binds NADPH/NADP+ and has a classical nucleotide binding fold. The two domains associate to form a deep cleft where the NADPH and FAD binding sites are juxtaposed. The structure displays sequence conserved motifs in the region surrounding the two dinucleotide binding sites, which are characteristic of the homologous enzymes. The folded over conformation of FAD in AvFPR is similar to that in EcFldR due to stacking of Phe255 on the adenine ring of FAD, but it differs from that in the FNR enzymes, which lack a homologous aromatic residue. The structure of AvFPR displays three unique features in the environment of the bound FAD. Two features may affect the rate of reduction of FAD: the absence of an aromatic residue stacked on the isoalloxazine ring in the NADPH binding site; and the interaction of a carbonyl group with N10 of the flavin. Both of these features are due to the substitution of a conserved C-terminal tyrosine residue with alanine (Ala254) in AvFPR. An additional unique feature may affect the interaction of AvFPR with its redox partner ferredoxin I (FdI). This is the extension of the C-terminus by three residues relative to EcFldR and by four residues relative to FNR. The C-terminal residue, Lys258, interacts with the AMP phosphate of FAD. Consequently, both phosphate groups are paired with a basic group due to the simultaneous interaction of the FMN

  6. Selective inhibition by chloramphenicol of pregnenolone-16. cap alpha. -carbonitrile-inducible rat liver cytochrome P-450 isozymes

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Graves, P.E.; Kaminsky, L.S.; Halpert, J.

    Pregnenolone-16 ..cap alpha..-carbonitrile (PCN) has been shown to induce, in male rats, cytochrome P-450 isozymes responsible for the formation of R-10-hydroxywarfarin and R-dehydrowarfarin. Antibodies to the major PCN-inducible isozyme (PB/PCN-E) inhibit both activities in microsomal preparations. Recently the authors have shown that PCN treatment of female rats also induces the formation of both R-warfarin metabolites. However, in both sexes chloramphenicol (CAP) treatment selectively inhibits only the rate of formation of the R-dehydrowarfarin. A decrease in microsomal P-450 content occurs after in vivo administration of CAP to PCN-treated rats of both sexes. This is in contrast to the lack of effectmore » of CAP on P-450 levels in phenobarbital-treated rats. Covalent binding of /sup 14/C-CAP to microsomal protein in vitro was increased 3 to 4-fold following PCN treatment. Chromatographic evidences suggests the presence of at least two PCN-induced isozymes of similar molecular weights in both male and female rat liver microsomes. These data are consistent with the multiplicity of PCN-inducible P-450 in rat liver.« less

  7. Cytochrome P450 CYP716A254 catalyzes the formation of oleanolic acid from β-amyrin during oleanane-type triterpenoid saponins biosynthesis in Anemone flaccida.

    PubMed

    Zhan, Chuansong; Ahmed, Shakeel; Hu, Sheng; Dong, Shuang; Cai, Qian; Yang, Tewu; Wang, Xuekui; Li, Xiaohua; Hu, Xuebo

    2018-01-01

    Anemone flaccida Fr. Shmidt (Ranunculaceae), known as 'Di Wu' in China, is a perennial herb which has long been used to treat arthritis. The rhizome of A. flaccida contains pharmacologically active components i.e. oleanane-type triterpenoid saponins. Oleanolic acid is natural triterpenoid in plants with diverse biological activities. The biosynthesis of oleanolic acid involves cyclization of 2,3-oxidosqualene to the oleanane-type triterpenoid skeleton, followed by a series of oxidation reactions catalyzed by cytochrome P450 monooxygenase (CYP450). Previously, we identified four possible cytochrome P450 genes belonging to CYP716A subfamily from the transcriptome of A. flaccida. In this study, we identified one of those genes "CYP716A254" encoding a cytochrome P450 monooxygenase from A. flaccida that catalyzes the conversion of the β-amyrin into oleanolic acid. The heterologous expression of CYP716A254 in yeast resulted in oxidation of β-amyrin at the C-18 position to oleanolic acid production. These results provide an important basis for further studies of oleanane-type triterpenoid saponins synthesis in A. flaccida. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. Androgen receptor antagonism drives cytochrome P450 17A1 inhibitor efficacy in prostate cancer

    PubMed Central

    Norris, John D.; Ellison, Stephanie J.; Baker, Jennifer G.; Stagg, David B.; Wardell, Suzanne E.; Park, Sunghee; Alley, Holly M.; Baldi, Robert M.; Yllanes, Alexander; Andreano, Kaitlyn J.; Stice, James P.; Lawrence, Scott A.; Eisner, Joel R.; Price, Douglas K.; Moore, William R.; Figg, William D.; McDonnell, Donald P.

    2017-01-01

    The clinical utility of inhibiting cytochrome P450 17A1 (CYP17), a cytochrome p450 enzyme that is required for the production of androgens, has been exemplified by the approval of abiraterone for the treatment of castration-resistant prostate cancer (CRPC). Recently, however, it has been reported that CYP17 inhibitors can interact directly with the androgen receptor (AR). A phase I study recently reported that seviteronel, a CYP17 lyase–selective inhibitor, ædemonstrated a sustained reduction in prostate-specific antigen in a patient with CRPC, and another study showed seviteronel’s direct effects on AR function. This suggested that seviteronel may have therapeutically relevant activities in addition to its ability to inhibit androgen production. Here, we have demonstrated that CYP17 inhibitors, with the exception of orteronel, can function as competitive AR antagonists. Conformational profiling revealed that the CYP17 inhibitor–bound AR adopted a conformation that resembled the unliganded AR (apo-AR), precluding nuclear localization and DNA binding. Further, we observed that seviteronel and abiraterone inhibited the growth of tumor xenografts expressing the clinically relevant mutation AR-F876L and that this activity could be attributed entirely to competitive AR antagonism. The results of this study suggest that the ability of CYP17 inhibitors to directly antagonize the AR may contribute to their clinical efficacy in CRPC. PMID:28463227

  9. Cytochrome b 5 reductase and the control of lipid metabolism and healthspan.

    PubMed

    Martin-Montalvo, Alejandro; Sun, Yaning; Diaz-Ruiz, Alberto; Ali, Ahmed; Gutierrez, Vincent; Palacios, Hector H; Curtis, Jessica; Siendones, Emilio; Ariza, Julia; Abulwerdi, Gelareh A; Sun, Xiaoping; Wang, Annie X; Pearson, Kevin J; Fishbein, Kenneth W; Spencer, Richard G; Wang, Miao; Han, Xianlin; Scheibye-Knudsen, Morten; Baur, Joe A; Shertzer, Howard G; Navas, Placido; Villalba, Jose Manuel; Zou, Sige; Bernier, Michel; de Cabo, Rafael

    2016-01-01

    Cytochrome b 5 reductases (CYB5R) are required for the elongation and desaturation of fatty acids, cholesterol synthesis and mono-oxygenation of cytochrome P450 enzymes, all of which are associated with protection against metabolic disorders. However, the physiological role of CYB5R in the context of metabolism, healthspan and aging remains ill-defined. We generated CYB5R-overexpressing flies (CYB5R-OE) and created a transgenic mouse line overexpressing CYB5R3 (CYB5R3-Tg) in the C57BL/6J background to investigate the function of this class of enzymes as regulators of metabolism and age-associated pathologies. Gender- and/or stage-specific induction of CYB5R, and pharmacological activation of CYB5R with tetrahydroindenoindole extended fly lifespan. Increased expression of CYB5R3 was associated with significant improvements in several metabolic parameters that resulted in modest lifespan extension in mice. Diethylnitrosamine-induced liver carcinogenesis was reduced in CYB5R3-Tg mice. Accumulation of high levels of long-chain polyunsaturated fatty acids, improvement in mitochondrial function, decrease in oxidative damage and inhibition of chronic pro-inflammatory pathways occurred in the transgenic animals. These results indicate that CYB5R represents a new target in the study of genes that regulate lipid metabolism and healthspan.

  10. Human skin levels of retinoic acid and cytochrome P-450-derived 4-hydroxyretinoic acid after topical application of retinoic acid in vivo compared to concentrations required to stimulate retinoic acid receptor-mediated transcription in vitro.

    PubMed Central

    Duell, E A; Aström, A; Griffiths, C E; Chambon, P; Voorhees, J J

    1992-01-01

    Metabolism of retinoic acid to a less active metabolite, 4-hydroxyretinoic acid, occurs via cytochrome P-450 isozyme(s). Effect of a pharmacological dose of retinoic acid on the level of retinoic acid in skin and on cytochrome P-450 activity was investigated. A cream containing 0.1% retinoic acid or cream alone was applied topically to adult human skin for four days under occlusion. Treated areas were removed by a keratome and a microsomal fraction was isolated from each biopsy. In vitro incubation of 3H-retinoic acid with microsomes from in vivo retinoic acid treated sites resulted in a 4.5-fold increase (P = 0.0001, n = 13) in its transformation to 4-hydroxyretinoic acid in comparison to in vitro incubations with microsomes from in vivo cream alone treated sites. This cytochrome P-450 mediated activity was oxygen- and NADPH-dependent and was inhibited 68% by 5 microM ketoconazole (P = 0.0035, n = 8) and 51% by carbon monoxide (P = 0.02, n = 6). Cotransfection of individual retinoic acid receptors (RARs) or retinoid X receptor-alpha (RXR-alpha) and a chloramphenicol acetyl transferase (CAT) reporter plasmid containing a retinoic acid responsive element into CV-1 cells was used to determine the ED50 values for stimulation of CAT activity by retinoic acid and its metabolites. Levels of all trans and 13-cis RA in RA-treated tissues were greater than the ED50 values determined for all three RARs with these compounds. Furthermore, the level of all trans RA was greater than the ED50 for RXR-alpha whereas the 4-OH RA level was greater than the ED50 for RAR-beta and RAR-gamma but less than for RAR-alpha and RXR-alpha. These data suggest that there are sufficient amounts of retinoic acid in treated skin to activate gene transcription over both RARs and RXR-alpha. PMID:1328295

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

    PubMed

    Zhang, Jing; Ji, Li; Liu, Weiping

    2015-08-17

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

  13. In silico prediction of cytochrome P450-mediated drug metabolism.

    PubMed

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

    2011-06-01

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

  14. Occupation of the cytochrome P450 substrate pocket by diverse compounds at general anesthesia concentrations.

    PubMed

    LaBella, F S; Stein, D; Queen, G

    1998-10-02

    Each of a diverse array of compounds, at concentrations reported to effect general anesthesia, when added to liver microsomes, forms a complex with cytochromes P450 to generate, with reference to a cuvette containing microsomes only, a characteristic absorbance-difference spectrum. This spectrum results from a change in the electron-spin state of the heme iron atom induced upon entry by the anesthetic molecule into the enzyme catalytic pocket. The difference spectrum, representing the anesthetic-P450 complex, is characteristic of substances that are substrates for the enzyme. For the group of compounds as a whole, the magnitudes of the absorbance-difference spectra vary only about twofold, although the anesthetic potencies vary by several orders of magnitude. The dissociation constants (Ks), calculated from absorbance data and representing affinities of the anesthetics for P450, agree closely with the respective EC50 (concentration that effects anesthesia in 50% of individuals) values, and with the respective Ki (concentration that inhibits P450 catalytic activities half-maximally) values reported by us previously. The absorbance complex resulting from the occupation of the catalytic pocket by endogenous substrates, androstenedione and arachidonic acid, is inhibited, competitively, by anesthetics. Occupation of and perturbation of the heme catalytic pocket by anesthetic, as monitored by the absorbance-difference spectrum, is rapidly reversible. The presumed in vivo consequences of perturbation by general anesthetics of heme proteins is suppression of the generation of chemical signals that determine cell sensitivity and response.

  15. Aromatic hydroxylation by cytochrome P450: model calculations of mechanism and substituent effects.

    PubMed

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

    2003-12-10

    The mechanism and selectivity of aromatic hydroxylation by cytochrome P450 enzymes is explored using new B3LYP density functional theory computations. The calculations, using a realistic porphyrin model system, show that rate-determining addition of compound I to an aromatic carbon atom proceeds via a transition state with partial radical and cationic character. Reactivity is shown to depend strongly on ring substituents, with both electron-withdrawing and -donating groups strongly decreasing the addition barrier in the para position, and it is shown that the calculated barrier heights can be reproduced by a new dual-parameter equation based on radical and cationic Hammett sigma parameters.

  16. Genetic variations in NADPH-CYP450 oxidoreductase in a Czech Slavic cohort.

    PubMed

    Tomková, Mária; Panda, Satya Prakash; Šeda, Ondřej; Baxová, Alice; Hůlková, Martina; Siler Masters, Bettie Sue; Martásek, Pavel

    2015-01-01

    Estimating polymorphic allele frequencies of the NADPH-CYP450 oxidoreductase (POR) gene in a Czech Slavic population. The POR gene was analyzed in 322 individuals from a control cohort by sequencing and high resolution melting analysis. We identified seven unreported SNP genetic variations, including two SNPs in the 5' flanking region (g.4965C>T and g.4994G>T), one intronic variant (c.1899-20C>T), one synonymous SNP (p.20Ala=) and three nonsynonymous SNPs (p.Thr29Ser, p.Pro384Leu and p.Thr529Met). The p.Pro384Leu variant exhibited reduced enzymatic activities compared with wild-type. New POR variant identification indicates the number of uncommon variants might be specific for each subpopulation being investigated, particularly germane to the singular role that POR plays in providing reducing equivalents to all CYP450s in the endoplasmic reticulum. Original submitted 15 September 2014; Revision submitted 17 November 2014.

  17. Differential transcription of cytochrome P450s and glutathione S transferases in DDT-susceptible and resistant Drosophila melanogaster strains in response to DDT and oxidative stress

    USDA-ARS?s Scientific Manuscript database

    Metabolic DDT resistance in Drosophila melanogaster has previously been associated with constitutive over-transcription of cytochrome P450s. Increased P450 activity has also been associated with increased oxidative stress. In contrast, over-transcription of glutathione S transferases (GSTs) has been...

  18. Effect of feeding quandong (Santalum acuminatum) oil to rats on tissue lipids, hepatic cytochrome P-450 and tissue histology.

    PubMed

    Jones, G P; Birkett, A; Sanigorski, A; Sinclair, A J; Hooper, P T; Watson, T; Rieger, V

    1994-06-01

    Quandong kernels are a traditional Aboriginal food item; they are rich in oil and contain large amounts of an unusual fatty acid, trans-11-octadecen-9-ynoic acid (santalbic acid), but it is not known whether this acid is absorbed and/or metabolized. The oil was fed at 12.6% of total energy content in semi-synthetic diets to groups of male Sprague-Dawley rats for 10 and 20 days. Santalbic acid was found in the lipids of plasma, adipose tissue, skeletal muscle, kidney, heart and liver but not in brain. Hepatic microsomal cytochrome P-450 activity in animals fed for 20 days was significantly higher (P < 0.05) than in controls. Histopathological examination did not reveal any lesions in the tissues of any animal fed quandong oil. The fact that santalbic acid was readily absorbed, widely distributed in tissues and was associated with an elevated level of hepatic cytochrome P-450 indicates that further studies are required to investigate whether or not there is a hazard associated with the human practice of consuming quandong kernels.

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

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Aoyama, Toshifumi; Yamano, Shigeru; Gelboin, H.V.

    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 cellsmore » 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.« less

  20. Imidacloprid is degraded by CYP353D1v2, a cytochrome P450 overexpressed in a resistant strain of Laodelphax striatellus.

    PubMed

    Elzaki, Mohammed Esmail Abdalla; Miah, Mohammad Asaduzzaman; Wu, Min; Zhang, Haomiao; Pu, Jian; Jiang, Ling; Han, Zhaojun

    2017-07-01

    Cytochrome P450s are associated with the metabolising of a wide range of compounds, including insecticides. CYP353D1v2 has been found to be overexpressed in an imidacloprid-resistant strain of Laodelphax striatellus. Thus, this study was conducted to express CYP353D1v2 in Sf9 cells as a recombinant protein, to assess its ability to metabolise imidacloprid. Western blot and carbon monoxide difference spectrum analysis indicated that the intact CYP353D1v2 protein had been successfully expressed in Sf9 insect cells. Catalytic activity tests with four traditional P450-activity-probing substrates found that the expressed CYP353D1v2 preferentially metabolised p-nitroanisole, ethoxycoumarin and ethoxyresorufin with specific activities of 32.70, 0.317 and 1.22 pmol min -1 pmol -1 protein respectively, but no activity to luciferin-H EGE. The enzyme activity for degrading imidacloprid was tested by measuring substrate depletion and formation of the metabolite. Kinetic parameters for imidacloprid were K m 5.99 ± 0.95 µm and k cat 0.03 ± 0.0004 min -1 . The chromatogram analysis showed clearly the NADPH-dependent depletion of imidacloprid and the formation of an unknown metabolite. The UPLC-MS mass spectrum demonstrated that the metabolite was an oxidative product of imidacloprid, 5-hydroxy-imidacloprid. These results suggest that CYP353D1v2 in L. striatellus is capable of degrading imidacloprid, and that enzyme activity can be evaluated well only by some traditional probing substrates. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

  1. Genomic and transcriptomic insights into the cytochrome P450 monooxygenase gene repertoire in the rice pest brown planthopper, Nilaparvata lugens.

    PubMed

    Lao, Shu-Hua; Huang, Xiao-Hui; Huang, Hai-Jian; Liu, Cheng-Wen; Zhang, Chuan-Xi; Bao, Yan-Yuan

    2015-11-01

    The cytochrome P450 monooxygenase (P450) gene family is one of the most abundant eukaryotic gene families that encode detoxification enzymes. In this study, we identified an abundance of P450 gene repertoire through genome- and transcriptome-wide analysis in the brown planthopper (Nilaparvata lugens), the most destructive rice pest in Asia. Detailed gene information including the exon-intron organization, size, transcription orientation and distribution in the genome revealed that many P450 loci were closely situated on the same scaffold, indicating frequent occurrence of gene duplications. Insecticide-response expression profiling revealed that imidacloprid significantly increased NlCYP6CS1v2, NLCYP4CE1v2, NlCYP4DE1, NlCYP417A1v2 and NlCYP439A1 expression; while triazophos and deltamethrin notably enhanced NlCYP303A1 expression. Expression analysis at the developmental stage showed the egg-, nymph-, male- and female-specific expression patterns of N. lugens P450 genes. These novel findings will be helpful for clarifying the P450 functions in physiological processes including development, reproduction and insecticide resistance in this insect species. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. Multi-step oxidations catalyzed by cytochrome P450 enzymes: Processive vs. distributive kinetics and the issue of carbonyl oxidation in chemical mechanisms

    PubMed Central

    Guengerich, F. Peter; Sohl, Christal D.; Chowdhury, Goutam

    2010-01-01

    Catalysis of sequential oxidation reactions is not unusual in cytochrome P450 (P450) reactions, not only in steroid metabolism but also with many xenobiotics. One issue is how processive/distributive these reactions are, i.e. how much do the “intermediate” products dissociate. Our work with human P450s 2E1, 2A6, and 19A1 on this subject has revealed a mixture of systems, surprisingly with a more distributive mechanism with an endogenous substrate (P450 19A1) than for some xenobiotics (P450s 2E1, 2A6). One aspect of this research involves carbonyl intermediates, and the choice of catalytic mechanism is linked to the hydration state of the aldehyde. The non-enzymatic rates of hydration and dehydration of carbonyls are not rapid and whether P450s catalyze the reversible hydration is unknown. If carbonyl hydration and dehydration are slow, the mechanism may be set by the carbonyl hydration status. PMID:20804723

  3. P450 monooxygenases (P450ome) of the model white rot fungus Phanerochaete chrysosporium.

    PubMed

    Syed, Khajamohiddin; Yadav, Jagjit S

    2012-11-01

    Phanerochaete chrysosporium, the model white rot fungus, has been the focus of research for the past about four decades for understanding the mechanisms and processes of biodegradation of the natural aromatic polymer lignin and a broad range of environmental toxic chemicals. The ability to degrade this vast array of xenobiotic compounds was originally attributed to its lignin-degrading enzyme system, mainly the extracellular peroxidases. However, subsequent physiological, biochemical, and/or genetic studies by us and others identified the involvement of a peroxidase-independent oxidoreductase system, the cytochrome P450 monooxygenase system. The whole genome sequence revealed an extraordinarily large P450 contingent (P450ome) with an estimated 149 P450s in this organism. This review focuses on the current status of understanding on the P450 monooxygenase system of P. chrysosproium in terms of pre-genomic and post-genomic identification, structural and evolutionary analysis, transcriptional regulation, redox partners, and functional characterization for its biodegradative potential. Future research on this catalytically diverse oxidoreductase enzyme system and its major role as a newly emerged player in xenobiotic metabolism/degradation is discussed.

  4. Docking and QSAR comparative studies of polycyclic aromatic hydrocarbons and other procarcinogen interactions with cytochromes P450 1A1 and 1B1.

    PubMed

    Gonzalez, J; Marchand-Geneste, N; Giraudel, J L; Shimada, T

    2012-01-01

    To obtain chemical clues on the process of bioactivation by cytochromes P450 1A1 and 1B1, some QSAR studies were carried out based on cellular experiments of the metabolic activation of polycyclic aromatic hydrocarbons and heterocyclic aromatic compounds by those enzymes. Firstly, the 3D structures of cytochromes 1A1 and 1B1 were built using homology modelling with a cytochrome 1A2 template. Using these structures, 32 ligands including heterocyclic aromatic compounds, polycyclic aromatic hydrocarbons and corresponding diols, were docked with LigandFit and CDOCKER algorithms. Binding mode analysis highlighted the importance of hydrophobic interactions and the hydrogen bonding network between cytochrome amino acids and docked molecules. Finally, for each enzyme, multilinear regression and artificial neural network QSAR models were developed and compared. These statistical models highlighted the importance of electronic, structural and energetic descriptors in metabolic activation process, and could be used for virtual screening of ligand databases. In the case of P450 1A1, the best model was obtained with artificial neural network analysis and gave an r (2) of 0.66 and an external prediction [Formula: see text] of 0.73. Concerning P450 1B1, artificial neural network analysis gave a much more robust model, associated with an r (2) value of 0.73 and an external prediction [Formula: see text] of 0.59.

  5. Selective aliphatic carbon-hydrogen bond activation of protected alcohol substrates by cytochrome P450 enzymes.

    PubMed

    Bell, Stephen G; Spence, Justin T J; Liu, Shenglan; George, Jonathan H; Wong, Luet-Lok

    2014-04-21

    Protected cyclohexanol and cyclohex-2-enol substrates, containing benzyl ether and benzoate ester moieties, were designed to fit into the active site of the Tyr96Ala mutant of cytochrome P450cam. The protected cyclohexanol substrates were efficiently and selectively hydroxylated by the mutant enzyme at the trans C-H bond of C-4 on the cyclohexyl ring. The selectivity of oxidation of the benzoate ester protected cyclohexanol could be altered by making alternative amino acid substitutions in the P450cam active site. The addition of the double bond in the cyclohexyl ring of the benzoate ester protected cyclohex-2-enol has a debilitative effect on the activity of the Tyr96Ala mutant with this substrate. However, the Phe87Ala/Tyr96Phe double mutant, which introduces space at a different location in the active site than the Tyr96Ala mutant, was able to efficiently hydroxylate the C-H bonds of 1-cyclohex-2-enyl benzoate at the allylic C-4 position. Mutations at Phe87 improved the selectivity of the oxidation of 1-phenyl-1-cyclohexylethylene to trans-4-phenyl-ethenylcyclohexanol (92%) when compared to single mutants at Tyr96 of P450cam.

  6. 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

  7. Catalytic strategy for carbon−carbon bond scission by the cytochrome P450 OleT

    PubMed Central

    Grant, Job L.; Mitchell, Megan E.; Makris, Thomas Michael

    2016-01-01

    OleT is a cytochrome P450 that catalyzes the hydrogen peroxide-dependent metabolism of Cn chain-length fatty acids to synthesize Cn-1 1-alkenes. The decarboxylation reaction provides a route for the production of drop-in hydrocarbon fuels from a renewable and abundant natural resource. This transformation is highly unusual for a P450, which typically uses an Fe4+−oxo intermediate known as compound I for the insertion of oxygen into organic substrates. OleT, previously shown to form compound I, catalyzes a different reaction. A large substrate kinetic isotope effect (≥8) for OleT compound I decay confirms that, like monooxygenation, alkene formation is initiated by substrate C−H bond abstraction. Rather than finalizing the reaction through rapid oxygen rebound, alkene synthesis proceeds through the formation of a reaction cycle intermediate with kinetics, optical properties, and reactivity indicative of an Fe4+−OH species, compound II. The direct observation of this intermediate, normally fleeting in hydroxylases, provides a rationale for the carbon−carbon scission reaction catalyzed by OleT. PMID:27555591

  8. The biodiversity of microbial cytochromes P450.

    PubMed

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

    2003-01-01

    The cytochrome P450 (CYP) superfamily of genes and proteins are well known for their involvement in pharmacology and toxicology, but also increasingly for their importance and diversity in microbes. The extent of diversity has only recently become apparent with the emergence of data from whole genome sequencing projects and the coming years will reveal even more information on the diversity in microbial eukaryotes. This review seeks to describe the historical development of these studies and to highlight the importance of the genes and proteins. CYPs are deeply involved in the development of strategies for deterrence and attraction as well as detoxification. As such, there is intense interest in pathways of secondary metabolism that include CYPs in oxidative tailoring of antibiotics, sometimes influencing potency as bioactive compounds. Further to this is interest in CYPs in metabolism of xenobiotics for use as carbon sources for microbial growth and as biotransformation agents or in bioremediation. CYPs are also current and potential drug targets; compounds inhibiting CYP are antifungal and anti-protozoan agents, and potentially similar compounds may be useful against some bacterial diseases such as tuberculosis. Of note is the diversity of CYP requirements within an organism, ranging from Escherichia coli that has no CYPs as in many bacteria, to Mycobacterium smegmatis that has 40 representing 1% of coding genes. The basidiomycete fungus Phanerochaete chrysosporium surprised all when it was found to contain a hundred or more CYPs. The functional genomic investigation of these orphan CYPs is a major challenge for the future.

  9. Interaction potential of Trigonella foenum graceum through cytochrome P450 mediated inhibition

    PubMed Central

    Ahmmed, Sk Milan; Mukherjee, Pulok K.; Bahadur, Shiv; Kar, Amit; Mukherjee, Kakali; Karmakar, Sanmoy; Bandyopadhyay, Arun

    2015-01-01

    Objective: The seeds of Trigonella foenum-graecum (TFG) (family: Leguminosae) are widely consumed both as a spice in food and Traditional Medicine in India. The present study was undertaken to evaluate the inhibitory effect of standardized extract of TFG and its major constituent trigonelline (TG) on rat liver microsome (RLM) and cytochrome P450 (CYP450) drug metabolizing isozymes (CYP3A4 and CYP2D6), which may indicate the possibility of a probable unwanted interaction. Materials and Methods: Reverse phase-high performance liquid chromatography method was developed to standardize the hydroalcoholic seed extract with standard TG. The inhibitory potential of the extract and TG was evaluated on RLM and CYP isozymes using CYP450-carbon monoxide (CYP450-CO) complex assay and fluorescence assay, respectively. Results: The content of TG in TFG was found to be 3.38% (w/w). The CYP-CO complex assay showed 23.32% inhibition on RLM. Fluorescence study revealed that the extract and the biomarker had some inhibition on CYP450 isozymes e.g. CYP3A4 and CYP2D6 (IC50 values of the extract: 102.65 ± 2.63–142.23 ± 2.61 µg/ml and TG: 168.73 ± 4.03–180.90 ± 2.49 µg/ml) which was very less compared to positive controls ketoconazole and quinidine. Inhibition potential of TFG was little higher than TG but very less compared to positive controls. Conclusions: From the present study, we may conclude that the TFG or TG has very less potential to inhibit the CYP isozymes (CYP3A4, CYP2D6), so administration of this plant extract or its biomarker TG may be safe. PMID:26600643

  10. A novel role of Drosophila cytochrome P450-4e3 in permethrin insecticide tolerance.

    PubMed

    Terhzaz, Selim; Cabrero, Pablo; Brinzer, Robert A; Halberg, Kenneth A; Dow, Julian A T; Davies, Shireen-A

    2015-12-01

    The exposure of insects to xenobiotics, such as insecticides, triggers a complex defence response necessary for survival. This response includes the induction of genes that encode key Cytochrome P450 monooxygenase detoxification enzymes. Drosophila melanogaster Malpighian (renal) tubules are critical organs in the detoxification and elimination of these foreign compounds, so the tubule response induced by dietary exposure to the insecticide permethrin was examined. We found that expression of the gene encoding Cytochrome P450-4e3 (Cyp4e3) is significantly up-regulated by Drosophila fed on permethrin and that manipulation of Cyp4e3 levels, specifically in the principal cells of the Malpighian tubules, impacts significantly on the survival of permethrin-fed flies. Both dietary exposure to permethrin and Cyp4e3 knockdown cause a significant elevation of oxidative stress-associated markers in the tubules, including H2O2 and lipid peroxidation byproduct, HNE (4-hydroxynonenal). Thus, Cyp4e3 may play an important role in regulating H2O2 levels in the endoplasmic reticulum (ER) where it resides, and its absence triggers a JAK/STAT and NF-κB-mediated stress response, similar to that observed in cells under ER stress. This work increases our understanding of the molecular mechanisms of insecticide detoxification and provides further evidence of the oxidative stress responses induced by permethrin metabolism. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

  11. Association of cytochrome P450 genetic polymorphisms with neoadjuvant chemotherapy efficacy in breast cancer patients

    PubMed Central

    2012-01-01

    Background The enzymes of the cytochrome P450 family (CYPs) play an important role in the metabolism of a great variety of anticancer agents; therefore, polymorphisms in genes encoding for metabolizing enzymes and drugs transporters can affect drug efficacy and toxicity. Methods The genetic polymorphisms of cytochrome P450 were studied in 395 patients with breast cancer by RLFP analysis. Results Here, we studied the association of functionally significant variant alleles of CYP3A4, CYP3A5, CYP2B6, CYP2C8, CYP2C9 and CYP2C19 with the clinical response to neoadjuvant chemotherapy in breast cancer patients. A significant correlation was observed between the CYP2C9*2 polymorphism and chemotherapy resistance (OR = 4.64; CI 95% = 1.01 – 20.91), as well as between CYP2C9*2 heterozygotes and chemotherapy resistance in women with nodal forms of breast cancer and a cancer hereditary load (OR = 15.50; CI 95% = 1.08 – 826.12) when the potential combined effects were examined. No significant association between chemotherapy resistance and the other examined genotypes and the potential combined clinical and tumour-related parameters were discovered. Conclusion In conclusion, CYP2C9*2 was associated with neoadjuvant chemotherapy resistance (OR = 4.64; CI 95% = 1.01 – 20.91) in the population of interest. PMID:22702493

  12. The human cytochrome P450 3A locus. Gene evolution by capture of downstream exons.

    PubMed

    Finta, C; Zaphiropoulos, P G

    2000-12-30

    Using a bacterial artificial chromosome (BAC) clone, we have mapped the human cytochrome P450 3A (CYP3A) locus containing the genes encoding for CYP3A4, CYP3A5 and CYP3A7. The genes lie in a head-to-tail orientation in the order of 3A4, 3A7 and 3A5. In both intergenic regions (3A4-3A7 and 3A7-3A5), we have detected several additional cytochrome P450 3A exons, forming two CYP3A pseudogenes. These pseudogenes have the same orientation as the CYP3A genes. To our surprise, a 3A7 mRNA species has been detected in which the exons 2 and 13 of one of the pseudogenes (the one that is downstream of 3A7) are spliced after the 3A7 terminal exon. This results in an mRNA molecule that consists of the 13 3A7 exons and two additional exons at the 3' end. The additional two exons originating from the pseudogene are in an altered reading frame and consequently have the capability to code a completely different amino acid sequence than the canonical CYP3A exons 2 and 13. These findings may represent a generalized evolutionary process with genes having the potential to capture neighboring sequences and use them as functional exons.

  13. NQO1 and CYP450 reductase decrease the systemic exposure of rifampicin-quinone and mediate its redox cycle in rats.

    PubMed

    Shi, Fuguo; Li, Xiaobing; Pan, Hong; Ding, Li

    2017-01-05

    Rifampicin (RIF) is used in regimens for infections caused by Mycobacteria accompanied by serious adverse reactions. Rifampicin-quinone (RIF-Q) is a major autoxidation product of RIF. It is not clear whether RIF-Q plays a role in RIF induced adverse reactions. Investigation of the systemic exposure of RIF-Q is helpful to better understand the role of RIF-Q in RIF induced adverse reactions. In this study, a simple and reproducible high performance liquid chromatography-mass spectrometry (LC-MS) method involving a procedure to prevent the RIF from oxidation for simultaneous quantification of RIF and RIF-Q in rat plasma has been developed and validated, and applied to elucidate the systemic exposure of RIF-Q in rats. The pharmacokinetics data showed that the systemic exposure of RIF-Q was very low (0.67% of RIF, AUC 0-24 ) in rats after oral administration of RIF. However, RIF-Q may undergo the redox cycle in vivo by the evidence that the majority of RIF-Q was reduced to RIF after an oral dose of RIF-Q. Pretreatment with the NAD(P)H: quinone oxidoreductase 1 (NQO1) specific inhibitor dicoumarol and/or cytochrome P450 reductase (CPR) inhibitor diphenyleneiodonium suppressed the redox cycle and significantly increased the systemic exposure of RIF-Q. The inhibitors also attenuated the redox cycle induced reactive oxygen species formation and cytotoxicity in RIF-Q-treated HepG2 cells. These results indicate that NQO1 and CPR play an important role in redox cycle of RIF-Q and may thus contribute to RIF-induced adverse reactions. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. The usage of a three-compartment model to investigate the metabolic differences between hepatic reductase null and wild-type mice.

    PubMed

    Hill, Lydia; Chaplain, Mark A J; Wolf, Roland; Kapelyukh, Yury

    2017-03-01

    The Cytochrome P450 (CYP) system is involved in 90% of the human body's interactions with xenobiotics and due to this, it has become an area of avid research including the creation of transgenic mice. This paper proposes a three-compartment model which is used to explain the drug metabolism in the Hepatic Reductase Null (HRN) mouse developed by the University of Dundee (Henderson, C. J., Otto, D. M. E., Carrie, D., Magnuson, M. A., McLaren, A. W., Rosewell, I. and Wolf, C. R. (2003) Inactivation of the hepatic cytochrome p450 system by conditional deletion of hepatic cytochrome p450 reductase. J. Biol. Chem. , 13480-13486). The model is compared with a two-compartment model using experimental data from studies using wild-type and HRN mice. This comparison allowed for metabolic differences between the two types of mice to be isolated. The three sets of drug data (Gefitinib, Midazolam and Thalidomide) showed that the transgenic mouse has a decreased rate of metabolism. © The authors 2015. Published by Oxford University Press on behalf of the Institute of Mathematics and its Applications. All rights reserved.

  15. Gravity Persistent Signal 1 (GPS1) reveals novel cytochrome P450s involved in gravitropism.

    PubMed

    Withers, John C; Shipp, Matthew J; Rupasinghe, Sanjeewa G; Sukumar, Poornima; Schuler, Mary A; Muday, Gloria K; Wyatt, Sarah E

    2013-01-01

    Gravity is an important environmental factor that affects growth and development of plants. In response to changes in gravity, directional growth occurs along the major axes and lateral branches of both shoots and roots. The gravity persistent signal (gps) mutants of Arabidopsis thaliana were previously identified as having an altered response to gravity when reoriented relative to the gravity vector in the cold, with the gps1 mutant exhibiting a complete loss of tropic response under these conditions. Thermal asymmetric interlaced (TAIL) PCR was used to identify the gene defective in gps1. Gene expression data, molecular modeling and computational substrate dockings, quantitative RT-PCR analyses, reporter gene fusions, and physiological analyses of knockout mutants were used to characterize the genes identified. Cloning of the gene defective in gps1 and genetic complementation revealed that GPS1 encodes CYP705A22, a cytochrome P450 monooxygenase (P450). CYP705A5, a closely related family member, was identified as expressed specifically in roots in response to gravistimulation, and a mutation affecting its expression resulted in a delayed gravity response, increased flavonol levels, and decreased basipetal auxin transport. Molecular modeling coupled with in silico substrate docking and diphenylboric acid 2-aminoethyl ester (DBPA) staining indicated that these P450s are involved in biosynthesis of flavonoids potentially involved in auxin transport. The characterization of two novel P450s (CYP705A22 and CYP705A5) and their role in the gravity response has offered new insights into the regulation of the genetic and physiological controls of plant gravitropism.

  16. Identification of cytochrome P450 2E1 as the predominant enzyme catalyzing human liver microsomal defluorination of sevoflurane, isoflurane, and methoxyflurane.

    PubMed

    Kharasch, E D; Thummel, K E

    1993-10-01

    Renal and hepatic toxicity of the fluorinated ether volatile anesthetics is caused by biotransformation to toxic metabolites. Metabolism also contributes significantly to the elimination pharmacokinetics of some volatile agents. Although innumerable studies have explored anesthetic metabolism in animals, there is little information on human volatile anesthetic metabolism with respect to comparative rates or the identity of the enzymes responsible for defluorination. The first purpose of this investigation was to compare the metabolism of the fluorinated ether anesthetics by human liver microsomes. The second purpose was to test the hypothesis that cytochrome P450 2E1 is the specific P450 isoform responsible for volatile anesthetic defluorination in humans. Microsomes were prepared from human livers. Anesthetic metabolism in microsomal incubations was measured by fluoride production. The strategy for evaluating the role of P450 2E1 in anesthetic defluorination involved three approaches: for a series of 12 human livers, correlation of microsomal defluorination rate with microsomal P450 2E1 content (measured by Western blot analysis), correlation of defluorination rate with microsomal P450 2E1 catalytic activity using marker substrates (para-nitrophenol hydroxylation and chlorzoxazone 6-hydroxylation), and chemical inhibition by P450 isoform-selective inhibitors. The rank order of anesthetic metabolism, assessed by fluoride production at saturating substrate concentrations, was methoxyflurane > sevoflurane > enflurane > isoflurane > desflurane > 0. There was a significant linear correlation of sevoflurane and methoxyflurane defluorination with antigenic P450 2E1 content (r = 0.98 and r = 0.72, respectively), but not with either P450 1A2 or P450 3A3/4. Comparison of anesthetic defluorination with either para-nitrophenol or chlorzoxazone hydroxylation showed a significant correlation for sevoflurane (r = 0.93, r = 0.95) and methoxyflurane (r = 0.78, r = 0

  17. Novel phacB-encoded cytochrome P450 monooxygenase from Aspergillus nidulans with 3-hydroxyphenylacetate 6-hydroxylase and 3,4-dihydroxyphenylacetate 6-hydroxylase activities.

    PubMed

    Ferrer-Sevillano, Francisco; Fernández-Cañón, José M

    2007-03-01

    Aspergillus nidulans catabolizes phenylacetate (PhAc) and 3-hydroxy-, 4-hydroxy-, and 3,4-dihydroxyphenylacetate (3-OH-PhAc, 4-OH-PhAc, and 3,4-diOH-PhAc, respectively) through the 2,5-dihydroxyphenylacetate (homogentisic acid) catabolic pathway. Using cDNA subtraction techniques, we isolated a gene, denoted phacB, which is strongly induced by PhAc (and its hydroxyderivatives) and encodes a new cytochrome P450 (CYP450). A disrupted phacB strain (delta phacB) does not grow on 3-hydroxy-, 4-hydroxy-, or 3,4-dihydroxy-PhAc. High-performance liquid chromatography and gas chromatography-mass spectrum analyses of in vitro reactions using microsomes from wild-type and several A. nidulans mutant strains confirmed that the phacB-encoded CYP450 catalyzes 3-hydroxyphenylacetate and 3,4-dihydroxyphenylacetate 6-hydroxylations to generate 2,5-dihydroxyphenylacetate and 2,4,5-trihydroxyphenylacetate, respectively. Both of these compounds are used as substrates by homogentisate dioxygenase. This cytochrome P450 protein also uses PhAc as a substrate to generate 2-OH-PhAc with a very low efficiency. The phacB gene is the first member of a new CYP450 subfamily (CYP504B).

  18. Novel phacB-Encoded Cytochrome P450 Monooxygenase from Aspergillus nidulans with 3-Hydroxyphenylacetate 6-Hydroxylase and 3,4-Dihydroxyphenylacetate 6-Hydroxylase Activities▿

    PubMed Central

    Ferrer-Sevillano, Francisco; Fernández-Cañón, José M.

    2007-01-01

    Aspergillus nidulans catabolizes phenylacetate (PhAc) and 3-hydroxy-, 4-hydroxy-, and 3,4-dihydroxyphenylacetate (3-OH-PhAc, 4-OH-PhAc, and 3,4-diOH-PhAc, respectively) through the 2,5-dihydroxyphenylacetate (homogentisic acid) catabolic pathway. Using cDNA subtraction techniques, we isolated a gene, denoted phacB, which is strongly induced by PhAc (and its hydroxyderivatives) and encodes a new cytochrome P450 (CYP450). A disrupted phacB strain (ΔphacB) does not grow on 3-hydroxy-, 4-hydroxy-, or 3,4-dihydroxy-PhAc. High-performance liquid chromatography and gas chromatography-mass spectrum analyses of in vitro reactions using microsomes from wild-type and several A. nidulans mutant strains confirmed that the phacB-encoded CYP450 catalyzes 3-hydroxyphenylacetate and 3,4-dihydroxyphenylacetate 6-hydroxylations to generate 2,5-dihydroxyphenylacetate and 2,4,5-trihydroxyphenylacetate, respectively. Both of these compounds are used as substrates by homogentisate dioxygenase. This cytochrome P450 protein also uses PhAc as a substrate to generate 2-OH-PhAc with a very low efficiency. The phacB gene is the first member of a new CYP450 subfamily (CYP504B). PMID:17189487

  19. Oxidation of Methyl and Ethyl Nitrosamines by Cytochromes P450 2E1 and 2B1

    PubMed Central

    Chowdhury, Goutam; Calcutt, M. Wade; Nagy, Leslie D.; Guengerich, F. Peter

    2012-01-01

    Cytochrome P450 (P450) 2E1 is the major enzyme that oxidizes N-nitrosodimethylamine (N,N-dimethylnitrosamine, DMN), a carcinogen and also a representative of some nitrosamines formed endogenously. Oxidation of DMN by rat or human P450 2E1 to HCHO showed a high apparent intrinsic kinetic deuterium isotope effect (KIE), ≥ 8. The KIE was not attenuated in non-competitive intermolecular experiments with rat liver microsomes (DV 12.5, D(V/K) 10.9, nomenclature of Northrop, D.B. (1982) Methods Enzymol. 87, 607–625) but was with purified human P450 2E1 (DV 3.3, D(V/K) 3.7), indicating that C-H bond breaking is partially rate-limiting with human P450 2E1. With N-nitrosodiethylamine (N,N-diethylnitrosamine, DEN), the intrinsic KIE was slightly lower and was not expressed (e.g., D(V/K) 1.2) in non-competitive intermolecular experiments. The same general pattern of KIEs was also seen in the D(V/K) results with DMN and DEN for the minor products resulting from the denitrosation reactions (CH3NH2, CH3CH2NH2, and NO2−). Experiments with deuterated N-nitroso-N-methyl,N-ethylamine demonstrated that the lower KIEs associated for ethyl compared to methyl oxidation could be distinguished within a single molecule. P450 2E1 oxidized DMN and DEN to aldehydes and then to the carboxylic acids. No kinetic lags were observed in acid formation; pulse-chase experiments with carrier aldehydes showed only limited equilibration with P450 2E1-bound aldehydes, indicative of processive reactions, as reported for P450 2A6 (Chowdhury, G. et al. (2010) J. Biol. Chem. 285, 8031–8044). These same features (no lag phase for HCO2H formation, lack of equilibration in pulse-chase assays) were also seen with (rat) P450 2B1, which has lower catalytic efficiency for DMN oxidation and a larger active site. Thus, the processivity of dialkylnitrosamine oxidation appears to be shared by a number of P450s. PMID:23186213

  20. Crystallization and preliminary X-ray analysis of the NADPH-dependent 3-quinuclidinone reductase from Rhodotorula rubra

    PubMed Central

    Takeshita, Daijiro; Kataoka, Michihiko; Miyakawa, Takuya; Miyazono, Ken-ichi; Uzura, Atsuko; Nagata, Koji; Shimizu, Sakayu; Tanokura, Masaru

    2009-01-01

    (R)-3-Quinuclidinol is a useful compound that is applicable to the synthesis of various pharmaceuticals. The NADPH-dependent carbonyl reductase 3-­quinuclidinone reductase from Rhodotorula rubra catalyzes the stereospecific reduction of 3-quinuclidinone to (R)-3-quinuclidinol and is expected to be utilized in industrial production of this alcohol. 3-Quinuclidinone reductase from R. rubra was expressed in Escherichia coli and purified using Ni-affinity and ion-exchange column chromatography. Crystals of the protein were obtained by the sitting-drop vapour-diffusion method using PEG 8000 as the precipitant. The crystals belonged to space group P41212, with unit-cell parameters a = b = 91.3, c = 265.4 Å, and diffracted X-rays to 2.2 Å resolution. The asymmetric unit contained four molecules of the protein and the solvent content was 48.4%. PMID:19478454

  1. Crystallization and Preliminary X-ray Analysis of Allene Oxide Synthase, Cytochrome P450 CYP74A2, from Parthenium argentatum

    USDA-ARS?s Scientific Manuscript database

    Oxylipins are oxygenated derivatives of fatty acids and pivotal signaling molecules in plants and animals. Allene oxide synthase (AOS) is a key cytochrome P450 CYP74 enzyme involved in the biosynthesis of plant oxylipin jasmonates to convert 13(S)-hydroperoxide to allene oxide. Guayule (Parthenium a...

  2. The CYP88A cytochrome P450, ent-kaurenoic acid oxidase, catalyzes three steps of the gibberellin biosynthesis pathway

    PubMed Central

    Helliwell, Chris A.; Chandler, Peter M.; Poole, Andrew; Dennis, Elizabeth S.; Peacock, W. James

    2001-01-01

    We have shown that ent-kaurenoic acid oxidase, a member of the CYP88A subfamily of cytochrome P450 enzymes, catalyzes the three steps of the gibberellin biosynthetic pathway from ent-kaurenoic acid to GA12. A gibberellin-responsive barley mutant, grd5, accumulates ent-kaurenoic acid in developing grains. Three independent grd5 mutants contain mutations in a gene encoding a member of the CYP88A subfamily of cytochrome P450 enzymes, defined by the maize Dwarf3 protein. Mutation of the Dwarf3 gene gives rise to a gibberellin-responsive dwarf phenotype, but the lesion in the gibberellin biosynthesis pathway has not been identified. Arabidopsis thaliana has two CYP88A genes, both of which are expressed. Yeast strains expressing cDNAs encoding each of the two Arabidopsis and the barley CYP88A enzymes catalyze the three steps of the GA biosynthesis pathway from ent-kaurenoic acid to GA12. Sequence comparison suggests that the maize Dwarf3 locus also encodes ent-kaurenoic acid oxidase. PMID:11172076

  3. Biotransformation of Hexahydro-1,3,5-trinitro-1,3,5-triazine Catalyzed by a NAD(P)H: Nitrate Oxidoreductase from Aspergillus niger

    DTIC Science & Technology

    2002-01-01

    Biotransformation of Hexahydro-1,3,5-trinitro-1,3,5-triazine Catalyzed by a NAD(P)H: Nitrate Oxidoreductase from Aspergillus niger B H A R A T B H U...reductase from Aspergillus niger catalyzed the biotransformation of RDX most effectively at pH 7.0 and 30 °C under anaerobic conditions using NADPH as...nitroreductase. We selected a nitrate reductase (EC 1.6.6.2) from a fungus Aspergillus niger to transform RDX under anaerobic condi- tions because nitrate

  4. Simultaneous pharmacokinetics evaluation of human cytochrome P450 probes, caffeine, warfarin, omeprazole, metoprolol and midazolam, in common marmosets (Callithrix jacchus).

    PubMed

    Uehara, Shotaro; Inoue, Takashi; Utoh, Masahiro; Toda, Akiko; Shimizu, Makiko; Uno, Yasuhiro; Sasaki, Erika; Yamazaki, Hiroshi

    2016-01-01

    1. Pharmacokinetics of human cytochrome P450 probes (caffeine, racemic warfarin, omeprazole, metoprolol and midazolam) composite, after single intravenous and oral administrations at doses of 0.20 and 1.0 mg kg(-1), respectively, to four male common marmosets were investigated. 2. The plasma concentrations of caffeine and warfarin decreased slowly in a monophasic manner but those of omeprazole, metoprolol and midazolam decreased extensively after intravenous and oral administrations, in a manner that approximated those as reported for pharmacokinetics in humans. 3. Bioavailabilities were ∼100% for caffeine and warfarin, but <25% for omeprazole and metoprolol. Bioavailability of midazolam was 4% in marmosets, presumably because of contribution of marmoset P450 3A4 expressed in small intestine and liver, with a high catalytic efficiency for midazolam 1'-hydroxylation as evident in the recombinant system. 4. These results suggest that common marmosets, despite their rapid clearance of some human P450 probe substrates, could be an experimental model for humans and that marmoset P450s have functional characteristics that differ from those of human and/or cynomolgus monkey P450s in some aspects, indicating their importance in modeling in P450-dependent drug metabolism studies in marmosets and of further studies.

  5. An in vitro evaluation of cytochrome P450 inhibition and P-glycoprotein interaction with goldenseal, Ginkgo biloba, grape seed, milk thistle, and ginseng extracts and their constituents.

    PubMed

    Etheridge, Amy S; Black, Sherry R; Patel, Purvi R; So, James; Mathews, James M

    2007-07-01

    Drug-herb interactions can result from the modulation of the activities of cytochrome P450 (P450) and/or drug transporters. The effect of extracts and individual constituents of goldenseal, Ginkgo biloba (and its hydrolyzate), grape seed, milk thistle, and ginseng on the activities of cytochrome P450 enzymes CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 in human liver microsomes were determined using enzyme-selective probe substrates, and their effect on human P-glycoprotein (Pgp) was determined using a baculovirus expression system by measuring the verapamil-stimulated, vanadate-sensitive ATPase activity. Extracts were analyzed by HPLC to standardize their concentration(s) of constituents associated with the pharmacological activity, and to allow comparison of their effects on P450 and Pgp with literature values. Many of the extracts/constituents exerted > or = 50 % inhibition of P450 activity. These include those from goldenseal (normalized to alkaloid content) inhibiting CYP2C8, CYP2D6, and CYP3A4 at 20 microM, ginkgo inhibiting CYP2C8 at 10 microM, grape seed inhibiting CYP2C9 and CYP3A4 at 10 microM, milk thistle inhibiting CYP2C8 at 10 microM, and ginsenosides F1 and Rh1 (but not ginseng extract) inhibiting CYP3A4 at 10 microM. Goldenseal extracts/constituents (20 microM, particularly hydrastine) and ginsenoside Rh1 stimulated ATPase at about half of the activity of the model substrate, verapamil (20 microM). The data suggest that the clearance of a variety of drugs may be diminished by concomitant use of these herbs via inhibition of P450 enzymes, but less so by Pgp-mediated effects.

  6. INDUCTION AND POST-TRANSCRIPTIONAL SUPPRESSION OF HEPATIC CYTOCHROME P450 1A1 BY 3,3',4,4'-TETRACHLOROBIPHENYL. (R827102)

    EPA Science Inventory

    Abstract

    <p>3,3',4,4'-Tetrachlorobiphenyl (TCB) can induce and inhibit cytochrome P450 1A1 (CYP1A1) in vertebrates. TCB may also suppress CYP1A1 protein levels, but the mechanism is unknown. This study examined transcriptional and translational aspects of hepatic ...

  7. Iron(IV)hydroxide pK(a) and the role of thiolate ligation in C-H bond activation by cytochrome P450.

    PubMed

    Yosca, Timothy H; Rittle, Jonathan; Krest, Courtney M; Onderko, Elizabeth L; Silakov, Alexey; Calixto, Julio C; Behan, Rachel K; Green, Michael T

    2013-11-15

    Cytochrome P450 enzymes activate oxygen at heme iron centers to oxidize relatively inert substrate carbon-hydrogen bonds. Cysteine thiolate coordination to iron is posited to increase the pK(a) (where K(a) is the acid dissociation constant) of compound II, an iron(IV)hydroxide complex, correspondingly lowering the one-electron reduction potential of compound I, the active catalytic intermediate, and decreasing the driving force for deleterious auto-oxidation of tyrosine and tryptophan residues in the enzyme's framework. Here, we report on the preparation of an iron(IV)hydroxide complex in a P450 enzyme (CYP158) in ≥90% yield. Using rapid mixing technologies in conjunction with Mössbauer, ultraviolet/visible, and x-ray absorption spectroscopies, we determine a pK(a) value for this compound of 11.9. Marcus theory analysis indicates that this elevated pK(a) results in a >10,000-fold reduction in the rate constant for oxidations of the protein framework, making these processes noncompetitive with substrate oxidation.

  8. [Peroxide modification of membranes and isomorphic composition of cytochrome P-450 of rat liver microsomes during antioxidant deficiency].

    PubMed

    Gubskiy, Iu I; Paramonova, G I; Boldeskul, A E; Primak, R G; Bogdanova, L A; Zadorina, O V; Litvinova, N V

    1992-01-01

    Lipid peroxidation (LPO), physico-chemical properties of the membranes and isoformic composition of microsomal cytochrome P-450 from the rat liver were studied under conditions of antioxidant insufficiency (AOI) which was modelled by exclusion of alpha-tocopherol from the animals' ration. An insignificant accumulation of microsomal diene conjugates and schiff bases against a sharp increase of the ability to the prooxidant stimulated LPO in vitro took place. A significant decrease of membrane lipid microviscosity and a change in surface properties of microsomal membranes of rats with AOI was determined. Absence of alpha-tocopherol in the ration was accompanied by a significant change in the content of separate isoforms of cytochrome P-450 exhibited in growth of a polypeptide with m. w. 54 kDa and the lowering of proteins with m. w. 48 and 50 kDa. Less intensive quenching of tryptophan fluorescence by acrylamide was also revealed, which testified to a lower accessibility of the quencher to membrane proteins or their fluorophore sites. Modification of lipid composition and of physicochemical properties of the rat liver membrane microsomes which was observed at AOI was significantly correlated by pretreatment with the antioxidant 4-methyl-2,6-ditretbutylphenol (ionol).

  9. Summary of Information on the Effects of Ionizing and Non-ionizing Radiation on Cytochrome P450 and Other Drug Metabolizing Enzymes and Transporters

    PubMed Central

    Rendic, Slobodan; Guengerich, F. Peter

    2014-01-01

    The present paper is an update of data on the effects of ionizing radiation (γ-rays, X-rays, high energy UV, fast neutron) caused by environmental pollution or clinical treatments and the effects of non-ionizing radiation (low energy UV) on the expression and/or activity of drug metabolism (e.g., cytochrome P450,, glutathione transferase), enzymes involved in oxidative stress (e.g., peroxidases, catalase,, aconitase, superoxide dismutase), and transporters. The data are presented in tabular form (Tables 1–3) and are a continuation of previously published summaries on the effects of drugs and other chemicals on cytochrome P450 enzymes (Rendic, S.; Di Carlo, F. Drug Metab. Rev., 1997, 29 (1–2), 413–580, Rendic, S. Drug Metab. Rev., 2002, 34 (1–2), 83–448) and of the data on the effects of diseases and environmental factors on the expression and/or activity of human cytochrome P450 enzymes and transporters (Guengerich, F.P.; Rendic, S. Curr. Drug Metab., 2010, 11(1), 1–3, Rendic, S.; Guengerich, F.P. Curr. Drug Metab., 2010, 11 (1), 4–84). The collective information is as presented by the cited author(s) in cases where several references are cited the latest published information is included. Remarks and conclusions suggesting clinically important impacts are highlighted, followed by discussion of the major findings. The searchable database is available as an Excel file (for information about file availability contact the corresponding author). PMID:22571481

  10. Cytochrome P-450 isoforms involved in carboxylic acid ester cleavage of Hantzsch pyridine ester of pranidipine.

    PubMed

    Kudo, S; Okumura, H; Miyamoto, G; Ishizaki, T

    1999-02-01

    Cytochrome P-450 (CYP) isoforms responsible for the cleavage of Hantzsch pyridine ester at the 3-position of pranidipine were studied in vitro using cDNA-expressed human CYP enzymes. CYP1A1, 1A2, 2D6, and 3A4 cleaved the ester with a catalytic activity of 5.5, 0. 93, 13.1, and 22.4 nmol/30 min/nmol P-450, respectively. CYP2A6, 2B6, 2C8, 2C9, 2C19, and 2E1 were not involved in the de-esterification. The Km and Vmax values for the de-esterification were 11.8 microM and 0.47 nmol/min/nmol P-450 in the CYP2D6-catalyzed reaction and 8. 7 microM and 0.84 nmol/min/nmol P-450 in the CYP3A4-catalyzed reaction. The intrinsic clearance (Vmax/Km) of the de-esterification by CYP3A4 was 2-fold greater than that by CYP2D6. Quinidine almost completely inhibited the CYP2D6-mediated de-esterification at the concentration of 1 x 10(-6) M. Ketoconazole and troleandomycin inhibited the CYP3A4-mediated reaction in a dose-related manner. The results indicate that although the multiple CYP isoforms can catalyze the de-esterification, CYP3A4 and 2D6 are the major isoforms.

  11. Cytochrome P450 binding studies of novel tacrine derivatives: Predicting the risk of hepatotoxicity.

    PubMed

    McEneny-King, Alanna; Osman, Wesseem; Edginton, Andrea N; Rao, Praveen P N

    2017-06-01

    The 1,2,3,4-tetrahydroacridine derivative tacrine was the first drug approved to treat Alzheimer's disease (AD). It is known to act as a potent cholinesterase inhibitor. However, tacrine was removed from the market due to its hepatotoxicity concerns as it undergoes metabolism to toxic quinonemethide species through the cytochrome P450 enzyme CYP1A2. Despite these challenges, tacrine serves as a useful template in the development of novel multi-targeting anti-AD agents. In this regard, we sought to evaluate the risk of hepatotoxicity in a series of C9 substituted tacrine derivatives that exhibit cholinesterase inhibition properties. The hepatotoxic potential of tacrine derivatives was evaluated using recombinant cytochrome (CYP) P450 CYP1A2 and CYP3A4 enzymes. Molecular docking studies were conducted to predict their binding modes and potential risk of forming hepatotoxic metabolites. Tacrine derivatives compound 1 (N-(3,4-dimethoxybenzyl)-1,2,3,4-tetrahydroacridin-9-amine) and 2 (6-chloro-N-(3,4-dimethoxybenzyl)-1,2,3,4-tetrahydroacridin-9-amine) which possess a C9 3,4-dimethoxybenzylamino substituent exhibited weak binding to CYP1A2 enzyme (1, IC 50 =33.0µM; 2, IC 50 =8.5µM) compared to tacrine (CYP1A2 IC 50 =1.5µM). Modeling studies show that the presence of a bulky 3,4-dimethoxybenzylamino C9 substituent prevents the orientation of the 1,2,3,4-tetrahydroacridine ring close to the heme-iron center of CYP1A2 thereby reducing the risk of forming hepatotoxic species. Crown Copyright © 2017. Published by Elsevier Ltd. All rights reserved.

  12. Does Compound I Vary Significantly between Isoforms of Cytochrome P450?

    PubMed Central

    2011-01-01

    The cytochrome P450 (CYP) enzymes are important in many areas, including pharmaceutical development. Subtle changes in the electronic structure of the active species, Compound I, have been postulated previously to account partly for the experimentally observed differences in reactivity between isoforms. Current predictive models of CYP metabolism typically assume an identical Compound I in all isoforms. Here we present a method to calculate the electronic structure and to estimate the Fe–O bond enthalpy of Compound I, and apply it to several human and bacterial CYP isoforms. Conformational flexibility is accounted for by sampling large numbers of structures from molecular dynamics simulations, which are subsequently optimized with density functional theory (B3LYP) based quantum mechanics/molecular mechanics. The observed differences in Compound I between human isoforms are small: They are generally smaller than the spread of values obtained for the same isoform starting from different initial structures. Hence, it is unlikely that the variation in activity between human isoforms is due to differences in the electronic structure of Compound I. A larger difference in electronic structure is observed between the human isoforms and P450cam and may be explained by the slightly different hydrogen-bonding environment surrounding the cysteinyl sulfur. The presence of substrate in the active site of all isoforms studied appears to cause a slight decrease in the Fe–O bond enthalpy, apparently due to displacement of water out of the active site, suggesting that Compound I is less stable in the presence of substrate. PMID:21863858

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

    PubMed

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

    2011-06-01

    The study describes cytochrome P450 1A1 (CYPA1) expression in the skin of different cetacean species (Megaptera novaeangliae, n=15; Stenella attenuata, n=7 and Stenella longirostris, n=24) from the Mozambique Channel island of Mayotte. Immunohistochemical examination was performed with a monoclonal antibody against scup cytochrome CYPA1. The sex was determined using a molecular approach consisting in the genotyping sex-specific genes. CYPA1 was detected at the junction between epidermis and blubber on dolphins only, mostly in the endothelial cells. Similar observation was obtained in the dermis of one M. novaeangliae. Immunohistochemical slides were scored to evaluate the expression of the CYPA1 and a higher expression was observed in S. longirostris, suggesting a higher exposure to pollutants for this species. The difference of expression between sexes was not significant. Copyright © 2011 Elsevier Ltd. All rights reserved.

  14. The effects of milk thistle (Silybum marianum) on human cytochrome P450 activity.

    PubMed

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

    2014-10-01

    Milk thistle (Silybum marianum) extracts are widely used as a complementary and alternative treatment of various hepatic conditions and a host of other diseases/disorders. The active constituents of milk thistle supplements are believed to be the flavonolignans contained within the extracts. In vitro studies have suggested that some milk thistle components may significantly inhibit specific cytochrome P450 (P450) enzymes. However, determining the potential for clinically significant drug interactions with milk thistle products has been complicated by inconsistencies between in vitro and in vivo study results. The aim of the present study was to determine the effect of a standardized milk thistle supplement on major P450 drug-metabolizing enzymes after a 14-day exposure period. CYP1A2, CYP2C9, CYP2D6, and CYP3A4/5 activities were measured by simultaneously administering the four probe drugs, caffeine, tolbutamide, dextromethorphan, and midazolam, to nine healthy volunteers before and after exposure to a standardized milk thistle extract given thrice daily for 14 days. The three most abundant falvonolignans found in plasma, following exposure to milk thistle extracts, were silybin A, silybin B, and isosilybin B. The concentrations of these three major constituents were individually measured in study subjects as potential perpetrators. The peak concentrations and areas under the time-concentration curves of the four probe drugs were determined with the milk thistle administration. Exposure to milk thistle extract produced no significant influence on CYP1A2, CYP2C9, CYP2D6, or CYP3A4/5 activities. Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.

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

    PubMed

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

    2014-09-01

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

  16. CYP101J2, CYP101J3, and CYP101J4, 1,8-Cineole-Hydroxylating Cytochrome P450 Monooxygenases from Sphingobium yanoikuyae Strain B2

    PubMed Central

    Unterweger, Birgit; Bulach, Dieter M.; Scoble, Judith; Midgley, David J.; Greenfield, Paul; Lyras, Dena; Johanesen, Priscilla

    2016-01-01

    ABSTRACT We report the isolation and characterization of three new cytochrome P450 monooxygenases: CYP101J2, CYP101J3, and CYP101J4. These P450s were derived from Sphingobium yanoikuyae B2, a strain that was isolated from activated sludge based on its ability to fully mineralize 1,8-cineole. Genome sequencing of this strain in combination with purification of native 1,8-cineole-binding proteins enabled identification of 1,8-cineole-binding P450s. The P450 enzymes were cloned, heterologously expressed (N-terminally His6 tagged) in Escherichia coli BL21(DE3), purified, and spectroscopically characterized. Recombinant whole-cell biotransformation in E. coli demonstrated that all three P450s hydroxylate 1,8-cineole using electron transport partners from E. coli to yield a product putatively identified as (1S)-2α-hydroxy-1,8-cineole or (1R)-6α-hydroxy-1,8-cineole. The new P450s belong to the CYP101 family and share 47% and 44% identity with other 1,8-cineole-hydroxylating members found in Novosphingobium aromaticivorans and Pseudomonas putida. Compared to P450cin (CYP176A1), a 1,8-cineole-hydroxylating P450 from Citrobacter braakii, these enzymes share less than 30% amino acid sequence identity and hydroxylate 1,8-cineole in a different orientation. Expansion of the enzyme toolbox for modification of 1,8-cineole creates a starting point for use of hydroxylated derivatives in a range of industrial applications. IMPORTANCE CYP101J2, CYP101J3, and CYP101J4 are cytochrome P450 monooxygenases from S. yanoikuyae B2 that hydroxylate the monoterpenoid 1,8-cineole. These enzymes not only play an important role in microbial degradation of this plant-based chemical but also provide an interesting route to synthesize oxygenated 1,8-cineole derivatives for applications as natural flavor and fragrance precursors or incorporation into polymers. The P450 cytochromes also provide an interesting basis from which to compare other enzymes with a similar function and expand the CYP101

  17. High-yield expression and purification of isotopically labeled cytochrome P450 monooxygenases for solid-state NMR spectroscopy

    PubMed Central

    Rupasinghe, Sanjeewa G.; Duan, Hui; Frericks Schmidt, Heather L.; Berthold, Deborah A.; Rienstra, Chad M.; Schuler, Mary A.

    2008-01-01

    Cytochrome P450 monooxygenases (P450s), which represent the major group of drug metabolizing enzymes in humans, also catalyze important synthetic and detoxicative reactions in insects, plants and many microbes. Flexibilities in their catalytic sites and membrane associations are thought to play central roles in substrate binding and catalytic specificity. To date, E. coli expression strategies for structural analysis of eukaryotic membrane-bound P450s by X-ray crystallography have necessitated full or partial removal of their N-terminal signal anchor domain (SAD) and, often, replacement of residues more peripherally associated with the membrane (such as the F-G loop region). Even with these modifications, investigations of P450 structural flexibility remain challenging with multiple single crystal conditions needed to identify spatial variations between substrate-free and different substrate-bound forms. To overcome these limitations, we have developed methods for the efficient expression of 13C- and 15N-labeled P450s and analysis of their structures by magic-angle spinning solid-state NMR (SSNMR) spectroscopy. In the presence of co-expressed GroEL and GroES chaperones, full-length (53 kDa) Arabidopsis 13C,15N-labeled CYP98A3 is expressed at yields of 2–4 mg per liter of minimal media without the necessity of generating side chain modifications or N-terminal deletions. Precipitated CYP98A3 generates high quality SSNMR spectra consistent with a homogeneous, folded protein. These data highlight the potential of these methodologies to contribute to the structural analysis of membrane-bound proteins. PMID:18005930

  18. The diheme cytochrome c4 from Vibrio cholerae is a natural electron donor to the respiratory cbb3 oxygen reductase

    PubMed Central

    Chang, Hsin-Yang; Ahn, Young; Pace, Laura A.; Lin, Myat T.; Lin, Yun-Hui; Gennis, Robert B.

    2010-01-01

    The respiratory chain of Vibrio cholerae contains three bd-type quinol oxygen reductases as well as one cbb3 oxygen reductase. The cbb3 oxygen reductase has been previously isolated and characterized, however the natural mobile electron donor(s) which shuttles electrons between the bc1 complex and the cbb3 oxygen reductase is not known. The most likely candidates are the diheme cytochrome c4 and mono-heme cytochrome c5, which have been previously shown to be present in the periplasm of aerobically grown cultures of V. cholerae. Both cytochromes c4 and c5 from V. cholerae have been cloned and expressed heterologously in E. coli. It is shown that reduced cytochrome c4 is a substrate for the purified cbb3 oxygen reductase and can support steady state oxygen reductase activity of at least 300 e−1/s. In contrast, reduced cytochrome c5 is not a good substrate for the cbb3 oxygen reductase. Surprisingly, the dependence of the oxygen reductase activity on the concentration of cytochrome c4 does not exhibit saturation. Global spectroscopic analysis of the time course of the oxidation of cytochrome c4 indicates that the apparent lack of saturation is due to the strong dependence of KM and Vmax on the concentration of oxidized cytochrome c4. Whether this is an artifact of the in vitro assay or has physiological significance remains unknown. Cyclic voltammetry was used to determine that the midpoint potentials of the two hemes in cytochrome c4 are 240 mV and 340 mV (vs SHE), similar to the electrochemical properties of other c4-type cytochromes. Genomic analysis shows a strong correlation between the presence of a c4-type cytochrome and a cbb3 oxygen reductase within the β- and γ- proteobacterial clades, suggesting that cytochrome c4 is the likely natural electron donor to the cbb3 oxygen reductases within these organisms. These would include the β-proteobacteria Neisseria meningitidis and Neisseria gonnorhoeae, in which the cbb3 oxygen reductases are the only terminal

  19. Effect of zolpidem on human cytochrome P450 activity, and on transport mediated by P-glycoprotein.

    PubMed

    von Moltke, Lisa L; Weemhoff, James L; Perloff, Michael D; Hesse, Leah M; Harmatz, Jerold S; Roth-Schechter, Barbara F; Greenblatt, David J

    2002-12-01

    The influence of high concentrations of zolpidem (100 microM, corresponding to approximately 200 times maximum therapeutic concentrations) on the activity of six human Cytochrome P450 (CYP) enzymes was evaluated in a model system using human liver microsomes. Zolpidem produced negligible or weak inhibition of human CYP1A2, 2B6, 2C9, 2C19, 2D6, and 3A. Transport of rhodamine 123, presumed to be mediated mainly by the energy-dependent efflux transport protein P-glycoprotein, was studied in a cell culture system using a human intestinal cell line. High concentrations of zolpidem (100 microM), exceeding the usual therapeutic range by more than 100-fold, produced only modest impairment of rhodamine 123 transport. The findings indicate that zolpidem is very unlikely to cause clinical drug interactions attributable to impairment of CYP activity or P-gp mediated transport. Copyright 2002 John Wiley & Sons, Ltd.

  20. Biallelic mutations in the ferredoxin reductase gene cause novel mitochondriopathy with optic atrophy.

    PubMed

    Peng, Yanyan; Shinde, Deepali N; Valencia, C Alexander; Mo, Jun-Song; Rosenfeld, Jill; Truitt Cho, Megan; Chamberlin, Adam; Li, Zhuo; Liu, Jie; Gui, Baoheng; Brockhage, Rachel; Basinger, Alice; Alvarez-Leon, Brenda; Heydemann, Peter; Magoulas, Pilar L; Lewis, Andrea M; Scaglia, Fernando; Gril, Solange; Chong, Shuk Ching; Bower, Matthew; Monaghan, Kristin G; Willaert, Rebecca; Plona, Maria-Renee; Dineen, Rich; Milan, Francisca; Hoganson, George; Powis, Zoe; Helbig, Katherine L; Keller-Ramey, Jennifer; Harris, Belinda; Anderson, Laura C; Green, Torrian; Sukoff Rizzo, Stacey J; Kaylor, Julie; Chen, Jiani; Guan, Min-Xin; Sellars, Elizabeth; Sparagana, Steven P; Gibson, James B; Reinholdt, Laura G; Tang, Sha; Huang, Taosheng

    2017-12-15

    Iron-sulfur (Fe-S) clusters are ubiquitous cofactors essential to various cellular processes, including mitochondrial respiration, DNA repair, and iron homeostasis. A steadily increasing number of disorders are being associated with disrupted biogenesis of Fe-S clusters. Here, we conducted whole-exome sequencing of patients with optic atrophy and other neurological signs of mitochondriopathy and identified 17 individuals from 13 unrelated families with recessive mutations in FDXR, encoding the mitochondrial membrane-associated flavoprotein ferrodoxin reductase required for electron transport from NADPH to cytochrome P450. In vitro enzymatic assays in patient fibroblast cells showed deficient ferredoxin NADP reductase activity and mitochondrial dysfunction evidenced by low oxygen consumption rates (OCRs), complex activities, ATP production and increased reactive oxygen species (ROS). Such defects were rescued by overexpression of wild-type FDXR. Moreover, we found that mice carrying a spontaneous mutation allelic to the most common mutation found in patients displayed progressive gait abnormalities and vision loss, in addition to biochemical defects consistent with the major clinical features of the disease. Taken together, these data provide the first demonstration that germline, hypomorphic mutations in FDXR cause a novel mitochondriopathy and optic atrophy in humans. © The Author 2017. Published by Oxford University Press.

  1. Cytochrome P450 and Lipoxygenase Metabolites on Renal Function

    PubMed Central

    Imig, John D.; Hye Khan, Md. Abdul

    2018-01-01

    Arachidonic acid metabolites have a myriad of biological actions including effects on the kidney to alter renal hemodynamics and tubular transport processes. Cyclooxygenase metabolites are products of an arachidonic acid enzymatic pathway that has been extensively studied in regards to renal function. Two lesser-known enzymatic pathways of arachidonic acid metabolism are the lipoxygenase (LO) and cytochrome P450 (CYP) pathways. The importance of LO and CYP metabolites to renal hemodynamics and tubular transport processes is now being recognized. LO and CYP metabolites have actions to alter renal blood flow and glomerular filtration rate. Proximal and distal tubular sodium transport and fluid and electrolyte homeostasis are also significantly influenced by renal CYP and LO levels. Metabolites of the LO and CYP pathways also have renal actions that influence renal inflammation, proliferation, and apoptotic processes at vascular and epithelial cells. These renal LO and CYP pathway actions occur through generation of specific metabolites and cell-signaling mechanisms. Even though the renal physiological importance and actions for LO and CYP metabolites are readily apparent, major gaps remain in our understanding of these lipid mediators to renal function. Future studies will be needed to fill these major gaps regarding LO and CYP metabolites on renal function. PMID:26756638

  2. Binding Specificity Determines the Cytochrome P450 3A4 Mediated Enantioselective Metabolism of Metconazole.

    PubMed

    Zhuang, Shulin; Zhang, Leili; Zhan, Tingjie; Lu, Liping; Zhao, Lu; Wang, Haifei; Morrone, Joseph A; Liu, Weiping; Zhou, Ruhong

    2018-01-25

    Cytochrome P450 3A4 (CYP3A4) is a promiscuous enzyme, mediating the biotransformations of ∼50% of clinically used drugs, many of which are chiral molecules. Probing the interactions between CYP3A4 and chiral chemicals is thus essential for the elucidation of molecular mechanisms of enantioselective metabolism. We developed a stepwise-restrained-molecular-dynamics (MD) method to model human CYP3A4 in a complex with cis-metconazole (MEZ) isomers and performed conventional MD simulations with a total simulation time of 2.2 μs to probe the molecular interactions. Our current study, which employs a combined experimental and theoretical approach, reports for the first time on the distinct conformational changes of CYP3A4 that are induced by the enantioselective binding of cis-MEZ enantiomers. CYP3A4 preferably metabolizes cis-RS MEZ over the cis-SR isomer, with the resultant enantiomer fraction for cis-MEZ increasing rapidly from 0.5 to 0.82. cis-RS MEZ adopts a more extended structure in the active pocket with its Cl atom exposed to the solvent, whereas cis-SR MEZ sits within the hydrophobic core of the active pocket. Free-energy-perturbation calculations indicate that unfavorable van der Waals interactions between the cis-MEZ isomers and the CYP3A4 binding pocket predominantly contribute to their binding-affinity differences. These results demonstrate that binding specificity determines the cytochrome P450 3A4 mediated enantioselective metabolism of cis-MEZ.

  3. Identification of human cytochrome P450 2D6 as major enzyme involved in the O-demethylation of the designer drug p-methoxymethamphetamine.

    PubMed

    Staack, Roland F; Theobald, Denis S; Paul, Liane D; Springer, Dietmar; Kraemer, Thomas; Maurer, Hans H

    2004-04-01

    p-Methoxymethamphetamine (PMMA) is a new designer drug, listed in many countries as a controlled substance. Several fatalities have been attributed to the abuse of this designer drug. Previous in vivo studies using Wistar rats had shown that PMMA was metabolized mainly by O-demethylation. The aim of the study presented here was to identify the human hepatic cytochrome P450 (P450) enzymes involved in the biotransformation of PMMA to p-hydroxymethamphetamine. Baculovirus-infected insect cell microsomes, pooled human liver microsomes (pHLMs), and CYP2D6 poor-metabolizer genotype human liver microsomes (PM HLMs) were used for this purpose. Only CYP2D6 catalyzed O-demethylation. The apparent K(m) and V(max) values in baculovirus-infected insect cell microsomes were 4.6 +/- 1.0 microM and 92.0 +/- 3.7 pmol/min/pmol P450, respectively, and 42.0 +/- 4.0 microM and 412.5 +/- 10.8 pmol/min/mg protein in pHLMs. Inhibition studies with 1 microM quinidine showed significant inhibition of the metabolite formation (67.2 +/- 0.6%; p < 0.0001), and comparison of the metabolite formation between pHLMs and PM HLMs revealed significantly lower metabolite formation in the incubations with PM HLMs (87.3 +/- 1.1%; p < 0.0001). According to these studies, CYP2D6 is the major P450 involved in O-demethylation of PMMA.

  4. N-Hydroxylation of dapsone by multiple enzymes of cytochrome P450: implications for inhibition of haemotoxicity.

    PubMed Central

    Gill, H J; Tingle, M D; Park, B K

    1995-01-01

    1. The adverse reactions associated with the administration of dapsone are believed to be caused by metabolism to its hydroxylamine. Previous reports suggest that CYP3A4 is responsible for this biotransformation [1]. 2. Data presented in this paper illustrate the involvement of more than one cytochrome P450 enzyme in dapsone hydroxylamine formation using human liver microsomes. Eadie-Hofstee plots demonstrated bi-phasic kinetics in several livers. No correlation could be established between hydroxylamine formation and CYP3A concentrations in six human livers (r = -0.47; P = 0.34). 3. Studies with low molecular weight inhibitors illustrate the importance of CYP2C9 and CYP3A in dapsone N-hydroxylation. 4. Differential sensitivity of dapsone N-hydroxylation to selective CYP inhibitors indicated that the contribution of individual CYP enzymes varies between livers. Selective inhibition ranged from 6.8 to 44.1% by 5 microM ketoconazole, and from 24.0 to 68.4% by 100 microM sulphaphenazole. The extent of inhibition, by either ketoconazole or sulphaphenazole was dependent on the CYP3A content of the liver. 5. The levels of expression of these cytochrome P450 enzymes may be an important determinant of individual susceptibility to the toxic effects of dapsone, and may influence the ability of an enzyme inhibitor to block dapsone toxicity in vivo. Because of the inability to produce complete inhibition, selective CYP inhibitors are unlikely to offer any clinical advantage over cimetidine in decreasing dapsone hydroxylamine formation in vivo. PMID:8703658

  5. In vitro metabolism of nobiletin, a polymethoxy-flavonoid, by human liver microsomes and cytochrome P450.

    PubMed

    Koga, Nobuyuki; Ohta, Chiho; Kato, Yoshihisa; Haraguchi, Koichi; Endo, Tetsuya; Ogawa, Kazunori; Ohta, Hideaki; Yano, Masamichi

    2011-11-01

    Cytochrome P450 enzymes (CYPs) in the liver metabolize drugs prior to excretion, with different enzymes acting at different molecular motifs. At present, the human CYPs responsible for the metabolism of the flavonoid, nobiletin (NBL), are unidentified. We investigated which enzymes were involved using human liver microsomes and 12 cDNA-expressed human CYPs. Human liver microsomes metabolized NBL to three mono-demethylated metabolites (4'-OH-, 7-OH- and 6-OH-NBL) with a relative ratio of 1:4.1:0.5, respectively, by aerobic incubation with nicotinamide adenine dinucleotide phosphate (NADPH). Of 12 human CYPs, CYP1A1, CYP1A2 and CYP1B1 showed high activity for the formation of 4'-OH-NBL. CYP3A4 catalyzed the formation of 7-OH-NBL with the highest activity and of 6-OH-NBL with lower activity. CYP3A5 also catalyzed the formation of both metabolites but considerably more slowly than CYP3A4. In contrast, seven CYPs (CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP2E1) were inactive for NBL. Both ketoconazole and troleandomycin (CYP3A inhibitors) almost completely inhibited the formation of 7-OH- and 6-OH-NBL. Similarly, α-naphthoflavone (CYP1A1 inhibitor) and furafylline (CYP1A2 inhibitor) significantly decreased the formation of 4'-OH-NBL. These results suggest that CYP1A2 and CYP3A4 are the key enzymes in human liver mediating the oxidative demethylation of NBL in the B-ring and A-ring, respectively.

  6. Intermediates in the reaction of substrate-free cytochrome P450cam with peroxy acetic acid.

    PubMed

    Schünemann, V; Jung, C; Trautwein, A X; Mandon, D; Weiss, R

    2000-08-18

    Freeze-quenched intermediates of substrate-free cytochrome 57Fe-P450(cam) in reaction with peroxy acetic acid as oxidizing agent have been characterized by EPR and Mossbauer spectroscopy. After 8 ms of reaction time the reaction mixture consists of approximately 90% of ferric low-spin iron with g-factors and hyperfine parameters of the starting material; the remaining approximately 10% are identified as a free radical (S' = 1/2) by its EPR and as an iron(IV) (S= 1) species by its Mossbauer signature. After 5 min of reaction time the intermediates have disappeared and the Mossbauer and EPR-spectra exhibit 100% of the starting material. We note that the spin-Hamiltonian analysis of the spectra of the 8 ms reactant clearly reveals that the two paramagnetic species, e.g. the ferryl (iron(IV)) species and the radical, are not exchanged coupled. This led to the conclusion that under the conditions used, peroxy acetic acid oxidized a tyrosine residue (probably Tyr-96) into a tyrosine radical (Tyr*-96), and the iron(III) center of substrate-free P450(cam) to iron(IV).

  7. Human reductive halothane metabolism in vitro is catalyzed by cytochrome P450 2A6 and 3A4.

    PubMed

    Spracklin, D K; Thummel, K E; Kharasch, E D

    1996-09-01

    The anesthetic halothane undergoes extensive oxidative and reductive biotransformation, resulting in metabolites that cause hepatotoxicity. Halothane is reduced anaerobically by cytochrome P450 (P450) to the volatile metabolites 2-chloro-1,1-difluoroethene (CDE) and 2-chloro-1,1,1-trifluoroethane (CTE). The purpose of this investigation was to identify the human P450 isoform(s) responsible for reductive halothane metabolism. CDE and CTE formation from halothane metabolism by human liver microsomes was determined by GC/MS analysis. Halothane metabolism to CDE and CTE under reductive conditions was completely inhibited by carbon monoxide, which implicates exclusively P450 in this reaction. Eadie-Hofstee plots of both CDE and CTE formation were nonlinear, suggesting multiple P450 isoform involvement. Microsomal CDE and CTE formation were each inhibited 40-50% by P450 2A6-selective inhibitors (coumarin and 8-methoxypsoralen) and 55-60% by P450 3A4-selective inhibitors (ketoconazole and troleandomycin). P450 1A-, 2B6-, 2C9/10-, and 2D6-selective inhibitors (7,8-benzoflavone, furafylline, orphenadrine, sulfaphenazole, and quinidine) had no significant effect on reductive halothane metabolism. Measurement of product formation catalyzed by a panel of cDNA-expressed P450 isoforms revealed that maximal rates of CDE formation occurred with P450 2A6, followed by P450 3A4. P450 3A4 was the most effective catalyst of CTE formation. Among a panel of 11 different human livers, there were significant linear correlations between the rate of CDE formation and both 2A6 activity (r = 0.64, p < 0.04) and 3A4 activity (r = 0.64, p < 0.03). Similarly, there were significant linear correlations between CTE formation and both 2A6 activity (r = 0.55, p < 0.08) and 3A4 activity (r = 0.77, p < 0.005). The P450 2E1 inhibitors 4-methylpyrazole and diethyldithiocarbamate inhibited CDE and CTE formation by 20-45% and 40-50%, respectively; however, cDNA-expressed P450 2E1 did not catalyze

  8. Characterization of human cytochrome P450s involved in the bioactivation of tri-ortho-cresyl phosphate (ToCP).

    PubMed

    Reinen, Jelle; Nematollahi, Leyla; Fidder, Alex; Vermeulen, Nico P E; Noort, Daan; Commandeur, Jan N M

    2015-04-20

    Tri-ortho-cresyl phosphate (ToCP) is a multipurpose organophosphorus compound that is neurotoxic and suspected to be involved in aerotoxic syndrome in humans. It has been reported that not ToCP itself but a metabolite of ToCP, namely, 2-(ortho-cresyl)-4H-1,2,3-benzodioxaphosphoran-2-one (CBDP), may be responsible for this effect as it can irreversibly bind to human butyrylcholinesterase (BuChE) and human acetylcholinesterase (AChE). The bioactivation of ToCP into CBDP involves Cytochrome P450s (P450s). However, the individual human P450s responsible for this bioactivation have not been identified yet. In the present study, we aimed to investigate the metabolism of ToCP by different P450s and to determine the inhibitory effect of the in vitro generated ToCP-metabolites on human BuChE and AChE. Human liver microsomes, rat liver microsomes, and recombinant human P450s were used for that purpose. The recombinant P450s 2B6, 2C18, 2D6, 3A4 and 3A5 showed highest activity of ToCP-bioactivation to BuChE-inhibitory metabolites. Inhibition experiments using pooled human liver microsomes indicated that P450 3A4 and 3A5 were mainly involved in human hepatic bioactivation of ToCP. In addition, these experiments indicated a minor role for P450 1A2. Formation of CBDP by in-house expressed recombinant human P450s 1A2 and 3A4 was proven by both LC-MS and GC-MS analysis. When ToCP was incubated with P450 1A2 and 3A4 in the presence of human BuChE, CBDP-BuChE-adducts were detected by LC-MS/MS which were not present in the corresponding control incubations. These results confirmed the role of human P450s 1A2 and 3A4 in ToCP metabolism and demonstrated that CBDP is the metabolite responsible for the BuChE inactivation. Interindividual differences at the level of P450 1A2 and 3A4 might play an important role in the susceptibility of humans in developing neurotoxic effects, such as aerotoxic syndrome, after exposure to ToCP.

  9. Biosynthesis of Sandalwood Oil: Santalum album CYP76F cytochromes P450 produce santalols and bergamotol.

    PubMed

    Diaz-Chavez, Maria L; Moniodis, Jessie; Madilao, Lufiani L; Jancsik, Sharon; Keeling, Christopher I; Barbour, Elizabeth L; Ghisalberti, Emilio L; Plummer, Julie A; Jones, Christopher G; Bohlmann, Jörg

    2013-01-01

    Sandalwood oil is one of the world's most highly prized essential oils, appearing in many high-end perfumes and fragrances. Extracted from the mature heartwood of several Santalum species, sandalwood oil is comprised mainly of sesquiterpene olefins and alcohols. Four sesquiterpenols, α-, β-, and epi-β-santalol and α-exo-bergamotol, make up approximately 90% of the oil of Santalum album. These compounds are the hydroxylated analogues of α-, β-, and epi-β-santalene and α-exo-bergamotene. By mining a transcriptome database of S. album for candidate cytochrome P450 genes, we cloned and characterized cDNAs encoding a small family of ten cytochrome P450-dependent monooxygenases annotated as SaCYP76F37v1, SaCYP76F37v2, SaCYP76F38v1, SaCYP76F38v2, SaCYP76F39v1, SaCYP76F39v2, SaCYP76F40, SaCYP76F41, SaCYP76F42, and SaCYP76F43. Nine of these genes were functionally characterized using in vitro assays and yeast in vivo assays to encode santalene/bergamotene oxidases and bergamotene oxidases. These results provide a foundation for production of sandalwood oil for the fragrance industry by means of metabolic engineering, as demonstrated with proof-of-concept formation of santalols and bergamotol in engineered yeast cells, simultaneously addressing conservation challenges by reducing pressure on supply of sandalwood from native forests.

  10. Identification and cloning of an NADPH-dependent hydroxycinnamoyl-CoA double bond reductase involved in dihydrochalcone formation in Malus×domestica Borkh.

    PubMed

    Ibdah, Mwafaq; Berim, Anna; Martens, Stefan; Valderrama, Andrea Lorena Herrera; Palmieri, Luisa; Lewinsohn, Efraim; Gang, David R

    2014-11-01

    The apple tree (Malus sp.) is an agriculturally and economically important source of food and beverages. Many of the health beneficial properties of apples are due to (poly)phenolic metabolites that they contain, including various dihydrochalcones. Although many of the genes and enzymes involved in polyphenol biosynthesis are known in many plant species, the specific reactions that lead to the biosynthesis of the dihydrochalcone precursor, p-dihydrocoumaroyl-CoA (3), are unknown. To identify genes involved in the synthesis of these metabolites, existing genome databases of the Rosaceae were screened for apple genes with significant sequence similarity to Arabidopsis alkenal double bond reductases. Herein described are the isolation and characterization of a Malus hydroxycinnamoyl-CoA double bond reductase, which catalyzed the NADPH-dependent reduction of p-coumaroyl-CoA and feruloyl-CoA to p-dihydrocoumaroyl-CoA and dihydroferuloyl-CoA, respectively. Its apparent Km values for p-coumaroyl-CoA, feruloyl-CoA and NADPH were 96.6, 92.9 and 101.3μM, respectively. The Malus double bond reductase preferred feruloyl-CoA to p-coumaroyl-CoA as a substrate by a factor of 2.1 when comparing catalytic efficiencies in vitro. Expression analysis of the hydroxycinnamoyl-CoA double bond reductase gene revealed that its transcript levels showed significant variation in tissues of different developmental stages, but was expressed when expected for involvement in dihydrochalcone formation. Thus, the hydroxycinnamoyl-CoA double bond reductase appears to be responsible for the reduction of the α,β-unsaturated double bond of p-coumaroyl-CoA, the first step of dihydrochalcone biosynthesis in apple tissues, and may be involved in the production of these compounds. Copyright © 2014 Elsevier Ltd. All rights reserved.

  11. Expansion of cytochrome P450 and cathepsin genes in the generalist herbivore brown marmorated stink bug.

    PubMed

    Bansal, Raman; Michel, Andy

    2018-01-18

    The brown marmorated stink bug (Halyomorpha halys) is an invasive pest in North America which causes severe economic losses on tree fruits, ornamentals, vegetables, and field crops. The H. halys is an extreme generalist and this feeding behaviour may have been a major contributor behind its establishment and successful adaptation in invasive habitats of North America. To develop an understanding into the mechanism of H. halys' generalist herbivory, here we specifically focused on genes putatively facilitating its adaptation on diverse host plants. We generated over 142 million reads via sequencing eight RNA-Seq libraries, each representing an individual H. halys adult. The de novo assembly contained 79,855 high quality transcripts, totalling 39,600,178 bases. Following a comprehensive transcriptome analysis, H. halys had an expanded suite of cytochrome P450 and cathepsin-L genes compared to other insects. Detailed characterization of P450 genes from the CYP6 family, known for herbivore adaptation on host plants, strongly hinted towards H. halys-specific expansions involving gene duplications. In subsequent RT-PCR experiments, both P450 and cathepsin genes exhibited tissue-specific or distinct expression patterns which supported their principal roles of detoxification and/or digestion in a particular tissue. Our analysis into P450 and cathepsin genes in H. halys offers new insights into potential mechanisms for understanding generalist herbivory and adaptation success in invasive habitats. Additionally, the large-scale transcriptomic resource developed here provides highly useful data for gene discovery; functional, population and comparative genomics as well as efforts to assemble and annotate the H. halys genome.

  12. Crude oil exposure results in oxidative stress-mediated dysfunctional development and reproduction in the copepod Tigriopus japonicus and modulates expression of cytochrome P450 (CYP) genes.

    PubMed

    Han, Jeonghoon; Won, Eun-Ji; Hwang, Dae-Sik; Shin, Kyung-Hoon; Lee, Yong Sung; Leung, Kenneth Mei-Yee; Lee, Su-Jae; Lee, Jae-Seong

    2014-07-01

    In this study, we investigated the effects of the water-accommodated fraction (WAF) of crude oil on the development and reproduction of the intertidal copepod Tigriopus japonicus through life-cycle experiments. Furthermore, we investigated the mechanisms underlying the toxic effects of WAF on this benthic organism by studying expression patterns of cytochrome P450 (CYP) genes. Development of T. japonicus was delayed and molting was interrupted in response to WAF exposure. Hatching rate was also significantly reduced in response to WAF exposure. Activities of antioxidant enzymes such as glutathione S-transferase (GST), glutathione reductase (GR), and catalase (CAT) were increased by WAF exposure in a concentration-dependent manner. These results indicated that WAF exposure resulted in oxidative stress, which in turn was associated with dysfunctional development and reproduction. To evaluate the involvement of cytochrome P450 (CYP) genes, we cloned the entire repertoire of CYP genes in T. japonicus (n=52) and found that the CYP genes belonged to five different clans (i.e., Clans 2, 3, 4, mitochondrial, and 20). We then examined expression patterns of these 52 CYP genes in response to WAF exposure. Three TJ-CYP genes (CYP3024A2, CYP3024A3, and CYP3027C2) belonging to CYP clan 3 were significantly induced by WAF exposure in a time- and concentration-dependent manner. We identified aryl hydrocarbon responsive elements (AhRE), xenobiotic responsive elements (XREs), and metal response elements (MRE) in the promoter regions of these three CYP genes, suggesting that these genes are involved in detoxification of toxicants. Overall, our results indicate that WAF can trigger oxidative stress and thus induce dysfunctional development and reproduction in the copepod T. japonicus. Furthermore, we identified three TJ-CYP genes that represent potential biomarkers of oil pollution. Copyright © 2014 Elsevier B.V. All rights reserved.

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

    PubMed

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

    2010-12-01

    This study aimed to investigate the biotransformation of cat liver microsomes in comparison to dogs and humans using a high throughput method with fluorescent substrates and classical inhibitors specific for certain isozymes of the human cytochrome P450 (CYP) enzyme family. The metabolic activities associated with CYP1A, CYP2B, CYP2C, CYP2D, CYP2E and CYP3A were measured. Cat liver microsomes metabolized all substrates selected for the assessment of cytochrome P450 activity. The activities associated with CYP3A and CYP2B were higher than the activities of the other measured CYPs. Substrate selectivity could be demonstrated by inhibition studies with α-naphthoflavone (CYP1A), tranylcypromine/quercetine (CYP2C), quinidine (CYP2D), diethyldithiocarbamic acid (CYP2E) and ketoconazole (CYP3A) respectively. Other prototypical inhibitors used for characterization of human CYP activities such as furafylline (CYP1A), tranylcypromine (CYP2B) and sulfaphenazole (CYP2C) did not show significant effects in cat and dog liver microsomes. Moreover, IC50-values of cat CYPs differed from dog and human CYPs underlining the interspecies differences. Gender differences were observed in the oxidation of 7-ethoxy-4-trifluoromethylcoumarin (CYP2B) and 3-[2-(N, N-diethyl-N-methylamino)ethyl]-7-methoxy-4-methylcoumarin (CYP2D), which were significantly higher in male cats than in females. Conversely, oxidation of the substrates dibenzylfluorescein (CYP2C) and 7-methoxy-4-trifluoromethylcoumarin (CYP2E) showed significant higher activities in females than in male cats. Overall CYP-activities in cat liver microsomes were lower than in those from dogs or humans, except for CYP2B. The presented difference between feline and canine CYP-activities are useful to establish dose corrections for feline patients of intensively metabolized drugs licensed for dogs or humans. © 2010 Blackwell Publishing Ltd.

  14. Active sites of two orthologous cytochromes P450 2E1: Differences revealed by spectroscopic methods

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Anzenbacherova, Eva; Hudecek, Jiri; Murgida, Daniel

    2005-12-09

    Cytochromes P450 2E1 of human and minipig origin were examined by absorption spectroscopy under high hydrostatic pressure and by resonance Raman spectroscopy. Human enzyme tends to denature to the P420 form more easily than the minipig form; moreover, the apparent compressibility of the heme active site (as judged from a redshift of the absorption maximum with pressure) is greater than that of the minipig counterpart. Relative compactness of the minipig enzyme is also seen in the Raman spectra, where the presence of planar heme conformation was inferred from band positions characteristic of the low-spin heme with high degree of symmetry.more » In this respect, the CYP2E1 seems to be another example of P450 conformational heterogeneity as shown, e.g., by Davydov et al. for CYP3A4 [Biochem. Biophys. Res. Commun. 312 (2003) 121-130]. The results indicate that the flexibility of the CYP active site is likely one of its basic structural characteristics.« less

  15. Influence of cytochrome P450 oxidoreductase genetic polymorphisms on CYP1A2 activity and inducibility by smoking.

    PubMed

    Dobrinas, Maria; Cornuz, Jacques; Pedrido, Leticia; Eap, Chin B

    2012-02-01

    Cytochrome P4501A2 (CYP1A2) presents a high interindividual variability in its activity and also in its inducibility by smoking. Cytochrome P450 oxidoreductase (POR) is an electron transfer protein that catalyzes the activity of several cytochromes P450. We aimed to study the influence of POR genetic polymorphisms on CYP1A2 activity while smoking and after smoking cessation, as well as on CYP1A2 inducibility. CYP1A2 activity was determined by the paraxanthine/caffeine ratio in 184 smokers and in 113 of these smokers who were abstinent during a 4-week period. Participants were genotyped for POR rs17148944G>A, rs10239977C>T, rs3815455C>T, rs2286823G>A, rs2302429G>A, and rs1057868C>T (POR*28) polymorphisms. While smoking, none of the tested POR polymorphisms showed a significant influence on CYP1A2 activity. After smoking cessation, significantly higher CYP1A2 activity was found in POR rs2302429A carriers (P=0.038) and in carriers of rs17148944G-rs10239977C-rs3815455T-rs2286823G-rs2302429A-rs1057868T haplotype (P=0.038), whereas carriers of POR rs2286823A (P=0.031) and of the rs17148944G-rs10239977C-rs3815455C-rs2286823A-rs2302429G-rs1057868C haplotype (P=0.031) had decreased CYP1A2 activity. In the complete regression model, only POR rs2302429G>A showed a significant effect (P=0.017). No influence of POR genotypes or haplotypes was observed on the inducibility of CYP1A2. POR genetic polymorphisms influence CYP1A2 basal but not induced activity and do not seem to influence CYP1A2 inducibility. Future work is warranted to identify other clinical and genetic factors that may explain the variability in CYP1A2 activity and inducibility by smoking.

  16. Effect of p-amino-diphenyl ethers on hepatic microsomal cytochrome P450.

    PubMed

    Jiang, Huidi; Xuan, Guida

    2003-09-01

    The present paper aims to investigate whether p-amino-2',4'-dichlorodiphenyl ether and p-amino-4'-methyldiphenyl ether are inhibitors as well as inducers of P450. Mice were given daily intraperitoneal (ip) injections of p-amino-2',4'-dichlorodiphenyl ether (0.25 mmol/kg) or p-amino-4'-methyldiphenyl ether (0.25 mmol/kg) for 4 days and tested at 24 h and 48 h after the last dose injection. The results showed the mice pentobarbital sleeping time was shorter and the P450 content of hepatic microsome increased significantly in the group pretreated with p-amino-4'-methyldiphenyl ether when compared with the control group, while in mice pretreated with p-amino-2',4'-dichlorodiphenyl ether the hepatic microsome P450 content increased but the pentobarbital sleeping time was extended in clear contrast to the control group. The sleeping time of the phenobarbital group (80 mg/kg daily ip injection for 4 days) was shortened at 24 h after the last injection with increased P450 content of hepatic microsome, but it showed no difference at 48 h. The zoxazolamine-paralysis times of mice treated with p-amino-2',4'-dichlorodiphenyl ether were longer than those of the control mice, while the same dose of zoxazolamine did not lead to paralysis in mice pretreated with BNF. p-Amino-2',4'-dichlorodiphenyl ether and p-amino-4'-methyldiphenyl ether inhibited the activity of 7-ethoxyresorufin O-deethylase from rat hepatic microsome induced by BNF in vitro by 70.0% and 50.1% respectively. These results suggest that p-amino-2',4'-dichlorodiphenyl ether and p-amino-4'-methyldiphenyl ether are inhibitors as well as inducers of P450.

  17. Marmoset Cytochrome P450 3A4 Ortholog Expressed in Liver and Small-Intestine Tissues Efficiently Metabolizes Midazolam, Alprazolam, Nifedipine, and Testosterone.

    PubMed

    Uehara, Shotaro; Uno, Yasuhiro; Nakanishi, Kazuyuki; Ishii, Sakura; Inoue, Takashi; Sasaki, Erika; Yamazaki, Hiroshi

    2017-05-01

    Common marmosets ( Callithrix jacchus ), small New World primates, are increasingly attracting attention as potentially useful animal models for drug development. However, characterization of cytochrome P450 (P450) 3A enzymes involved in the metabolism of a wide variety of drugs has not investigated in marmosets. In this study, sequence homology, tissue distribution, and enzymatic properties of marmoset P450 3A4 ortholog, 3A5 ortholog, and 3A90 were investigated. Marmoset P450 3A forms exhibited high amino acid sequence identities (88-90%) to the human and cynomolgus monkey P450 3A orthologs and evolutionary closeness to human and cynomolgus monkey P450 3A orthologs compared with other P450 3A enzymes. Among the five marmoset tissues examined, P450 3A4 ortholog mRNA was abundant in livers and small intestines where P450 3A4 ortholog proteins were immunologically detected. Three marmoset P450 3A proteins heterologously expressed in Escherichia coli membranes catalyzed midazolam 1'- and 4-hydroxylation, alprazolam 4-hydroxylation, nifedipine oxidation, and testosterone 6 β -hydroxylation, similar to cynomolgus monkey and human P450 3A enzymes. Among the marmoset P450 3A enzymes, P450 3A4 ortholog effectively catalyzed midazolam 1'-hydroxylation, comparable to microsomes from marmoset livers and small intestines. Correlation analyses with 23 individual marmoset liver microsomes suggested contributions of P450 3A enzymes to 1'-hydroxylation of both midazolam (human P450 3A probe) and bufuralol (human P450 2D6 probe), similar to cynomolgus monkey P450 3A enzymes. These results indicated that marmoset P450 3A forms had functional characteristics roughly similar to cynomolgus monkeys and humans in terms of tissue expression patterns and catalytic activities, suggesting marmosets as suitable animal models for P450 3A-dependent drug metabolism. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

  18. P450 AND METABOLISM IN TOXICOLOGY

    EPA Science Inventory

    The cytochromes P450 catalyze the initial phase of detoxification of many environmental chemicals, xenobiotic, drugs and the secondary metabolic product of plants. Plant secondary chemicals can be highly toxic, and they evolved in a coevolving plant - animal warfare - the plants ...

  19. Hydrogen Peroxide Inhibits Cytochrome P450 Epoxygenases

    PubMed Central

    Larsen, Brandon T.; Gutterman, David D.; Sato, Atsushi; Toyama, Kazuyoshi; Campbell, William B.; Zeldin, Darryl C.; Manthati, Vijay L.; Falck, John R.; Miura, Hiroto

    2008-01-01

    The cytochrome P450 epoxygenase (CYP)-derived metabolites of arachidonic acid the epoxyeicosatrienoic acids (EETs) and hydrogen peroxide (H2O2) both function as endothelium-derived hyperpolarizing factors (EDHFs) in the human coronary microcirculation. However, the relative importance of and potential interactions between these 2 vasodilators remain unexplored. We identified a novel inhibitory interaction between CYPs and H2O2 in human coronary arterioles, where EDHF-mediated vasodilatory mechanisms are prominent. Bradykinin induced vascular superoxide and H2O2 production in an endothelium-dependent manner and elicited a concentration-dependent dilation that was reduced by catalase but not by 14,15-epoxyeicosa-5(Z)-enoic acid (EEZE), 6-(2-propargyloxyphenyl)hexanoic acid, sulfaphenazole, or iberiotoxin. However, in the presence of catalase, an inhibitory effect of these compounds was unmasked. In a tandem-bioassay preparation, application of bradykinin to endothelium-intact donor vessels elicited dilation of downstream endothelium-denuded detectors that was partially inhibited by donor-applied catalase but not by detector-applied EEZE; however, EEZE significantly inhibited dilation in the presence of catalase. EET production by human recombinant CYP 2C9 and 2J2, 2 major epoxygenase isozymes expressed in human coronary arterioles, was directly inhibited in a concentration-dependent fashion by H2O2 in vitro, as observed by high-performance liquid chromatography (HPLC); however, EETs were not directly sensitive to oxidative modification. H2O2 inhibited dilation to arachidonic acid but not to 11,12-EET. These findings suggest that an inhibitory interaction exists between 2 EDHFs in the human coronary microcirculation. CYP epoxygenases are directly inhibited by H2O2, and this interaction may modulate vascular EET bioavailability. PMID:17975109

  20. 21-Aminosteroids prevent the down-regulation of hepatic cytochrome P450 induced by hypoxia and inflammation in conscious rabbits

    PubMed Central

    Galal, Ahmed; du Souich, Patrick

    1999-01-01

    This study was conducted to assess whether a 21-aminosteroid, U74389G, could prevent the down-regulation of hepatic cytochrome P450 (P450) induced by acute moderate hypoxia or an inflammatory reaction.The rabbits of two groups (n=6 per group) were subjected to acute moderate hypoxia (PaO2≈35 mmHg), one pre-treated with U74389G (3 mg kg−1 i.v. every 6 h, for 48 h). The rabbits of two other groups received 5 ml of turpentine s.c., one of them being pre-treated with U74389G (3 mg kg−1 i.v. every 6 h, for 72 h). The kinetics of theophylline (2.5 mg kg−1) were assessed to evaluate the activity of the P450. Once the rabbits were sacrificed, the P450 content and the amount of thiobarbituric acid reactive substances (TBARS), a marker of lipid peroxidation, were estimated in the liver.Compared with control rabbits, hypoxia and inflammation increased theophylline plasma concentrations, as a result of a decrease in theophylline systemic clearance (P<0.05). Both experimental conditions reduced hepatic content of P450 by 40–50% (P<0.05) and increased the amount of hepatic TBARS by around 50% (P<0.05). Pre-treatment with U74389G prevented the hypoxia- and inflammation-induced decrease in theophylline systemic clearance, the down-regulation of hepatic P450, and the increase in liver TBARS.It is concluded that in the rabbit, U74389G prevents hepatic P450 depression produced by acute moderate hypoxia and a turpentine-induced inflammatory reaction, possibly by eliciting a radical quenching antioxidant activity. PMID:10510447

  1. Polycyclic aromatic hydrocarbons and cytochrome P450 in HIV pathogenesis

    PubMed Central

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

    2015-01-01

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

  2. Preliminary characterization of a murine model for 1-bromopropane neurotoxicity: Role of cytochrome P450.

    PubMed

    Zong, Cai; Garner, C Edwin; Huang, Chinyen; Zhang, Xiao; Zhang, Lingyi; Chang, Jie; Toyokuni, Shinya; Ito, Hidenori; Kato, Masashi; Sakurai, Toshihiro; Ichihara, Sahoko; Ichihara, Gaku

    2016-09-06

    Neurotoxicity of 1-bromopropane (1-BP) has been reported in both human cases and animal studies. To date, neurotoxicity of 1-BP has been induced in rats but not in mice due to the lethal hepatotoxicity of 1-BP. Oxidization by cytochromes P450 and conjugation with glutathione (GSH) are two critical metabolism pathways of 1-BP and play important roles in toxicity of 1-BP. The aim of the present study was to establish a murine model of 1-BP neurotoxicity, by reducing the hepatotoxicity of 1-BP with 1-aminobenzotriazole (1-ABT); a commonly used nonspecific P450s inhibitor. The results showed that subcutaneous or intraperitoneal injection of 1-ABT at 50mg/kg body weight BID (100mg/kg BW/day) for 3days, inhibited about 92-96% of hepatic microsomal CYP2E1 activity, but only inhibited about 62-64% of CYP2E1 activity in brain microsomes. Mice treated with 1-ABT survived even after exposure to 1200ppm 1-BP for 4 weeks and histopathological studies showed that treatment with 1-ABT protected mice from 1-BP-induced hepatic necrosis, hepatocyte degeneration, and hemorrhage. After 4-week exposure to 1-BP, the brain weight of 1-ABT(+)/1200ppm 1-BP group was decreased significantly. In 1-ABT-treated groups, expression of hippocampal Ran protein and cerebral cortical GRP78 was dose-dependently increased by exposure to 1-BP. We conclude that the control of hepatic P450 activity allows the observation of effects of 1-BP on the murine brain at a higher concentration by reduction of hepatotoxicity. The study suggests that further experiments with liver-specific control of P450 activity using gene technology might provide better murine models for 1-bromopropane-induced neurotoxicity. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  3. Differential cytochrome content and reductase activity in Geospirillum barnesii strain SeS3

    USGS Publications Warehouse

    Stolz, J.F.; Gugliuzza, T.; Switzer, Blum J.; Oremland, R.; Martinez, Murillo F.

    1997-01-01

    The protein composition, cytochrome content, and reductase activity in the dissimilatory selenate-reducing bacterium Geospirillum barnesii strain SeS3, grown with thiosulfate, nitrate, selenate, or fumarate as the terminal electron acceptor, was investigated. Comparison of seven high-molecular-mass membrane proteins (105.3, 90.3, 82.6, 70.2, 67.4, 61.1, and 57.3 kDa) by SDS-PAGE showed that their detection was dependent on the terminal electron acceptor used. Membrane fractions from cells grown on thiosulfate contained a 70.2-kDa c-type cytochrome with absorbance maxima at 552, 522, and 421 nm. A 61.1-kDa c-type cytochrome with absorption maxima at 552, 523, and 423 nm was seen in membrane fractions from cells grown on nitrate. No c-type cytochromes were detected in membrane fractions of either selenate- or fumarate-grown cells. Difference spectra, however, revealed the presence of a cytochrome b554 (absorption maxima at 554, 523, and 422 nm) in membrane fractions from selenate-grown cells and a cytochrome b556 (absorption maxima at 556, 520, and 416 nm) in membrane fractions from fumarate-grown cells. Analysis of reductase activity in the different membrane fractions showed variability in substrate specificity. However, enzyme activity was greatest for the substrate on which the cells had been grown (e.g., membranes from nitrate-grown cells exhibited the greatest activity with nitrate). These results show that protein composition, cytochrome content, and reductase activity are dependent on the terminal electron acceptor used for growth.

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

    PubMed Central

    Miao, Yinglong; Baudry, Jerome

    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. PMID:21943431

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

    PubMed Central

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

    2014-01-01

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

  6. Analysis of Cytochrome P450 CYP119 Ligand-dependent Conformational Dynamics by Two-dimensional NMR and X-ray Crystallography*

    PubMed Central

    Basudhar, Debashree; Madrona, Yarrow; Kandel, Sylvie; Lampe, Jed N.; Nishida, Clinton R.; de Montellano, Paul R. Ortiz

    2015-01-01

    Defining the conformational states of cytochrome P450 active sites is critical for the design of agents that minimize drug-drug interactions, the development of isoform-specific P450 inhibitors, and the engineering of novel oxidative catalysts. We used two-dimensional 1H,15N HSQC chemical shift perturbation mapping of 15N-labeled Phe residues and x-ray crystallography to examine the ligand-dependent conformational dynamics of CYP119. Active site Phe residues were most affected by the binding of azole inhibitors and fatty acid substrates, in agreement with active site localization of the conformational changes. This was supported by crystallography, which revealed movement of the F-G loop with various azoles. Nevertheless, the NMR chemical shift perturbations caused by azoles and substrates were distinguishable. The absence of significant chemical shift perturbations with several azoles revealed binding of ligands to an open conformation similar to that of the ligand-free state. In contrast, 4-phenylimidazole caused pronounced NMR changes involving Phe-87, Phe-144, and Phe-153 that support the closed conformation found in the crystal structure. The same closed conformation is observed by NMR and crystallography with a para-fluoro substituent on the 4-phenylimidazole, but a para-chloro or bromo substituent engendered a second closed conformation. An open conformation is thus favored in solution with many azole ligands, but para-substituted phenylimidazoles give rise to two closed conformations that depend on the size of the para-substituent. The results suggest that ligands selectively stabilize discrete cytochrome P450 conformational states. PMID:25670859

  7. Analysis of Cytochrome P450 CYP119 Ligand-dependent Conformational Dynamics by Two-dimensional NMR and X-ray Crystallography

    DOE PAGES

    Basudhar, Debashree; Madrona, Yarrow; Kandel, Sylvie; ...

    2015-02-10

    Defining the conformational states of cytochrome P450 active sites is critical for the design of agents that minimize drug-drug interactions, the development of isoform-specific P450 inhibitors, and the engineering of novel oxidative catalysts. In this paper, we used two-dimensional 1H,15N HSQC chemical shift perturbation mapping of 15N-labeled Phe residues and x-ray crystallography to examine the ligand-dependent conformational dynamics of CYP119. Active site Phe residues were most affected by the binding of azole inhibitors and fatty acid substrates, in agreement with active site localization of the conformational changes. This was supported by crystallography, which revealed movement of the F-G loop withmore » various azoles. Nevertheless, the NMR chemical shift perturbations caused by azoles and substrates were distinguishable. The absence of significant chemical shift perturbations with several azoles revealed binding of ligands to an open conformation similar to that of the ligand-free state. In contrast, 4-phenylimidazole caused pronounced NMR changes involving Phe-87, Phe-144, and Phe-153 that support the closed conformation found in the crystal structure. The same closed conformation is observed by NMR and crystallography with a para-fluoro substituent on the 4-phenylimidazole, but a para-chloro or bromo substituent engendered a second closed conformation. An open conformation is thus favored in solution with many azole ligands, but para-substituted phenylimidazoles give rise to two closed conformations that depend on the size of the para-substituent. Finally, the results suggest that ligands selectively stabilize discrete cytochrome P450 conformational states.« less

  8. Analysis of Cytochrome P450 CYP119 Ligand-dependent Conformational Dynamics by Two-dimensional NMR and X-ray Crystallography

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Basudhar, Debashree; Madrona, Yarrow; Kandel, Sylvie

    Defining the conformational states of cytochrome P450 active sites is critical for the design of agents that minimize drug-drug interactions, the development of isoform-specific P450 inhibitors, and the engineering of novel oxidative catalysts. In this paper, we used two-dimensional 1H,15N HSQC chemical shift perturbation mapping of 15N-labeled Phe residues and x-ray crystallography to examine the ligand-dependent conformational dynamics of CYP119. Active site Phe residues were most affected by the binding of azole inhibitors and fatty acid substrates, in agreement with active site localization of the conformational changes. This was supported by crystallography, which revealed movement of the F-G loop withmore » various azoles. Nevertheless, the NMR chemical shift perturbations caused by azoles and substrates were distinguishable. The absence of significant chemical shift perturbations with several azoles revealed binding of ligands to an open conformation similar to that of the ligand-free state. In contrast, 4-phenylimidazole caused pronounced NMR changes involving Phe-87, Phe-144, and Phe-153 that support the closed conformation found in the crystal structure. The same closed conformation is observed by NMR and crystallography with a para-fluoro substituent on the 4-phenylimidazole, but a para-chloro or bromo substituent engendered a second closed conformation. An open conformation is thus favored in solution with many azole ligands, but para-substituted phenylimidazoles give rise to two closed conformations that depend on the size of the para-substituent. Finally, the results suggest that ligands selectively stabilize discrete cytochrome P450 conformational states.« less

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

    EPA Science Inventory

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

  10. Thermodynamics of interactions between mammalian cytochromes P450 and b5.

    PubMed

    Yablokov, Evgeny; Florinskaya, Anna; Medvedev, Alexei; Sergeev, Gennady; Strushkevich, Natallia; Luschik, Alexander; Shkel, Tatsiana; Haidukevich, Irina; Gilep, Andrei; Usanov, Sergey; Ivanov, Alexis

    2017-04-01

    Cytochromes P450 (CYPs) play an important role in the metabolism of xenobiotics and various endogenous substrates. Being a crucial component of the microsomal monooxygenase system, CYPs are involved in numerous protein-protein interactions. However, mechanisms underlying molecular interactions between components of the monooxygenase system still need better characterization. In this study thermodynamic parameters of paired interactions between mammalian CYPs and cytochromes b5 (CYB5) have been evaluated using a Surface Plasmon Resonance (SPR) based biosensor Biacore 3000. Analysis of 18 pairs of CYB5-CYP complexes formed by nine different isoforms of mammalian CYPs and two isoforms of human CYB5 has shown that thermodynamically these complexes can be subdivided into enthalpy-driven and entropy-driven groups. Formation of the enthalpy-driven complexes was observed in the case of microsomal CYPs allosterically regulated by CYB5 (CYB5A-CYP3A4, CYB5A-CYP3A5, CYB5A-CYP17A1). The entropy-driven complexes were formed when CYB5 had no effect on the CYP activity (CYB5A-CYP51A1, CYB5A-CYP1B1, CYB5B-CYP11A1). Results of this study suggest that such interactions determining protein clustering are indirectly linked to the monooxygenase functioning. Positive ΔH values typical for such interactions may be associated with displacement of the solvation shells of proteins upon clustering. CYB5-CYP complex formation accompanied by allosteric regulation of CYP activity by CYB5 is enthalpy-dependent. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Identification of a novel cytochrome P450 CYP321B1 gene from tobacco cutworm moth (Spodoptera litura) and RNA interference to evaluate its role in commonly used insecticides

    USDA-ARS?s Scientific Manuscript database

    BACKGROUND: Insect cytochrome P450 monooxygenases (CYPs or P450s) play an important role in detoxifying insecticides leading to resistance in insect populations. A polyphagous pest, Spodoptera litura (Lepidoptera, Noctuidae) has been shown to be resistant to a wide range of insecticides. In this stu...

  12. Prediction of Human Cytochrome P450 Inhibition Using a Multitask Deep Autoencoder Neural Network.

    PubMed

    Li, Xiang; Xu, Youjun; Lai, Luhua; Pei, Jianfeng

    2018-05-30

    Adverse side effects of drug-drug interactions induced by human cytochrome P450 (CYP450) inhibition is an important consideration in drug discovery. It is highly desirable to develop computational models that can predict the inhibitive effect of a compound against a specific CYP450 isoform. In this study, we developed a multitask model for concurrent inhibition prediction of five major CYP450 isoforms, namely, 1A2, 2C9, 2C19, 2D6, and 3A4. The model was built by training a multitask autoencoder deep neural network (DNN) on a large dataset containing more than 13 000 compounds, extracted from the PubChem BioAssay Database. We demonstrate that the multitask model gave better prediction results than that of single-task models, previous reported classifiers, and traditional machine learning methods on an average of five prediction tasks. Our multitask DNN model gave average prediction accuracies of 86.4% for the 10-fold cross-validation and 88.7% for the external test datasets. In addition, we built linear regression models to quantify how the other tasks contributed to the prediction difference of a given task between single-task and multitask models, and we explained under what conditions the multitask model will outperform the single-task model, which suggested how to use multitask DNN models more effectively. We applied sensitivity analysis to extract useful knowledge about CYP450 inhibition, which may shed light on the structural features of these isoforms and give hints about how to avoid side effects during drug development. Our models are freely available at http://repharma.pku.edu.cn/deepcyp/home.php or http://www.pkumdl.cn/deepcyp/home.php .

  13. Overcoming heterologous protein interdependency to optimize P450-mediated Taxol precursor synthesis in Escherichia coli

    PubMed Central

    Biggs, Bradley Walters; Lim, Chin Giaw; Sagliani, Kristen; Shankar, Smriti; Stephanopoulos, Gregory; Ajikumar, Parayil Kumaran

    2016-01-01

    Recent advances in metabolic engineering have demonstrated the potential to exploit biological chemistry for the synthesis of complex molecules. Much of the progress to date has leveraged increasingly precise genetic tools to control the transcription and translation of enzymes for superior biosynthetic pathway performance. However, applying these approaches and principles to the synthesis of more complex natural products will require a new set of tools for enabling various classes of metabolic chemistries (i.e., cyclization, oxygenation, glycosylation, and halogenation) in vivo. Of these diverse chemistries, oxygenation is one of the most challenging and pivotal for the synthesis of complex natural products. Here, using Taxol as a model system, we use nature’s favored oxygenase, the cytochrome P450, to perform high-level oxygenation chemistry in Escherichia coli. An unexpected coupling of P450 expression and the expression of upstream pathway enzymes was discovered and identified as a key obstacle for functional oxidative chemistry. By optimizing P450 expression, reductase partner interactions, and N-terminal modifications, we achieved the highest reported titer of oxygenated taxanes (∼570 ± 45 mg/L) in E. coli. Altogether, this study establishes E. coli as a tractable host for P450 chemistry, highlights the potential magnitude of protein interdependency in the context of synthetic biology and metabolic engineering, and points to a promising future for the microbial synthesis of complex chemical entities. PMID:26951651

  14. Overcoming heterologous protein interdependency to optimize P450-mediated Taxol precursor synthesis in Escherichia coli.

    PubMed

    Biggs, Bradley Walters; Lim, Chin Giaw; Sagliani, Kristen; Shankar, Smriti; Stephanopoulos, Gregory; De Mey, Marjan; Ajikumar, Parayil Kumaran

    2016-03-22

    Recent advances in metabolic engineering have demonstrated the potential to exploit biological chemistry for the synthesis of complex molecules. Much of the progress to date has leveraged increasingly precise genetic tools to control the transcription and translation of enzymes for superior biosynthetic pathway performance. However, applying these approaches and principles to the synthesis of more complex natural products will require a new set of tools for enabling various classes of metabolic chemistries (i.e., cyclization, oxygenation, glycosylation, and halogenation) in vivo. Of these diverse chemistries, oxygenation is one of the most challenging and pivotal for the synthesis of complex natural products. Here, using Taxol as a model system, we use nature's favored oxygenase, the cytochrome P450, to perform high-level oxygenation chemistry in Escherichia coli. An unexpected coupling of P450 expression and the expression of upstream pathway enzymes was discovered and identified as a key obstacle for functional oxidative chemistry. By optimizing P450 expression, reductase partner interactions, and N-terminal modifications, we achieved the highest reported titer of oxygenated taxanes (∼570 ± 45 mg/L) in E. coli. Altogether, this study establishes E. coli as a tractable host for P450 chemistry, highlights the potential magnitude of protein interdependency in the context of synthetic biology and metabolic engineering, and points to a promising future for the microbial synthesis of complex chemical entities.

  15. Effects of chronic exposure to tributyltin on tissue-specific cytochrome P450 1 regulation in juvenile common carp.

    PubMed

    Li, Zhi-Hua; Zhong, Li-Qiao; Mu, Wei-Na; Wu, Yan-Hua

    2016-01-01

    1. The purpose of this study was to compare tributyltin (TBT)-induced cytochrome P450 1 (CYP450 1) regulation in liver, gills and muscle of juvenile common carp (Cyprinus carpio). 2. Fish were exposed to sublethal concentrations of TBT (75, 0.75 and 7.5 μg/L) for 60 days. CYP450 1A was measured at the enzyme activity level as 7-ethoxyresorufin-O-deethylase (EROD) activity, as well as the mRNA expression of CYP450 1 family genes (CYP1A, CYP1B, CYP1C1 and CYP1C2) in fish tissues. 3. Based on the results, the liver displayed the highest absolute levels of EROD activity, both under nonexposed and exposed conditions. Additional, EROD activities and CYP1A gene levels showed a good correlation in all three organs. According to the mRNA expression of CYP450 1 family genes, it suggested that CYP1A was to accommodate most EROD activity in fish, but other CYP450 forms also involved in this proceeding. 4. Overall, the study revealed both similarities and differences in the concentration-dependent CYP450 1 responses of the three target organs, which could provide useful information to better understand the mechanisms of TBT-induced bio-toxicity.

  16. Mining of the Uncharacterized Cytochrome P450 Genes Involved in Alkaloid Biosynthesis in California Poppy Using a Draft Genome Sequence

    PubMed Central

    Hori, Kentaro; Yamada, Yasuyuki; Purwanto, Ratmoyo; Minakuchi, Yohei; Toyoda, Atsushi; Hirakawa, Hideki

    2018-01-01

    Abstract Land plants produce specialized low molecular weight metabolites to adapt to various environmental stressors, such as UV radiation, pathogen infection, wounding and animal feeding damage. Due to the large variety of stresses, plants produce various chemicals, particularly plant species-specific alkaloids, through specialized biosynthetic pathways. In this study, using a draft genome sequence and querying known biosynthetic cytochrome P450 (P450) enzyme-encoding genes, we characterized the P450 genes involved in benzylisoquinoline alkaloid (BIA) biosynthesis in California poppy (Eschscholzia californica), as P450s are key enzymes involved in the diversification of specialized metabolism. Our in silico studies showed that all identified enzyme-encoding genes involved in BIA biosynthesis were found in the draft genome sequence of approximately 489 Mb, which covered approximately 97% of the whole genome (502 Mb). Further analyses showed that some P450 families involved in BIA biosynthesis, i.e. the CYP80, CYP82 and CYP719 families, were more enriched in the genome of E. californica than in the genome of Arabidopsis thaliana, a plant that does not produce BIAs. CYP82 family genes were highly abundant, so we measured the expression of CYP82 genes with respect to alkaloid accumulation in different plant tissues and two cell lines whose BIA production differs to estimate the functions of the genes. Further characterization revealed two highly homologous P450s (CYP82P2 and CYP82P3) that exhibited 10-hydroxylase activities with different substrate specificities. Here, we discuss the evolution of the P450 genes and the potential for further genome mining of the genes encoding the enzymes involved in BIA biosynthesis. PMID:29301019

  17. Structural and Functional Characterization of a Cytochrome P450 2B4 F429H Mutant with an Axial Thiolate–Histidine Hydrogen Bond

    PubMed Central

    2015-01-01

    The structural basis of the regulation of microsomal cytochrome P450 (P450) activity was investigated by mutating the highly conserved heme binding motif residue, Phe429, on the proximal side of cytochrome P450 2B4 to a histidine. Spectroscopic, pre-steady-state and steady-state kinetic, thermodynamic, theoretical, and structural studies of the mutant demonstrate that formation of an H-bond between His429 and the unbonded electron pair of the Cys436 axial thiolate significantly alters the properties of the enzyme. The mutant lost >90% of its activity; its redox potential was increased by 87 mV, and the half-life of the oxyferrous mutant was increased ∼37-fold. Single-crystal electronic absorption and resonance Raman spectroscopy demonstrated that the mutant was reduced by a small dose of X-ray photons. The structure revealed that the δN atom of His429 forms an H-bond with the axial Cys436 thiolate whereas the εN atom forms an H-bond with the solvent and the side chain of Gln357. The amide of Gly438 forms the only other H-bond to the tetrahedral thiolate. Theoretical quantification of the histidine–thiolate interaction demonstrates a significant electron withdrawing effect on the heme iron. Comparisons of structures of class I–IV P450s demonstrate that either a phenylalanine or tryptophan is often found at the location corresponding to Phe429. Depending on the structure of the distal pocket heme, the residue at this location may or may not regulate the thermodynamic properties of the P450. Regardless, this residue appears to protect the thiolate from solvent, oxidation, protonations, and other deleterious reactions. PMID:25029089

  18. Cytochrome P450s--Their expression, regulation, and role in insecticide resistance.

    PubMed

    Liu, Nannan; Li, Ming; Gong, Youhui; Liu, Feng; Li, Ting

    2015-05-01

    P450s are known to be critical for the detoxification and/or activation of xenobiotics such as drugs and pesticides and overexpression of P450 genes can significantly affect the disposition of xenobiotics in the tissues of organisms, altering their pharmacological/toxicological effects. In insects, P450s play an important role in detoxifying exogenous compounds such as insecticides and plant toxins and their overexpression can result in increased levels of P450 proteins and P450 activities. This has been associated with enhanced metabolic detoxification of insecticides and has been implicated in the development of insecticide resistance in insects. Multiple P450 genes have been found to be co-overexpressed in individual insect species via several constitutive overexpression and induction mechanisms, which in turn are co-responsible for high levels of insecticide resistance. Many studies have also demonstrated that the transcriptional overexpression of P450 genes in resistant insects is regulated by trans and/or cis regulatory genes/factors. Taken together, these earlier findings suggest not only that insecticide resistance is conferred via multi-resistance P450 genes, but also that it is mediated through the interaction of regulatory genes/factors and resistance genes. This chapter reviews our current understanding of how the molecular mechanisms of P450 interaction/gene regulation govern the development of insecticide resistance in insects and our progress along the road to a comprehensive characterization of P450 detoxification-mediated insecticide resistance. Copyright © 2015 Elsevier Inc. All rights reserved.

  19. Biosynthesis of Sandalwood Oil: Santalum album CYP76F Cytochromes P450 Produce Santalols and Bergamotol

    PubMed Central

    Diaz-Chavez, Maria L.; Moniodis, Jessie; Madilao, Lufiani L.; Jancsik, Sharon; Keeling, Christopher I.; Barbour, Elizabeth L.; Ghisalberti, Emilio L.; Plummer, Julie A.; Jones, Christopher G.; Bohlmann, Jörg

    2013-01-01

    Abstract Sandalwood oil is one of the world’s most highly prized essential oils, appearing in many high-end perfumes and fragrances. Extracted from the mature heartwood of several Santalum species, sandalwood oil is comprised mainly of sesquiterpene olefins and alcohols. Four sesquiterpenols, α-, β-, and epi-β-santalol and α-exo-bergamotol, make up approximately 90% of the oil of Santalum album. These compounds are the hydroxylated analogues of α-, β-, and epi-β-santalene and α-exo-bergamotene. By mining a transcriptome database of S. album for candidate cytochrome P450 genes, we cloned and characterized cDNAs encoding a small family of ten cytochrome P450-dependent monooxygenases annotated as SaCYP76F37v1, SaCYP76F37v2, SaCYP76F38v1, SaCYP76F38v2, SaCYP76F39v1, SaCYP76F39v2, SaCYP76F40, SaCYP76F41, SaCYP76F42, and SaCYP76F43. Nine of these genes were functionally characterized using in vitro assays and yeast in vivo assays to encode santalene/bergamotene oxidases and bergamotene oxidases. These results provide a foundation for production of sandalwood oil for the fragrance industry by means of metabolic engineering, as demonstrated with proof-of-concept formation of santalols and bergamotol in engineered yeast cells, simultaneously addressing conservation challenges by reducing pressure on supply of sandalwood from native forests. PMID:24324844

  20. Emerging roles in plant defense for cis-jasmone-induced cytochrome P450 CYP81D11.

    PubMed

    Matthes, Michaela; Bruce, Toby; Chamberlain, Keith; Pickett, John; Napier, Johnathan

    2011-04-01

    cis-Jasmone is a volatile organic compound emitted constitutively by flowers or leaves of several plant species where it acts as an attractant for pollinators and as a chemical cue for host localisation (or avoidance) for insects. ( 1-3) It is also released by some plant species after feeding damage inflicted by herbivorous insects and in this case might serve as a chemical cue for parasitoids to guide them to their prey (so called "indirect defense"). ( 4,5) Moreover, we have recently shown that plants can perceive cis-jasmone and that it acts as a signaling molecule in A. thaliana, inducing a discrete and distinctive suite of genes, of which a large subset is putatively involved in metabolism and defense responses. ( 6) Cytochrome P450s feature prominently in these functional subsets and of these the highest fold change upon cis-jasmone treatment occurred with the cytochrome CYP81D11 (At3g28740). ( 6) Hence this gene was chosen for a more thorough analysis of the potential biological relevance of the cis-jasmone induced defense response. Although the precise function of CYP81D11 remains to be determined, we could previously demonstrate its involvement in the indirect defense response in Arabidopsis, as plants exposed to cis-jasmone ceased to be attractive to the aphid parasitoid Aphidius ervi when this P450 was inactivated by T-DNA insertion mutagenesis. ( 6) Here we report additional experiments which give further support to a role of CYP81D11 in the direct or indirect defense response of A. thaliana.