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Sample records for human cyp3a locus

  1. Phenotype-genotype variability in the human CYP3A locus as assessed by the probe drug quinine and analyses of variant CYP3A4 alleles

    International Nuclear Information System (INIS)

    Rodriguez-Antona, Cristina; Sayi, Jane G.; Gustafsson, Lars L.; Bertilsson, Leif; Ingelman-Sundberg, Magnus

    2005-01-01

    The human cytochrome P450 3A (CYP3A) enzymes, which metabolize 50% of currently used therapeutic drugs, exhibit great interindividual differences in activity that have a major impact on drug treatment outcome, but hitherto no genetic background importantly contributing to this variation has been identified. In this study we show that CYP3A4 mRNA and hnRNA contents with a few exceptions vary in parallel in human liver, suggesting that mechanisms affecting CYP3A4 transcription, such as promoter polymorphisms, are relevant for interindividual differences in CYP3A4 expression. Tanzanian (n = 143) healthy volunteers were phenotyped using quinine as a CYP3A probe and the results were used for association studies with CYP3A4 genotypes. Carriers of CYP3A4*1B had a significantly lower activity than those with CYP3A4*1 whereas no differences were seen for five other SNPs investigated. Nuclear proteins from the B16A2 hepatoma cells were found to bind with less affinity to the CYP3A4*1B element around -392 bp as compared to CYP3A4*1. The data indicate the existence of a genetic CYP3A4 polymorphism with functional importance for interindividual differences in enzyme expression

  2. Development of Murine Cyp3a Knockout Chimeric Mice with Humanized Liver.

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    Kato, Kota; Ohbuchi, Masato; Hamamura, Satoko; Ohshita, Hiroki; Kazuki, Yasuhiro; Oshimura, Mitsuo; Sato, Koya; Nakada, Naoyuki; Kawamura, Akio; Usui, Takashi; Kamimura, Hidetaka; Tateno, Chise

    2015-08-01

    We developed murine CYP3A knockout ko chimeric mice with humanized liver expressing human P450S similar to those in humans and whose livers and small intestines do not express murine CYP3A this: approach may overcome effects of residual mouse metabolic enzymes like Cyp3a in conventional chimeric mice with humanized liver, such as PXB-mice [urokinase plasminogen activator/severe combined immunodeficiency (uPA/SCID) mice repopulated with over 70% human hepatocytes] to improve the prediction of drug metabolism and pharmacokinetics in humans. After human hepatocytes were transplanted into Cyp3a KO/uPA/SCID host mice, human albumin levels logarithmically increased until approximately 60 days after transplantation, findings similar to those in PXB-mice. Quantitative real-time-polymerase chain reaction analyses showed that hepatic human P450s, UGTs, SULTs, and transporters mRNA expression levels in Cyp3a KO chimeric mice were also similar to those in PXB-mice and confirmed the absence of Cyp3a11 mRNA expression in mouse liver and intestine. Findings for midazolam and triazolam metabolic activities in liver microsomes were comparable between Cyp3a KO chimeric mice and PXB-mice. In contrast, these activities in the intestine of Cyp3a KO chimeric mice were attenuated compared with PXB-mice. Owing to the knockout of murine Cyp3a, hepatic Cyp2b10 and 2c55 mRNA levels in Cyp3a KO/uPA/SCID mice (without hepatocyte transplants) were 8.4- and 61-fold upregulated compared with PXB-mice, respectively. However, human hepatocyte transplantation successfully restored Cyp2b10 level nearly fully and Cyp2c55 level partly (still 13-fold upregulated) compared with those in PXB-mice. Intestinal Cyp2b10 and 2c55 were also repressed by human hepatocyte transplantation in Cyp3a KO chimeric mice. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

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

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    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.

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

    International Nuclear Information System (INIS)

    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.

  5. Tiamulin inhibits human CYP3A4 activity in an NIH/3T3 cell line stably expressing CYP3A4 cDNA.

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    De Groene, E M; Nijmeijer, S M; Horbach, G J; Witkamp, R F

    1995-09-07

    Tiamulin is an antibiotic frequently used in veterinary medicine. The drug has been shown to produce clinically important interactions with other compounds that are administered simultaneously. An NIH/3T3 cell line, stably expressing human cytochrome P450 (EC 1.14.14.1) cDNA (CYP3A4), was used to study the effect of tiamulin on CYP3A4 activity. The 6 beta-hydroxylation activity of testosterone, which is increased in CYP3A4-expressing cells compared to vector-transfected cells, showed reduced activity after incubation with 1 microM tiamulin and was completely reduced to background level after incubation with 2, 5 and 10 microM tiamulin. The CYP3A4-expressing cell line was used in combination with a shuttle vector containing the bacterial lacZ' gene to study the effect of tiamulin on CYP3A4-mediated mutagenicity of aflatoxin B1. The mutation frequency of aflatoxin B1 could be completely inhibited by tiamulin in CYP3A4-expressing cells, but no effect was observed on the mutation frequency of the direct mutagen ethylmethanesulphonate. Western blotting of homogenates of the CYP3A4-expressing cell line showed stabilization of CYP3A4 protein after incubation with tiamulin, supporting the hypothesis that the mechanism of inhibition is by binding of tiamulin to the cytochrome.

  6. Interactions of endosulfan and methoxychlor involving CYP3A4 and CYP2B6 in human HepaRG cells.

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    Savary, Camille C; Jossé, Rozenn; Bruyère, Arnaud; Guillet, Fabrice; Robin, Marie-Anne; Guillouzo, André

    2014-08-01

    Humans are usually exposed to several pesticides simultaneously; consequently, combined actions between pesticides themselves or between pesticides and other chemicals need to be addressed in the risk assessment. Many pesticides are efficient activators of pregnane X receptor (PXR) and/or constitutive androstane receptor (CAR), two major nuclear receptors that are also activated by other substrates. In the present work, we searched for interactions between endosulfan and methoxychlor, two organochlorine pesticides whose major routes of metabolism involve CAR- and PXR-regulated CYP3A4 and CYP2B6, and whose mechanisms of action in humans remain poorly understood. For this purpose, HepaRG cells were treated with both pesticides separately or in mixture for 24 hours or 2 weeks at concentrations relevant to human exposure levels. In combination they exerted synergistic cytotoxic effects. Whatever the duration of treatment, both compounds increased CYP3A4 and CYP2B6 mRNA levels while differently affecting their corresponding activities. Endosulfan exerted a direct reversible inhibition of CYP3A4 activity that was confirmed in human liver microsomes. By contrast, methoxychlor induced this activity. The effects of the mixture on CYP3A4 activity were equal to the sum of those of each individual compound, suggesting an additive effect of each pesticide. Despite CYP2B6 activity being unchanged and increased with endosulfan and methoxychlor, respectively, no change was observed with their mixture, supporting an antagonistic effect. Altogether, our data suggest that CAR and PXR activators endosulfan and methoxychlor can interact together and with other exogenous substrates in human hepatocytes. Their effects on CYP3A4 and CYP2B6 activities could have important consequences if extrapolated to the in vivo situation. Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.

  7. Dual-color fluorescence imaging to monitor CYP3A4 and CYP3A7 expression in human hepatic carcinoma HepG2 and HepaRG cells.

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    Saori Tsuji

    Full Text Available Human adult hepatocytes expressing CYP3A4, a major cytochrome P450 enzyme, are required for cell-based assays to evaluate the potential risk of drug-drug interactions caused by transcriptional induction of P450 enzymes in early-phase drug discovery and development. However, CYP3A7 is preferentially expressed in premature hepatoblasts and major hepatic carcinoma cell lines. The human hepatocellular carcinoma cell line HepaRG possesses a high self-renewal capacity and can differentiate into hepatic cells similar to human adult hepatocytes in vitro. Transgenic HepaRG cells, in which the expression of fluorescent reporters is regulated by 35 kb regulatory elements of CYP3A4, have a distinct advantage over human hepatocytes isolated by collagenase perfusion, which are unstable in culture. Thus, we created transgenic HepaRG and HepG2 cells by replacing the protein-coding regions of human CYP3A4 and CYP3A7 with enhanced green fluorescent protein (EGFP and DsRed reporters, respectively, in a bacterial artificial chromosome vector that included whole regulatory elements. The intensity of DsRed fluorescence was initially high during the proliferation of transgenic HepaRG cells. However, most EGFP-positive cells were derived from those in which DsRed fluorescence was extinguished. Comparative analyses in these transgenic clones showed that changes in the total fluorescence intensity of EGFP reflected fold changes in the mRNA level of endogenous CYP3A4. Moreover, CYP3A4 induction was monitored by the increase in EGFP fluorescence. Thus, this assay provides a real-time evaluation system for quality assurance of hepatic differentiation into CYP3A4-expressing cells, unfavourable CYP3A4 induction, and fluorescence-activated cell sorting-mediated enrichment of CYP3A4-expressing hepatocytes based on the total fluorescence intensities of fluorescent reporters, without the need for many time-consuming steps.

  8. Effect of Curcuma longa on CYP2D6- and CYP3A4-mediated metabolism of dextromethorphan in human liver microsomes and healthy human subjects.

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    Al-Jenoobi, Fahad Ibrahim; Al-Thukair, Areej A; Alam, Mohd Aftab; Abbas, Fawkeya A; Al-Mohizea, Abdullah M; Alkharfy, Khalid M; Al-Suwayeh, Saleh A

    2015-03-01

    Effect of Curcuma longa rhizome powder and its ethanolic extract on CYP2D6 and CYP3A4 metabolic activity was investigated in vitro using human liver microsomes and clinically in healthy human subjects. Dextromethorphan (DEX) was used as common probe for CYP2D6 and CYP3A4 enzymes. Metabolic activity of CYP2D6 and CYP3A4 was evaluated through in vitro study; where microsomes were incubated with NADPH in presence and absence of Curcuma extract. In clinical study phase-I, six healthy human subjects received a single dose (30 mg) of DEX syrup, and in phase-II DEX syrup was administered with Curcuma powder. The enzyme CYP2D6 and CYP3A4 mediated O- and N-demethylation of dextromethorphan into dextrorphan (DOR) and 3-methoxymorphinan (3-MM), respectively. Curcuma extract significantly inhibited the formation of DOR and 3-MM, in a dose-dependent and linear fashion. The 100 μg/ml dose of curcuma extract produced highest inhibition, which was about 70 % for DOR and 80 % for 3-MM. Curcuma significantly increases the urine metabolic ratio of DEX/DOR but the change in DEX/3-MM ratio was statistically insignificant. Present findings suggested that curcuma significantly inhibits the activity of CYP2D6 in in vitro as well as in vivo; which indicates that curcuma has potential to interact with CYP2D6 substrates.

  9. Porcine foetal and neonatal CYP3A liver expression

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    Marie Louise Hiort Hermann

    2011-05-01

    Full Text Available Human cytochrome P450 3A7 (CYP3A7 and cytochrome P450 3A4 (CYP3A4 are hepatic metabolising enzymes which participates in the biotransformation of endo- and exogenous substances in foetuses and neonates respectively. These CYP3A enzymes display an inverse relationship: CYP3A7 is the dominant enzyme in the foetal liver, whereas the expression of CYP3A4 is low. After parturition there is a shift in the expression, thus CYP3A7 is down regulated, while the level of CYP3A4 gradually increases and becomes the dominant metabolising CYP3A enzyme in the adult. The minipig is increasingly being used as a model for humans in biomedical studies, because of its many similarities with the human physiology and anatomy. The aim of this study was to examine whether, as in humans, a shift is seen in the hepatic expression of a CYP3A7- like enzyme to cytochrome P450 3A29 (CYP3A29 (an orthologue to the human CYP3A4 in minipigs. This was elucidated by examining the hepatic mRNA expression of CYP3A7 and CYP3A29 in 39 foetuses and newborn Göttingen minipigs using quantitative real time polymerase chain reaction (qPCR. Furthermore the immunochemical level of CYP3A7-LE and CYP3A29 was measured in liver microsomes using western blotting. The expression of CYP3A29 was approximately 9- fold greater in neonates compared to foetuses, and a similar difference was reflected on the immunochemical level. It was not possible to detect a significant level of foetal CYP3A7 mRNA, but immunoblotting showed a visible difference depending on age. This study demonstrates an increase in the expression of CYP3A29, the CYP3A4 orthologue in perinatal minipigs as in humans, which suggests that the minipig could be a good model when testing for human foetal toxicity towards CYP3A4 substrates.

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

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    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.

  11. Furocoumarins from grapefruit juice and their effect on human CYP 3A4 and CYP 1B1 isoenzymes.

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    Girennavar, Basavaraj; Poulose, Shibu M; Jayaprakasha, Guddadarangavvanahally K; Bhat, Narayan G; Patil, Bhimanagouda S

    2006-04-15

    Bioactive compounds present in grapefruit juice are known to increase the bioavailability of certain medications by acting as potent CYP 3A4 inhibitors. An efficient technique has been developed for isolation and purification of three furocoumarins. The isolated compounds have been tested for the inhibition of human CYP 1B1 isoform using specific substrates. Grapefruit juice was extracted with ethyl acetate (EtOAc) and the dried extract was loaded onto silica gel column chromatography. Further, column fractions were subjected to preparative HPLC to obtain three compounds. The purity of these compounds was analyzed by HPLC and structures were determined by NMR studies. The identified compounds, bergamottin, 6',7'-dihydroxybergamottin (DHB), and paradisin-A, were tested for their inhibitory effects on hydroxylase and O-dealkylase activities of human cytochrome P450 isoenzymes CYP 3A4 and CYP 1B1. Paradisin-A was found to be a potent CYP 3A4 inhibitor with an IC50 of 1.2 microM followed by DHB and bergamottin. All three compounds showed a substantial inhibitory effect on CYP 3A4 below 10 microM. Inhibitory effects on CYP 1B1 exhibited a greater variation due to the specificity of substrates. Paradisin A showed an IC50 of 3.56+/-0.12 microM for the ethoxy resorufin O-dealkylase (EROD) activity and 33.56+/-0.72 microM for the benzyloxy resorufin (BROD). DHB and bergamottin showed considerable variations for EROD and BROD activities with an IC50 of 7.17 microM and 13.86 microM, respectively.

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

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    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.

  13. Assessment of human pregnane X receptor involvement in pesticide-mediated activation of CYP3A4 gene.

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    Matsubara, Tsutomu; Noracharttiyapot, Wachiraporn; Toriyabe, Takayoshi; Yoshinari, Kouichi; Nagata, Kiyoshi; Yamazoe, Yasushi

    2007-05-01

    Assessment of foreign chemical inducibility on CYP3A4 is necessary to optimize drug therapies. The properties of chemicals such as pesticides, however, are not well investigated. In the present study, properties of various pesticides on human CYP3A4 induction have been tested using HepG2-derived cells stably expressing the CYP3A4 promoter/enhancer (3-1-10 cells) and the human pregnane X receptor (hPXR)-small interfering RNA (siRNA) system. Among the examined pesticides, 13 pesticides were observed to activate the CYP3A4 gene. Surprisingly, pyributicarb was found to increase the CYP3A4 reporter activity at 0.1 to 1 microM more strongly than typical CYP3A4 inducer rifampicin. Expression of hPXR-siRNA clearly diminished the pyributicarb-stimulated CYP3A4 reporter activity in 3-1-10 cells and decreased the endogenous CYP3A4 mRNA levels in HepG2 cells. Pyributicarb caused enhancement of CYP3A4-derived reporter activity in mouse livers introduced with hPXR by adenovirus. These results indicate pyributicarb as a potent activator of CYP3A4 gene, suggesting the existence of pesticides leading to CYP3A4 induction in our environment.

  14. CYP2B6, CYP2D6, and CYP3A4 catalyze the primary oxidative metabolism of perhexiline enantiomers by human liver microsomes.

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    Davies, Benjamin J; Coller, Janet K; Somogyi, Andrew A; Milne, Robert W; Sallustio, Benedetta C

    2007-01-01

    The cytochrome P450 (P450)-mediated 4-monohydroxylations of the individual enantiomers of the racemic antianginal agent perhexiline (PHX) were investigated in human liver microsomes (HLMs) to identify stereoselective differences in metabolism and to determine the contribution of the polymorphic enzyme CYP2D6 and other P450s to the intrinsic clearance of each enantiomer. The cis-, trans1-, and trans2-4-monohydroxylation rates of (+)- and (-)-PHX by human liver microsomes from three extensive metabolizers (EMs), two intermediate metabolizers (IMs), and two poor metabolizers (PMs) of CYP2D6 were measured with a high-performance liquid chromatography assay. P450 isoform-specific inhibitors, monoclonal antibodies directed against P450 isoforms, and recombinantly expressed human P450 enzymes were used to define the P450 isoform profile of PHX 4-monohydroxylations. The total in vitro intrinsic clearance values (mean +/- S.D.) of (+)- and (-)-PHX were 1376 +/- 330 and 2475 +/- 321, 230 +/- 225 and 482 +/- 437, and 63.4 +/- 1.6 and 54.6 +/- 1.2 microl/min/mg for the EM, IM, and PM HLMs, respectively. CYP2D6 catalyzes the formation of cis-OH-(+)-PHX and trans1-OH-(+)-PHX from (+)-PHX and cis-OH-(-)-PHX from (-)-PHX with high affinity. CYP2B6 and CYP3A4 each catalyze the trans1- and trans2-4-monohydroxylation of both (+)- and (-)-PHX with low affinity. Both enantiomers of PHX are subject to significant polymorphic metabolism by CYP2D6, although this enzyme exhibits distinct stereoselectivity with respect to the conformation of metabolites and the rate at which they are formed. CYP2B6 and CYP3A4 are minor contributors to the intrinsic P450-mediated hepatic clearance of both enantiomers of PHX, except in CYP2D6 PMs.

  15. In vitro modulatory effects of Terminalia arjuna, arjunic acid, arjunetin and arjungenin on CYP3A4, CYP2D6 and CYP2C9 enzyme activity in human liver microsomes

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    Alice Varghese

    2015-01-01

    Full Text Available Terminalia arjuna is a tree having an extensive medicinal potential in cardiovascular disorders. Triterpenoids are mainly responsible for cardiovascular properties. Alcoholic and aqueous bark extracts of T. arjuna, arjunic acid, arjunetin and arjungenin were evaluated for their potential to inhibit CYP3A4, CYP2D6 and CYP2C9 enzymes in human liver microsomes. We have demonstrated that alcoholic and aqueous bark extract of T. arjuna showed potent inhibition of all three enzymes in human liver microsomes with IC50 values less than 50 μg/mL. Arjunic acid, arjunetin and arjungenin did not show significant inhibition of CYP enzymes in human liver microsomes. Enzyme kinetics studies suggested that the extracts of arjuna showed reversible non-competitive inhibition of all the three enzymes in human liver microsomes. Our findings suggest strongly that arjuna extracts significantly inhibit the activity of CYP3A4, CYP2D6 and CYP2C9 enzymes, which is likely to cause clinically significant drug–drug interactions mediated via inhibition of the major CYP isozymes.

  16. Cytochrome P450 CYP3A in marsupials: cloning and identification of the first CYP3A subfamily member, isoform 3A70 from Eastern gray kangaroo (Macropus giganteus).

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    El-Merhibi, Adaweyah; Ngo, Suong N T; Marchant, Ceilidh L; Height, Tamara A; Stupans, Ieva; McKinnon, Ross A

    2012-09-15

    Australian marsupials are unique fauna that have evolved and adapted to unique environments and thus it is likely that their detoxification systems differ considerably from those of well-studied eutherian mammals. Knowledge of these processes in marsupials is therefore vital to understanding the consequences of exposure to xenobiotics. Cytochromes P450 (CYPs) are critically important in the oxidative metabolism of a diverse array of both xenobiotics and endogenous substrates. In this study we have cloned and characterized CYP3A70, the first identified member of the CYP3A gene subfamily from Eastern gray kangaroo (Macropus giganteus). A 1665 base pair kangaroo hepatic CYP3A complete cDNA, designated CYP3A70, was cloned by reverse transcription-polymerase chain reaction approaches, which encodes a protein of 506 amino acids. The CYP3A70 cDNA shares approximately 71% nucleotide and 65% amino acid sequence homology to human CYP3A4 and displays high sequence similarity to other published mammalian CYP3As from human, monkey, cow, pig, dog, rat, rabbit, mouse, hamster, and guinea pig. Transfection of the CYP3A70 cDNAs into 293T cells resulted in stable cell lines expressing a CYP3A immuno-reactive protein that was recognized by a goat anti-human CYP3A4 polyclonal antibody. The anti-human CYP3A4 antibody also detected immunoreactive proteins in liver microsomes from all test marsupials, including the kangaroo, koala, wallaby, and wombat, with multiple CYP3A immunoreactive bands observed in kangaroo and wallaby tissues. Relatively, very low CYP catalytic activity was detected for the kangaroo CYP3A70 cDNA-expressed proteins (19.6 relative luminescent units/μg protein), which may be due to low protein expression levels. Collectively, this study provides primary molecular data regarding the Eastern kangaroo hepatic CYP3A70 gene and enables further functional analyses of CYP3A enzymes in marsupials. Copyright © 2012 Elsevier B.V. All rights reserved.

  17. Cytochrome P450 CYP3A in marsupials: cloning and characterisation of the second identified CYP3A subfamily member, isoform 3A78 from koala (Phascolarctos cinereus).

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    El-Merhibi, Adaweyah; Ngo, Suong N T; Crittenden, Tamara A; Marchant, Ceilidh L; Stupans, Ieva; McKinnon, Ross A

    2011-11-01

    Cytochromes P450 (CYPs) are critically important in the oxidative metabolism of a diverse array of xenobiotics and endogenous substrates. Previously, we cloned and characterised the CYP2C, CYP4A, and CYP4B gene subfamilies from marsupials and demonstrated important species-differences in both activity and tissue expression of these CYP enzymes. Recently, we isolated the Eastern grey kangaroo CYP3A70. Here we have cloned and characterised the second identified member of marsupial CYP3A gene subfamily, CYP3A78 from the koala (Phascolarctos cinereus). In addition, we have examined the gender-differences in microsomal erythromycin N-demethylation activity (a CYP3A marker) and CYP3A protein expression across test marsupial species. Significant differences in hepatic erythromycin N-demethylation activity were observed between male and female koalas, with the activity detected in female koalas being 2.5-fold higher compared to that in male koalas (p<0.01). No gender-differences were observed in tammar wallaby or Eastern grey kangaroo. Immunoblot analysis utilising anti-human CYP3A4 antibody detected immunoreactive proteins in liver microsomes from all test male and female marsupials including the koala, tammar wallaby, and Eastern grey kangaroo, with no gender-differences detected across test marsupials. A 1610 bp koala hepatic CYP3A complete cDNA, designated CYP3A78, was cloned by reverse transcription-polymerase chain reaction approaches. It displays 64% nucleotide and 57% amino acid sequence identity to the Eastern grey kangaroo CYP3A70. The CYP3A78 cDNA encodes a protein of 515 amino acids, shares approximately 68% nucleotide and 56% amino acid sequence identity to human CYP3A4, and displays high sequence similarity to other published mammalian CYP3As from human, monkey, cow, pig, dog, rat, rabbit, mouse, hamster, and guinea pig. Collectively, this study provides primary molecular data regarding koala hepatic CYP3A78 gene and enables further functional analyses of CYP

  18. Foetal and adult human CYP3A isoforms in the bioactivation of organophosphorothionate insecticides.

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

    2006-12-15

    In humans organophosphorothionate pesticides (OPT) prenatal exposure has been demonstrated. Since OPT-induced neurodevelopmental effects may be due to in situ bioactivation by foetal enzymes, the catalytic activity of the foetal CYP3A7 toward chlorpyrifos (CPF), parathion (PAR), malathion (MAL) and fenthion (FEN) has been assessed by using recombinant enzymes. A comparison with the adult isoforms CYP3A4 and CYP3A5 has been also carried out. CYP3A7 was able to produce significant levels of oxon or sulfoxide from the four OPTs in the range of tested concentrations (0.05-200 microM). When the efficiencies of CYP3A isoforms were compared, the ranking, expressed as CLi values, were: CPF=3A4>3A5>3A7; PAR=3A4>3A7>3A5; MAL=3A4>3A7>3A5; FEN (sulfoxide formation)=3A4>3A5>3A7. The CYP3A5 efficiency appeared to be more dependent on the single insecticide than its related isozyme CYP3A4. Our results indicate that the levels of toxic metabolite formed in situ by CYP3A7 from CPF, MAL and PAR but not from FEN have the chance to inhibit acetylcholinesterase, following prenatal exposure to OPTs. However, due to the smaller weight of foetal liver, the contribution to total OPT biotransformation is relatively low. On the other hand, our results clearly indicate that at low CPF concentrations, the formation of the non-toxic metabolites is highly favoured in the foetus.

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

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    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.

  20. Indirubin, a component of Ban-Lan-Gen, activates CYP3A4 gene transcription through the human pregnane X receptor.

    Science.gov (United States)

    Kumagai, Takeshi; Aratsu, Yusuke; Sugawara, Ryosuke; Sasaki, Takamitsu; Miyairi, Shinichi; Nagata, Kiyoshi

    2016-04-01

    Ban-Lan-Gen is the common name for the dried roots of indigo plants, including Polygonum tinctorium, Isatis indigotica, Isatis tinctoria, and Strobilanthes cusia. Ban-Lan-Gen is frequently used as an anti-inflammatory and an anti-viral for the treatment of hepatitis, influenza, and various types of inflammation. One of the cytochrome P450 (CYP) enzymes, CYP3A4, is responsible for the metabolism of a wide variety of xenobiotics, including an estimated 60% of all clinically used drugs. In this study, we investigated the effect of Ban-Lan-Gen on the transcriptional activation of the CYP3A4 gene. Ban-Lan-Gen extract increased CYP3A4 gene reporter activity in a dose-dependent manner. Indirubin, one of the biologically active ingredients in the Ban-Lan-Gen, also dose-dependently increased CYP3A4 gene reporter activity. Expression of short hairpin RNA for the human pregnane X receptor (hPXR-shRNA) inhibited CYP3A4 gene reporter activity, and overexpression of human PXR increased indirubin- and rifampicin-induced CYP3A4 gene reporter activity. Furthermore, indirubin induced CYP3A4 mRNA expression in HepG2 cells. Taken together, these results indicate that indirubin, a component of Ban-Lan-Gen, activated CYP3A4 gene transcription through the activation of the human PXR. Copyright © 2016. Published by Elsevier Ltd.

  1. 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

  2. PXR-dependent induction of human CYP3A4 gene expression by organochlorine pesticides.

    Science.gov (United States)

    Coumoul, Xavier; Diry, Monique; Barouki, Robert

    2002-11-15

    OCP are xenobiotics which display various toxic effects on animal and human health. One of their effects is to bind and activate estrogen receptor alpha (ERalpha). We have previously studied the down-regulation of induced CYP1A1 (cytochrome P450) expression by this class of molecules in mammary carcinoma cells and shown the importance of ERalpha in this process. However, an alternative mechanism was suggested by those experiments in hepatoma cells. In this study, we have performed Northern blot and transient transfection assays in various cell lines and shown that OCP activate human pregnane X receptor (PXR) and subsequent CYP3A4 mRNA expression. This effect is mediated by the distal xenobiotic responsive element modulator of the promoter. The induction of CYP3A4 by OCP was dose-dependent within the 1-10 microM range. The data suggest that chronic exposure to OCP could alter a major metabolite pathway in human liver and putatively modify the pharmacokinetics of drugs and pollutants.

  3. CYP1A1, CYP3A5 and CYP3A7 polymorphisms and testicular cancer susceptibility.

    Science.gov (United States)

    Kristiansen, W; Haugen, T B; Witczak, O; Andersen, J M; Fosså, S D; Aschim, E L

    2011-02-01

    Testicular cancer (TC) incidence is increasing worldwide, but the aetiology remains largely unknown. An unbalanced level of oestrogens and androgens in utero is hypothesized to influence TC risk. Polymorphisms in genes encoding cytochrome P450 (CYP) enzymes involved in metabolism of reproductive hormones, such as CYP1A1, CYP3A5 and CYP3A7, may contribute to variability of an individual's susceptibility to TC. The aim of this case-control study was to investigate possible associations between different CYP genotypes and TC, as well as histological type of TC. The study comprised 652 TC cases and 199 controls of Norwegian Caucasian origin. Genotyping of the CYP1A1*2A (MspI), CYP1A1*2C (I462V), CYP1A1*4 (T461N), CYP3A5*3C (A6986G) and CYP3A7*2 (T409R) polymorphisms was performed using TaqMan allelic discrimination or sequencing. The CYP1A1*2A allele was associated with 44% reduced risk of TC with each polymorphic allele [odds ratio (OR) = 0.56, 95% confidence interval (CI) = 0.40-0.78, p(trend) = 0.001], whereas the CYP1A1*2C allele was associated with 56% reduced risk of TC with each polymorphic allele (OR = 0.44, 95% CI = 0.25-0.75, p(trend) = 0.003). The decreased risk per allele was significant for seminomas (OR = 0.46, 95% CI, 0.31-0.70, p(trend) < 0.001 and OR = 0.31, 95% CI = 0.14-0.66, p(trend) = 0.002, respectively), but only borderline significant for non-seminomas (OR = 0.65, 95% CI = 0.45-0.95, p(trend) = 0.027 and OR = 0.55, 95% CI = 0.30-1.01, p(trend) = 0.052, respectively). There were no statistically significant differences in the distribution of the CYP3A5*3C and CYP3A7*2 polymorphic alleles between TC cases and controls. This study suggests that polymorphisms in the CYP1A1 gene may contribute to variability of individual susceptibility to TC. © 2010 The Authors. International Journal of Andrology © 2010 European Academy of Andrology.

  4. Evidences for CYP3A4 autoactivation in the desulfuration of dimethoate by the human liver.

    Science.gov (United States)

    Buratti, Franca M; Testai, Emanuela

    2007-11-20

    Dimethoate (DIM) is an organophosphorothionate (OPT) pesticide used worldwide as a systemic insecticide and acaricide. It is characterized by low-to-moderate acute mammalian toxicity; similarly to the other OPT pesticides, its mode of action is mediated by the inhibition of acetylcholinesterase (AChE), exerted by its toxic metabolite dimethoate-oxon or omethoate (OME), which is also used as a direct acting pesticide. Human hepatic DIM bioactivation to the toxic metabolite OME has been characterized by using c-DNA expressed human CYPs and human liver microsomes (HLM) also in the presence of CYP-specific chemical inhibitors, with a method based on AChE inhibition. The obtained kinetic parameters and AChE IC(50) have been compared with those previously obtained with other OPTs, indicating a lower efficiency in DIM desulfuration reaction and a lower potency in inhibiting AChE. Results showed that, similarly to the other OPTs tested so far, at low DIM concentration OME formation is mainly catalysed by CYP1A2, while the role of 3A4 is relevant at high DIM levels. Differently from the other OPTs, DIM desulfuration reaction showed an atypical kinetic profile, likely due to CYP3A4 autoactivation. The sigmoidicity degree of the activity curve increased with the level of CYP3A4 in HLM or disappeared in the presence of a CYP3A4 chemical inhibitor. This atypical kinetic behaviour can be considered one of the possible explanations for the recent findings that among patients hospitalized following OPT intoxication, DIM ingestion gave different symptoms and more severe poisoning (23.1% of fatal cases versus total) than chlorpyrifos (8% of deaths), which has a lower LD(50) value. Since DIM-poisoned patients poorly responded to pralidoxime, the possibility to use CYP3A4 inhibitors could be considered as a complementary treatment.

  5. Temporal kinetics and concentration-response relationships for induction of CYP1A, CYP2B, and CYP3A in primary cultures of beagle dog hepatocytes.

    Science.gov (United States)

    Graham, Richard A; Tyler, Lindsey O; Krol, Wojciech L; Silver, Ivin S; Webster, Lindsey O; Clark, Philip; Chen, Liangfu; Banks, Troy; LeCluyse, Edward L

    2006-01-01

    Compared to other species, little information is available on the xenobiotic-induced regulation of cytochrome P450 enzymes in the beagle dog. Dogs are widely used in the pharmaceutical industry for many study types, including those that will impact decisions on compound progression. The purpose of this study was (1) to determine the temporal kinetics of drug-induced changes in canine CYP1A, CYP2B, and CYP3A mRNA and enzymatic activity, and (2) to characterize concentration-response relationships for CYP1A2, CYP2B11, and CYP3A12 using primary cultures of canine hepatocytes treated with beta-naphthoflavone (BNF), phenobarbital (PB), and rifampin (RIF), respectively. CYP1A1 and CYP1A2 mRNA exhibited maximal expression (12,700-fold and 206-fold, respectively) after 36 h of treatment with BNF. PB treatment, but not RIF treatment, caused maximal induction of CYP2B11 mRNA (149-fold) after 48 h of treatment. CYP3A12 and CYP3A26 mRNA levels were increased maximally after 72 h of treatment with PB and RIF (CYP3A12, 35-fold and 18-fold, and CYP3A26, 72-fold and 22-fold with PB and RIF treatment, respectively). Concentration-response relationships for BNF induced 7-ethoxyresorufin O-dealkylation (EROD) (EC(50) = 7.8 +/- 4.2 microM), PB induced 7-benzyloxyresorufin O-dealkylation (BROD) (EC(50) = 123 +/- 30 microM), and PB and RIF induced testosterone 6beta-hydroxylation (EC(50) = 132 +/- 28 microM and 0.98 +/- 0.16 microM) resembled the relationship for human CYP induction compared to that of rodent. Interestingly, RIF had no effect on CYP2B11 expression, which represents a species difference overlooked in previous investigations. Overall, the induction of dog CYP1A, CYP2B, and CYP3A exhibits characteristics that are intermediate to those of rodent and human. (c) 2006 Wiley Periodicals, Inc.

  6. Endosulfan induces CYP2B6 and CYP3A4 by activating the pregnane X receptor

    International Nuclear Information System (INIS)

    Casabar, Richard C.T.; Das, Parikshit C.; DeKrey, Gregory K.; Gardiner, Catherine S.; Cao Yan; Rose, Randy L.; Wallace, Andrew D.

    2010-01-01

    Endosulfan is an organochlorine pesticide commonly used in agriculture. Endosulfan has affects on vertebrate xenobiotic metabolism pathways that may be mediated, in part, by its ability to activate the pregnane X receptor (PXR) and/or the constitutive androstane receptor (CAR) which can elevate expression of cytochrome P450 (CYP) enzymes. This study examined the dose-dependency and receptor specificity of CYP induction in vitro and in vivo. The HepG2 cell line was transiently transfected with CYP2B6- and CYP3A4-luciferase promoter reporter plasmids along with human PXR (hPXR) or hCAR expression vectors. In the presence of hPXR, endosulfan-alpha exposure caused significant induction of CYP2B6 (16-fold) and CYP3A4 (11-fold) promoter activities over control at 10 μM. The metabolite endosulfan sulfate also induced CYP2B6 (12-fold) and CYP3A4 (6-fold) promoter activities over control at 10 μM. In the presence of hCAR-3, endosulfan-alpha induced CYP2B6 (2-fold) promoter activity at 10 μM, but not at lower concentrations. These data indicate that endosulfan-alpha significantly activates hPXR strongly and hCAR weakly. Using western blot analysis of human hepatocytes, the lowest concentrations at which CYP2B6 and CYP3A4 protein levels were found to be significantly elevated by endosulfan-alpha were 1.0 μM and 10 μM, respectively. In mPXR-null/hPXR-transgenic mice, endosulfan-alpha exposure (2.5 mg/kg/day) caused a significant reduction of tribromoethanol-induced sleep times by approximately 50%, whereas no significant change in sleep times was observed in PXR-null mice. These data support the role of endosulfan-alpha as a strong activator of PXR and inducer of CYP2B6 and CYP3A4, which may impact metabolism of CYP2B6 or CYP3A4 substrates.

  7. Endosulfan induces CYP2B6 and CYP3A4 by activating the pregnane X receptor.

    Science.gov (United States)

    Casabar, Richard C T; Das, Parikshit C; Dekrey, Gregory K; Gardiner, Catherine S; Cao, Yan; Rose, Randy L; Wallace, Andrew D

    2010-06-15

    Endosulfan is an organochlorine pesticide commonly used in agriculture. Endosulfan has affects on vertebrate xenobiotic metabolism pathways that may be mediated, in part, by its ability to activate the pregnane X receptor (PXR) and/or the constitutive androstane receptor (CAR) which can elevate expression of cytochrome P450 (CYP) enzymes. This study examined the dose-dependency and receptor specificity of CYP induction in vitro and in vivo. The HepG2 cell line was transiently transfected with CYP2B6- and CYP3A4-luciferase promoter reporter plasmids along with human PXR (hPXR) or hCAR expression vectors. In the presence of hPXR, endosulfan-alpha exposure caused significant induction of CYP2B6 (16-fold) and CYP3A4 (11-fold) promoter activities over control at 10 microM. The metabolite endosulfan sulfate also induced CYP2B6 (12-fold) and CYP3A4 (6-fold) promoter activities over control at 10 microM. In the presence of hCAR-3, endosulfan-alpha induced CYP2B6 (2-fold) promoter activity at 10 microM, but not at lower concentrations. These data indicate that endosulfan-alpha significantly activates hPXR strongly and hCAR weakly. Using western blot analysis of human hepatocytes, the lowest concentrations at which CYP2B6 and CYP3A4 protein levels were found to be significantly elevated by endosulfan-alpha were 1.0 microM and 10 microM, respectively. In mPXR-null/hPXR-transgenic mice, endosulfan-alpha exposure (2.5mg/kg/day) caused a significant reduction of tribromoethanol-induced sleep times by approximately 50%, whereas no significant change in sleep times was observed in PXR-null mice. These data support the role of endosulfan-alpha as a strong activator of PXR and inducer of CYP2B6 and CYP3A4, which may impact metabolism of CYP2B6 or CYP3A4 substrates. Copyright 2010 Elsevier Inc. All rights reserved.

  8. Identification of the UBP1 locus as a critical blood pressure determinant using a combination of mouse and human genetics

    DEFF Research Database (Denmark)

    Koutnikova, Hana; Laakso, Markku; Lu, Lu

    2009-01-01

    complementarities of mouse and human genetic approaches, identifies the UBP1 locus as a critical blood pressure determinant. UBP1 plays a role in cholesterol and steroid metabolism via the transcriptional activation of CYP11A, the rate-limiting enzyme in pregnenolone and aldosterone biosynthesis. We suggest......Hypertension is a major health problem of largely unknown genetic origins. To identify new genes responsible for hypertension, genetic analysis of recombinant inbred strains of mice followed by human association studies might prove powerful and was exploited in our current study. Using a set of 27...... recombinant BXD strains of mice we identified a quantitative trait locus (QTL) for blood pressure (BP) on distal chromosome 9. The association analysis of markers encompassing the syntenic region on human chromosome 3 gave in an additive genetic model the strongest association for rs17030583 C/T and rs2291897...

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

    Directory of Open Access Journals (Sweden)

    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.

  10. CAR/PXR provide directives for Cyp3a41 gene regulation differently from Cyp3a11.

    Science.gov (United States)

    Anakk, S; Kalsotra, A; Kikuta, Y; Huang, W; Zhang, J; Staudinger, J L; Moore, D D; Strobel, H W

    2004-01-01

    This study reports that Cyp3a41 gene contains 13 exons and is localized on the chromosome 5. CYP3A41 is a female-specific isoform that is predominantly expressed in the liver. Estrogen signaling is not responsible for its female specificity. CYP3A41 expression in kidney and brain is observed only in 50% of mice examined. PXR mediates dexamethasone-dependent suppression of CYP3A41. In contrast to CYP3A11, CYP3A41 expression is not induced by pregnenolone-16alpha-carbonitrile (PCN) in wild-type mice, but is significantly suppressed by PCN in PXR(-/-) mice. Phenobarbital and TCPOBOP induce CYP3A11 expression only in the presence of CAR, but have no effect on CYP3A41 expression. Immunoblot and erythromycin demethylase activity analysis reveal robust CYP3A induction after PCN treatment, which is poorly correlated to CYP3A41. These findings suggest a differential role for CAR/PXR in regulating individual CYP3A isoforms by previously characterized CYP3A inducers.

  11. Time-dependent inhibition of CYP3A4 by gallic acid in human liver microsomes and recombinant systems.

    Science.gov (United States)

    Pu, Qiang-Hong; Shi, Liang; Yu, Chao

    2015-03-01

    1.Gallic acid is a main polyphenol in various fruits and plants. Inhibitory characteristics of gallic acid on CYP3A4 were still unclear. The objective of this work is hence to investigate inhibitory characteristics of gallic acid on CYP3A4 using testosterone as the probe substrate in human liver microsomes (HLMs) and recombinant CYP3A4 (rCYP3A4) systems. 2.Gallic acid caused concentration-dependent loss of CYP3A4 activity with IC50 values of 615.2 μM and 669.5 μM in HLM and rCYP3A4 systems, respectively. IC50-shift experiments showed that pre-incubation with gallic acid in the absence of NADPH contributed to 12- or 14-fold reduction of IC50 in HLM and rCYP3A4 systems, respectively, supporting a time-dependent inhibition. In HLM, time-dependent inactivation variables KI and Kinact were 485.8 μM and 0.05 min(-1), respectively. 3.Compared with the presence of NADPH, pre-incubation of gallic acid in the absence of NADPH markedly increased its inhibitory effects in HLM and rCYP3A4 systems. Those results indicate that CYP3A4 inactivation by gallic acid was independent on NADPH and was mainly mediated its oxidative products. 4.In conclusion, we showed that gallic acid weakly and time-dependently inactivated CYP3A4 via its oxidative products.

  12. Interactions between CYP3A4 and Dietary Polyphenols

    Directory of Open Access Journals (Sweden)

    Loai Basheer

    2015-01-01

    Full Text Available The human cytochrome P450 enzymes (P450s catalyze oxidative reactions of a broad spectrum of substrates and play a critical role in the metabolism of xenobiotics, such as drugs and dietary compounds. CYP3A4 is known to be the main enzyme involved in the metabolism of drugs and most other xenobiotics. Dietary compounds, of which polyphenolics are the most studied, have been shown to interact with CYP3A4 and alter its expression and activity. Traditionally, the liver was considered the prime site of CYP3A-mediated first-pass metabolic extraction, but in vitro and in vivo studies now suggest that the small intestine can be of equal or even greater importance for the metabolism of polyphenolics and drugs. Recent studies have pointed to the role of gut microbiota in the metabolic fate of polyphenolics in human, suggesting their involvement in the complex interactions between dietary polyphenols and CYP3A4. Last but not least, all the above suggests that coadministration of drugs and foods that are rich in polyphenols is expected to stimulate undesirable clinical consequences. This review focuses on interactions between dietary polyphenols and CYP3A4 as they relate to structural considerations, food-drug interactions, and potential negative consequences of interactions between CYP3A4 and polyphenols.

  13. Mutation analysis of the human CYP3A4 gene 5' regulatory region: population screening using non-radioactive SSCP.

    Science.gov (United States)

    Hamzeiy, Hossein; Vahdati-Mashhadian, Nasser; Edwards, Helen J; Goldfarb, Peter S

    2002-03-20

    Human CYP3A4 is the major cytochrome P450 isoenzyme in adult human liver and is known to metabolise many xenobiotic and endogenous compounds. There is substantial inter-individual variation in the hepatic levels of CYP3A4. Although, polymorphic mutations have been reported in the 5' regulatory region of the CYP3A4 gene, those that have been investigated so far do not appear to have any effect on gene expression. To determine whether other mutations exist in this region of the gene, we have performed a new population screen on a panel of 101 human DNA samples. A 1140 bp section of the 5' proximal regulatory region of the CYP3A4 gene, containing numerous regulatory motifs, was amplified from genomic DNA as three overlapping segments. The 300 bp distal enhancer region at -7.9kb containing additional regulatory motifs was also amplified. Mutation analysis of the resulting PCR products was carried out using non-radioactive single strand conformation polymorphism (SSCP) and confirmatory sequencing of both DNA strands in those samples showing extra SSCP bands. In addition to detection of the previously reported CYP3A4*1B allele in nine subjects, three novel alleles were found: CYP3A4*1E (having a T-->A transversion at -369 in one subject), CYP3A4*1F (having a C-->G tranversion at -747 in 17 subjects) and CYP3A4*15B containing a nine-nucleotide insertion between -845 and -844 linked to an A-->G transition at -392 and a G-->A transition in exon 6 (position 485 in the cDNA) in one subject. All the novel alleles were heterozygous. No mutations were found in the upstream distal enhancer region. Our results clearly indicate that this rapid and simple SSCP approach can reveal mutant alleles in drug metabolising enzyme genes. Detection and determination of the frequency of novel alleles in CYP3A4 will assist investigation of the relationship between genotype, xenobiotic metabolism and toxicity in the CYP3A family of isoenzymes.

  14. Role of CYP2B6 and CYP3A4 in the in vitro N-dechloroethylation of (R)- and (S)-ifosfamide in human liver microsomes.

    Science.gov (United States)

    Granvil, C P; Madan, A; Sharkawi, M; Parkinson, A; Wainer, I W

    1999-04-01

    The central nervous system toxicity of ifosfamide (IFF), a chiral antineoplastic agent, is thought to be dependent on its N-dechloroethylation by hepatic cytochrome P-450 (CYP) enzymes. The purpose of this study was to identify the human CYPs responsible for IFF-N-dechloroethylation and their corresponding regio- and enantioselectivities. IFF exists in two enantiomeric forms, (R) - and (S)-IFF, which can be dechloroethylated at either the N2 or N3 positions, producing the corresponding (R,S)-2-dechloroethyl-IFF [(R, S)-2-DCE-IFF] and (R,S)-3-dechloroethyl-IFF [(R,S)-3-DCE-IFF]. The results of the present study suggest that the production of (R)-2-DCE-IFF and (S)-3-DCE-IFF from (R)-IFF is catalyzed by different CYPs as is the production of (S)-2-DCE-IFF and (R)-3-DCE-IFF from (S)-IFF. In vitro studies with a bank of human liver microsomes revealed that the sample-to-sample variation in the production of (S)-3-DCE-IFF from (R)-IFF and (S)-2-DCE-IFF from (S)-IFF was highly correlated with the levels of (S)-mephenytoin N-demethylation (CYP2B6), whereas (R)-2-DCE-IFF production from (R)-IFF and (R)-3-DCE-IFF production from (S)-IFF were both correlated with the activity of testosterone 6beta-hydroxylation (CYP3A4/5). Experiments with cDNA-expressed P-450 and antibody and chemical inhibition studies supported the conclusion that the formation of (S)-3-DCE-IFF and (S)-2-DCE-IFF is catalyzed primarily by CYP2B6, whereas (R)-2-DCE-IFF and (R)-3-DCE-IFF are primarily the result of CYP3A4/5 activity.

  15. Novel mutations of CYP3A4 in Chinese.

    Science.gov (United States)

    Hsieh, K P; Lin, Y Y; Cheng, C L; Lai, M L; Lin, M S; Siest, J P; Huang, J D

    2001-03-01

    Human cytochrome P450 3A4 is a major P450 enzyme in the liver and gastrointestinal tract. It plays important roles in the metabolism of a wide variety of drugs, some endogenous steroids, and harmful environmental contaminants. CYP3A4 exhibits a remarkable interindividual activity variation as high as 20-fold. To investigate whether the interindividual variation in CYP3A4 levels can be partly explained by genetic polymorphism, we analyzed DNA samples from 102 Chinese subjects by polymerase chain reaction (PCR)-single-strand conformation polymorphism analysis for novel point mutation in the CYP3A4 coding sequence and promoter region. Using PCR and directed sequencing method to establish the complete intron sequence of CYP3A4 from leukocytes, the complete genomic sequence from exon 1 through 13 of CYP3A4 was determined and published in the GenBank database (accession no. AF209389). CYP3A4-specific primers were designed accordingly. After PCR-single-strand conformation polymorphism and restriction fragment length polymorphism screening, we found three novel mutations; two are point mutations and one is insertion. The first variant allele (CYP3A4*4), an Ile118Val change, was found in 3 of 102 Chinese subjects. The next allele (CYP3A4*5), which causes a Pro218Arg amino acid change, was found in 2 of 102 subjects. We found an insertion in A(17776), designated as CYP3A4*6, which causes frame shift and an early stop codon in exon 9, in one heterozygous subject. We also investigated the CYP3A4 activity in these mutant subjects by measuring the morning spot urinary 6beta-hydroxycortisol to free cortisol ratio with the enzyme-linked immunosorbent assay method. When compared with healthy Chinese population data, the 6beta-hydroxycortisol to free cortisol ratio data suggested that these alleles (CYP3A4*4, CYP3A4*5, and CYP3A4*6) may decrease the CYP3A4 activity. Incidences of these mutations in Chinese subjects are rare. The prevalence of these point mutations in other ethnic

  16. Inhibitory Effects of Juices Prepared from Individual Vegetables on CYP3A4 Activity in Recombinant CYP3A4 and LS180 Cells.

    Science.gov (United States)

    Tsujimoto, Masayuki; Agawa, Chie; Ueda, Shinya; Yamane, Takayoshi; Kitayama, Haruna; Terao, Aya; Fukuda, Tomoya; Minegaki, Tetsuya; Nishiguchi, Kohshi

    2017-01-01

    Human intestinal absorption and drug metabolism vary to a large extent among individuals. For example, CYP3A4 activity has large individual variation that cannot be attributed to only genetic differences. Various flavonoids in vegetables, such as kaempferol and quercetin, possess inhibitory effects, and some vegetable and fruit juices have also been found to inhibit CYP3A4 activity. Therefore, differences in daily intake of flavonoid-containing vegetables may induce individual variation in intestinal bioavailability. To identify a vegetable that strongly inhibits CYP3A4, we investigated the effects of juices, prepared from individual vegetables, on CYP3A4 activity using recombinant CYP3A4 and LS180 cells in this study. Nine vegetable juices (cabbage, Japanese radish, onion, tomato, eggplant, carrot, Chinese cabbage, green pepper, and lettuce), were prepared and recombinant CYP3A4 and LS180 cells were used for evaluation of CYP3A4 activity. Metabolism to 6β-hydroxytestosterone by recombinant CYP3A4 was strongly inhibited by cabbage, onion, and green pepper juices, and cabbage and green pepper juices significantly inhibited CYP3A4 activity in a preincubation time-dependent manner. In addition, CYP3A4 activity in LS180 cells was significantly inhibited by cabbage and onion juices. In conclusion, this study showed that juices prepared from some individual vegetables could significantly inhibit CYP3A4 activity. Therefore, variation in the daily intake of vegetables such as cabbage and onion may be one of the factors responsible for individual differences in intestinal bioavailability.

  17. 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.

  18. Allosteric activation of midazolam CYP3A5 hydroxylase activity by icotinib - Enhancement by ketoconazole.

    Science.gov (United States)

    Zhuang, XiaoMei; Zhang, TianHong; Yue, SiJia; Wang, Juan; Luo, Huan; Zhang, YunXia; Li, Zheng; Che, JinJing; Yang, HaiYing; Li, Hua; Zhu, MingShe; Lu, Chuang

    2016-12-01

    Icotinib (ICO), a novel small molecule and a tyrosine kinase inhibitor, was developed and approved recently in China for non-small cell lung cancer. During screening for CYP inhibition potential in human liver microsomes (HLM), heterotropic activation toward CYP3A5 was revealed. Activation by icotinib was observed with CYP3A-mediated midazolam hydroxylase activity in HLM (∼40% over the baseline) or recombinant human CYP3A5 (rhCYP3A5) (∼70% over the baseline), but not in the other major CYPs including rhCYP3A4. When co-incubated with selective CYP3A4 inhibitor CYP3cide or monoclonal human CYP3A4 inhibitory antibody in HLM, the activation was extended to ∼60%, suggesting CYP3A5 might be the isozyme involved. Further, the relative activation was enhanced to ∼270% in rhCYP3A5 in the presence of ketoconazole. The activation was substrate and pathway dependent and observed only in the formation of 1'-OH-midazolam, and not 4-OH-midazolam, 6β-OH-testosterone, or oxidized nifedipine. The activation requires the presence of cytochrome b5 and it is only observed in the liver microsomes of dogs, monkeys, and humans, but not in rats and mice. Kinetic analyses of 1'-OH-midazolam formation showed that ICO increased the V max values in HLM and rhCYP3A5 with no significant changes in K m values. By adding CYP3cide with ICO to the incubation, the V max values increased 2-fold over the CYP3cide control. Addition of ketoconazole with ICO alone or ICO plus CYP3cide resulted in an increase in V max values and decrease in K m values compared to their controls. This phenomenon may be attributed to a new mechanism of CYP3A5 heterotropic activation, which warrants further investigation. Copyright © 2016 Elsevier Inc. All rights reserved.

  19. CYP1A1 induction and CYP3A4 inhibition by the fungicide imazalil in the human intestinal Caco-2 cells-comparison with other conazole pesticides.

    Science.gov (United States)

    Sergent, Thérèse; Dupont, Isabelle; Jassogne, Coralie; Ribonnet, Laurence; van der Heiden, Edwige; Scippo, Marie-Louise; Muller, Marc; McAlister, Dan; Pussemier, Luc; Larondelle, Yvan; Schneider, Yves-Jacques

    2009-02-10

    Imazalil (IMA) is a widely used imidazole-antifungal pesticide and, therefore, a food contaminant. This compound is also used as a drug (enilconazole). As intestine is the first site of exposure to ingested drugs and pollutants, we have investigated the effects of IMA, at realistic intestinal concentrations, on xenobiotic-metabolizing enzymes and efflux pumps by using Caco-2 cells, as a validated in vitro model of the human intestinal absorptive epithelium. For comparison, other conazole fungicides, i.e. ketoconazole, propiconazole and tebuconazole, were also studied. IMA induced cytochrome P450 (CYP) 1A1 activity to the same extent as benzo(a)pyrene (B(a)P) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), in a dose- and time-dependent manner. Cell-free aryl hydrocarbon receptor (AhR) binding assay and reporter gene assay suggested that IMA is not an AhR-ligand, implying that IMA-mediated induction should involve an AhR-independent pathway. Moreover, IMA strongly inhibited the CYP3A4 activity in 1,25-vitamin D(3)-induced Caco-2 cells. The other fungicides had weak or nil effects on CYP activities. Study of the apical efflux pump activities revealed that ketoconazole inhibited both P-glycoprotein (Pgp) and multidrug resistance-associated protein 2 (MRP-2) or breast cancer resistance protein (BCRP), whereas IMA and other fungicides did not. Our results imply that coingestion of IMA-contaminated food and CYP3A4- or CYP1A1-metabolizable drugs or chemicals could lead to drug bioavailability modulation or toxicological interactions, with possible adverse effects for human health.

  20. 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.

  1. Association between cytochrome CYP17A1, CYP3A4, and CYP3A43 polymorphisms and prostate cancer risk and aggressiveness in a Korean study population

    Directory of Open Access Journals (Sweden)

    Jun Hyun Han

    2015-04-01

    Full Text Available In this study, we evaluated genetic variants of the androgen metabolism genes CYP17A1, CYP3A4, and CYP3A43 to determine whether they play a role in the development of prostate cancer (PCa in Korean men. The study population included 240 pathologically diagnosed cases of PCa and 223 age-matched controls. Among the 789 single-nucleotide polymorphism (SNP database variants detected, 129 were reported in two Asian groups (Han Chinese and Japanese in the HapMap database. Only 21 polymorphisms of CYP17A1, CYP3A4, and CYP3A43 were selected based on linkage disequilibrium in Asians (r2 = 1, locations (SNPs in exons were preferred, and amino acid changes and were assessed. In addition, we performed haplotype analysis for the 21 SNPs in CYP17A1, CYP3A4, and CYP3A43 genes. To determine the association between genotype and haplotype distributions of patients and controls, logistic analyses were carried out, controlling for age. Twelve sequence variants and five major haplotypes were identified in CYP17A1. Five sequence variants and two major haplotypes were identified in CYP3A4. Four sequence variants and four major haplotypes were observed in CYP3A43. CYP17A1 haplotype-2 (Ht-2 (odds ratio [OR], 1.51; 95% confidence interval [CI], 1.04-2.18 was associated with PCa susceptibility. CYP3A4 Ht-2 (OR: 1.87; 95% CI: 1.02-3.43 was associated with PCa metastatic potential according to tumor stage. rs17115149 (OR: 1.96; 95% CI: 1.04-3.68 and CYP17A1 Ht-4 (OR: 2.01; 95% CI: 1.07-4.11 showed a significant association with histologic aggressiveness according to Gleason score. Genetic variants of CYP17A1 and CYP3A4 may play a role in the development of PCa in Korean men.

  2. Association between cytochrome CYP17A1, CYP3A4, and CYP3A43 polymorphisms and prostate cancer risk and aggressiveness in a Korean study population.

    Science.gov (United States)

    Han, Jun Hyun; Lee, Yong Seong; Kim, Hae Jong; Lee, Shin Young; Myung, Soon Chul

    2015-01-01

    In this study, we evaluated genetic variants of the androgen metabolism genes CYP17A1, CYP3A4, and CYP3A43 to determine whether they play a role in the development of prostate cancer (PCa) in Korean men. The study population included 240 pathologically diagnosed cases of PCa and 223 age-matched controls. Among the 789 single-nucleotide polymorphism (SNP) database variants detected, 129 were reported in two Asian groups (Han Chinese and Japanese) in the HapMap database. Only 21 polymorphisms of CYP17A1, CYP3A4, and CYP3A43 were selected based on linkage disequilibrium in Asians (r2 = 1), locations (SNPs in exons were preferred), and amino acid changes and were assessed. In addition, we performed haplotype analysis for the 21 SNPs in CYP17A1, CYP3A4, and CYP3A43 genes. To determine the association between genotype and haplotype distributions of patients and controls, logistic analyses were carried out, controlling for age. Twelve sequence variants and five major haplotypes were identified in CYP17A1. Five sequence variants and two major haplotypes were identified in CYP3A4. Four sequence variants and four major haplotypes were observed in CYP3A43. CYP17A1 haplotype-2 (Ht-2) (odds ratio [OR], 1.51; 95% confidence interval [CI], 1.04-2.18) was associated with PCa susceptibility. CYP3A4 Ht-2 (OR: 1.87; 95% CI: 1.02-3.43) was associated with PCa metastatic potential according to tumor stage. rs17115149 (OR: 1.96; 95% CI: 1.04-3.68) and CYP17A1 Ht-4 (OR: 2.01; 95% CI: 1.07-4.11) showed a significant association with histologic aggressiveness according to Gleason score. Genetic variants of CYP17A1 and CYP3A4 may play a role in the development of PCa in Korean men.

  3. A PXR reporter gene assay in a stable cell culture system: CYP3A4 and CYP2B6 induction by pesticides.

    Science.gov (United States)

    Lemaire, Géraldine; de Sousa, Georges; Rahmani, Roger

    2004-12-15

    A stable hepatoma cell line expressing the human pregnane X receptor (hPXR) and the cytochrome P4503A4 (CYP3A4) distal and proximal promoters plus the luciferase reporter gene was developed to assess the ability of several xenobiotic agents to induce CYP3A4 and CYP2B6. After selection for neomycin resistance, one clone, displaying high luciferase activity in response to rifampicin (RIF), was isolated and the stable expression of hPXR was confirmed by reverse transcription polymerase chain reaction (RT-PCR). Dose-response curves were generated by treating these cells with increasing concentrations of RIF, phenobarbital (PB), clotrimazole (CLOT) or 5beta-pregnane-3,20-dione (5beta-PREGN). The effective concentrations for half maximal response (EC50) were determined for each of these compounds. RIF was the most effective compound, with maximal luciferase activity induced at 10 microM. The agonist activities of PXR-specific inducers measured using our stable model were consistent with those measured in transient transfectants. The abilities of organochlorine (OC), organophosphate (OP) and pyrethroid pesticides (PY) to activate hPXR were also assessed and found to be consistent with the abilities of these compounds to induce CYP3A4 and CYP2B6 in primary culture of human hepatocytes. These results suggest that CYP3A4 and CYP2B6 regulation through PXR activation by persistent pesticides may have an impact on the metabolism of xenobiotic agents and endogenous steroid hormones. Our model provides a useful tool for studying hPXR activation and for identifying agents capable of inducing CYP3A4 and CYP2B6.

  4. Association of CYP3A4 and CYP3A5 polymorphisms with Iranian ...

    African Journals Online (AJOL)

    Elham Badavi

    2015-04-20

    Apr 20, 2015 ... cancer, CYP3A5 (P-value = 0.561) and CYP3A4 allele distribution. У 2015 The Authors. ... other hand, causes alternative splicing and blocks protein pro- ... Homozygote individuals for A/A genotype present only one peak.

  5. Haplotypes of CYP3A4 and their close linkage with CYP3A5 haplotypes in a Japanese population.

    Science.gov (United States)

    Fukushima-Uesaka, Hiromi; Saito, Yoshiro; Watanabe, Hidemi; Shiseki, Kisho; Saeki, Mayumi; Nakamura, Takahiro; Kurose, Kouichi; Sai, Kimie; Komamura, Kazuo; Ueno, Kazuyuki; Kamakura, Shiro; Kitakaze, Masafumi; Hanai, Sotaro; Nakajima, Toshiharu; Matsumoto, Kenji; Saito, Hirohisa; Goto, Yu-ichi; Kimura, Hideo; Katoh, Masaaki; Sugai, Kenji; Minami, Narihiro; Shirao, Kuniaki; Tamura, Tomohide; Yamamoto, Noboru; Minami, Hironobu; Ohtsu, Atsushi; Yoshida, Teruhiko; Saijo, Nagahiro; Kitamura, Yutaka; Kamatani, Naoyuki; Ozawa, Shogo; Sawada, Jun-ichi

    2004-01-01

    In order to identify single nucleotide polymorphisms (SNPs) and haplotype frequencies of CYP3A4 in a Japanese population, the distal enhancer and proximal promoter regions, all exons, and the surrounding introns were sequenced from genomic DNA of 416 Japanese subjects. We found 24 SNPs, including 17 novel ones: two in the distal enhancer, four in the proximal promoter, one in the 5'-untranslated region (UTR), seven in the introns, and three in the 3'-UTR. The most common SNP was c.1026+12G>A (IVS10+12G>A), with a 0.249 frequency. Four non-synonymous SNPs, c.554C>G (p.T185S, CYP3A4(*)16), c.830_831insA (p.E277fsX8, (*)6), c.878T>C (p.L293P, (*)18), and c.1088 C>T (p.T363M, (*)11) were found with frequencies of 0.014, 0.001, 0.028, and 0.002, respectively. No SNP was found in the known nuclear transcriptional factor-binding sites in the enhancer and promoter regions. Using these 24 SNPs, 16 haplotypes were unambiguously identified, and nine haplotypes were inferred by aid of an expectation-maximization-based program. In addition, using data from 186 subjects enabled a close linkage to be found between CYP3A4 and CYP3A5 SNPs, especially among the SNPs at c.1026+12 in CYP3A4 and c.219-237 (IVS3-237, a key SNP site for CYP3A5(*)3), c.865+77 (IVS9+77) and c.1523 in CYP3A5. This result suggested that CYP3A4 and CYP3A5 are within the same gene block. Haplotype analysis between CYP3A4 and CYP3A5 revealed several major haplotype combinations in the CYP3A4-CYP3A5 block. Our findings provide fundamental and useful information for genotyping CYP3A4 (and CYP3A5) in the Japanese, and probably Asian populations. Copyright 2003 Wiley-Liss, Inc.

  6. Endosulfan-alpha Induces CYP26 and CYP3A4 by Activating the Pregnane X Receptor But Not the Constitutive Androstane Receptor

    Science.gov (United States)

    2006-01-01

    CYP3A4 gene expression by organochlorine pesticides . Biochem Pharmacol 64:1513-1519. Dinham B (1993) The Pesticide Hazard. A Global Health and...Coumoul X, Diry M and Barouki R (2002) PXR-dependent induction of human CYP3A4 gene expression by organochlorine pesticides . Biochem Pharmacol 64:1513...system: CYP3A4 and CYP2B6 induction by pesticides . Biochem Pharmacol 68:2347-2358. 71 Nelson D (2003) Cytochrome P450 Homepage (http

  7. Genetic variation at CYP3A is associated with age at menarche and breast cancer risk

    DEFF Research Database (Denmark)

    Johnson, Nichola; Dudbridge, Frank; Orr, Nick

    2014-01-01

    INTRODUCTION: We have previously shown that a tag single nucleotide polymorphism (rs10235235), which maps to the CYP3A locus (7q22.1), was associated with a reduction in premenopausal urinary estrone glucuronide levels and a modest reduction in risk of breast cancer in women age <=50 years. METHO...

  8. 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.

    Science.gov (United States)

    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.

  9. 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.

  10. 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

    Energy Technology Data Exchange (ETDEWEB)

    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

  11. Association of CYP3A4 and CYP3A5 polymorphisms with Iranian ...

    African Journals Online (AJOL)

    Background: Polymorphisms of different gene have been reported to be associated with cancer including breast cancer. Hospitalization rate for breast cancer has increased over the years in Iran. Aim: The aim of this study was to examine whether polymorphisms in the CYP3A4 and. CYP3A5 genes affect the risk of ...

  12. Lentiviral transgenic microRNA-based shRNA suppressed mouse cytochromosome P450 3A (CYP3A expression in a dose-dependent and inheritable manner.

    Directory of Open Access Journals (Sweden)

    Yong Wang

    Full Text Available Cytochomosome P450 enzymes (CYP are heme-containing monooxygenases responsible for oxidative metabolism of many exogenous and endogenous compounds including drugs. The species difference of CYP limits the extent to which data obtained from animals can be translated to humans in pharmacodynamics or pharmacokinetics studies. Transgenic expression of human CYP in animals lacking or with largely reduced endogenous CYP counterparts is recognized as an ideal strategy to correct CYP species difference. CYP3A is the most abundant CYP subfamily both in human and mammals. In this study, we designed a microRNA-based shRNA (miR-shRNA simultaneously targeting four members of mouse CYP3A subfamily (CYP3A11, CYP3A16, CYP3A41 and CYP3A44, and transgenic mice expressing the designed miR-shRNA were generated by lentiviral transgenesis. Results showed that the CYP3A expression level in transgenic mice was markedly reduced compared to that in wild type or unrelated miR-shRNA transgenic mice, and was inversely correlated to the miR-shRNA expression level. The CYP3A expression levels in transgenic offspring of different generations were also remarkably lower compared to those of controls, and moreover the inhibition rate of CYP3A expression remained comparable over generations. The ratio of the targeted CYP3A transcriptional levels was comparable between knockdown and control mice of the same gender as detected by RT-PCR DGGE analysis. These data suggested that transgenic miR-shRNA suppressed CYP3A expression in a dose-dependent and inheritable manner, and transcriptional levels of the targeted CYP3As were suppressed to a similar extent. The observed knockdown efficacy was further confirmed by enzymatic activity analysis, and data showed that CYP3A activities in transgenic mice were markedly reduced compared to those in wild-type or unrelated miR-shRNA transgenic controls (1.11±0.71 vs 5.85±1.74, 5.9±2.4; P<0.01. This work laid down a foundation to further knock

  13. Epigenetic Regulation of Vitamin D 24-Hydroxylase/CYP24A1 in Human Prostate Cancer

    Science.gov (United States)

    Luo, Wei; Karpf, Adam R.; Deeb, Kristin K.; Muindi, Josephia R.; Morrison, Carl D.; Johnson, Candace S.; Trump, Donald L.

    2010-01-01

    Calcitriol, a regulator of calcium homeostasis with antitumor properties, is degraded by the product of the CYP24A1 gene which is downregulated in human prostate cancer by unknown mechanisms. We found that CYP24A1 expression is inversely correlated with promoter DNA methylation in prostate cancer cell lines. Treatment with the DNA methyltransferase inhibitor 5-aza-2′-deoxycytidine (DAC) activates CYP24A1 expression in prostate cancer cells. In vitro methylation of the CYP24A1 promoter represses its promoter activity. Furthermore, inhibition of histone deacetylases by trichostatin A (TSA) enhances the expression of CYP24A1 in prostate cancer cells. ChIP-qPCR reveals that specific histone modifications are associated with the CYP24A1 promoter region. Treatment with TSA increases H3K9ac and H3K4me2 and simultaneously decreases H3K9me2 at the CYP24A1 promoter. ChIP-qPCR assay reveals that treatment with DAC and TSA increases the recruitment of VDR to the CYP24A1 promoter. RT-PCR analysis of paired human prostate samples reveals that CYP24A1 expression is down-regulated in prostate malignant lesions compared to adjacent histologically benign lesions. Bisulfite pyrosequencing shows that CYP24A1 gene is hypermethylated in malignant lesions compared to matched benign lesions. Our findings indicate that repression of CYP24A1 gene expression in human prostate cancer cells is mediated in part by promoter DNA methylation and repressive histone modifications. PMID:20587525

  14. Modeling of drug-mediated CYP3A4 induction by using human iPS cell-derived enterocyte-like cells

    International Nuclear Information System (INIS)

    Negoro, Ryosuke; Takayama, Kazuo; Nagamoto, Yasuhito; Sakurai, Fuminori; Tachibana, Masashi; Mizuguchi, Hiroyuki

    2016-01-01

    Many drugs have potential to induce the expression of drug-metabolizing enzymes, particularly cytochrome P450 3A4 (CYP3A4), in small intestinal enterocytes. Therefore, a model that can accurately evaluate drug-mediated CYP3A4 induction is urgently needed. In this study, we overlaid Matrigel on the human induced pluripotent stem cells-derived enterocyte-like cells (hiPS-ELCs) to generate the mature hiPS-ELCs that could be applied to drug-mediated CYP3A4 induction test. By overlaying Matrigel in the maturation process of enterocyte-like cells, the gene expression levels of intestinal markers (VILLIN, sucrase-isomaltase, intestine-specific homeobox, caudal type homeobox 2, and intestinal fatty acid-binding protein) were enhanced suggesting that the enterocyte-like cells were maturated by Matrigel overlay. The percentage of VILLIN-positive cells in the hiPS-ELCs found to be approximately 55.6%. To examine the CYP3A4 induction potential, the hiPS-ELCs were treated with various drugs. Treatment with dexamethasone, phenobarbital, rifampicin, or 1α,25-dihydroxyvitamin D3 resulted in 5.8-fold, 13.4-fold, 9.8-fold, or 95.0-fold induction of CYP3A4 expression relative to that in the untreated controls, respectively. These results suggest that our hiPS-ELCs would be a useful model for CYP3A4 induction test. - Highlights: • The hiPS-ELCs were matured by Matrigel overlay. • The hiPS-ELCs expressed intestinal nuclear receptors, such as PXR, GR and VDR. • The hiPS-ELC is a useful model for the drug-mediated CYP3A4 induction test.

  15. Modeling of drug-mediated CYP3A4 induction by using human iPS cell-derived enterocyte-like cells

    Energy Technology Data Exchange (ETDEWEB)

    Negoro, Ryosuke [Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka 565-0871 (Japan); Takayama, Kazuo [Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka 565-0871 (Japan); The Keihanshin Consortium for Fostering the Next Generation of Global Leaders in Research (K-CONNEX), Kyoto University, Kyoto 606-8302 (Japan); Laboratory of Hepatocyte Regulation, National Institute of Biomedical Innovation, Health and Nutrition, Osaka 567-0085 (Japan); Nagamoto, Yasuhito [Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka 565-0871 (Japan); Laboratory of Hepatocyte Regulation, National Institute of Biomedical Innovation, Health and Nutrition, Osaka 567-0085 (Japan); Sakurai, Fuminori [Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka 565-0871 (Japan); Laboratory of Regulatory Sciences for Oligonucleotide Therapeutics, Clinical Drug Development Project, Graduate School of Pharmaceutical Sciences, Osaka University Osaka 565-0871 (Japan); Tachibana, Masashi [Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka 565-0871 (Japan); Mizuguchi, Hiroyuki, E-mail: mizuguch@phs.osaka-u.ac.jp [Laboratory of Biochemistry and Molecular Biology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka 565-0871 (Japan); Laboratory of Hepatocyte Regulation, National Institute of Biomedical Innovation, Health and Nutrition, Osaka 567-0085 (Japan); Global Center for Medical Engineering and Informatics, Osaka University, Osaka 565-0871 (Japan)

    2016-04-15

    Many drugs have potential to induce the expression of drug-metabolizing enzymes, particularly cytochrome P450 3A4 (CYP3A4), in small intestinal enterocytes. Therefore, a model that can accurately evaluate drug-mediated CYP3A4 induction is urgently needed. In this study, we overlaid Matrigel on the human induced pluripotent stem cells-derived enterocyte-like cells (hiPS-ELCs) to generate the mature hiPS-ELCs that could be applied to drug-mediated CYP3A4 induction test. By overlaying Matrigel in the maturation process of enterocyte-like cells, the gene expression levels of intestinal markers (VILLIN, sucrase-isomaltase, intestine-specific homeobox, caudal type homeobox 2, and intestinal fatty acid-binding protein) were enhanced suggesting that the enterocyte-like cells were maturated by Matrigel overlay. The percentage of VILLIN-positive cells in the hiPS-ELCs found to be approximately 55.6%. To examine the CYP3A4 induction potential, the hiPS-ELCs were treated with various drugs. Treatment with dexamethasone, phenobarbital, rifampicin, or 1α,25-dihydroxyvitamin D3 resulted in 5.8-fold, 13.4-fold, 9.8-fold, or 95.0-fold induction of CYP3A4 expression relative to that in the untreated controls, respectively. These results suggest that our hiPS-ELCs would be a useful model for CYP3A4 induction test. - Highlights: • The hiPS-ELCs were matured by Matrigel overlay. • The hiPS-ELCs expressed intestinal nuclear receptors, such as PXR, GR and VDR. • The hiPS-ELC is a useful model for the drug-mediated CYP3A4 induction test.

  16. In Vitro Biotransformation of Two Human CYP3A Probe Substrates and Their Inhibition during Early Zebrafish Development.

    Science.gov (United States)

    Verbueken, Evy; Alsop, Derek; Saad, Moayad A; Pype, Casper; Van Peer, Els M; Casteleyn, Christophe R; Van Ginneken, Chris J; Wilson, Joanna; Van Cruchten, Steven J

    2017-01-22

    At present, the zebrafish embryo is increasingly used as an alternative animal model to screen for developmental toxicity after exposure to xenobiotics. Since zebrafish embryos depend on their own drug-metabolizing capacity, knowledge of their intrinsic biotransformation is pivotal in order to correctly interpret the outcome of teratogenicity assays. Therefore, the aim of this in vitro study was to assess the activity of cytochrome P450 (CYP)-a group of drug-metabolizing enzymes-in microsomes from whole zebrafish embryos (ZEM) of 5, 24, 48, 72, 96 and 120 h post-fertilization (hpf) by means of a mammalian CYP substrate, i.e., benzyloxy-methyl-resorufin (BOMR). The same CYP activity assays were performed in adult zebrafish liver microsomes (ZLM) to serve as a reference for the embryos. In addition, activity assays with the human CYP3A4-specific Luciferin isopropyl acetal (Luciferin-IPA) as well as inhibition studies with ketoconazole and CYP3cide were carried out to identify CYP activity in ZLM. In the present study, biotransformation of BOMR was detected at 72 and 96 hpf; however, metabolite formation was low compared with ZLM. Furthermore, Luciferin-IPA was not metabolized by the zebrafish. In conclusion, the capacity of intrinsic biotransformation in zebrafish embryos appears to be lacking during a major part of organogenesis.

  17. Omeprazole and lansoprazole enantiomers induce CYP3A4 in human hepatocytes and cell lines via glucocorticoid receptor and pregnane X receptor axis.

    Science.gov (United States)

    Novotna, Aneta; Dvorak, Zdenek

    2014-01-01

    Benzimidazole drugs lansoprazole and omeprazole are used for treatment of various gastrointestinal pathologies. Both compounds cause drug-drug interactions because they activate aryl hydrocarbon receptor and induce CYP1A genes. In the current paper, we examined the effects of lansoprazole and omeprazole enantiomers on the expression of key drug-metabolizing enzyme CYP3A4 in human hepatocytes and human cancer cell lines. Lansoprazole enantiomers, but not omeprazole, were equipotent inducers of CYP3A4 mRNA in HepG2 cells. All forms (S-, R-, rac-) of lansoprazole and omeprazole induced CYP3A4 mRNA and protein in human hepatocytes. The quantitative profiles of CYP3A4 induction by individual forms of lansoprazole and omeprazole exerted enantiospecific patterns. Lansoprazole dose-dependently activated pregnane X receptor PXR in gene reporter assays, and slightly modulated rifampicin-inducible PXR activity, with similar potency for each enantiomer. Omeprazole dose-dependently activated PXR and inhibited rifampicin-inducible PXR activity. The effects of S-omeprazole were much stronger as compared to those of R-omeprazole. All forms of lansoprazole, but not omeprazole, slightly activated glucocorticoid receptor and augmented dexamethasone-induced GR transcriptional activity. Omeprazole and lansoprazole influenced basal and ligand inducible expression of tyrosine aminotransferase, a GR-target gene, in HepG2 cells and human hepatocytes. Overall, we demonstrate here that omeprazole and lansoprazole enantiomers induce CYP3A4 in HepG2 cells and human hepatocytes. The induction comprises differential interactions of omeprazole and lansoprazole with transcriptional regulators PXR and GR, and some of the effects were enantiospecific. The data presented here might be of toxicological and clinical importance, since the effects occurred in therapeutically relevant concentrations.

  18. In Vitro Evaluation of Reversible and Time-Dependent Inhibitory Effects of Kalanchoe crenata on CYP2C19 and CYP3A4 Activities.

    Science.gov (United States)

    Awortwe, Charles; Manda, Vamshi K; Avonto, Cristina; Khan, Shabana I; Khan, Ikhlas A; Walker, Larry A; Bouic, Patrick J; Rosenkranz, Bernd

    2015-01-01

    Kalanchoe crenata popularly known as "dog's liver" is used in most African countries for the treatment of chronic diseases such as diabetes, asthma and HIV/AIDS related infections. The evaluation of K. crenata for herb-drug interactions has not been reported. This study therefore aims to evaluate the risk of K. crenata for herb-drug interaction in vitro. Crude methanol and fractions of K. crenata were incubated and preincubated with recombinant human CYP2C19 and CYP3A4. Comparative studies were conducted in both human liver microsomes and recombinant human CYP to ascertain the inhibition profile of the crude extract and the various fractions. The cocktail approach of recombinant human CYPs was conducted to confirm the inhibition potential of the fractions in the presence of other CYPs. The results showed significant time-dependent inhibition of tested samples on CYP3A4 with crude methanol (39KC), fractions 45A, 45B and 45D given IC50 fold decrease of 3.29, 2.26, 1.91 and 1.49, respective. Time dependent kinetic assessment of 39KC and 45D showed KI and kinact values for 39KC as 1.77 µg/mL and 0.091 min(-1) while that of 45D were 6.45 µg/mL and 0.024 min(-1), respectively. Determination of kinact based on IC50 calculations yielded 0.015 and 0.04 min(-1) for 39KC and 45D, respectively. Cocktail approach exhibited fold decreases in IC50 for all test fractions on CYP3A4 within the ranges of 2.10 - 4.10. At least one phytoconstituent in the crude methanol extract of Kalanchoe crenata is a reversible and time-dependent inhibitor of CYP3A4.

  19. Effect of Garden Cress Seeds Powder and Its Alcoholic Extract on the Metabolic Activity of CYP2D6 and CYP3A4

    Directory of Open Access Journals (Sweden)

    Fahad I. Al-Jenoobi

    2014-01-01

    Full Text Available The powder and alcoholic extract of dried seeds of garden cress were investigated for their effect on metabolic activity of CYP2D6 and CYP3A4 enzymes. In vitro and clinical studies were conducted on human liver microsomes and healthy human subjects, respectively. Dextromethorphan was used as a common marker for measuring metabolic activity of CYP2D6 and CYP3A4 enzymes. In in vitro studies, microsomes were incubated with NADPH in presence and absence of different concentrations of seeds extract. Clinical investigations were performed in two phases. In phase I, six healthy female volunteers were administered a single dose of dextromethorphan and in phase II volunteers were treated with seeds powder for seven days and dextromethorphan was administered with last dose. The O-demethylated and N-demethylated metabolites of dextromethorphan were measured as dextrorphan (DOR and 3-methoxymorphinan (3-MM, respectively. Observations suggested that garden cress inhibits the formation of DOR and 3-MM metabolites. This inhibition of metabolite level was attributed to the inhibition of CYP2D6 and CYP3A4 activity. Garden cress decreases the level of DOR and 3-MM in urine and significantly increases the urinary metabolic ratio of DEX/DOR and DEX/3-MM. The findings suggested that garden cress seeds powder and ethanolic extract have the potential to interact with CYP2D6 and CYP3A4 substrates.

  20. Porcine foetal and neonatal CYP3A liver expression

    DEFF Research Database (Denmark)

    Hermann-Bank, Marie Louise; Skaanild, Mette Tingleff

    2011-01-01

    enzyme in the foetal liver, whereas the expression of CYP3A4 is low. After parturition there is a shift in the expression, thus CYP3A7 is down regulated, while the level of CYP3A4 gradually increases and becomes the dominant metabolising CYP3A enzyme in the adult. The minipig is increasingly being used......3A4) in minipigs. This was elucidated by examining the hepatic mRNA expression of CYP3A7 and CYP3A29 in 39 foetuses and newborn Göttingen minipigs using quantitative real time polymerase chain reaction (qPCR). Furthermore the immunochemical level of CYP3A7-LE and CYP3A29 was measured in liver...

  1. Genetic variation at CYP3A is associated with age at menarche and breast cancer risk: a case-control study.

    LENUS (Irish Health Repository)

    Johnson, Nichola

    2014-05-26

    We have previously shown that a tag single nucleotide polymorphism (rs10235235), which maps to the CYP3A locus (7q22.1), was associated with a reduction in premenopausal urinary estrone glucuronide levels and a modest reduction in risk of breast cancer in women age ≤50 years.

  2. Genetic variation at CYP3A is associated with age at menarche and breast cancer risk: a case-control study

    NARCIS (Netherlands)

    Johnson, N. (Nichola); F. Dudbridge (Frank); N. Orr (Nick); L.J. Gibson (Lorna); M. Jones (Michael); M. Schoemaker (Minouk); Folkerd, E.J. (Elizabeth J.); Haynes, B.P. (Ben P.); J.L. Hopper (John); M.C. Southey (Melissa); Dite, G.S. (Gillian S.); Apicella, C. (Carmel); M.K. Schmidt (Marjanka); A. Broeks (Annegien); L.J. van 't Veer (Laura); F. Atsma (Femke); K.R. Muir (K.); A. Lophatananon (Artitaya); P.A. Fasching (Peter); M.W. Beckmann (Matthias); A.B. Ekici (Arif); S.P. Renner (S.); E.J. Sawyer (Elinor); I.P. Tomlinson (Ian); M. Kerin (Michael); N. Miller (Nicola); B. Burwinkel (Barbara); Marme, F. (Frederik); A. Schneeweiss (Andreas); C. Sohn (Christof); P. Guénel (Pascal); T. Truong (Thérèse); Cordina, E. (Emilie); F. Menegaux (Florence); S.E. Bojesen (Stig); B.G. Nordestgaard (Børge); H. Flyger (Henrik); R.L. Milne (Roger); M.P. Zamora (Pilar); J.I. Arias Pérez (José Ignacio); J. Benítez (Javier); L. Bernstein (Leslie); H. Anton-Culver (Hoda); A. Ziogas (Argyrios); Clarke Dur, C. (Christina); Brenner, H. (Hermann); H. Mul̈ler (Heiko); Arndt, V. (Volker); A.K. Dieffenbach (Aida Karina); A. Meindl (Alfons); J. Heil (Joerg); C.R. Bartram (Claus); R.K. Schmutzler (Rita); H. Brauch (Hiltrud); C. Justenhoven (Christina); Y-D. Ko (Yon-Dschun); H. Nevanlinna (Heli); Muranen, T.A. (Taru A.); K. Aittomäki (Kristiina); C. Blomqvist (Carl); K. Matsuo (Keitaro); Dörk, T. (Thilo); N.V. Bogdanova (Natalia); Antonenkova, N.N. (Natalia N.); A. Lindblom (Annika); A. Mannermaa (Arto); V. Kataja (Vesa); V-M. Kosma (Veli-Matti); J.M. Hartikainen (J.); G. Chenevix-Trench (Georgia); J. Beesley (Jonathan); A.H. Wu (Anna); D. Van Den Berg (David); C.-C. Tseng (Chiu-Chen); Lambrechts, D. (Diether); D. Smeets (Dominiek); P. Neven (Patrick); H. Wildiers (Hans); J. Chang-Claude (Jenny); Rudolph, A. (Anja); Nickels, S. (Stefan); D. Flesch-Janys (Dieter); P. Radice (Paolo); P. Peterlongo (Paolo); B. Bonnani (Bernardo); V. Pensotti (Valeria); Couch, F.J. (Fergus J.); J.E. Olson (Janet); Wang, X. (Xianshu); Fredericksen, Z. (Zachary); V.S. Pankratz (Shane); Giles, G.G. (Graham G.); G. Severi (Gianluca); Baglietto, L. (Laura); C.A. Haiman (Christopher); J. Simard (Jacques); M.S. Goldberg (Mark); Labrèche, F. (France); M. Dumont (Martine); Soucy, P. (Penny); S.-H. Teo (Soo-Hwang); C.H. Yip (Cheng Har); S.-Y. Phuah (Sze-Yee); Cornes, B.K. (Belinda K.); Kristensen, V.N. (Vessela N.); G. Grenaker Alnæs (Grethe); A.-L. Borresen-Dale (Anne-Lise); W. Zheng (Wei); R. Winqvist (Robert); K. Pykäs (Katri); A. Jukkola-Vuorinen (Arja); Grip, M. (Mervi); I.L. Andrulis (Irene); J.A. Knight (Julia); Glendon, G. (Gord); A.-M. Mulligan (Anna-Marie); P. Devillee (Peter); J.D. Figueroa (Jonine); S.J. Chanock (Stephen); J. Lissowska (Jolanta); M.E. Sherman (Mark); P. Hall (Per); N. Schoof (Nils); M.J. Hooning (Maartje); A. Hollestelle (Antoinette); R.A. Oldenburg (Rogier); M.M.A. Tilanus-Linthorst (Madeleine); Liu, J. (Jianjun); A. Cox (Angela); I.W. Brock (Ian); M.W.R. Reed (Malcolm); S.S. Cross (Simon); W.J. Blot (William); L.B. Signorello (Lisa B.); P.D.P. Pharoah (Paul); A.M. Dunning (Alison); Shah, M. (Mitul); D. Kang (Daehee); Noh, D.-Y. (Dong-Young); Park, S.K. (Sue K.); Choi, J.-Y. (Ji-Yeob); Hartman, M. (Mikael); X. Miao; Lim, W.Y. (Wei Yen); A. Tang (Anthony); U. Hamann (Ute); A. Försti (Asta); T. Rud̈iger (Thomas); H.U. Ulmer (Hans); A. Jakubowska (Anna); J. Lubinski (Jan); Jaworska-Bieniek, K. (Katarzyna); Durda, K. (Katarzyna); Sangrajrang, S. (Suleeporn); Gaborieau, V. (Valerie); P. Brennan (Paul); McKay, J. (James); S. Slager (Susan); A.E. Toland (Amanda E.); C. Vachon (Celine); Yannoukakos, D. (Drakoulis); C.-Y. Shen (Chen-Yang); J-C. Yu (Jyh-Cherng); Huang, C.-S. (Chiun-Sheng); M.-F. Hou (Ming-Feng); A. González-Neira (Anna); D.C. Tessier (Daniel C.); D. Vincent (Daniel); F. Bacot (Francois); C. Luccarini (Craig); J. Dennis (Joe); K. Michailidou (Kyriaki); M.K. Bolla (Manjeet K.); J. Wang (Jinxia); D.F. Easton (Douglas); M. García-Closas (Montserrat); M. Dowsett (Mitch); A. Ashworth (Alan); A.J. Swerdlow (Anthony ); J. Peto (Julian); I. dos Santos Silva (Isabel); O. Fletcher (Olivia)

    2014-01-01

    textabstractINTRODUCTION: We have previously shown that a tag single nucleotide polymorphism (rs10235235), which maps to the CYP3A locus (7q22.1), was associated with a reduction in premenopausal urinary estrone glucuronide levels and a modest reduction in risk of breast cancer in women age ≤50

  3. Genetic variation at CYP3A is associated with age at menarche and breast cancer risk: A case-control study

    NARCIS (Netherlands)

    N. Johnson (Nichola); F. Dudbridge (Frank); N. Orr (Nick); L.J. Gibson (Lorna); M. Jones (Michael); M. Schoemaker (Minouk); Folkerd, E.J. (Elizabeth J.); Haynes, B.P. (Ben P.); J.L. Hopper (John); M.C. Southey (Melissa); G.S. Dite (Gillian); C. Apicella (Carmel); M.K. Schmidt (Marjanka); A. Broeks (Annegien); L.J. van 't Veer (Laura); F. Atsma (Femke); K.R. Muir (K.); A. Lophatananon (Artitaya); P.A. Fasching (Peter); M.W. Beckmann (Matthias); A.B. Ekici (Arif); S.P. Renner (S.); E.J. Sawyer (Elinor); I.P. Tomlinson (Ian); M. Kerin (Michael); N. Miller (Nicola); B. Burwinkel (Barbara); F. Marme (Federick); A. Schneeweiss (Andreas); C. Sohn (Christof); P. Guénel (Pascal); T. Truong (Thérèse); Cordina, E. (Emilie); F. Menegaux (Florence); S.E. Bojesen (Stig); B.G. Nordestgaard (Børge); H. Flyger (Henrik); R.L. Milne (Roger); M.P. Zamora (Pilar); J.I.A. Perez (Jose Ignacio Arias); J. Benítez (Javier); L. Bernstein (Leslie); H. Anton-Culver (Hoda); A. Ziogas (Argyrios); C.C. Dur (Christina Clarke); H. Brenner (Hermann); H. Mul̈ler (Heiko); V. Arndt (Volker); A.K. Dieffenbach (Aida Karina); A. Meindl (Alfons); J. Heil (Joerg); C.R. Bartram (Claus); R.K. Schmutzler (Rita); H. Brauch (Hiltrud); C. Justenhoven (Christina); Y-D. Ko (Yon-Dschun); H. Nevanlinna (Heli); T.A. Muranen (Taru); K. Aittomäki (Kristiina); C. Blomqvist (Carl); K. Matsuo (Keitaro); T. Dörk (Thilo); Bogdanova, N.V. (Natalia V.); N.N. Antonenkova (Natalia); A. Lindblom (Annika); A. Mannermaa (Arto); V. Kataja (Vesa); V-M. Kosma (Veli-Matti); J.M. Hartikainen (J.); G. Chenevix-Trench (Georgia); J. Beesley (Jonathan); A.H. Wu (Anna); D. Van Den Berg (David); C.-C. Tseng (Chiu-Chen); Lambrechts, D. (Diether); D. Smeets (Dominiek); P. Neven (Patrick); H. Wildiers (Hans); J. Chang-Claude (Jenny); A. Rudolph (Anja); S. Nickels (Stefan); D. Flesch-Janys (Dieter); P. Radice (Paolo); P. Peterlongo (Paolo); B. Bonnani (Bernardo); V. Pensotti (Valeria); F.J. Couch (Fergus); Olson, J.E. (Janet E.); X. Wang (X.); Fredericksen, Z. (Zachary); V.S. Pankratz (Shane); Giles, G.G. (Graham G.); G. Severi (Gianluca); Baglietto, L. (Laura); C.A. Haiman (Christopher A.); J. Simard (Jacques); M.S. Goldberg (Mark); F. Labrèche (France); M. Dumont (Martine); Soucy, P. (Penny); S.-H. Teo (Soo-Hwang); C.H. Yip (Cheng Har); S.-Y. Phuah (Sze-Yee); Cornes, B.K. (Belinda K.); V. Kristensen (Vessela); Alnæs, G.G. (Grethe Grenaker); A.-L. Borresen-Dale (Anne-Lise); W. Zheng (Wei); R. Winqvist (Robert); K. Pykäs (Katri); A. Jukkola-Vuorinen (Arja); M. Grip (Mervi); I.L. Andrulis (Irene); J.A. Knight (Julia); G. Glendon (Gord); A.-M. Mulligan (Anna-Marie); P. Devillee (Peter); J.D. Figueroa (Jonine); Chanock, S.J. (Stephen J.); J. Lissowska (Jolanta); M.E. Sherman (Mark); P. Hall (Per); N. Schoof (Nils); M.J. Hooning (Maartje); A. Hollestelle (Antoinette); R.A. Oldenburg (Rogier); M.M.A. Tilanus-Linthorst (Madeleine); Liu, J. (Jianjun); A. Cox (Angela); I.W. Brock (Ian); M.W.R. Reed (Malcolm); S.S. Cross (Simon); W.J. Blot (William); L.B. Signorello (Lisa B.); P.D.P. Pharoah (Paul); A.M. Dunning (Alison); M. Shah (Mitul); D. Kang (Daehee); D-Y. Noh (Dong-Young); S.K. Park (Sue K.); J.-Y. Choi (Ji-Yeob); J.M. Hartman (Joost); X. Miao; Lim, W.Y. (Wei Yen); A. Tang (Anthony); U. Hamann (Ute); A. Försti (Asta); T. Rud̈iger (Thomas); H.U. Ulmer (Hans); A. Jakubowska (Anna); J. Lubinski (Jan); Jaworska-Bieniek, K. (Katarzyna); Durda, K. (Katarzyna); S. Sangrajrang (Suleeporn); Gaborieau, V. (Valerie); P. Brennan (Paul); J.D. McKay (James); S. Slager (Susan); A.E. Toland (Amanda); Vachon, C. (Celine); D. Yannoukakos (Drakoulis); C.-Y. Shen (Chen-Yang); J-C. Yu (Jyh-Cherng); Huang, C.-S. (Chiun-Sheng); M.-F. Hou (Ming-Feng); A. González-Neira (Anna); D.C. Tessier (Daniel C.); D. Vincent (Daniel); F. Bacot (Francois); C. Luccarini (Craig); J. Dennis (Joe); K. Michailidou (Kyriaki); Bolla, M.K. (Manjeet K.); J. Wang (Jinxia); D.F. Easton (Douglas F.); M. García-Closas (Montserrat); M. Dowsett (Mitch); A. Ashworth (Alan); A.J. Swerdlow (Anthony ); J. Peto (Julian); I. dos Santos Silva (Isabel); O. Fletcher (Olivia)

    2014-01-01

    textabstractIntroduction: We have previously shown that a tag single nucleotide polymorphism (rs10235235), which maps to the CYP3A locus (7q22.1), was associated with a reduction in premenopausal urinary estrone glucuronide levels and a modest reduction in risk of breast cancer in women age ≤50

  4. Genomic variation in CYP3A4: type, frequencies and potential implications for pharmacogenetic understanding.

    OpenAIRE

    Creemer, O.

    2012-01-01

    The human cytochrome P450 3A subfamily metabolises endogenous substances and approximately half of all currently available drugs. There is marked inter-individual variation in hepatic expression of the major adult isoform, CYP3A4; the genetic component of this variability is estimated at 60-90% and, as yet, remains largely uncharacterised. Elucidation of genetic factors determining CYP3A4 activity would permit personalised dose-adjustment in therapies with CYP3A4 drug substrates. CYP3A4 genom...

  5. The Effect of CYP2B6, CYP2D6, and CYP3A4 Alleles on Methadone Binding: A Molecular Docking Study

    Directory of Open Access Journals (Sweden)

    Nik Nur Syazana Bt Nik Mohamed Kamal

    2013-01-01

    Full Text Available Current methadone maintenance therapy (MMT is yet to ensure 100% successful treatment as the optimum dosage has yet to be determined. Overdose leads to death while lower dose causes the opioid withdrawal effect. Single-nucleotide polymorphisms (SNP in cytochrome P450s (CYPs, the methadone metabolizers, have been showen to be the main factor for the interindividual variability of methadone clinical effects. In this study, we investigated the effect of SNPs in three major methadone metabolizers (CYP2B6, CYP2D6, and CYP3A4 on methadone binding affinity. Results showed that CYP2B6*11, CYP2B6*12, CYP2B6*18, and CYP3A4*12 have significantly higher binding affinity to R-methadone compared to wild type. S-methadone has higher binding affinity in CYP3A4*3, CYP3A4*11, and CYP3A4*12 compared to wild type. R-methadone was shown to be the active form of methadone; thus individuals with CYP alleles that binds better to R-methadone will have higher methadone metabolism rate. Therefore, a higher dosage of methadone is necessary to obtain the opiate effect compared to a normal individual and vice versa. These results provide an initial prediction on methadone metabolism rate for individuals with mutant type CYP which enables prescription of optimum methadone dosage for individuals with CYP alleles.

  6. In Vitro Biotransformation of Two Human CYP3A Probe Substrates and Their Inhibition during Early Zebrafish Development

    Directory of Open Access Journals (Sweden)

    Evy Verbueken

    2017-01-01

    Full Text Available At present, the zebrafish embryo is increasingly used as an alternative animal model to screen for developmental toxicity after exposure to xenobiotics. Since zebrafish embryos depend on their own drug-metabolizing capacity, knowledge of their intrinsic biotransformation is pivotal in order to correctly interpret the outcome of teratogenicity assays. Therefore, the aim of this in vitro study was to assess the activity of cytochrome P450 (CYP—a group of drug-metabolizing enzymes—in microsomes from whole zebrafish embryos (ZEM of 5, 24, 48, 72, 96 and 120 h post-fertilization (hpf by means of a mammalian CYP substrate, i.e., benzyloxy-methyl-resorufin (BOMR. The same CYP activity assays were performed in adult zebrafish liver microsomes (ZLM to serve as a reference for the embryos. In addition, activity assays with the human CYP3A4-specific Luciferin isopropyl acetal (Luciferin-IPA as well as inhibition studies with ketoconazole and CYP3cide were carried out to identify CYP activity in ZLM. In the present study, biotransformation of BOMR was detected at 72 and 96 hpf; however, metabolite formation was low compared with ZLM. Furthermore, Luciferin-IPA was not metabolized by the zebrafish. In conclusion, the capacity of intrinsic biotransformation in zebrafish embryos appears to be lacking during a major part of organogenesis.

  7. 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

  8. [Toxic effect of trichloroethylene on liver cells with CYP3A4 gene defect].

    Science.gov (United States)

    Liao, R Y; Liu, S

    2016-06-20

    To investigate the toxic effect of trichloroethylene on liver cells with CYP3A4 gene defect. The normal human liver cells (L02 cells) and liver cells with CYP3A4 gene defect were exposed to trichloroethylene at different doses (0.0, 0.4, 0.8, 1.6, 3.2, and 6.4 mmol/L). CCK8 assay and RT-qPCR were used to measure cell viability and changes in the expression of apoptosis genes and oncogenes. After being exposed to trichloroethylene at doses of 1.6, 3.2, and 6.4 mmol/L, the liver cells with CYP3A4 gene defect showed significantly higher cell viability than L02 cells (0.91±0.06/0.89±0.05/0.85±0.07 vs 0.80±0.04/0.73±0.06/0.67±0.07, Ptrichloroethylene groups showed significant increases in the expression of the apoptosis genes caspase-3, caspase-8, and caspase-9 (PTrichloroethylene exposure has a less effect on the expression of apoptosis genes and oncogenes in liver cells with CYP3A4 gene defect than in normal human liver cells, suggesting that CYP3A4 gene defect reduces the inductive effect of trichloroethylene on apoptosis genes and oncogenes.

  9. Arsenite and its metabolites, MMAIII and DMAIII, modify CYP3A4, PXR and RXR alpha expression in the small intestine of CYP3A4 transgenic mice

    International Nuclear Information System (INIS)

    Medina-Diaz, I.M.; Estrada-Muniz, E.; Reyes-Hernandez, O.D.; Ramirez, P.; Vega, L.; Elizondo, G.

    2009-01-01

    Arsenic is an environmental pollutant that has been associated with an increased risk for the development of cancer and several other diseases through alterations of cellular homeostasis and hepatic function. Cytochrome P450 (P450) modification may be one of the factors contributing to these disorders. Several reports have established that exposure to arsenite modifies P450 expression by decreasing or increasing mRNA and protein levels. Cytochrome P450 3A4 (CYP3A4), the predominant P450 expressed in the human liver and intestines, which is regulated mainly by the Pregnane X Receptor-Retinoid X Receptor alpha (PXR-RXR alpha) heterodimer, contributes to the metabolism of approximately half the drugs in clinical use today. The present study investigates the effect of sodium arsenite and its metabolites monomethylarsonous acid (MMA III ) and dimethylarsinous acid (DMA III ) on CYP3A4, PXR, and RXR alpha expression in the small intestine of CYP3A4 transgenic mice. Sodium arsenite treatment increases mRNA, protein and CYP3A4 activity in a dose-dependent manner. However, the increase in protein expression was not as marked as compared to the increase in mRNA levels. Arsenite treatment induces the accumulation of Ub-protein conjugates, indicating that the activation of this mechanism may explain the differences observed between the mRNA and protein expression of CYP3A4 induction. Treatment with 0.05 mg/kg of DMA III induces CYP3A4 in a similar way, while treatment with 0.05 mg/kg of MMA III increases mostly mRNA, and to a lesser degree, CYP3A4 activity. Sodium arsenite and both its metabolites increase PXR mRNA, while only DMA III induces RXR alpha expression. Overall, these results suggest that sodium arsenite and its metabolites induce CYP3A4 expression by increasing PXR expression in the small intestine of CYP3A4 transgenic mice.

  10. 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

  11. Association of CYP2B6, CYP3A5, and CYP2C19 genetic polymorphisms with sibutramine pharmacokinetics in healthy Korean subjects.

    Science.gov (United States)

    Kim, K A; Song, W K; Park, J Y

    2009-11-01

    We assessed the association of CYP2B6, CYP3A5, and CYP2C19 polymorphisms with sibutramine pharmacokinetics. Forty six healthy male subjects were enrolled, and their CYP2B6 (*4 and *6), CYP3A5 (*3), and CYP2C19 (*2, and *3) genotypes were analyzed. After a single 15-mg dose of sibutramine was administered, plasma concentrations of sibutramine and its metabolites, M1 and M2, were measured. CYP2B6 and CYP3A5 polymorphisms did not affect the pharmacokinetics of sibutramine and its metabolites. However, the CYP2C19 genotype substantially influenced plasma levels of sibutramine and its metabolites. The mean area under the curve (AUC) of sibutramine in CYP2C19 intermediate metabolizers (IMs; *1/*2 or *1/*3) and poor metabolizers (PMs; *2/*2, *2/*3)) was 18.5 and 252.2% higher, respectively, than the AUC in extensive metabolizers (EMs, *1/*1) (P sibutramine.

  12. Genetic variation at CYP3A is associated with age at menarche and breast cancer risk: a case-control study

    OpenAIRE

    Johnson, Nichola; Dudbridge, Frank; Orr, Nick; Gibson, Lorna; Jones, Michael E; Schoemaker, Minouk J; Folkerd, Elizabeth J; Haynes, Ben P; Hopper, John L; Southey, Melissa C; Dite, Gillian S; Apicella, Carmel; Schmidt, Marjanka K; Broeks, Annegien; Van?t Veer, Laura J

    2014-01-01

    Abstract Introduction We have previously shown that a tag single nucleotide polymorphism (rs10235235), which maps to the CYP3A locus (7q22.1), was associated with a reduction in premenopausal urinary estrone glucuronide levels and a modest reduction in risk of breast cancer in women age ≤50 years. Methods We further investigated the association of rs10235235 with b...

  13. 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...

  14. CYP1A1 and CYP1B1 in human lymphocytes as biomarker of exposure: effect of dioxin exposure and polymorphisms

    Energy Technology Data Exchange (ETDEWEB)

    Duursen, M. van; Sanderson, T.; Berg, M. van den [Inst. for Risk Assessment Sciences, Utrecht (Netherlands)

    2004-09-15

    There are several known genetic polymorphisms of the CYP1A1 and CYP1B1 genes. A polymorphism in the 3'-untranslated region of the CYP1A1 gene (CYP1A1 MspI or CYP1A1 m1) is often studied in relation with breast or lung cancer, but little is known about the functional effect of this polymorphism. An amino acid substitution in codon 432 (Val to Leu) of the CYP1B1 gene is associated with a lower catalytic activity of the enzyme. However, the involvement of these polymorphisms on the inducibility of CYP1A1 and CYP1B1 gene expression is unclear. CYP1A1 and CYP1B1 mRNA expression levels can be determined in peripheral blood lymphocytes. This makes them potential candidates for use as biomarker of exposure to environmental compounds. Interindividual variations in mRNA expression patterns, catalytic activity and polymorphisms are very important factors when CYP1A1 and CYP1B1 expression patterns are used as biomarker of exposure, but little is known about it. Spencer et al. showed a concentration-dependent increase of CYP1B1 mRNA in lymphocytes upon exposure in vitro to 2,3,7,8-tetrachloro-p-dibenzodioxin (TCDD), the most potent dioxin. Yet, only a few studies describe the in vivo correlation between polymorphisms, mRNA expression level and exposure to environmental factors. In this study, we wanted to obtain a better insight in the CYP1A1 and CYP1B1 mRNA expression and enzyme activity in human lymphocytes. We determined the constitutive CYP1A1 and CYP1B1 mRNA expression in lymphocytes of ten healthy volunteers and the variability in sensitivity toward enzyme induction by TCDD. Further, the CYP1A1 m1 and CYP1B1 Val432Leu polymorphisms were determined.

  15. Regulation of zebrafish CYP3A65 transcription by AHR2

    International Nuclear Information System (INIS)

    Chang, Chin-Teng; Chung, Hsin-Yu; Su, Hsiao-Ting; Tseng, Hua-Pin; Tzou, Wen-Shyong; Hu, Chin-Hwa

    2013-01-01

    CYP3A proteins are the most abundant CYPs in the liver and intestines, and they play a pivotal role in drug metabolism. In mammals, CYP3A genes are induced by various xenobiotics through processes mediated by PXR. We previously identified zebrafish CYP3A65 as a CYP3A ortholog that is constitutively expressed in gastrointestinal tissues, and is upregulated by treatment with dexamethasone, rifampicin or tetrachlorodibenzo-p-dioxin (TCDD). However, the underlying mechanism of TCDD-mediated CYP3A65 transcription is unclear. Here we generated two transgenic zebrafish, Tg(CYP3A65S:EGFP) and Tg(CYP3A65L:EGFP), which contain 2.1 and 5.4 kb 5′ flanking sequences, respectively, of the CYP3A65 gene upstream of EGFP. Both transgenic lines express EGFP in larval gastrointestinal tissues in a pattern similar to that of the endogenous CYP3A65 gene. Moreover, EGFP expression can be significantly induced by TCDD exposure during the larval stage. In addition, EGFP expression can be stimulated by kynurenine, a putative AHR ligand produced during tryptophan metabolism. AHRE elements in the upstream regulatory region of the CYP3A65 gene are indispensible for basal and TCDD-induced transcription. Furthermore, the AHR2 DNA and ligand-binding domains are required to mediate effective CYP3A65 transcription. AHRE sequences are present in the promoters of many teleost CYP3 genes, but not of mammalian CYP3 genes, suggesting that AHR/AHR2-mediated transcription is likely a common regulatory mechanism for teleost CYP3 genes. It may also reflect the different environments that terrestrial and aquatic organisms encounter. - Highlights: • Tg(CYP3A65:EGFP) and CYP3A65 exhibits identical expression pattern. • CYP3A65 can be significantly induced by TCDD or kynurenine. • The AHRE elements are required to mediate CYP3A65 transcription. • The AHR2 DNA and ligand-binding domains are required for CYP3A65 transcription. • AHRE elements are present in many teleost CYP3 genes, but not in

  16. Regulation of zebrafish CYP3A65 transcription by AHR2

    Energy Technology Data Exchange (ETDEWEB)

    Chang, Chin-Teng; Chung, Hsin-Yu; Su, Hsiao-Ting; Tseng, Hua-Pin [Institute of Bioscience and Biotechnology, National Taiwan Ocean University, Keelung, Taiwan (China); Tzou, Wen-Shyong [Institute of Bioscience and Biotechnology, National Taiwan Ocean University, Keelung, Taiwan (China); Center of Excellence for Marine Bioenvironment and Biotechnology, National Taiwan Ocean University, Keelung, Taiwan (China); Hu, Chin-Hwa, E-mail: chhu@mail.ntou.edu.tw [Institute of Bioscience and Biotechnology, National Taiwan Ocean University, Keelung, Taiwan (China); Center of Excellence for Marine Bioenvironment and Biotechnology, National Taiwan Ocean University, Keelung, Taiwan (China)

    2013-07-15

    CYP3A proteins are the most abundant CYPs in the liver and intestines, and they play a pivotal role in drug metabolism. In mammals, CYP3A genes are induced by various xenobiotics through processes mediated by PXR. We previously identified zebrafish CYP3A65 as a CYP3A ortholog that is constitutively expressed in gastrointestinal tissues, and is upregulated by treatment with dexamethasone, rifampicin or tetrachlorodibenzo-p-dioxin (TCDD). However, the underlying mechanism of TCDD-mediated CYP3A65 transcription is unclear. Here we generated two transgenic zebrafish, Tg(CYP3A65S:EGFP) and Tg(CYP3A65L:EGFP), which contain 2.1 and 5.4 kb 5′ flanking sequences, respectively, of the CYP3A65 gene upstream of EGFP. Both transgenic lines express EGFP in larval gastrointestinal tissues in a pattern similar to that of the endogenous CYP3A65 gene. Moreover, EGFP expression can be significantly induced by TCDD exposure during the larval stage. In addition, EGFP expression can be stimulated by kynurenine, a putative AHR ligand produced during tryptophan metabolism. AHRE elements in the upstream regulatory region of the CYP3A65 gene are indispensible for basal and TCDD-induced transcription. Furthermore, the AHR2 DNA and ligand-binding domains are required to mediate effective CYP3A65 transcription. AHRE sequences are present in the promoters of many teleost CYP3 genes, but not of mammalian CYP3 genes, suggesting that AHR/AHR2-mediated transcription is likely a common regulatory mechanism for teleost CYP3 genes. It may also reflect the different environments that terrestrial and aquatic organisms encounter. - Highlights: • Tg(CYP3A65:EGFP) and CYP3A65 exhibits identical expression pattern. • CYP3A65 can be significantly induced by TCDD or kynurenine. • The AHRE elements are required to mediate CYP3A65 transcription. • The AHR2 DNA and ligand-binding domains are required for CYP3A65 transcription. • AHRE elements are present in many teleost CYP3 genes, but not in

  17. CYP3A4 Mediates Oxidative Metabolism of the Synthetic Cannabinoid AKB-48.

    Science.gov (United States)

    Holm, Niels Bjerre; Nielsen, Line Marie; Linnet, Kristian

    2015-09-01

    Synthetic cannabinoid designer drugs have emerged as drugs of abuse during the last decade, and acute intoxication cases are documented in the scientific literature. Synthetic cannabinoids are extensively metabolized, but our knowledge of the involved enzymes is limited. Here, we investigated the metabolism of N-(1-adamantyl)-1-pentyl-1H-indazole-3-carboxamide (AKB-48), a compound identified in herbal blends from 2012 and onwards. We screened for metabolite formation using a panel of nine recombinant cytochrome P450 (CYP) enzymes (CYP1A2, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, and 3A4) and compared the formed metabolites to human liver microsomal (HLM) incubations with specific inhibitors against CYP2D6, 2C19, and 3A4, respectively. The data reported here demonstrate CYP3A4 to be the major CYP enzyme responsible for the oxidative metabolism of AKB-48, preferentially performing the oxidation on the adamantyl moiety. Genetic polymorphisms are likely not important with regard to toxicity given the major involvement of CYP3A4. Adverse drug-drug interactions (DDIs) could potentially occur in cases with co-intake of strong CYP3A4 inhibitors, e.g., HIV antivirals and azole antifungal agents.

  18. Human CYP2E1 mediates the formation of glycidamide from acrylamide

    Energy Technology Data Exchange (ETDEWEB)

    Settels, Eva; Appel, Klaus E. [Federal Institute for Risk Assessment, Center for Experimental Toxicology, Berlin (Germany); Bernauer, Ulrike; Gundert-Remy, Ursula [Federal Institute for Risk Assessment, Department of Safety of Substances and Preparations, Berlin (Germany); Palavinskas, Richard; Klaffke, Horst S. [Federal Institute for Risk Assessment, Center for Analytical Chemistry, Berlin (Germany)

    2008-10-15

    Regarding the cancer risk assessment of acrylamide (AA) it is of basic interest to know, as to what amount of the absorbed AA is metabolized to glycidamide (GA) in humans, compared to what has been observed in laboratory animals. GA is suspected of being the ultimate carcinogenic metabolite of AA. From experiments with CYP2E1-deficient mice it can be concluded that AA is metabolized to GA primarily by CYP2E1. We therefore examined whether CYP2E1 is involved in GA formation in non-rodent species with the focus on humans by using human CYP2E1 supersomes trademark, marmoset and human liver microsomes and in addition, genetically engineered V79 cells expressing human CYP2E1 (V79h2E1 cells). Special emphasis was placed on the analytical detection of GA, which was performed by gas chromatography/mass spectrometry. The results show that AA is metabolized to GA in human CYP2E1 supersomes trademark, in marmoset and human liver microsomes as well as in V79h2E1 cells. The activity of GA formation is highest in supersomes trademark; in human liver it is somewhat higher than in marmoset liver. A monoclonal CYP2E1 human selective antibody (MAB-2E1) and diethyldithiocarbamate (DDC) were used as specific inhibitors of CYP2E1. The generation of GA could be inhibited by MAB-2E1 to about 80% in V79h2E1 cells and to about 90% in human and marmoset liver microsomes. Also DDC led to an inhibition of about 95%. In conclusion, AA is metabolized to GA by human CYP2E1. Overall, the present work describes (1) the application and refinement of a sensitive methodology in order to determine low amounts of GA, (2) the applicability of genetically modified V79 cell lines in order to investigate specific questions concerning metabolism and (3) the involvement, for the first time, of human CYP2E1 in the formation of GA from AA. Further studies will compare the activities of GA formation in genetically engineered V79 cells expressing CYP2E1 from different species. (orig.)

  19. Molecular cloning and 3D model of first cytochrome P450 from CYP3A subfamily in saltwater crocodile (Crocodylus porosus).

    Science.gov (United States)

    Tabassum, Rabia

    2017-10-18

    Cytochrome P450s (CYPs) play critical role in oxidative metabolism of numerous xenobiotics and endogenous compounds. The first CYP3A subfamily member in saltwater crocodile has been cloned and modelled for three-dimensional (3D) structure. The full-length cDNA was obtained employing reverse transcription polymerase chain reaction (RT-PCR) strategy and rapid amplification of cDNA ends (RACE). The cDNA sequence of 1659 nucleotides includes 132 nucleotides from 5' untranslated region (UTR), an open reading frame of 1527 nucleotides encoding 509 amino acids designated as CYP3A163. The alignment of CYP3A163 sequence with CYP3A subfamily across the lineages exhibit the loss of 1 residue in birds and 7 residues in mammals in comparison to reptiles suggesting the adaptation processes during evolution. The amino acid identity of CYP3A163 with Alligator mississippiensis CYP3A77 and Homo sapiens CYP3A4 is 91% and 62% respectively. The 3D structure of CYP3A163 modelled using human CYP3A4 structure as a template with Phyre 2 software, represents high similarity with its functionally important motifs and catalytic domain. Both sequence and structure of CYP3A163 display the common and conserved features of CYP3A subfamily. Overall, this study provides primary molecular and structural data of CYP3A163 required to investigate the xenobiotic metabolism in saltwater crocodiles. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. Effects of triazole fungicides on androgenic disruption and CYP3A4 enzyme activity.

    Science.gov (United States)

    Lv, Xuan; Pan, Liumeng; Wang, Jiaying; Lu, Liping; Yan, Weilin; Zhu, Yanye; Xu, Yiwen; Guo, Ming; Zhuang, Shulin

    2017-03-01

    Triazole fungicides are widely used as broad-spectrum fungicides, non-steroidal antiestrogens and for various industrial applications. Their residues have been frequently detected in multiple environmental and human matrices. The increasingly reported toxicity incidents have led triazole fungicides as emerging contaminants of environmental and public health concern. However, whether triazole fungicides behave as endocrine disruptors by directly mimicking environmental androgens/antiandrogens or exerting potential androgenic disruption indirectly through the inhibition of cytochrome P450 (CYP450) enzyme activity is yet an unresolved question. We herein evaluated five commonly used triazole fungicides including bitertanol, hexaconazole, penconazole, tebuconazole and uniconazole for the androgenic and anti-androgenic activity using two-hybrid recombinant human androgen receptor (AR) yeast bioassay and comparatively evaluated their effects on enzymatic activity of CYP3A4 by P450-Glo™ CYP3A4 bioassay. All five fungicides showed moderate anti-androgenic activity toward human AR with the IC 50 ranging from 9.34 μM to 79.85 μM. The anti-androgenic activity remained no significant change after the metabolism mediated by human liver microsomes. These fungicides significantly inhibited the activity of CYP3A4 at the environmental relevant concentrations and the potency ranks as tebuconazole > uniconazole > hexaconazole > penconazole > bitertanol with the corresponding IC 50 of 0.81 μM, 0.93 μM, 1.27 μM, 2.22 μM, and 2.74 μM, respectively. We found that their anti-androgenic activity and the inhibition potency toward CYP3A4 inhibition was significantly correlated (R 2 between 0.83 and 0.97, p pesticides and structurally similar chemicals should fully consider potential androgenic disrupting effects and the influences on the activity of CYP450s. Copyright © 2016 Elsevier Ltd. All rights reserved.

  1. Identification of epoxybergamottin as a CYP3A4 inhibitor in grapefruit peel.

    Science.gov (United States)

    Wangensteen, H; Molden, E; Christensen, H; Malterud, K E

    2003-02-01

    The oral availability of many drugs metabolised by the enzyme cytochrome P(450) 3A4 (CYP3A4) is increased if co-administered with grapefruit juice. Extracts from grapefruit peel have also demonstrated inhibitory activity and, during commercial manufacturing of grapefruit juice, inhibitory components might be squeezed into the juice from the peel. Thus, the aim of this in vitro study was to identify CYP3A4 inhibitors in grapefruit peel. Grapefruit peel was extracted with diethyl ether, and the extract was further fractionated by normal-phase chromatography. Fractions demonstrating significant CYP3A4 inhibitory activity, as measured by the relative reduction in N-demethylation of diltiazem in transfected human liver epithelial cells, were subsequently separated by preparative thin-layer chromatography. Constituents of the fractions and isolated compounds were identified by nuclear magnetic resonance spectroscopy. Analysis of diltiazem and N-demethyl-diltiazem was performed using high-performance liquid chromatography. Of the identified components in grapefruit peel, only epoxybergamottin demonstrated a concentration-dependent inhibition of the CYP3A4-mediated N-demethylation of diltiazem. The IC(50) value was calculated to be 4.2+/-1.1 micro M. Coumarins without the furan ring and flavonoids isolated from grapefruit peel did not interfere with the metabolism of diltiazem. The results indicated the presence of other CYP3A4 inhibitors in grapefruit peel, but these agents were lost during the purification process excluding their identification. The furanocoumarin epoxybergamottin, present in grapefruit peel, is an inhibitor of CYP3A4. In commercial manufacturing of grapefruit juice, epoxybergamottin is possibly distributed into the juice. During manufacturing, however, epoxybergamottin may be hydrolysed to 6',7'-dihydroxybergamottin, which has been suggested as an important CYP3A4 inhibitor in grapefruit juice.

  2. Genotyping of TRIM5 locus in northern pig-tailed macaques (Macaca leonina, a primate species susceptible to Human Immunodeficiency Virus type 1 infection

    Directory of Open Access Journals (Sweden)

    Jiang Xue-Long

    2009-06-01

    Full Text Available Abstract Background The pig-tailed macaques are the only Old World monkeys known to be susceptible to human immunodeficiency virus type 1 (HIV-1 infection. We have previously reported that the TRIM5-Cyclophilin A (TRIMCyp fusion in pig-tailed macaques (Macaca nemestrina is dysfunctional in restricting HIV-1, which may explain why pig-tailed macaques are susceptible to HIV-1 infection. Similar results have also been reported by other groups. However, according to the current primate taxonomy, the previously reported M. nemestrina are further classified into three species, which all belong to the Macaca spp. This calls for the need to look into the previous studies in more details. Results The local species Northern pig-tailed macaque (M. leonina was analyzed for the correlation of TRIM5 structure and HIV-1 infection. Eleven M. leonina animals were analyzed, and all of them were found to possess TRIM5-CypA fusion at the TRIM5 locus. The transcripts encoding the dysfunctional TRIM5-CypA should result from the G-to-T mutation in the 3'-splicing site of intron 6. Polymorphism in the putative TRIMCyp recognition domain was observed. The peripheral blood mononuclear cells (PBMCs of M. leonina were susceptible to HIV-1 infection. Consistent with the previous results, expression of the M. leonina TRIMCyp in HeLa-T4 cells rendered the cells resistant to HIV-2ROD but not to SIVmac239 infection. Conclusion The susceptibility of M. leonina to HIV-1 infection is due to the dysfunctional TRIM5-CypA fusion in the TRIM5 locus. This finding should broaden our perspective in developing better HIV/AIDS non-human primate animal models.

  3. Association between cytochrome CYP17A1, CYP3A4, and CYP3A43 polymorphisms and prostate cancer risk and aggressiveness in a Korean study population

    OpenAIRE

    Han, Jun Hyun; Lee, Yong Seong; Kim, Hae Jong; Lee, Shin Young; Myung, Soon Chul

    2014-01-01

    In this study, we evaluated genetic variants of the androgen metabolism genes CYP17A1, CYP3A4, and CYP3A43 to determine whether they play a role in the development of prostate cancer (PCa) in Korean men. The study population included 240 pathologically diagnosed cases of PCa and 223 age-matched controls. Among the 789 single-nucleotide polymorphism (SNP) database variants detected, 129 were reported in two Asian groups (Han Chinese and Japanese) in the HapMap database. Only 21 polymorphisms o...

  4. Novel deletion alleles carrying CYP21A1P/A2 chimeric genes in Brazilian patients with 21-hydroxylase deficiency

    Directory of Open Access Journals (Sweden)

    Guerra-Júnior Gil

    2010-06-01

    Full Text Available Abstract Background Congenital adrenal hyperplasia due to 21-hydroxylase deficiency is caused by deletions, large gene conversions or mutations in CYP21A2 gene. The human gene is located at 6p21.3 within a locus containing the genes for putative serine/threonine Kinase RP, complement C4, steroid 21-hydroxylase CYP21 tenascin TNX, normally, in a duplicated cluster known as RCCX module. The CYP21 extra copy is a pseudogene (CYP21A1P. In Brazil, 30-kb deletion forming monomodular alleles that carry chimeric CYP21A1P/A2 genes corresponds to ~9% of disease-causing alleles. Such alleles are considered to result from unequal crossovers within the bimodular C4/CYP21 locus. Depending on the localization of recombination breakpoint, different alleles can be generated conferring the locus high degree of allelic variability. The purpose of the study was to investigate the variability of deleted alleles in patients with 21-hydroxylase deficiency. Methods We used different techniques to investigate the variability of 30-kb deletion alleles in patients with 21-hydroxylase deficiency. Alleles were first selected after Southern blotting. The composition of CYP21A1P/A2 chimeric genes was investigated by ASO-PCR and MLPA analyses followed by sequencing to refine the location of recombination breakpoints. Twenty patients carrying at least one allele with C4/CYP21 30-kb deletion were included in the study. Results An allele carrying a CYP21A1P/A2 chimeric gene was found unusually associated to a C4B/C4A Taq I 6.4-kb fragment, generally associated to C4B and CYP21A1P deletions. A novel haplotype bearing both p.P34L and p.H62L, novel and rare mutations, respectively, was identified in exon 1, however p.P30L, the most frequent pseudogene-derived mutation in this exon, was absent. Four unrelated patients showed this haplotype. Absence of p.P34L in CYP21A1P of normal controls indicated that it is not derived from pseudogene. In addition, the combination of different

  5. Enhancement of hepatic 4-hydroxylation of 25-hydroxyvitamin D3 through CYP3A4 induction in vitro and in vivo: implications for drug-induced osteomalacia.

    Science.gov (United States)

    Wang, Zhican; Lin, Yvonne S; Dickmann, Leslie J; Poulton, Emma-Jane; Eaton, David L; Lampe, Johanna W; Shen, Danny D; Davis, Connie L; Shuhart, Margaret C; Thummel, Kenneth E

    2013-05-01

    Long-term therapy with certain drugs, especially cytochrome P450 (P450; CYP)-inducing agents, confers an increased risk of osteomalacia that is attributed to vitamin D deficiency. Human CYP24A1, CYP3A4, and CYP27B1 catalyze the inactivation and activation of vitamin D and have been implicated in the adverse drug response. In this study, the inducibility of these enzymes and monohydroxylation of 25-hydroxyvitamin D3 (25OHD3) were evaluated after exposure to P450-inducing drugs. With human hepatocytes, treatment with phenobarbital, hyperforin, carbamazepine, and rifampin significantly increased the levels of CYP3A4, but not CYP24A1 or CYP27B1 mRNA. In addition, rifampin pretreatment resulted in an 8-fold increase in formation of the major metabolite of 25OHD3, 4β,25(OH)2D3. This inductive effect was blocked by the addition of 6',7'-dihydroxybergamottin, a selective CYP3A4 inhibitor. With human renal proximal tubular HK-2 cells, treatment with the same inducers did not alter CYP3A4, CYP24A1, or CYP27B1 expression. 24R,25(OH)2 D3 was the predominant monohydroxy metabolite produced from 25OHD3, but its formation was unaffected by the inducers. With healthy volunteers, the mean plasma concentration of 4β,25(OH)2D3 was increased 60% (p osteomalacia. Copyright © 2013 American Society for Bone and Mineral Research.

  6. Sex difference in induction of hepatic CYP2B and CYP3A subfamily enzymes by nicardipine and nifedipine in rats

    International Nuclear Information System (INIS)

    Konno, Yoshihiro; Sekimoto, Masashi; Nemoto, Kiyomitsu; Degawa, Masakuni

    2004-01-01

    Male and female of F344 rats were treated per os with nicardipine (Nic) and nifedipine (Nif), and changes in the levels of mRNA and protein of hepatic cytochrome P450 (P450) enzymes, CYP2B1, CYP2B2, CYP3A1, CYP3A2, CYP3A9, and CYP3A18 were examined. Furthermore, hepatic microsomal activities for pentoxyresorufin O-dealkylation (PROD) and nifedipine oxidation, which are mainly mediated by CYP2B and CYP3A subfamily enzymes, respectively, were measured. Analyses of RT-PCR and Western blotting revealed that Nic and Nif induced predominantly CYP3A and CYP2B enzymes, respectively. As for the gene activation of CYP2B enzymes, especially CYP2B1, Nif showed high capacity in both sexes of rats, whereas Nic did a definite capacity in the males but little in the females. Gene activations of CYP3A1, CYP3A2, and CYP3A18 by Nic occurred in both sexes of rats, although that of CYP3A9 did only in the male rats. Although gene activations of CYP3A1 and CYP3A2 by Nif were observed in both sexes of rats, a slight activation of the CYP3A9 gene occurred only in female rats, and the CYP3A18 gene activation, in neither male nor female rats. Thus, changes in levels of the mRNA or protein of CYP2B and CYP3A enzymes, especially CYP2B1 and CYP3A2, were closely correlated with those in hepatic PROD and nifedipine oxidation activities, respectively. The present findings demonstrate for the first time the sex difference in the Nic- and Nif-mediated induction of hepatic P450 enzymes in rats and further indicate that Nic and Nif show different specificities and sex dependencies in the induction of hepatic P450 enzymes

  7. Effect of botanical immunomodulators on human CYP3A4 inhibition: implications for concurrent use as adjuvants in cancer therapy.

    Science.gov (United States)

    Patil, Dada; Gautam, Manish; Gairola, Sunil; Jadhav, Suresh; Patwardhan, Bhushan

    2014-03-01

    Many botanical immunomodulators are used as adjuvants along with cancer chemotherapy. However, information on the impact of concurrent administration of such botanicals on pharmacokinetics of chemotherapy agents is inadequate. This study investigates inhibitory activities of 3 popular botanical adjuvants: ASPARAGUS RACEMOSU: (root aqueous extract; ARE), WITHANIA SOMNIFER: (root aqueous extract; WSE), and TINOSPORA CORDIFOLI: (stem aqueous extract, TCE) on human CYP3A4 isoenzyme, responsible for metabolism of several chemotherapy agents. . Testosterone 6-β hydroxylation was monitored using high-performance liquid chromatography as an indicator of CYP3A4 catalytic activities. Ketoconazole (positive control) and extracts were studied at their in vivo-relevant concentrations. TCE showed mild inhibition while no significant inhibitory activities were observed in WSE and ARE. TCE was further fractionated to obtain polar and nonpolar fractions. The nonpolar fraction showed significant CYP3A4 inhibition with IC50 13.06 ± 1.38 µg/mL. Major constituents of nonpolar fraction were identified using HPLC-DAD-MS profiling as berberine, jatrorrhizine, and palmatine, which showed IC50 values as 6.25 ± 0.30, 15.18 ± 1.59, and 15.53 ± 1.89 µg/mL, respectively. Our findings suggest that constituents of TCE extract especially protoberberine alkaloids have the potential to interact with cancer chemotherapy agents that are metabolized by CYP3A4 in vivo.

  8. Relative contributions of the major human CYP450 to the metabolism of icotinib and its implication in prediction of drug-drug interaction between icotinib and CYP3A4 inhibitors/inducers using physiologically based pharmacokinetic modeling.

    Science.gov (United States)

    Chen, Jia; Liu, Dongyang; Zheng, Xin; Zhao, Qian; Jiang, Ji; Hu, Pei

    2015-06-01

    Icotinib is an anticancer drug, but relative contributions of CYP450 have not been identified. This study was carried out to identify the contribution percentage of CYP450 to icotinib and use the results to develop a physiologically based pharmacokinetic (PBPK) model, which can help to predict drug-drug interaction (DDI). Human liver microsome (HLM) and supersome using relative activity factor (RAF) were employed to determine the relative contributions of the major human P450 to the net hepatic metabolism of icotinib. These values were introduced to develop a PBPK model using SimCYP. The model was validated by the observed data in a Phase I clinical trial in Chinese healthy subjects. Finally, the model was used to simulate the DDI with ketoconazole or rifampin. Final contribution of CYP450 isoforms determined by HLM showed that CYP3A4 provided major contributions to the metabolism of icotinib. The percentage contributions of the P450 to the net hepatic metabolism of icotinib were determined by HLM inhibition assay and RAF. The AUC ratio under concomitant use of ketoconazole and rifampin was 3.22 and 0.55, respectively. Percentage of contribution of CYP450 to icotinib metabolism was calculated by RAF. The model has been proven to fit the observed data and is used in predicting icotinib-ketoconazole/rifampin interaction.

  9. Polychlorinated biphenyl (PCB) induction of CYP3A4 enzyme activity in healthy Faroese adults

    DEFF Research Database (Denmark)

    Petersen, Maria Skaalum; Halling, Jónrit; Damkier, Per

    2007-01-01

    The CYP3A4 enzyme is, along with other cytochrome P450 enzymes, involved in the metabolism of environmental pollutants and is highly inducible by these substances. A commercial polychlorinated biphenyl (PCB) mixture, 1,1,1,-trichloro-2-(o-chlorophenyl), 2-(p'-chlorophenyl)ethane (o,p'-DDT) and 1......,1,-dichloro-2,2-bis (p-chlorophenyl)ethene (p,p'-DDE) are known to induce CYP3A4 activity through activation of nuclear receptors, such as the pregnane X receptor. However, this induction of CYP3A4 has not yet been investigated in humans. Thus, the aim of the study was to determine the variability of the CYP3......A4 phenotype in regard to increased concentrations of PCBs and other persistent organohalogen pollutants (POPs) in healthy Faroese adults. In 310 randomly selected Faroese residents aged 18-60 years, the CYP3A4 activity was determined based on the urinary 6beta-hydroxycortisol/cortisol (6beta...

  10. CYP2D6*4, CYP3A5*3 and ABCB1 3435T polymorphisms and drug-related falls in elderly people

    NARCIS (Netherlands)

    Blonk, Maren I.; van der Velde, Nathalie; van den Bemt, Patricia M. L. A.; van Schaik, Ron H. N.; van der Cammen, Tischa J. M.

    2010-01-01

    The objective of this study is to investigate the association between CYP2D6*4, CYP3A5*3 and ABCB1 3435T polymorphisms and drug-related falls. Multivariate logistic regression was performed in an existing database in order to study the association between falls history and CYP2D6*4, CYP3A5*3, ABCB1

  11. Evolutionary changes in gene expression, coding sequence and copy-number at the Cyp6g1 locus contribute to resistance to multiple insecticides in Drosophila.

    Directory of Open Access Journals (Sweden)

    Thomas W R Harrop

    Full Text Available Widespread use of insecticides has led to insecticide resistance in many populations of insects. In some populations, resistance has evolved to multiple pesticides. In Drosophila melanogaster, resistance to multiple classes of insecticide is due to the overexpression of a single cytochrome P450 gene, Cyp6g1. Overexpression of Cyp6g1 appears to have evolved in parallel in Drosophila simulans, a sibling species of D. melanogaster, where it is also associated with insecticide resistance. However, it is not known whether the ability of the CYP6G1 enzyme to provide resistance to multiple insecticides evolved recently in D. melanogaster or if this function is present in all Drosophila species. Here we show that duplication of the Cyp6g1 gene occurred at least four times during the evolution of different Drosophila species, and the ability of CYP6G1 to confer resistance to multiple insecticides exists in D. melanogaster and D. simulans but not in Drosophila willistoni or Drosophila virilis. In D. virilis, which has multiple copies of Cyp6g1, one copy confers resistance to DDT and another to nitenpyram, suggesting that the divergence of protein sequence between copies subsequent to the duplication affected the activity of the enzyme. All orthologs tested conferred resistance to one or more insecticides, suggesting that CYP6G1 had the capacity to provide resistance to anthropogenic chemicals before they existed. Finally, we show that expression of Cyp6g1 in the Malpighian tubules, which contributes to DDT resistance in D. melanogaster, is specific to the D. melanogaster-D. simulans lineage. Our results suggest that a combination of gene duplication, regulatory changes and protein coding changes has taken place at the Cyp6g1 locus during evolution and this locus may play a role in providing resistance to different environmental toxins in different Drosophila species.

  12. Increased expression of CYP4Z1 promotes tumor angiogenesis and growth in human breast cancer

    International Nuclear Information System (INIS)

    Yu, Wei; Chai, Hongyan; Li, Ying; Zhao, Haixia; Xie, Xianfei; Zheng, Hao; Wang, Chenlong; Wang, Xue; Yang, Guifang; Cai, Xiaojun; Falck, John R.; Yang, Jing

    2012-01-01

    Cytochrome P450 (CYP) 4Z1, a novel CYP4 family member, is over-expressed in human mammary carcinoma and associated with high-grade tumors and poor prognosis. However, the precise role of CYP4Z1 in tumor progression is unknown. Here, we demonstrate that CYP4Z1 overexpression promotes tumor angiogenesis and growth in breast cancer. Stable expression of CYP4Z1 in T47D and BT-474 human breast cancer cells significantly increased mRNA expression and production of vascular endothelial growth factor (VEGF)-A, and decreased mRNA levels and secretion of tissue inhibitor of metalloproteinase-2 (TIMP-2), without affecting cell proliferation and anchorage-independent cell growth in vitro. Notably, the conditioned medium from CYP4Z1-expressing cells enhanced proliferation, migration and tube formation of human umbilical vein endothelial cells, and promoted angiogenesis in the zebrafish embryo and chorioallantoic membrane of the chick embryo. In addition, there were lower levels of myristic acid and lauric acid, and higher contents of 20-hydroxyeicosatetraenoic acid (20-HETE) in CYP4Z1-expressing T47D cells compared with vector control. CYP4Z1 overexpression significantly increased tumor weight and microvessel density by 2.6-fold and 1.9-fold in human tumor xenograft models, respectively. Moreover, CYP4Z1 transfection increased the phosphorylation of ERK1/2 and PI3K/Akt, while PI3K or ERK inhibitors and siRNA silencing reversed CYP4Z1-mediated changes in VEGF-A and TIMP-2 expression. Conversely, HET0016, an inhibitor of the CYP4 family, potently inhibited the tumor-induced angiogenesis with associated changes in the intracellular levels of myristic acid, lauric acid and 20-HETE. Collectively, these data suggest that increased CYP4Z1 expression promotes tumor angiogenesis and growth in breast cancer partly via PI3K/Akt and ERK1/2 activation. -- Highlights: ► CYP4Z1 overexpression promotes human breast cancer growth and angiogenesis. ► The pro-angiogenic effects of CYP4Z1 have

  13. Increased expression of CYP4Z1 promotes tumor angiogenesis and growth in human breast cancer

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Wei [Department of Pharmacology, School of Medicine, Wuhan University, Wuhan 430071 (China); Chai, Hongyan [Center for Gene Diagnosis, Zhongnan Hospital, Wuhan University, Wuhan 430071 (China); Li, Ying; Zhao, Haixia; Xie, Xianfei; Zheng, Hao; Wang, Chenlong; Wang, Xue [Department of Pharmacology, School of Medicine, Wuhan University, Wuhan 430071 (China); Yang, Guifang [Department of Pathology, Zhongnan Hospital, Wuhan University, Wuhan 430071 (China); Cai, Xiaojun [Department of Ophthalmology, Zhongnan Hospital, Wuhan University, Wuhan 430071 (China); Falck, John R. [Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX 75390 (United States); Yang, Jing, E-mail: yangjingliu@yahoo.com.cn [Department of Pharmacology, School of Medicine, Wuhan University, Wuhan 430071 (China); Research Center of Food and Drug Evaluation, Wuhan University, Wuhan 430071 (China)

    2012-10-01

    Cytochrome P450 (CYP) 4Z1, a novel CYP4 family member, is over-expressed in human mammary carcinoma and associated with high-grade tumors and poor prognosis. However, the precise role of CYP4Z1 in tumor progression is unknown. Here, we demonstrate that CYP4Z1 overexpression promotes tumor angiogenesis and growth in breast cancer. Stable expression of CYP4Z1 in T47D and BT-474 human breast cancer cells significantly increased mRNA expression and production of vascular endothelial growth factor (VEGF)-A, and decreased mRNA levels and secretion of tissue inhibitor of metalloproteinase-2 (TIMP-2), without affecting cell proliferation and anchorage-independent cell growth in vitro. Notably, the conditioned medium from CYP4Z1-expressing cells enhanced proliferation, migration and tube formation of human umbilical vein endothelial cells, and promoted angiogenesis in the zebrafish embryo and chorioallantoic membrane of the chick embryo. In addition, there were lower levels of myristic acid and lauric acid, and higher contents of 20-hydroxyeicosatetraenoic acid (20-HETE) in CYP4Z1-expressing T47D cells compared with vector control. CYP4Z1 overexpression significantly increased tumor weight and microvessel density by 2.6-fold and 1.9-fold in human tumor xenograft models, respectively. Moreover, CYP4Z1 transfection increased the phosphorylation of ERK1/2 and PI3K/Akt, while PI3K or ERK inhibitors and siRNA silencing reversed CYP4Z1-mediated changes in VEGF-A and TIMP-2 expression. Conversely, HET0016, an inhibitor of the CYP4 family, potently inhibited the tumor-induced angiogenesis with associated changes in the intracellular levels of myristic acid, lauric acid and 20-HETE. Collectively, these data suggest that increased CYP4Z1 expression promotes tumor angiogenesis and growth in breast cancer partly via PI3K/Akt and ERK1/2 activation. -- Highlights: ► CYP4Z1 overexpression promotes human breast cancer growth and angiogenesis. ► The pro-angiogenic effects of CYP4Z1 have

  14. Influence of genetic variants of CYP2D6, CYP2C9, CYP2C19 and CYP3A4 on antiepileptic drug metabolism in pediatric patients with refractory epilepsy.

    Science.gov (United States)

    López-García, Miguel A; Feria-Romero, Iris A; Serrano, Héctor; Rayo-Mares, Darío; Fagiolino, Pietro; Vázquez, Marta; Escamilla-Núñez, Consuelo; Grijalva, Israel; Escalante-Santiago, David; Orozco-Suarez, Sandra

    2017-06-01

    Identified the polymorphisms of CYP2D6, CYP2C9, CYP2C19 and CYP3A4, within a rigorously selected population of pediatric patients with drug-resistant epilepsy. The genomic DNA of 23 drug-resistant epilepsy patients and 7 patients with good responses were analyzed. Ten exons in these four genes were genotyped, and the drug concentrations in saliva and plasma were determined. The relevant SNPs with pharmacogenomics relations were CYP2D6*2 (rs16947) decreased your activity and CYP2D6*4 (rs1065852), CYP2C19*2 (rs4244285) and CYP3A4*1B (rs2740574) by association with poor metabolizer. The strongest risk factors were found in the AA genotype and allele of SNP rs3892097 from the CYP2D6 gene, followed by the alleles A and T of SNPs rs2740574 and rs2687116, respectively from CYP3A4. The most important concomitance was between homozygous genotype AA of rs3892097 and genotype AA of rs2740574 with 78.3% in drug-resistant epilepsy patients as compared to 14.3% in control patients. The results demonstrated the important role of the CYP 3A4*1B allelic variant as risk factor for developing drug resistance and CYP2D6, CYP2C19 SNPs and haplotypes may affect the response to antiepileptic drugs. Copyright © 2017. Published by Elsevier Urban & Partner Sp. z o.o.

  15. Guanfu base A, an antiarrhythmic alkaloid of Aconitum coreanum, Is a CYP2D6 inhibitor of human, monkey, and dog isoforms.

    Science.gov (United States)

    Sun, Jianguo; Peng, Ying; Wu, Hui; Zhang, Xueyuan; Zhong, Yunxi; Xiao, Yanan; Zhang, Fengyi; Qi, Huanhuan; Shang, Lili; Zhu, Jianping; Sun, Yue; Liu, Ke; Liu, Jinghan; A, Jiye; Ho, Rodney J Y; Wang, Guangji

    2015-05-01

    Guanfu base A (GFA) is a novel heterocyclic antiarrhythmic drug isolated from Aconitum coreanum (Lèvl.) rapaics and is currently in a phase IV clinical trial in China. However, no study has investigated the influence of GFA on cytochrome P450 (P450) drug metabolism. We characterized the potency and specificity of GFA CYP2D inhibition based on dextromethorphan O-demethylation, a CYP2D6 probe substrate of activity in human, mouse, rat, dog, and monkey liver microsomes. In addition, (+)-bufuralol 1'-hydroxylation was used as a CYP2D6 probe for the recombinant form (rCYP2D6), 2D1 (rCYP2D1), and 2D2 (rCYP2D2) activities. Results show that GFA is a potent noncompetitive inhibitor of CYP2D6, with inhibition constant Ki = 1.20 ± 0.33 μM in human liver microsomes (HLMs) and Ki = 0.37 ± 0.16 μM for the human recombinant form (rCYP2D6). GFA is also a potent competitive inhibitor of CYP2D in monkey (Ki = 0.38 ± 0.12 μM) and dog (Ki = 2.4 ± 1.3 μM) microsomes. However, GFA has no inhibitory activity on mouse or rat CYP2Ds. GFA did not exhibit any inhibition activity on human recombinant CYP1A2, 2A6, 2C8, 2C19, 3A4, or 3A5, but showed slight inhibition of 2B6 and 2E1. Preincubation of HLMs and rCYP2D6 resulted in the inactivation of the enzyme, which was attenuated by GFA or quinidine. Beagle dogs treated intravenously with dextromethorphan (2 mg/ml) after pretreatment with GFA injection showed reduced CYP2D metabolic activity, with the Cmax of dextrorphan being one-third that of the saline-treated group and area under the plasma concentration-time curve half that of the saline-treated group. This study suggests that GFA is a specific CYP2D6 inhibitor that might play a role in CYP2D6 medicated drug-drug interaction. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

  16. Polymorphisms of CYP17A1, CYP19, and androgen in Brazilian women with uterine leiomyomas

    DEFF Research Database (Denmark)

    Rosa, Fabíola Encinas; Canevari, Renata de Azevedo; Ambrosio, Eliane Papa

    2008-01-01

    BACKGROUND: Uterine leiomyomas are common, benign, smooth muscle tumors representing a significant public health problem. The aim of this study was to investigate CYP17A1, CYP19, and androgen (AR) polymorphisms, their relative risks for uterine leiomyomas and possible associations with clinical...... parameters. METHODS: Uterine leiomyoma tissues and blood samples were obtained from 87 patients, as were peripheral blood samples from 68 control women. Clinical data were recorded in both groups. The CYP17A1 (rs743572) polymorphism was analyzed by PCR-RFLP, and the CYP19 [TTTA](n) repeat and AR [CAG...... were exclusive to the leiomyoma group. The LOH assay showed allele losses at AR locus in four informative tumors and X chromosome inactivation analysis revealed that these tumors retained the active allele. CONCLUSIONS: The overall lack of association between uterine leiomyomas with polymorphisms...

  17. CYP3A5 mRNA degradation by nonsense-mediated mRNA decay.

    Science.gov (United States)

    Busi, Florent; Cresteil, Thierry

    2005-09-01

    The total CYP3A5 mRNA level is significantly greater in carriers of the CYP3A5*1 allele than in CYP3A5*3 homozygotes. Most of the CYP3A5*3 mRNA includes an intronic sequence (exon 3B) containing premature termination codons (PTCs) between exons 3 and 4. Two models were used to investigate the degradation of CYP3A5 mRNA: a CYP3A5 minigene consisting of CYP3A5 exons and introns 3 to 6 transfected into MCF7 cells, and the endogenous CYP3A5 gene expressed in HepG2 cells. The 3'-untranslated region g.31611C>T mutation has no effect on CYP3A5 mRNA decay. Splice variants containing exon 3B were more unstable than wild-type (wt) CYP3A5 mRNA. Cycloheximide prevents the recognition of PTCs by ribosomes: in transfected MCF7 and HepG2 cells, cycloheximide slowed down the degradation of exon 3B-containing splice variants, suggesting the participation of nonsense-mediated decay (NMD). When PTCs were removed from pseudoexon 3B or when UPF1 small interfering RNA was used to impair the NMD mechanism, the decay of the splice variant was reduced, confirming the involvement of NMD in the degradation of CYP3A5 splice variants. Induction could represent a source of variability for CYP3A5 expression and could modify the proportion of splice variants. The extent of CYP3A5 induction was investigated after exposure to barbiturates or steroids: CYP3A4 was markedly induced in a pediatric population compared with untreated neonates. However, no effect could be detected in either the total CYP3A5 RNA, the proportion of splice variant RNA, or the protein level. Therefore, in these carriers, induction is unlikely to switch on the phenotypic CYP3A5 expression in carriers of CYP3A5*3/*3.

  18. Analysis of CYP3A4 genetic polymorphisms in Han Chinese.

    Science.gov (United States)

    Zhou, Qing; Yu, Xiaomin; Shu, Chang; Cai, Yimei; Gong, Wei; Wang, Xumin; Wang, Duen-mei; Hu, Songnian

    2011-06-01

    Our study aimed to comprehensively investigate the genetic polymorphisms of CYP3A4 in Han Chinese. We sequenced the gene regions of CYP3A4, including its promoter, exons, surrounding introns and 3' untranslated region (3'UTR), from 100 unrelated-healthy Han Chinese individuals. We detected 11 SNPs, three of which are novel. According to in silico functional prediction of novel variants, 20148 A>G in exon 10, resulting in substitution of Tyr319 with Cys (CYP3A4*21), may induce dramatic alteration of protein conformation, and 26908 G>A in 3'UTR may disrupt post-transcriptional regulation. We identified five alleles in Han Chinese, the allele frequencies of CYP3A4*1, *5, *6, *18 and *21 are 97, 0.5, 1, 1 and 0.5%, respectively. Haplotype inference revealed 14 haplotypes, of which the major haplotype CYP3A4*1A constitutes 59% of the total chromosomes. We also examined the possible role of natural selection in shaping the variation of CYP3A4 and confirmed a trend, consistent with the action of positive selection. We systematically screened the genetic polymorphisms of CYP3A4 in Han Chinese, highlighted possible functional impairment of the novel allele and summarized the distinct allele and haplotype frequency distribution, with an emphasis on detecting the footprint of recent positive selection on the CYP3A4 gene in Han Chinese.

  19. An impact of CYP3A4 *1B polymorphism on rifampicin metabolism

    Directory of Open Access Journals (Sweden)

    H. O. Poludenko

    2017-08-01

    Full Text Available Until now, the enzyme systems responsible for biotransformation of the antituberculous drug rifampicin remain unknown. The aim of research was an investigation of the candidate enzymes involved in the biotransformation of rifampicin using the computer system PASS and an experimental study concerning the effect of the polymorphism of the biotransformation gene CYP3A4 *1B on the level of rifampicin in the blood of patients with pulmonary tuberculosis (РTB. The probability (Pa of certain pharmacological activity and the effect on putative enzyme systems of the human body of rifampicin has been calculated by the PASS method. Polymerase chain reaction revealed the polymorphism of the CYP3A4 *1B gene among healthy volunteers as well as patients with РTB. With a high degree of probability, according to PASS calculations, it was predicted that rifampicin undergo metabolism with the CYP3A4 enzyme - probability (Ra were 0.891. According to the genotype CYP3A4 *1B, 95.3% of the healthy donors carried a homozygous wild-type gene (i.e., had high enzymatic activity - AA genotype; the rest 4.7% - were carriers of the heterozygous AG genotype (moderate enzyme activity.The polymorphism of CYP3A4 *1B genotypes and alleles in the south-west of Ukraine was close to the results obtained in European countries. 91.4% and 8.6% of the patients with РTB had AA and AG genotype, correspondently. Thus, among the patients with РTB, the AG genotype was more often observed than among healthy volunteers. There was no significant difference in rifampicin concentration among РTB-patients concerning CYP3A4 * 1B polymorphism.

  20. Genetic variation at CYP3A is associated with age at menarche and breast cancer risk: a case-control study

    OpenAIRE

    Johnson, N. (Nichola); Dudbridge, Frank; Orr, Nick; Gibson, Lorna; Jones, Michael; Schoemaker, Minouk; Folkerd, E.J. (Elizabeth J.); Haynes, B.P. (Ben P.); Hopper, John; Southey, Melissa; Dite, G.S. (Gillian S.); Apicella, C. (Carmel); Schmidt, Marjanka; Broeks, Annegien; Veer, Laura

    2014-01-01

    textabstractINTRODUCTION: We have previously shown that a tag single nucleotide polymorphism (rs10235235), which maps to the CYP3A locus (7q22.1), was associated with a reduction in premenopausal urinary estrone glucuronide levels and a modest reduction in risk of breast cancer in women age ≤50 years.METHODS: We further investigated the association of rs10235235 with breast cancer risk in a large case control study of 47,346 cases and 47,570 controls from 52 studies participating in the Breas...

  1. Genetic variation at CYP3A is associated with age at menarche and breast cancer risk: A case-control study

    OpenAIRE

    Johnson, Nichola; Dudbridge, Frank; Orr, Nick; Gibson, Lorna; Jones, Michael; Schoemaker, Minouk; Folkerd, E.J. (Elizabeth J.); Haynes, B.P. (Ben P.); Hopper, John; Southey, Melissa; Dite, Gillian; Apicella, Carmel; Schmidt, Marjanka; Broeks, Annegien; Veer, Laura

    2014-01-01

    textabstractIntroduction: We have previously shown that a tag single nucleotide polymorphism (rs10235235), which maps to the CYP3A locus (7q22.1), was associated with a reduction in premenopausal urinary estrone glucuronide levels and a modest reduction in risk of breast cancer in women age ≤50 years. Methods: We further investigated the association of rs10235235 with breast cancer risk in a large case control study of 47,346 cases and 47,570 controls from 52 studies participating in the Brea...

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

    International Nuclear Information System (INIS)

    Flueck, Christa E.; Mullis, Primus E.; Pandey, Amit V.

    2010-01-01

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

  3. CYP21A2 polymorphisms in patients with autoimmune Addison's disease, and linkage disequilibrium to HLA risk alleles.

    Science.gov (United States)

    Brønstad, Ingeborg; Skinningsrud, Beate; Bratland, Eirik; Løvås, Kristian; Undlien, Dag; Sverre Husebye, Eystein; Wolff, Anette Susanne Bøe

    2014-12-01

    Steroid 21-hydroxylase, encoded by CYP21A2, is the major autoantigen in autoimmune Addison's disease (AAD). CYP21A2 is located in the region of the HLA complex on chromosome 6p21.3, which harbours several risk alleles for AAD. The objective was to investigate whether CYP21A2 gene variants confer risk of AAD independently of other risk alleles in the HLA loci. DNA samples from 381 Norwegian patients with AAD and 340 healthy controls (HC) previously genotyped for the HLA-A, -B, -DRB1, and -DQB1 and MICA loci were used for genotyping of CYP21A2. Genotyping of CYP21A2 was carried out by direct sequencing. Linkage of CYP21A2 to the HLA loci was assessed using UNPHASED version 3.0.10 and PHASE version 2.1. Heterozygotes of the single-nucleotide polymorphisms (SNPs) rs397515394, rs6467, rs6474, rs76565726 and rs6473 were detected significantly more frequently in AAD patients compared with HC (P<0.005), but all SNPs were in a linkage disequilibrium (LD) with high-risk HLA-DRB1 haplotypes. rs6472C protected against AAD (odds ratio=0.15, 95% CI (0.08-0.30), P=3.8×10(-10)). This SNP was not in an LD with HLA loci (P=0.02), but did not increase protection when considering the effect of HLA-DRB1 alleles. Mutations causing congenital adrenal hyperplasia were found in heterozygosity in <1.5% of the cases in both groups. Genetic variants of CYP21A2 associated to AAD are in LD with the main AAD risk locus HLA-DRB1, and CYP21A2 does not constitute an independent susceptibility locus. © 2014 European Society of Endocrinology.

  4. The cytochrome P450 2AA gene cluster in zebrafish (Danio rerio): Expression of CYP2AA1 and CYP2AA2 and response to phenobarbital-type inducers

    Energy Technology Data Exchange (ETDEWEB)

    Kubota, Akira [Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543 (United States); Bainy, Afonso C.D. [Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543 (United States); Departamento de Bioquímica, CCB, Universidade Federal de Santa Catarina, Florianopolis, SC 88040-900 (Brazil); Woodin, Bruce R.; Goldstone, Jared V. [Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543 (United States); Stegeman, John J., E-mail: jstegeman@whoi.edu [Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543 (United States)

    2013-10-01

    The cytochrome P450 (CYP) 2 gene family is the largest and most diverse CYP gene family in vertebrates. In zebrafish, we have identified 10 genes in a new subfamily, CYP2AA, which does not show orthology to any human or other mammalian CYP genes. Here we report evolutionary and structural relationships of the 10 CYP2AA genes and expression of the first two genes, CYP2AA1 and CYP2AA2. Parsimony reconstruction of the tandem duplication pattern for the CYP2AA cluster suggests that CYP2AA1, CYP2AA2 and CYP2AA3 likely arose in the earlier duplication events and thus are most diverged in function from the other CYP2AAs. On the other hand, CYP2AA8 and CYP2AA9 are genes that arose in the latest duplication event, implying functional similarity between these two CYPs. A molecular model of CYP2AA1 showing the sequence conservation across the CYP2AA cluster reveals that the regions with the highest variability within the cluster map onto CYP2AA1 near the substrate access channels, suggesting differing substrate specificities. Zebrafish CYP2AA1 transcript was expressed predominantly in the intestine, while CYP2AA2 was most highly expressed in the kidney, suggesting differing roles in physiology. In the liver CYP2AA2 expression but not that of CYP2AA1, was increased by 1,4-bis [2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP) and, to a lesser extent, by phenobarbital (PB). In contrast, pregnenolone 16α-carbonitrile (PCN) increased CYP2AA1 expression, but not CYP2AA2 in the liver. The results identify a CYP2 subfamily in zebrafish that includes genes apparently induced by PB-type chemicals and PXR agonists, the first concrete in vivo evidence for a PB-type response in fish. - Highlights: • A tandemly duplicated cluster of ten CYP2AA genes was described in zebrafish. • Parsimony and duplication analyses suggest pathways to CYP2AA diversity. • Homology models reveal amino acid positions possibly related to functional diversity. • The CYP2AA locus does not share synteny with

  5. The cytochrome P450 2AA gene cluster in zebrafish (Danio rerio): Expression of CYP2AA1 and CYP2AA2 and response to phenobarbital-type inducers

    International Nuclear Information System (INIS)

    Kubota, Akira; Bainy, Afonso C.D.; Woodin, Bruce R.; Goldstone, Jared V.; Stegeman, John J.

    2013-01-01

    The cytochrome P450 (CYP) 2 gene family is the largest and most diverse CYP gene family in vertebrates. In zebrafish, we have identified 10 genes in a new subfamily, CYP2AA, which does not show orthology to any human or other mammalian CYP genes. Here we report evolutionary and structural relationships of the 10 CYP2AA genes and expression of the first two genes, CYP2AA1 and CYP2AA2. Parsimony reconstruction of the tandem duplication pattern for the CYP2AA cluster suggests that CYP2AA1, CYP2AA2 and CYP2AA3 likely arose in the earlier duplication events and thus are most diverged in function from the other CYP2AAs. On the other hand, CYP2AA8 and CYP2AA9 are genes that arose in the latest duplication event, implying functional similarity between these two CYPs. A molecular model of CYP2AA1 showing the sequence conservation across the CYP2AA cluster reveals that the regions with the highest variability within the cluster map onto CYP2AA1 near the substrate access channels, suggesting differing substrate specificities. Zebrafish CYP2AA1 transcript was expressed predominantly in the intestine, while CYP2AA2 was most highly expressed in the kidney, suggesting differing roles in physiology. In the liver CYP2AA2 expression but not that of CYP2AA1, was increased by 1,4-bis [2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP) and, to a lesser extent, by phenobarbital (PB). In contrast, pregnenolone 16α-carbonitrile (PCN) increased CYP2AA1 expression, but not CYP2AA2 in the liver. The results identify a CYP2 subfamily in zebrafish that includes genes apparently induced by PB-type chemicals and PXR agonists, the first concrete in vivo evidence for a PB-type response in fish. - Highlights: • A tandemly duplicated cluster of ten CYP2AA genes was described in zebrafish. • Parsimony and duplication analyses suggest pathways to CYP2AA diversity. • Homology models reveal amino acid positions possibly related to functional diversity. • The CYP2AA locus does not share synteny with

  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. Structural Basis of Human CYP51 Inhibition by Antifungal Azoles

    Energy Technology Data Exchange (ETDEWEB)

    Strushkevich, Natallia; Usanov, Sergey A.; Park, Hee-Won (Toronto); (IBC-Belarus)

    2010-09-22

    The obligatory step in sterol biosynthesis in eukaryotes is demethylation of sterol precursors at the C14-position, which is catalyzed by CYP51 (sterol 14-alpha demethylase) in three sequential reactions. In mammals, the final product of the pathway is cholesterol, while important intermediates, meiosis-activating sterols, are produced by CYP51. Three crystal structures of human CYP51, ligand-free and complexed with antifungal drugs ketoconazole and econazole, were determined, allowing analysis of the molecular basis for functional conservation within the CYP51 family. Azole binding occurs mostly through hydrophobic interactions with conservative residues of the active site. The substantial conformational changes in the B{prime} helix and F-G loop regions are induced upon ligand binding, consistent with the membrane nature of the protein and its substrate. The access channel is typical for mammalian sterol-metabolizing P450 enzymes, but is different from that observed in Mycobacterium tuberculosis CYP51. Comparison of the azole-bound structures provides insight into the relative binding affinities of human and bacterial P450 enzymes to ketoconazole and fluconazole, which can be useful for the rational design of antifungal compounds and specific modulators of human CYP51.

  8. Genotype-phenotype associations for common CYP3A4 and CYP3A5 variants in the basal and induced metabolism of midazolam in European- and African-American men and women.

    Science.gov (United States)

    Floyd, Michael D; Gervasini, Guillermo; Masica, Andrew L; Mayo, Gail; George, Alfred L; Bhat, Kolari; Kim, Richard B; Wilkinson, Grant R

    2003-10-01

    CYP3A activity in adults varies between individuals and it has been suggested that this has a genetic basis, possibly related to variant alleles in CYP3A4 and CYP3A5 genes. Accordingly, genotype-phenotype associations were investigated under constitutive and induced conditions. Midazolam's systemic and oral clearances, and the erythromycin breath test (ERBT) were determined in 57 healthy subjects: 23 (11 men, 12 women) European- and 34 (14 men, 20 women) African-Americans. Studies were undertaken in the basal state and after 14-15 days pretreatment with rifampin. DNA was characterized for the common polymorphisms CYP3A4*1B, CYP3A5*3, CYP3A5*6 and CYP3A5*7 by direct sequencing, and for exon 21 and exon 26 variants of MDR1 by allele-specific, real-time polymerase chain reaction. In 95% of subjects, the basal systemic clearance of midazolam was unimodally distributed and variability was less than four-fold whereas, in 98% of the study population, oral clearance varied five-fold. No population or sex-related differences were apparent. Similar findings were observed with the ERBT. Rifampin pretreatment markedly increased the systemic (two-fold) and oral clearance (16-fold) of midazolam, and the ERBT (two-fold) but the variabilities were unchanged. No associations were noted between these phenotypic measures and any of the studied genotypes, except for oral clearance and its fold-increase after rifampin. These were related to the presence of CYP3A4*1B and the inversely linked CYP3A5*3 polymorphism, with the extent of induction being approximately 50% greater in CYP3A5*3 homozygotes compared to wild-type subjects. In most healthy subjects, variability in intestinal and hepatic CYP3A activity, using midazolam as an in-vivo probe, is modest and common polymorphisms in CYP3A4 and CYP3A5 do not appear to have important functional significance.

  9. Reductive metabolism of oxymatrine is catalyzed by microsomal CYP3A4

    Directory of Open Access Journals (Sweden)

    Liu W

    2015-10-01

    Full Text Available Wenqin Liu,1,2,* Jian Shi,1,2,* Lijun Zhu,2 Lingna Dong,1 Feifei Luo,2 Min Zhao,2 Ying Wang,2 Ming Hu,2,3 Linlin Lu,2 Zhongqiu Liu1,2 1Department of Pharmaceutics, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou, Guangdong, People’s Republic of China; 2International Institute for Translational Chinese Medicine, Guangzhou University of Chinese Medicine, Guangzhou, Guangdong, People’s Republic of China; 3Department of Pharmacological and Pharmaceutical Sciences, College of Pharmacy, University of Houston, Houston, TX, USA *These authors contributed equally to this work Abstract: Oxymatrine (OMT is a pharmacologically active primary quinolizidine alkaloid with various beneficial and toxic effects. It is confirmed that, after oral administration, OMT could be transformed to the more toxic metabolite matrine (MT, and this process may be through the reduction reaction, but the study on the characteristics of this transformation is limited. The aim of this study was to investigate the characteristics of this transformation of OMT in the human liver microsomes (HLMs and human intestinal microsomes (HIMs and the cytochrome P450 (CYP isoforms involved in this transformation. The current studies demonstrated that OMT could be metabolized to MT rapidly in HLMs and HIMs and CYP3A4 greatly contributed to this transformation. All HLMs, HIMs, and CYP3A4 isoform mediated reduction reaction followed typical biphasic kinetic model, and Km, Vmax, and CL were significant higher in HLMs than those in HIMs. Importantly, different oxygen contents could significantly affect the metabolism of OMT, and with the oxygen content decreased, the formation of metabolite was increased, suggesting this transformation was very likely a reduction reaction. Results of this in vitro study elucidated the metabolic pathways and characteristics of metabolism of OMT to MT and would provide a theoretical basis and guidance for the safe application of OMT

  10. 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.

  11. Characterization of the genetic variation present in CYP3A4 in three South African populations.

    Science.gov (United States)

    Drögemöller, Britt; Plummer, Marieth; Korkie, Lundi; Agenbag, Gloudi; Dunaiski, Anke; Niehaus, Dana; Koen, Liezl; Gebhardt, Stefan; Schneider, Nicol; Olckers, Antonel; Wright, Galen; Warnich, Louise

    2013-01-01

    The CYP3A4 enzyme is the most abundant human cytochrome P450 (CYP) and is regarded as the most important enzyme involved in drug metabolism. Inter-individual and inter-population variability in gene expression and enzyme activity are thought to be influenced, in part, by genetic variation. Although Southern African individuals have been shown to exhibit the highest levels of genetic diversity, they have been under-represented in pharmacogenetic research to date. Therefore, the aim of this study was to identify genetic variation within CYP3A4 in three South African population groups comprising of 29 Khoisan, 65 Xhosa and 65 Mixed Ancestry (MA) individuals. To identify known and novel CYP3A4 variants, 15 individuals were randomly selected from each of the population groups for bi-directional Sanger sequencing of ~600 bp of the 5'-upstream region and all thirteen exons including flanking intronic regions. Genetic variants detected were genotyped in the rest of the cohort. In total, 24 SNPs were detected, including CYP3A4(*)12, CYP3A4(*)15, and the reportedly functional CYP3A4(*)1B promoter polymorphism, as well as two novel non-synonymous variants. These putatively functional variants, p.R162W and p.Q200H, were present in two of the three populations and all three populations, respectively, and in silico analysis predicted that the former would damage the protein product. Furthermore, the three populations were shown to exhibit distinct genetic profiles. These results confirm that South African populations show unique patterns of variation in the genes encoding xenobiotic metabolizing enzymes. This research suggests that population-specific genetic profiles for CYP3A4 and other drug metabolizing genes would be essential to make full use of pharmacogenetics in Southern Africa. Further investigation is needed to determine if the identified genetic variants influence CYP3A4 metabolism phenotype in these populations.

  12. A novel CYP1A1 gene polymorphism and the risk of head and neck ...

    African Journals Online (AJOL)

    Several polymorphisms in the CYP1A1 locus have been identified and their genotypes appear to exhibit population frequencies that depend on ethnicity. In this study, we assessed the role of CYP1A1 genotype in 388 head and neck cancer patients in Pakistani population via a case-control study. Polymerase chain reaction ...

  13. Role of genetic polymorphisms of CYP1A1, CYP3A5, CYP2C9, CYP2D6, and PON1 in the modulation of DNA damage in workers occupationally exposed to organophosphate pesticides

    International Nuclear Information System (INIS)

    Singh, Satyender; Kumar, Vivek; Vashisht, Kapil; Singh, Priyanka; Banerjee, Basu Dev; Rautela, Rajender Singh; Grover, Shyam Sunder; Rawat, Devendra Singh; Pasha, Syed Tazeen; Jain, Sudhir Kumar; Rai, Arvind

    2011-01-01

    Organophosphate pesticides (OPs) are primarily metabolized by several xenobiotic metabolizing enzymes (XMEs). Very few studies have explored genetic polymorphisms of XMEs and their association with DNA damage in pesticide-exposed workers. The present study was designed to determine the role of genetic polymorphisms of CYP1A1, CYP3A5, CYP2C9, CYP2D6, and PON1 in the modulation of DNA damage in workers occupationally exposed to OPs. We examined 284 subjects including 150 workers occupationally exposed to OPs and 134 normal healthy controls. The DNA damage was evaluated using the alkaline comet assay and genotyping was done using PCR–RFLP. The results revealed that the PONase activity toward paraoxonase and AChE activity was found significantly lowered in workers as compared to control subjects (p < 0.001). Workers showed significantly higher DNA damage compared to control subjects (14.37 ± 2.15 vs. 6.24 ± 1.37 tail% DNA, p < 0.001). Further, the workers with CYP2D6*3 PM and PON1 (QQ and MM) genotypes were found to have significantly higher DNA damage when compared to other genotypes (p < 0.05). In addition, significant increase in DNA damage was also observed in workers with concomitant presence of certain CYP2D6 and PON1 (Q192R and L55M) genotypes which need further extensive studies. In conclusion, the results indicate that the PON1 and CYP2D6 genotypes can modulate DNA damage elicited by some OPs possibly through gene-environment interactions. -- Highlights: ► Role of CYP1A1, CYP3A5, CYP2C, CYP2D6 and PON1 genotypes on DNA damage. ► Workers exposed to some OPs demonstrated increased DNA damage. ► CYP2D6 *3 PM and PON1 (Q192R and L55M) genotypes are associated with DNA damage. ► Concomitant presence of certain CYP2D6 and PON1 genotypes can increase DNA damage.

  14. CYP3A-mediated apoptosis of dauricine in cultured human bronchial epithelial cells and in lungs of CD-1 mice

    International Nuclear Information System (INIS)

    Jin, Hua; Shen, Shuijie; Chen, Xiaoyan; Zhong, Dafang; Zheng, Jiang

    2012-01-01

    Dauricine is the major bioactive component isolated from the root of Menispermum dauricum DC and has shown promising pharmacologic activities with a great potential for clinical use. Recently, we found that intraperitoneal exposure of dauricine produced selective pulmonary injury in mice. A quinone methide metabolite of dauricine was identified and is suggested to be associated with the pulmonary toxicity of dauricine. The present study evaluated the apoptotic effect of dauricine in cultured cells and mice, determined the change in cellular glutathione (GSH) contents after exposure to dauricine, investigated the role of GSH depletion in dauricine-induced cytotoxicity and apoptosis, and examined the role of CYP3A in dauricine-induced GSH depletion and apoptosis. Dauricine was found to induce apoptosis in NL-20 cells. Additionally, intraperitoneal administration of dauricine caused GSH depletion and apoptosis in lungs of mice. Treatment with ketoconazole, an inhibitor of CYP3A, reversed cellular GSH depletion in lungs of mice given dauricine and showed protective effect on dauricine-induced apoptosis in lungs of mice. This indicates that metabolic activation is involved in dauricine-induced GSH-depletion, cytotoxicity and apoptosis. The glutathione depletor L-buthionine sulfoximine showed potentiating effect on cytotoxicity and apoptosis induced by dauricine. We propose that dauricine is metabolized to a quinone methide intermediate which depletes cellular GSH, and the depletion of GSH may trigger and/or intensify the cytotoxicity and apoptosis induced by dauricine. -- Highlights: ► Dauricine induced apoptosis in lungs in mice and in cultured human pulmonary cells. ► Dauricine depleted cellular GSH in lungs of mice and in the human pulmonary cells. ► CYP3A subfamily mediated GSH depletion and apoptosis induced by dauricine. ► L-Buthionine sulfoximine potentiated dauricine-induced GSH depletion and apoptosis.

  15. CYP3A-mediated apoptosis of dauricine in cultured human bronchial epithelial cells and in lungs of CD-1 mice

    Energy Technology Data Exchange (ETDEWEB)

    Jin, Hua; Shen, Shuijie [Center for Developmental Therapeutics, Seattle Children' s Research Institute, Division of Gastroenterology and Hepatology, Department of Pediatrics, University of Washington, Seattle, WA 98101 (United States); Chen, Xiaoyan; Zhong, Dafang [Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, 201203 (China); Zheng, Jiang, E-mail: jiang.zheng@seattlechildrens.org [Center for Developmental Therapeutics, Seattle Children' s Research Institute, Division of Gastroenterology and Hepatology, Department of Pediatrics, University of Washington, Seattle, WA 98101 (United States)

    2012-06-15

    Dauricine is the major bioactive component isolated from the root of Menispermum dauricum DC and has shown promising pharmacologic activities with a great potential for clinical use. Recently, we found that intraperitoneal exposure of dauricine produced selective pulmonary injury in mice. A quinone methide metabolite of dauricine was identified and is suggested to be associated with the pulmonary toxicity of dauricine. The present study evaluated the apoptotic effect of dauricine in cultured cells and mice, determined the change in cellular glutathione (GSH) contents after exposure to dauricine, investigated the role of GSH depletion in dauricine-induced cytotoxicity and apoptosis, and examined the role of CYP3A in dauricine-induced GSH depletion and apoptosis. Dauricine was found to induce apoptosis in NL-20 cells. Additionally, intraperitoneal administration of dauricine caused GSH depletion and apoptosis in lungs of mice. Treatment with ketoconazole, an inhibitor of CYP3A, reversed cellular GSH depletion in lungs of mice given dauricine and showed protective effect on dauricine-induced apoptosis in lungs of mice. This indicates that metabolic activation is involved in dauricine-induced GSH-depletion, cytotoxicity and apoptosis. The glutathione depletor L-buthionine sulfoximine showed potentiating effect on cytotoxicity and apoptosis induced by dauricine. We propose that dauricine is metabolized to a quinone methide intermediate which depletes cellular GSH, and the depletion of GSH may trigger and/or intensify the cytotoxicity and apoptosis induced by dauricine. -- Highlights: ► Dauricine induced apoptosis in lungs in mice and in cultured human pulmonary cells. ► Dauricine depleted cellular GSH in lungs of mice and in the human pulmonary cells. ► CYP3A subfamily mediated GSH depletion and apoptosis induced by dauricine. ► L-Buthionine sulfoximine potentiated dauricine-induced GSH depletion and apoptosis.

  16. Distribution of Exogenous and Endogenous CYP3A Markers and Related Factors in Healthy Males and Females.

    Science.gov (United States)

    Lee, Jieon; Kim, Andrew HyoungJin; Yi, SoJeong; Lee, SeungHwan; Yoon, Seo Hyun; Yu, Kyung-Sang; Jang, In-Jin; Cho, Joo-Youn

    2017-07-01

    Cytochrome P450 (CYP) 3A is an important drug-metabolizing enzyme in humans. Assessing CYP3A activity is necessary for predicting therapeutic outcomes or the potential adverse events of various therapeutics. This study sought to evaluate the distribution of endogenous and exogenous markers reflecting hepatic CYP3A activity and related factors affecting its activity in healthy male and female. Each subject was given a single 1 mg dose of midazolam intravenously. Pharmacokinetics, pharmacometabolomics, and pharmacogenomics analyses were performed to evaluate CYP3A activity. Urinary and plasma steroids were quantified with gas chromatography coupled with triple-quadrupole mass spectrometry (GC-MS), and the concentrations of midazolam and its metabolites were quantified with liquid chromatography coupled with electrospray tandem mass spectrometry (LC-MS/MS). A total of 100 subjects completed this study. Midazolam clearance (MDZ CL) and the metabolic ratio (MDZ MR) were significantly correlated with 6β-OH-cortisol/cortisol and 6β-OH-cortisone/cortisone. MDZ CL, 6β-OH-cortisol/cortisol, and 6β-OH-cortisone/cortisone decreased with increasing age (Pearson r = -0.333, -0.329, and -0.528, respectively; P exogenous and endogenous markers showed decreased CYP3A activity with increasing age, which suggested that age could be a factor that significantly influences CYP3A activity.

  17. Alteration in the Expression of Cytochrome P450s (CYP1A1, CYP2E1, and CYP3A11 in the Liver of Mouse Induced by Microcystin-LR

    Directory of Open Access Journals (Sweden)

    Bangjun Zhang

    2015-03-01

    Full Text Available Microcystins (MCs are cyclic heptapeptide toxins and can accumulate in the liver. Cytochrome P450s (CYPs play an important role in the biotransformation of endogenous substances and xenobiotics in animals. It is unclear if the CYPs are affected by MCs exposure. The objective of this study was to evaluate the effects of microcystin-LR (MCLR on cytochrome P450 isozymes (CYP1A1, CYP2E1, and CYP3A11 at mRNA level, protein content, and enzyme activity in the liver of mice the received daily, intraperitoneally, 2, 4, and 8 µg/kg body weight of MCLR for seven days. The result showed that MCLR significantly decreased ethoxyresorufin-O-deethylase (EROD (CYP1A1 and erythromycin N-demthylase (ERND (CYP3A11 activities and increased aniline hydroxylase (ANH activity (CYP2E1 in the liver of mice during the period of exposure. Our findings suggest that MCLR exposure may disrupt the function of CYPs in liver, which may be partly attributed to the toxicity of MCLR in mice.

  18. 2,3,7,8-Tetrachlorodibenzo-p-dioxin modulates estradiol-induced aldehydic DNA lesions in human breast cancer cells through alteration of CYP1A1 and CYP1B1 expression.

    Science.gov (United States)

    Chen, Shou-Tung; Chen, Dar-Ren; Fang, Ju-Pin; Lin, Po-Hsiung

    2015-05-01

    Many genes responsible for the bioactivation of endogenous estrogen to reactive quinonoid metabolites, including cytochrome P450 (CYP) 1A1, 1A2, and 1B1, are well-known target genes of the aryl hydrocarbon receptor agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The purpose of this research was to investigate the roles of TCDD-mediated altered gene expression in the induction of aldehydic DNA lesions (ADLs) by 17β-estradiol (E2) in human MDA-MB-231 and MCF-7 breast cancer cells. We demonstrated that increases in the number of oxidant-mediated ADLs, including abasic sites and aldehydic base/sugar lesions, were detected in MDA-MB-231 cells exposed to E2. The DNA-damaging effects of E2 in MDA-MB-231 cells were prevented by pretreatment of cells with TCDD. In contrast, we did not observe statistically significant increases in the number of ADLs in MCF-7 cells exposed to E2. However, with TCDD pretreatment, an approximately twofold increase in the number of ADLs was detected in MCF-7 cells exposed to E2. TCDD pretreatment induces disparity in the disposition of E2 to reactive quinonoid metabolites and the subsequent formation of oxidative DNA lesions through alteration of CYP1A1 and CYP1B1 expression in human breast cancer cells.

  19. CYP3A5 polymorphisms in renal transplant recipients: influence on tacrolimus treatment

    Directory of Open Access Journals (Sweden)

    Chen L

    2018-03-01

    Full Text Available Lucy Chen,1 G V Ramesh Prasad2 1Kidney Transplant Program, St Michael’s Hospital, Toronto, ON, Canada; 2Division of Nephrology, St Michael’s Hospital, Toronto, ON, Canada Abstract: Tacrolimus is a commonly used immunosuppressant after kidney transplantation. It has a narrow therapeutic range and demonstrates wide interindividual variability in pharmacokinetics, leading to potential underimmunosuppression or toxicity. Genetic polymorphism in CYP3A5 enzyme expression contributes to differences in tacrolimus bioavailability between individuals. Individuals carrying one or more copies of the wild-type allele *1 express CYP3A5, which increases tacrolimus clearance. CYP3A5 expressers require 1.5 to 2-fold higher tacrolimus doses compared to usual dosing to achieve therapeutic blood concentrations. Individuals with homozygous *3/*3 genotype are CYP3A5 nonexpressers. CYP3A5 nonexpression is the most frequent phenotype in most ethnic populations, except blacks. Differences between CYP3A5 genotypes in tacrolimus disposition have not translated into differences in clinical outcomes, such as acute rejection and graft survival. Therefore, although genotype-based dosing may improve achievement of therapeutic drug concentrations with empiric dosing, its role in clinical practice is unclear. CYP3A5 genotype may predict differences in absorption of extended-release and immediate-release oral formulations of tacrolimus. Two studies found that CYP3A5 expressers require higher doses of tacrolimus in the extended-release formulation compared to immediate release. CYP3A5 genotype plays a role in determining the impact of interacting drugs, such as fluconazole, on tacrolimus pharmacokinetics. Evidence conflicts regarding the impact of CYP3A5 genotype on risk of nephrotoxicity associated with tacrolimus. Further study is required. Keywords: calcineurin inhibitor, graft, pharmacogenomics, kidney, genotype

  20. The mibefradil derivative NNC55-0396, a specific T-type calcium channel antagonist, exhibits less CYP3A4 inhibition than mibefradil.

    Science.gov (United States)

    Bui, Peter H; Quesada, Arnulfo; Handforth, Adrian; Hankinson, Oliver

    2008-07-01

    A novel mibefradil derivative, NNC55-0396, designed to be hydrolysis-resistant, was shown to be a selective T-type Ca(2+) channel inhibitor without L-type Ca(2+) channel efficacy. However, its effects on cytochromes P450 (P450s) have not previously been examined. We investigated the inhibitory effects of NNC55-0396 toward seven major recombinant human P450s--CYP3A4, CYP2D6, CYP1A2, CYP2C9, CYP2C8, CYPC19, and CYP2E1--and compared its effects with those of mibefradil and its hydrolyzed metabolite, Ro40-5966. Our results show that CYP3A4 and CYP2D6 are the two P450s most affected by mibefradil, Ro40-5966, and NNC55-0396. Mibefradil (IC(50) = 33 +/- 3 nM, K(i) = 23 +/- 0.5 nM) and Ro40-5966 (IC(50) = 30 +/- 7.8 nM, K(i) = 21 +/- 2.8 nM) have a 9- to 10-fold greater inhibitory activity toward recombinant CYP3A4 benzyloxy-4-trifluoromethylcoumarin-O-debenzylation activity than NNC55-0396 (IC(50) = 300 +/- 30 nM, K(i) = 210 +/- 6 nM). More dramatically, mibefradil (IC(50) = 566 +/- 71 nM, K(i) = 202 +/- 39 nM) shows 19-fold higher inhibition of CYP3A-associated testosterone 6beta-hydroxylase activity in human liver microsomes compared with NNC55-0396 (IC(50) = 11 +/- 1.1 microM, K(i) = 3.9 +/- 0.4 microM). Loss of testosterone 6beta-hydroxylase activity by recombinant CYP3A4 was shown to be time- and concentration-dependent with both compounds. However, NNC55-0396 (K(I) = 3.87 microM, K(inact) = 0.061/min) is a much less potent mechanism-based inhibitor than mibefradil (K(I) = 83 nM, K(inact) = 0.048/min). In contrast, NNC55-0396 (IC(50) = 29 +/- 1.2 nM, K(i) = 2.8 +/- 0.3 nM) and Ro40-5966 (IC(50) = 46 +/- 11 nM, K(i) = 4.5 +/- 0.02 nM) have a 3- to 4-fold greater inhibitory activity toward recombinant CYP2D6 than mibefradil (IC(50) = 129 +/- 21 nM, K(i) = 12.7 +/- 0.9 nM). Our results suggest that NNC55-0396 could be a more favorable T-type Ca(2+) antagonist than its parent compound, mibefradil, which was withdrawn from the market because of strong inhibition of CYP3A4.

  1. Characterization of the genetic variation present in CYP3A4 in three South African populations

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    Britt Ingrid Drögemöller

    2013-02-01

    Full Text Available TThe CYP3A4 enzyme is the most abundant human cytochrome P450 and is regarded as the most important enzyme involved in drug metabolism. Inter-individual and inter-population variability in gene expression and enzyme activity are thought to be influenced, in part, by genetic variation. Although Southern African individuals have been shown to exhibit the highest levels of genetic diversity, they have been under-represented in pharmacogenetic research to date. Therefore, the aim of this study was to identify genetic variation within CYP3A4 in three South African population groups comprising of 29 Khoisan, 65 Xhosa and 65 Mixed Ancestry individuals. To identify known and novel CYP3A4 variants, 15 individuals were randomly selected from each of the population groups for bi-directional Sanger sequencing of approximately 600 bp of the 5’-upstream region and all thirteen exons including flanking intronic regions. Genetic variants detected were genotyped in the rest of the cohort. In total, 24 SNPs were detected, including CYP3A4*12, CYP3A4*15, and the reportedly functional CYP3A4*1B promoter polymorphism, as well as two novel non-synonymous variants. These putatively functional variants, p.R162W and p.Q200H, were present in two of the three populations and all three populations, respectively, and in silico analysis predicted that the former would damage the protein product. Furthermore, the three populations were shown to exhibit distinct genetic profiles. These results confirm that South African populations show unique patterns of variation in the genes encoding xenobiotic metabolizing enzymes. This research suggests that population-specific genetic profiles for CYP3A4 and other drug metabolizing genes would be essential to make full use of pharmacogenetics in Southern Africa. Further investigation is needed to determine if the identified genetic variants influence CYP3A4 metabolism phenotype in these populations.

  2. Systematic screening for CYP3A4 genetic polymorphisms in a Han Chinese population.

    Science.gov (United States)

    Hu, Guo-Xin; Dai, Da-Peng; Wang, Hao; Huang, Xiang-Xin; Zhou, Xiao-Yang; Cai, Jie; Chen, Hao; Cai, Jian-Ping

    2017-03-01

    To systematically investigate the genetic polymorphisms of the CYP3A4 gene in a Han Chinese population. The promoter and exons of CYP3A4 gene in 1114 unrelated, healthy Han Chinese subjects were amplified and genotyped by direct sequencing. In total, five previously reported alleles (*1G, *4, *5, *18B and *23) were detected, of which one allele (*23) was reported for the first time in Han Chinese population. Additionally, seven novel exonic variants were also identified and designated as new alleles CYP3A4*28-*34. This study provides the most comprehensive data of CYP3A4 polymorphisms in Han Chinese population and detects the largest number of novel CYP3A4 alleles in one ethnic group.

  3. Inferring mechanisms of copy number change from haplotype structures at the human DEFA1A3 locus.

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    Black, Holly A; Khan, Fayeza F; Tyson, Jess; Al Armour, John

    2014-07-21

    The determination of structural haplotypes at copy number variable regions can indicate the mechanisms responsible for changes in copy number, as well as explain the relationship between gene copy number and expression. However, obtaining spatial information at regions displaying extensive copy number variation, such as the DEFA1A3 locus, is complex, because of the difficulty in the phasing and assembly of these regions. The DEFA1A3 locus is intriguing in that it falls within a region of high linkage disequilibrium, despite its high variability in copy number (n = 3-16); hence, the mechanisms responsible for changes in copy number at this locus are unclear. In this study, a region flanking the DEFA1A3 locus was sequenced across 120 independent haplotypes with European ancestry, identifying five common classes of DEFA1A3 haplotype. Assigning DEFA1A3 class to haplotypes within the 1000 Genomes project highlights a significant difference in DEFA1A3 class frequencies between populations with different ancestry. The features of each DEFA1A3 class, for example, the associated DEFA1A3 copy numbers, were initially assessed in a European cohort (n = 599) and replicated in the 1000 Genomes samples, showing within-class similarity, but between-class and between-population differences in the features of the DEFA1A3 locus. Emulsion haplotype fusion-PCR was used to generate 61 structural haplotypes at the DEFA1A3 locus, showing a high within-class similarity in structure. Structural haplotypes across the DEFA1A3 locus indicate that intra-allelic rearrangement is the predominant mechanism responsible for changes in DEFA1A3 copy number, explaining the conservation of linkage disequilibrium across the locus. The identification of common structural haplotypes at the DEFA1A3 locus could aid studies into how DEFA1A3 copy number influences expression, which is currently unclear.

  4. Impact of CYP2C8*3 polymorphism on in vitro metabolism of imatinib to N-desmethyl imatinib.

    Science.gov (United States)

    Khan, Muhammad Suleman; Barratt, Daniel T; Somogyi, Andrew A

    2016-01-01

    1. Imatinib is metabolized to N-desmethyl imatinib by CYPs 3A4 and 2C8. The effect of CYP2C8*3 genotype on N-desmethyl imatinib formation was unknown. 2. We examined imatinib N-demethylation in human liver microsomes (HLMs) genotyped for CYP2C8*3, in CYP2C8*3/*3 pooled HLMs and in recombinant CYP2C8 and CYP3A4 enzymes. Effects of CYP-selective inhibitors on N-demethylation were also determined. 3. A single-enzyme Michaelis-Menten model with autoinhibition best fitted CYP2C8*1/*1 HLM (n = 5) and recombinant CYP2C8 kinetic data (median ± SD Ki = 139 ± 61 µM and 149 µM, respectively). Recombinant CYP3A4 showed two-site enzyme kinetics with no autoinhibition. Three of four CYP2C8*1/*3 HLMs showed single-enzyme kinetics with no autoinhibition. Binding affinity was higher in CYP2C8*1/*3 than CYP2C8*1/*1 HLM (median ± SD Km = 6 ± 2 versus 11 ± 2 µM, P=0.04). CYP2C8*3/*3 (pooled HLM) also showed high binding affinity (Km = 4 µM) and single-enzyme weak autoinhibition (Ki = 449 µM) kinetics. CYP2C8 inhibitors reduced HLM N-demethylation by 47-75%, compared to 0-30% for CYP3A4 inhibitors. 4. In conclusion, CYP2C8*3 is a gain-of-function polymorphism for imatinib N-demethylation, which appears to be mainly mediated by CYP2C8 and not CYP3A4 in vitro in HLM.

  5. Role of genetic polymorphisms of CYP1A1, CYP3A5, CYP2C9, CYP2D6, and PON1 in the modulation of DNA damage in workers occupationally exposed to organophosphate pesticides

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    Singh, Satyender [Division of Biochemistry and Biotechnology, National Centre for Disease Control 22, Sham Nath Marg, Delhi-110054 (India); Kumar, Vivek [Environmental Biochemistry and Molecular Biology laboratory, Department of Biochemistry, University College of Medical Sciences and GTB Hospital, University of Delhi, Dilshad Garden, Delhi-110095 (India); Vashisht, Kapil; Singh, Priyanka [Division of Biochemistry and Biotechnology, National Centre for Disease Control 22, Sham Nath Marg, Delhi-110054 (India); Banerjee, Basu Dev, E-mail: banerjeebd@hotmail.com [Environmental Biochemistry and Molecular Biology laboratory, Department of Biochemistry, University College of Medical Sciences and GTB Hospital, University of Delhi, Dilshad Garden, Delhi-110095 (India); Rautela, Rajender Singh; Grover, Shyam Sunder; Rawat, Devendra Singh; Pasha, Syed Tazeen [Division of Biochemistry and Biotechnology, National Centre for Disease Control 22, Sham Nath Marg, Delhi-110054 (India); Jain, Sudhir Kumar [Centre for Epidemiology and Parasitic Diseases, National Centre for Disease Control 22, Sham Nath Marg, Delhi-110054 (India); Rai, Arvind [Division of Biochemistry and Biotechnology, National Centre for Disease Control 22, Sham Nath Marg, Delhi-110054 (India)

    2011-11-15

    Organophosphate pesticides (OPs) are primarily metabolized by several xenobiotic metabolizing enzymes (XMEs). Very few studies have explored genetic polymorphisms of XMEs and their association with DNA damage in pesticide-exposed workers. The present study was designed to determine the role of genetic polymorphisms of CYP1A1, CYP3A5, CYP2C9, CYP2D6, and PON1 in the modulation of DNA damage in workers occupationally exposed to OPs. We examined 284 subjects including 150 workers occupationally exposed to OPs and 134 normal healthy controls. The DNA damage was evaluated using the alkaline comet assay and genotyping was done using PCR-RFLP. The results revealed that the PONase activity toward paraoxonase and AChE activity was found significantly lowered in workers as compared to control subjects (p < 0.001). Workers showed significantly higher DNA damage compared to control subjects (14.37 {+-} 2.15 vs. 6.24 {+-} 1.37 tail% DNA, p < 0.001). Further, the workers with CYP2D6*3 PM and PON1 (QQ and MM) genotypes were found to have significantly higher DNA damage when compared to other genotypes (p < 0.05). In addition, significant increase in DNA damage was also observed in workers with concomitant presence of certain CYP2D6 and PON1 (Q192R and L55M) genotypes which need further extensive studies. In conclusion, the results indicate that the PON1 and CYP2D6 genotypes can modulate DNA damage elicited by some OPs possibly through gene-environment interactions. -- Highlights: Black-Right-Pointing-Pointer Role of CYP1A1, CYP3A5, CYP2C, CYP2D6 and PON1 genotypes on DNA damage. Black-Right-Pointing-Pointer Workers exposed to some OPs demonstrated increased DNA damage. Black-Right-Pointing-Pointer CYP2D6 *3 PM and PON1 (Q192R and L55M) genotypes are associated with DNA damage. Black-Right-Pointing-Pointer Concomitant presence of certain CYP2D6 and PON1 genotypes can increase DNA damage.

  6. Genomic and Transcriptomic Associations Identify a New Insecticide Resistance Phenotype for the Selective Sweep at the Cyp6g1 Locus of Drosophila melanogaster.

    Science.gov (United States)

    Battlay, Paul; Schmidt, Joshua M; Fournier-Level, Alexandre; Robin, Charles

    2016-08-09

    Scans of the Drosophila melanogaster genome have identified organophosphate resistance loci among those with the most pronounced signature of positive selection. In this study, the molecular basis of resistance to the organophosphate insecticide azinphos-methyl was investigated using the Drosophila Genetic Reference Panel, and genome-wide association. Recently released full transcriptome data were used to extend the utility of the Drosophila Genetic Reference Panel resource beyond traditional genome-wide association studies to allow systems genetics analyses of phenotypes. We found that both genomic and transcriptomic associations independently identified Cyp6g1, a gene involved in resistance to DDT and neonicotinoid insecticides, as the top candidate for azinphos-methyl resistance. This was verified by transgenically overexpressing Cyp6g1 using natural regulatory elements from a resistant allele, resulting in a 6.5-fold increase in resistance. We also identified four novel candidate genes associated with azinphos-methyl resistance, all of which are involved in either regulation of fat storage, or nervous system development. In Cyp6g1, we find a demonstrable resistance locus, a verification that transcriptome data can be used to identify variants associated with insecticide resistance, and an overlap between peaks of a genome-wide association study, and a genome-wide selective sweep analysis. Copyright © 2016 Battlay et al.

  7. CYP3A4*18: it is not rare allele in Japanese population.

    Science.gov (United States)

    Yamamoto, Takehito; Nagafuchi, Nobue; Ozeki, Takeshi; Kubota, Takahiro; Ishikawa, Hiroshi; Ogawa, Seishi; Yamada, Yasuhiko; Hirai, Hisamaru; Iga, Tatsuji

    2003-01-01

    We sequenced all 13 exons of the CYP3A4 gene derived from 48 Japanese subjects. One subject possess the 20070 T>C mutation in the exon 10 (result in leu293Pro substitution, namely CYP3A4(*)18), as heterozygote. Thus, we investigated the frequency of CYP3A4(*)18 in 118 Japanese population using polymerase chain reaction-restriction fragment length polymorphism with Msp I and determined that the frequency of the CYP3A4(*)18 allele was 1.3%.

  8. Global pharmacogenomics: distribution of CYP3A5 polymorphisms and phenotypes in the Brazilian population.

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    Guilherme Suarez-Kurtz

    Full Text Available The influence of self-reported "race/color", geographical origin and genetic ancestry on the distribution of three functional CYP3A5 polymorphisms, their imputed haplotypes and inferred phenotypes was examined in 909 healthy, adult Brazilians, self-identified as White, Brown or Black ("race/color" categories of the Brazilian census. The cohort was genotyped for CYP3A5*3 (rs776746, CYP3A5*6 (rs10264272 and CYP3A5*7 (rs41303343, CYP3A5 haplotypes were imputed and CYP3A5 metabolizer phenotypes were inferred according to the number of defective CYP3A5 alleles. Estimates of the individual proportions of Amerindian, African and European ancestry were available for the entire cohort. Multinomial log-linear regression models were applied to infer the statistical association between the distribution of CYP3A5 alleles, haplotypes and phenotypes (response variables, and self-reported Color, geographical region and ancestry (explanatory variables. We found that Color per se or in combination with geographical region associates significantly with the distribution of CYP3A5 variant alleles and CYP3A5 metabolizer phenotypes, whereas geographical region per se influences the frequency distribution of CYP3A5 variant alleles. The odds of having the default CYP3A5*3 allele and the poor metabolizer phenotype increases continuously with the increase of European ancestry and decrease of African ancestry. The opposite trend is observed in relation to CYP3A5*6, CYP3A5*7, the default CYP3A5*1 allele, and both the extensive and intermediate phenotypes. No significant effect of Amerindian ancestry on the distribution of CYP3A5 alleles or phenotypes was observed. In conclusion, this study strongly supports the notion that the intrinsic heterogeneity of the Brazilian population must be acknowledged in the design and interpretation of pharmacogenomic studies, and dealt with as a continuous variable, rather than proportioned in arbitrary categories that do not capture the

  9. Germ-line mutations at a mouse ESTR (Pc-3) locus and human microsatellite loci

    International Nuclear Information System (INIS)

    Ryo, Haruko; Nakajima, Hiroo; Nomura, Taisei

    2006-01-01

    We examined the use of the mouse Pc-3 ESTR (expanded simple tandem repeat) locus and 72 human microsatellite loci as potentially sensitive biomarkers for mutagenic exposures to germ cells in mice and humans respectively. In the mouse work, we treated male mice with TCDD (2, 3, 7, 8-tetrachlo-rodibenzo-p-dioxin; a chemical known to induce congenital anomalies in humans and mice) and, analysed the F 1 fetuses for Pc-3 mutations. Although the incidence of anomalies was higher in the TCDD group, there were no induced mutations. However, respiratory distress syndrome (RDS) was observed in 3 of 7 fetuses born to male mice which were treated with TCDD and which showed abnormal length of Pc-3 allele. In the human studies, the children of Chernobyl liquidators were examined for mutations at a total of 72 (31 autosomal, 1 X-linked and 40 Y-linked) microsatellite loci. This study was prompted by earlier findings of increases in microsatellite mutations in barn swallows and wheat in the highly contaminated areas after the Chernobyl accident. We examined 64 liquidator families (70 children) and 66 control families (70 children). However, no increases in mutation rates were found. The estimated mean dose to the liquidators was about 39 mSv and this might be one possible reason why no increases of mutations could be found. (author)

  10. Functional characterization of a first avian cytochrome P450 of the CYP2D subfamily (CYP2D49.

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    Hua Cai

    Full Text Available The CYP2D family members are instrumental in the metabolism of 20-25% of commonly prescribed drugs. Although many CYP2D isoforms have been well characterized in other animal models, research concerning the chicken CYP2Ds is limited. In this study, a cDNA encoding a novel CYP2D enzyme (CYP2D49 was cloned from the chicken liver for the first time. The CYP2D49 cDNA contained an open reading frame of 502 amino acids that shared 52%-57% identities with other CYP2Ds. The gene structure and neighboring genes of CYP2D49 are conserved and similar to those of human CYP2D6. Additionally, similar to human CYP2D6, CYP2D49 is un-inducible in the liver and expressed predominantly in the liver, kidney and small intestine, with detectable levels in several other tissues. Metabolic assays of the CYP2D49 protein heterologously expressed in E. coli and Hela cells indicated that CYP2D49 metabolized the human CYP2D6 substrate, bufuralol, but not debrisoquine. Moreover, quinidine, a potent inhibitor of human CYP2D6, only inhibited the bufuralol 1'-hydroxylation activity of CYP2D49 to a negligible degree. All these results indicated that CYP2D49 had functional characteristics similar to those of human CYP2D6 but measurably differed in the debrisoquine 4'-hydroxylation and quinidine inhibitory profile. Further structure-function investigations that employed site-directed mutagenesis and circular dichroism spectroscopy identified the importance of Val-126, Glu-222, Asp-306, Phe-486 and Phe-488 in keeping the enzymatic activity of CYP2D49 toward bufuralol as well as the importance of Asp-306, Phe-486 and Phe-488 in maintaining the conformation of CYP2D49 protein. The current study is only the first step in characterizing the metabolic mechanism of CYP2D49; further studies are still required.

  11. Polychlorinated biphenyl (PCB) induction of CYP3A4 enzyme activity in healthy Faroese adults

    International Nuclear Information System (INIS)

    Petersen, Maria Skaalum; Halling, Jonrit; Damkier, Per; Nielsen, Flemming; Grandjean, Philippe; Weihe, Pal; Brosen, Kim

    2007-01-01

    The CYP3A4 enzyme is, along with other cytochrome P450 enzymes, involved in the metabolism of environmental pollutants and is highly inducible by these substances. A commercial polychlorinated biphenyl (PCB) mixture, 1,1,1,-trichloro-2-(o-chlorophenyl), 2-(p'-chlorophenyl)ethane (o,p'-DDT) and 1,1,-dichloro-2,2-bis (p-chlorophenyl)ethene (p,p'-DDE) are known to induce CYP3A4 activity through activation of nuclear receptors, such as the pregnane X receptor. However, this induction of CYP3A4 has not yet been investigated in humans. Thus, the aim of the study was to determine the variability of the CYP3A4 phenotype in regard to increased concentrations of PCBs and other persistent organohalogen pollutants (POPs) in healthy Faroese adults. In 310 randomly selected Faroese residents aged 18-60 years, the CYP3A4 activity was determined based on the urinary 6β-hydroxycortisol/cortisol (6β-OHC/FC) ratio. POP exposures were assessed by measuring their concentrations in serum lipid. The results showed a unimodal distribution of the 6β-OHC/FC ratio with values ranging from 0.58 to 27.38. Women had a slightly higher 6β-OHC/FC ratio than men (p = 0.07). Confounder-adjusted multiple regression analysis showed significant associations between 6β-OHC/FC ratios and ΣPCB, PCB-TEQ and p,p'-DDE, o,p'-DDT and HCB, respectively, but the associations were statistically significant for men only

  12. Effects of CYP3A5, CYP2C19, and CYP2B6 on the clinical efficacy and adverse outcomes of sibutramine therapy: a crucial role for the CYP2B6*6 allele.

    Science.gov (United States)

    Hwang, In Cheol; Park, Ji Young; Ahn, Hong Yup; Kim, Kyoung Kon; Suh, Heuy Sun; Ko, Ki Dong; Kim, Kyoung-Ah

    2014-01-20

    Various cytochrome P450 isoforms modulate sibutramine activity and influence sibutramine plasma levels and pharmacokinetics. However, there are no available data to demonstrate the association of these polymorphisms with the clinical outcomes of sibutramine administration. This study was a sub-investigation of a 12-week, double-blind, placebo-controlled trial examining the additive effect of orlistat on sibutramine. The final analysis was restricted to 101 women who had fulfilled the protocol. We evaluated the effects of genetic polymorphisms of CYP3A5, CYP2C19 and CYP2B6 on the % weight loss and the occurrence of adverse events. The change of pulse rate from baseline value was affected by both CYP2B6 and CYP3A5 genetic polymorphisms (Psibutramine treatment. Copyright © 2013 Elsevier B.V. All rights reserved.

  13. Aspartame Administration and Insulin Treatment Altered Brain Levels of CYP2E1 and CYP3A2 in Streptozotocin-Induced Diabetic Rats.

    Science.gov (United States)

    Nosti-Palacios, Rosario; Gómez-Garduño, Josefina; Molina-Ortiz, Dora; Calzada-León, Raúl; Dorado-González, Víctor Manuel; Vences-Mejía, Araceli

    2014-07-01

    This study demonstrates that aspartame consumption and insulin treatment in a juvenile diabetic rat model leads to increase in cytochrome P450 (CYP) 2E1 and CYP3A2 isozymes in brain. Diabetes mellitus was induced in postweaned 21-day-old Wistar male rat by streptozotocin. Animals were randomly assigned to one of the following groups: untreated control, diabetic (D), D-insulin, D-aspartame, or the D-insulin + aspartame-treated group. Brain and liver tissue samples were used to analyze the activity of CYP2E1 and CYP3A2 and protein levels. Our results indicate that combined treatment with insulin and aspartame in juvenile diabetic rats significantly induced CYP2E1 in the cerebrum and cerebellum without modifying it in the liver, while CYP3A2 protein activity increased both in the brain and in the liver. The induction of CYP2E1 in the brain could have important in situ toxicological effects, given that this CYP isoform is capable of bioactivating various toxic substances. Additionally, CYP3A2 induction in the liver and brain could be considered a decisive factor in the variation of drug response and toxicity. © The Author(s) 2014.

  14. CYP3A5 Polymorphism In Serbian Paediatric Epileptic Patients On Carbamazepine Treatment

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    Milovanovic Dragana Dragas

    2015-06-01

    Full Text Available Carbamazepine exhibits significant inter-individual variability in its efficacy and safety, which leads to unpredictable therapy outcomes for the majority of patients. Although its complex biotransformation depends on CYP3A5 activity, evidence of association between carbamazepine treatment outcomes and CYP3A5 functional variations remains inconclusive. The aim of the present study was to investigate the distribution of two of the functionally important CYP3A5 variants *2 and *3 as well as their effects on carbamazepine dose requirements, plasma concentrations and clearance in a Serbian population. The study involved 40 paediatric epileptic patients on steady-state carbamazepine treatment. Genotyping was conducted using the PCR-RFLP method, and carbamazepine plasma concentrations were determined using the HPLC method. CYP3A5*2 and *3 polymorphisms were found at frequencies of 0.0% and 97.5%, respectively, which corresponds well to previously published data for Caucasians. No differences in CYP3A5*3 allele frequencies were detected among epileptic patients in comparison to healthy volunteers within similar ethnic populations (p>0.08, indicating that CYP3A5 polymorphism does not represent a risk factor for epilepsy development. There was an observed tendency towards lower dosage requirements (mean±SD: 15.06±4.45 mg/kg vs. 18.74±5.55 mg/kg; p=0.26, higher plasma concentrations (mean±SD: 0.45±0.13 mg/kg vs. 0.38±0.03 mg/kg; p=0.47 and lower clearance (mean±SD: 0.14±0.05 mg/kg vs. 0.15±0.01 mg/kg; p=0.79 of carbamazepine in homozygous carriers of CYP3A5*3/*3 compared to heterozygous CYP3A5*1A/*3 Serbians. Because these genotype groups did not differ significantly in terms of their carbamazepine pharmacokinetics parameters, the proposed effects of CYP3A5*3 on carbamazepine metabolism could not be confirmed.

  15. Genetic analysis of drug metabolizing phase-I enzymes CYP3A4 in Tibetan populations.

    Science.gov (United States)

    Liu, Lijun; Chang, Yu; Du, Shuli; Shi, Xugang; Yang, Hua; Kang, Longli; Jin, Tianbo; Yuan, Dongya; He, Yongjun

    2017-06-01

    The enzymatic activity of CYP3A4 results in broad interindividual variability in response to certain pharmacotherapies. The present study aimed to screen Tibetan volunteers for CYP3A4 genetic polymorphisms. Previous research has focussed on Han Chinese patients, while little is known about the genetic variation of CYP3A4 in the Tibetan populations. Here, we adopted DNA sequencing to investigate the promoter, exons and surrounding introns, and 3'-untranslated region of the CYP3A4 gene in 96 unrelated healthy Tibetan individuals.We identified 20 different CYP3A4 polymorphisms in the Tibetan population, including two novel variants (21824 A>G and 15580 G>C). In addition, we also determined the allele frequencies of CYP3A4*1A and CYP3A4*1H were 82.29% and 28.13%, respectively. CYP3A4*1P and *1G were relatively rare with frequencies of only 1.04% and 0.52%, respectively. Our results provide information on CYP3A4 polymorphisms in Tibetan individuals which may help to optimize pharmacotherapy effectiveness by providing personalized medicine to this ethnic group.

  16. CYP Suppression in Human Hepatocytes by Monomethyl Auristatin E, the Payload in Brentuximab Vedotin (Adcetris®), is Associated with Microtubule Disruption.

    Science.gov (United States)

    Wolenski, Francis S; Xia, Cindy Q; Ma, Bingli; Han, Tae H; Shyu, Wen C; Balani, Suresh K

    2018-06-01

    Monomethyl auristatin E (MMAE), the toxin linked to CD30-specific monoclonal antibody of Adcetris ® (brentuximab vedotin), is a potent anti-microtubule agent. Brentuximab vedotin has been approved for the treatment of relapsed or refractory Hodgkin lymphoma and anaplastic large cell lymphoma. Cytochrome P450 (CYP) induction assessment of MMAE was conducted in human hepatocytes to assess DDI potentials and its translation to clinic. MMAE was incubated at 1-1000 nM with cultured primary human hepatocytes for 72 h, and CYP1A2, CYP2B6, and CYP3A4 mRNA expression was assessed by quantitative reverse transcription-polymerase chain reaction and CYP-specific probe substrate by liquid chromatography coupled with mass spectrometry, along with microtubule disruption by immunofluorescence staining using anti-β-tubulin antibody and imaging. MMAE up to 10 nM had no significant effect on CYP1A2, CYP2B6, and CYP3A4 mRNA expression and activity, whereas at higher concentrations of 100- and 1000-nM MMAE, the CYP mRNA expression and activity were diminished substantially. Further investigation showed that the degree of CYP suppression was paralleled by that of microtubule disruption by MMAE, as measured by increase in the number of β-tubulin-positive aggregates. At the clinical dose, the concentration of MMAE was 7 nM which did not show any significant CYP suppression or microtubule disruption in hepatocytes. MMAE was not a CYP inducer in human hepatocytes. However, it caused a concentration-dependent CYP mRNA suppression and activity. The CYP suppression was associated with microtubule disruption, supporting the reports that intact microtubule architecture is required for CYP regulations. The absence of CYP suppression and microtubule disruption in vitro at the clinical plasma concentrations of MMAE (< 10 nM) explains the lack of pharmacokinetic drug interaction between brentuximab vedotin and midazolam, a sensitive CYP3A substrate, reported in patients.

  17. NAD(P)H:quinone oxidoreductase expression in Cyp1a-knockout and CYP1A-humanized mouse lines and its effect on bioactivation of the carcinogen aristolochic acid I

    Energy Technology Data Exchange (ETDEWEB)

    Levova, Katerina; Moserova, Michaela [Department of Biochemistry, Faculty of Science, Charles University, Prague (Czech Republic); Nebert, Daniel W. [Department of Environmental Health, University of Cincinnati Medical Center, Cincinnati (United States); Phillips, David H. [Analytical and Environmental Sciences Division, MRC-HPA Centre for Environment and Health, King' s College London, London (United Kingdom); Frei, Eva [Division of Preventive Oncology, National Center for Tumor Diseases, German Cancer Research Center (DKFZ), Heidelberg (Germany); Schmeiser, Heinz H. [Research Group Genetic Alterations in Carcinogenesis, German Cancer Research Center (DKFZ), Heidelberg (Germany); Arlt, Volker M. [Analytical and Environmental Sciences Division, MRC-HPA Centre for Environment and Health, King' s College London, London (United Kingdom); Stiborova, Marie, E-mail: stiborov@natur.cuni.cz [Department of Biochemistry, Faculty of Science, Charles University, Prague (Czech Republic)

    2012-12-15

    Aristolochic acid causes a specific nephropathy (AAN), Balkan endemic nephropathy, and urothelial malignancies. Using Western blotting suitable to determine protein expression, we investigated in several transgenic mouse lines expression of NAD(P)H:quinone oxidoreductase (NQO1)—the most efficient cytosolic enzyme that reductively activates aristolochic acid I (AAI). The mouse tissues used were from previous studies [Arlt et al., Chem. Res. Toxicol. 24 (2011) 1710; Stiborova et al., Toxicol. Sci. 125 (2012) 345], in which the role of microsomal cytochrome P450 (CYP) enzymes in AAI metabolism in vivo had been determined. We found that NQO1 levels in liver, kidney and lung of Cyp1a1(−/−), Cyp1a2(−/−) and Cyp1a1/1a2(−/−) knockout mouse lines, as well as in two CYP1A-humanized mouse lines harboring functional human CYP1A1 and CYP1A2 and lacking the mouse Cyp1a1/1a2 orthologs, differed from NQO1 levels in wild-type mice. NQO1 protein and enzymic activity were induced in hepatic and renal cytosolic fractions isolated from AAI-pretreated mice, compared with those in untreated mice. Furthermore, this increase in hepatic NQO1 enzyme activity was associated with bioactivation of AAI and elevated AAI-DNA adduct levels in ex vivo incubations of cytosolic fractions with DNA and AAI. In conclusion, AAI appears to increase its own metabolic activation by inducing NQO1, thereby enhancing its own genotoxic potential. Highlights: ► NAD(P)H:quinone oxidoreductase expression in Cyp1a knockout and humanized CYP1A mice ► Reductive activation of the nephrotoxic and carcinogenic aristolochic acid I (AAI) ► NAD(P)H:quinone oxidoreductase is induced in mice treated with AAI. ► Induced hepatic enzyme activity resulted in elevated AAI-DNA adduct levels.

  18. NAD(P)H:quinone oxidoreductase expression in Cyp1a-knockout and CYP1A-humanized mouse lines and its effect on bioactivation of the carcinogen aristolochic acid I

    International Nuclear Information System (INIS)

    Levova, Katerina; Moserova, Michaela; Nebert, Daniel W.; Phillips, David H.; Frei, Eva; Schmeiser, Heinz H.; Arlt, Volker M.; Stiborova, Marie

    2012-01-01

    Aristolochic acid causes a specific nephropathy (AAN), Balkan endemic nephropathy, and urothelial malignancies. Using Western blotting suitable to determine protein expression, we investigated in several transgenic mouse lines expression of NAD(P)H:quinone oxidoreductase (NQO1)—the most efficient cytosolic enzyme that reductively activates aristolochic acid I (AAI). The mouse tissues used were from previous studies [Arlt et al., Chem. Res. Toxicol. 24 (2011) 1710; Stiborova et al., Toxicol. Sci. 125 (2012) 345], in which the role of microsomal cytochrome P450 (CYP) enzymes in AAI metabolism in vivo had been determined. We found that NQO1 levels in liver, kidney and lung of Cyp1a1(−/−), Cyp1a2(−/−) and Cyp1a1/1a2(−/−) knockout mouse lines, as well as in two CYP1A-humanized mouse lines harboring functional human CYP1A1 and CYP1A2 and lacking the mouse Cyp1a1/1a2 orthologs, differed from NQO1 levels in wild-type mice. NQO1 protein and enzymic activity were induced in hepatic and renal cytosolic fractions isolated from AAI-pretreated mice, compared with those in untreated mice. Furthermore, this increase in hepatic NQO1 enzyme activity was associated with bioactivation of AAI and elevated AAI-DNA adduct levels in ex vivo incubations of cytosolic fractions with DNA and AAI. In conclusion, AAI appears to increase its own metabolic activation by inducing NQO1, thereby enhancing its own genotoxic potential. Highlights: ► NAD(P)H:quinone oxidoreductase expression in Cyp1a knockout and humanized CYP1A mice ► Reductive activation of the nephrotoxic and carcinogenic aristolochic acid I (AAI) ► NAD(P)H:quinone oxidoreductase is induced in mice treated with AAI. ► Induced hepatic enzyme activity resulted in elevated AAI-DNA adduct levels.

  19. Epigenetic Determinants of CYP1A1 Induction by the Aryl Hydrocarbon Receptor Agonist 3,3',4,4',5-Pentachlorobiphenyl (PCB 126

    Directory of Open Access Journals (Sweden)

    Sabine U. Vorrink

    2014-08-01

    Full Text Available Many enzymes involved in xenobiotic metabolism, including cytochrome P450 (CYP 1A1, are regulated by the aryl hydrocarbon receptor (AhR. 3,3',4,4',5-Penta chlorobiphenyl (PCB 126 is a potent ligand for AhR and can thus induce the expression of CYP1A1. Interestingly, we observed that human carcinoma cell lines derived from different types of epithelial cells displayed divergent degrees of CYP1A1 induction after exposure to PCB 126. Since epigenetic mechanisms are known to be involved in cell type-specific gene expression, we sought to assess the epigenetic determinants of CYP1A1 induction in these carcinoma cell lines. In contrast to HepG2 hepatocarcinoma cells, HeLa cervical carcinoma cells showed significantly lower levels of CYP1A1 mRNA expression following PCB 126 exposure. Our results show that the two cell lines maintained differences in the chromatin architecture along the CYP1A1 promoter region. Furthermore, treatment with the epigenetic modifiers, trichostatin A (TSA and 5-aza-2'-deoxycytidine (5-Aza-dC, significantly increased the expression of CYP1A1 after PCB 126 treatment in HeLa cells. However, we did not observe apparent differences in methylation levels or specific location of CpG DNA methylation between the two cell lines in the analyzed CYP1A1 promoter region. Taken together, our findings suggest that the differences in CYP1A1 expression between HepG2 and HeLa cells are due to differences in the chromatin architecture of the CYP1A1 promoter and thus establish a role of epigenetic regulation in cell-specific CYP1A1 expression.

  20. Effect of CYP3A perpetrators on ibrutinib exposure in healthy participants.

    Science.gov (United States)

    de Jong, Jan; Skee, Donna; Murphy, Joe; Sukbuntherng, Juthamas; Hellemans, Peter; Smit, Johan; de Vries, Ronald; Jiao, Juhui James; Snoeys, Jan; Mannaert, Erik

    2015-08-01

    Ibrutinib (PCI-32765), a potent covalent inhibitor of Bruton's tyrosine kinase, has shown efficacy against a variety of B-cell malignancies. Given the prominent role of CYP3A in ibrutinib metabolism, effect of coadministration of CYP3A perpetrators with ibrutinib was evaluated in healthy adults. Ibrutinib (120 mg [Study 1, fasted], 560 mg [studies 2 (fasted), and 3 (nonfasted)]) was given alone and with ketoconazole [Study 1; 400 mg q.d.], rifampin [Study 2; 600 mg q.d.], and grapefruit juice [GFJ, Study 3]. Lower doses of ibrutinib were used together with CYP3A inhibitors [Study 1: 40 mg; Study 3: 140 mg], as safety precaution. Under fasted condition, ketoconazole increased ibrutinib dose-normalized (DN) exposure [DN-AUClast: 24-fold; DN-C max: 29-fold], rifampin decreased ibrutinib exposure [C max: 13-fold; AUClast: 10-fold]. Under nonfasted condition, GFJ caused a moderate increase [DN-C max: 3.5-fold; DN-AUC: 2.2-fold], most likely through inhibition of intestinal CYP3A. Half-life was not affected by CYP perpetrators indicating the interaction was mainly on first-pass extraction. All treatments were well-tolerated.

  1. Stereoselective Inhibition of CYP2C19 and CYP3A4 by Fluoxetine and Its Metabolite: Implications for Risk Assessment of Multiple Time-Dependent Inhibitor Systems

    Science.gov (United States)

    Lutz, Justin D.; VandenBrink, Brooke M.; Babu, Katipudi N.; Nelson, Wendel L.; Kunze, Kent L.

    2013-01-01

    Recent guidance on drug-drug interaction (DDI) testing recommends evaluation of circulating metabolites. However, there is little consensus on how to quantitatively predict and/or assess the risk of in vivo DDIs by multiple time-dependent inhibitors (TDIs) including metabolites from in vitro data. Fluoxetine was chosen as the model drug to evaluate the role of TDI metabolites in DDI prediction because it is a TDI of both CYP3A4 and CYP2C19 with a circulating N-dealkylated inhibitory metabolite, norfluoxetine. In pooled human liver microsomes, both enantiomers of fluoxetine and norfluoxetine were TDIs of CYP2C19, (S)-norfluoxetine was the most potent inhibitor with time-dependent inhibition affinity constant (KI) of 7 μM, and apparent maximum time-dependent inhibition rate (kinact,app) of 0.059 min−1. Only (S)-fluoxetine and (R)-norfluoxetine were TDIs of CYP3A4, with (R)-norfluoxetine being the most potent (KI = 8 μM, and kinact,app = 0.011 min−1). Based on in-vitro-to-in-vivo predictions, (S)-norfluoxetine plays the most important role in in vivo CYP2C19 DDIs, whereas (R)-norfluoxetine is most important in CYP3A4 DDIs. Comparison of two multiple TDI prediction models demonstrated significant differences between them in in-vitro-to-in-vitro predictions but not in in-vitro-to-in-vivo predictions. Inclusion of all four inhibitors predicted an in vivo decrease in CYP2C19 (95%) and CYP3A4 (60–62%) activity. The results of this study suggest that adequate worst-case risk assessment for in vivo DDIs by multiple TDI systems can be achieved by incorporating time-dependent inhibition by both parent and metabolite via simple addition of the in vivo time-dependent inhibition rate/cytochrome P450 degradation rate constant (λ/kdeg) values, but quantitative DDI predictions will require a more thorough understanding of TDI mechanisms. PMID:23785064

  2. CYP24A1 inhibition facilitates the anti-tumor effect of vitamin D3 on colorectal cancer cells

    Science.gov (United States)

    Kósa, János P; Horváth, Péter; Wölfling, János; Kovács, Dóra; Balla, Bernadett; Mátyus, Péter; Horváth, Evelin; Speer, Gábor; Takács, István; Nagy, Zsolt; Horváth, Henrik; Lakatos, Péter

    2013-01-01

    AIM: The effects of vitamin D3 have been investigated on various tumors, including colorectal cancer (CRC). 25-hydroxyvitamin-D3-24-hydroxylase (CYP24A1), the enzyme that inactivates the active vitamin D3 metabolite 1,25-dihydroxyvitamin D3 (1,25-D3), is considered to be the main enzyme determining the biological half-life of 1,25-D3. During colorectal carcinogenesis, the expression and concentration of CYP24A1 increases significantly, suggesting that this phenomenon could be responsible for the proposed efficacy of 1,25-D3 in the treatment of CRC. The aim of this study was to investigate the anti-tumor effects of vitamin D3 on the human CRC cell line Caco-2 after inhibition of the cytochrome P450 component of CYP24A1 activity. METHODS: We examined the expression of CYP24A1 mRNA and the effects of 1,25-D3 on the cell line Caco-2 after inhibition of CYP24A1. Cell viability and proliferation were determined by means of sulforhodamine-B staining and bromodeoxyuridine incorporation, respectively, while cytotoxicity was estimated via the lactate dehydrogenase content of the cell culture supernatant. CYP24A1 expression was measured by real-time reverse transcription polymerase chain reaction. A number of tetralone compounds were synthesized to investigate their CP24A1 inhibitory activity. RESULTS: In response to 1,25-D3, CYP24A1 mRNA expression was enhanced significantly, in a time- and dose-dependent manner. Caco-2 cell viability and proliferation were not influenced by the administration of 1,25-D3 alone, but were markedly reduced by co-administration of 1,25-D3 and KD-35, a CYP24A1-inhibiting tetralone. Our data suggest that the mechanism of action of co-administered KD-35 and 1,25-D3 does not involve a direct cytotoxic effect, but rather the inhibition of cell proliferation. CONCLUSION: These findings demonstrate that the selective inhibition of CYP24A1 by compounds such as KD-35 may be a new approach for enhancement of the anti-tumor effect of 1,25-D3 on CRC. PMID

  3. 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....

  4. Up-regulatation of CYP3A expression through pregnent X receptor by praeruptorin D isolated from Peucedanum praeruptorum Dunn.

    Science.gov (United States)

    Huang, Ling; Huang, Min; Li, Yu-Hua; Li, Rui-Ming; Zeng, Yu; Kuang, Shao-Yi; Zhang, Li; Wang, Yi-Tao; Bi, Hui-Chang

    2013-07-09

    Qianhu, the dried roots of Peucedanum praeruptorum DUNN (Umbelliferae), is a well-known traditional Chinese medicinal herb which was officially listed in the Chinese Pharmacopoeia. Praeruptorin D (PD) is one of the major active constituents of Peucedanum praeruptorum Dunn (Qianhu). The Pregnane X receptor (PXR) is an orphan nuclear receptor and plays a pivotal role in the activation of human cytochrome P450 3A4 (CYP3A4) gene. The purpose of this study was to investigate the effect of PD on the PXR-mediated transactivation of CYP3A4, and thus to predict potential herb-drug interactions between PD, Qianhu, and the other co-administered drugs that metabolized by CYP3A4. The effect of PD on the Cyp3a11, mPXR mRNA expression in mice primary hepatocytes was measured using real-time PCR. The gene expression, protein expression, and catalytic activity of CYP3A4 in the LS174T cells after transfected with PXR expression plasmids were determined by real-time PCR, Western blot analysis, and LC-MS/MS based CYP3A4 substrate assay. The results revealed that the level of Cyp3a11 gene expression in mice primary hepatocytes was significantly increased by PD, but PD cannot induce the mPXR gene expression. On the other hand, CYP3A4 mRNA, protein expression and functional activity in PXR-over-expression LS174T cells were significantly increased by PD through PXR-mediated pathway; conversely, no significant change was found in the untransfected cells. These findings suggest that PD can significantly up-regulate CYP3A4 expression and activity via the PXR-mediated pathway and this should be taken into consideration to predict any potential herb-drug interactions when PD and Peucedanum praeruptorum Dunn are co-administered with other drugs. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  5. Investigation of the 5q33.3 longevity locus and age-related phenotypes

    DEFF Research Database (Denmark)

    Nygaard, Marianne; Thinggaard, Mikael; Christensen, Kaare

    2017-01-01

    A large meta-analysis recently found the 5q33.3 locus to be associated with survival to ≥ 90 years and lower all-cause mortality, thus suggesting it as a third human longevity locus alongside APOE and FOXO3A. The 5q33.3 locus has previously been associated with blood pressure regulation and cardi......A large meta-analysis recently found the 5q33.3 locus to be associated with survival to ≥ 90 years and lower all-cause mortality, thus suggesting it as a third human longevity locus alongside APOE and FOXO3A. The 5q33.3 locus has previously been associated with blood pressure regulation...... and cardiovascular diseases in middle-aged individuals. However, part of the influence on mortality appears to be independent of cardiovascular phenotypes, and the role of the 5q33.3 locus in longevity and survival is therefore still partly unknown. We investigated the association between the longevity......-associated variant rs2149954 on chromosome 5q33.3 and age-related phenotypes in two cohorts of 1,588 and 1,271 long-lived individuals (mean ages 93.1 and 95.9 years, respectively) as well as in 700 middle-aged and 677 elderly individuals (mean ages 52.5 and 78.7 years). Altogether, nominally significant associations...

  6. Evaluation of first-pass cytochrome P4503A (CYP3A) and P-glycoprotein activities using alfentanil and fexofenadine in combination.

    Science.gov (United States)

    Kharasch, Evan D; Walker, Alysa; Hoffer, Christine; Sheffels, Pamela

    2005-01-01

    Cytochrome P4503A (CYP3A) and P-glycoprotein (P-gp) are major determinants of oral bioavailability. Development of in vivo probe(s), for both CYP3A and P-gp, which could be administered in combination, is a current goal. Nevertheless, there is considerable overlap in CYP3A and P-gp substrate selectivities; there are few discrete probes. Alfentanil is a selective CYP3A probe but not a P-gp substrate. Fexofenadine is a P-gp probe but not a CYP3A substrate. This investigation tested the hypothesis that alfentanil and fexofenadine could be administered in combination to probe first-pass CYP3A and P-gp activities in humans. Two 3-way crossover studies were conducted in healthy volunteers. In the first protocol, subjects received oral alfentanil alone, fexofenadine alone, or fexofenadine 1 hour after alfentanil. In the second protocol, subjects abstained from citrus and apple products for 5 days and received fexofenadine alone, fexofenadine 1 hour after alfentanil, or alfentanil 4 hours after fexofenadine. An assay using solid-phase extraction and electrospray liquid chromatography/mass spectrometry was developed for the simultaneous quantification of plasma alfentanil and fexofenadine. In both protocols, alfentanil plasma concentrations and area under the concentration versus time curve (AUC) were unaffected by fexofenadine or meal composition. Fexofenadine given 1 hour after alfentanil and followed 1 hour later by a meal containing orange or apple juice had a somewhat lower AUC compared with fexofenadine alone (geometric mean ratio with and without the interacting drug = 0.73, 90% confidence interval [CI] = 0.59-1.04). Fexofenadine given 1 hour after alfentanil and followed 2 hours later by a meal not containing citrus or apple products had an AUC that was unchanged compared with fexofenadine alone (ratio = 0.91, 90% CI = 0.70-1.35). These results show that alfentanil disposition was not affected by fexofenadine. A dosing regimen was identified in which fexofenadine

  7. Hemodialysis does not alter in vitro hepatic CYP3A4 and CYP2D6 metabolic activity in uremic serum

    Directory of Open Access Journals (Sweden)

    Decker BS

    2013-12-01

    Full Text Available Brian S Decker,1,2 Kalisha D O'Neill,1,2 Mary A Chambers,1,2 James E Slaven,3 Zhangsheng Yu,3 David R Jones,2,4 Sharon M Moe1,21Division of Nephrology, 2Department of Medicine, 3Department of Biostatistics, 4Division of Clinical Pharmacology, Department of Medicine, School of Medicine, Indiana University, Indianapolis, IN, USAAbstract: There is a paucity of studies evaluating the change in liver metabolism in subjects receiving hemodialysis. The purpose of this study was to compare the effect of uremic toxins on hepatic cytochrome P450 (CYP3A4 and CYP2D6 metabolism before and after a 4-hour hemodialysis session. Midazolam and dextromethorphan were incubated with uremic serum collected from subjects before and after the 4-hour hemodialysis session. Analysis and quantification of the 1'-OH-midazolam and 4-OH-midazolam and dextrorphan metabolites were performed by high-pressure liquid chromatography/mass spectrometry. Statistical analysis using the Student's t-test (paired was used to compare the amount of metabolite formed. The mean amount of 1'-OH-midazolam, 4-OH-midazolam, and dextrorphan metabolites formed before and after hemodialysis did not significantly differ. There was no significant difference in CYP3A4 and CYP2D6 metabolic activity in uremic serum before and after hemodialysis.Keywords: hemodialysis, uremia, CYP3A4, CYP2D6, metabolism

  8. Polymorphisms in CYP1A1 and CYP3A5 Genes Contribute to the Variability in Granisetron Clearance and Exposure in Pregnant Women with Nausea and Vomiting.

    Science.gov (United States)

    Bustos, Martha L; Zhao, Yang; Chen, Huijun; Caritis, Steve N; Venkataramanan, Raman

    2016-12-01

    Nausea and vomiting affect up to 90% of pregnant women. Granisetron is a potent and highly selective serotonin receptor antagonist and is an effective antiemetic. Findings from a prior study in pregnant women demonstrated a large interindividual variability in granisetron exposure. Granisetron is primarily metabolized by the cytochrome P450 (CYP) enzymes CYP1A1 and CYP3A and is likely a substrate of the ABCB1 transporter. Single-nucleotide polymorphisms (SNPs) in CYP3A, CYP1A1, and ABCB1 can alter drug metabolism. This study evaluated the influence of polymorphisms in CYP3A4, CYP3A5, CYP1A1, and ABCB1 on the pharmacokinetic properties of granisetron in pregnant women. The study enrolled 16 pregnant women (gestational age of 12-19 wks). All patients had nausea and vomiting and were treated with granisetron 1 mg. Granisetron plasma concentrations were determined using liquid chromatography tandem-mass spectrometry. The patients' genotype was determined using TaqMan SNP Genotyping Assays. The Hardy-Weinberg equilibrium was assessed by comparing observed and expected genotype frequencies, using the exact test. Intravenous granisetron clearance was used as the dependent variable for analysis of associations. Of 16 patients, 25% were homozygous for the allele variant CYP3A5*3 and had a significantly lower granisetron clearance and increased area under the plasma concentration-versus-time curve (AUC) compared with nonhomozygous patients. Approximately one-third of patients (n=5) were carriers for the allele variant CYP1A1*2A and had a significantly higher granisetron clearance and decreased AUC. We did not find significant differences in the AUC or clearance for any SNPs in CYP3A4 and ABCB1 genes. Polymorphisms in CYP3A5 and CYP1A1 account for some of the variability in systemic clearance and exposure of granisetron in pregnant women. © 2016 Pharmacotherapy Publications, Inc.

  9. A search for new CYP3A4 variants as determinants of tacrolimus dose requirements in renal-transplanted patients.

    Science.gov (United States)

    Tavira, Beatriz; Coto, Eliecer; Diaz-Corte, Carmen; Alvarez, Victoria; López-Larrea, Carlos; Ortega, Francisco

    2013-08-01

    The CYP3A5*3 and CYP3A4*1B alleles have been related with tacrolimus (Tac) dose requirements. The rare CYP3A4*22 variant has also been associated with a significantly lower Tac dose. We genotyped the three single-nucleotide polymorphisms in 206 kidney-transplanted patients who received Tac as the primary immunosuppressor. CYP3A5*1 and CYP3A4*1B allele carriers received a significantly higher Tac dose (PCYP3A4*22 genotypes, either nominally or according to the CYP3A5 genotype (expressers vs. nonexpressers). Sequencing of CYP3A4 coding exons in a total of 15 patients revealed only one nonreported missense change (p.P227>T) in one patient. We concluded that CYP3A5*3 and CYP3A4*1B were the main determinants of the Tac dose-adjusted blood concentration in our cohort of renal-transplanted patients.

  10. Evaluation of 89 compounds for identification of substrates for cynomolgus monkey CYP2C76, a new bupropion/nifedipine oxidase.

    Science.gov (United States)

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

    2015-01-01

    Cynomolgus monkeys are widely used in preclinical studies during drug development because of their evolutionary closeness to humans, including their cytochrome P450s (P450s). Most cynomolgus monkey P450s are almost identical (≥90%) to human P450s; however, CYP2C76 has low sequence identity (approximately 80%) to any human CYP2Cs. Although CYP2C76 has no ortholog in humans and is partly responsible for species differences in drug metabolism between cynomolgus monkeys and humans, a broad evaluation of potential substrates for CYP2C76 has not yet been conducted. In this study, a screening of 89 marketed compounds, including human CYP2C and non-CYP2C substrates or inhibitors, was conducted to find potential CYP2C76 substrates. Among the compounds screened, 19 chemicals were identified as substrates for CYP2C76, including substrates for human CYP1A2 (7-ethoxyresorufin), CYP2B6 (bupropion), CYP2D6 (dextromethorphan), and CYP3A4/5 (dextromethorphan and nifedipine), and inhibitors for CYP2B6 (sertraline, clopidogrel, and ticlopidine), CYP2C8 (quercetin), CYP2C19 (ticlopidine and nootkatone), and CYP3A4/5 (troleandomycin). CYP2C76 metabolized a wide variety of the compounds with diverse structures. Among them, bupropion and nifedipine showed high selectivity to CYP2C76. As for nifedipine, CYP2C76 formed methylhydroxylated nifedipine, which was not produced by monkey CYP2C9, CYP2C19, or CYP3A4, as identified by mass spectrometry and estimated by a molecular docking simulation. This unique oxidative metabolite formation of nifedipine could be one of the selective marker reactions of CYP2C76 among the major CYP2Cs and CYP3As tested. These results suggest that monkey CYP2C76 contributes to bupropion hydroxylation and formation of different nifedipine oxidative metabolites as a result of its relatively large substrate cavity. Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.

  11. Expression of CYP2E1 in human nasopharynx and its metabolic effect in vitro.

    Science.gov (United States)

    Hou, De-Fu; Wang, Shui-Liang; He, Zhi-Min; Yang, Fang; Chen, Zhu-Chu

    2007-04-01

    It was evident that nitrosamines can act directly on target tissue and result in carcinogenesis. As has been shown, the carcinogenic activity of nitrosamines relied on its bioactivation by Cytochrome P450 2E1 (CYP2E1). In this study, we investigated the expression of CYP2E1 in Nasopharyngeal carcinoma (NPC) cells, embryonic nasopharyngeal epithelial tissue (ENET) specimens, and NPC biopsies by RT-PCR analysis. CYP2E1 was expressed in all NPC cell lines (6/6, including 7429) and ENET (6/6), and 80% of NPC biopsie (8/10). The fact that Human nasopharynx expresses CYP2E1 suggests that CYP2E1 may play an important role in the course of NPC by indirect carcinogens nitrosamines. To further evaluate the function of CYP2E1, the CYP2E1 was stably expressed in the cell line NIH 3T3/rtTA under a tetracycline-controlled transactivator. The expression of CYP2E1 was tightly regulated in a dose-dependent manner by Doxycycline (Dox) When the catalytic activity of CYP2E1 was assayed, the result showed that the generation of 6-hydroxychlorzoxazone (6-OH-CZ) from chlorzoxazone (CZ) was dose- and time-dependent on Dox addition to the medium. In the presence of 1 microg/ml Dox, the CZ 6-hydroxylase activity of the cell line was found to be 0.986 +/- 0.034 nmol/10(6) cells/h. The metabolic activation of Tet/3T3/2E1-6 cells was also assayed by N,N'-dinitrosopiperazine (DNP) cytotoxicity, and the viability of Tet/3T3/2E1-6 cells treated with Dox was lower than that of untreated cells with a significant difference between them in 80 and 160 microg/ml DNP (P ( 0.05, t test. This cell line will be useful not only to assess the metabolic characteristics of CYP2E1, but also will be useful to investigate the role of CYP2E1 in metabolic activation of carcinogenic nitrosamines in vitro.

  12. Midazolam microdose to determine systemic and pre-systemic metabolic CYP3A activity in humans.

    Science.gov (United States)

    Hohmann, Nicolas; Kocheise, Franziska; Carls, Alexandra; Burhenne, Jürgen; Haefeli, Walter E; Mikus, Gerd

    2015-02-01

    We aimed to establish a method to assess systemic and pre-systemic cytochrome P450 (CYP) 3A activity using ineffective microgram doses of midazolam. In an open, one sequence, crossover study, 16 healthy participants received intravenous and oral midazolam at microgram (0.001 mg intravenous and 0.003 mg oral) and regular milligram (1 mg intravenous and 3 mg oral) doses to assess the linearity of plasma and urine pharmacokinetics. Dose-normalized AUC and Cmax were 37.1 ng ml(-1 ) h [95% CI 35.5, 40.6] and 39.1 ng ml(-1) [95% CI 30.4, 50.2] for the microdose and 39.0 ng ml(-1 ) h [95% CI 36.1, 42.1] and 37.1 ng ml(-1) [95% CI 26.9, 51.3] for the milligram dose. CLmet was 253 ml min(-1) [95% CI 201, 318] vs. 278 ml min(-1) [95% CI 248, 311] for intravenous doses and 1880 ml min(-1) [95% CI 1590, 2230] vs. 2050 ml min(-1) [95% CI 1720, 2450] for oral doses. Oral bioavailability of a midazolam microdose was 23.4% [95% CI 20.0, 27.3] vs. 20.9% [95% CI 17.1, 25.5] after the regular dose. Hepatic and gut extraction ratios for microgram doses were 0.44 [95% CI 0.39, 0.49] and 0.53 [95% CI 0.45, 0.63] and compared well with those for milligram doses (0.43 [95% CI 0.37, 0.49] and 0.61 [95% CI 0.53, 0.70]). The pharmacokinetics of an intravenous midazolam microdose is linear to the applied regular doses and can be used to assess safely systemic CYP3A activity and, in combination with oral microdoses, pre-systemic CYP3A activity. © 2014 The British Pharmacological Society.

  13. High allele frequency of CYP2C9*3 (rs1057910) in a Negrito's subtribe population in Malaysia; Aboriginal people of Jahai.

    Science.gov (United States)

    Rosdi, Rasmaizatul Akma; Mohd Yusoff, Narazah; Ismail, Rusli; Soo Choon, Tan; Saleem, Mohamed; Musa, Nurfadhlina; Yusoff, Surini

    2016-09-01

    CYP2C9 gene polymorphisms modulate inter-individual variations in the human body's responses to various endogenous and exogenous drug substrates. To date, little is known about the CYP2C9 gene polymorphisms among the aboriginal populations of the world, including those in Malaysia. To characterise and compare the CYP2C9 polymorphisms (CYP2C9*2, CYP2C9*3, CYP2C9*4 and CYP2C9*5) between one of Malaysia's aboriginal populations, Jahai, with the national major ethnic, Malay. To also compare the allele frequencies from these two populations with available data of other aboriginal populations around the world. The extracted DNA of 155 Jahais and 183 Malays was genotyped for CYP2C9 polymorphisms using a nested multiplex allele-specific polymerase chain reaction technique. The results were confirmed by DNA direct sequencing. Genotyping results revealed that CYP2C9*2, CYP2C9*4 and CYP2C9*5 were absent in Jahais, while only the latter two were absent in Malays. The CYP2C9*3 allelic frequency in Jahais was 36.2%, making them the most frequent carriers of the allele thus far reported in any ethnic group from Southeast Asia. The high frequency of CYP2C9*3 and the absence of CYP2C9*2 in Jahais suggest that genetic drift may be occurring in this ethnic group. This is the first study to determine the CYP2C9 polymorphisms in an aboriginal population in Malaysia.

  14. Pregnane X receptor-dependent induction of the CYP3A4 gene by o,p'-1,1,1,-trichloro-2,2-bis (p-chlorophenyl)ethane.

    Science.gov (United States)

    Medina-Díaz, Irma M; Arteaga-Illán, Georgina; de León, Mario Bermudez; Cisneros, Bulmaro; Sierra-Santoyo, Adolfo; Vega, Libia; Gonzalez, Frank J; Elizondo, Guillermo

    2007-01-01

    CYP3A4, the predominant cytochrome P450 (P450) expressed in human liver and intestine, contributes to the metabolism of approximately half the drugs in clinical use today. CYP3A4 catalyzes the 6beta-hydroxylation of a number of steroid hormones and is involved in the bioactivation of environmental procarcinogens. The expression of CYP3A4 is affected by several stimuli, including environmental factors such as insecticides and pesticides. The o,p'-1,1,1,-trichloro-2,2-bis (p-chlorophenyl)ethane (DDT) isomer of DDT comprises approximately 20% of technical grade DDT, which is an organochloride pesticide. We have recently shown that o,p'-DDT exposure increases CYP3A4 mRNA levels in HepG2 cells. To determine the mechanism by which o,p'-DDT induces CYP3A4 expression, transactivation and electrophoretic mobility shift assays were carried out, revealing that o,p'-DDT activates the CYP3A4 gene promoter through the pregnane X receptor (PXR). CYP3A4 gene promoter activation resulted in both an increase in CYP3A4 mRNA levels and an increase in the total CYP3A4 activity in HepG2 cells. We also observed induction of CYP3A4 and mouse Cyp3a11 mRNA in the intestine of CYP3A4-transgenic mice after exposure to 1 mg/kg o,p'-DDT. At higher doses, a decrease of CYP3A4 inducibility was observed together with an increase in levels of interleukin 6 mRNA, a proinflammatory cytokine that strongly represses CYP3A4 transcription. The present study indicates that regulation of other genes under PXR control may be altered by o,p'-DDT exposure.

  15. Metabolic activity and mRNA levels of human cardiac CYP450s involved in drug metabolism.

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    Veronique Michaud

    2010-12-01

    Full Text Available Tissue-specific expression of CYP450s can regulate the intracellular concentration of drugs and explain inter-subject variability in drug action. The overall objective of our study was to determine in a large cohort of samples, mRNA levels and CYP450 activity expressed in the human heart.CYP450 mRNA levels were determined by RTPCR in left ventricular samples (n = 68 of explanted hearts from patients with end-stage heart failure. Samples were obtained from ischemic and non-ischemic hearts. In some instances (n = 7, samples were available from both the left and right ventricles. A technique for the preparation of microsomes from human heart tissue was developed and CYP450-dependent activity was determined using verapamil enantiomers as probe-drug substrates.Our results show that CYP2J2 mRNA was the most abundant isoform in all human heart left ventricular samples tested. Other CYP450 mRNAs of importance were CYP4A11, CYP2E1, CYP1A1 and CYP2C8 mRNAs while CYP2B6 and CYP2C9 mRNAs were present at low levels in only some of the hearts analyzed. CYP450 mRNAs did not differ between ischemic and non-ischemic hearts and appeared to be present at similar levels in the left and right ventricles. Incubation of verapamil with heart microsomes led to the formation of nine CYP450-dependent metabolites: a major finding was the observation that stereoselectivity was reversed compared to human liver microsomes, in which the R-enantiomer is metabolized to a greater extent.This study determined cardiac mRNA levels of various CYP450 isozymes involved in drug metabolism and demonstrated the prevalent expression of CYP2J2 mRNA. It revealed that cardiomyocytes can efficiently metabolize drugs and that cardiac CYP450s are highly relevant with regard to clearance of drugs in the heart. Our results support the claim that drug metabolism in the vicinity of a drug effector site can modulate drug effects.

  16. CYP3A isoforms in Ewing's sarcoma tumours: an immunohistochemical study with clinical correlation.

    Science.gov (United States)

    Zia, Hamid; Murray, Graeme I; Vyhlidal, Carrie A; Leeder, J Steven; Anwar, Ahmed E; Bui, Marilyn M; Ahmed, Atif A

    2015-04-01

    Ewing's sarcoma is an aggressive malignancy of bone and soft tissue with high incidence of metastasis and resistance to chemotherapy. Cytochrome P450 (CYP) monooxygenases are a family of enzymes that are involved in the metabolism of exogenous and endogenous compounds, including anti-cancer drugs, and have been implicated in the aggressive behaviour of various malignancies. Tumour samples and clinical information including age, sex, tumour site, tumour size, clinical stage and survival were collected from 36 adult and paediatric patients with Ewing's sarcoma family tumours. Tissue microarrays slides were processed for immunohistochemical labelling for CYP3A4, CYP3A5 and CYP3A7 using liver sections as positive control. The intensity of staining was scored as negative, low or high expression and was analysed statistically for any association with patients' clinical information. Four cases were later excluded due to inadequate viable tissue. CYP3A4 staining was present in 26 (81%) cases with high expression noted in 13 (40%) of 32 cases. High expression was significantly associated with distant metastases (P Ewing's sarcoma tumours and high CYP3A4 expression may be associated with metastasis. Additional studies are needed to further investigate the role of CYP3A4 in the prognosis of these tumours. © 2015 The Authors. International Journal of Experimental Pathology © 2015 International Journal of Experimental Pathology.

  17. Effect of Methamphetamine on Spectral Binding, Ligand Docking and Metabolism of Anti-HIV Drugs with CYP3A4

    Science.gov (United States)

    Ande, Anusha; Wang, Lei; Vaidya, Naveen K.; Li, Weihua; Kumar, Santosh; Kumar, Anil

    2016-01-01

    Cytochrome P450 3A4 (CYP3A4) is the major drug metabolic enzyme, and is involved in the metabolism of antiretroviral drugs, especially protease inhibitors (PIs). This study was undertaken to examine the effect of methamphetamine on the binding and metabolism of PIs with CYP3A4. We showed that methamphetamine exhibits a type I spectral change upon binding to CYP3A4 with δAmax and KD of 0.016±0.001 and 204±18 μM, respectively. Methamphetamine-CYP3A4 docking showed that methamphetamine binds to the heme of CYP3A4 in two modes, both leading to N-demethylation. We then studied the effect of methamphetamine binding on PIs with CYP3A4. Our results showed that methamphetamine alters spectral binding of nelfinavir but not the other type I PIs (lopinavir, atazanavir, tipranavir). The change in spectral binding for nelfinavir was observed at both δAmax (0.004±0.0003 vs. 0.0068±0.0001) and KD (1.42±0.36 vs.2.93±0.08 μM) levels. We further tested effect of methamphetamine on binding of 2 type II PIs; ritonavir and indinavir. Our results showed that methamphetamine alters the ritonavir binding to CYP3A4 by decreasing both the δAmax (0.0038±0.0003 vs. 0.0055±0.0003) and KD (0.043±0.0001 vs. 0.065±0.001 nM), while indinavir showed only reduced KD in presence of methamphetamine (0.086±0.01 vs. 0.174±0.03 nM). Furthermore, LC-MS/MS studies in high CYP3A4 human liver microsomes showed a decrease in the formation of hydroxy ritonavir in the presence of methamphetamine. Finally, CYP3A4 docking with lopinavir and ritonavir in the absence and presence of methamphetamine showed that methamphetamine alters the docking of ritonavir, which is consistent with the results obtained from spectral binding and metabolism studies. Overall, our results demonstrated differential effects of methamphetamine on the binding and metabolism of PIs with CYP3A4. These findings have clinical implication in terms of drug dose adjustment of antiretroviral medication, especially with ritonavir

  18. Metabolism of six CYP probe substrates in fetal hepatocytes

    Directory of Open Access Journals (Sweden)

    Abdul Naveed Shaik

    2016-06-01

    Full Text Available Cytochrome P-450 (CYP are the most common drug metabolizing enzymes and are abundantly expressed in liver apart from kidney, lungs, intestine, brain etc. Their expression levels change with physiological conditions and disease states. The expression of these CYPs is less in human foetus and neonates compared to adults, which results in lower clearance of xenobiotics in infants and neonates compared to adults. Hepatocytes are the cells which are largely used to study these CYPs. We have isolated hepatocytes from aborted foetus to study the metabolism of six probe substrates: phenacetin, diclofenac, S-mephenytoin, dextromethorphan, nifedipine and testosterone. The results obtained show the expression of various CYPs (CYP1A2, CYP2C19, CYP2C9, CYP2D6, and CYP3A4 in human foetus and their involvement in metabolism of CYP probe substrates.

  19. A locus for isolated cataract on human Xp.

    Science.gov (United States)

    Francis, P J; Berry, V; Hardcastle, A J; Maher, E R; Moore, A T; Bhattacharya, S S

    2002-02-01

    To genetically map the gene causing isolated X linked cataract in a large European pedigree. Using the patient registers at Birmingham Women's Hospital, UK, we identified and examined 23 members of a four generation family with nuclear cataract. Four of six affected males also had complex congenital heart disease. Pedigree data were collated and leucocyte DNA extracted from venous blood. Linkage analysis by PCR based microsatellite marker genotyping was used to identify the disease locus and mutations within candidate genes screened by direct sequencing. The disease locus was genetically refined to chromosome Xp22, within a 3 cM linkage interval flanked by markers DXS9902 and DXS999 (Zmax=3.64 at theta=0 for marker DXS8036). This is the first report of a locus for isolated inherited cataract on the X chromosome. The disease interval lies within the Nance-Horan locus suggesting allelic heterogeneity. The apparent association with congenital cardiac anomalies suggests a possible new oculocardiac syndrome.

  20. Influence of Donor and Recipient CYP3A4, CYP3A5, and ABCB1 Genotypes on Clinical Outcomes and Nephrotoxicity in Liver Transplant Recipients.

    Science.gov (United States)

    Debette-Gratien, Marilyne; Woillard, Jean-Baptiste; Picard, Nicolas; Sebagh, Mylène; Loustaud-Ratti, Véronique; Sautereau, Denis; Samuel, Didier; Marquet, Pierre

    2016-10-01

    This study investigated the influence of the CYP3A4*22, CYP3A5*3, and ABCB1 exons 12, 21, and 26 polymorphisms in donors and recipients on clinical outcomes and renal function in 170 liver transplant patients on cyclosporin A (CsA) or tacrolimus (Tac). Allelic discrimination assays were used for genotyping. Multivariate time-dependent Cox proportional hazard models, multiple linear regression using the generalized estimating equation and linear mixed-effect models were used for statistical analysis. Expression of CYP3A5 by either or both the donor and the recipient was significantly associated with lower Tac, but not CsA, dose-normalized trough levels. In the whole population, graft loss was only significantly associated with longer exposure to high calcineurin inhibitor (CNI) concentrations (hazard ratio, 6.93; 95% confidence interval, 2.13-22.55), P = 0.00129), whereas in the Tac subgroup, the risk of graft loss was significantly higher in recipient CYP3A5*1 expressers (hazard ratio, 3.39; 95% confidence interval, 1.52-7.58; P = 0.0028). Renal function was significantly associated with: (1) baseline modification of diet in renal disease (β = 0.51 ± 0.05; P < 0.0001); (2) duration of patient follow-up (per visit, β = -0.98 ± 0.22; P < 0.0001); and (3) CNI exposure (per quantile increase, β = -2.42 ± 0.59; P < 0.0001). No genetic factor was associated with patient survival, acute rejection, liver function test results, recurrence of viral or other initial liver disease, or renal function. This study confirms the effect of CYP3A5*3 on tacrolimus dose requirement in liver transplantation and shows unexpected associations between the type of, and exposure to, CNI and either chronic rejection or graft loss. None of the genetic polymorphisms studied had a noticeable impact on renal function degradation at 10 years.

  1. AhR Activation Underlies the CYP1A Autoinduction by A-998679 in Rats

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    Michael J. Liguori

    2012-10-01

    Full Text Available Xenobiotic-mediated induction of cytochrome P450 (CYP drug metabolizing enzymes (DMEs is frequently encountered in drug discovery and can influence disposition, pharmacokinetic, and toxicity profiles. The CYP1A subfamily of DMEs plays a central role in the biotransformation of several drugs and environmental chemicals. Autoinduction of drugs through CYP3A enzymes is a common mechanism for their enhanced clearance. However, autoinduction via CYP1A is encountered less frequently. In this report, an experimental compound, A-998679 (3-(5-pyridin-3-yl-1,2,4-oxadiazol-3-yl benzonitrile, was shown to enhance its own clearance via induction of CYP1A1 and CYP1A2. Rats were dosed for 5 days with 30, 100, and 200 mg/kg/day A-998679. During the dosing period, the compound’s plasma AUC decreased at 30 mg/kg (95% and 100 mg/kg (80%. Gene expression analysis and immunohistochemistry of the livers showed a large increase in the mRNA and protein levels of CYP1A, which was involved in the biotransformation of A-998679. Induction of CYP1A was confirmed in primary rat, human, and dog hepatocytes. The compound also weakly inhibited CYP1A2 in human liver microsomes. A-998679 activated the aryl hydrocarbon receptor (AhR in a luciferase gene reporter assay in HepG2 cells, upregulated expression of genes associated with AhR activation in rat liver, and enhanced nuclear migration of AhR in HepG2 cells. Collectively these results demonstrate that A-998679 is an AhR activator that induces CYP1A1 and CYP1A2 expression, resulting in an autoinduction phenomenon. The unique properties of A-998679, along with its novel structure distinct from classical polycyclic aromatic hydrocarbons, may warrant its further evaluation as a tool compound for use in studies involving AhR biology and CYP1A related mechanisms of drug metabolism and toxicity.

  2. Three new shRNA expression vectors targeting the CYP3A4 coding sequence to inhibit its expression

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    Siyun Xu

    2014-10-01

    Full Text Available RNA interference (RNAi is useful for selective gene silencing. Cytochrome P450 3A4 (CYP3A4, which metabolizes approximately 50% of drugs in clinical use, plays an important role in drug metabolism. In this study, we aimed to develop a short hairpin RNA (shRNA to modulate CYP3A4 expression. Three new shRNAs (S1, S2 and S3 were designed to target the coding sequence (CDS of CYP3A4, cloned into a shRNA expression vector, and tested in different cells. The mixture of three shRNAs produced optimal reduction (55% in CYP3A4 CDS-luciferase activity in both CHL and HEK293 cells. Endogenous CYP3A4 expression in HepG2 cells was decreased about 50% at both mRNA and protein level after transfection of the mixture of three shRNAs. In contrast, CYP3A5 gene expression was not altered by the shRNAs, supporting the selectivity of CYP3A4 shRNAs. In addition, HepG2 cells transfected with CYP3A4 shRNAs were less sensitive to Ginkgolic acids, whose toxic metabolites are produced by CYP3A4. These results demonstrate that vector-based shRNAs could modulate CYP3A4 expression in cells through their actions on CYP3A4 CDS, and CYP3A4 shRNAs may be utilized to define the role of CYP3A4 in drug metabolism and toxicity.

  3. Inhibition of protein kinase CK2 reduces CYP24A1 expression and enhances 1,25-dihydroxyvitamin D3 anti-tumor activity in human prostate cancer cells

    Science.gov (United States)

    Luo, Wei; Yu, Wei-Dong; Ma, Yingyu; Chernov, Mikhail; Trump, Donald L.; Johnson, Candace S.

    2013-01-01

    Vitamin D has broad range of physiological functions and anti-tumor effects. 24-hydroxylase, encoded by the CYP24A1 gene, is the key enzyme for degrading many forms of vitamin D including the most active form, 1,25D3. Inhibition of CYP24A1 enhances 1,25D3 anti-tumor activity. In order to isolate regulators of CYP24A1 expression in prostate cancer cells, we established a stable prostate cancer cell line PC3 with CYP24A1 promoter driving luciferase expression to screen a small molecular library for compounds that inhibit CYP24A1 promoter activity. From this screening, we identified, 4,5,6,7-tetrabromobenzimidazole (TBBz), a protein kinase CK2 selective inhibitor as a disruptor of CYP24A1 promoter activity. We show that TBBz inhibits CYP24A1 promoter activity induced by 1,25D3 in prostate cancer cells. In addition, TBBz downregulates endogenous CYP24A1 mRNA level in TBBz treated PC3 cells. Furthermore, siRNA-mediated CK2 knockdown reduces 1,25D3 induced CYP24A1 mRNA expression in PC3 cells. These results suggest that CK2 contributes to 1,25D3 mediated target gene expression. Lastly, inhibition of CK2 by TBBz or CK2 siRNA significantly enhanced 1,25D3 mediated anti-proliferative effect in vitro and in vivo in a xenograft model. In summary, our findings reveal that protein kinase CK2 is involved in the regulation of CYP24A1 expression by 1,25D3 and CK2 inhibitor enhances 1,25D3 mediated anti-tumor effect. PMID:23358686

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

    Science.gov (United States)

    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.

  5. 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

  6. Discovery of a novel dual fungal CYP51/human 5-lipoxygenase inhibitor: implications for anti-fungal therapy.

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    Eric K Hoobler

    Full Text Available We report the discovery of a novel dual inhibitor targeting fungal sterol 14α-demethylase (CYP51 or Erg11 and human 5-lipoxygenase (5-LOX with improved potency against 5-LOX due to its reduction of the iron center by its phenylenediamine core. A series of potent 5-LOX inhibitors containing a phenylenediamine core, were synthesized that exhibit nanomolar potency and >30-fold selectivity against the LOX paralogs, platelet-type 12-human lipoxygenase, reticulocyte 15-human lipoxygenase type-1, and epithelial 15-human lipoxygenase type-2, and >100-fold selectivity against ovine cyclooxygenase-1 and human cyclooxygnease-2. The phenylenediamine core was then translated into the structure of ketoconazole, a highly effective anti-fungal medication for seborrheic dermatitis, to generate a novel compound, ketaminazole. Ketaminazole was found to be a potent dual inhibitor against human 5-LOX (IC50 = 700 nM and CYP51 (IC50 = 43 nM in vitro. It was tested in whole blood and found to down-regulate LTB4 synthesis, displaying 45% inhibition at 10 µM. In addition, ketaminazole selectively inhibited yeast CYP51 relative to human CYP51 by 17-fold, which is greater selectivity than that of ketoconazole and could confer a therapeutic advantage. This novel dual anti-fungal/anti-inflammatory inhibitor could potentially have therapeutic uses against fungal infections that have an anti-inflammatory component.

  7. Genotyping and phenotyping of CYP2D6 and CYP3A isoenzymes in patients with alcohol use disorder: correlation with haloperidol plasma concentration.

    Science.gov (United States)

    Sychev, Dmitry A; Zastrozhin, Mikhail S; Miroshnichenko, Igor I; Baymeeva, Natalia V; Smirnov, Valery V; Grishina, Elena A; Ryzhikova, Kristina A; Mirzaev, Karin B; Markov, Dmitry D; Skryabin, Valentin Y; Snalina, Nataliya E; Nosikova, Polina G; Savchenko, Ludmila M; Bryun, Evgeny A

    2017-09-26

    Haloperidol is used for the treatment of alcohol use disorders in patients with signs of alcohol-related psychosis. Haloperidol therapy poses a high risk of adverse drug reactions (ADR). Contradictory data, which include the effects of genetic polymorphisms in genes encoding the elements of haloperidol biotransformation system on haloperidol metabolism rate and plasma drug concentration ratio, are described in patients with different genotypes. The primary objective of this study was to investigate the effects of CYP2D6 and CYP3A5 genetic polymorphisms on haloperidol equilibrium concentration in patients with alcohol use disorder. The study included 69 male patients with alcohol use disorder. Genotyping was performed using the allele-specific real-time PCR. CYP2D6 and CYP3A were phenotyped with HPLC-MS using the concentration of endogenous substrate of the enzyme and its urinary metabolites [6-hydroxy-1,2,3,4-tetrahydro-β-carboline(6-HO-THBC) to pinoline ratio for CYP2D6 and 6-β-hydroxycortisol to cortisol ratio for CYP3A]. The equilibrium plasma concentration was determined using LC-MS-MS. Results indicated that both C/D indexes and equilibrium concentration levels depend on CYP2D6 genetic polymorphism, but only in patients receiving haloperidol intramuscular injections [0.26 (0.09; 0.48) vs. 0.54 (0.44; 0.74), p=0.037]. The study demonstrates that CYP2D6 genetic polymorphism (1846G>A) can affect haloperidol concentration levels in patients with alcohol use disorder.

  8. Improved Predictions of Drug-Drug Interactions Mediated by Time-Dependent Inhibition of CYP3A.

    Science.gov (United States)

    Yadav, Jaydeep; Korzekwa, Ken; Nagar, Swati

    2018-05-07

    Time-dependent inactivation (TDI) of cytochrome P450s (CYPs) is a leading cause of clinical drug-drug interactions (DDIs). Current methods tend to overpredict DDIs. In this study, a numerical approach was used to model complex CYP3A TDI in human-liver microsomes. The inhibitors evaluated included troleandomycin (TAO), erythromycin (ERY), verapamil (VER), and diltiazem (DTZ) along with the primary metabolites N-demethyl erythromycin (NDE), norverapamil (NV), and N-desmethyl diltiazem (NDD). The complexities incorporated into the models included multiple-binding kinetics, quasi-irreversible inactivation, sequential metabolism, inhibitor depletion, and membrane partitioning. The resulting inactivation parameters were incorporated into static in vitro-in vivo correlation (IVIVC) models to predict clinical DDIs. For 77 clinically observed DDIs, with a hepatic-CYP3A-synthesis-rate constant of 0.000 146 min -1 , the average fold difference between the observed and predicted DDIs was 3.17 for the standard replot method and 1.45 for the numerical method. Similar results were obtained using a synthesis-rate constant of 0.000 32 min -1 . These results suggest that numerical methods can successfully model complex in vitro TDI kinetics and that the resulting DDI predictions are more accurate than those obtained with the standard replot approach.

  9. 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.

  10. The genes of all seven CYP3A isoenzymes identified in the equine genome are expressed in the airways of horses.

    Science.gov (United States)

    Tydén, E; Löfgren, M; Hakhverdyan, M; Tjälve, H; Larsson, P

    2013-08-01

    In the present study, we examined the gene expression of cytochrome P450 3A (CYP3A) isoenzymes in the tracheal and bronchial mucosa and in the lung of equines using TaqMan probes. The results show that all seven CYP3A isoforms identified in the equine genome, that is, CYP3A89, CYP3A93, CYP3A94, CYP3A95, CYP3A96, CYP3A97 and CYP3A129, are expressed in the airways of the investigated horses. Though in previous studies, CYP3A129 was found to be absent in equine intestinal mucosa and liver, this CYP3A isoform is expressed in the airways of horses. The gene expression of the CYP3A isoenzymes varied considerably between the individual horses studied. However, in most of the horses CYP3A89, CYP3A93, CYP3A96, CYP3A97 and CYP3A129 were expressed to a high extent, while CYP3A94 and CYP3A95 were expressed to a low extent in the different parts of the airways. The CYP3A isoenzymes present in the airways may play a role in the metabolic degradation of inhaled xenobiotics. In some instances, the metabolism may, however, result in bioactivation of the xenobiotics and subsequent tissue injury. © 2012 John Wiley & Sons Ltd.

  11. Investigation of the association between the GLC3A locus and normal tension glaucoma in Japanese patients by microsatellite analysis

    Directory of Open Access Journals (Sweden)

    M Kamio

    2009-01-01

    Full Text Available M Kamio1*, A Meguro1*, M Ota2, N Nomura1, K Kashiwagi3, F Mabuchi3, H Iijima3, K Kawase4, T Yamamoto4, M Nakamura5, A Negi5, T Sagara6, T Nishida6, M Inatani7, H Tanihara7, M Aihara8, M Araie8, T Fukuchi9, H Abe9, T Higashide10, K Sugiyama10, T Kanamoto11, Y Kiuchi11, A Iwase12, S Ohno13, H Inoko14, N Mizuki11Department of Ophthalmology, Yokohama City University School of Medicine, Yokohama, Kanagawa, Japan; 2Department of Legal Medicine, Shinshu University School of Medicine, Matsumoto, Nagano, Japan; 3Department of Ophthalmology, University of Yamanashi, Faculty of Medicine, Yamanashi, Japan; 4Department of Ophthalmology, Gifu University Graduate School of Medicine, Gifu, Japan; 5Department of Surgery, Division of Ophthalmology, Kobe University Graduate School of Medicine, Kobe, Hyogo, Japan; 6Department of Ophthalmology, Yamaguchi University Graduate School of Medicine, Ube, Yamaguchi, Japan; 7Department of Ophthalmology and Visual Science, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan; 8Department of Ophthalmology, University of Tokyo School of Medicine, Tokyo, Japan; 9Division of Ophthalmology and Visual Science, Graduated School of Medical and Dental Sciences, Niigata University, Niigata, Japan; 10Department of Ophthalmology and Visual Science, Kanazawa University Graduate School of Medical Science, Kanazawa, Ishikawa, Japan; 11Department of Ophthalmology and Visual Science, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan; 12Department of Ophthalmology, Tajimi Municipal Hospital, Tajimi, Gifu, Japan; 13Department of Ophthalmology, Hokkaido University School of Medicine, Sapporo, Japan; 14Department of Genetic Information, Division of Molecular Life Science, Tokai University School of Medicine, Isehara, Kanagawa, Japan; *These authors contributed equally to this reportPurpose: To investigate whether the GLC3A locus harboring the CYP1B1 gene is associated with normal tension glaucoma

  12. Comparative study of polymorphism frequencies of the CYP2D6, CYP3A5, CYP2C8 and IL-10 genes in Mexican and Spanish women with breast cancer.

    Science.gov (United States)

    Alcazar-González, Gregorio Antonio; Calderón-Garcidueñas, Ana Laura; Garza-Rodríguez, María Lourdes; Rubio-Hernández, Gabriela; Escorza-Treviño, Sergio; Olano-Martin, Estibaliz; Cerda-Flores, Ricardo Martín; Castruita-Avila, Ana Lilia; González-Guerrero, Juan Francisco; le Brun, Stéphane; Simon-Buela, Laureano; Barrera-Saldaña, Hugo Alberto

    2013-10-01

    Pharmacogenetic studies in breast cancer (BC) may predict the efficacy of tamoxifen and the toxicity of paclitaxel and capecitabine. We determined the frequency of polymorphisms in the CYP2D6 gene associated with activation of tamoxifen, and those of the genes CYP2C8, CYP3A5 and DPYD associated with toxicity of paclitaxel and capecitabine. We also included a IL-10 gene polymorphism associated with advanced tumor stage at diagnosis. Genomic DNAs from 241 BC patients from northeast Mexico were genotyped using DNA microarray technology. For tamoxifen processing, CYP2D6 genotyping predicted that 90.8% of patients were normal metabolizers, 4.2% ultrarapid, 2.1% intermediate and 2.9% poor metabolizers. For paclitaxel and the CYP2C8 gene, 75.3% were normal, 23.4% intermediate and 1.3% poor metabolizers. Regarding the DPYD gene, only one patient was a poor metabolizer. For the IL-10 gene, 47.1% were poor metabolizers. These results contribute valuable information towards personalizing BC chemotherapy in Mexican women.

  13. Influence of CYP3A5 genetic variation on everolimus maintenance dosing after cardiac transplantation.

    Science.gov (United States)

    Lesche, Dorothea; Sigurdardottir, Vilborg; Setoud, Raschid; Englberger, Lars; Fiedler, Georg M; Largiadèr, Carlo R; Mohacsi, Paul; Sistonen, Johanna

    2015-12-01

    Everolimus (ERL) has become an alternative to calcineurin inhibitors (CNIs) due to its renal-sparing properties, especially in heart transplant (HTx) recipients with kidney dysfunction. However, ERL dosing is challenging due to its narrow therapeutic window combined with high interindividual pharmacokinetic variability. Our aim was to evaluate the effect of clinical and genetic factors on ERL dosing in a pilot cohort of 37 HTx recipients. Variants in CYP3A5, CYP3A4, CYP2C8, POR, NR1I2, and ABCB1 were genotyped, and clinical data were retrieved from patient charts. While ERL trough concentration (C0 ) was within the targeted range for most patients, over 30-fold variability in the dose-adjusted ERL C0 was observed. Regression analysis revealed a significant effect of the non-functional CYP3A5*3 variant on the dose-adjusted ERL C0 (p = 0.031). ERL dose requirement was 0.02 mg/kg/d higher in patients with CYP3A5*1/*3 genotype compared to patients with CYP3A5*3/*3 to reach the targeted C0 (p = 0.041). ERL therapy substantially improved estimated glomerular filtration rate (28.6 ± 6.6 mL/min/1.73 m(2)) in patients with baseline kidney dysfunction. Everolimus pharmacokinetics in HTx recipients is highly variable. Our preliminary data on patients on a CNI-free therapy regimen suggest that CYP3A5 genetic variation may contribute to this variability. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  14. Controlled indole-3-acetaldoxime production through ethanol-induced expression of CYP79B2

    DEFF Research Database (Denmark)

    Mikkelsen, M.D.; Fuller, V.L.; Hansen, Bjarne Gram

    2009-01-01

    Indole-3-acetaldoxime (IAOx) is a key branching point between primary and secondary metabolism. IAOx serves as an intermediate in the biosynthesis of indole glucosinolates (I-GLSs), camalexin and the plant hormone indole-3-acetic acid (IAA). The cytochrome P450s CYP79B2 and CYP79B3 catalyze......OH)-inducible CYP79B2 construct into double (cyp79b2 cyp79b3) or triple (cyp79b2 cyp79b3 cyp83b1) mutant lines. We show EtOH-dependent induction of camalexin and identify a number of candidate IAA homeostasis- or defense-related genes by clustered microarray analysis. The transgenic mutant lines are thus promising...

  15. Inhibitory potency of 8-methoxypsoralen on cytochrome P450 2A6 (CYP2A6 allelic variants CYP2A6 15, CYP2A6 16, CYP2A6 21 and CYP2A6 22: differential susceptibility due to different sequence locations of the mutations.

    Directory of Open Access Journals (Sweden)

    Kai Hung Tiong

    Full Text Available Human cytochrome P450 2A6 (CYP2A6 is a highly polymorphic isoform of CYP2A subfamily. Our previous kinetic study on four CYP2A6 allelic variants (CYP2A6 15, CYP2A6 16, CYP2A6 21 and CYP2A6 22 have unveiled the functional significance of sequence mutations in these variants on coumarin 7-hydroxylation activity. In the present study, we further explored the ability of a typical CYP2A6 inhibitor, 8-methoxypsoralen (8-MOP, in inhibition of these alleles and we hypothesized that translational mutations in these variants are likely to give impact on 8-MOP inhibitory potency. The CYP2A6 variant and the wild type proteins were subjected to 8-MOP inhibition to yield IC50 values. In general, a similar trend of change in the IC50 and Km values was noted among the four mutants towards coumarin oxidation. With the exception of CYP2A6 16, differences in IC50 values were highly significant which implied compromised interaction of the mutants with 8-MOP. Molecular models of CYP2A6 were subsequently constructed and ligand-docking experiments were performed to rationalize experimental data. Our docking study has shown that mutations have induced enlargement of the active site volume in all mutants with the exception of CYP2A6 16. Furthermore, loss of hydrogen bond between 8-MOP and active site residue Asn297 was evidenced in all mutants. Our data indicate that the structural changes elicited by the sequence mutations could affect 8-MOP binding to yield differential enzymatic activities in the mutant CYP2A6 proteins.

  16. CYP3A4 expression in breast cancer and its association with risk factors in Mexican women.

    Science.gov (United States)

    Floriano-Sanchez, Esau; Rodriguez, Noemi Cardenas; Bandala, Cindy; Coballase-Urrutia, Elvia; Lopez-Cruz, Jaime

    2014-01-01

    In Mexico, breast cancer (BCa) is the leading type of cancer in women. Cytochrome P450 (CYP450) is a superfamily of major oxidative enzymes that metabolize carcinogens and many antineoplastic drugs. In addition, these enzymes have influence on tumor development and tumor response to therapy. In this report, we analyzed the protein expression in patients with BCa and in healthy women. Links with some clinic-pathological characteristic were also assessed. Immunohistochemical analyses were conducted on 48 sets of human breast tumors and normal breast tissues enrolled in Hospital Militar de Especialidades de la Mujer y Neonatologia and Hospital Central Militar, respectively, during the time period from 2010 to 2011. Informed consent was obtained from all participants. Statistical analysis was performed using χ2 or Fisher exact tests to estimate associations and the Mann Whitney U test for comparison of group means. We found a significant CYP3A4 overexpression in BCa stroma and gland regions in comparison with healthy tissue. A significant association between protein expression with smoking, alcoholism and hormonal contraceptives use was also observed. Additionally, we observed estrogen receptor (ER) and progesterone receptor (PR) positive association in BCa. We suggest that CYP3A4 expression promotes BCa development and can be used in the prediction of tumor response to different treatments. One therapeutic approach may thus be to block CYP3A4 function.

  17. The influence of CYP3A5 polymorphisms on haloperidol treatment in patients with alcohol addiction

    Directory of Open Access Journals (Sweden)

    Zastrozhin MS

    2017-12-01

    Full Text Available Mikhail Sergeevich Zastrozhin,1,2 Elena Anatolievna Grishina,1 Kristina Anatolievna Ryzhikova,1 Valery Valerievich Smirnov,3 Ludmila Mikhailovna Savchenko,1 Evgeny Alekseevich Bryun,1,2 Dmitry Alekseevich Sychev1 1Russian Medical Academy of Continuous Professional Education, Ministry of Health of the Russian Federation, 2Moscow Research and Practical Centre on Addictions, Moscow Department of Healthcare, 3National Research Center Institute of Immunology, Federal Medical-Biological Agency, Moscow, Russia Background: Isoenzymes CYP2D6 and CYP3A4, the activity of which varies widely, are involved in metabolism of haloperidol and may influence its profile of efficacy and safety.Objective: The primary aim of this study was to estimate the relationship between CYP3A5 gene polymorphism, activity of the CYP3A isoenzyme, and the risk of development of adverse drug reactions by haloperidol in patients with alcohol abuse.Methods: Sixty-six male alcohol-addicted patients participated in the study. The safety of haloperidol was evaluated by Udvalg for Kliniske Undersogelser Side Effect Rating Scale (UKU and Simpson–Angus Scale for extrapyramidal symptoms (SAS. The activity of CYP3A was evaluated by determining the concentrations of an endogenous substrate of this isoenzyme (cortisol and its urinary metabolite (6-beta-hydroxycortisol, 6-B-HC. Genotyping of CYP3A5*3 was performed by real-time polymerase chain reaction with allele-specific hybridization.Results: The frequency of A-allele occurrence in Russian population was very poor (2.27%. CYP3A5*3 polymorphism had no influence on safety profile indicators of haloperidol (UKU scale: p=0.55, SAS scale: p=0.64. In addition, there was no statistical significant difference between the values of indexes of the metabolic ratio (6-B-HC/cortisol in groups with different genotypes of CYP3A5*3: GG 5.00 (3.36; 6.39 vs AG 5.26 (2.10; 6.78 (p=0.902.Conclusion: The frequency of A-allele occurrence of CYP3A5*3 in Russian

  18. Nonylphenol-mediated CYP induction is PXR-dependent: The use of humanized mice and human hepatocytes suggests that hPXR is less sensitive than mouse PXR to nonylphenol treatment

    International Nuclear Information System (INIS)

    Mota, Linda C.; Barfield, Christina; Hernandez, Juan P.; Baldwin, William S.

    2011-01-01

    Nonylphenol (NP), a by-product of alkylphenol ethoxylates, is a pervasive surfactant that activates the xenosensing nuclear receptor, the pregnane X-receptor (PXR) in transactivation assays in vitro. We are interested in determining if NP activates PXR in vivo, determining if hPXR and mPXR act similarly, and investigating the role of PXR in protecting individuals from NP. Wild-type (WT), PXR-null, and humanized PXR (hPXR) mice were treated with NP at 0, 50 or 75 mg/kg/day for one week, and cytochrome P450 (CYP) induction, liver histopathology, and serum NP concentrations were examined. WT mice treated with NP showed induction of Cyp2b, and male-specific induction of Cyp2c and Cyp3a. CYPs were not induced in PXR-null mice, demonstrating that PXR is necessary for NP-mediated CYP induction. CAR-mediated CYP induction was not observed in the PXR-null mice despite previous data demonstrating that NP is also a CAR activator. hPXR mice only showed moderate Cyp induction, suggesting that hPXR is not as sensitive to NP as mPXR in vivo. NP-mediated Cyp3a induction from three human hepatocyte donors was not significant, confirming that hPXR is not very sensitive to NP-mediated CYP induction. Lastly, mice with PXR (mPXR and hPXR) showed lower NP serum concentrations than PXR-null mice treated with NP suggesting that PXR plays a role in decreasing liver toxicity by basally regulating phase I-III detoxification enzymes that promote the metabolism and elimination of NP. In summary, PXR is required for NP-mediated CYP-induction, mPXR mediates greater CYP induction than hPXR in vivo, and the presence of PXR, especially mPXR, is associated with altered histopathology and increased clearance of NP.

  19. Cyp1a reporter zebrafish reveals target tissues for dioxin

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Kun-Hee [Department of Biomedical Sciences, Chonnam National University Medical School, Gwangju (Korea, Republic of); Department of Microbiology, Chonnam National University Medical School, Gwangju (Korea, Republic of); Park, Hye-Jeong [Department of Biomedical Sciences, Chonnam National University Medical School, Gwangju (Korea, Republic of); Kim, Jin Hee [Department of Biomedical Sciences, Chonnam National University Medical School, Gwangju (Korea, Republic of); Department of Microbiology, Chonnam National University Medical School, Gwangju (Korea, Republic of); Kim, Suhyun [Graduate School of Medicine, Korea University, Ansan (Korea, Republic of); Williams, Darren R. [New Drug Targets Laboratory, School of Life Sciences, Gwangju Institute of Science and Technology, Gwangju (Korea, Republic of); Kim, Myeong-Kyu [Department of Neurology, Chonnam National University Medical School, Gwangju (Korea, Republic of); Jung, Young Do [Department of Biochemistry, Chonnam National University Medical School, Gwangju (Korea, Republic of); Teraoka, Hiroki [School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu (Japan); Park, Hae-Chul [Graduate School of Medicine, Korea University, Ansan (Korea, Republic of); Choy, Hyon E., E-mail: hyonchoy@chonnam.ac.kr [Department of Microbiology, Chonnam National University Medical School, Gwangju (Korea, Republic of); Shin, Boo Ahn, E-mail: bashin@chonnam.ac.kr [Department of Microbiology, Chonnam National University Medical School, Gwangju (Korea, Republic of); Choi, Seok-Yong, E-mail: zebrafish@chonnam.ac.kr [Department of Biomedical Sciences, Chonnam National University Medical School, Gwangju (Korea, Republic of); School of Biological Sciences and Technology, Chonnam National University, Gwangju (Korea, Republic of)

    2013-06-15

    Highlights: •2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is the most toxic anthropogenic substance ever identified. •Transgenic cyp1a reporter zebrafish reveals target tissues for TCDD. •The retinal bipolar cells, otic vesicle, lateral line, pancreas, cloaca and pectoral fin bud are novel targets in zebrafish for TCDD. •Our findings will further understanding of human health risks by TCDD. -- Abstract: 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is the unintentional byproduct of various industrial processes, is classified as human carcinogen and could disrupt reproductive, developmental and endocrine systems. Induction of cyp1a1 is used as an indicator of TCDD exposure. We sought to determine tissues that are vulnerable to TCDD toxicity using a transgenic zebrafish (Danio rerio) model. We inserted a nuclear enhanced green fluorescent protein gene (EGFP) into the start codon of a zebrafish cyp1a gene in a fosmid clone using DNA recombineering. The resulting recombineered fosmid was then used to generate cyp1a reporter zebrafish, embryos of which were exposed to TCDD. Expression pattern of EGFP in the reporter zebrafish mirrored that of endogenous cyp1a mRNA. In addition, exposure of the embryos to TCDD at as low as 10 pM for 72 h, which does not elicit morphological abnormalities of embryos, markedly increased GFP expression. Furthermore, the reporter embryos responded to other AhR ligands as well. Exposure of the embryos to TCDD revealed previously reported (the cardiovascular system, liver, pancreas, kidney, swim bladder and skin) and unreported target tissues (retinal bipolar cells, otic vesicle, lateral line, cloaca and pectoral fin bud) for TCDD. Transgenic cyp1a reporter zebrafish we have developed can further understanding of ecotoxicological relevance and human health risks by TCDD. In addition, they could be used to identify agonists of AhR and antidotes to TCDD toxicity.

  20. Coadministration of gemfibrozil and itraconazole has only a minor effect on the pharmacokinetics of the CYP2C9 and CYP3A4 substrate nateglinide

    Science.gov (United States)

    Niemi, Mikko; Backman, Janne T; Juntti-Patinen, Laura; Neuvonen, Mikko; Neuvonen, Pertti J

    2005-01-01

    Background and aims Gemfibrozil, and particularly its combination with itraconazole, greatly increases the area under the plasma concentration-time curve [AUC(0, ∞)] and response to the cytochrome P450 (CYP) 2C8 and 3A4 substrate repaglinide. In vitro, gemfibrozil is a more potent inhibitor of CYP2C9 than of CYP2C8. Our aim was to investigate the effects of the gemfibrozil-itraconazole combination on the pharmacokinetics and pharmacodynamics of another meglitinide analogue, nateglinide, which is metabolized by CYP2C9 and CYP3A4. Methods In a randomized crossover study with two phases, nine healthy subjects took 600 mg gemfibrozil and 100 mg itraconazole (first dose 200 mg) twice daily or placebo for 3 days. On day 3, they ingested a single 30-mg dose of nateglinide. Plasma nateglinide and blood glucose concentrations were measured for up to 12 h. Results During the gemfibrozil-itraconazole phase, the AUC(0, ∞) and Cmax of nateglinide were 47% (range 23–74%; P gemfibrozil and itraconazole had no effect on the formation of the M7 metabolite of nateglinide but impaired its elimination. The blood glucose response to nateglinide was not significantly changed by coadministration of gemfibrozil and itraconazole. Conclusions The combination of gemfibrozil and itraconazole has only a limited influence on the pharmacokinetics of nateglinide. This is in marked contrast to the substantial effect of this combination on the pharmacokinetics of repaglinide. The findings suggest that in vivo gemfibrozil, probably due to its metabolites, is a much more potent inhibitor of CYP2C8 than of CYP2C9. PMID:16042675

  1. Pharmacogenetic Variation at CYP2D6, CYP2C9, and CYP2C19: Population Genetic and Forensic Aspects

    OpenAIRE

    Sistonen, Johanna

    2008-01-01

    Pharmacogenetics deals with genetically determined variation in drug response. In this context, three phase I drug-metabolizing enzymes, CYP2D6, CYP2C9, and CYP2C19, have a central role, affecting the metabolism of about 20-30% of clinically used drugs. Since genes coding for these enzymes in human populations exhibit high genetic polymorphism, they are of major pharmacogenetic importance. The aims of this study were to develop new genotyping methods for CYP2D6, CYP2C9, and CYP2C19 that would...

  2. Comparison of chlorzoxazone one-sample methods to estimate CYP2E1 activity in humans

    DEFF Research Database (Denmark)

    Kramer, Iza; Dalhoff, Kim; Clemmesen, Jens O

    2003-01-01

    OBJECTIVE: Comparison of a one-sample with a multi-sample method (the metabolic fractional clearance) to estimate CYP2E1 activity in humans. METHODS: Healthy, male Caucasians ( n=19) were included. The multi-sample fractional clearance (Cl(fe)) of chlorzoxazone was compared with one...... estimates, Cl(est) at 3 h or 6 h, and MR at 3 h, can serve as reliable markers of CYP2E1 activity. The one-sample clearance method is an accurate, renal function-independent measure of the intrinsic activity; it is simple to use and easily applicable to humans.......-time-point clearance estimation (Cl(est)) at 3, 4, 5 and 6 h. Furthermore, the metabolite/drug ratios (MRs) estimated from one-time-point samples at 1, 2, 3, 4, 5 and 6 h were compared with Cl(fe). RESULTS: The concordance between Cl(est) and Cl(fe) was highest at 6 h. The minimal mean prediction error (MPE) of Cl...

  3. CYP3A5* 1 is an Inhibitory Factor for Lung Cancer in Taiwanese

    Directory of Open Access Journals (Sweden)

    Kun-Tu Yeh

    2003-05-01

    Full Text Available The expression of the cytochrome P450 CYP3A5 enzymes shows a wide variation across the general population and ethnic groups. This wide disparity implies interracial differences in drug clearance and susceptibility to diseases such as cancer. CYP3A5 polymorphisms were rapidly determined using polymerase chain reaction-restriction fragment length polymorphism analysis in 113 Taiwanese patients with hepatoma, 70 with cervical cancer, 92 with breast cancer, 82 with oral cancer, 90 with thyroid cancer, 133 with lung cancer, and 270 healthy controls. The allelic frequencies of CYP3A5*1 were 25% in hepatoma patients, 33% in cervical cancer patients, 31% in breast cancer patients, 22% in oral cancer patients, 23% in thyroid cancer patients, 20% in lung cancer patients, and 27% in healthy subjects. Lung cancer patients had a significantly lower frequency (20% of CYP3A5*1 expression than healthy controls (p = 0.028, odds ratio = 1.49, 95% confidence interval = 1.04-2.13, but there was no statistically significant difference between healthy controls and other cancers. We suggest that CYP3A5*1 may play an important role in individual predisposition to lung cancer in Taiwan.

  4. Overexpression of Populus trichocarpa CYP85A3 promotes growth and biomass production in transgenic trees.

    Science.gov (United States)

    Jin, Yan-Li; Tang, Ren-Jie; Wang, Hai-Hai; Jiang, Chun-Mei; Bao, Yan; Yang, Yang; Liang, Mei-Xia; Sun, Zhen-Cang; Kong, Fan-Jing; Li, Bei; Zhang, Hong-Xia

    2017-10-01

    Brassinosteroids (BRs) are essential hormones that play crucial roles in plant growth, reproduction and response to abiotic and biotic stress. In Arabidopsis, AtCYP85A2 works as a bifunctional cytochrome P450 monooxygenase to catalyse the conversion of castasterone to brassinolide, a final rate-limiting step in the BR-biosynthetic pathway. Here, we report the functional characterizations of PtCYP85A3, one of the three AtCYP85A2 homologous genes from Populus trichocarpa. PtCYP85A3 shares the highest similarity with AtCYP85A2 and can rescue the retarded-growth phenotype of the Arabidopsis cyp85a2-2 and tomato d x mutants. Constitutive expression of PtCYP85A3, driven by the cauliflower mosaic virus 35S promoter, increased the endogenous BR levels and significantly promoted the growth and biomass production in both transgenic tomato and poplar. Compared to the wild type, plant height, shoot fresh weight and fruit yield increased 50%, 56% and 43%, respectively, in transgenic tomato plants. Similarly, plant height and stem diameter increased 15% and 25%, respectively, in transgenic poplar plants. Further study revealed that overexpression of PtCYP85A3 enhanced xylem formation without affecting the composition of cellulose and lignin, as well as the cell wall thickness in transgenic poplar. Our finding suggests that PtCYP85A3 could be used as a potential candidate gene for engineering fast-growing trees with improved wood production. © 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.

  5. Characterization of polymorphisms of genes ADH2, ADH3, ALDH2 and CYP2E1 and relationship to the alcoholism in a Colombian population.

    Science.gov (United States)

    Méndez, Claudia; Rey, Mauricio

    2015-12-30

    Identify and characterize polymorphisms of genes ADH2, ADH3, ALDH2 and CYP2E1 in a Colombian population residing in the city of Bogotá and determine its possible relationship to the alcoholism. ADH2, ADH3, ALDH2, and CYP2E1 genotypes a population of 148 individuals with non-problematic alcohol and 65 individuals with alcoholism were determined with TaqMan probes and PCR-RFLP. DNA was obtained from peripheral blood white cells. Significant difference was found in family history of alcoholism and use of other psychoactive substances to compare alcoholics with controls. When allelic frequencies for each category (gender) were considered, frequency of A2 allele carriers in ADH2 was found higher in male patients than controls. In women, the relative frequency for c1 allele in CYP2E1 was lower in controls than alcoholics. The ALDH2 locus is monomorphic. No significant differences in allele distributions of the loci examined to compare two populations were observed, however when stratifying the same trend was found that these differences tended to be significant. This study allows us to conclude the positive association between family history of alcoholism and alcoholism suggesting that there is a favourable hereditary predisposition. Since substance dependence requires interaction of multiple genes, the combination of genotypes ADH2 * 2, CYP2E1 * 1 combined with genotype homozygous ALDH2 * 1 found in this study could be leading to the population to a potential risk to alcoholism.

  6. Effect of Genetic Variability in the CYP4F2, CYP4F11, and CYP4F12 Genes on Liver mRNA Levels and Warfarin Response

    Directory of Open Access Journals (Sweden)

    J. E. Zhang

    2017-05-01

    Full Text Available Genetic polymorphisms in the gene encoding cytochrome P450 (CYP 4F2, a vitamin K oxidase, affect stable warfarin dose requirements and time to therapeutic INR. CYP4F2 is part of the CYP4F gene cluster, which is highly polymorphic and exhibits a high degree of linkage disequilibrium, making it difficult to define causal variants. Our objective was to examine the effect of genetic variability in the CYP4F gene cluster on expression of the individual CYP4F genes and warfarin response. mRNA levels of the CYP4F gene cluster were quantified in human liver samples (n = 149 obtained from a well-characterized liver bank and fine mapping of the CYP4F gene cluster encompassing CYP4F2, CYP4F11, and CYP4F12 was performed. Genome-wide association study (GWAS data from a prospective cohort of warfarin-treated patients (n = 711 was also analyzed for genetic variations across the CYP4F gene cluster. In addition, SNP-gene expression in human liver tissues and interactions between CYP4F genes were explored in silico using publicly available data repositories. We found that SNPs in CYP4F2, CYP4F11, and CYP4F12 were associated with mRNA expression in the CYP4F gene cluster. In particular, CYP4F2 rs2108622 was associated with increased CYP4F2 expression while CYP4F11 rs1060467 was associated with decreased CYP4F2 expression. Interestingly, these CYP4F2 and CYP4F11 SNPs showed similar effects with warfarin stable dose where CYP4F11 rs1060467 was associated with a reduction in daily warfarin dose requirement (∼1 mg/day, Pc = 0.017, an effect opposite to that previously reported with CYP4F2 (rs2108622. However, inclusion of either or both of these SNPs in a pharmacogenetic algorithm consisting of age, body mass index (BMI, gender, baseline clotting factor II level, CYP2C9∗2 rs1799853, CYP2C9∗3 rs1057910, and VKORC1 rs9923231 improved warfarin dose variability only by 0.5–0.7% with an improvement in dose prediction accuracy of ∼1–2%. Although there is complex

  7. In vivo effects of chronic contamination with depleted uranium on CYP3A and associated nuclear receptors PXR and CAR in the rat

    International Nuclear Information System (INIS)

    Souidi, M.; Gueguen, Y.; Linard, C.; Dudoignon, N.; Grison, S.; Baudelin, C.; Marquette, C.; Gourmelon, P.; Aigueperse, J.; Dublineau, I.

    2005-01-01

    In addition to its natural presence at high concentrations in some areas, uranium has several civilian and military applications that could cause contamination of human populations, mainly through chronic ingestion. Reports describe the accumulation of this radionuclide in some organs (including the bone, kidney, and liver) after acute or chronic contamination and show that it produces chemical or radiological toxicity or both. The literature is essentially devoid of information about uranium-related cellular and molecular effects on metabolic functions such as xenobiotic detoxification. The present study thus evaluated rats chronically exposed to depleted uranium in their drinking water (1 mg/(rat day)) for 9 months. Our specific aim was to evaluate the hepatic and extrahepatic mRNA expression of CYP3A1/A2, CYP2B1, and CYP1A1 as well as of the nuclear receptors PXR, CAR, and RXR in these rats. CYP3A1 mRNA expression was significantly higher in the brain (200%), liver (300%), and kidneys (900%) of exposed rats compared with control rats, while CYP3A2 mRNA levels were higher in the lungs (300%) and liver (200%), and CYP2B1 mRNA expression in the kidneys (300%). Expression of CYP1A1 mRNA did not change significantly during this study. PXR mRNA levels increased in the brain (200%), liver (150%), and kidneys (200%). Uranium caused CAR mRNA expression in the lungs to double. Expression of RXR mRNA did not change significantly in the course of this study, nor did the hepatic activity of CYP2C, CYP3A, CYP2A, or CYP2B. Uranium probably affects the expression of drug-metabolizing CYP enzymes through the PXR and CAR nuclear receptors. These results suggest that the stimulating effect of uranium on these enzymes might lead to hepatic or extrahepatic toxicity (or both) during drug treatment and then affect the entire organism

  8. Expression of the vitamin D metabolizing enzyme CYP24A1 at the annulus of human spermatozoa may serve as a novel marker of semen quality

    DEFF Research Database (Denmark)

    Jensen, Martin Blomberg; Jørgensen, A; Nielsen, J E

    2012-01-01

    Vitamin D (VD) is important for male reproduction in mammals and the VD receptor (VDR) and VD-metabolizing enzymes are expressed in human spermatozoa. The VD-inactivating enzyme CYP24A1 titrates the cellular responsiveness to VD, is transcriptionally regulated by VD, and has a distinct expression...... at the sperm annulus. Here, we investigated if CYP24A1 expression serves as a marker for VD metabolism in spermatozoa, and whether CYP24A1 expression was associated with semen quality. We included 130 men (53 healthy young volunteers and 77 subfertile men) for semen analysis and immunocytochemical (ICC.......3%. Functional studies revealed that 1,25(OH)(2) D(3) increased [Ca(2+) ](i) and sperm motility in young healthy men, while 1,25(OH)(2) D(3) was unable to increase motility in subfertile patients. In conclusion, we suggest that CYP24A1 expression at the annulus may serve as a novel marker of semen quality...

  9. Inferring mechanisms of copy number change from haplotype structures at the human DEFA1A3 locus

    OpenAIRE

    Black, Holly A; Khan, Fayeza F; Tyson, Jess; Armour, John AL

    2014-01-01

    Background The determination of structural haplotypes at copy number variable regions can indicate the mechanisms responsible for changes in copy number, as well as explain the relationship between gene copy number and expression. However, obtaining spatial information at regions displaying extensive copy number variation, such as the DEFA1A3 locus, is complex, because of the difficulty in the phasing and assembly of these regions. The DEFA1A3 locus is intriguing in that it falls within a reg...

  10. A T-cell specific transcriptional enhancer element 3' of Cα in the human T-cell receptor α locus

    International Nuclear Information System (INIS)

    Ho, Icheng; Yang, Lihsuan; Morle, G.; Leiden, J.M.

    1989-01-01

    A transcriptional enhancer element has been identified 4.5 kilobases 3' of C α (constant region α chain) in the human T-cell receptor (TCR) α-chain locus. This enhancer is active on both a TCR V α (variable region α chain) promoter and the minimal simian virus 40 promoter in TCR α/β Jurkat and EL4 cells but is inactive on a V α promoter TCR γ/δ PEER and Molt-13 cells, clone 13 B cells, and HeLa fibroblasts. The enhancer has been localized to a 116-base-pair BstXI/Dra I restriction enzyme fragment, which lacks immunoglobulin octamer and κB enhancer motifs but does contain a consensus cAMP-response element (CRE). DNase I footprint analyses demonstrated that the minimal enhancer contains two binding sites for Jurkat nuclear proteins. One of these sites corresponds to the CRE, while the other does not correspond to a known transcriptional enhancer motif. These data support a model in which TCR α gene transcription is regulated by a unique set of cis-acting sequences and trans-acting factors, which are differentially active in cells of the TCR α/β lineage. In addition, the TCR α enhancer may play a role in activating oncogene expression in T-lymphoblastoid tumors that have previously been shown to display chromosomal translocations into the human TCR α locus

  11. Study of exon 12 polymorphism of the human thromboxane synthase (CYP5A1) gene in Egyptian stroke patients

    International Nuclear Information System (INIS)

    Soliman, S.E.T.; Zaater, M.K.

    2010-01-01

    Thromboxane synthase (CYP5A1) catalyzes the conversion of prostaglandin H2 to thromboxane A2, a potent mediator of platelet aggregation, vasoconstriction and bronchoconstriction. It has been implicated in the patho-physiological process of a variety of diseases, such as atherosclerosis, myocardial infarction, stroke and asthma. On the basis of the hypothesis that variations of the CYP5A1 gene may play an important role in human diseases, we performed screening for the prevalence of exon12 polymorphism of the human Thromboxane synthase (CYP5A1) gene among Egyptian normal and stroke patients. Using sequence-specific PCR, we examined the allelic prevalence in 70 Egyptian patients with ischemic strokes and in 70 controls. In addition, we compared the CYP5A1 allelic prevalence in 30 patients with stroke recurrence despite Aspirin use, in comparison with patients who have not experienced recurrent stroke while taking Aspirin. The frequencies of the CYP5A1*9 mutant (substitution of guanine by adenine near the heme-binding catalytic domain) and of the wild-type allele were 0.197(19.7%) and 0.803 (80.3%) respectively; they did not differ significantly between stroke patients and controls. The CYP5A1*9 mutant was significantly more prevalent among stroke patients with history of previous cerebrovascular attacks; even after adjusting for the common risk factors for cardiovascular disease (odds ratio (OR)1.73, 95%, confidence interval ( CI) 1.10-2.73; p=0.017). Among stroke patients, the presence of the CYP5A1 wild type allele was more frequent among the hypertensives (OR 1.68, 95% CI, 1.01-2.79; p=0.045), and less frequent among the diabetics (OR 0.55, 95%, CI 0.36-0.84; p=0.006). Also among stroke patients, the CYP5A1*9 mutant was significantly more prevalent among those, who failed secondary Aspirin prophylaxis compared to those with successful secondary Aspirin prophylaxis (OR 1.49, 95%, CI 1.06-2.11). This study provides evidence for high prevalence of the CYP5A1*9 mutant

  12. PSA and androgen-related gene (AR, CYP17, and CYP19) polymorphisms and the risk of adenocarcinoma at prostate biopsy

    DEFF Research Database (Denmark)

    dos Santos, Rodrigo Mattos; de Jesus, Carlos Márcio Nóbrega; Trindade Filho, José Carlos Souza

    2008-01-01

    The aim of the present study was to examine the impact of polymorphisms in prostate-specific antigen (PSA) and androgen-related genes (AR, CYP17, and CYP19) on prostate cancer (PCa) risk in selected high-risk patients who underwent prostate biopsy. Blood samples and prostate tissues were obtained......=0.0110) genotypes. Genetic instability at the AR locus leading to somatic mosaicism was detected in one PCa patient by comparing the length of AR CAG repeats in matched peripheral blood and prostate biopsy cores. Taken together, these findings suggest that the PSA genotype should be a clinically relevant biomarker...

  13. Translocations affecting human immunoglobulin heavy chain locus

    Directory of Open Access Journals (Sweden)

    Sklyar I. V.

    2014-03-01

    Full Text Available Translocations involving human immunoglobulin heavy chain (IGH locus are implicated in different leukaemias and lymphomas, including multiple myeloma, mantle cell lymphoma, Burkitt’s lymphoma and diffuse large B cell lymphoma. We have analysed published data and identified eleven breakpoint cluster regions (bcr related to these cancers within the IgH locus. These ~1 kbp bcrs are specific for one or several types of blood cancer. Our findings could help devise PCR-based assays to detect cancer-related translocations, to identify the mechanisms of translocations and to help in the research of potential translocation partners of the immunoglobulin locus at different stages of B-cell differentiation.

  14. 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)

    Science.gov (United States)

    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 ...

  15. Effect of ABCB1 (3435C>T) and CYP3A5 (6986A>G) genes ...

    African Journals Online (AJOL)

    Marwa Helal

    2016-11-30

    Nov 30, 2016 ... Tacrolimus (TAC) is the backbone of immunosuppressive drugs used worldwide ... the pharmacokinetics of tacrolimus and the polymorphisms of the. CYP3A5 and .... performance liquid chromatography tandem mass spectrometry. (LC/MS/MS ..... CYP3A5 polymorphism became the key factor. The deficient ...

  16. Characterization of histone H3K27 modifications in the β-globin locus

    International Nuclear Information System (INIS)

    Kim, Yea Woon; Kim, AeRi

    2011-01-01

    Research highlights: → The β-globin locus control region is hyperacetylated and monomethylated at histone H3K27. → Highly transcribed globin genes are marked by H3K27ac, but H3K27me2 is remarkable at silent globin genes in erythroid K562 cells. → Association of PRC2 subunits is comparable with H3K27me3 pattern. → Modifications of histone H3K27 are established in an enhancer-dependent manner. -- Abstract: Histone H3K27 is acetylated or methylated in the environment of nuclear chromatin. Here, to characterize the modification pattern of H3K27 in locus control region (LCR) and to understand the correlation of various H3K27 modifications with transcriptional activity of genes, we analyzed the human β-globin locus using the ChIP assay. The LCR of the human β-globin locus was enriched by H3K27ac and H3K27me1 in erythroid K562 cells. The highly transcribed globin genes were hyperacetylated at H3K27, but the repressed globin genes were highly dimethylated at this lysine in these cells. However, in non-erythroid 293FT cells, the β-globin locus was marked by a high level of H3K27me3. EZH2 and SUZ12, subunits of polycomb repressive complex 2, were comparably detected with the H3K27me3 pattern in K562 and 293FT cells. In addition, H3K27ac, H3K27me1 and H3K27me3 were established in an enhancer-dependent manner in a model minichromosomal locus containing an enhancer and its target gene. Taken together, these results show that H3K27 modifications have distinctive correlations with the chromatin state or transcription level of genes and are influenced by an enhancer.

  17. Ibrutinib Dosing Strategies Based on Interaction Potential of CYP3A4 Perpetrators Using Physiologically Based Pharmacokinetic Modeling.

    Science.gov (United States)

    de Zwart, L; Snoeys, J; De Jong, J; Sukbuntherng, J; Mannaert, E; Monshouwer, M

    2016-11-01

    Based on ibrutinib pharmacokinetics and potential sensitivity towards CYP3A4-mediated drug-drug interactions (DDIs), a physiologically based pharmacokinetic approach was developed to mechanistically describe DDI with various CYP3A4 perpetrators in healthy men under fasting conditions. These models were verified using clinical data for ketoconazole (strong CYP3A4 inhibitor) and used to prospectively predict and confirm the inducing effect of rifampin (strong CYP3A4 inducer); DDIs with mild (fluvoxamine, azithromycin) and moderate inhibitors (diltiazem, voriconazole, clarithromycin, itraconazole, erythromycin), and moderate (efavirenz) and strong CYP3A4 inducers (carbamazepine), were also predicted. Ketoconazole increased ibrutinib area under the curve (AUC) by 24-fold, while rifampin decreased ibrutinib AUC by 10-fold; coadministration of ibrutinib with strong inhibitors or inducers should be avoided. The ibrutinib dose should be reduced to 140 mg (quarter of maximal prescribed dose) when coadministered with moderate CYP3A4 inhibitors so that exposures remain within observed ranges at therapeutic doses. Thus, dose recommendations for CYP3A4 perpetrator use during ibrutinib treatment were developed and approved for labeling. © 2016 American Society for Clinical Pharmacology and Therapeutics.

  18. Vascular endothelial overexpression of human CYP2J2 (Tie2-CYP2J2 Tr) modulates cardiac oxylipin profiles and enhances coronary reactive hyperemia in mice

    Science.gov (United States)

    Hanif, Ahmad; Edin, Matthew L.; Zeldin, Darryl C.; Morisseau, Christophe; Falck, John R.

    2017-01-01

    Arachidonic acid is metabolized to epoxyeicosatrienoic acids (EETs) by cytochrome (CYP) P450 epoxygenases, and to ω-terminal hydroxyeicosatetraenoic acids (HETEs) by ω-hydroxylases. EETs and HETEs often have opposite biologic effects; EETs are vasodilatory and protect against ischemia/reperfusion injury, while ω-terminal HETEs are vasoconstrictive and cause vascular dysfunction. Other oxylipins, such as epoxyoctadecaenoic acids (EpOMEs), hydroxyoctadecadienoic acids (HODEs), and prostanoids also have varied vascular effects. Post-ischemic vasodilation in the heart, known as coronary reactive hyperemia (CRH), protects against potential damage to the heart muscle caused by ischemia. The relationship among CRH response to ischemia, in mice with altered levels of CYP2J epoxygenases has not yet been investigated. Therefore, we evaluated the effect of endothelial overexpression of the human cytochrome P450 epoxygenase CYP2J2 in mice (Tie2-CYP2J2 Tr) on oxylipin profiles and CRH. Additionally, we evaluated the effect of pharmacologic inhibition of CYP-epoxygenases and inhibition of ω-hydroxylases on CRH. We hypothesized that CRH would be enhanced in isolated mouse hearts with vascular endothelial overexpression of human CYP2J2 through modulation of oxylipin profiles. Similarly, we expected that inhibition of CYP-epoxygenases would reduce CRH, whereas inhibition of ω-hydroxylases would enhance CRH. Compared to WT mice, Tie2-CYP2J2 Tr mice had enhanced CRH, including repayment volume, repayment duration, and repayment/debt ratio (P iso-PGF2α (P < 0.05). Inhibition of CYP epoxygenases with MS-PPOH attenuated CRH (P < 0.05). Ischemia caused a decrease in mid-chain HETEs (5-, 11-, 12-, 15-HETEs P < 0.05) and HODEs (P < 0.05). These data demonstrate that vascular endothelial overexpression of CYP2J2, through changing the oxylipin profiles, enhances CRH. Inhibition of CYP epoxygenases decreases CRH, whereas inhibition of ω-hydroxylases enhances CRH. PMID:28328948

  19. Asynchronous DNA replication within the human β-globin gene locus

    International Nuclear Information System (INIS)

    Epner, E.; Forrester, W.C.; Groudine, M.

    1988-01-01

    The timing of DNA replication of the human β-globin gene locus has been studied by blot hybridization of newly synthesized BrdUrd-substituted DNA from cells in different stages of the S phase. Using probes that span >120 kilobases across the human β-globin gene locus, the authors show that the majority of this domain replicates in early S phase in the human erythroleukemia cell line K562 and in middle-to-late S phase in the lymphoid cell line Manca. However, in K562 cells three small regions display a strikingly different replication pattern than adjacent sequences. These islands, located in the inter-γ-globin gene region and approximately 20 kilobases 5' to the ε-globin gene and 20 kilobases 3' to the β-globin gene, replicate later and throughout S phase. A similar area is also present in the α-globin gene region in K562 cells. They suggest that these regions may represent sites of termination of replication forks

  20. Aryl hydrocarbon receptor activation and CYP1A induction by cooked food-derived carcinogenic heterocyclic amines in human HepG2 cell lines.

    Science.gov (United States)

    Sekimoto, Masashi; Sumi, Haruna; Hosaka, Takuomi; Umemura, Takashi; Nishikawa, Akiyoshi; Degawa, Masakuni

    2016-11-01

    The ability of nine cooked food-derived heterocyclic aromatic amines (HCAs), such as 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1), 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), 2-amino-6-methylpyrido[12-a:3',2'-d]imidazole (Glu-P-1), 2-amino-pyrido[12-a:3',2'-d]imidazole hydrochloride (Glu-P-2), 2-amino-9H-pyrido[2,3-b]indole (AαC), 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeAαC), 2-amino-3-methylimidazo[4,5-f]quinolone (IQ), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenyl-1H-imidazo[4,5-b]pyridine (PhIP), to activate human aryl hydrocarbon receptor (hAhR) was examined using a HepG2-A10 cell line, which has previously established from human hepatocarcinoma-derived HepG2 cells for use in hAhR-based luciferase reporter gene assays. Trp-P-1, Trp-P-2, AαC, MeAαC, IQ and MeIQx showed a definite ability to induce not only luciferase (hAhR activation) in HepG2-A10 cells but also cytochrome P450 (CYP)1A1/1A2 mRNAs in HepG2 cells, while such the ability of Glu-P-1, Glu-P-2, and PhIP was very low. In addition, all the HCAs examined, especially MeAαC and MeIQx, had a definite capacity for inhibiting the activity of ethoxyresorfin O-deethylase (CYP1As, especially CYP1A1). The present findings demonstrate that all the HCAs examined have the ability to activate hAhR and its target genes, and further confirm that these HCAs become good substrates for human CYP1A subfamily enzyme(s). Copyright © 2016 Elsevier Ltd. All rights reserved.

  1. UV-dependent production of 25-hydroxyvitamin D2 in the recombinant yeast cells expressing human CYP2R1

    International Nuclear Information System (INIS)

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

    2013-01-01

    Highlights: •We produce 25-hydroxyvitamin D in the recombinant yeast expressing human CYP2R1. •Vitamin D2 is produced in yeast from endogenous ergosterol with UV irradiation. •We produce 25-hydroxyvitamin D2 in the recombinant yeast without added substrate. -- Abstract: CYP2R1 is known to be a physiologically important vitamin D 25-hydroxylase. We have successfully expressed human CYP2R1 in Saccharomyces cerevisiae to reveal its enzymatic properties. In this study, we examined production of 25-hydroxylated vitamin D using whole recombinant yeast cells that expressed CYP2R1. When vitamin D 3 or vitamin D 2 was added to the cell suspension of CYP2R1-expressing yeast cells in a buffer containing glucose and β-cyclodextrin, the vitamins were converted into their 25-hydroxylated products. Next, we irradiated the cell suspension with UVB and incubated at 37 °C. Surprisingly, the 25-hydroxy vitamin D 2 was produced without additional vitamin D 2 . Endogenous ergosterol was likely converted into vitamin D 2 by UV irradiation and thermal isomerization, and then the resulting vitamin D 2 was converted to 25-hydroxyvitamin D 2 by CYP2R1. This novel method for producing 25-hydroxyvitamin D 2 without a substrate could be useful for practical purposes

  2. Increased CYP1A1 expression in human exfoliated urothelial cells of cigarette smokers compared to non-smokers

    Energy Technology Data Exchange (ETDEWEB)

    Doerrenhaus, Angelika; Roos, Peter H. [Institute for Occupational Physiology at the University Dortmund, Dortmund (Germany); Mueller, Tina [Institute for Occupational Physiology at the University Dortmund, Dortmund (Germany); University Dortmund, Department of Statistics, Mathematical Statistics with Applications in Biometrics, Dortmund (Germany)

    2007-01-15

    Polycyclic aromatic hydrocarbons, arylamines and nitrosamines, constituents of cigarette smoke, are known inducers of bladder cancer. The biochemical response of the target tissue, the bladder urothelium, following inhalation of cigarette smoke has not been studied so far. We used exfoliated transitional urothelial cells from human urine samples to analyze effects of smoking on induction of the cytochrome P450 enzyme CYP1A1. Samples of 40 subjects, including male and female smokers and non-smokers, were examined. A prerequisite for the immunofluorescence microscopic analysis of the cells was the enrichment of the urothelial cell population. This was achieved by a new method which is based on magnetic cell sorting exploiting specific binding of immobilized Griffonia simplicifolia lectin to the surface of urothelial cells. Immunostaining of the final cell preparation with a monoclonal antibody to CYP1A1 showed that about 6% of the urothelial cells of non-smokers stained positive for CYP1A1. However, this fraction of positive cells was more than 44% of the urothelial cells in samples from cigarette smokers. In spite of the individual variation, the difference was statistically significant. There were no gender-related differences in the portion of CYP1A1 expressing urothelial cells of smokers and non-smokers. In essence, we show for the first time that human urothelial cells respond to cigarette smoking by induction of CYP1A1. The approach opens new fields of mechanistic and biomarker research with respect to the pathogenetic processes of cancer development in the human bladder. (orig.)

  3. Dietary tocopherols inhibit PhIP-induced prostate carcinogenesis in CYP1A-humanized mice.

    Science.gov (United States)

    Chen, Jayson X; Li, Guangxun; Wang, Hong; Liu, Anna; Lee, Mao-Jung; Reuhl, Kenneth; Suh, Nanjoo; Bosland, Maarten C; Yang, Chung S

    2016-02-01

    Tocopherols, the major forms of vitamin E, exist as alpha-tocopherol (α-T), β-T, γ-T and δ-T. The cancer preventive activity of vitamin E is suggested by epidemiological studies, but recent large-scale cancer prevention trials with high dose of α-T yielded disappointing results. Our hypothesis that other forms of tocopherols have higher cancer preventive activities than α-T was tested, herein, in a novel prostate carcinogenesis model induced by 2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine (PhIP), a dietary carcinogen, in the CYP1A-humanized (hCYP1A) mice. Treatment of hCYP1A mice with PhIP (200 mg/kg b.w., i.g.) induced high percentages of mouse prostatic intraepithelial neoplasia (mPIN), mainly in the dorsolateral glands. Supplementation with a γ-T-rich mixture of tocopherols (γ-TmT, 0.3% in diet) significantly inhibited the development of mPIN lesions and reduced PhIP-induced elevation of 8-oxo-deoxyguanosine, COX-2, nitrotyrosine, Ki-67 and p-AKT, and the loss of PTEN and Nrf2. Further studies with purified δ-T, γ-T or α-T (0.2% in diet) showed that δ-T was more effective than γ-T or α-T in preventing mPIN formations and p-AKT elevation. These results indicate that γ-TmT and δ-T could be effective preventive agents of prostate cancer. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  4. Microsomal cytochrome P450-3A4 (CYP3A4) nanobiosensor for the determination of 2,4-dichlorophenol-An endocrine disruptor compound

    International Nuclear Information System (INIS)

    Hendricks, Nicolette R.; Waryo, Tesfaye T.; Arotiba, Omotayo; Jahed, Nazeem; Baker, Priscilla G.L.; Iwuoha, Emmanuel I.

    2009-01-01

    Cytochrome P450-3A4 (CYP3A4) is a monooxygenase enzyme that plays a major role in the detoxification of bioactive compounds and hydrophobic xenobiotics (e.g. medicines, drugs, environmental pollutants, food supplements and steroids). Physiologically the monooxygenation reactions of this class II, microsomal, b-type heme enzyme, usually requires cytochrome P450 reductase, NADPH. A novel CYP3A4 biosensor system that essentially simplified the enzymatic redox processes by allowing electron transfer between the electrode and the enzyme redox centre to occur, without any need for the physiological redox partners, was developed for the detection of 2,4-dichlorophenol (2,4-DCP), a priority environmental pollutant and an endocrine disruptor. The biosensor, GC/Naf-Co(Sep) 3+ /CYP3A4/Naf, was constructed by encapsulating CYP3A4 in a Nafion-cobalt (III) sepulchrate (Naf-Co(Sep) 3+ ) composite film on a glassy carbon (GC) electrode. The responses of the biosensor to 2,4-dichlorophenol, erythromycin (CYP3A4 native substrate) and ketoconazole (CYP 3A4 natural inhibitor) were studied by cyclic and square wave voltammetric techniques. The detection limit (DL) of the biosensor for 2,4-dichlorophenol was 0.043 μg L -1 , which is by an order of magnitude lower than the EU limit (0.3 μg L -1 ) for any pesticide compound in ground water. The biosensor's DL is lower than the U.S. Environmental Protection Agency's drinking water equivalent level (DWEL) value for 2,4-DCP, which is 2 μg L -1

  5. Imidacloprid is hydroxylated by Laodelphax striatellus CYP6AY3v2.

    Science.gov (United States)

    Wang, R; Zhu, Y; Deng, L; Zhang, H; Wang, Q; Yin, M; Song, P; Elzaki, M E A; Han, Z; Wu, M

    2017-10-01

    Laodelphax striatellus (Fallén) is one of the most destructive pests of rice, and has developed high resistance to imidacloprid. Our previous work indicated a strong association between imidacloprid resistance and the overexpression of a cytochrome P450 gene CYP6AY3v2 in a L. striatellus imidacloprid resistant strain (Imid-R). In this study, a transgenic Drosophila melanogaster line that overexpressed the L. striatellus CYP6AY3v2 gene was established and was found to confer increased levels of imidacloprid resistance. Furthermore, CYP6AY3v2 was co-expressed with D. melanogaster cytochrome P450 reductase (CPR) in Spodoptera frugiperda 9 (SF9) cells. A carbon monoxide difference spectra analysis indicated that CYP6AY3v2 was expressed predominately in its cytochrome P450 (P450) form, which is indicative of a good-quality functional enzyme. The recombinant CYP6AY3v2 protein efficiently catalysed the model substrate P-nitroanisole to p-nitrophenol with a maximum velocity (V max ) of 60.78 ± 3.93 optical density (mOD)/min/mg protein. In addition, imidacloprid itself was metabolized by the recombinant CYP6AY3v2/nicotinamide adenine dinucleotide 2'-phosphate reduced tetrasodium salt (NADPH) CPR microsomes in in vitro assays (catalytic constant (K cat ) = 0.34 pmol/min/pmol P450, michaelis constant (K m ) = 41.98 μM), and imidacloprid depletion and metabolite peak formation were with a time dependence. The data provided direct evidence that CYP6AY3v2 is capable of hydroxylation of imidacloprid and conferring metabolic resistance in L. striatellus. © 2017 The Royal Entomological Society.

  6. CYP3A5*3 and MDR1 C3435T are influencing factors of inter-subject variability in rupatadine pharmacokinetics in healthy Chinese volunteers.

    Science.gov (United States)

    Xiong, Yuqing; Yuan, Zhao; Yang, Jingzhi; Xia, Chunhua; Li, Xinhua; Huang, Shibo; Zhang, Hong; Liu, Mingyi

    2016-04-01

    Rupatadine (RUP) is an oral antihistamine and platelet-activating factor antagonist and is shown as the substrate of CYP3A5 and P-gp. The significant interindividual differences of CYP3A5 and P-gp often cause bioavailability differences of some clinical drugs. The present study is aimed to evaluate the effect of genetic polymorphisms of CYP3A5 and MDR1 on RUP pharmacokinetics in healthy male Chinese volunteer subjects. Blood samples were collected from 36 subjects before and after a single, oral RUP 10 mg dose. A PCR-RFLP assay was used to genotype CYP3A5*3 and assess MDR1 C3435T variation. A validated LC-MS/MS method quantified plasma RUP concentration. The relationship between RUP plasma concentration, pharmacokinetic parameters, and polymorphic alleles (CYP3A5 and MDR1) were assessed. Plasma RUP concentrations were lower for CYP3A5*1/*1 carriers than for CYP3A5*3/*3 and CYP3A5*1/*3 carriers. Mean C(max), AUC(0-t) and AUC(0-∞) were significantly lower, and the CLz and Vd were significantly higher in the CYP3A5 wild-type group, than in the CYP3A5 mutated group. MDR1 CT and MDR1 TT carriers had lower plasma RUP concentrations than MDR1 CC carriers. The mean C(max), AUC(0-t), AUC(0-∞) and T max were significantly lower in the TT group than in the CC and CT groups. The mean CLz was higher in the TT group than in the CC and CT groups, but not significantly. These results suggest that CYP3A5 and MDR1 may play a key role in the variability of RUP metabolism and transport, respectively. CYP3A5 and MDR1 polymorphisms may be the main explanation for the differences observed in RUP pharmacokinetics, and therefore may provide a rationale for safe and effective clinical use of RUP. Our research lays down a solid theory foundation to guide the safe and effective clinical use of RUP and a route to achieve individualized therapy.

  7. Efficacy of piroxicam for postoperative pain after lower third molar surgery associated with CYP2C8*3 and CYP2C9

    Directory of Open Access Journals (Sweden)

    Calvo AM

    2017-07-01

    Full Text Available Adriana Maria Calvo, Paulo Zupelari-Gonçalves, Thiago José Dionísio, Daniel Thomas Brozoski, Flávio Augusto Faria, Carlos Ferreira Santos Department of Biological Sciences, Bauru School of Dentistry, University of São Paulo, São Paulo, Brazil Objective: Nonsteroidal anti-inflammatory drugs (NSAIDs are metabolized by the cytochrome P450 enzymes (CYPs, predominantly CYP2C8 and CYP2C9. The aim of this study was to evaluate the possible association of polymorphisms in the CYP2C8*3 and CYP2C9 genes with the clinical efficacy of oral piroxicam (20 mg daily for 4 days after lower third molar surgeries with regard to postoperative pain, swelling, trismus, adverse reactions, need for rescue medication and the volunteer’s overall satisfaction. Materials and methods: For this purpose, 102 volunteers were genotyped for CYP2C8*3 and CYP2C9 polymorphisms. Briefly, genomic DNA was isolated from saliva collected from volunteers subjected to invasive lower third molar surgeries, and the preoperative, intraoperative and postoperative parameters were collected and analyzed. Results: An equal amount of piroxicam sufficiently managed postoperative pain and inflammatory symptoms, with visual analog pain scores typically <40 mm for all genotypes investigated. Furthermore, only two out of 102 volunteers heterozygous for CYP2C8*3 and CYP2C9*3 reported adverse side effects. Conclusion: In general, slow metabolizers of piroxicam, who were volunteers with mutant alleles, were indifferent from normal metabolizers with the wild-type alleles and therefore did not require specialized piroxicam doses to manage postoperative pain and inflammation. Keywords: piroxicam, lower third molar surgery, P450, CYP2C8, CYP2C9, pharmacogenetics 

  8. Microsomal cytochrome P450-3A4 (CYP3A4) nanobiosensor for the determination of 2,4-dichlorophenol-An endocrine disruptor compound

    Energy Technology Data Exchange (ETDEWEB)

    Hendricks, Nicolette R.; Waryo, Tesfaye T.; Arotiba, Omotayo; Jahed, Nazeem; Baker, Priscilla G.L. [SensorLab, Department of Chemistry, University of Western Cape, Moderddam Road, Bellville, Cape Town 7535 (South Africa); Iwuoha, Emmanuel I. [SensorLab, Department of Chemistry, University of Western Cape, Moderddam Road, Bellville, Cape Town 7535 (South Africa)], E-mail: eiwuoha@uwc.ac.za

    2009-02-28

    Cytochrome P450-3A4 (CYP3A4) is a monooxygenase enzyme that plays a major role in the detoxification of bioactive compounds and hydrophobic xenobiotics (e.g. medicines, drugs, environmental pollutants, food supplements and steroids). Physiologically the monooxygenation reactions of this class II, microsomal, b-type heme enzyme, usually requires cytochrome P450 reductase, NADPH. A novel CYP3A4 biosensor system that essentially simplified the enzymatic redox processes by allowing electron transfer between the electrode and the enzyme redox centre to occur, without any need for the physiological redox partners, was developed for the detection of 2,4-dichlorophenol (2,4-DCP), a priority environmental pollutant and an endocrine disruptor. The biosensor, GC/Naf-Co(Sep){sup 3+}/CYP3A4/Naf, was constructed by encapsulating CYP3A4 in a Nafion-cobalt (III) sepulchrate (Naf-Co(Sep){sup 3+}) composite film on a glassy carbon (GC) electrode. The responses of the biosensor to 2,4-dichlorophenol, erythromycin (CYP3A4 native substrate) and ketoconazole (CYP 3A4 natural inhibitor) were studied by cyclic and square wave voltammetric techniques. The detection limit (DL) of the biosensor for 2,4-dichlorophenol was 0.043 {mu}g L{sup -1}, which is by an order of magnitude lower than the EU limit (0.3 {mu}g L{sup -1}) for any pesticide compound in ground water. The biosensor's DL is lower than the U.S. Environmental Protection Agency's drinking water equivalent level (DWEL) value for 2,4-DCP, which is 2 {mu}g L{sup -1}.

  9. Unraveling of the polymorphic C lambda 2-C lambda 3 amplification and the Ke+Oz- polymorphism in the human Ig lambda locus

    NARCIS (Netherlands)

    M. van der Burg (Mirjam); B.H. Barendregt (Barbara); E.J. van Gastel-Mol (Ellen); T. Tümkaya (Talip); A.W. Langerak (Anton); J.J.M. van Dongen (Jacques)

    2002-01-01

    textabstractTwo polymorphisms of the human Ig(lambda) (IGL) locus have been described. The first polymorphism concerns a single, 2- or 3-fold amplification of 5.4 kb of DNA in the C(lambda)2-C(lambda)3 region. The second polymorphism is the Mcg(-)Ke(+)Oz(-) isotype, which has

  10. Biochemical and structural characterization of CYP109A2, a vitamin D3 25-hydroxylase from Bacillus megaterium.

    Science.gov (United States)

    Abdulmughni, Ammar; Jóźwik, Ilona K; Brill, Elisa; Hannemann, Frank; Thunnissen, Andy-Mark W H; Bernhardt, Rita

    2017-11-01

    Cytochrome P450 enzymes are increasingly investigated due to their potential application as biocatalysts with high regio- and/or stereo-selectivity and under mild conditions. Vitamin D 3 (VD 3 ) metabolites are of pharmaceutical importance and are applied for the treatment of VD 3 deficiency and other disorders. However, the chemical synthesis of VD 3 derivatives shows low specificity and low yields. In this study, cytochrome P450 CYP109A2 from Bacillus megaterium DSM319 was expressed, purified, and shown to oxidize VD 3 with high regio-selectivity. The in vitro conversion, using cytochrome P450 reductase (BmCPR) and ferredoxin (Fdx2) from the same strain, showed typical Michaelis-Menten reaction kinetics. A whole-cell system in B. megaterium overexpressing CYP109A2 reached 76 ± 5% conversion after 24 h and allowed to identify the main product by NMR analysis as 25-hydroxylated VD 3 . Product yield amounted to 54.9 mg·L -1 ·day -1 , rendering the established whole-cell system as a highly promising biocatalytic route for the production of this valuable metabolite. The crystal structure of substrate-free CYP109A2 was determined at 2.7 Å resolution, displaying an open conformation. Structural analysis predicts that CYP109A2 uses a highly similar set of residues for VD 3 binding as the related VD 3 hydroxylases CYP109E1 from B. megaterium and CYP107BR1 (Vdh) from Pseudonocardia autotrophica. However, the folds and sequences of the BC loops in these three P450s are highly divergent, leading to differences in the shape and apolar/polar surface distribution of their active site pockets, which may account for the observed differences in substrate specificity and the regio-selectivity of VD 3 hydroxylation. The atomic coordinates and structure factors have been deposited in the Protein Data Bank with accession code 5OFQ (substrate-free CYP109A2). Cytochrome P450 monooxygenase CYP109A2, EC 1.14.14.1, UniProt ID: D5DF88, Ferredoxin, UniProt ID: D5DFQ0, cytochrome P450

  11. UV-dependent production of 25-hydroxyvitamin D{sub 2} in the recombinant yeast cells expressing human CYP2R1

    Energy Technology Data Exchange (ETDEWEB)

    Yasuda, Kaori; Endo, Mariko; Ikushiro, Shinichi; Kamakura, Masaki [Department of Biotechnology, Faculty of Engineering, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398 (Japan); Ohta, Miho [Department of Food and Nutrition Management Studies, Faculty of Human Development, Soai University, 4-4-1 Nanko-naka, Suminoe-ku, Osaka 559-0033 (Japan); Sakaki, Toshiyuki, E-mail: tsakaki@pu-toyama.ac.jp [Department of Biotechnology, Faculty of Engineering, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398 (Japan)

    2013-05-03

    Highlights: •We produce 25-hydroxyvitamin D in the recombinant yeast expressing human CYP2R1. •Vitamin D2 is produced in yeast from endogenous ergosterol with UV irradiation. •We produce 25-hydroxyvitamin D2 in the recombinant yeast without added substrate. -- Abstract: CYP2R1 is known to be a physiologically important vitamin D 25-hydroxylase. We have successfully expressed human CYP2R1 in Saccharomyces cerevisiae to reveal its enzymatic properties. In this study, we examined production of 25-hydroxylated vitamin D using whole recombinant yeast cells that expressed CYP2R1. When vitamin D{sub 3} or vitamin D{sub 2} was added to the cell suspension of CYP2R1-expressing yeast cells in a buffer containing glucose and β-cyclodextrin, the vitamins were converted into their 25-hydroxylated products. Next, we irradiated the cell suspension with UVB and incubated at 37 °C. Surprisingly, the 25-hydroxy vitamin D{sub 2} was produced without additional vitamin D{sub 2}. Endogenous ergosterol was likely converted into vitamin D{sub 2} by UV irradiation and thermal isomerization, and then the resulting vitamin D{sub 2} was converted to 25-hydroxyvitamin D{sub 2} by CYP2R1. This novel method for producing 25-hydroxyvitamin D{sub 2} without a substrate could be useful for practical purposes.

  12. Modeling Chemical Interaction Profiles: I. Spectral Data-Activity Relationship and Structure-Activity Relationship Models for Inhibitors and Non-inhibitors of Cytochrome P450 CYP3A4 and CYP2D6 Isozymes

    Directory of Open Access Journals (Sweden)

    Richard D. Beger

    2012-03-01

    techniques, providing an independent estimator that can increase confidence in a structure-activity assessment. When modeling was applied to hazardous environmental chemicals, it was found that up to 20% of them may be substrates and up to 10% of them may be inhibitors of the CYP3A4 and CYP2D6 isoforms. The developed models provide a rare opportunity for the environmental health branch of the public health service to extrapolate to hazardous chemicals directly from human clinical data. Therefore, the pharmacological and environmental health branches are both expected to benefit from these reported models.

  13. Impact of inhalational exposure to ethanol fuel on the pharmacokinetics of verapamil, ibuprofen and fluoxetine as in vivo probe drugs for CYP3A, CYP2C and CYP2D in rats.

    Science.gov (United States)

    Cardoso, Juciane Lauren Cavalcanti; Lanchote, Vera Lucia; Pereira, Maria Paula Marques; Capela, Jorge Manuel Vieira; de Moraes, Natália Valadares; Lepera, José Salvador

    2015-10-01

    Occupational toxicology and clinical pharmacology integration will be useful to understand potential exposure-drug interaction and to shape risk assessment strategies in order to improve occupational health. The aim of the present study was to evaluate the effect of exposure to ethanol fuel on in vivo activities of cytochrome P450 (CYP) isoenzymes CYP3A, CYP2C and CYP2D by the oral administration of the probe drugs verapamil, ibuprofen and fluoxetine. Male Wistar rats exposed to filtered air or to 2000 ppm ethanol in a nose-only inhalation chamber during (6 h/day, 5 days/week, 6 weeks) received single oral doses of 10 mg/kg verapamil or 25 mg/kg ibuprofen or 10 mg/kg fluoxetine. The enantiomers of verapamil, norverapamil, ibuprofen and fluoxetine in plasma were analyzed by LC-MS/MS. The area under the curve plasma concentration versus time extrapolated to infinity (AUC(0-∞)) was calculated using the Gauss-Laguerre quadrature. Inhalation exposure to ethanol reduces the AUC of both verapamil (approximately 2.7 fold) and norverapamil enantiomers (>2.5 fold), reduces the AUC(0-∞) of (+)-(S)-IBU (approximately 2 fold) and inhibits preferentially the metabolism of (-)-(R)-FLU. In conclusion, inhalation exposure of ethanol at a concentration of 2 TLV-STEL (6 h/day for 6 weeks) induces CYP3A and CYP2C but inhibits CYP2D in rats. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. Contrasting exome constancy and regulatory region variation in the gene encoding CYP3A4: an examination of the extent and potential implications.

    Science.gov (United States)

    Creemer, Olivia J; Ansari-Pour, Naser; Ekong, Rosemary; Tarekegn, Ayele; Plaster, Christopher; Bains, Ripudaman K; Itan, Yuval; Bekele, Endashaw; Bradman, Neil

    2016-06-01

    CYP3A4 expression varies up to 100-fold among individuals, and, to date, genetic causes remain elusive. As a major drug-metabolizing enzyme, elucidation of such genetic causes would increase the potential for introducing personalized dose adjustment of therapies involving CYP3A4 drug substrates. The foetal CYP3A isoform, CYP3A7, is reported to be expressed in ∼10% of European adults and may thus contribute towards the metabolism of endogenous substances and CYP3A drug substrates. However, little is known about the distribution of the variant expressed in the adult. We resequenced the exons, flanking introns, regulatory elements and 3'UTR of CYP3A4 in five Ethiopian populations and incorporated data from the 1000 Genomes Project. Using bioinformatic analysis, we assessed likely consequences of observed CYP3A4 genomic variation. We also conducted the first extensive geographic survey of alleles associated with adult expression of CYP3A7 - that is, CYP3A7*1B and CYP3A7*1C. Ethiopia contained 60 CYP3A4 variants (26 novel) and more variants (>1%) than all non-African populations combined. No nonsynonymous mutation was found in the homozygous form or at more than 2.8% in any population. Seventy-nine per cent of haplotypes contained 3'UTR and/or regulatory region variation with striking pairwise population differentiation, highlighting the potential for interethnic variation in CYP3A4 expression. Conversely, coding region variation showed that significant interethnic variation is unlikely at the protein level. CYP3A7*1C was found at up to 17.5% in North African populations and in significant linkage disequilibrium with CYP3A5*3, indicating that adult expression of the foetal isoform is likely to be accompanied by reduced or null expression of CYP3A5.

  15. Population pharmacokinetic modelling to assess the impact of CYP2D6 and CYP3A metabolic phenotypes on the pharmacokinetics of tamoxifen and endoxifen

    NARCIS (Netherlands)

    ter Heine, Rob; Binkhorst, Lisette; de Graan, Anne Joy M; de Bruijn, Peter; Beijnen, Jos H; Mathijssen, Ron H J; Huitema, Alwin D R

    AIMS: Tamoxifen is considered a pro-drug of its active metabolite endoxifen. The major metabolic enzymes involved in endoxifen formation are CYP2D6 and CYP3A. There is considerable evidence that variability in activity of these enzymes influences endoxifen exposure and thereby may influence the

  16. Effect of a new functional CYP3A4 polymorphism on calcineurin inhibitors' dose requirements and trough blood levels in stable renal transplant patients.

    Science.gov (United States)

    Elens, Laure; van Schaik, Ron H; Panin, Nadtha; de Meyer, Martine; Wallemacq, Pierre; Lison, Dominique; Mourad, Michel; Haufroid, Vincent

    2011-10-01

    CYP3A4 is involved in the oxidative metabolism of many drugs and xenobiotics including the immunosuppressants tacrolimus (Tac) and cyclosporine (CsA). The objective of the study was to assess the potential influence of a new functional SNP in CYP3A4 on the pharmacokinetic parameters assessed by dose requirements and trough blood levels of both calcineurin inhibitors (CNI) in stable renal transplant patients. A total of 99 stable renal transplant patients receiving either Tac (n = 49) or CsA (n = 50) were genotyped for the CYP3A4 intron 6 C>T (rs35599367) and CYP3A5*3 SNPs. Trough blood levels ([Tac](0) or [CsA](0) in ng/ml), dose-adjusted [Tac](0) or [CsA](0) (ng/ml per mg/kg bodyweight) as well as doses (mg/kg bodyweight) required to achieve target concentrations were compared among patients according to allelic status for CYP3A4 and CYP3A5. Dose-adjusted concentrations were 2.0- and 1.6-fold higher in T-variant allele carriers for the CYP3A4 intron 6 C>T SNP compared with homozygous CC for Tac and CsA, respectively. When CYP3A4/CYP3A5 genotypes were combined, the difference was even more striking as the so-defined CYP3A poor metabolizer group presented dose-adjusted concentration 1.6- and 4.1-fold higher for Tac, and 1.5- and 2.2-fold higher for CsA than the intermediate metabolizer and extensive metabolizer groups, respectively. Multiple linear regression analysis revealed that, taken together, both CYP3A4 intron 6 and CYP3A5*3 SNPs explained more than 60 and 20% of the variability observed in dose-adjusted [Tac](0) and [CsA](0), respectively. The CYP3A4 intron 6 C>T polymorphism is associated with altered Tac and CsA metabolism. CYP3A4 intron 6 C>T along with CYP3A5*3 (especially for Tac) pharmacogenetic testing performed just before transplantation may help identifying patients at risk of CNI overexposure and contribute to limit CNI-related nephrotoxicity by refining the starting dose according to their genotype. Original submitted 5 May 2011; Revision

  17. Regional mapping of the phenylalanine hydroxylase gene and the phenylketonuria locus in the human genome

    Energy Technology Data Exchange (ETDEWEB)

    Lidsky, A.S.; Law, M.L.; Morse, H.G.; Kao, F.T.; Rabin, M.; Ruddle, F.H.; Woo, S.L.C.

    1985-09-01

    Phenylketonuria (PKU) is an autosomal recessive disorder of amino acid metabolism caused by a deficiency of the hepatic enzyme phenylalanine hydroxylase. To define the regional map position of the disease locus and the PAH gene on human chromosome 12, DNA was isolated from human-hamster somatic cell hybrids with various deletions of human chromosome 12 and was analyzed by Southern blot analysis using the human cDNA PAH clone as a hybridization probe. From these results, together with detailed biochemical and cytogenetic characterization of the hybrid cells, the region on chromosome 12 containing the human PAH gene has been defined as 12q14.3..-->..qter. The PAH map position on chromosome 12 was further localized by in situ hybridization of /sup 125/I-labeled human PAH cDNA to chromosomes prepared from a human lymphoblastoid cell line. Results of these experiments demonstrated that the region on chromosome 12 containing the PAH gene and the PKU locus in man is 12q22..-->..12q24.1. These results not only provide a regionalized map position for a major human disease locus but also can serve as a reference point for linkage analysis with other DNA markers on human chromosome 12.

  18. Regional mapping of the phenylalanine hydroxylase gene and the phenylketonuria locus in the human genome

    International Nuclear Information System (INIS)

    Lidsky, A.S.; Law, M.L.; Morse, H.G.; Kao, F.T.; Rabin, M.; Ruddle, F.H.; Woo, S.L.C.

    1985-01-01

    Phenylketonuria (PKU) is an autosomal recessive disorder of amino acid metabolism caused by a deficiency of the hepatic enzyme phenylalanine hydroxylase. To define the regional map position of the disease locus and the PAH gene on human chromosome 12, DNA was isolated from human-hamster somatic cell hybrids with various deletions of human chromosome 12 and was analyzed by Southern blot analysis using the human cDNA PAH clone as a hybridization probe. From these results, together with detailed biochemical and cytogenetic characterization of the hybrid cells, the region on chromosome 12 containing the human PAH gene has been defined as 12q14.3→qter. The PAH map position on chromosome 12 was further localized by in situ hybridization of 125 I-labeled human PAH cDNA to chromosomes prepared from a human lymphoblastoid cell line. Results of these experiments demonstrated that the region on chromosome 12 containing the PAH gene and the PKU locus in man is 12q22→12q24.1. These results not only provide a regionalized map position for a major human disease locus but also can serve as a reference point for linkage analysis with other DNA markers on human chromosome 12

  19. The Absence of CYP3A5*3 Is a Protective Factor to Anticonvulsants Hypersensitivity Reactions: A Case-Control Study in Brazilian Subjects.

    Science.gov (United States)

    Tanno, Luciana Kase; Kerr, Daniel Shikanai; dos Santos, Bernardo; Talib, Leda Leme; Yamaguti, Célia; Rodrigues, Helcio; Gattaz, Wagner Farid; Kalil, Jorge

    2015-01-01

    Although aromatic anticonvulsants are usually well tolerated, they can cause cutaneous adverse drug reactions in up to 10% of patients. The clinical manifestations of the antiepileptics-induced hypersensitivity reactions (AHR) vary from mild skin rashes to severe cutaneous drug adverse reactions which are related to high mortality and significant morbidity. Genetic polymorphisms in cytochrome P450 genes are associated with altered enzymatic activity and may contribute to the risk of AHR. Here we present a case-control study in which we genotyped SNPs of CYP2C19, 2C9 and 3A5 of 55 individuals with varying severities of AHR, 83 tolerant, and 366 healthy control subjects from São Paulo, Brazil. Clinical characterization was based on standardized scoring systems and drug patch test. All in vivo investigation followed the ENDA (European Network of Drug Allergy) recommendations. Genotype was determined by real time PCR using peripheral blood DNA as a template. Of all 504 subjects, 65% were females, 45% self-identified as Afro-American, 38% as Caucasian and 17% as having non-African mixed ascendancy. Amongst 55 subjects with AHR, 44 had severe cutaneous drug adverse reactions. Of the 46 drug patch tests performed, 29 (63%) were positive. We found a strong association between the absence of CYP3A5*3 and tolerant subjects when compared to AHR (p = 0.0002, OR = 5.28 [CI95% 2.09-14.84]). None of our groups presented positive association with CYP2C19 and 2C9 polymorphisms, however, both SNPs contributed to separation of cases and tolerants in a Classification and Regression Tree. Our findings indicate that drug metabolism genes can contribute in the tolerability of antiepileptics. CYP3A5*3 is the most prevalent CYP3A5 allele associated with reduced enzymatic function. The current study provides evidence that normal CYP3A5 activity might be a protective factor to aromatic antiepileptics-induced hypersensitivity reactions in Brazilian subjects.

  20. Effect of CYP2C9*3 gene polymorphism on lipid-lowering efficacy of ...

    African Journals Online (AJOL)

    Purpose: To investigate the frequency of gene CYP2C9*3 in Chinese populations, and to analyze the impact of CYP2C9*3 genetic polymorphism on the cholesterol-lowering effect of fluvastatin in a Chinese hyperlipidemic population. Methods: CYP2C9 genotype was determined by polymerase chain reaction - restriction ...

  1. Specific locus mutagenesis of human mammary epithelial cells by ultraviolet radiation

    International Nuclear Information System (INIS)

    Eldridge, S.R.; Gould, M.N.

    1991-01-01

    Tissue and locus specificity of mutation induction was studied in human mammary epithelial cells (HMEC). Primary HMEC from normal tissue, and immortalized HMEC (184B5) derived from normal HMEC, were cultured under identical conditions and exposed to 10J/m 2 ultraviolet (UV) radiation (254 nm peak wavelength), which produced approximately 50% mean survival in all cell strains and lines tested. UV radiation was found to induce mutations at the Na + -K + ATPase locus as determined by ouabain-resistance in both normal and immortalized HMEC. Mutation frequencies measured in these cells following UV exposure were similar to those reported for human diploid fibroblasts. Mutation induction was investigated at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus in normal and immortalized HMEC. Induced mutations at the HPRT locus as determined by 6-thioguanine resistance in normal primary HMEC were not observed following UV radiation. Mutation induction was observed at this locus UV-exposed immortalized HMEC. (author)

  2. Identification of a novel locus for a USH3 like syndrome combined with congenital cataract.

    Science.gov (United States)

    Dad, S; Østergaard, E; Thykjaer, T; Albrectsen, A; Ravn, K; Rosenberg, T; Møller, L B

    2010-10-01

    Usher syndrome (USH) is the most common genetic disease that causes both deafness and blindness. USH is divided into three types, USH1, USH2 and USH3, depending on the age of onset, the course of the disease, and on the degree of vestibular dysfunction. By homozygosity mapping of a consanguineous Danish family of Dutch descent, we have identified a novel locus for a rare USH3-like syndrome. The affected family members have a unique association of retinitis pigmentosa, progressive hearing impairment, vestibular dysfunction, and congenital cataract. The phenotype is similar, but not identical to that of USH3 patients, as congenital cataract has not been reported for USH3. By homozygosity mapping, we identified a 7.3 Mb locus on chromosome 15q22.2-23 with a maximum multipoint LOD score of 2.0. The locus partially overlaps with the USH1 locus, USH1H, a novel unnamed USH2 locus, and the non-syndromic deafness locus DFNB48. © 2010 John Wiley & Sons A/S.

  3. Strong synergistic induction of CYP1A1 expression by andrographolide plus typical CYP1A inducers in mouse hepatocytes

    Energy Technology Data Exchange (ETDEWEB)

    Jaruchotikamol, Atika [Department of Toxicology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194 (Japan); Jarukamjorn, Kanokwan [Department of Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen 40002 (Thailand); Sirisangtrakul, Wanna [Department of Toxicology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194 (Japan); Department of Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen 40002 (Thailand); Sakuma, Tsutomu; Kawasaki, Yuki [Department of Toxicology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194 (Japan); Nemoto, Nobuo [Department of Toxicology, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194 (Japan)

    2007-10-15

    The effects of andrographolide, the major diterpenoid constituent of Andrographis paniculata, on the expression of cytochrome P450 superfamily 1 members, including CYP1A1, CYP1A2, and CYP1B1, as well as on aryl hydrocarbon receptor (AhR) expression in primary cultures of mouse hepatocytes were investigated in comparison with the effects of typical CYP1A inducers, including benz[a]anthracene, {beta}-naphthoflavone, and 2,3,7,8-tetrachlorodibenzo-p-dioxin. Andrographolide significantly induced the expression of CYP1A1 and CYP1A2 mRNAs in a concentration-dependent manner, as did the typical CYP1A inducers, but did not induce that of CYP1B1 or AhR. Interestingly, andrographolide plus the typical CYP1A inducers synergistically induced CYP1A1 expression, and the synergism was blocked by an AhR antagonist, resveratrol. The CYP1A1 enzyme activity showed a similar pattern of induction. This is the first report that shows that andrographolide has a potency to induce CYP1A1 enzyme and indicates that andrographolide could be a very useful compound for investigating the regulatory mechanism of the CYP1A1 induction pathway. In addition, our findings suggest preparing advice for rational administration of A. paniculata, according to its ability to induce CYP1A1 expression.

  4. Strong synergistic induction of CYP1A1 expression by andrographolide plus typical CYP1A inducers in mouse hepatocytes

    International Nuclear Information System (INIS)

    Jaruchotikamol, Atika; Jarukamjorn, Kanokwan; Sirisangtrakul, Wanna; Sakuma, Tsutomu; Kawasaki, Yuki; Nemoto, Nobuo

    2007-01-01

    The effects of andrographolide, the major diterpenoid constituent of Andrographis paniculata, on the expression of cytochrome P450 superfamily 1 members, including CYP1A1, CYP1A2, and CYP1B1, as well as on aryl hydrocarbon receptor (AhR) expression in primary cultures of mouse hepatocytes were investigated in comparison with the effects of typical CYP1A inducers, including benz[a]anthracene, β-naphthoflavone, and 2,3,7,8-tetrachlorodibenzo-p-dioxin. Andrographolide significantly induced the expression of CYP1A1 and CYP1A2 mRNAs in a concentration-dependent manner, as did the typical CYP1A inducers, but did not induce that of CYP1B1 or AhR. Interestingly, andrographolide plus the typical CYP1A inducers synergistically induced CYP1A1 expression, and the synergism was blocked by an AhR antagonist, resveratrol. The CYP1A1 enzyme activity showed a similar pattern of induction. This is the first report that shows that andrographolide has a potency to induce CYP1A1 enzyme and indicates that andrographolide could be a very useful compound for investigating the regulatory mechanism of the CYP1A1 induction pathway. In addition, our findings suggest preparing advice for rational administration of A. paniculata, according to its ability to induce CYP1A1 expression

  5. Association of polymorphisms in CYP19A1 and CYP3A4 genes with lower urinary tract symptoms, prostate volume, uroflow and PSA in a population-based sample.

    Science.gov (United States)

    Berges, Richard; Gsur, Andrea; Feik, Elisabeth; Höfner, Klaus; Senge, Theodor; Pientka, Ludger; Baierl, Andreas; Michel, Martin C; Ponholzer, Anton; Madersbacher, Stephan

    2011-04-01

    The known importance of testosterone for the development of benign prostatic hyperplasia (BPH) prompted us to test the hypothesis whether polymorphisms of two genes (CYP19A1 and CYP3A4) involved in testosterone metabolism are associated with clinical BPH-parameters. A random sample of the population-based Herne lower urinary tract symptoms cohort was analysed. All these men underwent a detailed urological work-up. Two polymorphisms in the CYP19A1 gene [rs700518 in exon 4 (A57G); rs10046 at the 3'UTR(C268T)] and one in the 3'UTR of CYP3A4 [rs2740574 (A392G)] were determined by TaqMan assay from genomic DNA of peripheral blood. These polymorphisms were correlated to clinical and laboratory BPH-parameters. A total of 392 men (65.4 ± 7.0 years; 52-79 years) were analysed. Mean International Prostate Symptom Score (IPSS; 7.5), Q (max) (15.4 ml/s), prostate volume (31 ml) and prostate specific antigen (PSA) (1.8 ng/ml) indicated a typical elderly population. Both polymorphisms in the CYP19A1 gene were not correlated to age, IPSS, Q (max), prostate volume and post-void residual volume. Serum PSA was higher in men carrying the heterozygous rs10046 genotype (2.0 ± 0.1 ng/ml) than in those with the CC-genotype (1.7 ± 0.2 ng/ml, P = 0.012). Men carrying one a mutated allele of the CYP3A4 gene had smaller prostates (27.0 ± 2.0 vs. 32 ± 0.8 ml, P = 0.02) and lower PSA levels (1.6 ± 0.3 vs. 1.9 ± 0.1 ng/ml). The inconsistent associations observed herein and for other gene polymorphisms warrant further studies. In general, the data regarding the association of gene polymorphism to BPH-parameters suggest that this disease is caused by multiple rather than a single genetic variant. A rigorous patient selection based on anatomo-pathological and hormonal profile may possible reduce the number of confounders for future studies thus enabling a more detailed assessment of the association between genetic factors and BPH-parameters.

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

    Science.gov (United States)

    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.

  7. Generation of a human iPSC line from a patient with congenital glaucoma caused by mutation in CYP1B1 gene

    Directory of Open Access Journals (Sweden)

    Arantxa Bolinches-Amorós

    2018-04-01

    Full Text Available The human iPSC cell line, GLC-FiPS4F1 (ESi047-A, derived from dermal fibroblast from the patient with congenital glaucoma caused by the mutation of the gene CYP1B1, was generated by non-integrative reprogramming technology using OCT3/4, SOX2, CMYC and KLF4 reprogramming factors.

  8. DFNB79: reincarnation of a nonsyndromic deafness locus on chromosome 9q34.3.

    Science.gov (United States)

    Khan, Shahid Yar; Riazuddin, Saima; Shahzad, Mohsin; Ahmed, Nazir; Zafar, Ahmad Usman; Rehman, Atteeq Ur; Morell, Robert J; Griffith, Andrew J; Ahmed, Zubair M; Riazuddin, Sheikh; Friedman, Thomas B

    2010-01-01

    Genetic analysis of an inbred Pakistani family PKDF280, segregating prelingual severe to profound sensorineural hearing loss, provided evidence for a DFNB locus on human chromosome 9q34.3. Co-segregation of the deafness trait with marker D9SH159 was determined by a two-point linkage analysis (LOD score 9.43 at theta=0). Two additional large families, PKDF517 and PKDF741, co-segregate recessive deafness with markers linked to the same interval. Haplotype analyses of these three families refined the interval to 3.84 Mb defined by D9S1818 (centromeric) and D9SH6 (telomeric). This interval overlaps with the previously reported DFNB33 locus whose chromosomal map position has been recently revised and assigned to a new position on chromosome 10p11.23-q21.1. The nonsyndromic deafness locus on chromosome 9q segregating in family PKDF280 was designated DFNB79. We are currently screening the 113 candidate DFNB79 genes for mutations and have excluded CACNA1B, EDF1, PTGDS, EHMT1, QSOX2, NOTCH1, MIR126 and MIR602.

  9. Model based on GRID-derived descriptors for estimating CYP3A4 enzyme stability of potential drug candidates

    Science.gov (United States)

    Crivori, Patrizia; Zamora, Ismael; Speed, Bill; Orrenius, Christian; Poggesi, Italo

    2004-03-01

    A number of computational approaches are being proposed for an early optimization of ADME (absorption, distribution, metabolism and excretion) properties to increase the success rate in drug discovery. The present study describes the development of an in silico model able to estimate, from the three-dimensional structure of a molecule, the stability of a compound with respect to the human cytochrome P450 (CYP) 3A4 enzyme activity. Stability data were obtained by measuring the amount of unchanged compound remaining after a standardized incubation with human cDNA-expressed CYP3A4. The computational method transforms the three-dimensional molecular interaction fields (MIFs) generated from the molecular structure into descriptors (VolSurf and Almond procedures). The descriptors were correlated to the experimental metabolic stability classes by a partial least squares discriminant procedure. The model was trained using a set of 1800 compounds from the Pharmacia collection and was validated using two test sets: the first one including 825 compounds from the Pharmacia collection and the second one consisting of 20 known drugs. This model correctly predicted 75% of the first and 85% of the second test set and showed a precision above 86% to correctly select metabolically stable compounds. The model appears a valuable tool in the design of virtual libraries to bias the selection toward more stable compounds. Abbreviations: ADME - absorption, distribution, metabolism and excretion; CYP - cytochrome P450; MIFs - molecular interaction fields; HTS - high throughput screening; DDI - drug-drug interactions; 3D - three-dimensional; PCA - principal components analysis; CPCA - consensus principal components analysis; PLS - partial least squares; PLSD - partial least squares discriminant; GRIND - grid independent descriptors; GRID - software originally created and developed by Professor Peter Goodford.

  10. A case report of a patient carrying CYP2C9*3/4 genotype with extremely low warfarin dose requirement.

    Science.gov (United States)

    Lee, Soo Youn; Nam, Myung Hyun; Kim, June Soo; Kim, Jong Won

    2007-06-01

    We report a case of intolerance to warfarin dosing due to impaired drug metabolism in a patient with CYP2C9*3/*4. A 73-yr-old woman with atrial fibrilation was taking warfarin. She attained a high prothrombin time international normalized ratio (INR) at the standard doses during the induction of anticoagulation and extremely low dose of warfarin (6.5 mg/week) was finally chosen to reach the target INR. Genotyping for CYP2C9 revealed that this patient had a genotype CYP2C9*3/*4. This is the first Korean compound heterozygote for CYP2C9*3 and *4. This case suggests the clinical usefulness of pharmacogenetic testing for individualized dosage adjustments of warfarin.

  11. Evaluation of the Effects of S-Allyl-L-cysteine, S-Methyl-L-cysteine, trans-S-1-Propenyl-L-cysteine, and Their N-Acetylated and S-Oxidized Metabolites on Human CYP Activities.

    Science.gov (United States)

    Amano, Hirotaka; Kazamori, Daichi; Itoh, Kenji

    2016-01-01

    Three major organosulfur compounds of aged garlic extract, S-allyl-L-cysteine (SAC), S-methyl-L-cysteine (SMC), and trans-S-1-propenyl-L-cysteine (S1PC), were examined for their effects on the activities of five major isoforms of human CYP enzymes: CYP1A2, 2C9, 2C19, 2D6, and 3A4. The metabolite formation from probe substrates for the CYP isoforms was examined in human liver microsomes in the presence of organosulfur compounds at 0.01-1 mM by using liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Allicin, a major component of garlic, inhibited CYP1A2 and CYP3A4 activity by 21-45% at 0.03 mM. In contrast, a CYP2C9-catalyzed reaction was enhanced by up to 1.9 times in the presence of allicin at 0.003-0.3 mM. SAC, SMC, and S1PC had no effect on the activities of the five isoforms, except that S1PC inhibited CYP3A4-catalyzed midazolam 1'-hydroxylation by 31% at 1 mM. The N-acetylated metabolites of the three compounds inhibited the activities of several isoforms to a varying degree at 1 mM. N-Acetyl-S-allyl-L-cysteine and N-acetyl-S-methyl-L-cysteine inhibited the reactions catalyzed by CYP2D6 and CYP1A2, by 19 and 26%, respectively, whereas trans-N-acetyl-S-1-propenyl-L-cysteine showed weak to moderate inhibition (19-49%) of CYP1A2, 2C19, 2D6, and 3A4 activities. On the other hand, both the N-acetylated and S-oxidized metabolites of SAC, SMC, and S1PC had little effect on the reactions catalyzed by the five isoforms. These results indicated that SAC, SMC, and S1PC have little potential to cause drug-drug interaction due to CYP inhibition or activation in vivo, as judged by their minimal effects (IC 50 >1 mM) on the activities of five major isoforms of human CYP in vitro.

  12. Pharmacokinetics of diclofenac and inhibition of cyclooxygenases 1 and 2: no relationship to the CYP2C9 genetic polymorphism in humans

    Science.gov (United States)

    Kirchheiner, Julia; Meineke, Ingolf; Steinbach, Nadine; Meisel, Christian; Roots, Ivar; Brockmöller, Jürgen

    2003-01-01

    Aims The cytochrome P450 enzyme CYP2C9 catalyses the 4′-hydroxylation of the nonsteroidal analgesic drug diclofenac in humans. We studied the influences of the known amino acid variants, CYP2C9*2 (Arg144Cys) and CYP2C9*3 (Ile359Leu), on diclofenac pharmacokinetics after a 50-mg oral dose of diclofenac in healthy volunteers. As a surrogate marker of diclofenac activity, the ex vivo formation of prostaglandin E2 and thromboxane B2, which reflects COX-2 and COX-1 activity, was measured. Methods Genotyping was performed in 516 healthy volunteers to obtain 20 participants with all allelic combinations of the two CYP2C9 variants Arg144Cys (*2) and Ile359Leu (*3). Diclofenac and 4′-hydroxydiclofenac were quantified in plasma by reversed phase h.p.l.c. after oral intake of 50 mg diclofenac. Concentrations of thromboxane B2 (TxB2) and prostaglandin E2 (PGE2) were measured by immunoassays. Results There was no evidence of impaired metabolism of oral diclofenac in heterozygous and homozygous carriers of the CYP2C9 alleles *2 and *3 compared with the wild type (mean CL/F (95% CI) 20.5 (11, 30) l h−1 for *1/*1, 29.9 (19, 40) l h−1 for *1/*2, 30.0 (4, 56) l h−1 for *2/*2, 22.6 (12, 33) l h−1 for *1/*3, 23.5 (11, 37) l h−1 for *3/*3 and 37.3 (−15, 89) l h−1 in *2/*3). Furthermore, plasma concentrations of the metabolite 4′-hydroxydiclofenac were not lower in carriers of the CYP2C9 low-activity alleles *2 and *3 compared with carriers of the CYP2C9*1/*1 genotype. Marked diclofenac mediated inhibition of COX-1- and COX-2 activity was detected in all individuals independent of CYP2C9 genotype. Conclusions Polymorphisms of the CYP2C9 gene had no discernible effect on the pharmacokinetics and pharmacodynamics of diclofenac. The question of whether enzymes other than CYP2C9 play a major role in diclofenac 4′-hydroxylation in vivo or whether 4′-hydroxylation is not a rate-limiting step in diclofenac elimination in vivo, or whether the effect of the CYP2C9

  13. Possible involvement of pregnane X receptor–enhanced CYP24 expression in drug-induced osteomalacia

    Science.gov (United States)

    Pascussi, Jean Marc; Robert, Agnes; Nguyen, Minh; Walrant-Debray, Odile; Garabedian, Michèle; Martin, Pascal; Pineau, Thierry; Saric, Jean; Navarro, Fréderic; Maurel, Patrick; Vilarem, Marie Josè

    2005-01-01

    Vitamin D controls calcium homeostasis and the development and maintenance of bones through vitamin D receptor activation. Prolonged therapy with rifampicin or phenobarbital has been shown to cause vitamin D deficiency or osteomalacia, particularly in patients with marginal vitamin D stores. However, the molecular mechanism of this process is unknown. Here we show that these drugs lead to the upregulation of 25-hydroxyvitamin D3-24-hydroxylase (CYP24) gene expression through the activation of the nuclear receptor pregnane X receptor (PXR; NR1I2). CYP24 is a mitochondrial enzyme responsible for inactivating vitamin D metabolites. CYP24 mRNA is upregulated in vivo in mice by pregnenolone 16α-carbonitrile and dexamethasone, 2 murine PXR agonists, and in vitro in human hepatocytes by rifampicin and hyperforin, 2 human PXR agonists. Moreover, rifampicin increased 24-hydroxylase activity in these cells, while, in vivo in mice, pregnenolone 16α-carbonitrile increased the plasma concentration of 24,25-dihydroxyvitamin D3. Transfection of PXR in human embryonic kidney cells resulted in rifampicin-mediated induction of CYP24 mRNA. Analysis of the human CYP24 promoter showed that PXR transactivates the sequence between –326 and –142. We demonstrated that PXR binds to and transactivates the 2 proximal vitamin D–responsive elements of the human CYP24 promoter. These data suggest that xenobiotics and drugs can modulate CYP24 gene expression and alter vitamin D3 hormonal activity and calcium homeostasis through the activation of PXR. PMID:15630458

  14. Association of CYP3A polymorphisms with the pharmacokinetics of cyclosporine A in early post-renal transplant recipients in China

    Institute of Scientific and Technical Information of China (English)

    Xiang-guang MENG; Ji-ye YIN; Xiang-dong PENG; Qi PEI; Wei ZHANG; Guo WANG; Meng HE; Min LIU; Jing-ke YANG; Hong-hao ZHOU; Cheng-xian GUO; Guo-qing FENG; Ying-chun ZHAO; Bo-ting ZHOU; Jian-le HAN; Xin CHEN; Yong SHI; Hong-yao SHI

    2012-01-01

    Aim: To evaluate retrospectively the association of cytochrome P450 3A (CYP3A) and ATP-binding cassette sub-family B member 1(ABCB1) gene polymorphisms with the pharmacokinetics of cyclosporine A (CsA) in Chinese renal transplant patients.Methods: One hundred and twenty-six renal transplant patients were recruited.Blood samples were collected,and corresponding clinical indices were recorded on the seventh day after the procedure.The patients were genotyped for CYP3A4*1G,CYP3A5*3C,ABCB1 1236 C>T,ABCB1 2677 G>T/A,and ABCB1 3435 C>T polymorphisms.Whole blood trough concentrations of CsA at time zero (Co)were measured before the drug administration.A multiple regression model was developed to analyze the effects of genetic factors on the CsA dose-adjusted Co (Co/dose) based on several clinical indices.Results: The CYP3A5*3C polymorphism influenced the Co and Co/dose of CsA,which were significantly higher in patients with the GG genotype than in patients with the AA or GA genotypes.No significant differences were detected for other SNPs (CYP3A4*1G,ABCB1 1236 C>T,ABCB1 2677 G>T/A,and ABCB1 3435 C>T).In a univariate analysis using Pearson's correlation test,age,hemoglobin,blood urea nitrogen and blood creatinine levels were significantly correlated with the log-transformed CsA Co/dose.In the multiple regression model,CYP3A5*3C,age,hemoglobin and blood creatinine level were associated with the log-transformed CsA Co/dose.Conclusion: CYP3A5*3C correlates with the Co/dose of CsA on the seventh day after renal transplantation.The allele is a putative indicator for the optimal CsA dosage in the early phase of renal transplantation in the Chinese population.

  15. Characterization of the genetic variation present in CYP3A4 in three South African populations

    OpenAIRE

    Drögemöller, Britt; Plummer, Marieth; Korkie, Lundi; Agenbag, Gloudi; Dunaiski, Anke; Niehaus, Dana; Koen, Liezl; Gebhardt, Stefan; Schneider, Nicol; Olckers, Antonel; Wright, Galen; Warnich, Louise

    2013-01-01

    TThe CYP3A4 enzyme is the most abundant human cytochrome P450 and is regarded as the most important enzyme involved in drug metabolism. Inter-individual and inter-population variability in gene expression and enzyme activity are thought to be influenced, in part, by genetic variation. Although Southern African individuals have been shown to exhibit the highest levels of genetic diversity, they have been under-represented in pharmacogenetic research to date. Therefore, the aim of this study wa...

  16. Identification of a novel locus for a USH3 like syndrome combined with congenital cataract

    DEFF Research Database (Denmark)

    Dad, S.; Østergaard, Elsebet; Thykjær, T.

    2010-01-01

    Usher syndrome (USH) is the most common genetic disease that causes both deafness and blindness. USH is divided into three types, USH1, USH2 and USH3, depending on the age of onset, the course of the disease, and on the degree of vestibular dysfunction. By homozygosity mapping of a consanguineous...... Danish family of Dutch descent, we have identified a novel locus for a rare USH3-like syndrome. The affected family members have a unique association of retinitis pigmentosa, progressive hearing impairment, vestibular dysfunction, and congenital cataract. The phenotype is similar, but not identical...... to that of USH3 patients, as congenital cataract has not been reported for USH3. By homozygosity mapping, we identified a 7.3 Mb locus on chromosome 15q22.2-23 with a maximum multipoint LOD score of 2.0. The locus partially overlaps with the USH1 locus, USH1H, a novel unnamed USH2 locus, and the non-syndromic...

  17. Impact of the CYP2C8 *3 polymorphism on the drug-drug interaction between gemfibrozil and pioglitazone.

    Science.gov (United States)

    Aquilante, Christina L; Kosmiski, Lisa A; Bourne, David W A; Bushman, Lane R; Daily, Elizabeth B; Hammond, Kyle P; Hopley, Charles W; Kadam, Rajendra S; Kanack, Alexander T; Kompella, Uday B; Le, Merry; Predhomme, Julie A; Rower, Joseph E; Sidhom, Maha S

    2013-01-01

    The objective of this study was to determine the extent to which the CYP2C8*3 allele influences pharmacokinetic variability in the drug-drug interaction between gemfibrozil (CYP2C8 inhibitor) and pioglitazone (CYP2C8 substrate). In this randomized, two phase crossover study, 30 healthy Caucasian subjects were enrolled based on CYP2C8*3 genotype (n = 15, CYP2C8*1/*1; n = 15, CYP2C8*3 carriers). Subjects received a single 15 mg dose of pioglitazone or gemfibrozil 600 mg every 12 h for 4 days with a single 15 mg dose of pioglitazone administered on the morning of day 3. A 48 h pharmacokinetic study followed each pioglitazone dose and the study phases were separated by a 14 day washout period. Gemfibrozil significantly increased mean pioglitazone AUC(0,∞) by 4.3-fold (P gemfibrozil administration was significantly influenced by CYP2C8 genotype. Specifically, CYP2C8*3 carriers had a 5.2-fold mean increase in pioglitazone AUC(0,∞) compared with a 3.3-fold mean increase in CYP2C8*1 homozygotes (P = 0.02). CYP2C8*3 is associated with decreased pioglitazone plasma exposure in vivo and significantly influences the pharmacokinetic magnitude of the gemfibrozil-pioglitazone drug-drug interaction. Additional studies are needed to evaluate the impact of CYP2C8 genetics on the pharmacokinetics of other CYP2C8-mediated drug-drug interactions. © 2012 The Authors. British Journal of Clinical Pharmacology © 2012 The British Pharmacological Society.

  18. Impact of the CYP2C8 *3 polymorphism on the drug–drug interaction between gemfibrozil and pioglitazone

    Science.gov (United States)

    Aquilante, Christina L; Kosmiski, Lisa A; Bourne, David W A; Bushman, Lane R; Daily, Elizabeth B; Hammond, Kyle P; Hopley, Charles W; Kadam, Rajendra S; Kanack, Alexander T; Kompella, Uday B; Le, Merry; Predhomme, Julie A; Rower, Joseph E; Sidhom, Maha S

    2013-01-01

    AIM The objective of this study was to determine the extent to which the CYP2C8*3 allele influences pharmacokinetic variability in the drug–drug interaction between gemfibrozil (CYP2C8 inhibitor) and pioglitazone (CYP2C8 substrate). METHODS In this randomized, two phase crossover study, 30 healthy Caucasian subjects were enrolled based on CYP2C8*3 genotype (n = 15, CYP2C8*1/*1; n = 15, CYP2C8*3 carriers). Subjects received a single 15 mg dose of pioglitazone or gemfibrozil 600 mg every 12 h for 4 days with a single 15 mg dose of pioglitazone administered on the morning of day 3. A 48 h pharmacokinetic study followed each pioglitazone dose and the study phases were separated by a 14 day washout period. RESULTS Gemfibrozil significantly increased mean pioglitazone AUC(0,∞) by 4.3-fold (P gemfibrozil administration was significantly influenced by CYP2C8 genotype. Specifically, CYP2C8*3 carriers had a 5.2-fold mean increase in pioglitazone AUC(0,∞) compared with a 3.3-fold mean increase in CYP2C8*1 homozygotes (P= 0.02). CONCLUSION CYP2C8*3 is associated with decreased pioglitazone plasma exposure in vivo and significantly influences the pharmacokinetic magnitude of the gemfibrozil–pioglitazone drug-drug interaction. Additional studies are needed to evaluate the impact of CYP2C8 genetics on the pharmacokinetics of other CYP2C8-mediated drug–drug interactions. PMID:22625877

  19. Genome scan identifies a locus affecting gamma-globin expression in human beta-cluster YAC transgenic mice

    Energy Technology Data Exchange (ETDEWEB)

    Lin, S.D.; Cooper, P.; Fung, J.; Weier, H.U.G.; Rubin, E.M.

    2000-03-01

    Genetic factors affecting post-natal g-globin expression - a major modifier of the severity of both b-thalassemia and sickle cell anemia, have been difficult to study. This is especially so in mice, an organism lacking a globin gene with an expression pattern equivalent to that of human g-globin. To model the human b-cluster in mice, with the goal of screening for loci affecting human g-globin expression in vivo, we introduced a human b-globin cluster YAC transgene into the genome of FVB mice . The b-cluster contained a Greek hereditary persistence of fetal hemoglobin (HPFH) g allele resulting in postnatal expression of human g-globin in transgenic mice. The level of human g-globin for various F1 hybrids derived from crosses between the FVB transgenics and other inbred mouse strains was assessed. The g-globin level of the C3HeB/FVB transgenic mice was noted to be significantly elevated. To map genes affecting postnatal g-globin expression, a 20 centiMorgan (cM) genome scan of a C3HeB/F VB transgenics [prime] FVB backcross was performed, followed by high-resolution marker analysis of promising loci. From this analysis we mapped a locus within a 2.2 cM interval of mouse chromosome 1 at a LOD score of 4.2 that contributes 10.4% of variation in g-globin expression level. Combining transgenic modeling of the human b-globin gene cluster with quantitative trait analysis, we have identified and mapped a murine locus that impacts on human g-globin expression in vivo.

  20. Contrasting exome constancy and regulatory region variation in the gene encoding CYP3A4: an examination of the extent and potential implications.

    OpenAIRE

    Creemer, O. J.; Ansari-Pour, N.; Ekong, R.; Tarekegn, A.; Plaster, C.; Bains, R. K.; Itan, Y.; Bekele, E.; Bradman, N.

    2016-01-01

    OBJECTIVE: CYP3A4 expression varies up to 100-fold among individuals, and, to date, genetic causes remain elusive. As a major drug-metabolizing enzyme, elucidation of such genetic causes would increase the potential for introducing personalized dose adjustment of therapies involving CYP3A4 drug substrates. The foetal CYP3A isoform, CYP3A7, is reported to be expressed in ∼10% of European adults and may thus contribute towards the metabolism of endogenous substances and CYP3A drug substrates. H...

  1. Role of aryl hydrocarbon receptor polymorphisms on TCDD-mediated CYP1B1 induction and IgM suppression by human B cells

    Energy Technology Data Exchange (ETDEWEB)

    Kovalova, Natalia, E-mail: kovalova@msu.edu [Department of Pharmacology and Toxicology, Michigan State University, Lansing, MI 48824 (United States); Institute for Integrative Toxicology, Michigan State University, Lansing, MI 48824 (United States); Manzan, Maria, E-mail: ale.manzan@gmail.com [Institute for Integrative Toxicology, Michigan State University, Lansing, MI 48824 (United States); Crawford, Robert, E-mail: crawfo28@msu.edu [Institute for Integrative Toxicology, Michigan State University, Lansing, MI 48824 (United States); Kaminski, Norbert, E-mail: kamins11@msu.edu [Department of Pharmacology and Toxicology, Michigan State University, Lansing, MI 48824 (United States); Institute for Integrative Toxicology, Michigan State University, Lansing, MI 48824 (United States)

    2016-10-15

    Previous studies have demonstrated that most of the intraspecies variation in sensitivity to the toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), including suppression of antibody responses, in murine models is due to single nucleotide polymorphisms (SNPs) within the aryl hydrocarbon receptor (AhR) gene. The underlying reason for variation in sensitivity to TCDD-induced suppression of IgM responses among humans is not well understood, but is thought, in part, to be a result of different polymorphic forms of the AhR expressed by different individuals. In this study, the functional properties of six (P517S, R554K, V570I, V570I + P517S, R554K + V570I and P517S + R554K + V570I) human AhR variants were examined in the human B cell line, SKW 6.4. TCDD-induced Cyp1B1 and Cyp1A2 mRNA expression levels and Cyp1B1-regulated reporter gene activity, used for comparative purposes, were markedly lower in SKW cells containing the R554K SNP than in SKW-AHR{sup +} (control AhR) cells. Furthermore, all AhR variants were able to mediate TCDD-induced suppression of the IgM response; however, a combined P517S + R554K + V570I variant partially reduced sensitivity to TCDD-mediated suppression of IgM secretion. Collectively, our findings show that the R554K human AhR SNP alone altered sensitivity of human B cells to TCDD-mediated induction of Cyp1B1 and Cyp1A2. By contrast, attenuation of TCDD-induced IgM suppression required a combination of all three SNPs P517S, R554K, and V570I. - Highlights: • Mouse, rat and SKW-AHR{sup +} B cells have a similar window of sensitivity to TCDD. • R554K AhR SNP alters B cell sensitivity to TCDD-mediated Cyp1B1 and Cyp1A2 induction. • Combination of P517S, R554K, and V570I SNPs attenuates TCDD-induced IgM suppression.

  2. Insights into CYP2B6-mediated drug–drug interactions

    Directory of Open Access Journals (Sweden)

    William D. Hedrich

    2016-09-01

    Full Text Available Mounting evidence demonstrates that CYP2B6 plays a much larger role in human drug metabolism than was previously believed. The discovery of multiple important substrates of CYP2B6 as well as polymorphic differences has sparked increasing interest in the genetic and xenobiotic factors contributing to the expression and function of the enzyme. The expression of CYP2B6 is regulated primarily by the xenobiotic receptors constitutive androstane receptor (CAR and pregnane X receptor (PXR in the liver. In addition to CYP2B6, these receptors also mediate the inductive expression of CYP3A4, and a number of important phase II enzymes and drug transporters. CYP2B6 has been demonstrated to play a role in the metabolism of 2%–10% of clinically used drugs including widely used antineoplastic agents cyclophosphamide and ifosfamide, anesthetics propofol and ketamine, synthetic opioids pethidine and methadone, and the antiretrovirals nevirapine and efavirenz, among others. Significant inter-individual variability in the expression and function of the human CYP2B6 gene exists and can result in altered clinical outcomes in patients receiving treatment with CYP2B6-substrate drugs. These variances arise from a number of sources including genetic polymorphism, and xenobiotic intervention. In this review, we will provide an overview of the key players in CYP2B6 expression and function and highlight recent advances made in assessing clinical ramifications of important CYP2B6-mediated drug–drug interactions.

  3. 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.

  4. Hypoxia perturbs aryl hydrocarbon receptor signaling and CYP1A1 expression induced by PCB 126 in human skin and liver-derived cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Vorrink, Sabine U. [Interdisciplinary Graduate Program in Human Toxicology, The University of Iowa, Iowa City, IA (United States); Department of Radiation Oncology, The University of Iowa, Iowa City, IA (United States); Severson, Paul L. [Department of Pharmacology and Toxicology, The University of Arizona, Tucson, AZ (United States); Kulak, Mikhail V. [Department of Surgery, The University of Iowa, Iowa City, IA (United States); Futscher, Bernard W. [Department of Pharmacology and Toxicology, The University of Arizona, Tucson, AZ (United States); Domann, Frederick E., E-mail: frederick-domann@uiowa.edu [Interdisciplinary Graduate Program in Human Toxicology, The University of Iowa, Iowa City, IA (United States); Department of Radiation Oncology, The University of Iowa, Iowa City, IA (United States); Department of Surgery, The University of Iowa, Iowa City, IA (United States)

    2014-02-01

    The aryl hydrocarbon receptor (AhR) is an important mediator of toxic responses after exposure to xenobiotics including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and dioxin-like polychlorinated biphenyls (PCBs). Activation of AhR responsive genes requires AhR dimerization with the aryl hydrocarbon receptor nuclear translocator (ARNT), a heterodimeric partner also shared by the hypoxia-inducible factor-1α (HIF-1α) protein. TCDD-stimulated AhR transcriptional activity can be influenced by hypoxia; however, it less well known whether hypoxia interferes with AhR transcriptional transactivation in the context of PCB-mediated AhR activation in human cells. Elucidation of this interaction is important in liver hepatocytes which extensively metabolize ingested PCBs and experience varying degrees of oxygen tension during normal physiologic function. This study was designed to assess the effect of hypoxia on AhR transcriptional responses after exposure to 3,3′,4,4′,5-pentachlorobiphenyl (PCB 126). Exposure to 1% O{sub 2} prior to PCB 126 treatment significantly inhibited CYP1A1 mRNA and protein expression in human HepG2 and HaCaT cells. CYP1A1 transcriptional activation was significantly decreased upon PCB 126 stimulation under conditions of hypoxia. Additionally, hypoxia pre-treatment reduced PCB 126 induced AhR binding to CYP1 target gene promoters. Importantly, ARNT overexpression rescued cells from the inhibitory effect of hypoxia on XRE-luciferase reporter activity. Therefore, the mechanism of interference of the signaling crosstalk between the AhR and hypoxia pathways appears to be at least in part dependent on ARNT availability. Our results show that AhR activation and CYP1A1 expression induced by PCB 126 were significantly inhibited by hypoxia and hypoxia might therefore play an important role in PCB metabolism and toxicity. - Highlights: • Significant crosstalk exists between AhR and HIF-1α signaling. • Hypoxia perturbs PCB 126 induced AhR function and

  5. Sulforaphane inhibits CYP1A1 activity and promotes genotoxicity induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Fangxing, E-mail: fxyang@zju.edu.cn [MOE Key Laboratory of Environmental Remediation and Ecosystem Health, College of Environmental and Resource Sciences, Zhejiang University, Hangzhou, 310058 (China); Zhuang, Shulin [MOE Key Laboratory of Environmental Remediation and Ecosystem Health, College of Environmental and Resource Sciences, Zhejiang University, Hangzhou, 310058 (China); Zhang, Chao; Dai, Heping [State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, 430072 (China); Liu, Weiping, E-mail: wliu@zju.edu.cn [MOE Key Laboratory of Environmental Remediation and Ecosystem Health, College of Environmental and Resource Sciences, Zhejiang University, Hangzhou, 310058 (China)

    2013-06-15

    Increasing environmental pollution by carcinogens such as some of persistent organic pollutants (POPs) has prompted growing interest in searching for chemopreventive compounds which are readily obtainable. Sulforaphane (SFN) is isolated from cruciferous vegetables and has the potentials to reduce carcinogenesis through various pathways. In this study, we studied the effects of SFN on CYP1A1 activity and genotoxicity induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). The results showed that SFN inhibited TCDD-induced CYP1A1 activity in H4IIE cells by directly inhibiting CYP1A1 activity, probably through binding to aryl hydrocarbon receptor and/or CYP1A1 revealed by molecular docking. However, SFN promoted TCDD-induced DNA damage in yeast cells and reduced the viability of initiated yeast cells. Besides, it is surprising that SFN also failed to reduce genotoxicity induced by other genotoxic reagents which possess different mechanisms to lead to DNA damage. Currently, it is difficult to predict whether SFN has the potentials to reduce the risk of TCDD based on the conflicting observations in the study. Therefore, further studies should be urgent to reveal the function and mechanism of SFN in the stress of such POPs on human health. - Highlights: • Sulforaphane inhibited TCDD-induced CYP1A1 activity in H4IIE cells. • Sulforaphane may bind to aryl hydrocarbon receptor and/or CYP1A1. • Sulforaphane promoted TCDD-induced DNA damage in yeast cells. • Sulforaphane may promote DNA damage by DNA strand breaks or DNA alkylation.

  6. Effects of CYP2C9*1/*3 genotype on the pharmacokinetics of flurbiprofen in Korean subjects.

    Science.gov (United States)

    Lee, Yun-Jeong; Byeon, Ji-Yeong; Kim, Young-Hoon; Kim, Se-Hyung; Choi, Chang-Ik; Bae, Jung-Woo; Sohn, Uy-Dong; Jang, Choon-Gon; Lee, Jeongmi; Lee, Seok-Yong

    2015-06-01

    The aim of this study was to investigate the impact of CYP2C9*1/*3 genotype on the pharmacokinetics of flurbiprofen and its metabolite. The CYP2C9 genotypes were determined with the use of polymerase chain reaction and restriction fragment and DNA sequencing analysis in 358 healthy Koreans. Among them, twenty individuals with CYP2C9*1/*1 (n = 12) or CYP2C9*1/*3 (n = 8) genotypes received a single 40 mg oral dose of flurbiprofen. The plasma concentrations of flurbiprofen and its metabolite, 4'-hydroxyflurbiprofen were measured by HPLC. AUCinf of flurbiprofen was significantly higher and its clearance was significantly lower in the CYP2C9*1/*3 individuals than in those with CYP2C9*1/*1. The AUC ratio of 4'-hydroxyflurbiprofen to flurbiprofen was significantly lower in the CYP2C9*1/*3 individuals than in those with CYP2C9*1/*1. These results indicate that the individuals carrying of CYP2C9*3 have significant reduction in flurbiprofen metabolism. The clinical use of this information may allow for more efficient personalized pharmacotherapy.

  7. CYP2E1 Metabolism of Styrene Involves Allostery

    Science.gov (United States)

    Hartman, Jessica H.; Boysen, Gunnar

    2012-01-01

    We are the first to report allosterism during styrene oxidation by recombinant CYP2E1 and human liver microsomes. At low styrene concentrations, oxidation is inefficient because of weak binding to CYP2E1 (Ks = 830 μM). A second styrene molecule then binds CYP2E1 with higher affinity (Kss = 110 μM) and significantly improves oxidation to achieve a kcat of 6.3 nmol · min−1 · nmol CYP2E1−1. The transition between these metabolic cycles coincides with reported styrene concentrations in blood from exposed workers; thus, this CYP2E1 mechanism may be relevant in vivo. Scaled modeling of the in vitro-positive allosteric mechanism for styrene metabolism to its in vivo clearance led to significant deviations from the traditional model based on Michaelis-Menten kinetics. Low styrene levels were notably much less toxic than generally assumed. We interrogated the allosteric mechanism using the CYP2E1-specific inhibitor and drug 4-methylpyrazole, which we have shown binds two CYP2E1 sites. From the current studies, styrene was a positive allosteric effector on 4-methylpyrazole binding, based on a 10-fold increase in 4-methylpyrazole binding affinity from Ki 0.51 to Ksi 0.043 μM. The inhibitor was a negative allosteric effector on styrene oxidation, because kcat decreased 6-fold to 0.98 nmol · min−1 · nmol CYP2E1−1. Consequently, mixtures of styrene and other molecules can induce allosteric effects on binding and metabolism by CYP2E1 and thus mitigate the efficiency of their metabolism and corresponding effects on human health. Taken together, our elucidation of mechanisms for these allosteric reactions provides a powerful tool for further investigating the complexities of CYP2E1 metabolism of drugs and pollutants. PMID:22807108

  8. The inactivation of human CYP2E1 by phenethyl isothiocyanate, a naturally occurring chemopreventive agent, and its oxidative bioactivation.

    Science.gov (United States)

    Yoshigae, Yasushi; Sridar, Chitra; Kent, Ute M; Hollenberg, Paul F

    2013-04-01

    Phenethylisothiocyanate (PEITC), a naturally occurring isothiocyanate and potent cancer chemopreventive agent, works by multiple mechanisms, including the inhibition of cytochrome P450 (P450) enzymes, such as CYP2E1, that are involved in the bioactivation of carcinogens. PEITC has been reported to be a mechanism-based inactivator of some P450s. We describe here the possible mechanism for the inactivation of human CYP2E1 by PEITC, as well as the putative intermediate that might be involved in the bioactivation of PEITC. PEITC inactivated recombinant CYP2E1 with a partition ratio of 12, and the inactivation was not inhibited in the presence of glutathione (GSH) and not fully recovered by dialysis. The inactivation of CYP2E1 by PEITC is due to both heme destruction and protein modification, with the latter being the major pathway for inactivation. GSH-adducts of phenethyl isocyanate (PIC) and phenethylamine were detected during the metabolism by CYP2E1, indicating formation of PIC as a reactive intermediate following P450-catalyzed desulfurization of PEITC. Surprisingly, PIC bound covalently to CYP2E1 to form protein adducts but did not inactivate the enzyme. Liquid chromatography mass spectroscopy analysis of the inactivated CYP2E1 apo-protein suggests that a reactive sulfur atom generated during desulfurization of PEITC is involved in the inactivation of CYP2E1. Our data suggest that the metabolism of PEITC by CYP2E1 that results in the inactivation of CYP2E1 may occur by a mechanism similar to that observed with other sulfur-containing compounds, such as parathion. Digestion of the inactivated enzyme and analysis by SEQUEST showed that Cys 268 may be the residue modified by PIC.

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

    Directory of Open Access Journals (Sweden)

    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.

  10. CYP3A5*3 and ABCB1 61A>G Significantly Influence Dose-adjusted Trough Blood Tacrolimus Concentrations in the First Three Months Post-Kidney Transplantation.

    Science.gov (United States)

    Hu, Rong; Barratt, Daniel T; Coller, Janet K; Sallustio, Benedetta C; Somogyi, Andrew A

    2018-03-30

    Tacrolimus (TAC) is a first-line immunosuppressant used to prevent organ rejection after kidney transplantation. There is large inter-individual variability in its pharmacokinetics. Single nucleotide polymorphisms (SNPs) in genes encoding TAC metabolizing enzymes cytochromes P450 3A4/5 (CYP3A4/5), P-glycoprotein efflux transporter (ABCB1), their expression regulator pregnane X receptor (NR1I2) and CYP3A co-factor cytochrome P450 reductase (POR) have been studied for their effects on tacrolimus disposition. However, except for CYP3A5*3, controversies remain about their roles in predicting dose-adjusted trough blood TAC concentrations (C 0 /D). This study aimed to investigate the effects of ABCB1 (61A>G, 1199G>A, 1236C>T, 2677G>T and 3435C>T), CYP3A4*22, CYP3A5*3, NR1I2 (8055C>T, 63396C>T and -25385C>T) and POR*28 SNPs on TAC C 0 /D. In total, 165 kidney transplant recipients were included in this study. SNPs were genotyped by probe-based real-time polymerase chain reaction. Associations between log-transformed whole blood TAC C 0 /D (measured at 1 and 3 months post-transplant) and genotypes/haplotypes were assessed by linear mixed effects analysis, controlling for age, sex and haematocrit. It was observed that CYP3A5 expressors (*1/*1 + *1/*3) (p = 5.5 × 10 -16 ) and ABCB1 61G allele carriers (p = 0.001) had lower log-transformed TAC C 0 /D (56% and 26% lower geometric mean TAC C 0 /D, respectively) and accounted for approximately 30% and 4%, respectively, of log-transformed TAC C 0 /D variability in the first 3 months post-transplant. In conclusion, CYP3A5*3 is a major, and ABCB1 61A>G is a novel, although minor, genetic factor affecting TAC C 0 /D in kidney transplant recipients. © 2018 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).

  11. Kinetic analysis of human CYP24A1 metabolism of vitamin D via the C24-oxidation pathway.

    Science.gov (United States)

    Tieu, Elaine W; Tang, Edith K Y; Tuckey, Robert C

    2014-07-01

    CYP24A1 is the multicatalytic cytochrome P450 responsible for the catabolism of vitamin D via the C23- and C24-oxidation pathways. We successfully expressed the labile human enzyme in Escherichia coli and partially purified it in an active state that permitted detailed characterization of its metabolism of 1,25-dihydroxyvitamin D3 [1,25(OH)2 D3] and the intermediates of the C24-oxidation pathway in a phospholipid-vesicle reconstituted system. The C24-oxidation pathway intermediates, 1,24,25-trihydroxyvitamin D3, 24-oxo-1,25-dihydroxyvitamin D3, 24-oxo-1,23,25-trihydroxyvitamin D3 and tetranor-1,23-dihydroxyvitamin D3, were enzymatically produced from 1,25(OH)2 D3 using rat CYP24A1. Both 1,25(OH)2 D3 and 1,23-dihydroxy-24,25,26,27-tetranorvitamin D3 were found to partition strongly into the phospholipid bilayer when in aqueous medium. Changes to the phospholipid concentration did not affect the kinetic parameters for the metabolism of 1,25(OH)2 D3 by CYP24A1, indicating that it is the concentration of substrates in the membrane phase (mol substrate·mol phospholipid(-1) ) that determines their rate of metabolism. CYP24A1 exhibited Km values for the different C24-intermediates ranging from 0.34 to 15 mmol·mol phospholipid(-1) , with 24-oxo-1,23,25-trihydroxyvitamin D3 [24-oxo-1,23,25(OH)3 D3] displaying the lowest and 1,24,25-trihydroxyvitamin D3 [1,24,25(OH)3 D3] displaying the highest. The kcat values varied by up to 3.8-fold, with 1,24,25(OH)3 D3 displaying the highest kcat (34 min(-1) ) and 24-oxo-1,23,25(OH)3 D3 the lowest. The data show that the cleavage of the side chain of 24-oxo-1,23,25(OH)3 D3 occurs with the highest catalytic efficiency (kcat /Km ) and produces 1-hydroxy-23-oxo-24,25,26,27-tetranorvitamin D3 and not 1,23-dihydroxy-24,25,26,27-tetranorvitamin D3, as the primary product. These kinetic analyses also show that intermediates of the C24-oxidation pathway effectively compete with precursor substrates for binding to the active site of the

  12. Influencia de polimorfismos genéticos de CYP3A4/5 en la farmacocinética de levonorgestrel: estudio piloto

    Directory of Open Access Journals (Sweden)

    Iván Moreno

    2012-12-01

    Full Text Available Introducción. El levonorgestrel, un progestágeno sintético usado para endometriosis, dismenorrea y anticoncepción de emergencia, es rápida y completamente absorbido en el tubo digestivo. Su metabolismo es principalmente hepático, mediante las enzimas CYP3A4 y CYP3A5. Objetivo. El presente estudio tuvo como objetivo evaluar la asociación entre la farmacocinética de levonorgestrel y las variantes alélicas de CYP3A4*1B y CYP3A5*3. Materiales y métodos. En un grupo de 17 mujeres adultas sanas, que firmaron un consentimiento informado, se practicó genotipificación para CYP3A4*1B y CYP3A5*3 mediante PCR. Posteriormente, las voluntarias fueron sometidas a un estudio farmacocinético donde, luego de 12 horas de ayuno, recibieron una dosis de 0,75 mg de levonorgestrel. Se extrajeron muestras sanguíneas seriadas (0 a 24 horas y se determinaron las concentraciones de levonorgestrel mediante un método validado de UPLC-ms/ms, para luego obtener los parámetros farmacocinéticos. Todos los procedimientos consideraron los aspectos éticos de la Declaración de Helsinki y las buenas prácticas clínicas. Resultados. Las frecuencias genotípicas observadas para el grupo de estudio fueron 11,8 % para*1B/*1B; 5,8 % para *1/*1B, y 82,4 % para *1/*1 de CYP3A4*1B. Para CYP3A5*3, las frecuencias genotípicas fueron 70,5 % para *3/*3; 23,5 % para *1/*3, y 6,5 % para *1/*1. Se observa una interesante variabilidad entre las voluntarias que sugiere una relación con las variantes genéticas CYP3A, pero que no permite establecer una asociación estadísticamente significativa, presumiblemente debido albajo número de individuos homocigotos mutados de CYP3A4 y silvestres de CYP3A5. Conclusiones. Los polimorfismos genéticos podrían ser factores relevantes en la determinación de la variabilidad entre pacientes en las concentraciones plasmáticas de levonorgestrel, lo cual, sin embargo, no pudo ser establecido estadísticamente en este estudio. Por lo tanto

  13. The steroid metabolite 16(β)-OH-androstenedione generated by CYP21A2 serves as a substrate for CYP19A1.

    Science.gov (United States)

    Neunzig, J; Milhim, M; Schiffer, L; Khatri, Y; Zapp, J; Sánchez-Guijo, A; Hartmann, M F; Wudy, S A; Bernhardt, R

    2017-03-01

    The 21-hydroxylase (CYP21A2) is a steroidogenic enzyme crucial for the synthesis of mineralo- and glucocorticoids. It is described to convert progesterone as well as 17-OH-progesterone, through a hydroxylation at position C21, into 11-deoxycorticosterone (DOC) and 11-deoxycortisol (RSS), respectively. In this study we unraveled CYP21A2 to have a broader steroid substrate spectrum than assumed. Utilizing a reconstituted in vitro system, consisting of purified human CYP21A2 and human cytochrome P450 reductase (CPR) we demonstrated that CYP21A2 is capable to metabolize DOC, RSS, androstenedione (A4) and testosterone (T). In addition, the conversion of A4 rendered a product whose structure was elucidated through NMR spectroscopy, showing a hydroxylation at position C16-beta. The androgenic properties of this steroid metabolite, 16(β)-OH-androstenedione (16bOHA4), were investigated and compared with A4. Both steroid metabolites were shown to be weak agonists for the human androgen receptor. Moreover, the interaction of 16bOHA4 with the aromatase (CYP19A1) was compared to that of A4, indicating that the C16 hydroxyl group does not influence the binding with CYP19A1. In contrast, the elucidation of the kinetic parameters showed an increased K m and decreased k cat value resulting in a 2-fold decreased catalytic efficiency compared to A4. These findings were in accordance with our docking studies, revealing a similar binding conformation and distance to the heme iron of both steroids. Furthermore, the product of 16bOHA4, presumably 16-hydroxy-estrone (16bOHE1), was investigated with regard to its estrogenic activity, which was negligible compared to estradiol and estrone. Finally, 16bOHA4 was found to be present in a patient with 11-hydroxylase deficiency and in a patient with an endocrine tumor. Taken together, this study provides novel information on the steroid hormone biosynthesis and presents a new method to detect further potential relevant novel steroid metabolites

  14. A new CYP21A1P/CYP21A2 chimeric gene identified in an Italian woman suffering from classical congenital adrenal hyperplasia form

    Science.gov (United States)

    Concolino, Paola; Mello, Enrica; Minucci, Angelo; Giardina, Emiliano; Zuppi, Cecilia; Toscano, Vincenzo; Capoluongo, Ettore

    2009-01-01

    Background More than 90% of Congenital Adrenal Hyperplasia (CAH) cases are associated with mutations in the 21-hydroxylase gene (CYP21A2) in the HLA class III area on the short arm of chromosome 6p21.3. In this region, a 30 kb deletion produces a non functional chimeric gene with its 5' and 3' ends corresponding to CYP21A1P pseudogene and CYP21A2, respectively. To date, five different CYP21A1P/CYP21A2 chimeric genes have been found and characterized in recent studies. In this paper, we describe a new CYP21A1P/CYP21A2 chimera (CH-6) found in an Italian CAH patient. Methods Southern blot analysis and CYP21A2 sequencing were performed on the patient. In addition, in order to isolate the new CH-6 chimeric gene, two different strategies were used. Results The CYP21A2 sequencing analysis showed that the patient was homozygote for the g.655C/A>G mutation and heterozygote for the p.P30L missense mutation. In addition, the promoter sequence revealed the presence, in heterozygosis, of 13 SNPs generally produced by microconversion events between gene and pseudogene. Southern blot analysis showed that the woman was heterozygote for the classic 30-kb deletion producing a new CYP21A1P/CYP21A2 chimeric gene (CH-6). The hybrid junction site was located between the end of intron 2 pseudogene, after the g.656C/A>G mutation, and the beginning of exon 3, before the 8 bp deletion. Consequently, CH-6 carries three mutations: the weak pseudogene promoter region, the p.P30L and the g.655C/A>G splice mutation. Conclusion We describe a new CYP21A1P/CYP21A2 chimera (CH-6), associated with the HLA-B15, DR13 haplotype, in a young Italian CAH patient. PMID:19624807

  15. Impact of the herbal medicine Sophora flavescens on the oral pharmacokinetics of indinavir in rats: the involvement of CYP3A and P-glycoprotein.

    Directory of Open Access Journals (Sweden)

    Jia-Ming Yang

    Full Text Available Sophora flavescens is a Chinese medicinal herb used for the treatment of gastrointestinal hemorrhage, skin diseases, pyretic stranguria and viral hepatitis. In this study the herb-drug interactions between S. flavescens and indinavir, a protease inhibitor for HIV treatment, were evaluated in rats. Concomitant oral administration of Sophora extract (0.158 g/kg or 0.63 g/kg, p.o. and indinavir (40 mg/kg, p.o. in rats twice a day for 7 days resulted in a dose-dependent decrease of plasma indinavir concentrations, with 55%-83% decrease in AUC(0-∞ and 38%-78% reduction in C(max. The CL (Clearance/F (fraction of dose available in the systemic circulation increased up to 7.4-fold in Sophora-treated rats. Oxymatrine treatment (45 mg/kg, p.o. also decreased indinavir concentrations, while the ethyl acetate fraction of Sophora extract had no effect. Urinary indinavir (24-h was reduced, while the fraction of indinavir in faeces was increased after Sophora treatment. Compared to the controls, multiple dosing of Sophora extract elevated both mRNA and protein levels of P-gp in the small intestine and liver. In addition, Sophora treatment increased intestinal and hepatic mRNA expression of CYP3A1, but had less effect on CYP3A2 expression. Although protein levels of CYP3A1 and CYP3A2 were not altered by Sophora treatment, hepatic CYP3A activity increased in the Sophora-treated rats. All available data demonstrated that Sophora flavescens reduced plasma indinavir concentration after multiple concomitant doses, possibly through hepatic CYP3A activity and induction of intestinal and hepatic P-gp. The animal study would be useful for predicting potential interactions between natural products and oral pharmaceutics and understanding the mechanisms prior to human studies. Results in the current study suggest that patients using indinavir might be cautioned in the use of S. flavescens extract or Sophora-derived products.

  16. Direct sequencing and comprehensive screening of genetic polymorphisms on CYP2 family genes (CYP2A6, CYP2B6, CYP2C8, and CYP2E1) in five ethnic populations.

    Science.gov (United States)

    Kim, Jeong-Hyun; Cheong, Hyun Sub; Park, Byung Lae; Kim, Lyoung Hyo; Shin, Hee Jung; Na, Han Sung; Chung, Myeon Woo; Shin, Hyoung Doo

    2015-01-01

    Recently, CYP2A6, CYP2B6, CYP2C8, and CYP2E1 have been reported to play a role in the metabolic effect of pharmacological and carcinogenic compounds. Moreover, genetic variations of drug metabolism genes have been implicated in the interindividual variation in drug disposition and pharmacological response. To define the distribution of single nucleotide polymorphisms (SNPs) in these four CYP2 family genes and to discover novel SNPs across ethnic groups, 288 DNAs composed of 48 African-Americans, 48 European-Americans, 48 Japanese, 48 Han Chinese, and 96 Koreans were resequenced. A total of 143 SNPs, 26 in CYP2A6, 45 in CYP2B6, 29 in CYP2C8, and 43 in CYP2E1, were identified, including 13 novel variants. Notably, two SNPs in the regulatory regions, a promoter SNP rs2054675 and a nonsynonymous rs3745274 (p.172Q>H) in CYP2B6, showed significantly different minor allele frequencies (MAFs) among ethnic groups (minimum P = 4.30 × 10(-12)). In addition, rs2031920 in the promoter region of CYP2E1 showed a wide range of MAF between different ethnic groups, and even among other various ethnic groups based on public reports. Among 13 newly discovered SNPs in this study, 5 SNPs were estimated to have potential functions in further in silico analyses. Some differences in genetic variations and haplotypes of CYP2A6, CYP2B6, CYP2C8, and CYP2E1 were observed among populations. Our findings could be useful in further researches, such as genetic associations with drug responses.

  17. Caffeic acid phenethyl ester inhibits 3-MC-induced CYP1A1 expression through induction of hypoxia-inducible factor-1α

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Hyung Gyun [Department of Toxicology, College of Pharmacy, Chungnam National University, Daejeon (Korea, Republic of); Han, Eun Hee [Division of Life Science, Korea Basic Science Institute, Daejeon (Korea, Republic of); Im, Ji Hye; Lee, Eun Ji; Jin, Sun Woo [Department of Toxicology, College of Pharmacy, Chungnam National University, Daejeon (Korea, Republic of); Jeong, Hye Gwang, E-mail: hgjeong@cnu.ac.kr [Department of Toxicology, College of Pharmacy, Chungnam National University, Daejeon (Korea, Republic of)

    2015-09-25

    Caffeic acid phenethyl ester (CAPE), a natural component of propolis, is reported to have anticarcinogenic properties, although its precise chemopreventive mechanism remains unclear. In this study, we examined the effects of CAPE on 3-methylcholanthrene (3-MC)-induced CYP1A1 expression and activities. CAPE reduced the formation of the benzo[a]pyrene-DNA adduct. Moreover, CAPE inhibited 3-MC-induced CYP1A1 activity, mRNA expression, protein level, and promoter activity. CAPE treatment also decreased 3-MC-inducible xenobiotic-response element (XRE)-linked luciferase, aryl hydrocarbons receptor (AhR) transactivation and nuclear localization. CAPE induced hypoxia inducible factor-1α (HIF-1α) protein level and HIF-1α responsible element (HRE) transcriptional activity. CAPE-mediated HIF-1α reduced 3-MC-inducible CYP1A1 protein expression. Taken together, CAPE decreases 3-MC-mediated CYP1A1 expression, and this inhibitory response is associated with inhibition of AhR and HIF-1α induction. - Highlights: • CAPE reduced the formation of the benzo[a]pyrene-DNA adduct. • CAPE inhibited 3-MC-induced CYP1A1 expression. • CAPE induced HIF-1α induction. • CAPE-mediated HIF-1α reduced 3-MC-inducible CYP1A1 expression.

  18. Effects of chlorpyrifos on the transcription of CYP3A cDNA, activity of acetylcholinesterase, and oxidative stress response of goldfish (Carassius auratus).

    Science.gov (United States)

    Ma, Junguo; Liu, Yang; Niu, Daichun; Li, Xiaoyu

    2015-04-01

    Chlorpyrifos (CPF) is the widely used organophosphate pesticide in agriculture throughout the world. It has been found that CPF is relatively safe to human but highly toxic to fish. In this study, acute toxicity of CPF on goldfish was determined and then the transcription of goldfish cytochrome P450 (CYP) 3A was evaluated after 96 h of CPF exposure at concentrations of 15.3 [1/10 50% lethal concentration (LC50 )] or 51 μg L(-1) (1/3 LC50 ) of CPF. Meanwhile, the enzymatic activities of acetylcholinesterase (AChE), superoxide dismutase (SOD), and catalase (CAT), total antioxidant activity (T-AOC), and the contents of malondialdehyde (MDA) in the liver or brain of goldfish were also determined. The results of acute toxicity testing showed that the 96-h LC50 of CPF to the goldfish was 153 μg L(-1) . Moreover, a length sequence of 1243 bp CYP3A cDNA encoding for 413 amino acids from goldfish liver was cloned. Polymerase chain reaction results reveal that CPF exposure downregulates CYP 3A transcription in goldfish liver, suggesting that goldfish CYP 3A may be not involved in CPF bioactivation. Finally, the results of biochemical assays indicate that 96 h of CPF exposure remarkably inhibits AChE activity in fish liver or brain, alters hepatic antioxidant enzyme activities, decreases brain T-AOC, and causes lipid peroxidation in fish liver. These results suggest that oxidative stress might be involved in CPF toxicity on goldfish. Copyright © 2013 Wiley Periodicals, Inc.

  19. Impact of the CYP3A5*1 Allele on the Pharmacokinetics of Tacrolimus in Japanese Heart Transplant Patients.

    Science.gov (United States)

    Uno, Takaya; Wada, Kyoichi; Matsuda, Sachi; Terada, Yuka; Oita, Akira; Kawase, Atsushi; Takada, Mitsutaka

    2018-04-24

    Tacrolimus, a major immunosuppressant used after transplantation, is associated with large interindividual variation involving genetic polymorphisms in metabolic processes. A common variant of the cytochrome P450 (CYP) 3A5 gene, CYP3A5*3, affects blood concentrations of tacrolimus. However, tacrolimus pharmacokinetics at the early stage of transplantation have not been adequately studied in heart transplantation. We retrospectively examined the impact of the CYP3A5 genotype on tacrolimus pharmacokinetics at the early stage of heart transplantation. The tacrolimus pharmacokinetic profile was obtained from 65 patients during the first 5 weeks after heart transplantation. Differences in the patients' characteristics and tacrolimus pharmacokinetic parameters between the CYP3A5 expresser (*1/*1 or *1/*3 genotypes) and non-expresser (*3/*3 genotype) groups were assessed by the Chi-square test, Student's t test, or Mann-Whitney U test. The CYP3A5 *1/*1, *1/*3, and *3/*3 genotypes were detected in 5, 22, and 38 patients, respectively. All patients started clotrimazole therapy approximately 1 week after starting tacrolimus. Apparent clearance and dose/weight to reach the target trough concentration (C 0 ) were significantly higher in the expresser group than in the non-expresser group (0.32 vs. 0.19 L/h/kg, p = 0.0003; 0.052 vs. 0.034 mg/kg/day, p = 0.0002); there were no significant differences in the area under the concentration-time curve from 0 to 12 h (AUC 0-12 ) and concentrations at any sampling time point between the two groups. Similar concentration-time curves for tacrolimus were obtained in the expresser and non-expresser groups by dose adjustment based on therapeutic drug monitoring. These results demonstrate the importance of the CYP3A5 genotype in tacrolimus dose optimization based on therapeutic drug monitoring after heart transplantation.

  20. Regulated expression of genes inserted at the human chromosomal β-globin locus by homologous recombination

    International Nuclear Information System (INIS)

    Nandi, A.K.; Roginski, R.S.; Gregg, R.G.; Smithies, O.; Skoultchi, A.I.

    1988-01-01

    The authors have examined the effect of the site of integration on the expression of cloned genes introduced into cultured erythroid cells. Smithies et al. reported the targeted integration of DNA into the human β-globin locus on chromosome 11 in a mouse erythroleukemia-human cell hybrid. These hybrid cells can undergo erythroid differentiation leading to greatly increased mouse and human β-globin synthesis. By transfection of these hybrid cells with a plasmid carrying a modified human β-globin gene and a foreign gene composed of the coding sequence of the bacterial neomycin-resistance gene linked to simian virus 40 transcription signals (SVneo), cells were obtained in which the two genes are integrated at the β-globin locus on human chromosome 11 or at random sites. When they examined the response of the integrated genes to cell differentation, they found that the genes inserted at the β-globin locus were induced during differentiation, whereas randomly positioned copies were not induced. Even the foreign SVneo gene was inducible when it had been integrated at the β-globin locus. The results show that genes introduced at the β-globin locus acquire some of the regulatory properties of globin genes during erythroid differentiation

  1. CHRNA3 and CYP3A5*3 genotype, lung function and chronic obstructive pulmonary disease in the general population

    DEFF Research Database (Denmark)

    Kaur-Knudsen, Diljit; Bojesen, Stig E; Nordestgaard, Børge G

    2014-01-01

    OBJECTIVE: Genetic variations are most likely an additional risk factor besides tobacco smoking per se for the risk of chronic obstructive pulmonary disease (COPD). In this study, we compared genetic variants influencing the effect of smoking on COPD, that is, the effect of the well-known splicin.......1-2.2) for GOLD 3-4. This association could not be found in never-smokers. No association was found for CYP3A5*3. CONCLUSION: The CHRNA3 genotype is associated with decreased lung function and risk of COPD among ever-smokers, whereas this was not the case for CYP3A5*3....... were genotyped. Information on spirometry, hospital admissions and smoking behaviour was recorded. Endpoints were lung function and COPD. RESULTS: For CHRNA3, the percentage of forced expiratory volume in 1 s (FEV1%) predicted was 89.3, 90.6 and 92.4% in homozygous, heterozygous and noncarrier ever-smokers...... (P-trendsmokers was 1.5 [95...

  2. Cytochrome P450 CYP4DE1 and CYP6CW3v2 contribute to ethiprole resistance in Laodelphax striatellus (Fallén).

    Science.gov (United States)

    Elzaki, M E A; Zhang, W; Han, Z

    2015-06-01

    Laodelphax striatellus Fallén (Hemiptera: Delphacidae), a destructive pest of rice, has developed high resistance to multiple insecticides, threatening the success of pest management programmes. The present study investigated ethiprole resistance mechanisms in a field population that is highly resistant to ethiprole. That population was used to establish a laboratory population that was subjected to further selection to produce a resistant strain. Target genes were cloned and compared between the resistant and the susceptible strains, the role of detoxification enzymes was examined, and the relative expression levels of 71 detoxification enzyme genes were tested using quantitative real time (RT)-PCR. The laboratory selection enhanced the resistance from 107-fold to 180-fold. The Rdl-type target site mutation seldom occurred in the resistant strain and is unlikely to represent the major mechanism underlying the observed resistance. Of the three important detoxification enzymes, only P450 monooxygenase was found to be associated with ethiprole resistance. Moreover, two genes, CYP4DE1 and CYP6CW3v2, were found to be overexpressed in the resistant strain. Furthermore, gene-silencing via a double-stranded RNA feeding test was carried out, and the results showed that the mRNA levels of CYP4DE1 and CYP6CW3v2 were reduced in the resistant strain, whereas ethiprole susceptibility was increased. These results suggest that CYP4DE1 and CYP6CW3v2 play an important role in ethiprole resistance in L. striatellus. © 2015 The Royal Entomological Society.

  3. Up-regulation of the alligator CYP3A77 gene by toxaphene and dexamethasone and its short term effect on plasma testosterone concentrations

    International Nuclear Information System (INIS)

    Gunderson, M.P.; Kohno, S.; Blumberg, B.; Iguchi, T.; Guillette, L.J.

    2006-01-01

    In this study we describe an alligator hepatic CYP3A gene, CYP3A77, which is inducible by dexamethasone and toxaphene. CYP3A plays a broad role in biotransforming both exogenous compounds and endogenous hormones such as testosterone and estradiol. Alligators collected from sites in Florida that are contaminated with organochlorine compounds exhibit differences in sex steroid concentrations. Many organochlorine compounds induce CYP3A expression in other vertebrates; hence, CYP3A induction by organochlorine contaminants could increase biotransformation and clearance of sex steroids by CYP3A and provide a plausible mechanism for the lowering of endogenous sex steroid concentrations in alligator plasma. We used real time PCR to examine whether known and suspected CYP3A inducers (dexamethasone, metyrapone, rifampicin, and toxaphene) up-regulate steady state levels of hepatic CYP3A77 transcript to determine if induction patterns in female juvenile alligators are similar to those reported in other vertebrates and whether toxaphene, an organochlorine compound found in high concentrations in Lake Apopka alligators, induces this gene. Estrogen receptor α (ERα), estrogen receptor β (ERβ), androgen receptor (AR), glucocorticoid receptor (GR), progesterone receptor (PR), and steroid-xenobiotic receptor (SXR) transcripts were also measured to determine whether any of these nuclear receptors are also regulated by these compounds in alligators. Dexamethasone (4.2-fold) and toxaphene (3.5-fold) significantly induced CYP3A77 gene transcript, whereas rifampicin (2.8-fold) and metyrapone (2.1-fold) up-regulated ERβ after 24 h. None of the compounds significantly up-regulated AR, ERα, GR, PR, or SXR over this time period. Plasma testosterone (T) did not change significantly after 24 h in alligators from any of the treatment groups. Dexamethasone treated animals exhibited a strong relationship between the 24 h plasma T concentrations and CYP3A77 (R 2 = 0.9, positive) and SXR (R 2

  4. Methylation and Transcripts Expression at the Imprinted GNAS Locus in Human Embryonic and Induced Pluripotent Stem Cells and Their Derivatives

    Directory of Open Access Journals (Sweden)

    Virginie Grybek

    2014-09-01

    Full Text Available Data from the literature indicate that genomic imprint marks are disturbed in human pluripotent stem cells (PSCs. GNAS is an imprinted locus that produces one biallelic (Gsα and four monoallelic (NESP55, GNAS-AS1, XLsα, and A/B transcripts due to differential methylation of their promoters (DMR. To document imprinting at the GNAS locus in PSCs, we studied GNAS locus DMR methylation and transcript (NESP55, XLsα, and A/B expression in human embryonic stem cells (hESCs and human induced pluripotent stem cells (hiPSCs derived from two human fibroblasts and their progenies. Results showed that (1 methylation at the GNAS locus DMRs is DMR and cell line specific, (2 changes in allelic transcript expression can be independent of a change in allele-specific DNA methylation, and (3 interestingly, methylation at A/B DMR is correlated with A/B transcript expression. These results indicate that these models are valuable to study the mechanisms controlling GNAS methylation, factors involved in transcript expression, and possibly mechanisms involved in the pathophysiology of pseudohypoparathyroidism type 1B.

  5. An overview on the allelic variant of CYP2D6 genotype

    African Journals Online (AJOL)

    Yomi

    plants, fungi, slime molds, bacteria and archaea. More than 7700 distinct CYP sequences are known. ... SIGNIFICANCE OF CYTOCHROME P450 IN HUMANS. Human CYPs are primarily membrane-associated proteins, ..... Evolution of a highly polymorphic human cytochrome P450 gene cluster: CYP2D6. Genomics.

  6. Relevance of the ancestry for the variability of the Drug-Metabolizing Enzymes CYP2C9, CYP2C19 and CYP2D6 polymorphisms in a multiethnic Costa Rican population.

    Science.gov (United States)

    Céspedes-Garro, Carolina; Rodrigues-Soares, Fernanda; Jiménez-Arce, Gerardo; Naranjo, María-Eugenia G; Tarazona-Santos, Eduardo; Fariñas, Humberto; Barrantes, Ramiro; Llerena, Adrián

    2016-09-01

    CYP2C9, CYP2C19 and CYP2D6 metabolize around 40% of drugs and their genes vary across populations. The Costa Rican population has a trihybrid ancestry and its key geographic location turns it into a suitable scenario to evaluate interethnic differences across populations. This study aims to describe the diversity of CYP2C9, CYP2C19 and CYP2D6 polymorphisms in Costa Rican populations in the context of their ancestry. A total of 448 healthy individuals were included in the study: Bribri (n= 47), Cabécar (n= 27), Maleku (n= 16), Guaymí (n= 30), Huetar (n= 48), Chorotega (n= 41), Admixed/Mestizos from the Central Valley/Guanacaste (n= 189), and Afro-Caribbeans (n= 50) from Limón. CYP2C9 (alleles *2, *3, *6) and CYP2C19 (*2, *3, *4, *5, *17) genotypes were determined by Real-Time PCR. African, European and Native American ancestry were inferred using 87 ancestry informative markers. The frequency of the decreased activity allele CYP2C9*2 is lower in the self-reported Amerindian groups compared to the admixed population, and the highest frequencies of CYP2C19*2 (null activity) and the CYP2C19*17 (increased activity) were found in the self-reported Afro-Caribbean population. Moreover, a frequency of 0.7 % CYP2C9 gPMs in the Admixed population and a variable frequency of CYP2C19 gUMs (0.0-32.6 %, more prevalent in Afro-Caribbeans) in Costa Rican populations, was found. Finally, the following alleles were positively correlated with genomic African ancestry and negatively correlated with genomic Native American ancestry: CYP2D6*5 (null activity), CYP2D6*17 (decreased activity), CYP2D6*29 (decreased activity) and CYP2C19*17 (increased activity). No correlation for CYP2C9 polymorphisms and genomic ancestry was found. Further studies assessing the CYP2C9 and CYP2C19 sequence in these populations, preferentially by sequencing these genes, are warranted.

  7. Editor's Highlight: Complete Attenuation of Mouse Lung Cell Proliferation and Tumorigenicity in CYP2F2 Knockout and CYP2F1 Humanized Mice Exposed to Inhaled Styrene for up to 2 Years Supports a Lack of Human Relevance.

    Science.gov (United States)

    Cruzan, George; Bus, James S; Banton, Marcy I; Sarang, Satinder S; Waites, Robbie; Layko, Debra B; Raymond, James; Dodd, Darol; Andersen, Melvin E

    2017-10-01

    Styrene is a mouse-specific lung carcinogen, and short-term mode of action studies have demonstrated that cytotoxicity and/or cell proliferation, and genomic changes are dependent on CYP2F2 metabolism. The current study examined histopathology, cell proliferation, and genomic changes in CD-1, C57BL/6 (WT), CYP2F2(-/-) (KO), and CYP2F2(-/-) (CYP2F1, 2B6, 2A13-transgene) (TG; humanized) mice following exposure for up to 104 weeks to 0- or 120-ppm styrene vapor. Five mice per treatment group were sacrificed at 1, 26, 52, and 78 weeks. Additional 50 mice per treatment group were followed until death or 104 weeks of exposure. Cytotoxicity was present in the terminal bronchioles of some CD-1 and WT mice exposed to styrene, but not in KO or TG mice. Hyperplasia in the terminal bronchioles was present in CD-1 and WT mice exposed to styrene, but not in KO or TG mice. Increased cell proliferation, measured by KI-67 staining, occurred in CD-1 and WT mice exposed to styrene for 1 week, but not after 26, 52, or 78 weeks, nor in KO or TG mice. Styrene increased the incidence of bronchioloalveolar adenomas and carcinomas in CD-1 mice. No increase in lung tumors was found in WT despite clear evidence of lung toxicity, or, KO or TG mice. The absence of preneoplastic lesions and tumorigenicity in KO and TG mice indicates that mouse-specific CYP2F2 metabolism is responsible for both the short-term and chronic toxicity and tumorigenicity of styrene, and activation of styrene by CYP2F2 is a rodent MOA that is neither quantitatively or qualitatively relevant to humans. © The Author 2017. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  8. Physical map and one-megabase sequencing of the human immunoglobulin lambda locus

    Directory of Open Access Journals (Sweden)

    Geraldo A.S. Passos Jr.

    1998-06-01

    Full Text Available The human immunoglobulin lambda (IGL locus is located on chromosome 22q11.1-q11.2 and contains the genes responsible for the immunoglobulin lambda light chains. This locus was recently mapped (physical map and its 1-Mb DNA totally sequenced. In this review we focus on the characterization of the v-lambda genes, its chromosomal location, genomics and sequencing of the IGL locus.O locus IGL humano está localizado no cromosomo 22q11.1-q11.2 e contém os genes responsáveis pelas cadeias leves de imunoglobulina tipo lambda. Este locus foi recentemente mapeado (mapa físico e seu 1 Mb DNA totalmente sequenciado. Nesta revisão focamos os principais resultados de caracterização dos genes v-lambda, sua localização cromossômica, a genômica e seqüenciamento do locus IGL.

  9. The impact of CYP3A5*3 on risk and prognosis in childhood acute lymphoblastic leukemia

    DEFF Research Database (Denmark)

    Borst, Louise; Wallerek, Sandra; Dalhoff, Kim

    2011-01-01

    Objectives: Acute lymphoblastic leukemia (ALL) is the most common cancer in childhood; however, little is known of the molecular etiology and environmental exposures causing the disease. Cytochrome P450 3A5 (CYP3A5) plays a crucial role in the catalytic oxidation of endogenous metabolites and toxic...

  10. The impact of CYP3A5*3 on risk and prognosis in childhood acute lymphoblastic leukemia

    DEFF Research Database (Denmark)

    Borst, Louise; Wallerek, Sandra; Dalhoff, Kim Peder

    2011-01-01

    Objectives:  Acute lymphoblastic leukemia (ALL) is the most common cancer in childhood; however, little is known of the molecular etiology and environmental exposures causing the disease. Cytochrome P450 3A5 (CYP3A5) plays a crucial role in the catalytic oxidation of endogenous metabolites...

  11. CYP3A4 Mediates Oxidative Metabolism of the Synthetic Cannabinoid AKB-48

    DEFF Research Database (Denmark)

    Holm, Niels Bjerre; Nielsen, Line Marie; Linnet, Kristian

    2015-01-01

    the metabolism of N-(1-adamantyl)-1-pentyl-1H-indazole-3-carboxamide (AKB-48), a compound identified in herbal blends from 2012 and onwards. We screened for metabolite formation using a panel of nine recombinant cytochrome P450 (CYP) enzymes (CYP1A2, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, and 3A4) and compared...

  12. Regulation of CYP3A4 by pregnane X receptor: The role of nuclear receptors competing for response element binding

    Energy Technology Data Exchange (ETDEWEB)

    Istrate, Monica A., E-mail: monicai@scripps.edu [Dr. Margarete Fischer-Bosch-Institute of Clinical Pharmacology, Stuttgart, Germany, and University of Tuebingen, Auerbachstr. 112, D-70376 Stuttgart (Germany); Nussler, Andreas K., E-mail: nuessler@uchir.me.tum.de [Department of Traumatology, Technical University Munich, Ismaningerstr. 22, 81675 Munich (Germany); Eichelbaum, Michel, E-mail: michel.eichelbaum@ikp-stuttgart.de [Dr. Margarete Fischer-Bosch-Institute of Clinical Pharmacology, Stuttgart, Germany, and University of Tuebingen, Auerbachstr. 112, D-70376 Stuttgart (Germany); Burk, Oliver, E-mail: oliver.burk@ikp-stuttgart.de [Dr. Margarete Fischer-Bosch-Institute of Clinical Pharmacology, Stuttgart, Germany, and University of Tuebingen, Auerbachstr. 112, D-70376 Stuttgart (Germany)

    2010-03-19

    Induction of the major drug metabolizing enzyme CYP3A4 by xenobiotics contributes to the pronounced interindividual variability of its expression and often results in clinically relevant drug-drug interactions. It is mainly mediated by PXR, which regulates CYP3A4 expression by binding to several specific elements in the 5' upstream regulatory region of the gene. Induction itself shows a marked interindividual variability, whose underlying determinants are only partly understood. In this study, we investigated the role of nuclear receptor binding to PXR response elements in CYP3A4, as a potential non-genetic mechanism contributing to interindividual variability of induction. By in vitro DNA binding experiments, we showed that several nuclear receptors bind efficiently to the proximal promoter ER6 and distal xenobiotic-responsive enhancer module DR3 motifs. TR{alpha}1, TR{beta}1, COUP-TFI, and COUP-TFII further demonstrated dose-dependent repression of PXR-mediated CYP3A4 enhancer/promoter reporter activity in transient transfection in the presence and absence of the PXR inducer rifampin, while VDR showed this effect only in the absence of treatment. By combining functional in vitro characterization with hepatic expression analysis, we predict that TR{alpha}1, TR{beta}1, COUP-TFI, and COUP-TFII show a strong potential for the repression of PXR-mediated activation of CYP3A4 in vivo. In summary, our results demonstrate that nuclear receptor binding to PXR response elements interferes with PXR-mediated expression and induction of CYP3A4 and thereby contributes to the interindividual variability of induction.

  13. Avian cytochrome P450 (CYP 1-3 family genes: isoforms, evolutionary relationships, and mRNA expression in chicken liver.

    Directory of Open Access Journals (Sweden)

    Kensuke P Watanabe

    Full Text Available Cytochrome P450 (CYP of chicken and other avian species have been studied primarily with microsomes or characterized by cloning and protein expression. However, the overall existing isoforms in avian CYP1-3 families or dominant isoforms in avian xenobiotic metabolism have not yet been elucidated. In this study, we aimed to clarify and classify all of the existing isoforms of CYP1-3 in avian species using available genome assemblies for chicken, zebra finch, and turkey. Furthermore, we performed qRT-PCR assay to identify dominant CYP genes in chicken liver. Our results suggested that avian xenobiotic-metabolizing CYP genes have undergone unique evolution such as CYP2C and CYP3A genes, which have undergone avian-specific gene duplications. qRT-PCR experiments showed that CYP2C45 was the most highly expressed isoform in chicken liver, while CYP2C23b was the most highly induced gene by phenobarbital. Considering together with the result of further enzymatic characterization, CYP2C45 may have a dominant role in chicken xenobiotic metabolism due to the constitutive high expression levels, while CYP2C23a and CYP2C23b can be greatly induced by chicken xenobiotic receptor (CXR activators. These findings will provide not only novel insights into avian xenobiotic metabolism, but also a basis for the further characterization of each CYP gene.

  14. Effect of cyclophilin A on gene expression in human pancreatic cancer cells.

    Science.gov (United States)

    Li, Min; Wang, Hao; Li, Fei; Fisher, William E; Chen, Changyi; Yao, Qizhi

    2005-11-01

    We previously found that cyclophilin A (CypA) is overexpressed in human pancreatic cancer cells and stimulates cell proliferation through CD147. In this study, we further investigated the effect of CypA on gene expression of several key molecules that are involved in pancreatic cancer cell proliferation. Human pancreatic cancer cell lines (Panc-1, MIA PaCa-2, and BxPC-3) and human pancreatic ductal epithelial (HPDE) cells were used. The messenger RNA (mRNA) levels of CypA, CypB, CD147, neuropilins (NRPs), vascular endothelial growth factor (VEGF), and VEGF receptors upon the treatment of exogenous recombinant human CypA were determined by real-time reverse-transcription polymerase chain reaction. Exogenous human recombinant CypA reduced the mRNA levels of NRP-1 and VEGF, but not endogenous CypA, CypB, and CD147, in Panc-1, MIA PaCa-2, and BxPC-3 cells. In contrast, HPDE cells showed a decrease of endogenous CypA and CD147 mRNA, but not detectable changes of CypB, NRPs, and VEGF mRNA levels upon exogenous CypA treatment. These data show that exogenous CypA downregulates NRP-1 and VEGF expression in pancreatic cancer cells. This effect is different in normal HPDE cells. Thus, soluble CypA may affect cell growth of pancreatic cancer.

  15. Altered Protein Expression of Cardiac CYP2J and Hepatic CYP2C, CYP4A, and CYP4F in a Mouse Model of Type II Diabetes—A Link in the Onset and Development of Cardiovascular Disease?

    Directory of Open Access Journals (Sweden)

    Benoit Drolet

    2017-10-01

    Full Text Available Arachidonic acid can be metabolized by cytochrome P450 (CYP450 enzymes in a tissue- and cell-specific manner to generate vasoactive products such as epoxyeicosatrienoic acids (EETs-cardioprotective and hydroxyeicosatetraenoic acids (HETEs-cardiotoxic. Type II diabetes is a well-recognized risk factor for developing cardiovascular disease. A mouse model of Type II diabetes (C57BLKS/J-db/db was used. After sacrifice, livers and hearts were collected, washed, and snap frozen. Total proteins were extracted. Western blots were performed to assess cardiac CYP2J and hepatic CYP2C, CYP4A, and CYP4F protein expression, respectively. Significant decreases in relative protein expression of cardiac CYP2J and hepatic CYP2C were observed in Type II diabetes animals compared to controls (CYP2J: 0.80 ± 0.03 vs. 1.05 ± 0.06, n = 20, p < 0.001; (CYP2C: 1.56 ± 0.17 vs. 2.21 ± 0.19, n = 19, p < 0.01. In contrast, significant increases in relative protein expression of both hepatic CYP4A and CYP4F were noted in Type II diabetes mice compared to controls (CYP4A: 1.06 ± 0.09 vs. 0.18 ± 0.01, n = 19, p < 0.001; (CYP4F: 2.53 ± 0.22 vs. 1.10 ± 0.07, n = 19, p < 0.001. These alterations induced by Type II diabetes in the endogenous pathway (CYP450 of arachidonic acid metabolism may increase the risk for cardiovascular disease by disrupting the fine equilibrium between cardioprotective (CYP2J/CYP2C-generated and cardiotoxic (CYP4A/CYP4F-generated metabolites of arachidonic acid.

  16. Impact of CYP2C8*3 on paclitaxel clearance

    DEFF Research Database (Denmark)

    Bergmann, T K; Brasch-Andersen, C; Gréen, H

    2011-01-01

    The primary purpose of this study was to evaluate the effect of CYP2C8*3 and three genetic ABCB1 variants on the elimination of paclitaxel. We studied 93 Caucasian women with ovarian cancer treated with paclitaxel and carboplatin. Using sparse sampling and nonlinear mixed effects modeling, the in...... associations found for CYP2C8*4 (P=0.04) and ABCC1 g.7356253C>G (P=0.04).The Pharmacogenomics Journal advance online publication, 6 April 2010; doi:10.1038/tpj.2010.19....

  17. Genetic analysis of drug metabolizing phase-I enzymes CYP3A4 in ...

    Indian Academy of Sciences (India)

    LIJUN LIU1

    3Key Laboratory of High Altitude Environment and Gene Related to Disease of Tibet Ministry of Education,. Xizang Minzu University ..... basic profile of CYP3A4 in the Tibetan population, and can be used to determine optimal dosage ... 19), and The Project of Young Teachers' Innovative Support. Programme in Universities ...

  18. Newly identified CYP2C93 is a functional enzyme in rhesus monkey, but not in cynomolgus monkey.

    Directory of Open Access Journals (Sweden)

    Yasuhiro Uno

    Full Text Available Cynomolgus monkey and rhesus monkey are used in drug metabolism studies due to their evolutionary closeness and physiological resemblance to human. In cynomolgus monkey, we previously identified cytochrome P450 (P450 or CYP 2C76 that does not have a human ortholog and is partly responsible for species differences in drug metabolism between cynomolgus monkey and human. In this study, we report characterization of CYP2C93 cDNA newly identified in cynomolgus monkey and rhesus monkey. The CYP2C93 cDNA contained an open reading frame of 490 amino acids approximately 84-86% identical to human CYP2Cs. CYP2C93 was located in the genomic region, which corresponded to the intergenic region in the human genome, indicating that CYP2C93 does not correspond to any human genes. CYP2C93 mRNA was expressed predominantly in the liver among 10 tissues analyzed. The CYP2C93 proteins heterologously expressed in Escherichia coli metabolized human CYP2C substrates, diclofenac, flurbiprofen, paclitaxel, S-mephenytoin, and tolbutamide. In addition to a normal transcript (SV1, an aberrantly spliced transcript (SV2 lacking exon 2 was identified, which did not give rise to a functional protein due to frameshift and a premature termination codon. Mini gene assay revealed that the genetic variant IVS2-1G>T at the splice site of intron 1, at least partly, accounted for the exon-2 skipping; therefore, this genotype would influence CYP2C93-mediated drug metabolism. SV1 was expressed in 6 of 11 rhesus monkeys and 1 of 8 cynomolgus monkeys, but the SV1 in the cynomolgus monkey was nonfunctional due to a rare null genotype (c.102T>del. These results suggest that CYP2C93 can play roles as a drug-metabolizing enzyme in rhesus monkeys (not in cynomolgus monkeys, although its relative contribution to drug metabolism has yet to be validated.

  19. Metformin suppresses CYP1A1 and CYP1B1 expression in breast cancer cells by down-regulating aryl hydrocarbon receptor expression

    Energy Technology Data Exchange (ETDEWEB)

    Do, Minh Truong; Kim, Hyung Gyun; Tran, Thi Thu Phuong; Khanal, Tilak; Choi, Jae Ho [Department of Toxicology, College of Pharmacy, Chungnam National University, Daejeon (Korea, Republic of); Chung, Young Chul [Department of Food Science and Culinary, International University of Korea, Jinju (Korea, Republic of); Jeong, Tae Cheon, E-mail: taecheon@ynu.ac.kr [College of Pharmacy, Yeungnam University, Gyeongsan (Korea, Republic of); Jeong, Hye Gwang, E-mail: hgjeong@cnu.ac.kr [Department of Toxicology, College of Pharmacy, Chungnam National University, Daejeon (Korea, Republic of)

    2014-10-01

    Induction of cytochrome P450 (CYP) 1A1 and CYP1B1 by environmental xenobiotic chemicals or endogenous ligands through the activation of the aryl hydrocarbon receptor (AhR) has been implicated in a variety of cellular processes related to cancer, such as transformation and tumorigenesis. Here, we investigated the effects of the anti-diabetes drug metformin on expression of CYP1A1 and CYP1B1 in breast cancer cells under constitutive and inducible conditions. Our results indicated that metformin down-regulated the expression of CYP1A1 and CYP1B1 in breast cancer cells under constitutive and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced conditions. Down-regulation of AhR expression was required for metformin-mediated decreases in CYP1A1 and CYP1B1 expression, and the metformin-mediated CYP1A1 and CYP1B1 reduction is irrelevant to estrogen receptor α (ERα) signaling. Furthermore, we found that metformin markedly down-regulated Sp1 protein levels in breast cancer cells. The use of genetic and pharmacological tools revealed that metformin-mediated down-regulation of AhR expression was mediated through the reduction of Sp1 protein. Metformin inhibited endogenous AhR ligand-induced CYP1A1 and CYP1B1 expression by suppressing tryptophan-2,3-dioxygenase (TDO) expression in MCF-7 cells. Finally, metformin inhibits TDO expression through a down-regulation of Sp1 and glucocorticoid receptor (GR) protein levels. Our findings demonstrate that metformin reduces CYP1A1 and CYP1B1 expression in breast cancer cells by down-regulating AhR signaling. Metformin would be able to act as a potential chemopreventive agent against CYP1A1 and CYP1B1-mediated carcinogenesis and development of cancer. - Graphical abstract: Schematic of the CYP1A1 and CYP1B1 gene regulation by metformin. - Highlights: • Metformin inhibits CYP1A1 and CYP1B1 expression. • Metformin down-regulates the AhR signaling. • Metformin reduces Sp1 protein expression. • Metformin suppresses TDO expression.

  20. Metformin suppresses CYP1A1 and CYP1B1 expression in breast cancer cells by down-regulating aryl hydrocarbon receptor expression

    International Nuclear Information System (INIS)

    Do, Minh Truong; Kim, Hyung Gyun; Tran, Thi Thu Phuong; Khanal, Tilak; Choi, Jae Ho; Chung, Young Chul; Jeong, Tae Cheon; Jeong, Hye Gwang

    2014-01-01

    Induction of cytochrome P450 (CYP) 1A1 and CYP1B1 by environmental xenobiotic chemicals or endogenous ligands through the activation of the aryl hydrocarbon receptor (AhR) has been implicated in a variety of cellular processes related to cancer, such as transformation and tumorigenesis. Here, we investigated the effects of the anti-diabetes drug metformin on expression of CYP1A1 and CYP1B1 in breast cancer cells under constitutive and inducible conditions. Our results indicated that metformin down-regulated the expression of CYP1A1 and CYP1B1 in breast cancer cells under constitutive and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-induced conditions. Down-regulation of AhR expression was required for metformin-mediated decreases in CYP1A1 and CYP1B1 expression, and the metformin-mediated CYP1A1 and CYP1B1 reduction is irrelevant to estrogen receptor α (ERα) signaling. Furthermore, we found that metformin markedly down-regulated Sp1 protein levels in breast cancer cells. The use of genetic and pharmacological tools revealed that metformin-mediated down-regulation of AhR expression was mediated through the reduction of Sp1 protein. Metformin inhibited endogenous AhR ligand-induced CYP1A1 and CYP1B1 expression by suppressing tryptophan-2,3-dioxygenase (TDO) expression in MCF-7 cells. Finally, metformin inhibits TDO expression through a down-regulation of Sp1 and glucocorticoid receptor (GR) protein levels. Our findings demonstrate that metformin reduces CYP1A1 and CYP1B1 expression in breast cancer cells by down-regulating AhR signaling. Metformin would be able to act as a potential chemopreventive agent against CYP1A1 and CYP1B1-mediated carcinogenesis and development of cancer. - Graphical abstract: Schematic of the CYP1A1 and CYP1B1 gene regulation by metformin. - Highlights: • Metformin inhibits CYP1A1 and CYP1B1 expression. • Metformin down-regulates the AhR signaling. • Metformin reduces Sp1 protein expression. • Metformin suppresses TDO expression

  1. Inactivation of CYP2A6 by the Dietary Phenylpropanoid trans-Cinnamic Aldehyde (Cinnamaldehyde) and Estimation of Interactions with Nicotine and Letrozole

    OpenAIRE

    Chan, Jeannine; Oshiro, Tyler; Thomas, Sarah; Higa, Allyson; Black, Stephen; Todorovic, Aleksandar; Elbarbry, Fawzy; Harrelson, John P.

    2016-01-01

    Human exposure to trans-cinnamic aldehyde [t-CA; cinnamaldehyde; cinnamal; (E)-3-phenylprop-2-enal] is common through diet and through the use of cinnamon powder for diabetes and to provide flavor and scent in commercial products. We evaluated the likelihood of t-CA to influence metabolism by inhibition of P450 enzymes. IC50 values from recombinant enzymes indicated that an interaction is most probable for CYP2A6 (IC50 = 6.1 ?M). t-CA was 10.5-fold more selective for human CYP2A6 than for CYP...

  2. Influence of CYP3A5 and ABCB1 gene polymorphisms on calcineurin inhibitor-related neurotoxicity after hematopoietic stem cell transplantation.

    Science.gov (United States)

    Yanagimachi, Masakatsu; Naruto, Takuya; Tanoshima, Reo; Kato, Hiromi; Yokosuka, Tomoko; Kajiwara, Ryosuke; Fujii, Hisaki; Tanaka, Fumiko; Goto, Hiroaki; Yagihashi, Tatsuhiko; Kosaki, Kenjiro; Yokota, Shumpei

    2010-01-01

    One severe side effect of calcineurin inhibitors (CNIs: such as cyclosporine [CsA] and tacrolimus [FK506]) is neurotoxicity. CNIs are substrates for CYP3A5 and P-glycoprotein (P-gp), encoded by ABCB1 gene. In the present study, we hypothesized that genetic variability in CYP3A5 and ABCB1 genes may be associated with CNI-related neurotoxicity. The effects of the polymorphisms, such as CYP3A5 A6986G, ABCB1 C1236T, G2677T/A, and C3435T, associated with CNI-related neurotoxicity were evaluated in 63 patients with hematopoietic stem cell transplantation.   Of the 63 cases, 15 cases developed CNI-related neurotoxicity. In the CsA patient group (n = 30), age (p = 0.008), hypertension (p = 0.017), renal dysfunction (p < 0.001), ABCB1 C1236T (p < 0.001), and G2677T/A (p = 0.014) were associated with neurotoxicities. The CC genotype at ABCB1 C1236T was associated with it, but not significantly so (p = 0.07), adjusted for age, hypertension, and renal dysfunction. In the FK506 patient group (n = 33), CYP3A5 A6986G (p < 0.001), and ABCB1 C1236T (p = 0.002) were associated with neurotoxicity. At least one A allele at CYP3A5 A6986G (expressor genotype) was strongly associated with it according to logistic regression analysis (p = 0.01; OR, 8.5; 95% CI, 1.4-51.4).   The polymorphisms in CYP3A5 and ABCB1 genes were associated with CNI-related neurotoxicity. This outcome is probably because of CYP3A5 or P-gp functions or metabolites of CNIs. © 2009 John Wiley & Sons A/S.

  3. Somatic cell genotoxicity at the glycophorin A locus in humans

    International Nuclear Information System (INIS)

    Jensen, R.H.; Grant, S.G.; Langlois, R.G.; Bigbee, W.L.

    1990-01-01

    We have developed an assay for detecting variant erythrocytes that occur as a result of in vivo allele loss at the glycophorin A (GPA) locus on chromosome 4 in humans. This gene codes for an erythroid- specific cell surface glycoprotein, and with our assay we are able to detect rare variant erythrocytes that have lost expression of one of the two GPA alleles. Two distinctly different variant cell types are detected with this assay. One variant cell type (called N OE) is hemizygous. Our assay also detects homozygous variant erythrocytes that have lost expression of the GPA(M) allele and express the GPA(N) allele at twice the heterozygous level. The results of this assay are an enumeration of the frequency of N OE and NN variant cell types for each individual analyzed. These variant cell frequencies provide a measure of the amount of somatic cell genotoxicity that has occurred at the GPA locus. Such genotoxicity could be the result of (1) reactions of toxic chemicals to which the individual has been exposed, or (2) high energy radiation effects on erythroid precursor cells, or (3) errors in DNA replication or repair in these cells of the bone marrow. Thus, the GPA-based variant cell frequency can serve as a biodosimeter that indicates the amount of genotoxic exposure each individual has received. Because two very different kinds of variant cells are enumerated, different kinds of genotoxicity should be distinguishable. Results of the GPA somatic genotoxicity assay may also provide valuable information for cancer-risk estimation on each individual. 16 refs

  4. Dose-dependent interaction between gemfibrozil and repaglinide in humans: strong inhibition of CYP2C8 with subtherapeutic gemfibrozil doses.

    Science.gov (United States)

    Honkalammi, Johanna; Niemi, Mikko; Neuvonen, Pertti J; Backman, Janne T

    2011-10-01

    Gemfibrozil 1-O-β-glucuronide inactivates CYP2C8 irreversibly. We investigated the effect of gemfibrozil dose on CYP2C8 activity in humans using repaglinide as a probe drug. In a randomized, five-phase crossover study, 10 healthy volunteers ingested 0.25 mg of repaglinide 1 h after different doses of gemfibrozil or placebo. Concentrations of plasma repaglinide, gemfibrozil, their metabolites, and blood glucose were measured. A single gemfibrozil dose of 30, 100, 300, and 900 mg increased the area under the concentration-time curve of repaglinide 1.8-, 4.5-, 6.7-, and 8.3-fold (P Gemfibrozil pharmacokinetics was characterized by a slightly more than dose-proportional increase in the area under the curve of gemfibrozil and its glucuronide. The gemfibrozil-repaglinide interaction could be mainly explained by gemfibrozil 1-O-β-glucuronide concentration-dependent, mechanism-based inhibition of CYP2C8, with a minor contribution by competitive inhibition of organic anion-transporting polypeptide 1B1 at the highest gemfibrozil dose. The findings are consistent with ∼50% inhibition of CYP2C8 already with a single 30-mg dose of gemfibrozil and >95% inhibition with 900 mg. In clinical drug-drug interaction studies, a single 900-mg dose of gemfibrozil can be used to achieve nearly complete inactivation of CYP2C8.

  5. CYP24A1 exacerbated activity during diabetes contributes to kidney tubular apoptosis via caspase-3 increased expression and activation.

    Directory of Open Access Journals (Sweden)

    Alexandre Tourigny

    Full Text Available Decreases in circulating 25,hydroxyl-vitamin D3 (25 OH D3 and 1,25,dihydroxyl-vitamin D3 (1,25 (OH2 D3 have been extensively documented in patients with type 2 diabetes. Nevertheless, the molecular reasons behind this drop, and whether it is a cause or an effect of disease progression is still poorly understood. With the skin and the liver, the kidney is one of the most important sites for vitamin D metabolism. Previous studies have also shown that CYP24A1 (an enzyme implicated in vitamin D metabolism, might play an important role in furthering the progression of kidney lesions during diabetic nephropathy. In this study we show a link between CYP24A1 increase and senescence followed by apoptosis induction in the renal proximal tubules of diabetic kidneys. We show that CYP24A1 expression was increased during diabetic nephropathy progression. This increase derived from protein kinase C activation and increased H(2O(2 cellular production. CYP24A1 increase had a major impact on cellular phenotype, by pushing cells into senescence, and later into apoptosis. Our data suggest that control of CYP24A1 increase during diabetes has a beneficial effect on senescence induction and caspase-3 increased expression. We concluded that diabetes induces an increase in CYP24A1 expression, destabilizing vitamin D metabolism in the renal proximal tubules, leading to cellular instability and apoptosis, and thereby accelerating tubular injury progression during diabetic nephropathy.

  6. The enhanced atorvastatin hepatotoxicity in diabetic rats was partly attributed to the upregulated hepatic Cyp3a and SLCO1B1

    Science.gov (United States)

    Shu, Nan; Hu, Mengyue; Ling, Zhaoli; Liu, Peihua; Wang, Fan; Xu, Ping; Zhong, Zeyu; Sun, Binbin; Zhang, Mian; Li, Feng; Xie, Qiushi; Liu, Xiaodong; Liu, Li

    2016-01-01

    Liver injury is a common adverse effect of atorvastatin. This study aimed to investigate atorvastatin-induced hepatotoxicity in diabetic rats induced by high-fat diet combined with streptozotocin. The results showed that 40 mg/kg atorvastatin was lethal to diabetic rats, whose mean survival time was 6.2 days. Severe liver injury also occurred in diabetic rats treated with 10 mg/kg and 20 mg/kg atorvastatin. The in vitro results indicated that atorvastatin cytotoxicity in hepatocytes of diabetic rats was more severe than normal and high-fat diet feeding rats. Expressions and activities of hepatic Cyp3a and SLCO1B1 were increased in diabetic rats, which were highly correlated with hepatotoxicity. Antioxidants (glutathione and N-Acetylcysteine), Cyp3a inhibitor ketoconazole and SLCO1B1 inhibitor gemfibrozil suppressed cytotoxicity and ROS formation in primary hepatocytes of diabetic rats. In HepG2 cells, up-regulations of CYP3A4 and SLCO1B1 potentiated hepatotoxicity and ROS generation, whereas knockdowns of CYP3A4 and SLCO1B1 as well as CYP3A4/SLCO1B1 inhibitions showed the opposite effects. Phenobarbital pretreatment was used to induce hepatic Cyp3a and SLCO1B1 in rats. Phenobarbital aggravated atorvastatin-induced hepatotoxicity, while decreased plasma exposure of atorvastatin. All these findings demonstrated that the upregulations of hepatic Cyp3a and SLCO1B1 in diabetic rats potentiated atorvastatin-induced hepatotoxicity via increasing ROS formation. PMID:27624558

  7. Characterization of midazolam metabolism in locusts: The role of CYP3A4-like enzyme in the formation of 1'-OH and 4-OH midazolam

    DEFF Research Database (Denmark)

    Olsen, Line Rørbæk; Gabel-Jensen, Charlotte; Wubshet, Sileshi Gizachew

    2016-01-01

    ) were in the same range as reported in humans (in locusts: 7-23 and 33-85 µM for the formation of the 1'-OH and 4-OH metabolites, respectively). 3. The formation of hydroxylated metabolites could successfully be inhibited by co-administration of ketoconazole, a known CYP3A4/5 inhibitor. 4. Besides phase...

  8. PacCYP707A2 negatively regulates cherry fruit ripening while PacCYP707A1 mediates drought tolerance.

    Science.gov (United States)

    Li, Qian; Chen, Pei; Dai, Shengjie; Sun, Yufei; Yuan, Bing; Kai, Wenbin; Pei, Yuelin; He, Suihuan; Liang, Bin; Zhang, Yushu; Leng, Ping

    2015-07-01

    Sweet cherry is a non-climacteric fruit and its ripening is regulated by abscisic acid (ABA) during fruit development. In this study, four cDNAs (PacCYP707A1-4) encoding 8'-hydroxylase, a key enzyme in the oxidative catabolism of ABA, were identified in sweet cherry fruits using tobacco rattle virus-induced gene silencing (VIGS) and particle bombardment approaches. Quantitative real-time PCR confirmed significant down-regulation of target gene transcripts in VIGS-treated cherry fruits. In PacCYP707A2-RNAi-treated fruits, ripening and fruit colouring were promoted relative to control fruits, and both ABA accumulation and PacNCED1 transcript levels were up-regulated by 140%. Silencing of PacCYP707A2 by VIGS significantly altered the transcripts of both ABA-responsive and ripening-related genes, including the ABA metabolism-associated genes NCED and CYP707A, the anthocyanin synthesis genes PacCHS, PacCHI, PacF3H, PacDFR, PacANS, and PacUFGT, the ethylene biosynthesis gene PacACO1, and the transcription factor PacMYBA. The promoter of PacMYBA responded more strongly to PacCYP707A2-RNAi-treated fruits than to PacCYP707A1-RNAi-treated fruits. By contrast, silencing of PacCYP707A1 stimulated a slight increase in fruit colouring and enhanced resistance to dehydration stress compared with control fruits. These results suggest that PacCYP707A2 is a key regulator of ABA catabolism that functions as a negative regulator of fruit ripening, while PacCYP707A1 regulates ABA content in response to dehydration during fruit development. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  9. 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

  10. Biotransformation of the mineralocorticoid receptor antagonists spironolactone and canrenone by human CYP11B1 and CYP11B2: Characterization of the products and their influence on mineralocorticoid receptor transactivation.

    Science.gov (United States)

    Schiffer, Lina; Müller, Anne-Rose; Hobler, Anna; Brixius-Anderko, Simone; Zapp, Josef; Hannemann, Frank; Bernhardt, Rita

    2016-10-01

    Spironolactone and its major metabolite canrenone are potent mineralocorticoid receptor antagonists and are, therefore, applied as drugs for the treatment of primary aldosteronism and essential hypertension. We report that both compounds can be converted by the purified adrenocortical cytochromes P450 CYP11B1 and CYP11B2, while no conversion of the selective mineralocorticoid receptor antagonist eplerenone was observed. As their natural function, CYP11B1 and CYP11B2 carry out the final steps in the biosynthesis of gluco- and mineralocorticoids. Dissociation constants for the new exogenous substrates were determined by a spectroscopic binding assay and demonstrated to be comparable to those of the natural substrates, 11-deoxycortisol and 11-deoxycorticosterone. Metabolites were produced at preparative scale with a CYP11B2-dependent Escherichia coli whole-cell system and purified by HPLC. Using NMR spectroscopy, the metabolites of spironolactone were identified as 11β-OH-spironolactone, 18-OH-spironolactone and 19-OH-spironolactone. Canrenone was converted to 11β-OH-canrenone, 18-OH-canrenone as well as to the CYP11B2-specific product 11β,18-diOH-canrenone. Therefore, a contribution of CYP11B1 and CYP11B2 to the biotransformation of drugs should be taken into account and the metabolites should be tested for their potential toxic and pharmacological effects. A mineralocorticoid receptor transactivation assay in antagonist mode revealed 11β-OH-spironolactone as pharmaceutically active metabolite, whereas all other hydroxylation products negate the antagonist properties of spironolactone and canrenone. Thus, human CYP11B1 and CYP11B2 turned out to metabolize steroid-based drugs additionally to the liver-dependent biotransformation of drugs. Compared with the action of the parental drug, changed properties of the metabolites at the target site have been observed. Copyright © 2016 Elsevier Ltd. All rights reserved.

  11. Mutation at the Human D1S80 Minisatellite Locus

    Directory of Open Access Journals (Sweden)

    Kuppareddi Balamurugan

    2012-01-01

    Full Text Available Little is known about the general biology of minisatellites. The purpose of this study is to examine repeat mutations from the D1S80 minisatellite locus by sequence analysis to elucidate the mutational process at this locus. This is a highly polymorphic minisatellite locus, located in the subtelomeric region of chromosome 1. We have analyzed 90,000 human germline transmission events and found seven (7 mutations at this locus. The D1S80 alleles of the parentage trio, the child, mother, and the alleged father were sequenced and the origin of the mutation was determined. Using American Association of Blood Banks (AABB guidelines, we found a male mutation rate of 1.04×10-4 and a female mutation rate of 5.18×10-5 with an overall mutation rate of approximately 7.77×10-5. Also, in this study, we found that the identified mutations are in close proximity to the center of the repeat array rather than at the ends of the repeat array. Several studies have examined the mutational mechanisms of the minisatellites according to infinite allele model (IAM and the one-step stepwise mutation model (SMM. In this study, we found that this locus fits into the one-step mutation model (SMM mechanism in six out of seven instances similar to STR loci.

  12. Identification of novel CYP2D7-2D6 hybrids: non-functional and functional variants

    Directory of Open Access Journals (Sweden)

    Andrea Gaedigk

    2010-10-01

    Full Text Available Polymorphic expression of CYP2D6 contributes to the wide range of activity observed for this clinically important drug metabolizing enzyme. In this report we describe novel CYP2D7/2D6 hybrid genes encoding non-functional and functional CYP2D6 protein and a CYP2D7 variant that mimics a CYP2D7/2D6 hybrid gene. Five kb long PCR products encompassing the novel genes were entirely sequenced. A quantitative assay probing in different gene regions was employed to determine CYP2D6 and 2D7 copy number variations and the relative position of the hybrid genes within the locus was assessed by long-range PCR. In addition to the previously known CYP2D6*13 and *66 hybrids, we describe three novel non-functional CYP2D7-2D6 hybrids with gene switching in exon 2 (CYP2D6*79, intron 2 (CYP2D6*80 and intron 5 (CYP2D6*67. A CYP2D7-specific T-ins in exon 1 causes a detrimental frame shift. One subject revealed a CYP2D7 conversion in the 5’-flanking region of a CYP2D6*35 allele, was otherwise unaffected (designated CYP2D6*35B. Finally, three DNAs revealed a CYP2D7 gene with a CYP2D6-like region downstream of exon 9 (designated CYP2D7[REP6]. Quantitative copy number determination, sequence analyses and long-range PCR mapping were in agreement and excluded the presence of additional gene units. Undetected hybrid genes may cause over-estimation of CYP2D6 activity (CYP2D6*1/*1 vs *1/hybrid, etc, but may also cause results that may interfere with the genotype determination. Detection of hybrid events, ‘single’ and tandem, will contribute to more accurate phenotype prediction from genotype data.

  13. A novel locus for dilated cardiomyopathy maps to canine chromosome 8.

    Science.gov (United States)

    Werner, Petra; Raducha, Michael G; Prociuk, Ulana; Sleeper, Meg M; Van Winkle, Thomas J; Henthorn, Paula S

    2008-06-01

    Dilated cardiomyopathy (DCM), the most common form of cardiomyopathy, often leads to heart failure and sudden death. While a substantial proportion of DCMs are inherited, mutations responsible for the majority of DCMs remain unidentified. A genome-wide linkage study was performed to identify the locus responsible for an autosomal recessive inherited form of juvenile DCM (JDCM) in Portuguese water dogs using 16 families segregating the disease. Results link the JDCM locus to canine chromosome 8 with two-point and multipoint lod scores of 10.8 and 14, respectively. The locus maps to a 3.9-Mb region, with complete syntenic homology to human chromosome 14, that contains no genes or loci known to be involved in the development of any type of cardiomyopathy. This discovery of a DCM locus with a previously unknown etiology will provide a new gene to examine in human DCM patients and a model for testing therapeutic approaches for heart failure.

  14. Tributyltin modulates 3,3',4,4',5-pentachlorobiphenyl (PCB-126)-induced hepatic CYP1A activity in channel catfish, Ictalurus punctatus.

    Science.gov (United States)

    Rice, C D; Roszell, L E

    1998-10-09

    Many harbor estuaries and their tributaries are contaminated with halogenated aromatic hydrocarbons (HAHs) and polycyclic aromatic hydrocarbons (PAHs). Planar congeners of these two classes initiate their toxic effects, including reproductive, developmental, and immunological dysfunction, primarily through the cytosolic arylhydrocabon receptor (Ahr). However, only rarely are aquatic environments contaminated with Ahr-binding contaminants alone. Instead, most are impacted by a variety of pollutants in mixture. Tributyltin (TBT), a common antifouling biocide, is also found in many harbor estuaries and their tributaries. Several reports indicate that TBT inhibits the cytochrome P-4501A system of fish, at least in vitro, and our recent studies with rodents indicate that TBT potentiates PCB-induced CYP1A. However, the effects of TBT on xenobiotic-induced CYP1A activity in aquatic organisms has been virtually unexplored. To this end, channel catfish, Ictalurus punctatus, were exposed to 3,3'4,4',5-pentachlorobiphenyl (PCB-126, PeCB), TBT, or both in combination, with corn oil (CO) serving as the carrier control. Immunoreactive CYP1A protein and ethoxyresorufin O-deethylase (EROD) activity were measured after (1) a single dose of 0.01, 0. 1, or 1 mg/kg of each or both in combination, and (2) 6 injections of 0.017, 1.7, or 17 microg/kg of each (or in combination) given every 3 d over a 16-d period to yield a cumulative dose of 0.01, 0.1, or 1 mg/kg. As expected, PeCB alone, but not TBT, greatly induced these two CYP1A parameters. Low and middle doses of TBT (0.01 and 0.1 mg/kg), but not the high dose, potentiated PeCB-induced activity at these same doses. This effect of TBT was even more pronounced in the repeated exposure study. Furthermore, EROD activity did not always reflect CYP1A protein induction; enzyme activity was inhibited by TBT at doses that potentiated protein induction (0.01 and 0.1 mg/kg). In summary, TBT potentiates PeCB-induced CYP1A in channel catfish at

  15. Molecular analysis of the human β-globin locus activation region

    International Nuclear Information System (INIS)

    Forrester, W.C.; Novak, U.; Gelinas, R.; Groudine, M.

    1989-01-01

    Recently, DNA sequences containing four erythroid-specific DNase I hypersensitive sites within 20 kilobases 5' of the human ε-globin gene have been identified as an important cis-acting regulatory element, the locus activation region (LAR). Subfragments of the LAR, containing either all or only the two 5' or two 3' hypersensitive sites were linked to the human β-globin gene and analyzed for their effect on globin gene expression in stably transformed mouse erythroleukemia (MEL) cells. Constructs containing all four of the hypersensitive sites increase β-gobin mRNA levels 8- to 13-fold, while constructs with only the 5' or 3' sites increase globin expression to a lesser extent. No effect was seen when the constructs were assayed in 3T3 fibroblasts. All of the LAR derivatives form hypersensitive sites at the corresponding sequence position in MEL cells prior to and after induction of MEL cell differentiation. However, in 3T3 fibroblasts only the hypersensitive site corresponding to the previously described erythroid-specific -10.9 site was formed

  16. Racial Differences in CYP3A4 Genotype and Survival Among Men Treated on Radiation Therapy Oncology Group (RTOG) 9202: A Phase III Randomized Trial

    International Nuclear Information System (INIS)

    Roach, Mack; Silvio, Michelle de; Rebbick, Timothy; Grignon, David; Rotman, Marvin; Wolkov, Harvey; Fisher, Barbara; Hanks, Gerald; Shipley, William U.; Pollack, Alan; Sandler, Howard; Watkins-Bruner, Deborah Ph.D.

    2007-01-01

    Purpose: Inherited genotypes may explain the inferior outcomes of African American (AA) men with prostate cancer. To understand how variation in CYP3A4 correlated with outcomes, a retrospective examination of the CYP3A4*1B genotype was performed on men treated with Radiation Therapy Oncology Group (RTOG) 92-02. Methods and Materials: From 1,514 cases, we evaluated 56 (28.4%) of 197 AA and 54 (4.3%) of 1,274 European American (EA) patients. All patients received goserelin and flutamide for 2 months before and during RT (STAD-RT) ± 24 months of goserelin (long-term androgen deprivation plus radiation [LTAD-RT]). Events studied included overall survival and biochemical progression using American Society for Therapeutic Radiology and Oncology consensus guidelines. Results: There were no differences in outcome in patients in with or without CYP3A4 data. There was an association between race and CYP3A4 polymorphisms with 75% of EAs having the Wild Type compared to only 25% of AA men (p <0.0001). There was no association between CYP3A4 classification or race and survival or progression. Conclusions: The samples analyzed support previously reported observations about the distribution of CYP3A4*1B genotype by race, but race was not associated with poorer outcome. However, patient numbers were limited, and selection bias cannot be completely ruled out

  17. Overexpression of CYP3A4 in a COLO 205 Colon Cancer Stem Cell Model in vitro

    International Nuclear Information System (INIS)

    Olszewski, Ulrike; Liedauer, Richard; Ausch, Christoph; Thalhammer, Theresia; Hamilton, Gerhard

    2011-01-01

    Cancer stem cells (CSCs) seem to constitute a subpopulation of tumor cells that escape from chemotherapy and cause recurrent disease. Low proliferation rates, protection in a stem cell niche and overexpression of drug resistance proteins are considered to confer chemoresistance. We established an in vitro colon CSC-like model using the COLO 205 cell line, which revealed transiently increased expression of CD133 when transferred to serum-free stem cell culture medium. Assessment of global gene expression of COLO 205 cells under these conditions identified a set of upregulated genes including cytochrome P450 3A4 (CYP3A4) and aldehyde dehydrogenase 1A1 (ALDH1A1), as confirmed by real-time qPCR. ALDH1A1 is a CSC marker for certain tumor entities and confers resistance to cyclophosphamide. CYP3A4 is expressed in liver and colon and its overexpression seems particularly relevant in colon cancer, since it inactivates irinotecan and other xenobiotics, such as taxols and vinca alkaloids. In conclusion, this COLO 205 model provides evidence for CD133 induction concomitant with overexpression of CYP3A4, which, together with ATP-binding cassette, subfamily G, member 2 (ABCG2) and others, may have a role in chemoresistant colon CSCs and a negative impact on disease-free survival in colon cancer patients

  18. An independent occurrence of the chimeric P450 enzyme CYP337B3 of Helicoverpa armigera confers cypermethrin resistance in Pakistan.

    Science.gov (United States)

    Rasool, Akhtar; Joußen, Nicole; Lorenz, Sybille; Ellinger, Renate; Schneider, Bernd; Khan, Sher Afzal; Ashfaq, Muhammad; Heckel, David G

    2014-10-01

    The increasing resistance level of insect pest species is a major concern to agriculture worldwide. The cotton bollworm, Helicoverpa armigera, is one of the most important pest species due to being highly polyphagous, geographically widespread, and resistant towards many chemical classes of insecticides. We previously described the mechanism of fenvalerate resistance in Australian populations conferred by the chimeric cytochrome P450 monooxygenase CYP337B3, which arose by unequal crossing-over between CYP337B1 and CYP337B2. Here, we show that this mechanism is also present in the cypermethrin-resistant FSD strain from Pakistan. The Pakistani and the Australian CYP337B3 alleles differ by 18 synonymous and three nonsynonymous SNPs and additionally in the length and sequence of the intron. Nevertheless, the activity of both CYP337B3 proteins is comparable. We demonstrate that CYP337B3 is capable of metabolizing cypermethrin (trans- and especially cis-isomers) to the main metabolite 4'-hydroxycypermethrin, which exhibits no intrinsic toxicity towards susceptible larvae. In a bioassay, CYP337B3 confers a 7-fold resistance towards cypermethrin in FSD larvae compared to susceptible larvae from the Australian TWB strain lacking CYP337B3. Linkage analysis shows that presence of CYP337B3 accounts for most of the cypermethrin resistance in the FSD strain; up-regulation of other P450s in FSD plays no detectable role in resistance. The presence or absence of CYP337B3 can be easily detected by a simple PCR screen, providing a powerful tool to rapidly distinguish resistant from susceptible individuals in the field and to determine the geographical distribution of this resistance gene. Our results suggest that CYP337B3 evolved twice independently by unequal crossing-over between CYP337B2 and two different CYP337B1 alleles. Copyright © 2014 Elsevier Ltd. All rights reserved.

  19. 3'-Azido-3'-deoxythymidine (AZT) induces apoptosis and alters metabolic enzyme activity in human placenta

    International Nuclear Information System (INIS)

    Collier, Abby C.; Helliwell, Rachel J.A.; Keelan, Jeffrey A.; Paxton, James W.; Mitchell, Murray D.; Tingle, Malcolm D.

    2003-01-01

    The anti-HIV drug 3'-azido-3'-deoxythymidine (AZT) is the drug of choice for preventing maternal-fetal HIV transmission during pregnancy. Our aim was to assess the cytotoxic effects of AZT on human placenta in vitro. The mechanisms of AZT-induced effects were investigated using JEG-3 choriocarcinoma cells and primary explant cultures from term and first-trimester human placentas. Cytotoxicity measures included trypan blue exclusion, MTT, and reactive oxygen species (ROS) assays. Apoptosis was measured with an antibody specific to cleaved caspase-3 and by rescue of cells by the general caspase inhibitor Boc-D-FMK. The effect of AZT on the activities of glutathione-S-transferase, β-glucuronidase, UDP-glucuronosyl transferase, cytochrome P450 (CYP) 1A, and CYP reductase (CYPR) in the placenta was assessed using biochemical assays and immunoblotting. AZT increased ROS levels, decreased cellular proliferation rates, was toxic to mitochondria, and initiated cell death by a caspase-dependent mechanism in the human placenta in vitro. In the absence of serum, the effects of AZT were amplified in all the models used. AZT also increased the amounts of activity of GST, β-glucuronidase, and CYP1A, whereas UGT and CYPR were decreased. We conclude that AZT causes apoptosis in the placenta and alters metabolizing enzymes in human placental cells. These findings have implications for the safe administration of AZT in pregnancy with respect to the maintenance of integrity of the maternal-fetal barrier

  20. MAPK3 at the Autism-Linked Human 16p11.2 Locus Influences Precise Synaptic Target Selection at Drosophila Larval Neuromuscular Junctions.

    Science.gov (United States)

    Park, Sang Mee; Park, Hae Ryoun; Lee, Ji Hye

    2017-02-01

    Proper synaptic function in neural circuits requires precise pairings between correct pre- and post-synaptic partners. Errors in this process may underlie development of neuropsychiatric disorders, such as autism spectrum disorder (ASD). Development of ASD can be influenced by genetic factors, including copy number variations (CNVs). In this study, we focused on a CNV occurring at the 16p11.2 locus in the human genome and investigated potential defects in synaptic connectivity caused by reduced activities of genes located in this region at Drosophila larval neuromuscular junctions, a well-established model synapse with stereotypic synaptic structures. A mutation of rolled , a Drosophila homolog of human mitogen-activated protein kinase 3 ( MAPK3 ) at the 16p11.2 locus, caused ectopic innervation of axonal branches and their abnormal defasciculation. The specificity of these phenotypes was confirmed by expression of wild-type rolled in the mutant background. Albeit to a lesser extent, we also observed ectopic innervation patterns in mutants defective in Cdk2, Gα q , and Gp93, all of which were expected to interact with Rolled MAPK3. A further genetic analysis in double heterozygous combinations revealed a synergistic interaction between rolled and Gp93 . In addition, results from RT-qPCR analyses indicated consistently reduced rolled mRNA levels in Cdk2 , Gα q , and Gp93 mutants. Taken together, these data suggest a central role of MAPK3 in regulating the precise targeting of presynaptic axons to proper postsynaptic targets, a critical step that may be altered significantly in ASD.

  1. 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.

  2. Harman induces CYP1A1 enzyme through an aryl hydrocarbon receptor mechanism

    International Nuclear Information System (INIS)

    El Gendy, Mohamed A.M.; El-Kadi, Ayman O.S.

    2010-01-01

    Harman is a common compound in several foods, plants and beverages. Numerous studies have demonstrated its mutagenic, co-mutagenic and carcinogenic effects; however, the exact mechanism has not been fully identified. Aryl hydrocarbon receptor (AhR) is a transcription factor regulating the expression of the carcinogen-activating enzyme; cytochrome P450 1A1 (CYP1A1). In the present study, we examined the ability of harman to induce AhR-mediated signal transduction in human and rat hepatoma cells; HepG2 and H4IIE cells. Our results showed that harman significantly induced CYP1A1 mRNA in a time- and concentration-dependent manner. Similarly, harman significantly induced CYP1A1 at protein and activity levels in a concentration-dependent manner. Moreover, the AhR antagonist, resveratrol, inhibited the increase in CYP1A1 activity by harman. The RNA polymerase inhibitor, actinomycin D, completely abolished the CYP1A1 mRNA induction by harman, indicating a transcriptional activation. The role of AhR in CYP1A1 induction by harman was confirmed by using siRNA specific for human AhR. The ability of harman to induce CYP1A1 was strongly correlated with its ability to stimulate AhR-dependent luciferase activity and electrophoretic mobility shift assay. At post-transcriptional and post-translational levels, harman did not affect the stability of CYP1A1 at the mRNA and the protein levels, excluding other mechanisms participating in the obtained effects. We concluded that harman can directly induce CYP1A1 gene expression in an AhR-dependent manner and may represent a novel mechanism by which harman promotes mutagenicity, co-mutagenicity and carcinogenicity.

  3. The effects of Andrographis paniculata (Burm.f.) Nees extract and diterpenoids on the CYP450 isoforms' activities, a review of possible herb-drug interaction risks.

    Science.gov (United States)

    Tan, Mei Lan; Lim, Lin Ee

    2015-01-01

    Andrographis paniculata (Burm.f.) Nees is a popular medicinal plant and its components are used in various traditional product preparations. However, its herb-drug interactions risks remain unclear. This review specifically discusses the various published studies carried out to evaluate the effects of Andrographis paniculata (Burm.f.) Nees plant extracts and diterpenoids on the CYP450 metabolic enzyme and if the plant components pose a possible herb-drug interaction risk. Unfortunately, the current data are insufficient to indicate if the extracts or diterpenoids can be labeled as in vitro CYP1A2, CYP2C9 or CYP3A4 inhibitors. A complete CYP inhibition assay utilizing human liver microsomes and the derivation of relevant parameters to predict herb-drug interaction risks may be necessary for these isoforms. However, based on the current studies, none of the extracts and diterpenoids exhibited CYP450 induction activity in human hepatocytes or human-derived cell lines. It is crucial that a well-defined experimental design is needed to make a meaningful herb-drug interaction prediction.

  4. Inhibition of Cytochrome P450 (CYP3A4) Activity by Extracts from 57 Plants Used in Traditional Chinese Medicine (TCM)

    Science.gov (United States)

    Ashour, Mohamed L; Youssef, Fadia S; Gad, Haidy A; Wink, Michael

    2017-01-01

    Background: Herbal medicine is widely used all over the world for treating various health disorders. It is employed either alone or in combination with synthetic drugs or plants to be more effective. Objective: The assessment of the effect of both water and methanol extracts of 57 widely used plants from Traditional Chinese Medicine (TCM) against the main phase I metabolizing enzyme CYP3A4 in vitro for the first time. Materials and Methods: The inhibition of cytochrome P450 activity was evaluated using a luminescence assay. The principal component analysis (PCA) was used to correlate the inhibitory activity with the main secondary metabolites present in the plant extracts. Molecular modeling studies on CYP3A4 (PDB ID 4NY4) were carried out with 38 major compounds present in the most active plant extracts to validate the observed inhibitory effect. Results: Aqueous extracts of Acacia catechu, Andrographis paniculata, Arctium lappa, Areca catechu, Bupleurum marginatum, Chrysanthemum indicum, Dysosma versipellis, and Spatholobus suberectus inhibited CYP3A4 is more than 85% (at a dose of 100 μg/mL). The corresponding methanol extracts of A. catechu, A. paniculata, A. catechu, Mahonia bealei, and Sanguisorba officinalis inhibited the enzyme by more than 50%. Molecular modeling studies revealed that two polyphenols, namely hesperidin and rutin, revealed the highest fitting scores in the active sites of the CYP3A4 with binding energies equal to -74.09 and -71.34 kcal/mol, respectively. Conclusion: These results provide evidence that many TCM plants can inhibit CYP3A4, which might cause a potential interference with the metabolism of other concomitantly administered herbs or drugs. SUMMARY In this study, the inhibitory activity of the aqueous and methanol extracts of 57 widely used plants from Traditional Chinese Medicine (TCM) against the main phase I metabolizing enzyme CYP3A4 was tested in vitro for the first time.Aqueous extracts of Acacia catechu, Andrographis

  5. CYP2A6 and CYP2E1 polymorphisms in a Brazilian population living in Rio de Janeiro

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    A. Rossini

    2006-02-01

    Full Text Available Cytochrome P450 (CYP is a superfamily of enzymes involved in the metabolism of endogenous compounds and xenobiotics. CYP2A6 catalyzes the oxidation of nicotine and the activation of carcinogens such as aflatoxin B1 and nitrosamines. CYP2E1 metabolizes ethanol and other low-molecular weight compounds and can also activate nitrosamines. The CYP2A6 and CYP2E1 genes are polymorphic, altering their catalytic activities and susceptibility to cancer and other diseases. A number of polymorphisms described are ethnic-dependent. In the present study, we determined the genotype and allele frequencies of the main CYP2A6 and CYP2E1 polymorphisms in a group of 289 volunteers recruited at the Central Laboratory of Hospital Universitário Pedro Ernesto. They had been residing in the city of Rio de Janeiro for at least 6 months and were divided into two groups according to skin color (white and non-white. The alleles were determined by allele specific PCR (CYP2A6 or by PCR-RFLP (CYP2E1. The frequencies of the CYP2A6*1B and CYP2A6*2 alleles were 0.29 and 0.02 for white individuals and 0.24 and 0.01 for non-white individuals, respectively. The CYP2A6*5 allele was not found in the population studied. Regarding the CYP2E1*5B allele, we found a frequency of 0.07 in white individuals, which was statistically different (P < 0.05 from that present in non-white individuals (0.03. CYP2E1*6 allele frequency was the same (0.08 in both groups. The frequencies of CYP2A6*1B, CYP2A6*2 and CYP2E1*6 alleles in Brazilians are similar to those found in Caucasians and African-Americans, but the frequency of the CYP2E1*5B allele is higher in Brazilians.

  6. CYP2A6 and CYP2E1 polymorphisms in a Brazilian population living in Rio de Janeiro

    Directory of Open Access Journals (Sweden)

    Rossini A.

    2006-01-01

    Full Text Available Cytochrome P450 (CYP is a superfamily of enzymes involved in the metabolism of endogenous compounds and xenobiotics. CYP2A6 catalyzes the oxidation of nicotine and the activation of carcinogens such as aflatoxin B1 and nitrosamines. CYP2E1 metabolizes ethanol and other low-molecular weight compounds and can also activate nitrosamines. The CYP2A6 and CYP2E1 genes are polymorphic, altering their catalytic activities and susceptibility to cancer and other diseases. A number of polymorphisms described are ethnic-dependent. In the present study, we determined the genotype and allele frequencies of the main CYP2A6 and CYP2E1 polymorphisms in a group of 289 volunteers recruited at the Central Laboratory of Hospital Universitário Pedro Ernesto. They had been residing in the city of Rio de Janeiro for at least 6 months and were divided into two groups according to skin color (white and non-white. The alleles were determined by allele specific PCR (CYP2A6 or by PCR-RFLP (CYP2E1. The frequencies of the CYP2A6*1B and CYP2A6*2 alleles were 0.29 and 0.02 for white individuals and 0.24 and 0.01 for non-white individuals, respectively. The CYP2A6*5 allele was not found in the population studied. Regarding the CYP2E1*5B allele, we found a frequency of 0.07 in white individuals, which was statistically different (P < 0.05 from that present in non-white individuals (0.03. CYP2E1*6 allele frequency was the same (0.08 in both groups. The frequencies of CYP2A6*1B, CYP2A6*2 and CYP2E1*6 alleles in Brazilians are similar to those found in Caucasians and African-Americans, but the frequency of the CYP2E1*5B allele is higher in Brazilians.

  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. Functional characterization of CYP52G3 from Aspergillus oryzae and its application for bioconversion and synthesis of hydroxyl flavanone and steroids.

    Science.gov (United States)

    Uno, Tomohide; Yanase, Takeshi; Imaishi, Hiromasa

    2017-05-01

    Aspergillus oryzae is a fungus widely used in traditional Japanese fermentation industries. Cytochrome P450 (CYP) proteins are ubiquitously distributed in nature and display a broad range of enzymatic activities. A novel CYP52 (CYP52G3) gene was found in A. oryzae. In this study, we report the functional characterization of CYP52G3. The recombinant protein was expressed heterologously in Escherichia coli, and its membrane fraction isolated. CYP52G3 showed activities for 7-ethoxycoumarin and α-naphtoflavone. Furthermore, CYP52G3 hydroxylated flavanone at the 4' and 6 position and metabolized some hydroxyl-flavanones and steroids. Bioconversion experiments indicated that CYP52G3 could convert flavanone and testosterone in a synthetic medium. The conversion rates of flavanone and testosterone at 24 H were 50% and 70%, respectively. These results support that CYP52G3 could prove a useful enzyme for the efficient production of new compounds from flavonoids and steroids. © 2016 International Union of Biochemistry and Molecular Biology, Inc.

  9. A Family-Based Association Study of CYP11A1 and CYP11B1 Gene Polymorphisms With Autism in Chinese Trios.

    Science.gov (United States)

    Deng, Hong-Zhu; You, Cong; Xing, Yu; Chen, Kai-Yun; Zou, Xiao-Bing

    2016-05-01

    Autism spectrum disorder is a group of neurodevelopmental disorders with the higher prevalence in males. Our previous studies have indicated lower progesterone levels in the children with autism spectrum disorder, suggesting involvement of the cytochrome P-450scc gene (CYP11A1) and cytochrome P-45011beta gene (CYP11B1) as candidate genes in autism spectrum disorder. The aim of this study was to investigate the family-based genetic association between single-nucleotide polymorphisms, rs2279357 in the CYP11A1 gene and rs4534 and rs4541 in the CYP11B1 gene and autism spectrum disorder in Chinese children, which were selected according to the location in the coding region and 5' and 3' regions and minor allele frequencies of greater than 0.05 in the Chinese populations. The transmission disequilibrium test and case-control association analyses were performed in 100 Chinese Han autism spectrum disorder family trios. The genotype and allele frequency of the 3 single-nucleotide polymorphisms had no statistical difference between the children with autism spectrum disorder and their parents (P> .05). Transmission disequilibrium test analysis showed transmission disequilibrium of CYP11A1 gene rs2279357 single-nucleotide polymorphisms (χ(2)= 5.038,Pautism spectrum disorder exists within or near the CYP11A1 gene in the Han Chinese population. © The Author(s) 2015.

  10. Vinclozolin, a widely used fungizide, enhanced BaP-induced micronucleus formation in human derived hepatoma cells by increasing CYP1A1 expression.

    Science.gov (United States)

    Wu, Xin-Jiang; Lu, Wen-qing; Roos, Peter H; Mersch-Sundermann, Volker

    2005-10-15

    Vinclozolin, a widely used fungicide, can be identified as a residue in numerous vegetable and fruit samples. To get insight in its genetic toxicity, we investigated the genotoxic effect of vinclozolin in the human derived hepatoma cell line HepG2 using the micronucleus (MN) assay. Additionally, to evaluate the co- or anti-mutagenic potency of vinclozolin, we treated HepG2 cells with different concentrations of vinclozolin for 24 h. Subsequently, the cells were exposed to benzo[a]pyrene (BaP) for 1h. Exposure of HepG2 cells to 50-400 microM vinclozolin alone did not cause any induction of micronuclei. However, a pronounced co-mutagenic effect was observed. MN frequencies caused by BaP increased by 30.6%, 52.8% and 65.3% after pretreatment of the cell cultures with 50, 100 and 200 microM vinclozolin, respectively. The highest concentration (400 microM) of vinclozolin tested caused cytotoxicity. Therefore, micronuclei were not considered for that concentration. To clarify the mechanism of cogenotoxicity, we assayed cytochrome P450 1A1 (CYP1A1), which plays a pivotal role in activation of BaP. Cells exposed to vinclozolin led to significant increase of CYP1A1 expression in Western blot. The result suggested that induction of CYP1A1 by vinclozolin account for its enhancing effect on genotoxicity caused by BaP.

  11. 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

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

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    Maria eThomas

    2015-11-01

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

  13. Characterization of cytochrome P450 CYP109E1 from Bacillus megaterium as a novel vitamin D3 hydroxylase.

    Science.gov (United States)

    Abdulmughni, Ammar; Jóźwik, Ilona K; Putkaradze, Natalia; Brill, Elisa; Zapp, Josef; Thunnissen, Andy-Mark W H; Hannemann, Frank; Bernhardt, Rita

    2017-02-10

    In this study the ability of CYP109E1 from Bacillus megaterium to metabolize vitamin D 3 (VD 3 ) was investigated. In an in vitro system using bovine adrenodoxin reductase (AdR) and adrenodoxin (Adx 4-108 ), VD 3 was converted by CYP109E1 into several products. Furthermore, a whole-cell system in B. megaterium MS941 was established. The new system showed a conversion of 95% after 24h. By NMR analysis it was found that CYP109E1 catalyzes hydroxylation of VD 3 at carbons C-24 and C-25, resulting in the formation of 24(S)-hydroxyvitamin D 3 (24S(OH)VD 3 ), 25-hydroxyvitamin D 3 (25(OH)VD 3 ) and 24S,25-dihydroxyvitamin D 3 (24S,25(OH) 2 VD 3 ). Through time dependent whole-cell conversion of VD 3 , we identified that the formation of 24S,25(OH) 2 VD 3 by CYP109E1 is derived from VD 3 via the intermediate 24S(OH)VD 3 . Moreover, using docking analysis and site-directed mutagenesis, we identified important active site residues capable of determining substrate specificity and regio-selectivity. HPLC analysis of the whole-cell conversion with the I85A-mutant revealed an increased selectivity towards 25-hydroxylation of VD 3 compared with the wild type activity, resulting in an approximately 2-fold increase of 25(OH)VD 3 production (45mgl -1 day -1 ) compared to wild type (24.5mgl -1 day -1 ). Copyright © 2017 Elsevier B.V. All rights reserved.

  14. 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.

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

    DEFF Research Database (Denmark)

    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...

  16. Tacrolimus Updated Guidelines through popPK Modeling: How to Benefit More from CYP3A Pre-emptive Genotyping Prior to Kidney Transplantation

    Directory of Open Access Journals (Sweden)

    Jean-Baptiste Woillard

    2017-06-01

    Full Text Available Tacrolimus (Tac is a profoundly effective immunosuppressant that reduces the risk of rejection after solid organ transplantation. However, its use is hampered by its narrow therapeutic window along with its highly variable pharmacological (pharmacokinetic [PK] and pharmacodynamic [PD] profile. Part of this variability is explained by genetic polymorphisms affecting the metabolic pathway. The integration of CYP3A4 and CY3A5 genotype in tacrolimus population-based PK (PopPK modeling approaches has been proven to accurately predict the dose requirement to reach the therapeutic window. The objective of the present study was to develop an accurate PopPK model in a cohort of 59 kidney transplant patients to deliver this information to clinicians in a clear and actionable manner. We conducted a non-parametric non-linear effects PopPK modeling analysis in Pmetrics®. Patients were genotyped for the CYP3A4∗22 and CYP3A5∗3 alleles and were classified into 3 different categories [poor-metabolizers (PM, Intermediate-metabolizers (IM or extensive-metabolizers (EM]. A one-compartment model with double gamma absorption route described very accurately the tacrolimus PK. In covariate analysis, only CYP3A genotype was retained in the final model (Δ-2LL = -73. Our model estimated that tacrolimus concentrations were 33% IC95%[20–26%], 41% IC95%[36–45%] lower in CYP3A IM and EM when compared to PM, respectively. Virtually, we proved that defining different starting doses for PM, IM and EM would be beneficial by ensuring better probability of target concentrations attainment allowing us to define new dosage recommendations according to patient CYP3A genetic profile.

  17. Mechanisms of anaphylaxis in human low-affinity IgG receptor locus knock-in mice.

    Science.gov (United States)

    Gillis, Caitlin M; Jönsson, Friederike; Mancardi, David A; Tu, Naxin; Beutier, Héloïse; Van Rooijen, Nico; Macdonald, Lynn E; Murphy, Andrew J; Bruhns, Pierre

    2017-04-01

    Anaphylaxis can proceed through distinct IgE- or IgG-dependent pathways, which have been investigated in various mouse models. We developed a novel mouse strain in which the human low-affinity IgG receptor locus, comprising both activating (hFcγRIIA, hFcγRIIIA, and hFcγRIIIB) and inhibitory (hFcγRIIB) hFcγR genes, has been inserted into the equivalent murine locus, corresponding to a locus swap. We sought to determine the capabilities of hFcγRs to induce systemic anaphylaxis and identify the cell types and mediators involved. hFcγR expression on mouse and human cells was compared to validate the model. Passive systemic anaphylaxis was induced by injection of heat-aggregated human intravenous immunoglobulin and active systemic anaphylaxis after immunization and challenge. Anaphylaxis severity was evaluated based on hypothermia and mortality. The contribution of receptors, mediators, or cell types was assessed based on receptor blockade or depletion. The human-to-mouse low-affinity FcγR locus swap engendered hFcγRIIA/IIB/IIIA/IIIB expression in mice comparable with that seen in human subjects. Knock-in mice were susceptible to passive and active anaphylaxis, accompanied by downregulation of both activating and inhibitory hFcγR expression on specific myeloid cells. The contribution of hFcγRIIA was predominant. Depletion of neutrophils protected against hypothermia and mortality. Basophils contributed to a lesser extent. Anaphylaxis was inhibited by platelet-activating factor receptor or histamine receptor 1 blockade. Low-affinity FcγR locus-switched mice represent an unprecedented model of cognate hFcγR expression. Importantly, IgG-related anaphylaxis proceeds within a native context of activating and inhibitory hFcγRs, indicating that, despite robust hFcγRIIB expression, activating signals can dominate to initiate a severe anaphylactic reaction. Copyright © 2016 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights

  18. Acetaldehyde and parkinsonism: role of CYP450 2E1

    Directory of Open Access Journals (Sweden)

    Francesca eVaglini

    2013-06-01

    Full Text Available The present review update the relationship between acetaldehyde and parkinsonism with a specific focus on the role of P450 system and CYP 2E1 isozyme particularly.We have indicated that acetaldehyde is able to enhance the parkinsonism induced in mice by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, a neurotoxin able to damage the nigrostriatal dopaminergic pathway. Similarly diethyldithiocarbamate, the main metabolite of disulfiram, a drug widely used to control alcoholism, diallylsulfide and phenylisothiocyanate also markedly enhance the toxin-related parkinsonism. All these compounds are substrate/inhibitors of CYP450 2E1 isozyme. The presence of CYP 2E1 has been detected in the dopamine neurons of rodent Substantia Nigra, but a precise function of the enzyme has not been elucidated yet. By treating CYP 2E1 knockout mice with the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, the Substantia Nigra induced lesion was significantly reduced when compared with the lesion observed in wild-type animals. Several in vivo and in vitro studies led to the conclusion that CYP 2E1 may enhance the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine toxicity in mice by increasing free radical production inside the dopaminergic neurons. Acetaldehyde is a good substrate for CYP 2E1 enzyme as the other substrate-inhibitors and by this way may facilitate the susceptibility of dopaminergic neurons to toxic events. The literature suggests that ethanol and/or disulfiram may be responsible for toxic parkinsonism in human and it indicates that basal ganglia are the major targets of disulfiram toxicity. A very recent study reports that there are a decreased methylation of the CYP 2E1 gene and increased expression of CYP 2E1 mRNA in Parkinson’s Disease patient brains. This study suggests that epigenetic variants of this cytochrome contribute to the susceptibility, thus confirming multiples lines of evidence which indicate a link between environmental toxins and

  19. Downregulation of COX-2 and CYP 4A signaling by isoliquiritigenin inhibits human breast cancer metastasis through preventing anoikis resistance, migration and invasion

    Energy Technology Data Exchange (ETDEWEB)

    Zheng, Hao; Li, Ying [Department of Pharmacology, School of Medicine, Wuhan University, Wuhan 430071 (China); Wang, Yuzhong [Key Laboratory for Oral Biomedical Engineering of Ministry of Education, School and Hospital of Stomatology, Wuhan University, Wuhan 430079 (China); Zhao, Haixia [Department of Pharmacology, School of Medicine, Wuhan University, Wuhan 430071 (China); Zhang, Jing [Animal Experimental Center of Wuhan University, Wuhan 430071 (China); Chai, Hongyan [Center for Gene Diagnosis, Zhongnan Hospital, Wuhan University, Wuhan 430071 (China); Tang, Tian [Department of Oncology, Renmin Hospital of Wuhan University, Wuhan 430060 (China); Yue, Jiang [Department of Pharmacology, School of Medicine, Wuhan University, Wuhan 430071 (China); Guo, Austin M., E-mail: Austin_Guo@nymc.edu [Department of Pharmacology, School of Medicine, Wuhan University, Wuhan 430071 (China); Department of Pharmacology, New York Medical College, Valhalla, NY 10595 (United States); Yang, Jing, E-mail: yangjingliu2013@163.com [Department of Pharmacology, School of Medicine, Wuhan University, Wuhan 430071 (China)

    2014-10-01

    Flavonoids exert extensive in vitro anti-invasive and in vivo anti-metastatic activities. Anoikis resistance occurs at multiple key stages of the metastatic cascade. Here, we demonstrate that isoliquiritigenin (ISL), a flavonoid from Glycyrrhiza glabra, inhibits human breast cancer metastasis by preventing anoikis resistance, migration and invasion through downregulating cyclooxygenase (COX)-2 and cytochrome P450 (CYP) 4A signaling. ISL induced anoikis in MDA-MB-231 and BT-549 human breast cancer cells as evidenced by flow cytometry and the detection of caspase cleavage. Moreover, ISL inhibited the mRNA expression of phospholipase A2, COX-2 and CYP 4A and decreased the secretion of prostaglandin E{sub 2} (PGE{sub 2}) and 20-hydroxyeicosatetraenoic acid (20-HETE) in detached MDA-MB-231 cells. In addition, it decreased the levels of phospho-PI3K (Tyr{sup 458}), phospho-PDK (Ser{sup 241}) and phospho-Akt (Thr{sup 308}). Conversely, the exogenous addition of PGE{sub 2}, WIT003 (a 20-HETE analog) and an EP4 agonist (CAY10580) or overexpression of constitutively active Akt reversed ISL-induced anoikis. ISL exerted the in vitro anti-migratory and anti-invasive activities, whereas the addition of PGE{sub 2}, WIT003 and CAY10580 or overexpression of constitutively active Akt reversed the in vitro anti-migratory and anti-invasive activities of ISL in MDA-MB-231 cells. Notably, ISL inhibited the in vivo lung metastasis of MDA-MB-231 cells, together with decreased intratumoral levels of PGE{sub 2}, 20-HETE and phospho-Akt (Thr{sup 308}). In conclusion, ISL inhibits breast cancer metastasis by preventing anoikis resistance, migration and invasion via downregulating COX-2 and CYP 4A signaling. It suggests that ISL could be a promising multi-target agent for preventing breast cancer metastasis, and anoikis could represent a novel mechanism through which flavonoids may exert the anti-metastatic activities. - Highlights: • Isoliquiritigenin induces anoikis and suppresses

  20. CYP79 P450 monooxygenases in gymnosperms: CYP79A118 is associated with the formation of taxiphyllin in Taxus baccata.

    Science.gov (United States)

    Luck, Katrin; Jia, Qidong; Huber, Meret; Handrick, Vinzenz; Wong, Gane Ka-Shu; Nelson, David R; Chen, Feng; Gershenzon, Jonathan; Köllner, Tobias G

    2017-09-01

    Conifers contain P450 enzymes from the CYP79 family that are involved in cyanogenic glycoside biosynthesis. Cyanogenic glycosides are secondary plant compounds that are widespread in the plant kingdom. Their biosynthesis starts with the conversion of aromatic or aliphatic amino acids into their respective aldoximes, catalysed by N-hydroxylating cytochrome P450 monooxygenases (CYP) of the CYP79 family. While CYP79s are well known in angiosperms, their occurrence in gymnosperms and other plant divisions containing cyanogenic glycoside-producing plants has not been reported so far. We screened the transcriptomes of 72 conifer species to identify putative CYP79 genes in this plant division. From the seven resulting full-length genes, CYP79A118 from European yew (Taxus baccata) was chosen for further characterization. Recombinant CYP79A118 produced in yeast was able to convert L-tyrosine, L-tryptophan, and L-phenylalanine into p-hydroxyphenylacetaldoxime, indole-3-acetaldoxime, and phenylacetaldoxime, respectively. However, the kinetic parameters of the enzyme and transient expression of CYP79A118 in Nicotiana benthamiana indicate that L-tyrosine is the preferred substrate in vivo. Consistent with these findings, taxiphyllin, which is derived from L-tyrosine, was the only cyanogenic glycoside found in the different organs of T. baccata. Taxiphyllin showed highest accumulation in leaves and twigs, moderate accumulation in roots, and only trace accumulation in seeds and the aril. Quantitative real-time PCR revealed that CYP79A118 was expressed in plant organs rich in taxiphyllin. Our data show that CYP79s represent an ancient family of plant P450s that evolved prior to the separation of gymnosperms and angiosperms. CYP79A118 from T. baccata has typical CYP79 properties and its substrate specificity and spatial gene expression pattern suggest that the enzyme contributes to the formation of taxiphyllin in this plant species.

  1. Coffee inhibition of CYP3A4 in vitro was not translated to a grapefruit-like pharmacokinetic interaction clinically.

    Science.gov (United States)

    Dresser, George K; Urquhart, Brad L; Proniuk, Julianne; Tieu, Alvin; Freeman, David J; Arnold, John Malcolm; Bailey, David G

    2017-10-01

    Grapefruit can augment oral medication bioavailability through irreversible (mechanism-based) inhibition of intestinal CYP3A4. Supplementary data from our recent coffee-drug interaction clinical study showed some subjects had higher area under the plasma drug concentration - time curve (AUC) and plasma peak drug concentration (Cmax) of the CYP3A4 probe felodipine compared to aqueous control. It was hypothesized that coffee might interact like grapefruit in responsive individuals. Beans from six geographical locations were consistently brewed into coffee that was separated chromatographically to a methanolic fraction for in vitro inhibition testing of CYP3A4 metabolism of felodipine at 1% coffee strength. The effect of simultaneous incubation and 10-min preincubation with coffee fractions determined whether coffee had direct and mechanism-based inhibitory activity. A subsequent five-way randomized balanced controlled crossover clinical study evaluated the clinical pharmacokinetic interaction with single-dose felodipine. Grapefruit juice, water, or three of the in vitro tested coffees were ingested at 300 mL alone 1 h before and then with felodipine. In vitro, all six coffees decreased felodipine metabolism for both simultaneous and preincubation exposure compared to corresponding control. Five coffees demonstrated mechanism-based inhibition. Grapefruit increased felodipine AUC 0-8 (25 vs. 13 ng.h/mL, P coffees caused no change in these parameters compared to water. Despite high in vitro potency of CYP3A4 inhibition, the coffees did not cause a clinical pharmacokinetic interaction possibly from insufficient amount of inhibitor(s) in coffee reaching intestinal CYP3A4 during the absorption phase of felodipine. The results of this study highlight the need for follow-up clinical testing when in vitro results indicate the possibility of an interaction. © 2017 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd, British

  2. Strengthening Locus Standi in Human Rights Litigation in Zimbabwe ...

    African Journals Online (AJOL)

    MJM Venter

    2016-05-30

    May 30, 2016 ... approach is likely to impact on the right to access to justice and human rights protection. 2. Locus standi ... Committee on Economic, Social and Cultural Rights General Comment 9: The. Domestic .... animosity towards an accused, in bad faith and without any warrant, were to rule that the question raised by ...

  3. Evaluation of CYP1A1 and CYP2B1/2 m-RNA induction in rat liver slices using the NanoString technology: a novel tool for drug discovery lead optimization.

    Science.gov (United States)

    Palamanda, Jairam R; Kumari, Pramila; Murgolo, Nicholas; Benbow, Larry; Lin, Xinjie; Nomeir, Amin A

    2009-08-01

    Cytochrome P450 (CYP) induction in rodents and humans is considered a liability for new chemical entities (NCEs) in drug discovery. In particular, CYP1A1 and CYP2B1/2 have been associated with the induction of liver tumors in oncogenicity studies during safety evaluation studies of potential drugs. In our laboratory, real time PCR (Taqman) has been used to quantify the induction of rat hepatic CYP1A1 and CYP2B1/2 in precision -cut rat liver slices. A novel technology that does not require m-RNA isolation or RT-PCR, (developed by NanoString Technologies) has been investigated to quantify CYP1A1 and CYP2B1/2 induction in rat liver slices. Seventeen commercially available compounds were evaluated using both Taqman and NanoString technologies. Precision-cut rat liver slices were incubated with individual compounds for 24 hr at 37 degrees C in a humidified CO(2) incubator and CYP1A1 and CYP2B1/2 m-RNA were quantified. The results from the NanoString technology were similar to those of the Taqman(R) with a high degree of correlation for both CYP isoforms (r(2)>0.85). Therefore, NanoString provides an additional new technology to evaluate the induction of CYP1A1 and 2B1/2, as well as potentially other enzymes or transporters in rat liver slices.

  4. Effect of ketoconazole-mediated CYP3A4 inhibition on clinical pharmacokinetics of panobinostat (LBH589), an orally active histone deacetylase inhibitor

    NARCIS (Netherlands)

    A.P. Hamberg (Paul); M.M. Woo (Margaret M.); L.C. Chen (Lin-Chi); J. Verweij (Jaap); M.G. Porro; L. Zhao (Ling); W. Li (Weili); D.A.J. van der Biessen (Diane); H.S. Sharma (Hari); T. Hengelage (Thomas); M.J.A. de Jonge (Maja)

    2011-01-01

    textabstractPurpose: Panobinostat is partly metabolized by CYP3A4 in vitro. This study evaluated the effect of a potent CYP3A inhibitor, ketoconazole, on the pharmacokinetics and safety of panobinostat. Methods: Patients received a single panobinostat oral dose on day 1, followed by 4 days wash-out

  5. Cytochrome P-450 2D6 (CYP2D6) Genotype and Breast Cancer Recurrence in Tamoxifen-Treated Patients

    DEFF Research Database (Denmark)

    Ahern, Thomas P; Hertz, Daniel L; Damkier, Per

    2017-01-01

    -infiltrated tissues, all of which showed excellent CYP2D6 genotype agreement. We applied these concordance data to a quantitative bias analysis of the subset of the 31 studies that were based on genotypes from tumor-infiltrated tissue to examine whether genotyping errors substantially biased estimates of association...... genotyped DNA from tumor-infiltrated tissues, and their results may have been susceptible to germline genotype misclassification from loss of heterozygosity at the CYP2D6 locus. We systematically reviewed 6 studies of concordance between genotypes obtained from paired nonneoplastic and breast tumor...

  6. Mechanism of the Dual Activities of Human CYP17A1 and Binding to Anti-Prostate Cancer Drug Abiraterone Revealed by a Novel V366M Mutation Causing 17,20 Lyase Deficiency

    Directory of Open Access Journals (Sweden)

    Mónica Fernández-Cancio

    2018-04-01

    Full Text Available The CYP17A1 gene regulates sex steroid biosynthesis in humans through 17α-hydroxylase/17,20 lyase activities and is a target of anti-prostate cancer drug abiraterone. In a 46, XY patient with female external genitalia, together with a loss of function mutation S441P, we identified a novel missense mutation V366M at the catalytic center of CYP17A1 which preferentially impaired 17,20 lyase activity. Kinetic experiments with bacterially expressed proteins revealed that V366M mutant enzyme can bind and metabolize pregnenolone to 17OH-pregnenolone, but 17OH-pregnenolone binding and conversion to dehydroepiandrosterone (DHEA was impaired, explaining the patient’s steroid profile. Abiraterone could not bind and inhibit the 17α-hydroxylase activity of the CYP17A1-V366M mutant. Molecular dynamics (MD simulations showed that V366M creates a “one-way valve” and suggests a mechanism for dual activities of human CYP17A1 where, after the conversion of pregnenolone to 17OH-pregnenolone, the product exits the active site and re-enters for conversion to dehydroepiandrosterone. The V366M mutant also explained the effectiveness of the anti-prostate cancer drug abiraterone as a potent inhibitor of CYP17A1 by binding tightly at the active site in the WT enzyme. The V366M is the first human mutation to be described at the active site of CYP17A1 that causes isolated 17,20 lyase deficiency. Knowledge about the specificity of CYP17A1 activities is of importance for the development of treatments for polycystic ovary syndrome and inhibitors for prostate cancer therapy.

  7. Khellin and visnagin differentially modulate AHR signaling and downstream CYP1A activity in human liver cells.

    Directory of Open Access Journals (Sweden)

    Radim Vrzal

    Full Text Available Khellin and visnagin are two furanochromones that can be frequently found in ethnomedical formulations in Asia and the Middle East. Both compounds possess anti-inflammatory and analgesic properties, therefore modern medicine uses these compounds or structurally related derivatives for treatment of vitiligo, bronchial asthma and renal colics. Despite their frequent usage, the potential toxic properties of visnagin and khellin are not well characterized up-to-now. Many natural compounds modulate the expression and activity of cytochrome P450 1A1 (CYP1A1, which is well-known to bioactivate pro-carcinogens. The expression of this enzyme is controlled by the aryl hydrocarbon receptor (AHR, a ligand-activated transcription factor and regulator of drug metabolism. Here, we investigated the influence of both furanochromones on AHR signaling in human HepG2 hepatocarcinoma cells and primary human hepatocytes. Both compounds transactivated xenobiotic response element (XRE-driven reporter gene activity in a dose-dependent manner and induced CYP1A1 transcription in HepG2 cells and primary hepatocytes. The latter was abolished in presence of a specific AHR antagonist. CYP1A enzyme activity assays done in HepG2 cells and primary hepatocytes revealed an inhibition of enzyme activity by both furanochromones, which may become relevant regarding the metabolism of xenobiotics and co-administered therapeutic drugs. The observed induction of several other members of the AHR gene battery, whose gene products are involved in regulation of cell growth, differentiation and migration, indicates that a further toxicological characterization of visnagin and khelllin is urgently required in order to minimize potential drug-drug interactions and other toxic side-effects that may occur during therapeutic usage of these furanochromones.

  8. Newly Identified CYP2C93 Is a Functional Enzyme in Rhesus Monkey, but Not in Cynomolgus Monkey

    OpenAIRE

    Uno, Yasuhiro; Uehara, Shotaro; Kohara, Sakae; Iwasaki, Kazuhide; Nagata, Ryoichi; Fukuzaki, Koichiro; Utoh, Masahiro; Murayama, Norie; Yamazaki, Hiroshi

    2011-01-01

    Cynomolgus monkey and rhesus monkey are used in drug metabolism studies due to their evolutionary closeness and physiological resemblance to human. In cynomolgus monkey, we previously identified cytochrome P450 (P450 or CYP) 2C76 that does not have a human ortholog and is partly responsible for species differences in drug metabolism between cynomolgus monkey and human. In this study, we report characterization of CYP2C93 cDNA newly identified in cynomolgus monkey and rhesus monkey. The CYP2C9...

  9. A Markov chain model to evaluate the effect of CYP3A5 and ABCB1 polymorphisms on adverse events associated with tacrolimus in pediatric renal transplantation.

    Science.gov (United States)

    Sy, Sherwin K B; Heuberger, Jules; Shilbayeh, Sireen; Conrado, Daniela J; Derendorf, Hartmut

    2013-10-01

    The SNP A6986G of the CYP3A5 gene (*3) results in a non-functional protein due to a splicing defect whereas the C3435T was associated with variable expression of the ABCB1 gene, due to protein instability. Part of the large interindividual variability in tacrolimus efficacy and toxicity can be accounted for by these genetic factors. Seventy-two individuals were examined for A6986G and C3435T polymorphism using a PCR-RFLP-based technique to estimate genotype and allele frequencies in the Jordanian population. The association of age, hematocrit, platelet count, CYP3A5, and ABCB1 polymorphisms with tacrolimus dose- and body-weight-normalized levels in the subset of 38 pediatric renal transplant patients was evaluated. A Markov model was used to evaluate the time-dependent probability of an adverse event occurrence by CYP3A5 phenotypes and ABCB1 genotypes. The time-dependent probability of adverse event was about double in CYP3A5 non-expressors compared to the expressors for the first 12 months of therapy. The CYP3A5 non-expressors had higher corresponding normalized tacrolimus levels compared to the expressors in the first 3 months. The correlation trend between probability of adverse events and normalized tacrolimus concentrations for the two CYP3A5 phenotypes persisted for the first 9 months of therapy. The differences among ABCB1 genotypes in terms of adverse events and normalized tacrolimus levels were only observed in the first 3 months of therapy. The information on CYP3A5 genotypes and tacrolimus dose requirement is important in designing effective programs toward management of tacrolimus side effects particularly for the initial dose when tacrolimus blood levels are not available for therapeutic drug monitoring.

  10. Energy homeostasis targets chromosomal reconfiguration of the human GH1 locus.

    Science.gov (United States)

    Vakili, Hana; Jin, Yan; Cattini, Peter A

    2014-11-01

    Levels of pituitary growth hormone (GH), a metabolic homeostatic factor with strong lipolytic activity, are decreased in obese individuals. GH declines prior to the onset of weight gain in response to excess caloric intake and hyperinsulinemia; however, the mechanism by which GH is reduced is not clear. We used transgenic mice expressing the human GH (hGH) gene, GH1, to assess the effect of high caloric intake on expression as well as the local chromosome structure of the intact GH1 locus. Animals exposed to 3 days of high caloric intake exhibited hyperinsulinemia without hyperglycemia and a decrease in both hGH synthesis and secretion, but no difference in endogenous production of murine GH. Efficient GH1 expression requires a long-range intrachromosomal interaction between remote enhancer sequences and the proximal promoter region through "looping" of intervening chromatin. High caloric intake disrupted this interaction and decreased both histone H3/H4 hyperacetylation and RNA polymerase II occupancy at the GH1 promoter. Incorporation of physical activity muted the effects of excess caloric intake on insulin levels, GH1 promoter hyperacetylation, chromosomal architecture, and expression. These results indicate that energy homeostasis alters postnatal hGH synthesis through dynamic changes in the 3-dimensional chromatin structure of the GH1 locus, including structures required for cell type specificity during development.

  11. Updated listing of haplotypes at the human phenylalanine hydroxylase (PAH) locus

    Energy Technology Data Exchange (ETDEWEB)

    Eisensmith, R.C.; Woo, S.L.C. (Baylor College of Medicine, Houston, TX (United States))

    1992-12-01

    Analysis of mutant PAH chromosomes has identified approximately 60 different single-base substitutions and deletions within the PAH locus. Nearly all of these molecular lesions are in strong linkage disequilibrium with specific RFLP haplotypes in different ethnic populations. Thus, haplotype analysis is not only useful for diagnostic purposes but is proving to be a valuable tool in population genetic studies of the origin and spread of phenylketonuria alleles in human populations. PCR-based methods have been developed to detect six of the eight polymorphic restriction sites used for determination of RFLP haplotypes at the PAH locus. A table of the proposed expanded haplotypes is given.

  12. The effect of trimethoprim on CYP2C8 mediated rosiglitazone metabolism in human liver microsomes and healthy subjects

    Science.gov (United States)

    Hruska, M W; Amico, J A; Langaee, T Y; Ferrell, R E; Fitzgerald, S M; Frye, R F

    2005-01-01

    Aims Rosiglitazone, a thiazolidinedione antidiabetic medication used in the treatment of Type 2 diabetes mellitus, is predominantly metabolized by the cytochrome P450 (CYP) enzyme CYP2C8. The anti-infective drug trimethoprim has been shown in vitro to be a selective inhibitor of CYP2C8. The purpose of this study was to evaluate the effect of trimethoprim on the CYP2C8 mediated metabolism of rosiglitazone in vivo and in vitro. Methods The effect of trimethoprim on the metabolism of rosiglitazone in vitro was assessed in pooled human liver microsomes. The effect in vivo was determined by evaluating rosiglitazone pharmacokinetics in the presence and absence of trimethoprim. Eight healthy subjects (four men and four women) completed a randomized, cross-over study. Subjects received single dose rosiglitazone (8 mg) in the presence and absence of trimethoprim 200 mg given twice daily for 5 days. Results Trimethoprim inhibited rosiglitazone metabolism both in vitro and in vivo. Inhibition of rosiglitazone para-hydroxylation by trimethoprim in vitro was found to be competitive with apparent Ki and IC50 values of 29 µm and 54.5 µm, respectively. In the presence of trimethoprim, rosiglitazone plasma AUC was increased by 31% (P = 0.01) from 2774 ± 645 µg l−1 h to 3643 ± 1051 µg l−1 h (95% confidence interval (Cl) for difference 189, 1549), and half-life was increased by 27% (P = 0.006) from 3.3 ± 0.5 to 4.2 ± 0.8 h (95% Cl for difference 0.36, 1.5). Trimethoprim reduced the para-O-sulphate rosiglitazone/rosiglitazone and the N-desmethylrosiglitazone/rosiglitazone AUC(0–24) ratios by 22% and 38%, respectively. Conclusions These results indicate that trimethoprim is a competitive inhibitor of CYP2C8-mediated rosiglitazone metabolism in vitro and that trimethoprim administration increases plasma rosiglitazone concentrations in healthy subjects. PMID:15606443

  13. 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.

  14. CYP2D6 and CYP2C19 in Papua New Guinea: High frequency of previously uncharacterized CYP2D6 alleles and heterozygote excess.

    Science.gov (United States)

    von Ahsen, Nicolas; Tzvetkov, Mladen; Karunajeewa, Harin A; Gomorrai, Servina; Ura, Alice; Brockmöller, Jürgen; Davis, Timothy M E; Mueller, Ivo; Ilett, Kenneth F; Oellerich, Michael

    2010-08-18

    A high frequency of previously unknown CYP2D6 alleles have been reported in Oceania populations. Genetic and functional properties of these alleles remain unknown. We performed analyses of the genetic variability of CYP2D6 and CYP2C19 genes using AmpliChip genotyping in cohorts from two distinct Papua New Guinea (PNG) populations (Kunjingini, n=88; Alexishafen, n=84) focussing on the genetic characterisation of PNG-specific alleles by re-sequencing. Previously unknown CYP2D6 alleles have population frequencies of 24% (Kunjingini) and 12% (Alexishafen). An allele similar to CYP2D6*1, but carrying the 1661G>C substitution, was the second most frequent CYP2D6 allele (20% Kunjingini and 10% Alexishafen population frequency). Sequencing suggests the CYP2D6* 1661G>C allele originated from a cross-over between CYP2D6*1 and *2 and thus is predicted to confer fully active CYP2D6 enzyme. Two additional predicted full activity alleles [1661G>C;4180G>C] and 31G>A were found in the Kunjingini cohort (frequencies 3 c/c and 1%, respectively) and a novel predicted reduced activity allele [100C>T;1039C>T] was found in the Alexishafen cohort (frequency 2%). A high frequency of ultra-rapid (15%) and notably low frequencies of intermediate and poor CYP2D6 metabolizers (exogamy and recent introduction of alleles by migration that are yet to reach HWE in relatively isolated populations. The CYP2D6*1661 allele common in Oceania may be regarded as functionally equivalent to the full activity CYP2D6*1 allele.

  15. The NDE1 genomic locus can affect treatment of psychiatric illness through gene expression changes related to microRNA-484.

    Science.gov (United States)

    Bradshaw, Nicholas J; Ukkola-Vuoti, Liisa; Pankakoski, Maiju; Zheutlin, Amanda B; Ortega-Alonso, Alfredo; Torniainen-Holm, Minna; Sinha, Vishal; Therman, Sebastian; Paunio, Tiina; Suvisaari, Jaana; Lönnqvist, Jouko; Cannon, Tyrone D; Haukka, Jari; Hennah, William

    2017-11-01

    Genetic studies of familial schizophrenia in Finland have observed significant associations with a group of biologically related genes, DISC1 , NDE1 , NDEL1 , PDE4B and PDE4D , the 'DISC1 network'. Here, we use gene expression and psychoactive medication use data to study their biological consequences and potential treatment implications. Gene expression levels were determined in 64 individuals from 18 families, while prescription medication information has been collected over a 10-year period for 931 affected individuals. We demonstrate that the NDE1 SNP rs2242549 associates with significant changes in gene expression for 2908 probes (2542 genes), of which 794 probes (719 genes) were replicable. A significant number of the genes altered were predicted targets of microRNA-484 ( p = 3.0 × 10 -8 ), located on a non-coding exon of NDE1 Variants within the NDE1 locus also displayed significant genotype by gender interaction to early cessation of psychoactive medications metabolized by CYP2C19. Furthermore, we demonstrate that miR-484 can affect the expression of CYP2C19 in a cell culture system. Thus, variation at the NDE1 locus may alter risk of mental illness, in part through modification of miR-484, and such modification alters treatment response to specific psychoactive medications, leading to the potential for use of this locus in targeting treatment. © 2017 The Authors.

  16. Flavonoids exhibit diverse effects on CYP11B1 expression and cortisol synthesis

    Energy Technology Data Exchange (ETDEWEB)

    Cheng, Li-Chuan; Li, Lih-Ann, E-mail: lihann@nhri.org.tw

    2012-02-01

    CYP11B1 catalyzes the final step of cortisol biosynthesis. The effects of flavonoids on transcriptional expression and enzyme activity of CYP11B1 were investigated using the human adrenocortical H295R cell model. All tested nonhydroxylated flavones including 3′,4′-dimethoxyflavone, α-naphthoflavone, and β-naphthoflavone upregulated CYP11B1 expression and cortisol production, whereas apigenin and quercetin exhibited potent cytotoxicity and CYP11B1 repression at high concentrations. Nonhydroxylated flavones stimulated CYP11B1-catalyzed cortisol formation at transcriptional level. Resveratrol increased endogenous and substrate-supported cortisol production like nonhydroxylated flavones tested, but it had no effect on CYP11B1 gene expression and enzyme activity. Resveratrol appeared to alter cortisol biosynthesis at an earlier step. The Ad5 element situated in the − 121/− 106 region was required for basal and flavone-induced CYP11B1 expression. Overexpression of COUP-TFI did not improve the responsiveness of Ad5 to nonhydroxylated flavones. Although COUP-TFI overexpression increased CYP11B1 and CYP11B2 promoter activation, its effect was not mediated through the common Ad5 element. Treating cells with PD98059 (a flavone-type MEK1 inhibitor) increased CYP11B1 promoter activity, but not involving ERK signaling because phosphorylation of ERK1/2 remained unvarying throughout the course of treatment. Likewise, AhR was not responsible for the CYP11B1-modulating effects of flavonoids because inconsistency with their effects on AhR activation. 3′,4′-dimethoxyflavone and 8-Br-cAMP additively activated CYP11B1 promoter activity. H-89 reduced 3′,4′-dimethoxyflavone-induced CYP11B1 promoter activation but to a lesser extent as compared to its inhibition on cAMP-induced transactivation. Our data suggest that constant exposure to nonhydroxylated flavones raises a potential risk of high basal and cAMP-induced cortisol synthesis in consequence of increased CYP11B1

  17. Flavonoids exhibit diverse effects on CYP11B1 expression and cortisol synthesis

    International Nuclear Information System (INIS)

    Cheng, Li-Chuan; Li, Lih-Ann

    2012-01-01

    CYP11B1 catalyzes the final step of cortisol biosynthesis. The effects of flavonoids on transcriptional expression and enzyme activity of CYP11B1 were investigated using the human adrenocortical H295R cell model. All tested nonhydroxylated flavones including 3′,4′-dimethoxyflavone, α-naphthoflavone, and β-naphthoflavone upregulated CYP11B1 expression and cortisol production, whereas apigenin and quercetin exhibited potent cytotoxicity and CYP11B1 repression at high concentrations. Nonhydroxylated flavones stimulated CYP11B1-catalyzed cortisol formation at transcriptional level. Resveratrol increased endogenous and substrate-supported cortisol production like nonhydroxylated flavones tested, but it had no effect on CYP11B1 gene expression and enzyme activity. Resveratrol appeared to alter cortisol biosynthesis at an earlier step. The Ad5 element situated in the − 121/− 106 region was required for basal and flavone-induced CYP11B1 expression. Overexpression of COUP-TFI did not improve the responsiveness of Ad5 to nonhydroxylated flavones. Although COUP-TFI overexpression increased CYP11B1 and CYP11B2 promoter activation, its effect was not mediated through the common Ad5 element. Treating cells with PD98059 (a flavone-type MEK1 inhibitor) increased CYP11B1 promoter activity, but not involving ERK signaling because phosphorylation of ERK1/2 remained unvarying throughout the course of treatment. Likewise, AhR was not responsible for the CYP11B1-modulating effects of flavonoids because inconsistency with their effects on AhR activation. 3′,4′-dimethoxyflavone and 8-Br-cAMP additively activated CYP11B1 promoter activity. H-89 reduced 3′,4′-dimethoxyflavone-induced CYP11B1 promoter activation but to a lesser extent as compared to its inhibition on cAMP-induced transactivation. Our data suggest that constant exposure to nonhydroxylated flavones raises a potential risk of high basal and cAMP-induced cortisol synthesis in consequence of increased CYP11B1

  18. 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)).

  19. A recombinant CYP11B1 dependent Escherichia coli biocatalyst for selective cortisol production and optimization towards a preparative scale.

    Science.gov (United States)

    Schiffer, Lina; Anderko, Simone; Hobler, Anna; Hannemann, Frank; Kagawa, Norio; Bernhardt, Rita

    2015-02-25

    Human mitochondrial CYP11B1 catalyzes a one-step regio- and stereoselective 11β-hydroxylation of 11-deoxycortisol yielding cortisol which constitutes not only the major human stress hormone but also represents a commercially relevant therapeutic drug due to its anti-inflammatory and immunosuppressive properties. Moreover, it is an important intermediate in the industrial production of synthetic pharmaceutical glucocorticoids. CYP11B1 thus offers a great potential for biotechnological application in large-scale synthesis of cortisol. Because of its nature as external monooxygenase, CYP11B1-dependent steroid hydroxylation requires reducing equivalents which are provided from NADPH via a redox chain, consisting of adrenodoxin reductase (AdR) and adrenodoxin (Adx). We established an Escherichia coli based whole-cell system for selective cortisol production from 11-deoxycortisol by recombinant co-expression of the demanded 3 proteins. For the subsequent optimization of the whole-cell activity 3 different approaches were pursued: Firstly, CYP11B1 expression was enhanced 3.3-fold to 257 nmol∗L(-1) by site-directed mutagenesis of position 23 from glycine to arginine, which was accompanied by a 2.6-fold increase in cortisol yield. Secondly, the electron transfer chain was engineered in a quantitative manner by introducing additional copies of the Adx cDNA in order to enhance Adx expression on transcriptional level. In the presence of 2 and 3 copies the initial linear conversion rate was greatly accelerated and the final product concentration was improved 1.4-fold. Thirdly, we developed a screening system for directed evolution of CYP11B1 towards higher hydroxylation activity. A culture down-scale to microtiter plates was performed and a robot-assisted, fluorescence-based conversion assay was applied for the selection of more efficient mutants from a random library. Under optimized conditions a maximum productivity of 0.84 g cortisol∗L(-1)∗d(-1) was achieved, which

  20. Autophagy Protects against CYP2E1/Chronic Ethanol-Induced Hepatotoxicity

    Directory of Open Access Journals (Sweden)

    Yongke Lu

    2015-10-01

    Full Text Available Autophagy is an intracellular pathway by which lysosomes degrade and recycle long-lived proteins and cellular organelles. The effects of ethanol on autophagy are complex but recent studies have shown that autophagy serves a protective function against ethanol-induced liver injury. Autophagy was found to also be protective against CYP2E1-dependent toxicity in vitro in HepG2 cells which express CYP2E1 and in vivo in an acute alcohol/CYPE1-dependent liver injury model. The goal of the current report was to extend the previous in vitro and acute in vivo experiments to a chronic ethanol model to evaluate whether autophagy is also protective against CYP2E1-dependent liver injury in a chronic ethanol-fed mouse model. Wild type (WT, CYP2E1 knockout (KO or CYP2E1 humanized transgenic knockin (KI, mice were fed an ethanol liquid diet or control dextrose diet for four weeks. In the last week, some mice received either saline or 3-methyladenine (3-MA, an inhibitor of autophagy, or rapamycin, which stimulates autophagy. Inhibition of autophagy by 3-MA potentiated the ethanol-induced increases in serum transaminase and triglyceride levels in the WT and KI mice but not KO mice, while rapamycin prevented the ethanol liver injury. Treatment with 3-MA enhanced the ethanol-induced fat accumulation in WT mice and caused necrosis in the KI mice; little or no effect was found in the ethanol-fed KO mice or any of the dextrose-fed mice. 3-MA treatment further lowered the ethanol-decrease in hepatic GSH levels and further increased formation of TBARS in WT and KI mice, whereas rapamycin blunted these effects of ethanol. Neither 3-MA nor rapamycin treatment affected CYP2E1 catalytic activity or content or the induction CYP2E1 by ethanol. The 3-MA treatment decreased levels of Beclin-1 and Atg 7 but increased levels of p62 in the ethanol-fed WT and KI mice whereas rapamycin had the opposite effects, validating inhibition and stimulation of autophagy, respectively. These

  1. MDMA, methamphetamine, and CYP2D6 pharmacogenetics: what is clinically relevant?

    Directory of Open Access Journals (Sweden)

    Rafael eDe La Torre

    2012-11-01

    Full Text Available In vitro human studies show that the metabolism of most amphetamine-like psychostimulants is regulated by the polymorphic cytochrome P450 isozyme CYP2D6. Two compounds, methamphetamine and 3,4-methylenedioxymethamphetamine (MDMA, were selected as archetypes to discuss the translation and clinical significance of in vitro to in vivo findings. Both compounds were chosen based on their differential interaction with CYP2D6 and their high abuse prevalence in society. Methamphetamine behaves as both a weak substrate and competitive inhibitor of CYP2D6, while MDMA acts as a high affinity substrate and potent mechanism-based inhibitor (MBI of the enzyme. The MBI behavior of MDMA on CYP2D6 implies that subjects, irrespective of their genotype/phenotype, are phenocopied to the poor metabolizer phenotype. The fraction of metabolic clearance regulated by CYP2D6 for both drugs is substantially lower than expected from in vitro studies. Other isoenzymes of cytochrome P450 and a relevant contribution of renal excretion play a part in their clearance. These facts tune down the potential contribution of CYP2D6 polymorphism in the clinical outcomes of both substances. Globally, the clinical relevance of CYP2D6 polymorphism is lower than that predicted by in vitro studies.

  2. Selective inhibition of CYP2C8 by fisetin and its methylated metabolite, geraldol, in human liver microsomes.

    Science.gov (United States)

    Shrestha, Riya; Kim, Ju-Hyun; Nam, Wongshik; Lee, Hye Suk; Lee, Jae-Mok; Lee, Sangkyu

    2018-04-01

    Fisetin is a flavonol compound commonly found in edible vegetables and fruits. It has anti-tumor, antioxidant, and anti-inflammatory effects. Geraldol, the O-methyl metabolite of fisetin in mice, is reported to suppress endothelial cell migration and proliferation. Although the in vivo and in vitro effects of fisetin and its metabolites are frequently reported, studies on herb-drug interactions have not yet been performed. This study was designed to investigate the inhibitory effect of fisetin and geraldol on eight isoforms of human cytochrome P450 (CYP) by using cocktail assay and LC-MS/MS analysis. The selective inhibition of CYP2C8-catalyzed paclitaxel hydroxylation by fisetin and geraldol were confirmed in pooled human liver microsomes (HLMs). In addition, an IC 50 shift assay under different pre-incubation conditions confirmed that fisetin and geraldol shows a reversible concentration-dependent, but not mechanism-based, inhibition of CYP2C8. Moreover, Michaelis-Menten, Lineweaver-burk plots, Dixon and Eadie-Hofstee showed a non-competitive inhibition mode with an equilibrium dissociation constant of 4.1 μM for fisetin and 11.5 μM for geraldol, determined from secondary plot of the Lineweaver-Burk plot. In conclusion, our results indicate that fisetin showed selective reversible and non-competitive inhibition of CYP2C8 more than its main metabolite, geraldol, in HLMs. Copyright © 2018 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.

  3. The involvement of flavin-containing monooxygenase but not CYP3A4 in metabolism of itopride hydrochloride, a gastroprokinetic agent: comparison with cisapride and mosapride citrate.

    Science.gov (United States)

    Mushiroda, T; Douya, R; Takahara, E; Nagata, O

    2000-10-01

    The goals of the present study were to identify the enzyme responsible for metabolism of itopride hydrochloride (itopride) and to evaluate the likelihood of drug interaction involving itopride. In human liver microsomes, the involvement of flavin-containing monooxygenase in N-oxygenation, the major metabolic pathway of itopride, was indicated by the following results: inhibition by methimazole and thiourea, heat inactivation, and protection against heat inactivation by NADPH. When the effects of ketoconazole on the metabolism of itopride, cisapride, and mosapride citrate (mosapride) were examined using human liver microsomes, ketoconazole strongly inhibited the formation of the primary metabolites of cisapride and mosapride, but not itopride. Other cytochrome P450 (CYP) 3A4 inhibitors, cimetidine, erythromycin, and clarithromycin, also inhibited the metabolism of cisapride and mosapride. In an in vivo study, itopride (30 mg/kg), cisapride (1.5 mg/kg), or mosapride (3 mg/kg) was orally administered to male rats with or without oral pretreatment with ketoconazole (120 mg/kg) twice daily for 2 days. The ketoconazole pretreatment significantly increased the area under the serum concentration curve and the maximum serum concentration of cisapride and mosapride but had no significant effect on the pharmacokinetics of itopride. In addition, itopride did not inhibit five specific CYP-mediated reactions of human liver microsomes. These results suggest that itopride is unlikely to alter the pharmacokinetics of other concomitantly administered drugs.

  4. A Dual Reporter Mouse Model of the Human β-Globin Locus: Applications and Limitations

    NARCIS (Netherlands)

    P. Papadopoulos (Petros); L. Gutiérrez (Laura); R. van der Linden (Reinier); J. Kong-a-San (John); A. Maas (Alex); D.D. Drabek (Dubravka); G.P. Patrinos (George); J.N.J. Philipsen (Sjaak); F.G. Grosveld (Frank)

    2012-01-01

    textabstractThe human β-globin locus contains the β-like globin genes (i.e. fetal γ-globin and adult β-globin), which heterotetramerize with α-globin subunits to form fetal or adult hemoglobin. Thalassemia is one of the commonest inherited disorders in the world, which results in quantitative

  5. The relevance of chemical interactions with CYP17 enzyme activity: Assessment using a novel in vitro assay

    International Nuclear Information System (INIS)

    Roelofs, Maarke J.E.; Piersma, Aldert H.; Berg, Martin van den; Duursen, Majorie B.M. van

    2013-01-01

    The steroidogenic cytochrome P450 17 (CYP17) enzyme produces dehydroepiandrosterone (DHEA), which is the most abundant circulating endogenous sex steroid precursor. DHEA plays a key role in e.g. sexual functioning and development. To date, no rapid screening assay for effects on CYP17 is available. In this study, a novel assay using porcine adrenal cortex microsomes (PACMs) was described. Effects of twenty-eight suggested endocrine disrupting compounds (EDCs) on CYP17 activity were compared with effects in the US EPA validated H295R (human adrenocorticocarcinoma cell line) steroidogenesis assay. In the PACM assay DHEA production was higher compared with the H295R assay (4.4 versus 2.2 nmol/h/mg protein). To determine the additional value of a CYP17 assay, all compounds were also tested for interaction with CYP19 (aromatase) using human placental microsomes (HPMs) and H295R cells. 62.5% of the compounds showed enzyme inhibition in at least one of the microsomal assays. Only the cAMP inducer forskolin induced CYP17 activity, while CYP19 was induced by four test compounds in the H295R assay. These effects remained unnoticed in the PACM and HPM assays. Diethylstilbestrol and tetrabromobisphenol A inhibited CYP17 but not CYP19 activity, indicating different mechanisms for the inhibition of these enzymes. From our results it becomes apparent that CYP17 can be a target for EDCs and that this interaction differs from interactions with CYP19. Our data strongly suggest that research attention should focus on validating a specific assay for CYP17 activity, such as the PACM assay, that can be included in the EDC screening battery. - Highlights: ► DHEA, produced by CYP17, plays a key role in sexual functioning and development. ► No rapid screening assay for effects on CYP17 is available yet. ► A novel assay using porcine adrenal cortex microsomes (PACMs) was described. ► Endocrine disrupting compounds (EDCs) targeting CYP17 interact differently with CYP19. ► A

  6. The relevance of chemical interactions with CYP17 enzyme activity: Assessment using a novel in vitro assay

    Energy Technology Data Exchange (ETDEWEB)

    Roelofs, Maarke J.E., E-mail: m.j.e.roelofs@uu.nl [Endocrine Toxicology Research Group, Institute for Risk Assessment Sciences (IRAS), Utrecht University, P.O. Box 80.177, NL-3508 TD Utrecht (Netherlands); Center for Health Protection, National Institute for Public Health and the Environment (RIVM), P.O. Box 1, 3720 BA Bilthoven (Netherlands); Piersma, Aldert H. [Endocrine Toxicology Research Group, Institute for Risk Assessment Sciences (IRAS), Utrecht University, P.O. Box 80.177, NL-3508 TD Utrecht (Netherlands); Center for Health Protection, National Institute for Public Health and the Environment (RIVM), P.O. Box 1, 3720 BA Bilthoven (Netherlands); Berg, Martin van den; Duursen, Majorie B.M. van [Endocrine Toxicology Research Group, Institute for Risk Assessment Sciences (IRAS), Utrecht University, P.O. Box 80.177, NL-3508 TD Utrecht (Netherlands)

    2013-05-01

    The steroidogenic cytochrome P450 17 (CYP17) enzyme produces dehydroepiandrosterone (DHEA), which is the most abundant circulating endogenous sex steroid precursor. DHEA plays a key role in e.g. sexual functioning and development. To date, no rapid screening assay for effects on CYP17 is available. In this study, a novel assay using porcine adrenal cortex microsomes (PACMs) was described. Effects of twenty-eight suggested endocrine disrupting compounds (EDCs) on CYP17 activity were compared with effects in the US EPA validated H295R (human adrenocorticocarcinoma cell line) steroidogenesis assay. In the PACM assay DHEA production was higher compared with the H295R assay (4.4 versus 2.2 nmol/h/mg protein). To determine the additional value of a CYP17 assay, all compounds were also tested for interaction with CYP19 (aromatase) using human placental microsomes (HPMs) and H295R cells. 62.5% of the compounds showed enzyme inhibition in at least one of the microsomal assays. Only the cAMP inducer forskolin induced CYP17 activity, while CYP19 was induced by four test compounds in the H295R assay. These effects remained unnoticed in the PACM and HPM assays. Diethylstilbestrol and tetrabromobisphenol A inhibited CYP17 but not CYP19 activity, indicating different mechanisms for the inhibition of these enzymes. From our results it becomes apparent that CYP17 can be a target for EDCs and that this interaction differs from interactions with CYP19. Our data strongly suggest that research attention should focus on validating a specific assay for CYP17 activity, such as the PACM assay, that can be included in the EDC screening battery. - Highlights: ► DHEA, produced by CYP17, plays a key role in sexual functioning and development. ► No rapid screening assay for effects on CYP17 is available yet. ► A novel assay using porcine adrenal cortex microsomes (PACMs) was described. ► Endocrine disrupting compounds (EDCs) targeting CYP17 interact differently with CYP19. ► A

  7. Dried chicory root modifies the activity and expression of porcine hepatic CYP3A but not 2C – Effect of in vitro and in vivo exposure

    DEFF Research Database (Denmark)

    Rasmussen, Martin Krøyer; Zamaratskaia, Galia; Andersen, Bente

    2012-01-01

    Hepatic cytochrome P450 expression and activity are dependent on many factors, including dietary ingredients. In the present study, we investigated the in vivo and in vitro effect of chicory root on hepatic CYP3A and 2C in male pigs. Chicory feeding increased the expression of CYP3A29 mRNA but no......Hepatic cytochrome P450 expression and activity are dependent on many factors, including dietary ingredients. In the present study, we investigated the in vivo and in vitro effect of chicory root on hepatic CYP3A and 2C in male pigs. Chicory feeding increased the expression of CYP3A29 m......RNA but not CYP2C33. Correspondingly, CYP3A activity was increased by chicory feeding, while CYP2C activity was not affected. Additionally, the in vitro effect of chicory e