The Experts below are selected from a list of 375 Experts worldwide ranked by ideXlab platform
Tsutomu Shimada - One of the best experts on this subject based on the ideXlab platform.
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cytochrome p450 1b1 a target for inhibition in anticarcinogenesis strategies
Mutation Research, 2003Co-Authors: Peter F Guengerich, Elizabeth M J Gillam, Youngjin Chun, Tsutomu ShimadaAbstract:Cytochrome P450 (P450) 1B1 is expressed in a number of human tissues in which cancers occur (e.g. prostate, ovary, uterus, mammary gland). P450 1B1 activates many environmental mutagens and also catalyzes the 4-hydroxylation of estrogens, considered to be an important step in hormonal carcinogenesis. We have examined the activities of several of the major allelic variants of human P450 1B1 in these reactions. Another interest has been the development of chemical inhibitors of P450 1B1. 2-Ethynylpyrene and Alpha-Naphthoflavone preferentially inhibit human P450 1B1 compared to P450 1A1, which may be present in the same tissue sites. The natural product resveratrol is also an inhibitor of P450 1B1. Further studies with rhapontigenin and synthetic stilbenes led to the discovery of 2,4,3,5'-tetramethoxystilbene, a selective inhibitor of P450 1B1 relative to other P450s. Inhibition is competitive, with a K-i value of 3 nM, and the inhibitor is resistant to metabolism. In addition to blocking 17beta-estradiol 4-hydroxylation, this stilbene also inhibited the activation of heterocyclic amines to mutagens. 2,4,3',5'-Tetramethoxystilbene also suppressed expression of P450 1B1 and growth of human mammary tumor cells. 3,3',4',5,5'-Pentamethoxystilbene was a selective inhibitor of P450 1A1, showing mixed inhibition, and also suppressed P450 1A1 expression in HepG2 cells. Substituted stilbenes may be useful in preventing cancer caused by estrogens and xenobiotics. (C) 2002 Elsevier Science B.V. All rights reserved.
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bufuralol hydroxylation by cytochrome p450 2d6 and 1a2 enzymes in human liver microsomes
Molecular Pharmacology, 1994Co-Authors: Hiroshi Yamazaki, Zuyu Guo, M Persmark, M Mimura, K Inoue, F P Guengerich, Tsutomu ShimadaAbstract:Bufuralol 1'-hydroxylation is a prototypical reaction catalyzed by cytochrome P450 (P450) 2D6, an enzyme known to show debrisoquine/sparteine-type genetic polymorphism in humans. In the present study we further examined the roles of several human P450 enzymes, as well as P450 2D6, in the hydroxylation of (+/-)-bufuralol, using liver microsomes from several human samples and human P450 enzymes expressed in human lymphoblastoid cell lines or Escherichia coli. Kinetic analysis of bufuralol 1'-hydroxylation by liver microsomes showed that there were different Km and Vmax values in seven human samples examined; low Km values (approximately 0.05 mM) were observed in four samples (including sample HL-18), high Km values (approximately 0.25 mM) in two samples (including sample HL-67), and an intermediate Km value (approximately 0.1 mM) in one sample. Quinidine and anti-rat P450 2D1 antibody almost completely inhibited bufuralol 1'-hydroxylation in human sample HL-18 at a substrate concentration of 0.4 mM, whereas these effects were not so drastic when liver microsomes from human sample HL-67 were used. In contrast, a very low concentration (< 10 microM) of Alpha-Naphthoflavone or anti-human P450 1A2 antibody significantly inhibited bufuralol 1'-hydroxylation catalyzed by human sample HL-67, but not HL-18, with 0.4 mM bufuralol. When the relative contents of P450 2D6 and P450 1A2 in 20 human samples were determined, bufuralol 1'-hydroxylation in samples containing large amounts of P450 2D6 tended to be more sensitive to quinidine, whereas the P450 1A2-rich samples were highly susceptible to Alpha-Naphthoflavone. However, at low substrate concentrations bufuralol 1'-hydroxylation was shown to be catalyzed principally by P450 2D6, based on the inhibitory effects of anti-rat P450 2D1 antibody and quinidine, in both human samples HL-18 and HL-67. At least five other, minor, bufuralol products were formed by human liver microsomes, in addition to 1'-hydroxybufuralol. Two of them were identified as 4- and 6-hydroxybufuralol by 1H NMR spectroscopy and mass spectrometry. The formation of the 4- and 6-hydroxylated products was suggested to be catalyzed by P450 1A2, based on the results of correlation with P450 1A2 contents in 60 human samples and inhibition by anti-P450 1A2 and Alpha-Naphthoflavone. Purified recombinant P450 1A2 (expressed in E. coli) produced 1'-, 4-, and 6-hydroxybufuralol in a reconstituted system, although P450 2D6 (expressed in human lymphoblast cell lines) was found to catalyze only bufuralol 1'-hydroxylation.(ABSTRACT TRUNCATED AT 400 WORDS)
J R Bend - One of the best experts on this subject based on the ideXlab platform.
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differential in vivo effects of Alpha Naphthoflavone and beta Naphthoflavone on cyp1a1 and cyp2e1 in rat liver lung heart and kidney
Journal of Biochemical and Molecular Toxicology, 1999Co-Authors: Christopher J Sinal, Christopher D Webb, J R BendAbstract:Male Sprague-Dawley rats were treated intraperitoneally with corn oil, the aryl hydrocarbon receptor (AHR) agonist beta-Naphthoflavone (betaNF), or the relatively weak AHR agonist Alpha-Naphthoflavone (AlphaNF). Animals treated with betaNF experienced a significant loss (12%) of total body mass over 5 days and a dramatic elevation of CYP1A1 mRNA in all of the organs studied. Treatment with AlphaNF had no significant effect on body mass after 5 days and caused only minor increases of liver, kidney, and heart CYP1A1 mRNA. In contrast, lung CYP1A1 mRNA was increased by AlphaNF treatment to levels comparable to that seen with betaNF treatment. CYP2E1 mRNA levels were also elevated in liver, lung, kidney, and heart in response to betaNF treatment, whereas AlphaNF was without effect. Large increases of CYP1Al-dependent 7-ethoxyresorufin O-deethylation (EROD) activity occurred with microsomes prepared from the tissues of betaNF-treated animals. Comparatively small changes were associated with AlphaNF treatment, with the exception of lung, where EROD activity was increased to approximately 60% of that with betaNF treatment. CYP2E1-dependent p-nitrophenol hydroxylase (PNP) activity was also increased by betaNF treatment in microsomes prepared from kidney (3.1-fold), whereas AlphaNF was without effect. In contrast, AlphaNF or betaNF treatment caused significant decreases of lung microsomal PNP (72% and 27% of corn oil control, respectively) and 7-pentoxyresorufin O-deethylation (48% and 17% of corn oil control, respectively) activities, indicating that PNP activity may be catalyzed by P450 isoforms other than CYP2E1 in rat lung. We conclude that betaNF and AlphaNF have differential effects on the expression and catalytic activity of CYP1A1 and CYP2E1, depending upon the organ studied. These changes most likely occur as a result of the direct actions of these compounds as AHR agonists, in addition to secondary effects associated with AHR-mediated toxicity.
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bioactivation of arachidonic acid by the cytochrome p450 monooxygenases of guinea pig lung the orthologue of cytochrome p450 2b4 is solely responsible for formation of epoxyeicosatrienoic acids
Molecular Pharmacology, 1994Co-Authors: L C Knickle, J R BendAbstract:Guinea pig lung microsomes converted arachidonic acid (AA) to two classes of cytochrome P450 (P450)-dependent metabolites, 16- through 20-hydroxyeicosatetraenoic acids [(16-20)-OH-AA] and epoxyeicosatrienoic acids (EETs). The rate of formation of (16-20)-OH-AA was approximately 3-fold higher in microsomes from beta-Naphthoflavone-induced versus untreated animals. In microsomes from untreated or induced animals EETs, the major class of P450 metabolites in guinea pig lung, were formed in a regioselective manner, with 8,9-, 11,12-, and 14,15-regioisomers accounting for > or = 90% of the total EETs. With isozyme-selective inhibitors and inhibitory antibodies the role of individual pulmonary P450 isozymes in AA metabolism was examined. Metyrapone and SKF-525A (P450 2B selective) inhibited EET formation by > or = 85% with little effect on (16-20)-OH-AA formation. 1-Aminobenzotriazole (1 mM), a mechanism-based inhibitor with low isozyme selectivity, inhibited the formation of both classes of metabolites by > 95%, whereas N-Alpha-methylbenzyl-1-aminobenzotriazole (1 microM), a P450 2B-selective mechanism-based inhibitor, abolished EET formation with little effect on (16-20)-OH-AA formation. Antibodies to rabbit P450 2B4 also abolished EET formation without inhibiting the formation of (16-20)-OH-AA, whereas antibodies to rabbit P450 4B1 did not inhibit the formation of either class of metabolites. Alpha-Naphthoflavone (P450 1A1 selective in lung) did not inhibit the formation of either class of metabolites. These data demonstrate that the guinea pig orthologue of P450 2B4 is solely responsible for the bioactivation of AA to EETs in guinea pig lung and that a form of P450 other than a 2B, 4B, or 1A isozyme, which is inducible by beta-Naphthoflavone, is responsible for (16-20)-OH-AA formation.
George Rucci - One of the best experts on this subject based on the ideXlab platform.
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Alpha Naphthoflavone acts as an antagonist of 2 3 7 8 tetrachlorodibenzo p dioxin by forming an inactive complex with the ah receptor
Molecular Pharmacology, 1991Co-Authors: Thomas A Gasiewicz, George RucciAbstract:Alpha-Naphthoflavone (ANF) has previously been shown to compete with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) for binding to the Ah receptor under conditions in vitro. However, ANF also prevents TCDD-elicited cytochrome P450lA1 induction, immunosuppression, and down-regulation of the estrogen receptor in vivo and within intact isolated cells. These data suggest that ANF is a TCDD antagonist. This study investigated the ability of ANF to transform the Ah receptor contained in rat hepatic cytosol or mouse hepatoma cells to a form that recognizes the dioxin-responsive enhancer element (DRE) upstream of the cytochrome P450lA1 gene. Gel retardation analysis indicated that TCDD- or beta-Naphthoflavone (BNF)-bound receptor was able to bind to the DRE, whereas essentially no receptor-DRE complexes were observed using cytosol incubated with ANF concentrations as high as 1000 nM. Furthermore, an excess of ANF, when added to cytosol just before TCDD, blocked, in a concentration-dependent manner, the ability of TCDD to transform the receptor to a form that bound to the DRE. These studies indicated that ANF binds to the receptor and confers on it a conformation that cannot recognize the DNA recognition sequence contained in the DRE. Although an excess of the agonist 2,3,7,8-tetrachlorodibenzofuran (TCDF) readily reversed the inhibitory actions of ANF, ANF was unable to reverse the effects of TCDD, TCDF, or BNF on the receptor. These studies suggested that TCDD binding, unlike that of ANF, results in a receptor conformation that has higher affinity for the ligand. Treatment of mouse hepatoma Hepa 1c1c7 cells with TCDD or BNF resulted in receptor contained in nuclear extracts that bound to the DRE. Only a very minor ligand-dependent protein-DNA complex was detected when cells were treated with ANF. These data indicated that ANF acts as an antagonist of TCDD by directly binding to the Ah receptor and eliciting a protein conformation that has very low affinity for DNA.
John O Miners - One of the best experts on this subject based on the ideXlab platform.
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diazepam metabolism by human liver microsomes is mediated by both s mephenytoin hydroxylase and cyp3a isoforms
British Journal of Clinical Pharmacology, 1994Co-Authors: Tommy B Andersson, John O Miners, M E Veronese, Donald J. BirkettAbstract:Abstract 1. The primary metabolism of diazepam was studied in human liver microsomes in order to investigate the kinetics and to identify the cytochrome P450 (CYP) isoforms responsible for the formation of the main diazepam metabolites, temazepam and N-desmethyldiazepam. 2. The formation kinetics of both metabolites were atypical and consistent with the occurrence of substrate activation. A sigmoid Vmax model equivalent to the Hill equation was used to fit the data. The degree of sigmoidicity was greater for temazepam formation than for N-desmethyldiazepam formation, so that the ratio of desmethyldiazepam:temazepam formation increased as the substrate (diazepam) concentration decreased. 3. Alpha-Naphthoflavone activated both reactions but with a greater effect on temazepam formation than on N-desmethyldiazepam formation. In the presence of 25 microM Alpha-Naphthoflavone the kinetics for both pathways were approximated by Michaelis-Menten kinetics. 4. Studies with a series of CYP isoform selective inhibitors and with an inhibitory anti-CYP2C antibody indicated that temazepam formation was carried out mainly by CYP3A isoforms, whereas the formation of N-desmethyldiazepam was mediated by both CYP3A isoforms and S-mephenytoin hydroxylase.
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in vitro proguanil activation to cycloguanil by human liver microsomes is mediated by cyp3a isoforms as well as by s mephenytoin hydroxylase
British Journal of Clinical Pharmacology, 1994Co-Authors: Donald J. Birkett, F.j. Gonzalez, John O Miners, Tommy B Andersson, D. Rees, M E VeroneseAbstract:1. The activation of proguanil to cycloguanil by human liver microsomes was studied to define the cytochrome P450 (CYP) isoforms involved in this reaction. 2. Apparent Km values for proguanil ranged from 35 microM to 183 microM with microsomes from four human livers. 3. There was a 6.3-fold range of activity with microsomes from seventeen human livers. Rates of proguanil activation correlated significantly with CYP3A activities (benzo[a]pyrene metabolism, caffeine 8-oxidation and omeprazole sulphone formation) and CYP3A immunoreactive content. There was also a highly significant correlation with rates of hydroxyomeprazole formation. Correlations with activities selective for CYP1A2, CYP2C9/10 and CYP2E1, and with immunoreactive CYP1A2 content were not significant. 4. Proguanil activation was inhibited by R,S-mephenytoin, troleandomycin and by inhibitory anti-CYP3A antiserum and anti-CYP2C IgG and was activated by Alpha-Naphthoflavone. Inhibitors selective for CYP1A2, CYP2E1, CYP2A6 or CYP2C9/10 had little or no effect on proguanil activation. The extents of inhibition by R,S-mephenytoin, troleandomycin and the two antibodies varied with the immunoreactive CYP3A content of the microsomes used. 5. It is concluded that proguanil activation to cycloguanil by human liver microsomes is mediated both by S-mephenytoin hydroxylase and isoforms of the CYP3A subfamily. This has implications for the use of proguanil as an in vivo probe for the S-mephenytoin poor metaboliser phenotype.
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specificity of substrate and inhibitor probes for human cytochromes p450 1a1 and 1a2
Journal of Pharmacology and Experimental Therapeutics, 1993Co-Authors: Wongwiwat Tassaneeyakul, Donald J. Birkett, Maurice E Veronese, Michael E Mcmanus, Robert H Tukey, Linda C Quattrochi, H V Gelboin, John O MinersAbstract:Kinetic and inhibitor studies using cDNA-expressed enzymes and human liver microsomes have characterized the specificity of a range of cytochrome P450 (CYP) 1A substrate and inhibitor probes towards the two isoforms comprising this subfamily. Expressed CYP1A1 and CYP1A2 both catalyzed the O-deethylation of phenacetin, although the apparent Km was about 4-fold lower for CYP1A2 (25 vs. 108 microM). Phenacetin O-deethylation exhibited biphasic kinetics in human liver microsomes, and the apparent Km for the high-affinity component (9 +/- 6 microM) was consistent with the involvement of CYP1A2 in this reaction. The prototypic CYP1A xenobiotic inhibitor and substrate probes Alpha-Naphthoflavone, ellipticine, 7-ethoxycoumarin and 7-ethoxyresorufin all inhibited CYP1A1- and CYP1A2-mediated phenacetin O-deethylation as well as the high-affinity component of human liver phenacetin O-deethylase activity. Alpha-Naphthoflavone and 7-ethoxycoumarin were, however, approximately 10-fold more potent as inhibitors of CYP1A2 than CYP1A1. Other putative human CYP1A xenobiotic substrates and inhibitors, including caffeine, 5- and 8-methoxypsoralen, nifedipine, paraxanthine, propranolol and theophylline similarly inhibited CYP1A1- and 1A2-catalyzed phenacetin O-deethylation and the high-affinity human liver phenacetin O-deethylase. In contrast, the monoclonal antibody MAb 1-7-1, raised against 3-methylcholanthrene-inducible rat cytochromes 450, almost abolished CYP1A1-mediated phenacetin O-deethylation, but had no effect on human liver microsomal- or CYP1A2-catalyzed phenacetin dealkylation. Together with previous data, the results indicate that the majority of human CYP1A xenobiotic inhibitor and substrate probes are nonspecific in their recognition of CYP1A1 and CYP1A2, although selectivity is apparent for some compounds.
F.j. Gonzalez - One of the best experts on this subject based on the ideXlab platform.
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Assessment of specificity of eight chemical inhibitors using cDNA-expressed cytochromes P450
Xenobiotica, 2000Co-Authors: Yaozhang Sai, R. Dai, F.j. Gonzalez, Harry V. Gelboin, T. J. Yang, Kristopher W Krausz, Minshan ShouAbstract:1. The selectivity of eight chemical inhibitors has been extensively evaluated with 10 cDNA-expressed human cytochrome P450 isoforms (CYP). The results indicate that sulphaphenazole, quinidine and Alpha-Naphthoflavone are selective inhibitors of CYP2C9 (IC50 = 0.5-0.7 microM), CYP2D6 (0.3-0.4 microM) and CYP1A (0.05-5 microM) respectively on the basis of the IC50, which are much lower than those of other P450 isoforms (> 10-fold). 2. Ketoconazole exhibited potent inhibition of both CYP3A4-catalysed metabolism of phenanthrene, testosterone, diazepam (IC50 = 0.03-0.5 microM) and CYP1A1-catalysed deethylation of 7-ethoxycoumarin (0.33 microM). The selectivity of ketoconazole for other P450s was highly related to the concentration used. 3. Diethyldithiocarbamate, orphenadrine and furafylline were shown separately to be less selective inhibitors of CYP2E1, CYP2B6 and CYP1A isoforms by a broad range of IC50 that overlap those observed with other P450 isoforms. 4. Furafylline, quinidine and Alpha-Naphthoflavone activated CYP3A4-catalysed phenanthrene metabolism by 1.7-, 2- and 15-fold respectively. 5. The selectivity of orphenadrine and ketoconazole was further examined by using inhibitory monoclonal antibodies (MAb). Inhibitory MAb specific for the individual P450 isoforms may be of greater value than chemical inhibitors.
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evaluation of triacetyloleandomycin Alpha Naphthoflavone and diethyldithiocarbamate as selective chemical probes for inhibition of human cytochromes p450
Archives of Biochemistry and Biophysics, 1994Co-Authors: Thomas Chang, F.j. Gonzalez, David J WaxmanAbstract:Abstract A variety of chemicals, including triacetyloleandomycin (TAO), α-nasymphthoflavone (ANF), and diethyldithiocarbamate (DDC), are widely used as inhibitory probes for select individual human cytochrome P450 (CYP) enzymes, despite the fact that the selectivity of these inhibitors has not been rigorously evaluated. In the present study we take advantage of recent advances in cDNA-directed human P450 expression to evaluate directly the P450 form selectivity of TAO, ANF, and DDC, using a panel of 10 individual cDNA-expressed human P450s. Under experimental conditions known to yield maximal TAO complexation with P450 hemoproteins, TAO (20 μM) inhibited the catalytic activity of expressed CYPs 3A3, 3A4, and 3A5, whereas it did not affect CYPs 1A1, 1A2, 2A6, 2B6, 2C8, 2C9, or 2E1 activity. ANF inhibited not only CYPs 1A1 and 1A2 (IC 50 = 0.4-0.5 μM), but it was also similarly effective against CYPs 2C8 and 2C9. Increasing the concentration of ANF to 10 μM led to inhibition of CYP2A6 and CYP2B6. Although a previous study suggested that DDC is a selective inhibitor of CYP2E1, the present investigation shows that at concentrations required to inhibit CYP2E1 (IC 50 ≍ 125 μM when preincubated with NADPH), DDC also inhibited CYPs 1A1, 1A2, 2A6, 2B6, 2C8, 3A3, and 3A4. Decreasing the concentration of DDC to 10 μM, however, led to inhibition of CYP2A6 (65% inhibition) and CYP2B6 (50% inhibition), but none of the other p450s examined, including CYP2E1. Overall, these results establish that (a) TAO is a selective inhibitor of the human CYP3A subfamily; (b) ANF potently inhibits CYP2C8 and CYP2C9, in addition to CYPs 1A1 and 1A2; and (c) DDC cannot be employed as a diagnostic inhibitory probe for CYP2E1.
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in vitro proguanil activation to cycloguanil by human liver microsomes is mediated by cyp3a isoforms as well as by s mephenytoin hydroxylase
British Journal of Clinical Pharmacology, 1994Co-Authors: Donald J. Birkett, F.j. Gonzalez, John O Miners, Tommy B Andersson, D. Rees, M E VeroneseAbstract:1. The activation of proguanil to cycloguanil by human liver microsomes was studied to define the cytochrome P450 (CYP) isoforms involved in this reaction. 2. Apparent Km values for proguanil ranged from 35 microM to 183 microM with microsomes from four human livers. 3. There was a 6.3-fold range of activity with microsomes from seventeen human livers. Rates of proguanil activation correlated significantly with CYP3A activities (benzo[a]pyrene metabolism, caffeine 8-oxidation and omeprazole sulphone formation) and CYP3A immunoreactive content. There was also a highly significant correlation with rates of hydroxyomeprazole formation. Correlations with activities selective for CYP1A2, CYP2C9/10 and CYP2E1, and with immunoreactive CYP1A2 content were not significant. 4. Proguanil activation was inhibited by R,S-mephenytoin, troleandomycin and by inhibitory anti-CYP3A antiserum and anti-CYP2C IgG and was activated by Alpha-Naphthoflavone. Inhibitors selective for CYP1A2, CYP2E1, CYP2A6 or CYP2C9/10 had little or no effect on proguanil activation. The extents of inhibition by R,S-mephenytoin, troleandomycin and the two antibodies varied with the immunoreactive CYP3A content of the microsomes used. 5. It is concluded that proguanil activation to cycloguanil by human liver microsomes is mediated both by S-mephenytoin hydroxylase and isoforms of the CYP3A subfamily. This has implications for the use of proguanil as an in vivo probe for the S-mephenytoin poor metaboliser phenotype.