RÉSUMÉ
Derivatives of tirapazamine and other heteroaromatic N-oxides (ArNâO) exhibit tumoricidal, antibacterial, and antiprotozoal activities, which are typically attributed to bioreductive activation and free radical generation. In this work, we aimed to clarify the role of NAD(P)H:quinone oxidoreductase (NQO1) in ArNâO aerobic cytotoxicity. We synthesized 9 representatives of ArNâO with uncharacterized redox properties and examined their single-electron reduction by rat NADPH:cytochrome P-450 reductase (P-450R) and Plasmodium falciparum ferredoxin:NADP+ oxidoreductase (PfFNR), and by rat NQO1. NQO1 catalyzed both redox cycling and the formation of stable reduction products of ArNâO. The reactivity of ArNâO in NQO1-catalyzed reactions did not correlate with the geometric average of their activity towards P-450R- and PfFNR, which was taken for the parameter of their redox cycling efficacy. The cytotoxicity of compounds in murine hepatoma MH22a cells was decreased by antioxidants and the inhibitor of NQO1, dicoumarol. The multiparameter regression analysis of the data of this and a previous study (DOI: 10.3390/ijms20184602) shows that the cytotoxicity of ArNâO (n = 18) in MH22a and human colon carcinoma HCT-116 cells increases with the geometric average of their reactivity towards P-450R and PfFNR, and with their reactivity towards NQO1. These data demonstrate that NQO1 is a potentially important target of action of heteroaromatic N-oxides.
Sujet(s)
Antibactériens/pharmacologie , Antioxydants/pharmacologie , Antiprotozoaires/pharmacologie , N-oxydes cycliques/pharmacologie , Ferredoxine-NADP reductase/antagonistes et inhibiteurs , NADPH dehydrogenase (quinone)/antagonistes et inhibiteurs , NADPH-ferrihemoprotéine reductase/antagonistes et inhibiteurs , Aérobiose , Animaux , Antibactériens/synthèse chimique , Antioxydants/synthèse chimique , Antiprotozoaires/synthèse chimique , Lignée cellulaire tumorale , Survie cellulaire/effets des médicaments et des substances chimiques , N-oxydes cycliques/synthèse chimique , Dicoumarol/pharmacologie , Dosages enzymatiques , Antienzymes/pharmacologie , Ferredoxine-NADP reductase/composition chimique , Ferredoxine-NADP reductase/métabolisme , Cellules HCT116 , Hépatocytes/effets des médicaments et des substances chimiques , Hépatocytes/enzymologie , Hépatocytes/anatomopathologie , Humains , Cinétique , Souris , NADPH dehydrogenase (quinone)/composition chimique , NADPH dehydrogenase (quinone)/métabolisme , NADPH-ferrihemoprotéine reductase/composition chimique , NADPH-ferrihemoprotéine reductase/métabolisme , Oxydoréduction , Plasmodium falciparum/composition chimique , Plasmodium falciparum/enzymologie , Protéines de protozoaire/antagonistes et inhibiteurs , Protéines de protozoaire/composition chimique , Protéines de protozoaire/métabolisme , Rats , Tirapazamine/composition chimique , Tirapazamine/pharmacologieRÉSUMÉ
Exposure to polychlorinated biphenyls (PCBs) has been implicated in adverse human health effects, including developmental neurotoxicity. Several neurotoxic PCBs are chiral and undergo atropisomeric enrichment in vivo due to atropselective metabolism by cytochrome P450 enzymes. Here we study how the liver-specific deletion of the cytochrome P450 reductase ( cpr) gene alters the toxicokinetics of 2,2',3,3',6,6'-hexachlorobiphenyl (PCB 136) in mice. Male and female mice with a liver-specific deletion of cpr (KO) and congenic wild-type (WT) mice were exposed to a single oral dose of racemic PCB 136 (6.63 mg/kg). Levels and chiral signatures of PCB 136 and its hydroxylated metabolites were determined 1-48 h after PCB exposure in whole blood. Blood levels of PCB 136 were typically higher in M-WT compared to F-WT mice. At the later time points, F-KO mice had significantly higher PCB 136 levels than F-WT mice. 2,2',3',4,6,6'-Hexachlorobiphenyl-3-ol (3-150), 2,2',3,3',6,6'-hexachlorobiphenyl-4-ol (4-136), 2,2',3,3',6,6'-hexachlorobiphenyl-5-ol (5-136), and 4,5-dihydroxy-2,2',3,3',6,6'-hexachlorobiphenyl (4,5-136) were detected in blood, with 5-136 and 4-136 being major metabolites. At later time points, the sum of HO-PCB (∑HO-PCB) levels exceeded PCB 136 levels in the blood; however, higher ∑HO-PCB than PCB 136 levels were observed later in KO than WT mice. PCB 136 and its major metabolites displayed atropisomeric enrichment in a manner that depended on the time point, sex, and genotype. Toxicokinetic analysis revealed sex and genotype-dependent differences in toxicokinetic parameters for PCB 136 atropisomers and its metabolites. The results suggest that mice with a liver-specific deletion of the cpr gene can potentially be used to assess how an altered metabolism of neurotoxic PCB congeners affects neurotoxic outcomes following exposure of the offspring to PCBs via the maternal diet.
Sujet(s)
Foie/effets des médicaments et des substances chimiques , NADPH-ferrihemoprotéine reductase/antagonistes et inhibiteurs , Polychlorobiphényles/toxicité , Animaux , Cinétique , Foie/métabolisme , Souris , Souris knockout , Structure moléculaire , NADPH-ferrihemoprotéine reductase/déficit , NADPH-ferrihemoprotéine reductase/métabolisme , Polychlorobiphényles/composition chimique , Polychlorobiphényles/métabolismeRÉSUMÉ
Chimerogenesis involving cytochromes P450 is a successful approach to generate catalytically self-sufficient enzymes. However, the connection between the different functional modules should allow a certain degree of flexibility in order to obtain functional and catalytically efficient proteins. We previously applied the molecular Lego approach to develop a chimeric P450 3A4 enzyme linked to the reductase domain of P450 BM3 (BMR). Three constructs were designed with the connecting loop containing no glycine, 3 glycine or 5 glycine residues and showed a different catalytic activity and coupling efficiency. Here we investigate how the linker affects the ability of P450 3A4 to bind substrates and inhibitors. We measure the electron transfer rates and the catalytic properties of the enzyme also in the presence of ketoconazole as inhibitor. The data show that the construct 3A4-5GLY-BMR with the longest loop better retains the binding ability and cooperativity for testosterone, compared to P450 3A4. In both 3A4-3GLY-BMR and 3A4-5GLY-BMR, the substrate induces an increase in the first electron transfer rate and a shorter lag phase related to a domain rearrangements, when compared to the construct without Gly. These data are consistent with docking results and secondary structure predictions showing a propensity to form helical structures in the loop of the 3A4-BMR and 3A4-3GLY-BMR. All three chimeras retain the ability to bind the inhibitor ketoconazole and show an IC50 comparable with those reported for the wild type protein. 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.
Sujet(s)
Bacillus megaterium/génétique , Protéines bactériennes/composition chimique , Inhibiteurs du cytochrome P-450 CYP3A/composition chimique , Cytochrome P-450 CYP3A/composition chimique , Kétoconazole/composition chimique , NADPH-ferrihemoprotéine reductase/composition chimique , Protéines de fusion recombinantes/composition chimique , Bacillus megaterium/enzymologie , Protéines bactériennes/antagonistes et inhibiteurs , Protéines bactériennes/génétique , Protéines bactériennes/métabolisme , Sites de fixation , Cytochrome P-450 CYP3A/génétique , Cytochrome P-450 CYP3A/métabolisme , Inhibiteurs du cytochrome P-450 CYP3A/métabolisme , Expression des gènes , Humains , Kétoconazole/métabolisme , Cinétique , Ligands , Simulation de docking moléculaire , NADPH-ferrihemoprotéine reductase/antagonistes et inhibiteurs , NADPH-ferrihemoprotéine reductase/génétique , NADPH-ferrihemoprotéine reductase/métabolisme , Liaison aux protéines , Structure en hélice alpha , Structure en brin bêta , Ingénierie des protéines , Motifs et domaines d'intéraction protéique , Protéines de fusion recombinantes/génétique , Protéines de fusion recombinantes/métabolisme , Relation structure-activité , Spécificité du substrat , Testostérone/composition chimique , Testostérone/métabolismeRÉSUMÉ
Mammals incorporate a major proportion of absorbed iron as heme, which is catabolized by the heme oxygenase 1 (HO1)-NADPH-cytochrome P450 reductase (CPR) complex into biliverdin, carbon monoxide, and ferrous iron. Moreover, intestinal iron is incorporated as ferrous iron, which is transported via the iron importer, divalent metal transporter 1 (DMT1). Recently, we demonstrated that the iron chaperone poly(rC)-binding protein 2 (PCBP2) can directly receive ferrous iron from DMT1 or transfer iron to the iron exporter, ferroportin 1. To promote intracellular iron flux, an iron chaperone may be essential for receiving iron generated by heme catabolism, but this hypothesis is untested so far. Herein, we demonstrate that HO1 binds to PCBP2, but not to other PCBP family members, namely PCBP1, PCBP3, or PCBP4. Interestingly, HO1 formed a complex with either CPR or PCBP2, and it was demonstrated that PCBP2 competes with CPR for HO1 binding. Using PCBP2-deletion mutants, we demonstrated that the PCBP2 K homology 3 domain is important for the HO1/PCBP2 interaction. In heme-loaded cells, heme prompted HO1-CPR complex formation and decreased the HO1/PCBP2 interaction. Furthermore, in vitro reconstitution experiments with purified recombinant proteins indicated that HO1 could bind to PCBP2 in the presence of heme, whereas loading of PCBP2 with ferrous iron caused PCBP2 to lose its affinity for HO1. These results indicate that ferrous iron released from heme can be bound by PCBP2 and suggest a model for an integrated heme catabolism and iron transport metabolon.
Sujet(s)
Heme oxygenase-1/métabolisme , Hème/métabolisme , Fer/métabolisme , NADPH-ferrihemoprotéine reductase/métabolisme , Protéines de liaison à l'ARN/métabolisme , Substitution d'acide aminé , Sites de fixation , Fixation compétitive , Transport biologique , Lignée cellulaire , Délétion de gène , Heme oxygenase (decyclizing)/composition chimique , Heme oxygenase (decyclizing)/génétique , Heme oxygenase (decyclizing)/métabolisme , Heme oxygenase-1/antagonistes et inhibiteurs , Heme oxygenase-1/composition chimique , Heme oxygenase-1/génétique , Humains , Métalloporphyrines/métabolisme , Mutation , NADPH-ferrihemoprotéine reductase/antagonistes et inhibiteurs , NADPH-ferrihemoprotéine reductase/composition chimique , NADPH-ferrihemoprotéine reductase/génétique , Motifs et domaines d'intéraction protéique , Multimérisation de protéines , Transport des protéines , Interférence par ARN , Protéines de liaison à l'ARN/composition chimique , Protéines de liaison à l'ARN/génétique , Protéines de fusion recombinantes/composition chimique , Protéines de fusion recombinantes/métabolisme , Similitude structurale de protéines , Facteurs de transcription/composition chimique , Facteurs de transcription/génétique , Facteurs de transcription/métabolismeRÉSUMÉ
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.
Sujet(s)
Résistance aux insecticides/génétique , Insecticides/toxicité , Malathion/toxicité , NADPH-ferrihemoprotéine reductase/métabolisme , Tephritidae/effets des médicaments et des substances chimiques , Tephritidae/enzymologie , Animaux , Clonage moléculaire , Cytochromes c/métabolisme , Femelle , Mâle , NADPH-ferrihemoprotéine reductase/antagonistes et inhibiteurs , NADPH-ferrihemoprotéine reductase/génétique , Phylogenèse , Isoformes de protéines/classification , Isoformes de protéines/génétique , Isoformes de protéines/métabolisme , Interférence par ARN , ARN double brin/métabolisme , ARN messager/métabolisme , Analyse de séquence d'ADN , Cellules Sf9 , Spodoptera , TranscriptomeRÉSUMÉ
A complete cDNA encoding the NADPH-cytochrome P450 reductase (haCPR) and its genomic sequence from the cotton bollworm Helicoverpa armigera were cloned and sequenced. The open reading frame of haCPR codes for a protein of 687 amino acid residues with a calculated molecular mass of 77.4kDa. The haCPR gene spans over 11 kb and its coding region is interrupted by 11 introns. haCPR is ubiquitously expressed in various tissues and at various stages of development. Escherichia coli produced haCPR enzyme exhibited catalytic activity for NADPH-dependent reduction of cytochrome c, following Michaelis-Menten kinetics. The functionality of CPR was further demonstrated by its capacity to support cytochrome P450 (e.g. haCYP9A14 and chicken CYP1A5) mediated O-dealkylation activity of alkoxyresorufins. The flavoprotein-specific inhibitor (diphenyleneiodonium chloride, DPI) showed a potent inhibition to haCPR activity (IC50=1.69 µM). Inhibitory effect of secondary metabolites in the host plants (tannic acid, quercetin and gossypol) on CPR activity (with an IC50 value ranged from 15 to 90 µM) was also observed.
Sujet(s)
Papillons de nuit/génétique , NADPH-ferrihemoprotéine reductase/génétique , Séquence d'acides aminés , Animaux , Clonage moléculaire , Activation enzymatique , Antienzymes/pharmacologie , Femelle , Expression des gènes , Ordre des gènes , Concentration inhibitrice 50 , Cinétique , Mâle , Données de séquences moléculaires , Papillons de nuit/enzymologie , NADPH-ferrihemoprotéine reductase/antagonistes et inhibiteurs , NADPH-ferrihemoprotéine reductase/composition chimique , NADPH-ferrihemoprotéine reductase/métabolisme , Protéines recombinantes/génétique , Protéines recombinantes/isolement et purification , Protéines recombinantes/métabolisme , Alignement de séquences , Analyse de séquence d'ADN , Spécificité du substratRÉSUMÉ
Fluorescence-based assays for the cytochrome P450 BM3 monooxygenase from Bacillus megaterium address an attractive biotechnological challenge by facilitating enzyme engineering and the identification of potential substrates of this highly promising biocatalyst. In the current study, we used the scarcity of corresponding screening systems as an opportunity to evaluate a novel and continuous high-throughput assay for this unique enzyme. A set of nine catalytically diverse P450 BM3 variants was constructed and tested toward the native substrate-inspired fluorogenic substrate 12-(4-trifluoromethylcoumarin-7-yloxy)dodecanoic acid. Particularly high enzyme-mediated O-dealkylation yielding the fluorescent product 7-hydroxy-4-trifluoromethylcoumarin was observed with mutants containing the F87V substitution, with A74G/F87V showing the highest catalytic efficiency (0.458 min(-1)µM(-1)). To simplify the assay procedure and show its versatility, different modes of application were successfully demonstrated, including (i) the direct use of NADPH or its oxidized form NADP(+) along with diverse NADPH recycling systems for electron supply, (ii) the use of cell-free lysates and whole-cell preparations as the biocatalyst source, and (iii) its use for competitive inhibition screens to identify or characterize substrates and inhibitors. A detailed comparison with known, fluorescence-based P450 BM3 assays finally emphasizes the relevance of our contribution to the ongoing research.
Sujet(s)
Protéines bactériennes/antagonistes et inhibiteurs , Protéines bactériennes/métabolisme , Coumarines/composition chimique , Coumarines/métabolisme , Inhibiteurs des enzymes du cytochrome P-450/pharmacologie , Cytochrome P-450 enzyme system/métabolisme , Évaluation préclinique de médicament/méthodes , NADPH-ferrihemoprotéine reductase/antagonistes et inhibiteurs , NADPH-ferrihemoprotéine reductase/métabolisme , Alkylation , Biocatalyse , Coumarines/synthèse chimique , Colorants fluorescents/synthèse chimique , Colorants fluorescents/composition chimique , Colorants fluorescents/métabolisme , Tests de criblage à haut débit , Hydroxylation , Concentration inhibitrice 50 , Cinétique , NADP/métabolisme , Oxazines/composition chimique , Oxazines/métabolismeRÉSUMÉ
Acute myeloid leukemia (AML) cell lines can be driven to differentiate to monocyte-like cells by 1,25- dihydroxyvitamin D3 (1,25D) and to granulocyte-like cells by all-trans-retinoic acid (ATRA). Both compounds activate their specific intracellular receptors, vitamin D receptor (VDR) and retinoic acid receptors (RARs) respectively. Inside the cells 1,25D is degraded to calcitrioic acid by a mitochondrial enzyme CYP24A1, while ATRA is degraded to several polar metabolites by CYP26. NADPH-cytochrome P450 oxidoreductase (POR) is a membrane-bound enzyme required for electron transfer to cytochrome P450 (CYP), vital in the processes of the metabolism of drugs and steroid production in humans. In this paper we report that POR in AML cells, from both cell lines and patients, is upregulated by ATRA and by 1,25D at the level of mRNA and protein. Partial silencing of POR in HL60 cells resulted in augmented differentiation response to 1,25D.
Sujet(s)
Régulation de l'expression des gènes/effets des médicaments et des substances chimiques , Granulocytes/effets des médicaments et des substances chimiques , Monocytes/effets des médicaments et des substances chimiques , NADPH-ferrihemoprotéine reductase/génétique , Trétinoïne/pharmacologie , Vitamine D/analogues et dérivés , Différenciation cellulaire/effets des médicaments et des substances chimiques , Extinction de l'expression des gènes , Granulocytes/cytologie , Granulocytes/métabolisme , Cellules HL-60 , Humains , Monocytes/cytologie , Monocytes/métabolisme , NADPH-ferrihemoprotéine reductase/antagonistes et inhibiteurs , NADPH-ferrihemoprotéine reductase/métabolisme , Petit ARN interférent/génétique , Petit ARN interférent/métabolisme , Transduction du signal , Vitamine D/pharmacologieRÉSUMÉ
BACKGROUND: The involvement of oxidative stress in the pathophysiological process of alcohol-induced liver injury has been studied for decades. However, the role of microRNAs (miRNAs) targeting to oxidative stress genes in the pathogenesis of alcohol-induced liver injury has not yet been determined. The aim of this study was to identify the targeting of miR-214 to both glutathione reductase (GSR) and cytochrome P450 oxidoreductase (POR) genes and elucidate their impact on alcohol-induced oxidative stress in liver cells. METHODS: The miR-214 expression vector and reporter vectors of GSR and POR 3'-UTR were constructed. Human hepatoma cell (Bel7402), human embryonic kidney 293 cell (HEK293), and rat normal hepatocyte (BRL) were transfected and stimulated with ethanol (EtOH). Wistar rats were fed with EtOH for 4 weeks. The GSR and POR protein levels were detected by Western blot, and their activities were measured using the spectrophotometric method. The miR-214 expression was detected by real-time PCR. The index of oxidative stress including the total antioxidant capacity (T-AOC) and malondialdehyde (MDA) level was detected by commercial kits. RESULTS: miR-214 bound specifically to the GSR and POR 3'-UTR and repressed the expressions and activities of both GSR and POR. EtOH up-regulated the miR-214 expression, down-regulated the GSR and POR protein levels and activities, and induced the oxidative stress in human and rat liver cells. EtOH-fed Wistar rats further confirmed that alcohol up-regulates the miR-214 expression in liver and repressed both GSR and POR in vivo. CONCLUSIONS: These findings demonstrated a new mechanism by which the alcohol repressed the GSR and POR expression via up-regulation of miR-214 and in turn induced oxidative stress in liver cells.
Sujet(s)
Éthanol/administration et posologie , Glutathione reductase/antagonistes et inhibiteurs , Hépatocytes/métabolisme , microARN/physiologie , NADPH-ferrihemoprotéine reductase/antagonistes et inhibiteurs , Stress oxydatif/physiologie , Animaux , Lignée cellulaire tumorale , Régulation négative/effets des médicaments et des substances chimiques , Régulation négative/physiologie , Glutathione reductase/métabolisme , Cellules HEK293 , Hépatocytes/effets des médicaments et des substances chimiques , Hépatocytes/enzymologie , Humains , Mâle , NADPH-ferrihemoprotéine reductase/métabolisme , Stress oxydatif/effets des médicaments et des substances chimiques , Rats , Rat WistarRÉSUMÉ
OBJECTIVES: We previously established HepG2-GS-3A4, a cell line from hepatoblastoma with overexpression of human CYP3A4 and glutamine synthetase (GS). We further reported that these cells can be applied for screening inhibitors of CYP3A4 in vitro. The purpose of this study was to determine whether our CYP3A4-overexpresed cell could be applied to evaluate mechanisms of CYP3A4 inhibition by 6',7'-dihydroxybergamottin (DHB), which is one of the major furanocoumarins in grapefruit juice, by using these cells. METHODS: Nifedipine oxidation, activity and protein expression of NADPH-cytochrome reductase (POR) of HepG2-GS-3A4 cell were measured. CO-binding spectrumassay in microsomal fraction of the cells was also evaluated. KEY FINDINGS: DHB and ketoconazole, a well-known inhibitor of CYP3A4, inhibited nifedipine oxidation in a concentration-dependent manner. DHB at a concentration of 3.0 µm, sufficient to inhibit the nifedipine oxidation, decreased POR activity; however, ketoconazole at a concentration of 0.9 µm, sufficient to inhibit the oxidation, did not affect the activity. The expression of POR protein in HepG2-GS-3A4 cells was not changed by either DHB or ketoconazole. The expression of CYP3A4 mRNA and protein was not changed by the addition of DHB or ketoconazole. DHB also reduced the absorption rate at 450 nm in a CO-binding spectrum assay without alteration of the wavelength of maximum absorption. The mean absorption value at 450 nm slightly decreased with ketoconazole; however, the difference was not significant. CONCLUSIONS: We concluded that inhibition of CYP3A4 activity by DHB includes the inhibition of POR activity. HepG2-GS-3A4 might be a good tool to evaluate the mechanisms.
Sujet(s)
Citrus paradisi/composition chimique , Inhibiteurs du cytochrome P-450 CYP3A , Interactions aliments-médicaments , Furocoumarines/pharmacologie , Modèles biologiques , NADPH-ferrihemoprotéine reductase/antagonistes et inhibiteurs , Nifédipine/métabolisme , Cytochrome P-450 CYP3A/génétique , Relation dose-effet des médicaments , Antienzymes/pharmacologie , Cellules HepG2 , Humains , Kétoconazole/pharmacologie , Extraits de plantes/pharmacologie , ARN messager/métabolismeRÉSUMÉ
We describe herein an electrochemically driven drug metabolism strategy based on nanocomposites that integrate cyt P450 2C9 (CYP2C9) isozyme microsomes with cyt P450 reductase (CPR), indium tin oxide (ITO) nanoparticles and chitosan (CS). This novel bioelectronic system enables monitoring of the drug metabolism and enzyme inhibition.
Sujet(s)
Aryl hydrocarbon hydroxylases/antagonistes et inhibiteurs , Nanoparticules métalliques/composition chimique , Microsomes/métabolisme , NADPH-ferrihemoprotéine reductase/antagonistes et inhibiteurs , Préparations pharmaceutiques/métabolisme , Composés de l'étain/composition chimique , Aryl hydrocarbon hydroxylases/métabolisme , Carbone/composition chimique , Chitosane/composition chimique , Cytochrome P-450 CYP2C9 , Techniques électrochimiques , Électrodes , Antienzymes/composition chimique , Humains , NADPH-ferrihemoprotéine reductase/métabolismeRÉSUMÉ
Tannic acid (TA) inhibits nicotinamide adenine dinucleotide phosphate (NADPH)-cytochrome P450 reductase (CPR) activity, which is measured by reduction of cytochrome c, in rat liver microsomes (RLMs). In the current study, we noticed that TA directly reduces cytochrome c in the absence of microsomes, thus confounding the CPR activity assay. A method is presented that measures CPR activity in the presence of TA by subtracting the cytochrome c reduction in the absence of NADPH (TA effect) from that in the presence of NADPH (TA plus CPR effect). The method was used to determine the inhibitory effect of TA in RLMs, recombinant CPR enzyme, and primary hepatocytes. Additionally, application of TA in a study of role of CPR in a primary rat hepatocyte model of ischemia-reperfusion (IR) was investigated. TA showed concentration-dependent, complete inhibition of CPR with half maximal inhibitory concentration (IC(50) ) values of 58.2 µM in RLMs and 54.6 and 275 µM in primary rat hepatocytes in the absence and presence of serum in the medium, respectively. Additionally, inhibition of CPR by TA was associated with a significant reduction in reactive oxygen species and cell death after IR injury. These data may be useful in future studies using TA as an inhibitor of CPR in microsomes and primary hepatocytes.
Sujet(s)
Artéfacts , Hépatocytes/effets des médicaments et des substances chimiques , Microsomes du foie/effets des médicaments et des substances chimiques , NADPH-ferrihemoprotéine reductase/antagonistes et inhibiteurs , Lésion d'ischémie-reperfusion/enzymologie , Tanins/pharmacologie , Animaux , Dosage biologique/méthodes , Hypoxie cellulaire/effets des médicaments et des substances chimiques , Cellules cultivées , Milieux de culture sans sérum , Cytochromes c/métabolisme , Relation dose-effet des médicaments , Hépatocytes/enzymologie , Mâle , Microsomes du foie/enzymologie , Rats , Rat Sprague-Dawley , Espèces réactives de l'oxygène/métabolisme , Lésion d'ischémie-reperfusion/métabolismeRÉSUMÉ
Astaxanthin (Ax), a xanthophyll carotenoid, is reported to induce cytochrome P450 (CYP) 1A-dependent activity. CYP1A is one of the most important enzymes participating in phase I metabolism for chemicals, and it can activate various mutagens. To investigate the effect of Ax on the metabolic activation of a typical promutagen, benzo[a]pyrene by CYP1A, we orally administrated Ax-containing oil (100 mg Ax/kg body weight/day for 3 days) to male Wistar rats. In the treated rat liver, expression of CYP1A1 mRNA, protein, and its activity were significantly increased (5.5-, 8.5-, and 2.5-fold, respectively). In contrast, the activities of phase II enzymes (glutathione S-transferase and glucuronosyl-transferase) were not modulated by Ax-containing oil. As a consequence, the mutagenicity of benzo[a]pyrene was more enhanced in Ax-treated rats, compared with controls in the Ames assay. On the other hand, NADPH P450 reductase activity was decreased in liver microsomes from the treated group. This result suggests the possibility that Ax inhibits the electron supply necessary for CYP catalytic activities and decreases CYP1A activity indirectly. In conclusion, Ax-containing oil intake can alter CYP1A-dependent activities through two different mechanisms: (1) induction of CYP1A1 mRNA, protein expression, and activity; and (2) inhibition of the electron supply for the enzyme.
Sujet(s)
Antimutagènes/pharmacologie , Cytochrome P-450 CYP1A1/antagonistes et inhibiteurs , Antienzymes/pharmacologie , Fibrinolytiques/pharmacologie , NADPH-ferrihemoprotéine reductase/antagonistes et inhibiteurs , Animaux , Benzo[a]pyrène/toxicité , Cytochrome P-450 CYP1A1/biosynthèse , Cytochrome P-450 CYP1A1/génétique , Transport d'électrons/génétique , Régulation de l'expression des gènes codant pour des enzymes/effets des médicaments et des substances chimiques , Glucuronosyltransferase/antagonistes et inhibiteurs , Glucuronosyltransferase/génétique , Glucuronosyltransferase/métabolisme , Glutathione transferase/antagonistes et inhibiteurs , Glutathione transferase/génétique , Glutathione transferase/métabolisme , Foie/effets des médicaments et des substances chimiques , Foie/enzymologie , Mâle , Mutagènes/toxicité , NADPH-ferrihemoprotéine reductase/génétique , NADPH-ferrihemoprotéine reductase/métabolisme , ARN messager/métabolisme , Rats , Rat Wistar , Xanthophylles/pharmacologieRÉSUMÉ
To predict CYP4A-mediated reactions, we developed a two-dimensional template scoring system based on published data. The system predicts the order of occurrence among multiple oxidation sites, as well as the regioselectivity. The template has a linearly arranged honeycomb shape and an adjacent area. Molecules are overlaid on the template with the locations of the atoms restricted to the corners of hexagonal blocks. The overlaid conformers are then checked to determine whether they reside within the template area, and their position occupancy and position function scores are calculated. The position occupancy score is determined based on occupation of the respective positions on the template. The functional and steric properties are reflected in the position function score. The sum of these scores is compared among possible conformers, and the conformer with the highest total score is predicted to be preferentially metabolized. In the present study, prediction of sites of CYP4A-mediated oxidation and classification into substrates and non-substrates were performed for collected compounds, and agreement between predicted and experimental data exceeded 95% for substrates and non-substrates. The template scoring system can be easily linked to databases of two-dimensional chemical structures, and thus this system may be useful for drug development and studies of drug metabolism.
Sujet(s)
Cytochrome P-450 CYP4A/antagonistes et inhibiteurs , Cytochrome P-450 CYP4A/métabolisme , Isoenzymes/métabolisme , Mixed function oxygenases/métabolisme , NADPH-ferrihemoprotéine reductase/antagonistes et inhibiteurs , NADPH-ferrihemoprotéine reductase/métabolisme , Logiciel , Site allostérique , Animaux , Sites de fixation , Simulation numérique , Cytochrome P-450 CYP4A/composition chimique , Produits dermatologiques/composition chimique , Produits dermatologiques/métabolisme , Humains , Hydroxylation , Isotrétinoïne/composition chimique , Isotrétinoïne/métabolisme , Acides lauriques/composition chimique , Acides lauriques/métabolisme , Ligands , Mixed function oxygenases/antagonistes et inhibiteurs , Mixed function oxygenases/composition chimique , Structure moléculaire , NADPH-ferrihemoprotéine reductase/composition chimique , Oxydoréduction , Rats , Spécificité du substratRÉSUMÉ
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.
Sujet(s)
Lipidoses/enzymologie , Foie/enzymologie , NADPH-ferrihemoprotéine reductase/antagonistes et inhibiteurs , Animaux , Lignée cellulaire tumorale , Chromatographie gazeuse-spectrométrie de masse , Immunotransfert , Métabolisme lipidique/physiologie , Lipidoses/génétique , Lipidoses/métabolisme , Foie/métabolisme , Souris , Souris knockout , Microscopie de fluorescence , NADPH-ferrihemoprotéine reductase/génétique , NADPH-ferrihemoprotéine reductase/physiologie , Plasmides , Petit ARN interférent/génétique , Rats , RT-PCR , Sterol 14-demethylase/génétique , Sterol 14-demethylase/métabolisme , Sterol 14-demethylase/physiologie , Transfection , Triglycéride/génétique , Triglycéride/métabolismeRÉSUMÉ
Inhalation of vesicants including sulfur mustard can cause significant damage to the upper airways. This is the result of vesicant-induced modifications of proteins important in maintaining the integrity of the lung. Cytochrome P450s are the major enzymes in the lung mediating detoxification of sulfur mustard and its metabolites. NADPH cytochrome P450 reductase is a flavin-containing electron donor for cytochrome P450. The present studies demonstrate that the sulfur mustard analog, 2-chloroethyl ethyl sulfide (CEES), is a potent inhibitor of human recombinant cytochrome P450 reductase, as well as native cytochrome P450 reductase from liver microsomes of saline and beta-naphthoflavone-treated rats, and cytochrome P450 reductase from type II lung epithelial cells. Using rat liver microsomes from beta-naphthoflavone-treated rats, CEES was found to inhibit CYP 1A1 activity. This inhibition was overcome by microsomal cytochrome P450 reductase from saline-treated rats, which lack CYP 1A1 activity, demonstrating that the CEES inhibitory activity was selective for cytochrome P450 reductase. Cytochrome P450 reductase also generates reactive oxygen species (ROS) via oxidation of NADPH. In contrast to its inhibitory effects on the reduction of cytochrome c and CYP1A1 activity, CEES was found to stimulate ROS formation. Taken together, these data demonstrate that sulfur mustard vesicants target cytochrome P450 reductase and that this effect may be an important mechanism mediating oxidative stress and lung injury.
Sujet(s)
Irritants/toxicité , Gaz moutarde/analogues et dérivés , NADPH-ferrihemoprotéine reductase/antagonistes et inhibiteurs , Espèces réactives de l'oxygène/métabolisme , Animaux , Cellules cultivées , Cytochrome P-450 CYP1A1/antagonistes et inhibiteurs , Cytochrome P-450 CYP1A1/métabolisme , Gaz moutarde/toxicité , NADPH-ferrihemoprotéine reductase/métabolisme , RatsRÉSUMÉ
OBJECTIVE: Transforming growth factor beta (TGF-beta) is a potent inhibitor of 17alpha-hydroxylase/17,20 lyase activity and CYP17 gene expression. We investigated the mechanism how CYP17 is inhibited by TGF-beta in adrenocortical cells. METHODS: H295R cells were culture and incubated with TGF-beta, transcription inhibitor (DRB), activin receptor-like kinase 5 ALK5 (TbetaRII) inhibitor (SB431542), mitogen activated kinases inhibitors (PD98059 and SB203580), subsequently using reverse transcription and quantitative PCR (RT-qPCR) we determined CYP17 expression. RESULTS: TGF-beta significantly decreased the level of cytochrome P450c17 mRNA and this inhibitory effect of TGF-beta on CYP17 expression required activin receptor-like kinase 5 (ALK5) and on-going transcription. Mitogen activated kinases MEK1 and p38 MAPK are not involved it the inhibitory effect of TGF-beta on CYP17 expression. CONCLUSION: We concluded that the TGF-beta-dependent decrease of 17alpha-hydroxylase/17,20 lyase activity in the H295R cells is caused by inhibition of CYP17 transcription and is mediated by the ALK5 receptor.
Sujet(s)
Récepteur activine/physiologie , Steroid 17-alpha-hydroxylase/génétique , Facteur de croissance transformant bêta/pharmacologie , Cortex surrénal , Benzamides/pharmacologie , Lignée cellulaire , Colforsine/pharmacologie , Cytochromes b5/métabolisme , Dichlororibofuranosylbenzimidazole/pharmacologie , Dioxoles/pharmacologie , Humains , Imidazoles , MAP Kinase Kinase 1/physiologie , NADPH-ferrihemoprotéine reductase/antagonistes et inhibiteurs , NADPH-ferrihemoprotéine reductase/métabolisme , Protein-Serine-Threonine Kinases/antagonistes et inhibiteurs , Protein-Serine-Threonine Kinases/physiologie , Pyridines , ARN messager/métabolisme , Récepteur de type I du facteur de croissance transformant bêta , Récepteurs TGF-bêta/antagonistes et inhibiteurs , Récepteurs TGF-bêta/physiologie , Steroid 17-alpha-hydroxylase/antagonistes et inhibiteurs , p38 Mitogen-Activated Protein Kinases/physiologieRÉSUMÉ
NADPH-cytochrome P450 reductase (CPR) and cytochrome-b(5) (b(5)) together with NADH-b(5) reductase (b(5)R) play important roles in cytochrome P450 3A-mediated drug metabolism via electron transfer. However, it is not clear whether variability in expression of these accessory proteins contributes to the known interindividual variability in CYP3A activity. CPR and b(5) were measured in human liver microsomes (HLMs) by spectrophotometry and immunoblotting. HLMs from elderly (>or=46 years) male donors (n=11) averaged 27% (P=0.034) and 41% (P=0.011) lower CPR levels than young (Sujet(s)
Cytochrome P-450 enzyme system/métabolisme
, Cytochrome-B(5) reductase/métabolisme
, Microsomes du foie/enzymologie
, NADPH-ferrihemoprotéine reductase/métabolisme
, Vieillissement/métabolisme
, Cytochrome P-450 CYP3A
, Cytochrome-B(5) reductase/antagonistes et inhibiteurs
, Antienzymes/pharmacologie
, Femelle
, Humains
, Mâle
, NADPH-ferrihemoprotéine reductase/antagonistes et inhibiteurs
, Facteurs sexuels
, Acide lipoïque/pharmacologie
RÉSUMÉ
Tannic acid has been shown to decrease mutagenicity and/or carcinogenicity of several amine derivatives and polycyclic aromatic hydrocarbons in rodents. The purpose of this study was to evaluate the effect of tannic acid on cytochrome P450 (CYP)-catalyzed oxidations using rat liver microsomes (RLM) and human liver microsomes (HLM) as the enzyme sources. In RLM, tannic acid showed a non-selective inhibitory effect on 7-methoxyresorufin O-demethylation (MROD), 7-ethoxyresorufin O-deethylation (EROD), tolbutamide hydroxylation, p-nitrophenol hydroxylation and testosterone 6beta-hydroxylation activities with IC(50) values ranged from 14.9 to 27.4 microM. In HLM, tannic acid inhibited EROD, MROD and phenacetin O-deethylation activities with IC(50) values ranged from 5.1 to 7.5 microM, and diclofenac 4-hydroxylation, dextromethorphan O-demethylation, chlorzoxazone 6-hydroxylation and testosterone 6beta-hydroxylation with IC(50) values ranged from 20 to 77 microM. In baculovirus-insect cell-expressed human CYP 1A1 and 1A2, the IC(50) values of tannic acid for CYP 1A1- and 1A2-catalyzed EROD activities were 23.1 and 2.3 microM, respectively, indicating that tannic acid preferably inhibited the activity of CYP1A2. Tannic acid inhibited human CYP1A2 non-competitively with a Ki value of 4.8 microM. Tannic acid was also found to inhibit NADPH-CYP reductase in RLM and HLM with IC(50) values of 11.8 and 17.4 microM, respectively. These results suggested that the inhibition of CYP enzyme activities by tannic acid may be partially attributed to its inhibition of NADPH-CYP reductase activity.
Sujet(s)
Inhibiteurs des enzymes du cytochrome P-450 , Antienzymes/pharmacologie , Microsomes du foie/effets des médicaments et des substances chimiques , Microsomes du foie/enzymologie , NADPH-ferrihemoprotéine reductase/antagonistes et inhibiteurs , Tanins/pharmacologie , Animaux , Cytochrome P-450 enzyme system/métabolisme , Relation dose-effet des médicaments , Humains , Isoenzymes , Mâle , NADPH-ferrihemoprotéine reductase/effets des médicaments et des substances chimiques , Rats , Rat Sprague-Dawley , Spécificité d'espèceRÉSUMÉ
Aluminum and thallium may reach life-threatening levels in aquatic systems in the near future because of their extensive use in various industrial fields. It is therefore important to study the mechanism of toxicity of aluminum and thallium on fish enzymes. To this aim, the effects of aluminum and thallium on the activity of purified leaping mullet (Liza saliens) cytochrome P450 reductase, an essential component of the important cytochrome P450 system, have been studied. Results indicated that both metal ions strongly inhibited the NADPH-cytochrome P450 reductase. The IC50 values of AlCl3 and TlCl3 were estimated to be 34 microM and 3 microM, respectively. The Lineweaver-Burk plot and Dixon plot revealed that both metal ions noncompetitively inhibited the purified mullet cytochrome P450 reductase. The K(i) values of Al3+ and Tl3+ were calculated from Dixon plots as 8.9 and 5.6 microM, respectively. The inhibitory effects of Al3+ and Tl3+ on purified cytochrome P450 reductase were partially recovered by 1 mM EDTA. Additionally, tin and magnesium were shown to have no apparent effect on purified mullet cytochrome P450 reductase.