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Sample records for human p450 cyp1a2

  1. Genetic polymorphisms of cytochrome P450-1A2 (CYP1A2 among Emiratis.

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    Mohammad M Al-Ahmad

    Full Text Available Cytochrome P450 1A2 (CYP1A2 is one of the CYP450 mixed-function oxidase system that is of clinical importance due to the large number of drug interactions associated with its induction and inhibition. In addition, significant inter-individual differences in the elimination of drugs metabolized by CYP1A2 enzyme have been observed which are largely due to the highly polymorphic nature of CYP1A2 gene. However, there are limited studies on CYP1A2 phenotypes and CYP1A2 genotypes among Emiratis and thus this study was carried out to fill this gap. Five hundred and seventy six non-smoker Emirati subjects were asked to consume a soft drink containing caffeine (a non-toxic and reliable probe for predicting CYP1A2 phenotype and then provide a buccal swab along with a spot urine sample. Taq-Man Real Time PCR was used to determine the CYP1A2 genotype of each individual. Phenotyping was carried out by analyzing the caffeine metabolites using High Performance Liquid Chromatography (HPLC analysis. We found that 1.4%, 16.3% and 82.3% of the Emirati subjects were slow, intermediate and rapid CYP1A2 metabolizers, respectively. In addition, we found that 1.4% of the subjects were homozygote for derived alleles while 16.1% were heterozygote and 82.5% were homozygote for the ancestral allele. The genotype frequency of the ancestral allele, CYP1A2*1A/*1A, is the highest in this population, followed by CYP1A2 *1A/*1C and CYP1A2 *1A/*1K genotypes, with frequencies of 0.825, 0.102 and 0.058, respectively. The degree of phenotype/genotype concordance was equal to 81.6%. The CYP1A2*1C/*1C and CYP1A2*3/*3 genotypes showed significantly the lowest enzyme phenotypic activity. The frequency of slow activity CYP1A2 enzyme alleles is very low among Emiratis which correlates with the presence of low frequencies of derived alleles in CYP1A2 gene.

  2. Scutellarin inhibits cytochrome P450 isoenzyme 1A2 (CYP1A2) in rats.

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    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.

  3. Induction of CYP1A1, CYP1A2, and CYP1B1 mRNAs by nitropolycyclic aromatic hydrocarbons in various human tissue-derived cells: chemical-, cytochrome P450 isoform-, and cell-specific differences

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    Iwanari, M.; Nakajima, M.; Yokoi, T. [Div. of Drug Metabolism, Kanazawa Univ., Kanazawa (Japan); Kizu, R.; Hayakawa, K. [Lab. of Hygienic Chemistry, Kanazawa Univ., Kanazawa (Japan)

    2002-06-01

    Nitropolycyclic aromatic hydrocarbons (NPAHs) are found in diesel exhaust and ambient air. NPAHs as well as polycyclic aromatic hydrocarbons (PAHs) are known to have mutagenicity, carcinogenicity, and endocrine-disruptive effects. In the present study, the inducibility of the human cytochrome P450-1 (CYP1) family by NPAHs was compared with those produced by their parent PAHs and some reductive metabolites, amino-PAHs. Furthermore, to investigate the differences in the inducibility of the CYP1 family in human tissues, various human tissue-derived cell lines, namely HepG2 (hepatocellular carcinoma), ACHN (renal carcinoma), A549 (lung carcinoma), MCF-7 (breast carcinoma), LS-180 (colon carcinoma), HT-1197 (bladder carcinoma), HeLa (cervix of uterus adenocarcinoma), OMC-3 (ovarian carcinoma), and NEC14 (testis embryonal carcinoma), were treated with NPAHs, PAHs, or amino-PAHs. The mRNA levels of CYP1A1, CYP1A2, and CYP1B1 were determined with reverse transcription-polymerase chain reaction (RT-PCR). The cell lines were classified into two groups: CYP1 inducible cell lines, comprising HepG2, MCF-7, LS-180, and OMC-3 cells, and CYP1 non-inducible cell lines, comprising ACHN, A549, HT-1197, HeLa, and NEC14 cells. In inducible cell lines, the induction profile of chemical specificity was similar for CYP1A1, CYP1A2, and CYP1B1, although the extent of induction differed among the cell lines and for the CYP isoforms. Pyrene, 1-nitropyrene, 1-aminopyrene, 1,3-, 1,6-, and 1,8-dinitropyrenes slightly induced CYP1 mRNAs, but 1,3-dinitropyrene produced a 6-fold induction of CYP1A1 mRNA in MCF-7 cells. 2-Nitrofluoranthene and 3-nitrofluoranthene exhibited stronger inducibility than fluoranthene in the inducible cell lines. 6-Nitrochrysene induced CYP1 mRNAs to the same extent or more potently than chrysene. The induction potencies of 6-nitrobenzo[a]pyrene and 7-nitrobenz[a]anthracene were weaker than those of their parents benzo[a]pyrene and benz[a]anthracene, respectively. This

  4. The effect of lycopene on the total cytochrome P450, CYP1A2 and CYP2E1

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    Melva Louisa

    2009-12-01

    Full Text Available Aim: Some carotenoids such as canthaxantin, astaxanthin and beta apo-8’-carotenal were reported to have modulatoryeffect on the cytochrome P450. The present study was conducted to investigate the effects of lycopene, a nonprovitamin A carotenoid, on microsomal cytochrome P450, CYP1A2 and CYP2E1.Methods: Total cytochrome P450 levels, CYP1A2 and CYP2E1-catalyzed reactions (acetanilide 4-hydroxylation and p-nitrophenol hydroxylation were studied in the liver microsomes of male Sprague Dawley rats. Microsomes were prepared using differential centrifugation combined with calcium aggregation method. Lycopene was orally administered in the dosages of 0, 25, 50 or 100 mg/kgBW/day for 14 days in a repeated fashion. Data were analyzed using ANOVA test.Results: Total cytochrome P450 level and acetanilide 4-hydroxylase activity were unaffected by any of the treatments. The CYP2E1 probe enzyme (p-nitrophenol hydroxylase was significantly reduced by repeated administration of 100mg/ kgBW/day lycopene (7.88 + 2.04 vs 12.26 + 2.77 n mol/min/mg prot.Conclusion: The present results suggest that lycopene does not affect the total cytochrome P450 or CYP1A2 activity but it inhibits the activity of CYP2E1 (p-nitrophenol hydroxylase in the rat. (Med J Indones 2009; 18: 233-8Keywords: lycopene, cytochrome P450, CYP1A2, CYP2E1

  5. Caffeine raises the serum melatonin level in healthy subjects: an indication of melatonin metabolism by cytochrome P450(CYP)1A2.

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    Ursing, C; Wikner, J; Brismar, K; Röjdmark, S

    2003-05-01

    Caffeine is metabolized in the liver by cytochrome P450(CYP)1A2. Recent findings imply that this enzyme may also be of importance for the metabolism of human melatonin (MT). If caffeine and MT are metabolized by the same enzyme, one may expect to find different serum MT levels after ingestion of coffee compared with placebo. Although coffee is consumed by people all over the world, few studies have focused on whether caffeine actually affects serum MT levels in normal subjects. We decided to study that particular topic. For that purpose 12 healthy individuals were tested on two occasions, one week apart. On one of these occasions they were given a capsule containing 200 mg caffeine in the evening. On the other, they received placebo. The experimental order was randomized. Serum MT levels were determined every second hour between 22:00 h and 08:00 h, and the melatonin areas under the curve (MT-AUCs) were calculated. After caffeine the serum MT level rose from 0.09 +/- 0.03 nmol/l at 22:00 h to 0.48 +/- 0.07 nmol/l at 04:00 h. The corresponding rise after placebo was less prominent (from 0.06 +/- 0.01 to 0.35 +/- 0.06 nmol/l). This was reflected by the MT-AUC which was 32% larger after ingestion of caffeine compared with placebo (MT-AUC(caffeine) 3.16 +/- 0.44 nmol/l x h vs MT-AUC(placebo) 2.39 +/- 0.40 nmol/l x h; p coffee, augments the nocturnal serum MT level, which in turn supports the notion that cytochrome P450(CYP)1A2 is involved in the hepatic metabolism of human MT.

  6. Evaluation of toxic equivalency factors for induction of cytochromes P450 CYP1A1 and CYP1A2 enzyme activity by dioxin-like compounds

    International Nuclear Information System (INIS)

    Toyoshiba, Hiroyoshi; Walker, Nigel J.; Bailer, A. John; Portier, Christopher J.

    2004-01-01

    The toxic equivalency factor (TEF) method has been used to characterize the toxicity of human mixtures of dioxin-like compounds and is being considered for use with other classes of potentially toxic agents. TEFs are estimated by examining the relative potencies of the various congeners for a series of biological and toxicological effects. In this paper, we consider changes in activity for two enzymes, cytochrome P450 1A1 (CYP1A1)-associated 7-ethoxyresorufin-O-deethylase (EROD) and CYP1A2-associated acetanilide-4-hydroxylase (A4H) activity, resulting from exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 3,3',4,4',5-pentachlorobiphenyl (PCB), 2,3,4,7,8-pentachlorodibenzofuran (PeCDF) or a mixture of these agents. The ratio of median effective dose (ED 50 ) is one way to estimate the relative potencies, especially for gene expression and protein endpoints. ED 50 's were estimated with a nonlinear regression model in which dose-related changes in mean responses are described by a Hill function. ED 50 's along with other model parameters were estimated by fitting this model to a given data set. Significant differences in estimated model parameters were tested by likelihood ratio methods. The estimated parameters indicated that congener-specific dose-response shapes were significantly different, that additivity failed for these congeners, and that the ratios of ED 50 's did not predict the response seen for the mixture. These results indicate that for some biological responses, the use of a single relative potency factor (RPF) is not appropriate for the comparison of the dose response behavior of different dioxin-like congeners

  7. Effect of diethyldithiocarbamate (DDC) and ticlopidine on CYP1A2 activity and caffeine metabolism: an in vitro comparative study with human cDNA-expressed CYP1A2 and liver microsomes.

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    Kot, Marta; Daniel, Władysława A

    2009-01-01

    The aim of the present study was to test the effect of diethyldithiocarbamate (DDC), which is regarded as a cytochrome P450 (CYP) CYP2A6 and CYP2E1 inhibitor, and ticlopidine, an efficient CYP2B6, CYP2C19 and CYP2D6 inhibitor, on the activity of human CYP1A2 and the metabolism of caffeine (1-N-, 3-N- and 7-N-demethylation, and C-8-hydroxylation). The experiment was carried out in vitro using human cDNA-expressed CYP1A2 (Supersomes) and human pooled liver microsomes. The effects of DDC and ticlopidine were compared to those of furafylline (a strong CYP1A2 inhibitor). A comparative in vitro study provides clear evidence that ticlopidine and DDC, applied at concentrations that inhibit the above-mentioned CYP isoforms, potently (as compared to furafylline) inhibit human CYP1A2 and caffeine metabolism, in particular 1-N- and 3-N-demethylation.

  8. Polymorphisms in the cytochrome P450 genes CYP1A2, CYP1B1, CYP3A4, CYP3A5, CYP11A1, CYP17A1, CYP19A1 and colorectal cancer risk

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    Withey Laura

    2007-07-01

    Full Text Available Abstract Background Cytochrome P450 (CYP enzymes have the potential to affect colorectal cancer (CRC risk by determining the genotoxic impact of exogenous carcinogens and levels of sex hormones. Methods To investigate if common variants of CYP1A2, CYP1B1, CYP3A4, CYP3A5, CYP11A1, CYP17A1 and CYP19A1 influence CRC risk we genotyped 2,575 CRC cases and 2,707 controls for 20 single nucleotide polymorphisms (SNPs that have not previously been shown to have functional consequence within these genes. Results There was a suggestion of increased risk, albeit insignificant after correction for multiple testing, of CRC for individuals homozygous for CYP1B1 rs162558 and heterozygous for CYP1A2 rs2069522 (odds ratio [OR] = 1.36, 95% confidence interval [CI]: 1.03–1.80 and OR = 1.34, 95% CI: 1.00–1.79 respectively. Conclusion This study provides some support for polymorphic variation in CYP1A2 and CYP1B1 playing a role in CRC susceptibility.

  9. CYP1A1 and CYP1A2 expression: Comparing 'humanized' mouse lines and wild-type mice; comparing human and mouse hepatoma-derived cell lines

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    Uno, Shigeyuki; Endo, Kaori; Ishida, Yuji; Tateno, Chise; Makishima, Makoto; Yoshizato, Katsutoshi; Nebert, Daniel W.

    2009-01-01

    Human and rodent cytochrome P450 (CYP) enzymes sometimes exhibit striking species-specific differences in substrate preference and rate of metabolism. Human risk assessment of CYP substrates might therefore best be evaluated in the intact mouse by replacing mouse Cyp genes with human CYP orthologs; however, how 'human-like' can human gene expression be expected in mouse tissues? Previously a bacterial-artificial-chromosome-transgenic mouse, carrying the human CYP1A1 C YP1A2 locus and lacking the mouse Cyp1a1 and Cyp1a2 orthologs, was shown to express robustly human dioxin-inducible CYP1A1 and basal versus inducible CYP1A2 (mRNAs, proteins, enzyme activities) in each of nine mouse tissues examined. Chimeric mice carrying humanized liver have also been generated, by transplanting human hepatocytes into a urokinase-type plasminogen activator(+/+) s evere-combined-immunodeficiency (uPA/SCID) line with most of its mouse hepatocytes ablated. Herein we compare basal and dioxin-induced CYP1A mRNA copy numbers, protein levels, and four enzymes (benzo[a]pyrene hydroxylase, ethoxyresorufin O-deethylase, acetanilide 4-hydroxylase, methoxyresorufin O-demethylase) in liver of these two humanized mouse lines versus wild-type mice; we also compare these same parameters in mouse Hepa-1c1c7 and human HepG2 hepatoma-derived established cell lines. Most strikingly, mouse liver CYP1A1-specific enzyme activities are between 38- and 170-fold higher than human CYP1A1-specific enzyme activities (per unit of mRNA), whereas mouse versus human CYP1A2 enzyme activities (per unit of mRNA) are within 2.5-fold of one another. Moreover, both the mouse and human hepatoma cell lines exhibit striking differences in CYP1A mRNA levels and enzyme activities. These findings are relevant to risk assessment involving human CYP1A1 and CYP1A2 substrates, when administered to mice as environmental toxicants or drugs.

  10. Paracetamol-induced spindle disturbances in V79 cells with and without expression of human CYP1A2

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    Jensen, K G; Poulsen, H E; Doehmer, J

    1996-01-01

    Spindle disturbing effects in terms of c-mitosis and cytotoxicity of paracetamol were investigated in two Chinese hamster V79 cell lines, one of which (V79MZh1A2) was transfected with human CYP1A2. This enzyme catalyses the oxidative formation of the reactive paracetamol metabolite, NAPQI, believed...... to initiate hepatoxicity by covalent binding to proteins after overdose. In the native V79 cell line paracetamol increased c-mitosis frequency in a concentration dependent manner from 8.7 + or - 3.5% (control) to 66 + or - 18% at 20 mM. A significant increase to 13.3 + or - 3.5% was first seen at 2.5 m......M in the native cell line (Pparacetamol. At 5 mM paracetamol the c-mitosis frequency was 14.4 + or - 5.0% and 19.0 + or - 3...

  11. Cytochrome P450 isoform selectivity in human hepatic theobromine metabolism

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    Gates, Simon; Miners, John O

    1999-01-01

    Aims The plasma clearance of theobromine (TB; 3,7-dimethylxanthine) is known to be induced in cigarette smokers. To determine whether TB may serve as a model substrate for cytochrome P450 (CYP) 1A2, or possibly other isoforms, studies were undertaken to identify the individual human liver microsomal CYP isoforms responsible for the conversion of TB to its primary metabolites. Methods The kinetics of formation of the primary TB metabolites 3-methylxanthine (3-MX), 7-methylxanthine (7-MX) and 3,7-dimethyluric acid (3,7-DMU) by human liver microsomes were characterized using a specific hplc procedure. Effects of CYP isoform-selective xenobiotic inhibitor/substrate probes on each pathway were determined and confirmatory studies with recombinant enzymes were performed to define the contribution of individual isoforms to 3-MX, 7-MX and 3,7-DMU formation. Results The CYP1A2 inhibitor furafylline variably inhibited (0–65%) 7-MX formation, but had no effect on other pathways. Diethyldithiocarbamate and 4-nitrophenol, probes for CYP2E1, inhibited the formation of 3-MX, 7-MX and 3,7-DMU by ≈55–60%, 35–55% and 85%, respectively. Consistent with the microsomal studies, recombinant CYP1A2 and CYP2E1 exhibited similar apparent Km values for 7-MX formation and CYP2E1 was further shown to have the capacity to convert TB to both 3-MX and 3,7-DMU. Conclusions Given the contribution of multiple isoforms to 3-MX and 7-MX formation and the negligible formation of 3,7-DMU in vivo, TB is of little value as a CYP isoform-selective substrate in humans. PMID:10215755

  12. Inhibition of the human liver microsomal and human cytochrome P450 1A2 and 3A4 metabolism of estradiol by deployment-related and other chemicals.

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    Usmani, Khawja A; Cho, Taehyeon M; Rose, Randy L; Hodgson, Ernest

    2006-09-01

    Cytochromes P450 (P450s) are major catalysts in the metabolism of xenobiotics and endogenous substrates such as estradiol (E2). It has previously been shown that E2 is predominantly metabolized in humans by CYP1A2 and CYP3A4 with 2-hydroxyestradiol (2-OHE2) the major metabolite. This study examines effects of deployment-related and other chemicals on E2 metabolism by human liver microsomes (HLM) and individual P450 isoforms. Kinetic studies using HLM, CYP3A4, and CYP1A2 showed similar affinities (Km) for E2 with respect to 2-OHE2 production. Vmax and CLint values for HLM are 0.32 nmol/min/mg protein and 7.5 microl/min/mg protein; those for CYP3A4 are 6.9 nmol/min/nmol P450 and 291 microl/min/nmol P450; and those for CYP1A2 are 17.4 nmol/min/nmol P450 and 633 microl/min/nmol P450. Phenotyped HLM use showed that individuals with high levels of CYP1A2 and CYP3A4 have the greatest potential to metabolize E2. Preincubation of HLM with a variety of chemicals, including those used in military deployments, resulted in varying levels of inhibition of E2 metabolism. The greatest inhibition was observed with organophosphorus compounds, including chlorpyrifos and fonofos, with up to 80% inhibition for 2-OHE2 production. Carbaryl, a carbamate pesticide, and naphthalene, a jet fuel component, inhibited ca. 40% of E2 metabolism. Preincubation of CYP1A2 with chlorpyrifos, fonofos, carbaryl, or naphthalene resulted in 96, 59, 84, and 87% inhibition of E2 metabolism, respectively. Preincubation of CYP3A4 with chlorpyrifos, fonofos, deltamethrin, or permethrin resulted in 94, 87, 58, and 37% inhibition of E2 metabolism. Chlorpyrifos inhibition of E2 metabolism is shown to be irreversible.

  13. Inhibitory and inductive effects of Phikud Navakot extract on human cytochrome P450.

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    Chiangsom, Abhiruj; Lawanprasert, Somsong; Oda, Shingo; Kulthong, Kornphimol; Luechapudiporn, Rataya; Yokoi, Tsuyoshi; Maniratanachote, Rawiwan

    2016-06-01

    Effects of the hydroethanolic extract of Phikud Navakot (PN), a Thai traditional remedy, on human cytochrome P450s (CYPs) were investigated in vitro. Selective substrates of CYPs were used to investigate the effects and kinetics of PN on CYP inhibition using human liver microsomes. Primary human hepatocytes were used to assess the inductive effects of PN on CYP enzyme activities and protein expressions. The results showed that PN inhibited the activities of CYP1A2, CYP2C9, CYP2D6, and CYP3A4 with half maximal inhibitory concentration (IC50) values of 13, 62, 67, and 88 μg/mL, respectively. Meanwhile, it had no effect on the activities of CYP2C19 and CYP2E1 (IC50 > 1 mg/mL). PN exhibited competitive inhibition of CYP1A2 (Ki = 34 μg/mL), mixed type inhibition of CYP2C9 and CYP2D6 (Ki = 80 and 12 μg/mL, respectively), and uncompetitive inhibition of CYP3A4 (Ki = 150 μg/mL). PN did not have an inductive effect on CYP1A2, CYP2C9, CYP2C19 and CYP3A4 in primary human hepatocytes, which is an advantageous characteristic of the extract. However the extract may cause herb-drug interactions via inhibition of CYP1A2, CYP2C9, CYP2D6 and CYP3A4, and precautions should be taken when PN is coadministered with drugs that are metabolized by these CYP enzymes. Copyright © 2016 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.

  14. The impact of Cytochrome P450 CYP1A2, CYP2C9, CYP2C19 and CYP2D6 genes on suicide attempt and suicide risk-a European multicentre study on treatment-resistant major depressive disorder.

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    Höfer, Peter; Schosser, Alexandra; Calati, Raffaella; Serretti, Alessandro; Massat, Isabelle; Kocabas, Neslihan Aygun; Konstantinidis, Anastasios; Linotte, Sylvie; Mendlewicz, Julien; Souery, Daniel; Zohar, Joseph; Juven-Wetzler, Alzbeta; Montgomery, Stuart; Kasper, Siegfried

    2013-08-01

    Recently published data have reported associations between cytochrome P450 metabolizer status and suicidality. The aim of our study was to investigate the role of genetic polymorphisms of the cytochrome P450 genes on suicide risk and/or a personal history of suicide attempts. Two hundred forty-three major depressive disorder patients were collected in the context of a European multicentre resistant depression study and treated with antidepressants at adequate doses for at least 4 weeks. Suicidality was assessed using the Mini International Neuropsychiatric Interview and the Hamilton Rating Scale for Depression (HAM-D). Treatment response was defined as HAM-D ≤ 17 and remission as HAM-D ≤ 7 after 4 weeks of treatment with antidepressants at adequate dose. Genotyping was performed for all relevant variations of the CYP1A2 gene (*1A, *1F, *1C, *1 J, *1 K), the CYP2C9 gene (*2, *3), the CYP2C19 gene (*2, *17) and the CYP2D6 gene (*3, *4, *5, *6, *9, *19, *XN). No association between both suicide risk and personal history of suicide attempts, and the above mentioned metabolic profiles were found after multiple testing corrections. In conclusion, the investigated cytochrome gene polymorphisms do not seem to be associated with suicide risk and/or a personal history of suicide attempts, though methodological and sample size limitations do not allow definitive conclusions.

  15. Expression of a Human Cytochrome P450 in Yeast Permits Analysis of Pathways for Response to and Repair of Aflatoxin-Induced DNA Damage†

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    Guo, Yingying; Breeden, Linda L.; Zarbl, Helmut; Preston, Bradley D.; Eaton, David L.

    2005-01-01

    Aflatoxin B1 (AFB1) is a human hepatotoxin and hepatocarcinogen produced by the mold Aspergillus flavus. In humans, AFB1 is primarily bioactivated by cytochrome P450 1A2 (CYP1A2) and 3A4 to a genotoxic epoxide that forms N7-guanine DNA adducts. A series of yeast haploid mutants defective in DNA repair and cell cycle checkpoints were transformed with human CYP1A2 to investigate how these DNA adducts are repaired. Cell survival and mutagenesis following aflatoxin B1 treatment was assayed in str...

  16. Inhibition selectivity of grapefruit juice components on human cytochromes P450.

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    Tassaneeyakul, W; Guo, L Q; Fukuda, K; Ohta, T; Yamazoe, Y

    2000-06-15

    Five compounds including furanocoumarin monomers (bergamottin, 6', 7'-dihydroxybergamottin (DHB)), furanocoumarin dimers (4-¿¿6-hydroxy-71-¿(1-hydroxy-1-methyl)ethyl-4-methyl-6-(7-oxo-7H- furo¿3,2-g1benzopyran-4-yl)-4-hexenyl]oxy]-3,7-dimethyl- 2-octenyl]oxy]-7H-furo[3,2-g]¿1benzopyran-7-one (GF-I-1) and 4-¿¿6-hydroxy-7¿¿4-methyl-1-(1-methylethenyl)-6-(7-oxo-7H-furo¿3, 2-g1benzopyran-4-yl)-4-hexenylŏxy-3, 7-dimethyl-2-octenylŏxy-7H-furo¿3,2-g1benzopyran-7-one (GF-I-4)), and a sesquiterpene nootkatone have been isolated from grapefruit juice and screened for their inhibitory effects toward human cytochrome P450 (P450) forms using selective substrate probes. Addition of ethyl acetate extract of grapefruit juice into an incubation mixture resulted in decreased activities of CYP3A4, CYP1A2, CYP2C9, and CYP2D6. All four furanocoumarins clearly inhibited CYP3A4-catalyzed nifedipine oxidation in concentration- and time-dependent manners, suggesting that these compounds are mechanism-based inhibitors of CYP3A4. Of the furanocoumarins investigated, furanocoumarin dimers, GF-I-1 and GF-I-4, were the most potent inhibitors of CYP3A4. Inhibitor concentration required for half-maximal rate of inactivation (K(I)) values for bergamottin, DHB, GF-I-1, and GF-I-4 were calculated, respectively, as 40.00, 5. 56, 0.31, and 0.13 microM, whereas similar values were observed on their inactivation rate constant at infinite concentration of inhibitor (k(inact), 0.05-0.08 min(-1)). Apparent selectivity toward CYP3A4 does occur with the furanocoumarin dimers. In contrast, bergamottin showed rather stronger inhibitory effect on CYP1A2, CYP2C9, CYP2C19, and CYP2D6 than on CYP3A4. DHB inhibited CYP3A4 and CYP1A2 activities at nearly equivalent potencies. Among P450 forms investigated, CYP2E1 was the least sensitive to the inhibitory effect of furanocoumarin components. A sesquiterpene nootkatone has no significant effect on P450 activities investigated except for CYP2A6 and CYP2C19

  17. A COMPARISON OF THE METABOLISM OF METHOXYRESORUFIN, ACETANILIDE AND CAFFIENE IN RAT AND HUMAN CYP1A2 SUPERSOMES AND THEIR INHIBITION BY 2, 3, 7, 8-TETRACHLORODIBENZO-P-DIOXIN (TCDD)

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    A COMPARISON OF THE METABOLISM OF METHOXYRESORUFIN, ACETANILIDE AND CAFFIENE IN RAT AND HUMAN CYP1A2 SUPERSOMES AND THEIR INHIBITION BY 2,3,7,8-TETRACHLORODIBENZO-P-DIOXIN (TCDD). DF Staskal1, DG Ross2, LS Birnbaum2 and MJ DeVito2 1Curriculum In Toxicology, UNC-CH, Chapel Hill ...

  18. Metabolism of ethylbenzene by human liver microsomes and recombinant human cytochrome P450s (CYP).

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    Sams, Craig; Loizou, George D; Cocker, John; Lennard, Martin S

    2004-03-07

    The enzyme kinetics of the initial hydroxylation of ethylbenzene to form 1-phenylethanol were determined in human liver microsomes. The individual cytochrome P450 (CYP) forms catalysing this reaction were identified using selective inhibitors and recombinant preparations of hepatic CYPs. Production of 1-phenylethanol in hepatic microsomes exhibited biphasic kinetics with a high affinity, low Km, component (mean Km = 8 microM; V(max) = 689 pmol/min/mg protein; n = 6 livers) and a low affinity, high Km, component (Km = 391 microM; V(max) = 3039 pmol/min/mg protein; n = 6). The high-affinity component was inhibited 79%-95% (mean 86%) by diethyldithiocarbamate, and recombinant CYP2E1 was shown to metabolise ethylbenzene with low Km (35 microM), but also low (max) (7 pmol/min/pmol P450), indicating that this isoform catalysed the high-affinity component. Recombinant CYP1A2 and CYP2B6 exhibited high V(max) (88 and 71 pmol/min/pmol P450, respectively) and high Km (502 and 219 microM, respectively), suggesting their involvement in catalysing the low-affinity component. This study has demonstrated that CYP2E1 is the major enzyme responsible for high-affinity side chain hydroxylation of ethylbenzene in human liver microsomes. Activity of this enzyme in the population is highly variable due to induction or inhibition by physiological factors, chemicals in the diet or some pharmaceuticals. This variability can be incorporated into the risk assessment process to improve the setting of occupational exposure limits and guidance values for biological monitoring.

  19. Inhibition of Human Cytochrome P450 Enzymes by Allergen Removed Rhus verniciflua Stoke Standardized Extract and Constituents

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    Hyunsik Jung

    2014-01-01

    Full Text Available Objective. Potential interactions between herbal extracts and the cytochrome P450 (CYP system lead to serious adverse events or decreased drug efficacy. Rhus verniciflua stoke (RVS and its constituents have been reported to have various pharmacological properties. We evaluated the inhibitory potential of RVS and its constituents on the major CYP isoforms. Methods. The effects of allergen removed RVS (aRVS standardized extract and major components, fustin and fisetin isolated from aRVS, were evaluated on CYP1A2, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 isoenzyme activity by a luminescent CYP recombinant human enzyme assay. Results. The aRVS extract showed relative potent inhibitory effects on the CYP2C9 (IC50, <0.001 μg/mL, CYP2C19 (IC50, 9.68 μg/mL, and CYP1A2 (IC50, 10.0 μg/mL. However, it showed weak inhibition on CYP3A4 and CYP2D6. Fustin showed moderate inhibitory effects on the CYP2C19 (IC50, 64.3 μg/mL and weak inhibition of the other CYP isoforms similar to aRVS. Fisetin showed potent inhibitory effects on CYP2C9, CYP2C19, and CYP1A2. Fisetin showed moderate inhibition of CYP2D6 and weak inhibition of CYP3A4. Conclusions. These results indicate that aRVS, a clinically available herbal medicine, could contribute to herb-drug interactions when orally coadministered with drugs metabolized by CYP2C9, CYP2C19, and CYP1A2.

  20. Human cytochrome P450 and personalized medicine.

    Science.gov (United States)

    Chen, Qi; Wei, Dongqing

    2015-01-01

    Personalized medicine has become a hot topic ascribed to the development of Human Genome Project. And currently, bioinformatics methodology plays an essential role in personal drug design. Here in this review we mainly focused on the basic introduction of the SNPs of human drug metabolic enzymes and their relationships with personalized medicine. Some common bioinformatics analysis methods and latest progresses and applications in personal drug design have also been discussed. Thus bioinformatics studies on SNPs of human CYP450 genes will contribute to indicate the most possible genes that are associated with human diseases and relevant therapeutic targets, identify and predict the drug efficacy and adverse drug response, investigate individual gene specific properties and then provide personalized and optimal clinic therapies.

  1. Identification of human cytochrome P450s as autoantigens.

    Science.gov (United States)

    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.

  2. Effect of zolpidem on human cytochrome P450 activity, and on transport mediated by P-glycoprotein.

    Science.gov (United States)

    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.

  3. In vitro inhibitory mechanisms and molecular docking of 1'-S-1'-acetoxychavicol acetate on human cytochrome P450 enzymes.

    Science.gov (United States)

    Haque, A K M Mahmudul; Leong, Kok Hoong; Lo, Yoke Lin; Awang, Khalijah; Nagoor, Noor Hasima

    2017-07-15

    The compound, 1'-S-1'-acetoxychavicol acetate (ACA), isolated from the rhizomes of a Malaysian ethno-medicinal plant, Alpinia conchigera Griff. (Zingiberaceae), was previously shown to have potential in vivo antitumour activities. In the development of a new drug entity, potential interactions of the compound with the cytochrome P450 superfamily metabolizing enzymes need to be ascertain. The concomitant use of therapeutic drugs may cause potential drug-drug interactions by decreasing or increasing plasma levels of the administered drugs, leading to a suboptimal clinical efficacy or a higher risk of toxicity. Thus, evaluating the inhibitory potential of a new chemical entity, and to clarify the mechanism of inhibition and kinetics in the various CYP enzymes is an important step to predict drug-drug interactions. This study was designed to assess the potential inhibitory effects of Alpinia conchigera Griff. rhizomes extract and its active constituent, ACA, on nine c-DNA expressed human cytochrome P450s (CYPs) enzymes using fluorescent CYP inhibition assay. The half maximal inhibitory concentration (IC 50 ) of Alpinia conchigera Griff. rhizomes extract and ACA was determined for CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C19, CYP2D6, CYP2E1, CYP3A4 and CYP3A5. A. conchigera extract only moderately inhibits on CYP3A4 (IC 50 = 6.76 ± 1.88µg/ml) whereas ACA moderately inhibits the activities of CYP1A2 (IC 50 = 4.50 ± 0.10µM), CYP2D6 (IC 50 = 7.50 ± 0.17µM) and CYP3A4 (IC 50 = 9.50 ± 0.57µM) while other isoenzymes are weakly inhibited. In addition, mechanism-based inhibition studies reveal that CYP1A2 and CYP3A4 exhibited non-mechanism based inhibition whereas CYP2D6 showed mechanism-based inhibition. Lineweaver-Burk plots depict that ACA competitively inhibited both CYP1A2 and CYP3A4, with a K i values of 2.36 ± 0.03 µM and 5.55 ± 0.06µM, respectively, and mixed inhibition towards CYP2D6 with a K i value of 4.50 ± 0.08µ

  4. In vitro inhibitory effects of plumbagin, the promising antimalarial candidate, on human cytochrome P450 enzymes.

    Science.gov (United States)

    Sumsakul, Wiriyaporn; Chaijaroenkul, Wanna; Na-Bangchang, Kesara

    2015-11-01

    To investigate the propensity of plumbagin to inhibit the three isoforms of human cytochrome P450 (CYP), i.e., CYP1A2, CYP2C19, and CYP3A4 using human liver microsomes in vitro. Inhibitory effects of plumbagin on the three human CYP isoforms were investigated using pooled human liver microsomes. Phenacetin O-deethylation, omeprazole hydroxylation and nifedipine oxidation were used as selective substrates for CYP1A2, CYP2C19 and CYP3A4 activities, respectively. Concentrations of paracetamol, 5-hydroxyomeprazole, and oxidized nifedipine were determined in microsomal incubation mixture using high-performance liquid chromatography. Plumbagin showed significant inhibitory effects on all CYP isoforms, but with the most potent activity on CYP2C19-mediated omeprazole hydroxylation. The IC50 (concentration that inhibits enzyme activity by 50%) values of plumbagin and nootkatone (selective inhibitor) for CYP2C19 were (0.78 ± 0.01) and (27.31 ± 0.66) μM, respectively. The inhibitory activities on CYP1A2-mediated phenacetin O-deethylation and CYP3A4-mediated nifedipine oxidation were moderate. The IC50 values of plumbagin and α-naphthoflavone (selective inhibitor) for CYP1A2 were (1.39 ± 0.01) and (0.02 ± 0.36) μM, respectively. The corresponding IC50 values of plumbagin and ketoconazole (selective inhibitor) for CYP3A4 were (2.37 ± 0.10) and (0.18 ± 0.06) μM, respectively. Clinical relevance of the interference of human drug metabolizing enzymes should be aware of for further development scheme of plumbagin as antimalarial drug when used in combination with other antimalarial drugs which are metabolized by these CYP isoforms. Copyright © 2015 Hainan Medical College. Production and hosting by Elsevier B.V. All rights reserved.

  5. Interaction of rocuronium with human liver cytochromes P450

    OpenAIRE

    Anzenbacherova, Eva; Spicakova, Alena; Jourova, Lenka; Ulrichova, Jitka; Adamus, Milan; Bachleda, Petr; Anzenbacher, Pavel

    2015-01-01

    Rocuronium is a neuromuscular blocking agent acting as a competitive antagonist of acetylcholine. Results of an inhibition of eight individual liver microsomal cytochromes P450 (CYP) are presented. As the patients are routinely premedicated with diazepam, possible interaction of diazepam with rocuronium has been also studied. Results indicated that rocuronium interacts with human liver microsomal CYPs by binding to the substrate site. Next, concentration dependent inhibition of liver micro...

  6. Inhibitory effects of cytostatically active 6-aminobenzo[c]phenanthridines on cytochrome P450 enzymes in human hepatic microsomes.

    Science.gov (United States)

    Zebothsen, Inga; Kunze, Thomas; Clement, Bernd

    2006-07-01

    Besides assays for the evaluation of efficacy new drug candidates have to undergo extensive testings for enhancement of pharmaceutical drug safety and optimization of application. The objective of the present work was to investigate the pharmacokinetic drug drug interaction potential for the cytostatically active 6-aminobenzo[c]phenanthridines BP-11 (6-amino-11,12-dihydro-11-(4-hydroxy-3,5-dimethoxyphenyl)benzo[c]phenanthridine) and BP-D7 (6-amino-11-(3,4,5-trimethoxyphenyl)benzo[c]phenanthridine) in vitro through incubation with human hepatic microsomes and marker substrates. For these studies the cytochrome P-450 isoenzymes and corresponding marker substrates recommended by the EMEA (The European Agency for the Evaluation of Medicinal Products) were chosen. In detail these selective substrates were caffeine (CYP1A2), coumarin (CYP2A6), tolbutamide (CYP2C9), S-(+)-mephenytoin (CYP2C19), dextromethorphane (CYP2D6), chlorzoxazone (CYP2E1) and testosterone (CYP3A4). Incubations with each substrate were carried out without a possible inhibitor and in the presence of a benzo[c]phenanthridine or a selective inhibitor at varying concentrations. Marker activities were determined by HPLC (high performance liquid chromatography). For the isoenzymes showing more than 50% inhibition by the addition of 20 microM BP-11 or BP-D7 additional concentrations of substrate and inhibitor were tested for a characterization of the inhibition. The studies showed a moderate risk for BP-11 for interactions with the cytochrome P-450 isoenzymes CYP1A2, CYP2C9, CYP2D6 and CYP3A4. BP-D7, the compound with the highest cytotstatic efficacy, showed only a moderate risk for interactions with drugs, also metabolized by CYP3A4.

  7. Coupled motions direct electrons along human microsomal P450 Chains.

    Directory of Open Access Journals (Sweden)

    Christopher R Pudney

    2011-12-01

    Full Text Available Protein domain motion is often implicated in biological electron transfer, but the general significance of motion is not clear. Motion has been implicated in the transfer of electrons from human cytochrome P450 reductase (CPR to all microsomal cytochrome P450s (CYPs. Our hypothesis is that tight coupling of motion with enzyme chemistry can signal "ready and waiting" states for electron transfer from CPR to downstream CYPs and support vectorial electron transfer across complex redox chains. We developed a novel approach to study the time-dependence of dynamical change during catalysis that reports on the changing conformational states of CPR. FRET was linked to stopped-flow studies of electron transfer in CPR that contains donor-acceptor fluorophores on the enzyme surface. Open and closed states of CPR were correlated with key steps in the catalytic cycle which demonstrated how redox chemistry and NADPH binding drive successive opening and closing of the enzyme. Specifically, we provide evidence that reduction of the flavin moieties in CPR induces CPR opening, whereas ligand binding induces CPR closing. A dynamic reaction cycle was created in which CPR optimizes internal electron transfer between flavin cofactors by adopting closed states and signals "ready and waiting" conformations to partner CYP enzymes by adopting more open states. This complex, temporal control of enzyme motion is used to catalyze directional electron transfer from NADPH→FAD→FMN→heme, thereby facilitating all microsomal P450-catalysed reactions. Motions critical to the broader biological functions of CPR are tightly coupled to enzyme chemistry in the human NADPH-CPR-CYP redox chain. That redox chemistry alone is sufficient to drive functionally necessary, large-scale conformational change is remarkable. Rather than relying on stochastic conformational sampling, our study highlights a need for tight coupling of motion to enzyme chemistry to give vectorial electron

  8. Human HepaRG Cells can be Cultured in Hanging-drop Plates for Cytochrome P450 Induction and Function Assays.

    Science.gov (United States)

    Murayama, Norie; Usui, Takashi; Slawny, Nicky; Chesné, Christophe; Yamazaki, Hiroshi

    2015-01-01

    Recent guidance/guidelines for industry recommend that cytochrome P450 induction can be assessed using human hepatocyte enzyme activity and/or mRNA levels to evaluate potential drug- drug interactions. To evaluate time-dependent cytochrome P450 induction precisely, induction of CYP1A2, CYP2B6, and CYP3A4 mRNA was confirmed (>2-fold) by the treatment with omeprazole, phenobarbital, and rifampicin, respectively, for 24 or 48 h on day 3 from the start of culture. After 24 h, the fold induction of CYP1A2 with 3.6 and 1.8x10(4) HepaRG cells per well was lower than that for 7.2x10(4) cells. CYP1A2 induction levels at 24 h were higher than those after 48 h. In contrast, higher CYP2B6 inductions were confirmed after 48 h exposure than after 24 h, independent of the number of cells per well. To help reduce the use of human cryopreserved hepatocytes, typical P450-dependent enzyme activities were investigated in human HepaRG cells cultured in commercial hanging-drop plates. Newly designed 96-well hanging-drop plates were capable of maintaining human CYP3A-dependent midazolam hydroxylation activities for up to 4 days using only 10% of the recommended initial 7.2x10(4) cells per well. Favorable HepaRG function using hanging-drop plates was confirmed by detecting 1'- hydroxymidazolam O-glucuronide on day 3, suggesting an improvement over traditional control plates in which this metabolite can be detected for 24-well plates. These results suggest that the catalytic function and/or induction of CYP1A2, CYP2B6, and CYP3A4 can be readily assessed with reduced numbers of starting HepaRG cells cultured in three-dimensional cultures in drops prepared with hanging-drop plates.

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

    Directory of Open Access Journals (Sweden)

    Ju-Hyun Kim

    2017-03-01

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

  10. Interaction of rocuronium with human liver cytochromes P450.

    Science.gov (United States)

    Anzenbacherova, Eva; Spicakova, Alena; Jourova, Lenka; Ulrichova, Jitka; Adamus, Milan; Bachleda, Petr; Anzenbacher, Pavel

    2015-02-01

    Rocuronium is a neuromuscular blocking agent acting as a competitive antagonist of acetylcholine. Results of an inhibition of eight individual liver microsomal cytochromes P450 (CYP) are presented. As the patients are routinely premedicated with diazepam, possible interaction of diazepam with rocuronium has been also studied. Results indicated that rocuronium interacts with human liver microsomal CYPs by binding to the substrate site. Next, concentration dependent inhibition of liver microsomal CYP3A4 down to 42% (at rocuronium concentration 189 μM) was found. This effect has been confirmed with two CYP3A4 substrates, testosterone (formation of 6β-hydroxytestosterone) and diazepam (temazepam formation). CYP2C9 and CYP2C19 activities were inhibited down to 75-80% (at the same rocuronium concentration). Activities of other microsomal CYPs have not been inhibited by rocuronium. To prove the possibility of rocuronium interaction with other drugs (diazepam), the effect of rocuronium on formation of main diazepam metabolites, temazepam (by CYP3A4) and desmethyldiazepam, (also known as nordiazepam; formed by CYP2C19) in primary culture of human hepatocytes has been examined. Rocuronium has caused inhibition of both reactions by 20 and 15%, respectively. The results open a possibility that interactions of rocuronium with drugs metabolized by CYP3A4 (and possibly also CYP2C19) may be observed. Copyright © 2014 Japanese Pharmacological Society. Production and hosting by Elsevier B.V. All rights reserved.

  11. Gene-Environment Interaction in Parkinson's Disease: Coffee, ADORA2A, and CYP1A2.

    Science.gov (United States)

    Chuang, Yu-Hsuan; Lill, Christina M; Lee, Pei-Chen; Hansen, Johnni; Lassen, Christina F; Bertram, Lars; Greene, Naomi; Sinsheimer, Janet S; Ritz, Beate

    2016-01-01

    Drinking caffeinated coffee has been reported to provide protection against Parkinson's disease (PD). Caffeine is an adenosine A2A receptor (encoded by the gene ADORA2A) antagonist that increases dopaminergic neurotransmission and Cytochrome P450 1A2 (gene: CYP1A2) metabolizes caffeine; thus, gene polymorphisms in ADORA2A and CYP1A2 may influence the effect coffee consumption has on PD risk. In a population-based case-control study (PASIDA) in Denmark (1,556 PD patients and 1,606 birth year- and gender-matched controls), we assessed interactions between lifetime coffee consumption and 3 polymorphisms in ADORA2A and CYP1A2 for all subjects, and incident and prevalent PD cases separately using logistic regression models. We also conducted a meta-analysis combining our results with those from previous studies. We estimated statistically significant interactions for ADORA2A rs5760423 and heavy vs. light coffee consumption in incident (OR interaction = 0.66 [95% CI 0.46-0.94], p = 0.02) but not prevalent PD. We did not observe interactions for CYP1A2 rs762551 and rs2472304 in incident or prevalent PD. In meta-analyses, PD associations with daily coffee consumption were strongest among carriers of variant alleles in both ADORA2A and CYP1A2. We corroborated results from a previous report that described interactions between ADORA2A and CYP1A2 polymorphisms and coffee consumption. Our results also suggest that survivor bias may affect results of studies that enroll prevalent PD cases. © 2017 S. Karger AG, Basel.

  12. Potent inhibition of cytochrome P450 2B6 by sibutramine in human liver microsomes.

    Science.gov (United States)

    Bae, Soo Hyeon; Kwon, Min Jo; Choi, Eu Jin; Zheng, Yu Fen; Yoon, Kee Dong; Liu, Kwang-Hyeon; Bae, Soo Kyung

    2013-09-05

    The present study was performed to evaluate the potency and specificity of sibutramine as an inhibitor of the activities of nine human CYP isoforms in liver microsomes. Using a cocktail assay, the effects of sibutramine on specific marker reactions of the nine CYP isoforms were measured in human liver microsomes. Sibutramine showed potent inhibition of CYP2B6-mediated bupropion 6-hydroxylation with an IC50 value of 1.61μM and Ki value of 0.466μM in a competitive manner at microsomal protein concentrations of 0.25mg/ml; this was 3.49-fold more potent than the typical CYP2B6 inhibitor thio-TEPA (Ki=1.59μM). In addition, sibutramine slightly inhibited CYP2C19 activity (Ki=16.6μM, noncompetitive inhibition) and CYP2D6 activity (Ki=15.7μM, noncompetitive inhibition). These observations indicated 35.6- and 33.7-fold decreases in inhibition potency, respectively, compared with that of CYP2B6 by sibutramine. However, no inhibition of CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2D6, or CYP2E1 activities was observed. In addition, the CYP2B6 inhibitory potential of sibutramine was enhanced at a lower microsomal protein concentration of 0.05mg/ml. After 30min preincubation of human liver microsomes with sibutramine in the presence of NADPH, no shift in IC50 was observed in terms of inhibition of the activities of the nine CYPs, suggesting that sibutramine is not a time-dependent inactivator. These observations suggest that sibutramine is a selective and potent inhibitor of CYP2B6 in vitro, whereas inhibition of other CYPs is substantially lower. These in vitro data support the use of sibutramine as a well-known inhibitor of CYP2B6 for routine screening of P450 reversible inhibition when human liver microsomes are used as the enzyme source. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  13. Hexachlorobenzene stimulates uroporphyria in low affinity AHR mice without increasing CYP1A2

    International Nuclear Information System (INIS)

    Gorman, Nadia; Trask, Heidi S.; Robinson, Susan W.; Sinclair, Jacqueline F.; Gerhard, Glenn S.; Smith, Andrew G.; Sinclair, Peter R.

    2007-01-01

    Hexachlorobenzene (HCB), a weak ligand of the aryl hydrocarbon receptor (AHR), causes hepatic uroporphyrin (URO) accumulation (uroporphyria) in humans and animals. CYP1A2 has been shown to be necessary in the development of uroporphyria in mice. Using mice expressing the low affinity form of the AH receptor (AHRd), we investigated whether the enhancement of uroporphyria by HCB involves an obligatory increase in CYP1A2 as measured by specific enzyme assays and immunoblotting. We compared the ability of HCB, in combination with iron dextran and the porphyrin precursor, 5-aminolevulinate (ALA), to cause uroporphyria in a strain of mice (C57BL/6) which expresses the high affinity form of the receptor (AHRb 1 ), with three strains of mice (SWR and two 129 sublines) expressing the low affinity AHRd. In C57BL/6 mice, HCB-enhanced uroporphyria was associated with a doubling of CYP1A2. HCB treatment produced uroporphyria in iron-loaded mice expressing AHRd, even though there was little or no increase in CYP1A2. Cyp1a2(-/-) mice in a 129 background were completely resistant to HCB-induced uroporphyria, and female Hfe(-/-) 129 mice, in which the levels of hepatic CYP1A2 were half of those of the male levels, responded poorly. The effect of exogenous iron, administered in the form of iron dextran, on HCB enhancement of uroporphryia could be replicated utilizing the endogenous hepatic iron accumulated in 129 Hfe(-/-) mice. In conclusion, some minimal basal expression of CYP1A2 is essential for HCB-mediated enhancement of uroporphyria, but increases in CYP1A2 above that level are not essential

  14. Immunohistochemical detection of cytochrome P450 isoenzymes in cultured human epidermal cells.

    Science.gov (United States)

    Van Pelt, F N; Meierink, Y J; Blaauboer, B J; Weterings, P J

    1990-12-01

    We used specific monoclonal antibodies (MAb) to human cytochrome P450 isoenzymes to determine the presence of these proteins in human epidermal cells. Two MAb (P450-5 and P450-8) recognize major forms of hepatic cytochrome P450 involved in biotransformation of xenobiotics. A third MAb, to cytochrome P450-9, is not fully characterized. The proteins were determined by the indirect immunoperoxidase technique after fixation with methanol and acetone. Biopsy materials for cultured keratinocytes, i.e., foreskin and hair follicles, contained the two major forms of cytochrome P450. In cultured keratinocytes derived from hair follicles the proteins were undetectable, whereas the keratinocytes derived from foreskin continued to express the two major forms of hepatic cytochrome P450. Cultured human fibroblasts and a human keratinocyte cell line (SVK14) showed staining similar to that of the foreskin keratinocytes. Cytochrome P450-9 was detectable only in human hepatocytes. The results indicate that, under the culture conditions applied, cultured human foreskin cells and the cell line SVK14 continue to express specific cytochrome P450 isoenzymes in culture, in contrast to hair follicle keratinocytes.

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

    Science.gov (United States)

    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.

  16. In vivo assessment of botanical supplementation on human cytochrome P450 phenotypes: Citrus aurantium, Echinacea purpurea, milk thistle, and saw palmetto.

    Science.gov (United States)

    Gurley, Bill J; Gardner, Stephanie F; Hubbard, Martha A; Williams, D Keith; Gentry, W Brooks; Carrier, Julie; Khan, Ikhlas A; Edwards, David J; Shah, Amit

    2004-11-01

    Phytochemical-mediated modulation of cytochrome P450 (CYP) activity may underlie many herb-drug interactions. Single-time point phenotypic metabolic ratios were used to determine whether long-term supplementation of Citrus aurantium , Echinacea purpurea , milk thistle (Silybum marianum), or saw palmetto (Serenoa repens) extracts affected CYP1A2, CYP2D6, CYP2E1, or CYP3A4 activity. Twelve healthy volunteers (6 women, 6 men) were randomly assigned to receive C aurantium , E purpurea , milk thistle, or saw palmetto for 28 days. For each subject, a 30-day washout period was interposed between each supplementation phase. Probe drug cocktails of midazolam and caffeine, followed 24 hours later by chlorzoxazone and debrisoquin (INN, debrisoquine), were administered before (baseline) and at the end of supplementation. Presupplementation and postsupplementation phenotypic trait measurements were determined for CYP3A4, CYP1A2, CYP2E1, and CYP2D6 by use of 1-hydroxymidazolam/midazolam serum ratios (1-hour sample), paraxanthine/caffeine serum ratios (6-hour sample), 6-hydroxychlorzoxazone/chlorzoxazone serum ratios (2-hour sample), and debrisoquin urinary recovery ratios (8-hour collection), respectively. The content of purported "active" phytochemicals was determined for each supplement. Comparisons of presupplementation and postsupplementation phenotypic ratios suggested that these particular supplements had no significant effect on CYP1A2, CYP2D6, CYP2E1, or CYP3A4 activity. Phytochemical profiles indicated that C aurantium was devoid of the CYP3A4 inhibitor 6',7'-dihydroxybergamottin. Quantities of fatty acids, flavonolignans, and cichoric acid were consistent with label claims for saw palmetto, milk thistle, and E purpurea , respectively. Botanical supplements containing C aurantium , milk thistle, or saw palmetto extracts appear to pose a minimal risk for CYP-mediated herb-drug interactions in humans. Although the effects of E purpurea on CYP activity were minor, further

  17. Mangifera indica L. extract and mangiferin modulate cytochrome P450 and UDP-glucuronosyltransferase enzymes in primary cultures of human hepatocytes.

    Science.gov (United States)

    Rodeiro, Idania; José Gómez-Lechón, M; Perez, Gabriela; Hernandez, Ivones; Herrera, José Alfredo; Delgado, Rene; Castell, José V; Teresa Donato, M

    2013-05-01

    The aqueous stem bark extract of Mangifera indica L. (MSBE) has been reported to have antioxidant, anti-inflammatory and analgesic properties. In previous studies, we showed that MSBE and mangiferin, its main component, lower the activity of some cytochrome P-450 (P450) enzymes in rat hepatocytes and human liver microsomes. In the present study, the effects of MSBE and mangiferin on several P450 enzymes and UDP-glucuronosyltransferases (UGTs) in human-cultured hepatocytes have been examined. After hepatocytes underwent a 48-h treatment with sub-cytotoxic concentrations of the products (50-250 µg/mL), a concentration-dependent decrease of the activity of the five P450 enzymes measured (CYP1A2, 2A6, 2C9, 2D6 and 3A4) was observed. For all the activities, a reduction of at least 50% at the highest concentration (250 µg/mL) was observed. In addition, UGT activities diminished. MSBE considerably reduced UGT1A9 activity (about 60% at 250 µg/mL) and lesser effects on the other UGTs. In contrast, 250 µg/mL mangiferin had greater effects on UGT1A1 and 2B7 than on UGT1A9 (about 55% vs. 35% reduction, respectively). Quantification of specific mRNAs revealed reduced CYP3A4 and 3A5 mRNAs content, and an increase in CYP1A1, CYP1A2, UGT1A1 and UGT1A9 mRNAs. No remarkable effects on the CYP2A6, 2B6, 2C9, 2C19, 2D6 and 2E1 levels were observed. Our results suggest that the activity and/or expression of major P450 and UGT enzymes is modulated by MSBE and that potential herb-drugs interactions could arise after a combined intake of this extract with conventional medicines. Therefore, the potential safety risks of this natural product derived by altering the ADMET properties of co-administered drugs should be examined. Copyright © 2012 John Wiley & Sons, Ltd.

  18. Inhibitory Effects of Dimethyllirioresinol, Epimagnolin A, Eudesmin, Fargesin, and Magnolin on Cytochrome P450 Enzyme Activities in Human Liver Microsomes

    Directory of Open Access Journals (Sweden)

    Ju-Hyun Kim

    2017-05-01

    Full Text Available Magnolin, epimagnolin A, dimethyllirioresinol, eudesmin, and fargesin are pharmacologically active tetrahydrofurofuranoid lignans found in Flos Magnoliae. The inhibitory potentials of dimethyllirioresinol, epimagnolin A, eudesmin, fargesin, and magnolin on eight major human cytochrome P450 (CYP enzyme activities in human liver microsomes were evaluated using liquid chromatography–tandem mass spectrometry to determine the inhibition mechanisms and inhibition potency. Fargesin inhibited CYP2C9-catalyzed diclofenac 4’-hydroxylation with a Ki value of 16.3 μM, and it exhibited mechanism-based inhibition of CYP2C19-catalyzed [S]-mephenytoin 4’-hydroxylation (Ki, 3.7 μM; kinact, 0.102 min−1, CYP2C8-catalyzed amodiaquine N-deethylation (Ki, 10.7 μM; kinact, 0.082 min−1, and CYP3A4-catalyzed midazolam 1’-hydroxylation (Ki, 23.0 μM; kinact, 0.050 min−1 in human liver microsomes. Fargesin negligibly inhibited CYP1A2-catalyzed phenacetin O-deethylation, CYP2A6-catalyzed coumarin 7-hydroxylation, CYP2B6-catalyzed bupropion hydroxylation, and CYP2D6-catalyzed bufuralol 1’-hydroxylation at 100 μM in human liver microsomes. Dimethyllirioresinol weakly inhibited CYP2C19 and CYP2C8 with IC50 values of 55.1 and 85.0 μM, respectively, without inhibition of CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2D6, and CYP3A4 activities at 100 μM. Epimagnolin A, eudesmin, and magnolin showed no the reversible and time-dependent inhibition of eight major CYP activities at 100 μM in human liver microsomes. These in vitro results suggest that it is necessary to investigate the potentials of in vivo fargesin-drug interaction with CYP2C8, CYP2C9, CYP2C19, and CYP3A4 substrates.

  19. Organophosphorothionate pesticides inhibit the bioactivation of imipramine by human hepatic cytochrome P450s

    International Nuclear Information System (INIS)

    Di Consiglio, Emma; Meneguz, Annarita; Testai, Emanuela

    2005-01-01

    The drug-toxicant interaction between the antidepressant imipramine (IMI) and three organophosphorothionate pesticides (OPTs), to which humans may be chronically and simultaneously exposed, has been investigated in vitro. Concentrations of IMI (2-400 μM) and OPTs (≤10 μM) representative of actual human exposure have been tested with recombinant human CYPs and human liver microsomes (HLM). The different CYPs involved in IMI demethylation to the pharmacologically active metabolite desipramine (DES) were CYP2C19 > CYP1A2 > CYP3A4. The OPTs significantly inhibited (up to >80%) IMI bioactivation catalyzed by the recombinant CYPs tested, except CYP2D6, and by HLM; the inhibition was dose-dependent and started at low pesticide concentrations (0.25-2.5 μM). The OPTs, having lower K m values, efficiently competed with IMI for the enzyme active site, as in the case of CYP2C19. However, with CYP1A2 and CYP3A4, a time- and NADPH-dependent mechanism-based inactivation also occurred, consistently with irreversible inhibition due to the release of the sulfur atom, binding to the active CYP during OPT desulfuration. At low IMI and OPT concentrations, lower IC50 values have been obtained with recombinant CYP1A2 (0.7-1.1 μM) or with HLM rich in 1A2-related activity (2-10.8 μM). The K i values (2-14 μM), independent on substrate concentrations, were quite low and similar for the three pesticides. Exposure to OPTs during IMI therapeutic treatments may lead to decreased DES formation, resulting in high plasma levels of the parent drug, eventual impairment of its pharmacological action and possible onset of adverse drug reactions (ADRs)

  20. Advances in molecular modeling of human cytochrome P450 polymorphism.

    Science.gov (United States)

    Martiny, Virginie Y; Miteva, Maria A

    2013-11-01

    Cytochrome P450 (CYP) is a supergene family of metabolizing enzymes involved in the phase I metabolism of drugs and endogenous compounds. CYP oxidation often leads to inactive drug metabolites or to highly toxic or carcinogenic metabolites involved in adverse drug reactions (ADR). During the last decade, the impact of CYP polymorphism in various drug responses and ADR has been demonstrated. Of the drugs involved in ADR, 56% are metabolized by polymorphic phase I metabolizing enzymes, 86% among them being CYP. Here, we review the major CYP polymorphic forms, their impact for drug response and current advances in molecular modeling of CYP polymorphism. We focus on recent studies exploring CYP polymorphism performed by the use of sequence-based and/or protein-structure-based computational approaches. The importance of understanding the molecular mechanisms related to CYP polymorphism and drug response at the atomic level is outlined. © 2013.

  1. Two mutant alleles of the human cytochrome P-450dbl gene (P450C2D1) associated with genetically deficient metabolism of debrisoquine and other drugs

    International Nuclear Information System (INIS)

    Skoda, R.C.; Gonzalez, F.L.; Demierre, A.; Meyer, R.A.

    1988-01-01

    The debrisoquine polymorphism is a clinically important genetic defect of drug metabolism affecting 5-10% of individuals in Caucasian populations. It is inherited as an autosomal recessive trait. A full-length cDNA for human cytochrome P-450db1, the deficient enzyme (also designated P450IID1 for P450 family II subfamily D isozyme 1), has recently been cloned. Leukocyte DNA from extensive metabolizers (EMs) or poor metabolizers (PMs) of debrisoquine was examined by Southern analysis. Two polymorphic restriction fragments were associated with the PM phenotype when DNAs from 24 unrelated PM and 29 unrelated EM individuals were probed with P-450db1 cDNA after digestion with Xba I restriction endonuclease and Southern blotting. Seventy-five percent of PMs had either the 44-kb or the 11.5-kb fragment or both. Segregation of these restriction fragment length polymorphisms in the families of six PM probands demonstrated that each of the two fragments is allelic with the 29-kb fragment present in all EM individuals and suggests that they identify two independent mutated alleles of the P-450db1 gene (designated P450C2D1). The Xba I 44-kb fragment and 11.5-kb fragment were in linkage disequilibrium with restriction fragment length polymorphisms generated by four and five additional restriction endonucleases, respectively, which can be used to identify the same mutant alleles for the P-450db1 gene

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

    Science.gov (United States)

    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. The effects of gender, age, ethnicity, and liver cirrhosis on cytochrome P450 enzyme activity in human liver microsomes and inducibility in cultured human hepatocytes

    International Nuclear Information System (INIS)

    Parkinson, Andrew; Mudra, Daniel R.; Johnson, Cory; Dwyer, Anne; Carroll, Kathleen M.

    2004-01-01

    We have measured cytochrome P450 (CYP) activity in nearly 150 samples of human liver microsomes and 64 samples of cryopreserved human hepatocytes, and we have performed induction studies in over 90 preparations of cultured human hepatocytes. We have analyzed these data to examine whether the expression of CYP enzyme activity in liver microsomes and isolated hepatocytes or the inducibility of CYP enzymes in cultured hepatocytes is influenced by the gender, age, or ethnicity of the donor (the latter being limited to Caucasians, African Americans, and Hispanics due to a paucity of livers from Asian donors). In human liver microsomes, there were no statistically significant differences (P > 0.05) in CYP activity as a function of age, gender, or ethnicity with one exception. 7-Ethoxyresorufin O-dealkylase (CYP1A2) activity was greater in males than females, which is consistent with clinical observation. Liver microsomal testosterone 6β-hydroxylase (CYP3A4) activity was slightly greater in females than males, but the difference was not significant. However, in cryopreserved human hepatocytes, the gender difference in CYP3A4 activity (females = twice males) did reach statistical significance, which supports the clinical observation that females metabolize certain CYP3A4 substrates faster than do males. Compared with those from Caucasians and African Americans, liver microsomes from Hispanics had about twice the average activity of CYP2A6, CYP2B6, and CYP2C8 and half the activity of CYP1A2, although this apparent ethnic difference may be a consequence of the relatively low number of Hispanic donors. Primary cultures of hepatocytes were treated with β-naphthoflavone, an inducer of CYP1A2, phenobarbital or rifampin, both of which induce CYP2B6, CYP2C9, CYP2C19, and CYP3A4, albeit it to different extents. Induction of these CYP enzymes in freshly cultured hepatocytes did not appear to be influenced by the gender or age of the donor. Furthermore, CYP3A4 induction in

  4. In vitro investigation of cytochrome P450-mediated metabolism of dietary flavonoids

    DEFF Research Database (Denmark)

    Breinholt, Vibeke; Offord, E.A.; Brouwer, C.

    2002-01-01

    Human and mouse liver microsomes And membranes isolated from Escherichia coli, which expressed cytochrome P450 (CYP) 1A2, 3A4 2C9 or 2D6, were used to investigate CYP-mediated metabolism of five selected dietary flavonoids. In human and mouse liver microsomes kaempferol, apigenin and naringenin...... were hydroxylated at the 3'-position to yield their corresponding analogs quercetin, luteolin and eriodietyol, whereas hesperetin and tamarixetin were demethylated at the 4'-position to yield eriodictyol and quercetin. respectively, Microsomal flavonoid metabolism as potently inhibited by the CYP1A2...... inhibitors. fluvoxamine and alpha-naphthoflavone. Recombinant CYP1A2 as capable of metabolizing all five investigated flavonoids. CYP3A4 recombinant protein did not catalyze hesperetin demethylation. but showed similar metabolic profiles for the remaining compounds, as did human microsomes and recombinant...

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

    Science.gov (United States)

    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.

  6. Prototype Systems Containing Human Cytochrome P450 for High-Throughput Real-Time Detection of DNA Damage by Compounds That Form DNA-Reactive Metabolites.

    Science.gov (United States)

    Brito Palma, Bernardo; Fisher, Charles W; Rueff, José; Kranendonk, Michel

    2016-05-16

    The formation of reactive metabolites through biotransformation is the suspected cause of many adverse drug reactions. Testing for the propensity of a drug to form reactive metabolites has increasingly become an integral part of lead-optimization strategy in drug discovery. DNA reactivity is one undesirable facet of a drug or its metabolites and can lead to increased risk of cancer and reproductive toxicity. Many drugs are metabolized by cytochromes P450 in the liver and other tissues, and these reactions can generate hard electrophiles. These hard electrophilic reactive metabolites may react with DNA and may be detected in standard in vitro genotoxicity assays; however, the majority of these assays fall short due to the use of animal-derived organ extracts that inadequately represent human metabolism. The current study describes the development of bacterial systems that efficiently detect DNA-damaging electrophilic reactive metabolites generated by human P450 biotransformation. These assays use a GFP reporter system that detects DNA damage through induction of the SOS response and a GFP reporter to control for cytotoxicity. Two human CYP1A2-competent prototypes presented here have appropriate characteristics for the detection of DNA-damaging reactive metabolites in a high-throughput manner. The advantages of this approach include a short assay time (120-180 min) with real-time measurement, sensitivity to small amounts of compound, and adaptability to a microplate format. These systems are suitable for high-throughput assays and can serve as prototypes for the development of future enhanced versions.

  7. Inhibitory Effects of Trapping Agents of Sulfur Drug Reactive Intermediates against Major Human Cytochrome P450 Isoforms

    Directory of Open Access Journals (Sweden)

    Jasleen K. Sodhi

    2017-07-01

    Full Text Available In some cases, the formation of reactive species from the metabolism of xenobiotics has been linked to toxicity and therefore it is imperative to detect potential bioactivation for candidate drugs during drug discovery. Reactive species can covalently bind to trapping agents in in vitro incubations of compound with human liver microsomes (HLM fortified with β-nicotinamide adenine dinucleotide phosphate (NADPH, resulting in a stable conjugate of trapping agent and reactive species, thereby facilitating analytical detection and providing evidence of short-lived reactive metabolites. Since reactive metabolites are typically generated by cytochrome P450 (CYP oxidation, it is important to ensure high concentrations of trapping agents are not inhibiting the activities of CYP isoforms. Here we assessed the inhibitory properties of fourteen trapping agents against the major human CYP isoforms (CYP1A2, 2C9, 2C19, 2D6 and 3A. Based on our findings, eleven trapping agents displayed inhibition, three of which had IC50 values less than 1 mM (2-mercaptoethanol, N-methylmaleimide and N-ethylmaleimide (NEM. Three trapping agents (dimedone, N-acetyl-lysine and arsenite did not inhibit CYP isoforms at concentrations tested. To illustrate effects of CYP inhibition by trapping agents on reactive intermediate trapping, an example drug (ticlopidine and trapping agent (NEM were chosen for further studies. For the same amount of ticlopidine (1 μM, increasing concentrations of the trapping agent NEM (0.007–40 mM resulted in a bell-shaped response curve of NEM-trapped ticlopidine S-oxide (TSO-NEM, due to CYP inhibition by NEM. Thus, trapping studies should be designed to include several concentrations of trapping agent to ensure optimal trapping of reactive metabolites.

  8. Bioconversion of deoxypodophyllotoxin into epipodophyllotoxin in E-coli using human cytochrome P450 3A4

    NARCIS (Netherlands)

    Vasilev, Nikolay P.; Julsing, Mattijs K.; Koulman, Albert; Clarkson, Cailean; Woerdenbag, Herman J.; Ionkova, Iliana; Bos, Rein; Jaroszewski, Jerzy W.; Kayser, Oliver; Quax, Wim J.

    2006-01-01

    Biotransformation of deoxypodophyllotoxin to epipodophyllotoxin by three major human hepatic enzymes, CYP1A2, CYP2C9 and CYP3A4, heterologously expressed in E coli DH5 alpha, was investigated. It was shown that CYP3A4 catalysed the hydroxylation of deoxypodophyllotoxin into epipodophyllotoxin in

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

    Science.gov (United States)

    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.

  10. Coffee, ADORA2A, and CYP1A2: the caffeine connection in Parkinson's disease.

    Science.gov (United States)

    Popat, R A; Van Den Eeden, S K; Tanner, C M; Kamel, F; Umbach, D M; Marder, K; Mayeux, R; Ritz, B; Ross, G W; Petrovitch, H; Topol, B; McGuire, V; Costello, S; Manthripragada, A D; Southwick, A; Myers, R M; Nelson, L M

    2011-05-01

    In 1-methyl-4-phenyl 1,2,3,6-tetrahydropyridine animal models of Parkinson's disease (PD), caffeine protects neurons by blocking the adenosine receptor A2A (ADORA2A). Caffeine is primarily metabolized by cytochrome P450 1A2 (CYP1A2). Our objective was to examine whether ADORA2A and CYP1A2 polymorphisms are associated with PD risk or modify the caffeine-PD association. Parkinson's Epidemiology and Genetic Associations Studies in the United States (PEGASUS) included five population-based case-control studies. One laboratory genotyped four ADORA2A and three CYP1A2 polymorphisms in 1325 PD cases and 1735 age- and sex-matched controls. Information regarding caffeine (coffee) consumption and other lifestyle factors came from structured in-person or telephone interviews. Odds ratios (OR) and 95% confidence intervals (CI) were estimated using logistic regression. Two ADORA2A polymorphisms were inversely associated with PD risk - rs71651683, a 5' variant (adjusted allelic OR = 0.51, 95% CI 0.33-0.80, permutation-adjusted P = 0.015) and rs5996696, a promoter region variant (adjusted OR for AC and CC genotypes compared with the AA wild-type genotype were 0.76 (95% CI 0.57-1.02) and 0.37 (95% CI 0.13-1.01), respectively (permutation-adjusted P for trend = 0.04). CYP1A2 polymorphisms were not associated with PD risk; however, the coffee-PD association was strongest among subjects homozygous for either variant allele rs762551 (P(interaction) = 0.05) or rs2470890 (P(interaction) = 0.04). In this consortium study, two ADORA2A polymorphisms were inversely associated with PD risk, but there was weak evidence of interaction with coffee consumption. In contrast, the coffee-PD association was strongest among slow metabolizers of caffeine who were homozygous carriers of the CYP1A2 polymorphisms. © 2011 The Author(s). European Journal of Neurology © 2011 EFNS.

  11. Gene-environment interaction in Parkinson’s disease: coffee, ADORA2A, and CYP1A2

    Science.gov (United States)

    Chuang, Yu-Hsuan; Lill, Christina M.; Lee, Pei-Chen; Hansen, Johnni; Lassen, Christina Funch; Bertram, Lars; Greene, Naomi; Sinsheimer, Janet S.; Ritz, Beate

    2017-01-01

    Background and purpose Drinking caffeinated coffee has been reported to protect against Parkinson’s disease (PD). Caffeine is an adenosine A2A receptor (encoded by the gene ADORA2A) antagonist that increases dopaminergic neurotransmission and Cytochrome P450 1A2 (gene: CYP1A2) metabolizes caffeine, thus gene polymorphisms in ADORA2A and CYP1A2 may influence the effect coffee consumption has on PD risk. Methods In a population-based case control study (PASIDA) in Denmark (1,556 PD patients and 1,606 birth year- and sex- matched controls), we assessed interactions between lifetime coffee consumption and three polymorphisms in ADORA2A and CYP1A2 for all subjects and incident and prevalent PD cases separately using logistic regression models. We also conducted a meta-analysis combining our results with those from previous studies. Results We estimated statistically significant interactions for ADORA2A rs5760423 and heavy vs. light coffee consumption in incident (OR interaction=0.66 [0.46–0.94], p=0.02) but not prevalent PD. We did not observe interactions for CYP1A2 rs762551 and rs2472304 in incident or prevalent PD. In meta-analyses, PD associations with daily coffee consumption were strongest among carriers of variant alleles in both ADORA2A and CYP1A2. Conclusion We corroborated results from a previous report that described interactions between ADORA2A and CYP1A2 polymorphisms and coffee consumption. Our results also suggest that survivor bias may affect results of studies that enrol prevalent PD cases. PMID:28135712

  12. Human cytochrome P450 enzymes of importance for the bioactivation of methyleugenol to the proximate carcinogen 1′-hydroxymethyleugenol

    NARCIS (Netherlands)

    Jeurissen, S.M.F.; Bogaards, J.J.P.; Boersma, M.G.; Horst, J.P.F. ter; Awad, H.M.; Fiamegos, Y.C.; Beek, T.A. van; Alink, G.M.; Sudhölter, E.J.R.; Cnubben, N.H.P.; Rietjens, I.M.C.M.

    2006-01-01

    In vitro studies were performed to elucidate the human cytochrome P450 enzymes involved in the bioactivation of methyleugenol to its proximate carcinogen 1′-hydroxymethyleugenol. Incubations with Supersomes, expressing individual P450 enzymes to a high level, revealed that P450 1A2, 2A6, 2C9, 2C19,

  13. Phytoremediation of the organic Xenobiotic simazine by p450-1a2 transgenic Arabidopsis thaliana plants.

    Science.gov (United States)

    Azab, Ehab; Hegazy, Ahmad K; El-Sharnouby, Mohamed E; Abd Elsalam, Hassan E

    2016-01-01

    The potential use of human P450-transgenic plants for phytoremediation of pesticide contaminated soils was tested in laboratory and greenhouse experiments. The transgenic P450 CYP1A2 gene Arabidopsis thaliana plants metabolize number of herbicides, insecticides and industrial chemicals. The P450 isozymes CYP1A2 expressed in A. thaliana were examined regarding the herbicide simazine (SIM). Transgenic A. thaliana plants expressing CYP1A2 gene showed significant resistance to SIM supplemented either in plant growth medium or sprayed on foliar parts. The results showed that SIM produces harmful effect on both rosette diameter and primary root length of the wild type (WT) plants. In transgenic A. thaliana lines, the rosette diameter and primary root length were not affected by SIM concentrations used in this experiment. The results indicate that CYP1A2 can be used as a selectable marker for plant transformation, allowing efficient selection of transgenic lines in growth medium and/or in soil-grown plants. The transgenic A. thaliana plants exhibited a healthy growth using doses of up to 250 μmol SIM treatments, while the non-transgenic A. thaliana plants were severely damaged with doses above 50 μmol SIM treatments. The transgenic A. thaliana plants can be used as phytoremediator of environmental SIM contaminants.

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

    International Nuclear Information System (INIS)

    Yoshida, Nobutaka; Osawa, Yoshio

    1991-01-01

    A simple and efficient method is described for the purification of microsomal aromatase cytochrome P-450 from human placenta. The enzyme was solubilized with Emulgen 913 and sodium cholate and subjected to chromatography on a column of Sepharose 4B couples with a specific monoclonal antibody, followed by hydroxyapatite column chromatography. The specific cytochrome P-450 content of purified aromatase was 13.1 (12-14.8) nmol/mg of protein. Aromatase assays were carried out with reconstituted systems of bovine liver P-450 reductase and dilauroyl-L-α-phosphatidylcholine with [1β- 3 H,4- 14 C]androstenedione as substrate. The total recovery of purified aromatase activity was 32.2%, and P-450 recovery was 17.6%. The very high K m value for 16α-hydroxytestosterone aromatization gives a reasonable indication that estriol is not the directly aromatized product in the fetoplacental unit of human pregnancy. The aromatase P-450 was subjected to SDS-polyacrylamide gel electrophoresis in increasing quantities. Silver stain detection techniques indicated a single band having a molecular mass of 55 kDa with greater than 97% purity. The stability analysis showed a half-life of over 4 years on storage at -80C

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

    Science.gov (United States)

    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.

  16. Study on the cytochrome P-450- and glutathione-dependent biotransformation of trichloroethylene in humans

    NARCIS (Netherlands)

    Bloemen, L. J.; Monster, A. C.; Kezic, S.; Commandeur, J. N.; Veulemans, H.; Vermeulen, N. P.; Wilmer, J. W.

    2001-01-01

    To investigate in humans the contribution of the cytochrome P-450- and glutathione-dependent biotransformation of trichloroethylene (TRI) under controlled repeated exposure in volunteers, and under occupational conditions. Volunteers were exposed to TRI, using repeated 15 min exposures at 50 and 100

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

    NARCIS (Netherlands)

    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.

    2015-01-01

    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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2013-01-25

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

  19. CYP1A2 rs762551 polymorphism contributes to cancer susceptibility: a meta-analysis from 19 case-control studies

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    Wang Hongge

    2012-11-01

    Full Text Available Abstract Background Genetic polymorphism (rs762551A>C in gene encoding cytochrome P450 1A2 (CYP1A2 has been shown to influence the inducibility of CYP1A2 expression and thus might be associated with risk of several types of human cancer. However, the results of previous studies on the associations of this polymorphism with risk of cancer are not all consistent. To clarify the potential contribution of CYP1A2 rs762551 to cancer risk, we performed a meta-analysis of the published case–control studies. Methods We used PubMed, Embase, OVID, ScienceDirect, and Chinese National Knowledge Infrastructure databases to identify the related publications for this meta-analysis. The pooled odds ratio (OR and 95% confidence interval (CI were calculated using random effect model to evaluate the association of rs762551 with cancer risk. A χ2-based Q-test was used to examine the heterogeneity assumption and the funnel plot and Egger’s test were used to examine the potential publication bias. The leave-one-out sensitivity analysis was conducted to determine whether our assumptions or decisions have a major effect on the results of the review. Results Our analysis of 19 eligible case–control studies showed a significant association between rs762551C variant with risk of cancer in the genetic model of CC versus AA (OR = 1.30, 95% CI = 1.02-1.64 and the dominant model (OR = 1.19, 95% CI = 1.04-1.36. In subgroup analysis based on ethnicity, the rs762551CC genotype was associated with increased cancer risk (OR = 1.29, 95% CI = 1.27-1.63 in co-dominate model and OR = 1.17, 95% CI = 1.02-1.34 in dominant model in Caucasians, but not in Asians and the mixed population. Conclusion These results suggested that CYP1A2 rs762551 polymorphism is likely to be associated with susceptibility to cancer in Caucasians.

  20. Similar substrate specificity of cynomolgus monkey cytochrome P450 2C19 to reported human P450 2C counterpart enzymes by evaluation of 89 drug clearances.

    Science.gov (United States)

    Hosaka, Shinya; Murayama, Norie; Satsukawa, Masahiro; Uehara, Shotaro; Shimizu, Makiko; Iwasaki, Kazuhide; Iwano, Shunsuke; Uno, Yasuhiro; Yamazaki, Hiroshi

    2015-12-01

    Cynomolgus monkeys are used widely in preclinical studies as non-human primate species. The amino acid sequence of cynomolgus monkey cytochrome P450 (P450 or CYP) 2C19 is reportedly highly correlated to that of human CYP2C19 (92%) and CYP2C9 (93%). In the present study, 89 commercially available compounds were screened to find potential substrates for cynomolgus monkey CYP2C19. Of 89 drugs, 34 were metabolically depleted by cynomolgus monkey CYP2C19 with relatively high rates. Among them, 30 compounds have been reported as substrates or inhibitors of, either or both, human CYP2C19 and CYP2C9. Several compounds, including loratadine, showed high selectivity to cynomolgus monkey CYP2C19, and all of these have been reported as human CYP2C19 and/or CYP2C9 substrates. In addition, cynomolgus monkey CYP2C19 formed the same loratadine metabolite as human CYP2C19, descarboethoxyloratadine. These results suggest that cynomolgus monkey CYP2C19 is generally similar to human CYP2C19 and CYP2C9 in its substrate recognition functionality. Copyright © 2015 John Wiley & Sons, Ltd.

  1. Identification of putative substrates for cynomolgus monkey cytochrome P450 2C8 by substrate depletion assays with 22 human P450 substrates and inhibitors.

    Science.gov (United States)

    Hosaka, Shinya; Murayama, Norie; Satsukawa, Masahiro; Uehara, Shotaro; Shimizu, Makiko; Iwasaki, Kazuhide; Iwano, Shunsuke; Uno, Yasuhiro; Yamazaki, Hiroshi

    2016-07-01

    Cynomolgus monkeys are widely used in drug developmental stages as non-human primate models. Previous studies used 89 compounds to investigate species differences associated with cytochrome P450 (P450 or CYP) function that reported monkey specific CYP2C76 cleared 19 chemicals, and homologous CYP2C9 and CYP2C19 metabolized 17 and 30 human CYP2C9 and/or CYP2C19 substrates/inhibitors, respectively. In the present study, 22 compounds selected from viewpoints of global drug interaction guidances and guidelines were further evaluated to seek potential substrates for monkey CYP2C8, which is highly homologous to human CYP2C8 (92%). Amodiaquine, montelukast, quercetin and rosiglitazone, known as substrates or competitive inhibitors of human CYP2C8, were metabolically depleted by recombinant monkey CYP2C8 at relatively high rates. Taken together with our reported findings of the slow eliminations of amodiaquine and montelukast by monkey CYP2C9, CYP2C19 and CYP2C76, the present results suggest that these at least four chemicals may be good marker substrates for monkey CYP2C8. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

  2. Analysis of cellular responses to aflatoxin B1 in yeast expressing human cytochrome P450 1A2 using cDNA microarrays

    International Nuclear Information System (INIS)

    Guo Yingying; Breeden, Linda L.; Fan, Wenhong; Zhao Lueping; Eaton, David L.; Zarbl, Helmut

    2006-01-01

    Aflatoxin B1 (AFB 1 ) is a potent human hepatotoxin and hepatocarcinogen produced by the mold Aspergillus flavus. In human, AFB 1 is bioactivated by cytochrome P450 (CYP450) enzymes, primarily CYP1A2, to the genotoxic epoxide that forms N 7 -guanine DNA adducts. To characterize the transcriptional responses to genotoxic insults from AFB 1 , a strain of Saccharomyces cerevisiae engineered to express human CYP1A2 was exposed to doses of AFB 1 that resulted in minimal lethality, but substantial genotoxicity. Flow cytometric analysis demonstrated a dose and time dependent S phase delay under the same treatment conditions, indicating a checkpoint response to DNA damage. Replicate cDNA microarray analyses of AFB 1 treated cells showed that about 200 genes were significantly affected by the exposure. The genes activated by AFB 1 -treatment included RAD51, DUN1 and other members of the DNA damage response signature reported in a previous study with methylmethane sulfonate and ionizing radiation [A.P. Gasch, M. Huang, S. Metzner, D. Botstein, S.J. Elledge, P.O. Brown, Genomic expression responses to DNA-damaging agents and the regulatory role of the yeast ATR homolog Mec1p, Mol. Biol. Cell 12 (2001) 2987-3003]. However, unlike previous studies using highly cytotoxic doses, environmental stress response genes [A.P. Gasch, P.T. Spellman, C.M. Kao, O. Carmel-Harel, M.B. Eisen, G. Storz, D. Botstein, P.O. Brown, Genomic expression programs in the response of yeast cells to environmental changes, Mol. Biol. Cell 11 (2000) 4241-4257] were largely unaffected by our dosing regimen. About half of the transcripts affected are also known to be cell cycle regulated. The most strongly repressed transcripts were those encoding the histone genes and a group of genes that are cell cycle regulated and peak in M phase and early G1. These include most of the known daughter-specific genes. The rapid and coordinated repression of histones and M/G1-specific transcripts cannot be explained by

  3. Analysis of cellular responses to aflatoxin B{sub 1} in yeast expressing human cytochrome P450 1A2 using cDNA microarrays

    Energy Technology Data Exchange (ETDEWEB)

    Guo Yingying [Departmental of Environmental and Occupational Health Sciences, University of Washington, Seattle, WA (United States); Fred Hutchinson Cancer Research Center, Seattle, WA (United States); Breeden, Linda L. [Fred Hutchinson Cancer Research Center, Seattle, WA (United States); Fan, Wenhong [Fred Hutchinson Cancer Research Center, Seattle, WA (United States); Zhao Lueping [Fred Hutchinson Cancer Research Center, Seattle, WA (United States); Eaton, David L. [Departmental of Environmental and Occupational Health Sciences, University of Washington, Seattle, WA (United States); Fred Hutchinson Cancer Research Center, Seattle, WA (United States); Zarbl, Helmut [Departmental of Environmental and Occupational Health Sciences, University of Washington, Seattle, WA (United States) and Fred Hutchinson Cancer Research Center, Seattle, WA (United States)]. E-mail: hzarbl@fhcrc.org

    2006-01-29

    Aflatoxin B1 (AFB{sub 1}) is a potent human hepatotoxin and hepatocarcinogen produced by the mold Aspergillus flavus. In human, AFB{sub 1} is bioactivated by cytochrome P450 (CYP450) enzymes, primarily CYP1A2, to the genotoxic epoxide that forms N{sup 7}-guanine DNA adducts. To characterize the transcriptional responses to genotoxic insults from AFB{sub 1}, a strain of Saccharomyces cerevisiae engineered to express human CYP1A2 was exposed to doses of AFB{sub 1} that resulted in minimal lethality, but substantial genotoxicity. Flow cytometric analysis demonstrated a dose and time dependent S phase delay under the same treatment conditions, indicating a checkpoint response to DNA damage. Replicate cDNA microarray analyses of AFB{sub 1} treated cells showed that about 200 genes were significantly affected by the exposure. The genes activated by AFB{sub 1}-treatment included RAD51, DUN1 and other members of the DNA damage response signature reported in a previous study with methylmethane sulfonate and ionizing radiation [A.P. Gasch, M. Huang, S. Metzner, D. Botstein, S.J. Elledge, P.O. Brown, Genomic expression responses to DNA-damaging agents and the regulatory role of the yeast ATR homolog Mec1p, Mol. Biol. Cell 12 (2001) 2987-3003]. However, unlike previous studies using highly cytotoxic doses, environmental stress response genes [A.P. Gasch, P.T. Spellman, C.M. Kao, O. Carmel-Harel, M.B. Eisen, G. Storz, D. Botstein, P.O. Brown, Genomic expression programs in the response of yeast cells to environmental changes, Mol. Biol. Cell 11 (2000) 4241-4257] were largely unaffected by our dosing regimen. About half of the transcripts affected are also known to be cell cycle regulated. The most strongly repressed transcripts were those encoding the histone genes and a group of genes that are cell cycle regulated and peak in M phase and early G1. These include most of the known daughter-specific genes. The rapid and coordinated repression of histones and M/G1-specific

  4. Application of the relative activity factor approach in scaling from heterologously expressed cytochromes p450 to human liver microsomes: studies on amitriptyline as a model substrate.

    Science.gov (United States)

    Venkatakrishnan, K; von Moltke, L L; Greenblatt, D J

    2001-04-01

    The relative activity factor (RAF) approach is being increasingly used in the quantitative phenotyping of multienzyme drug biotransformations. Using lymphoblast-expressed cytochromes P450 (CYPs) and the tricyclic antidepressant amitriptyline as a model substrate, we have tested the hypothesis that the human liver microsomal rates of a biotransformation mediated by multiple CYP isoforms can be mathematically reconstructed from the rates of the biotransformation catalyzed by individual recombinant CYPs using the RAF approach, and that the RAF approach can be used for the in vitro-in vivo scaling of pharmacokinetic clearance from in vitro intrinsic clearance measurements in heterologous expression systems. In addition, we have compared the results of two widely used methods of quantitative reaction phenotyping, namely, chemical inhibition studies and the prediction of relative contributions of individual CYP isoforms using the RAF approach. For the pathways of N-demethylation (mediated by CYPs 1A2, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4) and E-10 hydroxylation (mediated by CYPs 2B6, 2D6, and 3A4), the model-predicted biotransformation rates in microsomes from a panel of 12 human livers determined from enzyme kinetic parameters of the recombinant CYPs were similar to, and correlated with the observed rates. The model-predicted clearance via N-demethylation was 53% lower than the previously reported in vivo pharmacokinetic estimates. Model-predicted relative contributions of individual CYP isoforms to the net biotransformation rate were similar to, and correlated with the fractional decrement in human liver microsomal reaction rates by chemical inhibitors of the respective CYPs, provided the chemical inhibitors used were specific to their target CYP isoforms.

  5. Pi-pi Stacking Mediated Cooperative Mechanism for Human Cytochrome P450 3A4

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    Botao Fa

    2015-04-01

    Full Text Available Human Cytochrome P450 3A4 (CYP3A4 is an important member of the cytochrome P450 superfamily with responsibility for metabolizing ~50% of clinical drugs. Experimental evidence showed that CYP3A4 can adopt multiple substrates in its active site to form a cooperative binding model, accelerating substrate metabolism efficiency. In the current study, we constructed both normal and cooperative binding models of human CYP3A4 with antifungal drug ketoconazoles (KLN. Molecular dynamics simulation and free energy calculation were then carried out to study the cooperative binding mechanism. Our simulation showed that the second KLN in the cooperative binding model had a positive impact on the first one binding in the active site by two significant pi-pi stacking interactions. The first one was formed by Phe215, functioning to position the first KLN in a favorable orientation in the active site for further metabolism reactions. The second one was contributed by Phe304. This pi-pi stacking was enhanced in the cooperative binding model by the parallel conformation between the aromatic rings in Phe304 and the dioxolan moiety of the first KLN. These findings can provide an atomic insight into the cooperative binding in CYP3A4, revealing a novel pi-pi stacking mechanism for drug-drug interactions.

  6. Inhibitory Effects of Aschantin on Cytochrome P450 and Uridine 5′-diphospho-glucuronosyltransferase Enzyme Activities in Human Liver Microsomes

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    Soon-Sang Kwon

    2016-04-01

    Full Text Available Aschantin is a bioactive neolignan found in Magnolia flos with antiplasmodial, Ca2+-antagonistic, platelet activating factor-antagonistic, and chemopreventive activities. We investigated its inhibitory effects on the activities of eight major human cytochrome P450 (CYP and uridine 5′-diphospho-glucuronosyltransferase (UGT enzymes of human liver microsomes to determine if mechanistic aschantin–enzyme interactions were evident. Aschantin potently inhibited CYP2C8-mediated amodiaquine N-de-ethylation, CYP2C9-mediated diclofenac 4′-hydroxylation, CYP2C19-mediated [S]-mephenytoin 4′-hydroxylation, and CYP3A4-mediated midazolam 1′-hydroxylation, with Ki values of 10.2, 3.7, 5.8, and 12.6 µM, respectively. Aschantin at 100 µM negligibly inhibited CYP1A2-mediated phenacetin O-de-ethylation, CYP2A6-mediated coumarin 7-hydroxylation, CYP2B6-mediated bupropion hydroxylation, and CYP2D6-mediated bufuralol 1′-hydroxylation. At 200 µM, it weakly inhibited UGT1A1-catalyzed SN-38 glucuronidation, UGT1A6-catalyzed N-acetylserotonin glucuronidation, and UGT1A9-catalyzed mycophenolic acid glucuronidation, with IC50 values of 131.7, 144.1, and 71.0 µM, respectively, but did not show inhibition against UGT1A3, UGT1A4, or UGT2B7 up to 200 µM. These in vitro results indicate that aschantin should be examined in terms of potential interactions with pharmacokinetic drugs in vivo. It exhibited potent mechanism-based inhibition of CYP2C8, CYP2C9, CYP2C19, and CYP3A4.

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

    Science.gov (United States)

    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.

  8. The human cytochrome P450 3A locus. Gene evolution by capture of downstream exons.

    Science.gov (United States)

    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.

  9. Reduction of Aromatic and Heterocyclic Aromatic N-Hydroxylamines by Human Cytochrome P450 2S1

    Science.gov (United States)

    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

  10. Water pipe (Shisha, Hookah, Arghile) Smoking and Secondhand Tobacco Smoke Effects on CYP1A2 and CYP2A6 Phenotypes as Measured by Caffeine Urine Test.

    Science.gov (United States)

    Yılmaz, Şenay Görücü; Llerena, Adrián; De Andrés, Fernando; Karakaş, Ümit; Gündoğar, Hasan; Erciyas, Kamile; Kimyon, Sabit; Mete, Alper; Güngör, Kıvanç; Özdemir, Vural

    2017-03-01

    Public policies to stop or reduce cigarette smoking and exposure to secondhand smoke and associated diseases have yielded successful results over the past decade. Yet, the growing worldwide popularity of another form of tobacco consumption, water pipe smoking, has received relatively less attention. To the best of our knowledge, no study to date has evaluated the effects of water pipe smoking on cytochrome P450 (CYP450) activities and drug interaction potential in humans, whereas only limited information is available on the impact of secondhand smoke on drug metabolism. In a sample of 99 healthy volunteers (28 water pipe smokers, 30 secondhand tobacco smoke exposed persons, and 41 controls), we systematically compared CYP1A2 and CYP2A6 enzyme activities in vivo using caffeine urine test. The median self-reported duration of water pipe smoking was 7.5 h/week and 3 years of exposure in total. The secondhand smoke group had a median of 14 h of self-reported weekly exposure to tobacco smoke indoor where a minimum of five cigarettes were smoked/hour for a total of 3.5 years (median). Analysis of variance did not find a significant difference in CYP1A2 and CYP2A6 activities among the three study groups (p > 0.05). Nor was there a significant association between the extent of water pipe or secondhand smoke exposure and the CYP1A2 and CYP2A6 activities (p > 0.05). Further analysis in a subsample with smoke exposure more than the median values also did not reveal a significant difference from the controls. Although we do not rule out an appreciable possible impact of water pipe smoke and secondhand smoke on in vivo activities of these two drug metabolism pathways, variability in smoke constituents from different tobacco consumption methods (e.g., water pipe) might affect drug metabolism in ways that might differ from that of cigarette smoke. Further studies in larger prospective samples are recommended to evaluate water pipe and secondhand tobacco smoke effects

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

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    Alexandr N Simonov

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

  12. Relative contribution of rat cytochrome P450 isoforms to the metabolism of caffeine: the pathway and concentration dependence.

    Science.gov (United States)

    Kot, Marta; Daniel, Władysława A

    2008-04-01

    The aim of the present study was to estimate the relative contribution of rat P450 isoforms to the metabolism of caffeine and to assess the usefulness of caffeine as a marker substance for estimating the activity of P450 in rat liver and its potential for pharmacokinetic interactions in pharmacological experiments. The results obtained using rat cDNA-expressed P450s indicated that 8-hydroxylation was the main oxidation pathway of caffeine (70%) in the rat. CYP1A2 was found to be a key enzyme catalyzing 8-hydroxylation (72%) and substantially contributing to 3-N-demethylation (47%) and 1-N-demethylation (37.5%) at a caffeine concentration of 0.1mM (relevant to "the maximum therapeutic concentration in humans"). Furthermore, CYP2C11 considerably contributed to 3-N-demethylation (31%). The CYP2C subfamily (66%) - mainly CYP2C6 (27%) and CYP2C11 (29%) - played a major role in catalyzing 7-N-demethylation. At higher substrate concentrations, the contribution of CYP1A2 to the metabolism of caffeine decreased in favor of CYP2C11 (N-demethylations) and CYP3A2 (mainly 8-hydroxylation). The obtained results were confirmed with liver microsomes (inhibition and correlation studies). Therefore, caffeine may be used as a marker substance for assessing the activity of CYP1A2 in rats, using 8-hydroxylation (but not 3-N-demethylation-like in humans); moreover, caffeine may also be used to simultaneously, preliminarily estimate the activity of CYP2C using 7-N-demethylation as a marker reaction. Hence caffeine pharmacokinetics in rats may be changed by drugs affecting the activity of CYP1A2 and/or CYP2C, e.g. by some antidepressants.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-03-15

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

  14. Characterization of the human cytochrome P450 enzymes involved in the metabolism of dihydrocodeine

    Science.gov (United States)

    Kirkwood, L. C.; Nation, R. L.; Somogyi, A. A.

    1997-01-01

    Aims Using human liver microsomes from donors of the CYP2D6 poor and extensive metabolizer genotypes, the role of individual cytochromes P-450 in the oxidative metabolism of dihydrocodeine was investigated. Methods The kinetics of formation of N- and O-demethylated metabolites, nordihydrocodeine and dihydromorphine, were determined using microsomes from six extensive and one poor metabolizer and the effects of chemical inhibitors selective for individual P-450 enzymes of the 1A, 2A, 2C, 2D, 2E and 3A families and of LKM1 (anti-CYP2D6) antibodies were studied. Results Nordihydrocodeine was the major metabolite in both poor and extensive metabolizers. Kinetic constants for N-demethylation derived from the single enzyme Michaelis-Menten model did not differ between the two groups. Troleandomycin and erythromycin selectively inhibited N-demethylation in both extensive and poor metabolizers. The CYP3A inducer, α-naphthoflavone, increased N-demethylation rates. The kinetics of formation of dihydromorphine in both groups were best described by a single enzyme Michaelis-Menten model although inhibition studies in extensive metabolizers suggested involvement of two enzymes with similar Km values. The kinetic constants for O-demethylation were significantly different in extensive and poor metabolizers. The extensive metabolizers had a mean intrinsic clearance to dihydromorphine more than ten times greater than the poor metabolizer. The CYP2D6 chemical inhibitors, quinidine and quinine, and LKM1 antibodies inhibited O-demethylation in extensive metabolizers; no effect was observed in microsomes from a poor metabolizer. Conclusions CYP2D6 is the major enzyme mediating O-demethylation of dihydrocodeine to dihydromorphine. In contrast, nordihydrocodeine formation is predominantly catalysed by CYP3A. PMID:9431830

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2013-02-15

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

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

    International Nuclear Information System (INIS)

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

    2013-01-01

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

  17. Lack of evidence for metabolism of p-phenylenediamine by human hepatic cytochrome P450 enzymes

    International Nuclear Information System (INIS)

    Stanley, Lesley A.; Skare, Julie A.; Doyle, Edward; Powrie, Robert; D'Angelo, Diane; Elcombe, Clifford R.

    2005-01-01

    p-Phenylenediamine (PPD) is a widely used ingredient in permanent hair dyes; however, little has been published on its metabolism, especially with respect to hepatic cytochrome P450 (CYP)-mediated oxidation. This is regarded as a key step in the activation of carcinogenic arylamines that ultimately leads to the development of bladder cancer. Most epidemiology studies show no significant association between personal use of hair dyes and bladder cancer, but one recent study reported an increased risk of bladder cancer in women who were frequent users of permanent hair dyes. The aim of the present study was to use intact human hepatocytes, human liver microsomes, and heterologously expressed human CYPs to determine whether PPD is metabolised by hepatic CYPs to form an N-hydroxylamine. p-Phenylenediamine was N-acetylated by human hepatocytes to form N-acetylated metabolites, but there was no evidence for the formation of mono-oxygenated metabolites or for enzyme-mediated covalent binding of 14 C-PPD to microsomal protein. In contrast, 2-aminofluorene underwent CYP-mediated metabolism to ≥4 different hydroxylated metabolites. The lack of evidence for hepatic CYP-mediated metabolism of PPD is inconsistent with the hypothesis that this compound plays a causal role in the development of bladder cancer via a mode of action involving hepatic metabolism to an N-hydroxyarylamine

  18. DrugMetZ DB: an anthology of human drug metabolizing Chytochrome P450 enzymes.

    Science.gov (United States)

    Antony, Tresa Remya Thomas; Nagarajan, Shanthi

    2006-11-14

    Understandings the basics of Cytochrome P450 (P450 or CYP) will help to discern drug metabolism. CYP, a super-family of heme-thiolate proteins, are found in almost all living organisms and is involved in the biotransformation of a diverse range of xenobiotics, therapeutic drugs and toxins. Here, we describe DrugMetZ DB, a database for CYP metabolizing drugs. The DB is implemented in MySQL, PHP and HTML. www.bicpu.edu.in/DrugMetZDB/

  19. Metabolism of agrochemicals and related environmental chemicals based on cytochrome P450s in mammals and plants.

    Science.gov (United States)

    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.

  20. Comparison of xenobiotic-metabolising human, porcine, rodent, and piscine cytochrome P450

    International Nuclear Information System (INIS)

    Burkina, Viktoriia; Rasmussen, Martin Krøyer; Pilipenko, Nadezhda; Zamaratskaia, Galia

    2017-01-01

    Highlights: • The percent identity of porcine, murine and piscine CYPs was compared with human CYPs. • Main similarities and differences were reviewed. • Understanding of molecular mechanisms of CYP system will provide further insights into the CYP regulatory processes, and responses to different factors. - Abstract: Cytochrome P450 proteins (CYP450s) are present in most domains of life and play a critical role in the metabolism of endogenous compounds and xenobiotics. The effects of exposure to xenobiotics depend heavily on the expression and activity of drug-metabolizing CYP450s, which is determined by species, genetic background, age, gender, diet, and exposure to environmental pollutants. Numerous reports have investigated the role of different vertebrate CYP450s in xenobiotic metabolism. Model organisms provide powerful experimental tools to investigate Phase I metabolism. The aim of the present review is to compare the existing data on human CYP450 proteins (1–3 families) with those found in pigs, mice, and fish. We will highlight differences and similarities and identify research gaps which need to be addressed in order to use these species as models that mimic human traits. Moreover, we will discuss the roles of nuclear receptors in the cellular regulation of CYP450 expression in select organisms.

  1. Influence of the urine flow rate on some caffeine metabolite ratios used to assess CYP1A2 activity.

    Science.gov (United States)

    Sinués, Blanca; Fanlo, Ana; Bernal, María Luisa; Mayayo, Esteban; Soriano, María Antonia; Martínez-Ballarin, Enrique

    2002-12-01

    Five established metabolite ratios (MRs) to measure P450 CYP1A2 activity--MR1 (17X + 17U)/137X, MR2 (AFMU + 1X + 1U)/17U, MR3 (17X/137X), MR4 (AFMU + 1X + 1U + 17X + 17U)/137X, and MR5 (AFMU + 1X + 1U)/17X--were calculated in urine 4-5 hours after caffeine intake. First, to assess the potential of omeprazole to induce CYP1A2 activity, a caffeine test was performed in 27 subjects on two occasions: before and after 14 days on omeprazole (20 mg/day). Samples of urine were analyzed by high-performance liquid chromatography (HPLC) to quantify caffeine and metabolites used to calculate the different caffeine MRs. MR1, MR3, and MR4 were enhanced after treatment; the percentage of change was inversely associated with that of the urine flow, with r values of -0.48, -0.49, and -0.47, respectively. However, MR2 or MR5 were not modified. To determine the reason for these contradictory results, the authors analyzed data of metabolites, ratios, and their components (numerators and denominators) from 152 subjects (who underwent one caffeine test) and their relationship with the urinary flow. Caffeine concentration in urine was the only compound nondependent on the urine flow. Consistently, ratios containing caffeine (MR1, MR3, and MR4) were highly influenced by the rate of urine excretion, since the flow dependence of their numerators is not canceled out by that of caffeine in their denominators. The dependency of the caffeine excretion on renal factors may explain the opposite results found with the different ratios in the aforementioned prospective study of drug interaction, the absence of closer correlations of the five MRs to each other, the discrepancies about the type of frequency distribution of the different MRs (either normal or multimodal), and the higher sensitivity of MR2 to detect gender differences in CYP1A2 activity found in this study. In summary, the data clearly emphasize the need for a strict control of the liquid intake to avoid high urine flows when MRs

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

    International Nuclear Information System (INIS)

    Ingelman-Sundberg, Magnus

    2005-01-01

    Currently, 57 active cytochrome P450 (CYP) genes and 58 pseudogenes are known to be present in the human genome. Among the genes discovered by initiatives in the human genome project are CYP2R1, CYP2W1, CYP2S1, CYP2U1 and CYP3A43, the latter apparently encoding a pseudoenzyme. The function, polymorphism and regulation of these genes are still to be discovered to a great extent. The polymorphism of drug metabolizing CYPs is extensive and influences the outcome of drug therapy causing lack of response or adverse drug reactions. The basis for the differences in the global distribution of the polymorphic variants is inactivating gene mutations and subsequent genetic drift. However, polymorphic alleles carrying multiple active gene copies also exist and are suggested in case of CYP2D6 to be caused by positive selection due to development of alkaloid resistance in North East Africa about 10,000-5000 BC. The knowledge about the CYP genes and their polymorphisms is of fundamental importance for effective drug therapy and for drug development as well as for understanding metabolic activation of carcinogens and other xenobiotics. Here, a short review of the current knowledge is given

  3. Comparative study of hop-containing products on human cytochrome p450-mediated metabolism.

    Science.gov (United States)

    Foster, Brian C; Kearns, Nikia; Arnason, John T; Saleem, Ammar; Ogrodowczyk, Carolina; Desjardins, Suzanne

    2009-06-10

    Thirty-five national and international brands of beer were examined for their potential to affect human cytochrome P450 (CYP)-mediated metabolism. They represented the two main categories of beer, ales and lagers, and included a number of specialty products including bitter (porter, stout), coffee, ice, wheat, Pilsner, and hemp seed. Aliquots were examined for nonvolatile soluble solids, effect on CYP metabolism and P-glycoprotein (Pgp) transport, and major alpha- and beta-hop acids. Wide variance was detected in contents of alcohol, nonvolatile suspended solids, and hop acids and in the potential to affect CYP-mediated metabolism and Pgp-mediated efflux transport. Many of the products affected CYP2C9-mediated metabolism, and only two (NRP 306 and 307) markedly affected CYP3A4; hence, some products have the capacity to affect drug safety. CYP3A4, CYP3A5, CYP3A7, and CYP19 (aromatase) inhibition to the log concentration of beta-acid content was significant with r(2) > 0.37, suggesting that these components can account for some of the variation in inhibition of CYP metabolism.

  4. Dynamics of water molecules in the active-site cavity of human cytochromes P450

    DEFF Research Database (Denmark)

    Rydberg, Patrik; Rod, Thomas Holm; Olsen, Lars

    2007-01-01

    We have studied the dynamics of water molecules in six crystal structures of four human cytochromes P450, 2A6, 2C8, 2C9, and 3A4, with molecular dynamics simulations. In the crystal structures, only a few water molecules are seen and the reported sizes of the active-site cavity vary a lot....... In the simulations, the cavities are completely filled with water molecules, although with approximately 20% lower density than in bulk water. The 2A6 protein differs from the other three in that it has a very small cavity with only two water molecules and no exchange with the surroundings. The other three proteins...... channels, through which there is a quite frequent exchange of water molecules (one molecule is exchanged every 30-200 ps), except in 2A6. Most of the channels are observed also in the crystal structures, but two to three channels in each protein open only during the simulations. There are no water...

  5. Genetic polymorphism of human cytochrome P-450 (S)-mephenytoin 4-hydroxylase. Studies with human autoantibodies suggest a functionally altered cytochrome P-450 isozyme as cause of the genetic deficiency

    International Nuclear Information System (INIS)

    Meier, U.T.; Meyer, U.A.

    1987-01-01

    The metabolism of the anticonvulsant mephenytoin is subject to a genetic polymorphism. In 2-5% of Caucasians and 18-23% of Japanese subjects a specific cytochrome P-450 isozyme, P-450 meph, is functionally deficient or missing. The authors have accumulated evidence that autoimmune antibodies observed in sera of patients with tienilic acid induced hepatitis (anti-liver kidney microsome 2 or anti-LKM2 antibodies) specifically recognize the cytochrome P-450 involved in the mephrenytoin hydroxylation polymorphism. This is demonstrated by immunoinhibition and immunoprecipitation of microsomal (S)-mephenytoin 4-hydroxylation activity and by the recognition by anti-LKM2 antibodies of a single [ 125 I]-protein band on immunoblots of human liver microsomes after sodium dodecyl sulfate-polyacrylamide gel electrophoresis or isoelectric focusing. The cytochrome P-450 recognized by anti-LKM2 antibodies was immunopurified from microsomes derived from livers of extensive (EM) or poor metabolizers (PM) of (S)-mephenytoin. Comparison of the EM-type cytochrome P-450 to that isolated from PM livers revealed no difference in regard to immuno-cross-reactivity, molecular weight, isoelectric point, relative content in microsomes, two-dimensional tryptic peptide maps, one-dimensional peptide maps with three proteases, amino acid composition, and amino-terminal protein sequence. Finally, the same protein was precipitated from microsomes prepared from the liver biopsy of a subject phenotyped in vivo as a poor metabolizer of mephenytoin. These data strongly suggest that the mephenytoin hydroxylation deficiency is caused by a minor structural change leading to a functionally altered cytochrome P-450 isozyme

  6. P450 oxidoreductase deficiency: a disorder of steroidogenesis with multiple clinical manifestations.

    Science.gov (United States)

    Miller, Walter L

    2012-10-23

    Cytochrome P450 enzymes catalyze the biosynthesis of steroid hormones and metabolize drugs. There are seven human type I P450 enzymes in mitochondria and 50 type II enzymes in endoplasmic reticulum. Type II enzymes, including both drug-metabolizing and some steroidogenic enzymes, require electron donation from a two-flavin protein, P450 oxidoreductase (POR). Although knockout of the POR gene causes embryonic lethality in mice, we discovered human POR deficiency as a disorder of steroidogenesis associated with the Antley-Bixler skeletal malformation syndrome and found mild POR mutations in phenotypically normal adults with infertility. Assay results of mutant forms of POR using the traditional but nonphysiologic assay (reduction of cytochrome c) did not correlate with patient phenotypes; assays based on the 17,20 lyase activity of P450c17 (CYP17) correlated with clinical phenotypes. The POR sequence in 842 normal individuals revealed many polymorphisms; amino acid sequence variant A503V is encoded by ~28% of human alleles. POR A503V has about 60% of wild-type activity in assays with CYP17, CYP2D6, and CYP3A4, but nearly wild-type activity with P450c21, CYP1A2, and CYP2C19. Activity of a particular POR variant with one P450 enzyme will not predict its activity with another P450 enzyme: Each POR-P450 combination must be studied individually. Human POR transcription, initiated from an untranslated exon, is regulated by Smad3/4, thyroid receptors, and the transcription factor AP-2. A promoter polymorphism reduces transcription to 60% in liver cells and to 35% in adrenal cells. POR deficiency is a newly described disorder of steroidogenesis, and POR variants may account for some genetic variation in drug metabolism.

  7. Polymerase chain reaction amplification fails to detect aromatase cytochrome P450 transcripts in normal human endometrium or decidua.

    Science.gov (United States)

    Bulun, S E; Mahendroo, M S; Simpson, E R

    1993-06-01

    It has been proposed that the biosynthesis of estrogens by the human endometrium may be of physiological significance during the menstrual cycle. Local estrogen production was also suggested to be important in the development of endometrial cancer; however, the presence or absence of aromatase enzyme activity in normal human endometrium is controversial. To address this issue, we used a sensitive technique capable of detecting mRNA transcripts present in only very low copy number. The polymerase chain reaction linked to reverse transcription (RT-PCR) was used to evaluate the presence or absence of aromatase cytochrome P450 (P450arom) transcripts in endometrial tissues (n = 7) and endometrial stromal cells (n = 9) under various culture conditions. RNA was isolated from four proliferative and three secretory tissue samples and from cultured endometrial stromal cells isolated from seven proliferative and two secretory endometria. Five sets of cultures were treated with medroxyprogesterone acetate (MPA), estradiol (E2), and forskolin. Additionally, RNA was isolated from decidualized endometrium obtained from a patient with tubal pregnancy. A single stranded cDNA was synthesized from total RNA using Moloney murine leukemia virus reverse transcriptase and a P450arom-specific oligonucleotide. The single stranded cDNA was used as a template for PCR and was amplified for 20-35 cycles using P450arom-specific primers. RNA from adipose tissue and placenta was amplified to provide positive controls, whereas myometrial RNA was used as a negative control. In two experiments involving two endometrial tissues and three sets of cells in culture, a rat P450arom cRNA was coamplified in each sample as an internal control to demonstrate that the remote possibility of RT-PCR failures in individual test samples cannot account for our negative results. By Southern or slot blot hybridization of the amplified fragments using human and rat P450arom-specific probes, we found no evidence for

  8. Radical Intermediates in the Catalytic Oxidation of Hydrocarbons by Bacterial and Human Cytochrome P450 Enzymes†

    OpenAIRE

    Jiang, Yongying; He, Xiang; Ortiz de Montellano, Paul R.

    2006-01-01

    Cytochromes P450cam and P450BM3 oxidize α- and β-thujone into multiple products, including 7-hydroxy-α-(or β-)thujone, 7,8-dehydro-α-(or β-)thujone, 4-hydroxy-α-(or β-)thujone, 2-hydroxy α-(or β-)thujone, 5-hydroxy-5-isopropyl-2-methyl-2-cyclohexen-1-one, 4,10-dehydrothujone, and carvacrol. Quantitative analysis of the 4-hydroxylated isomers and the ring opened product indicates that the hydroxylation proceeds via a radical mechanism with a radical recombination rate ranging from 0.7 ± 0.3 × ...

  9. Cytochrome P450 2A13 enhances the sensitivity of human bronchial epithelial cells to aflatoxin B1-induced DNA damage

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Xuejiao [Key Lab of Modern Toxicology of Ministry of Education, School of Public Health, Nanjing Medical University, 818 East Tiangyuan Rd., Nanjing 211166 (China); Jiaojiang District Center for Disease Control and Prevention, 518 Jingdong Rd., Taizhou 318000 (China); Zhang, Zhan; Wang, Xichen; Wang, Yun; Zhang, Xiaoming; Lu, Huiyuan [Key Lab of Modern Toxicology of Ministry of Education, School of Public Health, Nanjing Medical University, 818 East Tiangyuan Rd., Nanjing 211166 (China); Wang, Shou-Lin, E-mail: wangshl@njmu.edu.cn [Key Lab of Modern Toxicology of Ministry of Education, School of Public Health, Nanjing Medical University, 818 East Tiangyuan Rd., Nanjing 211166 (China)

    2013-07-15

    Cytochrome P450 2A13 (CYP2A13) mainly expresses in human respiratory system and mediates the metabolic activation of aflatoxin B1 (AFB1). Our previous study suggested that CYP2A13 could increase the cytotoxic and apoptotic effects of AFB1 in immortalized human bronchial epithelial cells (BEAS-2B). However, the role of CYP2A13 in AFB1-induced DNA damage is unclear. Using BEAS-2B cells that stably express CYP2A13 (B-2A13), CYP1A2 (B-1A2), and CYP2A6 (B-2A6), we compared their effects in AFB1-induced DNA adducts, DNA damage, and cell cycle changes. BEAS-2B cells that were transfected with vector (B-vector) were used as a control. The results showed that AFB1 (5–80 nM) dose- and time-dependently induced DNA damage in B-2A13 cells. AFB1 at 10 and 80 nM significantly augmented this effect in B-2A13 and B-1A2 cells, respectively. B-2A6 cells showed no obvious DNA damage, similar to B-vector cells and the vehicle control. Similarly, compared with B-vector, B-1A2 or B-2A6 cells, B-2A13 cells showed more sensitivity in AFB1-induced γH2AX expression, DNA adduct 8-hydroxy-deoxyguanosine formation, and S-phase cell-cycle arrest. Furthermore, AFB1 activated the proteins related to DNA damage responses, such as ATM, ATR, Chk2, p53, BRCA1, and H2AX, rather than the proteins related to DNA repair. These effects could be almost completely inhibited by 100 μM nicotine (a substrate of CYP2A13) or 1 μM 8-methoxypsoralen (8-MOP; an inhibitor of CYP enzyme). Collectively, these findings suggest that CYP2A13 plays an important role in low-concentration AFB1-induced DNA damage, possibly linking environmental airborne AFB1 to genetic injury in human respiratory system. - Highlights: • CYP2A13 plays a critical role in low concentration of AFB1-induced DNA damage. • B-2A13 cells were more sensitive to AFB1 than B-1A2 cells and B-2A6 cells. • AFB1 dose- and time-dependently induced DNA damage in B-2A13 cells • AFB1-induced DNA adducts and damage can be inhibited by nicotine and 8

  10. Cytochrome P450 2A13 enhances the sensitivity of human bronchial epithelial cells to aflatoxin B1-induced DNA damage

    International Nuclear Information System (INIS)

    Yang, Xuejiao; Zhang, Zhan; Wang, Xichen; Wang, Yun; Zhang, Xiaoming; Lu, Huiyuan; Wang, Shou-Lin

    2013-01-01

    Cytochrome P450 2A13 (CYP2A13) mainly expresses in human respiratory system and mediates the metabolic activation of aflatoxin B1 (AFB1). Our previous study suggested that CYP2A13 could increase the cytotoxic and apoptotic effects of AFB1 in immortalized human bronchial epithelial cells (BEAS-2B). However, the role of CYP2A13 in AFB1-induced DNA damage is unclear. Using BEAS-2B cells that stably express CYP2A13 (B-2A13), CYP1A2 (B-1A2), and CYP2A6 (B-2A6), we compared their effects in AFB1-induced DNA adducts, DNA damage, and cell cycle changes. BEAS-2B cells that were transfected with vector (B-vector) were used as a control. The results showed that AFB1 (5–80 nM) dose- and time-dependently induced DNA damage in B-2A13 cells. AFB1 at 10 and 80 nM significantly augmented this effect in B-2A13 and B-1A2 cells, respectively. B-2A6 cells showed no obvious DNA damage, similar to B-vector cells and the vehicle control. Similarly, compared with B-vector, B-1A2 or B-2A6 cells, B-2A13 cells showed more sensitivity in AFB1-induced γH2AX expression, DNA adduct 8-hydroxy-deoxyguanosine formation, and S-phase cell-cycle arrest. Furthermore, AFB1 activated the proteins related to DNA damage responses, such as ATM, ATR, Chk2, p53, BRCA1, and H2AX, rather than the proteins related to DNA repair. These effects could be almost completely inhibited by 100 μM nicotine (a substrate of CYP2A13) or 1 μM 8-methoxypsoralen (8-MOP; an inhibitor of CYP enzyme). Collectively, these findings suggest that CYP2A13 plays an important role in low-concentration AFB1-induced DNA damage, possibly linking environmental airborne AFB1 to genetic injury in human respiratory system. - Highlights: • CYP2A13 plays a critical role in low concentration of AFB1-induced DNA damage. • B-2A13 cells were more sensitive to AFB1 than B-1A2 cells and B-2A6 cells. • AFB1 dose- and time-dependently induced DNA damage in B-2A13 cells • AFB1-induced DNA adducts and damage can be inhibited by nicotine and 8

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

    Science.gov (United States)

    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.

  12. INTERACTION OF AROMATIC CYTOKININS WITH HUMAN LIVER MICROSOMAL CYTOCHROMES P450

    Czech Academy of Sciences Publication Activity Database

    Anzenbacherová, E.; Janalík, J.; Popa, Igor; Strnad, Miroslav; Anzenbacher, P.

    2005-01-01

    Roč. 149, č. 2 (2005), s. 349-351 ISSN 1213-8118 Institutional research plan: CEZ:AV0Z50380511 Keywords : Cytokinins * Cyclin dependent kinase inhibitor * Cytochrome P450 Subject RIV: CE - Biochemistry http://publib.upol.cz/~obd/fulltext/Biomed/2005/2/349.pdf

  13. Genetic polymorphisms of drug-metabolizing cytochrome P450 enzymes in cynomolgus and rhesus monkeys and common marmosets in preclinical studies for humans.

    Science.gov (United States)

    Uno, Yasuhiro; Uehara, Shotaro; Yamazaki, Hiroshi

    2017-12-23

    Cynomolgus monkeys (Macaca fascicularis, Old World Monkeys) and common marmosets (Callithrix jacchus, New World Monkeys) have been widely, and expectedly, used as non-human primate models in drug development studies. Major drug-metabolizing cytochrome P450 (P450) enzymes information is now available that supports these primate species as animal models, and it is established that multiple forms of cynomolgus monkey and common marmoset P450 enzymes have generally similar substrate recognition functionality to human P450 enzymes. This research update provides information on genetic polymorphisms of P450 enzymes in cynomolgus monkey and common marmoset like human P450 enzymes. Information on rhesus monkeys (Macaca mulatta), another macaque species used in drug metabolism studies, is also included for comparison. Among a variety of cynomolgus monkey P450 variants investigated, typical examples include individual pharmacokinetic data for efavirenz and R-warfarin associated with cynomolgus monkey P450 2C9 (formerly 2C43) and 2C19 (2C75) variants, respectively, and for R-omeprazole and S-warfarin associated with marmoset P450 2C19 variants. These findings provide a foundation for understanding the individual pharmacokinetic and toxicological results in non-human primates as preclinical models and will help to further support understanding of molecular mechanisms of human P450 function. In addition to these polymorphic P450 enzymes, effects of aging on some drug clearances mediated by cynomolgus monkey and common marmoset P450 enzymes were found in elder animals or animals pretreated with rifampicin. This review describes genetic and acquired individual differences in cynomolgus monkey and common marmoset P450 enzymes involved in drug oxidation associated with pharmacological and/or toxicological effects. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Metabolism of bilirubin by human cytochrome P450 2A6

    Energy Technology Data Exchange (ETDEWEB)

    Abu-Bakar, A' edah, E-mail: a.abubakar@uq.edu.au [The University of Queensland, National Research Centre for Environmental Toxicology (Entox), 4072 Brisbane, Queensland (Australia); Arthur, Dionne M. [The University of Queensland, National Research Centre for Environmental Toxicology (Entox), 4072 Brisbane, Queensland (Australia); Cooperative Research Centre for Contamination Assessment and Remediation of the Environment, Adelaide (Australia); Wikman, Anna S. [The University of Queensland, National Research Centre for Environmental Toxicology (Entox), 4072 Brisbane, Queensland (Australia); Department of Pharmaceutical Biosciences, Uppsala University, SE-75123 Uppsala (Sweden); Rahnasto, Minna; Juvonen, Risto O.; Vepsäläinen, Jouko; Raunio, Hannu [School of Pharmacy, Faculty of Health Sciences, University of Eastern Finland, POB 1627, 70211 Kuopio (Finland); Ng, Jack C. [The University of Queensland, National Research Centre for Environmental Toxicology (Entox), 4072 Brisbane, Queensland (Australia); Cooperative Research Centre for Contamination Assessment and Remediation of the Environment, Adelaide (Australia); Lang, Matti A. [The University of Queensland, National Research Centre for Environmental Toxicology (Entox), 4072 Brisbane, Queensland (Australia)

    2012-05-15

    The mouse cytochrome P450 (CYP) 2A5 has recently been shown to function as hepatic “Bilirubin Oxidase” (Abu-Bakar, A., et al., 2011. Toxicol. Appl. Pharmacol. 257, 14–22). To date, no information is available on human CYP isoforms involvement in bilirubin metabolism. In this paper we provide novel evidence for human CYP2A6 metabolising the tetrapyrrole bilirubin. Incubation of bilirubin with recombinant yeast microsomes expressing the CYP2A6 showed that bilirubin inhibited CYP2A6-dependent coumarin 7-hydroxylase activity to almost 100% with an estimated K{sub i} of 2.23 μM. Metabolite screening by a high-performance liquid chromatography/electrospray ionisation mass spectrometry indicated that CYP2A6 oxidised bilirubin to biliverdin and to three other smaller products with m/z values of 301, 315 and 333. Molecular docking analyses indicated that bilirubin and its positively charged intermediate interacted with key amino acid residues at the enzyme's active site. They were stabilised at the site in a conformation favouring biliverdin formation. By contrast, the end product, biliverdin was less fitting to the active site with the critical central methylene bridge distanced from the CYP2A6 haem iron facilitating its release. Furthermore, bilirubin treatment of HepG2 cells increased the CYP2A6 protein and activity levels with no effect on the corresponding mRNA. Co-treatment with cycloheximide (CHX), a protein synthesis inhibitor, resulted in increased half-life of the CYP2A6 compared to cells treated only with CHX. Collectively, the observations indicate that the CYP2A6 may function as human “Bilirubin Oxidase” where bilirubin is potentially a substrate and a regulator of the enzyme. -- Highlights: ► Human CYP2A6 interacts with bilirubin with a high affinity. ► Bilirubin docking to the CYP2A6 active site is more stable than biliverdin docking. ► Recombinant CYP2A6 microsomes metabolised bilirubin to biliverdin. ► Bilirubin increased the hepatic

  15. Metabolism of bilirubin by human cytochrome P450 2A6

    International Nuclear Information System (INIS)

    Abu-Bakar, A'edah; Arthur, Dionne M.; Wikman, Anna S.; Rahnasto, Minna; Juvonen, Risto O.; Vepsäläinen, Jouko; Raunio, Hannu; Ng, Jack C.; Lang, Matti A.

    2012-01-01

    The mouse cytochrome P450 (CYP) 2A5 has recently been shown to function as hepatic “Bilirubin Oxidase” (Abu-Bakar, A., et al., 2011. Toxicol. Appl. Pharmacol. 257, 14–22). To date, no information is available on human CYP isoforms involvement in bilirubin metabolism. In this paper we provide novel evidence for human CYP2A6 metabolising the tetrapyrrole bilirubin. Incubation of bilirubin with recombinant yeast microsomes expressing the CYP2A6 showed that bilirubin inhibited CYP2A6-dependent coumarin 7-hydroxylase activity to almost 100% with an estimated K i of 2.23 μM. Metabolite screening by a high-performance liquid chromatography/electrospray ionisation mass spectrometry indicated that CYP2A6 oxidised bilirubin to biliverdin and to three other smaller products with m/z values of 301, 315 and 333. Molecular docking analyses indicated that bilirubin and its positively charged intermediate interacted with key amino acid residues at the enzyme's active site. They were stabilised at the site in a conformation favouring biliverdin formation. By contrast, the end product, biliverdin was less fitting to the active site with the critical central methylene bridge distanced from the CYP2A6 haem iron facilitating its release. Furthermore, bilirubin treatment of HepG2 cells increased the CYP2A6 protein and activity levels with no effect on the corresponding mRNA. Co-treatment with cycloheximide (CHX), a protein synthesis inhibitor, resulted in increased half-life of the CYP2A6 compared to cells treated only with CHX. Collectively, the observations indicate that the CYP2A6 may function as human “Bilirubin Oxidase” where bilirubin is potentially a substrate and a regulator of the enzyme. -- Highlights: ► Human CYP2A6 interacts with bilirubin with a high affinity. ► Bilirubin docking to the CYP2A6 active site is more stable than biliverdin docking. ► Recombinant CYP2A6 microsomes metabolised bilirubin to biliverdin. ► Bilirubin increased the hepatic CYP2A6

  16. Determination of the human cytochrome P450 monooxygenase catalyzing the enantioselective oxidation of 2,2',3,5',6-pentachlorobiphenyl (PCB 95) and 2,2',3,4,4',5',6-heptachlorobiphenyl (PCB 183).

    Science.gov (United States)

    Nagayoshi, Haruna; Kakimoto, Kensaku; Konishi, Yoshimasa; Kajimura, Keiji; Nakano, Takeshi

    2017-10-17

    2,2',3,5',6-Pentachlorobiphenyl (PCB 95) and 2,2',3,4,4',5',6-heptachlorobiphenyl (PCB 183) possess axial chirality and form the aS and aR enantiomers. The enantiomers of these congeners have been reported to accumulate in the human body enantioselectively via unknown mechanisms. In this study, we determined the cytochrome P450 (CYP) monooxygenase responsible for the enantioselective oxidization of PCB 95 and PCB 183, using a recombinant human CYP monooxygenase. We evaluated 13 CYP monooxygenases, namely CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2C8, CYP2C19, CYP2E1, CYP2J2, CYP3A4, CYP3A5, CYP4F2, and aromatase (CYP19), and revealed that CYP2A6 preferably oxidizes aS-PCB 95 enantioselectively; however, it did not oxidize PCB 183. The enantiomer composition was elevated from 0.5 (racemate) to 0.54. In addition, following incubation with CYP2A6, the enantiomer fraction (EF) of PCB 95 demonstrated a time-dependent increase.

  17. Estimation of the binding modes with important human cytochrome P450 enzymes, drug interaction potential, pharmacokinetics, and hepatotoxicity of ginger components using molecular docking, computational, and pharmacokinetic modeling studies.

    Science.gov (United States)

    Qiu, Jia-Xuan; Zhou, Zhi-Wei; He, Zhi-Xu; Zhang, Xueji; Zhou, Shu-Feng; Zhu, Shengrong

    2015-01-01

    Ginger is one of the most commonly used herbal medicines for the treatment of numerous ailments and improvement of body functions. It may be used in combination with prescribed drugs. The coadministration of ginger with therapeutic drugs raises a concern of potential deleterious drug interactions via the modulation of the expression and/or activity of drug-metabolizing enzymes and drug transporters, resulting in unfavorable therapeutic outcomes. This study aimed to determine the molecular interactions between 12 main active ginger components (6-gingerol, 8-gingerol, 10-gingerol, 6-shogaol, 8-shogaol, 10-shogaol, ar-curcumene, β-bisabolene, β-sesquiphelandrene, 6-gingerdione, (-)-zingiberene, and methyl-6-isogingerol) and human cytochrome P450 (CYP) 1A2, 2C9, 2C19, 2D6, and 3A4 and to predict the absorption, distribution, metabolism, excretion, and toxicity (ADMET) of the 12 ginger components using computational approaches and comprehensive literature search. Docking studies showed that ginger components interacted with a panel of amino acids in the active sites of CYP1A2, 2C9, 2C19, 2D6, and 3A4 mainly through hydrogen bond formation, to a lesser extent, via π-π stacking. The pharmacokinetic simulation studies showed that the [I]/[Ki ] value for CYP2C9, 2C19, and 3A4 ranged from 0.0002 to 19.6 and the R value ranged from 1.0002 to 20.6 and that ginger might exhibit a high risk of drug interaction via inhibition of the activity of human CYP2C9 and CYP3A4, but a low risk of drug interaction toward CYP2C19-mediated drug metabolism. Furthermore, it has been evaluated that the 12 ginger components possessed a favorable ADMET profiles with regard to the solubility, absorption, permeability across the blood-brain barrier, interactions with CYP2D6, hepatotoxicity, and plasma protein binding. The validation results showed that there was no remarkable effect of ginger on the metabolism of warfarin in humans, whereas concurrent use of ginger and nifedipine exhibited a

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

    Energy Technology Data Exchange (ETDEWEB)

    Jan, Yi-Hua [Department of Environmental and Occupational Medicine, Rutgers Robert Wood Johnson Medical School, Piscataway, NJ (United States); Richardson, Jason R., E-mail: jricha3@eohsi.rutgers.edu [Department of Environmental and Occupational Medicine, Rutgers Robert Wood Johnson Medical School, Piscataway, NJ (United States); Baker, Angela A. [Department of Environmental and Occupational Medicine, Rutgers Robert Wood Johnson Medical School, Piscataway, NJ (United States); Mishin, Vladimir [Department of Pharmacology and Toxicology, Rutgers University, Piscataway, NJ (United States); Heck, Diane E. [Department of Environmental Health Science, New York Medical College, Valhalla, NY (United States); Laskin, Debra L. [Department of Pharmacology and Toxicology, Rutgers University, Piscataway, NJ (United States); Laskin, Jeffrey D., E-mail: jlaskin@eohsi.rutgers.edu [Department of Environmental and Occupational Medicine, Rutgers Robert Wood Johnson Medical School, Piscataway, NJ (United States)

    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 (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.

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

    International Nuclear Information System (INIS)

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

    2015-01-01

    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 (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.

  20. Development of an on-line high performance liquid chromatography detection system for human cytochrome P450 1A2 inhibitors in extracts of natural products

    NARCIS (Netherlands)

    Jeurissen, S.M.F.; Claassen, F.W.; Havlik, J.; Bouwmans, E.E.; Cnubben, N.H.P.; Sudhölter, E.J.R.; Rietjens, I.M.C.M.; Beek, T.A. van

    2007-01-01

    An on-line HPLC screening method for detection of inhibitors of human cytochrome P450 1A2 in extracts was developed. HPLC separation of extracts is connected to a continuous methoxyresorufin-O-demethylation (MROD) assay in which recombinant human P450 1A2 converts methoxyresorufin to its fluorescent

  1. [Modeling of a three-dimensional structure of cytochrome P-450 1A2 and search for its new ligands].

    Science.gov (United States)

    Belkina, N V; Skvortsov, V S; Ivanov, A S; Archakov, A I

    1998-01-01

    The substances inhibiting cytochrome P450 1A2 (CYP1A2) represent a perspective class of new drugs, which application in clinical practice can become the important part in preventive maintenance in oncology. The present work is devoted to computer modelling of 3-D structure of CYP1A2 and searching of new inhibitors by database mining. The modelling of CYP1A2 was done based on homology with 4 bacterial cytochromes P450 with known 3-D structure. For optimization of CYP1A2 active site structure the models of its complexes with characteristic substrates (caffeine and 7-ethoxyresorufin) were designed. These complexes were optimized by molecular dynamics simulation in water. The models of 24 complexes of CYP1A2 with known ligands with known Kd were designed by means of DockSearch and LeapFrog programs. 3D-QSAR model with good predictive force was created based on these complexes. On a final stage the search of knew CYP1A2 ligands in testing database (more than 23.000 substances from database Maybridge and 112 known CYP1A2 ligands from database Metabolite, MDL) was executed. 680 potential ligands of CYP1A2 with Kd values, comparable with known ones were obtained. This number has included 73 compounds from 112 known ligands, introduced in tested database as the internal control.

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

    Science.gov (United States)

    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

  3. Comparative study of hops-containing products on human cytochrome P450-mediated metabolism.

    Science.gov (United States)

    Foster, Brian C; Arnason, John T; Saleem, Ammar; Tam, Teresa W; Liu, Rui; Mao, Jingqin; Desjardins, Suzanne

    2011-05-11

    The potential for 15 different ales (6), ciders (2 apple and 1 pear), and porters (6) and 2 non-alcoholic products to affect cytochrome P450 (CYP)-mediated biotransformation and P-glycoprotein-mediated efflux of rhodamine was examined. As in our previous study, a wide range of recovered nonvolatile suspended solids dry weights were noted. Aliquots were also found to have varying effects on biotransformation and efflux. Distinct differences in product ability to affect the safety and efficacy of therapeutic products confirmed our initial findings that some porters (stouts) have a potential to affect the safety and efficacy of health products metabolized by CYP2D6 and CYP3A4 isozymes. Most products, except 2 of the ciders and the 2 non-alcoholic products, also have the potential to affect the safety of CYP2C9 metabolized medications and supplements. Further studies are required to determine the clinical significance of these findings.

  4. Metabolic Pathway of Icotinib In Vitro: The Differential Roles of CYP3A4, CYP3A5, and CYP1A2 on Potential Pharmacokinetic Drug-Drug Interaction.

    Science.gov (United States)

    Zhang, TianHong; Zhang, KeRong; Ma, Li; Li, Zheng; Wang, Juan; Zhang, YunXia; Lu, Chuang; Zhu, Mingshe; Zhuang, XiaoMei

    2018-04-01

    Icotinib is the first self-developed small molecule drug in China for targeted therapy of non-small cell lung cancer. To date, systematic studies on the pharmacokinetic drug-drug interaction of icotinib were limited. By identifying metabolite generated in human liver microsomes and revealing the contributions of major cytochromes P450 (CYPs) in the formation of major metabolites, the aim of the present work was to understand the mechanisms underlying pharmacokinetic and pharmacological variability in clinic. A liquid chromatography/UV/high-resolution mass spectrometer method was developed to characterize the icotinib metabolites. The formation of 6 major metabolites was studied in recombinant CYP isozymes and human liver microsomes with specific inhibitors to identify the CYPs responsible for icotinib metabolism. The metabolic pathways observed in vitro are consistent with those observed in human. Results demonstrated that the metabolites are predominantly catalyzed by CYP3A4 (77%∼87%), with a moderate contribution from CYP3A5 (5%∼15%) and CYP1A2 (3.7%∼7.5%). The contribution of CYP2C8, 2C9, 2C19, and 2D6 is insignificant. Based on our observations, to minimize drug-drug interaction risk in clinic, coprescription of icotinib with strong CYP3A inhibitors or inducers must be weighed. CYP1A2, a highly inducible enzyme in the smoking population, may also represent a determinant of pharmacokinetic and pharmacological variability of icotinib, especially in lung cancer patients with smoking history. Copyright © 2018 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

  5. The Flavin-Containing Reductase Domain of Cytochrome P450 BM3 Acts as a Surrogate for Mammalian NADPH-P450 Reductase.

    Science.gov (United States)

    Park, Seon-Ha; Kang, Ji-Yeon; Kim, Dong-Hyun; Ahn, Taeho; Yun, Chul-Ho

    2012-11-01

    Cytochrome P450 BM3 (CYP102A1) from Bacillus megaterium is a self-sufficient monooxygenase that consists of a heme domain and FAD/FMN-containing reductase domain (BMR). In this report, the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) by BMR was evaluated as a method for monitoring BMR activity. The electron transfer proceeds from NADPH to BMR and then to BMR substrates, MTT and CTC. MTT and CTC are monotetrazolium salts that form formazans upon reduction. The reduction of MTT and CTC followed classical Michaelis-Menten kinetics (kcat =4120 min(-1), Km =77 μM for MTT and kcat =6580 min(-1), Km =51 μM for CTC). Our continuous assay using MTT and CTC allows the simple, rapid measurement of BMR activity. The BMR was able to metabolize mitomycin C and doxorubicin, which are anticancer drug substrates for CPR, producing the same metabolites as those produced by CPR. Moreover, the BMR was able to interact with CYP1A2 and transfer electrons to promote the oxidation reactions of substrates by CYP1A2 and CYP2E1 in humans. The results of this study suggest the possibility of the utilization of BMR as a surrogate for mammalian CPR.

  6. Simultaneous quantification of the abundance of several cytochrome P450 and uridine 5'-diphospho-glucuronosyltransferase enzymes in human liver microsomes using multiplexed targeted proteomics.

    Science.gov (United States)

    Achour, Brahim; Russell, Matthew R; Barber, Jill; Rostami-Hodjegan, Amin

    2014-04-01

    Cytochrome P450 (P450) and uridine 5'-diphospho-glucuronosyltransferase (UGT) enzymes mediate a major proportion of phase I and phase II metabolism of xenobiotics. In vitro-in vivo extrapolation (IVIVE) of hepatic clearance in conjunction with physiologically-based pharmacokinetics (PBPK) has become common practice in drug development. However, prediction of xenobiotic kinetics in virtual populations requires knowledge of both enzyme abundances and the extent to which these correlate. A multiplexed quantification concatemer (QconCAT) strategy was used in this study to quantify the expression of several P450 and UGT enzymes simultaneously and to establish correlations between various enzyme abundances in 24 individual liver samples (ages 27-66, 14 male). Abundances were comparable to previously reported values, including CYP2C9 (40.0 ± 26.0 pmol mg(-1)), CYP2D6 (11.9 ± 13.2 pmol mg(-1)), CYP3A4 (68.1 ± 52.3 pmol mg(-1)), UGT1A1 (33.6 ± 34.0 pmol mg(-1)), and UGT2B7 (82.9 ± 36.1 pmol mg(-1)), expressed as mean ± S.D. Previous reports of correlations in expression of various P450 (CYP3A4/CYP3A5*1/*3, CYP2C8/CYP2C9, and CYP3A4/CYP2B6) were confirmed. New correlations were demonstrated between UGTs [including UGT1A6/UGT1A9 (r(s) = 0.82, P enzymes were shown to be correlated [including CYP1A2/UGT2B4 (r(s) = 0.67, P = 0.0002)]. The expression of CYP3A5 in individuals with *1/*3 genotype (n = 11) was higher than those with *3/*3 genotype (n = 10) (P history of smoking or alcohol use on enzyme expression was observed; however, expression of several enzymes declined with age. The correlation matrix produced for the first time by this study can be used to generate more realistic virtual populations with respect to abundance of various enzymes.

  7. Evaluation of the Effects of Mitragyna speciosa Alkaloid Extract on Cytochrome P450 Enzymes Using a High Throughput Assay

    Directory of Open Access Journals (Sweden)

    Raja Elina Raja Aziddin

    2011-08-01

    Full Text Available The extract from Mitragyna speciosa has been widely used as an opium substitute, mainly due to its morphine-like pharmacological effects. This study investigated the effects of M. speciosa alkaloid extract (MSE on human recombinant cytochrome P450 (CYP enzyme activities using a modified Crespi method. As compared with the liquid chromatography-mass spectrometry method, this method has shown to be a fast and cost-effective way to perform CYP inhibition studies. The results indicated that MSE has the most potent inhibitory effect on CYP3A4 and CYP2D6, with apparent half-maximal inhibitory concentration (IC50 values of 0.78 µg/mL and 0.636 µg/mL, respectively. In addition, moderate inhibition was observed for CYP1A2, with an IC50 of 39 µg/mL, and weak inhibition was detected for CYP2C19. The IC50 of CYP2C19 could not be determined, however, because inhibition was < 50%. Competitive inhibition was found for the MSE-treated CYP2D6 inhibition assay, whereas non-competitive inhibition was shown in inhibition assays using CYP3A4, CYP1A2 and CYP2C19. Quinidine (CYP2D6, ketoconazole (CYP3A4, tranylcypromine (CYP2C19 and furafylline (CYP1A2 were used as positive controls throughout the experiments. This study shows that MSE may contribute to an herb-drug interaction if administered concomitantly with drugs that are substrates for CYP3A4, CYP2D6 and CYP1A2.

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

    Science.gov (United States)

    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.

  9. Inhibition of Procarcinogen Activating Enzyme CYP1A2 Activity and Free Radical Formation by Caffeic Acid and its Amide Analogues.

    Science.gov (United States)

    Narongchai, Paitoon; Niwatananun, Kanokporn; Narongchai, Siripun; Kusirisin, Winthana; Jaikang, Churdsak

    2016-01-01

    Caffeic acid (CAF) and its amide analogues, ethyl 1-(3',4'-dihydroxyphenyl) propen amide (EDPA), phenethyl 1-(3',4'-dihydroxyphenyl) propen amide (PEDPA), phenmethyl 1- (3',4'-dihydroxyphenyl) propen amide (PMDPA) and octyl 1-(3',4'-dihydroxyphenyl) propen amide (ODPA) were investigated for the inhibition of procarcinogen activating enzyme. CYP1A2 and scavenging activity on formation of nitric oxide, superoxide anion, DPPH radical and hydroxyl radical. It was found that they inhibited CYP1A2 enzyme by uncompetitive inhibition. Apparent Ki values of CAF, EDPA, PEDPA, PMDPA and ODPA were 0.59, 0.39, 0.45, 0.75 and 0.80 µM, respectively suggesting potent inhibitors of CYP1A2. Moreover, they potentially scavenged nitric oxide radical with IC 50 values of 0.12, 0.22, 0.28, 0.22 and 0.51 mM, respectively. The IC50 values of superoxide anion scavenging were 0.20, 0.22, 0.44, 2.18 and 2.50 mM, respectively. 1, 1- diphenyl-2- picrylhydrazyl (DPPH) radical-scavenging ability, shown as IC50 values, were 0.41, 0.29, 0.30, 0.89 and 0.84 mM, respectively. Moreover, the hydroxyl radical scavenging in vitro model was shown as IC50 values of 23.22, 21.06, 17.10, 17.21 and 15.81 µM, respectively. From our results, caffeic acid and its amide analogues are in vitro inhibitors of human CYP1A2 catalytic activity and free radical formation. They may be useful to be developed as potential chemopreventive agents that block CYP1A2-mediated chemical carcinogenesis.

  10. Subgrouping of patients with oral lichen planus according to cytochrome P450 enzyme phenotype and genotype

    DEFF Research Database (Denmark)

    Kragelund, Camilla; Jensen, Siri Beier; Hansen, Claus

    2014-01-01

    Objective. This study aimed to determine if the activity of the environmentally influenced cytochrome P450 enzyme CYP1A2, alone or in combination with CYP2D6*4 genotype, discriminates subgroups of oral lichen planus (OLP) according to lifestyle factors and clinical manifestations. Study Design...

  11. Comparison of CYP1A2 and NAT2 phenotypes between black and white smokers.

    Science.gov (United States)

    Muscat, Joshua E; Pittman, Brian; Kleinman, Wayne; Lazarus, Philip; Stellman, Steven D; Richie, John P

    2008-10-01

    The lower incidence rate of transitional cell carcinoma of the urinary bladder in blacks than in whites may be due to racial differences in the catalytic activity of enzymes that metabolize carcinogenic arylamines in tobacco smoke. To examine this, we compared cytochrome P4501A2 (CYP1A2) and N-acetyltransferase-2 activities (NAT2) in black and white smokers using urinary caffeine metabolites as a probe for enzyme activity in a community-based study of 165 black and 183 white cigarette smokers. The paraxanthine (1,7-dimethylxanthine, 17X)/caffeine (trimethylxanthine, 137X) ratio or [17X+1,7-dimethyluric acid (17U)]/137X ratio was used as an indicator of CYP1A2 activity. The 5-acetyl-amino-6-formylamino-3-methyluracil (AFMU)/1-methylxanthine (1X) ratio indicated NAT2 activity. The odds ratio for the slow NAT2 phenotype associated with black race was 0.4; 95% confidence intervals 0.2-0.7. The putative combined low risk phenotype (slow CYP1A2/rapid NAT2) was more common in blacks than in whites (25% vs. 15%, Pwhites.

  12. The structures of three metabolites of the algal hepatotoxin okadaic acid produced by oxidation with human cytochrome P450

    Science.gov (United States)

    Liu, Li; Guoa, Fujiang; Crain, Sheila; Quilliam, Michael A.; Wang, Xiaotang; Rein, Kathleen S.

    2012-01-01

    Four metabolites of okadaic acid were generated by incubation with human recombinant cytochrome P450 3A4. The structures of two of the four metabolites have been determined by MS/MS experiments and 1D and 2D NMR methods using 94 and 133 μg of each metabolite. The structure of a third metabolite was determined by oxidation to a metabolite of known structure. Like okadaic acid, the metabolites are inhibitors of protein phosphatase PP2A. Although one of the metabolites does have an α,β unsaturated carbonyl with the potential to form adducts with an active site cysteine, all of the metabolites are reversible inhibitors of PP2A. PMID:22608922

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

    Directory of Open Access Journals (Sweden)

    Diana Campelo

    2017-10-01

    Full Text Available 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.

  14. Cytochrome P450 humanised mice

    Directory of Open Access Journals (Sweden)

    Gonzalez Frank J

    2004-05-01

    Full Text Available Abstract 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.

  15. Cytochrome P450 humanised mice

    Science.gov (United States)

    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

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

    OpenAIRE

    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...

  17. Estimation of the binding modes with important human cytochrome P450 enzymes, drug interaction potential, pharmacokinetics, and hepatotoxicity of ginger components using molecular docking, computational, and pharmacokinetic modeling studies

    Directory of Open Access Journals (Sweden)

    Qiu JX

    2015-02-01

    Full Text Available Jia-Xuan Qiu,1,2 Zhi-Wei Zhou,3 Zhi-Xu He,4 Xueji Zhang,5 Shu-Feng Zhou,3 Shengrong Zhu11Department of Stomatology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, People’s Republic of China; 2Department of Oral and Maxillofacial Surgery, The First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi, People’s Republic of China; 3Department of Pharmaceutical Sciences, College of Pharmacy, University of South Florida, Tampa, FL, USA; 4Guizhou Provincial Key Laboratory for Regenerative Medicine, Stem Cell and Tissue Engineering Research Center and Sino-US Joint Laboratory for Medical Sciences, Guiyang Medical University, Guiyang, Guizhou, People’s Republic of China; 5Research Center for Bioengineering and Sensing Technology, University of Science and Technology Beijing, Beijing, People’s Republic of ChinaAbstract: Ginger is one of the most commonly used herbal medicines for the treatment of numerous ailments and improvement of body functions. It may be used in combination with prescribed drugs. The coadministration of ginger with therapeutic drugs raises a concern of potential deleterious drug interactions via the modulation of the expression and/or activity of drug-metabolizing enzymes and drug transporters, resulting in unfavorable therapeutic outcomes. This study aimed to determine the molecular interactions between 12 main active ginger components (6-gingerol, 8-gingerol, 10-gingerol, 6-shogaol, 8-shogaol, 10-shogaol, ar-curcumene, ß-bisabolene, ß-sesquiphelandrene, 6-gingerdione, (--zingiberene, and methyl-6-isogingerol and human cytochrome P450 (CYP 1A2, 2C9, 2C19, 2D6, and 3A4 and to predict the absorption, distribution, metabolism, excretion, and toxicity (ADMET of the 12 ginger components using computational approaches and comprehensive literature search. Docking studies showed that ginger components interacted with a panel of amino acids in the active sites of CYP1A

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

    Science.gov (United States)

    Wanwimolruk, Sompon; Phopin, Kamonrat; Prachayasittikul, Virapong

    2014-01-01

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

  19. Pharmacokinetics and Differential Regulation of Cytochrome P450 Enzymes in Type 1 Allergic Mice.

    Science.gov (United States)

    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. Environmentally persistent free radical-containing particulate matter competitively inhibits metabolism by cytochrome P450 1A2

    Energy Technology Data Exchange (ETDEWEB)

    Reed, James R., E-mail: rreed@lsuhsc.edu [Department of Pharmacology and Experimental Therapeutics and Stanley S. Scott Cancer Center, Louisiana State University Health Sciences Center, 533 Bolivar St., New Orleans, LA 70112 (United States); Cruz, Albert Leo N. dela, E-mail: adelac2@tigers.lsu.edu [Department of Environmental Sciences and LSU Superfund Research Center, Louisiana State University A& M College, Baton Rouge, LA 70803 (United States); Lomnicki, Slawo M., E-mail: slomni1@lsu.edu [Department of Environmental Sciences and LSU Superfund Research Center, Louisiana State University A& M College, Baton Rouge, LA 70803 (United States); Backes, Wayne L., E-mail: wbacke@lsuhsc.edu [Department of Pharmacology and Experimental Therapeutics and Stanley S. Scott Cancer Center, Louisiana State University Health Sciences Center, 533 Bolivar St., New Orleans, LA 70112 (United States)

    2015-12-01

    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 reductase 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. • PM

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

    Science.gov (United States)

    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.

  2. Modulation of the interaction between human P450 3A4 and B. megaterium reductase via engineered loops.

    Science.gov (United States)

    Castrignanò, Silvia; D'Avino, Serena; Di Nardo, Giovanna; Catucci, Gianluca; Sadeghi, Sheila J; Gilardi, Gianfranco

    2018-01-01

    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 IC 50 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. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Cytochrome P450s and molecular epidemiology

    Science.gov (United States)

    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.

  4. Computational Identification of the Paralogs and Orthologs of Human Cytochrome P450 Superfamily and the Implication in Drug Discovery

    Directory of Open Access Journals (Sweden)

    Shu-Ting Pan

    2016-06-01

    Full Text Available The human cytochrome P450 (CYP superfamily consisting of 57 functional genes is the most important group of Phase I drug metabolizing enzymes that oxidize a large number of xenobiotics and endogenous compounds, including therapeutic drugs and environmental toxicants. The CYP superfamily has been shown to expand itself through gene duplication, and some of them become pseudogenes due to gene mutations. Orthologs and paralogs are homologous genes resulting from speciation or duplication, respectively. To explore the evolutionary and functional relationships of human CYPs, we conducted this bioinformatic study to identify their corresponding paralogs, homologs, and orthologs. The functional implications and implications in drug discovery and evolutionary biology were then discussed. GeneCards and Ensembl were used to identify the paralogs of human CYPs. We have used a panel of online databases to identify the orthologs of human CYP genes: NCBI, Ensembl Compara, GeneCards, OMA (“Orthologous MAtrix” Browser, PATHER, TreeFam, EggNOG, and Roundup. The results show that each human CYP has various numbers of paralogs and orthologs using GeneCards and Ensembl. For example, the paralogs of CYP2A6 include CYP2A7, 2A13, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 2F1, 2J2, 2R1, 2S1, 2U1, and 2W1; CYP11A1 has 6 paralogs including CYP11B1, 11B2, 24A1, 27A1, 27B1, and 27C1; CYP51A1 has only three paralogs: CYP26A1, 26B1, and 26C1; while CYP20A1 has no paralog. The majority of human CYPs are well conserved from plants, amphibians, fishes, or mammals to humans due to their important functions in physiology and xenobiotic disposition. The data from different approaches are also cross-validated and validated when experimental data are available. These findings facilitate our understanding of the evolutionary relationships and functional implications of the human CYP superfamily in drug discovery.

  5. Pilot Study on Genetic Polymorphisms CYP1A2*1F on Asthma Patients and Nonasthma in Indonesia

    Directory of Open Access Journals (Sweden)

    Doddy de Queljoe

    2015-03-01

    Full Text Available Genetic polymorphisms of CYP1A2 is related to the theophylline metabolism that may affect drug levels in the blood, which can also affect incidence of adverse drug reaction (ADR and clinical outcomes of asthma therapy. The frequency of CYP1A2 polymorphism is known to vary among ethnic. Allegedly the Indonesian population has high frequency of gene variants of CYP1A2*1F. This study aims to determine the profile of CYP1A2*1F gene polymorphism in a sample of nonasthma and asthma in Indonesia with other populations based on the literature. Data were taken on January–June 2014. Blood samples were obtained from 29 nonasthma samples and 16 patients with asthma. After extraction of genomic DNA, CYP1A2*1F gene polymorphisms determined by PCR-RFLP. The results of this study indicate that the CYP1A2*1F gene polymorphism in nonasthma samples was 10.35% (3/29 for C/C, 37.93% (11/29 for the C/A, and 51.72% (15/29 for A/A. The asthmatics genotype have a frequency distribution of C/A genotype of 81.25% (13/16 and A/A of 18.75% (3/16. There was no significant difference (p=0.276 allele frequencies between samples of nonasthma and asthma patients. The frequency of CYP1A2*1F gene in Indonesian population is higher than the population of Egypt, Japan, and UK, but lower compared to Malaysia. It can be concluded that there is no difference in frequency.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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.

  9. Coffee consumption and CYP1A2 genotype in relation to bone mineral density of the proximal femur in elderly men and women: a cohort study

    Directory of Open Access Journals (Sweden)

    Lind Lars

    2010-02-01

    Full Text Available Abstract Background Drinking coffee has been linked to reduced calcium conservation, but it is less clear whether it leads to sustained bone mineral loss and if individual predisposition for caffeine metabolism might be important in this context. Therefore, the relation between consumption of coffee and bone mineral density (BMD at the proximal femur in men and women was studied, taking into account, for the first time, genotypes for cytochrome P450 1A2 (CYP1A2 associated with metabolism of caffeine. Methods Dietary intakes of 359 men and 358 women (aged 72 years, participants of the Prospective Investigation of the Vasculature in Uppsala Seniors (PIVUS, were assessed by a 7-day food diary. Two years later, BMD for total proximal femur, femoral neck and trochanteric regions of the proximal femur were measured by Dual-energy X-ray absorptiometry (DXA. Genotypes of CYP1A2 were determined. Adjusted means of BMD for each category of coffee consumption were calculated. Results Men consuming 4 cups of coffee or more per day had 4% lower BMD at the proximal femur (p = 0.04 compared with low or non-consumers of coffee. This difference was not observed in women. In high consumers of coffee, those with rapid metabolism of caffeine (C/C genotype had lower BMD at the femoral neck (p = 0.01 and at the trochanter (p = 0.03 than slow metabolizers (T/T and C/T genotypes. Calcium intake did not modify the relation between coffee and BMD. Conclusion High consumption of coffee seems to contribute to a reduction in BMD of the proximal femur in elderly men, but not in women. BMD was lower in high consumers of coffee with rapid metabolism of caffeine, suggesting that rapid metabolizers of caffeine may constitute a risk group for bone loss induced by coffee.

  10. Coffee consumption and CYP1A2 genotype in relation to bone mineral density of the proximal femur in elderly men and women: a cohort study.

    Science.gov (United States)

    Hallström, Helena; Melhus, Håkan; Glynn, Anders; Lind, Lars; Syvänen, Ann-Christine; Michaëlsson, Karl

    2010-02-22

    Drinking coffee has been linked to reduced calcium conservation, but it is less clear whether it leads to sustained bone mineral loss and if individual predisposition for caffeine metabolism might be important in this context. Therefore, the relation between consumption of coffee and bone mineral density (BMD) at the proximal femur in men and women was studied, taking into account, for the first time, genotypes for cytochrome P450 1A2 (CYP1A2) associated with metabolism of caffeine. Dietary intakes of 359 men and 358 women (aged 72 years), participants of the Prospective Investigation of the Vasculature in Uppsala Seniors (PIVUS), were assessed by a 7-day food diary. Two years later, BMD for total proximal femur, femoral neck and trochanteric regions of the proximal femur were measured by Dual-energy X-ray absorptiometry (DXA). Genotypes of CYP1A2 were determined. Adjusted means of BMD for each category of coffee consumption were calculated. Men consuming 4 cups of coffee or more per day had 4% lower BMD at the proximal femur (p = 0.04) compared with low or non-consumers of coffee. This difference was not observed in women. In high consumers of coffee, those with rapid metabolism of caffeine (C/C genotype) had lower BMD at the femoral neck (p = 0.01) and at the trochanter (p = 0.03) than slow metabolizers (T/T and C/T genotypes). Calcium intake did not modify the relation between coffee and BMD. High consumption of coffee seems to contribute to a reduction in BMD of the proximal femur in elderly men, but not in women. BMD was lower in high consumers of coffee with rapid metabolism of caffeine, suggesting that rapid metabolizers of caffeine may constitute a risk group for bone loss induced by coffee.

  11. Spaceflight Effects on Cytochrome P450 Content in Mouse Liver.

    Directory of Open Access Journals (Sweden)

    Natalia Moskaleva

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

  12. Identification of human cytochrome P450 and UGT enzymes involved in the metabolism of ferulic acid, a major bioactive component in traditional Chinese medicines.

    Science.gov (United States)

    Zhuang, Xiao-Mei; Chen, Lin; Tan, Yan; Yang, Hai-Ying; Lu, Chuang; Gao, Yue; Li, Hua

    2017-09-01

    Ferulic acid (FA) is an active component of herbal medicines. One of the best documented activities of FA is its antioxidant property. Moreover, FA exerts antiallergic, anti-inflammatory, and hepatoprotective effects. However, the metabolic pathways of FA in humans remain unclear. To identify whether human CYP or UGT enzymes are involved in the metabolism of FA, reaction phenotyping of FA was conducted using major CYP-selective chemical inhibitors together with individual CYP and UGT Supersomes. The CYP- and/or UGT-mediated metabolism kinetics were examined simultaneously or individually. Relative activity factor and total normalized rate approaches were used to assess the relative contributions of each major human CYPs towards the FA metabolism. Incubations of FA with human liver microsomes (HLM) displayed NADPH- and UDPGA-dependent metabolism with multiple CYP and UGT isoforms involved. CYPs and UGTs contributed equally to the metabolism of FA in HLM. Although CYP1A2 and CYP3A4 appeared to be the major contributors in the CYP-mediated clearance, their contributions to the overall clearance are still minor (medicines because multiple phase I and phase II enzymes are involved in its metabolism. Copyright © 2017 China Pharmaceutical University. Published by Elsevier B.V. All rights reserved.

  13. Trapping of cis-2-butene-1,4-dial to measure furan metabolism in human liver microsomes by cytochrome P450 enzymes.

    Science.gov (United States)

    Gates, Leah A; Lu, Ding; Peterson, Lisa A

    2012-03-01

    Furan is a liver toxicant and carcinogen in rodents. It is classified as a possible human carcinogen, but the human health effects of furan exposure remain unknown. The oxidation of furan by cytochrome P450 (P450) enzymes is necessary for furan toxicity. The product of this reaction is the reactive α,β-unsaturated dialdehyde, cis-2-butene-1,4-dial (BDA). To determine whether human liver microsomes metabolize furan to BDA, a liquid chromatography/tandem mass spectrometry method was developed to detect and quantify BDA by trapping this reactive metabolite with N-acetyl-l-cysteine (NAC) and N-acetyl-l-lysine (NAL). Reaction of NAC and NAL with BDA generates N-acetyl-S-[1-(5-acetylamino-5-carboxypentyl)-1H-pyrrol-3-yl]-l-cysteine (NAC-BDA-NAL). Formation of NAC-BDA-NAL was quantified in 21 different human liver microsomal preparations. The levels of metabolism were comparable to that observed in F-344 rat and B6C3F1 mouse liver microsomes, two species known to be sensitive to furan-induced toxicity. Studies with recombinant human liver P450s indicated that CYP2E1 is the most active human liver furan oxidase. The activity of CYP2E1 as measured by p-nitrophenol hydroxylase activity was correlated to the extent of NAC-BDA-NAL formation in human liver microsomes. The formation of NAC-BDA-NAL was blocked by CYP2E1 inhibitors but not other P450 inhibitors. These results suggest that humans are capable of oxidizing furan to its toxic metabolite, BDA, at rates comparable to those of species sensitive to furan exposure. Therefore, humans may be susceptible to furan's toxic effects.

  14. Enantioselective N-demethylation and hydroxylation of sibutramine in human liver microsomes and recombinant cytochrome p-450 isoforms.

    Science.gov (United States)

    Shinde, Dhananjay D; Kim, Min-Jung; Jeong, Eun-Sook; Kim, Yang-Weon; Lee, Ji-Woo; Shin, Jae-Gook; Kim, Dong-Hyun

    2014-01-01

    The enantioselective metabolism of sibutramine was examined using human liver microsomes (HLM) and recombinant cytochrome P-450 (CYP) isoforms. This drug is metabolized to N-mono-desmethyl- (M1) and N,N-di-desmethylsibutramine (M2), and subsequent hydroxylation results in hydroxyl M1 (HM1) and hydroxyl M2 (HM2). No significant difference was noted in formation of M1from sibutramine between R- and S-sibutramine in HLM. However, S-enantiomers of M1 and M2 were preferentially metabolized to M2, HM1, and HM2compared to R-enantiomers in HLM, and intrinsic clearance (Clint) ratios of S-enantiomers/R-enantiomers were 1.97, 4.83, and 9.94 for M2, HM1, and HM2, respectively. CYP3A4 and CYP3A5 were only involved in the formation of M1, whereas CYP2B6 and CYP2C19 were responsible for all metabolic reactions of sibutramine. CYP2C19 and CYP3A5 displayed catalytic preference for S-sibutramine to S-M1, whereas CYP2B6 and CYP3A4 showed little or no stereoselectivity in metabolism of sibutramine to M1. In the case of M2 formation, CYP2B6 metabolized S-M1 more rapidly than R-M1 with a Clint ratio of 2.14. However, CYP2C19 catalyzed less S-M1 than R-M1 and the Clint ratio of S-M1 to R-M1 was 0.65. The most significant enantioselectivity was observed in formation of HM1 from M1, and HM2 from M2. CYP2B6 and CYP2C19 exhibited preferential catalysis of formation of hydroxyl metabolites from S-enantiomers rather than R-enantiomers. These results indicate that S-sibutramine was more rapidly metabolized by CYP isoforms than R-sibutramine, and that enantioselective metabolism needs to be considered in drug interactions involving sibutramine and co-administered drugs.

  15. A High-Throughput (HTS) Assay for Enzyme Reaction Phenotyping in Human Recombinant P450 Enzymes Using LC-MS/MS.

    Science.gov (United States)

    Li, Xiaofeng; Suhar, Tom; Glass, Lateca; Rajaraman, Ganesh

    2014-03-03

    Enzyme reaction phenotyping is employed extensively during the early stages of drug discovery to identify the enzymes responsible for the metabolism of new chemical entities (NCEs). Early identification of metabolic pathways facilitates prediction of potential drug-drug interactions associated with enzyme polymorphism, induction, or inhibition, and aids in the design of clinical trials. Incubation of NCEs with human recombinant enzymes is a popular method for such work because of the specificity, simplicity, and high-throughput nature of this approach for phenotyping studies. The availability of a relative abundance factor and calculated intersystem extrapolation factor for the expressed recombinant enzymes facilitates easy scaling of in vitro data, enabling in vitro-in vivo extrapolation. Described in this unit is a high-throughput screen for identifying enzymes involved in the metabolism of NCEs. Emphasis is placed on the analysis of the human recombinant enzymes CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2B6, and CYP3A4, including the calculation of the intrinsic clearance for each. Copyright © 2014 John Wiley & Sons, Inc. All rights reserved.

  16. Computational prediction of binding affinity for CYP1A2-ligand complexes using empirical free energy calculations

    DEFF Research Database (Denmark)

    Poongavanam, Vasanthanathan; Olsen, Lars; Jørgensen, Flemming Steen

    2010-01-01

    , and methods based on statistical mechanics. In the present investigation, we started from an LIE model to predict the binding free energy of structurally diverse compounds of cytochrome P450 1A2 ligands, one of the important human metabolizing isoforms of the cytochrome P450 family. The data set includes both...... substrates and inhibitors. It appears that the electrostatic contribution to the binding free energy becomes negligible in this particular protein and a simple empirical model was derived, based on a training set of eight compounds. The root mean square error for the training set was 3.7 kJ/mol. Subsequent......Predicting binding affinities for receptor-ligand complexes is still one of the challenging processes in computational structure-based ligand design. Many computational methods have been developed to achieve this goal, such as docking and scoring methods, the linear interaction energy (LIE) method...

  17. Species-related exposure of phase II metabolite gemfibrozil 1-O-β-glucuronide between human and mice: A net induction of mouse P450 activity was revealed.

    Science.gov (United States)

    Luo, Min; Dai, Manyun; Lin, Hante; Xie, Minzhu; Lin, Jiao; Liu, Aiming; Yang, Julin

    2017-12-01

    Gemfibrozil is a fibrate drug used widely for dyslipidemia associated with atherosclerosis. Clinically, both gemfibrozil and its phase II metabolite gemfibrozil 1-O-β-glucuronide (gem-glu) are involved in drug-drug interaction (DDI). But the DDI risk caused by gem-glu between human and mice has not been compared. In this study, six volunteers were recruited and took a therapeutic dose of gemfibrozil for 3 days for examination of the gemfibrozil and gem-glu level in human. Male mice were fed a gemfibrozil diet (0.75%) for 7 days, following which a cocktail-based inhibitory DDI experiment was performed. Plasma samples and liver tissues from mice were collected for determination of gemfibrozil, gem-glu concentration and cytochrome p450 enzyme (P450) induction analysis. In human, the molar ratio of gem-glu/gemfibrozil was 15% and 10% at the trough concentration and the concentration at 1.5 h after the 6th dose. In contrast, this molar ratio at steady state in mice was 91%, demonstrating a 6- to 9-fold difference compared with that in human. Interestingly, a net induction of P450 activity and in vivo inductive DDI potential in mice was revealed. The P450 activity was not inhibited although the gem-glu concentration was high. These data suggested species difference of relative gem-glu exposure between human and mice, as well as a net inductive DDI potential of gemfibrozil in mouse model. Copyright © 2017 John Wiley & Sons, Ltd.

  18. Human Cytochrome P450 3A4 as a Biocatalyst: Effects of the Engineered Linker in Modulation of Coupling Efficiency in 3A4-BMR Chimeras.

    Science.gov (United States)

    Degregorio, Danilo; D'Avino, Serena; Castrignanò, Silvia; Di Nardo, Giovanna; Sadeghi, Sheila J; Catucci, Gianluca; Gilardi, Gianfranco

    2017-01-01

    Human liver cytochrome P450 3A4 is the main enzyme involved in drug metabolism. This makes it an attractive target for biocatalytic applications, such as the synthesis of pharmaceuticals and drug metabolites. However, its poor solubility, stability and low coupling have limited its application in the biotechnological context. We previously demonstrated that the solubility of P450 3A4 can be increased by creating fusion proteins between the reductase from Bacillus megaterium BM3 (BMR) and the N-terminally modified P450 3A4 (3A4-BMR). In this work, we aim at increasing stability and coupling efficiency by varying the length of the loop connecting the two domains to allow higher inter-domain flexibility, optimizing the interaction between the domains. Starting from the construct 3A4-BMR containing the short linker Pro-Ser-Arg, two constructs were generated by introducing a 3 and 5 glycine hinge (3A4-3GLY-BMR and 3A4-5GLY-BMR). The three fusion proteins show the typical absorbance at 450 nm of the reduced heme-CO adduct as well as the correct incorporation of the FAD and FMN cofactors. Each of the three chimeric proteins were more stable than P450 3A4 alone. Moreover, the 3A4-BMR-3-GLY enzyme showed the highest NADPH oxidation rate in line with the most positive reduction potential. On the other hand, the 3A4-BMR-5-GLY fusion protein showed a V max increased by 2-fold as well as a higher coupling efficiency when compared to 3A4-BMR in the hydroxylation of the marker substrate testosterone. This protein also showed the highest rate value of cytochrome c reduction when this external electron acceptor is used to intercept electrons from BMR to P450. The data suggest that the flexibility and the interaction between domains in the chimeric proteins is a key parameter to improve turnover and coupling efficiency. These findings provide important guidelines in engineering catalytically self-sufficient human P450 for applications in biocatalysis.

  19. Gene structure of CYP3A4, an adult-specific form of cytochrome P450 in human livers, and its transcriptional control.

    Science.gov (United States)

    Hashimoto, H; Toide, K; Kitamura, R; Fujita, M; Tagawa, S; Itoh, S; Kamataki, T

    1993-12-01

    CYP3 A4 is the adult-specific form of cytochrome P450 in human livers [Komori, M., Nishio, K., Kitada, M., Shiramatsu, K., Muroya, K., Soma, M., Nagashima, K. & Kamataki, T. (1990) Biochemistry 29, 4430-4433]. The sequences of three genomic clones for CYP3A4 were analyzed for all exons, exon-intron junctions and the 5'-flanking region from the major transcription site to nucleotide position -1105, and compared with those of the CYP3A7 gene, a fetal-specific form of cytochrome P450 in humans. The results showed that the identity of 5'-flanking sequences between CYP3A4 and CYP3A7 genes was 91%, and that each 5'-flanking region had characteristic sequences termed as NFSE (P450NF-specific element) and HFLaSE (P450HFLa specific element), respectively. A basic transcription element (BTE) also lay in the 5'-flanking region of the CYP3A4 gene as seen in many CYP genes [Yanagida, A., Sogawa, K., Yasumoto, K. & Fujii-Kuriyama, Y. (1990) Mol. Cell. Biol. 10, 1470-1475]. The BTE binding factor (BTEB) was present in both adult and fetal human livers. To examine the transcriptional activity of the CYP3A4 gene, DNA fragments in the 5'-flanking region of the gene were inserted in front of the simian virus 40 promoter and the chloramphenicol acetyltransferase structural gene, and the constructs were transfected in HepG2 cells. The analysis of the chloramphenicol acetyltransferase activity indicated that (a) specific element(s) which could bind with a factor(s) in livers was present in the 5'-flanking region of the CYP3A4 gene to show the transcriptional activity.

  20. Glutathione S-transferase M1 and T1, CYP1A2-2467T/delT ...

    African Journals Online (AJOL)

    The present study investigated the impact of metabolic gene polymorphisms in modulating lung cancer risk susceptibility. Gene polymorphisms encoding Cytochrome 1A2 (CYP1A2) and Glutathione-S-transferases (GSTT1 and GSTM1) are involved in the bioactivation and detoxification of tobacco carcinogens and may ...

  1. Polymorphism in cytochrome P450 1A2 and their interaction with risk factors in determining risk of squamous cell lung carcinoma in men.

    Science.gov (United States)

    Singh, Arvind P; Pant, Mohan C; Ruwali, Munindra; Shah, Parag P; Prasad, Rajendra; Mathur, Neeraj; Parmar, Devendra

    The present case-control study was carried out to investigate the association of functionally important polymorphisms of cytochrome P450 1A2 (CYP1A2) involved in the metabolic activation of tobacco derived procarcinogens with squamous cell carcinoma (SCC) of lung in North Indian men. The study consisted of 200 male cases with SCC of lung and an equal number of age and sex matched healthy controls. Our data showed that variant genotype of CYP1A2*1D and CYP1A2*1F were significantly associated with increased susceptibility to SCC of lung. Likewise, GSTM1 null genotype was found to be over represented in patients when compared to controls. Haplotype analysis revealed that haplotype, G-Tdel-T-C was significantly associated with risk to SCC of lung. Moreover, a significant increase in the risk to SCC of lung in the cases carrying combination of variant genotype of CYP1A2 with either CYP1A1 or GSTM1 have shown that gene-gene interactions may play an important role in squamous cell lung cancer risk. Our data also revealed that smokers or tobacco chewers carrying variant alleles of either CYP1A2*1D or CYP1A2*1F were at increased risk to SCC of lung, further demonstrating that CYP1A2 genotypes interact with environmental risk factors in enhancing the risk to squamous cell lung carcinoma.

  2. The cytochrome p450 homepage.

    Science.gov (United States)

    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.

  3. Roles of different forms of cytochrome P450 in the activation of the promutagen 6-aminochrysene to genotoxic metabolites in human liver microsomes.

    Science.gov (United States)

    Yamazaki, H; Mimura, M; Oda, Y; Inui, Y; Shiraga, T; Iwasaki, K; Guengerich, F P; Shimada, T

    1993-07-01

    We reported previously that the potent mutagen 6-aminochrysene is catalyzed principally by rat liver microsomal P4501A and P4502B enzymes to reactive metabolites that induce umu gene expression in O-acetyltransferase-over-expressing strain Salmonella typhimurium NM2009; the proposal was made that there are different mechanisms in the formation of reactive N-hydroxylated and diolepoxide metabolites by P450 enzymes (Yamazaki, H. and Shimada, T., Biochem. Pharmacol., 44, 913-920, 1992). Here we further examined the roles of human liver P450 enzymes and the mechanism of activation of 6-aminochrysene by rat and human P450 enzymes in the Salmonella tester strains. Liver microsomes from 18 different human samples catalyzed activation of 6-aminochrysene more efficiently in S. typhimurium NM2009 than in the original strain of S. typhimurium TA1535/pSK1002. The rates of 6-aminochrysene activation in 18 human liver samples showed good correlation to the contents of P4502B6 as well as contents of P4503A4 and the respective mono-oxygenase activities catalyzed by P4503A4. Among purified P450 enzymes examined, P4501A2 as well as P4503A4 were highly active in transforming 6-amino-chrysene to reactive metabolites, suggesting the involvement of different human P450 enzymes in the reaction. Four human samples that contained relatively high levels of particular P450 enzymes in their microsomes were selected and used for further characterization. Liver microsomes from human samples HL-13 and HL-4 that contained the highest levels of P4502B6 and P4503A4 respectively, were sensitive to the respective antibodies raised against monkey P4502B and human P4503A4; the activity in sample HL-16 having the highest level of P4501A2 was inhibited by anti-P4501A2 IgG. alpha-Naphthoflavone enhanced the activation of 6-aminochrysene very significantly in human liver microsomes enriched in P4503A4 and P4502B6 enzymes. Pentachlorophenol, an inhibitor of acetyltransferase activity, suppressed the

  4. The effects of commercial preparations of herbal supplements commonly used by women on the biotransformation of fluorogenic substrates by human cytochromes P450.

    Science.gov (United States)

    Ho, Shirley H Y; Singh, Mohini; Holloway, Alison C; Crankshaw, Denis J

    2011-07-01

    The study set out to determine the potential for commercially available preparations of black cohosh (Actaea racemosa), chaste tree berry (Vitex agnus-castus), crampbark (Viburnum opulus) and false unicorn (Chamaelirium luteum) to inhibit the major human drug metabolizing enzymes CYP1A2, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 as well as CYP1A1 which activates some carcinogens. In vitro microplate-based assays using cDNA-expressed CYP450 isoforms and fluorogenic substrates were used. Components of the commercial herbal preparations interfered with the assays and limited the concentration ranges that could be tested. Nevertheless, the fluorogenic assays were robust, reproducible and easy to perform and thus are still useful for initial screening for potential herb-drug interactions. None of the preparations affected CYPs 1A1 or 2C9 at the concentrations tested but all preparations inhibited some of the enzymes with potencies around 1 μg/mL. The three most potent interactions were: chaste tree berry and CYP2C19 (IC₅₀) 0.22 μg/mL); chaste tree berry and CYP3A4 (IC₅₀) 0.3 μg/mL); black cohosh and CYP2C19 (IC₅₀) 0.37 μg/mL,). Thus, the study successfully identified the potential for the commercial herbal preparations to inhibit human drug metabolizing enzymes. Whether this potential translates into clinically significant herb-drug interactions can only be confirmed by appropriate in vivo studies. Copyright © 2011 John Wiley & Sons, Ltd.

  5. Human cytochrome P450 2B6 genetic variability in Botswana: a case of haplotype diversity and convergent phenotypes

    KAUST Repository

    Tawe, Leabaneng

    2018-03-14

    Identification of inter-individual variability for drug metabolism through cytochrome P450 2B6 (CYP2B6) enzyme is important for understanding the differences in clinical responses to malaria and HIV. This study evaluates the distribution of CYP2B6 alleles, haplotypes and inferred metabolic phenotypes among subjects with different ethnicity in Botswana. A total of 570 subjects were analyzed for CYP2B6 polymorphisms at position 516 G > T (rs3745274), 785 A > G (rs2279343) and 983 T > C (rs28399499). Samples were collected in three districts of Botswana where the population belongs to Bantu (Serowe/Palapye and Chobe) and San-related (Ghanzi) ethnicity. The three districts showed different haplotype composition according to the ethnic background but similar metabolic inferred phenotypes, with 59.12%, 34.56%, 2.10% and 4.21% of the subjects having, respectively, an extensive, intermediate, slow and rapid metabolic profile. The results hint at the possibility of a convergent adaptation of detoxifying metabolic phenotypes despite a different haplotype structure due to the different genetic background. The main implication is that, while there is substantial homogeneity of metabolic inferred phenotypes among the country, the response to drugs metabolized via CYP2B6 could be individually associated to an increased risk of treatment failure and toxicity. These are important facts since Botswana is facing malaria elimination and a very high HIV prevalence.

  6. Human cytochrome P450 2B6 genetic variability in Botswana: a case of haplotype diversity and convergent phenotypes

    KAUST Repository

    Tawe, Leabaneng; Motshoge, Thato; Ramatlho, Pleasure; Mutukwa, Naledi; Muthoga, Charles Waithaka; Dongho, Ghyslaine Bruna Djeunang; Martinelli, Axel; Peloewetse, Elias; Russo, Gianluca; Quaye, Isaac Kweku; Paganotti, Giacomo Maria

    2018-01-01

    Identification of inter-individual variability for drug metabolism through cytochrome P450 2B6 (CYP2B6) enzyme is important for understanding the differences in clinical responses to malaria and HIV. This study evaluates the distribution of CYP2B6 alleles, haplotypes and inferred metabolic phenotypes among subjects with different ethnicity in Botswana. A total of 570 subjects were analyzed for CYP2B6 polymorphisms at position 516 G > T (rs3745274), 785 A > G (rs2279343) and 983 T > C (rs28399499). Samples were collected in three districts of Botswana where the population belongs to Bantu (Serowe/Palapye and Chobe) and San-related (Ghanzi) ethnicity. The three districts showed different haplotype composition according to the ethnic background but similar metabolic inferred phenotypes, with 59.12%, 34.56%, 2.10% and 4.21% of the subjects having, respectively, an extensive, intermediate, slow and rapid metabolic profile. The results hint at the possibility of a convergent adaptation of detoxifying metabolic phenotypes despite a different haplotype structure due to the different genetic background. The main implication is that, while there is substantial homogeneity of metabolic inferred phenotypes among the country, the response to drugs metabolized via CYP2B6 could be individually associated to an increased risk of treatment failure and toxicity. These are important facts since Botswana is facing malaria elimination and a very high HIV prevalence.

  7. An in-vitro cocktail assay for assessing compound-mediated inhibition of six major cytochrome P450 enzymes

    Directory of Open Access Journals (Sweden)

    Jing-Jing Wang

    2014-08-01

    Full Text Available An efficient screening assay was developed and validated for simultaneous assessment of compound-mediated inhibition of six major human cytochrome P450 (CYP enzymes. This method employed a cocktail of six probe substrates (i.e., phenacetin, amodiaquine, diclofenac, S-mephenytoin, dextromethorphan and midazolam for CYP1A2, 2C8, 2C9, 2C19, 2D6 and 3A4, respectively as well as individual prototypical inhibitors of the six CYP enzymes in human liver microsomes under optimized incubation conditions. The corresponding marker metabolites (i.e., acetaminophen, N-desethylamodiaquine, 4-OH-diclofenac, 4-OH-S-mephenytoin, dextrorphan and 1-OH-midazolam in the incubates were quantified using LC–MS/MS methods either by an internal standard (IS calibration curve or a simplified analyte-to-IS peak area ratio approach. The results showed that the IC50 values determined by the cocktail approach were in good agreement with those obtained by the individual substrate approach as well as those reported in the literature. Besides, no remarkable difference was observed between the two quantification approaches. In conclusion, this new cocktail assay can be used for reliable screening of compound-mediated CYP inhibition. Keywords: LC–MS/MS, Cytochrome P450, Cocktail-probe, Inhibition assessment, Drug screenning

  8. Expression of cytochrome P450 regulators in cynomolgus macaque.

    Science.gov (United States)

    Uno, Yasuhiro; Yamazaki, Hiroshi

    2017-09-11

    1. Cytochrome P450 (P450) regulators including nuclear receptors and transcription factors have not been fully investigated in cynomolgus macaques, an important species used in drug metabolism studies. In this study, we analyzed 17 P450 regulators by sequence and phylogenetic analysis, and tissue expression. 2. Gene and genome structures of 17 P450 regulators were similar to the human orthologs, and the deduced amino acid sequences showed high sequence identities (92-95%) and more closely clustered in a phylogenetic tree, with the human orthologs. 3. Many of the P450 regulator mRNAs were preferentially expressed in the liver, kidney, and/or jejunum. Among the P450 regulator mRNAs, PXR was most abundant in the liver and jejunum, and HNF4α in the kidney. In the liver, the expression of most P450 regulator mRNAs did not show significant differential expression (>2.5-fold) between cynomolgus macaques bred in Cambodia, China, and Indonesia, or rhesus macaques. 4. By correlation analysis, most of the P450 regulators were significantly (p < 0.05) correlated to other P450 regulators, and many of them were also significantly (p < 0.05) correlated with P450s. 5. These results suggest that 17 P450 regulators of cynomolgus macaques had similar molecular characteristics to the human orthologs.

  9. Characterization of the Ala62Pro polymorphic variant of human cytochrome P450 1A1 using recombinant protein expression

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Seung Heon; Kang, Sukmo [College of Veterinary Medicine, BK21plus Program for Creative Veterinary Science Research, and Research Institute for Veterinary Science, Seoul National University, Seoul (Korea, Republic of); Dong, Mi Sook [School of Life Sciences and Biotechnology, Korea University, Seoul (Korea, Republic of); Park, Jung-Duck [College of Medicine, Chung-Ang University, Seoul (Korea, Republic of); Park, Jinseo; Rhee, Sangkee [College of Agriculture of Life Science, Seoul National University, Seoul (Korea, Republic of); Ryu, Doug-Young, E-mail: dyryu@snu.ac.kr [College of Veterinary Medicine, BK21plus Program for Creative Veterinary Science Research, and Research Institute for Veterinary Science, Seoul National University, Seoul (Korea, Republic of)

    2015-06-15

    Cytochrome P450 (CYP) 1A1 is a heme-containing enzyme involved in detoxification of hydrophobic pollutants. Its Ala62Pro variant has been identified previously. Ala62 is located in α-helix A of CYP1A1. Residues such as Pro and Gly are α-helix breakers. In this study, the Ala62Pro variant was characterized using heterologous expression. E. coli expressing the Ala62Pro variant, and the purified variant protein, had lower CYP (i.e. holoenzyme) contents than their wild-type (WT) equivalents. The CYP variant from E. coli and mammalian cells exhibited lower 7-ethoxyresorufin O-dealkylation (EROD) and benzo[a]pyrene hydroxylation activities than the WT. Enhanced supplementation of a heme precursor during E. coli culture did not increase CYP content in E. coli expressing the variant, but did for the WT. As for Ala62Pro, E. coli expressing an Ala62Gly variant had a lower CYP content than the WT counterpart, but substitution of Ala62 with α-helix-compatible residues such as Ser and Val partially recovered the level of CYP produced. Microsomes from mammalian cells expressing Ala62Pro and Ala62Gly variants exhibited lower EROD activities than those expressing the WT or Ala62Val variant. A region harboring α-helix A has interactions with another region containing heme-interacting residues. Site-directed mutagenesis analyses suggest the importance of interactions between the two regions on holoenzyme expression. Together, these findings suggest that the Ala62Pro substitution leads to changes in protein characteristics and function of CYP1A1 via structural disturbance of the region where the residue is located. - Highlights: • Ala62 is located in α-helix A of the carcinogen-metabolizing enzyme CYP1A1. • Pro acts as an α-helix breaker. • A variant protein of CYP1A1, Ala62Pro, had lower heme content than the wild-type. • The variant of CYP1A1 had lower enzyme activities than the wild-type.

  10. Linear Interaction Energy Based Prediction of Cytochrome P450 1A2 Binding Affinities with Reliability Estimation.

    Directory of Open Access Journals (Sweden)

    Luigi Capoferri

    Full Text Available Prediction of human Cytochrome P450 (CYP binding affinities of small ligands, i.e., substrates and inhibitors, represents an important task for predicting drug-drug interactions. A quantitative assessment of the ligand binding affinity towards different CYPs can provide an estimate of inhibitory activity or an indication of isoforms prone to interact with the substrate of inhibitors. However, the accuracy of global quantitative models for CYP substrate binding or inhibition based on traditional molecular descriptors can be limited, because of the lack of information on the structure and flexibility of the catalytic site of CYPs. Here we describe the application of a method that combines protein-ligand docking, Molecular Dynamics (MD simulations and Linear Interaction Energy (LIE theory, to allow for quantitative CYP affinity prediction. Using this combined approach, a LIE model for human CYP 1A2 was developed and evaluated, based on a structurally diverse dataset for which the estimated experimental uncertainty was 3.3 kJ mol-1. For the computed CYP 1A2 binding affinities, the model showed a root mean square error (RMSE of 4.1 kJ mol-1 and a standard error in prediction (SDEP in cross-validation of 4.3 kJ mol-1. A novel approach that includes information on both structural ligand description and protein-ligand interaction was developed for estimating the reliability of predictions, and was able to identify compounds from an external test set with a SDEP for the predicted affinities of 4.6 kJ mol-1 (corresponding to 0.8 pKi units.

  11. The participation of human hepatic P450 isoforms, flavin-containing monooxygenases and aldehyde oxidase in the biotransformation of the insecticide fenthion

    International Nuclear Information System (INIS)

    Leoni, Claudia; Buratti, Franca M.; Testai, Emanuela

    2008-01-01

    Although fenthion (FEN) is widely used as a broad spectrum insecticide on various crops in many countries, very scant data are available on its biotransformation in humans. In this study the in vitro human hepatic FEN biotransformation was characterized, identifying the relative contributions of cytochrome P450 (CYPs) and/or flavin-containing monooxygenase (FMOs) by using single c-DNA expressed human enzymes, human liver microsomes and cytosol and CYP/FMO-specific inhibitors. Two major metabolites, FEN-sulfoxide and FEN-oxon (FOX), are formed by some CYPs although at very different levels, depending on the relative CYP hepatic content. Formation of further oxidation products and the reduction of FEN-sulfoxide back to FEN by the cytosolic aldehyde oxidase enzyme were ruled out. Comparing intrinsic clearance values, FOX formation seemed to be favored and at low FEN concentrations CYP2B6 and 1A2 are mainly involved in its formation. At higher levels, a more widespread CYP involvement was evident, as in the case of FEN-sulfoxide, although a higher efficiency of CYP2C family was suggested. Hepatic FMOs were able to catalyze only sulfoxide formation, but at low FEN concentrations hepatic FEN sulfoxidation is predominantly P450-driven. Indeed, the contribution of the hepatic isoforms FMO 3 and FMO 5 was generally negligible, although at high FEN concentrations FMO's showed activities comparable to the active CYPs, accounting for up to 30% of total sulfoxidation. Recombinant FMO 1 showed the highest efficiency with respect to CYPs and the other FMOs, but it is not expressed in the adult human liver. This suggests that FMO 1 -catalysed sulfoxidation may represent the major extra-hepatic pathway of FEN biotransformation

  12. ANTIBODIES TO BENZO[A]PYRENE AND POLYMORPHISMS OF CYP1A1*2A, CYP1A2*1F, GSTT1, AND GSTM1 GENES IN HEALTHY MEN AND LUNG CANCER PATIENTS

    Directory of Open Access Journals (Sweden)

    A. N. Glushkov

    2016-01-01

    Full Text Available Some genetic polymorphisms of CYP and GST enzymes metabolizing low-molecular weight xenobiotics may represent endogenous risk factors for carcinogenesis. However, possible relationships between the enzyme activities, amounts of carcinogen adducts and synthesis of anticarcinogen antibodies in humans (including cancer patients are still poorly studied. The purpose of this study was to identify possible associations between occurrence of antibodies against benzo[a]pyrene, and frequency of genetic polymorphisms of CYP1A1*2A, CYP1A2*1F, GSTT1, GSTM1 in healthy men and in lung cancer patients. Materials and methods. We have examined 203 men with non-small cell lung cancer and 267 apparently healthy donors without respiratory diseases. A non-competitive solid phase immunoassay of antibodies to benzo[a]pyrene was performed. Analysis of polymorphic loci within CYP1A1 (rs4646903, CYP1A2 (rs762551, GSTP1 (rs1695, rs1138272 was performed by means of real-time PCR using TaqMan technology. Null-alleles of GSTM1 (del, GSTT1 (del genes were detected by multiplex PCR with real-time fluorescent assay. Results. Among the lung cancer patients, the proportion of cases with a high level of IgG antibodies to benzo[a]pyrene in carriers of GSTT1+ and GSTM1+ in conjunction with the CYP1A2*1F C allele was significantly greater than in AA homozygotes CYP1A2*1F. The risk of lung cancer was increased to 5.5 in carriers of CYP1A2*1F C allele combined with GSTT1+ and GSTM1+ at high levels of IgG antibodies to benzo [a] pyrene. In healthy male donors, we have not found differences between the incidence of low and high levels of IgG anti-benzo[a]pyrene antibodies in the carriers of certain CYP1A1*2A, CYP1A2*1F, GSTT1 and GSTM1 genotypes. Conclusions. We have first reported a relationship between CYP1 and GST gene polymorphisms and specific immune response to chemical carcinogens in lung cancer patients. Immunoassays of IgG antibodies to benzo[a]pyrene combined with molecular

  13. The CYP1A2 -163C>A polymorphism does not alter the effects of caffeine on basketball performance.

    Science.gov (United States)

    Puente, Carlos; Abián-Vicén, Javier; Del Coso, Juan; Lara, Beatriz; Salinero, Juan José

    2018-01-01

    The aim of this investigation was to analyze the influence of the genetic variations of the -163C>A polymorphism of the CYP1A2 gene on the ergogenic effects of caffeine in elite basketball players. Nineteen elite basketball players (10 men and 9 women) ingested 3 mg⋅kg-1 of caffeine or a placebo 60 min before performing 10 repetitions of the following series: the Abalakov jump test followed by the Change-of-Direction and Acceleration Test (CODAT). The players then competed in a 20-min simulated basketball game. Self-perceived performance and side effects were recorded by questionnaires after the trials. The effects of caffeine on basketball performance were established according to players' CYP1A2 genotype (rs762551): AA homozygotes (n = 10) and C-allele carriers (n = 9). In the 10 repetitions, caffeine increased Abalakov jump height by a mean of 2.9±3.6% in AA homozygotes (p = 0.03) while this effect did not reach statistical significance for C-allele carriers (2.3 ± 6.8%; p = 0.33). Caffeine did not affect sprint time in the CODAT test in either genotype group but it increased the number of impacts performed during the simulated game in both AA homozygotes (4.1 ± 5.3%; p = 0.02) and C-allele carriers (3.3 ± 3.2%; p = 0.01). During the 24 h following the test, AA homozygotes tended to experience increased insomnia with caffeine while C-allele carriers did not present this effect. The remaining variables were unaffected by the genotype. The CYP1A2 -163C>A polymorphism minimally altered the ergogenicity derived from the consumption of a moderate dose of caffeine in elite basketball players.

  14. The CYP1A2 -163C>A polymorphism does not alter the effects of caffeine on basketball performance

    Science.gov (United States)

    Puente, Carlos; Abián-Vicén, Javier; Lara, Beatriz; Salinero, Juan José

    2018-01-01

    Purpose The aim of this investigation was to analyze the influence of the genetic variations of the -163C>A polymorphism of the CYP1A2 gene on the ergogenic effects of caffeine in elite basketball players. Methods Nineteen elite basketball players (10 men and 9 women) ingested 3 mg⋅kg-1 of caffeine or a placebo 60 min before performing 10 repetitions of the following series: the Abalakov jump test followed by the Change-of-Direction and Acceleration Test (CODAT). The players then competed in a 20-min simulated basketball game. Self-perceived performance and side effects were recorded by questionnaires after the trials. The effects of caffeine on basketball performance were established according to players’ CYP1A2 genotype (rs762551): AA homozygotes (n = 10) and C-allele carriers (n = 9). Results In the 10 repetitions, caffeine increased Abalakov jump height by a mean of 2.9±3.6% in AA homozygotes (p = 0.03) while this effect did not reach statistical significance for C-allele carriers (2.3 ± 6.8%; p = 0.33). Caffeine did not affect sprint time in the CODAT test in either genotype group but it increased the number of impacts performed during the simulated game in both AA homozygotes (4.1 ± 5.3%; p = 0.02) and C-allele carriers (3.3 ± 3.2%; p = 0.01). During the 24 h following the test, AA homozygotes tended to experience increased insomnia with caffeine while C-allele carriers did not present this effect. The remaining variables were unaffected by the genotype. Conclusion The CYP1A2 -163C>A polymorphism minimally altered the ergogenicity derived from the consumption of a moderate dose of caffeine in elite basketball players. PMID:29668752

  15. CYP1A2 Genotype Variations Do Not Modify the Benefits and Drawbacks of Caffeine during Exercise: A Pilot Study

    Directory of Open Access Journals (Sweden)

    Juan J. Salinero

    2017-03-01

    Full Text Available Previous investigations have determined that some individuals have minimal or even ergolytic performance effects after caffeine ingestion. The aim of this study was to analyze the influence of the genetic variations of the CYP1A2 gene on the performance enhancement effects of ingesting a moderate dose of caffeine. In a double-blind randomized experimental design, 21 healthy active participants (29.3 ± 7.7 years ingested 3 mg of caffeine per kg of body mass or a placebo in testing sessions separated by one week. Performance in the 30 s Wingate test, visual attention, and side effects were evaluated. DNA was obtained from whole blood samples and the CYP1A2 polymorphism was analyzed (rs762551. We obtained two groups: AA homozygotes (n = 5 and C-allele carriers (n = 16. Caffeine ingestion increased peak power (682 ± 140 vs. 667 ± 137 W; p = 0.008 and mean power during the Wingate test (527 ± 111 vs. 518 ± 111 W; p < 0.001 with no differences between AA homozygotes and C-allele carriers (p > 0.05. Reaction times were similar between caffeine and placebo conditions (276 ± 31 vs. 269 ± 71 milliseconds; p = 0.681 with no differences between AA homozygotes and C-allele carriers. However, 31.3% of the C-allele carriers reported increased nervousness after caffeine ingestion, while none of the AA homozygotes perceived this side effect. Genetic variations of the CYP1A2 polymorphism did not affect the ergogenic effects and drawbacks derived from the ingestion of a moderate dose of caffeine.

  16. CYP1A2 Genotype Variations Do Not Modify the Benefits and Drawbacks of Caffeine during Exercise: A Pilot Study.

    Science.gov (United States)

    Salinero, Juan J; Lara, Beatriz; Ruiz-Vicente, Diana; Areces, Francisco; Puente-Torres, Carlos; Gallo-Salazar, César; Pascual, Teodoro; Del Coso, Juan

    2017-03-11

    Previous investigations have determined that some individuals have minimal or even ergolytic performance effects after caffeine ingestion. The aim of this study was to analyze the influence of the genetic variations of the CYP1A2 gene on the performance enhancement effects of ingesting a moderate dose of caffeine. In a double-blind randomized experimental design, 21 healthy active participants (29.3 ± 7.7 years) ingested 3 mg of caffeine per kg of body mass or a placebo in testing sessions separated by one week. Performance in the 30 s Wingate test, visual attention, and side effects were evaluated. DNA was obtained from whole blood samples and the CYP1A2 polymorphism was analyzed (rs762551). We obtained two groups: AA homozygotes ( n = 5) and C-allele carriers ( n = 16). Caffeine ingestion increased peak power (682 ± 140 vs. 667 ± 137 W; p = 0.008) and mean power during the Wingate test (527 ± 111 vs. 518 ± 111 W; p 0.05). Reaction times were similar between caffeine and placebo conditions (276 ± 31 vs. 269 ± 71 milliseconds; p = 0.681) with no differences between AA homozygotes and C-allele carriers. However, 31.3% of the C-allele carriers reported increased nervousness after caffeine ingestion, while none of the AA homozygotes perceived this side effect. Genetic variations of the CYP1A2 polymorphism did not affect the ergogenic effects and drawbacks derived from the ingestion of a moderate dose of caffeine.

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

    Science.gov (United States)

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

    2013-01-01

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

  18. In vitro cytochrome P450 inhibition potential of methylenedioxy-derived designer drugs studied with a two-cocktail approach.

    Science.gov (United States)

    Dinger, Julia; Meyer, Markus R; Maurer, Hans H

    2016-02-01

    In vitro cytochrome P450 (CYP) inhibition assays are common approaches for testing the inhibition potential of drugs for predicting potential interactions. In contrast to marketed medicaments, drugs of abuse, particularly the so-called novel psychoactive substances, were not tested before distribution and consumption. Therefore, the inhibition potential of methylenedioxy-derived designer drugs (MDD) of different drug classes such as aminoindanes, amphetamines, benzofurans, cathinones, piperazines, pyrrolidinophenones, and tryptamines should be elucidated. The FDA-preferred test substrates, split in two cocktails, were incubated with pooled human liver microsomes and analysed after protein precipitation using LC-high-resolution-MS/MS. IC50 values were determined of MDD showing more than 50 % inhibition in the prescreening. Values were calculated by plotting the relative metabolite concentration formed over the logarithm of the inhibitor concentration. All MDD showed inhibition against CYP2D6 activity and most of them in the range of the clinically relevant CYP2D6 inhibitors quinidine and fluoxetine. In addition, the beta-keto compounds showed inhibition of the activity of CYP2B6, 5,6-MD-DALT of CYP1A2 and CYP3A, and MDAI of CYP2A6, all in the range of clinically relevant inhibitors. In summary, all MDD showed inhibition of the activity of CYP2D6, six of CYP1A2, three of CYP2A6, 13 of CYP2B6, two of CYP2C9, six of CYP2C19, one of CYP2E1, and six of CYP3A. These results showed that the CYP inhibition by MDD might be clinically relevant, but further studies are needed for final conclusions.

  19. Genome-wide meta-analysis identifies regions on 7p21 (AHR and 15q24 (CYP1A2 as determinants of habitual caffeine consumption.

    Directory of Open Access Journals (Sweden)

    Marilyn C Cornelis

    2011-04-01

    Full Text Available We report the first genome-wide association study of habitual caffeine intake. We included 47,341 individuals of European descent based on five population-based studies within the United States. In a meta-analysis adjusted for age, sex, smoking, and eigenvectors of population variation, two loci achieved genome-wide significance: 7p21 (P = 2.4 × 10(-19, near AHR, and 15q24 (P = 5.2 × 10(-14, between CYP1A1 and CYP1A2. Both the AHR and CYP1A2 genes are biologically plausible candidates as CYP1A2 metabolizes caffeine and AHR regulates CYP1A2.

  20. Characterization of human cytochrome P450s involved in the bioactivation of tri-Ortho-Cresyl phosphate (ToCP)

    NARCIS (Netherlands)

    Reinen, Jelle; Nematollahi, Leyla; Fidder, Alex; Vermeulen, Nico P E; Noort, Daan; Commandeur, Jan N M

    2015-01-01

    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

  1. Recombinant Saccharomyces cerevisiae expressing P450 in artificial digestive systems : A model for biodetoxication in the human digestive environment

    NARCIS (Netherlands)

    Blanquet, S.; Meunier, J.P.; Minekus, M.; Marol-Bonnin, S.; Alric, M.

    2003-01-01

    The use of genetically engineered microorganisms such as bacteria or yeasts as live vehicles to carry out bioconversion directly in the digestive environment is an important challenge for the development of innovative biodrugs. A system that mimics the human gastrointestinal tract was combined with

  2. Novel extrahepatic cytochrome P450s

    International Nuclear Information System (INIS)

    Karlgren, Maria; Miura, Shin-ichi; Ingelman-Sundberg, Magnus

    2005-01-01

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

  3. Pharmacokinetic study of isocorynoxeine metabolites mediated by cytochrome P450 enzymes in rat and human liver microsomes.

    Science.gov (United States)

    Zhao, Lizhu; Zang, Bin; Qi, Wen; Chen, Fangfang; Wang, Haibo; Kano, Yoshihiro; Yuan, Dan

    2016-06-01

    Isocorynoxeine (ICN) is one of the major bioactive tetracyclic oxindole alkaloids found in Uncaria rhynchophylla (Miq.) Jacks. that is widely used for the treatment of hypertension, vascular dementia, and stroke. The present study was undertaken to assess the plasma pharmacokinetic characteristics of major ICN metabolites, and the role of simulated gastric and intestinal fluid (SGF and SIF), human and rat liver microsomes (HLMs and RLMs), and seven recombinant human CYP enzymes in the major metabolic pathway of ICN. A rapid, sensitive and accurate UHPLC/Q-TOF MS method was validated for the simultaneous determination of ICN and its seven metabolites in rat plasma after oral administration of ICN at 40mg/kg. It was found that 18.19-dehydrocorynoxinic acid (DCA) and 5-oxoisocorynoxeinic acid (5-O-ICA) were both key and predominant metabolites, rather than ICN itself, due to the rapid and extensive metabolism of ICN in vivo. The further study indicated that ICN was mainly metabolized in human or rat liver, and CYPs 2C19, 3A4 and 2D6 were the major enzymes responsible for the biotransformation of ICN to DCA and 5-O-ICA in human. These findings are of significance in understanding of the pharmacokinetic nature of tetracyclic oxindole alkaloids, and provide helpful information for the clinical co-administration of the herbal preparations containing U. rhynchophylla with antihypertensive drugs that are mainly metabolized by CYP3A4 and CYP2C19. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. Effects of oral anorexiant sibutramine on the expression of cytochromes P450s in human hepatocytes and cancer cell lines.

    Science.gov (United States)

    Vrzal, Radim; Knoppová, Barbora; Bachleda, Petr; Dvořák, Zdeněk

    2013-12-01

    Sibutramine is a serotonin-norepinephrine reuptake inhibitor that was used for weight-loss management in obese patients. Even though it was officially withdrawn from the market in 2010, it is still present in some tainted weight-loss pills (as reported by US Food and Drug Administration). Thus, it is still reasonable to study the effects of this compound. The aim of this work was to investigate the potential of sibutramine to induce CYP1A1/CY3A4 in human cancer cell lines and CYP1A1/2, CYP2A6, CYP2B6, and CYP3A4 in human hepatocytes, a competent model of metabolically active cells. The levels of mRNA and protein of CYP1A1/1A2/3A4/2A6/2B6 were compared with the typical inducers, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and rifampicin (RIF) for CYP1A1/2 and for other CYPs, respectively. The mRNA and protein levels of all genes in either cancer cell lines or human hepatocytes were induced when treated with typical inducers but not with sibutramine. © 2013 Wiley Periodicals, Inc.

  5. Cytochrome P450-mediated metabolism of tumour promoters modifies the inhibition of intercellular communication: a modified assay for tumour promotion

    DEFF Research Database (Denmark)

    Vang, Ole; Wallin, H.; Doehmer, J.

    1993-01-01

    The role of metabolism of tumour promoters on the inhibition of intercellular communication was investigated in a modified V79 metabolic cooperation system. V79 cells, which stably express different rat cytochrome P450 enzymes (CYP1A1, CYP1A2 or CYP2B1), were used in the metabolic cooperation assay...... B1 and 4-nitrobiphenyl, did not inhibit metabolic cooperation in either V79 cells expressing or cells not expressing cytochrome P450. We conclude that cytochrome P450-associated metabolism plays an important role in the inhibition of gap junctional intercellular communication of some tumour...... promoters. The modified metabolic cooperation assay presented here is valuable for detecting some inhibitory chemicals which have been 'false negative' in previous assays for gap junctional intercellular communication. The assay also discloses that cytochrome P450 metabolism alters intercellular...

  6. cDNA cloning and initial characterization of CYP3A43, a novel human cytochrome P450.

    Science.gov (United States)

    Domanski, T L; Finta, C; Halpert, J R; Zaphiropoulos, P G

    2001-02-01

    The RACE amplification technology was used on a novel CYP3A-like exon 1 sequence detected during the reverse transcriptase/polymerase chain reaction analysis of human CYP3A gene expression. This resulted in the identification of cDNAs encompassing the complete coding sequence of a new member of the CYP3A gene subfamily, CYP3A43. Interestingly, the majority of the cDNAs identified were characterized by alternative splicing events such as exon skipping and complete or partial intron inclusion. CYP3A43 expression was detected in liver, kidney, pancreas, and prostate. The amino acid sequence is 75% identical to that of CYP3A4 and CYP3A5 and 71% identical to CYP3A7. CYP3A43 differs from CYP3A4 at six amino acid residues, found within the putative substrate recognition sites of CYP3A4, that are known to be determinants of substrate selectivity. The N terminus of CYP3A43 was modified for efficient expression of the protein in Escherichia coli, and a 6X histidine tag was added at the C terminus to facilitate purification. CYP3A43 gave a reduced carbon monoxide difference spectra with an absorbance maximum at 450 nm. The level of heterologous expression was significantly lower than that observed for CYP3A4 and CYP3A5. Immunoblot analyses revealed that CYP3A43 comigrates with CYP3A4 in polyacrylamide gel electrophoresis but does separate from CYP3A5. Monooxygenase assays were performed under a variety of conditions, several of which yielded reproducible, albeit low, testosterone hydroxylase activity. The findings from this study demonstrate that there is a novel CYP3A member expressed in human tissues, although its relative contribution to drug metabolism has yet to be ascertained.

  7. Induction of cytochrome P450 1A by cow milk-based formula: a comparative study between human milk and formula.

    Science.gov (United States)

    Xu, Haibo; Rajesan, Ratheishan; Harper, Patricia; Kim, Richard B; Lonnerdal, Bo; Yang, Mingdong; Uematsu, Satoko; Hutson, Janine; Watson-MacDonell, Jo; Ito, Shinya

    2005-09-01

    During the treatment of neonatal apnea, formula-fed infants, compared to breastfed infants, show nearly three-fold increase in clearance of caffeine, a substrate of cytochrome P450 1A (CYP1A) and in part CYP3A4. However, human milk is known to contain higher concentrations of environmental pollutants than infant formula, which are potent CYP1A inducers. To gain insight into the mechanism underlying this apparent contradiction, we characterized CYP1A and CYP3A4 induction by human milk and cow milk-based infant formula. The mRNA and protein expression of CYP1A1/1A2 were significantly induced by cow milk-based formula, but not by human milk, in HepG2 cells. Luciferase reporter assay demonstrated that cow milk-based formula but not human milk activated aryl hydrocarbon receptor (AhR) significantly. The cotreatment of 3,4-dimethoxyflavone, an AhR antagonist, abolished the formula-induced CYP1A expression. In addition, AhR activation by dibenzo[a,h]anthracene, a potent AhR agonist, was significantly suppressed by infant formula and even more by human milk. In contrast, CYP3A4 mRNA expression was only mildly induced by formula and human milk. Consistently, neither formula nor human milk substantially activated pregnane X receptor (PXR). Effects of whey and soy protein-based formulas on the AhR-CYP1A and the PXR-CYP3A4 pathways were similar to those of cow milk-based formula. In conclusion, infant formula, but not human milk, enhances in vitro CYP1A expression via an AhR-mediated pathway, providing a potential mechanistic basis for the increased caffeine elimination in formula-fed infants.

  8. Assessment of inhibitory effects on major human cytochrome P450 enzymes by spasmolytics used in the treatment of overactive bladder syndrome.

    Science.gov (United States)

    Dahlinger, Dominik; Aslan, Sevinc; Pietsch, Markus; Frechen, Sebastian; Fuhr, Uwe

    2017-07-01

    The objective of this study was to examine the inhibitory potential of darifenacin, fesoterodine, oxybutynin, propiverine, solifenacin, tolterodine and trospium chloride on the seven major human cytochrome P450 enzymes (CYP) by using a standardized and validated seven-in-one cytochrome P450 cocktail inhibition assay. An in vitro cocktail of seven highly selective probe substrates was incubated with human liver microsomes and varying concentrations of the seven test compounds. The major metabolites of the probe substrates were simultaneously analysed using a validated liquid chromatography tandem mass spectrometry (LC-MS/MS) method. Enzyme kinetics were estimated by determining IC 50 and K i values via nonlinear regression. Obtained K i values were used for predictions of potential clinical impact of the inhibition using a static mechanistic prediction model. In this study, 49 IC 50 experiments were conducted. In six cases, IC 50 values lower than the calculated threshold for drug-drug interactions (DDIs) in the gut wall were observed. In these cases, no increase in inhibition was determined after a 30 min preincubation. Considering a typical dosing regimen and applying the obtained K i values of 0.72 µM (darifenacin, 15 mg daily) and 7.2 µM [propiverine, 30 mg daily, immediate release (IR)] for the inhibition of CYP2D6 yielded a predicted 1.9-fold and 1.4-fold increase in the area under the curve (AUC) of debrisoquine (CYP2D6 substrate), respectively. Due to the inhibition of the particular intestinal CYP3A4, the obtained K i values of 14 µM of propiverine (30 mg daily, IR) resulted in a predicted doubling of the AUC for midazolam (CYP3A4 substrate). In vitro / in vivo extrapolation based on pharmacokinetic data and the conducted screening experiments yielded similar effects of darifenacin on CYP2D6 and propiverine on CYP3A4 as obtained in separately conducted in vivo DDI studies. As a novel finding, propiverine was identified to potentially inhibit CYP2D6 at

  9. Cytochrome P450c17 (steroid 17α-hydroxylase/17,20 lyase): cloning of human adrenal and testis cDNAs indicates the same gene is expressed in both tissues

    International Nuclear Information System (INIS)

    Chung, B.; Picado-Leonard, J.; Haniu, M.; Bienkowski, M.; Hall, P.F.; Shively, J.E.; Miller, W.L.

    1987-01-01

    P450c17 is the single enzyme mediating both 17α-hydroxylase (steroid 17α-monooxygenase, EC 1.14.99.9) and 17,20 lyase activities in the synthesis of steroid hormones. It has been suggested that different P450c17 isozymes mediate these activities in the adrenal gland and testis. The authors sequenced 423 of the 509 amino acids (83%) of the porcine adrenal enzyme; based on this partial sequence, a 128-fold degenerate 17-mer was synthesized and used to screen a porcine adrenal cDNA library. This yielded a 380-base cloned cDNA, which in turn was used to isolate several human adrenal cDNAs. The longest of these, λ hac 17-2, is 1754 base pairs long and includes the full-length coding region, the complete 3'-untranslated region, and 41 bases of the 5'-untranslated region. This cDNA encodes a protein of 508 amino acids having a predicted molecular weight of 57,379.82. High-stringency screening of a human testicular cDNA library yielded a partial clone containing 1303 identical bases. RNA gel blots and nuclease S1-protection experiments confirm that the adrenal and testicular P450c17 mRNAs are indistinguishable. These data indicate that the testis possesses a P450c17 identical to that in the adrenal. The human amino acid sequence is 66.7% homologous to the corresponding regions of the porcine sequence, and the human cDNA and amino acid sequences are 80.1 and 70.3% homologous, respectively, to bovine adrenal P450c17 cDNA. Both comparisons indicate that a central region comprising amino acid residues 160-268 is hypervariable among these species of P450c17

  10. Traditional Preparations and Methanol Extracts of Medicinal Plants from Papua New Guinea Exhibit Similar Cytochrome P450 Inhibition

    Directory of Open Access Journals (Sweden)

    Erica C. Larson

    2016-01-01

    Full Text Available The hypothesis underlying this current work is that fresh juice expressed from Papua New Guinea (PNG medicinal plants (succus will inhibit human Cytochrome P450s (CYPs. The CYP inhibitory activity identified in fresh material was compared with inhibition in methanol extracts of dried material. Succus is the most common method of traditional medicine (TM preparation for consumption in PNG. There is increasing concern that TMs might antagonize or complicate drug therapy. We have previously shown that methanol extracts of commonly consumed PNG medicinal plants are able to induce and/or inhibit human CYPs in vitro. In this current work plant succus was prepared from fresh plant leaves. Inhibition of three major CYPs was determined using human liver microsomes and enzyme-selective model substrates. Of 15 species tested, succus from 6/15 was found to inhibit CYP1A2, 7/15 inhibited CYP3A4, and 4/15 inhibited CYP2D6. Chi-squared tests determined differences in inhibitory activity between succus and methanol preparations. Over 80% agreement was found. Thus, fresh juice from PNG medicinal plants does exhibit the potential to complicate drug therapy in at risk populations. Further, the general reproducibility of these findings suggests that methanol extraction of dried material is a reasonable surrogate preparation method for fresh plant samples.

  11. A high-throughput inhibition screening of major human cytochrome P450 enzymes using an in vitro cocktail and liquid chromatography-tandem mass spectrometry.

    Science.gov (United States)

    Qin, Chong-Zhen; Ren, Xian; Tan, Zhi-Rong; Chen, Yao; Yin, Ji-Ye; Yu, Jing; Qu, Jian; Zhou, Hong-Hao; Liu, Zhao-Qian

    2014-02-01

    A sensitive and high-throughput inhibition screening liquid chromatography-mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous quantification of five probe metabolites (7-hydroxycoumarin, CYP2A6; 4-hydroxytolbutamide, CYP2C9; 4'-hydroxymephenytoin, CYP2C19; α-hydroxymetoprolol, CYP2D6; and 1-hydroxymidazolam, CYP3A4) for in vitro cytochrome P450 activity determination in human liver microsome and recombinant. All the metabolites and the internal standard, tramadol, were separated on a Waters 2695 series liquid chromatograph with a Phenomenex Luna C18 column (150 × 2.0 mm, 5 µm). Quality control samples and a positive control CYP inhibitor were included in the method. The IC50 values determined for typical CYP inhibitors were reproducible and in agreement with the literature. The method was selective and showed good accuracy (99.13-103.37%), and inter-day (RSD high-quality and -throughput cocktail provides suitable information in drug discovery and screening for new drug entities. Copyright © 2013 John Wiley & Sons, Ltd.

  12. Enhanced phytoremediation of mixed heavy metal (mercury)-organic pollutants (trichloroethylene) with transgenic alfalfa co-expressing glutathione S-transferase and human P450 2E1.

    Science.gov (United States)

    Zhang, Yuanyuan; Liu, Junhong; Zhou, Yuanming; Gong, Tingyun; Wang, Jing; Ge, Yinlin

    2013-09-15

    Soil contamination is a global environmental problem and many efforts have been made to find efficient remediation methods over the last decade. Moreover, remediation of mixed contaminated soils are more difficult. In the present study, transgenic alfalfa plants pKHCG co-expressing glutathione S-transferase (GST) and human P450 2E1 (CYP2E1) genes were used for phytoremediation of mixed mercury (Hg)-trichloroethylene (TCE) contaminants. Simultaneous expression of GST and CYP2E1 may produce a significant synergistic effect, and leads to improved resistance and accumulation to heavy metal-organic complex contaminants. Based on the tolerance and accumulation assays, pKHCG transgenic plants were more resistant to Hg/TCE complex pollutants and many folds higher in Hg/TCE-accumulation than the non-transgenic control plants in mixed contaminated soil. It is confirmed that GST and CYP2E1 co-expression may be a useful strategy to help achieve mixed heavy metal-organic pollutants phytoremediation. Copyright © 2013 Elsevier B.V. All rights reserved.

  13. Membrane-bound human orphan cytochrome P450 2U1: Sequence singularities, construction of a full 3D model, and substrate docking.

    Science.gov (United States)

    Ducassou, Lionel; Dhers, Laura; Jonasson, Gabriella; Pietrancosta, Nicolas; Boucher, Jean-Luc; Mansuy, Daniel; André, François

    2017-09-01

    Human cytochrome P450 2U1 (CYP2U1) is an orphan CYP that exhibits several distinctive characteristics among the 57 human CYPs with a highly conserved sequence in almost all living organisms. We compared its protein sequence with those of the 57 human CYPs and constructed a 3D structure of a full-length CYP2U1 model bound to a POPC membrane. We also performed docking experiments of arachidonic acid (AA) and N-arachidonoylserotonin (AS) in this model. The protein sequence of CYP2U1 displayed two unique characteristics when compared to those of the human CYPs, the presence of a longer N-terminal region upstream of the putative trans-membrane helix (TMH) containing 8 proline residues, and of an insert of about 20 amino acids containing 5 arginine residues between helices A' and A. Its N-terminal part upstream of TMH involved an additional short terminal helix, in a manner similar to what was reported in the crystal structure of Saccharomyces cerevisiae CYP51. Our model also showed a specific interaction between the charged residues of insert AA' and phosphate groups of lipid polar heads, suggesting a possible role of this insert in substrate recruitment. Docking of AA and AS in this model showed these substrates in channel 2ac, with the terminal alkyl chain of AA or the indole ring of AS close to the heme, in agreement with the reported CYP2U1-catalyzed AA and AS hydroxylation regioselectivities. This model should be useful to find new endogenous or exogenous CYP2U1 substrates and to interpret the regioselectivity of their hydroxylation. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  14. Relation of Transcriptional Factors to the Expression and Activity of Cytochrome P450 and UDP-Glucuronosyltransferases 1A in Human Liver: Co-Expression Network Analysis.

    Science.gov (United States)

    Zhong, Shilong; Han, Weichao; Hou, Chuqi; Liu, Junjin; Wu, Lili; Liu, Menghua; Liang, Zhi; Lin, Haoming; Zhou, Lili; Liu, Shuwen; Tang, Lan

    2017-01-01

    Cytochrome P450 (CYPs) and UDP-glucuronosyltransferases (UGTs) play important roles in the metabolism of exogenous and endogenous compounds. The gene transcription of CYPs and UGTs can be enhanced or reduced by transcription factors (TFs). This study aims to explore novel TFs involved in the regulatory network of human hepatic UGTs/CYPs. Correlations between the transcription levels of 683 key TFs and CYPs/UGTs in three different human liver expression profiles (n = 640) were calculated first. Supervised weighted correlation network analysis (sWGCNA) was employed to define hub genes among the selected TFs. The relationship among 17 defined TFs, CYPs/UGTs expression, and activity were evaluated in 30 liver samples from Chinese patients. The positive controls (e.g., PPARA, NR1I2, NR1I3) and hub TFs (NFIA, NR3C2, and AR) in the Grey sWGCNA Module were significantly and positively associated with CYPs/UGTs expression. And the cancer- or inflammation-related TFs (TEAD4, NFKB2, and NFKB1) were negatively associated with mRNA expression of CYP2C9/CYP2E1/UGT1A9. Furthermore, the effect of NR1I2, NR1I3, AR, TEAD4, and NFKB2 on CYP450/UGT1A gene transcription translated into moderate influences on enzyme activities. To our knowledge, this is the first study to integrate Gene Expression Omnibus (GEO) datasets and supervised weighted correlation network analysis (sWGCNA) for defining TFs potentially related to CYPs/UGTs. We detected several novel TFs involved in the regulatory network of hepatic CYPs and UGTs in humans. Further validation and investigation may reveal their exact mechanism of CYPs/UGTs regulation.

  15. SNP genetic polymorphisms of MDR-1, CYP1A2 and CYPB11 genes in four canine breeds upon toxicological evaluation.

    Science.gov (United States)

    Gagliardi, Rosa; Llambí, Silvia; Arruga, M Victoria

    2015-01-01

    The fields of pharmacogenetics and pharmacogenomics have become increasingly promising regarding the clinical application of genetic data to aid in prevention of adverse reactions. Specific screening tests can predict which animals express modified proteins or genetic sequences responsible for adverse effects associated with a drug. Among the genetic variations that have been investigated in dogs, the multidrug resistance gene (MDR) is the best studied. However, other genes such as CYP1A2 and CYP2B11 control the protein syntheses involved in the metabolism of many drugs. In the present study, the MDR-1, CYP1A2 and CYP2B11 genes were examined to identify SNP polymorphisms associated with these genes in the following four canine breeds: Uruguayan Cimarron, Border Collie, Labrador Retriever and German Shepherd. The results revealed that several SNPs of the CYP1A2 and CYP2B11 genes are potential targets for drug sensitivity investigations.

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

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Yue-Ming; Lin, Wenwei; Chai, Sergio C.; Wu, Jing; Ong, Su Sien [Department of Chemical Biology and Therapeutics, St. Jude Children' s Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105 (United States); Schuetz, Erin G. [Department of Pharmaceutical Sciences, St. Jude Children' s Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105 (United States); Chen, Taosheng, E-mail: taosheng.chen@stjude.org [Department of Chemical Biology and Therapeutics, St. Jude Children' s Research Hospital, 262 Danny Thomas Place, Memphis, TN 38105 (United States)

    2013-10-01

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

  17. Altered cytochrome P450 activities and expression levels in the liver and intestines of the monosodium glutamate-induced mouse model of human obesity.

    Science.gov (United States)

    Tomankova, Veronika; Liskova, Barbora; Skalova, Lenka; Bartikova, Hana; Bousova, Iva; Jourova, Lenka; Anzenbacher, Pavel; Ulrichova, Jitka; Anzenbacherova, Eva

    2015-07-15

    Cytochromes P450 (CYPs) are enzymes present from bacteria to man involved in metabolism of endogenous and exogenous compounds incl. drugs. Our objective was to assess whether obesity leads to changes in activities and expression of CYPs in the mouse liver, small intestine and colon. An obese mouse model with repeated injection of monosodium glutamate (MSG) to newborns was used. Controls were treated with saline. All mice were sacrificed at 8 months. In the liver and intestines, levels of CYP mRNA and proteins were analyzed using RT-PCR and Western blotting. Activities of CYP enzymes were measured with specific substrates of human orthologous forms. At the end of the experiment, body weight, plasma insulin and leptin levels as well as the specific content of hepatic CYP enzymes were increased in obese mice. Among CYP enzymes, hepatic CYP2A5 activity, protein and mRNA expression increased most significantly in obese animals. Higher activities and protein levels of hepatic CYP2E1 and 3A in the obese mice were also found. No or a weak effect on CYPs 2C and 2D was observed. In the small intestine and colon, no changes of CYP enzymes were detected except for increased expression of CYP2E1 and decreased expression of CYP3A mRNAs in the colon of the obese mice. Results of our study suggest that the specific content and activities of some liver CYP enzymes (especially CYP2A5) can be increased in obese mice. Higher activity of CYP2A5 (CYP2A6 human ortholog) could lead to altered metabolism of drug substrates of this enzyme (valproic acid, nicotine, methoxyflurane). Copyright © 2015 Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

    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.

  19. Systemic uptake of miconazole during vaginal suppository use and effect on CYP1A2 and CYP3A4 associated enzyme activities in women

    DEFF Research Database (Denmark)

    Kjærstad, Mia Birkhøj; Nielsen, Flemming; Nøhr-Jensen, Lene

    2010-01-01

    To investigate if the ordinary use of a vaginal suppository containing miconazole results in systemic absorption that is sufficient to affect the activities of CYP1A2 and CYP3A4, which are major drug- and steroid-metabolising enzymes.......To investigate if the ordinary use of a vaginal suppository containing miconazole results in systemic absorption that is sufficient to affect the activities of CYP1A2 and CYP3A4, which are major drug- and steroid-metabolising enzymes....

  20. One-electron oxidation of diclofenac by human cytochrome P450s as a potential bioactivation mechanism for formation of 2'-(glutathion-S-yl)-deschloro-diclofenac.

    NARCIS (Netherlands)

    Boerma, J.S.; Vermeulen, N.P.E.; Commandeur, J.N.M.

    2014-01-01

    Reactive metabolites have been suggested to play a role in the idiosyncratic hepatotoxicity observed with diclofenac (DF). By structural identification of the GSH conjugates formed after P450-catalyzed bioactivation of DF, it was shown that three types of reactive intermediates were formed:

  1. Development of an in vitro cytochrome P450 cocktail inhibition assay for assessing the inhibition risk of drugs of abuse.

    Science.gov (United States)

    Dinger, Julia; Meyer, Markus R; Maurer, Hans H

    2014-10-01

    Drugs of abuse are not tested for cytochrome P450 (CYP) inhibition potential before distribution. Therefore, a cocktail assay should be developed for testing the inhibition potential for all relevant CYPs. The following CYP test substrates and selective inhibitors were incubated in pooled human liver microsomes: phenacetin (alpha-naphthoflavone for CYP1A2), coumarin (tranylcypromine, CYP2A6), bupropion (sertraline, CYP2B6), amodiaquine (trimethoprim, CYP2C8), diclofenac (sulfaphenazole, CYP2C9), omeprazole (fluconazole, CYP2C19), dextromethorphan (quinidine, CYP2D6), chlorzoxazone (clomethiazole, CYP2E1), testosterone (verapamil, CYP3A). Samples were analyzed after protein precipitation using a Thermo Fisher Q-Exactive LC-high-resolution-MS/MS. The IC50 values were calculated by plotting the concentration of the formed metabolite, relative to the control sample, over the logarithm of the inhibitor concentration. They were determined either for single substrate or the cocktail incubation. Unfortunately, the cocktail assay had to be split because of interferences during incubation caused by substrates or metabolites, but the mixture of both incubates could be analyzed in one analytical run. The IC50 values determined in the single substrate or both cocktail incubations were comparable among themselves and with published data. In conclusion, the new inhibition cocktail assay was reproducible and applicable for testing the inhibition potential of drugs of abuse as exemplified for 2,5-dimethoxy-4-iodo-amfetamine (DOI). Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  2. Changes in cytochrome P450 gene expression and enzyme activity induced by xenobiotics in rabbits in vivo and in vitro

    Directory of Open Access Journals (Sweden)

    Orsolya Palócz

    2017-06-01

    Full Text Available As considerable inter-species differences exist in xenobiotic metabolism, developing new pharmaceutical therapies for use in different species is fraught with difficulties. For this reason, very few medicines have been registered for use in rabbits, despite their importance in inter alia meat and fur production. We have developed a rapid and sensitive screening system for drug safety in rabbits based on cytochrome P450 enzyme assays, specifically CYP1A1, CYP1A2 and CYP3A6, employing an adaptation of the luciferin-based clinical assay currently used in human drug screening. Short-term (4-h cultured rabbit primary hepatocytes were treated with a cytochrome inducer (phenobarbital and 2 inhibitors (alpha-naphthoflavone and ketoconazole. In parallel, and to provide verification, New Zealand white rabbits were dosed with 80 mg/kg phenobarbital or 40 mg/kg ketoconazole for 3 d. Ketoconazole significantly increased CYP3A6 gene expression and decreased CYP3A6 activity both in vitro and in vivo. CYP1A1 activity was decreased by ketoconazole in vitro and increased in vivo. This is the first report of the inducer effect of ketoconazole on rabbit cytochrome isoenzymes in vivo. Our data support the use of a luciferin-based assay in short-term primary hepatocytes as an appropriate tool for xenobiotic metabolism assays and short-term toxicity testing in rabbits.

  3. One-electron oxidation of diclofenac by human cytochrome P450s as a potential bioactivation mechanism for formation of 2'-(glutathion-S-yl)-deschloro-diclofenac.

    Science.gov (United States)

    Boerma, Jan Simon; Vermeulen, Nico P E; Commandeur, Jan N M

    2014-01-25

    Reactive metabolites have been suggested to play a role in the idiosyncratic hepatotoxicity observed with diclofenac (DF). By structural identification of the GSH conjugates formed after P450-catalyzed bioactivation of DF, it was shown that three types of reactive intermediates were formed: p-benzoquinone imines, o-imine methide and arene-oxide. Recently, detection of 2'-(glutathion-S-yl)-deschloro-diclofenac (DDF-SG), resulting from chlorine substitution, suggested the existence of a fourth type of P450-dependent reactive intermediate whose inactivation by GSH is completely dependent on presence of glutathione S-transferase. In this study, fourteen recombinant cytochrome P450s and three flavin-containing monooxygenases were tested for their ability to produce oxidative DF metabolites and their corresponding GSH conjugates. Concerning the hydroxymetabolites and their GSH conjugates, results were consistent with previous studies. Unexpectedly, all tested recombinant P450s were able to form DDF-SG to almost similar extent. DDF-SG formation was found to be partially independent of NADPH and even occurred by heat-inactivated P450. However, product formation was fully dependent on both GSH and glutathione-S-transferase P1-1. DDF-SG formation was also observed in reactions with horseradish peroxidase in absence of hydrogen peroxide. Because DDF-SG was not formed by free iron, it appears that DF can be bioactivated by iron in hemeproteins. This was confirmed by DDF-SG formation by other hemeproteins such as hemoglobin. As a mechanism, we propose that DF is subject to heme-dependent one-electron oxidation. The resulting nitrogen radical cation, which might activate the chlorines of DF, then undergoes a GST-catalyzed nucleophilic aromatic substitution reaction in which the chlorine atom of the DF moiety is replaced by GSH. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  4. Monocrotophos Induces the Expression of Xenobiotic Metabolizing Cytochrome P450s (CYP2C8 and CYP3A4) and Neurotoxicity in Human Brain Cells.

    Science.gov (United States)

    Tripathi, Vinay Kumar; Kumar, Vivek; Pandey, Ankita; Vatsa, Pankhi; Dhasmana, Anupam; Singh, Rajat Pratap; Appikonda, Sri Hari Chandan; Hwang, Inho; Lohani, Mohtashim

    2017-07-01

    Expression of various cytochrome P450s (CYPs) in mammalian brain cells is well documented. However, such studies are hampered in neural/glial cells of human origin due to nonavailability of human brain cells. To address this issue, we investigated the expression and inducibility of CYP2C8 and CYP3A4 and their responsiveness against cyclophosphamide (CPA) and organophosphorus pesticide monocrotophos (MCP), a known developmental neurotoxicant in human neural (SH-SY5Y) and glial (U373-MG) cell lines. CPA induced significant expression of CYP2C8 and CYP3A4 in both types of cells in a time-dependent manner. Neural cell line exhibited relatively higher constitutive and inducible expression of CYPs than the glial cell line. MCP exposure alone could not induce the significant expression of CYPs, whereas the cells preexposed to CPA showed a significant response to MCP. Similar to the case of CPA induced expressions, neural cells were found to be more vulnerable than glial cells. Our data indicate differential expressions of CYPs in cultured human neural and glial cell lines. The findings were synchronized with protein ligand docking studies, which showed a significant modulatory capacity of MCP by strong interaction with CYP regulators-CAR and PXR. Similarly, the known CYP inducer CPA has also shown significant high docking scores with the two studied CYP regulators. We also observed a significant induction in reactive oxygen species (ROS), lipid peroxides (LPO), micronucleus (MN), chromosomal aberration (CA), and reduction in reduced glutathione (GSH) and catalase following the exposure of MCP. Moreover, the expressions of apoptotic markers such as caspase-3, caspase-9, Bax, and p53 were significantly upregulated, whereas the levels of antiapoptotic marker, Bcl2, was downregulated after the exposure of MCP in both cell lines. These findings confirm the involvement of ROS-mediated oxidative stress, which subsequently triggers apoptosis pathways in both human neural (SH-SY5Y

  5. Congener-specific metabolism and sequestration of dioxin-like compounds by cytochrome P450 1A induced in the liver of crows from Tokyo, Japan

    Energy Technology Data Exchange (ETDEWEB)

    Watanabe, M.; Iwata, H.; Tanabe, S. [Ehime Univ., Matsuyama (Japan); Yoneda, K.; Hashimoto, T. [Japan Wildlife Research Center, Tokyo (Japan)

    2004-09-15

    Jungle crow (JC; Corvus macrorhynchos) is a useful bioindicator for monitoring contaminants in urban areas, because this species is residential, occupies a same habitat as human, and feeds variety of foods including domestic waste and garbage. Therefore, JCs may accumulate environmental contaminants such as polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs) and coplanar polychlorinated biphenyls (Co-PCBs), which are released by human activities. Induction of cytochrome P450 (CYP) 1A is a responsive mechanism elicited by exposure to dioxinlike compounds including PCDDs/DFs and Co-PCBs. Toxicokinetic behavior of dioxin-like compounds in organisms is controlled by excretion, metabolism and absorption. These processes are, at least partly, dependent on CYP1A expression in addition to chemical structure and number of chlorine substitution of each congener. Low chlorinated congeners such as 2378-T{sub 4}CDD, 2378- T{sub 4}CDF, 12378-P{sub 5}CDD and 33'44'-PCB were easily metabolized by CYP1A1/2 in rat liver microsomes. PCDDs/DFs accumulate in hepatic tissue to a greater extent than adipose tissue in rats and mice. Recent study using transgenic CYP1A2 knockout mice demonstrated that CYP1A2 is responsible for the sequestration of 2378-T{sub 4}CDD and 23478-P{sub 5}CDF in hepatic tissue. Therefore, CYP1A is considered as a key factor responsible for toxicokinetics of dioxin-like compounds. However, there's no comprehensive data on the contribution of CYP1A to the toxicokinetics of dioxin-like congeners in wild populations. In this study, we investigated contamination levels of PCDDs/DFs and Co-PCBs in liver and breast muscle of JCs from Tokyo, Japan, and interactions of dioxin-like congeners with hepatic CYP to elucidate congener-specific toxicokinetics related to CYP expression in JC.

  6. CYP2J2 and CYP2C19 are the major enzymes responsible for metabolism of albendazole and fenbendazole in human liver microsomes and recombinant P450 assay systems.

    Science.gov (United States)

    Wu, Zhexue; Lee, Doohyun; Joo, Jeongmin; Shin, Jung-Hoon; Kang, Wonku; Oh, Sangtaek; Lee, Do Yup; Lee, Su-Jun; Yea, Sung Su; Lee, Hye Suk; Lee, Taeho; Liu, Kwang-Hyeon

    2013-11-01

    Albendazole and fenbendazole are broad-spectrum anthelmintics that undergo extensive metabolism to form hydroxyl and sulfoxide metabolites. Although CYP3A and flavin-containing monooxygenase have been implicated in sulfoxide metabolite formation, the enzymes responsible for hydroxyl metabolite formation have not been identified. In this study, we used human liver microsomes and recombinant cytochrome P450s (P450s) to characterize the enzymes involved in the formation of hydroxyalbendazole and hydroxyfenbendazole from albendazole and fenbendazole, respectively. Of the 10 recombinant P450s, CYP2J2 and/or CYP2C19 was the predominant enzyme catalyzing the hydroxylation of albendazole and fenbendazole. Albendazole hydroxylation to hydroxyalbendazole is primarily mediated by CYP2J2 (0.34 μl/min/pmol P450, which is a rate 3.9- and 8.1-fold higher than the rates for CYP2C19 and CYP2E1, respectively), whereas CYP2C19 and CYP2J2 contributed to the formation of hydroxyfenbendazole from fenbendazole (2.68 and 1.94 μl/min/pmol P450 for CYP2C19 and CYP2J2, respectively, which are rates 11.7- and 8.4-fold higher than the rate for CYP2D6). Correlation analysis between the known P450 enzyme activities and the rate of hydroxyalbendazole and hydroxyfenbendazole formation in samples from 14 human liver microsomes showed that albendazole hydroxylation correlates with CYP2J2 activity and fenbendazole hydroxylation correlates with CYP2C19 and CYP2J2 activities. These findings were supported by a P450 isoform-selective inhibition study in human liver microsomes. In conclusion, our data for the first time suggest that albendazole hydroxylation is primarily catalyzed by CYP2J2, whereas fenbendazole hydroxylation is preferentially catalyzed by CYP2C19 and CYP2J2. The present data will be useful in understanding the pharmacokinetics and drug interactions of albendazole and fenbendazole in vivo.

  7. Nicotine Component of Cigarette Smoke Extract (CSE) Decreases the Cytotoxicity of CSE in BEAS-2B Cells Stably Expressing Human Cytochrome P450 2A13.

    Science.gov (United States)

    Ji, Minghui; Zhang, Yudong; Li, Na; Wang, Chao; Xia, Rong; Zhang, Zhan; Wang, Shou-Lin

    2017-10-13

    Cytochrome P450 2A13 (CYP2A13), an extrahepatic enzyme mainly expressed in the human respiratory system, has been reported to mediate the metabolism and toxicity of cigarette smoke. We previously found that nicotine inhibited 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) metabolism by CYP2A13, but its influence on other components of cigarette smoke remains unclear. The nicotine component of cigarette smoke extract (CSE) was separated, purified, and identified using high-performance liquid chromatography (HPLC) and ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS), splitting CSE into a nicotine section (CSE-N) and nicotine-free section (CSE-O). Cell viability and apoptosis by Cell Counting Kit-8 (CCK-8) and flow cytometry assays were conducted on immortalized human bronchial epithelial (BEAS-2B) cells stably expressing CYP2A13 (B-2A13) or vector (B-V), respectively. Interestingly, CSE and CSE-O were toxic to BEAS-2B cells whereas CSE-N showed less cytotoxicity. CSE-O was more toxic to B-2A13 cells than to B-V cells (IC 50 of 2.49% vs. 7.06%), which was flatted by 8-methoxypsoralen (8-MOP), a CYP inhibitor. CSE-O rather than CSE or CSE-N increased apoptosis of B-2A13 cells rather than B-V cells. Accordingly, compared to CSE-N and CSE, CSE-O significantly changed the expression of three pairs of pro- and anti-apoptotic proteins, Bcl-2 Associated X Protein/B cell lymphoma-2 (Bax/Bcl-2), Cleaved Poly (Adenosine Diphosphate-Ribose) Polymerase/Poly (Adenosine Diphosphate-Ribose) Polymerase (C-PARP/PARP), and C-caspase-3/caspase-3, in B-2A13 cells. In addition, recombination of CSE-N and CSE-O (CSE-O/N) showed similar cytotoxicity and apoptosis to the original CSE. These results demonstrate that the nicotine component decreases the metabolic activation of CYP2A13 to CSE and aids in understanding the critical role of CYP2A13 in human respiratory diseases caused by cigarette smoking.

  8. Chemical-induced coordinated and reciprocal changes in heme metabolism, cytochrome P450 synthesis and others in the liver of humans and rodents.

    Science.gov (United States)

    Yoshida, Takemi; Ashino, Takashi; Kobayashi, Yasuna

    2016-01-01

    A wide variety of drugs and chemicals have been shown to produce induction and inhibition of heme-metabolizing enzymes, and of drug-metabolizing enzymes, including cytochrome P450s (P450s, CYPs), which consist of many molecular species with lower substrate specificity. Such chemically induced enzyme alterations are coordinately or reciprocally regulated through the same and/or different signal transductions. From the toxicological point of view, these enzymatic changes sometimes exacerbate inherited diseases, such as precipitation of porphyrogenic attacks, although the induction of these enzymes is dependent on the animal species in response to the differences in the stimuli of the liver, where they are also metabolized by P450s. Since P450s are hemoproteins, their induction and/or inhibition by chemical compounds could be coordinately accompanied by heme synthesis and/or inhibition. This review will take a retrospective view of research works carried out in our department and current findings on chemical-induced changes in hepatic heme metabolism in many places, together with current knowledge. Specifically, current beneficial aspects of induction of heme oxygenase-1, a rate-limiting heme degradation enzyme, and its relation to reciprocal and coordinated changes in P450s, with special reference to CYP2A5, in the liver are discussed. Mechanistic studies are also summarized in relation to current understanding on these aspects. Emphasis is also paid to an example of a single chemical compound that could cause various changes by mediating multiple signal transduction systems. Current toxicological studies have been developing by utilizing a sophisticated "omics" technology and survey integrated changes in the tissues produced by the administration of a chemical, even in time- and dose-dependent manners. Toxicological studies are generally carried out step by step to determine and elucidate mechanisms produced by drugs and chemicals. Such approaches are correct

  9. Expression of cytochromes P450 1A1 and 1B1 in human lung from smokers, non-smokers, and ex-smokers

    International Nuclear Information System (INIS)

    Kim, James H.; Sherman, Mark E.; Curriero, Frank C.; Guengerich, F. Peter; Strickland, Paul T.; Sutter, Thomas R.

    2004-01-01

    Cytochromes P450 1A1 and 1B1 are known to bioactivate procarcinogens such as polycyclic aromatic hydrocarbons (PAHs) found in cigarette smoke and are inducible via an Ah receptor-mediated mechanism. The aim of this study was to examine the levels of expression of CYP1A1 and CYP1B1 in samples of lung from smokers (n = 18), non-smokers (n = 7), and ex-smokers (n = 7). Using immunoglobulin preparations of highly specific polyclonal antibodies and immunoblot analysis of microsomes from lung tissues, we determined the specific content for CYP1A1 and CYP1B1. For CYP1A1, we found median expression levels of 15.5 pmol/mg microsomal protein in smokers, 6.0 pmol/mg microsomal protein in non-smokers, and 19.0 pmol/mg microsomal protein in ex-smokers. The difference in median expression levels of smokers and ex-smokers compared to non-smokers was statistically significant. For CYP1B1, we found median expression levels of 1.8 pmol/mg microsomal protein in smokers, 1.0 pmol/mg microsomal protein in non-smokers, and 4.4 pmol/mg microsomal protein in ex-smokers. The difference in median expression levels between ex-smokers and non-smokers was statistically significant. These results suggest that levels of expression of CYP1A1 and CYP1B1 protein in lung tissues from smokers and ex-smokers are quantitatively greater than in non-smokers. By immunohistochemical analysis, we demonstrated the expression of CYP1A1 and CYP1B1 in normal human alveolar type I and II cells, ciliated columnar epithelial cells lining bronchoalveolar airways, and alveolar macrophages. These results confirm that CYP1A1 is expressed in normal human lung, appears to be induced in smokers, and show interindividual variation; the similar characteristics of CYP1B1 are demonstrated

  10. The influence of CYP1A2 genotype in the blood pressure response to caffeine ingestion is affected by physical activity status and caffeine consumption level.

    Science.gov (United States)

    Soares, Rogerio Nogueira; Schneider, Augusto; Valle, Sandra Costa; Schenkel, Paulo Cavalheiro

    2018-03-06

    This study aimed to investigate whether the influence of CYP1A2 genotype in the blood pressure (BP) response to caffeine ingestion was affected by physical activity status and habitual caffeine consumption. Thirty-seven participants (19-50 years old) took place in the study and were categorized according to i) genotype: CYP1A2 (AA) "fast metabolizer", and CYP1A2 (AC) "slow metabolizer"; ii) physical activity level: sedentary (S) and physically active (A); and iii) caffeine consumption level: non-habitual caffeine consumer (NC) and habitual heavy caffeine consumer (C). All groups had BP assessed before (basal) and 1 hourh after (post) caffeine ingestion (6 mg·kg -1 ). It was observed that AC genotype individuals had increased basal-DBP and post-caffeine SBP when compared to AA individuals. Additionally, acute caffeine ingestion increased SBP only in the AC group. It was also found that physical activity only modulated the BP responses to acute caffeine ingestion in AC individuals. Furthermore, the results indicated that the habitual heavy caffeine consumers AC individuals had increased basal-DBP when compared to the AA ones. Our results suggest that the influence of CYP1A2 genotype in the basal and post-caffeine BP response to caffeine ingestion is modified by physical activity status and caffeine consumption level. Copyright © 2018 Elsevier Inc. All rights reserved.

  11. Coffee and tea consumption, genotype-based CYP1A2 and NAT2 activity and colorectal cancer risk—Results from the EPIC cohort study

    NARCIS (Netherlands)

    Dik, V.K.; Bueno-de-Mesquita, H.B.; Oijen, van M.G.C.T.; Siersema, P.D.; Uiterwaal, C.S.P.M.; Gils, van C.H.; Duijnhoven, van F.J.B.

    2014-01-01

    Coffee and tea contain numerous antimutagenic and antioxidant components and high levels of caffeine that may protect against colorectal cancer (CRC). We investigated the association between coffee and tea consumption and CRC risk and studied potential effect modification by CYP1A2 and NAT2

  12. Dose-dependent inhibition of CYP1A2, CYP2C19 and CYP2D6 by citalopram, fluoxetine, fluvoxamine and paroxetine

    DEFF Research Database (Denmark)

    Jeppesen, U; Gram, L F; Vistisen, K

    1996-01-01

    OBJECTIVE: The purpose of this pharmacokinetic study was to investigate the dose-dependent inhibition of model substrates for CYP2D6, CYP2C19 and CYP1A2 by four marketed selective serotonin reuptake inhibitors (SSRIs): citalopram, fluoxetine, fluvoxamine and paroxetine. METHODS: The study...

  13. 7,12-Dimethylbenzanthracene induces apoptosis in RL95-2 human endometrial cancer cells: Ligand-selective activation of cytochrome P450 1B1

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Ji Young [Department of Anatomy and Cell Biology, College of Medicine, Dong-A University, Busan 602-714 (Korea, Republic of); Medical Research Science Center, Dong-A University, Busan 602-714 (Korea, Republic of); Lee, Seung Gee [Department of Anatomy and Cell Biology, College of Medicine, Dong-A University, Busan 602-714 (Korea, Republic of); Mitochondria Hub Regulation Center, Dong-A University, Busan 602-714 (Korea, Republic of); Chung, Jin-Yong [Department of Anatomy and Cell Biology, College of Medicine, Dong-A University, Busan 602-714 (Korea, Republic of); Medical Research Science Center, Dong-A University, Busan 602-714 (Korea, Republic of); Kim, Yoon-Jae [Department of Anatomy and Cell Biology, College of Medicine, Dong-A University, Busan 602-714 (Korea, Republic of); Mitochondria Hub Regulation Center, Dong-A University, Busan 602-714 (Korea, Republic of); Park, Ji-Eun [Department of Anatomy and Cell Biology, College of Medicine, Dong-A University, Busan 602-714 (Korea, Republic of); Medical Research Science Center, Dong-A University, Busan 602-714 (Korea, Republic of); Oh, Seunghoon [Department of Physiology, College of Medicine, Dankook University, Cheonan 330-714 (Korea, Republic of); Lee, Se Yong [Department of Obstetrics and Gynecology, Busan Medical Center, Busan 611-072 (Korea, Republic of); Choi, Hong Jo [Department of General Surgery, College of Medicine, Dong-A University, Busan 602-714 (Korea, Republic of); Yoo, Young Hyun, E-mail: yhyoo@dau.ac.kr [Department of Anatomy and Cell Biology, College of Medicine, Dong-A University, Busan 602-714 (Korea, Republic of); Mitochondria Hub Regulation Center, Dong-A University, Busan 602-714 (Korea, Republic of); Medical Research Science Center, Dong-A University, Busan 602-714 (Korea, Republic of); and others

    2012-04-15

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

  14. Regulation of rabbit lung cytochrome P-450 prostaglandin omega-hydroxylase (P-450/sub PG-omega/) during pregnancy

    International Nuclear Information System (INIS)

    Muerhoff, A.S.; Williams, D.E.; Jackson, V.; Leithauser, M.T.; Waterman, M.R.; Johnson, E.F.; Masters, B.S.S.

    1987-01-01

    The mechanism of induction during pregnancy of a rabbit lung prostaglandin omega-hydroxylase cytochrome P-450 has been investigated. This activity has been demonstrated to be induced over 100-fold in 28-day pregnant rabbits, as compared to nonpregnant rabbits. The induction is reflected by an increase in the amount of P-450/sub PG-omega/ protein as measured by Western blotting. P-450/sub PG-omega/ microsomal protein increases throughout gestation concomitant with an increase in PGE 1 omega-hydroxylase activity. Elucidation of the level of induction involved extraction of RNA from rabbit lungs obtained at various days of gestation followed by in vitro translation of the RNA in the presence of 35 S-methionine. Immunoprecipitation of newly synthesized P-450 and analysis of the immunoisolates by SDS-PAGE, autoradiography and densitometry of the P-450/sub PG-omega/ band revealed that the P-450/sub PG-omega/ mRNA levels followed the gestational time-dependent increase observed for both PGE 1 omega-hydroxylase activity and P-450/sub PG-omega/ protein, i.e., a gradual increase peaking at 28-days, dropping precipitously to near control levels following parturition. These data suggest that control of P-450/sub PG-omega expression occurs at the transcriptional level. Western blots of human lung bronchioloalveolar-carcinoma cell lines NCL-H322 and NCL-H358 utilizing a guinea pig IgG to P-450/sub PG-omega/ detect a cross-reactive species

  15. Cytochromes P450 are Expressed in Proliferating Cells in Barrett's Metaplasia

    Directory of Open Access Journals (Sweden)

    Steven J. Hughes

    1999-06-01

    Full Text Available The expression of cytochromes P450 (CYP in Barrett's esophagus and esophageal squamous mucosa was investigated. Esophagectomy specimens from 23 patients were examined for CYP expression of CYP1A2, CYP3A4, CYP2C9/10, and CYP2E1 by immunohistochemical analysis, and the expression of CYP1A1, CYP3A4, CYP1B1, CYP2E1, and CYP2C9/10 in these tissues was further confirmed by reverse transcription polymerase chain reaction. Immunohistochemical analysis of esophageal squamous mucosa (n = 12 showed expression of CYP1A2, CYP3A4, CYP2E1, and CYP2C9/10 proteins, but it was noted that cells within the basal proliferative zone did not express CYPs. Immunohistochemical analysis of Barrett's esophagus (n = 13 showed expression of CYP1A2, CYP3A4, CYP2E1, and CYP2C9/10 that was prominent in the basal glandular regions, which are areas containing a high percentage of actively proliferating cells. Immunohistochemical staining for both proliferating cell nuclear antigen and the CYPs further supported the colocalization of CYP expression to areas of active cell proliferation in Barrett's esophagus, whereas in the esophageal squamous epithelium, CYP expression is limited to cells that are not proliferating. RT-PCR with amplification product sequence analysis confirmed CYP1A1, CYP3A4, CYP1B1, CYP2E1, and CYP2C9/10 mRNA expression in Barrett's esophagus. These data suggest that the potential ability of cells in Barrett's esophagus to both activate carcinogens and proliferate may be important risk factors affecting carcinogenesis in this metaplastic tissue.

  16. Evaluation of herb-drug interaction of a polyherbal Ayurvedic formulation through high throughput cytochrome P450 enzyme inhibition assay.

    Science.gov (United States)

    Pandit, Subrata; Kanjilal, Satyajyoti; Awasthi, Anshumali; Chaudhary, Anika; Banerjee, Dipankar; Bhatt, B N; Narwaria, Avinash; Singh, Rahul; Dutta, Kakoli; Jaggi, Manu; Singh, Anu T; Sharma, Neena; Katiyar, Chandra Kant

    2017-02-02

    Arishtas are Ayurvedic formulation made with decoction of herbs. Arjunarishta formulation is being used in Ayurveda for cardio-protective activity. Ashwagandharishta formulation possesses antioxidant, anti-atherosclerotic and anti-stress properties. Ridayarishta, a novel empirical formulation was prepared using combination of selected ingredients from these two formulations to support healthy heart functions and to reduce stress. Aim of the Study was to investigate herb-drug interaction (HDI) of Ridayarishta formulation through human hepatic cytochrome P450 (CYP450) enzyme inhibition assay. Ridayarishta formulation was phyto-chemically standardized against arjunolic acid, arjunetin, berberine, piperine, resveratrol and withaferin-A using high performance thin layer chromatography (HPTLC) analysis. The formulation was standardized with respect to ethanol by gas chromatographic (GC) analysis. HDI was evaluated with Ridayarishta formulation and amlodipine besilate, atenolol, atorvastatin, metformin, glipizide glimepiride cocktail using high throughput CYP450 enzyme inhibition assay; against CYP1A2, 2C19, 2D6 and 3A4 isozymes. Contents of arjunolic acid, arjunetin, berberine, piperine, resveratrol and withaferin-A in Ridayarishta formulation were found to be 1.76±0.12, 1.51±0.09, 1.85±0.05, 3.2±0.12, 1.21±0.08, and 2.16±0.09ppm, respectively. Quantity of ethanol in Ridayarishta was found to be 7.95±0.023% (V/V). Ridayarishta showed significantly higher (Pdrugs showed significantly (P<0.001and P<0.01) less or negligible HDI. Ridayarishta formulation alone and cocktail with amlodipine besilate, atenolol, atorvastatin, metformin, glipizide, glimepiride had negligible or insignificant effect on CYP450 inhibition. It may be concluded that consumption of Ridayarishta along with selective cardio protective, antihypertensive and anti-diabetic conventional medicine is safe with negligible or without any significant CYP450 (CYP1A2, 2C19, 2D6 and 3A4) inhibition mediated

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

    Science.gov (United States)

    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

  18. Wpływ pojedynczej dawki paracetamolu i/lub N-acetylocysteiny na szczury przewlekle eksponowane na trichloroetylen. III. Wpływ na wybrane izoformy cytochromu P450 w wątrobie

    Directory of Open Access Journals (Sweden)

    Andrzej Plewka

    2012-12-01

    Full Text Available Background: In case of overdose of paracetamol the ability of hepatic biotransformation is saturated and accumulation of toxic metabolite – NAPQI takes place. Main CYP isoforms considered to be responsible for bioactivation of APAP and promoting the same liver intoxication are CYP2E1, CYP1A2, CYP3A4 and in animals 2B1/2 isoforms additionally. Purpose of this work was examination of paracetamol influence and/or trichloroethylene on the composition of hepatic cytochrome P450 isoforms. Materials and method: Tests were carried out on rats which were treated with trichloroethylene, paracetamol and/or N-acetylcysteine. In the microsomal fraction content of three isoforms of cytochrome P450 i.e. CYP2E1, CYP2B1/2 and CYP1A2 were determined. Results: Paracetamol slightly stimulated CYP2B1/2 lowering simultaneously level of CYP1A2. Trichloroethylene stimulated CYP2B1/2. N-acetylcysteine stimulated all tested P450 isoforms. N-acetylcysteine given together with examinated xenobiotics induced studied P450 isoforms. Conclusions: N-acetylcysteine demonstrated a protective effect on studied CYP isoforms especially when was given upon termination of xenobiotics exposure

  19. High-Throughput Cytochrome P450 Cocktail Inhibition Assay for Assessing Drug-Drug and Drug-Botanical Interactions.

    Science.gov (United States)

    Li, Guannan; Huang, Ke; Nikolic, Dejan; van Breemen, Richard B

    2015-11-01

    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. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

  20. Dietary effect on mixed function P450 1A2 activity assayed by estimation of caffeine metabolism in man.

    Science.gov (United States)

    Kall, M A; Clausen, J

    1995-10-01

    Two studies were performed in order to evaluate cytochrome P450 1A2 mediated caffeine metabolism during different nutritional conditions. 1. In the first study, 23 healthy male non-smokers, mean age 25, changed from a customary mixed diet to a standard diet in 6 days. The 6 day's standard diet was based on bread, potatoes, rice and boiled meat. Thus, broccoli, cabbage and other cruciferous vegetables, spinach, leeks, onion, parsley, grapefruit, toasted bread, fried and charcoal grilled food, smoked fish and meat, ham and sausages were avoided. 2. In the second study, 33 healthy non-smoking subjects, 24 men and nine women mean age 25 years, volunteered. The study was designed to compare a customary home dietary period with the 6 day period of low dietary P450 induction and with a 5 day supplementary dietary period, i.e. ingestion of known dietary inducers. None of the women were using oral contraceptives or were pregnant during the experimental period. In the period of diet supplementation, the volunteers received charcoal grilled hamburger as a supplement to the standard low induction diet for lunch for 5 days. The hamburgers were made with 150 g beef (18-20% fat) and were grilled on charcoal for 10 min on each side until they were 'well done'. In the present study P450 1A2 activity was estimated from the caffeine metabolic ratio, the so-called CYP 1A2 index:(AFMU + 1-MX + 1-MU/ 17 -DMU) of the caffeine metabolites formed after oral ingestion of 200 mg caffeine. Urine was collected 4-8 h after caffeine ingestion in study 1 and 5 h after caffeine ingestion in study 2. In study 1 the CYP 1A2 index decreased from 4.28 +/- 0.98 in the customary home dietary period to 3.87 +/- 0.69 in the standard dietary period corresponding to 10.6% (P hamburgers as a dietary supplement, the CYP 1A2 index increased to almost the same level as in the customary home dietary period. The index increased to 4.45 +/- 1.57 in the whole group of volunteers, corresponding to a 14.1% (P < 0

  1. Identification of cytochrome P450s involved in the metabolism of 6-benzyl-1-benzyloxymethyl-5-iodouracil (W-1) using human recombinant enzymes and rat liver microsomes in vitro.

    Science.gov (United States)

    Lu, Ying-Yuan; Cheng, Hai-Xu; Wang, Xin; Wang, Xiao-Wei; Liu, Jun-Yi; Li, Pu; Lou, Ya-Qing; Li, Jun; Lu, Chuang; Zhang, Guo-Liang

    2017-08-01

    1. The aim of this study was to identify the hepatic metabolic enzymes, which involved in the biotransformation of 6-benzyl-1-benzyloxymethyl-5-iodouracil (W-1), a novel non-nucleoside reverse transcriptase inhibitor (NNRTI) in rat and human in vitro. 2. The parent drug of W-1 was incubated with rat liver microsomes (RLMs) or recombinant CYPs (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, and CYP3A5, respectively) in the presence or absence of nicotinamide adeninedinucleotide phosphate (NADPH)-regenerating system. The metabolites of W-1 were analyzed with liquid chromatography-ion trap-time of flight-mass spectrometry (LC-IT-TOF-MS). 3. The parent drug of W-1 was metabolized in a NADPH-dependent manner in RLMs. The kinetic parameters of prototype W-1 including K m , V max , and CL int were 2.3 μM, 3.3 nmol/min/mg protein, and 1.4 mL/min/mg protein, respectively. Two metabolites M1 and M2 were observed in shorter retention times (2.988 and 3.188 min) with a higher molecular ion at m/z 463.0160 (both M1 and M2) than that of the W-1 parent drug (6.158 min with m/z 447.0218). The CYP selective inhibition and recombinant enzymes also showed that two hydroxyl metabolites M1 and M2 are mainly mediated by CYP2C19 and CYP3A4. 4. The identification of CYPs involved in W-1 biotransformation is important to understand and minimize, if possible, the potential of drug-drug interactions.

  2. Cytochrome P450-mediated metabolic engineering

    DEFF Research Database (Denmark)

    Renault, Hugues; Bassard, Jean-Étienne André; Hamberger, Björn Robert

    2014-01-01

    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...

  3. The effect of lycopene on cytochrome P450 isoenzymes and P-glycoprotein by using human liver microsomes and Caco-2 cell monolayer model.

    Science.gov (United States)

    Kong, Lingti; Song, Chunli; Ye, Linhu; Xu, Jian; Guo, Daohua; Shi, Qingping

    2018-01-11

    Lycopene is widely used as a dietary supplement. However, the effects of lycopene on cytochrome P450 (CYP) enzymes or P-glycoprotein (P-gp) are not comprehensive. The present study was performed to investigate the effects of lycopene on the CYP enzymes and P-gp activity. A cocktail method was used to evaluate the activities of CYP3A4, CYP2C9, CYP2C19, CYP2D6 and CYP2E1. Caco-2 cell monolayer model was carried out to assay lycopene on P-gp activity. The results indicated that lycopene had a moderate inhibitory effect on CYP2E1, with IC50 value of 43.65 μM, whereas no inhibitory effects on CYP3A4, CYP2C19, CYP2D6 and CYP2E1, with IC50 values all over 100 μM. In addition, lycopene showed almost no inhibitory effect on rhodamine-123 efflux and uptake (p > .05), indicated no effects on P-gp activity. In conclusion, there should be required attention when lycopene are coadministered with other drugs that are metabolised by CYP2E1.

  4. Upregulation of microRNA-320 decreases the risk of developing steroid-induced avascular necrosis of femoral head by inhibiting CYP1A2 both in vivo and in vitro.

    Science.gov (United States)

    Wei, Ji-Hua; Luo, Qun-Qiang; Tang, Yu-Jin; Chen, Ji-Xia; Huang, Chun-Lan; Lu, Ding-Gui; Tang, Qian-Li

    2018-06-20

    Steroid-induced avascular necrosis of femoral head (SANFH) occurs frequently in patients receiving high-dose steroid treatment for these underlying diseases. The target of this study is to investigate the effect of microRNA-320 (miR-320) on SANFH by targeting CYP1A2. CYP1A2 expression was detected using immunohistochemistry. Specimens were collected from patients with SANFH and femoral neck fracture. Seventy rats were assigned into seven groups. The targeting relationship between miR-320 and CYP1A2 was verified by bioinformatics website and dual luciferase reporter gene assay. RT-qPCR and Western blot analysis were used to detect miR-320 and CYP1A2 expressions. The enzymatic activity of CYP1A2 was detected by fluorescence spectrophotometry. Hemorheology and microcirculation were measured in rats. MiR-320 expression decreased and CYP1A2 expression and enzymatic activity increased in SANFH patients compared to those with femoral neck fracture. CYP1A2 was the target gene of miR-320. Hemorheology and microcirculation results showed that up-regulated expression of CYP1A2 promoted the development of SANFH while increased expression of miR-320 inhibited the development of SANFH. Compared with the SANFH group, the SANFH + miR-320 mimic group showed increased miRNA-320 expression, and decreased CYP1A2 expression and enzymatic activity. Opposite results were found in the SANFH + miR-320 inhibitor group. The SANFH + miR-320 inhibitor + pCR-CYP1A2_KO group showed decreased miRNA-320 expression and the SANFH + pCR-CYP1A2_KO group showed decreased CYP1A2 expression and enzymatic activity. Our findings provide evidences that miR-320 might inhibit the development of SANFH by targeting CYP1A2. Copyright © 2018 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

    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.

  6. CYP1A2 – a novel genetic marker for early aromatase inhibitor response in the treatment of breast cancer patients

    International Nuclear Information System (INIS)

    Simonsson, Maria; Veerla, Srinivas; Markkula, Andrea; Rose, Carsten; Ingvar, Christian; Jernström, Helena

    2016-01-01

    Endocrine resistance is a major obstacle to optimal treatment effect in breast cancer. Some genetic markers have been proposed to predict response to aromatase inhibitors (AIs) but the data is insufficient. The aim of the study was to find new genetic treatment predictive markers of AIs. The ongoing population-based BC-blood study in Lund, Sweden includes women with primary breast cancer. This paper is based on AI-treated patients with estrogen receptor positive tumors who underwent breast cancer surgery in 2002–2008. First, an exploratory analysis of 1931 SNPs in 227 genes involved in absorption, distribution, metabolism, and elimination of multiple medications, using DMET™ chips, was conducted in a subset of the cohort with last follow-up in December 31 st 2011 (13 cases, 11 controls). Second, selected SNPs from the first analysis were re-analyzed concerning risk for early breast cancer events in the extended cohort of 201 AI-treated with last follow-up in June 30 th 2014. Clinical data were obtained from medical records and population registries. Only CYP1A2 rs762551 C-allele was significantly associated with increased risk for early events in the 24 patients (P = 0.0007) and in the extended cohort, adjusted Hazard ratio (HR) 2.22 (95 % CI 1.03–4.80). However, the main prognostic impact was found within five years, adjusted HR 7.88 (95 % CI 2.13–29.19). The impact of the CYP1A2 rs762551 C-allele was modified by a functional polymorphism in the regulator gene AhR Arg554Lys (G > A). Compared to patients who were homozygous for the major allele in both genes (CYP1A2 A/A and AhR G/G), a 9-fold risk for early events was found in patients who had at least one minor allele in both genes, adjusted HR 8.95 (95 % CI 2.55–31.35), whereas patients with at least one minor allele in either but not both genes had a 3-fold risk for early events, adjusted HR 2.81 (95 % CI 1.07–7.33). The impact of CYP1A2 rs762551 C-allele was also modified by the CYP19A1 rs4646 C

  7. 20-Hydroxycholecalciferol, product of vitamin D3 hydroxylation by P450scc, decreases NF-kappaB activity by increasing IkappaB alpha levels in human keratinocytes.

    Directory of Open Access Journals (Sweden)

    Zorica Janjetovic

    Full Text Available The side chain of vitamin D3 is hydroxylated in a sequential manner by cytochrome P450scc (CYP11A1 to form 20-hydroxycholecalciferol, which can induce growth arrest and differentiation of both primary and immortalized epidermal keratinocytes. Since nuclear factor-kappaB (NF-kappaB plays a pivotal role in the regulation of cell proliferation, differentiation and apoptosis, we examined the capability of 20-hydroxycholecalciferol to modulate the activity of NF-kappaB, using 1,25-dihydroxycholecalciferol (calcitriol as a positive control. 20-hydroxycholecalciferol inhibits the activation of NFkappaB DNA binding activity as well as NF-kappaB-driven reporter gene activity in keratinocytes. Also, 20-hydroxycholecalciferol induced significant increases in the mRNA and protein levels of the NF-kappaB inhibitor protein, IkappaB alpha, in a time dependent manner, while no changes in total NF-kappaB-p65 mRNA or protein levels were observed. Another measure of NF-kappaB activity, p65 translocation from the cytoplasm into the nucleus was also inhibited in extracts of 20-hydroxycholecalciferol treated keratinocytes. Increased IkappaB alpha was concomitantly observed in cytosolic extracts of 20-hydroxycholecalciferol treated keratinocytes, as determined by immunoblotting and immunofluorescent staining. In keratinocytes lacking vitamin D receptor (VDR, 20-hydroxycholecalciferol did not affect IkappaB alpha mRNA levels, indicating that it requires VDR for its action on NF-kappaB activity. Comparison of the effects of calcitrol, hormonally active form of vitamin D3, with 20-hydrocholecalciferol show that both agents have a similar potency in inhibiting NF-kappaB. Since NF-kappaB is a major transcription factor for the induction of inflammatory mediators, our findings indicate that 20-hydroxycholecalciferol may be an effective therapeutic agent for inflammatory and hyperproliferative skin diseases.

  8. Cytochrome P450 humanised mice

    OpenAIRE

    Gonzalez Frank J

    2004-01-01

    Abstract 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 transfo...

  9. CYP1A1, CYP1A2, SULT1A1 AND SULT1E1 ALLELIC POLYMORPHISM IN CASE OF GENITAL ENDOMETRIOSIS

    Directory of Open Access Journals (Sweden)

    Konstantin Sergeevich Kublinskiy

    2016-02-01

    Up-to-date molecular and genetic analyses reveal that women predisposed to genital endometriosis possess Allele G and Genotypes AG and GG of the polymorphic option A-4889G of the CYP1A1 gene and Allele A and Genotypes CA and AA of the polymorphic option C-734A of the CYP1A2 gene. The polymorphism of the promoter regions of the SULT1A1 (G-638A and SULT1E1 (C-174T genes is not associated with genital endometriosis in women.

  10. Epidermal CYP2 family cytochromes P450

    International Nuclear Information System (INIS)

    Du Liping; Hoffman, Susan M.G.; Keeney, Diane S.

    2004-01-01

    Skin is the largest and most accessible drug-metabolizing organ. In mammals, it is the competent barrier that protects against exposure to harmful stimuli in the environment and in the systemic circulation. Skin expresses many cytochromes P450 that have critical roles in exogenous and endogenous substrate metabolism. Here, we review evidence for epidermal expression of genes from the large CYP2 gene family, many of which are expressed preferentially in extrahepatic tissues or specifically in epithelia at the environmental interface. At least 13 CYP2 genes (CYP2A6, 2A7, 2B6, 2C9, 2C18, 2C19, 2D6, 2E1, 2J2, 2R1, 2S1, 2U1, and 2W1) are expressed in skin from at least some human individuals, and the majority of these genes are expressed in epidermis or cultured keratinocytes. Where epidermal expression has been localized in situ by hybridization or immunocytochemistry, CYP2 transcripts and proteins are most often expressed in differentiated keratinocytes comprising the outer (suprabasal) cell layers of the epidermis and skin appendages. The tissue-specific transcriptional regulation of CYP2 genes in the epidermis, and in other epithelia that interface with the environment, suggests important roles for at least some CYP2 gene products in the production and disposition of molecules affecting competency of the epidermal barrier

  11. Evaluation of the in vitro and in vivo metabolic pathway and cytochrome P450 inhibition/induction profile of Huperzine A.

    Science.gov (United States)

    Lin, Ping-Ping; Li, Xue-Ning; Yuan, Fei; Chen, Wei-Li; Yang, Meng-Jie; Xu, Hong-Rong

    2016-11-11

    Huperzine A (HupA), one of the reversible and selective acetylcholinesterase inhibitors derived from Chinese herb Huperzia Serrata, possesses affirmative action of ameliorating cognitive dysfunction of Alzheimer's disease. Up to now, the effects of HupA on human cytochrome P450s (CYPs) have not been fully elucidated. The purpose of the present study was to clarify the metabolic pathway of HupA in vitro and in vivo, and to evaluate the CYPs inhibition/induction profile of HupA in vitro. The catalytic activity of CYP enzymes (CYP1A2, 2A6, 2C9, 2C19, 2D6, 2E1 and 3A4) was measured by the quantification of specific enzyme substrates using validated liquid chromatography-tandem mass spectrometry (LC/MS/MS) methods. The in vivo metabolic pathway evaluation was performed in an open, single-dose pharmacokinetic study of HupA in fourteen elderly subjects, with urine collecting at certain intervals. In human liver microsomes, HupA (10 ng/mL) was not metabolized within 90 min, and it showed negligible inhibition against these CYP isoforms within 0.2-100 ng/mL. In human liver hepatocytes, the activities of CYP1A2 and CYP3A4 were not significantly altered when incubated at 2 or 20 ng/mL of HupA. After oral administration of 0.1 mg HupA, the total proportion of HupA excreted through urine was relatively high, accounting to 35± 9% at the limited time period of 48 h. These results suggest that HupA is substantially excreted by kidney unchanged rather than metabolized by human liver, and is unlikely to cause clinically relevant drug-drug interaction (DDI) when co-administrated with drugs that are metabolized by CYP isoenzyme system. Copyright © 2016 Elsevier Inc. All rights reserved.

  12. Roles of Human CYP2A6 and Monkey CYP2A24 and 2A26 Cytochrome P450 Enzymes in the Oxidation of 2,5,2',5'-Tetrachlorobiphenyl.

    Science.gov (United States)

    Shimada, Tsutomu; Kakimoto, Kensaku; Takenaka, Shigeo; Koga, Nobuyuki; Uehara, Shotaro; Murayama, Norie; Yamazaki, Hiroshi; Kim, Donghak; Guengerich, F Peter; Komori, Masayuki

    2016-12-01

    2,5,2',5'-Tetrachlorobiphenyl (TCB) induced type I binding spectra with cytochrome P450 (P450) 2A6 and 2A13, with K s values of 9.4 and 0.51 µM, respectively. However, CYP2A6 oxidized 2,5,2',5'-TCB to form 4-hydroxylated products at a much higher rate (∼1.0 minute -1 ) than CYP2A13 (∼0.02 minute -1 ) based on analysis by liquid chromatography-tandem mass spectrometry. Formation of 4-hydroxy-2,5,2',5'-TCB by CYP2A6 was greater than that of 3-hydroxy-2,5,2',5'-TCB and three other hydroxylated products. Several human P450 enzymes, including CYP1A1, 1A2, 1B1, 2B6, 2D6, 2E1, 2C9, and 3A4, did not show any detectable activities in oxidizing 2,5,2',5'-TCB. Cynomolgus monkey CYP2A24, which shows 95% amino acid identity to human CYP2A6, catalyzed 4-hydroxylation of 2,5,2',5'-TCB at a higher rate (∼0.3 minute -1 ) than CYP2A26 (93% identity to CYP2A6, ∼0.13 minute -1 ) and CYP2A23 (94% identity to CYP2A13, ∼0.008 minute -1 ). None of these human and monkey CYP2A enzymes were catalytically active in oxidizing other TCB congeners, such as 2,4,3',4'-, 3,4,3',4'-, and 3,5,3',5'-TCB. Molecular docking analysis suggested that there are different orientations of interaction of 2,5,2',5'-TCB with the active sites (over the heme) of human and monkey CYP2A enzymes, and that ligand interaction energies (U values) of bound protein-ligand complexes show structural relationships of interaction of TCBs and other ligands with active sites of CYP2A enzymes. Catalytic differences in human and monkey CYP2A enzymes in the oxidation of 2,5,2',5'-TCB are suggested to be due to amino acid changes at substrate recognition sites, i.e., V110L, I209S, I300F, V365M, S369G, and R372H, based on the comparison of primary sequences. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

  13. Oxygen and xenobiotic reductase activities of cytochrome P450.

    NARCIS (Netherlands)

    Goeptar, A.R.; Scheerens, H.; Vermeulen, N.P.E.

    1995-01-01

    The oxygen reductase and xenobiotic reductase activities of cytochrome P450 (P450) are reviewed. During the oxygen reductase activity of P450, molecular oxygen is reduced to superoxide anion radicals (O

  14. Biotransformation of the sesquiterpene (+)-valencene by cytochrome P450cam and P450BM-3.

    Science.gov (United States)

    Sowden, Rebecca J; Yasmin, Samina; Rees, Nicholas H; Bell, Stephen G; Wong, Luet-Lok

    2005-01-07

    The sesquiterpenoids are a large class of naturally occurring compounds with biological functions and desirable properties. Oxidation of the sesquiterpene (+)-valencene by wild type and mutants of P450cam from Pseudomonas putida, and of P450BM-3 from Bacillus megaterium, have been investigated as a potential route to (+)-nootkatone, a fine fragrance. Wild type P450cam did not oxidise (+)-valencene but the mutants showed activities up to 9.8 nmol (nmol P450)(-1) min(-1), with (+)-trans-nootkatol and (+)-nootkatone constituting >85% of the products. Wild type P450BM-3 and mutants had higher activities (up to 43 min(-1)) than P450cam but were much less selective. Of the many products, cis- and trans-(+)-nootkatol, (+)-nootkatone, cis-(+)-valencene-1,10-epoxide, trans-(+)-nootkaton-9-ol, and (+)-nootkatone-13S,14-epoxide were isolated from whole-cell reactions and characterised. The selectivity patterns suggest that (+)-valencene has one binding orientation in P450cam but multiple orientations in P450BM-3.

  15. Pharmacokinetic Effects of Isavuconazole Coadministration With the Cytochrome P450 Enzyme Substrates Bupropion, Repaglinide, Caffeine, Dextromethorphan, and Methadone in Healthy Subjects

    OpenAIRE

    Yamazaki, Takao; Desai, Amit; Goldwater, Ronald; Han, David; Howieson, Corrie; Akhtar, Shahzad; Kowalski, Donna; Lademacher, Christopher; Pearlman, Helene; Rammelsberg, Diane; Townsend, Robert

    2016-01-01

    Abstract This report describes phase 1 clinical trials performed to assess interactions of oral isavuconazole at the clinically targeted dose (200 mg, administered as isavuconazonium sulfate 372 mg, 3 times a day for 2 days; 200 mg once daily [QD] thereafter) with single oral doses of the cytochrome P450 (CYP) substrates: bupropion hydrochloride (CYP2B6; 100?mg; n = 24), repaglinide (CYP2C8/CYP3A4; 0.5 mg; n = 24), caffeine (CYP1A2; 200 mg; n = 24), dextromethorphan hydrobromide (CYP2D6/CYP3A...

  16. CYP1A2 and NAT2 phenotyping and 3-aminobiphenyl and 4-aminobiphenyl hemoglobin adduct levels in smokers and non-smokers

    International Nuclear Information System (INIS)

    Sarkar, Mohamadi; Stabbert, Regina; Kinser, Robin D.; Oey, Jan; Rustemeier, Klaus; Holt, Klaus von; Schepers, Georg; Walk, Roger A.; Roethig, Hans J.

    2006-01-01

    Some aromatic amines are considered to be putative bladder carcinogens. Hemoglobin (Hb) adducts of 3-aminobiphenyl (3-ABP) and 4-aminobiphenyl (4-ABP) have been used as biomarkers of exposure to aromatic amines from cigarette smoke. One of the goals of this study was to determine intra- and inter-individual variability in 3-ABP and 4-ABP Hb adducts and to explore the predictability of ABP Hb adduct levels based on caffeine phenotyping. The study was conducted in adult smokers (S, n = 65) and non-smokers (NS, n 65). The subjects were phenotyped for CYP1A2 and NAT2 using urinary caffeine metabolites. Blood samples were collected twice within 6 weeks and adducts measured by GC/MS. The levels of 4-ABP Hb adducts were significantly (p < 0.0001) greater in S (34.5 ± 21.06 pg/g Hb) compared to NS (6.3 ± 3.02 pg/g Hb). The levels of 3-ABP Hb adducts were below the limit of quantification (BLOQ) in most (82%) of the NS and about 10-fold lower in S (3.6 ± 3.29 pg/g Hb) compared to 4-ABP Hb adducts. No differences were observed in the adduct levels between weeks 1 and 6 in the smokers, suggesting that a single sample would be adequate to monitor cigarette smoke exposure. The regression model developed with CYP1A2, NAT2 phenotype and number of cigarettes smoked (NCIG) accounted for 47% of the variability in 3-ABP adducts, whereas 32% variability in 4-ABP adducts was accounted by CYP1A2 and NCIG. The ratio of 4-ABP Hb adducts in adult S:NS was ∼ 5:1, whereas 3-ABP Hb adducts levels were BLOQ in some S, exhibited large interindividual variability (∼ 91% compared to 57% for 4-ABP Hb) and poor dose response relationship. Therefore, 4-ABP Hb adduct levels may be a more useful biomarker of aminobiphenyl exposure from cigarette smoke

  17. Differential Expression of Cytochrome P450 Enzymes in Normal and Tumor Tissues from Childhood Rhabdomyosarcoma

    Science.gov (United States)

    Molina-Ortiz, Dora; Camacho-Carranza, Rafael; González-Zamora, José Francisco; Shalkow-Kalincovstein, Jaime; Cárdenas-Cardós, Rocío; Ností-Palacios, Rosario; Vences-Mejía, Araceli

    2014-01-01

    Intratumoral expression of genes encoding Cytochrome P450 enzymes (CYP) might play a critical role not only in cancer development but also in the metabolism of anticancer drugs. The purpose of this study was to compare the mRNA expression patterns of seven representative CYPs in paired tumor and normal tissue of child patients with rabdomyosarcoma (RMS). Using real time quantitative RT-PCR, the gene expression pattern of CYP1A1, CYP1A2, CYP1B1, CYP2E1, CYP2W1, CYP3A4, and CYP3A5 were analyzed in tumor and adjacent non-tumor tissues from 13 child RMS patients. Protein concentration of CYPs was determined using Western blot. The expression levels were tested for correlation with the clinical and pathological data of the patients. Our data showed that the expression levels of CYP1A1 and CYP1A2 were negligible. Elevated expression of CYP1B1 mRNA and protein was detected in most RMS tumors and adjacent normal tissues. Most cancerous samples exhibit higher levels of both CYP3A4 and CYP3A5 compared with normal tissue samples. Expression of CYP2E1 mRNA was found to be significantly higher in tumor tissue, however no relation was found with protein levels. CYP2W1 mRNA and/or protein are mainly expressed in tumors. In conclusion, we defined the CYP gene expression profile in tumor and paired normal tissue of child patients with RMS. The overexpression of CYP2W1, CYP3A4 and CYP3A5 in tumor tissues suggests that they may be involved in RMS chemoresistance; furthermore, they may be exploited for the localized activation of anticancer prodrugs. PMID:24699256

  18. Albendazole metabolism in patients with neurocysticercosis: antipyrine as a multifunctional marker drug of cytochrome P450

    Directory of Open Access Journals (Sweden)

    M.P. Marques

    2002-02-01

    Full Text Available The present study investigates the isoform(s of cytochrome P450 (CYP involved in the metabolism of albendazole sulfoxide (ASOX to albendazole sulfone (ASON in patients with neurocysticercosis using antipyrine as a multifunctional marker drug. The study was conducted on 11 patients with neurocysticercosis treated with a multiple dose regimen of albendazole for 8 days (5 mg/kg every 8 h. On the 5th day of albendazole treatment, 500 mg antipyrine was administered po. Blood and urine samples were collected up to 72 h after antipyrine administration. Plasma concentrations of (+-ASOX, (--ASOX and ASON were determined by HPLC using a chiral phase column and detection by fluorescence. The apparent clearance (CL/f of ASON and of the (+ and (--ASOX enantiomers were calculated and compared to total antipyrine clearance (CL T and the clearance for the production of the three major antipyrine metabolites (CLm. A correlation (P<=0.05 was obtained only between the CL T of antipyrine and the CL/f of ASON (r = 0.67. The existence of a correlation suggests the involvement of CYP isoforms common to the metabolism of antipyrine and of ASOX to ASON. Since the CL T of antipyrine is a general measure of CYP enzymes but with a slight to moderate weight toward CYP1A2, we suggest the involvement of this enzyme in ASOX to ASON metabolism in man. The study supports the establishment of a specific marker drug of CYP1A2 in the study of the in vivo metabolism of ASOX to ASON.

  19. Carbon monoxide inhibits omega-oxidation of leukotriene B4 by human polymorphonuclear leukocytes: evidence that catabolism of leukotriene B4 is mediated by a cytochrome P-450 enzyme.

    Science.gov (United States)

    Shak, S; Goldstein, I M

    1984-09-17

    Carbon monoxide significantly inhibits omega-oxidation of exogenous leukotriene B4 to 20-OH-leukotriene B4 and 20-COOH-leukotriene B4 by unstimulated polymorphonuclear leukocytes as well as omega-oxidation of leukotriene B4 that is generated when cells are stimulated with the calcium ionophore, A23187. Inhibition of omega-oxidation by carbon monoxide is concentration-dependent, completely reversible, and specific. Carbon monoxide does not affect synthesis of leukotriene B4 by stimulated polymorphonuclear leukocytes or other cell functions (i.e., degranulation, superoxide anion generation). These findings suggest that a cytochrome P-450 enzyme in human polymorphonuclear leukocytes is responsible for catabolizing leukotriene B4 by omega-oxidation.

  20. The interindividual differences in the 3-demthylation of caffeine alias CYP1A2 is determined by both genetic and environmental factors

    DEFF Research Database (Denmark)

    Rasmussen, Birgitte B; Brix, Thomas H; Kyvik, Kirsten O

    2002-01-01

    . The mean (+/- SD) caffeine ratio was 5.9 +/- 3.4. The caffeine ratio was statistically significantly higher in men compared to women, in smoking men and women compared to non-smoking persons of the same gender and in women not taking oral contraceptives compared with women on oral contraceptives. Thus, we...... confirmed that CYP1A2 is more active in men than in women, that it is induced by smoking and inhibited by oral contraceptives. In the subsequent analysis of heritability, we included 49 monozygotic twin pairs and 34 same gender dizygotic twin pairs concordant for non-smoking and non-use of oral...... contraceptives. The intraclass correlation coefficient was 0.798 (95% confidence interval, 0.696-0.900) and 0.394 (95% confidence interval, 0.109-0.680) in the monozygotic and dizygotic twins, respectively. The correlation was statistically significantly higher (P = 0.0015) in the former compared with the latter...

  1. Caffeine intake and abstract reasoning among 1374 unselected men and women from general population. Role of the -163C>A polymorphism of CYP1A2 gene.

    Science.gov (United States)

    Casiglia, Edoardo; Tikhonoff, Valérie; Albertini, Federica; Favaro, Jacopo; Montagnana, Martina; Danese, Elisa; Finatti, Francesco; Benati, Marco; Mazza, Alberto; Dal Maso, Lucia; Spinella, Paolo; Palatini, Paolo

    2017-08-01

    The possible effect of caffeine as an enhancer of cognitive performance, particularly that on abstract reasoning, has never been studied in an epidemiological setting, especially in relation to -163C>A polymorphism of CYP1A2 gene, largely controlling caffeine metabolism. Aim of this study was to ascertain whether in general population free chronic caffeine intake modifies abstract reasoning, and if this effect is influenced by the above mentioned genotype, by age, schooling, ethanol intake and smoking habits. We studied 1374 unselected men and women aged 51 ± 15 years (range 18-89) from a general population. Daily caffeine intake deriving from coffee, tea, chocolate or cola was calculated from an anamnestic questionnaire and from a 7-day dietary diary. Abstract reasoning was measured in the frame of a neuropsychological assessment as the ability to find a concept linking two words indicating objects or actions and explaining how they were connected. In age-schooling-adjusted linear regression, the higher the caffeine intake, the better the abstraction score. Abstract reasoning depended on caffeine in the -163C>A CC homozygous only (so-called slow metabolizers), where it was higher in the 3rd tertile of caffeine intake. Age and ethanol reduced while smoking and schooling enhanced this association. The interaction term between caffeine and the -163C>A polymorphism was accepted in linear regressions. Caffeine consumption resulted innocuous for the A-carriers (so-called fast metabolizers). In general population, a positive association between caffeine intake and abstract reasoning exists in the CC homozygous of the -163C>A polymorphism of CYP1A2 gene. Copyright © 2017 European Society for Clinical Nutrition and Metabolism. Published by Elsevier Ltd. All rights reserved.

  2. Genome-wide association analysis of coffee drinking suggests association with CYP1A1/CYP1A2 and NRCAM.

    Science.gov (United States)

    Amin, N; Byrne, E; Johnson, J; Chenevix-Trench, G; Walter, S; Nolte, I M; Vink, J M; Rawal, R; Mangino, M; Teumer, A; Keers, J C; Verwoert, G; Baumeister, S; Biffar, R; Petersmann, A; Dahmen, N; Doering, A; Isaacs, A; Broer, L; Wray, N R; Montgomery, G W; Levy, D; Psaty, B M; Gudnason, V; Chakravarti, A; Sulem, P; Gudbjartsson, D F; Kiemeney, L A; Thorsteinsdottir, U; Stefansson, K; van Rooij, F J A; Aulchenko, Y S; Hottenga, J J; Rivadeneira, F R; Hofman, A; Uitterlinden, A G; Hammond, C J; Shin, S-Y; Ikram, A; Witteman, J C M; Janssens, A C J W; Snieder, H; Tiemeier, H; Wolfenbuttel, B H R; Oostra, B A; Heath, A C; Wichmann, E; Spector, T D; Grabe, H J; Boomsma, D I; Martin, N G; van Duijn, C M

    2012-11-01

    Coffee consumption is a model for addictive behavior. We performed a meta-analysis of genome-wide association studies (GWASs) on coffee intake from 8 Caucasian cohorts (N=18 176) and sought replication of our top findings in a further 7929 individuals. We also performed a gene expression analysis treating different cell lines with caffeine. Genome-wide significant association was observed for two single-nucleotide polymorphisms (SNPs) in the 15q24 region. The two SNPs rs2470893 and rs2472297 (P-values=1.6 × 10(-11) and 2.7 × 10(-11)), which were also in strong linkage disequilibrium (r(2)=0.7) with each other, lie in the 23-kb long commonly shared 5' flanking region between CYP1A1 and CYP1A2 genes. CYP1A1 was found to be downregulated in lymphoblastoid cell lines treated with caffeine. CYP1A1 is known to metabolize polycyclic aromatic hydrocarbons, which are important constituents of coffee, whereas CYP1A2 is involved in the primary metabolism of caffeine. Significant evidence of association was also detected at rs382140 (P-value=3.9 × 10(-09)) near NRCAM-a gene implicated in vulnerability to addiction, and at another independent hit rs6495122 (P-value=7.1 × 10(-09))-an SNP associated with blood pressure-in the 15q24 region near the gene ULK3, in the meta-analysis of discovery and replication cohorts. Our results from GWASs and expression analysis also strongly implicate CAB39L in coffee drinking. Pathway analysis of differentially expressed genes revealed significantly enriched ubiquitin proteasome (P-value=2.2 × 10(-05)) and Parkinson's disease pathways (P-value=3.6 × 10(-05)).

  3. Human cytochrome-P450 enzymes metabolize N-(2-methoxyphenyl)hydroxylamine, a metabolite of the carcinogens o-anisidine and o-nitroanisole, thereby dictating its genotoxicity.

    Science.gov (United States)

    Naiman, Karel; Martínková, Markéta; Schmeiser, Heinz H; Frei, Eva; Stiborová, Marie

    2011-12-24

    N-(2-Methoxyphenyl)hydroxylamine is a component in the human metabolism of two industrial and environmental pollutants and bladder carcinogens, viz. 2-methoxyaniline (o-anisidine) and 2-methoxynitrobenzene (o-nitroanisole), and it is responsible for their genotoxicity. Besides its capability to form three deoxyguanosine adducts in DNA, N-(2-methoxyphenyl)-hydroxylamine is also further metabolized by hepatic microsomal enzymes. To investigate its metabolism by human hepatic microsomes and to identify the major microsomal enzymes involved in this process are the aims of this study. N-(2-Methoxyphenyl)hydroxylamine is metabolized by human hepatic microsomes predominantly to o-anisidine, one of the parent carcinogens from which N-(2-methoxyphenyl)hydroxylamine is formed, while o-aminophenol and two N-(2-methoxyphenyl)hydroxylamine metabolites, whose exact structures have not been identified as yet, are minor products. Selective inhibitors of microsomal CYPs, NADPH:CYP reductase and NADH:cytochrome-b(5) reductase were used to characterize human liver microsomal enzymes reducing N-(2-methoxyphenyl)hydroxylamine to o-anisidine. Based on these studies, we attribute the main activity for this metabolic step in human liver to CYP3A4, 2E1 and 2C (more than 90%). The enzymes CYP2D6 and 2A6 also partake in this N-(2-methoxyphenyl)hydroxylamine metabolism in human liver, but only to ∼6%. Among the human recombinant CYP enzymes tested in this study, human CYP2E1, followed by CYP3A4, 1A2, 2B6 and 2D6, were the most efficient enzymes metabolizing N-(2-methoxyphenyl)hydroxylamine to o-anisidine. The results found in this study indicate that genotoxicity of N-(2-methoxyphenyl)hydroxylamine is dictated by its spontaneous decomposition to nitrenium/carbenium ions generating DNA adducts, and by its susceptibility to metabolism by CYP enzymes. Copyright © 2011 Elsevier B.V. All rights reserved.

  4. High Expression of UGT1A1/1A6 in Monkey Small Intestine: Comparison of Protein Expression Levels of Cytochromes P450, UDP-Glucuronosyltransferases, and Transporters in Small Intestine of Cynomolgus Monkey and Human.

    Science.gov (United States)

    Akazawa, Takanori; Uchida, Yasuo; Miyauchi, Eisuke; Tachikawa, Masanori; Ohtsuki, Sumio; Terasaki, Tetsuya

    2018-01-02

    Cynomolgus monkeys have been widely used for the prediction of drug absorption in humans. The purpose of this study was to clarify the regional protein expression levels of cytochromes P450 (CYPs), UDP-glucuronosyltransferases (UGTs), and transporters in small intestine of cynomolgus monkey using liquid chromatography-tandem mass spectrometry, and to compare them with the corresponding levels in human. UGT1A1 in jejunum and ileum were >4.57- and >3.11-fold and UGT1A6 in jejunum and ileum were >16.1- and >8.57-fold, respectively, more highly expressed in monkey than in human. Also, jejunal expression of monkey CYP3A8 (homologue of human CYP3A4) was >3.34-fold higher than that of human CYP3A4. Among apical drug efflux transporters, BCRP showed the most abundant expression in monkey and human, and the expression levels of BCRP in monkey and human were >1.74- and >1.25-fold greater than those of P-gp and >2.76- and >4.50-fold greater than those of MRP2, respectively. These findings should be helpful to understand species differences of the functions of CYPs, UGTs, and transporters between monkey and human. The UGT1A1/1A6 data would be especially important because it is difficult to identify isoforms responsible for species differences of intestinal glucuronidation by means of functional studies due to overlapping substrate specificity.

  5. Cytochrome P450 2C8 and flavin-containing monooxygenases are involved in the metabolism of tazarotenic acid in humans.

    Science.gov (United States)

    Attar, Mayssa; Dong, Dahai; Ling, Kah-Hiing John; Tang-Liu, Diane D-S

    2003-04-01

    Upon oral administration, tazarotene is rapidly converted to tazarotenic acid by esterases. The main circulating agent, tazarotenic acid is subsequently oxidized to the inactive sulfoxide metabolite. Therefore, alterations in the metabolic clearance of tazarotenic acid may have significant effects on its systemic exposure. The objective of this study was to identify the human liver microsomal enzymes responsible for the in vitro metabolism of tazarotenic acid. Tazarotenic acid was incubated with 1 mg/ml pooled human liver microsomes, in 100 mM potassium phosphate buffer (pH 7.4), at 37 degrees C, over a period of 30 min. The microsomal enzymes that may be involved in tazarotenic acid metabolism were identified through incubation with microsomes containing cDNA-expressed human microsomal isozymes. Chemical inhibition studies were then conducted to confirm the identity of the enzymes potentially involved in tazarotenic acid metabolism. Reversed-phase high performance liquid chromatography was used to quantify the sulfoxide metabolite, the major metabolite of tazarotenic acid. Upon incubation of tazarotenic acid with microsomes expressing CYP2C8, flavin-containing monooxygenase 1 (FMO1), or FMO3, marked formation of the sulfoxide metabolite was observed. The involvement of these isozymes in tazarotenic acid metabolism was further confirmed by inhibition of metabolite formation in pooled human liver microsomes by specific inhibitors of CYP2C8 or FMO. In conclusion, the in vitro metabolism of tazarotenic acid to its sulfoxide metabolite in human liver microsomes is mediated by CYP2C8 and FMO.

  6. Investigation of cytochrome P450 inhibitory properties of maslinic acid, a bioactive compound from Olea europaea L., and its structure-activity relationship.

    Science.gov (United States)

    Sun, Min; Tang, Yu; Ding, Tonggui; Liu, Mingyao; Wang, Xin

    2015-01-15

    Maslinic acid (MA), the main pentacyclic triterpene of Olea europaea L. fruit, possesses a variety of pharmacological actions, including hypoglycemic, antioxidant, cardioprotective and antitumoral activities. Despite its importance, little is known about its effects on the cytochrome P450 (CYP) activity in both humans and animals. Therefore, the aim of this study was to investigate the effects of MA on the CYP 1A2, 2C9/11, 2D1/6, 2E1 and 3A2/4 activities by human and rat liver microsomes and specific CYP isoforms. In humans, MA only weakly inhibited CYP3A4 activity in human liver microsomes and specific CYP3A4 isoform with IC50 value at 46.1 and 62.3µM, respectively. In rats, MA also exhibited weak inhibition on CYP2C11, CYP2E1 and CYP3A2 activities with IC50 values more than 100µM. Enzyme kinetic studies showed that the MA was not only a competitive inhibitor of CYP3A4 in humans, but also a competitive inhibitor of CYP2C11 and 3A2 in rats, with Ki of 18.4, 98.7 and 66.3µM, respectively. Moreover, the presence of hydroxyl group at C-2 position of triterpenic acid in MA compared with oleanolic acid could magnify its competitive inhibition on human CYP3A4 activity. The relatively high Ki values of MA would have a low potential to cause the possible toxicity and drug interactions involving CYP enzymes, thus suggesting a sufficient safety for its putative use as a nutraceutical taken together with drugs. Copyright © 2014 Elsevier GmbH. All rights reserved.

  7. Protective effect of tea polyphenols against paracetamol-induced hepatotoxicity in mice is significantly correlated with cytochrome P450 suppression.

    Science.gov (United States)

    Chen, Xia; Sun, Chang-Kai; Han, Guo-Zhu; Peng, Jin-Yong; Li, Ying; Liu, Yan-Xia; Lv, Yuan-Yuan; Liu, Ke-Xin; Zhou, Qin; Sun, Hui-Jun

    2009-04-21

    To investigate the hepatoprotective activity of tea polyphenols (TP) and its relation with cytochrome P450 (CYP450) expression in mice. Hepatic CYP450 and CYPb(5) levels were measured by UV-spectrophotometry in mice 2 d after intraperitoneal TP (25, 50 and 100 mg/kg per day). Then the mice were intragastricly pre-treated with TP (100, 200 and 400 mg/kg per day) for six days before paracetamol (1000 mg/kg) was given. Their acute mortality was compared with that of control mice. The mice were pre-treated with TP (100, 200, and 400 mg/kg per day) for five days before paracetamol (500 mg/kg) was given. Hepatic CYP2E1 and CYP1A2 protein and mRNA expression levels were evaluated by Western blotting, immunohistochemical staining and transcriptase-polymerase chain reaction. The hepatic CYP450 and CYPb(5) levels in mice of TP-treated groups (100, 200 and 400 mg/kg per day) were decreased in a dose-dependent manner compared with those in the negative control mice. TP significantly attenuated the paracetamol-induced hepatic injury and dramatically reduced the mortality of paracetamol-treated mice. Furthermore, TP reduced CYP2E1 and CYP1A2 expression at both protein and mRNA levels in a dose-dependent manner. TP possess potential hepatoprotective properties and can suppress CYP450 expression.

  8. Protection by Nigella sativa against carbon tetrachloride-induced downregulation of hepatic cytochrome P450 isozymes in rats.

    Science.gov (United States)

    Ibrahim, Zein S; Ishizuka, Mayumi; Soliman, Mohamed; ElBohi, Khlood; Sobhy, Wageh; Muzandu, Kaampwe; Elkattawy, Azza M; Sakamoto, Kentaro Q; Fujita, Shoichi

    2008-11-01

    Nigella sativa (family Ranunculaceae) is an annual plant that has been traditionally used on the Indian subcontinent and in Middle Eastern countries. In this study, we investigated the effect of N. sativa oil on the drug-metabolizing cytochrome P450 (CYP) enzymes and whether it has a protective effect against the acute hepatotoxicity of CCl4. Intraperitoneal injection of rats with CCl4 drastically decreased CYP2E1, CYP2B, CYP3A2, CYP2C11, and CYP1A2 mRNA and protein expressions. Oral administration of 1 ml/kg N. sativa oil every day for one week prior to CCl4 injection alleviated CCl4-induced suppression of CYP2B, CYP3A2, CYP2C11, and CYP1A2. Moreover, CCl4 increased iNOS and TNFalpha mRNA, while N. sativa oil administration for one week prior to CCl4 injection downregulated the CCl4-induced iNOS mRNA and up-regulated IL-10 mRNA. These results indicate that N. sativa oil administration has a protective effect against the CCl4-mediated suppression of hepatic CYPs and that this protective effect is partly due to the downregulation of NO production and up-regulation of the anti-inflammatory IL-10.

  9. Isolation of CYP3A5P cDNA from human liver: a reflection of a novel cytochrome P-450 pseudogene.

    Science.gov (United States)

    Schuetz, J D; Guzelian, P S

    1995-03-14

    We have isolated, from a human liver cDNA library, a 1627 bp CYP3A5 cDNA variant (CYP3A5P) that contains several large insertions, deletions, and in-frame termination codons. By comparison with the genomic structure of other CYP3A genes, the major insertions in CYP3A5P cDNA demarcate the inferred sites of several CYP3A5 exons. The segments inserted in CYP3A5P have no homology with splice donor acceptor sites. It is unlikely that CYP3A5P cDNA represents an artifact of the cloning procedures since Southern blot analysis of human genomic DNA disclosed that CYP3A5P cDNA hybridized with a DNA fragment distinct from fragments that hybridized with either CYP3A5, CYP3A3 or CYP3A4. Moreover, analysis of adult human liver RNA on Northern blots hybridized with a CYP3A5P cDNA fragment revealed the presence of an mRNA with the predicted size of CYP3A5P. We conclude that CYP3A5P cDNA was derived from a separate gene, CYP3A5P, most likely a pseudogene evolved from CYP3A5.

  10. Arecoline inhibits the 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced cytochrome P450 1A1 activation in human hepatoma cells

    International Nuclear Information System (INIS)

    Chang, Eddy Essen; Miao Zhifeng; Lee, W.-J.; Chao, H.-R.; Li, Lih-Ann; Wang, Y.-F.; Ko, Y.-C.; Tsai, F.-Y.; Yeh, S.C.; Tsou, T.-C.

    2007-01-01

    In the present study, we investigated the effect of arecoline, a major areca nut alkaloid, on the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced activation of cytochrome P4501A1 (CYP1A1) in a human hepatoma cell line Huh-7. We treated Huh-7 cells with 10 nM TCDD in the presence of different concentrations of arecoline (50-300 μM). Our results indicated that arecoline attenuated the TCDD-induced CYP1A1 enzyme activation with an inhibitory effect on cell proliferation. By using real-time RT-PCR, we demonstrated that arecoline inhibited the TCDD-induced activations of CYP1A1 and AhR repressor (AhRR) mRNA expression in a similar pattern. Our results revealed that arecoline inhibited AhR mRNA expression with no direct effect on CYP1A1 enzyme activity. Therefore, in our present study, the observed inhibitory effect of arecoline on CYP1A1 activation was not due to the up-regulation of AhRR or direct inhibitory effect on CYP1A1. Taken together, here we have demonstrated that arecoline attenuates the TCDD-induced CYP1A1 activation mainly via down-regulation of AhR expression in human hepatoma cells, suggesting the possible involvement of arecoline in the AhR-mediated metabolism of environmental toxicants in liver

  11. Genotypes for the cytochrome P450 enzymes CYP2D6 and CYP2C19 in human longevitY

    DEFF Research Database (Denmark)

    Bathum, L; Andersen-Ranberg, K; Boldsen, J

    1998-01-01

    (PCR). The CYP2D6*5 alleles were identified with a long PCR method. For CYP2C19 we identified the alleles CYP2C19*1, CYP2C19*2 and CYP2C19*3 with an oligonucleotide ligation assay. RESULTS: The four alleles for CYP2D6 did not occur in Hardy-Weinberg proportions. The frequency of poor metabolism...... was slightly higher (10.2%) than expected [7.7%; odds ratio (OR) = 1.36 (0.75-2.40)]. The genotypes for CYP2C19 occur in Hardy-Weinberg proportions. The frequency of poor metabolism (3.8%) was not significantly different from a young control group [3.1%; OR = 1.21 (0.26-5.75)]. CONCLUSION: CYP2D6 could play...... a role in human longevity due to the lack of Hardy-Weinberg proportions. If CYP2D6 only plays a role in longevity by protecting the poor metabolizers from cancer, we should expect a rise in the frequency in these genotypes in Denmark from 7.7% among young adults to 10-11% among very old people. We found...

  12. Guidelines for development and implementation of biocatalytic P450 processes

    DEFF Research Database (Denmark)

    Lundemo, Marie Therese; Woodley, John

    2015-01-01

    in order to apply and implement them in industrial processes, both from a biological and process perspective. Indeed, a combined approach of host selection and cell engineering, integrated with process engineering, is suggested as the most effective route to implementation.......Biocatalytic reactions performed by cytochrome P450 monooxygenases are interesting in pharmaceutical research since they are involved in human drug metabolism. Furthermore, they are potentially interesting as biocatalysts for synthetic chemistry because of the exquisite selectivity of the chemistry...... they undertake. For example, selective hydroxylation can be undertaken on a highly functionalized molecule without the need for functional group protection. Recent progress in the discovery of novel P450s as well as protein engineering of these enzymes strongly encourages further development of their application...

  13. Effect of Flavonoids on Glutathione Level, Lipid Peroxidation and Cytochrome P450 CYP1A1 Expression in Human Laryngeal Carcinoma Cell Lines

    Directory of Open Access Journals (Sweden)

    Lidija Vuković

    2007-01-01

    Full Text Available Flavonoids are phytochemicals exhibiting a wide range of biological activities, among which are antioxidant activity, the ability to modulate activity of several enzymes or cell receptors and possibility to interfere with essential biochemical pathways. Using human laryngeal carcinoma HEp2 cells and their drug-resistant CK2 subline, we examined the effect of five flavonoids, three structurally related flavons (quercetin, fisetin, and myricetin, one flavonol (luteolin and one glycosilated flavanone (naringin for: (i their ability to inhibit mitochondrial dehydrogenases as an indicator of cytotoxic effect, (ii their influence on glutathione level, (iii antioxidant/prooxidant effects and influence on cell membrane permeability, and (iv effect on expression of cytochrome CYP1A1. Cytotoxic action of the investigated flavonoids after 72 hours of treatment follows this order: luteolin>quercetin>fisetin>naringin>myricetin. Our results show that CK2 were more resistant to toxic concentrations of flavonoids as compared to parental cells. Quercetin increased the total GSH level in both cell lines. CK2 cells are less perceptible to lipid peroxidation and damage caused by free radicals. Quercetin showed prooxidant effect in both cell lines, luteolin only in HEp2 cells, whereas other tested flavonoids did not cause lipid peroxidation in the tested cell lines. These data suggest that the same compound, quercetin, can act as a prooxidant, but also, it may prevent damage in cells caused by free radicals, due to the induction of GSH, by forming less harmful complex. Quercetin treatment damaged cell membranes in both cell lines. Fisetin caused higher cell membrane permeability only in HEp2 cells. However, these two compounds did not enhance the damage caused by hydrogen peroxide. Quercetin, naringin, myricetin and fisetin increased the expression of CYP1A1 in both cell lines, while luteolin decreased basal level of CYP1A1 only in HEp2 cells. In conclusion, small

  14. Cytochromes P450 for natural product biosynthesis in Streptomyces: sequence, structure, and function.

    Science.gov (United States)

    Rudolf, Jeffrey D; Chang, Chin-Yuan; Ma, Ming; Shen, Ben

    2017-08-30

    Covering: up to January 2017Cytochrome P450 enzymes (P450s) are some of the most exquisite and versatile biocatalysts found in nature. In addition to their well-known roles in steroid biosynthesis and drug metabolism in humans, P450s are key players in natural product biosynthetic pathways. Natural products, the most chemically and structurally diverse small molecules known, require an extensive collection of P450s to accept and functionalize their unique scaffolds. In this review, we survey the current catalytic landscape of P450s within the Streptomyces genus, one of the most prolific producers of natural products, and comprehensively summarize the functionally characterized P450s from Streptomyces. A sequence similarity network of >8500 P450s revealed insights into the sequence-function relationships of these oxygen-dependent metalloenzymes. Although only ∼2.4% and structurally characterized, respectively, the study of streptomycete P450s involved in the biosynthesis of natural products has revealed their diverse roles in nature, expanded their catalytic repertoire, created structural and mechanistic paradigms, and exposed their potential for biomedical and biotechnological applications. Continued study of these remarkable enzymes will undoubtedly expose their true complement of chemical and biological capabilities.

  15. Fungal Cytochrome P450s and the P450 Complement (CYPome of Fusarium graminearum

    Directory of Open Access Journals (Sweden)

    Jiyoung Shin

    2018-03-01

    Full Text Available Cytochrome P450s (CYPs, heme-containing monooxygenases, play important roles in a wide variety of metabolic processes important for development as well as biotic/trophic interactions in most living organisms. Functions of some CYP enzymes are similar across organisms, but some are organism-specific; they are involved in the biosynthesis of structural components, signaling networks, secondary metabolisms, and xenobiotic/drug detoxification. Fungi possess more diverse CYP families than plants, animals, or bacteria. Various fungal CYPs are involved in not only ergosterol synthesis and virulence but also in the production of a wide array of secondary metabolites, which exert toxic effects on humans and other animals. Although few studies have investigated the functions of fungal CYPs, a recent systematic functional analysis of CYP genes in the plant pathogen Fusarium graminearum identified several novel CYPs specifically involved in virulence, asexual and sexual development, and degradation of xenobiotics. This review provides fundamental information on fungal CYPs and a new platform for further metabolomic and biochemical studies of CYPs in toxigenic fungi.

  16. Flower colour and cytochromes P450.

    Science.gov (United States)

    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.

  17. Prediction of cytochrome P450 mediated metabolism

    DEFF Research Database (Denmark)

    Olsen, Lars; Oostenbrink, Chris; Jørgensen, Flemming Steen

    2015-01-01

    Cytochrome P450 enzymes (CYPs) form one of the most important enzyme families involved in the metabolism of xenobiotics. CYPs comprise many isoforms, which catalyze a wide variety of reactions, and potentially, a large number of different metabolites can be formed. However, it is often hard...... to rationalize what metabolites these enzymes generate. In recent years, many different in silico approaches have been developed to predict binding or regioselective product formation for the different CYP isoforms. These comprise ligand-based methods that are trained on experimental CYP data and structure...

  18. Effect of a New Prokinetic Agent DA-9701 Formulated with Corydalis Tuber and Pharbitidis Semen on Cytochrome P450 and UDP-Glucuronosyltransferase Enzyme Activities in Human Liver Microsomes

    Directory of Open Access Journals (Sweden)

    Hye Young Ji

    2012-01-01

    Full Text Available DA-9701 is a new botanical drug composed of the extracts of Corydalis tuber and Pharbitidis semen, and it is used as an oral therapy for the treatment of functional dyspepsia in Korea. The inhibitory potentials of DA-9701 and its component herbs, Corydalis tuber and Pharbitidis semen, on the activities of seven major human cytochrome P450 (CYP enzymes and four UDP-glucuronosyltransferase (UGT enzymes in human liver microsomes were investigated using liquid chromatography-tandem mass spectrometry. DA-9701 and Corydalis tuber extract slightly inhibited UGT1A1-mediated etoposide glucuronidation, with 50% inhibitory concentration (IC50 values of 188 and 290 μg/mL, respectively. DA-9701 inhibited CYP2D6-catalyzed bufuralol 1′-hydroxylation with an inhibition constant (Ki value of 6.3 μg/mL in a noncompetitive manner. Corydalis tuber extract competitively inhibited CYP2D6-mediated bufuralol 1′-hydroxylation, with a Ki value of 3.7 μg/mL, whereas Pharbitidis semen extract showed no inhibition. The volume in which the dose could be diluted to generate an IC50 equivalent concentration (volume per dose index value of DA-9701 for inhibition of CYP2D6 activity was 1.16 L/dose, indicating that DA-9701 may not be a potent CYP2D6 inhibitor. Further clinical studies are warranted to evaluate the in vivo extent of the observed in vitro interactions.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  1. Monoclonal antibodies to drosophila cytochrome P-450's

    International Nuclear Information System (INIS)

    Sundseth, S.S.; Kennel, S.J.; Waters, L.C.

    1987-01-01

    Hybridomas producing monoclonal antibodies were prepared by the fusion of SP2/0 myeloma cells and spleen cells from a female BALB/c mouse immunized by cytochrome P-450-A and P-450-B purified from Drosophila Hikone-R (BG) microsomes. P-450-A and P-450-B are electrophoretically distinct subsets of Drosophila P-450. P-450-A is ubiquitous among strains tested, while P-450-B is present in only a few strains displaying unique enzyme activities and increased insecticide resistance. The Oregon-R strain contains only cytochromes P-450-A and is susceptible to insecticides. The authors Hikone-R (BG) strain expresses both cytochromes P-450-A and P-450-B and is insecticide resistant. Antibody producing hybridomas were detected in a solid-phase radioimmunoassay (RIA) by binding to Hikone-R (BG) or Oregon-R microsomes. Four independent hybridomas were identified as producing monoclonal antibodies that recognized proteins in the P-450 complex by immunoblot experiments. Three monoclonal antibodies recognized P-450-A proteins, while one monoclonal antibody bound predominantly P-450-B. This monoclonal antibody also recognized southern armyworm (Spodoptera eridania, Cramer) microsomal proteins

  2. In vitro study of modulatory effects of extracts of Strobilanthes Crispus on human cDNA-expressed cytochrome P450 2A6 (CYP2A6) and CYP3A4

    Energy Technology Data Exchange (ETDEWEB)

    Pan, Y.; Hsu, C.J.; Koh, R.I.; Ong, C.E.; Chieng, J.Y.

    2016-07-01

    Aim: Cytochrome P450 (CYP) 2A6 and CYP3A4 play important roles in biotransformation of endogenous substances as well as xenobiotics. Strobilanthes crispus (L.) Blume (S. crispus) has been found to have anti-cancer activities and this was suggested to be due to inhibition of enzymes involved in metabolic activation of procarcinogens. The purpose of this study was to look into the potential inhibitory effects of various extracts (aqueous, hexane, chloroform, ethyl acetate, and methanol) of S. crispus from leaf and stem on human cDNA-expressed CYP2A6 and CYP3A4 activities. Methods: The activity of CYP2A6 was examined via a fluorescence-based 7-hydroxylase coumarin assay. Meanwhile, high performance liquid chromatography (HPLC)-based testosterone 6β-hydroxylase assay was established to assess CYP3A4 activity. Results: It was shown that none of the extracts from both leaf and stem potently inhibited CYP2A6 and CYP3A4 activities with IC50values above 100μg/ml. Conclusion: The anticancer potency of S. crispus is unlikely due to the modulation of CYP2A6 and CYP3A4 activities, while other mechanisms might be involved and merits further investigation. On the other hand, potential drug-herb interactions occurring between CYP2A6 and CYP3A4 substrates and S. crispus preparations is relatively low, which requires further investigations via in vivo animal as well as clinical studies.

  3. Geneva cocktail for cytochrome p450 and P-glycoprotein activity assessment using dried blood spots.

    Science.gov (United States)

    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.

  4. Effects of Pristane on Cytochrome P450 Isozyme Expression in Rat Tissues

    Directory of Open Access Journals (Sweden)

    Marvin A. Cuchens

    2005-04-01

    Full Text Available Chemical carcinogenesis studies are powerful tools to obtain information on potential mechanisms of chemical factors for malignancies. In this study Western blot analyses, using monoclonal antibodies specific for three different cytochrome P450 (CYP isozymes (CYP1A1, CYP1A2 and CYP2B, were employed to examine the effect(s of 3-methylcholanthrene and/or pristane (2,6,10,14-tetramethylpentadecane on the basal and inducible levels of expression of CYP proteins within Copenhagen rat tissues. Pristane exposure led to tissue specific differences in the CYP isozymes expressed and elicited increased CYP protein expression over 3-methylcholanthrene induced levels in microsomes isolated from liver, Peyer's Patches, and thymus. Within the context of the chemical carcinogenesis model employed in this study, these observations correlated with the induction of B-cell malignancies by low doses of 3-methylcholanthrene and of thymic lymphomas by a high 3-methylcholanthrene dose. The data suggest that pristane treatment affects CYP isozyme expression. This pristane-mediated effect clearly could be a contributing factor in the chemical carcinogenesis of the previously observed lymphoid malignancies, and a possible basis for the tumor enhancing effects of pristane.

  5. Inhibitory effects of kale ingestion on metabolism by cytochrome P450 enzymes in rats.

    Science.gov (United States)

    Yamasaki, Izumi; Yamada, Masayoshi; Uotsu, Nobuo; Teramoto, Sachiyuki; Takayanagi, Risa; Yamada, Yasuhiko

    2012-01-01

    Kale (Brassica oleracea L. var acephala DC) is a leafy green vegetable belonging to the cabbage family (Brassicaceae) that contains a large amount of health-promoting phytochemicals. There are any reports about the effects of kale ingestion on the chemoprevention function and mechanism, but the interactions between kale and drugs have not been researched. We investigated the effects of kale intake on cytochrome P450 (CYP) metabolism by using cocktail probe drugs, including midazolam (for CYP3A4), caffeine (for CYP1A2), dextromethorphan (for CYP2D6), tolbutamide (for CYP2C9), omeprazole (for CYP2C19), and chlorzoxazone (for CYP2E1). Cocktail drugs were administered into rats treated with kale and cabbage (2000 mg/kg) for a week. The results showed that kale intake induced a significant increase in plasma levels and the AUC of midazolam, caffeine, and dextromethorphan. In addition, the plasma concentration and AUC of omeprazole tended to increase. Additionally, no almost differences in the mRNA expression levels of CYP enzymes in the liver were observed. In conclusion, kale ingestion was considered to have an inhibitory effect on the activities of CYP3A4, 1A2, 2D6, and 2C19 for a reason competitive inhibition than inhibitory changes in the mRNA expressions.

  6. Carbonated soft drinks alter hepatic cytochrome P450 isoform expression in Wistar rats.

    Science.gov (United States)

    Alkhedaide, Adel; Soliman, Mohamed Mohamed; Ibrahim, Zein Shaban

    2016-11-01

    The aim of the current study was to examine the effects of chronic consumption of soft drinks (SDs) on hepatic oxidative stress and cytochrome P450 enzymes (CYPs) expression in the livers of Wistar rats. For 3 consecutive months, the rats had free access to three different soft drinks, Coca-Cola, Pepsi-Cola and 7-UP. The rats were subsequently compared with control group rats that had consumed water. Blood and hepatic tissue samples were assayed for the changes in antioxidants, liver function biomarkers and hepatic gene expression for different isoforms of hepatic CYP. The results indicated that SD consumption (SDC) decreased serum antioxidant levels and increased malondialdehyde secretion, and increased liver biomarkers (glutamate pyruvate transaminase and glutamate oxaloacetate). SD induced alterations in mRNA expression of hepatic antioxidants and cytochrome isoforms. The expression of peroxidase, catalase, CYP1A2, CYP3A2 and CYP2C11 in the liver were upregulated following SDC. By contrast, CYP2B1 was downregulated after 3 months of SDC in liver tissue samples. Thus, the present findings indicate that SDs induced oxidative stress in the liver of Wistar rats and for the first time, to the best of our knowledge, indicate that SDC disrupts hepatic CYP enzymes that may affect drug metabolism. Therefore, drug-dosing programs should be carefully designed to take these novel findings into consideration for the treatment of diseases.

  7. Impact of fasting followed by short-term exposure to interleukin-6 on cytochrome P450 mRNA in mice.

    Science.gov (United States)

    Rasmussen, Martin Krøyer; Bertholdt, Lærke; Gudiksen, Anders; Pilegaard, Henriette; Knudsen, Jakob G

    2018-01-05

    The gene expression of the cytochrome P450 (CYP) enzyme family is regulated by numerous factors. Fasting has been shown to induce increased hepatic CYP mRNA in both humans and animals. However, the coordinated regulation of CYP, CYP-regulating transcription factors, and transcriptional co-factors in the liver linking energy metabolism to detoxification has never been investigated. Interleukin-6 (IL-6) has been suggested to be released during fasting and has been shown to regulate CYP expression. The present study investigated the hepatic mRNA content of selected CYP, AhR, CAR, PXR and PPARα in mice fasted for 18h and subsequently exposed to IL-6. Furthermore, the impact of fasting on PGC-1α, HNF-4α, SIRT1 and SIRT3 mRNA was examined. Fasting induced a marked increase in Cyp2b10, Cyp2e1 and Cyp4a10 mRNA, while CYP1a1, Cyp1a2, Cyp2a4 and Cyp3a11 mRNA levels remained unchanged. In accordance, the mRNA levels of CAR and PPARα were also increased with fasting. The PGC-1α, SIRT1 and SIRT3 mRNA levels were also increased after fasting, while the HNF-4α mRNA levels remained unchanged. In mice subjected to IL-6 injection, the fasting-induced PXR, PPARα and PGC-1α mRNA responses were lower than after saline injection. In conclusion, fasting was demonstrated to be a strong inducer of hepatic CYP mRNA as well as selected transcription factors controlling the expression of the investigated CYP. Moreover, the mRNA levels of transcriptional co-factors acting as energy sensors and co-factors for CYP regulation was also increased in the liver, suggesting crosstalk at the molecular level between regulation of energy metabolism and detoxification. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. A drug-drug interaction study to assess the effect of the CYP1A2 inhibitor fluvoxamine on the pharmacokinetics of dovitinib (TKI258) in patients with advanced solid tumors

    NARCIS (Netherlands)

    de Weger, Vincent A; Goel, Sanjay; von Moos, Roger; Schellens, Jan H M; Mach, Nicholas; Tan, Eugene; Anand, Suraj; Scott, Jeffrey W; Lassen, Ulrik N

    PURPOSE: Dovitinib is an orally available multi tyrosine kinase inhibitor which inhibits VEGFR 1-3, FGFR 1-3, and PDGFR. This study was performed to investigate the potential drug-drug interaction of dovitinib with the CYP1A2 inhibitor fluvoxamine in patients with advanced solid tumors. METHODS:

  9. A drug-drug interaction study to assess the effect of the CYP1A2 inhibitor fluvoxamine on the pharmacokinetics of dovitinib (TKI258) in patients with advanced solid tumors

    DEFF Research Database (Denmark)

    de Weger, Vincent A; Goel, Sanjay; von Moos, Roger

    2018-01-01

    PURPOSE: Dovitinib is an orally available multi tyrosine kinase inhibitor which inhibits VEGFR 1-3, FGFR 1-3, and PDGFR. This study was performed to investigate the potential drug-drug interaction of dovitinib with the CYP1A2 inhibitor fluvoxamine in patients with advanced solid tumors. METHODS: ...

  10. Sequencing and characterization of mixed function monooxygenase genes CYP1A1 and CYP1A2 of Mink (Mustela vison) to facilitate study of dioxin-like compounds

    International Nuclear Information System (INIS)

    Zhang Xiaowei; Moore, Jeremy N.; Newsted, John L.; Hecker, Markus; Zwiernik, Matthew J.; Jones, Paul D.; Bursian, Steven J.

    2009-01-01

    As part of an ongoing effort to understand aryl hydrocarbon receptor (AhR) mediated toxicity in mink, cDNAs encoding for CYP1A1 and the CYP1A2 mixed function monooxygenases were cloned and characterized. In addition, the effects of selected dibenzofurans on the expression of these genes and the presence of their respective proteins (P4501A) were investigated, and then correlated with the catalytic activities of these proteins as measured by ethoxyresorufin O-deethylase (EROD) and methoxyresorufin O-deethylase (MROD) activities. The predicted protein sequences for CYP1A1 and CYP1A2 comprise 517 and 512 amino acid residues, respectively. The phylogenetic analysis of the mink CYP1As with protein sequences of other mammals revealed high sequence homology with sea otter, seals and the dog, with amino acid identities ranging from 89 to 95% for CYP1A1 and 81 to 93% for CYP1A2. Since exposure to both 2,3,7,8-Tetrachlorodibenzofuran (TCDF) and 2,3,4,7,8-Pentachlorodibenzofuran (PeCDF) resulted in dose-dependent increases of CYP1A1 mRNA, CYP1A2 mRNA and CYP1A protein levels an underlying AhR-mediated mechanism is suggested. The up-regulation of CYP1A mRNA in liver was more consistent to the sum adipose TEQ concentration than to the liver TEQ concentration in minks treated with TCDF or PeCDF. The result suggested that the hepatic-sequestered fraction of PeCDF was biologically inactive to the induction of CYP1A1 and CYP1A2

  11. Effects of electro-acupuncture on ovarian P450arom, P450c17α and mRNA expression induced by letrozole in PCOS rats.

    Science.gov (United States)

    Sun, Jie; Jin, Chunlan; Wu, Huangan; Zhao, Jimeng; Cui, Yunhua; Liu, Huirong; Wu, Lingxiang; Shi, Yin; Zhu, Bing

    2013-01-01

    Hyperandrogenism is a core factor in the series of reproductive and endocrine metabolic disorders involved in polycystic ovary syndrome (PCOS). Abnormalities in enzymatic activity and the expression of ovarian granular cell layer P450arom and theca cell P450c17α can lead to an atypical environment of local ovarian hormones, including excessive androgen levels. Rat models prepared with letrozole exhibit similar endocrine and histological changes to those that occur in human PCOS. We used such a model to study the role of electro-acupuncture (EA) in regulating ovarian P450arom and P450c17α enzymatic activity and mRNA expression in PCOS rats. Female Sprague Dawley (SD) rats aged 42 days were randomly divided into 3 groups (control, PCOS, and PCOS EA) consisting of 10 rats each. The PCOS and PCOS EA groups were administered a gavage of 1.0 mg/kg(-1) of letrozole solution once daily for 21 consecutive days. Beginning in the ninth week, the PCOS EA group was administered low-frequency EA treatment daily for 14 consecutive days. After the treatment, we obtained the following results. The estrous cycles were restored in 8 of the 10 rats in the PCOS EA group, and their ovarian morphologies and ultrastructures normalized. The peripheral blood measurements (with ELISA) showed significantly decreased androgens (i.e., androstenedione and testosterone) with significantly increased estrogens (i.e., estrone, estradiol) and increased P450arom with decreased P450C17α. Immunohistochemistry and Western blotting methods showed enhanced expression of ovarian granular cell layer P450arom as well as decreased expression of theca cell layer P450C17α. Fluorescence quantitative PCR methods showed enhanced expression of ovarian granular cell layer P450arom mRNA as well as decreased expression of theca cell layer P450C17α mRNA. These results may help explain the effects of electro-acupuncture in changing the local ovarian hyperandrogenic environment and improving reproductive and

  12. Effects of Electro-Acupuncture on Ovarian P450arom, P450c17α and mRNA Expression Induced by Letrozole in PCOS Rats

    Science.gov (United States)

    Wu, Huangan; Zhao, Jimeng; Cui, Yunhua; Liu, Huirong; Wu, Lingxiang; Shi, Yin; Zhu, Bing

    2013-01-01

    Hyperandrogenism is a core factor in the series of reproductive and endocrine metabolic disorders involved in polycystic ovary syndrome (PCOS). Abnormalities in enzymatic activity and the expression of ovarian granular cell layer P450arom and theca cell P450c17α can lead to an atypical environment of local ovarian hormones, including excessive androgen levels. Rat models prepared with letrozole exhibit similar endocrine and histological changes to those that occur in human PCOS. We used such a model to study the role of electro-acupuncture (EA) in regulating ovarian P450arom and P450c17α enzymatic activity and mRNA expression in PCOS rats. Female Sprague Dawley (SD) rats aged 42 days were randomly divided into 3 groups (control, PCOS, and PCOS EA) consisting of 10 rats each. The PCOS and PCOS EA groups were administered a gavage of 1.0 mg/kg−1 of letrozole solution once daily for 21 consecutive days. Beginning in the ninth week, the PCOS EA group was administered low-frequency EA treatment daily for 14 consecutive days. After the treatment, we obtained the following results. The estrous cycles were restored in 8 of the 10 rats in the PCOS EA group, and their ovarian morphologies and ultrastructures normalized. The peripheral blood measurements (with ELISA) showed significantly decreased androgens (i.e., androstenedione and testosterone) with significantly increased estrogens (i.e., estrone, estradiol) and increased P450arom with decreased P450C17α. Immunohistochemistry and Western blotting methods showed enhanced expression of ovarian granular cell layer P450arom as well as decreased expression of theca cell layer P450C17α. Fluorescence quantitative PCR methods showed enhanced expression of ovarian granular cell layer P450arom mRNA as well as decreased expression of theca cell layer P450C17α mRNA. These results may help explain the effects of electro-acupuncture in changing the local ovarian hyperandrogenic environment and improving reproductive and

  13. Effects of electro-acupuncture on ovarian P450arom, P450c17α and mRNA expression induced by letrozole in PCOS rats.

    Directory of Open Access Journals (Sweden)

    Jie Sun

    Full Text Available Hyperandrogenism is a core factor in the series of reproductive and endocrine metabolic disorders involved in polycystic ovary syndrome (PCOS. Abnormalities in enzymatic activity and the expression of ovarian granular cell layer P450arom and theca cell P450c17α can lead to an atypical environment of local ovarian hormones, including excessive androgen levels. Rat models prepared with letrozole exhibit similar endocrine and histological changes to those that occur in human PCOS. We used such a model to study the role of electro-acupuncture (EA in regulating ovarian P450arom and P450c17α enzymatic activity and mRNA expression in PCOS rats. Female Sprague Dawley (SD rats aged 42 days were randomly divided into 3 groups (control, PCOS, and PCOS EA consisting of 10 rats each. The PCOS and PCOS EA groups were administered a gavage of 1.0 mg/kg(-1 of letrozole solution once daily for 21 consecutive days. Beginning in the ninth week, the PCOS EA group was administered low-frequency EA treatment daily for 14 consecutive days. After the treatment, we obtained the following results. The estrous cycles were restored in 8 of the 10 rats in the PCOS EA group, and their ovarian morphologies and ultrastructures normalized. The peripheral blood measurements (with ELISA showed significantly decreased androgens (i.e., androstenedione and testosterone with significantly increased estrogens (i.e., estrone, estradiol and increased P450arom with decreased P450C17α. Immunohistochemistry and Western blotting methods showed enhanced expression of ovarian granular cell layer P450arom as well as decreased expression of theca cell layer P450C17α. Fluorescence quantitative PCR methods showed enhanced expression of ovarian granular cell layer P450arom mRNA as well as decreased expression of theca cell layer P450C17α mRNA. These results may help explain the effects of electro-acupuncture in changing the local ovarian hyperandrogenic environment and improving reproductive

  14. CHARACTERIZATION OF THE ALKANE-INDUCIBLE CYTOCHROME P450 (P450ALK) GENE FROM THE YEAST CANDIDA TROPICALIS: IDENTIFICATION OF A NEW P450 FAMILY

    Science.gov (United States)

    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 ...

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

    DEFF Research Database (Denmark)

    Bavishi, Krutika; Laursen, Tomas; Martinez, Karen Laurence

    2016-01-01

    Direct electrochemistry of cytochrome P450 containing systems has primarily focused on investigating enzymes from microbes and animals for bio-sensing applications. Plant P450s receive electrons from NADPH P450 oxidoreductase (POR) to orchestrate the bio-synthesis of a plethora of commercially...... was electro-catalytically active while the P450s generated hydrogen peroxide (H2O2). These nanodisc-based investigations lay the prospects and guidelines for construction of a simplified platform to perform mediator-free, direct electrochemistry of non-engineered cytochromes P450 under native-like conditions...

  16. The biodiversity of microbial cytochromes P450.

    Science.gov (United States)

    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.

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

    OpenAIRE

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

    2012-01-01

    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. O...

  18. The expression of xenobiotic-metabolizing enzymes in human prostate and in prostate epithelial cells (PECs) derived from primary cultures.

    Science.gov (United States)

    Al-Buheissi, S Z; Cole, K J; Hewer, A; Kumar, V; Bryan, R L; Hudson, D L; Patel, H R; Nathan, S; Miller, R A; Phillips, D H

    2006-06-01

    Dietary heterocyclic amines (HCAs) are carcinogenic in rodent prostate requiring activation by enzymes such as cytochrome P450 (CYP) and N-acetyltransferase (NAT). We investigated by Western blotting and immunohistochemistry the expression of CYP1A1, CYP1A2, and NAT1 in human prostate and in prostate epithelial cells (PECs) derived from primary cultures and tested their ability to activate the dietary carcinogen 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and its N-hydroxy metabolite (N-OH-IQ) to DNA-damaging moieties. Western blotting identified CYP1A1, CYP1A2, and NAT1. Immunohistochemistry localized NAT1 to the cytoplasm of PECs. Inter-individual variation was observed in the expression levels of CYP1A1, 1A2, and NAT1 (11, 75, and 35-fold, respectively). PECs expressed CYP1A1 and NAT1 but not CYP1A2. When incubated with IQ or N-OH-IQ, PECs formed DNA adducts indicating their ability to metabolically activate these compounds. Prostate cells possess the capacity to activate dietary carcinogens. PECs may provide a useful model system to study their role in prostate carcinogenesis.

  19. Effect of strychnine hydrochloride on liver cytochrome P450 mRNA expression and monooxygenase activities in rat

    Directory of Open Access Journals (Sweden)

    Qian Gao

    2011-08-01

    Full Text Available Strychnos nux-vomica L. has been frequently used in traditional Chinese medicine but has high acute toxicity. It is commonly taken with Glycyrrhizae radix to decrease its toxicity but the mechanism of this interaction is unknown. In this work, the mRNA expression and the activity of four cytochrome P450 (CYP enzymes representative of four subfamilies (CYP1A, CYP3A, CYP2C and CYP2E were determined ex vivo in rat livers from groups of Wistar rats orally administered strychnine hydrochloride (SH at three doses (0.1, 0.3 and 0.9 mg/kg/day alone and, at the highest dose, in combination with glycyrrhetinic acid (GA, 25 mg/kg/day or liquiritin (LQ, 20 mg/kg/day once a day for 7 consecutive days. Compared to control, the mRNA expressions of CYP3A1, 1A2 and 2E1 were higher in rats receiving the highest dose of SH but lower for CYP3A1 and CYP2E1 in rats receiving the SH+GA and SH+LQ combinations. CYP2E1 activity was higher and CYP2C, CYP3A and CYP1A2 activities were lower in rats receiving the highest dose of SH. In contrast CYP1A2 and CYP2C activities were higher and CYP2E1 and CYP3A activities lower in rats receiving the SH+GA combination. CYP2E1 and CYP3A activities were also lower in rats receiving the SH+LQ combination. The results show that treatment with SH for 7 days affects the expression and the activity of CYP enzymes and that coadministration of GA and LQ modulates these effects. This modulation may explain the role of Glycyrrhizae radix in reducing the acute toxicity of Strychnos nux-vomica L.CYPs enzymes.

  20. Theoretical study on the N-demethylation mechanism of theobromine catalyzed by P450 isoenzyme 1A2.

    Science.gov (United States)

    Tao, Jing; Kang, Yuan; Xue, Zhiyu; Wang, Yongting; Zhang, Yan; Chen, Qiu; Chen, Zeqin; Xue, Ying

    2015-09-01

    Theobromine, a widely consumed pharmacological active substance, can cause undesirable muscle stiffness, nausea and anorexia in high doses ingestion. The main N-demethylation metabolic mechanism of theobromine catalyzed by P450 isoenzyme 1A2 (CYP1A2) has been explored in this work using the unrestricted hybrid density functional method UB3LYP in conjunction with the LACVP(Fe)/6-31G (H, C, N, O, S, Cl) basis set. Single-point calculations including empirical dispersion corrections were carried out at the higher 6-311++G** basis set. Two N-demethylation pathways were characterized, i.e., 3-N and 7-N demethylations, which involve the initial N-methyl hydroxylation to form carbinolamines and the subsequent carbinolamines decomposition to yield monomethylxanthines and formaldehydes. Our results have shown that the rate-limiting N-methyl hydroxylation occurs via a hydrogen atom transfer (HAT) mechanism, which proceeds in a spin-selective mechanism (SSM) in the gas phase. The carbinolamines generated are prone to decomposition via the contiguous heteroatom-assisted proton-transfer. Strikingly, 3-N demethylation is more favorable than 7-N demethylation due to its lower free energy barrier and 7-methylxanthine therefore is the optimum product reported for the demethylation of theobromine catalyzed by CYP1A2, which are in good agreement with the experimental observation. This work has first revealed the detail N-demethylation mechanisms of theobromine at the theoretical level. It can offer more significant information for the metabolism of purine alkaloid. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. Monkey liver cytochrome P450 2C9 is involved in caffeine 7-N-demethylation to form theophylline.

    Science.gov (United States)

    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.

  2. The Role of Cytochromes P450 in Infection

    Directory of Open Access Journals (Sweden)

    Elisavet Stavropoulou

    2018-01-01

    Full Text Available Cytochromes are expressed in many different tissues of the human body. They are found mostly in intestinal and hepatic tissues. Cytochromes P450 (CYPs are enzymes that oxidize substances using iron and are able to metabolize a large variety of xenobiotic substances. CYP enzymes are linked to a wide array of reactions including and O-dealkylation, S-oxidation, epoxidation, and hydroxylation. The activity of the typical P450 cytochrome is influenced by a variety of factors, such as genus, environment, disease state, herbicide, alcohol, and herbal medications. However, diet seems to play a major role. The mechanisms of action of dietary chemicals, macro- and micronutrients on specific CYP isoenzymes have been extensively studied. Dietary modulation has effects upon the metabolism of xenobiotics. Cytochromes harbor intra- or interindividual and intra- or interethnic genetic polymorphisms. Bacteria were shown to express CYP-like genes. The tremendous metabolic activity of the microbiota is associated to its abundant pool of CYP enzymes, which catalyze phase I and II reactions in drug metabolism. Disease states, intestinal disturbances, aging, environmental toxic effects, chemical exposures or nutrition modulate the microbial metabolism of a drug before absorption. A plethora of effects exhibited by most of CYP enzymes can resemble those of proinflammatory cytokines and IFNs. Moreover, they are involved in the initiation and persistence of pathologic pain by directly activating sensory neurons and inflammatory cytokines.

  3. Insect P450 inhibitors and insecticides: challenges and opportunities.

    Science.gov (United States)

    Feyereisen, René

    2015-06-01

    P450 enzymes are encoded by a large number of genes in insects, often over a hundred. They play important roles in insecticide metabolism and resistance, and growing numbers of P450 enzymes are now known to catalyse important physiological reactions, such as hormone metabolism or cuticular hydrocarbon synthesis. Ways to inhibit P450 enzymes specifically or less specifically are well understood, as P450 inhibitors are found as drugs, as fungicides, as plant growth regulators and as insecticide synergists. Yet there are no P450 inhibitors as insecticides on the market. As new modes of action are constantly needed to support insecticide resistance management, P450 inhibitors should be considered because of their high potential for insect selectivity, their well-known mechanisms of action and the increasing ease of rational design and testing. © 2014 Society of Chemical Industry.

  4. Ligand Access Channels in Cytochrome P450 Enzymes: A Review

    Directory of Open Access Journals (Sweden)

    Philippe Urban

    2018-05-01

    Full Text Available Quantitative structure-activity relationships may bring invaluable information on structural elements of both enzymes and substrates that, together, govern substrate specificity. Buried active sites in cytochrome P450 enzymes are connected to the solvent by a network of channels exiting at the distal surface of the protein. This review presents different in silico tools that were developed to uncover such channels in P450 crystal structures. It also lists some of the experimental evidence that actually suggest that these predicted channels might indeed play a critical role in modulating P450 functions. Amino acid residues at the entrance of the channels may participate to a first global ligand recognition of ligands by P450 enzymes before they reach the buried active site. Moreover, different P450 enzymes show different networks of predicted channels. The plasticity of P450 structures is also important to take into account when looking at how channels might play their role.

  5. Cytochrome P450 monooxygenases and insecticide resistance in insects.

    OpenAIRE

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

    1998-01-01

    Cytochrome P450 monooxygenases are involved in many cases of resistance of insects to insecticides. Resistance has long been associated with an increase in monooxygenase activities and with an increase in cytochrome P450 content. However, this increase does not always account for all of the resistance. In Drosophila melanogaster, we have shown that the overproduction of cytochrome P450 can be lost by the fly without a corresponding complete loss of resistance. These results prompted the seque...

  6. Effect of Ginkgo biloba extract on procarcinogen-bioactivating human CYP1 enzymes: Identification of isorhamnetin, kaempferol, and quercetin as potent inhibitors of CYP1B1

    International Nuclear Information System (INIS)

    Chang, Thomas K.H.; Chen Jie; Yeung, Eugene Y.H.

    2006-01-01

    In the present study, we investigated the effect of Ginkgo biloba extracts and some of its individual constituents on the catalytic activity of human cytochrome P450 enzymes CYP1B1, CYP1A1, and CYP1A2. G. biloba extract of known abundance of terpene trilactones and flavonol glycosides inhibited 7-ethoxyresorufin O-dealkylation catalyzed by human recombinant CYP1B1, CYP1A1, and CYP1A2, and human liver microsomes, with apparent K i values of 2 ± 0.3, 5 ± 0.5, 16 ± 1.4, and 39 ± 1.2 μg/ml (mean ± SE), respectively. In each case, the mode of inhibition was of the mixed type. Bilobalide, ginkgolides A, B, C, and J, quercetin 3-O-rutinoside, kaempferol 3-O-rutinoside, and isorhamentin 3-O-rutinoside were not responsible for the inhibition of CYP1 enzymes by G. biloba extract, as determined by experiments with these individual chemicals at the levels present in the extract. In contrast, the aglycones of quercetin, kaempferol, and isorhamentin inhibited CYP1B1, CYP1A1, and CYP1A2. Among the three flavonol aglycones, isorhamentin was the most potent in inhibiting CYP1B1 (apparent K i = 3 ± 0.1 nM), whereas quercetin was the least potent in inhibiting CYP1A2 (apparent K i 418 ± 50 nM). The mode of inhibition was competitive, noncompetitive, or mixed, depending on the enzyme and the flavonol. G. biloba extract also reduced benzo[a]pyrene hydroxylation, and the effect was greater with CYP1B1 than with CYP1A1 as the catalyst. Overall, our novel findings indicate that G. biloba extract and the flavonol aglycones isorhamnetin, kaempferol, and quercetin preferentially inhibit the in vitro catalytic activity of human CYP1B1

  7. Are there differences in the catalytic activity per unit enzyme of recombinantly expressed and human liver microsomal cytochrome P450 2C9? A systematic investigation into inter-system extrapolation factors.

    Science.gov (United States)

    Crewe, H K; Barter, Z E; Yeo, K Rowland; Rostami-Hodjegan, A

    2011-09-01

    The 'relative activity factor' (RAF) compares the activity per unit of microsomal protein in recombinantly expressed cytochrome P450 enzymes (rhCYP) and human liver without separating the potential sources of variation (i.e. abundance of enzyme per mg of protein or variation of activity per unit enzyme). The dimensionless 'inter-system extrapolation factor' (ISEF) dissects differences in activity from those in CYP abundance. Detailed protocols for the determination of this scalar, which is used in population in vitro-in vivo extrapolation (IVIVE), are currently lacking. The present study determined an ISEF for CYP2C9 and, for the first time, systematically evaluated the effects of probe substrate, cytochrome b5 and methods for assessing the intrinsic clearance (CL(int) ). Values of ISEF for S-warfarin, tolbutamide and diclofenac were 0.75 ± 0.18, 0.57 ± 0.07 and 0.37 ± 0.07, respectively, using CL(int) values derived from the kinetic values V(max) and K(m) of metabolite formation in rhCYP2C9 + reductase + b5 BD Supersomes™. The ISEF values obtained using rhCYP2C9 + reductase BD Supersomes™ were more variable, with values of 7.16 ± 1.25, 0.89 ± 0.52 and 0.50 ± 0.05 for S-warfarin, tolbutamide and diclofenac, respectively. Although the ISEF values obtained from rhCYP2C9 + reductase + b5 for the three probe substrates were statistically different (p system, with the intrinsic clearance calculated from full kinetic data is recommended for generation of the CYP2C9 ISEF. Furthermore, as ISEFs have been found to be sensitive to differences in accessory proteins, rhCYP system specific ISEFs are recommended. Copyright © 2011 John Wiley & Sons, Ltd.

  8. 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

    Science.gov (United States)

    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

  9. 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.

    Science.gov (United States)

    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.

  10. The molecular evolution of cytochrome P450 genes within and between drosophila species.

    Science.gov (United States)

    Good, Robert T; Gramzow, Lydia; Battlay, Paul; Sztal, Tamar; Batterham, Philip; Robin, Charles

    2014-04-20

    We map 114 gene gains and 74 gene losses in the P450 gene family across the phylogeny of 12 Drosophila species by examining the congruence of gene trees and species trees. Although the number of P450 genes varies from 74 to 94 in the species examined, we infer that there were at least 77 P450 genes in the ancestral Drosophila genome. One of the most striking observations in the data set is the elevated loss of P450 genes in the Drosophila sechellia lineage. The gain and loss events are not evenly distributed among the P450 genes-with 30 genes showing no gene gains or losses whereas others show as many as 20 copy number changes among the species examined. The P450 gene clades showing the fewest number of gene gain and loss events tend to be those evolving with the most purifying selection acting on the protein sequences, although there are exceptions, such as the rapid rate of amino acid replacement observed in the single copy phantom (Cyp306a1) gene. Within D. melanogaster, we observe gene copy number polymorphism in ten P450 genes including multiple cases of interparalog chimeras. Nonallelic homologous recombination (NAHR) has been associated with deleterious mutations in humans, but here we provide a second possible example of an NAHR event in insect P450s being adaptive. Specifically, we find that a polymorphic Cyp12a4/Cyp12a5 chimera correlates with resistance to an insecticide. Although we observe such interparalog exchange in our within-species data sets, we have little evidence of it between species, raising the possibility that such events may occur more frequently than appreciated but are masked by subsequent sequence change. © The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  11. CYP1A2*1C, CYP2E1*5B, and GSTM1 polymorphisms are predictors of risk and poor outcome in head and neck squamous cell carcinoma patients

    DEFF Research Database (Denmark)

    Olivieri, Eloisa Helena Ribeiro; da Silva, Sabrina Daniela; Mendonça, Fernando Fernandes

    2009-01-01

    is performed by glutathione S-transferases (GSTs). It has been suggested that genetic alterations, such as polymorphisms, play an important role in tumorigenesis and HNSCC progression. The aim of this study was to investigate CYP1A1, CYP1A2, CYP2E1, GSTM1, and GSTT1 polymorphisms as risk factors in HNSCC...... and their association with clinicopathologic data. The patients comprised 153 individuals with HNSCC (cases) and 145 with no current or previous diagnosis of cancer (controls). Genotyping of the single nucleotide polymorphisms (SNPs) of the CYP1A1, CYP1A2, and CYP2E1 genes was performed by PCR-RFLP and the GSTM1...... for determining the parameters associated with tumor progression and poor outcomes in HNSCC....

  12. LKM-1 autoantibodies recognize a short linear sequence in P450IID6, a cytochrome P-450 monooxygenase.

    OpenAIRE

    Manns, M P; Griffin, K J; Sullivan, K F; Johnson, E F

    1991-01-01

    LKM-1 autoantibodies, which are associated with autoimmune chronic active hepatitis, recognize P450IID6, a cytochrome P-450 monooxygenase. The reactivities of 26 LKM-1 antisera were tested with a panel of deletion mutants of P450IID6 expressed in Escherichia coli. 22 sera recognize a 33-amino acid segment of P450IID6, and 11 of these recognize a shorter segment, DPAQPPRD. PAQPPR is also found in IE175 of herpes simplex virus type 1 (HSV-1). Antibodies for HSV-1 proteins were detected by ELISA...

  13. Curcumin Prevents Aflatoxin B1 Hepatoxicity by Inhibition of Cytochrome P450 Isozymes in Chick Liver

    Directory of Open Access Journals (Sweden)

    Ni-Ya Zhang

    2016-11-01

    Full Text Available This study was designed to establish if Curcumin (CM alleviates Aflatoxin B1 (AFB1-induced hepatotoxic effects and to determine whether alteration of the expression of cytochrome P450 (CYP450 isozymes is involved in the regulation of these effects in chick liver. One-day-old male broilers (n = 120 were divided into four groups and used in a two by two factorial trial in which the main factors included supplementing AFB1 (< 5 vs. 100 μg/kg and CM (0 vs. 150 mg/kg in a corn/soybean-based diet. Administration of AFB1 induced liver injury, significantly decreasing albumin and total protein concentrations and increasing alanine aminotransferase and aspartate aminotransferase activities in serum, and induced hepatic histological lesions at week 2. AFB1 also significantly decreased hepatic glutathione peroxidase, catalase, and glutathione levels, while increasing malondialdehyde, 8-hydroxydeoxyguanosine, and exo-AFB1-8,9-epoxide (AFBO-DNA concentrations. In addition, the mRNA and/or activity of enzymes responsible for the bioactivation of AFB1 into AFBO—including CYP1A1, CYP1A2, CYP2A6, and CYP3A4—were significantly induced in liver microsomes after 2-week exposure to AFB1. These alterations induced by AFB1 were prevented by CM supplementation. Conclusively, dietary CM protected chicks from AFB1-induced liver injury, potentially through the synergistic actions of increased antioxidant capacities and inhibition of the pivotal CYP450 isozyme-mediated activation of AFB1 to toxic AFBO.

  14. Regional specificity in deltamethrin induced cytochrome P450 expression in rat brain

    International Nuclear Information System (INIS)

    Yadav, Sanjay; Johri, Ashu; Dhawan, Alok; Seth, Prahlad K.; Parmar, Devendra

    2006-01-01

    Oral administration of deltamethrin (5 mg/kg x 7 or 15 or 21 days) was found to produce a time-dependent increase in the mRNA expression of xenobiotic metabolizing cytochrome P450 1A1 (CYP1A1), 1A2 and CYP2B1, 2B2 isoenzymes in rat brain. RT-PCR studies further showed that increase in the mRNA expression of these CYP isoenzymes observed after 21 days of exposure was region specific. Hippocampus exhibited maximum increase in the mRNA expression of CYP1A1, which was followed by pons-medulla, cerebellum and hypothalamus. The mRNA expression of CYP2B1 also exhibited maximum increase in the hypothalamus and hippocampus followed by almost similar increase in midbrain and cerebellum. In contrast, mRNA expression of CYP1A2 and CYP2B2, the constitutive isoenzymes exhibited relatively higher increase in pons-medulla, cerebellum and frontal cortex. Immunoblotting studies carried out with polyclonal antibody raised against rat liver CYP1A1/1A2 or CYP2B1/2B2 isoenzymes also showed increase in immunoreactivity comigrating with CYP1A1/1A2 or 2B1/2B2 in the microsomal fractions isolated from hippocampus, hypothalamus and cerebellum of rat treated with deltamethrin. Though the exact relationship of the xenobiotic metabolizing CYPs with the physiological function of the brain is yet to be clearly understood, the increase in the mRNA expression of the CYPs in the brain regions that regulate specific brain functions affected by deltamethrin have further indicated that modulation of these CYPs could be associated with the various endogenous functions of the brain

  15. Computational identification of putative cytochrome P450 genes in ...

    African Journals Online (AJOL)

    Chattha

    Economically, legumes represent the second most important family of crop plants after Poacea (grass family), accounting for ... further characterization of P450 genes with both known and unknown functions. MATERIALS AND METHODS ..... Cytochrome P450. In: Somerville CR, Meyerowitz EM (eds) .The Arabidopsis book,.

  16. Identification of bottlenecks for P450 biotransformation processes

    DEFF Research Database (Denmark)

    Andersson, Marie Therese; Törnvall, Ulrika; Tufvesson, Pär

    Cytochrome P450 monooxygenases (P450 or CYP) is a group of heme-containing enzymes hydroxylating non-activated hydrocarbons in a stereospecific manner, something that is hard to achieve via classical chemistry. The importance of these reactions can be stressed by the hydroxylation of steroids, bu...... biotransformation process identifying the limiting parameters and defining relevant targets....

  17. The SMARTCyp cytochrome P450 metabolism prediction server

    DEFF Research Database (Denmark)

    Rydberg, Patrik; Gloriam, David Erik Immanuel; Olsen, Lars

    2010-01-01

    The SMARTCyp server is the first web application for site of metabolism prediction of cytochrome P450-mediated drug metabolism.......The SMARTCyp server is the first web application for site of metabolism prediction of cytochrome P450-mediated drug metabolism....

  18. Differences in hepatic microsomal cytochrome P-450 isoenzyme induction by pyrazole, chronic ethanol, 3-methylcholanthrene, and phenobarbital in high alcohol sensitivity (HAS) and low alcohol sensitivity (LAS) rats.

    Science.gov (United States)

    Lucas, D; Ménez, J F; Berthou, F; Cauvin, J M; Deitrich, R A

    1992-10-01

    High and low alcohol sensitivity (HAS and LAS) rats have been selected for their differences in ethanol-induced sleep time. Liver monooxygenase activities were studied in HAS and LAS rats before and after treatments with known inducers such as chronic ethanol, pyrazole, 3-methylcholanthrene (3-MC) and phenobarbital (PB) to determine whether the selection procedure also selected for differences in the cytochrome P-450 (P-450) inducibility. This previously has been shown with long sleep (LS) and short sleep (SS) mice, which were selected using a similar criterion. 3-MC and PB, in conjunction with chronic ethanol treatment, were used in order to evaluate the interactions of ethanol with these inducers. Prior to treatment, total P-450 content was slightly lower in LAS than in HAS rats. However, both lines displayed the same microsomal monooxygenase activities related to different P-450 isozymes. This was demonstrated by ethoxyresorufin deethylation (EROD) for cytochrome P-450 1A1 (CYP1A1), acetanilide hydroxylation (ACET) for CYP1A2, pentoxyresorufin dealkylation (PROD) for CYP2B, 1-butanol oxidation (BUTAN) and N-nitrosodimethylamine demethylation (NDMA) for CYP2E1. After the different treatments, HAS rats did not differ from LAS rats in their CYP2E1 inducibility. However, pyrazole, PB and 3-MC treatment led to differences in CYP1A and CYP2B monooxygenase activities between the two lines. The enhancement of PROD by pyrazole treatment was less prominent in LAS (1.7-fold of the control value) than in HAS rats (3.8-fold).(ABSTRACT TRUNCATED AT 250 WORDS)

  19. A Panel of Cytochrome P450 BM3 Variants To Produce Drug Metabolites and Diversify Lead Compounds

    Science.gov (United States)

    Sawayama, Andrew M.; Chen, Michael M. Y.; Kulanthaivel, Palaniappan; Kuo, Ming-Shang; Hemmerle, Horst; Arnold, Frances H.

    2011-01-01

    Here we demonstrate that a small panel of variants of cytochrome P450 BM3 from Bacillus megaterium covers the breadth of reactivity of human P450s by producing 12 of 13 mammalian metabolites for two marketed drugs, verapamil and astemizole, and one research compound. The most active enzymes support preparation of individual metabolites for preclinical bioactivity and toxicology evaluations. Underscoring their potential utility in drug lead diversification, engineered P450 BM3 variants also produce novel metabolites by catalyzing reactions at carbon centers beyond those targeted by animal and human P450s. Production of a specific metabolite can be improved by directed evolution of the enzyme catalyst. Some variants are more active on the more hydrophobic parent drug than on its metabolites, which limits production of multiply-hydroxylated species, a preference that appears to depend on the evolutionary history of the P450 variant. PMID:19774562

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

    Science.gov (United States)

    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...

  1. Evaluation of cytochrome P-450 concentration in Saccharomyces cerevisiae strains

    Directory of Open Access Journals (Sweden)

    Míriam Cristina Sakuragui Matuo

    2010-09-01

    Full Text Available Saccharomyces cerevisiae has been widely used in mutagenicity tests due to the presence of a cytochrome P-450 system, capable of metabolizing promutagens to active mutagens. There are a large number of S. cerevisiae strains with varying abilities to produce cytochrome P-450. However, strain selection and ideal cultivation conditions are not well defined. We compared cytochrome P-450 levels in four different S. cerevisiae strains and evaluated the cultivation conditions necessary to obtain the highest levels. The amount of cytochrome P-450 produced by each strain varied, as did the incubation time needed to reach the maximum level. The highest cytochrome P-450 concentrations were found in media containing fermentable sugars. The NCYC 240 strain produced the highest level of cytochrome P-450 when grown in the presence of 20 % (w/v glucose. The addition of ethanol to the media also increased cytochrome P-450 synthesis in this strain. These results indicate cultivation conditions must be specific and well-established for the strain selected in order to assure high cytochrome P-450 levels and reliable mutagenicity results.Linhagens de Saccharomyces cerevisiae tem sido amplamente empregadas em testes de mutagenicidade devido à presença de um sistema citocromo P-450 capaz de metabolizar substâncias pró-mutagênicas à sua forma ativa. Devido à grande variedade de linhagens de S. cerevisiae com diferentes capacidades de produção de citocromo P-450, torna-se necessária a seleção de cepas, bem como a definição das condições ideais de cultivo. Neste trabalho, foram comparados os níveis de citocromo P-450 em quatro diferentes linhagens de S. cerevisiae e avaliadas as condições de cultivo necessárias para obtenção de altas concentrações deste sistema enzimático. O maior nível enzimático foi encontrado na linhagem NCYC 240 em presença de 20 % de glicose (p/v. A adição de etanol ao meio de cultura também produziu um aumento na s

  2. The different metabolism of morusin in various species and its potent inhibition against UDP-glucuronosyltransferase (UGT) and cytochrome p450 (CYP450) enzymes.

    Science.gov (United States)

    Shi, Xianbao; Yang, Shuman; Zhang, Gang; Song, Yonggui; Su, Dan; Liu, Yali; Guo, Feng; Shan, Lina; Cai, Jiqun

    2016-01-01

    1. The aim of this study was to investigate the inhibitory effect of morusin on Glucuronosyltransferase (UGT) isoforms and cytochrome P450 enzymes (CYP450s). We also investigated the metabolism of morusin in human, rat, dog, monkey, and minipig liver microsomes. 2. 100 μM of morusin exhibited strong inhibition on all UGTs and CYP450s. The half inhibition concentration (IC50) values for CYP3A4, CYP1A2, CYP2C9, CYP2E1, UGT1A6, UGT1A7, and UGT1A8 were 2.13, 1.27, 3.18, 9.28, 4.23, 0.98, and 3.00 μM, and the inhibition kinetic parameters (Ki) were 1.34, 1.16, 2.98, 6.23, 4.09, 0.62, and 2.11 μM, respectively. 3. Metabolism of morusin exhibited significant species differences. The quantities of M1 from minipig, monkey, dog, and rat were 7.8, 11.9, 2.0, and 6.3-fold of human levels. The Km values in HLMs, RLMs, MLMs, DLMs, and PLMs were 7.84, 22.77, 14.32, 9.13, and 22.83 μM, and Vmax for these species were 0.09, 1.23, 1.43, 0.15, and 0.75 nmol/min/mg, respectively. CLint (intrinsic clearance) values (Vmax/Km) for morusin obeyed the following order: monkey > rat > minipig > dog > human. CLH (hepatic clearance) values for humans, dogs, and rats were calculated to be 8.28, 17.38, and 35.12 mL/min/kg body weight, respectively. 4. This study provided vital information to understand the inhibitory potential and metabolic behavior of morusin among various species.

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

    Science.gov (United States)

    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

  4. Fast prediction of cytochrome P450 mediated drug metabolism

    DEFF Research Database (Denmark)

    Rydberg, Patrik Åke Anders; Poongavanam, Vasanthanathan; Oostenbrink, Chris

    2009-01-01

    Cytochrome P450 mediated metabolism of drugs is one of the major determinants of their kinetic profile, and prediction of this metabolism is therefore highly relevant during the drug discovery and development process. A new rule-based method, based on results from density functional theory...... calculations, for predicting activation energies for aliphatic and aromatic oxidations by cytochromes P450 is developed and compared with several other methods. Although the applicability of the method is currently limited to a subset of P450 reactions, these reactions describe more than 90...

  5. Artificial Self-Sufficient P450 in Reversed Micelles

    Directory of Open Access Journals (Sweden)

    Teruyuki Nagamune

    2010-04-01

    Full Text Available Cytochrome P450s are heme-containing monooxygenases that require electron transfer proteins for their catalytic activities. They prefer hydrophobic compounds as substrates and it is, therefore, desirable to perform their reactions in non-aqueous media. Reversed micelles can stably encapsulate proteins in nano-scaled water pools in organic solvents. However, in the reversed micellar system, when multiple proteins are involved in a reaction they can be separated into different micelles and it is then difficult to transfer electrons between proteins. We show here that an artificial self-sufficient cytochrome P450, which is an enzymatically crosslinked fusion protein composed of P450 and electron transfer proteins, showed micelle-size dependent catalytic activity in a reversed micellar system. Furthermore, the presence of thermostable alcohol dehydrogenase promoted the P450-catalyzed reaction due to cofactor regeneration.

  6. Triterpene Structural Diversification by Plant Cytochrome P450 Enzymes

    Directory of Open Access Journals (Sweden)

    Sumit Ghosh

    2017-11-01

    Full Text Available Cytochrome P450 monooxygenases (P450s represent the largest enzyme family of the plant metabolism. Plants typically devote about 1% of the protein-coding genes for the P450s to execute primary metabolism and also to perform species-specific specialized functions including metabolism of the triterpenes, isoprene-derived 30-carbon compounds. Triterpenes constitute a large and structurally diverse class of natural products with various industrial and pharmaceutical applications. P450-catalyzed structural modification is crucial for the diversification and functionalization of the triterpene scaffolds. In recent times, a remarkable progress has been made in understanding the function of the P450s in plant triterpene metabolism. So far, ∼80 P450s are assigned biochemical functions related to the plant triterpene metabolism. The members of the subfamilies CYP51G, CYP85A, CYP90B-D, CYP710A, CYP724B, and CYP734A are generally conserved across the plant kingdom to take part in plant primary metabolism related to the biosynthesis of essential sterols and steroid hormones. However, the members of the subfamilies CYP51H, CYP71A,D, CYP72A, CYP81Q, CYP87D, CYP88D,L, CYP93E, CYP705A, CYP708A, and CYP716A,C,E,S,U,Y are required for the metabolism of the specialized triterpenes that might perform species-specific functions including chemical defense toward specialized pathogens. Moreover, a recent advancement in high-throughput sequencing of the transcriptomes and genomes has resulted in identification of a large number of candidate P450s from diverse plant species. Assigning biochemical functions to these P450s will be of interest to extend our knowledge on triterpene metabolism in diverse plant species and also for the sustainable production of valuable phytochemicals.

  7. Quantum Mechanics/Molecular Mechanics Modeling of Drug Metabolism: Mexiletine N-Hydroxylation by Cytochrome P450 1A2.

    Science.gov (United States)

    Lonsdale, Richard; Fort, Rachel M; Rydberg, Patrik; Harvey, Jeremy N; Mulholland, Adrian J

    2016-06-20

    The mechanism of cytochrome P450(CYP)-catalyzed hydroxylation of primary amines is currently unclear and is relevant to drug metabolism; previous small model calculations have suggested two possible mechanisms: direct N-oxidation and H-abstraction/rebound. We have modeled the N-hydroxylation of (R)-mexiletine in CYP1A2 with hybrid quantum mechanics/molecular mechanics (QM/MM) methods, providing a more detailed and realistic model. Multiple reaction barriers have been calculated at the QM(B3LYP-D)/MM(CHARMM27) level for the direct N-oxidation and H-abstraction/rebound mechanisms. Our calculated barriers indicate that the direct N-oxidation mechanism is preferred and proceeds via the doublet spin state of Compound I. Molecular dynamics simulations indicate that the presence of an ordered water molecule in the active site assists in the binding of mexiletine in the active site, but this is not a prerequisite for reaction via either mechanism. Several active site residues play a role in the binding of mexiletine in the active site, including Thr124 and Phe226. This work reveals key details of the N-hydroxylation of mexiletine and further demonstrates that mechanistic studies using QM/MM methods are useful for understanding drug metabolism.

  8. Biodegradation of Cosmetics Products: A Computational Study of Cytochrome P450 Metabolism of Phthalates

    Directory of Open Access Journals (Sweden)

    Fabián G. Cantú Reinhard

    2017-11-01

    Full Text Available Cytochrome P450s are a broad class of enzymes in the human body with important functions for human health, which include the metabolism and detoxification of compounds in the liver. Thus, in their catalytic cycle, the P450s form a high-valent iron(IV-oxo heme cation radical as the active species (called Compound I that reacts with substrates through oxygen atom transfer. This work discusses the possible degradation mechanisms of phthalates by cytochrome P450s in the liver, through computational modelling, using 2-ethylhexyl-phthalate as a model substrate. Phthalates are a type of compound commonly found in the environment from cosmetics usage, but their biodegradation in the liver may lead to toxic metabolites. Experimental studies revealed a multitude of products and varying product distributions among P450 isozymes. To understand the regio- and chemoselectivity of phthalate activation by P450 isozymes, we focus here on the mechanisms of phthalate activation by Compound I leading to O-dealkylation, aliphatic hydroxylation and aromatic hydroxylation processes. We set up model complexes of Compound I with the substrate and investigated the reaction mechanisms for products using the density functional theory on models and did a molecular mechanics study on enzymatic structures. The work shows that several reaction barriers in the gas-phase are close in energy, leading to a mixture of products. However, when we tried to dock the substrate into a P450 isozyme, some of the channels were inaccessible due to unfavorable substrate positions. Product distributions are discussed under various reaction conditions and rationalized with valence bond and thermodynamic models.

  9. Regulation of the cytochrome P450 2A genes

    International Nuclear Information System (INIS)

    Su Ting; Ding Xinxin

    2004-01-01

    Cytochrome P450 monooxygenases of the CYP2A subfamily play important roles in xenobiotic disposition in the liver and in metabolic activation in extrahepatic tissues. Many of the CYP2A transcripts and enzymes are inducible by xenobiotic compounds, and the expression of at least some of the CYP2A genes is influenced by physiological status, such as circadian rhythm, and pathological conditions, such as inflammation, microbial infection, and tumorigenesis. Variability in the expression of the CYP2A genes, which differs by species, animal strain, gender, and organ, may alter the risks of chemical toxicity for numerous compounds that are CYP2A substrates. The mechanistic bases of these variabilities are generally not well understood. However, recent studies have yielded interesting findings in several areas, such as the role of nuclear factor 1 in the tissue-selective expression of CYP2A genes in the olfactory mucosa (OM); the roles of constitutive androstane receptor, pregnane X receptor (PXR), and possibly, peroxisome proliferator-activated receptors in transcriptional regulation of the Cyp2a5 gene; and the involvement of heterogeneous nuclear ribonucleoprotein A1 in pyrazole-induced stabilization of CYP2A5 mRNA. The aims of this minireview are to summarize current knowledge of the regulation of the CYP2A genes in rodents and humans, and to stimulate further mechanistic studies that will ultimately improve our ability to determine, and to understand, these variabilities in humans

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

    Science.gov (United States)

    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.

  11. Cytochromes P450: History, Classes, Catalytic Mechanism, and Industrial Application.

    Science.gov (United States)

    Cook, D J; Finnigan, J D; Cook, K; Black, G W; Charnock, S J

    Cytochromes P450, a family of heme-containing monooxygenases that catalyze a diverse range of oxidative reactions, are so-called due to their maximum absorbance at 450nm, ie, "Pigment-450nm," when bound to carbon monoxide. They have appeal both academically and commercially due to their high degree of regio- and stereoselectivity, for example, in the area of active pharmaceutical ingredient synthesis. Despite this potential, they often exhibit poor stability, low turnover numbers and typically require electron transport protein(s) for catalysis. P450 systems exist in a variety of functional domain architectures, organized into 10 classes. P450s are also divided into families, each of which is based solely on amino acid sequence homology. Their catalytic mechanism employs a very complex, multistep catalytic cycle involving a range of transient intermediates. Mutagenesis is a powerful tool for the development of improved biocatalysts and has been used extensively with the archetypal Class VIII P450, BM3, from Bacillus megaterium, but with the increasing scale of genomic sequencing, a huge resource is now available for the discovery of novel P450s. © 2016 Elsevier Inc. All rights reserved.

  12. Repellents inhibit P450 enzymes in Stegomyia (Aedes aegypti.

    Directory of Open Access Journals (Sweden)

    Gloria Isabel Jaramillo Ramirez

    Full Text Available The primary defence against mosquitoes and other disease vectors is often the application of a repellent. Despite their common use, the mechanism(s underlying the activity of repellents is not fully understood, with even the mode of action of DEET having been reported to be via different mechanisms; e.g. interference with olfactory receptor neurones or actively detected by olfactory receptor neurones on the antennae or maxillary palps. In this study, we discuss a novel mechanism for repellence, one of P450 inhibition. Thirteen essential oil extracts from Colombian plants were assayed for potency as P450 inhibitors, using a kinetic fluorometric assay, and for repellency using a modified World Health Organisation Pesticide Evaluations Scheme (WHOPES arm-in cage assay with Stegomyia (Aedes aegypti mosquitoes. Bootstrap analysis on the inhibition analysis revealed a significant correlation between P450-inhibition and repellent activity of the oils.

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

    OpenAIRE

    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...

  14. Evaluación de la actividad de la enzima citocromo P-450 1A2 en una población colombiana

    Directory of Open Access Journals (Sweden)

    Fanny Cuesta González

    2000-02-01

    Full Text Available

    Introducción: La enzima citocromo P450 1A2 (CYP1A2, presente en tejidos hepáticos humanos, participa en el metabolismo de fármacos tales como Clozapina, Imipramina, Teofilina y Cafeína. Estudios realizados en diferentes poblaciones han mostrado una distribución bimodal y a veces trimodal en la actividad de esta enzima. Este comportamiento ha sido relacionado con variaciones tanto del gen codificante como de las regiones reguladoras, aunque hasta el momento los resultados no son concluyentes. Además del metabolismo de fármacos, la CYP1A2 interviene en la activación de sustancias procarcinogénicas tales como N-nitrosaminas, aminas heterocíclicas y aromáticas; es conocida la correlación entre una actividad incrementada de esta enzima (metabolizadores rápidos con una mayor exposición al riesgo de cáncer.

    Debido a la baja incidencia de efectos secundarios y la facilidad en la medición de los metabolitos en orina, la Cafeína es utilizada como fármaco prueba para la determinación de la actividad enzimática CYP1A2, calculando la relación entre las concentraciones molares de sus metabolitos (Actividad CYP1A2 = AFMU+MU+MX/17DMU.

    En el presente estudio se evaluará la actividad de la enzima CYP1A2 en una muestra de individuos sanos, con el fin de describir su comportamiento y compararlo con el encontrado en otras poblaciones, las cuales tienen una influencia genética y ambiental diferente a la de la población colombiana.

    Metodología: Una vez recolectadas las muestras de orina, se separarán de ellas los

  15. Characterization CYP1A2, CYP2C9, CYP2C19 and CYP2D6 polymorphisms using HRMA in Psychiatry patients with schizophrenia and bipolar disease for personalized medicine.

    Science.gov (United States)

    Yenilmez, Ebru Dundar; Tamam, Lut; Karaytug, Onur; Tuli, Abdullah

    2018-06-19

    The interindividual genetic variations in drug metabolizing enzymes effects the impact and toxicity in plenty of drugs. The CYP1A2, CYP2C9, CYP2C19 and CYP2D6 gene polymorphisms characterized using high resolution melting analysis (HRMA) in follow-up patients in psychiatry clinic as a preliminary preparation for personalized medicine. Genotyping of CYP1A2*1F, CYP2C9 *2, *3, CYP2C19 *2, *3 and *17 and CYP2D6 *3, *4 was conducted in 101 patients using HRMA. Genotype and allele frequencies of the CYP variants were found to be in equilibrium with the Hardy-Weinberg equation. The frequency of the CYP1A2*1F allele in schizophrenia and bipolar disease was 0.694 and 0.255, respectively. The CYP2C9 allele frequencies were 0.087 (CYP2C9*2), and 0.549 (CYP2C9*3) for bipolar; 0.278 (CYP2C9*2) and 0.648 (CYP2C9*3) in schizophrenias. The CYP2C19*2 and *17 allele frequencies was 0.111 and 0.185 in schizophrenia and variant *2 was 0.117 and variant *17 was 0.255 in bipolar group. The frequency of the CYP2D6*3 allele was 0.027 in schizophrenias. The frequencies for the CYP2D6*4 variant was 0.092 and 0.096 in schizophrenia and bipolar groups, respectively. The knowledge in pharmacogenomics and also the developments in molecular genetics are growing rapidly. In the future this can be expected to provide new methodologies in the prediction of the activity in drug metabolizing enzymes. The HRMA is a rapid and useful technique to identify the genotypes for drug dosage adjustment before therapy in psychiatry patients. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  16. Protein and DNA technologies for functional expression of membrane-associated cytochromes P450 in bacterial cell factories

    DEFF Research Database (Denmark)

    Vazquez Albacete, Dario

    450 engineering guidelines and serves as platform to improve performance of microbial cells, thereby boosting recombinant production of complex plant P450-derived biochemicals. The knowledge generated, could guide future reconstruction of functional plant metabolic pathways leading to high valuable...... potential as medicines, fuels or food for humans. Plants conquered different environments thereby developing adaptation strategies based on the biosynthesis of a myriad of compounds. Unfortunately they are present in small amounts in plants and are too complex and to produce by organic chemical synthesis....... In most of biosynthetic pathways leading to these chemicals the cytochrome P450 enzyme family (P450s) is responsible for their final functionalization. However, the membrane-bound nature of P450s, makes their expression in microbial hosts a challenge. In order to meet the global demand for these natural...

  17. Pharmacokinetic Effects of Isavuconazole Coadministration With the Cytochrome P450 Enzyme Substrates Bupropion, Repaglinide, Caffeine, Dextromethorphan, and Methadone in Healthy Subjects.

    Science.gov (United States)

    Yamazaki, Takao; Desai, Amit; Goldwater, Ronald; Han, David; Howieson, Corrie; Akhtar, Shahzad; Kowalski, Donna; Lademacher, Christopher; Pearlman, Helene; Rammelsberg, Diane; Townsend, Robert

    2017-01-01

    This report describes phase 1 clinical trials performed to assess interactions of oral isavuconazole at the clinically targeted dose (200 mg, administered as isavuconazonium sulfate 372 mg, 3 times a day for 2 days; 200 mg once daily [QD] thereafter) with single oral doses of the cytochrome P450 (CYP) substrates: bupropion hydrochloride (CYP2B6; 100 mg; n = 24), repaglinide (CYP2C8/CYP3A4; 0.5 mg; n = 24), caffeine (CYP1A2; 200 mg; n = 24), dextromethorphan hydrobromide (CYP2D6/CYP3A4; 30 mg; n = 24), and methadone (CYP2B6/CYP2C19/CYP3A4; 10 mg; n = 23). Compared with each drug alone, coadministration with isavuconazole changed the area under the concentration-time curves (AUC ∞ ) and maximum concentrations (C max ) as follows: bupropion, AUC ∞ reduced 42%, C max reduced 31%; repaglinide, AUC ∞ reduced 8%, C max reduced 14%; caffeine, AUC ∞ increased 4%, C max reduced 1%; dextromethorphan, AUC ∞ increased 18%, C max increased 17%; R-methadone, AUC ∞ reduced 10%, C max increased 3%; S-methadone, AUC ∞ reduced 35%, C max increased 1%. In all studies, there were no deaths, 1 serious adverse event (dextromethorphan study; perioral numbness, numbness of right arm and leg), and adverse events leading to study discontinuation were rare. Thus, isavuconazole is a mild inducer of CYP2B6 but does not appear to affect CYP1A2-, CYP2C8-, or CYP2D6-mediated metabolism. © 2016 The Authors. Clinical Pharmacology in Drug Development Published by Wiley Periodicals, Inc. on behalf of The American College of Clinical Pharmacology.

  18. Colonization by non-pathogenic bacteria alters mRNA expression of cytochromes P450 in originally germ-free mice.

    Science.gov (United States)

    Jourová, L; Anzenbacher, P; Lišková, B; Matušková, Z; Hermanová, P; Hudcovic, T; Kozáková, H; Hrnčířová, L; Anzenbacherová, E

    2017-11-01

    Gut microbiota provides a wide range of beneficial function for the host and has an immense effect on the host's health state. It has also been shown that gut microbiome is often involved in the biotransformation of xenobiotics; however, the molecular mechanisms of the interaction between the gut bacteria and the metabolism of drugs by the host are still unclear. To investigate the effect of microbial colonization on messenger RNA (mRNA) expression of liver cytochromes P450 (CYPs), the main drug-metabolizing enzymes, we used germ-free (GF) mice, lacking the intestinal flora and mice monocolonized by non-pathogenic bacteria Lactobacillus plantarum NIZO2877 or probiotic bacteria Escherichia coli Nissle 1917 compared to specific pathogen-free (SPF) mice. Our results show that the mRNA expression of Cyp1a2 and Cyp2e1 was significantly increased, while the expression of Cyp3a11 mRNA was decreased under GF conditions compared to the SPF mice. The both bacteria L. plantarum NIZO2877 and E. coli Nissle 1917 given to the GF mice decreased the level of Cyp1a2 mRNA and normalized it to the control level. On the other hand, the colonization by these bacteria had no effect on the expression of Cyp3a11 mRNA in the liver of the GF mice (which remained decreased). Surprisingly, monocolonization with chosen bacterial strains has shown a different effect on the expression of Cyp2e1 mRNA in GF mice. Increased level of Cyp2e1 expression observed in the GF mice was found also in mice colonized by L. plantarum NIZO2877 ; however, the colonization with probiotic E. coli Nissle 1917 caused a decrease in Cyp2e1 expression and partially restored the SPF mice conditions.

  19. Rational redesign of the biodegradative enzyme cytochrome P450 cam:

    International Nuclear Information System (INIS)

    Ornstein, R.; Paulsen, M.; Bass, M.; Arnold, G.

    1991-03-01

    Cytochromes P450, a superfamily of monooxygenase enzymes present in all kingdoms of living organisms, are very versatile with respect to substrate range and catalytic functionality. Many recalcitrant halogenated hydrocarbons, on DOE sites and throughout the nation, result in serious environmental impact. Cytochromes P450 have been shown to be catalytically capable of, at least partial, dehalogenation of some such compounds. Clearly, however, their active site stereochemistry and related functional components are not well suited for this role because the rates of dehalogenation are generally rather modest. The evolution of modified active site and access channel structures may proceed very slowly if multiple genetic changes are simultaneously required for enzyme adaptation. Since each mutational event is by itself a rare event, a basic premise of our research is that designing multiple changes into an enzyme may be more timely than waiting for them to occur biologically either via natural selection or under laboratory-controlled conditions. Starting with available high-resolution x-ray crystal structures, molecular modeling and molecular dynamics simulations have been used to probe the basic structure/function principles and conformational fluctuations of the biodegradative enzyme, cytochrome P450cam (camphor hydroxylase from Pseudomonas putida) and active site mutants, to provide the fundamental understanding necessary for rational engineering of the enzyme for modified substrate specificity. In the present paper, we review our progress to data, in the area of molecular dynamics simulations and active site redesign of P450cam. 36 refs., 2 figs

  20. Computational identification of putative cytochrome P450 genes in ...

    African Journals Online (AJOL)

    In this work, a computational study of expressed sequence tags (ESTs) of soybean was performed by data mining methods and bio-informatics tools and as a result 78 putative P450 genes were identified, including 57 new ones. These genes were classified into five clans and 20 families by sequence similarities and among ...

  1. Assembly of dynamic P450-mediated metabolons - order versus chaos

    DEFF Research Database (Denmark)

    Bassard, Jean-Étienne André; Møller, Birger Lindberg; Laursen, Tomas

    2017-01-01

    PURPOSE OF REVIEW: We provide an overview of the current knowledge on cytochrome P450-mediated metabolism organized as metabolons and factors that facilitate their stabilization. Essential parameters will be discussed including those that are commonly disregarded using the dhurrin metabolon from ...

  2. Multivariate Modeling of Cytochrome P450 Enzymes for 4 ...

    African Journals Online (AJOL)

    Conclusion: Apart from insights into important molecular properties for CYP inhibition, the findings may also guide further investigations of novel drug candidates that are unlikely to inhibit multiple CYP sub-types. Keywords: Antimalarial, Chloroquine, Cytochrome P450, Genetic algorithm-based multiple linear regression, ...

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

    Science.gov (United States)

    Coelho, Alexandra; Fraichard, Stephane; Le Goff, Gaëlle; Faure, Philippe; Artur, Yves; Ferveur, Jean-François; Heydel, Jean-Marie

    2015-01-01

    Caffeine (1, 3, 7-trimethylxanthine), an alkaloid produced by plants, has antioxidant and insecticide properties that can affect metabolism and cognition. In vertebrates, the metabolites derived from caffeine have been identified, and their functions have been characterized. However, the metabolites of caffeine in insects remain unknown. Thus, using radiolabelled caffeine, we have identified some of the primary caffeine metabolites produced in the body of Drosophila melanogaster males, including theobromine, paraxanthine and theophylline. In contrast to mammals, theobromine was the predominant metabolite (paraxanthine in humans; theophylline in monkeys; 1, 3, 7-trimethyluric acid in rodents). A transcriptomic screen of Drosophila flies exposed to caffeine revealed the coordinated variation of a large set of genes that encode xenobiotic-metabolizing proteins, including several cytochromes P450s (CYPs) that were highly overexpressed. Flies treated with metyrapone—an inhibitor of CYP enzymes—showed dramatically decreased caffeine metabolism, indicating that CYPs are involved in this process. Using interference RNA genetic silencing, we measured the metabolic and transcriptomic effect of three candidate CYPs. Silencing of CYP6d5 completely abolished theobromine synthesis, whereas CYP6a8 and CYP12d1 silencing induced different consequences on metabolism and gene expression. Therefore, we characterized several metabolic products and some enzymes potentially involved in the degradation of caffeine. In conclusion, this pioneer approach to caffeine metabolism in insects opens novel perspectives for the investigation of the physiological effects of caffeine metabolites. It also indicates that caffeine could be used as a biomarker to evaluate CYP phenotypes in Drosophila and other insects. PMID:25671424

  4. Cytochrome P450-dependent metabolism of caffeine in Drosophila melanogaster.

    Directory of Open Access Journals (Sweden)

    Alexandra Coelho

    Full Text Available Caffeine (1, 3, 7-trimethylxanthine, an alkaloid produced by plants, has antioxidant and insecticide properties that can affect metabolism and cognition. In vertebrates, the metabolites derived from caffeine have been identified, and their functions have been characterized. However, the metabolites of caffeine in insects remain unknown. Thus, using radiolabelled caffeine, we have identified some of the primary caffeine metabolites produced in the body of Drosophila melanogaster males, including theobromine, paraxanthine and theophylline. In contrast to mammals, theobromine was the predominant metabolite (paraxanthine in humans; theophylline in monkeys; 1, 3, 7-trimethyluric acid in rodents. A transcriptomic screen of Drosophila flies exposed to caffeine revealed the coordinated variation of a large set of genes that encode xenobiotic-metabolizing proteins, including several cytochromes P450s (CYPs that were highly overexpressed. Flies treated with metyrapone--an inhibitor of CYP enzymes--showed dramatically decreased caffeine metabolism, indicating that CYPs are involved in this process. Using interference RNA genetic silencing, we measured the metabolic and transcriptomic effect of three candidate CYPs. Silencing of CYP6d5 completely abolished theobromine synthesis, whereas CYP6a8 and CYP12d1 silencing induced different consequences on metabolism and gene expression. Therefore, we characterized several metabolic products and some enzymes potentially involved in the degradation of caffeine. In conclusion, this pioneer approach to caffeine metabolism in insects opens novel perspectives for the investigation of the physiological effects of caffeine metabolites. It also indicates that caffeine could be used as a biomarker to evaluate CYP phenotypes in Drosophila and other insects.

  5. Human Metabolism and Interactions of Deployment-Related Chemicals

    Science.gov (United States)

    2008-08-01

    stimulated by CPO (Fig. 6). Coincidently, α- naphthoflavone inhibited CYP1A2 metabolism of flavonoids /23/ and stimulated CYP3A4 metabolism of...by flavonoids of benzo[a]pyrene hydroxylation by cytochrome P-450 isozymes from rabbit liver microsomes, J. Biol. Chem. 1981; 256: 10897-10901. 17...P450-mediated metabolism of dietary flavonoids , Food Chem. Toxicol. 2002; 40: 609-616. 24. Cameron MD, Wen B, Allen KE, Roberts AG, Schuman JT

  6. Vinclozolin modulates hepatic cytochrome P450 isoforms during pregnancy.

    Science.gov (United States)

    de Oca, Félix Genoveva García-Montes; López-González, Ma de Lourdes; Escobar-Wilches, Derly Constanza; Chavira-Ramírez, Roberto; Sierra-Santoyo, Adolfo

    2015-06-01

    Vinclozolin (V) is classified as a potent endocrine disruptor. The aim of the present study was to determine the effects of V on rat liver CYP regulation and on serum levels of testosterone and estradiol during pregnancy. Pregnancy decreased the liver total CYP content by 65%, enzyme activities of MROD, PROD, and PNPH, and testosterone hydroxylation activities, as well as the protein content of CYP2A and 3A. V exposure remarkably induced the protein content and enzyme activities of CYP1A, 2A, 2B and 3A subfamilies. Testosterone and estradiol were affected in an opposite manner, provoking a 3.5-fold increase in the estradiol/testosterone ratio. These results suggest that V could regulate the hepatic CYP expression through interaction with receptors and coactivators involved in its expression and may play an important role in hormonal balance during pregnancy. In addition, the results may also contribute to understanding the toxicity of V by in utero exposure. Copyright © 2015 Elsevier Inc. All rights reserved.

  7. Cytochrome P450IID6 recognized by LKM1 antibody is not exposed on the surface of hepatocytes.

    Science.gov (United States)

    Yamamoto, A M; Mura, C; De Lemos-Chiarandini, C; Krishnamoorthy, R; Alvarez, F

    1993-06-01

    LKM1 autoantibody, directed against P450IID6, is accepted as a marker of a particular type of autoimmune hepatitis, but its role in the pathogenesis of the disease is controversial. Localization of P450IID6 on the cell surface of rat hepatocytes was previously reported, suggesting that membrane-bound P450IID6 could be the target of LKM1 antibodies, thus allowing immune lysis of hepatocytes. The objective of the present study was to determine, using various methods, the cell localization of P450IID6 in human and rat hepatocytes. Incubation of rat and human hepatocytes with LKM1-positive serum showed slight, if any, cell membrane staining using immunofluorescence, immunoperoxidase and immunoelectron microscopic studies. No staining of the plasma membrane of human hepatocytes was observed when incubations were carried out with immunoaffinity-purified antibody directed against peptide 254-271, the main epitope of P450IID6 recognized by all LKM1 sera tested. Chinese hamster ovary cells, transfected with the complete P450IID6 cDNA and incubated with the supernatant from a B cell lymphoblastoid cell line prepared with the lymphocytes of a LKM1-positive patient, did not show any staining of the cell surface by immunofluorescence. Incubation of rat microsomal fraction vesicles with LKM1-positive serum, followed by protein A-gold immunoelectron microscopy, displayed a staining of almost all vesicles, confirming that P450IID6 is present on the cytoplasmic side of the microsomal membrane, which makes it unable to be expressed on the cell surface even if it were transported from the endoplasmic reticulum (ER). Sulpho NHS Biotin labelling of rat hepatocyte cell membranes did not show the presence of a 50-kD molecule that could have reacted with LKM1 antibody. DNA sequencing of exon 1 of the CYP2D6 gene of a patient positive for LKM1 antibody did not show any difference from that of the normal published sequence of the gene. This does not favour an alteration of the NH2 terminal

  8. Cloning of cDNA encoding steroid 11β-hydroxylase (P450c11)

    International Nuclear Information System (INIS)

    Chua, S.C.; Szabo, P.; Vitek, A.; Grzeschik, K.H.; John, M.; White, P.C.

    1987-01-01

    The authors have isolated bovine and human adrenal cDNA clones encoding the adrenal cytochrome P-450 specific for 11β-hydroxylation (P450c11). A bovine adrenal cDNA library constructed in the bacteriophage λ vector gt10 was probed with a previously isolated cDNA clone corresponding to part of the 3' untranslated region of the 4.2-kilobase (kb) mRNA encoding P450c11. Several clones with 3.2-kb cDNA inserts were isolated. Sequence analysis showed that they overlapped the original probe by 300 base pairs (bp). Combined cDNA and RNA sequence data demonstrated a continuous open reading frame of 1509 bases. P450c11 is predicted to contain 479 amino acid residues in the mature protein in addition to a 24-residue amino-terminal mitochondrial signal sequence. A bovine clone was used to isolate a homologous clone with a 3.5-kb insert from a human adrenal cDNA library. A region of 1100 bp was 81% homologous to 769 bp of the coding sequence of the bovine cDNA except for a 400-bp segment presumed to be an unprocessed intron. Hybridization of the human cDNA to DNA from a panel of human-rodent somatic cell hybrid lines and in situ hybridization to metaphase spreads of human chromosomes localized the gene to the middle of the long arm of chromosome 8. These data should be useful in developing reagents for heterozygote detection and prenatal diagnosis of 11β-hydroxylase deficiency, the second most frequent cause of congenital adrenal hyperplasia

  9. Identifying Cytochrome P450 Functional Networks and Their Allosteric Regulatory Elements

    Science.gov (United States)

    2013-12-03

    based on physiochemical features not captured by residue co-evolution. In all the networks we characterized, it was evident that some residues were...corresponding iron and sulphur related parameters, were obtained from Bathelt et al. [46]. These parameters are based on QM/MM calculations and have been...2007) Adaptations for the oxidation of polycyclic aromatic hydrocarbons exhibited by the structure of human P450 1A2. J Biol Chem 282: 14348-14355. doi

  10. In vivo cytochrome P450 activity alterations in diabetic nonalcoholic steatohepatitis mice

    OpenAIRE

    Li, Hui; Clarke, John D.; Dzierlenga, Anika L.; Bear, John; Goedken, Michael J.; Cherrington, Nathan J.

    2016-01-01

    Nonalcoholic steatohepatitis (NASH) has been identified as a source of significant interindividual variation in drug metabolism. A previous ex vivo study demonstrated significant changes in hepatic Cytochrome P450 (CYP) activity in human NASH. This study evaluated the in vivo activities of multiple CYP isoforms simultaneously in prominent diabetic NASH mouse models. The pharmacokinetics of CYP selective substrates: caffeine, losartan, and omeprazole changed significantly in a diabetic NASH mo...

  11. Role of cytochrome P450 in drug interactions

    Directory of Open Access Journals (Sweden)

    Bibi Zakia

    2008-10-01

    Full Text Available Abstract Drug-drug interactions have become an important issue in health care. It is now realized that many drug-drug interactions can be explained by alterations in the metabolic enzymes that are present in the liver and other extra-hepatic tissues. Many of the major pharmacokinetic interactions between drugs are due to hepatic cytochrome P450 (P450 or CYP enzymes being affected by previous administration of other drugs. After coadministration, some drugs act as potent enzyme inducers, whereas others are inhibitors. However, reports of enzyme inhibition are very much more common. Understanding these mechanisms of enzyme inhibition or induction is extremely important in order to give appropriate multiple-drug therapies. In future, it may help to identify individuals at greatest risk of drug interactions and adverse events.

  12. Expanding P450 catalytic reaction space through evolution and engineering

    Science.gov (United States)

    McIntosh, John A.; Farwell, Christopher C.; Arnold, Frances H.

    2014-01-01

    Advances in protein and metabolic engineering have led to wider use of enzymes to synthesize important molecules. However, many desirable transformations are not catalyzed by any known enzyme, driving interest in understanding how new enzymes can be created. The cytochrome P450 enzyme family, whose members participate in xenobiotic metabolism and natural products biosynthesis, catalyzes an impressive range of difficult chemical reactions that continues to grow as new enzymes are characterized. Recent work has revealed that P450-derived enzymes can also catalyze useful reactions previously accessible only to synthetic chemistry. The evolution and engineering of these enzymes provides an excellent case study for how to genetically encode new chemistry and expand biology’s reaction space. PMID:24658056

  13. Differentially regulated NADPH: cytochrome p450 oxidoreductases in parsely

    International Nuclear Information System (INIS)

    Koopmann, E.; Hahlbrock, K.

    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

  14. Menadione Suppresses Benzo(αpyrene-Induced Activation of Cytochromes P450 1A: Insights into a Possible Molecular Mechanism.

    Directory of Open Access Journals (Sweden)

    Yulia A Sidorova

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

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

    Science.gov (United States)

    Sidorova, Yulia A; Perepechaeva, Maria L; Pivovarova, Elena N; Markel, Arkady L; Lyakhovich, Vyacheslav V; Grishanova, Alevtina Y

    2016-01-01

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

  16. Role of brain cytochrome P450 mono-oxygenases in bilirubin oxidation-specific induction and activity.

    Science.gov (United States)

    Gambaro, Sabrina E; Robert, Maria C; Tiribelli, Claudio; Gazzin, Silvia

    2016-02-01

    In the Crigler-Najjar type I syndrome, the genetic absence of efficient hepatic glucuronidation of unconjugated bilirubin (UCB) by the uridine 5'-diphospho-glucuronosyltransferase1A1 (UGT1A1) enzyme produces the rise of UCB level in blood. Its entry to central nervous system could generate toxicity and neurological damage, and even death. In the past years, a compensatory mechanism to liver glucuronidation has been indicated in the hepatic cytochromes P450 enzymes (Cyps) which are able to oxidize bilirubin. Cyps are expressed also in the central nervous system, the target of bilirubin toxicity, thus making them theoretically important to confer a protective activity toward bilirubin accumulation and neurotoxicity. We therefore investigated the functional induction (mRNA, EROD/MROD) and the ability to oxidize bilirubin of Cyp1A1, 1A2, and 2A3 in primary astrocytes cultures obtained from two rat brain region (cortex: Cx and cerebellum: Cll). We observed that Cyp1A1 was the Cyp isoform more easily induced by beta-naphtoflavone (βNF) in both Cx and Cll astrocytes, but oxidized bilirubin only after uncoupling by 3, 4,3',4'-tetrachlorobiphenyl (TCB). On the contrary, Cyp1A2 was the most active Cyp in bilirubin clearance without uncoupling, but its induction was confined only in Cx cells. Brain Cyp2A3 was not inducible. In conclusion, the exposure of astrocytes to βNF plus TCB significantly enhanced Cyp1A1 mediating bilirubin clearance, improving cell viability in both regions. These results may be a relevant groundwork for the manipulation of brain Cyps as a therapeutic approach in reducing bilirubin-induced neurological damage.

  17. Modulation of expression and activity of cytochrome P450s and alteration of praziquantel kinetics during murine schistosomiasis

    Directory of Open Access Journals (Sweden)

    Mara A Gotardo

    2011-03-01

    Full Text Available In this study, we investigated the expression and activity of liver cytochrome P450s (CYPs and praziquantel (PZQ kinetics in mice infected with Schistosoma mansoni. Swiss Webster (SW mice of both genders were infected (100 cercariae on postnatal day 10 and killed on post-infection days (PIDs 30 or 55. Non-infected mice of the same age and sex served as controls. Regardless of mouse sex, infection depressed the activities of CYP1A [ethoxy/methoxy-resorufin-O-dealkylases (EROD/MROD], 2B9/10 [pentoxy/benzyloxy-resorufin-O-dealkylases (PROD, BROD], 2E1 [p-nitrophenol-hydroxylase (PNPH] and 3A11 [erythromycin N-demethylase (END] on PID 55 but not on PID 30. On PID 55, infection decreased liver CYP mRNA levels (real-time reverse transcription-polymerase chain reaction. On PID 30, whereas mRNA levels remained unaltered in males, they were depressed in females. Plasma PZQ (200 and 400 mg/kg body weight intraperitoneally levels were measured (high-performance liquid chromatography at different post-treatment intervals. In males and females, infection delayed the PZQ clearance on PID 55, but not on PID 30. Therefore, it can be concluded that schistosomiasis down-modulated CYP expression and activity and delayed PZQ clearance on PID 55, when a great number of parasite eggs were lodged in the liver. On PID 30, when egg-laying was initiated by the worms, no change of CYP expression and activity was found, except for a depression of CYP1A2 and 3A11 mRNAs in female mice.

  18. Influence of polyhalogenated aromatic hydrocarbons on the induction, activity, and stabilization of cytochrome P450

    International Nuclear Information System (INIS)

    Voorman, R.

    1987-01-01

    In the course of experiments evaluating the metabolism of polybrominated biphenyls by cytochrome P450 isozymes induced by 3,4,5,3',4',5'-hexabromobiphenyl (HBB), it was discovered that the inducer remained closely associated with cytochrome P450d. Subsequent purification of cytochromes from HBB treated rates revealed a 0.5:1 association of HBB to cytochrome P450d but virtually none with cytochrome P450c or cytochrome b5. Immunochemical quantitation of cytochrome P450d in the same microsomes yielded a ratio of P450d:HBB that approached unity. Measurement of cytochrome P450d estradiol 2-hydroxylase indicated non-competitive or mixed type inhibition caused by HBB at a concentration of 10-1000 nM. Inhibition was specific to cytochrome P450d since estradiol 2-hydroxylase catalyzed by cytochrome P450h was unaffected by HBB. The ability of HCB and isosafrole to stabilize cytochrome P450d, and thus indirectly influence regulation of the enzyme, was evaluated by treating rats with a dose of TCDD sufficient to produce maximum induction of cytochromes P450c and P450d via the Ah receptor, yet insufficient to bind to the enzyme. Subsequent treatment of these animals with HCB or isosafrole and a radiolabeled amino acid, revealed a significant increase in cytochrome P450d specific content relative to cytochrome P450c and significant retention of the radiolabel in P450d relative to rats treated only with TCDD

  19. P450XXI (steroid 21-hydroxylase) gene deletions are not found in family studies of congenital adrenal hyperplasia

    International Nuclear Information System (INIS)

    Matteson, K.J.; Phillips, J.A. III; Miller, W.L.; Chung, B.C.; Orlando, P.J.; Frisch, H.; Ferrandez, A.; Burr, I.M.

    1987-01-01

    Congenital adrenal hyperplasia (CAH) is a common genetic disorder due to defective 21-hydroxylation of steroid hormones. The human P450XXIA2 gene encodes cytochrome P450c21 [steroid 21-monooxygenase (steroid 21-hydroxylase)], which mediates 21-hydroxylation. The P450XXIA2 gene may be distinguished from the duplicated P450XXIA1 pseudogene by cleavage with the restriction endonuclease Taq I, with the XXIA2 gene characterized by a 3.7-kilobase (kb) fragment and the XXIA1 pseudogene characterized by a 3.2-kb fragment. Restriction endonuclease mapping by several laboratories has suggested that deletion of the P450XXIA2 gene occurs in about 25% of patients with CAH, as their genomic DNA lacks detectable 3.7-kb Taq I fragments. The authors have cloned human P450c21 cDNA and used it to study genomic DNA prepared from 51 persons in 10 families, each of which includes 2 or more persons with CAH. After Taq I digestion, apparent deletions are seen in 7 of the 20 alleles of the probands; using EcoRI, apparent deletions are seen in 9 of the 20 alleles. However, the apparently deleted alleles seen with Taq I do not coincide with those seen with EcoRI. Furthermore, studies with Bgl II, EcoRI, Kpn I, and Xba I yield normal patterns with at least two enzymes in all cases. Since all probands yielded normal patterns with at least two of the five enzymes used, they conclude that the P450XXIA2 gene deletions widely reported in CAH patients probably represent gene conversions, unequal crossovers,or polymorphisms rather than simple gene deletions

  20. Food Polyphenol Apigenin Inhibits the Cytochrome P450 Monoxygenase Branch of the Arachidonic Acid Cascade.

    Science.gov (United States)

    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.

  1. A web-based resource for the Arabidopsis P450, cytochromes b5, NADPH-cytochrome P450 reductases, and family 1 glycosyltransferases (http://www.P450.kvl.dk).

    Science.gov (United States)

    Paquette, Suzanne M; Jensen, Kenneth; Bak, Søren

    2009-12-01

    Gene and genome duplication is a key driving force in evolution of plant diversity. This has resulted in a number of large multi-gene families. Two of the largest multi-gene families in plants are the cytochromes P450 (P450s) and family 1 glycosyltransferases (UGTs). These two families are key players in evolution, especially of plant secondary metabolism, and in adaption to abiotic and biotic stress. In the model plant Arabidopsis thaliana there are 246 and 112 cytochromes P450 and UGTs, respectively. The Arabidopsis P450, cytochromes b(5), NADPH-cytochrome P450 reductases, and family 1 glycosyltransferases website (http://www.P450.kvl.dk) is a sequence repository of manually curated sequences, multiple sequence alignments, phylogenetic trees, sequence motif logos, 3D structures, intron-exon maps, and customized BLAST datasets.

  2. Induction of P450 3A1/2 and 2C6 by gemfibrozil in Sprague-Dawley rats.

    Science.gov (United States)

    Liu, Aiming; Yang, Julin; Zhao, Xin; Jiao, Xiaolan; Zhao, Weihong; Ma, Qing; Tang, Zhiyuan; Dai, Renke

    2011-01-01

    Fibrates are a group of peroxisome proliferator-activated receptor α agonists used in the treatment of dyslipidemia; however, they have been reported to cause species-related hepatocarcinogenesis and clinical myotoxicity. Gemfibrozil is one of the most commonly used fibrates, and it shows the highest risk for myotoxicity among the fibrates. The inhibitory drug-drug interaction mechanism associated with gemfibrozil has been explored recently, and the induction of human P450 3A4 and 2C8 has been reported. In this study, in vivo induction of rat P450 by gemfibrozil was studied in Sprague-Dawley rats. After the rats were dosed with gemfibrozil by oral gavage, microsomes were prepared. The metabolic activities of P450 3A1/2, 2C6, and 2D2 were assayed using probe substrates, and the systemic concentration of gemfibrozil during its administration was determined. P450 3A1/2 and 2C6 activities were induced 32-77% in the rats by gemfibrozil when the exposure concentration was in the clinical range. These data indicate that the inducibility of homologous P450 isoforms by gemfibrozil is similar in Sprague-Dawley rats and in humans. Inductive drug-drug interactions and inhibitory actions are involved in the co-administration of gemfibrozil with other drugs, which suggests the relevance for a fibrate-toxicology investigation.

  3. Regulation of rat liver cytochrome P450j, a high affinity N-nitrosodimethylamine demethylase (NDMAD)

    International Nuclear Information System (INIS)

    Thomas, P.E.; Bandiera, S.; Maines, S.L.; Ryan, D.E.; Levin, W.

    1987-01-01

    Purified IgG from sera of rabbits immunized with homogeneous P450j was absorbed to produce monospecific anti-P450j. Results using anti-P450j in ELISA show that rat liver microsomal P450j content decreases between 3 and 6 wks of age in both sexes. Several xenobiotics (Aroclor 1254, mirex and 3-methylcholanthrene) repressed P450j levels when administered to male rats. In contrast, hepatic levels of P450j were induced by isoniazid, dimethylsulfoxide, pyrazole, 4-methylpyrazole, ethanol and chemically-induced diabetes. P450j levels were measurable in kidney, whereas this isozyme was barely detectable in lung, ovaries and testes; however, extra-hepatic P450j was inducible by isoniazid. Between 80-90% of microsomal NDMAD was inhibited by anti-P450j whether the microsomes were isolated from untreated rats or animals administered inducers or repressors of P450j. Results obtained with the reconstituted system suggest that the remaining microsomal NDMAD resistant to antibody inhibition is the result of the inaccessibility of a certain proportion of P450j due to interference by NADPH-P450 reductase. P450j content and NDMAD activity correlated well in microsomes from rats of all treatment groups. The evidence indicates that P450j is the primary, and possibly only, microsomal catalyst of NDMAD at substrate concentrations relevant to hepatocarcinogenesis induced by NDMA

  4. Homology modelling of Drosophila cytochrome P450 enzymes associated with insecticide resistance.

    Science.gov (United States)

    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.

  5. Molecular cloning of P450 aromatase from the leopard gecko and its expression in the ovary.

    Science.gov (United States)

    Endo, Daisuke; Park, Min Kyun

    2005-07-01

    In this study, we identified the cDNA of P450 aromatase in the leopard gecko, a lizard with temperature-dependent sex determination. The cDNA encodes a putative protein of 505 amino acids. The deduced amino acid sequence of leopard gecko aromatase cDNA showed 80% identity with that of turtles, 70% with humans and 77% with chickens. This is the first report of the identification of P450 aromatase cDNA in squamata species. It has been reported that this gene is expressed in different layers of cells in the ovary of mammalian species and avian species. Thus, we also investigated cells expressing the mRNA of this gene in the ovary of the leopard gecko by RT-PCR and in situ hybridization. The mRNA expression of leopard gecko P450 aromatase was localized in both the thecal and granulosa cell layers in the ovary. The expression in thecal and granulosa cell layers was examined in the largest follicle, second largest follicle and third largest follicle by RT-PCR. A higher level of mRNA expression was observed in the granulosa cell layer of the second largest follicle than in other cell layers. This result may reflect the characteristics of follicles in species with automonochronic ovulation.

  6. In vitro inhibitory effects of pulvinic acid derivatives isolated from Chinese edible mushrooms, Boletus calopus and Suillus bovinus, on cytochrome P450 activity.

    Science.gov (United States)

    Huang, Yu-Ting; Onose, Jun-ichi; Abe, Naoki; Yoshikawa, Kunie

    2009-04-23

    Increasing attention has been focused on food-drug interactions. We have investigated the inhibitory effect of Chinese edible mushrooms, Boletus calopus and Suillus bovinus, on cytochrome P450 (CYP) 1A2, 2C9, 2D6, and 3A4, the main drug-metabolizing enzymes. Three pulvinic acid derivatives, atromentic acid (1), variegatic acid (2), and xerocomic acid (3), isolated from Boletus calopus and Suillus bovinus, revealed nonspecific inhibitory effects on all four CYPs. Using these compounds, the maximum IC50 values obtained with CYP3A4 in vitro were atromentic acid (1), 65.1+/-3.9 microM; variegatic acid (2), 2.2+/-0.1 microM; and xerocomic acid (3), 2.4+/-0.1 microM. Variegatic acid (2) and xerocomic acid (3) were effective inhibitors, comparable to cimetidine, dicoumarol, erythromycin, safrole, and uniconazole. Variegatic acid (2) and xerocomic acid (3) efficiently reduced ferryl myoglobin in CYPs. Reduction of ferryl heme to ferric heme is likely the mechanism of the nonspecific inhibitory effects of these compounds on CYPs.

  7. Effect of Short-Term Fasting on Systemic Cytochrome P450-Mediated Drug Metabolism in Healthy Subjects: A Randomized, Controlled, Crossover Study Using a Cocktail Approach.

    Science.gov (United States)

    Lammers, Laureen A; Achterbergh, Roos; van Schaik, Ron H N; Romijn, Johannes A; Mathôt, Ron A A

    2017-10-01

    Short-term fasting can alter drug exposure but it is unknown whether this is an effect of altered oral bioavailability and/or systemic clearance. Therefore, the aim of our study was to assess the effect of short-term fasting on oral bioavailability and systemic clearance of different drugs. In a randomized, controlled, crossover trial, 12 healthy subjects received a single administration of a cytochrome P450 (CYP) probe cocktail, consisting of caffeine (CYP1A2), metoprolol (CYP2D6), midazolam (CYP3A4), omeprazole (CYP2C19) and warfarin (CYP2C9), on four occasions: an oral (1) and intravenous (2) administration after an overnight fast (control) and an oral (3) and intravenous (4) administration after 36 h of fasting. Pharmacokinetic parameters of the probe drugs were analyzed using the nonlinear mixed-effects modeling software NONMEM. Short-term fasting increased systemic caffeine clearance by 17% (p = 0.04) and metoprolol clearance by 13% (p < 0.01), whereas S-warfarin clearance decreased by 19% (p < 0.01). Fasting did not affect bioavailability. The study demonstrates that short-term fasting alters CYP-mediated drug metabolism in a non-uniform pattern without affecting oral bioavailability.

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

    OpenAIRE

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

    1981-01-01

    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...

  9. Classification of cytochrome P450 1A2 inhibitors and noninhibitors by machine learning techniques

    NARCIS (Netherlands)

    Vasanthanathan, P.; Taboureau, O.; Oostenbrink, C.; Vermeulen, N.P.; Olsen, L.; Jorgensen, F.S.

    2009-01-01

    The cytochrome P450 (P450) superfamily plays an important role in the metabolism of drug compounds, and it is therefore highly desirable to have models that can predict whether a compound interacts with a specific isoform of the P450s. In this work, we provide in silico models for classification of

  10. Metabolic stereoselectivity of cytochrome P450 3A4 towards deoxypodophyllotoxin : In silico predictions and experimental validation

    NARCIS (Netherlands)

    Julsing, Mattijs K.; Vasilev, Nikolay P.; Schneidman-Duhovny, Dina; Muntendarn, Remco; Woerdenbag, Herman J.; Quax, Wim J.; Wolfson, Haim J.; Ionkova, Iliana; Kayser, Oliver

    Deoxypodophyllotoxin is stereoselectively converted into epipodophyllotoxin by recombinant human cytochrome P450 3A4 (CY-P3A4). Further kinetic analysis revealed that the Michaelis-Menten K(m) and V(max) for hydroxylation of deoxypodophyllotoxin by CYP3A4 at C7 position were 1.93 mu M and 1.48

  11. Cytochrome P450 polymorphism and postoperative cognitive dysfunction

    DEFF Research Database (Denmark)

    Steinmetz, J; Jespersgaard, Cathrine; Dalhoff, Kim Peder

    2012-01-01

    neuropsychological testing at one week had POCD, and 24 out of 307 (7.8%) had POCD at three months. None of the examined CYP2C19, 2D6 alleles, or various phenotypes were significantly associated with POCD. CONCLUSION: Polymorphisms in CYP2C19, or 2D6 genes do not seem to be related to the occurrence of cognitive......BACKGROUND:The etiology of postoperative cognitive dysfunction (POCD) remains unclear but toxicity of anesthetic drugs and their metabolites could be important. We aimed to assess the possible association between POCD after propofol anesthesia and various phenotypes owing to polymorphisms...... in cytochrome P450 encoding genes. METHODS:We included patients who underwent non-cardiac surgery under total intravenous anesthesia with propofol. POCD was identified using a neuropsychological test-battery administered preoperatively, one week, and three months after surgery. Genotyping of CYP2C19*2, *3, CYP2...

  12. P450 Pharmacogenetics in Indigenous North American Populations

    Directory of Open Access Journals (Sweden)

    Lindsay M. Henderson

    2018-02-01

    Full Text Available Indigenous North American populations, including American Indian and Alaska Native peoples in the United States, the First Nations, Métis and Inuit peoples in Canada and Amerindians in Mexico, are historically under-represented in biomedical research, including genomic research on drug disposition and response. Without adequate representation in pharmacogenetic studies establishing genotype-phenotype relationships, Indigenous populations may not benefit fully from new innovations in precision medicine testing to tailor and improve the safety and efficacy of drug treatment, resulting in health care disparities. The purpose of this review is to summarize and evaluate what is currently known about cytochrome P450 genetic variation in Indigenous populations in North America and to highlight the importance of including these groups in future pharmacogenetic studies for implementation of personalized drug therapy.

  13. Autoantibodies against Cytochrome P450 Side-Chain Cleavage Enzyme in Dogs (Canis lupus familiaris) Affected with Hypoadrenocorticism (Addison's Disease).

    Science.gov (United States)

    Boag, Alisdair M; Christie, Michael R; McLaughlin, Kerry A; Syme, Harriet M; Graham, Peter; Catchpole, Brian

    2015-01-01

    Canine hypoadrenocorticism likely arises from immune-mediated destruction of adrenocortical tissue, leading to glucocorticoid and mineralocorticoid deficiency. In humans with autoimmune Addison's disease (AAD) or autoimmune polyendocrine syndrome (APS), circulating autoantibodies have been demonstrated against enzymes associated with adrenal steroid synthesis. The current study investigates autoantibodies against steroid synthesis enzymes in dogs with spontaneous hypoadrenocorticism. Coding regions of canine CYP21A2 (21-hydroxylase; 21-OH), CYP17A1 (17-hydroxylase; 17-OH), CYP11A1 (P450 side-chain cleavage enzyme; P450scc) and HSD3B2 (3β hydroxysteroid dehydrogenase; 3βHSD) were amplified, cloned and expressed as 35S-methionine radiolabelled recombinant protein. In a pilot study, serum samples from 20 dogs with hypoadrenocorticism and four unaffected control dogs were screened by radio-immunoprecipitation assay. There was no evidence of reactivity against 21-OH, 17-OH or 3βHSD, but five dogs with hypoadrenocorticism showed immunoreactivity to P450scc compared with controls. Serum samples were subsequently obtained from 213 dogs diagnosed with hypoadrenocorticism and 110 dogs from a hospital control population. Thirty control dogs were randomly selected to establish a threshold for antibody positivity (mean + 3 × standard deviation). Dogs with hypoadrenocorticism were more likely to be P450scc autoantibody positive than hospital controls (24% vs. 1.2%, respectively; p = 0.0016). Sex was significantly associated with the presence of P450scc autoantibodies in the case population, with 30% of females testing positive compared with 17% of males (p = 0.037). Significant associations with breed (p = 0.015) and DLA-type (DQA1*006:01 allele; p = 0.017) were also found. This cross-sectional study indicates that P450scc autoantibodies are present in a proportion of dogs affected with hypoadrenocorticism.

  14. Autoantibodies against Cytochrome P450 Side-Chain Cleavage Enzyme in Dogs (Canis lupus familiaris Affected with Hypoadrenocorticism (Addison's Disease.

    Directory of Open Access Journals (Sweden)

    Alisdair M Boag

    Full Text Available Canine hypoadrenocorticism likely arises from immune-mediated destruction of adrenocortical tissue, leading to glucocorticoid and mineralocorticoid deficiency. In humans with autoimmune Addison's disease (AAD or autoimmune polyendocrine syndrome (APS, circulating autoantibodies have been demonstrated against enzymes associated with adrenal steroid synthesis. The current study investigates autoantibodies against steroid synthesis enzymes in dogs with spontaneous hypoadrenocorticism. Coding regions of canine CYP21A2 (21-hydroxylase; 21-OH, CYP17A1 (17-hydroxylase; 17-OH, CYP11A1 (P450 side-chain cleavage enzyme; P450scc and HSD3B2 (3β hydroxysteroid dehydrogenase; 3βHSD were amplified, cloned and expressed as 35S-methionine radiolabelled recombinant protein. In a pilot study, serum samples from 20 dogs with hypoadrenocorticism and four unaffected control dogs were screened by radio-immunoprecipitation assay. There was no evidence of reactivity against 21-OH, 17-OH or 3βHSD, but five dogs with hypoadrenocorticism showed immunoreactivity to P450scc compared with controls. Serum samples were subsequently obtained from 213 dogs diagnosed with hypoadrenocorticism and 110 dogs from a hospital control population. Thirty control dogs were randomly selected to establish a threshold for antibody positivity (mean + 3 × standard deviation. Dogs with hypoadrenocorticism were more likely to be P450scc autoantibody positive than hospital controls (24% vs. 1.2%, respectively; p = 0.0016. Sex was significantly associated with the presence of P450scc autoantibodies in the case population, with 30% of females testing positive compared with 17% of males (p = 0.037. Significant associations with breed (p = 0.015 and DLA-type (DQA1*006:01 allele; p = 0.017 were also found. This cross-sectional study indicates that P450scc autoantibodies are present in a proportion of dogs affected with hypoadrenocorticism.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2011-09-09

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

  16. Deletion of P399E401 in NADPH cytochrome P450 oxidoreductase results in partial mixed oxidase deficiency

    International Nuclear Information System (INIS)

    Flueck, Christa E.; Mallet, Delphine; Hofer, Gaby; Samara-Boustani, Dinane; Leger, Juliane; Polak, Michel; Morel, Yves; Pandey, Amit V.

    2011-01-01

    Highlights: → Mutations in human POR cause congenital adrenal hyperplasia. → We are reporting a novel 3 amino acid deletion mutation in POR P399 E 401del. → POR mutation P399 E 401del decreased P450 activities by 60-85%. → Impairment of steroid metabolism may be caused by multiple hits. → 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 E 401del in two unrelated Turkish patients with 46,XX disorder of sexual development. Recombinant POR proteins were produced in yeast and tested for their ability to support steroid metabolizing P450 activities. In comparison to wild-type POR, the P399 E 401del protein was found to decrease catalytic efficiency of 21-hydroxylation of progesterone by 68%, 17α-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 E 401 revealed reduced stability and flexibility of the mutant. In conclusion, P399 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 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.

  17. Mode of Antifungal Drugs Interaction with Cytochrome P- 450

    Directory of Open Access Journals (Sweden)

    M- Mahmodian

    1991-07-01

    Full Text Available Computer was used to identify the interactions of substrates and antifungal drugs with the enzyme, Cytochrome P-450; and then Molplot.bas computer program was applied to get three dimensional figures of 5-hydroxy camphor.oxidation products of camphor analogues, and antifungal drugs.Cartesian characteristics of atoms building molecules, are taken from Buildz. for program, which can calculate X,Y,Z coordinates of atoms by Zmatrix data. The other program which can calculate X,Y,Z coordinates, using fractional characteristics, is the Coord, for program that, gives our cartesian characteristics of the atoms of molecule, then by using these data, we obtain three dimensional figures and distance between active atoms in compounds under consideration. Results show that distance between two oxygen atoms in 5-exo-hydroxy- camphor and the other compounds obtained from oxidation of camphor analogues, with the distance of two oxygen atoms in antifungal compounds under discussion are equal. Therefore, we can conclude that, the antifungal molecule also interacts with enzyme's active site, by its own sites, in a similar manner to the 5-hydroxy camphor molecule, which is:"n1. Nitrogen atom (N of Imidazole and Triazole ring in antifungal molecule with Iron atom in heam molecule belonging to Cytochrome P-450 enzyme, are coordinated."n2. The other atoms such as : 0,S or N in structure of the antifungal drug are coordinated with hydrogen atom of hydroxyl group belong ing to Tyr-96 in the structure of enzyme, forming hydrogen bonding.

  18. Role of cytochrome P450 IA2 in acetanilide 4-hydroxylation as determined with cDNA expression and monoclonal antibodies.

    Science.gov (United States)

    Liu, G; Gelboin, H V; Myers, M J

    1991-02-01

    The role of P450 IA2 in the hydroxylation of acetanilide was examined using an inhibitory monoclonal antibody (MAb) 1-7-1 and vaccinia cDNA expression producing murine P450 IA1 (mIA1), murine P450 IA2 (mIA2), or human P450 IA2 (hIA2). Acetanilide hydroxylase (AcOH) activity was measured using an HPLC method with more than 500-fold greater sensitivity than previously described procedures. This method, which does not require the use of radioactive acetanilide, was achieved by optimizing both the gradient system and the amount of enzyme needed to achieve detection by uv light. MAb 1-7-1 inhibits up to 80% of the AcOH activity in both rat liver microsomes and cDNA expressed mouse and human P450 IA2. MAb 1-7-1, which recognizes both P450 IA1 and P450 IA2, completely inhibits the aryl hydrocarbon hydroxylase (AHH) activity of cDNA expressed in IA1. The inhibition of only 80% of the AHH activity present in MC liver microsomes by MAb 1-7-1 suggests that additional P450 forms are contributing to the overall AHH activity present in methylcholanthrene (MC)-liver microsomes as MAb 1-7-1 almost completely inhibits the AHH activity of expressed mIA1. Maximal inhibition of IA2 by 1-7-1 results in an 80% decrease in acetanilide hydroxylase activity in both liver microsomes and expressed mouse and human IA2. The capacity of MAb 1-7-1 to produce identical levels of inhibition of acetanilide hydroxylase activity in rat MC microsomes (80%) and in expressed mouse (81%) and human P450 IA2 (80%) strongly suggests that P450 IA2 is the major and perhaps the only enzyme responsible for the metabolism of acetanilide. These results demonstrate the complementary utility of monoclonal antibodies and cDNA expression for defining the contribution of specific P450 enzymes to the metabolism of a given substrate. This complementary approach allows for a more precise determination of the inhibitory capacity of MAb with respect to the metabolic capacity of the target P450.

  19. Cytochrome P450 levels are altered in patients with esophageal squamous-cell carcinoma

    DEFF Research Database (Denmark)

    Bergheim, I.; Wolfgarten, E.; Bollschweiler, E.

    2007-01-01

    AIM: To investigate the role of cytochrome P450 (CYP) in the carcinogenesis of squamous-cell carcinoma (SCC) in human esophagus by determining expression patterns and protein levels of representative CYPs in esophageal tissue of patients with SCC and controls. METHODS: mRNA expression of CYP2E1...... tissue (e.g. CYP2C8, CYP3A4, CYP3A5, and CYP2E1) between SCC patients and healthy subjects and may contribute to the development of SCC in the esophagus....

  20. R-warfarin clearances from plasma associated with polymorphic cytochrome P450 2C19 and simulated by individual physiologically based pharmacokinetic models for 11 cynomolgus monkeys.

    Science.gov (United States)

    Utoh, Masahiro; Kusama, Takashi; Miura, Tomonori; Mitsui, Marina; Kawano, Mirai; Hirano, Takahiro; Shimizu, Makiko; Uno, Yasuhiro; Yamazaki, Hiroshi

    2018-02-01

    1. Cynomolgus monkey cytochrome P450 2C19 (formerly known as P450 2C75), homologous to human P450 2C19, has been identified as R-warfarin 7-hydroxylase. In this study, simulations of R-warfarin clearance in individual cynomolgus monkeys genotyped for P450 2C19 p.[(Phe100Asn; Ala103Val; Ile112Leu)] were performed using individual simplified physiologically based pharmacokinetic (PBPK) modeling. 2. Pharmacokinetic parameters and absorption rate constants, volumes of the systemic circulation, and hepatic intrinsic clearances for individual PBPK models were estimated for eleven cynomolgus monkeys. 3. One-way ANOVA revealed significant effects of the genotype (p warfarin among the homozygous mutant, heterozygous mutant, and wild-type groups. R-Warfarin clearances in individual cynomolgus monkeys genotyped for P450 2C19 were simulated by simplified PBPK modeling. The modeled hepatic intrinsic clearances were significantly associated with the P450 2C19 genotypes. The liver microsomal elimination rates of R-warfarin for individual animals after in vivo administration showed significant reductions associated with the genotype (p warfarin and related medicines associated with polymorphic P450 2C19 in individual cynomolgus monkeys, thereby facilitating calculation of the fraction of hepatic clearance.

  1. In Situ Proteolysis for Crystallization of Membrane Bound Cytochrome P450 17A1 and 17A2 Proteins from Zebrafish.

    Science.gov (United States)

    Lei, Li; Egli, Martin

    2016-04-01

    Fish and human cytochrome P450 (P450) 17A1 catalyze both steroid 17α-hydroxylation and 17α,20-lyase reactions. Fish P450 17A2 catalyzes only 17α-hydroxylation. Both enzymes are microsomal-type P450s, integral membrane proteins that bind to the membrane through their N-terminal hydrophobic segment, the signal anchor sequence. The presence of this N-terminal region renders expression of full-length proteins challenging or impossible. For some proteins, variable truncation of the signal anchor sequence precludes expression or results in poor expression levels. To crystallize P450 17A1 and 17A2 in order to gain insight into their different activities, we used an alternative N-terminal sequence to boost expression together with in situ proteolysis. Key features of our approach to identify crystallizable P450 fragments were the use of an N-terminal leader sequence, a screen composed of 12 proteases to establish optimal cleavage, variations of protease concentration in combination with an SDS-PAGE assay, and analysis of the resulting fragments using Edman sequencing. Described in this unit are protocols for vector preparation, expression, purification, and in situ proteolytic crystallization of two membrane-bound P450 proteins. Copyright © 2016 John Wiley & Sons, Inc.

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

    International Nuclear Information System (INIS)

    Polusani, Srikanth R.; Kar, Rekha; Riquelme, Manuel A.; Masters, Bettie Sue; Panda, Satya P.

    2011-01-01

    Highlights: → Humans with severe forms of cytochrome P450 oxidoreductase (CYPOR) mutations show bone defects as observed in Antley-Bixler Syndrome. → 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. → Knockdown of CYPOR decreased Gap Junctional Intercellular Communication and hemichannel activity. → Knockdown of CYPOR decreased Cx43 in mouse primary calvarial osteoblasts. → Decreased Cx43 expression was observed at the transcriptional level. -- Abstract: Cytochrome P450 oxidoreductase (CYPOR) is a microsomal electron-transferring enzyme containing both FAD and FMN as co-factors, which provides the reducing equivalents to various redox partners, such as cytochromes P450 (CYPs), heme oxygenase (HO), cytochrome b 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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Polusani, Srikanth R.; Kar, Rekha; Riquelme, Manuel A.; Masters, Bettie Sue [The University of Texas Health Science Center at San Antonio, Department of Biochemistry, San Antonio, TX 78229 (United States); Panda, Satya P., E-mail: panda@uthscsa.edu [The University of Texas Health Science Center at San Antonio, Department of Biochemistry, San Antonio, TX 78229 (United States)

    2011-08-05

    Highlights: {yields} Humans with severe forms of cytochrome P450 oxidoreductase (CYPOR) mutations show bone defects as observed in Antley-Bixler Syndrome. {yields} First report showing knockdown of CYPOR in osteoblasts decreased Connexin 43 (Cx43) protein levels. Cx43 is known to play an important role in bone modeling. {yields} Knockdown of CYPOR decreased Gap Junctional Intercellular Communication and hemichannel activity. {yields} Knockdown of CYPOR decreased Cx43 in mouse primary calvarial osteoblasts. {yields} Decreased Cx43 expression was observed at the transcriptional level. -- Abstract: Cytochrome P450 oxidoreductase (CYPOR) is a microsomal electron-transferring enzyme containing both FAD and FMN as co-factors, which provides the reducing equivalents to various redox partners, such as cytochromes P450 (CYPs), heme oxygenase (HO), cytochrome b{sub 5} and squalene monooxygenase. Human patients with severe forms of CYPOR mutation show bone defects such as cranio- and humeroradial synostoses and long bone fractures, known as Antley-Bixler-like Syndrome (ABS). To elucidate the role of CYPOR in bone, we knocked-down CYPOR in multiple osteoblast cell lines using RNAi technology. In this study, knock-down of CYPOR decreased the expression of Connexin 43 (Cx43), known to play a critical role in bone formation, modeling, and remodeling. Knock-down of CYPOR also decreased Gap Junction Intercellular Communication (GJIC) and hemichannel activity. Promoter luciferase assays revealed that the decrease in expression of Cx43 in CYPOR knock-down cells was due to transcriptional repression. Primary osteoblasts isolated from bone specific Por knock-down mice calvariae confirmed the findings in the cell lines. Taken together, our study provides novel insights into the regulation of gap junction function by CYPOR and suggests that Cx43 may play an important role(s) in CYPOR-mediated bone defects seen in patients.

  4. Intestinal cytochromes P450 regulating the intestinal microbiota and its probiotic profile

    Directory of Open Access Journals (Sweden)

    Eugenia Elefterios Venizelos Bezirtzoglou

    2012-09-01

    Full Text Available Cytochromes P450 (CYPs enzymes metabolize a large variety of xenobiotic substances. In this vein, a plethora of studies were conducted to investigate their role, as cytochromes are located in both liver and intestinal tissues. The P450 profile of the human intestine has not been fully characterized. Human intestine serves primarily as an absorptive organ for nutrients, although it has also the ability to metabolize drugs. CYPs are responsible for the majority of phase I drug metabolism reactions. CYP3A represents the major intestinal CYP (80% followed by CYP2C9. CYP1A is expressed at high level in the duodenum, together with less abundant levels of CYP2C8-10 and CYP2D6. Cytochromes present a genetic polymorphism intra- or interindividual and intra- or interethnic. Changes in the pharmacokinetic profile of the drug are associated with increased toxicity due to reduced metabolism, altered efficacy of the drug, increased production of toxic metabolites, and adverse drug interaction. The high metabolic capacity of the intestinal flora is due to its enormous pool of enzymes, which catalyzes reactions in phase I and phase II drug metabolism. Compromised intestinal barrier conditions, when rupture of the intestinal integrity occurs, could increase passive paracellular absorption. It is clear that high microbial intestinal charge following intestinal disturbances, ageing, environment, or food-associated ailments leads to the microbial metabolism of a drug before absorption. The effect of certain bacteria having a benefic action on the intestinal ecosystem has been largely discussed during the past few years by many authors. The aim of the probiotic approach is to repair the deficiencies in the gut flora and establish a protective effect. There is a tentative multifactorial association of the CYP (P450 cytochrome role in the different diseases states, environmental toxic effects or chemical exposures and nutritional status.

  5. Conformational changes of the NADPH-dependent cytochrome P450 reductase in the course of electron transfer to cytochromes P450

    DEFF Research Database (Denmark)

    Laursen, Tomas; Jensen, Kenneth; Møller, Birger Lindberg

    2011-01-01

    The NADPH-dependent cytochrome P450 reductase (CPR) is a key electron donor to eucaryotic cytochromes P450 (CYPs). CPR shuttles electrons from NADPH through the FAD and FMN-coenzymes into the iron of the prosthetic heme-group of the CYP. In the course of these electron transfer reactions, CPR und...... to serve as an effective electron transferring "nano-machine"....

  6. One-electron reduction of mitomycin c by rat liver : role of cytochrome P-450 and NADPH-cytochrome P-450 reductase

    NARCIS (Netherlands)

    Vromans, R M; Van de Straat, R; Groeneveld, M.; Vermeulen, N P

    1. The role of cytochrome P-450 in the one-electron reduction of mitomycin c was studied in rat hepatic microsomal systems and in reconstituted systems of purified cytochrome P-450. Formation of H2O2 from redox cycling of the reduced mitomycin c in the presence of O2 and the alkylation of

  7. Isolation of insecticide resistance-related forms of cytochrome P-450 from Drosophila melanogaster.

    OpenAIRE

    Sundseth, S S; Nix, C E; Waters, L C

    1990-01-01

    Significant purification of the ubiquitous cytochrome P-450-A and the strain-specific P-450-B from Drosophila melanogaster has been achieved by sequential chromatography on octylamino-agarose, DEAE-cellulose and hydroxyapatite. Preparations of P-450-A (specific contents of 7-9 nmol/mg) were homogeneous as determined by SDS/polyacrylamide-gel electrophoresis (PAGE) analysis. Preparations enriched for P-450-B (specific contents of 4-7 nmol/mg) contained significant amounts of P-450-A but were e...

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

    Science.gov (United States)

    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 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

  9. Metabolism of 7-ethoxycoumarin, flavanone and steroids by cytochrome P450 2C9 variants.

    Science.gov (United States)

    Uno, Tomohide; Nakano, Ryosuke; Kanamaru, Kengo; Takenaka, Shinji; Uno, Yuichi; Imaishi, Hiromasa

    2017-11-01

    CYP2C9 is a human microsomal cytochrome P450c (CYP). Much of the variation in CYP2C9 levels and activity can be attributed to polymorphisms of this gene. Wild-type CYP2C9 and mutants were coexpressed with NADPH-cytochrome P450 reductase in Escherichia coli. The hydroxylase activities toward 7-ethoxycoumarin, flavanone and steroids were examined. Six CYP2C9 variants showed Soret peaks (450 nm) typical of P450 in reduced CO-difference spectra. CYP2C9.38 had the highest 7-ethoxycoumarin de-ethylase activity. All the CYP2C9 variants showed lower flavanone 6-hydroxylation activities than CYP2C9.1 (the wild-type). CYP2C9.38 showed higher activities in testosterone 6β-hydroxylation, progesterone 6β-/16α-hydroxylation, estrone 11α-hydroxylation and estradiol 6α-hydroxylation than CYP2C9.1. CYP2C9.40 showed higher testosterone 17-oxidase activity than CYP2C9.1; CYP2C9.8 showed higher estrone 16α-hydroxylase activity and CYP2C9.12 showed higher estrone 11α-hydroxylase activity. CYP2C9.9 and CYP2C9.10 showed similar activities to CYP2C9.1. These results indicate that the substrate specificity of CYP2C9.9 and CYP2C9.10 was not changed, but CYP2C9.8, CYP2C9.12 and CYP2C9.40 showed different substrate specificity toward steroids compared with CYP2C9.1; and especially CYP2C9.38 displayed diverse substrate specificities towards 7-ethoxycoumarin and steroids. Copyright © 2017 John Wiley & Sons, Ltd.

  10. Identification and phylogenetic analysis of novel cytochrome P450 1A genes from ungulate species.

    Science.gov (United States)

    Darwish, Wageh Sobhy; Kawai, Yusuke; Ikenaka, Yoshinori; Yamamoto, Hideaki; Muroya, Tarou; Ishizuka, Mayumi

    2010-09-01

    As part of an ongoing effort to understand the biological response of wild and domestic ungulates to different environmental pollutants such as dioxin-like compounds, cDNAs encoding for CYP1A1 and CYP1A2 were cloned and characterized. Four novel CYP1A cDNA fragments from the livers of four wild ungulates (elephant, hippopotamus, tapir and deer) were identified. Three fragments from hippopotamus, tapir and deer were classified as CYP1A2, and the other fragment from elephant was designated as CYP1A1/2. The deduced amino acid sequences of these fragment CYP1As showed identities ranging from 76 to 97% with other animal CYP1As. The phylogenetic analysis of these fragments showed that both elephant and hippopotamus CYP1As made separate branches, while tapir and deer CYP1As were located beside that of horse and cattle respectively in the phylogenetic tree. Analysis of dN/dS ratio among the identified CYP1As indicated that odd toed ungulate CYP1A2s were exposed to different selection pressure.

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

    Energy Technology Data Exchange (ETDEWEB)

    Johri, Ashu; Yadav, Sanjay; Dhawan, Alok [Developmental Toxicology Division, Industrial Toxicology Research Centre, P. O. Box 80, M. G. Marg, Lucknow-226 001, U. P. (India); Parmar, Devendra [Developmental Toxicology Division, Industrial Toxicology Research Centre, P. O. Box 80, M. G. Marg, Lucknow-226 001, U. P. (India)

    2007-12-15

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

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

    International Nuclear Information System (INIS)

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

    2007-01-01

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

  13. Pyrethroid insecticide lambda-cyhalothrin induces hepatic cytochrome P450 enzymes, oxidative stress and apoptosis in rats.

    Science.gov (United States)

    Martínez, María-Aránzazu; Ares, Irma; Rodríguez, José-Luis; Martínez, Marta; Roura-Martínez, David; Castellano, Victor; Lopez-Torres, Bernardo; Martínez-Larrañaga, María-Rosa; Anadón, Arturo

    2018-08-01

    This study aimed to examine in rats the effects of the Type II pyrethroid lambda-cyhalothrin on hepatic microsomal cytochrome P450 (CYP) isoform activities, oxidative stress markers, gene expression of proinflammatory, oxidative stress and apoptosis mediators, and CYP isoform gene expression and metabolism phase I enzyme PCR array analysis. Lambda-cyhalothrin, at oral doses of 1, 2, 4 and 8mg/kg bw for 6days, increased, in a dose-dependent manner, hepatic activities of ethoxyresorufin O-deethylase (CYP1A1), methoxyresorufin O-demethylase (CYP1A2), pentoxyresorufin O-depentylase (CYP2B1/2), testosterone 7α- (CYP2A1), 16β- (CYP2B1), and 6β-hydroxylase (CYP3A1/2), and lauric acid 11- and 12-hydroxylase (CYP4A1/2). Similarly, lambda-cyhalothrin (4 and 8mg/kg bw, for 6days), in a dose-dependent manner, increased significantly hepatic CYP1A1, 1A2, 2A1, 2B1, 2B2, 2E1, 3A1, 3A2 and 4A1 mRNA levels and IL-1β, NFκB, Nrf2, p53, caspase-3 and Bax gene expressions. PCR array analysis showed from 84 genes examined (P1.5), changes in mRNA levels in 18 genes: 13 up-regulated and 5 down-regulated. A greater fold change reversion than 3-fold was observed on the up-regulated ALDH1A1, CYP2B2, CYP2C80 and CYP2D4 genes. Ingenuity Pathway Analysis (IPA) groups the expressed genes into biological mechanisms that are mainly related to drug metabolism. In the top canonical pathways, Oxidative ethanol degradation III together with Fatty Acid α-oxidation may be significant pathways for lambda-cyhalothrin. Our results may provide further understanding of molecular aspects involved in lambda-cyhalothrin-induced liver injury. Copyright © 2018. Published by Elsevier B.V.

  14. Active sites of two orthologous cytochromes P450 2E1: Differences revealed by spectroscopic methods

    International Nuclear Information System (INIS)

    Anzenbacherova, Eva; Hudecek, Jiri; Murgida, Daniel; Hildebrandt, Peter; Marchal, Stephane; Lange, Reinhard; Anzenbacher, Pavel

    2005-01-01

    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. 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

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

    Energy Technology Data Exchange (ETDEWEB)

    Reed, James R., E-mail: rreed@lsuhsc.edu [Department of Pharmacology and Experimental Therapeutics, Louisiana State University Health Science Center, 533 Bolivar St., New Orleans, LA 70112 (United States); The Stanley S. Scott Cancer Center, Louisiana State University Health Science Center, 533 Bolivar St., New Orleans, LA 70112 (United States); Cawley, George F.; Ardoin, Taylor G. [Department of Pharmacology and Experimental Therapeutics, Louisiana State University Health Science Center, 533 Bolivar St., New Orleans, LA 70112 (United States); The Stanley S. Scott Cancer Center, Louisiana State University Health Science Center, 533 Bolivar St., New Orleans, LA 70112 (United States); Dellinger, Barry; Lomnicki, Slawomir M.; Hasan, Farhana; Kiruri, Lucy W. [Department of Chemistry, Louisiana State University, Baton Rouge, LA 70803 (United States); Backes, Wayne L. [Department of Pharmacology and Experimental Therapeutics, Louisiana State University Health Science Center, 533 Bolivar St., New Orleans, LA 70112 (United States); The Stanley S. Scott Cancer Center, Louisiana State University Health Science Center, 533 Bolivar St., New Orleans, LA 70112 (United States)

    2014-06-01

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

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

    International Nuclear Information System (INIS)

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

    2014-01-01

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

  17. Clinical Pharmacogenetics of Cytochrome P450-Associated Drugs in Children

    Directory of Open Access Journals (Sweden)

    Ida Aka

    2017-11-01

    Full Text Available Cytochrome P450 (CYP enzymes are commonly involved in drug metabolism, and genetic variation in the genes encoding CYPs are associated with variable drug response. While genotype-guided therapy has been clinically implemented in adults, these associations are less well established for pediatric patients. In order to understand the frequency of pediatric exposures to drugs with known CYP interactions, we compiled all actionable drug–CYP interactions with a high level of evidence using Clinical Pharmacogenomic Implementation Consortium (CPIC data and surveyed 10 years of electronic health records (EHR data for the number of children exposed to CYP-associated drugs. Subsequently, we performed a focused literature review for drugs commonly used in pediatrics, defined as more than 5000 pediatric patients exposed in the decade-long EHR cohort. There were 48 drug–CYP interactions with a high level of evidence in the CPIC database. Of those, only 10 drugs were commonly used in children (ondansetron, oxycodone, codeine, omeprazole, lansoprazole, sertraline, amitriptyline, citalopram, escitalopram, and risperidone. For these drugs, reports of the drug–CYP interaction in cohorts including children were sparse. There are adequate data for implementation of genotype-guided therapy for children for three of the 10 commonly used drugs (codeine, omeprazole and lansoprazole. For the majority of commonly used drugs with known CYP interactions, more data are required to support pharmacogenomic implementation in children.

  18. Polycyclic aromatic hydrocarbons and cytochrome P450 in HIV pathogenesis

    Science.gov (United States)

    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

  19. Cytochrome P450s--Their expression, regulation, and role in insecticide resistance.

    Science.gov (United States)

    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.

  20. P450 reductase and cytochrome b5 interactions with cytochrome P450: Effects on house fly CYP6A1 catalysis

    OpenAIRE

    Murataliev, Marat B.; Guzov, Victor M.; Walker, F. Ann; Feyereisen, René

    2008-01-01

    The interactions of protein components of the xenobiotic-metabolizing cytochrome P450 system, CYP6A1, P450 reductase, and cytochrome b5 from the house fly (Musca domestica) have been characterized. CYP6A1 activity is determined by the concentration of the CYP6A1-P450 reductase complex, regardless of which protein is present in excess. Both holo- and apo-b5 stimulated CYP6A1 heptachlor epoxidase and steroid hydroxylase activities and influenced the regioselectivity of testosterone hydroxylatio...

  1. El citocromo P-450 y la respuesta terapéutica a los antimaláricos Cytochrome P-450 and the response to antimalarial drugs

    Directory of Open Access Journals (Sweden)

    Valentina Guzmán

    2006-01-01

    dealt with only four antimalarial drugs: amodiaquine, chloroquine, mefloquine, and proguanil. RESULTS: 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 antimalarial drugs, the distribution in the population of the genetic alleles linked to the enzymes involved in their metabolism, the contribution of these genetic mutations to therapeutic failure, and the possibility of predicting the response to antimalarial therapy. CONCLUSIONS: The therapeutic response to antimalarial drugs is a multifactorial process that is poorly understood, so that it is not possible to ascribe to a specific phenotype or genotype a role in the response to antimalarial therapy. Attention should be given to biological and social factors, such as diet, nutritional status, and inflammatory and infectious processes that are often present in areas where malaria is endemic.

  2. Understanding uncoupling in the multiredox centre P450 3A4-BMR model system.

    Science.gov (United States)

    Degregorio, Danilo; Sadeghi, Sheila J; Di Nardo, Giovanna; Gilardi, Gianfranco; Solinas, Sandro P

    2011-01-01

    Understanding the uncoupling at the haem active site and/or at the level of multidomain electron transfer is an important element in cytochrome P450 chemistry. Here a chimeric model system consisting of human cytochrome P450 3A4 and the soluble reductase domain of CYP102A1 from Bacillus megaterium (BMR) is used to study the relationship between electron transfer and the coupling efficiency in substrate monoxygenation. Several regulatory features were considered. FAD and FMN added to apoenzyme in oversaturating concentrations influence neither formaldehyde production nor coupling efficiency. The optimal conditions of coupling efficiency depended only on the NADPH concentration. The pH (8.0) and ionic strength (50 mM potassium phosphate) were found to modulate the level of coupling, indicating an influence over the formation of a productive interaction between the BMR and the haem domain. Overall, uncoupling is found to be an intrinsic property of the haem domain, and the covalent linkage of the reductase in a single polypeptide chain has little influence over the activity coupled to product formation.

  3. Bioactivation and Regioselectivity of Pig Cytochrome P450 3A29 towards Aflatoxin B1

    Directory of Open Access Journals (Sweden)

    Jun Wu

    2016-09-01

    Full Text Available Due to unavoidable contaminations in feedstuff, pigs are easily exposed to aflatoxin B1 (AFB1 and suffer from poisoning, thus the poisoned products potentially affect human health. Heretofore, the metabolic process of AFB1 in pigs remains to be clarified, especially the principal cytochrome P450 oxidases responsible for its activation. In this study, we cloned CYP3A29 from pig liver and expressed it in Escherichia coli, and its activity has been confirmed with the typical P450 CO-reduced spectral characteristic and nifedipine-oxidizing activity. The reconstituted membrane incubation proved that the recombinant CYP3A29 was able to oxidize AFB1 to form AFB1-exo-8,9-epoxide in vitro. The structural basis for the regioselective epoxidation of AFB1 by CYP3A29 was further addressed. The T309A mutation significantly decreased the production of AFBO, whereas F304A exhibited an enhanced activation towards AFB1. In agreement with the mutagenesis study, the molecular docking simulation suggested that Thr309 played a significant role in stabilization of AFB1 binding in the active center through a hydrogen bond. In addition, the bulk phenyl group of Phe304 potentially imposed steric hindrance on the binding of AFB1. Our study demonstrates the bioactivation of pig CYP3A29 towards AFB1 in vitro, and provides the insight for understanding regioselectivity of CYP3A29 to AFB1.

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

    Science.gov (United States)

    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.

  5. CW EPR parameters reveal cytochrome P450 ligand binding modes.

    Science.gov (United States)

    Lockart, Molly M; Rodriguez, Carlo A; Atkins, William M; Bowman, Michael K

    2018-06-01

    Cytochrome P450 (CYP) monoxygenses utilize heme cofactors to catalyze oxidation reactions. They play a critical role in metabolism of many classes of drugs, are an attractive target for drug development, and mediate several prominent drug interactions. Many substrates and inhibitors alter the spin state of the ferric heme by displacing the heme's axial water ligand in the resting enzyme to yield a five-coordinate iron complex, or they replace the axial water to yield a nitrogen-ligated six-coordinate iron complex, which are traditionally assigned by UV-vis spectroscopy. However, crystal structures and recent pulsed electron paramagnetic resonance (EPR) studies find a few cases where molecules hydrogen bond to the axial water. The water-bridged drug-H 2 O-heme has UV-vis spectra similar to nitrogen-ligated, six-coordinate complexes, but are closer to "reverse type I" complexes described in older liteature. Here, pulsed and continuous wave (CW) EPR demonstrate that water-bridged complexes are remarkably common among a range of nitrogenous drugs or drug fragments that bind to CYP3A4 or CYP2C9. Principal component analysis reveals a distinct clustering of CW EPR spectral parameters for water-bridged complexes. CW EPR reveals heterogeneous mixtures of ligated states, including multiple directly-coordinated complexes and water-bridged complexes. These results suggest that water-bridged complexes are under-represented in CYP structural databases and can have energies similar to other ligation modes. The data indicates that water-bridged binding modes can be identified and distinguished from directly-coordinated binding by CW EPR. Copyright © 2018 Elsevier Inc. All rights reserved.

  6. Regulation of cytochrome P-450 monooxygenases in the mouse

    International Nuclear Information System (INIS)

    Kelley, M.F.

    1986-01-01

    Recently, the compound 1,4-bis[2-(3,4-dichloropyridyloxy)] benzene (TCPOBOP) has been identified as a highly potent phenobabital-like agonist in mice. This finding has led to the suggestion that a receptor-mediated process may govern the induction of cytochrome P-450 monooxygenases by phenobarbital and phenobarbital-like agonists. This dissertation examines: (1) the effects of structural alterations of the TCPOBOP molecule on enzyme induction activity, (2) the induction response to phenobarbital and TCPOBOP among inbred mouse strains, (3) the spectrum of monooxygenase activities induced by phenobarbital and TCPOBOP compared to 3-methylcholanthrene, isosafrole and pregnenolone 16α-carbonitrile (PCN) and (4) the binding of [ 3 H] TCPOBOP in hepatic cytosol. Changes in the structure of the pyridyloxy or benzene rings markedly affect enzyme induction activity and provide additional indirect evidence for a receptor-mediated response. An evaluation of monooxygenase induction by TCPOBOP for 27 inbred mouse strains and by phenobarbital for 15 inbred mouse strains failed to identify a strain which was completely nonresponsive to these compounds, although several strains exhibited decreased responsiveness for select monooxygenase reactions. TCPOBOP, PCN and phenobarbital were all found to significantly increase the rate of hydroxylation of testosterone at the 2α-, 6β- and 15β- positions but only TCPOBOP and phenobarbital dramatically increased the rate of pentoxyresorufin O-dealkylation. The results demonstrates that TCPOBOP most closely resembles phenobarbital in its mode of monooxygenase induction in mice. Sucrose density gradient analysis of [ 3 H] TCPOBOP-hepatic cytosol incubations failed to identify specific, saturable binding of [ 3 H] TCPOBOP to cytosolic marcomolecular elements

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

    International Nuclear Information System (INIS)

    Liu, Senyan; Yao, Yunyi; Lu, Shijun; Aldous, Kenneth; Ding, Xinxin; Mei, Changlin; Gu, Jun

    2013-01-01

    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 with 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

  8. Cytochrome P450 1A1 (CYP1A1) protects against nonalcoholic fatty liver disease caused by Western diet containing benzo[a]pyrene in mice.

    Science.gov (United States)

    Uno, Shigeyuki; Nebert, Daniel W; Makishima, Makoto

    2018-03-01

    The Western diet contributes to nonalcoholic fatty liver disease (NAFLD) pathogenesis. Benzo[a]pyrene (BaP), a prototypical environmental pollutant produced by combustion processes, is present in charcoal-grilled meat. Cytochrome P450 1A1 (CYP1A1) metabolizes BaP, resulting in either detoxication or metabolic activation in a context-dependent manner. To elucidate a role of CYP1A1-BaP in NAFLD pathogenesis, we compared the effects of a Western diet, with or without oral BaP treatment, on the development of NAFLD in Cyp1a1(-/-) mice versus wild-type mice. A Western diet plus BaP induced lipid-droplet accumulation in liver of Cyp1a1(-/-) mice, but not wild-type mice. The hepatic steatosis observed in Cyp1a1(-/-) mice was associated with increased cholesterol, triglyceride and bile acid levels. Cyp1a1(-/-) mice fed Western diet plus BaP had changes in expression of genes involved in bile acid and lipid metabolism, and showed no increase in Cyp1a2 expression but did exhibit enhanced Cyp1b1 mRNA expression, as well as hepatic inflammation. Enhanced BaP metabolic activation, oxidative stress and inflammation may exacerbate metabolic dysfunction in liver of Cyp1a1(-/-) mice. Thus, Western diet plus BaP induces NAFLD and hepatic inflammation in Cyp1a1(-/-) mice in comparison to wild-type mice, indicating a protective role of CYP1A1 against NAFLD pathogenesis. Copyright © 2018 Elsevier Ltd. All rights reserved.

  9. The Effect of 5-Aminolevulinic Acid on Cytochrome P450-Mediated Prodrug Activation.

    Directory of Open Access Journals (Sweden)

    Mai Miura

    Full Text Available Of late, numerous prodrugs are widely used for therapy. The hemeprotein cytochrome P450 (CYP catalyzes the activation of prodrugs to form active metabolites. Therefore, the activation of CYP function might allow the use of lower doses of prodrugs and decrease toxicity. We hypothesized that the addition of 5-aminolevulinic acid (ALA, a precursor in the porphyrin biosynthetic pathway, enhances the synthesis of heme, leading to the up-regulation of CYP activity. To test this hypothesis, we treated a human gastric cancer cell line with ALA and determined the effect on CYP-dependent prodrug activation. For this purpose, we focused on the anticancer prodrug tegafur, which is converted to its active metabolite 5-fluorouracil (5-FU mainly by CYP2A6. We show here that ALA increased CYP2A6-dependent tegafur activation, suggesting that ALA elevated CYP activity and potentiated the activation of the prodrug.

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

    NARCIS (Netherlands)

    Oetari, S.; Sudibyo, M.; Commandeur, J.N.M.; Samhoedi, R.; Vermeulen, N.P.E.

    1996-01-01

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

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

    Science.gov (United States)

    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...

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Dragic Vukomanovic

    2017-01-01

    Full Text Available 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.

  15. Relationship between hydrocarbon structure and induction of P450: effects on protein levels and enzyme activities.

    Science.gov (United States)

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

    1993-12-01

    1. Treatment of male rat with the small aromatic hydrocarbons, benzene, toluene, ethylbenzene, n-propylbenzene, m-xylene, and p-xylene increased several P450-dependent activities, with ethylbenzene, m-xylene, and n-propylbenzene producing the greatest response. Hydrocarbon treatment differentially affected toluene metabolism, producing a response dependent on the metabolite monitored. In untreated rats, benzyl alcohol was the major hydroxylation product of toluene metabolism, comprising > 99% of the total metabolites formed. Hydrocarbon treatment increased the overall rate of toluene metabolism by dramatically increasing the amount of aromatic hydroxylation. Ethylbenzene, n-propylbenzene and m-xylene were the most effective inducers of aromatic hydroxylation of toluene. In contrast, production of the major toluene metabolite benzyl alcohol was increased only after treatment with m-xylene. 2. P450 2B1/2B2 levels were induced by each of the hydrocarbons examined, with the magnitude of induction increasing with increasing hydrocarbon size. P450 1A1 was also induced after hydrocarbon exposure; however, the degree of induction was smaller than that observed for P450 2B1/2B2. P450 2C11 levels were suppressed after treatment with benzene, ethylbenzene and n-propylbenzene. 3. Taken together these results display two induction patterns. The first generally corresponds to changes in the P450 2B subfamily, where activities (e.g. the aromatic hydroxylations of toluene) were most effectively induced by ethylbenzene, n-propylbenzene and m-xylene. In the second, induction was observed only after m-xylene treatment, a pattern that was found when the metabolism of the substrate was catalysed by both the P450 2B subfamily and P450 2C11. Hydrocarbons that both induced P450 2B1/2B2 and suppressed P450 2C11 (such as ethylbenzene and n-propylbenzene) showed little change in activities catalysed by both isozymes (e.g. aliphatic hydroxylation of toluene, and aniline hydroxylation

  16. A comparative study of P450 gene expression in field and laboratory Musca domestica L. strains

    DEFF Research Database (Denmark)

    Højland, Dorte Heidi; Vagn Jensen, Karl-Martin; Kristensen, Michael

    2014-01-01

    BACKGROUND The housefly is a global pest that has developed resistance to most insecticides applied for its control. Resistance has been associated with cytochrome P450 monooxygenases (P450s). The authors compare the expression of six genes possibly associated with insecticide resistance in three...... unselected strains: a multiresistant strain (791a), a neonicotinoid-resistant strain (766b) and a new field strain (845b). RESULTS CYP4G2 was highly expressed throughout the range of strains and proved to be the one of the most interesting expression profiles of all P450s analysed. CYP6G4 was expressed up...... to 11-fold higher in 766b than in WHO-SRS. Significant differences between expression of P450 genes between F1 flies from 845b and established laboratory strains were shown. In general, P450 gene expression in 845b was 2–14-fold higher than in the reference strain (P

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-02-17

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

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

    International Nuclear Information System (INIS)

    Kaspera, Rüdiger; Sahele, Tariku; Lakatos, Kyle; Totah, Rheem A.

    2012-01-01

    Highlights: ► Cytochrome P450BM-3 reduced aldehydes to alcohols efficiently (k cat ∼ 25 min −1 ). ► Reduction is a direct hydride transfer from R-NADP 2 H to the carbonyl moiety. ► P450 domain variants enhance reduction through potential allosteric/redox interactions. ► Novel reaction will have implications for metabolism of xenobiotics. -- Abstract: Cytochrome P450BM-3 catalyzed the reduction of lipophilic aldehydes to alcohols efficiently. A k cat of ∼25 min −1 was obtained for the reduction of methoxy benzaldehyde with wild type P450BM-3 protein which was higher than in the isolated reductase domain (BMR) alone and increased in specific P450-domain variants. The reduction was caused by a direct hydride transfer from preferentially R-NADP 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 2 H but not D 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.

  19. Cloning, purification, crystallization and preliminary X-ray analysis of a chimeric NADPH-cytochrome P450 reductase

    International Nuclear Information System (INIS)

    Aigrain, Louise; Pompon, Denis; Truan, Gilles; Moréra, Solange

    2009-01-01

    A 2.5 Å resolution data set was collected from a crystal of a soluble chimeric form of NADPH-cytochrome P450 reductase (CPR) produced using a fusion gene composed of the yeast FMN and the human FAD domains. The chimeric protein was crystallized in a modified conformation compared with the previously solved structures. NADPH-cytochrome P450 reductase (CPR) is the favoured redox partner of microsomal cytochromes P450. This protein is composed of two flavin-containing domains (FMN and FAD) connected by a structured linker. An active CPR chimera consisting of the yeast FMN and human FAD domains has been produced, purified and crystallized. The crystals belonged to the monoclinic space group C2 and contained one molecule per asymmetric unit. Molecular replacement was performed using the published rat and yeast structures as search models. The initial electron-density maps revealed that the chimeric enzyme had crystallized in a conformation that differed from those of previously solved structures

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  2. Classification of Cytochrome P450 1A2 Inhibitors and Non-Inhibitors by Machine Learning Techniques

    DEFF Research Database (Denmark)

    Vasanthanathan, Poongavanam; Taboureau, Olivier; Oostenbrink, Chris

    2009-01-01

    of CYP1A2 inhibitors and non-inhibitors. Training and test sets consisted of about 400 and 7000 compounds, respectively. Various machine learning techniques, like binary QSAR, support vector machine (SVM), random forest, kappa nearest neighbors (kNN), and decision tree methods were used to develop...

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-01-18

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

  4. An extensive (co-expression analysis tool for the cytochrome P450 superfamily in Arabidopsis thaliana

    Directory of Open Access Journals (Sweden)

    Provart Nicholas J

    2008-04-01

    Full Text Available Abstract Background Sequencing of the first plant genomes has revealed that cytochromes P450 have evolved to become the largest family of enzymes in secondary metabolism. The proportion of P450 enzymes with characterized biochemical function(s is however very small. If P450 diversification mirrors evolution of chemical diversity, this points to an unexpectedly poor understanding of plant metabolism. We assumed that extensive analysis of gene expression might guide towards the function of P450 enzymes, and highlight overlooked aspects of plant metabolism. Results We have created a comprehensive database, 'CYPedia', describing P450 gene expression in four data sets: organs and tissues, stress response, hormone response, and mutants of Arabidopsis thaliana, based on public Affymetrix ATH1 microarray expression data. P450 expression was then combined with the expression of 4,130 re-annotated genes, predicted to act in plant metabolism, for co-expression analyses. Based on the annotation of co-expressed genes from diverse pathway annotation databases, co-expressed pathways were identified. Predictions were validated for most P450s with known functions. As examples, co-expression results for P450s related to plastidial functions/photosynthesis, and to phenylpropanoid, triterpenoid and jasmonate metabolism are highlighted here. Conclusion The large scale hypothesis generation tools presented here provide leads to new pathways, unexpected functions, and regulatory networks for many P450s in plant metabolism. These can now be exploited by the community to validate the proposed functions experimentally using reverse genetics, biochemistry, and metabolic profiling.

  5. Structural and Kinetic Studies of Novel Cytochrome P450 Small-Alkane Hydroxylases

    Energy Technology Data Exchange (ETDEWEB)

    Arnold, Frances H.

    2012-02-27

    The goals of this project are to investigate (1) the kinetics and stabilities of engineered cytochrome P450 (P450) small alkane hydroxylases and their evolutionary intermediates, (2) the structural basis for catalytic proficiency on small alkanes of these engineered P450s, and (3) the changes in redox control resulting from protein engineering. To reach these goals, we have established new methods for determining the kinetics and stabilities of multicomponent P450s such as CYP153A6. Using these, we were able to determine that CYP153A6 is proficient for hydroxylation of alkanes as small as ethane, an activity that has never been observed previously in any natural P450. To elucidate the structures of the engineered P450s, we obtained x-ray diffraction data for two variants in the P450PMO (propane monooxygenase) lineage and a preliminary structure for the most evolved variant. This structure shows changes in the substrate binding regions of the enzyme and a reduction in active site volume that are consistent with the observed changes in substrate specificity from fatty acids in the native enzyme to small alkanes in P450PMO. We also constructed semi-rational designed libraries mutating only residues in the enzyme active site that in one round of mutagenesis and screening produced variants that achieved nearly half of the activity of the most evolved enzymes of the P450PMO lineage. Finally, we found that changes in redox properties of the laboratory-evolved P450 alkane hydroxylases did not reflect the improvement in their electron transfer efficiency. The heme redox potential remained constant throughout evolution, while activity increased and coupling efficiency improved from 10% to 90%. The lack of correlation between heme redox potential and enzyme activity and coupling efficiency led us to search for other enzyme properties that could be better predictors for activity towards small alkanes, specifically methane. We investigated the oxidation potential of the radical

  6. "Possible involvement of the long terminal repeat of transposable element 17.6 in regulating expression of an insecticide resistance-associated P450 gene in Drosophila.".

    OpenAIRE

    Waters, L C; Zelhof, A C; Shaw, B J; Ch'ang, L Y

    1992-01-01

    P450-A and P450-B are electrophoretically defined subsets of cytochrome P450 enzymes in Drosophila melanogaster. P450-A is present among all strains tested, whereas expression of P450-B is associated with resistance to insecticides. Monoclonal antibodies were used to obtain cDNA clones for an enzyme from each P450 subset (i.e., P450-A1 and P450-B1). The P450-B1 cDNA was sequenced and shown to code for a P450 of 507 amino acids. Its gene has been named CYP6A2. Comparative molecular analyses of...

  7. Enzyme induction and cytotoxicity in human hepatocytes by chlorpyrifos and N,N-diethyl-m-toluamide (DEET).

    Science.gov (United States)

    Das, Parikshit C; Cao, Yan; Rose, Randy L; Cherrington, Nathan; Hodgson, Ernest

    2008-01-01

    Xenobiotics, including drugs and environmental chemicals, can influence cytochrome P450 (CYP) levels by altering the transcription of CYP genes. To minimize potential drug-pesticide and pesticide-pesticide interactions it is important to evaluate the potential of pesticides to induce CYP isoforms and to cause cytotoxicity in humans. The present study was designed to examine chlorpyrifos and DEET mediated induction of CYP isoforms and also to characterize their potential cytotoxic effects on primary human hepatocytes. DEET significantly induced CYP3A4, CYP2B6, CYP2A6 and CYP1A2 mRNA expression while chlorpyrifos induced CYP1A1, CYP1A2 and CYP3A4 mRNA, and to a lesser extent, CYP1B1 and CYP2B6 mRNA in primary human hepatocytes. Chlorpyrifos and DEET also mediated the expression of CYP isoforms, particularly CYP3A4, CYP2B6 and CYP1A1, as shown by CYP3A4-specific protein expression, testosterone metabolism and CYP1Al-specific activity assays. DEET is a mild, while chlorpyrifos is a relatively potent, inducer of adenylate kinase and caspase-3/7, an indicator of apoptosis, while inducing 15-20% and 25-30% cell death, respectively. Therefore, DEET and chlorpyrifos mediated induction of CYP mRNA and functional CYP isoforms together with their cytotoxic potential in human hepatocytes suggests that exposure to chlorpyrifos and/or DEET should be considered in human health impact analysis.

  8. Cytochrome P450 from Plants: Platforms for Valuable ...

    African Journals Online (AJOL)

    diverse secondary metabolites with biological properties. ... enzymes actively involved in the biosynthesis of secondary ... toxic to humans and is an approved food additive commonly used in ..... fungus with medicinal and nutritional properties,.

  9. Clinical, genetic, and enzymatic characterization of P450 oxidoreductase deficiency in four patients.

    LENUS (Irish Health Repository)

    Sahakitrungruang, Taninee

    2009-12-01

    P450 oxidoreductase (POR) deficiency causes disordered steroidogenesis; severe mutations cause genital ambiguity in both sexes plus the Antley-Bixler skeletal malformation syndrome, whereas mild mutations can cause adult infertility.

  10. An expression tag toolbox for microbial production of membrane bound plant cytochromes P450

    DEFF Research Database (Denmark)

    Vazquez Albacete, Dario; Cavaleiro, Mafalda; Christensen, Ulla

    2017-01-01

    of the intermediate and the final product of the pathway. Finally, the effect of a robustly performing expression tag was explored with a library of 49 different P450s from medicinal plants and nearly half of these were improved in expression by more than 2-fold. The developed toolbox serves as platform to tune P450...... tag chimeras of the model plant P450 CYP79A1 in different Escherichia coli strains. Using a high-throughput screening platform based on C-terminal GFP fusions, we identify several highly expressing and robustly performing chimeric designs. Analysis of long-term cultures by flow cytometry showed...... homogeneous populations for some of the conditions. Three chimeric designs were chosen for a more complex combinatorial assembly of a multigene pathway consisting of two P450s and a redox partner. Cells expressing these recombinant enzymes catalysed the conversion of the substrate to highly different ratios...

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  13. Role of cytochrome P450 genotype in the steps toward personalized drug therapy

    Directory of Open Access Journals (Sweden)

    Cavallari LH

    2011-11-01

    Full Text Available Larisa H Cavallari1,2, Hyunyoung Jeong1,2, Adam Bress11Department of Pharmacy Practice, 2Department of Biopharmaceutical Sciences, College of Pharmacy, University of Illinois at Chicago, Chicago, IL, USAAbstract: Genetic polymorphism for cytochrome 450 (P450 enzymes leads to interindividual variability in the plasma concentrations of many drugs. In some cases, P450 genotype results in decreased enzyme activity and an increased risk for adverse drug effects. For example, individuals with the CYP2D6 loss-of-function genotype are at increased risk for ventricular arrhythmia if treated with usual does of thioridazine. In other cases, P450 genotype may influence the dose of a drug required to achieve a desired effect. This is the case with warfarin, with lower doses often necessary in carriers of a variant CYP2C9*2 or *3 allele to avoid supratherapeutic anticoagulation. When a prodrug, such as clopidogrel or codeine, must undergo hepatic biotransformation to its active form, a loss-of-function P450 genotype leads to reduced concentrations of the active drug and decreased drug efficacy. In contrast, patients with multiple CYP2D6 gene copies are at risk for opioid-related toxicity if treated with usual doses of codeine-containing analgesics. At least 25 drugs contain information in their US Food and Drug Administration-approved labeling regarding P450 genotype. The CYP2C9, CYP2C19, and CYP2D6 genes are the P450 genes most often cited. To date, integration of P450 genetic information into clinical decision making is limited. However, some institutions are beginning to embrace routine P450 genotyping to assist in the treatment of their patients. Genotyping for P450 variants may carry less risk for discrimination compared with genotyping for disease-associated variants. As such, P450 genotyping is likely to lead the way in the clinical implementation of pharmacogenomics. This review discusses variability in the CYP2C9, CYP2C19, and CYP2D6 genes and the

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

    Science.gov (United States)

    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.

  15. Export of Cytochrome P450 105D1 to the Periplasmic Space of Escherichia coli

    OpenAIRE

    Kaderbhai, Mustak A.; Ugochukwu, Cynthia C.; Kelly, Steven L.; Lamb, David C.

    2001-01-01

    CYP105D1, a cytochrome P450 from Streptomyces griseus, was appended at its amino terminus to the secretory signal of Escherichia coli alkaline phosphatase and placed under the transcriptional control of the native phoA promoter. Heterologous expression in E. coli phosphate-limited medium resulted in abundant synthesis of recombinant CYP105D1 that was translocated across the bacterial inner membrane and processed to yield authentic, heme-incorporated P450 within the periplasmic space. Cell ext...

  16. Cytochrome P-450 complex formation in rat liver by the antibiotic tiamulin.

    Science.gov (United States)

    Witkamp, R F; Nijmeijer, S M; van Miert, A S

    1996-01-01

    Tiamulin is a semisynthetic diterpene antibiotic frequently used in farm animals. The drug has been shown to produce clinically important--often lethal--interactions with other compounds. It has been suggested that this is caused by a selective inhibition of oxidative drug metabolism via the formation of a cytochrome P-450 metabolic intermediate complex. In the present study, rats were treated orally for 6 days with tiamulin at two different doses: 40 and 226 mg/kg of body weight. For comparison, another group received 300 mg of triacetyloleandomycin (TAO) per kg, which is equivalent to the 226-mg/kg tiamulin group. Subsequently, microsomal P-450 contents, P-450 enzyme activities, metabolic intermediate complex spectra, and P-450 apoprotein concentrations were assessed. In addition, effects on individual microsomal P-450 activities were studied in control microsomes at different tiamulin and substrate concentrations. In the rats treated with tiamulin, a dose-dependent complex formation as evidenced by its absorption spectrum and an increase in cytochrome P-4503A1/2 contents as assessed by Western blotting (immunoblotting) were found. The effects were comparable to those of TAO. Tiamulin induced microsomal P-450 content, testosterone 6 beta-hydroxylation rate, erythromycin N-demethylation rate, and the ethoxyresorufin O-deethylation activity. Other activities were not affected or decreased. When tiamulin was added to microsomes of control rats, the testosterone 6 beta-hydroxylation rate and the erythromycin N-demethylation were strongly inhibited. It is concluded that tiamulin is a potent and selective inducer-inhibitor of cytochrome P-450. Though not belonging to the macrolides, the compound produces an effect on P-450 similar to those of TAO and related compounds.

  17. Chemoenzymatic elaboration of monosaccharides using engineered cytochrome P450_(BM3) demethylases

    OpenAIRE

    Lewis, Jared C.; Bastian, Sabine; Bennett, Clay S.; Fu, Yu; Mitsuda, Yuuichi; Chen, Mike M.; Greenberg, William A.; Wong, Chi-Huey; Arnold, Frances H.

    2009-01-01

    Polysaccharides comprise an extremely important class of biopolymers that play critical roles in a wide range of biological processes, but the synthesis of these compounds is challenging because of their complex structures. We have developed a chemoenzymatic method for regioselective deprotection of monosaccharide substrates using engineered Bacillus megaterium cytochrome P450 (P450_(BM3)) demethylases that provides a highly efficient means to access valuable intermediate...

  18. Evolution of NADPH-cytochrome P450 oxidoreductases (POR) in Apiales - POR 1 is missing

    DEFF Research Database (Denmark)

    Andersen, Trine Bundgaard; Hansen, Niels Bjørn; Laursen, Tomas

    2016-01-01

    The NADPH-dependent cytochrome P450 oxidoreductase (POR) is the obligate electron donor to eukaryotic microsomal cytochromes P450 enzymes. The number of PORs within plant species is limited to one to four isoforms, with the most common being two PORs per plant. These enzymes provide electrons to ...... (available from the SRA at NCBI). All three genes were shown to be functional upon reconstitution into nanodiscs, confirming that none of the isoforms are pseudogenes....

  19. Cytochrome P-450 dependent ethanol oxidation. Kinetic isotope effects and absence of stereoselectivity

    International Nuclear Information System (INIS)

    Ekstroem, G.; Norsten, C.; Cronholm, T.; Ingelman-Sundberg, M.

    1987-01-01

    Deuterium isotope effects [/sup D/(V/K)] and stereoselectivity of ethanol oxidation in cytochrome P-450 containing systems and in the xanthine-xanthine oxidase system were compared with those of yeast alcohol dehydrogenase. The isotope effects were determined by using both a noncompetitive method, including incubation of unlabeled of [1,1- 2 H 2 ] ethanol at various concentrations, and a competitive method, where 1:1 mixtures of [1- 13 C]- and [ 2 H 6 ] ethanol or [2,2,2- 2 H 3 ]- and [1,1- 2 H 2 ] ethanol were incubated and the acetaldehyde formed was analyzed by gas chromatography/mass spectrometry. The /sup D/(V/K) isotope effects of the cytochrome P-450 dependent ethanol oxidation were about 4 with liver microsomes from imidazole-, phenobarbital- or acetone-treated rabbits or with microsomes from acetone- or ethanol-treated rats. Similar isotope effects were reached with reconstituted membranes containing the rabbit ethanol-inducible cytochrome P-450 (LMeb), whereas control rat microsomes and membranes containing rabbit phenobarbital-inducible P-450 LM 2 oxidized the alcohol with /sup D/(V/K) of about 2.8 and 1.8, respectively. Addition of Fe/sup III/EDTA either to microsomes from phenobarbital-treated rabbits or to membranes containing P-450 LMeb significantly lowered the isotope effect. Incubations of all cytochrome P-450 containing systems of the xanthine-xanthine oxidase systems with (1R)- and (1S)-[1- 2 H] ethanol, revealed, taking the isotope effects into account, that 44-66% of the ethanol oxidized had lost the 1-pro-R hydrogen. The data indicate that cytochrome P-450 dependent ethanol oxidation is not stereospecific and that cleavage of the C 1 -H bond appears to be a rate-determining step in the catalysis by the ethanol-inducible form of P-450. The contribution of hydroxyl radicals in ethanol oxidation by the various enzymic systems is discussed

  20. Microbial P450 Enzymes in Bioremediation and Drug Discovery: Emerging Potentials and Challenges.

    Science.gov (United States)

    Bhattacharya, Sukanta S; Yadav, Jagjit S

    2018-01-01

    Cytochrome P450 enzymes are a structurally conserved but functionally diverse group of heme-containing mixed function oxidases found across both prokaryotic and eukaryotic forms of the microbial world. Microbial P450s are known to perform diverse functions ranging from the synthesis of cell wall components to xenobiotic/drug metabolism to biodegradation of environmental chemicals. Conventionally, many microbial systems have been reported to mimic mammalian P450-like activation of drugs and were proposed as the in-vitro models of mammalian drug metabolism. Recent reports suggest that native or engineered forms of specific microbial P450s from these and other microbial systems could be employed for desired specific biotransformation reactions toward natural and synthetic (drug) compounds underscoring their emerging potential in drug improvement and discovery. On the other hand, microorganisms particularly fungi and actinomycetes have been shown to possess catabolic P450s with unusual potential to degrade toxic environmental chemicals including persistent organic pollutants (POPs). Wood-rotting basidiomycete fungi in particular have revealed the presence of exceptionally large P450 repertoire (P450ome) in their genomes, majority of which are however orphan (with no known function). Our pre- and post-genomic studies have led to functional characterization of several fungal P450s inducible in response to exposure to several environmental toxicants and demonstration of their potential in bioremediation of these chemicals. This review is an attempt to summarize the postgenomic unveiling of this versatile enzyme superfamily in microbial systems and investigation of their potential to synthesize new drugs and degrade persistent pollutants, among other biotechnological applications. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

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

    Science.gov (United States)

    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.

  2. Third international symposium: Cytochrome P450 biodiversity. Final report, January 1, 1995--December 31, 1995

    Energy Technology Data Exchange (ETDEWEB)

    Loper, J.C.

    1997-03-01

    The Symposium was held on October 8-12, 1995 at the Marine Biological Laboratory in Woods Hole Massachusetts. Other international symposia promote cytochrome P450 research but have a primary focus on mammalian systems. This symposium is exclusively devoted to research in other organisms, and major topics reflect the distribution and dominance of non-mammalian species in the biosphere. The five sessions focused on basic mechanism, regulation, biodiversity, host-parasite interactions, and practical applications. 170 Scientists contributed 38 oral presentations and 91 posters, with a truly international composition of the symposium. Practical applications were a recurring feature, linking reports on mechanism and regulation to studies on the engineering of substrate specificity, microorganisms to degrade halogenated hydrocarbons and herbicides, and the production of in vitro P450 electrochemical bioreactors. At the time of the symposium there were 477 cytochrome P450 sequences in the database. Expansion of the known plant P450 genes was reported, with 20 new plant P450 families added in the last 3 years. Of these only 5 families have a physiological function associated with them. A growing number of identified invertebrate P450s was documented, where in insects, the forms identified are primarily involved in inducible xenobiotic metabolism and detoxification of toxic plant substances.

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Lehlohonolo Benedict Qhanya

    Full Text Available 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

  5. Regulation of P450 oxidoreductase by gonadotropins in rat ovary and its effect on estrogen production

    Directory of Open Access Journals (Sweden)

    Uesaka Miki

    2008-12-01

    Full Text Available Abstract Background P450 oxidoreductase (POR catalyzes electron transfer to microsomal P450 enzymes. Its deficiency causes Antley-Bixler syndrome (ABS, and about half the patients with ABS have ambiguous genitalia and/or impaired steroidogenesis. POR mRNA expression is up-regulated when mesenchymal stem cells (MSCs differentiate into steroidogenic cells, suggesting that the regulation of POR gene expression is important for steroidogenesis. In this context we examined the regulation of POR expression in ovarian granulosa cells by gonadotropins, and its possible role in steroidogenesis. Methods Changes in gene expression in MSCs during differentiation into steroidogenic cells were examined by DNA microarray analysis. Changes in mRNA and protein expression of POR in the rat ovary or in granulosa cells induced by gonadotropin treatment were examined by reverse transcription-polymerase chain reaction and western blotting. Effects of transient expression of wild-type or mutant (R457H or V492E POR proteins on the production of estrone in COS-7 cells were examined in vitro. Effects of POR knockdown were also examined in estrogen producing cell-line, KGN cells. Results POR mRNA was induced in MSCs following transduction with the SF-1 retrovirus, and was further increased by cAMP treatment. Expression of POR mRNA, as well as Cyp19 mRNA, in the rat ovary were induced by equine chorionic gonadotropin and human chorionic gonadotropin. POR mRNA and protein were also induced by follicle stimulating hormone in primary cultured rat granulosa cells, and the induction pattern was similar to that for aromatase. Transient expression of POR in COS-7 cells, which expressed a constant amount of aromatase protein, greatly increased the rate of conversion of androstenedione to estrone, in a dose-dependent manner. The expression of mutant POR proteins (R457H or V492E, such as those found in ABS patients, had much less effect on aromatase activity than expression of wild

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

    Directory of Open Access Journals (Sweden)

    Martin Krøyer Rasmussen

    2014-09-01

    Full Text Available 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.

  7. Expression induction of P450 genes by imidacloprid in Nilaparvata lugens: A genome-scale analysis.

    Science.gov (United States)

    Zhang, Jianhua; Zhang, Yixi; Wang, Yunchao; Yang, Yuanxue; Cang, Xinzhu; Liu, Zewen

    2016-09-01

    The overexpression of P450 monooxygenase genes is a main mechanism for the resistance to imidacloprid, a representative neonicotinoid insecticide, in Nilaparvata lugens (brown planthopper, BPH). However, only two P450 genes (CYP6AY1 and CYP6ER1), among fifty-four P450 genes identified from BPH genome database, have been reported to play important roles in imidacloprid resistance until now. In this study, after the confirmation of important roles of P450s in imidacloprid resistance by the synergism analysis, the expression induction by imidacloprid was determined for all P450 genes. In the susceptible (Sus) strain, eight P450 genes in Clade4, eight in Clade3 and two in Clade2 were up-regulated by imidacloprid, among which three genes (CYP6CS1, CYP6CW1 and CYP6ER1, all in Clade3) were increased to above 4.0-fold and eight genes to above 2.0-fold. In contrast, no P450 genes were induced in Mito clade. Eight genes induced to above 2.0-fold were selected to determine their expression and induced levels in Huzhou population, in which piperonyl butoxide showed the biggest effects on imidacloprid toxicity among eight field populations. The expression levels of seven P450 genes were higher in Huzhou population than that in Sus strain, with the biggest differences for CYP6CS1 (9.8-fold), CYP6ER1 (7.7-fold) and CYP6AY1 (5.1-fold). The induction levels for all tested genes were bigger in Sus strain than that in Huzhou population except CYP425B1. Screening the induction of P450 genes by imidacloprid in the genome-scale will provide an overall view on the possible metabolic factors in the resistance to neonicotinoid insecticides. The further work, such as the functional study of recombinant proteins, will be performed to validate the roles of these P450s in imidacloprid resistance. Copyright © 2015 Elsevier B.V. All rights reserved.

  8. HPLC Determination of Caffeine and Paraxanthine in Urine: An Assay for Cytochrome P450 1A2 Activity

    Science.gov (United States)

    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…

  9. Effect of low-dose ritonavir (100 mg twice daily) on the activity of cytochrome P450 2D6 in healthy volunteers

    NARCIS (Netherlands)

    Aarnoutse, Rob E; Kleinnijenhuis, Johanneke; Koopmans, Peter P; Touw, Daan J; Wieling, Jaap; Hekster, Yechiel A; Burger, David M

    2005-01-01

    OBJECTIVE: In the treatment of human immunodeficiency virus infection, the protease inhibitor ritonavir is used in a low dose (100 mg twice daily) to inhibit cytochrome P450 (CYP) 3A4 and thereby increase plasma concentrations of coadministered protease inhibitors. When applied in a therapeutic dose

  10. Effect of low-dose ritonavir (100 mg twice daily) on the activity of cytochrome P450 2D6 in healthy volunteers.

    NARCIS (Netherlands)

    Aarnoutse, R.E.; Kleinnijenhuis, J.; Koopmans †, P.P.; Touw, D.J.; Wieling, J.; Hekster, Y.A.; Burger, D.M.

    2005-01-01

    OBJECTIVE: In the treatment of human immunodeficiency virus infection, the protease inhibitor ritonavir is used in a low dose (100 mg twice daily) to inhibit cytochrome P450 (CYP) 3A4 and thereby increase plasma concentrations of coadministered protease inhibitors. When applied in a therapeutic dose

  11. Evaluation of a novel high-throughput assay for cytochrome P450 2D6 using 7-methoxy-4-(aminomethyl)-coumarin

    NARCIS (Netherlands)

    Venhorst, J.; Onderwater, R C; Meerman, J H; Vermeulen, N P; Commandeur, J N

    2000-01-01

    We recently reported on the design, synthesis and characterisation of a novel and selective substrate of human cytochrome P450 2D6 (CYP2D6), 7-methoxy-4-(aminomethyl)-coumarin (MAMC). Here, we describe a high-throughput microplate reader assay, which makes use of MAMC as a fluorescent probe for

  12. Evaluation of a novel high-throughput assay for Cytochrome P450 2D6 using 7-Methoxy-4-(Aminomethyl)-Coumarin.

    NARCIS (Netherlands)

    Venhorst, J.; Onderwater, R.C.A.; Meerman, J.H.N.; Commandeur, J.N.M.; Vermeulen, N.P.E.

    2000-01-01

    We recently reported on the design, synthesis and characterisation of a novel and selective substrate of human cytochrome P450 2D6 (CYP2D6), 7-methoxy-4-(aminomethyl)-coumarin (MAMC). Here, we describe a high-throughput microplate reader assay, which makes use of MAMC as a fluorescent probe for

  13. Cytochrome P450-mediated metabolism of the synthetic cannabinoids UR-144 and XLR-11

    DEFF Research Database (Denmark)

    Nielsen, Line Marie; Holm, Niels Bjerre; Olsen, Lars

    2016-01-01

    In recent years, synthetic cannabinoids have emerged in the illicit drug market, in particular via the Internet, leading to abuse of these drugs. There is currently limited knowledge about the specific enzymes involved in the metabolism of these drugs. In this study, we investigated the cytochrome...... of UR-144 and XLR-11, while inhibition of the other CYP enzymes in HLM had only minor effects. Thus, CYP3A4 is the major contributor to the CYP mediated metabolism of UR-144 and XLR-11 with minor contributions from CYP1A2. Users of UR-144 and XLR-11 are thus subject to the influence of potential drug-drug...... interactions, if they are concomitantly medicated with CYP3A4 inducers (e.g. some antiepileptics) or inhibitors (e.g. some antifungal drugs). Copyright © 2015 John Wiley & Sons, Ltd....

  14. Hepatic Metabolism of Sakuranetin and Its Modulating Effects on Cytochrome P450s and UDP-Glucuronosyltransferases

    Directory of Open Access Journals (Sweden)

    Hyesoo Jeong

    2018-06-01

    Full Text Available Sakuranetin (SKN, found in cherry trees and rice, is a flavanone with various pharmacological activities. It is biosynthesized from naringenin in rice or cherry trees, and the metabolism of SKN has been studied in non-human species. The present study aimed to investigate the metabolic pathways of SKN in human liver microsomes and identify the phase I and phase II metabolites, as well as evaluate the potential for drug–herb interactions through the modulation of drug metabolizing enzymes (DMEs. HPLC-DAD and HPLC-electrospray mass spectrometry were used to study the metabolic stability and identify the metabolites from human liver microsomes incubated with SKN. The potential of SKN to inhibit the DMEs was evaluated by monitoring the formation of a DME-specific product. The cytochrome P450 2B6 and 3A4-inductive effects were studied using promoter reporter assays in human hepatocarcinoma cells. The major pathways for SKN metabolism include B-ring hydroxylation, 5-O-demethylation, and conjugation with glutathione or glucuronic acid. The phase I metabolites were identified as naringenin and eriodictyol. SKN was found to be a UDP-glucuronosyltransferases (UGT 1A9 inhibitor, whereas it induced transactivation of the human pregnane X receptor-mediated cytochrome P450 (CYP 3A4 gene.

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

    Science.gov (United States)

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

    2016-01-01

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

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

    International Nuclear Information System (INIS)

    Estabrook, Ronald W.

    2005-01-01

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

  17. Metabolic imidacloprid resistance in the brown planthopper, Nilaparvata lugens, relies on multiple P450 enzymes.

    Science.gov (United States)

    Zhang, Yixi; Yang, Yuanxue; Sun, Huahua; Liu, Zewen

    2016-12-01

    Target insensitivity contributing to imidacloprid resistance in Nilaparvata lugens has been reported to occur either through point mutations or quantitative change in nicotinic acetylcholine receptors (nAChRs). However, the metabolic resistance, especially the enhanced detoxification by P450 enzymes, is the major mechanism in fields. From one field-originated N. lugens population, an imidacloprid resistant strain G25 and a susceptible counterpart S25 were obtained to analyze putative roles of P450s in imidacloprid resistance. Compared to S25, over-expression of twelve P450 genes was observed in G25, with ratios above 5.0-fold for CYP6AY1, CYP6ER1, CYP6CS1, CYP6CW1, CYP4CE1 and CYP425B1. RNAi against these genes in vivo and recombinant tests on the corresponding proteins in vitro revealed that four P450s, CYP6AY1, CYP6ER1, CYP4CE1 and CYP6CW1, played important roles in imidacloprid resistance. The importance of the four P450s was not equal at different stages of resistance development based on their over-expression levels, among which CYP6ER1 was important at all stages, and that the others might only contribute at certain stages. The results indicated that, to completely reflect roles of P450s in insecticide resistances, their over-expression in resistant individuals, expression changes at the stages of resistance development, and catalytic activities against insecticides should be considered. In this study, multiple P450s, CYP6AY1, CYP6ER1, CYP4CE1 and CYP6CW1, have proven to be important in imidacloprid resistance. Copyright © 2016 Elsevier Ltd. All rights reserved.

  18. Cytochrome P-450-catalyzed desaturation of valproic acid in vitro. Species differences, induction effects, and mechanistic studies

    International Nuclear Information System (INIS)

    Rettie, A.E.; Boberg, M.; Rettenmeier, A.W.; Baillie, T.A.

    1988-01-01

    The cytochrome P-450-mediated desaturation of valproic acid (VPA) to its hepatotoxic metabolite, 2-n-propyl-4-pentenoic acid (4-ene-VPA), was examined in liver microsomes from rats, mice, rabbits and humans. The highest substrate turnover was found with microsomes from rabbits (44.2 +/- 2.7 pmol of product/nmol P-450/15 min), while lower activities were observed in preparations from human, mouse, and rat liver, in that order. Pretreatment of animals with phenobarbital led to enhanced rates of formation of 4-ene-VPA in vitro and yielded induction ratios for desaturation ranging from 2.5 to 8.4, depending upon the species. Comparative studies in the rat showed that phenobarbital is a more potent inducer of olefin formation than either phenytoin or carbamazepine. The mechanism of the desaturation reaction was studied by inter- and intramolecular deuterium isotope effect experiments, which demonstrated that removal of a hydrogen atom from the subterminal C-4 position of VPA is rate limiting in the formation of both 4-ene- and 4-hydroxy-VPA. Hydroxylation at the neighboring C-5 position, on the other hand, was highly sensitive to deuterium substitution at that site, but not to deuteration at C-4. Based on these findings, it is proposed that 4-ene- and 4-hydroxy-VPA are products of a common P-450-dependent metabolic pathway, in which a carbon-centered free radical at C-4 serves as the key intermediate. 5-Hydroxy-VPA, in contrast, derives from an independent hydroxylation reaction

  19. Low dose trichloroethylene alters cytochrome P450 - 2C subfamily expression in the developing chick heart

    Science.gov (United States)

    Makwana, Om; Ahles, Lauren; Lencinas, Alejandro; Selmin, Ornella I.; Runyan, Raymond B.

    2013-01-01

    Trichloroethylene (TCE) is an organic solvent and common environmental contaminant. TCE exposure is associated with heart defects in humans and animal models. Primary metabolism of TCE in adult rodent models is by specific hepatic cytochrome P450 enzymes (Lash et al., 2000). As association of TCE exposure with cardiac defects is in exposed embryos prior to normal liver development, we investigated metabolism of TCE in the early embryo. Developing chick embryos were dosed in ovo with environmentally relevant doses of TCE (8 ppb and 800 ppb) and RNA was extracted from cardiac and extra-cardiac tissue (whole embryo without heart). Real time PCR showed upregulation of CYP2H1 transcripts in response to TCE exposure in the heart. No detectable cytochrome expression was found in extra-cardiac tissue. As seen previously, the dose response was non-monotonic and 8ppb elicited stronger upregulation than 800 ppb. Immunostaining for CYP2C subfamily expression confirmed protein expression and showed localization in both myocardium and endothelium. TCE exposure increased protein expression in both tissues. These data demonstrate that the earliest embryonic expression of phase I detoxification enzymes is in the developing heart. Expression of these CYPs is likely to be relevant to the susceptibility of the developing heart to environmental teratogens. PMID:22855351

  20. Insights on Cytochrome P450 Enzymes and Inhibitors Obtained Through QSAR Studies

    Directory of Open Access Journals (Sweden)

    Maryam Foroozesh

    2012-08-01

    Full Text Available The cytochrome P450 (CYP superfamily of heme enzymes play an important role in the metabolism of a large number of endogenous and exogenous compounds, including most of the drugs currently on the market. Inhibitors of CYP enzymes have important roles in the treatment of several disease conditions such as numerous cancers and fungal infections in addition to their critical role in drug-drug interactions. Structure activity relationships (SAR, and three-dimensional quantitative structure activity relationships (3D-QSAR represent important tools in understanding the interactions of the inhibitors with the active sites of the CYP enzymes. A comprehensive account of the QSAR studies on the major human CYPs 1A1, 1A2, 1B1, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4 and a few other CYPs are detailed in this review which will provide us with an insight into the individual/common characteristics of the active sites of these enzymes and the enzyme-inhibitor interactions.