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Sample records for human liver microsomal

  1. Metabolism and covalent binding of [14C]toluene by human and rat liver microsomal fractions and liver slices.

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    Chapman, D E; Moore, T J; Michener, S R; Powis, G

    1990-01-01

    The in vitro metabolism of [14C]toluene by liver microsomes and liver slices from male Fischer F344 rats and human subjects has been compared. Rat liver microsomes produced only benzyl alcohol from toluene. Liver microsomes from human subjects metabolized toluene to benzyl alcohol, benzaldehyde, and benzoic acid. Liver microsomes from one human donor also produced p-cresol and o-cresol. The overall rate of toluene metabolism by human liver microsomes was 9-fold greater than by rat liver microsomes. Human liver microsomal metabolism of benzyl alcohol to benzaldehyde required NADPH and was inhibited by carbon monoxide and high pH (pH 10). but was not inhibited by ADP-ribose or sodium azide. These results suggest that cytochrome P-450, rather than alcohol dehydrogenase, was responsible for the metabolism of benzyl alcohol to benzaldehyde. Human and rat liver slices metabolized toluene to hippuric acid and benzoic acid. The overall rate of toluene metabolism by human liver slices was 1.3-fold greater than by rat liver slices. Cresols and cresol conjugates were not detected in human or rat liver slice incubations. Covalent binding of [14C]toluene to human liver microsomes and slices was 21-fold and 4-fold greater than to the comparable rat liver preparations. Covalent binding did not occur in the absence of NADPH, was significantly decreased by coincubation with cysteine, glutathione, or superoxide dismutase, and was unaffected by coincubation with lysine. Protease and ribonuclease digestion decreased the amount of toluene covalently bound to human liver microsomes by 78% and 27% respectively. Acid washing of human liver microsomes had no effect on covalent binding.(ABSTRACT TRUNCATED AT 250 WORDS)

  2. Development of in silico models for human liver microsomal stability

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    Lee, Pil H.; Cucurull-Sanchez, Lourdes; Lu, Jing; Du, Yuhua J.

    2007-12-01

    We developed highly predictive classification models for human liver microsomal (HLM) stability using the apparent intrinsic clearance (CLint, app) as the end point. HLM stability has been shown to be an important factor related to the metabolic clearance of a compound. Robust in silico models that predict metabolic clearance are very useful in early drug discovery stages to optimize the compound structure and to select promising leads to avoid costly drug development failures in later stages. Using Random Forest and Bayesian classification methods with MOE, E-state descriptors, ADME Keys, and ECFP_6 fingerprints, various highly predictive models were developed. The best performance of the models shows 80 and 75% prediction accuracy for the test and validation sets, respectively. A detailed analysis of results will be shown, including an assessment of the prediction confidence, the significant descriptors, and the application of these models to drug discovery projects.

  3. UPLC/MS MS data of testosterone metabolites in human and zebrafish liver microsomes and whole zebrafish larval microsomes

    Directory of Open Access Journals (Sweden)

    Moayad Saad

    2018-02-01

    Full Text Available This article represents data regarding a study published in Toxicology in vitro entitled “ in vitro CYP-mediated drug metabolism in the zebrafish (embryo using human reference compounds” (Saad et al., 2017 [1]. Data were acquired with ultra-performance liquid chromatography – accurate mass mass spectrometry (UPLC-amMS. A full spectrum scan was conducted for the testosterone (TST metabolites from the microsomal stability assay in zebrafish and humans. The microsomal proteins were extracted from adult zebrafish male (MLM and female (FLM livers, whole body homogenates of 96 h post fertilization larvae (EM and a pool of human liver microsomes from 50 donors (HLM. Data are expressed as the abundance from the extracted ion chromatogram of the metabolites.

  4. Metabolism of diazepam and related benzodiazepines by human liver microsomes.

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    Hooper, W D; Watt, J A; McKinnon, G E; Reilly, P E

    1992-01-01

    The metabolism of diazepam has been studied in vitro using microsomal preparations from five human livers. An HPLC method was developed for the assay of diazepam, its congeners and its metabolites. Various methods for the incorporation of diazepam into the incubation medium were explored. It was shown that the use of organic solvents or small quantities of hydrochloric acid enhanced the solubility of this substrate. However all of the organic solvents tested were associated with substantial (around 50%) inhibition of metabolism of diazepam by both major pathways (N-demethylation and C3-hydroxylation). The use of hydrochloric acid gave satisfactory solubilization of diazepam, but not of pinazepam, prazepam or halazepam. Detailed metabolic studies were conducted only for diazepam, using neither hydrochloric acid nor organic solvents in the incubation medium. Formation of N-desmethyl-diazepam increased approximately linearly with diazepam concentration to 200 microM, and did not show saturation. Formation of temazepam gave a curved profile over the same range of diazepam concentrations, suggestive of a sigmoidal relationship. Michaelis-Menten parameters could not be determined for either reaction, but intrinsic clearances for N-demethylation varied over a 6-fold range. Diazepam N-demethylation was apparently promoted by the inclusion of temazepam in the incubation medium, while C3-hydroxylation of diazepam was enhanced in the presence of N-desmethyldiazepam. Mephenytoin in the incubation mixture had no effect on diazepam metabolism by either pathway. The present studies have defined some of the methodological problems inherent in in vitro metabolic studies with benzodiazepines, and have shed further light on the metabolism of diazepam in vitro by human liver.

  5. Isoflavones modulate the glucuronidation of estradiol in human liver microsomes

    National Research Council Canada - National Science Library

    Pfeiffer, Erika; Treiling, Christian R; Hoehle, Simone I; Metzler, Manfred

    2005-01-01

    ... by the endogenous hormone 17beta-estradiol (E2). In the present study, we have examined if daidzein and genistein as well as several structurally related isoflavones are able to modulate the in vitro glucuronidation of E2 in human hepatic microsomes...

  6. Transesterification of a series of 12 parabens by liver and small-intestinal microsomes of rats and humans.

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    Fujino, Chieri; Watanabe, Yoko; Uramaru, Naoto; Kitamura, Shigeyuki

    2014-02-01

    Hydrolytic transformation of parabens (4-hydroxybenzoic acid esters; used as antibacterial agents) to 4-hydroxybenzoic acid and alcohols by tissue microsomes is well-known both in vitro and in vivo. Here, we investigated transesterification reactions of parabens catalyzed by rat and human microsomes, using a series of 12 parabens with C1-C12 alcohol side chains. Transesterification of parabens by rat liver and small-intestinal microsomes occurred in the presence of alcohols in the microsomal incubation mixture. Among the 12 parabens, propylparaben was most effectively transesterified by rat liver microsomes with methanol or ethanol, followed by butylparaben. Relatively low activity was observed with longer-side-chain parabens. In contrast, small-intestinal microsomes exhibited higher activity towards moderately long side-chain parabens, and showed the highest activity toward octylparaben. When parabens were incubated with liver or small-intestinal microsomes in the presence of C1-C12 alcohols, ethanol and decanol were most effectively transferred to parabens by rat liver microsomes and small-intestinal microsomes, respectively. Human liver and small-intestinal microsomes also exhibited significant transesterification activities with different substrate specificities, like rat microsomes. Carboxylesterase isoforms, CES1b and CES1c, and CES2, exhibited significant transesterification activity toward parabens, and showed similar substrate specificity to human liver and small-intestinal microsomes, respectively. Copyright © 2013 Elsevier Ltd. All rights reserved.

  7. Assessment of in silico models for fraction of unbound drug in human liver microsomes.

    Science.gov (United States)

    Gao, Hua; Steyn, Stefanus J; Chang, George; Lin, Jing

    2010-05-01

    Fraction of unbound drug in human liver microsome (fu(mic)) incubation media is an important parameter for accurate assessment of hepatic intrinsic clearance and drug-drug interactions. In recent years, there have been considerable advances in understanding structure-microsomal binding relationships. This review highlights the in silico modeling techniques for fu(mic) including physicochemical properties-based modeling, pharmacophore feature-based classification modeling and more complex statistical method-based modeling. The application of these modeling techniques to the understanding of the structure-binding relationships is also discussed. The reader will gain an understanding of the modeling techniques used for prediction of binding to human liver microsomes (fu(mic)). The reader will also understand the molecular structure-microsomal protein binding relationships. In all of these models, lipophilicity is the most important molecular property underlying the structure-binding relationship. Other molecular properties such as charge type (positive vs negative) and hydrogen bonding are also important factors for microsomal binding. The predictive accuracy of fu(mic) models in the high lipophilicity and tight microsomal binding ranges still needs to be further improved. However, in silico models are still valuable tools to aid chemical library design and prioritize multiple chemical series, which could provide efficiency and decrease knowledge cycle times in drug discovery.

  8. CHARACTERIZATION OF HUMAN LIVER MICROSOMAL UDP-GLYCOSYLTRANSFERASES USING PHOTOAFFINITY ANALOGS

    NARCIS (Netherlands)

    LITTLE, JM; DRAKE, RR; VONK, R; KUIPERS, F; LESTER, R; RADOMINSKA, A

    The photoaffinity analogs [beta-P-32]5-azido-UDP-glucuronic acid ([P-32]5N3UDP-GlcUA) and [beta-P-32]5-azido-UDP-glucose ([P-32]5N(3)UDP-Glc) were used to characterize UDP-glycosyl-transferases of microsomes prepared from human liver. Photoincorporation of both probes into proteins in the 50- to

  9. Effects of Eupatilin and Jaceosidin on Cytochrome P450 Enzyme Activities in Human Liver Microsomes

    Directory of Open Access Journals (Sweden)

    Ji Hyun Jeong

    2010-09-01

    Full Text Available Eupatilin and jaceosidin are bioactive flavones found in the medicinal herbs of the genus Artemisia. These bioactive flavones exhibit various antioxidant, antiinflammatory, antiallergic, and antitumor activities. The inhibitory potentials of eupatilin and jaceosidin on the activities of seven major human cytochrome P450 enzymes in human liver microsomes were investigated using a cocktail probe assay. Eupatilin and jaceosidin potently inhibited CYP1A2-catalyzed phenacetin O-deethylation with 50% inhibitory concentration (IC50 values of 9.4 mM and 5.3 mM, respectively, and CYP2C9-catalyzed diclofenac 4-hydroxylation with IC50 values of 4.1 mM and 10.2 mM, respectively. Eupatilin and jaceosidin were also found to moderately inhibit CYP2C19-catalyzed [S]-mephenytoin 4¢-hydroxylation, CYP2D6-catalyzed bufuralol 1¢-hydroxylation, and CYP2C8-catalyzed amodiaquine N-deethylation. Kinetic analysis of human liver microsomes showed that eupatilin is a competitive inhibitor of CYP1A2 with a Ki value of 2.3 mM and a mixed-type inhibitor of CYP2C9 with a Ki value of 1.6 mM. Jaceosidin was shown to be a competitive inhibitor of CYP1A2 with a Ki value of 3.8 mM and a mixed-type inhibitor of CYP2C9 with Ki value of 6.4 mM in human liver microsomes. These in vitro results suggest that eupatilin and jaceosidin should be further examined for potential pharmacokinetic drug interactions in vivo due to inhibition of CYP1A2 and CYP2C9.

  10. Metabolism of Ginger Component [6]-Shogaol in Liver Microsomes from Mouse, Rat, Dog, Monkey, and Human

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    Chen, Huadong; Soroka, Dominique; Zhu, Yingdong; Sang, Shengmin

    2013-01-01

    Scope There are limited data on the metabolism of [6]-shogaol, a major bioactive component of ginger. This study demonstrates metabolism of [6]-shogaol in liver microsomes from mouse, rat, dog, monkey, and human. Methods and results The in vitro metabolism of [6]-shogaol was compared among five species using liver microsomes from mouse, rat, dog, monkey, and human. Following incubations with [6]-shogaol, three major reductive metabolites 1-(4'-hydroxy-3'-methoxyphenyl)-4-decen-3-ol (M6), 1-(4′-hydroxy-3′-methoxyphenyl)-decan-3-ol (M9), and 1-(4'-hydroxy-3'-methoxyphenyl)-decan-3-one (M11), as well as two new oxidative metabolites (1E, 4E)-1-(4'-hydroxy-3'-methoxyphenyl)-deca-1,4-dien-3-one (M14) and (E)-1-(4'-hydroxy-3'-methoxyphenyl)-dec-1-en-3-one (M15) were found in all species. The kinetic parameters of M6 in liver microsomes from each respective species were quantified using Michaelis-Menten theory. A broad CYP-450 inhibitor, 1-aminobenzotriazole, precluded the formation of oxidative metabolites M14 and M15, and 18β-glycyrrhetinic acid, an aldo-keto reductase inhibitor, eradicated the formation of the reductive metabolites M6, M9, and M11 in all species. Metabolites M14 and M15 were tested for cancer cell growth inhibition and induction of apoptosis and both showed substantial activity, with M14 displaying greater potency than [6]-shogaol. Conclusion We conclude that [6]-shogaol is metabolized extensively in mammalian species mouse, rat, dog, monkey, and human, and that there are significant interspecies differences to consider when planning pre-clinical trials towards [6]-shogaol chemoprevention. PMID:23322474

  11. Biotransformation of Flavokawains A, B, and C, Chalcones from Kava (Piper methysticum), by Human Liver Microsomes.

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    Zenger, Katharina; Agnolet, Sara; Schneider, Bernd; Kraus, Birgit

    2015-07-22

    The in vitro metabolism of flavokawains A, B, and C (FKA, FKB, FKC), methoxylated chalcones from Piper methysticum, was examined using human liver microsomes. Phase I metabolism and phase II metabolism (glucuronidation) as well as combined phase I+II metabolism were studied. For identification and structure elucidation of microsomal metabolites, LC-HRESIMS and NMR techniques were applied. Major phase I metabolites were generated by demethylation in position C-4 or C-4' and hydroxylation predominantly in position C-4, yielding FKC as phase I metabolite of FKA and FKB, helichrysetin as metabolite of FKA and FKC, and cardamonin as metabolite of FKC. To an even greater extent, flavokawains were metabolized in the presence of uridine diphosphate (UDP) glucuronic acid by microsomal UDP-glucuronosyl transferases. For all flavokawains, monoglucuronides (FKA-2'-O-glucuronide, FKB-2'-O-glucuronide, FKC-2'-O-glucuronide, FKC-4-O-glucuronide) were found as major phase II metabolites. The dominance of generated glucuronides suggests a role of conjugated chalcones as potential active compounds in vivo.

  12. Differential inhibition of aflatoxin B1 oxidation by gestodene action on human liver microsomes.

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    Kim, B R; Oh, H S; Kim, D H

    1997-11-01

    Human cytochrome P450 (P450) 3A is known to be involved in the formation of both aflatoxin B1-exo-8,9-epoxide (exo-epoxidation) and aflatoxin Q1 (3 alpha-hydroxylation). Gestodene, a known inactivator of P450 3A4, inhibited the formation of AFB1 metabolites in a variety of ways depending on the incubation condition. Preincubation of gestodene with human liver microsomes prior to the addition of AFB1 inhibited both exo-epoxidation and 3 alpha-hydroxylation whereas simultaneous incubation of gestodene with AFB1 only inhibited 3 alpha-hydroxylation. These results suggest that two independent substrate binding sites exist in P450 3A4, and AFB1 binds to both of the binding sites. Gestodene selectively binds to one of the binding sites leading to the formation of AFQ1, whereas it does not affect the formation of exo-epoxide via the other binding site.

  13. Inhibition of in vitro metabolism of testosterone in human, dog and horse liver microsomes to investigate species differences.

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    Zielinski, Jana; Mevissen, Meike

    2015-04-01

    Testosterone hydroxylation was investigated in human, canine and equine liver microsomes and in human and canine single CYPs. The contribution of the CYP families 1, 2 and 3 was studied using chemical inhibitors. Testosterone metabolites were analyzed by HPLC. The metabolites androstenedione, 6β- and 11β-hydroxytestosterone were found in microsomes of all species, but the pattern of metabolites varied within species. Androstenedione was more prominent in the animal species, and an increase over time was seen in equines. Testosterone hydroxylation was predominantly catalyzed by the CYP3A subfamily in all three species. While CYP2C9 did not metabolise testosterone, the canine ortholog CYP2C21 produced androstenedione. Quercetin significantly inhibited 6β- and 11β-hydroxytestosterone in all species investigated, suggesting that CYP2C8 is involved in testosterone metabolism, whereas sulfaphenazole significantly inhibited the formation of 6β- and 11β-hydroxytestosterone in human microsomes, at 60 min in equine microsomes, but not in canine microsomes. A contribution of CYP2B6 in testosterone metabolism was only found in human and equine microsomes. Inhibition of 17β-hydroxysteroid dehydrogenase 2 indicated its involvement in androstenedione formation in humans, increased androstenedione formation was found in equines and no involvement in canines. These findings provide improved understanding of differences in testosterone biotransformation in animal species. Copyright © 2014 Elsevier Ltd. All rights reserved.

  14. Metabolism of lysergic acid diethylamide (LSD) to 2-oxo-3-hydroxy LSD (O-H-LSD) in human liver microsomes and cryopreserved human hepatocytes.

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    Klette, K L; Anderson, C J; Poch, G K; Nimrod, A C; ElSohly, M A

    2000-10-01

    The metabolism of lysergic acid diethylamide (LSD) to 2-oxo-3-hydroxy lysergic acid diethylamide (O-H-LSD) was investigated in liver microsomes and cyropreserved hepatocytes from humans. Previous studies have demonstrated that O-H-LSD is present in human urine at concentrations 16-43 times greater than LSD, the parent compound. Additionally, these studies have determined that O-H-LSD is not generated during the specimen extraction and analytical processes or due to parent compound degradation in aqueous urine samples. However, these studies have not been conclusive in demonstrating that O-H-LSD is uniquely produced during in vivo metabolism. Phase I drug metabolism was investigated by incubating human liver microsomes and cryopreserved human hepatocytes with LSD. The reaction was quenched at various time points, and the aliquots were extracted using liquid partitioning and analyzed by liquid chromatography-mass spectrometry. O-H-LSD was positively identified in all human liver microsomal and human hepatocyte fractions incubated with LSD. In addition, O-H-LSD was not detected in any microsomal or hepatocyte fraction not treated with LSD nor in LSD specimens devoid of microsomes or hepatocytes. This study provides definitive evidence that O-H-LSD is produced as a metabolic product following incubation of human liver microsomes and hepatocytes with LSD.

  15. Cytochrome P450 isoenzymes in rat and human liver microsomes associate with the metabolism of total coumarins in Fructus Cnidii.

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    Hu, Xiao; Huang, Wei; Yang, Yuan

    2015-12-01

    Fructus Cnidii (Cnidium) is isolated from the dry and ripe fruit of Cnidium monnier (L.) Cuss (umbelifera), an annual herb. It is demonstrated that the active constituents of Fructus Cnidii are coumarins, known as Total Coumarins of Cnidium Monnier (TCCM). Osthole (Ost) and imperatorin (Imp) are the most active constituents of TCCM which are usually regarded as the quality indicators of medicinal Fructus Cnidii. The aim is to study the metabolism of Fructus Cnidii effective monomer osthole and imperatorin in vitro by liver microsomes. CYP3A4 inhibitor ketoconazole, CYP2D6 inhibitor qunidine, CYP2C8 inhibitor trimethoprim, CYP2C9 inhibitor sulfaphenazole, and CYP1A2 inhibitor α-naphthoflavone were used to investigate the metabolism from incubation time, substrate concentration and liver microsomal concentration, respectively. The concentration of liver microsomes was 0.2 mg/ml. Ost (0.8/3.2/12.8 uM) was incubated at 37 °C for 20 min while Imp (1.6/6.4/19.2 uM) was incubated for 30 min. Qunidine, trimethoprim and α-naphthoflavone could significantly inhibit the disappearance of Imp; meanwhile ketoconazole, sulfaphenazole and qunidine could inhibit the disappearance of Ost. CYP1A, CYP2C are involved in the metabolism of Imp and CYP3A mediates the metabolism of Ost in rat liver microsomes. In human liver microsomes, CYP1A2, CYP2C8, CYP2D6 are involved in the metabolism of Imp; CYP3A4 is involved in the metabolism of Ost at all the tested concentrations of Ost, while CYP2C9, CYP2D6 mediate the metabolism at high concentration of Ost.

  16. Formation of the Thiol Conjugates and Active Metabolite of Clopidogrel by Human Liver Microsomes

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    Lau, Wei C.; Hollenberg, Paul F.

    2012-01-01

    We reported previously the formation of a glutathionyl conjugate of the active metabolite (AM) of clopidogrel and the covalent modification of a cysteinyl residue of human cytochrome P450 2B6 in a reconstituted system (Mol Pharmacol 80:839–847, 2011). In this work, we extended our studies of the metabolism of clopidogrel to human liver microsomes in the presence of four reductants, namely, GSH, l-Cys, N-acetyl-l-cysteine (NAC), and ascorbic acid. Our results demonstrated that formation of the AM was greatly affected by the reductant used and the relative amounts of the AM formed were increased in the following order: NAC (17%) clopidogrel. It was observed that the AM was slowly converted to the thiol conjugate, with a half-life of ∼10 h. Addition of dithiothreitol to the reaction mixture reversed the conversion, which resulted in a decrease in AM-thiol conjugate levels and a concomitant increase in AM levels, whereas addition of NAC led to the formation of AM-NAC and a concomitant decrease in AM-GSH levels. These results not only confirm that the AM is formed through oxidative opening of the thiolactone ring but also suggest the existence of an equilibrium between the AM, the thiol conjugates, and the reductants. These factors may affect the effective concentrations of the AM in vivo. PMID:22584220

  17. Determination of fluoxetine and its metabolite norfluoxetine in human liver microsomes by reversed-phase HPLC in vitro.

    Science.gov (United States)

    Liu, Z Q; Cheng, Z N; Wang, W; Tan, Z R; Ou-Yang, D S; Zhou, H H

    2000-11-01

    A high-performance liquid chromatography (HPLC) method was developed for the determination of fluoxetine (FLU) and its metabolite norfluoxetine (N-FLU) in human liver microsomes in vitro. An incubation buffer containing human liver microsomes, NADPH-generating system, and FLU, after termination of enzyme reaction and addition of nortriptyline (NOR) as internal standard (IS), was extracted with n-hexane/acetonitrile, and separated on a reversed-phase ODS column. Detection was achieved at 226 nm by ultraviolet detector (UV). The limit of detection was 5 micrograms/L for both FLU and N-FLU. No potential interference was found. The method provides recoveries of up to 94%-104% and acceptable coefficients of variation were found for both within-run (< 7.8%) and day to day (< 9.1%) assays. This method is rapid, sensitive, and simple for studying the metabolism of FLU and N-FLU.

  18. Transporter-mediated uptake of UDP-glucuronic acid by human liver microsomes: assay conditions, kinetics, and inhibition.

    Science.gov (United States)

    Rowland, Andrew; Mackenzie, Peter I; Miners, John O

    2015-01-01

    This study characterized the kinetics, variability, and factors that affect UDP-glucuronic acid (UDP-GlcUA) uptake by human liver microsomes (HLM). Biphasic kinetics were observed for UDP-GlcUA uptake by HLM. Uptake affinities (assessed as Kd) of the high- and low-affinity components differed by more than an order of magnitude (13 ± 6 vs. 374 ± 175 µM), but were comparable in terms of the maximal rate of uptake, with mean Vmax values differing less than 2.3-fold (56 ± 26 vs. 131 ± 35 pmol/min per mg). Variability in total intrinsic transporter activity (Uint) for microsomal UDP-GlcUA uptake across 12 livers was less than 4-fold. Experiments performed to optimize the conditions for microsomal UDP-GlcUA uptake demonstrated that both components were trans-stimulated by preloading (luminal addition) with an alternate UDP-sugar, and essentially abolished by the thiol-alkylating agent N-ethylmaleimide. Furthermore, interaction studies undertaken with a panel of drugs, alternate UDP-sugars, and glucuronide conjugates, at low (2.5 μM) and high (1000 μM) UDP-GlcUA concentrations, demonstrated that both components were inhibited to varying extents. Notably, the nucleoside analogs zidovudine, stavudine, lamivudine, and acyclovir inhibited both the high- and low- affinity components of microsomal UDP-GlcUA uptake by >45% at an inhibitor concentration of 100 μM. Taken together, these data demonstrate that human liver microsomal UDP-GlcUA uptake involves multiple protein-mediated components, and raises the possibility of impaired in vivo glucuronidation activity resulting from inhibition of UDP-GlcUA uptake into the endoplasmic reticulum membrane by drugs and other compounds. Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.

  19. Metabolism of aildenafil in vivo in rats and in vitro in mouse, rat, dog, and human liver microsomes.

    Science.gov (United States)

    Li, Yan; Wu, Linan; Gu, Yuan; Si, Duanyun; Liu, Changxiao

    2014-06-01

    Aildenafil, 1-{[3-(6, 7-dihydro-1-methyl-7-oxo-3-propyl-1H-pyrazolo [4, 3-d] primidin-5-yl)-4-ethoxyphenyl] sulfonyl}-cis-3, 5-dimethylpiperazine, a phosphodiesterase type V enzyme inhibitor (PDE5I), is under development for treatment of erectile dysfunction (ED). The purpose of this study was to elucidate metabolism of aildenafil in vivo in rats and in vitro in mouse, rat, dog, and human liver microsomes. Thirty-one phase I metabolites have been found by LTQ/Orbitrap hybrid mass spectrometry in rat urine, faeces, and bile after oral administration. Major biotransformation pathways of aildenafil included N-dealkylation of the piperazine ring, hydroxylation and dehydrogenation, aliphatic hydroxylation and loss of alkyl group of piperazine ring. Minor pathways involved hydroxylation on the phenyl ring, pyrazole N-demethylation, O-deethylation, loss of piperazine ring (cleavage of N-S bond) and dehydrogenation on the piperazine ring. Similar metabolic pathways of aildenafil were observed in the incubations of liver microsomes from mouse, rat, and dog as well as from human. The depletion rate of parent drug in mouse and rat liver microsomes was significantly different from that in human liver microsomes. The cytochrome P450 reaction phenotyping analysis was conducted using isozyme-specific inhibitors. The results indicated that CYP3A was the main isoenzyme involved in oxidative metabolism of aildenafil. Overall, these in vitro and in vivo findings should provide valuable information on possible metabolic behaviours of aildenafil in humans. Copyright © 2013 John Wiley & Sons, Ltd.

  20. Identification of the metabolites of episesamin in rat bile and human liver microsomes.

    Science.gov (United States)

    Tomimori, Namino; Nakai, Masaaki; Ono, Yoshiko; Kitagawa, Yoshinori; Kiso, Yoshinobu; Shibata, Hiroshi

    2012-01-01

    Episesamin is an isomer of sesamin, resulting from the refining process of non-roasted sesame seed oil. Episesamin has two methylendioxyphenyl groups on exo and endo faces of the bicyclic skeleton. The side methylendioxyphenyl group was metabolized by cytochrome-P450. Seven metabolites of episesamin were found in rat bile after treatment with glucuronidase/arylsulfatase and were identified using NMR and MS. The seven metabolites were (7α,7'β,8α,8'α)-3,4-dihydroxy-3',4'-methylenedioxy-7,9':7',9-diepoxylignane (EC-1-1), (7α,7'β,8α,8'α)-3,4-methylenedioxy-3',4'-dihydroxy-7,9':7',9-diepoxylignane (EC-1-2) and (7α,7'β,8α,8'α)-3,4:3',4'-bis(dihydroxy)-7,9':7',9-diepoxylignane (EC-2), (7α,7'β,8α,8'α)-3-methoxy-4-hydroxy-3',4'-methylenedioxy-7,9':7',9-diepoxylignane (EC-1m-1), (7α,7'β,8α,8'α)-3,4-methylenedioxy-3'-methoxy-4'-hydroxy-7,9':7',9-diepoxylignane (EC-1m-2), (7α,7'β,8α,8'α)-3-methoxy-4-hydroxy-3',4'-dihydroxy-7,9':7',9-diepoxylignane (EC-2m-1) and (7α,7'β,8α,8'α)-3,4-dihydroxy-3'-methoxy-4'-hydroxy-7,9':7',9-diepoxylignane (EC-2m-2). EC-1-1, EC-1-2 and EC-2 were also identified as metabolites of episesamin in human liver microsomes. These results suggested that similar metabolic pathways of episesamin could be proposed in rats and humans.

  1. Human Liver Microsomal Cytochrome P450 3A Enzymes Involved in Thalidomide 5-Hydroxylation and Formation of a Glutathione Conjugate

    Science.gov (United States)

    Chowdhury, Goutam; Murayama, Norie; Okada, Yusuke; Uno, Yasuhiro; Shimizu, Makiko; Shibata, Norio; Guengerich, F. Peter; Yamazaki, Hiroshi

    2013-01-01

    (R)-Thalidomide was oxidized to 5-hydroxythalidomide and 5’-hydroxythalidomide by NADPH-fortified liver microsomes from humans and monkeys. (R)-Thalidomide was hydroxylated more efficiently than (S)-thalidomide. Recombinant human P450s 3A4, 3A5, and 3A7 and monkey P450s 3A8 and 3A5 (co-expressed with NADPH-P450 reductase in bacterial membranes) also catalyzed (R)-thalidomide 5-hydroxylation. Purified human P450s 2C19, 3A4, and 3A5 mediated (R)-thalidomide 5-hydroxylation at similar rates in reconstituted systems. P450 2C19 showed a rather non-saturable substrate-velocity curve; however, P450s 3A4 and 3A5 showed sigmoidal curves. P450 also oxidized 5-hydroxythalidomide to an epoxide or dihydroxy compound. Liquid chromatography-mass spectrometry analysis revealed formation of a glutathione conjugate from (R)- and (S)-5-hydroxythalidomide, catalyzed by liver microsomal P450s 3A4 and 3A5 in the presence of glutathione (assigned as a conjugate of 5-hydroxythalidomide formed on the phenyl ring). These results indicate that human P450s 3A4 and 3A5 mediate thalidomide 5-hydroxylation and further oxidation leading to a glutathione conjugate, which may be of relevance in the pharmacological and toxicological actions of thalidomide. PMID:20443640

  2. Stereoselective Metabolism of Bupropion by Cytochrome P4502B6 (CYP2B6) and Human Liver Microsomes

    Science.gov (United States)

    Coles, Rebecka; Kharasch, Evan D.

    2013-01-01

    Purpose Hydroxylation of the antidepressant and smoking deterrent drug bupropion is a clinically important bioactivation and elimination pathway. Bupropion hydroxylation is catalyzed selectively by cytochrome P4502B6 (CYP2B6). CYP2B6-catalyzed bupropion hydroxylation has been used as an in vitro and in vivo phenotypic probe for CYP2B6 activity and CYP2B6 drug interactions. Bupropion is chiral, used clinically as a racemate, and disposition is stereoselective. Nevertheless, it is unknown whether CYP2B6-catalyzed bupropion hydroxylation is stereoselective. Methods Hydroxylation of racemic bupropion by recombinant CYP2B6 and human liver microsomes was evaluated using a stereoselective assay. Results At therapeutic concentrations, hydroxylation of (S)-bupropion was 3-fold and 1.5-greater than (R)-bupropion, respectively, by recombinant CYP2B6 and human liver microsomes. In vitro intrinsic clearances were likewise different for bupropion enantiomers. Conclusions Stereoselective bupropion hydroxylation may have implications for the therapeutic efficacy of bupropion as an antidepressant or smoking cessation therapy, and for the use of bupropion as an in vivo phenotypic probe for CYP2B6 activity. PMID:18219560

  3. Raloxifene glucuronidation in liver and intestinal microsomes of humans and monkeys: contribution of UGT1A1, UGT1A8 and UGT1A9.

    Science.gov (United States)

    Kishi, Naoki; Takasuka, Akane; Kokawa, Yuki; Isobe, Takashi; Taguchi, Maho; Shigeyama, Masato; Murata, Mikio; Suno, Manabu; Hanioka, Nobumitsu

    2016-01-01

    1. Raloxifene is an antiestrogen that has been marketed for the treatment of osteoporosis, and is metabolized into 6- and 4'-glucuronides by UDP-glucuronosyltransferase (UGT) enzymes. In this study, the in vitro glucuronidation of raloxifene in humans and monkeys was examined using liver and intestinal microsomes and recombinant UGT enzymes (UGT1A1, UGT1A8 and UGT1A9). 2. Although the K(m) and CL(int) values for the 6-glucuronidation of liver and intestinal microsomes were similar between humans and monkeys, and species differences in Vmax values (liver microsomes, humans > monkeys; intestinal microsomes, humans  UGT1A8 >UGT1A9 for humans, and UGT1A8 > UGT1A1 > UGT1A9 for monkeys. The activities of 4'-glucuronidation were UGT1A8 > UGT1A1 > UGT1A9 in humans and monkeys. 4. These results demonstrated that the profiles for the hepatic and intestinal glucuronidation of raloxifene by microsomes were moderately different between humans and monkeys.

  4. Gas chromatographic-mass spectrometric analysis of hydroxylamine for monitoring the metabolic hydrolysis of metalloprotease inhibitors in rat and human liver microsomes.

    Science.gov (United States)

    Peng, S X; Strojnowski, M J; Hu, J K; Smith, B J; Eichhold, T H; Wehmeyer, K R; Pikul, S; Almstead, N G

    1999-03-05

    A gas chromatographic-mass spectrometric (GC-MS) method was developed for the analysis of hydroxylamine (HA) in supernatants obtained from liver microsomes. HA monitoring was used to determine the metabolic hydrolysis of two hydroxamic acid-based matrix metalloprotease inhibitors in rat and human liver microsomes. The hydrolysis of the hydroxamic acids to their corresponding carboxylic acids releases HA as a common metabolic product. HA was derivatized to acetone oxime by addition of acetone to the liver microsomal supernatant, followed by direct injection of the supernatant into the GC-MS, with detection of the oxime by selected-ion-monitoring. The method is simple, reproducible, and sensitive for the determination of the hydrolysis of hydroxamic acid compounds, where hydrolysis is the major metabolic pathway. The methodology can be used for rank ordering and selecting hydroxamic acid analogs based on their susceptibility to hydrolysis.

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

    Directory of Open Access Journals (Sweden)

    Ju-Hyun Kim

    2017-03-01

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

  6. Metabolism-mediated interaction potential of standardized extract of Tinospora cordifolia through rat and human liver microsomes.

    Science.gov (United States)

    Bahadur, Shiv; Mukherjee, Pulok K; Milan Ahmmed, S K; Kar, Amit; Harwansh, Ranjit K; Pandit, Subrata

    2016-01-01

    Tinospora cordifolia is used for treatment of several diseases in Indian system of medicine. In the present study, the inhibition potential of T. cordifolia extracts and its constituent tinosporaside to cause herb-drug interactions through rat and human liver cytochrome enzymes was evaluated. Bioactive compound was quantified through reverse phase high-performance liquid chromatography, to standardize the plant extracts and interaction potential of standardized extract. Interaction potential of the test sample was evaluated through cytochrome P450-carbon monoxide complex (CYP450-CO) assay with pooled rat liver microsome. Influence on individual recombinant human liver microsomes such as CYP3A4, CYP2D6, CYP2C9, and CYP1A2 isozymes was analyzed through fluorescence microplate assay, and respective IC50 values were determined. The content of tinosporaside was found to be 1.64% (w/w) in T. cordifolia extract. Concentration-dependent inhibition was observed through T. cordifolia extract. Observed IC50 (μg/ml) value was 136.45 (CYP3A4), 144.37 (CYP2D6), 127.55 (CYP2C9), and 141.82 (CYP1A2). Tinosporaside and extract showed higher IC50 (μg/ml) value than the known inhibitors. T. cordifolia extract showed significantly less interaction potential and indicates that the selected plant has not significant herb-drug interactions relating to the inhibition of major CYP450 isozymes. Plant extract showed significantly higher IC50 value than respective positive inhibitors against CYP3A4, 2D6, 2C9, and 1A2 isozymes. Consumption of T. cordifolia may not cause any adverse effects when consumed along with other xenobiotics.

  7. Effects of Vernonia cinerea Compounds on Drug-metabolizing Cytochrome P450s in Human Liver Microsomes.

    Science.gov (United States)

    Pouyfung, Phisit; Sarapusit, Songklod; Rongnoparut, Pornpimol

    2017-12-01

    Vernonia cinerea has been widely used in traditional medicines for various diseases and shown to aid in smoking abstinence and has anticancer properties. V. cinerea bioactive compounds, including flavonoids and hirsutinolide-type sesquiterpene lactones, have shown an inhibition effect on the nicotine-metabolizing cytochrome P450 2A6 (CYP2A6) enzyme and hirsutinolides reported suppressing cancer growth. In this study, V. cinerea ethanol extract and its bioactive compounds, including four flavonoids and four hirsutinolides, were investigated for an inhibitory effect on human liver microsomal CYPs 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4 using cocktail inhibition assays combined with LC-MS/MS analysis. Among tested flavonoids, chrysoeriol was more potent in inhibition on CYP2A6 and CYP1A2 than other liver CYPs, with better binding efficiency toward CYP2A6 than CYP1A2 (Ki values in competitive mode of 1.93 ± 0.05 versus 3.39 ± 0.21 μM, respectively). Hirsutinolides were prominent inhibitors of CYP2A6 and CYP2D6, with IC50 values of 12-23 and 15-41 μM, respectively. These hirsutinolides demonstrated time-dependent inhibition, an indication of mechanism-based inactivation, toward CYP2A6. Quantitative prediction of microsomal metabolism of these flavonoids and hirsutinolides, including half-lives and hepatic clearance rate, was examined. These findings may have implications for further in vivo studies of V. cinerea. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

  8. In vitro Phase I and Phase II metabolism of α-pyrrolidinovalerophenone (α-PVP), methylenedioxypyrovalerone (MDPV) and methedrone by human liver microsomes and human liver cytosol.

    Science.gov (United States)

    Negreira, Noelia; Erratico, Claudio; Kosjek, Tina; van Nuijs, Alexander L N; Heath, Ester; Neels, Hugo; Covaci, Adrian

    2015-07-01

    The aim of the present study was to identify the in vitro Phase I and Phase II metabolites of three new psychoactive substances: α-pyrrolidinovalerophenone (α-PVP), methylenedioxypyrovalerone (MDPV), and methedrone, using human liver microsomes and human liver cytosol. Accurate-mass spectra of metabolites were obtained using liquid chromatography-quadrupole time-of-flight mass spectrometry. Six Phase I metabolites of α-PVP were identified, which were formed involving reduction, hydroxylation, and pyrrolidine ring opening reactions. The lactam compound was the major metabolite observed for α-PVP. Two glucuronidated metabolites of α-PVP, not reported in previous in vitro studies, were further identified. MDPV was transformed into 10 Phase I metabolites involving reduction, hydroxylation, and loss of the pyrrolidine ring. Also, six glucuronidated and two sulphated metabolites were detected. The major metabolite of MDPV was the catechol metabolite. Methedrone was transformed into five Phase I metabolites, involving N- and O-demethylation, hydroxylation, and reduction of the ketone group. Three metabolites of methedrone are reported for the first time. In addition, the contribution of individual human CYP enzymes in the formation of the detected metabolites was investigated.

  9. Metabolism of UV-filter benzophenone-3 by rat and human liver microsomes and its effect on endocrine-disrupting activity

    Energy Technology Data Exchange (ETDEWEB)

    Watanabe, Yoko, E-mail: y-watanabe@nichiyaku.ac.jp [Graduate School of Biomedical and Health Sciences, Hiroshima University, Kasumi 1-2-3, Minami-ku, Hiroshima 734-8553 (Japan); Nihon Pharmaceutical University, Komuro 10281, Ina-machi, Saitama 362-0806 (Japan); Kojima, Hiroyuki; Takeuchi, Shinji [Hokkaido Institute of Public Health, Kita-19, Nishi-12, Kita-ku, Sapporo 060-0819 (Japan); Uramaru, Naoto [Nihon Pharmaceutical University, Komuro 10281, Ina-machi, Saitama 362-0806 (Japan); Sanoh, Seigo [Graduate School of Biomedical and Health Sciences, Hiroshima University, Kasumi 1-2-3, Minami-ku, Hiroshima 734-8553 (Japan); Sugihara, Kazumi [Faculty of Pharmaceutical Science, Hiroshima International University, Koshingai 5-1-1, Kure, Hiroshima 737-0112 (Japan); Kitamura, Shigeyuki [Nihon Pharmaceutical University, Komuro 10281, Ina-machi, Saitama 362-0806 (Japan); Ohta, Shigeru [Graduate School of Biomedical and Health Sciences, Hiroshima University, Kasumi 1-2-3, Minami-ku, Hiroshima 734-8553 (Japan)

    2015-01-15

    Benzophenone-3 (2-hydroxy-4-methoxybenzophenone; BP-3) is widely used as sunscreen for protection of human skin and hair from damage by ultraviolet (UV) radiation. In this study, we examined the metabolism of BP-3 by rat and human liver microsomes, and the estrogenic and anti-androgenic activities of the metabolites. When BP-3 was incubated with rat liver microsomes in the presence of NADPH, 2,4,5-trihydroxybenzophenone (2,4,5-triOH BP) and 3-hydroxylated BP-3 (3-OH BP-3) were newly identified as metabolites, together with previously detected metabolites 5-hydroxylated BP-3 (5-OH BP-3), a 4-desmethylated metabolite (2,4-diOH BP) and 2,3,4-trihydroxybenzophenone (2,3,4-triOH BP). In studies with recombinant rat cytochrome P450, 3-OH BP-3 and 2,4,5-triOH BP were mainly formed by CYP1A1. BP-3 was also metabolized by human liver microsomes and CYP isoforms. In estrogen reporter (ER) assays using estrogen-responsive CHO cells, 2,4-diOH BP exhibited stronger estrogenic activity, 2,3,4-triOH BP exhibited similar activity, and 5-OH BP-3, 2,4,5-triOH BP and 3-OH BP-3 showed lower activity as compared to BP-3. Structural requirements for activity were investigated in a series of 14 BP-3 derivatives. When BP-3 was incubated with liver microsomes from untreated rats or phenobarbital-, 3-methylcholanthrene-, or acetone-treated rats in the presence of NADPH, estrogenic activity was increased. However, liver microsomes from dexamethasone-treated rats showed decreased estrogenic activity due to formation of inactive 5-OH BP-3 and reduced formation of active 2,4-diOH BP. Anti-androgenic activity of BP-3 was decreased after incubation with liver microsomes. - Highlights: • Metabolic modification of the endocrine-disrupting activity of BP-3 was examined. • 2,4,5-TriOH BP and 3-OH BP-3 were identified as new BP-3 metabolites. • 2,4-DiOH BP and 2,3,4-triOH BP exhibited high or similar estrogenic activities. • Estrogenic activity of BP-3 was enhanced by incubation with rat liver

  10. Effect of Honokiol on Cytochrome P450 and UDP-Glucuronosyltransferase Enzyme Activities in Human Liver Microsomes

    Directory of Open Access Journals (Sweden)

    Yong Yeon Cho

    2013-09-01

    Full Text Available Honokiol is a bioactive component isolated from the medicinal herbs Magnolia officinalis and Magnolia grandiflora that has antioxidative, anti-inflammatory, antithrombotic, and antitumor activities. The inhibitory potentials of honokiol on eight major human cytochrome P450 (CYP enzymes 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4, and four UDP-glucuronosyltransferases (UGTs 1A1, 1A4, 1A9, and 2B7 in human liver microsomes were investigated using liquid chromatography-tandem mass spectrometry. Honokiol strongly inhibited CYP1A2-mediated phenacetin O-deethylation, CYP2C8-mediated amodiaquine N-deethylation, CYP2C9-mediated diclofenac 4-hydroxylation, CYP2C19-mediated [S]-mephenytoin 4-hydroxylation, and UGT1A9-mediated propofol glucuronidation with Ki values of 1.2, 4.9, 0.54, 0.57, and 0.3 μM, respectively. Honokiol also moderately inhibited CYP2B6-mediated bupropion hydroxylation and CYP2D6-mediated bufuralol 1'-hydroxylation with Ki values of 17.5 and 12.0 μM, respectively. These in vitro results indicate that honokiol has the potential to cause pharmacokinetic drug interactions with other co-administered drugs metabolized by CYP1A2, CYP2C8, CYP2C9, CYP2C19, and UGT1A9.

  11. Different effects of dihydropyridine calcium channel antagonists on CYP3A4 enzyme of human liver microsomes.

    Science.gov (United States)

    Xia, Zongling; Wang, Mingli; Zou, Sulan; Chen, Rong

    2012-09-01

    The present study investigated inhibitory effects of 1,4-dihydropyridines (1,4-DHPs) calcium channel antagonists (1,4-DHP-CCAs) on cytochromeP450 3A4 (CYP3A4) of human liver microsomes and further explored importance of 1,4-DHPs molecular structural descriptors. Partial Least Squares method was applied to probe the quantitative relationships between the 1,4-DHPs molecular structural descriptors and its inhibitory actions, which demonstrated that different 1,4-DHP-CCAs could inhibit CYP3A4 enzyme's activity differently. The K (i) values of nicardipine, lercandipine, cilnidipine, nitrendipine, lacidipine, nifedipine, felodipine were 10.13, 10.17, 11.44, 23.90, 29.34, 29.06 and 32.64 μmol L⁻¹, respectively. It is suggested that the 1,4-DHPs molecular structural descriptors are the most important for its inhibitory effects based on the quantitative structure-activity relationship (QSAR) formula. The LogP was positively correlated to the K (i), whereas molecular weight and molecule volume were negatively correlated. It is concluded that analysis of K (i) of 1,4-DHPs derivatives on the CYP3A4 activity may apply for the QSAR formula at the initial stage of clinical application of new drugs.

  12. Reversible formation of fatty acid esters of budesonide, an antiasthma glucocorticoid, in human lung and liver microsomes.

    Science.gov (United States)

    Tunek, A; Sjödin, K; Hallström, G

    1997-11-01

    Microsomes from human lung and liver catalyze the formation of fatty acid esters of budesonide, a glucocorticoid used for inhalation treatment of asthma. The conjugation was dependent on coenzyme A and ATP. Addition of free fatty acids to the incubations affected the pattern of metabolites, but ester formation was observed also without such addition. Budesonide oleate, palmitate, linoleate, palmitoleate, and arachidonate were identified as metabolites. The fatty acid conjugates of budesonide were shown to be substrates for lipase in vitro, thus budesonide is regainable from the conjugates. The data suggest that an equilibrium between budesonide and these pharmacologically inactive lipoidal conjugates will be established in tissues at repeated exposure to budesonide. Since the fatty acid conjugates most likely will be retained intracellularly for a longer time than unchanged budesonide, the duration of tissue exposure to budesonide will depend partly on the rate of lipase-catalyzed hydrolysis of the conjugates. The findings in this study provide a possible explanation for the efficacy of budesonide in mild asthmatics also when inhaled once daily.

  13. Identification of metabolites of meisoindigo in rat, pig and human liver microsomes by UFLC-MS/MS.

    Science.gov (United States)

    Huang, Meng; Ho, Paul C

    2009-04-15

    3-(1,2-Dihydro-2-oxo-3H-indol-3-ylidene)-1,3-dihydro-1-methyl-2H-indol-2-one, abbreviated as meisoindigo, has been a routine therapeutic agent in the clinical treatment of chronic myelogenous leukemia in China since the 1980s. To gain an understanding of the interspecies differences in the metabolism of meisoindigo, the relevant metabolism studies were carried out for the first time in rat, pig and human liver microsomes of different genders by ultra fast liquid chromatography/tandem mass spectrometry (UFLC-MS/MS). The qualitative metabolite identification was accomplished by multiple reaction monitoring (MRM) in combination with Enhanced Product Ion (EPI). The semi-quantitative metabolic stability and metabolite formation were simultaneously measured by MRM. The in vitro metabolic pathways of meisoindigo in three species were proposed as 3,3' double bond reduction, followed by N-demethylation, and reduction followed by phenyl mono-oxidation. Two novel metabolic pathways involving direct phenyl mono-oxidation without reduction in the three species, and direct N-demethylation without reduction in only pig and human, were also proposed. It may be noted that the two metabolites formed after reduction followed by phenyl mono-oxidation at positions 4, 5, 6 or 7, as well as one metabolite formed from direct phenyl mono-oxidation at either of the two phenyl rings without reduction were found to be uniquely present in human. The in vitro t(1/2) and in vitro CL(int) values of meisoindigo were calculated. Statistical analysis showed there were no significant differences in the metabolic stability profiles of meisoindigo among three species, and gender effect on the metabolic stability of meisoindigo was negligible. Formation profiles of the most significant reductive metabolites were obtained in the three species.

  14. Stimulation of tolbutamide hydroxylation by acetone and acetonitrile in human liver microsomes and in a cytochrome P-450 2C9-reconstituted system.

    Science.gov (United States)

    Palamanda, J; Feng, W W; Lin, C C; Nomeir, A A

    2000-01-01

    Organic solvents are often used to solubilize lipophilic new chemical entities before their addition to in vitro test systems such as microsomal stability or cytochrome P-450 (CYP) inhibition. However, the effect of these organic solvents on the test systems is not usually characterized. This study was initiated to evaluate the effect of acetonitrile and acetone, in addition to other organic solvents, on the tolbutamide hydroxylation activity of CYP2C9 in both human liver microsomes and a CYP2C9-reconstituted system. Both acetonitrile and acetone significantly stimulated the NADPH-dependent tolbutamide hydroxylation by nearly 2- to 3-fold in human liver microsomes and CYP2C9-reconstituted system when incubated at 2 and 4% final solvent concentrations. When cumene hydroperoxide was used instead of NADPH, both acetone and acetonitrile significantly inhibited tolbutamide hydroxylation. This NADPH-dependent stimulatory effect was further evaluated by examining the effect of a series of other organic solvents with different carbon chain lengths and various functional groups, including hydroxyl, ketone, and aldehyde. Unlike acetone, two other ketone-containing solvents, methyl ethyl ketone (2-butanone) and diethyl ketone (3-pentanone) failed to significantly enhance tolbutamide hydroxylation. Other solvents tested, including methanol, ethanol, propanol, 1-butanol, 2-butanol, 1-pentanol, 2-pentanol, acetaldehyde, and dimethyl sulfoxide significantly inhibited NADPH-dependent tolbutamide hydroxylation. Overall, the stimulatory effect of both acetonitrile and acetone on tolbutamide hydroxylation was found to be primarily due to a consistent increase in V(max), whereas K(m) was unchanged in both human liver microsomes and the reconstituted CYP2C9 system. These data suggest that acetone and acetonitrile stimulate NADPH-mediated tolbutamide hydroxylation via the CYP reductase and not by modifying the affinity of tolbutamide for the CYP2C9 enzyme.

  15. Enzymatic activity in turkey, duck, quail and chicken liver microsomes against four human cytochrome P450 prototype substrates and aflatoxin B1

    Directory of Open Access Journals (Sweden)

    Hansen W. Murcia

    2011-10-01

    Full Text Available Cytochrome P450 enzymes (CYP are a group of monooxygenases able to biotransform several kinds of xenobiotics including aflatoxin B1 (AFB1, a highly toxic mycotoxin. These enzymes have been widely studied in humans and others mammals, but there is not enough information in commercial poultry species about their biochemical characteristics or substrate specificity. The aim of the present study was to identify CYPs from avian liver microsomes with the use of prototype substrates specific for human CYP enzymes and AFB1. Biochemical characterization was carried out in vitro and biotransformation products were detected by high-performance liquid chromatography (HPLC. Enzymatic constants were calculated and comparisons between turkey, duck, quail and chicken activities were done. The results demonstrate the presence of four avian ortholog enzyme activities possibly related with a CYP1A1, CYP1A2, CYP2A6 (activity not previously identified and CYP3A4 poultry orthologs, respectively. Large differences in enzyme kinetics specific for prototype substrates were found among the poultry species studied. Turkey liver microsomes had the highest affinity and catalytic rate for AFB1 whereas chicken enzymes had the lowest affinity and catalytic rate for the same substrate. Quail and duck microsomes showed intermediate values. These results correlate well with the known in vivo sensitivity for AFB1 except for the duck. A high correlation coefficient between 7-ethoxyresorufin-Odeethylase (EROD and 7-methoxyresorufin- O-deethylase (MROD activities was found in the four poultry species, suggesting that these two enzymatic activities might be carried out by the same enzyme. The results of the present study indicate that four prototype enzyme activities are present in poultry liver microsomes, possibly related with the presence of three CYP avian orthologs. More studies are needed in order to further characterize these enzymes.

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

    Science.gov (United States)

    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.

  17. Identification of cytochrome P450 2E1 as the predominant enzyme catalyzing human liver microsomal defluorination of sevoflurane, isoflurane, and methoxyflurane.

    Science.gov (United States)

    Kharasch, E D; Thummel, K E

    1993-10-01

    Renal and hepatic toxicity of the fluorinated ether volatile anesthetics is caused by biotransformation to toxic metabolites. Metabolism also contributes significantly to the elimination pharmacokinetics of some volatile agents. Although innumerable studies have explored anesthetic metabolism in animals, there is little information on human volatile anesthetic metabolism with respect to comparative rates or the identity of the enzymes responsible for defluorination. The first purpose of this investigation was to compare the metabolism of the fluorinated ether anesthetics by human liver microsomes. The second purpose was to test the hypothesis that cytochrome P450 2E1 is the specific P450 isoform responsible for volatile anesthetic defluorination in humans. Microsomes were prepared from human livers. Anesthetic metabolism in microsomal incubations was measured by fluoride production. The strategy for evaluating the role of P450 2E1 in anesthetic defluorination involved three approaches: for a series of 12 human livers, correlation of microsomal defluorination rate with microsomal P450 2E1 content (measured by Western blot analysis), correlation of defluorination rate with microsomal P450 2E1 catalytic activity using marker substrates (para-nitrophenol hydroxylation and chlorzoxazone 6-hydroxylation), and chemical inhibition by P450 isoform-selective inhibitors. The rank order of anesthetic metabolism, assessed by fluoride production at saturating substrate concentrations, was methoxyflurane > sevoflurane > enflurane > isoflurane > desflurane > 0. There was a significant linear correlation of sevoflurane and methoxyflurane defluorination with antigenic P450 2E1 content (r = 0.98 and r = 0.72, respectively), but not with either P450 1A2 or P450 3A3/4. Comparison of anesthetic defluorination with either para-nitrophenol or chlorzoxazone hydroxylation showed a significant correlation for sevoflurane (r = 0.93, r = 0.95) and methoxyflurane (r = 0.78, r = 0

  18. Rosiglitazone Metabolism in Human Liver Microsomes Using a Substrate Depletion Method

    OpenAIRE

    Bazargan, Maryam; Foster, David J R; Davey, Andrew K.; Muhlhausler, Beverly S.

    2017-01-01

    Background Elimination of rosiglitazone in humans is via hepatic metabolism. The existing studies suggest that CYP2C8 is the major enzyme responsible, with a minor contribution from CYP2C9; however, other studies suggest the involvement of additional cytochrome P450 enzymes and metabolic pathways. Thus a full picture of rosiglitazone metabolism is unclear. Objective This study aimed to improve the current understanding of potential drug?drug interactions and implications for therapy by evalua...

  19. Characterization of in vitro glucuronidation clearance of a range of drugs in human kidney microsomes: comparison with liver and intestinal glucuronidation and impact of albumin.

    Science.gov (United States)

    Gill, Katherine L; Houston, J Brian; Galetin, Aleksandra

    2012-04-01

    Previous studies have shown the importance of the addition of albumin for characterization of hepatic glucuronidation in vitro; however, no reports exist on the effects of albumin on renal or intestinal microsomal glucuronidation assays. This study characterized glucuronidation clearance (CL(int, UGT)) in human kidney, liver, and intestinal microsomes in the presence and absence of bovine serum albumin (BSA) for seven drugs with differential UDP-glucuronosyltransferase (UGT) 1A9 and UGT2B7 specificity, namely, diclofenac, ezetimibe, gemfibrozil, mycophenolic acid, naloxone, propofol, and telmisartan. The impact of renal CL(int, UGT) on accuracy of in vitro-in vivo extrapolation (IVIVE) of glucuronidation clearance was investigated. Inclusion of 1% BSA for acidic drugs and 2% for bases/neutral drugs in incubations was found to be suitable for characterization of CL(int, UGT) in different tissues. Although BSA increased CL(int, UGT) in all tissues, the extent was tissue- and drug-dependent. Scaled CL(int, UGT) in the presence of BSA ranged from 2.22 to 207, 0.439 to 24.4, and 0.292 to 23.8 ml · min(-1) · g tissue(-1) in liver, kidney, and intestinal microsomes. Renal CL(int, UGT) (per gram of tissue) was up to 2-fold higher in comparison with that for liver for UGT1A9 substrates; in contrast, CL(int, UGT) for UGT2B7 substrates represented approximately one-third of hepatic estimates. Scaled renal CL(int, UGT) (in the presence of BSA) was up to 30-fold higher than intestinal glucuronidation for the drugs investigated. Use of in vitro data obtained in the presence of BSA and inclusion of renal clearance improved the IVIVE of glucuronidation clearance, with 50% of drugs predicted within 2-fold of observed values. Characterization and consideration of kidney CL(int, UGT) is particularly important for UGT1A9 substrates.

  20. Metabolism of novel opioid agonists U-47700 and U-49900 using human liver microsomes with confirmation in authentic urine specimens from drug users.

    Science.gov (United States)

    Krotulski, Alex J; Mohr, Amanda L A; Papsun, Donna M; Logan, Barry K

    2017-06-13

    Recently, the number of adverse events, including death, involving novel opioids has continued to increase, providing additional and sustained challenges for forensic and medical communities. Identification of emerging novel opioids can be challenging, compounded by detection windows and unknown metabolic profiles. In this study, human liver microsomes were used for the generation of in vitro metabolic profiles of U-47700 and U-49900. Generated metabolites were analyzed via a SCIEX TripleTOF® 5600+ quadrupole time-of-flight mass spectrometer and resulting data files were processing using MetabolitePilot™. Characterized metabolites were verified in vivo by analysis of authentic human urine specimens collected after analytically confirmed cases of overdose involving U-47700 or U-49900. In total, four metabolites were identified and present in urine specimens for U-47700, and five metabolites for U-49900. N-Desmethyl-U-47700 was determined to be the primary metabolite of U-47700. Parent U-47700 was identified in all urine specimens. N-Desmethyl-U-47700 and N,N-didesmethyl-U-47700 were structurally confirmed for the first time during this study following acquisition of standard reference material. N-Desethyl-U-49900 was determined to be the primary metabolite of U-49900 following microsomal incubations, while N,N-didesethyl-N-desmethyl-U-49900 was the most abundant in a urine specimen. Similarities in metabolic transformation were identified between U-47700 and U-49900, resulting in a common metabolite and isomeric species. These phenomena should be considered in cases involving U-47700 or U-49900. This study is the first to map the metabolic profiles of U-47700 and U-49900 using human liver microsomes, as well as the first to report any literature involving U-49900 and analysis of case specimens. Copyright © 2017 John Wiley & Sons, Ltd.

  1. Evaluation of the stereoselective biotransformation of permethrin in human liver microsomes: contributions of cytochrome P450 monooxygenases to the formation of estrogenic metabolites.

    Science.gov (United States)

    Lavado, Ramon; Li, Jiwen; Rimoldi, John M; Schlenk, Daniel

    2014-04-21

    Permethrin (PM) is a pyrethroid insecticide that exists as 4 enantiomers. Biotransformation of PM to estrogen receptor agonists (3-phenoxybenzyl alcohol (PBOH) and 3-(4'-hydroxyphenoxy)-benzyl alcohol (3,4 PBOH)) has been shown to be stereoselective in other vertebrate species. This study evaluated the biotransformation of PM enantiomers in human liver microsomes and with recombinant CYP3A4 and CYP2C19. PBOH and 3,4 PBOH were the only metabolites detected from in vitro incubations including each of the 4 enantiomers of PM with 1R-trans PM having the most efficient NADPH-catalyzed biotransformation to both metabolites. Coincubation with the CYP inhibitor ketoconazole and time course experiments with liver microsomes and recombinant CYP2C19 and CYP3A4 indicated CYP-catalyzed stereoselective cleavage of the ester followed by 4-hydoxylation to 3,4' PBOH. These data indicate potential dispositional differences may occur with PM enantiomers and a shift in putative molecular targets. While cleavage of pyrethroid esters lead to detoxification of the acute neurological effects, formation of the benzyl alcohol and hydroxylated metabolite may lead to estrogenic responses, since each of these metabolites are estrogen receptor ligands. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  2. 3-aminobenzanthrone, a human metabolite of the environmental pollutant 3-nitrobenzanthrone, forms DNA adducts after metabolic activation by human and rat liver microsomes: evidence for activation by cytochrome P450 1A1 and P450 1A2.

    Science.gov (United States)

    Arlt, Volker M; Hewer, Alan; Sorg, Bernd L; Schmeiser, Heinz H; Phillips, David H; Stiborova, Marie

    2004-08-01

    3-Nitrobenzanthrone (3-NBA) is a suspected human carcinogen found in diesel exhaust and ambient air pollution. The main metabolite of 3-NBA, 3-aminobenzanthrone (3-ABA), was recently detected in the urine of salt mining workers occupationally exposed to diesel emissions. Determining the capability of humans to metabolize 3-ABA and understanding which human enzymes are involved in its activation are important in the assessment of individual susceptibility. We compared the ability of eight human hepatic microsomal samples to catalyze DNA adduct formation by 3-ABA. Using the (32)P-postlabeling method, we found that all hepatic microsomes were competent to activate 3-ABA. DNA adduct patterns with multiple adducts, qualitatively similar to those formed in vivo in rats treated with 3-ABA, were observed. These patterns were also similar to those formed by the nitroaromatic counterpart 3-NBA and which derive from reductive metabolites of 3-NBA bound to purine bases in DNA. The role of specific cytochrome P450s (P450s) in the human hepatic microsomal samples in 3-ABA activation was investigated by correlating the P450-linked catalytic activities in each microsomal sample with the level of DNA adducts formed by the same microsomes. On the basis of this analysis, most of the hepatic microsomal activation of 3-ABA was attributable to P450 1A1 and 1A2 enzyme activity. Inhibition of DNA adduct formation in human liver microsomes by alpha-naphthoflavone and furafylline, inhibitors of P450 1A1 and 1A2, and P450 1A2 alone, respectively, supported this finding. Using recombinant human P450 1A1 and 1A2 expressed in Chinese hamster V79 cells and microsomes of baculovirus-transfected insect cells (Supersomes), we confirmed the participation of these enzymes in the formation of 3-ABA-derived DNA adducts. Moreover, essentially the same DNA adduct pattern found in microsomes was detected in metabolically competent human lymphoblastoid MCL-5 cells expressing P450 1A1 and 1A2. Using rat

  3. In vitro bioactivation of 3-(N-phenylamino)propane-1,2-diol by human and rat liver microsomes and recombinant P450 enzymes. Implications for toxic oil syndrome

    NARCIS (Netherlands)

    Martinez-Cabot, A.; Morato, A.; Commandeur, J.N.M.; Vermeulen, N.P.E.; Messeguer, A.

    2007-01-01

    Toxic oil syndrome (TOS) was a massive food-borne intoxication that occurred in Spain in 1981. Epidemiological studies imputed 3-(N-phenylamino) propane-1,2-diol (PAP) derivatives as the toxic agents. The in vitro bioactivation of PAP by rat and human liver microsomes was studied. In both cases,

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

    Directory of Open Access Journals (Sweden)

    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.

  5. Strategy for Hepatotoxicity Prediction Induced by Drug Reactive Metabolites Using Human Liver Microsome and Online 2D-Nano-LC-MS Analysis.

    Science.gov (United States)

    Zhuo, Yue; Wu, Jian-Lin; Yan, Xiaojing; Guo, Ming-Quan; Liu, Ning; Zhou, Hua; Liu, Liang; Li, Na

    2017-12-19

    Hepatotoxicity is a leading cause of drug withdrawal from the market; thus, the assessment of potential drug induced liver injury (DILI) in preclinical trials is necessary. More and more research has shown that the covalent modification of drug reactive metabolites (RMs) for cellular proteins is a possible reason for DILI. Unfortunately, so far no appropriate method can be employed to evaluate this kind of DILI due to the low abundance of RM-protein adducts in complex biological samples. In this study, we proposed a mechanism-based strategy to solve this problem using human liver microsomes (HLMs) and online 2D nano-LC-MS analysis. First, RM modification patterns and potential modified AA residues are determined using HLM and model amino acids (AAs) by UHPLC-Q-TOF-MS. Then, a new online 2D-nano-LC-Q-TOF-MS method is established and applied to separate the digested modified microsomal peptides from high abundance peptides followed by identification of RM-modified proteins using Mascot, in which RM modification patterns on specific AA residues are added. Finally, the functions and relationship with hepatotoxicity of the RM-modified proteins are investigated using ingenuity pathway analysis (IPA) to predict the possible DILI. Using this strategy, 21 proteins were found to be modified by RMs of toosendanin, a hepatotoxic drug with complex structure, and some of them have been reported to be associated with hepatotoxicity. This strategy emphasizes the identification of drug RM-modified proteins in complex biological samples, and no pretreatment is required for the drugs. Consequently, it may serve as a valuable method to predict potential DILI, especially for complex compounds.

  6. In Vitro Enhancement of Carvedilol Glucuronidation by Amiodarone-Mediated Altered Protein Binding in Incubation Mixture of Human Liver Microsomes with Bovine Serum Albumin.

    Science.gov (United States)

    Sekimoto, Makoto; Takamori, Toru; Nakamura, Saki; Taguchi, Masato

    2016-01-01

    Carvedilol is mainly metabolized in the liver to O-glucuronide (O-Glu). We previously found that the glucuronidation activity of racemic carvedilol in pooled human liver microsomes (HLM) was increased, R-selectively, in the presence of amiodarone. The aim of this study was to clarify the mechanisms for the enhancing effect of amiodarone on R- and S-carvedilol glucuronidation. We evaluated O-Glu formation of R- and S-carvedilol enantiomers in a reaction mixture of HLM including 0.2% bovine serum albumin (BSA). In the absence of amiodarone, glucuronidation activity of R- and S-carvedilol for 25 min was 0.026, and 0.51 pmol/min/mg protein, and that was increased by 6.15 and 1.60-fold in the presence of 50 µM amiodarone, respectively. On the other hand, in the absence of BSA, or when BSA was replaced with human serum albumin, no enhancing effect of amiodarone on glucuronidation activity was observed, suggesting that BSA played a role in the mechanisms for the enhancement of glucuronidation activity. Unbound fraction of S-carvedilol in the reaction mixture was greater than that of R-carvedilol in the absence of amiodarone. Also, the addition of amiodarone caused a greater increase of unbound fraction of R-carvedilol than that of S-carvedilol. These results suggest that the altered protein binding by amiodarone is a key mechanism for R-selective stimulation of carvedilol glucuronidation.

  7. Identification of Uridine 5'-Diphosphate-Glucuronosyltransferases Responsible for the Glucuronidation of Mirabegron, a Potent and Selective β3-Adrenoceptor Agonist, in Human Liver Microsomes.

    Science.gov (United States)

    Konishi, Kentaro; Tenmizu, Daisuke; Takusagawa, Shin

    2017-11-21

    Mirabegron is cleared by multiple mechanisms, including drug-metabolizing enzymes. One of the most important clearance pathways is direct glucuronidation. In humans, M11 (O-glucuronide), M13 (carbamoyl-glucuronide), and M14 (N-glucuronide) have been identified, of which M11 is one of the major metabolites in human plasma. The objective of this study was to identify the uridine 5'-diphosphate (UDP)-glucuronosyltransferase (UGT) isoform responsible for the direct glucuronidation of mirabegron using human liver microsomes (HLMs) and recombinant human UGTs (rhUGTs). Reaction mixtures contained 1-1000 μM mirabegron, 8 mM MgCl2, alamethicin (25 μg/mL), 50 mM Tris-HCl buffer (pH 7.5), human liver microsome (HLM) or rhUGT (1.0 mg protein/mL), and 2 mM UDP-glucuronic acid in a total volume of 200 μL for 120 min at 37 °C. HLMs from 16 individuals were used for the correlation study, and mefenamic acid and propofol were used for the inhibition study. Regarding M11 formation, rhUGT2B7 showed high activity among the rhUGTs tested (11.3 pmol/min/mg protein). This result was supported by the correlation between M11 formation activity and UGT2B7 marker enzyme activity (3-glucuronidation of morphine, r 2 = 0.330, p = 0.020) in individual HLMs; inhibition by mefenamic acid in pooled HLMs (IC50 = 22.8 μM); and relatively similar K m values between pooled HLMs and rhUGT2B7 (1260 vs. 486 μM). Regarding M13 and M14 formation, rhUGT1A3 and rhUGT1A8 showed high activity among the rhUGTs tested, respectively. UGT2B7 is the main catalyst of M11 formation in HLMs. Regarding M13 and M14 formation, UGT1A3 and UGT1A8 are strong candidates for glucuronidation, respectively.

  8. Biotransformation of the Flame Retardant 1,2-Dibromo-4-(1,2-dibromoethyl)cyclohexane (TBECH) in Vitro by Human Liver Microsomes.

    Science.gov (United States)

    Nguyen, Khanh-Hoang; Abou-Elwafa Abdallah, Mohamed; Moehring, Thomas; Harrad, Stuart

    2017-09-19

    The technical mixture of 1,2-dibromo-4-(1,2-dibromoethyl)cyclohexane (TBECH or DBE-DBCH) and the pure β-TBECH isomer were subjected to in vitro biotransformation by human liver microsomes (HLM). After 60 min of incubation, 5 potential metabolites of TBECH were identified in microsomal assays of both the TBECH mixture and β-TBECH using ultraperformance liquid chromatography-Q-Exactive Orbitrap mass spectrometry. These include mono- and dihydroxylated TBECH and mono- and dihydroxylated TriBECH as well as an α-oxidation metabolite bromo-(1,2-dibromocyclohexyl)-acetic acid. Our results indicate potential hepatic biotransformation of TBECH via cyctochrome P450-catalyzed hydroxylation, debromination, and α-oxidation. Kinetic studies revealed that the formation of monohydroxy-TBECH, dihydroxy-TBECH, and monohydroxy-TriBECH were best fitted to a Michaelis-Menten enzyme kinetic model. Respective estimated Vmax values (maximum metabolic rate) for these metabolites were 11.8 ± 4, 0.6 ± 0.1, and 10.1 ± 0.8 pmol min(-1) mg protein(-1) in TBECH mixture and 4992 ± 1340, 14.1 ± 4.9, and 66.1 ± 7.3 pmol min(-1) mg protein(-1) in β-TBECH. This indicates monohydroxy-TBECH as the major metabolite of TBECH by in vitro HLM-based assay. The estimated in vitro intrinsic clearance (Clint) of TBECH mixture was slower (P < 0.05) than that of pure β-TBECH. While the formation of monohydroxy-TBECH may reduce the bioaccumulation potential and provide a useful biomarker for monitoring TBECH exposure, further studies are required to fully understand the levels and toxicological implications of the identified metabolites.

  9. Microsome composition-based model as a mechanistic tool to predict nonspecific binding of drugs in liver microsomes.

    Science.gov (United States)

    Poulin, Patrick; Haddad, Sami

    2011-10-01

    The purpose of this study was to investigate the ability of the microsome composition-based model to predict the unbound fraction determined in vitro in microsomal incubation system (fuinc ). Another objective was to make a comparative assessment between the proposed mechanistic method and three empirical methods published in the literature, namely the models of Austin et al. (2002, Drug Metab Dispos 30:1497-1503), Turner et al. [2007, Drug Metab Rev 38(S1):162], and Halifax and Houston (2006, Drug Metab Rev 34:724-726), which are based solely on physicochemical properties. The assessment was confined by the availability of measured fuinc data in rat and human at diverse microsomal protein concentrations for 132 compounds. The proposed microsome composition-based model can be viewed as a combination of two distinct processes, namely the nonspecific binding to neutral lipids and the ionic binding to acidic phospholipids. Across methods, the maximum success rate in predicting fuinc of all compounds was 98%, 91%, and 84% with predictions falling within threefold, twofold, and 1.5-fold error of the observed fuinc , respectively. The statistical analyses suggest that the prediction models are more effective at computing fuinc (i) for rat as compared with human, and (ii) for acids and neutral drugs as compared with strong basic drugs. In addition, on the basis of the comparisons made using all datasets, the method that made use of microsome composition data compares well with those methods that relied solely on physicochemistry. The sensitivity analysis demonstrated the importance of the compound properties and physiological parameters reflective of specific mechanistic determinants relevant to prediction of fuinc values of drugs. Overall, the results obtained with our proposed model demonstrate a significant step toward the development of a generic and mechanistic model of fuinc for liver microsomes, which should provide rationale extrapolation procedures of hepatic

  10. In vitro assessment of CYP1A2 and 2C9 inhibition potential of Withania somnifera and Centella asiatica in human liver microsomes.

    Science.gov (United States)

    Savai, Jay; Varghese, Alice; Pandita, Nancy; Chintamaneni, Meena

    2015-06-01

    Several herbal drugs and allopathic medicines when co-administered can lead to severe herb-drug interactions. Hence, this study was undertaken in order to assess the in vitro inhibition potential of Withania somnifera and Centella asiatica with cytochrome P450 (CYP) 1A2 and 2C9 enzyme using human liver microsomes. Inhibitory potential of crude extracts of both the medicinal plants along with their principal phytoconstituents were investigated using selective probe substrate technique. IC50, Ki values and mode of inhibition were determined. The results of the study revealed that W. somnifera showed no significant interaction with both the isoforms of CYP. However, ethanolic extract of C. asiatica significantly inhibited both CYP1A2 (IC50 value - 42.23±3.65 μg/mL/Ki value - 14.93±4.59 μg/mL) and 2C9 enzyme (IC50 value - 48.41±4.64 μg/mL/Ki value - 23.89±3.14 μg/mL) in a competitive manner. The flavonoids, quercetin and kaempferol showed potent (IC50 values less than 10 μM) inhibition of CYP1A2 activity with no significant inhibition of CYP2C9 enzyme. Thus, these findings of the study might be helpful for safe and effective use of C. asiatica in clinical practice. However, its in vivo interaction study in humans is still warranted.

  11. Effects of tanshinones from Salvia miltiorrhiza on CYP2C19 activity in human liver microsomes: enzyme kinetic and molecular docking studies.

    Science.gov (United States)

    Hu, Tao; Zhou, Xuelin; Wang, Lin; Or, Penelope M Y; Yeung, John H K; Kwan, Yiu Wa; Cho, Chi Hin

    2015-03-25

    This study aimed to investigate the effects of five tanshinones, the lipophilic components from Danshen (Salvia miltiorrhiza), on CYP2C19 activity in pooled human liver microsomes (HLMs). The effects of tanshinones on CYP2C19 activity were compared by enzyme inhibition study using omeprazole 5-hydroxylation in pooled HLMs. The inhibition constant (Ki) values and inhibition modes of effective tanshinones were evaluated by enzyme kinetic study. Molecular docking analysis was used to simulate the binding conformations of tanshinones to the active cavity of human CYP2C19. Dihydrotanshinone and miltirone showed potent inhibitory effects on CYP2C19 activity in a concentration-dependent manner. Tanshinone I showed weaker inhibitory effect, whereas tanshinone IIA and cryptotanshinone had no inhibitory effect. Further enzyme kinetic study showed that the inhibition by dihydrotanshinone and miltirone was a mixed type. The effects of tanshinones were also confirmed by a molecular docking study. Besides, the ethanol extract of Danshen also showed a mixed type of inhibition, whereas the water extract had no inhibitory effect. The current findings demonstrate the inhibition of CYP2C19 activity by the ethanol extract of Danshen and its components tanshinones, implicating the potential herb-drug interactions between Danshen and therapeutic agents metabolized by CYP2C19 in clinical practice. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  12. Role of specific cytochrome P450 isoforms in the conversion of phenoxypropoxybiguanide analogs in human liver microsomes to potent antimalarial dihydrotriazines.

    Science.gov (United States)

    Diaz, Damaris S; Kozar, Michael P; Smith, Kirsten S; Asher, Constance O; Sousa, Jason C; Schiehser, Guy A; Jacobus, David P; Milhous, Wilbur K; Skillman, Donald R; Shearer, Todd W

    2008-02-01

    Phenoxypropoxybiguanides, such as PS-15, are antimalarial prodrugs analogous to the relationship of proguanil and its active metabolite cycloguanil. Unlike cycloguanil, however, WR99210, the active metabolite of PS-15, has retained in vitro potency against newly emerging antifolate-resistant malaria parasites. Recently, in vitro metabolism of a new series of phenoxypropoxybiguanide analogs has examined the production of the active triazine metabolites by human liver microsomes. The purpose of this investigation was to elucidate the primary cytochrome P450 isoforms involved in the production of active metabolites in the current lead candidate. By using expressed human recombinant isoform preparations, specific chemical inhibitors, and isoform-specific inhibitory antibodies, the primary cytochrome P450 isoforms involved in the in vitro metabolic activation of JPC-2056 were elucidated. Unlike proguanil, which is metabolized primarily by CYP2C19, the results indicate that CYP3A4 plays a more important role in the metabolism of both PS-15 and JPC-2056. Whereas CYP2D6 appears to play a major role in the metabolism of PS-15 to WR99210, it appears less important in the conversion of JPC-2056 to JPC-2067. These results are encouraging, considering the prominence of CYP2C19 and CYP2D6 polymorphisms in certain populations at risk for contracting malaria, because the current clinical prodrug candidate from this series may be less dependent on these enzymes for metabolic activation.

  13. Characterization and identification of eight designer benzodiazepine metabolites by incubation with human liver microsomes and analysis by a triple quadrupole mass spectrometer.

    Science.gov (United States)

    El Balkhi, Souleiman; Chaslot, Maxime; Picard, Nicolas; Dulaurent, Sylvain; Delage, Martine; Mathieu, Olivier; Saint-Marcoux, Franck

    2017-07-01

    Designer benzodiazepines (DBZDs) have become of particular importance in the past few years. The metabolite monitoring of DBZD in biological fluids could be of great interest in clinical and forensic toxicology. However, DBZD metabolites are not known or not commercially available. The identification of some DBZD metabolites has been mostly explored by self-administration studies or by in vitro studies followed by high-resolution mass spectrometry. The question arose whether a unit resolution instrument could be efficient enough to allow the identification of DBZD metabolites. In this study, we used an in vitro experiment where eight DBZDs (diclazepam, flubromazepam, etizolam, deschloroetizolam, flubromazolam, nifoxipam, meclonazepam and clonazolam) were incubated with human liver microsomes (HLMs) and metabolite identification was carried out by using a UHPLC coupled to a QTRAP triple quadrupole linear iontrap tandem mass spectrometer system. Post-mortem samples obtained from a real poisoning case, involving deschloroetizolam and diclazepam, were also analysed and discussed. Our study using HLM allowed the identification of 26 metabolites of the 8 DBZDs. These were denitro-, mono- or di-hydroxylated and desmethyl metabolites. In the forensic case, diclazepam was not detected whereas its metabolites (lormetazepam and lorazepam) were present at high concentrations in urine. We also identified hydroxy-deschloroetizolam in urine, while the parent compound was not detected in this matrix. This supports the approach that LC coupled to a simple QTRAP could be used by laboratories to identify other not-known/not-commercialized new psychoactive substance (NPS) metabolites.

  14. Effect of thiols on lipid peroxidation in rat liver microsomes

    NARCIS (Netherlands)

    Haenen, G R; Vermeulen, N P; Timmerman, H; Bast, A

    1989-01-01

    The stimulatory or inhibitory effects of various thiol compounds on in vitro lipid peroxidation by iron-ascorbate in rat liver microsomes were determined. Glutathione had no measurable pro-oxidant capacity, in contrast, it protected against lipid peroxidation. N-Acetyl l-cysteine and

  15. Fenproporex N-dealkylation to amphetamine--enantioselective in vitro studies in human liver microsomes as well as enantioselective in vivo studies in Wistar and Dark Agouti rats.

    Science.gov (United States)

    Kraemer, Thomas; Pflugmann, Thomas; Bossmann, Michael; Kneller, Nicole M; Peters, Frank T; Paul, Liane D; Springer, Dietmar; Staack, Roland F; Maurer, Hans H

    2004-09-01

    Fenproporex (FP) is known to be N-dealkylated to R(-)-amphetamine (AM) and S(+)-amphetamine. Involvement of the polymorphic cytochrome P450 (CYP) isoform CYP2D6 in metabolism of such amphetamine precursors is discussed controversially in literature. In this study, the human hepatic CYPs involved in FP dealkylation were identified using recombinant CYPs and human liver microsomes (HLM). These studies revealed that not only CYP2D6 but also CYP1A2, CYP2B6 and CYP3A4 catalyzed this metabolic reaction for both enantiomers with slight preference for the S(+)-enantiomer. Formation of amphetamine was not significantly changed by quinidine and was not different in poor metabolizer HLM compared to pooled HLM. As in vivo experiments, blood levels of R(-)-amphetamine and S(+)-amphetamine formed after administration of FP were determined in female Dark Agouti rats (fDA), a model of the human CYP2D6 poor metabolizer phenotype (PM), male Dark Agouti rats (mDA), an intermediate model, and in male Wistar rats (WI), a model of the human CYP2D6 extensive metabolizer phenotype. Analysis of the plasma samples showed that fDA exhibited significantly higher plasma levels of both amphetamine enantiomers compared to those of WI. Corresponding plasma levels in mDA were between those in fDA and WI. Furthermore, pretreatment of WI with the CYP2D inhibitor quinine resulted in significantly higher amphetamine plasma levels, which did not significantly differ from those in fDA. The in vivo studies suggested that CYP2D6 is not crucial to the N-dealkylation but to another metabolic step, most probably to the ring hydroxylation. Further studies are necessary for elucidating the role of CYP2D6 in FP hydroxylation.

  16. Liver microsomal cytochromes P-450 and azoreductase activity.

    Science.gov (United States)

    Fujita, S; Peisach, J

    1978-07-10

    Hepatic microsomal azoreductase activity with amaranth (3-hydroxy-4[(4-sulfo-1-naphthalenyl)azo]-2,7-naphthalenedisulfonic acid trisodium salt) as a substrate is proportional to the levels of microsomal cytochrome P-450 from control or phenobarbital-pretreated rats and mice or cytochrome P-448 from 3-methylchol-anthrene-pretreated animals. In the "inducible" C57B/6J strain of mice, 3-methylcholanthrene and phenobarbital pretreatment cause an increase in cytochrome P-448 and P-450 levels, respectively, which is directly proportional to the increase of azoreductase activity. However, in the "noninducible" DBA/2J strain of mice, only phenobarbital treatment causes the increase both in cytochrome P-450 levels and azoreductase activity, while 3-methylcholanthrene has no effect. These experiments suggest that the P-450 type cytochromes are responsible for azoreductase activity in liver microsomes.

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

    Directory of Open Access Journals (Sweden)

    Soon-Sang Kwon

    2016-04-01

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

  18. In vitro oxidative metabolism of cajaninstilbene Acid by human liver microsomes and hepatocytes: involvement of cytochrome p450 reaction phenotyping, inhibition, and induction studies.

    Science.gov (United States)

    Hua, Xin; Peng, Xiao; Tan, Shengnan; Li, Chunying; Wang, Wei; Luo, Meng; Fu, Yujie; Zu, Yuangang; Smyth, Hugh

    2014-10-29

    Cajaninstilbene acid (CSA, 3-hydroxy-4-prenyl-5-methoxystilbene-2-carboxylic acid), an active constituent of pigeonpea leaves, an important tropical crop, is known for its clinical effects in the treatment of diabetes, hepatitis, and measles and its potential antitumor effect. In this study, the effect of the cytochrome P450 isozymes on the activity of CSA was investigated. Two hydroxylation metabolites were identified in the study. The reaction phenotype study showed that CYP3A4, CYP2C9, and CYP1A2 were the major cytochrome P450 isozymes in the metabolism of CSA. The metabolic food-drug interaction potential was also evaluated in vitro. The effect of CSA inhibition/induction of enzymatic activities of seven drug-metabolizing CYP450 isozymes in vitro was estimated by high-performance liquid chromatography and liquid chromatography-tandem mass spectrometry analytical techniques. CSA showed different inhibitory effects on different isozymes. CSA reversibly inhibited CYP3A4 and CYP2C9 activities in human liver microsomes with IC50 values of 28.3 and 31.3 μM, respectively, but exhibited no inhibition activities to CYP1A2, CYP2A6, CYP2C19, CYP2D6, and CYP2E1. CSA showed a weak effect on CYP450 enzymes in a time-dependent manner. CSA did not substantially induce CYP1A2, CYP2A6, CYP2B6, CYP2E1, CYP2C9, CYP2C19, CYP2D6, or CYP3A4 at concentrations up to 30 μM in primary human hepatocytes. The results of our experiments may be helpful to predict clinically significant food-drug interactions when other drugs are administered in combination with CSA.

  19. Investigation of CYP3A4 and CYP2D6 Interactions of Withania somnifera and Centella asiatica in Human Liver Microsomes.

    Science.gov (United States)

    Savai, Jay; Varghese, Alice; Pandita, Nancy; Chintamaneni, Meena

    2015-05-01

    Withania somnifera is commonly used as a rejuvenator, whereas Centella asiatica is well known for its anxiolytic and nootropic effects. The present study aims at investigating the effect of crude extracts and principal phytoconstituents of both the medicinal plants with CYP3A4 and CYP2D6 enzyme activity in human liver microsomes (HLM). Phytoconstituents were quantified in the crude extracts of both the medicinal plants using reverse phase HPLC. Crude extracts and phytoconstituents of W. somnifera showed no significant interaction with both CYP3A4 and CYP2D6 enzymes in HLM. Of the crude extracts of C. asiatica screened in vitro, methanolic extract showed potent noncompetitive inhibition of only CYP3A4 enzyme (Ki-64.36 ± 1.82 µg/mL), whereas ethanol solution extract showed potent noncompetitive inhibition of only CYP2D6 enzyme (Ki-36.3 ± 0.44 µg/mL). The flavonoids, quercetin, and kaempferol showed potent (IC50 values less than 100 μM) inhibition of CYP3A4 activity, whereas quercetin alone showed potent inhibition of CYP2D6 activity in HLM. Because methanolic extract of C. asiatica showed a relatively high percentage content of quercetin and kaempferol than ethanol solution extract, the inhibitory effect of methanolic extract on CYP3A4 enzyme activity could be attributed to the flavonoids. Thus, co-administration of the alcoholic extracts of C. asiatica with drugs that are substrates of CYP3A4 and CYP2D6 enzymes may lead to undesirable herb-drug interactions in humans. Copyright © 2015 John Wiley & Sons, Ltd.

  20. Inhibitory Effect of Selaginellins from Selaginella tamariscina (Beauv. Spring against Cytochrome P450 and Uridine 5′-Diphosphoglucuronosyltransferase Isoforms on Human Liver Microsomes

    Directory of Open Access Journals (Sweden)

    Jae-Kyung Heo

    2017-09-01

    Full Text Available Selaginella tamariscina (Beauv. has been used for traditional herbal medicine for treatment of cancer, hepatitis, and diabetes in the Orient. Numerous bioactive compounds including alkaloids, flavonoids, lignans, and selaginellins have been identified in this medicinal plant. Among them, selaginellins having a quinone methide unit and an alkylphenol moiety have been known to possess anticancer, antidiabetic, and neuroprotective activity. Although there have been studies on the biological activities of selaginellins, their modulatory potential of cytochrome P450 (P450 and uridine 5′-diphosphoglucuronosyltransferase (UGT activities have not been previously evaluated. In this study, we investigated the drug interaction potential of two selaginellins on ten P450 isoforms (CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 2J2 and 3A and six UGT isoforms (UGT1A1, 1A3, 1A4, 1A6, 1A9 and 2B7 using human liver microsomes and liquid chromatography-tandem mass spectrometry. Selaginellin and selaginellin M had high inhibitory potential for CYP2C8-mediated amodiaquine O-demethylation with IC50 values of 0.5 and 0.9 μM, respectively. Selaginellin and selaginellin M also showed medium inhibitory potential against CYP2C9, CYP2J2, UGT1A1, and UGT1A3 (1 μM < IC50 < 5 μM. These two selaginellins had low inhibitory potential against CYP1A2, CYP2A6, CYP2E1, and UGT1A6 (IC50 > 25 μM. This information might be helpful to predict possible drug interaction potential of between selaginellins and co-administered drugs.

  1. Metabolic profiling of five flavonoids from Dragon's Blood in human liver microsomes using high-performance liquid chromatography coupled with high resolution mass spectrometry.

    Science.gov (United States)

    Li, Yujuan; Zhang, Yushi; Wang, Rui; Wei, Lizhong; Deng, Yulin; Ren, Wei

    2017-05-01

    Although much is known about the pharmacological activities of Dragon's Blood (DB, a traditional Chinese herb), its metabolism in human liver microsomes (HLMs) and the cytochrome P450 (CYP) enzymes has not been studied. This study aims to identify the metabolic profile of five flavonoids (loureirin A, loureirin B, loureirin C, 7,4'-dihydroxyflavone and 5,7,4'-trihydroxyflavanone) from DB in HLMs as well as the CYP enzymes that are involved in the metabolism of them. High-resolution mass spectrometry was used to characterize the structures of their metabolites and 10 cDNA-expressed CYP enzymes (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4 and CYP3A5) were used to verify which isozymes mediate in the metabolism of the metabolites. Totally, 29 metabolites including 10 metabolites of loureirin A, 10 metabolites of loureirin B, 4 metabolites of loureirin C, 2 metabolites of 7,4'-dihydroxyflavone and 3 metabolites of 5,7,4'-trihydroxyflavanone were elucidated and identified on the basis of the high-resolution MS(n) data. The metabolic profile of the five flavonoids in HLMs involved hydroxylation, oxidation and demethylation. Among them, hydroxylation was the predominant biotransformation of the five flavonoids in HLMs, occurring in combination with other metabolic reactions. Assay with recombinant P450s revealed that CYP2C9 and CYP2C19 played an important role in the hydroxylation of flavonoids in HLMs. To the best of our knowledge, this is the first in vitro evaluation of the metabolic profile of loureirin A, loureirin B, loureirin C, 7,4'-dihydroxyflavone and 5,7,4'-trihydroxyflavanone in HLMs. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Effect of polyphenolic compounds from Solanum torvum on plasma lipid peroxidation, superoxide anion and cytochrome P450 2E1 in human liver microsomes.

    Science.gov (United States)

    Kusirisin, Winthana; Jaikang, Churdsak; Chaiyasut, Chaiyavat; Narongchai, Paitoon

    2009-11-01

    Previous studies presented evidence that plants contain antioxidants that have free radical-scavenging properties. Overproduction of free radicals leads to oxidative stress, a factor associated with a variety of diseases, such as diabetes. Cytochrome P450 2E1 enzymes (CYP2E1) are involved in drug metabolism in the liver and metabolism of DNA-reaction generating intra-mitochondrial ROS, which leads to micro- and macro-vascular pathology in diabetes. Plant-based chemicals can affect CYP2E1 enzymes and related defense mechanisms, possibly leading to protection against oxidative stress. We investigated the effect of Solanum torvum (ST) extracts on the inhibition of CYP2E1 activity in human liver microsomes. ST extract was analyzed for antioxidant activity by the ABTS method. Polyphenolic compounds were measured by the total phenol content using the Folin-Ciocalteau reagent. Flavonoid and tannin content were analyzed by standard methods. Oxidative stress was evaluated by measuring lipid peroxidation by TBARS and superoxide anion scavenging levels in plasma from diabetic patients. Results showed that 10 mg/ml of ST had CYP2E1 catalytic inhibiting activity (57.16 %). The IC50 value of CYP2E1 catalytic inhibiting activity level was 5.14 mg/ml by concentration in a dependent manner. One gram of concentrated ST extract had an antioxidant activity index of 3.68 mg of trolox and 360.53 mg of ascorbic acid equivalent. Effects on free radical-scavenging, as measured by TBARS and superoxide anion, showed IC50 values of 20.60 and 10.26 microg/ml, respectively. Polyphenolic compounds found included phenol, flavonoid and tannin, measuring 160.30, 104.36 and 65.91 mg/g, respectively. These results imply that ST is a natural source of polyphenolic antioxidants, which have cytochrome P450 2E1 enzyme inhibiting and free radical scavenging properties, as related to lipid peroxidation and superoxide anion activity. ST could potentially be used for reducing oxidative stress in diabetes

  3. In vitro biotransformation of flavonoids by rat liver microsomes

    DEFF Research Database (Denmark)

    Nielsen, S. E.; Breinholt, V.; Justesen, U.

    1998-01-01

    1. Sixteen naturally occurring flavonoids were investigated as substrates for cytochrome P450 in uninduced and Aroclor 1254-induced rat liver microsomes. Naringenin, hesperetin, chrysin, apigenin, tangeretin, kaempferol, galangin and tamarixetin were all metabolized extensively by induced rat liver...... pathway leading to the corresponding 3',4'-dihydroxylated flavonoids either by hydroxylation or demethylation. Structural requirements for microsomal hydroxylation appeared to be a single or no hydroxy group on the B-ring of the flavan nucleus. The presence of two or more hydroxy groups on the B......-ring seemed to prevent further hydroxylation. The results indicate that demethylation only occurs in the B-ring when the methoxy group is positioned at C-4'-, and not at the C-3'-position. 3. The CYP1A isozymes were found to be the main enzymes involved in flavonoid hydroxylation, whereas other cytochrome P...

  4. Sex-dependent genetic markers of CYP3A4 expression and activity in human liver microsomes.

    Science.gov (United States)

    Schirmer, Markus; Rosenberger, Albert; Klein, Kathrin; Kulle, Bettina; Toliat, Mohammad R; Nürnberg, Peter; Zanger, Ulrich M; Wojnowski, Leszek

    2007-05-01

    To find genetic markers of the individual cytochrome P450 (CYP)3A expression. A large collection of liver samples phenotyped for CYP3A expression and activity was genotyped for CYP3A variants. Data were analyzed for associations between CYP3A phenotypes and genotypes, and for evidence of recent selection. We report associations between the hepatic CYP3A4 protein expression level, as well as its enzymatic activity, measured as verapamil N-dealkylation, and genetic polymorphisms from two regions within the CYP3A gene cluster. One region is defined by several variants, mostly located within CYP3A7, the other by a single nucleotide polymorphism in intron 7 of CYP3A4. The effects of these single nucleotide polymorphisms are sex-dependent. For example, female carriers of T alleles of the single nucleotide polymorphism rs4646437C>T in CYP3A4 intron 7 have, respectively, 5.1-fold and 2.7-fold higher expression and activity compared with male T-carriers, but only 2.2-fold and 1.4-fold higher expression and activity compared with males of genotype CC. A regression analysis indicates that the impact of these single nucleotide polymorphisms in men goes beyond the previously reported sex effect. The rs4646437C undergoes positive selection in Caucasians, as evidenced by its relative extended haplotype homozygosity value located within the uppermost percentile of a genome-wide test set of haplotypes in the same 5% frequency bin. Our findings reconcile the apparent contradiction between the evidence for the influence of the individual genetic makeup on CYP3A4 expression and activity suggested by clinical studies, and the failure to identify the responsible gene variants.

  5. The Role of CYP2C8 and CYP2C9 Genotypes in Losartan-Dependent Inhibition of Paclitaxel Metabolism in Human Liver Microsomes.

    Science.gov (United States)

    Mukai, Yuji; Senda, Asuna; Toda, Takaki; Eliasson, Erik; Rane, Anders; Inotsume, Nobuo

    2016-06-01

    The aim of the present study was to further investigate a previously identified metabolic interaction between losartan and paclitaxel, which is one of the marker substrates of CYP2C8, by using human liver microsomes (HLMs) from donors with different CYP2C8 and CYP2C9 genotypes. Although CYP2C8 and CYP2C9 exhibit genetic linkage, previous studies have yet to determine whether losartan or its active metabolite, EXP-3174 which is specifically generated by CYP2C9, is responsible for CYP2C8 inhibition. Concentrations of 6α-hydroxypaclitaxel and EXP-3174 were measured by high-performance liquid chromatography after incubations with paclitaxel, losartan or EXP-3174 in HLMs from seven donors with different CYP2C8 and CYP2C9 genotypes. The half maximal inhibitory concentration (IC50 ) values were not fully dependent on CYP2C8 genotypes. Although the degree of inhibition was small, losartan significantly inhibited the production of 6α-hydroxypaclitaxel at a concentration of 1 μmol/L in only HL20 with the CYP2C8*3/*3 genotype. HLMs with either CYP2C9*2/*2 or CYP2C9*1/*3 exhibited a lower losartan intrinsic clearance (Vmax /Km ) than other HLMs including those with CYP2C9*1/*1 and CYP2C9*1/*2. Significant inhibition of 6α-hydroxypaclitaxel formation by EXP-3174 could only be found at levels that were 50 times higher (100 μmol/L) than the maximum concentration generated in the inhibition study using losartan. These results suggest that the metabolic interaction between losartan and paclitaxel is dependent on losartan itself rather than its metabolite and that the CYP2C8 inhibition by losartan is not affected by the CYP2C9 genotype. Further study is needed to define the effect of CYP2C8 genotypes on losartan-paclitaxel interaction. © 2015 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).

  6. Ethosuximide is primarily metabolized by CYP3A when incubated with isolated rat liver microsomes.

    Science.gov (United States)

    Sarver, J G; Bachmann, K A; Zhu, D; Klis, W A

    1998-01-01

    The cytochrome P450 (CYP) subfamily responsible for ethosuximide metabolism was investigated by HPLC assay of ethosuximide incubations with isolated rat liver microsomes from control rats and from rats treated with inducing agents to enrich hepatic microsomes in selected CYP isoforms. Inducing agents included beta-naphthoflavone (BNF, CYP1A inducer), phenobarbital (PB, CYP2B/2C/3A), isoniazid (INH, CYP2E1), clotrimazole (CTZ, CYP3A), clofibrate (CLO, CYP4A), and an imidazole CTZ-analog known as CDD3543 (CYP3A). Incubations with BNF, INH, CTZ, and control microsomes showed significantly (pCTZ microsomes vs. BNF, INH, and control microsomes at 10, 30, 60, and 120 min incubation. Ethosuximide metabolite levels generated by CTZ microsomes at 120 min were 36.5 times those of control microsomes. Correspondingly, ethosuximide concentrations were significantly (pCTZ microsomes compared with BNF, INH, and control microsomes at 60 and 120 min. Sixty-minute incubations with all microsome groups exhibited significantly (pCTZ (11.8x control) and PB (9.6x control) microsomes vs. all other groups. Antibody inhibition experiments demonstrated ethosuximide metabolite levels for PB microsomes were not affected by CYP2B1 antibodies, whereas CYP3A2 antibodies reduced metabolite levels for both PB and CTZ microsomes by over 80%. These results indicate CYP3A is primarily responsible for ethosuximide metabolism in rats.

  7. Studies on the transverse localization of lysophospholipase II in bovine liver microsomes by immunological techniques

    NARCIS (Netherlands)

    Moonen, H.; Bosch, H. van den

    1979-01-01

    1. 1. Lysophospholipase activity solubilized from bovine liver microsomes could be precipitated for more than 80% by antibodies evoked in rabbits against the purified bovine liver lysophospholipase II. 2. 2. After solubilization of the microsomes in 1.5% sodium deoxycholate, an immunoprecipitate

  8. Quantitative Characterization of Major Hepatic UDP-Glucuronosyltransferase Enzymes in Human Liver Microsomes: Comparison of Two Proteomic Methods and Correlation with Catalytic Activity.

    Science.gov (United States)

    Achour, Brahim; Dantonio, Alyssa; Niosi, Mark; Novak, Jonathan J; Fallon, John K; Barber, Jill; Smith, Philip C; Rostami-Hodjegan, Amin; Goosen, Theunis C

    2017-10-01

    Quantitative characterization of UDP-glucuronosyltransferase (UGT) enzymes is valuable in glucuronidation reaction phenotyping, predicting metabolic clearance and drug-drug interactions using extrapolation exercises based on pharmacokinetic modeling. Different quantitative proteomic workflows have been employed to quantify UGT enzymes in various systems, with reports indicating large variability in expression, which cannot be explained by interindividual variability alone. To evaluate the effect of methodological differences on end-point UGT abundance quantification, eight UGT enzymes were quantified in 24 matched liver microsomal samples by two laboratories using stable isotope-labeled (SIL) peptides or quantitative concatemer (QconCAT) standard, and measurements were assessed against catalytic activity in seven enzymes ( n = 59). There was little agreement between individual abundance levels reported by the two methods; only UGT1A1 showed strong correlation [Spearman rank order correlation (Rs) = 0.73, P quantitative proteomic data should be validated against catalytic activity whenever possible. In addition, metabolic reaction phenotyping exercises should consider spurious abundance-activity correlations to avoid misleading conclusions. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

  9. On the metabolism of the amphetamine-derived antispasmodic drug mebeverine: gas chromatography-mass spectrometry studies on rat liver microsomes and on human urine.

    Science.gov (United States)

    Kraemer, T; Bickeboeller-Friedrich, J; Maurer, H H

    2000-03-01

    We describe gas chromatography-mass spectrometry studies of the metabolism of the antispasmodic drug mebeverine [Duspatal, (MB)]. MB is the veratric acid (VA) ester of 4-¿ethyl-[2-(4-methoxyphenyl)-1-methylethyl]amino¿butan-1-ol (MB-OH), which is an N-substituted ethylamphetamine derivative. The metabolites were first identified in rat liver microsome incubates and then detected in urine samples of volunteers through the use of electron impact and positive chemical ionization gas chromatography-mass spectrometry. Urinary conjugates were enzymatically cleaved before analysis. The following phase I metabolites of MB could be identified: VA, O-demethyl VA (vanillic and/or isovanillic acid), O-bisdemethyl VA (protocatechuic acid), MB-OH, hydroxy MB-OH, O-demethyl MB-OH, O-demethyl-hydroxy MB-OH, N-desethyl MB-OH, N-desethyl-O-demethyl MB-OH, N-de(hydroxybutyl) MB-OH (methoxy-ethylamphetamine), N-de(hydroxybutyl)-O-demethyl MB-OH (hydroxy-ethylamphetamine), and N-bisdealkyl MB-OH (p-methoxy-amphetamine, known as the designer drug PMA). The following, partly overlapping metabolic pathways of MB could be postulated: ester hydrolysis, O-demethylation, ring hydroxylation, N-deethylation, and N-de(hydroxybutylation). The latter pathway led to ethylamphetamine derivatives and bisdealkylation led to PMA, which are substances of forensic interest. The metabolites containing alcoholic or phenolic hydroxy groups were partly excreted into urine as conjugates.

  10. The effect of alcoholic cirrhosis on the activities of microsomal aldrin epoxidase, 7-ethoxycoumarin O-de-ethylase and epoxide hydrolase, and on the concentrations of reduced glutathione in human liver.

    Science.gov (United States)

    Woodhouse, K W; Williams, F M; Mutch, E; Wright, P; James, O F; Rawlins, M D

    1983-01-01

    Activities of the microsomal mono-oxygenases 7-ethoxycoumarin O-de-ethylase (EOC) and aldrin epoxidase (AE), together with microsomal epoxide hydrolase (EH) activity and concentrations of reduced glutathione (GSH) have been measured in liver from patients with alcoholic cirrhosis and in normals. Activities of both mono-oxygenases were significantly reduced in alcoholic cirrhosis. EOC activity (pmol 7-OH coumarin formed/mg microsomal protein/min) was 108.0 +/- 10.6 (n = 8) in normals and 60.9 +/- 11.6 (n = 8) in alcoholic cirrhosis (P less than 0.01). AE activity (pmol dieldrin formed/mg microsomal protein/min) was 58.9 +/- 9.5 (n = 11) in normal liver biopsies and 29.9 +/- 8.6 (n = 9) in alcoholic cirrhosis (P less than 0.05). Microsomal EH activity (nmol styrene glycol formed/mg microsomal protein/min) was similar in normals (39.2 +/- 4.4, n = 11) and alcoholic cirrhosis (40.5 +/- 9.1, n = 6). GSH concentrations (microgram GSH/g liver tissue) were lower (P less than 0.01) in alcoholic cirrhosis (792 +/- 73, n = 10) compared to normals (1182 +/- 76, n = 6). PMID:6603231

  11. Differences in metabolite-mediated toxicity of tamoxifen in rodents versus humans elucidated with DNA/microsome electro-optical arrays and nanoreactors.

    Science.gov (United States)

    Zhao, Linlin; Krishnan, Sadagopan; Zhang, Yun; Schenkman, John B; Rusling, James F

    2009-02-01

    Tamoxifen, a therapeutic and chemopreventive breast cancer drug, was chosen as a model compound because of acknowledged species specific toxicity differences. Emerging approaches utilizing electro-optical arrays and nanoreactors based on DNA/microsome films were used to compare metabolite-mediated toxicity differences of tamoxifen in rodents versus humans. Hits triggered by liver enzyme metabolism were first provided by arrays utilizing a DNA damage end point. The arrays feature thin-film spots containing an electrochemiluminescent (ECL) ruthenium polymer ([Ru(bpy)(2)PVP(10)](2+); PVP, polyvinylpyridine), DNA, and liver microsomes. When DNA damage resulted from reactions with tamoxifen metabolites, it was detected by an increase in light from the oxidation of the damaged DNA by the ECL metallopolymer. The slope of ECL generation versus enzyme reaction time correlated with the rate of DNA damage. An approximate 2-fold greater ECL turnover rate was observed for spots with rat liver microsomes compared to that with human liver microsomes. These results were supported by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of reaction products using nanoreactors featuring analogous films on silica nanoparticles, allowing the direct measurement of the relative formation rate for alpha-(N(2)-deoxyguanosinyl)tamoxifen. We observed 2-5-fold more rapid formation rates for three major metabolites, i.e., alpha-hydroxytamoxifen, 4-hydroxytamoxifen, and tamoxifen N-oxide, catalyzed by rat liver microsomes compared to human liver microsomes. Comparable formation rates were observed for N-desmethyl tamoxifen with rat and human liver microsomes. A better detoxifying capacity for human liver microsomes than rat liver microsomes was confirmed utilizing glucuronyltransferase in microsomes together with UDP-glucuronic acid. Taken together, lower genotoxicity and higher detoxication rates presented by human liver microsomes correlate with the lower risk of tamoxifen in

  12. Evaluation of metabolism dependent inhibition of CYP2B6 mediated bupropion hydroxylation in human liver microsomes by monoamine oxidase inhibitors and prediction of potential as perpetrators of drug interaction.

    Science.gov (United States)

    Nirogi, Ramakrishna; Palacharla, Raghava Choudary; Mohammed, Abdul Rasheed; Manoharan, Arunkumar; Ponnamaneni, Ranjith Kumar; Bhyrapuneni, Gopinadh

    2015-03-25

    The objective of the study was to evaluate the metabolism dependent inhibition of CYP2B6 catalyzed bupropion hydroxylation in human liver microsomes by monoamine oxidase (MAO) inhibitors and to predict the drug-drug interaction potential of monoamine oxidase inhibitors as perpetrators of drug interaction. Human liver microsomal CYP2B6 activities were investigated using bupropion hydroxylation as probe substrate marker. The results from single point time dependent inhibition and shift assays suggest that clorgyline, pargyline, phenelzine, and selegiline were metabolism based inhibitors of CYP2B6. In IC50 shift assays, clorgyline, pargyline, phenelzine and selegiline are metabolism based inhibitors of CYP2B6 with fold shit of 3.0-, 3.7-, 2.9-, and 11.4-fold respectively. The inactivation of clorgyline was characterized by KI value of 2.5 ± 0.3 and k(inact) value of 0.045 ± 0.001 min(-1). Phenelzine inactivated CYP2B6 with KI and k(inact) values of 44.9 ± 6.9 μM and 0.085 ± 0.003 min(-1) respectively. Inactivation of selegiline was characterized with KI and k(inact) values of 22.0 ± 3.3 and 0.074 ± 0.002 min(-1) respectively. The inactivation caused by these inhibitors was not reversed by dialysis indicating irreversible inhibition. Based on the mechanistic static model, selegiline showed an increase in the area under the curve (AUC) of efavirenz and bupropion by 1.01-fold. Phenelzine predicted to cause an increase in the AUC of efavirenz and bupropion by 9.4- and 2.4-fold respectively considering unbound hepatic inlet concentrations of phenelzine. In conclusion, the results from this study demonstrated that MAO inhibitors can inactivate human liver microsomal CYP2B6. The likelihood of drug interaction when selegiline co-administered with CYP2B6 substrates is remote. Caution is required while co-administering phenelzine with substrates that are exclusively metabolized by CYP2B6 enzyme and substrates that have narrow therapeutic index. Copyright © 2015

  13. In Vitro Glucuronidation of Ochratoxin A by Rat Liver Microsomes

    Directory of Open Access Journals (Sweden)

    Zheng Han

    2013-12-01

    Full Text Available Ochratoxin A (OTA, one of the most toxic mycotoxins, can contaminate a wide range of food and feedstuff. To date, the data on its conjugates via glucuronidation request clarification and consolidation. In the present study, the combined approaches of ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS, UHPLC-Orbitrap-high resolution mass spectrometry (HRMS and liquid chromatography-multiple stage mass spectrometry (LC-MSn were utilized to investigate the metabolic profile of OTA in rat liver microsomes. Three conjugated products of OTA corresponding to amino-, phenol- and acyl-glucuronides were identified, and the related structures were confirmed by hydrolysis with β-glucuronidase. Moreover, OTA methyl ester, OTα and OTα-glucuronide were also found in the reaction solution. Based on these results, an in vitro metabolic pathway of OTA has been proposed for the first time.

  14. Alteration of rat liver microsomal monooxygenase activities by gasoline treatment

    Energy Technology Data Exchange (ETDEWEB)

    Brady, J.F.; Xiao Fang; Gapac, J.M.; Ning, S.M.; Yang, C.S. (Rutgers - the State Univ., Piscataway, NJ (USA). Dept. of Chemical Biology and Pharmacognosy)

    1990-11-01

    Previous work has shown an increase in rat liver enzyme activities after chronic exposure to gasoline vapor. In the present study, male Sprague-Dawley rats were pretreated with unleaded gasoline at 1 and 5 ml/kg, i.p., and selected hepatic microsomal monooxygenase activities were determined at 18, 48, and 72 h. At 18 h, moderate increases were observed in P450 content (1.3-fold), cytochrome c-reductase activity (1.25-fold), and in N-nitrosodimethylamine demethylation rate (1.25- to 1.6-fold). Pentoxyresorufin dealkylase activity (an activity displayed primarily by P450IIB1) was significantly elevated at 18 and 48 h (30- to 60-fold), and ethoxyresorufin dealkylase activity (an activity displayed by P450 IA1) was elevated (2- to 4-fold). Immunoblot analysis revealed no change in P450IIE1 at these time points, but an elevation in P450IIB1 in agreement with the pentoxyresorufin dealkylase activity measurements. (orig.).

  15. Stereoselective degradation of tebuconazole in rat liver microsomes.

    Science.gov (United States)

    Shen, Zhigang; Zhu, Wentao; Liu, Donghui; Xu, Xinyuan; Zhang, Ping; Zhou, Zhiqiang

    2012-01-01

    The aim of this study was to assess the stereoselectivity of two tebuconazole [(RS)-1-p-chlorophenyl-4,4-dimethyl-3-(1H-1,2,4-triazol-1-ylmethyl)pentan-3-ol] enantiomers in in vitro system (rat liver microsomes). The analytes were extracted with acetic ether and concentrations were determined by high performance liquid chromatography (HPLC) with a cellulose tris(3,5-dimethylphenylcarbamate)-based chiral stationary phase. The degradation of rac-tebuconazole (15 μM) followed first-order kinetics, and the degradation of the S-tebuconazole (t(1/2) = 22.31 min) was faster than that of the R-tebuconazole (t(1/2) = 48.76 min), but no significant difference between the enantiomers was found in the respective incubation (7.5 μM for each). Kinetic assays showed that the K(m) was different between the two enantiomers (K(mR) = 14.83 ± 2.19, K(mS) = 12.23 ± 2.72). The interaction results revealed that there was competitive inhibition between S- and R-form, and there was a significant difference between the IC(50) of R- to S-tebuconazole and S- to R-tebuconazole (IC(50R/S)/IC(50S/R) = 4.98). Copyright © 2011 Wiley Periodicals, Inc.

  16. Neonicotinoid insecticides: reduction and cleavage of imidacloprid nitroimine substituent by liver microsomal and cytosolic enzymes.

    Science.gov (United States)

    Schulz-Jander, Daniel A; Leimkuehler, William M; Casida, John E

    2002-09-01

    The major insecticide imidacloprid (IMI) is known to be metabolized by human cytochrome P450 3A4 with NADPH by imidazolidine hydroxylation and dehydrogenation to give 5-hydroxy-imidacloprid and the olefin, respectively, and by nitroimine reduction and cleavage to yield the nitrosoimine, guanidine, and urea derivatives. More extensive metabolism by human or rabbit liver microsomes with NADPH or rabbit liver cytosol without added cofactor reduces the IMI N-nitro group to an N-amino substituent, i.e., the corresponding hydrazone. A major metabolite on incubation of IMI in the human microsome-NADPH system is tentatively assigned by LC/MS as a 1,2,4-triazol-3-one derived from the hydrazone; the same product is obtained on reaction of the hydrazone with ethyl chloroformate. The hydrazone and proposed triazolone are considered here together (referred to as the hydrazone) for quantitation. Only a portion of the microsomal reduction and cleavage of the nitroimine substituent is attributable to a CYP450 enzyme. The cytosolic enzyme conversion to the hydrazone is inhibited by added cofactors (NAD > NADH > NADP > NADPH) and enhanced by an argon instead of an air atmosphere. The responsible cytosolic enzyme(s) does not appear to be DT-diaphorase (which is inhibited by several neonicotinoids), aldose reductase, aldehyde reductase, or xanthine oxidase. However, the cytosolic metabolism of IMI is inhibited by several aldo-keto-reductase inhibitors (i.e., alrestatin, EBPC, Ponalrestat, phenobarbital, and quercetin). Other neonicotinoids with nitroimine, nitrosoimine, and nitromethylene substituents are probably also metabolized by "neonicotinoid nitro reductase(s)" since they serve as competitive substrates for [(3)H]IMI metabolism.

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

    Energy Technology Data Exchange (ETDEWEB)

    Reed, James R., E-mail: rreed@lsuhsc.edu [Department of Pharmacology and Experimental Therapeutics, Louisiana State University Health Science Center, 533 Bolivar St., New Orleans, LA 70112 (United States); The Stanley S. Scott Cancer Center, Louisiana State University Health Science Center, 533 Bolivar St., New Orleans, LA 70112 (United States); Cawley, George F.; Ardoin, Taylor G. [Department of Pharmacology and Experimental Therapeutics, Louisiana State University Health Science Center, 533 Bolivar St., New Orleans, LA 70112 (United States); The Stanley S. Scott Cancer Center, Louisiana State University Health Science Center, 533 Bolivar St., New Orleans, LA 70112 (United States); Dellinger, Barry; Lomnicki, Slawomir M.; Hasan, Farhana; Kiruri, Lucy W. [Department of Chemistry, Louisiana State University, Baton Rouge, LA 70803 (United States); Backes, Wayne L. [Department of Pharmacology and Experimental Therapeutics, Louisiana State University Health Science Center, 533 Bolivar St., New Orleans, LA 70112 (United States); The Stanley S. Scott Cancer Center, Louisiana State University Health Science Center, 533 Bolivar St., New Orleans, LA 70112 (United States)

    2014-06-01

    Combustion processes generate particulate matter that affects human health. When incineration fuels include components that are highly enriched in aromatic hydrocarbons (especially halogenated varieties) and redox-active metals, ultrafine particulate matter containing air-stable, environmentally persistent free radicals (EPFRs) is generated. The exposure to fine EPFRs (less than 2.5 μm in diameter) has been shown to negatively influence pulmonary and cardiovascular functions in living organisms. The goal of this study was to determine if these EPFRs have a direct effect on cytochrome P450 function. This was accomplished by direct addition of the EPFRs to rat liver microsomal preparations and measurement of several P450 activities using form-selective substrates. The EPFRs used in this study were formed by heating vapors from an organic compound (either monochlorophenol (MCP230) or 1,2-dichlorobenzene (DCB230)) and 5% copper oxide supported on silica (approximately 0.2 μm in diameter) to 230 °C under vacuum. Both types of EPFRs (but not silica, physisorbed silica, or silica impregnated with copper oxide) dramatically inhibited the activities of CYP1A, CYP2B, CYP2E1, CYP2D2 and CYP3A when incubated at concentrations less than 0.1 mg/ml with microsomes and NADPH. Interestingly, at the same concentrations, the EPFRs did not inhibit HO-1 activity or the reduction of cytochrome c by NADPH-cytochrome P450 reductase. CYP2D2-selective metabolism by rat liver microsomes was examined in more detail. The inhibition of CYP2D2-selective metabolism by both DCB230- and MCP230-EPFRs appeared to be largely noncompetitive and was attenuated in the presence of catalase suggesting that reactive oxygen species may be involved in the mechanism of inhibition. - Highlights: • Combustion of organic pollutants generates long-lived particulate radicals (EPFRs). • EPFRs inhibit metabolism by all cytochromes P450 tested in rat liver microsomes. • EPFR-mediated inhibition is related to

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

    Directory of Open Access Journals (Sweden)

    Hye Young Ji

    2012-01-01

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

  19. Induction of liver microsomal UDP-glucuronyltransferase in the rat administered with a plant phenol, eugenol.

    Science.gov (United States)

    Yokota, H; Yuasa, A

    1990-02-01

    UDP-glucuronyltransferase activity toward xenobiotics in rat liver microsomes was increased about 2.6-fold by administration of a eugenol (4-allyl-2-methoxyphenol). Km value of the induced enzyme toward UDP-glucuronic acid, however, did not change. Immunoblotting analysis revealed that the amount of UDP-glucuronyltransferase protein was increased in the microsomes of eugenol-treated rat liver. In vitro translation assay showed that the level of translatable mRNA encoding this enzyme increased in the liver. These results indicate that mRNA specific for production of UDP-glucuronyltransferase has accumulated, presumably by de novo synthesis in response to a plant phenol, eugenol.

  20. Enzymatic denitrification of 2-nitropropane in uninduced mouse liver microsomes.

    Science.gov (United States)

    Marker, E K; Kulkarni, A P

    1985-08-01

    Hepatic microsomes from 5 strains of untreated mice were tested for the ability to enzymatically cleave the nitro group from 2-nitropropane (2NP). All strains showed significant NADPH-dependent nitrite release at pH 7.6 and pH 8.8. Statistical differences in nitrite-releasing activity between strains were found between BALB and PL/J and ATH strains at pH 7.6. At pH 8.8, BIO.M differed from CD-1 and BALB. These results are in contrast to a report of little or no denitrification activity in uninduced rats and suggest that the 2NP microsomal metabolism may be of greater importance than previously thought.

  1. Phosphonate O-deethylation of [4-(4-bromo-2-cyano-phenylcarbamoyl) benzyl]-phosphonic acid diethyl ester, a lipoprotein lipase-promoting agent, catalyzed by cytochrome P450 2C8 and 3A4 in human liver microsomes.

    Science.gov (United States)

    Morioka, Yujiro; Otsu, Makiko; Naito, Shinsaku; Imai, Teruko

    2002-03-01

    NO-1886 ([4-(4-bromo-2-cyano-phenylcarbamoyl) benzyl]-phosphonic acid diethyl ester) increases lipoprotein lipase activity, resulting in a reduction in plasma triglycerides and an increase in high-density lipoprotein cholesterol. The metabolism of NO-1886 in human liver was investigated in the present study. Ester cleavage of NO-1886 from diethyl phosphonate to monoethyl phosphonate was the major metabolic pathway catalyzed by cytochrome P450. In addition, the minor metabolic pathway in human liver was the hydrolysis of the amide bond of NO-1886 by a specific cytosolic esterase. Eadie-Hofstee plots of phosphonate O-deethylation of NO-1886 in human liver microsomes showed a biphasic curve, indicating low- and high-K(m) components. Inhibition experiments with chemical inhibitors and antibodies against various cytochrome P450 isoforms suggested the involvement of CYP2C8 and CYP3A in the phosphonate O-deethylation. Recombinant CYP3A4 and CYP2C8 expressed in baculovirus-infected insect cells and human lymphoblastoid cells exhibited a high activity for phosphonate O-deethylation of NO-1886. The recombinant cytochrome P450 enzymes indicated that CYP2C8 and CYP3A4 were responsible for the low- and high-K(m) components in human liver microsomes, respectively. The selectivity of CYP2C8 in catalyzing phosphonate O-deethylation indicates that coadministration of drugs that are metabolized by the same enzyme requires careful consideration.

  2. Beluga whale liver microsomal cytochrome P4501A (CYP1A) enzymes

    Energy Technology Data Exchange (ETDEWEB)

    Bullock, P.L.; Addison, R. [Inst. of Ocean Sciences, Sidney, British Columbia (Canada); Lockhart, L.; Metner, D. [Dept. of Fisheries and Oceans, Winnipeg, Manitoba (Canada)

    1995-12-31

    Beluga whale (Delphinapterus leucas) liver from the Canadian arctic was analyzed for the presence of CYP1A enzymes, as part of current studies on biomarkers for environmental contamination. CYP1A1-associated 7-ethoxyresorufin O-dealkylase activity (EROD) varied 13 fold among sixteen male whale liver microsomal samples and 31 fold among five females. Similarly, the rate of 7-methoxyresorufin O-dealkylation (MROD) varied 7 fold and 3 fold in microsomal samples from males and females, respectively. Furthermore, 7-pentoxyresorufin O-dealkylase activity (PROD) varied 10 fold in both sexes. None of these enzyme activities were sexually differentiated, and EROD and MROD were inhibited by {alpha}-naphthoflavone. There was very good correlation between EROD and MROD (r{sup 2} = .894), EROD and PROD (r{sup 2} = .909), but MROD and PROD were not as well correlated (r{sup 2} = 785). On Western immunoblots, a single band was recognized in Beluga whale liver microsomes by a polygonal antibody raised against an oligopeptide related to trout CYP1A1. This antibody also recognized purified rat CYP1A1 (56 kDa) and stained only one band (56 kDa) in liver microsomes isolated from male rats treated with {beta}-naphthoflavone. The interindividual variation in EROD paralleled differences in the amount of whale liver microsomal protein that cross-reacted with the anti-peptide antibody. The results suggest that Beluga whale liver contains at least one CYP1A enzyme which catalyzes the 0-dealkylation of 7-ethoxy, 7-methoxy and 7-pentoxyresorufin and has a molecular weight less than that of rat CYP1A1, but similar to rat CYP1A2 (52 kDa).

  3. Evidence for two enzymatic pathways for omega-oxidation of docosanoic acid in rat liver microsomes

    NARCIS (Netherlands)

    Sanders, Robert-Jan; Ofman, Rob; Valianpour, Fredoen; Kemp, Stephan; Wanders, Ronald J. A.

    2005-01-01

    We studied the omega-oxidation of docosanoic acid (C22:0) in rat liver microsomes. C22:0 and 22-hydroxy-docosanoic acid (omega-hydroxy-C22:0) were used as substrates, and the reaction products were analyzed by electrospray ionization mass spectrometry. In the presence of NADPH, omega-oxidation of

  4. Rat liver microsomal cytochrome P450-dependent oxidation of 3,5-disubstituted analogues of paracetamol

    NARCIS (Netherlands)

    Bessems, J.G.M.; Koppele, J.M. te; Dijk, P.A. van; Stee, L.L.P. van; Commandeur, J.N.M.; Vermeulen, N.P.E.

    1996-01-01

    1. The cytochrome P450-dependent binding of paracetamol and a series of 3,5-disubstituted paracetamol analogues (R = -F, -Cl, -Br, -I, -C(H)3, -C2H5, -iC3H7) have been determined with β-naphthoflavone (βNF)-induced rat liver microsomes and produced reverse type I spectral changes. K(s,app) varied

  5. Comparison of cytochrome P450 inhibition assays for drug discovery using human liver microsomes with LC-MS, rhCYP450 isozymes with fluorescence, and double cocktail with LC-MS.

    Science.gov (United States)

    Di, Li; Kerns, Edward H; Li, Susan Q; Carter, Guy T

    2007-04-20

    The disparity of IC(50)s from CYP450 inhibition assays used to assess drug-drug interaction potential was investigated, in order to have evidence for selecting a reliable in vitro CYP450 inhibition assay to support drug discovery. Three assays were studied: individual rhCYP isozymes and corresponding coumarin derivative-probe substrates with fluorescent detection, human liver microsomes (HLM) and cocktail drug-probe substrates with LC-MS detection, and double cocktail rhCYP isozymes mix and drug-probe mix with LC-MS detection. Data comparisons showed that the rhCYP-fluorescent assay and the cocktail assay with HLM-LC-MS had weak correlation. Detection method and probe substrates were shown to not be the major cause of the disparity in IC(50)s. However, the enzyme source and composition (HLM versus, rhCYP) caused disparity in IC(50)s. Specifically, the high concentrations of CYP isozymes often used with HLM-based assays produced high probe substrate conversion and test compound metabolism, which should both contribute to artificially higher IC(50)s. Non-specific binding of substrate to higher concentration proteins and lipids in the HLM-based assays should also contribute to higher IC(50)s. The modified double cocktail assay was found to overcome limitations of the other two assays. It uses an rhCYP isozymes mix, drug-probe substrate mix, low protein concentration, and LC-MS detection. The double cocktail assay is sensitive, selective, and high throughout for use in drug discovery to provide an early alert to potential toxicity with regard to drug-drug interaction, prioritize chemical series, and guide structural modification to circumvent CYP450 inhibition.

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

    Science.gov (United States)

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

    2017-08-01

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

  7. Monitoring by HPLC of Chamomile Flavonoids Exposed to Rat Liver Microsomal Metabolism

    Science.gov (United States)

    Petroianu, Georg; Szőke, Éva; Kalász, Huba; Szegi, Péter; Laufer, Rudolf; Benkő, Bernadett; Darvas, Ferenc; Tekes, Kornélia

    2009-01-01

    Three major flavonoid chamomile components (quercetin, apigenin-7-O-glucoside and rutin) were subjected to oxidative metabolism by cytochrome P-450 of rat liver microsomal preparations. Changes over time in their respective concentrations were followed using reversed-phase HPLC with UV detection. No clean-up had to be applied as only the specific flavonoid had to be separated from the background components originating from the rat liver microsome. Neither the concentration of apigenin-7-O-glucoside nor that of the diglycoside rutin decreased during one hour of exposure to rat microsomal treatment. In contrast, the concentration of quercetin, a lipophilic aglycon, decreased. Our analytical HPLC results complement the in silico calculated lipophilicity (logP) of these compounds; the relatively high lipophilicity of quercetin appears to predispose it to oxidative metabolism in order to decrease its fat solubility. In contrast the much less lipophilic compounds apigenin-7-O-glucoside and rutin were resistant in vitro to microsomal treatment. PMID:19707521

  8. Microsomal EROD data of fish liver sample assay from species collected in the Salt and Gila Rivers, Arizona

    Science.gov (United States)

    Nicks, Diane

    2017-01-01

    This dataset includes microsomal ERDO data from an assay done with liver samples from several fish species that are found in Arizona at sites that are being assessed for PBDE contamination. The data was created in September and October 2016.

  9. Characterization of metronidazole by human liver microsomes

    DEFF Research Database (Denmark)

    Loft, Steffen; Otton, S. Victoria; Lennard, Martin L.

    1991-01-01

    Farmakologi, Cytokrom P450, metronidazol, enzymkinetik, lever mikrosomer, fremmedstofmetabolisme......Farmakologi, Cytokrom P450, metronidazol, enzymkinetik, lever mikrosomer, fremmedstofmetabolisme...

  10. Specific metabolic pathway in vitro of pinazepam and diazepam by liver microsomal enzymes of different animal species.

    Science.gov (United States)

    Comi, V; Fossati, A; Gervasi, G B

    1977-04-01

    The metabolic pathway of Pinazepam and Diazepam in vitro was studied with rat, guinea pig and dog liver microsomes using a chromatographic and spectrophotometric technique. Two main pathways were observed, N1-dealkylation and C3-hydroxylation. N1-dealkylation was shown to be the predominant reaction for Pinazepam in all the animal species studied, while C3-hydroxylation was the major metabolic pathway for Diazepam in the rat. No oxazepam was found when Pinazepam and Diazepam were incubated with liver microsomes.

  11. Cumene hydroperoxide-supported denitrification of 2-nitropropane in uninduced mouse liver microsomes.

    Science.gov (United States)

    Marker, E K; Kulkarni, A P

    1986-01-01

    Cumene hydroperoxide supported oxidative denitrification of 2-nitropropane was investigated in uninduced mouse liver microsomes. The cytochrome P-450 peroxygenase catalyzed reaction resulted in the production of nitrite and acetone. Several lines of evidence suggested the involvement of multiple forms of cytochrome P-450. Acetone production was at least two times greater than nitrite release possibly due to sequestration of nitrite in the reaction mixtures.

  12. Effects of quinolones on liver microsome cytochrome P450 in rats

    Directory of Open Access Journals (Sweden)

    Yi ZHANG

    2012-11-01

    Full Text Available Objective  To study and compare the effects of fluoroquinolones (levofloxacin, gatifloxacin, moxifloxacin and pazufloxacin on the enzyme system of liver microsome cytochrome P450 in rat. Methods  Thirty male Wistar rats were equally assigned into five groups: control group, levofloxacin (LV group, gatifloxacin (GT group, moxifloxacin (MX group and pazufloxacin (PZ group. Each drug was consecutively administered by tail vein injection for 7 days in a dosage of 120 mg/(kg•d. Liver microsomes were prepared by differential centrifugation, the concentration of protein in the liver microsome was measured by Lowry method, the content and activity of cy tochrome P450 were detected by spectrophotometric determination, and the results were analyzed by one-way ANOVA. Results  Compared with control group, the weight of liver in MX group and GT group was significantly reduced (P 0.05. Assay of aminopyrine-N-demethylase activity showed that the difference in enzyme activity was statistically significant between the control group and groups LV, GT and MX (P < 0.01. Erythromycin-N-demethylase activity measurement revealed that the enzyme activity was lowered in GT group and slightly elevated in MX group, and the difference was statistically significant compared with that of control group (P < 0.01. Measurement of activity of rat liver microsomal CYP450 enzyme system subfamily showed that the BROD activity increased in LV, MX and PZ groups (P < 0.01, and slightly decreased in GT group as compared with control group (P < 0.05. The PROD activity increased in GT group, but decreased in PZ group (P < 0.01. The EROD activity increased in all the four groups (P < 0.01. Conclusions  The four fluoroquinolones have some effects on the enzyme system of liver microsome cytochrome P450 in rats, but the effects may be different (enhancement or attenuation of the enzymatic activity depending on the enzymes, and the extent of the decrease of effect is in the

  13. Effect of chronic alcohol drinking on rat liver microsomal nitroreductive metabolism of nifurtimox and benznidazole.

    Science.gov (United States)

    de Mecca, M M; Bartel, L C; Castro, J A

    2013-12-01

    Nifurtimox (Nfx) and benznidazole (Bz) have serious toxic side effects. Manufacturers warn about significant adverse effects when simultaneous alcohol consumption is being made, but its mechanism is not known. The levels and toxicity of these drugs are linked to their liver microsomal nitroreduction to reactive metabolites. In this study, we analyzed whether alcohol drinking enhanced those nitroreductive processes. Male and female Sprague-Dawley rats, 5-6 weeks old (125-150 g body weight) were used. They were fed ad libitum for 28 days with Lieber and De Carli control or alcohol regular liquid diets. The rats were separated into two dietary groups: ethanol and control group. Both were pair fed with the respective diet. Their liver microsomes were isolated and the nicotinamide adenine dinucleotide phosphate-dependent nitroreduction of Nfx and Bz were determined. Alcohol drinking significantly induced microsomal nitroreduction of these drugs in male rats (11% for Nfx and 41% for Bz) but not in females. The activity observed in the alcohol-induced male rats was 100% inhibited by diphenyleneiodonium and attributable to P450 reductase. Inductive effects of alcohol drinking on nitroreductive activation of both drugs might be only partially involved in the harmful interactions described.

  14. Inducible bilirubin-degrading system in the microsomal fraction of rat liver.

    Science.gov (United States)

    De Matteis, F; Trenti, T; Gibbs, A H; Greig, J B

    1989-06-01

    The hypothesis that treatment of Gunn rats with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) activates an alternative pathway of bilirubin disposal, involving an induced form of cytochrome P-450 [Proc. Natl. Acad. Sci. USA 75:682-685 (1978)], has been investigated by studying the mechanisms of bilirubin oxidation in chemical model systems and in liver microsomal systems in vitro. Hematin, copper sulfate, and the iron chelate of EDTA were all active in promoting degradation of bilirubin in the presence of hydrogen peroxide. Evidence was obtained for a microsomal bilirubin-degrading system that could be induced in the liver by treating either rats or chick embryos with TCDD, beta-naphthoflavone, or 3,4,3',4'-tetrachlorobiphenyl (3,4-TCB) in vivo. The activity of this system required NADPH and oxygen and was markedly stimulated by addition of 3,4-TCB (a planar polyhalogenated biphenyl) and much less markedly by the nonplanar analogue 2,4,2',4'-tetrachlorobiphenyl. These two biphenyls were also inhibitory towards the 7-ethoxyresorufin O-deethylase activity of the induced microsomes and here again the nonplanar analogue was markedly less active. Dose-response experiments for stimulation of bilirubin breakdown and inhibition of 7-ethoxyresorufin O-deethylase activity after addition of 3,4-TCB in vitro showed both effects to be caused by similar concentrations of the biphenyl. These results suggest that a polyhalogenated chemical may interact with TCDD-induced microsomes, inhibit their monooxygenase activity, and stimulate production of a bilirubin-degrading species.

  15. Soy isoflavone metabolism in cats compared with other species: Urinary metabolite concentrations and glucuronidation by liver microsomes

    OpenAIRE

    Redmon, Joanna M.; Shrestha, Binu; Cerundolo, Rosario; Court, Michael H.

    2015-01-01

    Soybean is a common source of protein in many pet foods. Slow glucuronidation of soy-derived isoflavones in cats has been hypothesized to result in accumulation with adverse health consequences. Here we evaluated species’ differences in soy isoflavone glucuronidation using urine samples from cats and dogs fed a soy-based diet and liver microsomes from cats compared with microsomes from 12 other species.Significant concentrations of conjugated (but not unconjugated) genistein, daidzein, and gl...

  16. Effect of rociverine on P450-dependent monooxygenases and its N-deethylation metabolism in rat liver microsomes.

    Science.gov (United States)

    Menicagli, S; Lippi, A; Criscuoli, M; Gervasi, P G

    1993-03-09

    Rociverine [2-(diethylamino)-1-methylethyl cis-1-hydroxy [bicyclohexyl]-2-carboxylate] citrate (ROC) is an antispasmodic agent therapeutically active in humans at doses of 0.5-1 mg/kg. This study investigated the effect of acute administration of the drug on hepatic microsomal cytochrome P450 (P450)-catalysed drug metabolism. Only high doses (> or = 100 mg/kg) of ROC were able to induce in rats the hepatic microsomal pentoxyresorufin O-depenthylase (PROD) and 16 beta-testosterone hydroxylase activities both associated with P4502B1/2 and the erythromycin N-dimethylase (ErD) and 2 beta-testosterone hydroxylase activities both dependent on P4503A1/2. However, at 100 and 200 mg/kg of ROC, the 16 beta-testosterone hydroxylase and PROD were the most induced activities, suggesting that P4502B1/2 are the isoforms most sensitive to ROC induction. Accordingly, ROC treatment enhanced, in a dose-dependent manner, the amount of P4502B1/2 and 3A1/2 in microsomes as assayed by western blotting. The northern blot analysis of ROC-treated rat liver showed that the P4502B1/2 induction appears to be regulated at the mRNA level as in the induction by phenobarbital (PB). The oxidative metabolism of ROC with hepatic microsomes from control or PB- and ROC-induced rats resulted in a N-deethyl ROC derivative (major metabolite) and an unknown minor ROC derivative. The kinetic parameters for the N-deethylation of ROC were studied with purified P4502B1 and with microsomes from control or rats treated with various inducers (phenobarbital, ethanol, beta-naphthoflavone, dexamethasone and rociverine). It was found that phenobarbital-, dexamethasone- and rociverine-induced microsomes deethylated ROC with a Vmax about five times higher than that (0.9 nmol/min/mg protein) of control microsomes, although with a similar affinity (Km approximately 0.3 mM). In a reconstituted system, the purified P4502B1 metabolized ROC with a high deethylation rate (22 nmol/min/nmol P450). Moreover, the ROC deethylation

  17. Effect of cholesterol feeding on tissue lipid perioxidation, glutathione peroxidase activity and liver microsomal functions in rats and guinea pigs

    NARCIS (Netherlands)

    TSAI, A. C.; THIE, G. M.; Lin, C. R.

    1977-01-01

    The effect of cholesterol feeding on liver and aortic nonenzymatic lipid peroxidation and glutathione peroxidase activities, and on liver microsomal NADPH-dependent lipid peroxidation, codeine hydroxylation and cytochrome P-450 levels was examined in rats and guinea pigs. One percent cholesterol was

  18. Cytochrome P-450-mediated denitrification of 2-nitropropane in mouse liver microsomes.

    Science.gov (United States)

    Marker, E K; Kulkarni, A P

    1986-09-01

    Enzymatic denitrification of 2-nitropropane (2NP) was investigated in an NADPH-dependent hepatic microsomal system from male CD1 mice. The involvement of cytochrome P-450 (P-450) as the catalyst in 2NP denitrification was revealed by the induction of nitrite-releasing activity following phenobarbital (PB) pretreatment, by a decrease in activity with carbon tetrachloride pretreatment, by the inhibition of the reaction with classical P-450 inhibitors, and by the observation of a type I binding spectrum. Under optimal conditions, two pH-dependent peaks of activity were observed at pH 7.6 and pH 8.8, each with its own optimal substrate concentration. Inhibition of the reaction by metyrapone and carbon monoxide (CO) (among others) produced differential responses dependent on pH. These results, along with two pH optima and two substrate optima, suggested the involvement of multiple P-450 isozymes. Average specific activities were 8.05 nmoles of nitrite released per minute per milligram microsomal protein at pH 7.6 and 6.44 nmoles of nitrite released per minute per milligram microsomal protein at pH 8.8. Acetone was identified as the second product of the reaction by gas chromatography/mass spectrometry (GC/MS). Stoichiometry studies indicated that the acetone production was slightly less than expected (about 70%) from nitrite release. Up to 25% residual activity was observed under anaerobic conditions. These results suggested that though the predominant reaction mechanism was oxidative, oxygen-independent metabolism of 2NP also occurred to some extent. In contrast to the reported lack of activity in untreated rat, the observed denitrification in uninduced mouse liver microsomes was significant and suggested that major species-specific differences exist in the in vitro metabolism of 2NP.

  19. Oxidation of R- and S-omeprazole stereoselectively mediated by liver microsomal cytochrome P450 2C19 enzymes from cynomolgus monkeys and common marmosets.

    Science.gov (United States)

    Uehara, Shotaro; Kawano, Mirai; Murayama, Norie; Uno, Yasuhiro; Utoh, Masahiro; Inoue, Takashi; Sasaki, Erika; Yamazaki, Hiroshi

    2016-11-15

    Racemic omeprazole has been used for clinically treating gastric acid-related diseases and also as a typical human cytochrome P450 (P450) 2C19 probe substrate in preclinical studies. S-Omeprazole has been developed as a single enantiomer medicine, which has been reported not to be associated with polymorphic human P450 2C19 phenotypes. In this study, 5-hydroxylation and sulfoxidation activities, with respect to stereoselective R- and S-omeprazole oxidations by liver microsomes from experimental animals including non-human primates and humans, were investigated in vitro. Liver microsomes from humans, cynomolgus monkeys, and mice preferentially mediated R-omeprazole 5-hydroxylations, however those from marmosets, minipigs, dogs, and rats preferentially mediated S-omeprazole 5-hydroxylations. High catalytic activities were observed for recombinant human P450 2C19 in R-omeprazole 5-hydroxlations, cynomolgus monkey P450 2C19 in both R- and S-omeprazole 5-hydroxlations, and marmoset P450 2C19 in S-omeprazole 5-hydroxlations. On the other hand, human, cynomolgus monkey, and marmoset P450 3A enzymes preferentially mediated S-omeprazole sulfoxidations. Correlation and kinetic analyses revealed a high affinity of polymorphic cynomolgus monkey and marmoset liver microsomal P450 2C19 enzymes with respect to R- and S-omeprazole 5-hydroxylations, respectively, and a high capacity of cynomolgus monkey and marmoset liver microsomal P450 3A4 for omeprazole 5-hydroxylations and sulfoxidations. R-and S-omeprazole 5-hydroxylation activities in cynomolgus monkey and marmoset liver microsomes were significantly different among wild-type, heterozygous, and homozygous animals genotyped for cynomolgus monkey P450 2C19 p.[(Phe100Asn; Ala103Val; Ile112Leu)] and for marmoset P450 2C19 p.[(Phe7Leu; Ser254Leu; Ile469Thr)], respectively. The results of this study demonstrate polymorphic cynomolgus monkey and marmoset P450 2C19-dependent omeprazole oxidation activities with individual variations

  20. [Metabolism of nicousamide in rat and human liver in vitro].

    Science.gov (United States)

    Sheng, Li; Hu, Jin-ping; Chen, Hui; Li, Yan

    2008-09-01

    This paper is aimed to study the metabolic kinetics of nicousamide in rat liver microsomes and cytosol and to identify the major metabolite and drug metabolizing enzymes involved in the metabolism of nicousamide in rat and human liver microsomes by selective inhibitors in vitro. The concentration of nicousamide was determined by HPLC-UV method. The metabolite of nicousamide in rat and human liver microsomes was isolated and identified by LC-MS/MS. The major metabolite of nicousamide in rat and human liver microsomes was identified to be 3-(3'-carboxy-4'-hydroxy-anilino-carbo-)-6-amino-7-hydroxy-8-methyl-coumarin (M1). The metabolite of nicousamide in rat plasma, urine, bile and liver was consistent with M1. The metabolism of nicousamide can be catalyzed by several reductases, including CYP450 reductases, cytochrome b5 reductases and CYP2C6 in rat liver microsomes, as well as xanthine oxidase and DT-diaphorase in rat liver cytosol.

  1. Cadmium exposure decreases androgen-dependent metabolism of acetohexamide in liver microsomes of male rats through its testicular toxicity

    Energy Technology Data Exchange (ETDEWEB)

    Shimada, H. [Kumamoto Univ. (Japan). Faculty of Education; Yamaguchi, S.; Murata, H.; Otagiri, M.; Imamura, Y. [Kumamoto Univ. (Japan). Faculty of Pharmaceutical Sciences

    2002-02-01

    Administration of cadmium (Cd) at a dose of 1.23 mg/kg (2.0 mg/kg as CdCl2) markedly decreased the activity of an enzyme (acetohexamide reductase) catalysing the ketone-reduction of acetohexamide, an oral antidiabetic drug, in liver microsomes of male rats. However, the decreased enzyme activity was increased by repeated treatment with testosterone propionate (TP). When male rats were castrated and TP was given to the castrated ones, a similar decrease and increase, as described above, were observed in the microsomal enzyme activity. Cd exposure to male rats induced haemorrhage and atrophy of the testes and significantly diminished serum testosterone levels. There was no possibility that Cd accumulated in liver microsomes of male rats causing direct inhibition of the microsomal enzyme activity. We conclude that Cd exposure decreases androgen-dependent metabolism of acetohexamide in liver microsomes of male rats through its testicular toxicity. Cd exposure had no effect on acetohexamide reductase activity in liver cytosol of male rats. (orig.)

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

    Directory of Open Access Journals (Sweden)

    Christopher R Pudney

    2011-12-01

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

  3. Photoaffinity labeling of rat liver microsomal morphine UDP-glucuronosyltransferase by ( sup 3 H)flunitrazepam

    Energy Technology Data Exchange (ETDEWEB)

    Thomassin, J.; Tephly, T.R. (Univ. of Iowa, Iowa City (USA))

    1990-09-01

    Benzodiazepines have been shown to competitively inhibit morphine glucuronidation in rat and human hepatic microsomes. Flunitrazepam exerted a potent competitive inhibition of rat hepatic morphine UDP-glucuronosyltransferase (UDPGT) activity (Ki = 130 microM). It has no effect on the activity of p-nitrophenol, 17 beta-hydroxysteroid, 3 alpha-hydroxysteroid, or 4-hydroxybiphenyl UDPGTs. Because flunitrazepam is an effective photoaffinity label for benzodiazepine receptors, studied were performed in solubilized rat hepatic microsomes and with partially purified preparations of morphine UDPGT to determine the enhancement of flunitrazepam inhibition and binding to morphine UDPGT promoted by exposure to UV light. Under UV light, flunitrazepam inhibition was markedly enhanced. UV light exposure also led to a marked increase in binding of (3H)flunitrazepam to microsomal protein, which was protected substantially by preincubation with morphine. Testosterone, androsterone, and UDP-glucuronic acid did not protect against UV-enhanced flunitrazepam binding, and morphine did not reverse flunitrazepam binding once binding had occurred. As morphine UDPGT was purified, a good correlation was found between the increases in specific activity of morphine UDPGT and flunitrazepam binding to protein. Chromatofocusing chromatography showed that flunitrazepam bound only to fractions containing active morphine UDPGT, and no binding to 4-hydroxybiphenyl UDPGT was observed. Fluorography of a sodium dodecyl sulfate-polyacrylamide electrophoresis gel of solubilized hepatic microsomes that had been treated with (3H) flunitrazepam under UV light revealed a band with a monomeric molecular weight between 54,000 and 58,000. This monomeric molecular weight compares favorably with the reported monomeric molecular weight of homogeneous morphine UDPGT (56,000).

  4. Purification and characterization of an acetone-inducible cytochrome P-450 from hamster liver microsomes.

    Science.gov (United States)

    Puccini, P; Menicagli, S; Longo, V; Santucci, A; Gervasi, P G

    1992-11-01

    A form of cytochrome P-450 has been purified to electrophoretic homogeneity from the hepatic microsomes of Syrian golden hamsters treated with acetone. This P-450 form, designated ha P-450j, had an M(r) of approximately 55,000, bound dimethyl sulphoxide and exhibited a CO-reduced absorbance maximum at 451 nm. The absolute spectra of its oxidized form indicated that ha P-450j was predominantly in the low-spin state. In a reconstituted system, ha P-450j showed relatively low catalytic activities towards 7-ethoxycoumarin, 7-ethoxyresorufin, aminopyrine, ethylmorphine and benzphetamine, whereas it catalysed the oxidation of aniline, acetone and thiobenzamide with a high catalytic-centre activity. In addition, ha P-450j catalysed at a high rate the high-affinity component of dimethylnitrosamine N-demethylase; in contrast, only the low-affinity component of diethylnitrosamine N-de-ethylase was efficiently catalysed. The addition of cytochrome b5 to the reconstitution system decreased the Km value for dimethylnitrosamine N-demethylase by a factor of 5 and increased the Vmax. value, and slightly enhanced the other activities. Thiobenzamide and diethyldithiocarbamate were found to be the most effective inhibitors of the ha-P-450j-dependent aniline hydroxylation. Polyclonal antibodies against rat P-450j recognized ha P-450j in immunoblots of control and treated hamster liver microsomes. Treatment of hamsters with acetone increased the apparent abundance of ha P-450j in microsomes, whereas phenobarbital and beta-naphthoflavone did not induce it. Analysis of N-terminal amino acid sequences demonstrated that ha P-450j has a high degree of sequence identity with rat P-450j. All the evidence presented in this study indicates that ha P-450j could represent the hamster orthologue of the previously described CYP2E1(s) of other species.

  5. Effect of repeated exposure to aniline, nitrobenzene, and benzene on liver microsomal metabolism in the rat.

    Science.gov (United States)

    Wiśniewska-Knypl, J M; Jablońska, J K; Piotrowski, J K

    1975-02-01

    Exposure of rats to aniline at daily doses of 50 mg/kg of body weight over a month stimulated the microsomal metabolism as manifested by (1) acceleration of p-hydroxylation of anilin and N-demethylation of aminopyrine in 9-000 times g postmitochondrial supernatant of the liver, (2) shortening the sleeping time after hexobarbital, and (3) reduction of the antipyretic effect of phenacetin. In the rats exposed to nitrobenzene in a similar manner to aniline, nitroreduction of nitrobenzene and p-hydroxylation of aniline remained unaffected; the antipyretic effect of phenacetin was decreased, whereas hexobarbital sleeping time remained unchanged. Exposure of rats to benzene (50 mg/kg of body weight daily for a month) had no effect on the rate of hydroxylation of benzene and N-demethylation of aminopyrine. In benzene-exposed rats hexobarbital sleeping time was prolonged whereas the antipyretic effect of phenacetin was unaffected. Microsomal metabolism of aniline, nitrobenzene, and benzene was stimulated and inhibited when the rats were pretreated with phenobarbital and SKF 525-A, respectively.

  6. Purification and characterization of a 4-hydroxybiphenyl UDP-glucuronosyltransferase from rat liver microsomes

    Energy Technology Data Exchange (ETDEWEB)

    Styczynski, B.; Green, M.; Coffman, B.; Puig, J.; Tephly, T. (Univ. of Iowa, Iowa City (United States))

    1991-03-11

    A phenobarbital-inducible rat liver microsomal UDP-glucuronosyltransferase (4-HBP UDPGT) which catalyzes the glucuronidation of 4-hydroxybiphenyl has been purified to homogeneity. The apparent subunit molecular weight of this protein is 52,500 as determined by SDS-PAGE. 4-HBP UDPGT was shown to react with 4-hydroxybiphenyl, p-nitrophenol and 4-methylumbelliferone, but did not react with morphine, testosteron or chloramphenicol. Upon treatment with Endoglycosidase H, the 4-HBP UDPGT underwent about a 2,000 dalton decrease in subunit molecular weight, suggesting that this protein in N-glycosylated. Western blot analysis has revealed immunorecognition of 4-HBP UDPGT by antibodies raised in rabbit against rat 3{alpha}- and 17{beta}-hydroxysteroid UDPGTs. Additionally, the authors have obtained the N-terminal amino acid sequence of 4-HBP UDPGT. These data provide evidence which suggests that this protein is different from another UDPGT previously shown to react with 4-hydroxybiphenyl, testosterone and chloramphenicol.

  7. Microsomal prediction of in vivo clearance of CYP2C9 substrates in humans.

    Science.gov (United States)

    Carlile, D J; Hakooz, N; Bayliss, M K; Houston, J B

    1999-06-01

    To assess the utility of human hepatic microsomes for predicting in vivo intrinsic clearance (CLint ) via the use of four cytochrome P450 2C9 substrates: phenytoin, tolbutamide (S)-ibuprofen (two pathways) and diclofenac, and to examine the role of exogenous albumin within the microsomal incubation. V max, Km and CLint (defined as V max/Km ratio) were estimated under initial rate conditions for five pathways of metabolism in a bank of 15 human hepatic microsomal samples and were scaled to in vivo units using the microsomal protein index. Non-metabolic related binding in microsomes was measured for phenytoin and tolbutamide in the presence and absence of albumin. Microsomal CLint values differed by over two orders of magnitude, with the means ranging from 0.18 (phenytoin) to 40.70 (diclofenac) microl min-1 mg-1 microsomal protein. When these data were scaled and compared with published in vivo studies a similar rank order was obtained, however, the actual CLint tended to be underpredicted. While the in vivo unbound Km for phenytoin, 1-5 micron is substantially lower than the value determined in microsomes based on total concentrations (56 micron), correction for the in vitro binding reduces this value to 20 micron and 6 micron in the absence and presence of albumin, respectively. Similar trends were seen with tolbutamide Km. An appreciation of the utility of in vitro prediction can be best achieved when the range of CLint values predicted from the individual hepatic microsomal samples are compared with the range of individual in vivo CLint values reported in the literature. The degree of underprediction is less evident using the range than the mean data and no consistent advantage in adding albumin to the incubation media is apparent.

  8. Participation of cytochrome P-450 in the reduction of nitro compounds by rat liver microsomes.

    Science.gov (United States)

    Harada, N; Omura, T

    1980-05-01

    1. The subcellular distribution of nitrobenzene reduction activity in rat liver cells indicated the existence of two different enzyme systems, one localized in microsomes and the other localized in cytosol. The activity in the cytosol was mainly attributable to xanthine oxidase, judging from its substrate specificity and the inhibition by allopurinol. 2. The participation of the microsomal electron transport system in nitrobenzene reduction was examined by using antibodies against four components of the system, NADPH-cytochrome c reductase (fpT), NADH-cytochrome b5 reductase (fpD), cytochrome b5, and cytochrome P-450. Both NADH- and NADPH-dependent nitrobenzene reduction activities were strongly inhibited by anti-fpT IG and also by anti-P450 IG, but not inhibited by anti-fpD IG or anti-b5 IG. The reduction of nitrosobenzene and phenylhydroxylamine, which are supposed to be the intermediates of nitrobenzene reduction, was also examined, and it was found that NADH- and NADPH-dependent reduction of both compounds were strongly inhibited by anti-fpT IG and anti-P450 IG, but not by anti-fpD IG or anti-b5 IG. 3. Reconstruction experiments using purified NADPH-cytochrome P-450 reductase and cytochrome P-450 were also carried out and it was confirmed that the reduction of nitrobenzene, nitrosobenzene, and phenylhydroxylamine to aniline could be effected by these two components. 4. Nitrobenzene reduction by microsomes exhibited a short initial time lag and was activated by the addition of purified NADPH-cytochrome c reductase, whereas nitrosobenzene and phenylhydroxylamine reductions did not show any initial time lag and were not activated by the reductase. These observations suggest that the reduction of nitrobenzene to an intermediate, possibly nitrosobenzene or phenylhydroxylamine, limits the rate of aniline formation, and such an initial step of nitrobenzene reduction can be catalyzed by NADPH-cytochrome c reductase alone. Cytochrome P-450 is essential at least in the

  9. Activity of liver microsomal enzymes during the chronic phase of murine schistosomiasis

    Directory of Open Access Journals (Sweden)

    F.P. Conte

    2007-05-01

    Full Text Available The effects of schistosomiasis on microsomal enzymes were studied on post-infection day 90 when accumulated damage and fibrosis are most intense but granulomatous reaction around the eggs harbored in the liver is smaller than during the earlier phases. Swiss Webster (SW and DBA/2 mice of either sex (N = 12 per sex per group were infected with 100 Schistosoma mansoni cercariae on postnatal day 10 and killed on post-infection day 90. Cytochrome P-450 (CYP concentration and alkoxyresorufin-O-dealkylases (EROD, MROD, BROD, and PROD, p-nitrophenol-hydroxylase (PNPH, coumarin-7-hydroxylase (COH, and UDP-glucuronosyltransferase (UGT activities were measured in hepatic microsomes. Age-matched mice of the same sex and strain were used as controls. In S. mansoni-infected mice, CYP1A- and 2B-mediated activities (control = 100% were reduced in SW (EROD: male (M 36%, female (F 38%; MROD: M 38%, F 39%; BROD: M 46%, F 19%; PROD: M 50%, F 28% and DBA/2 mice (EROD: M 64%, F 58%; MROD: M 60%; BROD: F 49%; PROD: M 73% while PNPH (CYP2E1 was decreased in SW (M 31%, F 38% but not in DBA/2 mice. COH did not differ between infected and control DBA/2 and UGT, a phase-2 enzyme, was not altered by infection. In conclusion, chronic S. mansoni infection reduced total CYP content and all CYP-mediated activities evaluated in SW mice, including those catalyzed by CYP2E1 (PNPH, CYP1A (EROD, MROD and 2B (BROD, PROD. In DBA/2 mice, however, CYP2A5- and 2E1-mediated activities remained unchanged while total CYP content and activities mediated by other CYP isoforms were depressed during chronic schistosomiasis.

  10. Grapefruit juice, lyophilized grapefruit juice, and powdered whole grapefruit inhibit cytochrome P450-mediated triazolam hydroxylation by beagle dog liver microsomes.

    Science.gov (United States)

    Hanley, M J; Cerundolo, R; Radwanski, N; Court, M H

    2010-04-01

    Coadministration of grapefruit juice (GFJ) has been proposed to enhance the systemic availability and decrease the required dose of drugs such as cyclosporine that are extensively metabolized in the intestine and liver. Although GFJ inhibits human cytochrome P450 (CYP) 3A, effects on dog CYP have not yet been reported. Consequently, we determined whether GFJ inhibits triazolam hydroxylation by Beagle dog liver microsomes (DLM) using human liver microsomes (HLM) as positive control. Results were compared with the effects of lyophilized GFJ and commercially-available powdered grapefruit capsules, which may be more convenient dosage forms. GFJ inhibited alpha-hydroxytriazolam formation in both DLM and HLM with similar IC(50) (inhibitor concentration producing a 50% decrease in reaction velocity) values of 0.56% and 0.52% (v/v), respectively. Lyophilized GFJ and powdered grapefruit also inhibited DLM alpha-hydroxytriazolam formation with IC(50) values of 0.76 and 1.2 mg/mL, respectively. Consistent with mechanism-based enzyme inhibition, preincubation of DLM with any of the grapefruit products for 20 min resulted in significant enhancement of inhibition of triazolam alpha-hydroxylation by 8-20%. The results indicate that 16 g of lyophilized GFJ or 23 g of powdered grapefruit would be equivalent to dosing 100 mL of GFJ. In vivo pharmacokinetic interaction studies are needed to confirm these in vitro findings.

  11. Microsomal glucosidases of rat liver. Partial purification and inhibition by disaccharides.

    Science.gov (United States)

    Ugalde, R A; Staneloni, R J; Leloir, L F

    1980-12-01

    Further work on microsomal glucosidases of rat liver has confirmed that at least two enzymes are involved in the removal of glucose from the glucose-containing oligosaccharide. One acts on the oligosaccharide containing three glucose residues and another on the oligosaccharide which has one or two glucoses. The glucosidase which acts on (Glc)2(Man)9(GlcNAc)2 could be purified with a Concanavalin-A--Sepharose column following by electrofocusing. This purified preparation was active on the oligosaccharide containing one or two glucoses. Heat inactivation and inhibition by disaccharides was parallel for both activities. Inhibition of the glucosidase active on (Glc)3(Man)9(GlcNAC)2 was obtained with kojibiose which has an alpha 1-2 linkage, while the glucosidase acting on (Glc)1-2(Man)9-(GlcNAc)2 was inhibited by nigerose (alpha 1-3 linkage), maltose (alpha 1-4 linkage) and glucose at a higher concentration. None of the beta anomers inhibited. These results are consistent with an alpha configuration of the three glucoses of the dolichyl-diphosphate-linked oligosaccharide. Kojibiose was found to inhibit glucosidase action not only on the free oligosaccharide but also on protein-bound one.

  12. Liver and lung microsomal metabolism of the tobacco alkaloid beta-nicotyrine.

    Science.gov (United States)

    Shigenaga, M K; Kim, B H; Caldera-Munoz, P; Cairns, T; Jacob, P; Trevor, A J; Castagnoli, N

    1989-01-01

    The in vitro metabolic fate of beta-nicotyrine has been examined in rabbit lung and liver microsomal preparations as part of an effort to characterize the formation of potentially reactive metabolic species that may contribute to the toxic properties of tobacco products. HPLC analysis revealed the formation of an unstable metabolite which displayed HPLC-MS/MS characteristics consistent with the structure 1-methyl-5-(3-pyridyl)-3-pyrrolin-2-one. Attempted synthesis of this pyrrolinone, however, resulted in the isolation of the isomeric 1-methyl-5-(3-pyridyl)-2-pyrrolin-2-one. The HPLC, diode array UV, and mass spectral characteristics of this delta 4,5-isomer proved to be identical with those of the metabolite derived from beta-nicotyrine. Studies in D2O suggest that the 2- and 3-pyrrolinones are in equilibrium in aqueous solution. The metabolite undergoes autoxidation, possibly via radical intermediates, to yield 1-methyl-5-(3-pyridyl)-5-hydroxy-3-pyrrolin-2-one.

  13. Characterization of the metabolic interaction between trihalomethanes and chloroacetic acids using rat liver microsomes.

    Science.gov (United States)

    St-Pierre, Annie; Krishnan, Kannan; Tardif, Robert

    2005-02-27

    The aim of this study was to investigate the in vitro metabolism of trihalomethanes (THMs) in the presence of trichloroacetic acid (TCA), dichloracetic acid (DCA), monochloroacetic acid (MCA), and 4-methylpyrazole (4-MP) using liver microsomes from male Sprague-Dawley rats. Using the vial equilibration technique, initial experiments were carried out with starting concentrations of approximately 40 ppm THMs and 12-22 mM chloroacetic acids. The results indicated a mutual metabolic inhibition between THMs present as binary or quaternary mixtures. Although DCA and MCA had no influence on THMs, TCA produced a marked inhibition of the metabolism of all THMs: chloroform (CHCl3) (55%), bromodichloromethane (BDCM) (34%), dibromochloromethane (DBCM) (30%), and bromoform (TBM) (23%). The presence of 4-MP also reduced THM metabolism, the importance of which decreased in the following order: CHCl3 > BDCM > DBCM = TBM. In further vial equilibration experiments, using 9-140 ppm as starting concentrations of THMs, enzyme kinetic parameters (i.e., Michaelis constant, K(m), and maximum velocity, V(max)) were determined both in the absence and in the presence of TCA (12.2 mM). Results are consistent with a competitive inhibition between TCA and CHCl3, whereas the metabolic inhibition of BDCM and TMB by TCA was non-competitive. As for DBCM, results suggest a more complex pattern of inhibition. These results suggest that CYP2E1 is involved in the metabolism of THMs as well as in the metabolic interaction between THMs and TCA.

  14. Aromatic hydroxylation of methylenedioxybenzene (MDB) and methylenedioxymethamphetamine (MDMA) by rabbit liver microsomes.

    Science.gov (United States)

    Kumagai, Y; Schmitz, D A; Cho, A K

    1992-04-01

    1. Metabolites formed during incubation of methylenedioxybenzene (MDB) and methylenedioxymethamphetamine (MDMA) with rabbit liver microsomes were examined by h.p.l.c.-electrochemical detection and g.l.c.-mass spectrometry. 2. The trifluoroacetyl derivative of metabolite M-1, obtained from MDB, had a molecular ion at m/z 234 and was identified as 3,4-methylenedioxy-6-hydroxybenzene (sesamol) by comparison with authentic material. 3. The trifluoroacetyl derivative of metabolite M-2, obtained from MDMA, exhibited a molecular ion at m/z 401. Experiments with the deuterium substituted variants of MDMA indicated that the product was hydroxylated on the aromatic ring. 4. The formation of these hydroxylated metabolites required NADPH and was inhibited by carbon monoxide, indicating the possible participation of cytochrome P-450. Phenobarbital (PB) induction caused a marked enhancement of MDP hydroxylase activity whereas MDMA hydroxylation was not affected. 5. The aromatic hydroxylation of MDB and MDMA was also observed in a reconstituted system with cytochrome P-450 isozyme IIB4.

  15. UDP-Glucuronosyltransferases 1A6 and 1A9 are the Major Isozymes Responsible for the 7-O-Glucuronidation of Esculetin and 4-Methylesculetin in Human Liver Microsomes.

    Science.gov (United States)

    Zhu, Lijun; Lu, Linlin; Zeng, Shan; Luo, Feifei; Dai, Peimin; Wu, Peng; Wang, Ying; Liu, Liang; Hu, Ming; Liu, Zhongqiu

    2015-07-01

    Esculetin (6,7-dihydroxycoumarin, ET) and 4-methylesculetin (6,7-dihydroxy-4-methylcoumarin, 4-ME) are typical coumarin derivatives that are attracting considerable attention because of their wide spectrum of biologic activities, but their metabolism remains unknown. This study aimed to elucidate the in vitro UDP-glucuronosyltransferase (UGT) metabolism characteristics of ET and 4-ME. 7-O-monoglucuronide esculetin (ET-G) and 7-O-monoglucuronide 4-methylesculetin (4-ME-G) were identified by liquid chromatography-mass spectrometry (LC-MS) and (1)H-nuclear magnetic resonance ((1)HNMR) when ET or 4-ME was incubated with human liver (HLM) in the presence of UDP-glucuronic acid. Screening assays with 12 human expressed UGTs demonstrated that the formations of ET-G and 4-ME-G were almost exclusively catalyzed by UGT1A6 and UGT1A9. Phenylbutazone and carvacrol (UGT1A6 and UGT1A9 chemical inhibitors, respectively) at different concentrations (50, 100, and 200 μM) significantly inhibited the formation of glucuronidates of ET and 4-ME in HLM, UGT1A6, and UGT1A9 when the concentrations of ET and 4-ME ranged from 10 to 300 μM (P < 0.05). Clearance rates of ET in HLM, HIM, UGT1A6, and UGT1A9 were 0.54, 0.16, 0.69, and 0.14 ml/min/mg, respectively. Corresponding clearance rates values of 4-ME were 0.59, 0.03, 0.14, and 0.04 ml/min/mg, respectively. In conclusion, 7-O-monoglucuronidation by UGT1A6 and UGT1A9 was the predominant UGT metabolic pathway for both ET and 4-ME in vitro. The liver is probably the major contributor to the glucuronidation metabolism of ET and 4-ME. ET showed more rapid metabolism than 4-ME in glucuronidation. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

  16. Inhibition of cytochrome P450 enzymes by saturated and unsaturated fatty acids in human liver microsomes, characterization of enzyme kinetics in the presence of bovine serum albumin (0.1 and 1.0% w/v) and in vitro - in vivo extrapolation of hepatic clearance.

    Science.gov (United States)

    Palacharla, Raghava Choudary; Uthukam, Venkatesham; Manoharan, Arunkumar; Ponnamaneni, Ranjith Kumar; Padala, Nagasurya Prakash; Boggavarapu, Rajesh Kumar; Bhyrapuneni, Gopinadh; Ajjala, Devender Reddy; Nirogi, Ramakrishna

    2017-04-01

    The objective of the study was to determine the effect of fatty acids on CYP enzymes and the effect of BSA on intrinsic clearance of probe substrates. The inhibitory effect of thirteen fatty acids including saturated, mono-unsaturated and polyunsaturated fatty acids on CYP enzymes, kinetic parameters and intrinsic clearance values of nine CYP marker probe substrate reactions in the absence and presence of BSA (0.1 and 1.0% w/v) were characterized in human liver microsomes. The results demonstrate that most of the unsaturated fatty acids showed marked inhibition towards CYP2C8 mediated amodiaquine N-deethylation followed by inhibition of CYP2C9 and CYP2B6 mediated activities. The addition of 0.1% BSA in the incubation markedly improved the unbound intrinsic clearance values of probe substrates by reducing the Km values with little or no effect on maximal velocity. The addition of BSA (0.1 and 1.0% w/v) did not influence the unbound intrinsic clearance of marker reactions for CYP2A6, and CYP3A4 enzymes. The addition of 0.1% w/v BSA is sufficient to determine the intrinsic clearance of marker probe reactions by metabolite formation approach. The predicted hepatic clearance values for the substrates using the well-stirred model, in the presence of BSA (0.1% BSA), are comparable to the in vivo hepatic clearance values. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Saponin permeabilization of rough microsomes from rat liver reveals a novel prothrombin pool.

    Science.gov (United States)

    Tollefsen, S; Wierød, L; Skotte, A; Rob, J A; Helgeland, L

    2001-06-15

    Saponin permeabilization of rough microsomes in the presence of high salt revealed a novel pool of prothrombin associated by ionic interactions to the microsomal membrane. The lumenal content was obtained by treating rough microsomes with 0.32% saponin in a low salt (0.05 M KCl) buffer. By a subsequent treatment with 0.32% saponin in a slightly alkaline high salt buffer a fraction of peripherally associated membrane prothrombin was released from rough microsomes. Finally, the membrane-bound fraction was solubilized with 2.5% Triton X-100. The lumenal content fraction, the peripherally membrane-associated and the membrane-bound fraction from normal rats contained 55%, 29% and 16% of the total rough microsomal prothrombin, respectively. The corresponding fractions from warfarin-treated rats contained 86%, 5% and 9% of the total prothrombin. Following (14)C-gamma-carboxylation of intact microsomes for 30 min, the novel membrane-associated and the membrane-bound pool contained 42% and 33%, respectively, of labeled prothrombin. A similar distribution was found with warfarin-treated rats.

  18. Soy isoflavone metabolism in cats compared with other species: Urinary metabolite concentrations and glucuronidation by liver microsomes

    Science.gov (United States)

    Redmon, Joanna M.; Shrestha, Binu; Cerundolo, Rosario; Court, Michael H.

    2016-01-01

    Soybean is a common source of protein in many pet foods. Slow glucuronidation of soy-derived isoflavones in cats has been hypothesized to result in accumulation with adverse health consequences. Here we evaluated species’ differences in soy isoflavone glucuronidation using urine samples from cats and dogs fed a soy-based diet and liver microsomes from cats compared with microsomes from 12 other species.Significant concentrations of conjugated (but not unconjugated) genistein, daidzein, and glycitein, and the gut microbiome metabolites, dihydrogenistein and dihydrodaidzein were found in cat and dog urine samples. Substantial amounts of conjugated equol were also found in cat urine but not in dog urine.β-glucuronidase treatment showed that all these compounds were significantly glucuronidated in dog urine while only daidzein (11%) and glycitein (37%) showed any glucuronidation in cat urine suggesting that alternate metabolic pathways including sulfation predominate in cats.Glucuronidation rates of genistein, daidzein, and equol by cat livers were consistently ranked within the lowest three out of 13 species’ livers evaluated. Ferret and mongoose livers were also ranked in the lowest four species.Our results demonstrate that glucuronidation is a minor pathway for soy isoflavone metabolism in cats compared with most other species. PMID:26366946

  19. P-NITROPHENOL METABOLISM BY JAPANESE MEDAKA (ORYZIAS LATIPES) LIVER MICROSOMES AND S-9 FRACTION: ADDITIONAL EVIDENCE FOR THE EXISTENCE OF A CYP2E1-LIKE ISOFORM IN TELEOSTS

    Science.gov (United States)

    Liver microsomes and S-9 fraction of Japanese medaka (Oryzias latipes) metabolized the CYP2E1 specific substrate, p-nitrophenol (PNP), to a single hydroxylated product, 4-nitrocatechol. The use of liver S-9 fraction proved to be a viable alternative to liver microsomes and allowe...

  20. A comparative study of precision cut liver slices, hepatocytes, and liver microsomes from the Wistar rat using metronidazole as a model substance

    DEFF Research Database (Denmark)

    Sidelmann, U. G.; Cornett, Claus; Tjornelund, J.

    1996-01-01

    1. Metronidazole is metabolized by rat liver in vitro models to form a hydroxy metabolite, an acetic acid metabolite, a glucuronic acid conjugate, and a sulphate conjugate. 2. Four different in vitro systems for investigation of drug metabolism based on liver preparations from the male Wistar rat...... have been investigated. 3. An incubation system where liver slices are incubated in 12-well culture plates was evaluated with respect to metabolism of metronidazole. Optimal viability was observed for a time period of up to 24 h. The Michaelis-Menten parameters for the metabolism of metronidazole......, whereas the intrinsic clearance with respect to formation of the glucuronic acid conjugate was lower in slices compared with hepatocytes. 4. The metabolism of metronidazole in liver slices, in hepatocytes in primary monolayer culture, in hepatocytes incubated in suspension, and in liver microsomes...

  1. Elucidation of the metabolites of the novel psychoactive substance 4-methyl-N-ethyl-cathinone (4-MEC) in human urine and pooled liver microsomes by GC-MS and LC-HR-MS/MS techniques and of its detectability by GC-MS or LC-MS(n) standard screening approaches.

    Science.gov (United States)

    Helfer, Andreas G; Turcant, Alain; Boels, David; Ferec, Séverine; Lelièvre, Bénédicte; Welter, Jessica; Meyer, Markus R; Maurer, Hans H

    2015-05-01

    4-methyl-N-ethcathinone (4-MEC), the N-ethyl homologue of mephedrone, is a novel psychoactive substance of the beta-keto amphetamine (cathinone) group. The aim of the present work was to study the phase I and phase II metabolism of 4-MEC in human urine as well as in pooled human liver microsome (pHLM) incubations. The urine samples were worked up with and without enzymatic cleavage, the pHLM incubations by simple deproteinization. The metabolites were separated and identified by gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-high resolution-tandem mass spectrometry (LC-HR-MS/MS). Based on the metabolites identified in urine and/or pHLM, the following metabolic pathways could be proposed: reduction of the keto group, N-deethylation, hydroxylation of the 4-methyl group followed by further oxidation to the corresponding 4-carboxy metabolite, and combinations of these steps. Glucuronidation could only be observed for the hydroxy metabolite. These pathways were similar to those described for the N-methyl homologue mephedrone and other related drugs. In pHLM, all phase I metabolites with the exception of the N-deethyl-dihydro isomers and the 4-carboxy-dihydro metabolite could be confirmed. Glucuronides could not be formed under the applied conditions. Although the taken dose was not clear, an intake of 4-MEC should be detectable in urine by the GC-MS and LC-MS(n) standard urine screening approaches at least after overdose. Copyright © 2014 John Wiley & Sons, Ltd.

  2. Oxidative denitrification of 2-nitropropane and propane-2-nitronate by mouse liver microsomes: lack of correlation with hepatocytotoxic potential.

    Science.gov (United States)

    Dayal, R; Goodwin, B; Linhart, I; Mynett, K; Gescher, A

    1991-01-01

    2-Nitropropane (2-NP) is an industrial chemical with hepatotoxic and genotoxic properties. It exists in chemical equilibrium with propane-2-nitronate, which is much more genotoxic than 2-NP. In this work the link between toxicity and metabolism of 2-NP and its nitronate was investigated. To that end 2-NP or propane-2-nitronate were incubated with murine hepatic microsomes at concentrations of up to 10 mM, and generation of nitrite was measured as product of metabolic oxidation of the two species. Under the acidic reaction conditions of the colorimetric nitrite assay propane-2-nitronate decomposed chemically to nitrite. Therefore an ion-pair HPLC assay at neutral pH was developed which enabled determination of nitrite formed from the nitronate. The rate of metabolic nitrite generation from propane-2-nitronate was 5-10-fold that obtained with 2-NP. Metabolism of either species to nitrite was dependent on the presence in the incubate of viable microsomes and of NADPH, and it was inhibited in the presence of carbon monoxide or the cytochrome P-450 inhibitor SKF525A. Acetone could also be measured as a metabolite of 2-NP. Optical difference spectra were recorded in mixtures of propane-2-nitronate with liver microsomes from phenobarbital-pretreated rats. The spectral dissociation constant was found to be 30 mM, which compares with 10 mM reported for 2-NP. 2-NP and propane 2-nitronate were incubated with mouse hepatocytes in suspension and cytotoxicity was determined by measurement of leakage of cellular lactate dehydrogenase into the medium. Both species were hardly toxic, as concentrations of 20 mM were required to elicit significant damage to the cells. The results demonstrate that propane-2-nitronate, like 2-NP, undergoes microsomal oxidative denitrification, probably catalysed by cytochrome P-450. Metabolism of both species occurs at markedly different rates, but the difference in metabolism is not reflected by a difference in hepatocytotoxic potential.

  3. Oxidative one-carbon cleavage of the octyl side chain of olanexidine, a novel antimicrobial agent, in dog liver microsomes.

    Science.gov (United States)

    Umehara, K; Shimokawa, Y; Koga, T; Ohtani, T; Miyamoto, G

    2004-01-01

    1. The oxidative one-carbon cleavage reaction in the octyl side chain of olanexidine [1-(3,4-dichlorobenzyl)-5-octylbiguanide], a new potent biguanide antiseptic, was characterized in dog liver microsomes. 2. Olanexidine was initially biotransformed to a monohydroxylated metabolite, 8-[5-(3,4-dichlorobenzyl)-1-biguanidino]-2-octanol (DM-215), and DM-215 was subsequently oxidized to the diol derivative, 8-[5-(3,4-dichlorobenzyl)-1-biguanidino]-1,2-octandiol (DM-220). DM-220 was further biotransformed to 2-hydroxy aldehyde derivative, 2-hydroxy carboxylic acid derivative, and an oxidative C-1-C-2 bond cleavage metabolite, 7-[5-(3,4-dichlorobenzyl)-1-biguanidino] heptanoic acid [DM-223 (C7), a seven-carbon chain derivative], after incubation with dog liver microsomes. 3. DM-223 formation required NADPH as a cofactor and was inhibited by quinidine and quinine, relatively selective inhibitors of CYP2D subfamilies in dogs. 4. The results suggest that the one-carbon fragment of the octyl side chain of olanexidine could be removed by the oxidative C-C bond cleavage with the possible involvement of cytochrome P450 systems such as CYP2D subfamily. This oxidative C-C bond cleavage reaction by cytochrome P450s could play an important role in the removal of one-carbon fragment of other drugs or endogenous compounds containing aliphatic chains.

  4. Age-related changes in O-deethylase and aldrin epoxidase activity in mouse skin and liver microsomes.

    Science.gov (United States)

    Williams, D; Woodhouse, K

    1996-09-01

    The metabolism of three model substrates for the cytochrome P450 dependent mono-oxygenase enzyme system (P450-MMO) was studied in microsomes isolated from skin and liver of young adult and senescent C57B1/6J mice. The substrates chosen were aldrin (AE), 7-ethoxycoumarin (EOC), and 7-ethoxyresorufin (EOR). Both EOC and EOR activities were lower in senescent skin. By contrast, no-age related changes were seen in senescent liver. AE was similar in young and old, in both tissues. We suggest that some important age-related differences in cutaneous xenobiotic metabolism do occur, but that these are not mirrored by hepatic differences, and are substrate specific. Previous work from these laboratories would also suggest significant species differences.

  5. Prediction of hepatic microsomal intrinsic clearance and human clearance values for drugs.

    Science.gov (United States)

    Nikolic, Katarina; Agababa, Danica

    2009-10-01

    Twenty-nine drugs of different structures were used in theoretical QSAR analysis of human hepatic microsomal intrinsic clearance (in vitro T(1/2) and in vitro CL'(int)) and whole body clearance (CL(blood)). The examined compounds demonstrated a wide range of scaled intrinsic clearance values. Constitutional, geometrical, physico-chemical and electronic descriptors were computed for the examined structures by use of the Marvin Sketch 5.1.3_2, the Chem3D Ultra 7.0.0 and the Dragon 5.4 program. Partial least squares regression (PLSR), has been applied for selection of the most relevant molecular descriptors and development of quantitative structure-activity relationship (QSAR) model for human hepatic microsomal intrinsic clearance (in vitro T(1/2)). Optimal QSAR models with nine and ten variables, R(2)>0.808 and cross-validation parameter q(pre)(2)>0.623, were selected and compared. Since the microsomal in vitro T(1/2) data can be used for calculation of in vitro CL'(int) and in vivo CL(blood), the developed QSAR model will enable one to analyze the kinetics of cytochrome P450-mediated reactions in term of intrinsic clearance and whole body clearance. A comparison is made between predictions produced from the QSAR analysis and experimental data, and there appears to be generally satisfactory correlations with the literature values for intrinsic clearance data.

  6. GLUCURONIDATION OF LABETALOL AT THE 2 HYDROXY POSITIONS BY BOVINE LIVER-MICROSOMES - ISOLATION, PURIFICATION, AND STRUCTURE ELUCIDATION OF THE GLUCURONIDES OF LABETALOL

    NARCIS (Netherlands)

    NIEMEIJER, NR; GERDING, TK; DEZEEUW, RA

    1991-01-01

    Glucuronidation is known to be a major metabolic pathway for labetalol. As the drug contains a phenolic and an alcoholic hydroxy group, in principle two regio isomeric glucuronides can be formed. By incubating the substrate labetalol with bovine liver microsomes, in the presence of the co-substrate

  7. Effects of antidepressant drugs on the activity of cytochrome P-450 measured by caffeine oxidation in rat liver microsomes.

    Science.gov (United States)

    Danie, W A; Syrek, M; Ryłko, Z; Wójcikowski, J

    2001-01-01

    Caffeine is a marker drug for testing the activity of CYP1A2 (3-N-demethylation) in humans and rats. Moreover, it is also a relatively specific substrate of CYP3A (8-hydroxylation). In the case of 1-N- and in particular 7-N-demethylation of caffeine, apart from CYP1A2, other cytochrome P-450 isoenzymes play a considerable role. The aim of the present study was to investigate the influence of imipramine, amitriptyline and fluoxetine on cytochrome P-450 activity measured by caffeine oxidation in rat liver microsomes. The obtained results showed that imipramine exerted a most potent inhibitory effect on caffeine metabolism. Imipramine decreased the rate of 3-N-, 1-N- and 7-N-demethylations, and 8-hydroxylation of caffeine, the effect on 3-N-demethylation being most pronounced (Ki = 33 microM). Amitriptyline showed distinct inhibition of 3-N- and 1-N-demethylation of caffeine, though its effect was less potent than in the case of imipramine (Ki = 57 and 61 pM, respectively). The influence of amitriptyline on 8-hydroxylation and especially on 7-N-demethylation of caffeine was weaker (Ki = 108 and 190 pM, respectively) than on 3-N- or 1-N-demethylation, suggesting a narrower spectrum of cytochrome P-450 inhibition by amitriptyline than by imipramine, involving mainly the subfamily CYP1A2, and--to a lesser degree--CYP3A. In contrast to the tested tricyclic antidepressants, fluoxetine did not exert any considerable effect on the 3-N- or 1-N-demethylation of caffeine (Ki = 152 and 196 microM, respectively), which indicates its low affinity for CYP1A2. However, fluoxetine displayed a clear inhibitory effect on caffeine 7-N-demethylation (Ki = 72 microM), the reaction which is catalyzed mainly by other than CYP1A2 isoenzymes. Fluoxetine diminished markedly the 8-hydroxylation of the marker drug; as reflected by Ki values, the potency of inhibition of rat CYP3A by fluoxetine was similar to that of imipramine (Ki = 40 and 45 microM, respectively). In summary, CYP1A2 was

  8. Purification and characterization of an acetone-inducible cytochrome P-450 from hamster liver microsomes.

    OpenAIRE

    Puccini, P; Menicagli, S; Longo, V.; Santucci, A.; Gervasi, P. G.

    1992-01-01

    A form of cytochrome P-450 has been purified to electrophoretic homogeneity from the hepatic microsomes of Syrian golden hamsters treated with acetone. This P-450 form, designated ha P-450j, had an M(r) of approximately 55,000, bound dimethyl sulphoxide and exhibited a CO-reduced absorbance maximum at 451 nm. The absolute spectra of its oxidized form indicated that ha P-450j was predominantly in the low-spin state. In a reconstituted system, ha P-450j showed relatively low catalytic activitie...

  9. Investigation of glycosylation processes in mitochondria and microsomal membranes from human skeletal muscle.

    Science.gov (United States)

    Gasnier, F; Lerme, F; Rousson, R; Roussouly, P; Vaganay, E; Louisot, P; Gateau-Roesch, O

    1991-05-31

    Glycoconjugates are directly involved in major skeletal muscle functions. As little is known about glycosylation processes in muscle, we investigated glycoconjugate synthesis in subcellular fractions from human skeletal muscle tissue. Mitochondria and microsomal membranes were prepared from muscle biopsies by thorough mechanical disruption and differential centrifugations. This procedure resulted in the isolation of intact mitochondria (1 mg protein/g muscle) and of a microsomal fraction (1.5 mg protein/g muscle). Glycosyltransferases were studied in both subcellular fractions using either dolichylmonophosphate as a polyprenic acceptor or chemically modified fetuin as a glycoprotein substrate. Our results provide evidence for high rates of glycosylation in muscle. The highest activities were obtained with GDP-mannose: dilichylmonophosphate mannosyltransferase, a key enzyme in glycosylation process (220 pmol/mg per h in mitochondria and 1,550 pmol/mg per h in microsomal membranes). Substantial individual variations were observed for dolichol pathway glycosyltransferases but low individual variations were found for glycosyltransferases involved in maturation of glycoproteins. The role which glycosylation defects may play in muscle dysfunction has yet to be defined.

  10. In vitro metabolism of isoline, a pyrrolizidine alkaloid from Ligularia duciformis, by rodent liver microsomal esterase and enhanced hepatotoxicity by esterase inhibitors.

    Science.gov (United States)

    Tang, Jun; Akao, Teruaki; Nakamura, Norio; Wang, Zheng-Tao; Takagawa, Kiyoshi; Sasahara, Masakiyo; Hattori, Masao

    2007-10-01

    Isoline, a major retronecine-type pyrrolizidine alkaloid (PA) from the Chinese medicinal herb Ligularia duciformis, was suggested to be the most toxic known PA. Its in vitro metabolism was thus examined in rat and mouse liver microsomes, and its toxicity was compared with that of clivorine and monocrotaline after i.p. injection in mice. Isoline was more rapidly metabolized by both microsomes than clivorine and monocrotaline and converted to two polar metabolites M1 and M2, which were spectroscopically determined to be bisline (a deacetylated metabolite of isoline) and bisline lactone, respectively. Both metabolites were formed in the presence or absence of an NADPH-generating system with liver microsomes but not cytosol. Their formation was completely inhibited by the esterase inhibitors, triorthocresyl phosphate (TOCP) and phenylmethylsulfonyl fluoride, but not at all or partially by cytochrome P450 (P450) inhibitors, alpha-naphthoflavone and proadifen (SKF 525A), respectively. These results demonstrated that both metabolites were produced by microsomal esterase(s) but not P450 isozymes. The esterase(s) involved showed not only quite different activities but also responses to different inhibitors in rat and mouse liver microsomes, suggesting that different key isozyme(s) or combinations might be responsible for the deacetylation of isoline. Isoline injected i.p. into mice induced liver-specific toxicity that was much greater than that with either clivorine or monocrotaline, as judged by histopathology as well as serum alanine aminotransferase and aspartate aminotransferase levels. Isoline-induced hepatotoxicity was remarkably enhanced by the esterase inhibitor TOCP but was reduced by the P450 inhibitor SKF 525A, indicating that rodent hepatic esterase(s) played a principal role in the detoxification of isoline via rapid deacetylation in vivo.

  11. Effects of hypo- and hyperthyroidism on rat liver microsomal long-chain fatty acyl-CoA synthetase and hydrolase

    Energy Technology Data Exchange (ETDEWEB)

    Dang, A.Q.; Faas, F.H.; Carter, W.J.

    1986-05-01

    The effects of hyperthyroidism (hyperT/sub 3/), (tri-iodothryonine (T/sub 3/) injected rats), and hypothyroidism (hypoT/sub 3/) (thyroidectomized rats) on the activation of fatty acids by a microsomal long-chain fatty acyl-CoA (LCA-CoA) synthetase and the degradation of LCA-CoA by a microsomal LCA-CoA hydrolase was determined. MAS was assayed by measuring the (1-/sup 14/C)-palmitate or -1-/sup 14/C) oleate incorporated into its water soluble CoA ester. MAH was assayed spectrophotomerically by following the reduction of 5',5'-dithiobis-(2-nitrobenzoic acid) by the CoA released from palmitoyl-CoA or oleoyl-CoA. Enzyme activities are given as mean (nmoles/mg/min) +/- SEM. MAS activities were decreased 36-44% (p < 0.01) in both hypoT/sub 3/ and hyperT/sub 3/ (controls = 101 +/- 4 (n = 11, (1-/sup 14/C)-palmitate) of 72 +/- 2 (n = 5,(1-/sup 14/C)oleate)). These decreases may contribute to the decreased triacelyglycerol (TG) and phospholipid contents in the hyperT/sub 3/ liver and the decreased clearance rate of plasma TG in the hypoT/sub 3/. MAH was decreased 27-42% (p<0.01) only in hypoT/sub 3/ (controls = 77 +/- 3 (n = 11, palmitoyl-CoA) or 45 +/- 1 (n = 5, oleoyl-CoA)). This decrease was corrected by T/sub 3/ treatment. Since the decreased MAH would increase the availability of LCA-CoA, it may contribute to the increased TG synthesis in hypoT/sub 3/.

  12. Prediction of human drug clearance by multiple metabolic pathways: integration of hepatic and intestinal microsomal and cytosolic data.

    Science.gov (United States)

    Cubitt, Helen E; Houston, J Brian; Galetin, Aleksandra

    2011-05-01

    The current study assesses hepatic and intestinal glucuronidation, sulfation, and cytochrome P450 (P450) metabolism of raloxifene, quercetin, salbutamol, and troglitazone using different in vitro systems. The fraction metabolized by conjugation and P450 metabolism was estimated in liver and intestine, and the importance of multiple metabolic pathways on accuracy of clearance prediction was assessed. In vitro intrinsic sulfation clearance (CL(int, SULT)) was determined in human intestinal and hepatic cytosol and compared with hepatic and intestinal microsomal glucuronidation (CL(int, UGT)) and P450 clearance (CL(int, CYP)) expressed per gram of tissue. Hepatic and intestinal cytosolic scaling factors of 80.7 mg/g liver and 18 mg/g intestine were estimated from published data. Scaled CL(int, SULT) ranged between 0.7 and 11.4 ml · min(-1) · g(-1) liver and 0.1 and 3.3 ml · min(-1) · g(-1) intestine (salbutamol and quercetin were the extremes). Salbutamol was the only compound with a high extent of sulfation (51 and 28% of total CL(int) for liver and intestine, respectively) and also significant renal clearance (26-57% of observed plasma clearance). In contrast, the clearance of quercetin was largely accounted for by glucuronidation. Drugs metabolized by multiple pathways (raloxifene and troglitazone) demonstrated improved prediction of intravenous clearance using data from all hepatic pathways (44-86% of observed clearance) compared with predictions based only on the primary pathway (22-36%). The assumption of no intestinal first pass resulted in underprediction of oral clearance for raloxifene, troglitazone, and quercetin (3-22% of observed, respectively). Accounting for the intestinal contribution to oral clearance via estimated intestinal availability improved prediction accuracy for raloxifene and troglitazone (within 2.5-fold of observed). Current findings emphasize the importance of both hepatic and intestinal conjugation for in vitro-in vivo extrapolation

  13. 2,2',3,3',6,6'-Hexachlorobiphenyl (PCB 136) is Enantioselectively Oxidized to Hydroxylated Metabolites by Rat Liver Microsomes

    Science.gov (United States)

    Wu, Xianai; Pramanik, Ananya; Duffel, Michael W.; Hrycay, Eugene G.; Bandiera, Stelvio M.; Lehmler, Hans-Joachim; Kania-Korwel, Izabela

    2011-01-01

    Developmental exposure to multiple-ortho substituted polychlorinated biphenyls (PCBs) causes adverse neurodevelopmental outcomes in laboratory animals and humans by mechanisms involving the sensitization of Ryanodine receptors (RyRs). In the case of PCB 136, the sensitization of RyR is enantiospecific, with only (-)-PCB 136 being active. However, the role of enantioselective metabolism in the developmental neurotoxicity of PCB 136 is poorly understood. The present study employed hepatic microsomes from phenobarbital (PB-), dexamethasone (DEX-) and corn oil (VEH-)treated male Sprague-Dawley rats to investigate the hypothesis that PCB 136 atropisomers are enantioselectively metabolized by P450 enzymes to potentially neurotoxic, hydroxylated PCB 136 metabolites. The results demonstrated the time- and isoform-dependent formation of three metabolites, with 5-OH-PCB 136 (2,2',3,3',6,6'-hexachlorobiphenyl-5-ol) being the major metabolite. The formation of 5-OH-PCB 136 increased with the activity of P450 2B enzymes in the microsomal preparation, which is consistent with PCB 136 metabolism by rat P450 2B1. The minor metabolite 4-OH-PCB 136 (2,2',3,3',6,6'-hexachlorobiphenyl-4-ol) was produced by a currently unidentified P450 enzymes. An enantiomeric enrichment of (-)-PCB 136 was observed in microsomal incubations due to the preferential metabolism of (+)-PCB 136 to the corresponding 5-OH-PCB 136 (2,2',3,3',6,6'-hexachlorobiphenyl-5-ol) atropisomer. 4-OH-PCB 136 displayed an enrichment of the atropisomer formed from (-)-PCB 136; however, the enrichment of this metabolite atropisomer didn't affect the enantiomeric enrichment of the parent PCB because 4-OH-PCB 136 is only a minor metabolite. Although the formation of 5- and 4-OH-PCB 136 atropisomers increased with time, the enantioselective formation of the OH-PCB metabolites resulted in constant enantiomeric enrichment, especially at later incubation times. These observations not only demonstrate that the chiral signatures of

  14. [Changes in the activity of enzymes of heme synthesis and catabolism, and level of microsomal hemoproteins during the liver acute intoxication by thioacetamide].

    Science.gov (United States)

    Kharimov, Kh Ia; Inoiatova, F Kh; Dolimova, M A

    2001-01-01

    Thioacetamide administration to rats (20 mg/100 g) caused the development of toxic hepatitis which was accompanied by the increase of hepatic ALA-synthase and D-ALA that led to accumulating free porphyrines in the liver. At the same time an increase in activity of heme oxigenase was also found. A decrease in heme synthesis correlated with a decrease in content of cytochrome P450 and b5 in microsomal hepatic fraction of experimental animals.

  15. Microsomal metabolism of trenbolone acetate metabolites ...

    Science.gov (United States)

    Trenbolone acetate (TBA) is a synthetic growth promoter widely used in animal agriculture, and its metabolites are suspected endocrine disrupting compounds in agriculturally impacted receiving waters. However, beyond the three widely recognized TBA metabolites (17-trenbolone, 17-trenbolone and trendione), little is known about other metabolites formed in vivo and subsequently discharged into the environment, with some evidence suggesting these unknown metabolites comprise a majority of the TBA mass dosed to the animal. Here, we explored the metabolism of the three known TBA metabolites using rat liver microsome studies. All TBA metabolites are transformed into a complex mixture of monohydroxylated products. Based on product characterization, the majority are more polar than the parent metabolites but maintain their characteristic trienone backbone. A minor degree of interconversion between known metabolites was also observed, as were higher order hydroxylated products with a greater extent of reaction. Notably, the distribution and yield of products were generally comparable across a series of variably induced rat liver microsomes, as well as during additional studies with human and bovine liver microsomes. Bioassays conducted with mixtures of these transformation products suggest that androgen receptor (AR) binding activity is diminished as a result of the microsomal treatment, suggesting that the transformation products are generally less potent than

  16. Effect of oxygen tension on the generation of F2-isoprostanes and malondialdehyde in peroxidizing rat liver microsomes.

    Science.gov (United States)

    Longmire, A W; Swift, L L; Roberts, L J; Awad, J A; Burk, R F; Morrow, J D

    1994-03-29

    Although numerous methods have been developed for the detection of lipid peroxidation, it is generally recognized that most of these lack specificity and/or sensitivity, particularly when applied to in vivo situations. We have reported recently that a series of prostaglandin F2-like compounds, termed F2-isoprostanes, are formed in vivo from the free radical catalyzed peroxidation of arachidonic acid and appear to be a useful marker of oxidant stress. Because of formation of other products of lipid peroxidation, such as alkanes and malondialdehyde (MDA), are affected by oxygen tension, which may influence their usefulness as markers of oxidant stress, we carried out a systematic study of the generation of F2-isoprostanes at various oxygen concentrations and compared these changes with the generation of MDA. The disappearance of the F2-isoprostane precursor, arachidonic acid, was used as a reference measure. Rat liver microsomes were peroxidized using an iron-ascorbate system. The incubations were carried out in sealed flasks at 37 degrees under N2 and various concentrations of O2 up to 100%. F2-isoprostanes were quantified by mass spectrometry and MDA by the thiobarbituric acid reaction. Microsomal fatty acids were measured by gas chromatography. Both MDA and F2-isoprostane formation increased in a time-dependent manner up to 15 min. Their formation correlated with a loss of polyunsaturated fatty acid and with an increase in O2 tension up to 21% O2. At oxygen tensions above 21%, MDA generation continued to increase, while F2-isoprostane generation and arachidonic acid loss did not. Levels of MDA and F2-isoprostanes increased a maximum of 65 and 9.4 times baseline values, respectively. These studies, therefore, define factors that influence the formation of F2-isoprostanes in an in vitro model of lipid peroxidation. Further, they demonstrate that higher O2 tensions do not block formation of F2-isoprostanes and validate their usefulness for assessing lipid

  17. Dietary saturated and monounsaturated fats protect against acute acetaminophen hepatotoxicity by altering fatty acid composition of liver microsomal membrane in rats.

    Science.gov (United States)

    Hwang, Jinah; Chang, Yun-Hee; Park, Jung Hwa; Kim, Soo Yeon; Chung, Haeyon; Shim, Eugene; Hwang, Hye Jin

    2011-10-20

    Dietary polyunsaturated fats increase liver injury in response to ethanol feeding. We evaluated the effect of dietary corn oil (CO), olive oil (OO), and beef tallow (BT) on fatty acid composition of liver microsomal membrane and acute acetaminophen hepatotoxicity. Male Sprague-Dawley rats were fed 15% (wt/wt) CO, OO or BT for 6 weeks. After treatment with acetaminophen (600 mg/kg), samples of plasma and liver were taken for analyses of the fatty acid composition and toxicity. Treatment with acetaminophen significantly elevated levels of plasma GOT and GPT as well as hepatic TBARS but reduced hepatic GSH levels in CO compared to OO and BT groups. Acetaminophen significantly induced protein expression of cytochrome P450 2E1 in the CO group. In comparison with the CO diet, lower levels of linoleic acid, higher levels of oleic acids and therefore much lower ratios of linoleic to oleic acid were detected in rats fed OO and BT diets. Dietary OO and BT produces similar liver microsomal fatty acid composition and may account for less severe liver injury after acetaminophen treatment compared to animals fed diets with CO rich in linoleic acid. These findings imply that types of dietary fat may be important in the nutritional management of drug-induced hepatotoxicity.

  18. The novel antifungal agent PLD-118 is neither metabolized by liver microsomes nor inhibits cytochrome P450 in vitro

    NARCIS (Netherlands)

    Parnham, M.J.; Bogaards, J.J.P.; Schrander, F.; Schut, M.W.; Orešković, K.; Mildner, B.

    2005-01-01

    PLD-118 is a novel, oral antifungal drug, under development for the treatment of Candida infections. Possible metabolism of PLD-118 by rat, dog and human S9 liver homogenates and inhibition of human cytochrome P450 (CYP) enzymes were investigated. PLD-118 (10 and 100 μm) incubated for 0-60 min with

  19. In vitro characterization of the oxidative cleavage of the octyl side chain of olanexidine, a novel antimicrobial agent, in dog liver microsomes.

    Science.gov (United States)

    Umehara, K; Kudo, S; Hirao, Y; Morita, S; Ohtani, T; Uchida, M; Miyamoto, G

    2000-12-01

    The metabolism of olanexidine [1-(3,4-dichlorobenzyl)-5-octylbiguanide], a new potent biguanide antiseptic, was investigated in dog liver microsomes to characterize the enzyme(s) catalyzing the biotransformation of olanexidine to C-C bond cleavage metabolites. Olanexidine was initially biotransformed to monohydroxylated metabolite 2-octanol (DM-215), and DM-215 was subsequently oxidized to diol derivatives threo-2,3-octandiol (DM-221) and erythro-2,3-octandiol (DM-222). Diols were further biotransformed to a ketol derivative and C-C bond cleavage metabolite (DM-210, hexanoic acid derivative), an in vivo end product, in the incubation with dog liver microsomes. The formations of DM-215, DM-221, DM-222, and DM-210 followed Michaelis-Menten kinetics, and Eadie-Hofstee analysis of the metabolite formation activity confirmed single-enzyme Michaelis-Menten kinetics. The K(m) and V(max) values for the formation of DM-210 appeared to be 2.42 microM and 26.6 pmol/min/mg in the oxidation of DM-221 and 2.48 microM and 30.2 pmol/min/mg in the oxidation of DM-222. The intrinsic clearance (V(max)/K(m)) of the C-C bond cleavage reactions was essentially the same with either DM-221 or DM-222 as substrate. These oxidative reactions were significantly inhibited by quinidine, a selective inhibitor of CYP2D subfamilies, indicating the metabolic C-C bond cleavage of the octyl side chain of olanexidine to likely be mediated via the CYP2D subfamily in dog liver microsomes. This aliphatic C-C bond cleavage by cytochrome P450s may play an important role in the metabolism of other drugs or endogenous compounds possessing aliphatic chains.

  20. Metabolism of methiocarb and carbaryl by rat and human livers and plasma, and effect on their PXR, CAR and PPARα activities.

    Science.gov (United States)

    Fujino, Chieri; Tamura, Yuki; Tange, Satoko; Nakajima, Hiroyuki; Sanoh, Seigo; Watanabe, Yoko; Uramaru, Naoto; Kojima, Hiroyuki; Yoshinari, Kouichi; Ohta, Shigeru; Kitamura, Shigeyuki

    2016-01-01

    The oxidative, reductive, and hydrolytic metabolism of methiocarb and the hydrolytic metabolism of carbaryl by liver microsomes and plasma of rats or humans were examined. The effects of the metabolism of methiocarb and carbaryl on their nuclear receptor activities were also examined. When methiocarb was incubated with rat liver microsomes in the presence of NADPH, methiocarb sulfoxide, and a novel metabolite, methiocarb sulfone were detected. Methiocarb sulfoxide was oxidized to the sulfone by liver microsomes and reduced back to methiocarb by liver cytosol. Thus, the interconversion between methiocarb and the sulfoxide was found to be a new metabolic pathway for methiocarb by liver microsomes. The product of methiocarb hydrolysis, which is methylthio-3,5-xylenol (MX), was also oxidized to sulfoxide form by rat liver microsomes. The oxidations were catalyzed by human flavin-containing monooxygenase isoform (FMO1). CYP2C19, which is a human cytochrome P450 (CYP) isoform, catalyzed the sulfoxidations of methiocarb and MX, while CYP1A2 also exhibited oxidase activity toward MX. Methiocarb and carbaryl were not enzymatically hydrolyzed by the liver microsomes, but they were mainly hydrolyzed by plasma and albumin to MX and 1-naphthol, respectively. Both methiocarb and carbaryl exhibited PXR and PPARα agonistic activities; however, methiocarb sulfoxide and sulfone showed markedly reduced activities. In fact, when methiocarb was incubated with liver microsomes, the receptor activities were decreased. In contrast, MX and 1-naphthol showed nuclear receptor activities equivalent to those of their parent carbamates. Thus, the hydrolysis of methiocarb and carbaryl and the oxidation of methiocarb markedly modified their nuclear receptor activities.

  1. Affinity of drugs for cytochrome P-450 determined by inhibition of p-nitrophenetole O-deethylation by rat liver microsomes

    DEFF Research Database (Denmark)

    Jørgensen, L; Johansen, Torben

    1983-01-01

    The rate of conversion of p-nitrophenetole to p-nitrophenol by rat liver microsomes was studied. Inhibition of the reaction by CO and by SKF 525A and the absolute dependence on NADPH and oxygen indicate that cytochrome P-450 catalyzes the reaction. The apparent Km for oxygen was 0.07 microM. Furt...... microM) and chloramphenicol (Ki, 20 microM), whereas a mixed-type inhibition by isoniazid was observed (Ki, 1,3 mM and Kii, 10,6 mM)....

  2. Protective properties of butanolic extract of the Calendula officinalis L. (marigold) against lipid peroxidation of rat liver microsomes and action as free radical scavenger.

    Science.gov (United States)

    Cordova, Clarissa A S; Siqueira, Ionara R; Netto, Carlos A; Yunes, Rosendo A; Volpato, Ana M; Cechinel Filho, Valdir; Curi-Pedrosa, Rozangela; Creczynski-Pasa, Tânia B

    2002-01-01

    Calendula officinalis (marigold) has many pharmacological properties. It is used for the treatment of skin disorders, pain and also as a bactericide, antiseptic and anti-inflammatory. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are known to participate in the pathogenesis of various human diseases and may be involved in the conditions which C. officinalis is used to treat. The aim of this study was to investigate the relationship between the beneficial properties of this plant and its antioxidant action. The butanolic fraction (BF) was studied because it is non-cytotoxic and is rich in a variety of bioactive metabolites including flavonoids and terpenoids. Superoxide radicals (O(2)(*-)) and hydroxyl radicals (HO(*)) are observed in decreasing concentrations in the presence of increasing concentrations of BF with IC(50) values of 1.0 +/- 0.09 mg/ml and 0.5 +/- 0.02 mg/ml, respectively, suggesting a possible free radical scavenging effect. Lipid peroxidation in liver microsomes induced by Fe(2+)/ascorbate was 100% inhibited by 0.5 mg/ml of BF (IC(50) = 0.15 mg/ml). Its total reactive antioxidant potential (TRAP) (in microM Trolox equivalents) was 368.14 +/- 23.03 and its total antioxidant reactivity (TAR) was calculated to be 249.19 +/- 14.5 microM. The results obtained suggest that the butanolic fraction of C. officinalis possesses a significant free radical scavenging and antioxidant activity and that the proposed therapeutic efficacy of this plant could be due, in part, to these properties.

  3. In vitro metabolism of cis- and trans-permethrin by rat liver microsomes, and its effect on estrogenic and anti-androgenic activities.

    Science.gov (United States)

    Tange, Satoko; Fujimoto, Nariaki; Uramaru, Naoto; Sugihara, Kazumi; Ohta, Shigeru; Kitamura, Shigeyuki

    2014-05-01

    Permethrin is a widely applied broad-spectrum pyrethroid insecticide that consists of a mixture of cis- and trans-isomers. We examined the changes of estrogenic and anti-androgenic activities resulting from metabolism of the isomers. Both cis- and trans-permethrin were hydrolyzed to 3-phenoxybenzyl alcohol (PBAlc) by rat liver microsomes, but the extent of hydrolysis of trans-permethrin was much greater than that of the cis-isomer. In the presence of NADPH, PBAlc was further transformed to 4'-hydroxylated PBAlc (4'-OH PBAlc), 3-phenoxybenzaldehyde (PBAld) and 3-phenoxybenzoic acid (PBAcid). cis-Permethrin, but not trans-permethrin, also afforded its 4'-hydroxylated derivative (4'-OH cis-permethrin). trans-Permethrin was an anti-androgen, but also showed weak estrogenic activity, while cis-permethrin was a weak estrogen and a weak anti-androgen. After incubation with rat liver microsomes in the presence of NADPH, cis-permethrin but not trans-permethrin was metabolically activated for estrogenic activity. On the other hand, estrogenic activity of trans-permethrin was not changed, but its anti-androgenic activity was enhanced after incubation. 4'-OH PBAlc and PBAlc showed estrogenic activity, while PBAld and PBAlc showed anti-androgenic activity. PBAcid showed neither activity. 4'-OH cis-permethrin showed both estrogenic and anti-androgenic activities. Overall, our results indicate that permethrin is metabolically activated for estrogenic and anti-androgen activities, and the microsomal transformation of permethrin to 4'-OH cis-permethrin, 4'-OH PBAlc and PBAlc contributes to the both metabolic activations. Copyright © 2014. Published by Elsevier B.V.

  4. Determination of species-difference in microsomal metabolism of amitriptyline using a predictive MRM-IDA-EPI method.

    Science.gov (United States)

    Lee, Ji-Yoon; Lee, Sang Yoon; Lee, KiHo; Oh, Soo Jin; Kim, Sang Kyum

    2015-03-05

    We investigated to compare species differences in amitriptyline (AMI) metabolism among mouse, rat, dog, and human liver microsomes. We developed a method for simultaneous determination of metabolic stability and metabolite profiling using predictive multiple reaction monitoring information-dependent acquisition-enhanced product ion (MRM-IDA-EPI) scanning. In the cofactor-dependent microsomal metabolism study, AMI was metabolized more rapidly in rat and human liver microsomes incubated with NADPH than UDPGA. AMI incubated with NADPH+UDPGA in rat, dog, or mouse liver microsomes disappeared rapidly with a half-life of 3.5, 8.4, or 9.2 min, respectively, but slowly in human liver microsomes with a half-life of 96 min. In total, 9, 10, 11, and 6 putative metabolites of AMI were detected in mouse, rat, dog, and human liver microsomes, respectively, based on mass spectrometric analyses. Kinetic analysis of metabolites in liver microsomes from each species over 120 min showed common metabolic routes of AMI, such as N-demethylation, hydroxylation, and glucuronidation, and subtle interspecies differences in AMI metabolism. The main metabolic routes in mouse, rat, dog, and human liver microsomes were hydroxylation followed by glucuronide conjugation, methyl hydroxylation, and N-demethylation, respectively. The MRM-IDA-EPI method can provide quantitative and qualitative information about metabolic stability and metabolite profiling simultaneously. Moreover, time course analysis of metabolites can not only eliminate false identification of metabolites, but also provide a rationale for proposed metabolic pathways. The MRM-IDA-EPI method combined with time course analysis of metabolites is useful for investigating drug metabolism at the early drug discovery stage. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  5. Hepatocellular necrosis, fibrosis and microsomal activity determine the hepatic pharmacokinetics of basic drugs in right-heart-failure-induced liver damage.

    Science.gov (United States)

    Li, Peng; Robertson, Thomas A; Zhang, Qian; Fletcher, Linda M; Crawford, Darrell H G; Weiss, Michael; Roberts, Michael S

    2012-06-01

    To explore how liver damage arising from cardio-hepatic syndromes in RHF affect the hepatic pharmacokinetics of basic drugs. The hepatic pharmacokinetics of five selected basic drugs with different physicochemical properties were studied in IPRL from control rats and rats with RHF. Hepatic pharmacokinetic modelling was performed with a two-phase physiologically-based organ pharmacokinetic model with the vascular space and dispersion evaluated with the MID technique. The liver damage arising from RHF was assessed by changes in liver biochemistry and histopathology. The expression of various CYP isoforms was evaluated by real-time RT-PCR analysis. Four of the five basic drugs had a significantly lower E in RHF rat livers compared to the control rat livers. Hepatic pharmacokinetic analysis showed that both the CL int and PS were significantly decreased in the RHF rat livers. Stepwise regression analysis showed that the alterations in the pharmacokinetic parameters (E, CL int and PS) can be correlated to the observed histopathological changes (NI, CYP concentration and FI) as well as to the lipophilicity of the basic drugs (logP app). Serious hepatocellular necrosis and fibrosis induced by RHF affects both hepatic microsomal activity and hepatocyte wall permeability, leading to significant impairment in the hepatic pharmacokinetics of basic drugs.

  6. The identification of lobeglitazone metabolites in rat liver microsomes and the kinetics of the in vivo formation of the major metabolite M1 in rats.

    Science.gov (United States)

    Lee, Jong-Hwa; Ahn, Sung Hoon; Maeng, Han-Joo; Lee, Wooin; Kim, Dae-Duk; Chung, Suk-Jae

    2015-11-10

    The objective of this study was to elucidate the chemical structure of the metabolites derived from lobeglitazone (LB) during its incubation with rat liver microsomes and to characterize the kinetics of formation of the major metabolite M1 in vivo. Using high performance liquid chromatography coupled with a hybrid quadrupole linear ion trap, the metabolites were derived from LB during its incubation with rat liver microsomes. From various fragmentation patterns obtained from the metabolites, LB was biotransformed into 5 metabolites in the incubation, in which demethylation and hydroxylation appeared to be the principle metabolic pathways in vitro; Amongst the five primary metabolites, M1, a demethylated derivative of LB, appeared to be the major metabolite of LB, based on a comparison on the peak intensities in the ion chromatogram. In a study of the in vivo kinetics of formation of M1 in rats, the rate of formation of M1 from LB was determined to be 0.252 and 0.216mL/min/kg at doses of 0.5mg/kg and 2mg/kg of LB, respectively, suggesting that the kinetics of M1 formation were linear in the dose range tested. Considering the fact that LB is primarily eliminated by hepatic metabolism in rats, the formation of M1 accounts for approximately 7.50-9.76% of the overall elimination of LB in rats. Copyright © 2015 Elsevier B.V. All rights reserved.

  7. Report on Liver Cell Transplantation Using Human Fetal Liver Cells.

    Science.gov (United States)

    Pietrosi, Giada; Chinnici, Cinzia

    2017-01-01

    In an era of organ shortage, human fetuses donated after medically indicated abortion could be considered a potential liver donor for hepatic cell isolation. We investigated transplantation of fetal liver cells as a strategy to support liver functionality in end-stage liver disease. Here, we report our protocol of human fetal liver cells (hFLC) isolation in fetuses from 17 to 22 gestational weeks, and our clinical procedure of hFLC transplantation through the splenic artery.

  8. Changes in the plasmatic membrane characteristics during microsomal monooxygenase induction in the liver of adult and old rats.

    Science.gov (United States)

    Frolkis, V V; Kobzar, A L; Paramonova, G I

    1995-05-12

    The experiments on adult (6-8 months) and old (24-26 months) male Wistar rats have shown that treatment of animals with phenobarbital results in a significant increase in hepatic microsomal enzyme content, plasmatic membrane Na+, K(+)-ATPase activities and the elevation of hepatocyte membrane potential value. It is presumed that the changes in plasmatic membrane characteristics during microsomal monooxygenase induction are related to the synthesis of specific intracellular factors (invertors). This assumption was verified by the experiments with 'cellular hybrid' system (cytosol--plasmatic membranes). Using this cross-systems, it was shown that the hepatocyte cytosol of rats treated with phenobarbital produced Na+, K(+)-ATPase activity. The extent of Na+, K(+)-ATPase activation was essentially lower when cytosol derived from old rat hepatocytes was used. The presence of specific factors that activated Na+, K(+)-ATPase in hepatocyte plasmatic membrane was also discovered in blood serum of induced adult and old rats.

  9. Development of monoclonal antibodies to human microsomal epoxide hydrolase and analysis of “preneoplastic antigen”-like molecules

    Energy Technology Data Exchange (ETDEWEB)

    Duan, Hongying [Department of Microbiology, Faculty of Medicine, Saitama Medical University, Moroyama-cho, Iruma-gun, Saitama 350-0495 (Japan); Yoshimura, Kazunori [Department of Physiology, Faculty of Medicine, Saitama Medical University, Moroyama-cho, Iruma-gun, Saitama 350-0495 (Japan); Kobayashi, Nobuharu; Sugiyama, Kazuo [Department of Microbiology, Faculty of Medicine, Saitama Medical University, Moroyama-cho, Iruma-gun, Saitama 350-0495 (Japan); Sawada, Jun-ichi; Saito, Yoshiro [Division of Biochemistry and Immunochemistry, National Institute of Health Sciences, Kamiyoga 1-18-1, Setagaya-ku, Tokyo 158-8501 (Japan); Morisseau, Christophe; Hammock, Bruce D. [Department of Entomology and Cancer Center, University of California, Davis, One Shields Avenue, Davis, CA 95616-8584 (United States); Akatsuka, Toshitaka, E-mail: akatsuka@saitama-med.ac.jp [Department of Microbiology, Faculty of Medicine, Saitama Medical University, Moroyama-cho, Iruma-gun, Saitama 350-0495 (Japan)

    2012-04-01

    Microsomal epoxide hydrolase (mEH) is a drug metabolizing enzyme which resides on the endoplasmic reticulum (ER) membrane and catalyzes the hydration of reactive epoxide intermediates that are formed by cytochrome P450s. mEH is also thought to have a role in bile acid transport on the plasma membrane of hepatocytes. It is speculated that efficient execution of such multiple functions is secured by its orientation and association with cytochrome P450 enzymes on the ER membrane and formation of a multiple transport system on the plasma membrane. In certain disease status, mEH loses its association with the membrane and can be detected as distinct antigens in the cytosol of preneoplastic foci of liver (preneoplastic antigen), in the serum in association with hepatitis C virus infection (AN antigen), or in some brain tumors. To analyze the antigenic structures of mEH in physiological and pathological conditions, we developed monoclonal antibodies against different portions of mEH. Five different kinds of antibodies were obtained: three, anti-N-terminal portions; one anti-C-terminal; and one, anti-conformational epitope. By combining these antibodies, we developed antigen detection methods which are specific to either the membrane-bound form or the linearized form of mEH. These methods detected mEH in the culture medium released from a hepatocellular carcinoma cell line and a glioblastoma cell line, which was found to be a multimolecular complex with a unique antigenic structure different from that of the membrane-bound form of mEH. These antibodies and antigen detection methods may be useful to study pathological changes of mEH in various human diseases. -- Highlights: ► Monoclonal antibodies against different portions of mEH were developed. ► They discriminate between the membrane-bound and the linearized forms of mEH. ► We analyze the antigenic structure of the altered form of mEH in tumor cells. ► Preneoplastic antigen is a multimolecular complex of mEH with

  10. Use of the human monocytic leukemia THP-1 cell line and co-incubation with microsomes to identify and differentiate hapten and prohapten sensitizers.

    Science.gov (United States)

    Chipinda, Itai; Ruwona, Tinashe B; Templeton, Steven P; Siegel, Paul D

    2011-02-27

    Consumer and medical products can contain leachable chemical allergens which can cause skin sensitization. Recent efforts have been directed at the development of non-animal based tests such as in vitro cell activation assays for the identification of skin sensitizers. Prohapten identification by in vitro assays is still problematic due to the lack of prohapten bioactivation. The present study evaluated the effect of hapten and prohapten exposure on cell surface markers expression (CD86, CD54 and CD40) in the human monocytic leukemia, THP-1, cell line. Upregulation of activation and costimulatory markers are key events in the allergic sensitization process and have been reported to serve as indicators of skin sensitization. Cells were exposed to the prohaptens benzo(a)pyrene (BaP), 7,12-dimethylbenz(a)anthracene (DMBA), carvone oxime (COx), cinnamic alcohol (CA) and isoeugenol (IEG) at concentrations ranging from 1 to 10 μM for 24 and 48 h. The direct-binding haptens dinitrochlorobenzene (DNCB), benzoquinone (BQ), hydroxylethyl acrylate (HEA) and benzylbromide (BB) were used as positive controls. Cells were also exposed to the irritants sodium dodecyl sulfate (SDS) and sulfanilamide (SFA). Bioactivation of prohaptens was achieved by adding aroclor-induced rat liver microsomes (S9) to the cell cultures. Consistent upregulation of surface expressions of CD86, CD54 (ICAM-1) and CD40 was observed in THP-1 cells treated with direct-acting haptens (±S9) or prohapten (+S9). Upregulation of these markers was not observed after exposure to skin irritants or prohaptens in the absence of exogenously added S9. In conclusion, modification of in vitro cell culture assays to include co-incubation with microsomes enhances identification of prohaptens and allows them to be clearly distinguished from direct-binding haptens. Published by Elsevier Ireland Ltd.

  11. Characterization of ethanol-inducible human liver N-nitrosodimethylamine demethylase

    Energy Technology Data Exchange (ETDEWEB)

    Wrighton, S.A.; Thomas P.E.; Molowa, D.T.; Haniu, M.; Shively, J.E.; Maines, S.L.; Watkins, P.B.; Parker, G.; Mendez-Picon, G.; Levin, W.; Guzelian, P.S.

    1986-11-04

    Through the use of monospecific antibodies directed against hepatic cytochrome P-450j, an enzyme induced in rats treated with ethanol or isoniazid, we have purified from human liver the related cytochrome P-450 termed HLj. HLj resembles rat P-450j and P-450 LM3a, the homologous cytochrome in rabbit liver, in its NH/sub 2/-terminal amino acid sequence, in being in highest concentration in liver microsome samples prepared from two patients intoxicated by ethanol and one patient given isoniazid, and in catalyzing the metabolic activation of the procarcinogen N-nitrosodimethylamine. Furthermore, each of nine human liver RNA samples contained a species of mRNA hybridizable to a cloned HLj cDNA. We conclude that HLj is related by structure, function, and some regulator characteristics to rat P-450j and rabbit P-450 LM3a, cytochromes critical for metabolism of several clinically relevant cytotoxic and carcinogenic agents.

  12. Gas chromatography-mass spectrometric study of 19-oxygenation of the aromatase inhibitor 19-methylandrostenedione with human placental microsomes.

    Science.gov (United States)

    Numazawa, Mitsuteru; Nagaoka, Masao; Handa, Wakako; Yamada, Akane

    2006-06-01

    To gain insight into the catalytic function of aromatase, we studied 19-oxygenation of 19-methyl-substituted derivative of the natural substrate androstenedione (AD), compound 1, with human placental aromatase by use of gas chromatography-mass spectrometry (GC-MS). Incubation of the 19-methyl derivative 1 with human placental microsomes in the presence of NADPH under an aerobic condition did not yield a detectable amount of [19S]19-hydroxy product 2 or its [19R]-isomer 3 when the product was analyzed as the bis-methoxime-trimethylsilyl (TMS) derivative by GC-MS; moreover, the production of estrogen was not detected as the bis-TMS derivative of estradiol (detection limit: about 3 ng and 10 pg per injection for the 19-ol and estradiol, respectively). The results reveal that the 19-methyl steroid 1 does not serve as a substrate of aromatase, although it does serve as a powerful inhibitor of the enzyme.

  13. Influence of Sulforaphane Metabolites on Activities of Human Drug-Metabolizing Cytochrome P450 and Determination of Sulforaphane in Human Liver Cells.

    Science.gov (United States)

    Vanduchova, Alena; Tomankova, Veronika; Anzenbacher, Pavel; Anzenbacherova, Eva

    2016-12-01

    The influence of metabolites of sulforaphane, natural compounds present in broccoli (Brassica oleracea var. botrytis italica) and in other cruciferous vegetables, on drug-metabolizing cytochrome P450 (CYP) enzymes in human liver microsomes and possible entry of sulforaphane into human hepatic cells were investigated. Metabolites studied are compounds derived from sulforaphane by the mercapturic acid pathway (conjugation with glutathione and by following reactions), namely sulforaphane glutathione and sulforaphane cysteine conjugates and sulforaphane-N-acetylcysteine. Their possible effect on four drug-metabolizing CYP enzymes, CYP3A4 (midazolam 1'-hydroxylation), CYP2D6 (bufuralol 1'-hydroxylation), CYP1A2 (7-ethoxyresorufin O-deethylation), and CYP2B6 (7-ethoxy-4-(trifluoromethyl)coumarin O-deethylation), was tested. Inhibition of four prototypical CYP activities by sulforaphane metabolites was studied in pooled human liver microsomes. Sulforaphane metabolites did not considerably affect biological function of drug-metabolizing CYPs in human liver microsomes except for CYP2D6, which was found to be inhibited down to 73-78% of the original activity. Analysis of the entry of sulforaphane into human hepatocytes was done by cell disruption by sonication, methylene chloride extraction, and modified high-performance liquid chromatography method. The results have shown penetration of sulforaphane into the human hepatic cells.

  14. The role and regulation of the peroxisome proliferator activated receptor alpha in human liver.

    Science.gov (United States)

    Kersten, Sander; Stienstra, Rinke

    2017-05-01

    The peroxisome proliferator-activated receptor α (PPARα) is a ligand-activated transcription factor that is abundantly expressed in liver. PPARα is activated by fatty acids and various other lipid species, as well as by a class of chemicals referred to as peroxisome proliferators. Studies in mice have shown that PPARα serves as the master regulator of hepatic lipid metabolism during fasting. In addition, PPARα suppresses inflammation and the acute phase response. Comparatively little is known about PPARα in human liver. Here, an overview is provided of the role and regulation of PPARα in human liver. The main outcomes are: 1) the level of PPARA mRNA expression in human and mouse liver is similar. 2) Expression of PPARA in human liver is reduced in patients with non-alcoholic steatohepatitis or infected with the hepatitis C virus. 3) PPARα in human liver is able to effectively induce the expression of numerous genes involved in numerous lipid metabolic pathways, including microsomal, peroxisomal and mitochondrial fatty acid oxidation, fatty acid binding and activation, fatty acid elongation and desaturation, synthesis and breakdown of triglycerides and lipid droplets, lipoprotein metabolism, gluconeogenesis, bile acid metabolism, and various other metabolic pathways and genes. 4) PPARα activation in human liver causes the down-regulation of a large number of genes involved in various immunity-related pathways. 5) Peroxisome proliferators do not promote tumour formation in human liver as opposed to mouse liver because of structural and functional differences between human and mouse PPARα. 6) In addition to helping to correct dyslipidemia, PPARα agonists may hold promise as a therapy for patients with cholestatic liver diseases, non-alcoholic fatty liver disease, and/or type 2 diabetes. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  15. In vitro screening of reversible and time-dependent inhibition on CYP3A by TM208 and TM209 in rat liver microsomes

    Directory of Open Access Journals (Sweden)

    Miaoran Ning

    2012-04-01

    Full Text Available TM208 and TM209, dithiocarbamate derivatives with potential anti-cancer effects, were evaluated in reversible and time-dependent cytochrome P450 (CYP 3A inhibition assays in rat liver microsomes using testosterone as probe substrate. Both compounds were found to be weak reversible inhibitors and moderate mechanism-based inhibitors of rat CYP3A. For reversible inhibition on rat CYP3A, the Ki values of competitive inhibition model were 12.10±1.75 and 13.94±1.31 μM, respectively. For time-dependent inhibition, the inactivation constants (Kl were 31.93±12.64 and 32.91±15.58 μM, respectively, and the maximum inactivation rates (kinact were 0.03497±0.0069 and 0.07259±0.0172 min−1 respectively. These findings would provide useful in vitro information for future in vivo DDI studies on TM208 or TM209.

  16. Stereoselective sulfoxidation of the pesticide methiocarb by flavin-containing monooxygenase and cytochrome P450-dependent monooxygenases of rat liver microsomes. Anticholinesterase activity of the two sulfoxide enantiomers.

    Science.gov (United States)

    Buronfosse, T; Moroni, P; Benoît, E; Rivière, J L

    1995-08-01

    Evidence based on thermal lability and enzyme inhibition data suggests that the sulfoxidation of methiocarb (an N-methylcarbamate insecticide) by rat liver microsomes is catalyzed by flavin-containing monooxygenase(s) (FMO) and by cytochrome(s) P450 (P450). In control rats, the relative proportion is ca. 50% P450:50% FMO. Stereoselective formation of methiocarb sulfoxide from the corresponding sulfide has also been examined to compare the enantioselectivity of the two different enzyme systems. Only the FMO-dependent sulfoxidation presents a high stereoselectivity with an enantiomeric excess of 88% in favor of the (A)-enantiomer. Pretreatment of rats with different P450 inducers such as phenobarbital, 3-methylcholanthrene, dexamethasone, and pyrazole did not affect, or decreased, the rate of methiocarb sulfoxidation. Stereoselectivity of the reaction was modified, mainly because of changes in the relative involvement of FMO and P450 in sulfoxidase activity in pretreated animals. The acetylcholinesterase inhibition properties of methiocarb and its main metabolites were also investigated. Racemic methiocarb sulfoxide was slightly less inhibitory (Ki = 0.216 microM-1.min-1) than methiocarb, but a 10-fold difference was observed between the bimolecular rate constants found for the two sulfoxides produced (0.054 and 0.502 microM-1.min-1 for the (A) and (B) enantiomers, respectively).

  17. Mice with humanized liver endothelium

    NARCIS (Netherlands)

    el Filali, E.

    2014-01-01

    The only curative treatment option for a large proportion of patients suffering from a liver disorder is liver transplantation. The use of ex vivo genetically modified autologous liver cells instead of whole liver transplantation could overcome the problem of donor scarcity. Even though clinical

  18. Inhibition of rat microsomal lipid peroxidation by the oral administration of D002

    Directory of Open Access Journals (Sweden)

    Menéndez R.

    2000-01-01

    Full Text Available The effect of D002, a defined mixture of higher primary alcohols purified from bee wax, on in vivo and in vitro lipid peroxidation was studied. The extent of lipid peroxidation was measured on the basis of the levels of thiobarbituric acid reactive substances (TBARS. When D002 (5-100 mg/kg body weight was administered orally to rats for two weeks, a partial inhibition of the in vitro enzymatic and non-enzymatic lipid peroxidation was observed in liver and brain microsomes. Maximal protection (46% occurred at a dose of 25 mg/kg. D002 behaved differently depending on both the presence of NADPH and the integrity of liver microsomes, which suggests that under conditions where microsomal metabolism was favored the protective effect of D002 was increased. D002 (25 mg/kg also completely inhibited carbon tetrachloride- and toluene-induced in vivo lipid peroxidation in liver and brain. Also, D002 significantly lowered in a dose-dependent manner the basal level of TBARS in liver (19-40% and brain (28-44% microsomes. We conclude that the oral administration of D002 (5, 25 and 100 mg/kg for two weeks protected rat liver and brain microsomes against microsomal lipid peroxidation in vitro and in vivo. Thus, D002 could be useful as a dietary natural antioxidant supplement. More studies are required before these data can be extrapolated to the recommendation for the use of D002 as a dietary antioxidant supplement for humans.

  19. Chimeric mice with humanized liver: Application in drug metabolism and pharmacokinetics studies for drug discovery.

    Science.gov (United States)

    Naritomi, Yoichi; Sanoh, Seigo; Ohta, Shigeru

    2017-11-09

    Predicting human drug metabolism and pharmacokinetics (PK) is key to drug discovery. In particular, it is important to predict human PK, metabolite profiles and drug-drug interactions (DDIs). Various methods have been used for such predictions, including in vitro metabolic studies using human biological samples, such as hepatic microsomes and hepatocytes, and in vivo studies using experimental animals. However, prediction studies using these methods are often inconclusive due to discrepancies between in vitro and in vivo results, and interspecies differences in drug metabolism. Further, the prediction methods have changed from qualitative to quantitative to solve these issues. Chimeric mice with humanized liver have been developed, in which mouse liver cells are mostly replaced with human hepatocytes. Since human drug metabolizing enzymes are expressed in the liver of these mice, they are regarded as suitable models for mimicking the drug metabolism and PK observed in humans; therefore, these mice are useful for predicting human drug metabolism and PK. In this review, we discuss the current state, issues, and future directions of predicting human drug metabolism and PK using chimeric mice with humanized liver in drug discovery. Copyright © 2017 The Japanese Society for the Study of Xenobiotics. Published by Elsevier Ltd. All rights reserved.

  20. Stoichiometries of transferrin receptors 1 and 2 in human liver.

    Science.gov (United States)

    Chloupková, Maja; Zhang, An-Sheng; Enns, Caroline A

    2010-01-15

    Mutations in either the hereditary hemochromatosis protein, HFE, or transferrin receptor 2, TfR2, result in a similarly severe form of the most common type of iron overload disease called hereditary hemochromatosis. Models of the interactions between HFE, TfR1, and TfR2 imply that these proteins are present in different molar concentrations in the liver, where they control expression of the iron regulatory hormone, hepcidin, in response to body iron loading. The aim of this study was to determine in vivo levels of mRNA by quantitative RT-PCR and concentrations of these proteins by quantitative immunoblotting in human liver tissues. The level of TfR2 mRNA was 21- and 63-fold higher than that of TfR1 and HFE, respectively. Molar concentration of TfR2 protein was the highest and determined to be 1.95 nmol/g protein in whole cell lysates and 10.89 nmol/g protein in microsomal membranes. Molar concentration of TfR1 protein was 4.5- and 6.1-fold lower than that of TfR2 in whole cell lysates and membranes, respectively. The level of HFE protein was below 0.53 nmol/g of total protein. HFE is thus present in substoichiometric concentrations with respect to both TfR1 and TfR2 in human liver tissue. This finding supports a model, in which availability of HFE is limiting for formation of complexes with TfR1 or TfR2. Copyright 2009 Elsevier Inc. All rights reserved.

  1. Aromatization of 16alpha-hydroxyandrostenedione by human placental microsomes: effect of preincubation with suicide substrates of androstenedione aromatization.

    Science.gov (United States)

    Numazawa, Mitsuteru; Tachibana, Mii; Mutsumi, Ayako; Yoshimura, Akiko; Osawa, Yoshio

    2002-06-01

    Estrogen synthase (aromatase) catalyzes the aromatization of androstenedione (AD) as well as 16alpha-hydroxyandrostenedione (16alpha-OHAD) leading to estrone and estriol, respectively. We found that several steroid analogs including 4-hydroxyandrostenedione (1), 6-oxoandrostenedione (6-oxoAD, 2) and its 19-hydroxy analog (3), 10beta-acetoxyestr-5-ene-7,17-dione (4), androst-5-ene-4,7,17-trione (5), and 17alpha-ethynyl-19-norteststerone (6), which are known suicide inactivators of AD aromatization, are not effective in inactivating 16alpha-OHAD aromatization in a time-dependent manner. The compounds were tested with the use of human placental microsomes and 1beta-tritiated-16alpha-OHAD as the substrate. The results of the tritium water method of 16alpha-OHAD aromatization was confirmed by the gas chromatography-mass spectrometry (GC-MS) method of estriol formation. The 1beta-tritiated-AD was used to measure AD aromatization as a positive control for these experiments. The compounds were tested at concentrations up to 40-fold higher than the K(i)'s determined for inhibition of AD aromatization. These studies suggest that differences exist in the binding site structures responsible for aromatization of 16alpha-OHAD and AD.

  2. Human microsomal carbonyl reducing enzymes in the metabolism of xenobiotics: well-known and promising members of the SDR superfamily.

    Science.gov (United States)

    Skarydová, Lucie; Wsól, Vladimír

    2012-05-01

    The best known, most widely studied enzyme system in phase I biotransformation is cytochrome P450 (CYP), which participates in the metabolism of roughly 9 of 10 drugs in use today. The main biotransformation isoforms of CYP are associated with the membrane of the endoplasmatic reticulum (ER). Other enzymes that are also active in phase I biotransformation are carbonyl reducing enzymes. Much is known about the role of cytosolic forms of carbonyl reducing enzymes in the metabolism of xenobiotics, but their microsomal forms have been mostly poorly studied. The only well-known microsomal carbonyl reducing enzyme taking part in the biotransformation of xenobiotics is 11β-hydroxysteroid dehydrogenase 1, a member of the short-chain dehydrogenase/reductase superfamily. Physiological roles of microsomal carbonyl reducing enzymes are better known than their participation in the metabolism of xenobiotics. This review is a summary of the fragmentary information known about the roles of the microsomal forms. Besides 11β-hydroxysteroid dehydrogenase 1, it has been reported, so far, that retinol dehydrogenase 12 participates only in the detoxification of unsaturated aldehydes formed upon oxidative stress. Another promising group of microsomal biotransformation carbonyl reducing enzymes are some members of 17β-hydroxysteroid dehydrogenases. Generally, it is clear that this area is, overall, quite unexplored, but carbonyl reducing enzymes located in the ER have proven very interesting. The study of these enzymes could shed new light on the metabolism of several clinically used drugs or they could become an important target in connection with some diseases.

  3. Human urine and plasma concentrations of bisphenol A extrapolated from pharmacokinetics established in in vivo experiments with chimeric mice with humanized liver and semi-physiological pharmacokinetic modeling.

    Science.gov (United States)

    Miyaguchi, Takamori; Suemizu, Hiroshi; Shimizu, Makiko; Shida, Satomi; Nishiyama, Sayako; Takano, Ryohji; Murayama, Norie; Yamazaki, Hiroshi

    2015-06-01

    The aim of this study was to extrapolate to humans the pharmacokinetics of estrogen analog bisphenol A determined in chimeric mice transplanted with human hepatocytes. Higher plasma concentrations and urinary excretions of bisphenol A glucuronide (a primary metabolite of bisphenol A) were observed in chimeric mice than in control mice after oral administrations, presumably because of enterohepatic circulation of bisphenol A glucuronide in control mice. Bisphenol A glucuronidation was faster in mouse liver microsomes than in human liver microsomes. These findings suggest a predominantly urinary excretion route of bisphenol A glucuronide in chimeric mice with humanized liver. Reported human plasma and urine data for bisphenol A glucuronide after single oral administration of 0.1mg/kg bisphenol A were reasonably estimated using the current semi-physiological pharmacokinetic model extrapolated from humanized mice data using algometric scaling. The reported geometric mean urinary bisphenol A concentration in the U.S. population of 2.64μg/L underwent reverse dosimetry modeling with the current human semi-physiological pharmacokinetic model. This yielded an estimated exposure of 0.024μg/kg/day, which was less than the daily tolerable intake of bisphenol A (50μg/kg/day), implying little risk to humans. Semi-physiological pharmacokinetic modeling will likely prove useful for determining the species-dependent toxicological risk of bisphenol A. Copyright © 2015 Elsevier Inc. All rights reserved.

  4. Comparative Study of Human Liver Ferritin and Chicken Liver by Moessbauer Spectroscopy. Preliminary Results

    Energy Technology Data Exchange (ETDEWEB)

    Oshtrakh, M. I. [Ural State Technical University - UPI, Division of Applied Biophysics, Faculty of Physical Techniques and Devices for Quality Control (Russian Federation); Milder, O. B.; Semionkin, V. A. [Ural State Technical University - UPI, Faculty of Experimental Physics (Russian Federation); Prokopenko, P. G. [Russian State Medical University, Faculty of Biochemistry (Russian Federation); Malakheeva, L. I. [Simbio Holding, Science Consultation Department (Russian Federation)

    2004-12-15

    A comparative study of normal human liver ferritin and livers from normal chicken and chicken with Marek disease was made by Moessbauer spectroscopy. Small differences of quadrupole splitting and isomer shift were found for human liver ferritin and chicken liver. Moessbauer parameters for liver from normal chicken and chicken with Marek disease were the same.

  5. Aromatization of androstenedione and 16alpha-hydroxyandrostenedione in human placental microsomes. Kinetic analysis of inhibition by the 19-oxygenated and 3-deoxy analogs.

    Science.gov (United States)

    Numazawa, Mitsuteru; Watari, Yoko; Komatsu, Sachiko; Yamashita, Kouwa; Nagaoka, Masao

    2008-11-01

    Inhibition of aromatase activity in human placental microsomes with androstenedione (AD) (1a) and its 19-oxygenated derivatives 1b and 1c, their 16alpha-hydroxy compounds 2 and 3, and 3-deoxyandrost-4-ene compounds 5 and 6 was studied using [1beta-(3)H]AD as a substrate and compared to that with [1beta-(3)H]16alpha-hydroxyandrostenedione (16-OHAD). AD series of steroids, compounds 1, inhibited competitively [1beta-(3)H]AD aromatization whereas other 16alpha-hydroxy steroids 2, 3, 5, and 6 inhibited AD aromatization in a non-competitive manner. On the other hand, all of 16-OHAD series, compounds 2, blocked the [1beta-(3)H]16-OHAD aromatization in a competitive manner whereas the AD series steroids 1 as well as the 3-deoxy-16alpha-hydroxy-17-one steroids 5 and 3-deoxy-16alpha,17beta-diol steroids 6 inhibited 16-OHAD aromatization non-competitively. 3-carbonyl and 16alpha-hydroxy functions of 16-OHAD play a critical role of selection of the 16-OHAD binding site. The results suggest that the AD derivatives 1 are kinetically aromatized at a different site from the 16-OHAD derivatives 2. Physical and/or chemical environments around the aromatase protein in the microsomal membrane may play a significant role in the expression of the substrate specificity, and the present results do not exclude the idea that the placental microsomes have a single binding site.

  6. AGE WISE HISTOMORPHOLOGICAL CHANGES IN HUMAN LIVER

    Directory of Open Access Journals (Sweden)

    Tribeni

    2015-11-01

    Full Text Available CONTEXT: Hepato cellular carcinoma (HCC results in between 2.5 lakhs to 1million deaths globally per annum. Liver transplantation nowadays is a well accepted treatment option for end-stage liver disease and acute liver failure. AIMS: Keeping this concept in view, a study was conducted in the Guwahati Zone of Northeast India, to compare the histomorphological features of the human liver in different age groups. SETTING AND DESIGN: Apparently healthy livers were obtained from 21 subjects on whom medicolegal post-mortems had been performed. Their ages varied from newborn to 90 years. Subjects were divided into 3 groups. 7 specimens were taken from each group. (1 Pediatric (2 Adult (3 Old age. METHODS AND MATERIALS: In all the above age groups, immediately after removal of the livers, they were washed in normal saline, dried with blotting paper and weighed in an electronic weighing machine. Sections of liver were fixed, processed, cut and stained with Harris Haematoxylin and Eosin stain. RESULTS: The liver loses weight from 50 years onwards. There appears to be racial and environmental differences in the change in liver weight in old age. Autopsy studies show a diminution of nearly 46% in liver weight between the 3rd and 10th decades of life. The liver decreases in size with age. The hepatocytes are radially disposed in the liver lobule. They are piled up, forming a layer one cell thick (except in young children in a fashion similar to the bricks of a wall. These plates are directed from the periphery of the lobule to its centre and anastomose freely forming a complex labyrinthine and sponge-like structure. CONCLUSIONS: From the findings in the present study it can be concluded that: 1. Nowadays, the measurement of liver volume has gained practical use in relation to liver transplantation. 2. We have compared the histomorphology of adult liver with a child. The findings in both the groups are very similar. This feature is important, since in

  7. EFFECTS OF THIOL ANTIOXIDANTS ON THE ATROPSELECTIVE OXIDATION OF 2,2′,3,3′,6,6′-HEXACHLOROBIPHENYL (PCB 136) BY RAT LIVER MICROSOMES

    Science.gov (United States)

    Wu, Xianai; Lehmler, Hans-Joachim

    2015-01-01

    Chiral polychlorinated biphenyl (PCB) congeners, such as PCB 136, are atropselectively metabolized to various hydroxylated PCB metabolites (HO-PCBs). The present study investigates the effect of two thiol antioxidants, glutathione and N-acetyl-cysteine (NAC), on profiles and chiral signatures of PCB 136 and its HO-PCB metabolites in rat liver microsomal incubations. Liver microsomes prepared from rats pretreated with phenobarbital were incubated with PCB 136 (5 μM) in the presence of the respective antioxidant (0–10 mM), and levels and chiral signatures of PCB 136 and its HO-PCB metabolites were determined. Three metabolites, 5-136 (2,2′,3,3′,6,6′-hexachlorobiphenyl-5-ol), 4-136 (2,2′,3,3′,6,6′-hexachlorobiphenyl-4-ol) and 4,5-136 (2,2′,3,3′,6,6′-hexachlorobiphenyl-4,5-diol), were detected in all incubations, with 5-136 being the major metabolite. Compared to microsomal incubations without antioxidant, levels of 4,5-136 increased with increasing antioxidant concentration, whereas levels of PCB 136 and both mono-HO-PCBs were not affected by the presence of either antioxidant. PCB 136, 4-136 and 5-136 displayed significant atropisomeric enrichment; however, the direction and extent of the atropisomeric enrichment was not altered in the presence of an antioxidant. Because 4,5-136 can either be conjugated to a sulfate or glucuronide metabolite that is readily excreted or further oxidized a potentially toxic PCB 136 quinone, the effect of both thiol antioxidants on 4,5-136 formation suggests that disruptions of glutathione homeostasis may alter the balance between both metabolic pathways and, thus, PCB 136 toxicity in vivo. PMID:26155892

  8. Characterization of phytanic acid omega-hydroxylation in human liver microsomes

    NARCIS (Netherlands)

    Komen, J. C.; Duran, M.; Wanders, R. J. A.

    2005-01-01

    Phytanic acid is a 3-methyl branched-chain fatty acid which originates from dietary sources. Since the 3-methyl group blocks regular beta-oxidation, it is broken down by peroxisomal alpha-oxidation. Adult Refsum disease patients accumulate phytanic acid as a result of an impairment in peroxisomal

  9. The Effect of Gender on the Rate of Metabolism of Midazolam in Humans Using Liver Microsomes

    Science.gov (United States)

    1997-05-01

    Sample chromatogram showing 4-hydroxy midazolam, midazolam, 1’-hydroxymidazolam, and Lorazepam 4 2 Figure 3. Representative curve depicting the...Cochrane, 1984). Other drugs that depress the central nervous system, such as narcotics or alcohol , can potentiate the sedative effect of MDZ...as a probe. She found that females had on average 24% more CYP3A activity than males, regardless of age, smoking history, alcohol use, or body

  10. Monoclonal antibodies reveal multiple forms of expression of human microsomal epoxide hydrolase

    Energy Technology Data Exchange (ETDEWEB)

    Duan, Hongying; Takagi, Akira [Department of Microbiology, Faculty of Medicine, Saitama Medical University, Moroyama-cho, Iruma-gun, Saitama 350-0495 (Japan); Kayano, Hidekazu [Department of Pathology, Faculty of Medicine, Saitama Medical University, Moroyama-cho, Iruma-gun, Saitama 350-0495 (Japan); Koyama, Isamu [Department of Digestive and General Surgery, Saitama International Medical Center, Faculty of Medicine, Saitama Medical University, 1397-1 Yamane, Hidaka, Saitama 350-1298 (Japan); Morisseau, Christophe; Hammock, Bruce D. [Department of Entomology and Cancer Center, University of California, Davis, One Shields Avenue, Davis, CA 95616-8584 (United States); Akatsuka, Toshitaka, E-mail: akatsuka@saitama-med.ac.jp [Department of Microbiology, Faculty of Medicine, Saitama Medical University, Moroyama-cho, Iruma-gun, Saitama 350-0495 (Japan)

    2012-04-01

    In a previous study, we developed five kinds of monoclonal antibodies against different portions of human mEH: three, anti-N-terminal; one, anti-C-terminal; one, anti-conformational epitope. Using them, we stained the intact and the permeabilized human cells of various kinds and performed flow cytometric analysis. Primary hepatocytes and peripheral blood mononuclear cells (PBMC) showed remarkable differences. On the surface, hepatocytes exhibited 4 out of 5 epitopes whereas PBMC did not show any of the epitopes. mEH was detected inside both cell types, but the most prominent expression was observed for the conformational epitope in the hepatocytes and the two N-terminal epitopes in PBMC. These differences were also observed between hepatocyte-derived cell lines and mononuclear cell-derived cell lines. In addition, among each group, there were several differences which may be related to the cultivation, the degree of differentiation, or the original cell subsets. We also noted that two glioblastoma cell lines reveal marked expression of the conformational epitope on the surface which seemed to correlate with the brain tumor-associated antigen reported elsewhere. Several cell lines also underwent selective permeabilization before flow cytometric analysis, and we noticed that the topological orientation of mEH on the ER membrane in those cells was in accordance with the previous report. However, the orientation on the cell surface was inconsistent with the report and had a great variation between the cells. These findings show the multiple mode of expression of mEH which may be possibly related to the multiple roles that mEH plays in different cells. -- Highlights: ► We examine expression of five mEH epitopes in human cells. ► Remarkable differences exist between hepatocytes and PBMC. ► mEH expression in cell lines differs depending on several factors. ► Some glioblastoma cell lines reveal marked surface expression of mEH. ► Topology of mEH on the cell

  11. Obesity accelerates epigenetic aging of human liver.

    Science.gov (United States)

    Horvath, Steve; Erhart, Wiebke; Brosch, Mario; Ammerpohl, Ole; von Schönfels, Witigo; Ahrens, Markus; Heits, Nils; Bell, Jordana T; Tsai, Pei-Chien; Spector, Tim D; Deloukas, Panos; Siebert, Reiner; Sipos, Bence; Becker, Thomas; Röcken, Christoph; Schafmayer, Clemens; Hampe, Jochen

    2014-10-28

    Because of the dearth of biomarkers of aging, it has been difficult to test the hypothesis that obesity increases tissue age. Here we use a novel epigenetic biomarker of aging (referred to as an "epigenetic clock") to study the relationship between high body mass index (BMI) and the DNA methylation ages of human blood, liver, muscle, and adipose tissue. A significant correlation between BMI and epigenetic age acceleration could only be observed for liver (r = 0.42, P = 6.8 × 10(-4) in dataset 1 and r = 0.42, P = 1.2 × 10(-4) in dataset 2). On average, epigenetic age increased by 3.3 y for each 10 BMI units. The detected age acceleration in liver is not associated with the Nonalcoholic Fatty Liver Disease Activity Score or any of its component traits after adjustment for BMI. The 279 genes that are underexpressed in older liver samples are highly enriched (1.2 × 10(-9)) with nuclear mitochondrial genes that play a role in oxidative phosphorylation and electron transport. The epigenetic age acceleration, which is not reversible in the short term after rapid weight loss induced by bariatric surgery, may play a role in liver-related comorbidities of obesity, such as insulin resistance and liver cancer.

  12. In vitro biotransformation of tris(2-butoxyethyl) phosphate (TBOEP) in human liver and serum

    Energy Technology Data Exchange (ETDEWEB)

    Van den Eede, Nele, E-mail: nele.vandeneede@uantwerpen.be [Toxicological Center, Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk, Antwerp (Belgium); Erratico, Claudio [Toxicological Center, Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk, Antwerp (Belgium); Exarchou, Vassiliki [Natural Products & Food Research and Analysis (NatuRA), Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk, Antwerp (Belgium); Maho, Walid; Neels, Hugo [Toxicological Center, Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk, Antwerp (Belgium); Covaci, Adrian, E-mail: adrian.covaci@uantwerpen.be [Toxicological Center, Department of Pharmaceutical Sciences, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk, Antwerp (Belgium)

    2015-04-15

    Tris(2-butoxyethyl) phosphate (TBOEP) is a plasticizer present in indoor dust, reaching levels of several micrograms per gram. Such levels could lead to significant daily exposure of adults and children. Currently, no toxicokinetic data are available to estimate TBOEP clearance in humans after uptake and therefore, one objective of this study was to investigate intrinsic clearance of TBOEP by human liver microsome (HLM) and serum enzymes. Another objective was to generate information to identify and prioritize several metabolites of TBOEP for investigation of human exposure by biomonitoring. 1D and 2D-NMR methodologies were successfully applied on a mixture of the metabolites to confirm the structure of 3-HO-TBOEP (bis(2-butoxyethyl) 3-hydroxyl-2-butoxyethyl phosphate) and to tentatively assign structures to 1-HO-TBOEP and 2-HO-TBOEP. HO-TBOEP isomers and bis(2-butoxyethyl) phosphate (BBOEP), bis(2-butoxyethyl) hydroxyethyl phosphate (BBOEHEP) were further monitored by liquid chromatography–tandem mass spectrometry. Rates of formation of BBOEHEP and HO-TBOEP metabolites by liver enzymes were best described by the Michaelis–Menten model. Apparent K{sub m} values for BBOEHEP, 3-HO-TBOEP, and sum of 1- and 2-HO-TBOEP isomer formation were 152, 197 and 148 μM, respectively. Apparent V{sub max} values for the formation of BBOEHEP, 3-HO-TBOEP, and the sum of 1- and 2-HO-TBOEP isomers were 2560, 643, and 254 pmol/min/mg protein, respectively. No detectable formation of BBOEP occurred with liver or serum enzymes. Our findings indicate that intrinsic clearance of TBOEP is mainly catalyzed by oxidative enzymes in the liver and that its major in vitro metabolite is BBOEHEP. These findings can be applied in human biomonitoring studies and risk assessment. - Highlights: • First steps in the elucidation of TBOEP toxicokinetics • Quantification of TBOEP metabolites in human serum and liver microsomes • No detectable formation of BBOEP occurred with liver or serum

  13. Liver Effects of Clinical Drugs Differentiated in Human Liver Slices

    Directory of Open Access Journals (Sweden)

    Alison E. M. Vickers

    2017-03-01

    Full Text Available Drugs with clinical adverse effects are compared in an ex vivo 3-dimensional multi-cellular human liver slice model. Functional markers of oxidative stress and mitochondrial function, glutathione GSH and ATP levels, were affected by acetaminophen (APAP, 1 mM, diclofenac (DCF, 1 mM and etomoxir (ETM, 100 μM. Drugs targeting mitochondria more than GSH were dantrolene (DTL, 10 μM and cyclosporin A (CSA, 10 μM, while GSH was affected more than ATP by methimazole (MMI, 500 μM, terbinafine (TBF, 100 μM, and carbamazepine (CBZ 100 μM. Oxidative stress genes were affected by TBF (18%, CBZ, APAP, and ETM (12%–11%, and mitochondrial genes were altered by CBZ, APAP, MMI, and ETM (8%–6%. Apoptosis genes were affected by DCF (14%, while apoptosis plus necrosis were altered by APAP and ETM (15%. Activation of oxidative stress, mitochondrial energy, heat shock, ER stress, apoptosis, necrosis, DNA damage, immune and inflammation genes ranked CSA (75%, ETM (66%, DCF, TBF, MMI (61%–60%, APAP, CBZ (57%–56%, and DTL (48%. Gene changes in fatty acid metabolism, cholestasis, immune and inflammation were affected by DTL (51%, CBZ and ETM (44%–43%, APAP and DCF (40%–38%, MMI, TBF and CSA (37%–35%. This model advances multiple dosing in a human ex vivo model, plus functional markers and gene profile markers of drug induced human liver side-effects.

  14. Decellularized human liver as a natural 3D-scaffold for liver bioengineering and transplantation

    Science.gov (United States)

    Mazza, Giuseppe; Rombouts, Krista; Rennie Hall, Andrew; Urbani, Luca; Vinh Luong, Tu; Al-Akkad, Walid; Longato, Lisa; Brown, David; Maghsoudlou, Panagiotis; Dhillon, Amar P.; Fuller, Barry; Davidson, Brian; Moore, Kevin; Dhar, Dipok; De Coppi, Paolo; Malago, Massimo; Pinzani, Massimo

    2015-01-01

    Liver synthetic and metabolic function can only be optimised by the growth of cells within a supportive liver matrix. This can be achieved by the utilisation of decellularised human liver tissue. Here we demonstrate complete decellularization of whole human liver and lobes to form an extracellular matrix scaffold with a preserved architecture. Decellularized human liver cubic scaffolds were repopulated for up to 21 days using human cell lines hepatic stellate cells (LX2), hepatocellular carcinoma (Sk-Hep-1) and hepatoblastoma (HepG2), with excellent viability, motility and proliferation and remodelling of the extracellular matrix. Biocompatibility was demonstrated by either omental or subcutaneous xenotransplantation of liver scaffold cubes (5 × 5 × 5 mm) into immune competent mice resulting in absent foreign body responses. We demonstrate decellularization of human liver and repopulation with derived human liver cells. This is a key advance in bioartificial liver development. PMID:26248878

  15. Discovery of a Novel Microsomal Epoxide Hydrolase-Catalyzed Hydration of a Spiro Oxetane.

    Science.gov (United States)

    Li, Xue-Qing; Hayes, Martin A; Grönberg, Gunnar; Berggren, Kristina; Castagnoli, Neal; Weidolf, Lars

    2016-08-01

    Oxetane moieties are increasingly being used by the pharmaceutical industry as building blocks in drug candidates because of their pronounced ability to improve physicochemical parameters and metabolic stability of drug candidates. The enzymes that catalyze the biotransformation of the oxetane moiety are, however, not well studied. The in vitro metabolism of a spiro oxetane-containing compound AZD1979 [(3-(4-(2-oxa-6-azaspiro[3.3]heptan-6-ylmethyl)phenoxy)azetidin-1-yl)(5-(4-ethoxyphenyl)-1,3,4-oxadiazol-2-yl)methanone] was studied and one of its metabolites, M1, attracted our interest because its formation was NAD(P)H independent. The focus of this work was to elucidate the structure of M1 and to understand the mechanism(s) of its formation. We established that M1 was formed via hydration and ring opening of the oxetanyl moiety of AZD1979. Incubations of AZD1979 using various human liver subcellular fractions revealed that the hydration reaction leading to M1 occurred mainly in the microsomal fraction. The underlying mechanism as a hydration, rather than an oxidation reaction, was supported by the incorporation of (18)O from H2 (18)O into M1. Enzyme kinetics were performed probing the formation of M1 in human liver microsomes. The formation of M1 was substantially inhibited by progabide, a microsomal epoxide hydrolase inhibitor, but not by trans-4-[4-(1-adamantylcarbamoylamino)cyclohexyloxy]benzoic acid, a soluble epoxide hydrolase inhibitor. On the basis of these results, we propose that microsomal epoxide hydrolase catalyzes the formation of M1. The substrate specificity of microsomal epoxide hydrolase should therefore be expanded to include not only epoxides but also the oxetanyl ring system present in AZD1979. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

  16. Activation and detoxification metabolism of urban air pollutants 2-nitrobenzanthrone and carcinogenic 3-nitrobenzanthrone by rat and mouse hepatic microsomes.

    Science.gov (United States)

    Stiborova, Marie; Cechova, Tereza; Borek-Dohalska, Lucie; Moserova, Michaela; Frei, Eva; Schmeiser, Heinz H; Paca, Jan; Arlt, Volker M

    2012-01-01

    2-Nitrobenzanthrone (2-NBA) has recently been detected in ambient air particulate matter. Its isomer 3-nitrobenzanthrone (3-NBA) is a potent mutagen and suspected human carcinogen identified in diesel exhaust. Understanding which enzymes are involved in metabolism of these toxicants is important in the assessment of individual susceptibility. Here, metabolism of 2-NBA and 3-NBA by rat and mouse hepatic microsomes containing cytochromes P450 (CYPs), their reductase (NADPH:CYP reductase), and NADH:cytochrome b5 reductase was investigated under anaerobic and aerobic conditions. In addition, using the same microsomal systems, 2-NBA and 3-NBA were evaluated to be enzymatically activated under anaerobic conditions to species generating 2-NBA- and 3-NBA-derived DNA adducts. High performance liquid chromatography (HPLC) with ultraviolet (UV) detection was employed for the separation and characterization of 2-NBA and 3-NBA metabolites formed by hepatic microsomes of rats and mice under the anaerobic and aerobic conditions. Microsomal systems isolated from the liver of the control (untreated) rats and rats pretreated with Sudan I, β-naphthoflavone (β-NF), phenobarbital (PB), ethanol and pregnenolon 16α-carbonitrile (PCN), the inducers of cytochromes P450 (CYP) 1A1, 1A1/2, 2B, 2E1 and 3A, respectively, were used in this study. Microsomes of mouse models, a control mouse line (wild-type, WT) and Hepatic Cytochrome P450 Reductase Null (HRN) mice with deleted gene of NADPH:CYP reductase in the liver, thus absenting this enzyme in their livers, were also employed. To detect and quantify the 2-NBA- and 3-NBA-derived DNA adducts, the 32P postlabeling technique was used. Both reductive metabolite of 3-NBA, 3-aminobenzanthrone (3-ABA), found to be formed predominantly under the anaerobic conditions, and two 3-NBA oxidative metabolites, whose structures have not yet been investigated, were formed by several microsomal systems used in the study. Whereas a 3-NBA reductive metabolite

  17. Dataset of protein species from human liver

    Directory of Open Access Journals (Sweden)

    Stanislav Naryzhny

    2017-06-01

    Full Text Available This article contains data related to the research article entitled “Zipf׳s law in proteomics” (Naryzhny et al., 2017 [1]. The protein composition in the human liver or hepatocarcinoma (HepG2 cells extracts was estimated using a filter-aided sample preparation (FASP protocol. The protein species/proteoform composition in the human liver was determined by two-dimensional electrophoresis (2-DE followed by Electrospray Ionization Liquid Chromatography-Tandem Mass Spectrometry (ESI LC-MS/MS. In the case of two-dimensional electrophoresis (2-DE, the gel was stained with Coomassie Brilliant Blue R350, and image analysis was performed with ImageMaster 2D Platinum software (GE Healthcare. The 96 sections in the 2D gel were selected and cut for subsequent ESI LC-MS/MS and protein identification. If the same protein was detected in different sections, it was considered to exist as different protein species/proteoforms. A list of human liver proteoforms detected in this way is presented.

  18. Bisphenol-A glucuronidation in human liver and breast: identification of UDP-glucuronosyltransferases (UGTs) and influence of genetic polymorphisms.

    Science.gov (United States)

    Street, Christina M; Zhu, Zhaohui; Finel, Moshe; Court, Michael H

    2017-01-01

    1. Bisphenol-A is a ubiquitous environmental contaminant that is primarily metabolized by glucuronidation and associated with various human diseases including breast cancer. Here we identified UDP-glucuronosyltransferases (UGTs) and genetic polymorphisms responsible for interindividual variability in bisphenol-A glucuronidation in human liver and breast. 2. Hepatic UGTs showing the highest bisphenol-A glucuronidation activity included UGT2B15 and UGT1A9. Relative activity factor normalization indicated that UGT2B15 contributes >80% of activity at bisphenol-A concentrations under 5 μM, while UGT1A9 contributes up to 50% of activity at higher concentrations. 3. Bisphenol-A glucuronidation by liver microsomes (46 donors) ranged from 0.25 to 4.3 nmoles/min/mg protein. Two-fold higher glucuronidation (p = 0.018) was observed in UGT1A9 *22/*22 livers compared with *1/*1 and *1/*22 livers. However, no associations were observed for UGT2B15*2 or UGT1A1*28 genotypes. 4. Bisphenol-A glucuronidation by breast microsomes (15 donors) ranged from bisphenol-A was highest for UGT1A1, followed by UGT2B4, UGT1A9, UGT1A10, UGT2B7 and UGT2B15. Bisphenol-A glucuronidation was over 10-fold lower in breast tissues with the UGT1A1*28 allele compared with tissues without this allele (p = 0.006). 5. UGT2B15 and UGT1A9 contribute to glucuronidation variability in liver, while UGT1A1 is important in breast.

  19. Acyl-CoA: cholesterol acyltransferase and 3-hydroxy-3-methylglutaryl-CoA reductase in carp-liver microsomes: effect of cold acclimation on enzyme activities and on hepatic and plasma lipid composition.

    Science.gov (United States)

    Teichert, T; Wodtke, E

    1992-12-02

    Hepatic microsomal activities of acyl-CoA:cholesterol acyltransferase (ACAT) and 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, rate-limiting enzymes in cholesterol esterification and cholesterol synthesis, and the concentration sand compartmentalization of esterified and unesterified cholesterol, were studied in carp acclimated to 10 and 30 degrees C. Irrespective of acclimation temperature, carp-liver ACAT is characterized by an apparent Km-value for oleoyl-CoA of 11-15 microM and displays an optimum activity at pH 7.4. The enzyme activity is reduced approx. 2-fold upon preincubation of microsomes with alkaline phosphatase. Arrhenius plots of ACAT-activity are curvilinear, with curvatures considerably affected by the acclimation temperature of the fish. Carp HMG-CoA reductase has been characterized previously by Teichert and Wodtke ((1987) Biochim. Biophys. Acta 920, 161-170). When measured at 30 degrees C, ACAT activities from 30 degrees C- and 10 degrees C-acclimated carp are identical (approx. 6 pmol/min per mg protein), whilst 'expressed' HMG-CoA reductase activity (18.1 +/- 12.2 pmol/min per mg protein for 30 degrees C-acclimated carp vs. 159.8 +/- 106.6 pmol/min per mg protein for 10 degrees C-acclimated carp) is enhanced 9-fold in the cold environment. This disparity indicates that cold-acclimation results in a massive increase in the capacity for hepatic cholesterol synthesis relative to hepatic cholesterol esterification. At the same time, hepatic compositional analysis reveals identical contents of unesterified cholesterol in either groups of carp but significantly decreased (3-fold) amounts in cholesterol ester (and also in triacylglycerol, 4-fold) in cold-acclimated carp. Moreover, microsomal fractions display lower cholesterol to phospholipid ratios in the cold. In contrast, concentrations of either cholesterol fractions (and of triacylglycerols) in plasma--the mobile compartment for lipoprotein transport--do not differ in cold- and warm

  20. Molecular Structure of Human-Liver Glycogen.

    Directory of Open Access Journals (Sweden)

    Bin Deng

    Full Text Available Glycogen is a highly branched glucose polymer which is involved in maintaining blood-sugar homeostasis. Liver glycogen contains large composite α particles made up of linked β particles. Previous studies have shown that the binding which links β particles into α particles is impaired in diabetic mice. The present study reports the first molecular structural characterization of human-liver glycogen from non-diabetic patients, using transmission electron microscopy for morphology and size-exclusion chromatography for the molecular size distribution; the latter is also studied as a function of time during acid hydrolysis in vitro, which is sensitive to certain structural features, particularly glycosidic vs. proteinaceous linkages. The results are compared with those seen in mice and pigs. The molecular structural change during acid hydrolysis is similar in each case, and indicates that the linkage of β into α particles is not glycosidic. This result, and the similar morphology in each case, together imply that human liver glycogen has similar molecular structure to those of mice and pigs. This knowledge will be useful for future diabetes drug targets.

  1. Induction of human microsomal prostaglandin E synthase 1 by activated oncogene RhoA GTPase in A549 human epithelial cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Choi, Hye Jin [Laboratory of Systems Mucosal Biomodulation, Department of Microbiology and Immunology, Pusan National University School of Medicine, Yangsan (Korea, Republic of); Lee, Dong-Hyung [Department of Obstetrics and Gynecology, Medical Research Institute, Pusan National University, Busan (Korea, Republic of); Park, Seong-Hwan; Kim, Juil; Do, Kee Hun [Laboratory of Systems Mucosal Biomodulation, Department of Microbiology and Immunology, Pusan National University School of Medicine, Yangsan (Korea, Republic of); An, Tae Jin; Ahn, Young Sup; Park, Chung Berm [Department of Herbal Crop Research, NIHHS, RDA, Eumseong (Korea, Republic of); Moon, Yuseok, E-mail: moon@pnu.edu [Laboratory of Systems Mucosal Biomodulation, Department of Microbiology and Immunology, Pusan National University School of Medicine, Yangsan (Korea, Republic of); Medical Research Institute and Research Institute for Basic Sciences, Pusan National University, Busan (Korea, Republic of)

    2011-09-30

    Highlights: {yields} As a target of oncogene RhoA-linked signal, a prostaglandin metabolism is assessed. {yields} RhoA activation increases PGE{sub 2} levels and its metabolic enzyme mPGES-1. {yields} RhoA-activated NF-{kappa}B and EGR-1 are positively involved in mPGES-1 induction. -- Abstract: Oncogenic RhoA GTPase has been investigated as a mediator of pro-inflammatory responses and aggressive carcinogenesis. Among the various targets of RhoA-linked signals, pro-inflammatory prostaglandin E{sub 2} (PGE{sub 2}), a major prostaglandin metabolite, was assessed in epithelial cancer cells. RhoA activation increased PGE{sub 2} levels and gene expression of the rate-limiting PGE{sub 2} producing enzymes, cyclooxygenase-2 and microsomal prostaglandin E synthase 1 (mPGES-1). In particular, human mPGES-1 was induced by RhoA via transcriptional activation in control and interleukin (IL)-1{beta}-activated cancer cells. To address the involvement of potent signaling pathways in RhoA-activated mPGES-1 induction, various signaling inhibitors were screened for their effects on mPGES-1 promoter activity. RhoA activation enhanced basal and IL-1{beta}-mediated phosphorylated nuclear factor-{kappa}B and extracellular signal-regulated kinase1/2 proteins, all of which were positively involved in RhoA-induced gene expression of mPGES-1. As one potent down-stream transcription factor of ERK1/2 signals, early growth response gene 1 product also mediated RhoA-induced gene expression of mPGES-1 by enhancing transcriptional activity. Since oncogene-triggered PGE{sub 2} production is a critical modulator of epithelial tumor cells, RhoA-associated mPGES-1 represents a promising chemo-preventive or therapeutic target for epithelial inflammation and its associated cancers.

  2. Cellular distribution and handling of liver-targeting preparations in human livers studied by a liver lobe perfusion.

    Science.gov (United States)

    Melgert, B N; Olinga, P; Weert, B; Slooff, M J; Meijer, D K; Poelstra, K; Groothuis, G M

    2001-06-01

    We developed and tested a novel method for perfusing parts of human liver to study uptake and handling of drug-targeting preparations. These preparations, designed for the treatment of liver fibrosis in man, have been extensively studied in animals, but little is known about the uptake and handling by human livers. Human liver tissue was obtained from livers procured from multiorgan donors and from cirrhotic livers of patients. To assess tissue viability, perfusate glutamate-oxalacetate-transaminase (GOT), glutamate-pyruvate-transaminase (GPT), and lactate dehydrogenase (LDH) levels were determined. To assess tissue functionality, the uptake of taurocholic acid and phase I and II metabolism of lidocaine and 7-hydroxycoumarin were determined. Uptake of a drug-targeting preparation was studied with Dexa(10)-HSA, which is designed for targeting of dexamethasone to nonparenchymal cells in the liver. During a 90-min perfusion period, no elevation of either GOT, GPT, or LDH was found. Both healthy control livers and cirrhotic livers showed phase I and II drug metabolism and functional taurocholic acid uptake. Studies with Dexa(10)-HSA revealed that 60 min after administration, 40% of the dose had been taken up by control livers and only 5% by cirrhotic livers. In control livers, Kupffer and endothelial cells had taken up Dexa(10)-HSA, whereas in cirrhotic livers only Kupffer cells were responsible for the uptake. Viability parameters and liver function tests clearly showed the applicability of this method. In the perfusion set-up, we showed uptake of the drug-targeting preparation Dexa(10)-HSA by healthy and cirrhotic human liver tissue, although the distribution patterns differed. This demonstrates the need to study new concepts in (diseased) human tissue.

  3. Recent developments on human cell lines for the bioartificial liver

    NARCIS (Netherlands)

    Hoekstra, R.; Chamuleau, R. A. F. M.

    2002-01-01

    Most bioartificial liver (BAL) devices contain porcine primary hepatocytes as their biological component. However, alternatives are needed due to xenotransplantation associated risks. Human liver cell lines have excellent growth characteristics and are therefore candidates for application in BAL

  4. HUMAN LIVER SLICES EXPRESS THE SAME LIDOCAINE BIOTRANSFORMATION RATE AS ISOLATED HUMAN HEPATOCYTES

    NARCIS (Netherlands)

    OLINGA, P; MEIJER, DKF; SLOOFF, MJH; GROOTHUIS, GMM; Merema, M.T.

    1993-01-01

    In order to investigate whether liver slices are a valuable tool for the assessment of drug metabolism in human liver, we compared the phase I metabolism of lidocaine in human liver slices and hepatocytes prepared from three human livers. Lidocaine is mainly metabolised to monoethylglycinexylidide

  5. Safety of frozen liver for human consumption

    Directory of Open Access Journals (Sweden)

    Ghada A.K. Kirrella

    2017-07-01

    Full Text Available The objective of this study was to ensure and evaluate the safety of imported frozen beef liver traded in supermarkets of Kafr El-Sheikh Governorate, Egypt, through detection of Salmonella typhimurium, Salmonella enteritidies, Escherichia coli O157:H7, antibiotic residues, and aflatoxin B1 residue. Fifty samples of imported frozen liver were randomly collected from different shops at Kafr El-Sheikh Governorate for isolation of S. typhimurium, S. enteritidies, and E. coli O157:H7. The results revealed that for both microorganisms 4% of the examined samples presumed to contain Salmonella and E. coli O157:H7 organisms, according to the colonial character on Harlequin Salmonella ABC agar media and Harlequin SMAC-BCIG agar media. According to biochemical and serological identifications, both organisms could not be detected in the examined samples. A total of 29 (58% samples were positive for antibiotic residues, using the Premi test (a broad-spectrum screening test for the detection of antibiotic residues in meat at or below the maximum residue limits. In addition, aflatoxin B1 was detected in one (2% samples with a concentration of 1.1 μg/kg. The results reflect that there was good hygiene practice for handling and preparation of frozen liver while selling to consumers. However, a high percentage of antibiotic residues reflect ignorance of withdrawal time before slaughtering of animals as well as misuse of antibiotics in veterinary fields. Furthermore, aflatoxin B1 residue was detected in examined frozen liver samples at a concentration below the maximum residual level, which is not enough to cause threat to humans, but it is enough to cause problem if it is eaten regularly reflect contamination of animal feed with aflatoxins.

  6. Approach for in vivo protein binding of 5-n-butyl-pyrazolo[1,5-a]pyrimidine bioactivated in chimeric mice with humanized liver by two-dimensional electrophoresis with accelerator mass spectrometry.

    Science.gov (United States)

    Yamazaki, Hiroshi; Kuribayashi, Shunji; Inoue, Tae; Tateno, Chise; Nishikura, Yasufumi; Oofusa, Ken; Harada, Daisuke; Naito, Shinsaku; Horie, Toru; Ohta, Shigeru

    2010-01-01

    Drug development of a potential analgesic agent 5-n-butyl-7-(3,4,5-trimethoxybenzoylamino)pyrazolo[1,5-a]pyrimidine was withdrawn because of its limited hepatotoxic effects in humans that could not be predicted from regulatory animal or in vitro studies. In vivo formation of glutathione conjugates and covalent binding of a model compound 5-n-butyl-pyrazolo[1,5-a]pyrimidine were investigated in the present study after intravenous administration to chimeric mice with a human or rat liver because of an interesting capability of human cytochrome P450 1A2 in forming a covalently bound metabolite in vitro. Rapid distribution and elimination of radiolabeled 5-n-butyl-pyrazolo[1,5-a]pyrimidine in plasma or liver fractions were seen in chimeric mice after intravenous administration. However, similar covalent binding in liver was detected over 0.17-24 h after intravenous administration. Radio-LC analyses revealed that the chimeric mice with humanized liver preferentially gave the 3-hydroxylated metabolite and its glutathione conjugate in the plasma and liver. On the contrary, chimeric mice with a rat liver had some rat-specific metabolites in vivo. Analyses by electrophoresis with accelerator mass spectrometry of in vivo radiolabeled liver proteins in chimeric mice revealed that bioactivated 5-n-butyl-pyrazolo[1,5-a]pyrimidine bound nonspecifically to a variety of microsomal proteins including human P450 1A2 as well as cytosolic proteins in the livers from chimeric mice with humanized liver. These results suggest that the hepatotoxic model compound 5-n-butyl-pyrazolo[1,5-a]pyrimidine was activated by human liver microsomal P450 1A2 to reactive intermediate(s) in vivo in humanized chimeric mice and could relatively nonspecifically bind to biomolecules such as P450 1A2 and other proteins.

  7. New IVIVE method for the prediction of total human clearance and relative elimination pathway contributions from in vitro hepatocyte and microsome data.

    Science.gov (United States)

    Riede, Julia; Poller, Birk; Umehara, Ken-ichi; Huwyler, Jörg; Camenisch, Gian

    2016-04-30

    Total human clearance is a key determinant for the pharmacokinetic behavior of drug candidates. Our group recently introduced the Extended Clearance Model (ECM) as an accurate in vitro-in vivo extrapolation (IVIVE) method for the prediction of hepatic clearance. Yet, knowledge about relative elimination pathway contributions is needed in order to predict the total human clearance of drug candidates. In the present work, a training set of 18 drug compounds was used to describe the affiliations between in vitro sinusoidal uptake clearance and the fractional contributions of hepatic (metabolic and biliary) or renal clearance to overall in vivo elimination. By means of these quantitative relationships and using a validation set of 10 diverse drug molecules covering different (sub)classes of the Extended Clearance Concept Classification System (ECCCS), the relative contributions of elimination pathways were calculated and demonstrated to well correlate with human reference data. Likewise, ECM- and pathway-based predictions of total clearances from both data sets demonstrated a strong correlation with the observed clinical values with 26 out of 28 compounds within a three-fold deviation. Hence, total human clearance and relative contributions of elimination pathways were successfully predicted by the presented method using solely hepatocyte and microsome in vitro data. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. Age-Dependent Human Hepatic Carboxylesterase 1 (Ces1) ...

    Science.gov (United States)

    Human hepatic carboxylesterase 1 and 2 (CES1 and CES2) are important for ester- and amide- bond containing pharmaceutical and environmental chemical disposition. Despite concern regarding juvenile sensitivity to such compounds, CES1 and CES2 ontogeny has not been well characterized. To define human hepatic microsomal and cytosolic CES1 and CES2 expression during early postnatal life, microsomal and cytosolic fractions were prepared using liver samples from subjects without liver disease [N=165, 1d-18 yrs]. Proteins were fractionated, detected and quantitated by western blotting. Median microsomal CES1 was lower among samples from subjects mg microsomal protein, respectively; pmg cytosolic protein, respectively; p values mg microsomal protein, respectively (p<0.001, both), whereas for cytosolic CES2, only the youngest age group differed from the two older g

  9. Microsomal lipid peroxidation as a mechanism of cellular damage. [Dissertation

    Energy Technology Data Exchange (ETDEWEB)

    Kornbrust, D.J.

    1979-01-01

    The NADPH/iron-dependent peroxidation of lipids in rat liver microsomes was found to be dependent on the presence of free ferrous ion and maintains iron in the reduced Fe/sup 2 +/ state. Chelation of iron by EDTA inhibited peroxidation. Addition of iron, after preincubation of microsomes in the absence of iron, did not enhance the rate of peroxidation suggesting that iron acts by initiating peroxidative decomposition of membrane lipids rather than by catalyzing the breakdown of pre-formed hydroperoxides. Liposomes also underwent peroxidation in the presence of ferrous iron at a rate comparable to intact microsomes and was stimulated by ascorbate. Carbon tetrachloride initiated lipid peroxidation in the absence of free metal ions. Rates of in vitro lipid peroxidation of microsomes and homogenates were found to vary widely between different tissues and species. The effects of paraquat on lipid peroxidation was also studied. (DC)

  10. Metabolism of bupropion by baboon hepatic and placental microsomes

    Science.gov (United States)

    Wang, Xiaoming; Abdelrahman, Doaa R.; Fokina, Valentina M.; Hankins, Gary D.V.; Ahmed, Mahmoud S.; Nanovskaya, Tatiana N.

    2011-01-01

    The aim of this investigation was to determine the biotransformation of bupropion by baboon hepatic and placental microsomes, identify the enzyme(s) catalyzing the reaction(s) and determine its kinetics. Bupropion was metabolized by baboon hepatic and placental microsomes to hydroxybupropion (OH-BUP), threo- (TB) and erythrohydrobupropion (EB). OH-bupropion was the major metabolite formed by hepatic microsomes (Km 36 ± 6 µM, Vmax 258 ± 32 pmol mg protein−1 min−1), however the formation of OH-BUP by placental microsomes was below the limit of quantification. The apparent Km values of bupropion for the formation of TB and EB by hepatic and placental microsomes were similar. The selective inhibitors of CYP2B6 (ticlopidine and phencyclidine) and monoclonal antibodies raised against human CYP2B6 isozyme caused 80% inhibition of OH-BUP formation by baboon hepatic microsomes. The chemical inhibitors of aldo-keto reductases (flufenamic acid), carbonyl reductases (menadione), and 11β-hydroxysteroid dehydrogenases (18β-glycyrrhetinic acid) significantly decreased the formation of TB and EB by hepatic and placental microsomes. Data indicate that CYP2B of baboon hepatic microsomes is responsible for biotransformation of bupropion to OH-BUP, while hepatic and placental short chain dehydrogenases/reductases and to a lesser extent aldo-keto reductases are responsible for the reduction of bupropion to TB and EB. PMID:21570381

  11. Catalysis of nitro-aci tautomerism of the genotoxicant 2-nitropropane by cytosol from rodent and human liver.

    Science.gov (United States)

    Kohl, C; Gescher, A

    1996-01-05

    2-Nitropropane (2-NP) is a genotoxicant and hepatocarcinogen in rodents. Conversion to propane 2-nitronate (P2N), the anion of the tautomeric aci form of 2-NP, seems to be a pivotal part of the mechanism by which 2-NP causes its toxicity. We tested the hypothesis that the tautomeric equilibrium is influenced by enzymes in the liver, the target organ of 2-NP toxicity. Rat or mouse hepatocytes were incubated with 2-NP, P2N or the 2-NP isotopomer 2-deutero 2-nitropropane (2H-2-NP), which equilibrates with P2N much more slowly than 2-NP. Tautomers were analyzed by HPLC. The rates of conversion of 2-NP to P2N expressed as nmol P2N x (10(6) cells/ml)-1 x min-1 were 4.0 and 4.2 in the presence of hepatocytes from rats or mice, respectively, and 2.6 in the absence of cells. Production of 2-NP to P2N expressed as nmol 2-NP x (10(6) cells/ml)-1 x min-1 was increased from 6.1 in the absence of cells to 11.9 or 9.9 in the presence of hepatocytes from rats or mice, respectively. The rate of formation of P2N from 2H-2-NP as compared to 2-NP was characterised by a primary isotope effect of 3.4 and 3.8 in hepatocytes from rats and mice, respectively, contrasting with a value of 9.6 measured in medium omitting cells. When 2-NP was incubated with subfractions of rodent or human liver homogenate, production of P2N by cytosol was between 7.3 (mouse liver) and 28.1 times (human liver) higher than that observed in microsomes. Similarly generation of 2-NP from P2N by cytosol exceeded that in microsomes by a factor of two. Tautomerism in heat-activated cytosol, mitochondria or microsomes was not different from that in buffer only. The results suggest that the nitro-aci tautomerism of secondary nitroalkanes is catalysed by a hepatic enzyme which resides predominantly in the cytosol and may thus contribute to the generation of the toxic species via which 2-NP exerts its toxicity.

  12. In vitro inhibitory effects of pristimerin on human liver cytochrome P450 enzymes.

    Science.gov (United States)

    Hao, Xiaoyi; Yuan, Jianlei; Xu, Yansen; Wang, Zhao; Hou, Jianzhang; Hu, Tao

    2017-04-07

    1. Pristimerin (PTM) is a biological component isolated from Chinese herbal plant Celastrus and Maytenus spp and it possesses numerous pharmacological activities. However, whether PTM affects the activity of human liver cytochrome P450 (CYP) enzymes remains unclear. 2. In this study, the inhibitory effects of PTM on the eight human liver CYP isoforms (i.e., 1A2, 3A4, 2A6, 2E1, 2D6, 2C9, 2C19, and 2C8) were investigated in vitro using human liver microsomes (HLMs). 3. The results showed that PTM inhibited the activity of CYP1A2, 3A4, and 2C9, with IC50 values of 21.74, 15.88, and 16.58 μM, respectively, but that other CYP isoforms were not affected. Enzyme kinetic studies showed that PTM was not only a non-competitive inhibitor of CYP3A4, but also a competitive inhibitor of CYP1A2 and 2C9, with Ki values of 7.33, 11.60, and 8.09 μM, respectively. In addition, PTM is a time-dependent inhibitor for CYP3A4 with Kinact/KI value of 0.049/11.62 μM-1min-1. 4. The in vitro studies of PTM with CYP isoforms indicate that PTM has the potential to cause pharmacokinetic drug interactions with other co-administered drugs metabolized by CYP1A2, 3A4, and 2C9. Further clinical studies are needed to evaluate the significance of this interaction.

  13. In vitro metabolism of benzo[a]pyrene-7,8-dihydrodiol and dibenzo[def,p]chrysene-11,12 diol in rodent and human hepatic microsomes

    Energy Technology Data Exchange (ETDEWEB)

    Smith, Jordan N.; Mehinagic, Denis; Nag, Subhasree; Crowell, Susan R.; Corley, Richard A.

    2017-03-01

    Polycyclic aromatic hydrocarbons (PAHs) are contaminants that are ubiquitously found in the environment, produced through combustion of organic matter or petrochemicals, and many of which are procarcinogens. The prototypic PAH, benzo[a]pyrene (B[a]P) and the highly carcinogenic dibenzo[def,p]chrysene (DBC) are metabolically activated by isoforms of the P450 enzyme superfamily producing benzo[a]pyrene-7,8-dihydrodiol (B[a]P diol), dibenzo[def,p]chrysene-11,12 diol (DBC diol). Each of these diols can be further metabolized by cytochrome P450 enzymes to highly reactive diol-epoxide metabolites that readily react with DNA or by phase II conjugation facilitating excretion. To complement prior in vitro metabolism studies with parent B[a]P and DBC, both phase I metabolism and phase II glucuronidation of B[a]P diol and DBC diol were measured in hepatic microsomes from female B6129SF1/J mice, male Sprague-Dawley rats, and female humans. Metabolic parameters, including intrinsic clearance and Michaelis-Menten kinetics were calculated from substrate depletion data. Mice and rats demonstrated similar B[a]P diol phase I metabolic rates. Compared to rodents, human phase I metabolism of B[a]P diol demonstrated lower overall metabolic capacity, lower intrinsic clearance at higher substrate concentrations (>0.14 µM), and higher intrinsic clearance at lower substrate concentrations (<0.07 µM). Rates of DBC diol metabolism did not saturate in mice or humans and were highest overall in mice. Higher affinity constants and lower capacities were observed for DBC diol glucuronidation compared to B[a]P diol glucuronidation; however, intrinsic clearance values for these compounds were consistent within each species. Kinetic parameters reported here will be used to extend physiologically based pharmacokinetic (PBPK) models to include the disposition of B[a]P and DBC metabolites in animal models and humans to support future human health risk assessments.

  14. Transcription of the Human Microsomal Epoxide Hydrolase Gene (EPHX1 Is Regulated by PARP-1 and Histone H1.2. Association with Sodium-Dependent Bile Acid Transport.

    Directory of Open Access Journals (Sweden)

    Hui Peng

    Full Text Available Microsomal epoxide hydrolase (mEH is a bifunctional protein that plays a central role in the metabolism of numerous xenobiotics as well as mediating the sodium-dependent transport of bile acids into hepatocytes. These compounds are involved in cholesterol homeostasis, lipid digestion, excretion of xenobiotics and the regulation of several nuclear receptors and signaling transduction pathways. Previous studies have demonstrated the critical role of GATA-4, a C/EBPα-NF/Y complex and an HNF-4α/CAR/RXR/PSF complex in the transcriptional regulation of the mEH gene (EPHX1. Studies also identified heterozygous mutations in human EPHX1 that resulted in a 95% decrease in mEH expression levels which was associated with a decrease in bile acid transport and severe hypercholanemia. In the present investigation we demonstrate that EPHX1 transcription is significantly inhibited by two heterozygous mutations observed in the Old Order Amish population that present numerous hypercholanemic subjects in the absence of liver damage suggesting a defect in bile acid transport into the hepatocyte. The identity of the regulatory proteins binding to these sites, established using biotinylated oligonucleotides in conjunction with mass spectrometry was shown to be poly(ADP-ribosepolymerase-1 (PARP-1 bound to the EPHX1 proximal promoter and a linker histone complex, H1.2/Aly, bound to a regulatory intron 1 site. These sites exhibited 71% homology and may represent potential nucleosome positioning domains. The high frequency of the H1.2 site polymorphism in the Amish population results in a potential genetic predisposition to hypercholanemia and in conjunction with our previous studies, further supports the critical role of mEH in mediating bile acid transport into hepatocytes.

  15. Cellular distribution and handling of liver-targeting preparations in human livers studied by a liver lobe perfusion

    NARCIS (Netherlands)

    Melgert, BN; Olinga, P; Weert, B; Slooff, MJH; Meijer, DKF; Poelstra, K; Groothuis, GMM

    We developed and tested a novel method for perfusing parts of human liver to study uptake and handling of drug-targeting preparations. These preparations, designed for the treatment of liver fibrosis in man, have been extensively studied in animals, but little is known about the uptake and handling

  16. Kinetic analysis of reversible inhibition of 16alpha-hydroxyandrostenedione aromatization in human placental microsomes by suicide substrates of androstenedione aromatization.

    Science.gov (United States)

    Numazawa, Mitsuteru; Mutsumi, Ayako; Tachibana, Mii; Yoshimura, Akiko

    2003-06-01

    To gain insight into the catalytic function of aromatase and its substrate specificity, we studied reversible inhibition of 16alpha-hydroxyandrostenedione (16alpha-OHAD) aromatization in human placental microsomes by several suicide substrates of androstenedione (AD) aromatization, including 4-hydroxyAD (1), 6-oxoAD (2) and its 19-hydroxy analogue 3, androst-5-ene-4,7,17-trione (4), and 10beta-acetoxyandrost-5-en-7,17-dione (5) that, in contrast, do not cause a suicide inactivation of 16alpha-OHAD aromatization. All inhibitors examined blocked 16alpha-OHAD aromatization in a competitive manner with apparent K(i) values ranging from 0.50 to 980 nM. The relative K(i) values between inhibitors 1-5 obtained in the 16alpha-OHAD aromatization experiments were markedly different from those obtained in the AD aromatization experiments. The results predict that all inhibitors examined bind to the 16alpha-OHAD binding site in a manner that does not cause suicide inactivation of 16alpha-OHAD aromatization. These findings would be useful for understanding the active (binding) site structure as well as the catalytic function of aromatase.

  17. Assessment of Liver and Renal Functions of Asymptomatic Human ...

    African Journals Online (AJOL)

    Winniecure), used in our institute for the treatment of Human Immuno Deficiency Virus (HIV) infection, on liver and renal functions of individuals undergoing therapy. A total of 100 asymptomatic Human Immuno Deficiency Virus (HIV) seropositive ...

  18. Mathematical model of liver regeneration in human live donors.

    Science.gov (United States)

    Periwal, V; Gaillard, J R; Needleman, L; Doria, C

    2014-05-01

    Liver regeneration after injury occurs in many mammals. Rat liver regenerates after partial hepatectomy over a period of 2 weeks while human liver regeneration takes several months. Notwithstanding this enormous difference in time-scales, with new data from five human live liver transplant donors, we show that a mathematical model of rat liver regeneration can be transferred to human, with all biochemical interactions and signaling unchanged. Only six phenomenological parameters need change, and three of these parameter changes are rescalings of rate constants by the ratio of human lifespan to rat lifespan. Data from three donor subjects with approximately equal resections were used to fit the three parameters and the data from the other two donor subjects was used to independently verify the fit. © 2013 Wiley Periodicals, Inc.

  19. Susceptibility of human liver cells to porcine endogenous retrovirus.

    Science.gov (United States)

    Lin, Xinzi; Qi, Lin; Li, Zhiguo; Chi, Hao; Lin, Wanjun; Wang, Yan; Jiang, Zesheng; Pan, Mingxin; Gao, Yi

    2013-12-01

    The risk of porcine endogenous retrovirus infection is a major barrier for pig-to-human xenotransplant. Porcine endogenous retrovirus, present in porcine cells, can infect many human and nonhuman primate cells in vitro, but there is no evidence available about in vitro infection of human liver cells. We investigated the susceptibility of different human liver cells to porcine endogenous retrovirus. The supernatant from a porcine kidney cell line was added to human liver cells, including a normal hepatocyte cell line (HL-7702 cells), primary hepatocytes (Phh cells), and a liver stellate cell line (Lx-2 cells), and to human embryonic kidney cells as a reference control. Expression of the porcine endogenous retrovirus antigen p15E in the human cells was evaluated with polymerase chain reaction, reverse transcription-polymerase chain reaction, and Western blot. The porcine endogenous retrovirus antigen p15E was not expressed in any human liver cells (HL-7702, Phh, or Lx-2 cells) that had been exposed to supernatants from porcine kidney cell lines. Porcine endogenous retrovirus-specific fragments were amplified in human kidney cells. Human liver cells tested were not susceptible to infection by porcine endogenous retrovirus. Therefore, not all human cells are susceptible to porcine endogenous retrovirus.

  20. LiverWiki: a wiki-based database for human liver.

    Science.gov (United States)

    Chen, Tao; Li, Mansheng; He, Qiang; Zou, Lei; Li, Youhuan; Chang, Cheng; Zhao, Dongyan; Zhu, Yunping

    2017-10-13

    Recent advances in omics technology have produced a large amount of liver-related data. A comprehensive and up-to-date source of liver-related data is needed to allow biologists to access the latest data. However, current liver-related data sources each cover only a specific part of the liver. It is difficult for them to keep pace with the rapid increase of liver-related data available at those data resources. Integrating diverse liver-related data is a critical yet formidable challenge, as it requires sustained human effort. We present LiverWiki, a first wiki-based database that integrates liver-related genes, homolog genes, gene expressions in microarray datasets and RNA-Seq datasets, proteins, protein interactions, post-translational modifications, associated pathways, diseases, metabolites identified in the metabolomics datasets, and literatures into an easily accessible and searchable resource for community-driven sharing. LiverWiki houses information in a total of 141,897 content pages, including 19,787 liver-related gene pages, 17,077 homolog gene pages, 50,251 liver-related protein pages, 36,122 gene expression pages, 2067 metabolites identified in the metabolomics datasets, 16,366 disease-related molecules, and 227 liver disease pages. Other than assisting users in searching, browsing, reviewing, refining the contents on LiverWiki, the most important contribution of LiverWiki is to allow the community to create and update biological data of liver in visible and editable tables. This integrates newly produced data with existing knowledge. Implemented in mediawiki, LiverWiki provides powerful extensions to support community contributions. The main goal of LiverWiki is to provide the research community with comprehensive liver-related data, as well as to allow the research community to share their liver-related data flexibly and efficiently. It also enables rapid sharing new discoveries by allowing the discoveries to be integrated and shared immediately

  1. Fructose and galactose enhance postexercise human liver glycogen synthesis.

    OpenAIRE

    Décombaz Jacques; Jentjens Roy; Ith Michael; Scheurer Eva; Buehler Tania; Jeukendrup Asker; Boesch Chris

    2011-01-01

    PURPOSE Both liver and muscle glycogen stores play a fundamental role in exercise and fatigue but the effect of different CHO sources on liver glycogen synthesis in humans is unclear. The aim was to compare the effect of maltodextrin (MD) drinks containing galactose fructose or glucose on postexercise liver glycogen synthesis. METHODS In this double blind triple crossover randomized clinical trial 10 well trained male cyclists performed three experimental exercise sessions separated by at ...

  2. Human liver chimeric mouse model based on diphtheria toxin-induced liver injury.

    Science.gov (United States)

    Ren, Xiao-Nan; Ren, Rong-Rong; Yang, Hua; Qin, Bo-Yin; Peng, Xiu-Hua; Chen, Li-Xiang; Li, Shun; Yuan, Meng-Jiao; Wang, Chao; Zhou, Xiao-Hui

    2017-07-21

    To establish an inducible liver injury mouse model and transplant human hepatocytes to obtain liver-humanized mice. We crossed three mouse strains, including albumin (Alb)-cre transgenic mice, inducible diphtheria toxin receptor (DTR) transgenic mice and severe combined immune deficient (SCID)-beige mice, to create Alb-cre/DTR/SCID-beige (ADSB) mice, which coincidentally harbor Alb-cre and DTR transgenes and are immunodeficient. As the Cre expression is driven by the liver-specific promoter Alb (encoding ALB), the DTR stop signal flanked by two loxP sites can be deleted in the ADSB mice, resulting in DTR expression in the liver. ADSB mice aged 8-10 wk were injected intraperitoneally (i.p.) with diphtheria toxin (DT) and liver damage was assessed by serum alanine aminotransferase (ALT) level. Two days later, mouse livers were sampled for histological analysis, and human hepatocytes were transplanted into the livers on the same day. A human ALB enzyme-linked immunosorbent assay was performed 7, 14, 21 and 28 d after transplantation. Human CD68 immunohistochemistry was performed 30 and 90 d after transplantation. We crossed Alb-cre with DTR and SCID-beige mice to obtain ADSB mice. These mice were found to have liver damage 4 d after i.p. injection of 2.5 ng/g bodyweight DT. Bodyweight began to decrease on day 2, increased on day 7, and was lowest on day 4 (range, 10.5%-13.4%). Serum ALT activity began to increase on day 2 and reached a peak value of 289.7 ± 16.2 IU/mL on day 4, then returned to background values on day 7. After transplantation of human liver cells, peripheral blood human ALB level was 1580 ± 454.8 ng/mL (range, 750.2-3064.9 ng/mL) after 28 d and Kupffer cells were present in the liver at 30 d in ADSB mice. Human hepatocytes were successfully repopulated in the livers of ADSB mice. The inducible mouse model of humanized liver in ADSB mice may have functional applications, such as hepatocyte transplantation, hepatic regeneration and drug metabolism.

  3. Human placental alkaline phosphatase in liver and intestine.

    OpenAIRE

    Garattini, E; Margolis, J; Heimer, E; Felix, A.; Udenfriend, S

    1985-01-01

    Three distinct forms of human alkaline phosphatase, presumably isozymes, are known, each apparently associated with a specific tissue. These are placental, intestinal, and liver (kidney and bone). We have used a specific immunoassay and HPLC to show that placental alkaline phosphatase is also present in extracts of liver and intestine in appreciable amounts.

  4. Genotoxicity and antioxidant activity of five Agrimonia and Filipendula species plant extracts evaluated by comet and micronucleus assays in human lymphocytes and Ames Salmonella/microsome test.

    Science.gov (United States)

    Pukalskienė, Milda; Slapšytė, Gražina; Dedonytė, Veronika; Lazutka, Juozas Rimantas; Mierauskienė, Jūratė; Venskutonis, Petras Rimantas

    2017-12-18

    The species of Agrimonia and Filipendula have been traditionally used in folk medicine as anti-inflammatory herbs. This study extends the knowledge on bioactivities of F. palmata, A. eupatoria, A. procera, F. ulmaria and F. vulgaris by comprehensive characterization of their methanolic extracts. Antioxidant properties of extracts were evaluated by DPPH• (2,2-diphenyl-1-picrylhydrazyl), ABTS•+ 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) scavenging and oxygen radical absorbance capacities (ORAC). Genotoxicity of extracts was tested using alkaline single-cell gel electrophoresis (comet) and cytokinesis-block micronucleus assays in human lymphocytes in vitro and the Ames Salmonella/microsome test. All investigated Agrimonia and Filipendula extracts possessed strong antioxidant activity, which was comparable with that of a standard antioxidant trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid). Thirty five compounds belonging to the classes of phenolic acids, flavonoids, phenylpropanoids and ellagitanins were detected by ultra-performance liquid chromatography - mass spectrometry (UPLC-Q-TOF-MS). Agrimonia and Filipendula extracts induced an increase in a DNA damage in the comet assay expressed as mean percentage of DNA in the comet tail. However, these extracts did not produce reverse mutation in bacterial cells in the Ames test and were not genotoxic in the micronucleus test. However, a slight though significant decrease of nuclear division index values was determined. In general, this study proved that Agrimonia and Filipendula species are a good source of bioactive compounds; their extracts may be classified as non-mutagenic and non-clastogenic in vitro under conditions of the current study. Consequently, the plants may be a promising material for nutraceuticals and natural medicines. Copyright © 2017. Published by Elsevier Ltd.

  5. Methanolic extract of Boswellia serrata exhibits anti-cancer activities by targeting microsomal prostaglandin E synthase-1 in human colon cancer cells.

    Science.gov (United States)

    Ranjbarnejad, Tayebeh; Saidijam, Massoud; Moradkhani, Shirin; Najafi, Rezvan

    2017-07-01

    Colorectal cancer (CRC) is the most common cancer. A proper method to reduce mortality of CRC is chemoprevention to prevent initiation and promotion of intestinal tumorgenesis. One of the promising and developing chemopreventive agents is natural compounds found in plants. Frankincense, the resin extract from the Boswellia specious, has been used in traditional and modern medicine for treating various diseases with very minimal side effects. In the current study, we investigated the anti-cancer activity of methanolic extract of Boswellia serrata (B. serrata) on HT-29 human colon cancer cells. HT-29 cells were treated with different concentrations of B. serrata and cell viability was assessed by MTT assay. mRNA expression of microsomal prostaglandin E synthase-1 (mPGES-1), vascular endothelial growth factor (VEGF), C-X-C chemokine receptor type 4 (CXCR4), matrix metalloproteinase-2 (MMP-2), MMP-9 and hypoxia-inducible factor-1 (HIF-1) were examined by quantitative real-time PCR. Apoptosis was evaluated by the proportion of sub-G1 cells. Prostaglandin E2 (PGE2) level and caspase 3 activity were determined by ELISA assay. Tube formation potential and HT-29 cells migration were assessed using three-dimensional vessel formation assay and scratch test. B. serrata extract considerably decreased the expression of mPGES-1, VEGF, CXCR4, MMP-2, MMP-9 and HIF-1. The caspase 3 activity and percent of cells in sub-G1 phase were increased by B. serrata extract. Cell viability, PGE2 generation, in vitro tube formation and cell migration were decreased significantly in B. serrata-treated HT-29 compared to the control group. Our findings suggest that B. serrata extract inhibits proliferation, angiogenesis and migration and induces apoptosis in HT-29 cells by inhibiting of mPGES-1 and decreasing the PGE2 level and its downstream targets. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Inhibition of microsomal oxidation of alcohols and of hydroxyl-radical-scavenging agents by the iron-chelating agent desferrioxamine.

    OpenAIRE

    Cederbaum, A I; Dicker, E

    1983-01-01

    Rat liver microsomes (microsomal fractions) catalyse the oxidation of straight-chain aliphatic alcohols and of hydroxyl-radical-scavenging agents during NADPH-dependent electron transfer. The iron-chelating agent desferrioxamine, which blocks the generation of hydroxyl radicals in other systems, was found to inhibit the following microsomal reactions: production of formaldehyde from either dimethyl sulphoxide or 2-methylpropan-2-ol (t-butylalcohol); generation of ethylene from 4-oxothiomethyl...

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2013-02-15

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

  8. Binding of uteroglobin to microsomes and plasmatic membranes.

    Science.gov (United States)

    Diaz González, K; Nieto, A

    1995-03-20

    Microsomes and plasmatic membranes from rat liver bind radioactive uteroglobin (UG) in vitro with high affinity (Kd = 1.7 x 10(-10) M. The binding is saturable and specific and dependent on previous reduction of UG with dithiothreitol. Microsomes from rat spleen or lung or from rabbit endometrium also possess a similar ability. Binding capacity is not affected by previous treatment of microsomes with phospholipase A2 or peptide-N-glycosidase F but is lost after brief treatment with trypsin. The complex formed between UG and the binding component can be solubilized from microsomes with 5 mM CHAPS and it elutes with an apparent Mr of 90,000 in a Sephacryl 200 column. The complex is resistant to 8 M urea but is completely dissociated by Triton X-100. The UG-binding protein(s) has been partially purified from solubilized microsomes and membranes by affinity chromatography. The results are discussed in relation to a possible physiological effect of UG on cellular membranes.

  9. Simultaneous characterization of progenitor cell compartments in adult human liver.

    Science.gov (United States)

    Porretti, Laura; Cattaneo, Alessandra; Colombo, Federico; Lopa, Raffaella; Rossi, Giorgio; Mazzaferro, Vincenzo; Battiston, Carlo; Svegliati-Baroni, Gianluca; Bertolini, Francesco; Rebulla, Paolo; Prati, Daniele

    2010-01-01

    The human liver is a complex tissue consisting of epithelial, endothelial, hematopoietic, and mesenchymal elements that probably derive from multiple lineage-committed progenitors, but no comprehensive study aimed at identifying and characterizing intrahepatic precursors has yet been published. Cell suspensions for this study were obtained by enzymatic digestion of liver specimens taken from 20 patients with chronic liver disease and 13 multiorgan donors. Stem and progenitor cells were first isolated, amplified, and characterized ex vivo according to previously validated methods, and then optimized flow cytometry was used to assess their relative frequencies and characterize their immunophenotypes in the clinical specimens. Stem and progenitor cells committed to hematopoietic, endothelial, epithelial, and mesenchymal lineages were clearly identifiable in livers from both healthy and diseased subjects. Within the mononuclear liver cell compartment, epithelial progenitors [epithelial cell adhesion molecule (EpCAM)(+)/CD49f(+)/CD29(+)/CD45(-)] accounted for 2.7-3.5% whereas hematopoietic (CD34(+)/CD45(+)), endothelial [vascular endothelial growth factor-2 (KDR)(+)/CD146(+)/CD45(-)], and mesenchymal [CD73(+)/CD105(+)/CD90 (Thy-1)(+)/CD45 (-)] stem cells and progenitors accounted for smaller fractions (0.02-0.6%). The patients' livers had higher percentages of hematopoietic and endothelial precursors than those of the donors. In conclusion, we identified and characterized precursors committed to four different lineages in adult human liver. We also optimized a flow cytometry approach that will be useful in exploring the contribution of these cells to the pathogenesis of liver disease.

  10. Conversion of the HIV protease inhibitor nelfinavir to a bioactive metabolite by human liver CYP2C19.

    Science.gov (United States)

    Hirani, Vandana N; Raucy, Judy L; Lasker, Jerome M

    2004-12-01

    Antiretroviral therapy for human immunodeficiency virus (HIV) infection includes treatment with both reverse transcriptase inhibitors and protease inhibitors, which markedly suppress viral replication and circulating HIV RNA levels. Cytochrome P450 (P450) enzymes in human liver, chiefly CYP3A4, play a pivotal role in protease inhibitor biotransformation, converting these agents to largely inactive metabolites. However, the protease inhibitor nelfinavir (Viracept) is metabolized mainly to nelfinavir hydroxy-t-butylamide (M8), which exhibits potent antiviral activity, and to other minor products (termed M1 and M3) that are inactive. Since indirect evidence suggests that CYP2C19 underlies M8 formation, we examined the role of this inducible, polymorphic P450 enzyme in nelfinavir t-butylamide hydroxylation by human liver. Rates of microsomal M8 formation were 50.6 +/- 28.3 pmol of product formed/min/nmol P450 (n = 5 subjects), whereas kinetic analysis of the reaction revealed a KM of 21.6 microM and a Vmax of 24.6 pmol/min/nmol P450. In reconstituted systems, CYP2C19 catalyzed nelfinavir t-butylamide hydroxylation at a turnover rate of 2.2 min(-1), whereas CYP2C9, CYP2C8, and CYP3A4 were inactive toward nelfinavir. Polyclonal anti-CYP2C9 (cross-reactive with CYP2C19) and monoclonal anti-CYP2C19 completely inhibited microsomal M8 production, whereas monoclonal CYP2C9 and polyclonal CYP3A4 antibodies were without effect. Similarly, the CYP2C19 substrate omeprazole strongly inhibited (75%) hepatic nelfinavir t-butylamide hydroxylation at a concentration of only 12.5 microM. Our study shows that CYP2C19 underlies formation in human liver of M8, a bioactive nelfinavir metabolite. The inducibility of CYP2C19 by agents (e.g., rifampicin) often taken concurrently with nelfinavir, together with this P450's known polymorphic nature, may thus be important determinants of nelfinavir's antiviral potency.

  11. Functional Blood Progenitor Markers in Developing Human Liver Progenitors

    Directory of Open Access Journals (Sweden)

    Orit Goldman

    2016-08-01

    Full Text Available In the early fetal liver, hematopoietic progenitors expand and mature together with hepatoblasts, the liver progenitors of hepatocytes and cholangiocytes. Previous analyses of human fetal livers indicated that both progenitors support each other's lineage maturation and curiously share some cell surface markers including CD34 and CD133. Using the human embryonic stem cell (hESC system, we demonstrate that virtually all hESC-derived hepatoblast-like cells (Hep cells transition through a progenitor stage expressing CD34 and CD133 as well as GATA2, an additional hematopoietic marker that has not previously been associated with human hepatoblast development. Dynamic expression patterns for CD34, CD133, and GATA2 in hepatoblasts were validated in human fetal livers collected from the first and second trimesters of gestation. Knockdown experiments demonstrate that each gene also functions to regulate hepatic fate mostly in a cell-autonomous fashion, revealing unprecedented roles of fetal hematopoietic progenitor markers in human liver progenitors.

  12. Effectiveness of human cytochrome P450 3A4 present in liposomal and microsomal nanoparticles in formation of covalent DNA adducts by ellipticine.

    Science.gov (United States)

    Sulc, Miroslav; Mrizova, Iveta; Cerna, Tereza; Frei, Eva; Eckschlager, Tomas; Adam, Vojtech; Kopeckova, Katerina; Stiborova, Marie

    2016-12-18

    Ellipticine is an anticancer agent that functions through multiple mechanisms participating in cell cycle arrest and initiation of apoptosis. This drug forms covalent DNA adducts after its enzymatic activation with cytochrome P450 (CYP), which is one of the most important ellipticine DNA-damaging mechanisms of its cytotoxic effects. The improvements of cancer treatment are the major challenge in oncology research. Nanotransporters (nanoparticles) are promising approaches to target tumor cells, frequently leading to improve drug therapeutic index. Ellipticine has already been prepared in nanoparticle forms. However, since its anticancer efficiency depends on the CYP3A4-mediated metabolism in cancer cells, the aim of our research is to develop nanoparticles containing this enzyme that can be transported to tumor cells, thereby potentiating ellipticine cytotoxicity. The CYP3A4 enzyme encapsulated into two nanoparticle forms, liposomes and microsomes, was tested to activate ellipticine to its reactive species forming covalent DNA adducts. Ellipticine-derived DNA adducts were determined by the 32P-postlabeling method. The CYP3A4 enzyme both in the liposome and microsome nanoparticle forms was efficient to activate ellipticine to species forming DNA adducts. Two DNA adducts, which are formed from ellipticine metabolites 12-hydroxy- and 13-hydroxyellipticine generated by its oxidation by CYP3A4, were formed by both CYP3A4 nanoparticle systems. A higher effectiveness of CYP3A4 in microsomal than in liposomal nanoparticles to form ellipticine-DNA adducts was found. Further testing in a suitable cancer cell model is encouraged to investigate whether the DNA-damaging effects of ellipticine after its activation by CYP3A4 nanoparticle forms are appropriate for active targeting of this enzyme to specific cancer cells.

  13. Characterization of human liver enzymes involved in the biotransformation of boceprevir, a hepatitis C virus protease inhibitor.

    Science.gov (United States)

    Ghosal, Anima; Yuan, Yuan; Tong, Wei; Su, Ai-Duen; Gu, Chunyan; Chowdhury, Swapan K; Kishnani, Narendra S; Alton, Kevin B

    2011-03-01

    Boceprevir (SCH 503034), a protease inhibitor, is under clinical development for the treatment of human hepatitis C virus infections. In human liver microsomes, formation of oxidative metabolites after incubations with [(14)C]boceprevir was catalyzed by CYP3A4 and CYP3A5. In addition, the highest turnover was observed in recombinant CYP3A4 and CYP3A5. After a single radiolabeled dose to human, boceprevir was subjected to two distinct pathways, namely cytochrome P450-mediated oxidation and ketone reduction. Therefore, attempts were made to identify the enzymes responsible for the formation of carbonyl-reduced metabolites. Human liver S9 and cytosol converted ∼ 28 and ∼ 68% of boceprevir to M28, respectively, in the presence of an NADPH-generating system. Screening of boceprevir with recombinant human aldo-keto reductases (AKRs) revealed that AKR1C2 and AKR1C3 exhibited catalytic activity with respect to the formation of M+2 metabolites (M28 and M31). The formation of M28 was inhibited by 100 μM flufenamic acid (80.3%), 200 μM mefenamic acid (83.7%), and 100 μM phenolphthalein (86.1%), known inhibitors of AKRs, suggesting its formation through carbonyl reduction pathway. Formation of M28 was also inhibited by 100 μM diazepam (75.1%), 1 mM ibuprofen (70%), and 200 μM diflunisal (89.4%). These data demonstrated that CYP3A4 and CYP3A5 are primarily responsible for the formation of oxidative metabolites and the formation of M28 and M31, the keto-reduced metabolites, are most likely mediated by AKR1C2 and AKR1C3. Because the biotransformation and clearance of boceprevir involves two different enzymatic pathways, boceprevir is less likely to be a victim of significant drug-drug interaction with concomitant medication affecting either of these pathways.

  14. PVA matches human liver in needle-tissue interaction.

    Science.gov (United States)

    de Jong, Tonke L; Pluymen, Loes H; van Gerwen, Dennis J; Kleinrensink, Gert-Jan; Dankelman, Jenny; van den Dobbelsteen, John J

    2017-05-01

    Medical phantoms can be used to study needle-tissue interaction and to train medical residents. The purpose of this research is to study the suitability of polyvinyl alcohol (PVA) as a liver tissue mimicking material in terms of needle-tissue interaction. Insertions into ex-vivo human livers were used for reference. Six PVA samples were created by varying the mass percentage of PVA to water (4m% and 7m%) and the number of freeze-thaw cycles (1, 2 and 3 cycles, 16hours of freezing at -19°C, 8hours of thawing). The inner needle of an 18 Gauge trocar needle with triangular tip was inserted 13 times into each of the samples, using an insertion velocity of 5 mm/s. In addition, 39 insertions were performed in two ex-vivo human livers. Axial forces on the needle were captured during insertion and retraction and characterized by friction along the needle shaft, peak forces, and number of peak forces per unit length. The concentration of PVA and the number of freeze-thaw cycles both influenced the mechanical interaction between needle and specimen. Insertions into 4m% PVA phantoms with 2 freeze-thaw cycles were comparable to human liver in terms of estimated friction along the needle shaft and the number of peak forces. Therefore, these phantoms are considered to be suitable liver mimicking materials for image-guided needle interventions. The mechanical properties of PVA hydrogels can be influenced in a controlled manner by varying the concentration of PVA and the number of freeze-thaw cycles, to mimic liver tissue characteristics. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. Human precision-cut liver slices as a model to test antifibrotic drugs in the early onset of liver fibrosis

    NARCIS (Netherlands)

    Westra, Inge M.; Mutsaers, Henricus A. M.; Luangmonkong, Theerut; Hadi, Mackenzie; Oosterhuis, Dorenda; de Jong, Koert P.; Groothuis, Geny M. M.; Olinga, Peter

    Liver fibrosis is the progressive accumulation of connective tissue ultimately resulting in loss of organ function. Currently, no effective antifibrotics are available due to a lack of reliable human models. Here we investigated the fibrotic process in human precision-cut liver slices (PCLS) and

  16. PLASMID DNA DAMAGE CAUSED BY METHYLATED ARSENICALS, ASCORBIC ACID AND HUMAN LIVER FERRITIN

    Science.gov (United States)

    PLASMID DNA DAMAGE CAOUSED BY METHYLATED ARSENICALS, ASCORBIC ACID AND HUMAN LIVER FERRITINABSTRACT Both dimethylarsinic acid (DMA(V)) and dimethylarsinous acid (DMA(III)) release iron from human liver ferritin (HLF) with or without the presence of ascorbic acid. ...

  17. Biomechanic study of the human liver during a frontal deceleration.

    Science.gov (United States)

    Cheynel, Nicolas; Serre, Thierry; Arnoux, Pierre-Jean; Baque, Patrick; Benoit, Laurent; Berdah, Stephane-Victor; Brunet, Christian

    2006-10-01

    Mechanisms of hepatic injury remain poorly understood. Surgical literature reports some speculative theories that have never been proved. The aim of this study was to examine the behavior of the liver during brutal frontal deceleration. Six trunks, removed from human cadavers, underwent free falls at 4, 6, and 8 meters per second (mps). Accelerometers were positioned in the two lobes of the liver, in front of the vertebra L2, and in the retro hepatic inferior vena cava. Relative motions of the lobes of the liver and of the two other anatomic marks were observed. In parallel, numerical simulations of this experiment have been performed using a finite element model. In the direction of impact, the vertebra L2 had no considerable displacement with the inferior vena cava. There was a noteworthy displacement between the two hepatic lobes. The left hepatic lobe had a large relative displacement with the vertebra L2 and the inferior vena cava. The right hepatic lobe was more stable with the vertebra L2 and the inferior vena cava. Numerical simulation of the same protocol underlined a rotation effect of the liver to the left around the axis of the inferior vena cava. These results support the surgical data. They highlight a crucial zone and explain how dramatic lacerations between the two lobes of the liver can occur.

  18. Thy-1 (CD90)-Positive Hepatic Progenitor Cells, Hepatoctyes, and Non-parenchymal Liver Cells Isolated from Human Livers.

    Science.gov (United States)

    Weiss, Thomas S; Dayoub, Rania

    2017-01-01

    In response to liver injury, hepatic cells, especially hepatocytes, can rapidly proliferate to repair liver damage. Additionally, it was shown that under certain circumstances liver resident cells with progenitor capabilities are involved in liver cell proliferation and differentiation. These hepatic progenitor cells (HPCs), known as oval cells in rodents, are derived from the canals of Hering, which are located in the periportal region of the liver. Regarding to different cell niches, which were defined for human HPCs, several markers have been used to identify these cells such as CD34, c-kit, OV-6, and Thy-1 (CD90). The latter was shown to be expressed on HPCs in human liver tissue with histological signs of regeneration. In this chapter we describe a detailed method for the isolation of Thy-1 positive cells from human resected liver tissue. Based on a procedure for isolating primary human hepatocytes and non-parenchymal cells (NPCs) we expanded this protocol to additional enzymatic dissociation, filtration, and centrifugation steps. This results in a bile duct cell enriched fraction of NPCs from which Thy-1 (CD90) positive cells were purified by Thy-1 positivity selection using MACS technique. Bipotential progenitor cells from human liver resections can be isolated using Thy-1 and was shown to be a suitable tool for the enrichment of liver resident progenitor cells for xenotransplantation.

  19. Obesity accelerates epigenetic aging of human liver

    OpenAIRE

    Horvath, S; Erhart, W.; Brosch, M; Ammerpohl, O.; von Schonfels, W.; Ahrens, M.; Heits, N; Bell, J.T.; Tsai, P.-C.; Spector, T. D.; Deloukas, P; Siebert, R; Sipos, B.; Becker, T.; Rocken, C.

    2014-01-01

    Because obese people are at an increased risk of many age-related diseases, it is a plausible hypothesis that obesity increases the biological age of some tissues and cell types. However, it has been difficult to detect such an accelerated aging effect because it is unclear how to measure tissue age. Here we use a recently developed biomarker of aging (known as “epigenetic clock”) to study the relationship between epigenetic age and obesity in several human tissues. We report an unexpectedly ...

  20. Assessment of a dry extract from milk thistle (Silybum marianum) for interference with human liver cytochrome-P450 activities.

    Science.gov (United States)

    Doehmer, Johannes; Weiss, Gabriele; McGregor, Gerard P; Appel, Kurt

    2011-02-01

    The effect of a standardised dry extract from Silybum marianum (HEPAR-PASC®) on the enzyme kinetics of cytochrome-P450 isoenzymes (CYP) was investigated with primary human hepatocytes and human liver microsomes in order to assess the potential for drug-drug interactions. A cytotoxic effect on hepatocytes was observed at concentrations at and above 50 μg/ml. The EC(50) value was calculated to be 72.0 μg/ml. Therefore, the chosen test concentrations for CYP induction on human hepatocytes were 50, 10, and 1.5 μg/ml, which allowed for interpretation of the clinical significance of the data with a range of 50-1-fold c(max) at maximal recommended doses. No induction was observed at the lowest concentration of 1.5 μg/ml, which is close to c(max). The extract did not induce CYP 3A4 at any of the tested concentrations. A low or marginal induction of 1A2, 2B6, and 2E1 at the maximum concentration of 50 μg/ml was observed. CYP inhibition on human microsomes was tested at concentrations of 150, 15, and 1.5 μg/ml. No or minor CYP inhibition was observed for all CYPs tested at the lowest concentration of 1.5 μg/ml, i.e. CYPs 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4. At concentrations of 15 and 150 μg/ml the extract significantly inhibited CYP 2B6, 2C8, 2C9, 2C19, 2E1, and 3A4. In these cases, K(i) values were determined. All K(i) values exceeded c(max) by at least a factor of 10-fold. According to FDA regulations 1>c(max)/K(i)>0.1 indicates, that drug-drug interactions are possible for CYPs 2C8, and 2C9, but not likely, and are remote for CYPs 2C19, 2D6, and 3A4. Copyright © 2010 Elsevier Ltd. All rights reserved.

  1. Usage analysis of human serum albumin in patients with liver cancer and liver cirrhosis after hepatectomy

    Directory of Open Access Journals (Sweden)

    HUANG Donghai

    2015-06-01

    Full Text Available ObjectiveTo analyze the usage of human serum albumin in patients with liver cancer and liver cirrhosis after hepatectomy. MethodsA total of 121 patients with liver cancer and liver cirrhosis who received hepatectomy in our hospital from January 2012 to January 2014 were divided into control group (n=60 and observation group (n=61. Both groups received human serum albumin in addition to the routine treatment for liver protection. The observation group was given intravenous drip of 5% human serum albumin within 48 h after surgery. The plasma albumin concentrations of patients were measured at 48 h after surgery, and if the concentration was <35 g/L, the patients would be given 20% human serum albumin until the concentration was ≥35 g/L. The control group was given intravenous drip of 20% human serum albumin within 48 h after surgery until the plasma albumin concentration was ≥35 g/L. The amounts of used human serum albumin and plasma were recorded for both groups. The urine volume, abdominal drainage volume, central venous pressure (CVP, mean arterial pressure (MAP, and thromboelastogram (TEG R and K values were measured at 1, 3, 7, and 10 days after surgery. The liver function indices before and after surgery and the indocyanine green retention rate at 15 minutes (ICG R15 at 7 days after surgery were measured. Comparison of continuous data between the two groups was made by t test, while comparison of categorical data was made by chisquare test. Results(1 There were no significant differences in age, sex, Child-Pugh classification, surgical approach, intraoperative blood loss, occlusion time of the first porta hepatis, and operation time between the two groups (P>0.05. But there were significant differences in the amounts of used human serum albumin and plasma and the length of hospital stay between the two groups (P<0.05. (2 There were significant differences in daily urine volume, CVP, MAP, abdominal drainage volume, and interstitial

  2. Human fetal liver cells for regulated ex vivo erythropoietin gene therapy

    Directory of Open Access Journals (Sweden)

    Ebtisam El Filali

    2014-01-01

    Full Text Available Possible risks and lack of donor livers limit application of liver transplantation. Liver cell transplantation is, at this moment, not a feasible alternative because engraftment in the liver is poor. Furthermore, there is also shortage of cells suitable for transplantation. Fetal liver cells are able to proliferate in cell culture and could therefore present an alternative source of cells for transplantation. In this study, we investigated the utility of human fetal liver cells for therapeutic protein delivery. We transplanted human fetal liver cells in immunodeficient mice but were not able to detect engraftment of human hepatocytes. In contrast, transplantation of human adult hepatocytes led to detectable engraftment of hepatocytes in murine liver. Transplantation of fetal liver cells did lead to abundant reconstitution of murine liver with human endothelium, indicating that endothelial cells are the most promising cell type for ex vivo liver cell gene therapy. Human liver endothelial cells were subsequently transduced with a lentiviral autoregulatory erythropoietin expression vector. After transplantation in immunodeficient mice, these cells mediated long-term regulation of murine hematocrits. Our study shows the potential of human liver endothelial cells for long-term regulated gene therapy.

  3. Expression of ATP7B in normal human liver

    Directory of Open Access Journals (Sweden)

    D Fanni

    2009-06-01

    Full Text Available ATP7B is a copper transporting P-type ATPase, also known as Wilson disease protein, which plays a key role in copper distribution inside cells. Recent experimental data in cell culture have shown that ATP7B putatively serves a dual function in hepatocytes: when localized to the Golgi apparatus, it has a biosynthetic role, delivering copper atoms to apoceruloplasmin; when the hepatocytes are under copper stress, ATP7B translocates to the biliary pole to transport excess copper out of the cell and into the bile canaliculus for subsequent excretion from the body via the bile. The above data on ATP7B localization have been mainly obtained in tumor cell systems in vitro. The aim of the present work was to assess the presence and localization of the Wilson disease protein in the human liver. We tested immunoreactivity for ATP7B in 10 human liver biopsies, in which no significant pathological lesion was found using a polyclonal antiserum specific for ATP7B. In the normal liver, immunoreactivity for ATP7B was observed in hepatocytes and in biliary cells. In the hepatocytes, immunoreactivity for ATP7B was observed close to the plasma membrane, both at the sinusoidal and at the biliary pole. In the biliary cells, ATP7B was localized close to the cell membrane, mainly concentrated at the basal pole of the cells. The data suggest that, in human liver, ATP7B is localized to the plasma membrane of both hepatocytes and biliary epithelial cells.

  4. Cellular distribution and handling of liver-targeting preparations in human livers studied by a liver lobe perfusion (vol 29, pg 361, 2001)

    NARCIS (Netherlands)

    Melgert, BN; Olinga, P; Weert, B; Slooff, MJH; Meijer, DKF; Poelstra, K; Groothuis, GMM

    We developed and tested a novel method for perfusing parts of human liver to study uptake and handling of drug-targeting preparations. These preparations, designed for the treatment of liver fibrosis in man, have been extensively studied in animals, but little is known about the uptake and handling

  5. Metabolomic Modularity Analysis (MMA) to Quantify Human Liver Perfusion Dynamics.

    Science.gov (United States)

    Sridharan, Gautham Vivek; Bruinsma, Bote; Bale, Shyam Sundhar; Swaminathan, Anandh; Saeidi, Nima; Yarmush, Martin L; Uygun, Korkut

    2017-11-13

    Large-scale -omics data are now ubiquitously utilized to capture and interpret global responses to perturbations in biological systems, such as the impact of disease states on cells, tissues, and whole organs. Metabolomics data, in particular, are difficult to interpret for providing physiological insight because predefined biochemical pathways used for analysis are inherently biased and fail to capture more complex network interactions that span multiple canonical pathways. In this study, we introduce a nov-el approach coined Metabolomic Modularity Analysis (MMA) as a graph-based algorithm to systematically identify metabolic modules of reactions enriched with metabolites flagged to be statistically significant. A defining feature of the algorithm is its ability to determine modularity that highlights interactions between reactions mediated by the production and consumption of cofactors and other hub metabolites. As a case study, we evaluated the metabolic dynamics of discarded human livers using time-course metabolomics data and MMA to identify modules that explain the observed physiological changes leading to liver recovery during subnormothermic machine perfusion (SNMP). MMA was performed on a large scale liver-specific human metabolic network that was weighted based on metabolomics data and identified cofactor-mediated modules that would not have been discovered by traditional metabolic pathway analyses.

  6. Effect of ethanol on CHCl3 metabolism in hepatic microsomes from Osborne-Mendel rats.

    Science.gov (United States)

    Testai, E; Gemma, S; Gervasi, P; Menicagli, S; Vittozzi, L

    1994-11-01

    The treatment of Osborne-Mendel rats with ethanol in drinking water for 2 weeks resulted in a 3-fold increase of hepatic microsomal hydroxylation of both p-nitrophenol and aniline, two substrates considered highly selective for P4502E1. No other forms of P450 seemed to be affected. These results, confirmed by the immunoblot analysis of microsomal protein, showed an induction of P4502E1. The levels of total covalent binding to microsomal phospholipid due to 14CHCl3 reactive intermediates in ethanol-pretreated microsomes were identical to those measured in microsomes from untreated rats at any pO2. The distribution of radioactivity obtained after transmethylation of the adducts of 14CHCl3 intermediates with microsomal phospholipids (PL) indicated that binding to fatty acyl chains (due to .CHCl2 radicals) increased with decreasing pO2. On the contrary, the binding to polar heads due to phosgene decreased. The ethanol treatment did not affect binding to either PL moieties. These results indicated that, in our experimental conditions, the in vitro production of both oxidative and reductive intermediates of CHCl3 in the liver of Osborne-Mendel rats were not influenced by ethanol consumption.

  7. Human Liver Infection in a Dish: Easy-To-Build 3D Liver Models for Studying Microbial Infection.

    Science.gov (United States)

    Petropolis, Debora B; Faust, Daniela M; Tolle, Matthieu; Rivière, Lise; Valentin, Tanguy; Neuveut, Christine; Hernandez-Cuevas, Nora; Dufour, Alexandre; Olivo-Marin, Jean-Christophe; Guillen, Nancy

    2016-01-01

    Human liver infection is a major cause of death worldwide, but fundamental studies on infectious diseases affecting humans have been hampered by the lack of robust experimental models that accurately reproduce pathogen-host interactions in an environment relevant for the human disease. In the case of liver infection, one consequence of this absence of relevant models is a lack of understanding of how pathogens cross the sinusoidal endothelial barrier and parenchyma. To fill that gap we elaborated human 3D liver in vitro models, composed of human liver sinusoidal endothelial cells (LSEC) and Huh-7 hepatoma cells as hepatocyte model, layered in a structure mimicking the hepatic sinusoid, which enable studies of key features of early steps of hepatic infection. Built with established cell lines and scaffold, these models provide a reproducible and easy-to-build cell culture approach of reduced complexity compared to animal models, while preserving higher physiological relevance compared to standard 2D systems. For proof-of-principle we challenged the models with two hepatotropic pathogens: the parasitic amoeba Entamoeba histolytica and hepatitis B virus (HBV). We constructed four distinct setups dedicated to investigating specific aspects of hepatic invasion: 1) pathogen 3D migration towards hepatocytes, 2) hepatocyte barrier crossing, 3) LSEC and subsequent hepatocyte crossing, and 4) quantification of human hepatic virus replication (HBV). Our methods comprise automated quantification of E. histolytica migration and hepatic cells layer crossing in the 3D liver models. Moreover, replication of HBV virus occurs in our virus infection 3D liver model, indicating that routine in vitro assays using HBV or others viruses can be performed in this easy-to-build but more physiological hepatic environment. These results illustrate that our new 3D liver infection models are simple but effective, enabling new investigations on infectious disease mechanisms. The better

  8. Expression of neural cell adhesion molecule in human liver development and in congenital and acquired liver diseases.

    Science.gov (United States)

    Libbrecht, L; Cassiman, D; Desmet, V; Roskams, T

    2001-09-01

    In the liver, neural cell adhesion molecule (NCAM) is a marker of immature cells committed to the biliary lineage and is expressed by reactive bile ductules in human liver diseases. We investigated the possible role of NCAM in the development of intrahepatic bile ducts and aimed at determining whether immature biliary cells can contribute to the repair of damaged bile ducts in chronic liver diseases. Therefore, we performed immunohistochemistry for NCAM and bile duct cell markers cytokeratin 7 and cytokeratin 19 on frozen sections of 85 liver specimens taken from 14 fetuses, 10 donor livers, 18 patients with congenital liver diseases characterized by ductal plate malformations (DPMs), and 43 cirrhotic explant livers. Duplicated ductal plates and incorporating bile ducts during development showed a patchy immunoreactivity for NCAM, while DPMs were continuously positive for NCAM. Bile ducts showing complete or patchy immunoreactivity for NCAM were found in cirrhotic livers, with higher frequency in biliary than in posthepatitic cirrhosis. Our results suggest that NCAM may have a function in the development of the intrahepatic bile ducts and that NCAM-positive immature biliary cells can contribute to the repair of damaged bile ducts in chronic liver diseases.

  9. Metabolism of α-thujone in human hepatic preparations in vitro.

    Science.gov (United States)

    Abass, Khaled; Reponen, Petri; Mattila, Sampo; Pelkonen, Olavi

    2011-02-01

    This study aims to characterize the metabolism of α-thujone in human liver preparations in vitro and to identify the role of cytochrome P450 (CYP) and possibly other enzymes catalyzing α-thujone biotransformations. With a liquid chromatography-mass spectrometry (LC-MS) method developed for measuring α-thujone and four potential metabolites, it was demonstrated that human liver microsomes produced two major (7- and 4-hydroxy-thujone) and two minor (2-hydroxy-thujone and carvacrol) metabolites. Glutathione and cysteine conjugates were detected in human liver homogenates, but not quantified. No glucuronide or sulphate conjugates were detected. Major hydroxylations accounted for more than 90% of the primary microsomal metabolism of α-thujone. Screening of α-thujone metabolism with CYP recombinant enzymes indicated that CYP2A6 was principally responsible for the major 7- and 4-hydroxylation reactions, although CYP3A4 and CYP2B6 participated to a lesser extent and CYP3A4 and CYP2B6 catalyzed minor 2-hydroxylation. Based on the intrinsic efficiencies of different recombinant CYP enzymes and average abundances of these enzymes in human liver microsomes, CYP2A6 was calculated to be the most active enzyme in human liver microsomes, responsible for 70-80% of the metabolism on average. Inhibition screening indicated that α-thujone inhibited both CYP2A6 and CYP2B6, with 50% inhibitory concentration values of 15.4 and 17.5 µM, respectively.

  10. The revised human liver cytochrome P450 "Pie": absolute protein quantification of CYP4F and CYP3A enzymes using targeted quantitative proteomics.

    Science.gov (United States)

    Michaels, Scott; Wang, Michael Zhuo

    2014-08-01

    The CYP4F subfamily of enzymes has been identified recently to be involved in the metabolism of endogenous compounds (arachidonic acid and leukotriene B4), nutrients (vitamins K1 and E), and xenobiotics (pafuramidine and fingolimod). CYP4F2 and CYP4F3B are reported to be expressed in the human liver. However, absolute concentrations of these enzymes in human liver microsomes (HLMs) and their interindividual variability have yet to be determined because of the lack of specific antibodies. Here, an liquid chromatography with tandem mass spectrometry (LC-MS/MS)-based targeted quantitative proteomic approach was employed to determine the absolute protein concentrations of CYP4F2 and CYP4F3B compared with CYP3A in two panels of HLMs (n = 31). As a result, the human hepatic cytochrome P450 (P450) "pie" has been revised to include the contribution of CYP4F enzymes, which amounts to 15% of the total hepatic cytochrome P450 enzymes. CYP4F3B displayed low interindividual variability (3.3-fold) in the HLM panels whereas CYP4F2 displayed large variability (21-fold). However, CYP4F2 variability decreased to 3.4-fold if the two donors with the lowest expression were excluded. In contrast, CYP3A exhibited 29-fold interindividual variability in the same HLM panels. The proposed marker reaction for CYP4F enzymes pafuramidine/DB289 M1 formation did not correlate with CYP4F protein content, suggesting alternate metabolic pathways for DB289 M1 formation in HLMs. In conclusion, CYP4F enzymes are highly expressed in the human liver and their physiologic and pharmacologic roles warrant further investigation. Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.

  11. All-Trans-Retinoic Acid Enhances Mitochondrial Function in Models of Human Liver

    OpenAIRE

    Tripathy, Sasmita; Chapman, John D.; Han, Chang Y; Hogarth, Cathryn A.; Arnold, Samuel L. M.; Onken, Jennifer; Kent, Travis; Goodlett, David R.; Isoherranen, Nina

    2016-01-01

    All-trans-retinoic acid (atRA) is the active metabolite of vitamin A. The liver is the main storage organ of vitamin A, but activation of the retinoic acid receptors (RARs) in mouse liver and in human liver cell lines has also been shown. Although atRA treatment improves mitochondrial function in skeletal muscle in rodents, its role in modulating mitochondrial function in the liver is controversial, and little data are available regarding the human liver. The aim of this study was to determin...

  12. Some properties of the fatty alcohol oxidation system and reconstitution of microsomal oxidation activity in intestinal mucosa.

    Science.gov (United States)

    Ichihara, K; Kusunose, E; Noda, Y; Kusunose, M

    1986-10-03

    This paper describes the metabolism of fatty alcohols by microsomal and cytosolic fractions from intestinal mucosa. Microsomes of rabbit intestinal mucosa had a high activity of [1-14C]dodecanol oxidation as did those of liver. The intestinal cytosolic fraction also exhibited oxidation activity to a lesser extent than the microsomes did. The reaction product was determined as lauric acid using thin-layer chromatography. Laurylaldehyde was detected as another product, when semicarbazide was added to the incubation system. Cyclodextrins exhibited a stimulation effect similarly to bovine serum albumin on the microsomal activity. We have compared the stimulatory effects of dimethyl-beta-cyclodextrin, beta-cyclodextrin, gamma-cyclodextrin and alpha-cyclodextrin, which decrease in that order. Effects of NAD+ and dodecanol concentrations, pH and pyrazole on microsomal activity were compared with those on cytosolic activity. Dodecanol oxidation activity was solubilized and reconstituted with a fatty alcohol dehydrogenase and a fatty aldehyde dehydrogenase separated from the intestinal microsomes. These findings indicate that both the dehydrogenases participate in microsomal oxidation of fatty alcohols to fatty acids with fatty aldehydes as intermediates in the reaction.

  13. Solubilisation, purification and reconstitution of hepatic microsomal azoreductase activity.

    Science.gov (United States)

    Mallett, A K; King, L J; Walker, R

    1985-02-01

    Microsomal NADPH-cytochrome c (P-450) reductase and cytochrome P-450 were purified from the livers of phenobarbitone-treated rats. Purified NADPH-cytochrome c (P-450) reductase effected the NADPH-dependent reduction of FMN and FAD under anaerobic conditions in a non-enzymic manner, but was unable to reduce directly the azo dye, amaranth. In the presence of FMN, the purified reductase effected reduction of amaranth through the production of reduced FMN. Incorporation of NADPH-cytochrome c (P-450) reductase into the microsomal fraction increased the azoreductase activity of liver preparations from phenobarbitone-treated rats, but had no effect on azoreductase activity in preparations from control animals. Azoreductase activity was reconstituted into dilauroyl phosphatidylcholine vesicles containing purified cytochrome P-450 and purified NADPH-cytochrome c (P-450) reductase. In the absence of supplementary FMN, amaranth reduction was completely dependent upon all three components, but in the presence of FMN, the omission of any one component failed to abolish completely azoreductase activity.

  14. Age dependence of rat liver function measurements

    DEFF Research Database (Denmark)

    Fischer-Nielsen, A; Poulsen, H E; Hansen, B A

    1989-01-01

    Changes in the galactose elimination capacity, the capacity of urea-N synthesis and antipyrine clearance were studied in male Wistar rats at the age of 8, 20 and 44 weeks. Further, liver tissue concentrations of microsomal cytochrome P-450, microsomal protein and glutathione were measured. All...... liver function measurements increased from the age of 8 to 44 weeks when expressed in absolute values. In relation to body weight, these function measurements were unchanged or reduced from week 8 to week 20. At week 44, galactose elimination capacity and capacity of urea-N synthesis related to body...... weight were increased by 10% and 36%, respectively, and antipyrine plasma clearance was reduced to 50%. Liver tissue concentrations of microsomal cytochrome P-450 and microsomal protein increased with age when expressed in absolute values, but were unchanged per g liver, i.e., closely related to liver...

  15. Obligatory role of cytochrome b5 in the microsomal metabolism of methoxyflurane.

    Science.gov (United States)

    Canova-Davis, E; Chiang, J Y; Waskell, L

    1985-06-01

    Cytochrome b5 has recently been shown to be required in the reconstituted cytochrome P-450 system for the metabolism of the volatile anesthetic methoxyflurane [E. Canova-Davis and L. A. Waskell, J. biol. Chem. 259, 2541 (1984)]. To determine whether this observation in the reconstituted system was merely dependent on the particular ratios of the various components or some other fortuitous, unknown factor, or whether cytochrome b5 plays a role in the liver microsomal metabolism of methoxyflurane, the following studies were undertaken. Antibody to rabbit holocytochrome b5 was raised in guinea pigs. The antibody to cytochrome b5 was able to inhibit 75% of the microsomal metabolism of methoxyflurane. This same antibody also inhibited methoxyflurane metabolism in the reconstituted system. When the antibody to cytochrome b5 was treated with purified cytochrome b5 before addition to the microsomes, it did not inhibit methoxyflurane metabolism. Furthermore, the antibody to cytochrome b5 did not inhibit the microsomal metabolism of benzphetamine. This suggests that cytochrome b5 was required for the microsomal metabolism of methoxyflurane. It is possible that cytochrome b5 functioned in the metabolism of methoxyflurane by retaining a specific conformation of cytochrome P-450 and not by transferring the second electron to cytochrome P-450. To explore this possibility, cytochrome b5 was reconstituted with Mn3+-protoporphyrin IX. The Mn3+-protoporphyrin IX derivative retained the conformation of cytochrome b5 but not its electron transfer properties. This manganese derivative of cytochrome b5 was unable to stimulate the metabolism of methoxyflurane. The study demonstrated that cytochrome b5 was obligatory for the microsomal metabolism of methoxyflurane, whereas it was not required for the microsomal N-demethylation of benzphetamine. Moreover, the heme moiety of cytochrome b5 functioned to transfer electrons in this reaction.

  16. Effect of the Human Amniotic Membrane on Liver Regeneration in Rats

    Science.gov (United States)

    Sipahi, Mesut; Şahin, Sevinç; Arslan, Ergin; Börekci, Hasan; Metin, Bayram; Cantürk, Nuh Zafer

    2015-01-01

    Introduction. Operations are performed for broader liver surgery indications for a better understanding of hepatic anatomy/physiology and developments in operation technology. Surgery can cure some patients with liver metastasis of some tumors. Nevertheless, postoperative liver failure is the most feared complication causing mortality in patients who have undergone excision of a large liver mass. The human amniotic membrane has regenerative effects. Thus, we investigated the effects of the human amniotic membrane on regeneration of the resected liver. Methods. Twenty female Wistar albino rats were divided into control and experimental groups and underwent a 70% hepatectomy. The human amniotic membrane was placed over the residual liver in the experimental group. Relative liver weight, histopathological features, and biochemical parameters were assessed on postoperative day 3. Results. Total protein and albumin levels were significantly lower in the experimental group than in the control group. No difference in relative liver weight was observed between the groups. Hepatocyte mitotic count was significantly higher in the experimental group than in the control group. Hepatic steatosis was detected in the experimental group. Conclusion. Applying the amniotic membrane to residual liver adversely affected liver regeneration. However, mesenchymal stem cell research has the potential to accelerate liver regeneration investigations. PMID:26457000

  17. Effect of the Human Amniotic Membrane on Liver Regeneration in Rats

    Directory of Open Access Journals (Sweden)

    Mesut Sipahi

    2015-01-01

    Full Text Available Introduction. Operations are performed for broader liver surgery indications for a better understanding of hepatic anatomy/physiology and developments in operation technology. Surgery can cure some patients with liver metastasis of some tumors. Nevertheless, postoperative liver failure is the most feared complication causing mortality in patients who have undergone excision of a large liver mass. The human amniotic membrane has regenerative effects. Thus, we investigated the effects of the human amniotic membrane on regeneration of the resected liver. Methods. Twenty female Wistar albino rats were divided into control and experimental groups and underwent a 70% hepatectomy. The human amniotic membrane was placed over the residual liver in the experimental group. Relative liver weight, histopathological features, and biochemical parameters were assessed on postoperative day 3. Results. Total protein and albumin levels were significantly lower in the experimental group than in the control group. No difference in relative liver weight was observed between the groups. Hepatocyte mitotic count was significantly higher in the experimental group than in the control group. Hepatic steatosis was detected in the experimental group. Conclusion. Applying the amniotic membrane to residual liver adversely affected liver regeneration. However, mesenchymal stem cell research has the potential to accelerate liver regeneration investigations.

  18. Extensive double humanization of both liver and hematopoiesis in FRGN mice.

    Science.gov (United States)

    Wilson, Elizabeth M; Bial, J; Tarlow, Branden; Bial, G; Jensen, B; Greiner, D L; Brehm, M A; Grompe, M

    2014-11-01

    Preclinical research in animals often fails to adequately predict the outcomes observed in human patients. Chimeric animals bearing individual human tissues have been developed to provide improved models of human-specific cellular processes. Mice transplanted with human hematopoietic stem cells can be used to study human immune responses, infections of blood cells and processes of hematopoiesis. Animals with humanized livers are useful for modeling hepatotropic infections as well as drug metabolism and hepatotoxicity. However, many pathophysiologic processes involve both the liver and the hematolymphoid system. Examples include hepatitis C/HIV co-infection, immune mediated liver diseases, liver injuries with inflammation such as steatohepatitis and alcoholic liver disease. We developed a robust protocol enabling the concurrent double-humanization of mice with mature hepatocytes and human blood. Immune-deficient, fumarylacetoacetate hydrolase (Fah(-/-)), Rag2(-/-) and Il2rg(-/-) deficient animals on the NOD-strain background (FRGN) were simultaneously co-transplanted with adult human hepatocytes and hematopoietic stem cells after busulfan and Ad:uPA pre-conditioning. Four months after transplantation the average human liver repopulation exceeded 80% and hematopoietic chimerism also was high (40-80% in bone marrow). Importantly, human macrophages (Kupffer cells) were present in the chimeric livers. Double-chimeric FRGN mice will serve as a new model for disease processes that involve interactions between hepatocytes and hematolymphoid cells. Copyright © 2014. Published by Elsevier B.V.

  19. PLASMID DNA DAMAGE CAUSED BY METHYLATED ARSENICALS, ASCORBIC ACID AND HUMAN LIVER FERRITIN

    Science.gov (United States)

    Plasmid DNA damage caused by methylated arsenicals, ascorbic acid and human liver ferritin. Arsenic causes cancer in human skin, urinary bladder, lung, liver and kidney and is a significant world-wide public health problem. Although the metabolism of inorganic arsenic is ...

  20. Human-scale whole-organ bioengineering for liver transplantation: a regenerative medicine approach.

    Science.gov (United States)

    Yagi, Hiroshi; Fukumitsu, Ken; Fukuda, Kazumasa; Kitago, Minoru; Shinoda, Masahiro; Obara, Hideaki; Itano, Osamu; Kawachi, Shigeyuki; Tanabe, Minoru; Coudriet, Gina M; Piganelli, Jon D; Gilbert, Thomas W; Soto-Gutierrez, Alejandro; Kitagawa, Yuko

    2013-01-01

    At this time, the only definitive treatment of hepatic failure is liver transplantation. However, transplantation has been limited by the severely limited supply of human donor livers. Alternatively, a regenerative medicine approach has been recently proposed in rodents that describe the production of three-dimensional whole-organ scaffolds for assembly of engineered complete organs. In the present study, we describe the decellularization of porcine livers to generate liver constructs at a scale that can be clinically relevant. Adult ischemic porcine livers were successfully decellularized using a customized perfusion protocol, the decellularization process preserved the ultrastructural extracellular matrix components, functional characteristics of the native microvascular and the bile drainage network of the liver, and growth factors necessary for angiogenesis and liver regeneration. Furthermore, isolated hepatocytes engrafted and reorganized in the porcine decellularized livers using a human-sized organ culture system. These results provide proof-of-principle for the generation of a human-sized, three-dimensional organ scaffold as a potential structure for human liver grafts reconstruction for transplantation to treat liver disease.

  1. Oxidized low-density lipoprotein-induced periodontal inflammation is associated with the up-regulation of cyclooxygenase-2 and microsomal prostaglandin synthase 1 in human gingival epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Nagahama, Yu [Department of Periodontology, School of Dentistry, Showa University Dental Hospital, Tokyo (Japan); Department of Biological Chemistry, Showa University School of Pharmacy, Tokyo (Japan); Obama, Takashi [Department of Biological Chemistry, Showa University School of Pharmacy, Tokyo (Japan); Usui, Michihiko [Department of Periodontology, School of Dentistry, Showa University Dental Hospital, Tokyo (Japan); Kanazawa, Yukari [Department of Biological Chemistry, Showa University School of Pharmacy, Tokyo (Japan); Iwamoto, Sanju [Department of Biochemistry, Showa University School of Medicine, Tokyo (Japan); Suzuki, Kazushige [Department of Periodontology, School of Dentistry, Showa University Dental Hospital, Tokyo (Japan); Miyazaki, Akira [Department of Biochemistry, Showa University School of Medicine, Tokyo (Japan); Yamaguchi, Tomohiro [Department of Biological Chemistry, Showa University School of Pharmacy, Tokyo (Japan); Yamamoto, Matsuo [Department of Periodontology, School of Dentistry, Showa University Dental Hospital, Tokyo (Japan); Itabe, Hiroyuki, E-mail: h-itabe@pharm.showa-u.ac.jp [Department of Biological Chemistry, Showa University School of Pharmacy, Tokyo (Japan)

    2011-10-07

    Highlights: {yields} OxLDL-induced responses in human gingival epithelial cells were studied. {yields} OxLDL enhanced the production of IL-8, IL-1{beta} and PGE{sub 2} in Ca9-22 cells. {yields} An NF-{kappa}B inhibitor suppressed the expression of COX-2 and mPGES1 induced by oxLDL. {yields} Unlike the case in macrophages, oxLDL did not increase the CD36 level. -- Abstract: Periodontitis is characterized by chronic gingival tissue inflammation, and inflammatory mediators such as IL-8 and prostaglandin E{sub 2} (PGE{sub 2}) are associated with disease progression. Previously we showed that oxidatively modified low-density lipoprotein (oxLDL) was present in gingival crevicular fluid. In this study, the role of oxLDL in the gingival epithelial cell inflammatory response was further investigated using Ca9-22 cells and primary human oral keratinocytes (HOK). Treatment of Ca9-22 cells and HOK with oxLDL induced an up-regulation of IL-8 and the PGE{sub 2}-producing enzymes, cyclooxygenase-2 and microsomal PGE{sub 2} synthase-1. These responses induced by oxLDL were significantly suppressed by a nuclear factor-kappa B (NF-{kappa}B) inhibitor. However, unlike the result in macrophages, oxLDL did not lead to an increase in CD36 expression in these two cells. These results suggest that oxLDL elicits gingival epithelial cell inflammatory responses through an activation of the NF-{kappa}B pathway. These data suggest a mechanistic link between periodontal disease and lipid metabolism-related disorders, including atherosclerosis.

  2. Photoacoustic physio-chemical analysis of liver conditions in animal and human subjects

    Science.gov (United States)

    Wang, Xueding; Xu, Guan; Tian, Chao; Wan, Shanshan; Welling, Theodore H.; Lok, Anna S. F.; Rubin, Jonathan M.

    2016-03-01

    Non-alcoholic fatty liver disease (NAFLD) is a common liver disease affecting 30% of the population in the United States. Biopsy is the gold standard for diagnosing NAFLD. Liver histology assesses the amount of fat, and determines type and extent of cell injury, inflammation and fibrosis. However, liver biopsy is invasive and is limited by sampling error. Current radiological diagnostic modalities can evaluate the 'physical' morphology in liver by quantifying the backscattered US signals, but cannot interrogate the 'histochemical' components forming these backscatterers. For example, ultrasound (US) imaging can detect the presence of fat but cannot differentiate steatosis alone from steatohepatitis. Our previous study of photoacoustic physiochemical analysis (PAPCA) has demonstrated that this method can characterize the histological changes in livers during the progression of NAFLD in animal models. In this study, we will further validate PAPCA with human livers. Ex vivo human liver samples with steatosis, fibrosis and cirrhosis will be scanned using optical illumination at wavelengths of 680-1700 nm and compared to histology results. In vivo study on human subjects with confirmed steatosis is planned using our PA-ultrasound (US) parallel imaging system based on Verasonic US imaging flatform with an L7-4 probe. 10 mJ/cm2 per pulse optical energy at 755 nm will be delivered to the skin surface, which is under the safety limit of American National Standard Institute. Preliminary study with ex vivo human tissue has demonstrated the potential of the proposed approach in differentiating human liver conditions.

  3. Human liver endothelial cells, but not macrovascular or microvascular endothelial cells, engraft in the mouse liver

    NARCIS (Netherlands)

    Filali, Ebtisam El; Hiralall, Johan K.; van Veen, Henk A.; Stolz, Donna B.; Seppen, Jurgen

    2013-01-01

    Liver cell transplantation has had limited clinical success so far, partly due to poor engraftment of hepatocytes. Instead of hepatocytes. other cell types, such as endothelial cells, could be used in ex vivo liver gene therapy. The goal of the present study was to compare the grafting and

  4. In utero transplantation of fetal liver stem cells in humans.

    Science.gov (United States)

    Touraine, J L

    1991-01-01

    Following 15 years experience in postnatal fetal liver transplantation (FLT), we have developed a new therapeutical method, namely the in utero transplantation of stem cells from the human fetal liver. This early transplant takes advantage of the immunological tolerance that exists in young fetal recipients. The three fetuses that we treated were 28, 26, and 12 weeks of age (weeks after fecundation). The first two patients had immunodeficiencies, the third one had thalassemia major. Donor cells were obtained from 7- to 10-week-old fetuses, with conditions approved by the National Committee for Bioethics. Donors and recipients were not matched. The fetal cells were infused through the umbilical vein of the first two patients and injected intraperitoneally into the third one, under ultrasonic visualization. The first patient, born in 1988, has evidence of engraftment and reconstitution of cell-mediated immunity: initially 10% than 26% of lymphocytes of donor origin (with distinct phenotype), T cell responses to tetanus toxoid and candida antigens. This child, who had bare lymphocyte syndrome, has no clinical manifestation of the disease and lives normally at home. The second child was born in 1989 and it is too early for a thorough evaluation of the immunological effects of the transplant, although donor cell engraftment has been proven (Y chromosome in this female patient). The third patient has also evidence of donor cell take (Y chromosome in a female patient) but the effect on thalassemia has not yet been fully analyzed (donor hemoglobin present in small quantity). In all three cases, no side effect of any kind developed in the mother nor in the fetus.(ABSTRACT TRUNCATED AT 250 WORDS)

  5. The histogenesis of regenerative nodules in human liver cirrhosis.

    Science.gov (United States)

    Lin, Wey-Ran; Lim, Siew-Na; McDonald, Stuart A C; Graham, Trevor; Wright, Victoria L; Peplow, Claire L; Humphries, Adam; Kocher, Hemant M; Wright, Nicholas A; Dhillon, Amar P; Alison, Malcolm R

    2010-03-01

    Here, we investigate the clonality and cells of origin of regenerative nodules in human liver cirrhosis using mitochondrial DNA (mtDNA) mutations as markers of clonal expansion. Mutated cells are identified phenotypically by deficiency in the entirely mtDNA encoded cytochrome c oxidase (CCO) enzyme by histochemical and immunohistochemical methods. Nodules were classified as either CCO-deficient or CCO-positive, and among 526 nodules from 10 cases, 18% were homogeneously CCO-deficient, whereas only 3% had a mixed phenotype. From frozen sections, hepatocytes were laser-capture microdissected from several sites within individual CCO-deficient nodules. Mutations were identified by polymerase chain reaction sequencing of the entire mtDNA genome. In all cases except for one, the nodules were monoclonal in nature, possessing up to four common mutations in all hepatocytes in a given nodule. Moreover, the identification of identical mutations in hepatic progenitor cells abutting CCO-deficient nodules proves that nodules can have their origins from such cells. These data support a novel pathway for the monoclonal derivation of human cirrhotic regenerative nodules from hepatic progenitor cells.

  6. The 24-hour normothermic machine perfusion of discarded human liver grafts.

    Science.gov (United States)

    Vogel, Thomas; Brockmann, Jens G; Quaglia, Alberto; Morovat, Alireza; Jassem, Wayel; Heaton, Nigel D; Coussios, Constantin C; Friend, Peter J

    2017-02-01

    Donor organ shortage necessitates use of less than optimal donor allografts for transplantation. The current cold storage preservation technique fails to preserve marginal donor grafts sufficiently. Evidence from large animal experiments suggests superiority of normothermic machine preservation (NMP) of liver allografts. In this study, we analyze discarded human liver grafts that underwent NMP for the extended period of 24 hours. Thirteen human liver grafts which had been discarded for transplantation were entered into this study. Perfusion was performed with an automated device using an oxygenated, sanguineous perfusion solution at normothermia. Automated control was incorporated for temperature-, flow-, and pressure-regulation as well as oxygenation. All livers were perfused for 24 hours; parameters of biochemical and synthetic liver function as well as histological parameters of liver damage were analyzed. Livers were stratified for expected viability according to the donor's medical history, procurement data, and their macroscopic appearance. Normothermic perfusion preservation of human livers for 24 hours was shown to be technically feasible. Human liver grafts, all of which had been discarded for transplantation, showed levels suggesting organ viability with respect to metabolic and synthetic liver function (to varying degrees). There was positive correlation between instantly available perfusion parameters and generally accepted predictors of posttransplant graft survival. In conclusion, NMP is feasible reliably for periods of at least 24 hours, even in highly suboptimal donor organs. Potential benefits include not only viability testing (as suggested in recent clinical implementations), but also removal of the time constraints associated with the utilization of high-risk livers, and recovery of ischemic and other preretrieval injuries (possibly by enabling therapeutic strategies during NMP). Liver Transplantation 23 207-220 2017 AASLD. © 2016 by the

  7. Reductase and oxidase activity of rat liver cytochrome P450 with 2,3,5,6-tetramethylbenzoquinone as substrate

    NARCIS (Netherlands)

    Goeptar, A R; te Koppele, J.M.; Neve, E P; Vermeulen, N P

    1992-01-01

    The main objective of the present study was to investigate the proposed role of cytochrome P450 in the reductive metabolism of quinones as well as in the formation of reduced oxygen species in liver microsomes from phenobarbital (PB-microsomes) and beta-naphthoflavone (beta NF-microsomes) pretreated

  8. Genotoxicity-related chemistry of human metabolites of benzo[ghi]perylene (B[ghi]P) investigated using electro-optical arrays and DNA/microsome biocolloid reactors with LC-MS/MS.

    Science.gov (United States)

    Pan, Shenmin; Li, Dandan; Zhao, Linlin; Schenkman, John B; Rusling, James F

    2013-08-19

    There is limited and sometimes contradictory information about the genotoxicity of the polycyclic aromatic hydrocarbon benzo[ghi]perylene (B[ghi]P). Using recently developed metabolic toxicity screening arrays and a biocolloid reactor-LC-MS/MS approach, both featuring films of DNA and human metabolic enzymes, we demonstrated the relatively low reactivity of metabolically activated B[ghi]P toward DNA. Electro-optical toxicity screening arrays showed that B[ghi]P metabolites damage DNA at a 3-fold lower rate than benzo[a]pyrene (B[a]P), whose metabolites have a strong and well-understood propensity for DNA damage. Metabolic studies using magnetic bead biocolloid reactors coated with microsomal enzymes in 96-well plates showed that cyt P450s 1A1 and 1B1 provide high activity for B[ghi]P and B[a]P conversion. Consistent with published results, the major metabolism of B[ghi]P involved oxidations at 3,4 and 11,12 positions, leading to the formation of B[ghi]P 3,4-oxide and B[ghi]P 3,4,11,12-bisoxide. B[ghi]P 3,4-oxide was synthesized and reacted with deoxyadenosine at N6 and N7 positions and with deoxyguanosine at the N2 position. B[ghi]P 3,4-oxide is hydrolytically unstable and transforms into the 3,4-diol or converts to 3- or 4-hydroxy B[ghi]P. LC-MS/MS of reaction products from the magnetic biocolloid reactor particles coated with DNA and human enzymes revealed for the first time that a major DNA adduct results from the reaction between B[ghi]P 3,4,11,12-bisoxide and deoxyguanosine. Results also demonstrated 5-fold lower formation rates of the major DNA adduct for B[ghi]P metabolites compared to B[a]P. Overall, results from both the electro-optical array and biocolloid reactor-LC-MS/MS consistently suggest a lower human genotoxicity profile of B[ghi]P than B[a]P.

  9. Three-dimensional reconstructions of intrahepatic bile duct tubulogenesis in human liver

    DEFF Research Database (Denmark)

    Vestentoft, Peter S; Jelnes, Peter; Hopkinson, Branden M

    2011-01-01

    BACKGROUND: During liver development, intrahepatic bile ducts are thought to arise by a unique asymmetric mode of cholangiocyte tubulogenesis characterized by a series of remodeling stages. Moreover, in liver diseases, cells lining the Canals of Hering can proliferate and generate new hepatic...... tissue. The aim of this study was to develop protocols for three-dimensional visualization of protein expression, hepatic portal structures and human hepatic cholangiocyte tubulogenesis. RESULTS: Protocols were developed to digitally visualize portal vessel branching and protein expression of hepatic...... in normal liver and in the extensive ductular reactions originating from intrahepatic bile ducts and branching into the parenchyma of the acetaminophen intoxicated liver. In the developing human liver, three-dimensional reconstructions using multiple marker proteins confirmed that the human intrahepatic...

  10. Metabolites of 5F-AKB-48, a synthetic cannabinoid receptor agonist, identified in human urine and liver microsomal preparations using liquid chromatography high-resolution mass spectrometry

    DEFF Research Database (Denmark)

    Holm, Niels Bjerre; Pedersen, Anders Just; Dalsgaard, Petur Weihe

    2015-01-01

    New types of synthetic cannabinoid designer drugs are constantly introduced to the illicit drug market to circumvent legislation. Recently, N-​(1-Adamant​yl)-​1-​(5-​fluoropentyl)-​1H-​indazole-​3-​carboxamide (5F-AKB-48), also known as 5F-APINACA, was identified as an adulterant in herbal products...

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

    Science.gov (United States)

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

    2018-01-11

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

  12. Cell sources for in vitro human liver cell culture models.

    Science.gov (United States)

    Zeilinger, Katrin; Freyer, Nora; Damm, Georg; Seehofer, Daniel; Knöspel, Fanny

    2016-09-01

    In vitro liver cell culture models are gaining increasing importance in pharmacological and toxicological research. The source of cells used is critical for the relevance and the predictive value of such models. Primary human hepatocytes (PHH) are currently considered to be the gold standard for hepatic in vitro culture models, since they directly reflect the specific metabolism and functionality of the human liver; however, the scarcity and difficult logistics of PHH have driven researchers to explore alternative cell sources, including liver cell lines and pluripotent stem cells. Liver cell lines generated from hepatomas or by genetic manipulation are widely used due to their good availability, but they are generally altered in certain metabolic functions. For the past few years, adult and pluripotent stem cells have been attracting increasing attention, due their ability to proliferate and to differentiate into hepatocyte-like cells in vitro However, controlling the differentiation of these cells is still a challenge. This review gives an overview of the major human cell sources under investigation for in vitro liver cell culture models, including primary human liver cells, liver cell lines, and stem cells. The promises and challenges of different cell types are discussed with a focus on the complex 2D and 3D culture approaches under investigation for improving liver cell functionality in vitro Finally, the specific application options of individual cell sources in pharmacological research or disease modeling are described. © 2016 by the Society for Experimental Biology and Medicine.

  13. Volumetric Growth of the Liver in the Human Fetus: An Anatomical, Hydrostatic, and Statistical Study

    Directory of Open Access Journals (Sweden)

    Michał Szpinda

    2015-01-01

    Full Text Available Using anatomical, hydrostatic, and statistical methods, liver volumes were assessed in 69 human fetuses of both sexes aged 18–30 weeks. No sex differences were found. The median of liver volume achieved by hydrostatic measurements increased from 6.57 cm3 at 18–21 weeks through 14.36 cm3 at 22–25 weeks to 20.77 cm3 at 26–30 weeks, according to the following regression: y = −26.95 + 1.74 × age ± Z  × (−3.15 + 0.27 × age. The median of liver volume calculated indirectly according to the formula liver volume = 0.55 × liver length × liver transverse diameter × liver sagittal diameter increased from 12.41 cm3 at 18–21 weeks through 28.21 cm3 at 22–25 weeks to 49.69 cm3 at 26–30 weeks. There was a strong relationship (r=0.91, p<0.001 between the liver volumes achieved by hydrostatic (x and indirect (y methods, expressed by y = −0.05 + 2.16x  ± 7.26. The liver volume should be calculated as follows liver volume = 0.26 × liver length × liver transverse diameter × liver sagittal diameter. The age-specific liver volumes are of great relevance in the evaluation of the normal hepatic growth and the early diagnosis of fetal micro- and macrosomias.

  14. In vitro metabolism of testosterone in the horse liver and involvement of equine CYPs 3A89, 3A94 and 3A95.

    Science.gov (United States)

    Schmitz, A; Zielinski, J; Dick, B; Mevissen, M

    2014-08-01

    Testosterone (TES) 6-β-hydroxylation is a significant metabolic step in the biotransformation of TES in human liver microsomes and reflects cytochrome P450 (CYP) 3A4/5 specific metabolic activity. Several CYP3A enzymes have been annotated in the horse genome, but functional characterization is missing. This descriptive study investigates TES metabolism in the horse liver in vitro and the qualitative contribution of three CYP3A isoforms of the horse. Metabolism of TES was investigated by using equine hepatocyte primary cultures and liver microsomes. Chemical inhibitors were used to determine the CYPs involved in TES biotransformation in equine microsomes. Single CYPs 3A89, 3A94, and 3A95, recombinantly expressed in V79 hamster lung fibroblasts, were incubated with TES and the fluorescent metabolite 7-benzyloxy-4-trifluoromethylcoumarin (BFC). The effect of ketoconazole and troleandomycin was evaluated on single CYPs. Testosterone metabolites were analyzed by HPLC and confirmed by GC/MS. In hepatocyte primary cultures, the most abundant metabolite was androstenedione (AS), whereas in liver microsomes, 6-β-hydroxytestosterone showed the largest peak. Formation of 6-β-hydroxytestosterone and 11-β-hydroxytestosterone in liver microsomes was inhibited by ketoconazole, troleandomycin, and quercetin. Equine recombinant CYP3A95 catalyzed 11-β-hydroxylation of testosterone (TES). Metabolism of BFC was significantly inhibited by ketoconazole in CYP3A95, whereas troleandomycin affected the activities of CYP3A94 and CYP3A95. Both inhibitors had no significant effect on CYP3A89. Metabolic reactions and effects of inhibitors differed between the equine CYP3A isoforms investigated. This has to be considered in future in vitro studies. © 2014 John Wiley & Sons Ltd.

  15. Activation of the microsomal glutathione-S-transferase and reduction of the glutathione dependent protection against lipid peroxidation by acrolein

    NARCIS (Netherlands)

    Haenen, G R; Vermeulen, N P; Tai Tin Tsoi, J N; Ragetli, H M; Timmerman, H; Blast, A

    1988-01-01

    Allyl alcohol is hepatotoxic. It is generally believed that acrolein, generated out of allyl alcohol by cytosolic alcohol dehydrogenase, is responsible for this toxicity. The effect of acrolein in vitro and in vivo on the glutathione (GSH) dependent protection of liver microsomes against lipid

  16. Stable liver-specific expression of human IDOL in humanized mice raises plasma cholesterol.

    Science.gov (United States)

    Ibrahim, Salam; Somanathan, Suryanarayan; Billheimer, Jeffrey; Wilson, James M; Rader, Daniel J

    2016-05-01

    IDOL (inducible degrader of the low-density lipoprotein receptor, LDLR) is an E3 ubiquitin ligase that promotes the ubiquitination and degradation of the LDLR. IDOL is a potential therapeutic target for the development of a novel class of low-density lipoprotein cholesterol (LDL-C)-lowering therapies. In an attempt to develop a mouse model for testing IDOL inhibitors, we examined the effects of adeno-associated virus (AAV)-mediated stable expression of human IDOL in the livers of mice 'humanized' with regard to lipoprotein metabolism. Using a liver-specific AAV serotype 8 (AAV8)-mediated delivery, AAV-hIDOL produced a dose-dependent increase in LDL-C levels and a decrease in liver LDLR protein. Furthermore, we expressed hIDOL in a 'humanized' mouse model of heterozygous familial hypercholesterolaemia (LDLR(+/-)/Apobec1(-/-)/hApoB-Tg, LAhB). In this model, total cholesterol (TC) and LDL-C levels were increased by ∼60% starting from 1 week and were sustainable for at least 3 weeks post-injection. Finally, we demonstrate that the effects caused by hIDOL expression are LDLR- dependent given the unchanged plasma lipids in LAhB mice lacking LDLR. In conclusion, our study demonstrates a dose-dependent physiological effect of human IDOL on LDL metabolism in mice. This provides a potential model for preclinical testing of IDOL inhibitors for reduction of LDL-C levels. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2016. For permissions please email: journals.permissions@oup.com.

  17. Characterization of Liver-Specific Functions of Human Fetal Hepatocytes in Culture.

    Science.gov (United States)

    Chinnici, Cinzia Maria; Timoneri, Francesca; Amico, Giandomenico; Pietrosi, Giada; Vizzini, Giovanni; Spada, Marco; Pagano, Duilio; Gridelli, Bruno; Conaldi, Pier Giulio

    2015-01-01

    This study was designed to assess liver-specific functions of human fetal liver cells proposed as a potential source for hepatocyte transplantation. Fetal liver cells were isolated from livers of different gestational ages (16-22 weeks), and the functions of cell preparations were evaluated by establishing primary cultures. We observed that 20- to 22-week-gestation fetal liver cell cultures contained a predominance of cells with hepatocytic traits that did not divide in vitro but were functionally competent. Fetal hepatocytes performed liver-specific functions at levels comparable to those of their adult counterpart. Moreover, exposure to dexamethasone in combination with oncostatin M promptly induced further maturation of the cells through the acquisition of additional functions (i.e., ability to store glycogen and uptake of indocyanine green). In some cases, particularly in cultures obtained from fetuses of earlier gestational ages (16-18 weeks gestation), cells with mature hepatocytic traits proved to be sporadic, and the primary cultures were mainly populated by clusters of proliferating cells. Consequently, the values of liver-specific functions detected in these cultures were low. We observed that a low cell density culture system rapidly prompted loss of the mature hepatocytic phenotype with downregulations of all the liver-specific functions. We found that human fetal liver cells can be cryopreserved without significant loss of viability and function and evaluated up to 1 year in storage in liquid nitrogen. They might, therefore, be suitable for cell banking and allow for the transplantation of large numbers of cells, thus improving clinical outcomes. Overall, our results indicate that fetal hepatocytes could be used as a cell source for hepatocyte transplantation. Fetal liver cells have been used so far to treat end-stage liver disease. Additional studies are needed to include these cells in cell-based therapies aimed to treat liver failure and inborn

  18. CD13 is a therapeutic target in human liver cancer stem cells

    National Research Council Canada - National Science Library

    Haraguchi, Naotsugu; Ishii, Hideshi; Mimori, Koshi; Tanaka, Fumiaki; Ohkuma, Masahisa; Kim, Ho Min; Akita, Hirofumi; Takiuchi, Daisuke; Hatano, Hisanori; Nagano, Hiroaki; Barnard, Graham F; Doki, Yuichiro; Mori, Masaki

    2010-01-01

    .... Here, we have demonstrated that CD13 is a marker for semiquiescent CSCs in human liver cancer cell lines and clinical samples and that targeting these cells might provide a way to treat this disease. CD13...

  19. Cadmium Chloride Induces DNA Damage and Apoptosis of Human Liver Carcinoma Cells via Oxidative Stress

    National Research Council Canada - National Science Library

    Skipper, Anthony; Sims, Jennifer N; Yedjou, Clement G; Tchounwou, Paul B

    2016-01-01

    ... mechanisms of action are not fully elucidated. In this research, we hypothesized that oxidative stress plays a key role in cadmium chloride-induced toxicity, DNA damage, and apoptosis of human liver carcinoma (HepG₂) cells...

  20. Side Population Cells Derived from Adult Human Liver Generate Hepatocyte-like Cells In Vitro

    OpenAIRE

    HUSSAIN, SUNNY ZAHEED; Strom, Stephen C.; Kirby, Martha R.; Burns, Sean; Langemeijer, Saskia; Ueda, Takahiro; HSIEH, MATTHEW; Tisdale, John F.

    2005-01-01

    We sought to determine whether hepatic side population (SP) cells derived from adult human liver possess the potential of a novel candidate hepatic stem cell. Human cadaveric donor liver was subjected to collagenase perfusion and hepatocytes were separated from nonparenchymal cells by differential centrifugation. SP cells were isolated from the nonparenchymal portion after Hoechst 33342 staining. Since CD45 is a panleukocyte antigen, CD45-negative SP cells were separated from the vast majorit...

  1. Long term culture of genome-stable bipotent progenitor cells from adult human liver

    OpenAIRE

    Huch, Meritxell; Gehart, Helmuth; van, Boxtel Ruben; Hamer, Karien; Blokzijl, Francis; Verstegen, Monique MA; Ellis, Ewa; van, Wenum Martien; Fuchs, Sabine A; de, Ligt Joep; van, de Wetering Marc; Sasaki, Nobuo; Boers, Susanne J; Kemperman, Hans; de, Jonge Jeroen

    2014-01-01

    Despite the enormous replication potential of the human liver, there are currently no culture systems available that sustain hepatocyte replication and/or function in vitro. We have shown previously that single mouse Lgr5+ liver stem cells can be expanded as epithelial organoids in vitro and can be differentiated into functional hepatocytes in vitro and in vivo. We now describe conditions allowing long-term expansion of adult bile duct-derived bipotent progenitor cells from human liver. The e...

  2. The prevalence of Antithyroglobulin and Antithyroid Microsomal ...

    African Journals Online (AJOL)

    Plasma levels of antithyroglobulin {TG} and microsomal thyroid peroxidase {TPO} autoantibodies were determined using the ELISA methods, in 87 euthyroid women. These were made up of 44 control women which included 8{18%} nulligravidae, 18{41%} non pregnant multiparous and 18{41%}, pregnant subjects.

  3. ACBP and cholesterol differentially alter fatty acyl CoA utilization by microsomal ACAT.

    Science.gov (United States)

    Chao, Hsu; Zhou, Minglong; McIntosh, Avery; Schroeder, Friedhelm; Kier, Ann B

    2003-01-01

    Microsomal acyl CoA:cholesterol acyltransferase (ACAT) is stimulated in vitro and/or in intact cells by proteins that bind and transfer both substrates, cholesterol, and fatty acyl CoA. To resolve the role of fatty acyl CoA binding independent of cholesterol binding/transfer, a protein that exclusively binds fatty acyl CoA (acyl CoA binding protein, ACBP) was compared. ACBP contains an endoplasmic reticulum retention motif and significantly colocalized with acyl-CoA cholesteryl acyltransferase 2 (ACAT2) and endoplasmic reticulum markers in L-cell fibroblasts and hepatoma cells, respectively. In the presence of exogenous cholesterol, ACAT was stimulated in the order: ACBP > sterol carrier protein-2 (SCP-2) > liver fatty acid binding protein (L-FABP). Stimulation was in the same order as the relative affinities of the proteins for fatty acyl CoA. In contrast, in the absence of exogenous cholesterol, these proteins inhibited microsomal ACAT, but in the same order: ACBP > SCP-2 > L-FABP. The extracellular protein BSA stimulated microsomal ACAT regardless of the presence or absence of exogenous cholesterol. Thus, ACBP was the most potent intracellular fatty acyl CoA binding protein in differentially modulating the activity of microsomal ACAT to form cholesteryl esters independent of cholesterol binding/transfer ability.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1987-12-15

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

  5. Analysis of bile acid-induced regulation of FXR target genes in human liver slices

    NARCIS (Netherlands)

    Jung, Diana; Elferink, Marieke G. L.; Stellaard, Frans; Groothuis, Geny M. M.

    Information about the role of nuclear receptors has rapidly increased over the last decade. However, details about their role in human are lacking. Owing to species differences, a powerful human in vitro system is needed. This study uses for the first time precision-cut human liver slices in the

  6. Pooled human liver preparations, HepaRG, or HepG2 cell lines for metabolism studies of new psychoactive substances? A study using MDMA, MDBD, butylone, MDPPP, MDPV, MDPB, 5-MAPB, and 5-API as examples.

    Science.gov (United States)

    Richter, Lilian H J; Flockerzi, Veit; Maurer, Hans H; Meyer, Markus R

    2017-09-05

    Metabolism studies play an important role in clinical and forensic toxicology. Because of potential species differences in metabolism, human samples are best suitable for elucidating metabolism. However, in the case of new psychoactive substances (NPS), human samples of controlled studies are not available. Primary human hepatocytes have been described as gold standard for in vitro metabolism studies, but there are some disadvantages such as high costs, limited availability, and variability of metabolic enzymes. Therefore, the aim of our study was to investigate and compare the metabolism of six methylenedioxy derivatives (MDMA, MDBD, butylone, MDPPP, MDPV, MDPB) and two bioisosteric analogues (5-MAPB, 5-API) using pooled human liver microsomes (pHLM) combined with cytosol (pHLC) or pooled human liver S9 fraction (pS9) all after addition of co-substrates for six phase I and II reactions. In addition, HepaRG and HepG2 cell lines were used. Results of the different in vitro tools were compared to each other, to corresponding published data, and to metabolites identified in human urine after consumption of MDMA, MDPV, or 5-MAPB. Incubations with pHLM plus pHLC showed similar results as pS9. A more cost efficient model for prediction of targets for toxicological screening procedures in human urine should be identified. As expected, the incubations with HepaRG provided better results than those with HepG2 concerning number and signal abundance of the metabolites. Due to easy handling without special equipment, incubations with pooled liver preparations should be the most suitable alternative to find targets for toxicological screening procedures for methylenedioxy derivatives and bioisosteric analogues. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. Decreased hepatotoxic bile acid composition and altered synthesis in progressive human nonalcoholic fatty liver disease

    Energy Technology Data Exchange (ETDEWEB)

    Lake, April D. [University of Arizona, Department of Pharmacology and Toxicology, Tucson, AZ 85721 (United States); Novak, Petr [Biology Centre ASCR, Institute of Plant Molecular Biology, Ceske Budejovice 37001 (Czech Republic); Shipkova, Petia; Aranibar, Nelly; Robertson, Donald; Reily, Michael D. [Pharmaceutical Candidate Optimization, Bristol-Myers Squibb Co., Princeton, NJ 08543 (United States); Lu, Zhenqiang [The Arizona Statistical Consulting Laboratory, University of Arizona, Tucson, AZ 85721 (United States); Lehman-McKeeman, Lois D. [Pharmaceutical Candidate Optimization, Bristol-Myers Squibb Co., Princeton, NJ 08543 (United States); Cherrington, Nathan J., E-mail: cherrington@pharmacy.arizona.edu [University of Arizona, Department of Pharmacology and Toxicology, Tucson, AZ 85721 (United States)

    2013-04-15

    Bile acids (BAs) have many physiological roles and exhibit both toxic and protective influences within the liver. Alterations in the BA profile may be the result of disease induced liver injury. Nonalcoholic fatty liver disease (NAFLD) is a prevalent form of chronic liver disease characterized by the pathophysiological progression from simple steatosis to nonalcoholic steatohepatitis (NASH). The hypothesis of this study is that the ‘classical’ (neutral) and ‘alternative’ (acidic) BA synthesis pathways are altered together with hepatic BA composition during progression of human NAFLD. This study employed the use of transcriptomic and metabolomic assays to study the hepatic toxicologic BA profile in progressive human NAFLD. Individual human liver samples diagnosed as normal, steatosis, and NASH were utilized in the assays. The transcriptomic analysis of 70 BA genes revealed an enrichment of downregulated BA metabolism and transcription factor/receptor genes in livers diagnosed as NASH. Increased mRNA expression of BAAT and CYP7B1 was observed in contrast to decreased CYP8B1 expression in NASH samples. The BA metabolomic profile of NASH livers exhibited an increase in taurine together with elevated levels of conjugated BA species, taurocholic acid (TCA) and taurodeoxycholic acid (TDCA). Conversely, cholic acid (CA) and glycodeoxycholic acid (GDCA) were decreased in NASH liver. These findings reveal a potential shift toward the alternative pathway of BA synthesis during NASH, mediated by increased mRNA and protein expression of CYP7B1. Overall, the transcriptomic changes of BA synthesis pathway enzymes together with altered hepatic BA composition signify an attempt by the liver to reduce hepatotoxicity during disease progression to NASH. - Highlights: ► Altered hepatic bile acid composition is observed in progressive NAFLD. ► Bile acid synthesis enzymes are transcriptionally altered in NASH livers. ► Increased levels of taurine and conjugated bile acids

  8. Assessment of mutagenic potential of pyrolysis biochars by Ames Salmonella/mammalian-microsomal mutagenicity test.

    Science.gov (United States)

    Anjum, Reshma; Krakat, Niclas; Toufiq Reza, M; Klocke, Michael

    2014-09-01

    Biochar is of raising interest in sustainable biomass utilization concepts. Particularly biochar derived from pyrolysis attaches important agricultural capacities mandatory for an improved carbon sequestration, soil fertility and amelioration, respectively. In fact, large scale field trials and commercial business with biochar materials have already been started but still only few are known about the mutagenic potential of biochars produced. In this study hemp bedding and wood pellet biomass were used for biochar production by pyrolysis. The total concentrations of polycyclic aromatic hydrocarbons (PAHs) were 34.9µgg(-1) of dry mass and 33.7µgg(-1) of dry mass for hemp biochar and wood biochar, respectively. The concentration of PAHs in tar produced during wood carbonization was 17.4µgg(-1). The concentrations of phenolic compounds were 55µgg(-1) and 8.3µgg(-1) for hemp and wood biochar, respectively. Salmonella/microsomal mutagenicity tests (i.e. Ames test) revealed a maximum mutagenicity for hemp biochar extracts with strains TA97, TA98 and TA100 in the presence and absence of liver microsomal fractions, respectively. Wood biochar and tar extract exhibited maximum mutagenicity with strains TA98 and T100 both in the presence and absence of liver microsomal fraction. The reversion of the applied tester strains increased in the presence and absence of liver microsomal fractions with an increasing dose of hemp biochar extract up to 2µl per plate and decreased at a concentration of 2.5µl per plate. For wood biochar and tar extracts, reversion of tester strains increased both in the presence and absence of S9 at extract concentrations of 4µl per plate and declined at a dose of 8µl per plate. By this study a significant higher mutagenic potential for hemp biochar compared to wood biochar and tar could be observed suggesting careful application in soil melioration. Copyright © 2014 Elsevier Inc. All rights reserved.

  9. Cloning and characterization of human liver cytosolic beta-glycosidase

    NARCIS (Netherlands)

    De Graaf, M; Van Veen, IC; Van Der Meulen-Muileman, IH; Gerritsen, WR; Pinedo, HM; Haisma, HJ

    2001-01-01

    Cytosolic beta -glucosidase (EC 3.2.1.21) from mammalian liver is a member of the family 1 glycoside hydrolases and is known for its ability to hydrolyse a range of beta -D-glycosides. including beta -D-glucoside acid beta -D-galactoside. We therefore refer to this enzyme as cytosolic beta

  10. Michaëlis--Menten kinetics of phenazone elimination in the perfused pig liver

    DEFF Research Database (Denmark)

    Andreasen, B; Tonnesen, K; Rabol, A

    1977-01-01

    followed saturation kinetics (concentrations 0.1-12 mmol x 1(-1) and the maximal elimination rate (Vmax) was on average 102 mumol x min-1 x kg-1 liver and the Michaëlis-constant (Km) of 2.6 mmol x 1(-1). Estimates of Vmax and Km for the microsomal phenazone hydroxylase activity measured in liver biopsies......The purpose of the present study was to define the elimination kinetics of phenazone (NFN) in the isolated perfused pig liver. In five experiments phenazone was administered as constant infusion to obtain steady-state periods over a wide range of concentrations. The elimination of phenazone...... found to be considerably lower than in the perfused liver. The hepatic elimination of phenazone during perfusion of pig liver at phenazone concentrations corresponding to human therapeutic doses follows first-order kinetics....

  11. Proteomic analysis of tyrosine phosphorylation during human liver transplantation

    Directory of Open Access Journals (Sweden)

    Boutros Tarek

    2007-01-01

    Full Text Available Abstract Background Ischemia-reperfusion (I/R causes a dramatic reprogramming of cell metabolism during liver transplantation and can be linked to an alteration of the phosphorylation level of several cellular proteins. Over the past two decades, it became clear that tyrosine phosphorylation plays a pivotal role in a variety of important signalling pathways and was linked to a wide spectrum of diseases. Functional profiling of the tyrosine phosphoproteome during liver transplantation is therefore of great biological significance and is likely to lead to the identification of novel targets for drug discovery and provide a basis for novel therapeutic strategies. Results Using liver biopsies collected during the early phases of organ procurement and transplantation, we aimed at characterizing the global patterns of tyrosine phosphorylation during hepatic I/R. A proteomic approach, based on the purification of tyrosine phosphorylated proteins followed by their identification using mass spectrometry, allowed us to identify Nck-1, a SH2/SH3 adaptor, as a potential regulator of I/R injury. Using immunoblot, cell fractionation and immunohistochemistry, we demonstrate that Nck-1 phosphorylation, expression and localization were affected in liver tissue upon I/R. In addition, mass spectrometry identification of Nck-1 binding partners during the course of the transplantation also suggested a dynamic interaction between Nck-1 and actin during I/R. Conclusion Taken together, our data suggest that Nck-1 may play a role in I/R-induced actin reorganization, which was previously reported to be detrimental for the hepatocytes of the transplanted graft. Nck-1 could therefore represent a target of choice for the design of new organ preservation strategies, which could consequently help to reduce post-reperfusion liver damages and improve transplantation outcomes.

  12. Studies on adenosine triphosphate transphosphorylases. Human isoenzymes of adenylate kinase: isolation and physicochemical comparison of the crystalline human ATP-AMP transphosphorylases from muscle and liver.

    Science.gov (United States)

    Kuby, S A; Fleming, G; Frischat, A; Cress, M C; Hamada, M

    1983-02-10

    Procedures are described for the isolation, in crystalline form, of the adenylate kinases from autopsy samples of human muscle and from human liver. Weight average molecular weights were determined by sedimentation equilibrium to be 22,000 (+/- 700) and 25,450 (+/- 160) for the human muscle and liver isoenzymes, respectively. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, their molecular weights were estimated to be 21,700 and 26,500 for the muscle and liver enzymes, respectively. Both isoenzymes are accordingly monomeric proteins in their native state. Amino acid analyses are reported here for the normal human liver, calf liver, and rabbit liver adenylate kinases and compared with the normal human muscle, calf muscle, and rabbit muscle myokinases. The liver types as a group and the muscle types as a group show a great deal of homology, but some distinct differences are evident between the liver and muscle enzyme groups, especially in the number of residues of His, Pro, half-cystine, and the presence of tryptophan in the liver enzymes. The normal human liver adenylate kinase, as isolated in this report, has proved to be similar in its properties, if not identical, to the adenylate kinase isolated directly from human liver mitochondria (Hamada, M., Sumida, M., Okuda, H., Watanabe, T., Nojima, M., and Kuby, S. A. (1982) J. Biol. Chem. 257, 13120-13128). Therefore, the liver-type adenylate kinase may be considered a mitochondrial type.

  13. Human Adipose Tissue Derived Stem Cells Promote Liver Regeneration in a Rat Model of Toxic Injury

    Directory of Open Access Journals (Sweden)

    Eva Koellensperger

    2013-01-01

    Full Text Available In the light of the persisting lack of donor organs and the risks of allotransplantations, the possibility of liver regeneration with autologous stem cells from adipose tissue (ADSC is an intriguing alternative. Using a model of a toxic liver damage in Sprague Dawley rats, generated by repetitive intraperitoneal application of retrorsine and allyl alcohol, the ability of human ADSC to support the restoration of liver function was investigated. A two-thirds hepatectomy was performed, and human ADSC were injected into one remaining liver lobe in group 1 (n = 20. Injection of cell culture medium performed in group 2 (n = 20 served as control. Cyclosporine was applied to achieve immunotolerance. Blood samples were drawn weekly after surgery to determine liver-correlated blood values. Six and twelve weeks after surgery, animals were sacrificed and histological sections were analyzed. ADSC significantly raised postoperative albumin (P < 0.017, total protein (P < 0.031, glutamic oxaloacetic transaminase (P < 0.001, and lactate dehydrogenase (P < 0.04 levels compared to injection of cell culture medium alone. Transplanted cells could be found up to twelve weeks after surgery in histological sections. This study points towards ADSC being a promising alternative to hepatocyte or liver organ transplantation in patients with severe liver failure.

  14. Lysosomal degradation of cell organelles. II. Ultrastructural analysis of uptake and digestion of intravenously injected microsomes and ribosomes by Kupffer cells.

    Science.gov (United States)

    Glaumann, H; Berezesky, I K; Ericsson, J L; Trump, B F

    1975-09-01

    Rough and smooth microsomes, "mixed" or total microsomes, and ribosomes were isolated from one single rat liver and subsequently injected intravenously into a series of inbred rats. The uptake and the degradation of the injected organelles by Kupffer cells were followed by means of electron microscopic analysis. By 1 minute after injection, microsomes were seen attached to the surface of Kupffer cells separated by a gap of 200 to 300 A. No attachment to hepatocytes, fat-storing cells, or endothelial cells was seen. By 5 and 10 minutes, most microsomes were phagocytosed and sequestered in large numbers within single membrane-enclosed vacuoles or phagosomes. The engulfment proceeded by two mechanisms: (1) most frequently, flaplike processes of cytoplasm embraced aggregates of microsomes, concomitant with the formation of indention of the cytoplasm; (2) occasionally, single microsomal profiles were taken up by bristle-coated endocytic vacuoles. Ribosomes were also seen penetrating into the wormlike structures (micropinocytosis vermiformis) at the cell surface. At 30 minutes after injection, clear signs of alteration were noted starting with vesicle aggregation, clumping, and elongation of the microsomal profiles. The ribosomes were quickly stripped from their microsomal membranes and marginated to the inside of the vacuoles but separated from the limiting membrane by a distance of 200 to 300 A. By 1 and 2 hours, disruption of the vesicles into membrane fragments and formation of dense material in and between the profiles occurred. By 8 hours it was difficult to recognize the degradation products as membrane derivatives. The digestive vacuoles retained their size at this time interval. Typical pentalaminar structures were observed. By 14 to 24 hours the digestive vacuoles became electron lucent and appeared to shrink, and in addition to containing various types of granular material, many were laden with lipid-like droplets presumed to be conglomerates of phospholipid

  15. Infection with the carcinogenic human liver fluke, Opisthorchis viverrini

    Science.gov (United States)

    Smout, Michael J.; Sripa, Banchob; Laha, Thewarach; Mulvenna, Jason; Gasser, Robin B.; Young, Neil D.; Bethony, Jeffrey M.; Brindley, Paul J.; Loukas, Alex

    2013-01-01

    Summary Throughout Southeast Asia there is a strikingly high incidence of cholangiocarcinoma (CCA - hepatic cancer of the bile duct epithelium), particularly in people from rural settings in Laos and Northeast Thailand who are infected with the liver fluke, Opisthorchis viverrini, one of only three carcinogenic eukaryotic pathogens. More ubiquitous carcinogenic microbes, such as Helicobacter pylori, induce cancer in less than 1% of infected people, while as many as one-sixth of people with opisthorchiasis will develop CCA. The mechanisms by which O. viverrini causes cancer are multi-factorial, involving mechanical irritation from the activities and movements of the flukes, immunopathology, dietary nitrosamines and the secretion of parasite proteins that promote a tumourigenic environment. Genomic and proteomic studies of the liver fluke secretome have accelerated the discovery of parasite proteins with known/potential roles in pathogenesis and tumourigenesis, establishing a framework towards understanding, and ultimately preventing, the morbidity and mortality attributed to this highly carcinogenic parasite. PMID:21311794

  16. A new human 3D-liver model unravels the role of galectins in liver infection by the parasite Entamoeba histolytica.

    Directory of Open Access Journals (Sweden)

    Debora B Petropolis

    2014-09-01

    Full Text Available Investigations of human parasitic diseases depend on the availability of appropriate in vivo animal models and ex vivo experimental systems, and are particularly difficult for pathogens whose exclusive natural hosts are humans, such as Entamoeba histolytica, the protozoan parasite responsible for amoebiasis. This common infectious human disease affects the intestine and liver. In the liver sinusoids E. histolytica crosses the endothelium and penetrates into the parenchyma, with the concomitant initiation of inflammatory foci and subsequent abscess formation. Studying factors responsible for human liver infection is hampered by the complexity of the hepatic environment and by the restrictions inherent to the use of human samples. Therefore, we built a human 3D-liver in vitro model composed of cultured liver sinusoidal endothelial cells and hepatocytes in a 3D collagen-I matrix sandwich. We determined the presence of important hepatic markers and demonstrated that the cell layers function as a biological barrier. E. histolytica invasion was assessed using wild-type strains and amoebae with altered virulence or different adhesive properties. We showed for the first time the dependence of endothelium crossing upon amoebic Gal/GalNAc lectin. The 3D-liver model enabled the molecular analysis of human cell responses, suggesting for the first time a crucial role of human galectins in parasite adhesion to the endothelial cells, which was confirmed by siRNA knockdown of galectin-1. Levels of several pro-inflammatory cytokines, including galectin-1 and -3, were highly increased upon contact of E. histolytica with the 3D-liver model. The presence of galectin-1 and -3 in the extracellular medium stimulated pro-inflammatory cytokine release, suggesting a further role for human galectins in the onset of the hepatic inflammatory response. These new findings are relevant for a better understanding of human liver infection by E. histolytica.

  17. Nifurtimox biotransformation to reactive metabolites or nitrite in liver subcellular fractions and model systems.

    Science.gov (United States)

    Montalto de Mecca, M; Diaz, E G; Castro, J A

    2002-11-15

    Liver microsomal (mic); nuclei (N) and mitochondria (mit) anaerobically nitroreduce Nifurtimox (Nfx) in the presence of NADPH generating system. Simultaneous formation of small amounts of nitrite was observed in microsomes and nuclei but not in mitochondria. The microsomal nitroreductase activity was enhanced by the presence of flavine-adenine-dinucleotide disodium salt (FAD), was not inhibited by CO and was significantly inhibited by diphenyleneiodonium (DPI). In the microsomal NADPH-dependent fraction nitrite formation was null in the presence of FAD, DPI and under air and was partially inhibited by pure CO. Pure human cytochrome P450 reductase in the presence of NADPH significantly nitroreduced Nfx and produced small amounts of nitrite. The nitroreductive process was significantly enhanced by FAD but the nitrite formation became null. FAD itself was able to chemically nitroreduce Nfx without production of nitrite. NADPH generating system enhanced the FAD nitroreductive effect and led to small production of nitrite. Formation of reactive metabolites and nitric oxide during Nfx metabolism might contribute to its toxicity.

  18. Cell Pleomorphism and Cytoskeleton Disorganization in Human Liver Cancer.

    Science.gov (United States)

    Cheng, Chiung-Chi; Lai, Yen-Chang Clark; Lai, Yih-Shyong; Chao, Wei-Ting; Tseng, Yu-Hui; Hsu, Yung-Hsiang; Chen, You-Yin; Liu, Yi-Hsiang

    Nucleoskeleton maintains the framework of a cell nucleus that is required for a variety of nuclear functions. However, the nature of nucleoskeleton structure has not been yet clearly elucidated due to microscopy visualization limitations. Plectin, a nuclear pore-permeable component of cytoskeleton, exhibits a role of cross-linking between cytoplasmic intermediate filaments and nuclear lamins. Presumably, plectin is also a part of nucleoskeleton. Previously, we demonstrated that pleomorphism of hepatoma cells is the consequence of cytoskeletal changes mediated by plectin deficiency. In this study, we applied a variety of technologies to detect the cytoskeletons in liver cells. The images of confocal microscopy did not show the existence of plectin, intermediate filaments, microfilaments and microtubules in hepatic nuclei. However, in the isolated nuclear preparation, immunohistochemical staining revealed positive results for plectin and cytoskeletal proteins that may contribute to the contamination derived from cytoplasmic residues. Therefore, confocal microscopy provides a simple and effective technology to observe the framework of nucleoskeleton. Accordingly, we verified that cytoskeletons are not found in hepatic cell nuclei. Furthermore, the siRNA-mediated knockdown of plectin in liver cells leads to collapsed cytoskeleton, cell transformation and pleomorphic nuclei. Plectin and cytoskeletons were not detected in the nuclei of liver cells compared to the results of confocal microscopy. Despite the absence of nuclear plectin and cytoskeletal filaments, the evidence provided support that nuclear pleomorphism of cancer cells is correlated with the cytoplasmic disorganization of cytoskeleton. Copyright © 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.

  19. Squamous cell carcinoma antigen in human liver carcinogenesis.

    Science.gov (United States)

    Guido, M; Roskams, T; Pontisso, P; Fassan, M; Thung, S N; Giacomelli, L; Sergio, A; Farinati, F; Cillo, U; Rugge, M

    2008-04-01

    Squamous cell carcinoma antigen (SCCA) is a serine protease inhibitor that can be overexpressed in hepatocellular carcinoma (HCC) at both molecular and protein level, but no data are available on its expression in pre-malignant stages. To assess SCCA expression by immunohistochemistry in HCC and its nodular precursors in cirrhotic livers. 55 nodules from 42 explanted livers were evaluated: 7 large regenerative nodules (LRNs), 7 low-grade dysplastic nodules (LG-DNs), 10 high-grade DNs (HG-DNs), and 31 HCC. SCCA expression was semiquantitatively scored on a four-tiered scale. SCCA hepatocyte immunostaining was always restricted to the cytoplasm, mainly exhibiting a granular pattern. Stain intensity varied, ranging from weak to very strong. Within the nodules, positive cells were unevenly distributed, either scattered or in irregular clusters. The prevalence of SCCA expression was 29% in LRNs, 100% in DNs and 93% in HCC. A significant difference emerged in both prevalence and score for LRNs versus LG-DNs (pHCC (p = 0.000). A barely significant difference (p = 0.49) was observed between LG-DNs and HG-DNs, while no difference in SCCA expression was detected between HG-DNs and HCC. Cirrhotic tissue adjacent to the nodules was positive in 96% of cases, with a significant difference in the score (p = 0.000) between hepatocytes adjacent to HCC and those surrounding LRNs. This study provides the first evidence that aberrant SCCA expression is an early event in liver cell carcinomatous transformation.

  20. Systemic chimerism in human female recipients of male livers

    Science.gov (United States)

    Starzl, Thomas E.; Trucco, Massimo; Zeevi, Adriana; Kocova, Mirjana; Ildstad, Suzanne; Demetris, Antony J.; Ramos, Hector; Rudert, William A.; Ricordi, Camillo; Murase, Noriko

    2011-01-01

    We have previously reported data from clinical and laboratory animal observations which suggest that organ tolerance after transplantation depends on a state of balanced lymphodendritic cell chimerism between the host and donor graft. We have sought further evidence to support this hypothesis by investigating HLA-mismatched liver allograft recipients. 9 of 9 female recipients of livers from male donors had chimerism in their allografts and extrahepatic tissues, according to in-situ hybridisation and molecular techniques 10 to 19 years post-transplantation. In 8 women with good graft function, evidence of the Y chromosome was found in the blood (6/8), skin (8/8), and lymph nodes (7/8). A ninth patient whose transplant failed after 12 years from recurrent chronic viral hepatitis had chimerism in her lymph nodes, skin, jejunum, and aorta at the time of retransplantation. Although cell migration is thought to take place after all types of transplantation, the large population of migratory cells in, and the extent of their seeding from, hepatic grafts may explain the privileged tolerogenicity of the liver compared with other organs. PMID:1357298

  1. In vitro functionality of human fetal liver cells and clonal derivatives under proliferative conditions

    NARCIS (Netherlands)

    Deurholt, Tanja; ten Bloemendaal, Lysbeth; Chhatta, Aniska A.; van Wijk, Albert C. W. A.; Weijer, Kees; Seppen, Jurgen; Elferink, Ronald P. J. Oude; Chamuleau, Robert A. F. M.; Hoekstra, Ruurdtje

    2006-01-01

    Mature human hepatocytes are not suitable for large-scale in vitro applications that rely on hepatocyte function, due to their limited availability and insufficient proliferation capacity in vitro. In contrast, human fetal liver cells (HFLC) can be easily expanded in vitro. In this study we

  2. Effect of extracellular vesicles of human adipose tissue on insulin signaling in liver and muscle cells

    NARCIS (Netherlands)

    Kranendonk, Mariëtte E G; Visseren, Frank L J; van Herwaarden, Joost A; Nolte-'t Hoen, Esther N M; de Jager, Wilco; Wauben, Marca H M; Kalkhoven, Eric; Nolte - t Hoen, Esther

    2014-01-01

    OBJECTIVE: Insulin resistance (IR) is a key mechanism in obesity-induced cardiovascular disease. To unravel mechanisms whereby human adipose tissue (AT) contributes to systemic IR, the effect of human AT-extracellular vesicles (EVs) on insulin signaling in liver and muscle cells was determined.

  3. Hepatocytic Differentiation Potential of Human Fetal Liver Mesenchymal Stem Cells: In Vitro and In Vivo Evaluation

    Directory of Open Access Journals (Sweden)

    Hoda El-Kehdy

    2016-01-01

    Full Text Available In line with the search of effective stem cell population that would progress liver cell therapy and because the rate and differentiation potential of mesenchymal stem cells (MSC decreases with age, the current study investigates the hepatogenic differentiation potential of human fetal liver MSCs (FL-MSCs. After isolation from 11-12 gestational weeks’ human fetal livers, FL-MSCs were shown to express characteristic markers such as CD73, CD90, and CD146 and to display adipocytic and osteoblastic differentiation potential. Thereafter, we explored their hepatocytic differentiation potential using the hepatogenic protocol applied for adult human liver mesenchymal cells. FL-MSCs differentiated in this way displayed significant features of hepatocyte-like cells as demonstrated in vitro by the upregulated expression of specific hepatocytic markers and the induction of metabolic functions including CYP3A4 activity, indocyanine green uptake/release, and glucose 6-phosphatase activity. Following transplantation, naive and differentiated FL-MSC were engrafted into the hepatic parenchyma of newborn immunodeficient mice and differentiated in situ. Hence, FL-MSCs appeared to be interesting candidates to investigate the liver development at the mesenchymal compartment level. Standardization of their isolation, expansion, and differentiation may also support their use for liver cell-based therapy development.

  4. A Nonhuman Primate Model of Human Radiation-Induced Venocclusive Liver Disease and Hepatocyte Injury

    Energy Technology Data Exchange (ETDEWEB)

    Yannam, Govardhana Rao [Department of Surgery, University of Nebraska Medical Center, Omaha, Nebraska (United States); Han, Bing [Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania (United States); Department of Hepatobiliary Surgery, First Affiliated Hospital of Xi' an Jiaotong University, Xi' an, Shaanxi (China); Setoyama, Kentaro [Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania (United States); Yamamoto, Toshiyuki [Department of Surgery, University of Nebraska Medical Center, Omaha, Nebraska (United States); Ito, Ryotaro; Brooks, Jenna M. [Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania (United States); Guzman-Lepe, Jorge [Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania (United States); Department of Pathology, Children' s Hospital of Pittsburgh, Pittsburgh, Pennsylvania (United States); Galambos, Csaba [Department of Pathology, Children' s Hospital of Pittsburgh, Pittsburgh, Pennsylvania (United States); Fong, Jason V. [Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania (United States); Deutsch, Melvin; Quader, Mubina A. [Department of Radiation Oncology, Children' s Hospital of Pittsburgh, Pittsburgh, Pennsylvania (United States); Yamanouchi, Kosho [Department of Radiation Oncology, Albert Einstein College of Medicine, Bronx, New York (United States); Marion Bessin Liver Research Center, Albert Einstein College of Medicine, Bronx, New York (United States); Kabarriti, Rafi; Mehta, Keyur [Department of Radiation Oncology, Albert Einstein College of Medicine, Bronx, New York (United States); Soto-Gutierrez, Alejandro [Department of Pathology, Children' s Hospital of Pittsburgh, Pittsburgh, Pennsylvania (United States); McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania (United States); and others

    2014-02-01

    Background: Human liver has an unusual sensitivity to radiation that limits its use in cancer therapy or in preconditioning for hepatocyte transplantation. Because the characteristic veno-occlusive lesions of radiation-induced liver disease do not occur in rodents, there has been no experimental model to investigate the limits of safe radiation therapy or explore the pathogenesis of hepatic veno-occlusive disease. Methods and Materials: We performed a dose-escalation study in a primate, the cynomolgus monkey, using hypofractionated stereotactic body radiotherapy in 13 animals. Results: At doses ≥40 Gy, animals developed a systemic syndrome resembling human radiation-induced liver disease, consisting of decreased albumin, elevated alkaline phosphatase, loss of appetite, ascites, and normal bilirubin. Higher radiation doses were lethal, causing severe disease that required euthanasia approximately 10 weeks after radiation. Even at lower doses in which radiation-induced liver disease was mild or nonexistent, latent and significant injury to hepatocytes was demonstrated by asialoglycoprotein-mediated functional imaging. These monkeys developed hepatic failure with encephalopathy when they received parenteral nutrition containing high concentrations of glucose. Histologically, livers showed central obstruction via an unusual intimal swelling that progressed to central fibrosis. Conclusions: The cynomolgus monkey, as the first animal model of human veno-occlusive radiation-induced liver disease, provides a resource for characterizing the early changes and pathogenesis of venocclusion, for establishing nonlethal therapeutic dosages, and for examining experimental therapies to minimize radiation injury.

  5. Acetanilide 4-hydroxylase and acetanilide 2-hydroxylase activity in hepatic microsomes from induced mice.

    Science.gov (United States)

    Lewandowski, M; Chui, Y C; Levi, P; Hodgson, E

    1991-02-01

    A simple and sensitive method for the separation of 14C-labelled acetanilide, 4-hydroxyacetanilide, 3-hydroxyacetanilide and 2-hydroxyacetanilide was developed using thin-layer chromatography. This separation is the basis for the assay of acetanilide 4-hydroxylase and acetanilide 2-hydroxylase activity in liver microsomes from DBA2/N male mice that had been treated with phenobarbital, 3-methylcholanthrene, isosafrole or n-butylbenzodioxole. Microsomes were incubated with [14C]acetanilide and extracted with benzene and ethyl acetate. The extract was applied to silica gel plates and developed with a hexane/isopropanol/ammonium hydroxide/water solvent system. The radiolabelled phenolic metabolites and the parent compound were detected using a Berthold Automatic TLC Linear Analyzer. Although the 4-hydroxylated metabolite was the primary product detected, this method can be used to detect other phenolic metabolites.

  6. Predictive value of animal models for human cytochrome P450 (CYP)-mediated metabolism: a comparative study in vitro.

    Science.gov (United States)

    Turpeinen, M; Ghiciuc, C; Opritoui, M; Tursas, L; Pelkonen, O; Pasanen, M

    2007-12-01

    One major challenge in drug development is defining of the optimal animal species to serve as a model of metabolism in man. The study compared the hepatic drug metabolism characteristics of humans and six widely used experimental animal species. Classical in vitro model enzyme assays with known human cytochrome P450 (CYP) enzyme selectivity were employed and optimized to target human hepatic CYP forms. The profile of CYP activities best resembling the human was seen in mouse followed by monkey, minipig, and dog liver microsomes, with rats displaying the most divergent. The widest interindividual variability was found in CYP3A-mediated midazolam -hydroxylase, and omeprazole sulphoxidase activities in human and monkey liver microsomes. These data demonstrate that if hepatic xenobiotic-metabolizing characteristics were to be the sole reason for the selection of animal species for toxicity studies, then the rat might not be the most appropriate model to mimic human CYP activity patterns.

  7. Reelin expression in human liver of patients with chronic hepatitis C infection

    Directory of Open Access Journals (Sweden)

    Simone Carotti

    2017-03-01

    Full Text Available Reelin is a secreted extracellular glycoprotein that plays a critical role during brain development. Several studies have described Reelin expression in hepatic stellate cells of the human liver. In order to investigate the possible role of Reelin in the process of hepatic fibrogenesis, in this study we investigated Reelin expression in the liver tissue of patients infected with the Hepatitis C Virus (HCV. On this basis, Reelin expression was analysed by immunohistochemistry during liver biopsies of 81 patients with HCV-related chronic hepatitis. A Knodell score was used to stage liver fibrosis. Hepatic stellate cells/myofibroblast immunohistochemical markers (CRBP-1, alpha-SMA were also evaluated. As further confirmed by co-localization experiments (Reelin +CRBP-1, Reelin protein was expressed by hepatic stellate cells/myofibroblasts, and a significant positive correlation was found between Reelin expression and the stage of liver fibrosis (P=0.002. Moreover, Reelin correlated with CRBP-1 positive cells (P=0.002, but not with alpha-SMA, suggesting that Reelin should not be regarded as a marker of hepatic stellate cells/myofibroblasts differentiation but rather as a functional protein expressed during some phases of liver fibrosis. Furthermore, Disabled-1 (Dab1, a Reelin adaptor protein, was expressed in cells of ductular reaction suggesting a paracrine role for Reelin with regards these elements. In conclusion, Reelin was expressed by human hepatic stellate cells/myofibroblasts and the number of these cells increased significantly in the lobule as the liver fibrosis progressed, suggesting a role for Reelin in the activation of hepatic stellate cells/myofibroblasts during liver injury. Reelin may potentially be incorporated into liver injury evaluations in combination with other histological data.

  8. Metabolism of Hydroxyalkyl Derivatives of 3-Hethylcholanthrene by Liver Microsomes

    Science.gov (United States)

    1991-04-05

    the interaction of 7,8- epoxido substrates with epoxide hydrolase. Our findings suggest that 1-OH-3MC 7,8-epoxides werc not good substrates for the...derived fi^m 9,10-diol- 7,8- epoxidos (anti and syn isomers) of 3-OHMC (37-51%) and 1-OH-3-OHMC (29-35%). In tiiat smdy, Osborne et al prepared ^H...induced cytochrome P-450c isozymes catalyze die formations of 9,10- epoxidos . However, tiiese fonned epoxides may not be good substrates to be

  9. Liver microsomal fraction is known to participate in:

    African Journals Online (AJOL)

    Abdullahi Balarabe

    Table 2 shows the effect of the phytochemicals contained in the various food materials on the activity of the endogenous antioxidant enzymes such as. Glutathione S-transferases (GST), Glutathione. Reductase (GR), Catalase (CAT), cytosolic superoxide dismutase (SOD1) and Mitochondrial superoxide dismutase (SOD2).

  10. Identification of metabolites of gardenin A in rat liver microsomes ...

    African Journals Online (AJOL)

    Tropical Journal of Pharmaceutical Research February 2017; 16 (2): 421-427 ... Pharmacotherapy Group, Faculty of Pharmacy, University of Benin, Benin ... helpful for better comprehension of the metabolism of GA and its pharmacological effects. ... International Pharmaceutical Abstract, Chemical Abstracts, Embase, Index ...

  11. Identification of metabolites of gardenin A in rat liver microsomes ...

    African Journals Online (AJOL)

    initiated by addition of NADPH (1 mM). The reaction was terminated by adding 1 mL of ice- cold acetonitrile, vortexing and centrifuging at. 15,000 rpm for 10 min at 4 °C. Aliquots of the supernatant were subjected to UHPLC-LTQ-. Orbitrap MS to identify the metabolites. Blank samples were of the same composition as the.

  12. Identification of metabolites of gardenin A in rat liver microsomes ...

    African Journals Online (AJOL)

    Then 5 μL of the sample was injected into UHPLC-LTQ- orbitrap mass spectrometer. The metabolites of GA were tentatively identified based on accurate mass measurements, fragmentation patterns, chromatographic retention times, and bibliography data. Results: A total of 12 metabolites were detected and identified.

  13. Phases I-II Matched Case-Control Study of Human Fetal Liver Cell Transplantation for Treatment of Chronic Liver Disease

    National Research Council Canada - National Science Library

    Pietrosi, Giada; Vizzini, Giovanni; Gerlach, Jorg; Chinnici, Cinzia; Luca, Angelo; Amico, Giandomenico; D'amato, Monica; Conaldi, Pier Giulio; Petri, Sergio Li; Spada, Marco; Tuzzolino, Fabio; Alio, Luigi; Schmelzer, Eva; Gridelli, Bruno

    2015-01-01

    Fetal hepatocytes have a high regenerative capacity. The aim of the study was to assess treatment safety and clinical efficacy of human fetal liver cell transplantation through splenic artery infusion...

  14. Stoichiometries of Transferrin Receptors 1 and 2 in Human Liver

    OpenAIRE

    Chloupková, Maja; Zhang, An-Sheng; Enns, Caroline A.

    2009-01-01

    Mutations in either the hereditary hemochromatosis protein, HFE, or transferrin receptor 2, TfR2, result in a similarly severe form of the most common type of iron overload disease called hereditary hemochromatosis. Models of the interactions between HFE, TfR1, and TfR2 imply that these proteins are present in different molar concentrations in the liver, where they control expression of the iron regulatory hormone, hepcidin, in response to body iron loading. The aim of this study was to deter...

  15. Human primary liver cancer-derived organoid cultures for disease modeling and drug screening.

    Science.gov (United States)

    Broutier, Laura; Mastrogiovanni, Gianmarco; Verstegen, Monique Ma; Francies, Hayley E; Gavarró, Lena Morrill; Bradshaw, Charles R; Allen, George E; Arnes-Benito, Robert; Sidorova, Olga; Gaspersz, Marcia P; Georgakopoulos, Nikitas; Koo, Bon-Kyoung; Dietmann, Sabine; Davies, Susan E; Praseedom, Raaj K; Lieshout, Ruby; IJzermans, Jan N M; Wigmore, Stephen J; Saeb-Parsy, Kourosh; Garnett, Mathew J; van der Laan, Luc Jw; Huch, Meritxell

    2017-12-01

    Human liver cancer research currently lacks in vitro models that can faithfully recapitulate the pathophysiology of the original tumor. We recently described a novel, near-physiological organoid culture system, wherein primary human healthy liver cells form long-term expanding organoids that retain liver tissue function and genetic stability. Here we extend this culture system to the propagation of primary liver cancer (PLC) organoids from three of the most common PLC subtypes: hepatocellular carcinoma (HCC), cholangiocarcinoma (CC) and combined HCC/CC (CHC) tumors. PLC-derived organoid cultures preserve the histological architecture, gene expression and genomic landscape of the original tumor, allowing for discrimination between different tumor tissues and subtypes, even after long-term expansion in culture in the same medium conditions. Xenograft studies demonstrate that the tumorogenic potential, histological features and metastatic properties of PLC-derived organoids are preserved in vivo. PLC-derived organoids are amenable for biomarker identification and drug-screening testing and led to the identification of the ERK inhibitor SCH772984 as a potential therapeutic agent for primary liver cancer. We thus demonstrate the wide-ranging biomedical utilities of PLC-derived organoid models in furthering the understanding of liver cancer biology and in developing personalized-medicine approaches for the disease.

  16. In vitro metabolism of aflatoxin B2 by animal and human liver.

    Science.gov (United States)

    Roebuck, B D; Siegel, W G; Wogan, G N

    1978-04-01

    The metabolism of aflatoxin B2 by postmitochondrial supernatant fractions of duck, rat, mouse, and human livers was studied in an in vitro system. Duck liver had a much higher level of activity than had tissues from other species. Postmitochondrial supernatant equivalent to 0.2 g whole liver metabolized 40 to 80% of the initial substrate in 30 min, compared to less than 6% for the other species. Among several metabolites formed by duck liver, aflatoxin B1 was produced in amounts equivalent to 2 to 8% of the initial substrate, and metabolites having chromatographic properties postualted for aflatoxicols 1 and 2 and aflatoxins M1 and M2 were also formed in small amounts. In contrast, rat, mouse, and human liver preparations produced no detectable aflatoxin B1 and only small amounts of compounds thought to be aflatoxins Q2 and P2. The greater susceptibility of duck liver to the toxicity of aflatoxin B2 may be attributable to its ability to form aflatoxin B2 may be attributable to its ability to form aflatoxin B1, which could then be activated through further metabolism.

  17. Long-Term Culture of Genome-Stable Bipotent Stem Cells from Adult Human Liver

    OpenAIRE

    Huch Meritxell; Gehart Helmuth; van Boxtel Ruben; Hamer Karien; Blokzijl Francis; Verstegen Monique M. A.; Ellis Ewa; van Wenum Martien; Fuchs Sabine A.; de Ligt Joep; van de Wetering Marc; Sasaki Nobuo; Boers Susanne J.; Kemperman Hans; de Jonge Jeroen

    2015-01-01

    Summary Despite the enormous replication potential of the human liver, there are currently no culture systems available that sustain hepatocyte replication and/or function in?vitro. We have shown previously that single mouse Lgr5+ liver stem cells can be expanded as epithelial organoids in?vitro and can be differentiated into functional hepatocytes in?vitro and in?vivo. We now describe conditions allowing long-term expansion of adult bile duct-derived bipotent progenitor cells from human live...

  18. Effect of ultrasound frequency on the Nakagami statistics of human liver tissues.

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    Po-Hsiang Tsui

    Full Text Available The analysis of the backscattered statistics using the Nakagami parameter is an emerging ultrasound technique for assessing hepatic steatosis and fibrosis. Previous studies indicated that the echo amplitude distribution of a normal liver follows the Rayleigh distribution (the Nakagami parameter m is close to 1. However, using different frequencies may change the backscattered statistics of normal livers. This study explored the frequency dependence of the backscattered statistics in human livers and then discussed the sources of ultrasound scattering in the liver. A total of 30 healthy participants were enrolled to undergo a standard care ultrasound examination on the liver, which is a natural model containing diffuse and coherent scatterers. The liver of each volunteer was scanned from the right intercostal view to obtain image raw data at different central frequencies ranging from 2 to 3.5 MHz. Phantoms with diffuse scatterers only were also made to perform ultrasound scanning using the same protocol for comparisons with clinical data. The Nakagami parameter-frequency correlation was evaluated using Pearson correlation analysis. The median and interquartile range of the Nakagami parameter obtained from livers was 1.00 (0.98-1.05 for 2 MHz, 0.93 (0.89-0.98 for 2.3 MHz, 0.87 (0.84-0.92 for 2.5 MHz, 0.82 (0.77-0.88 for 3.3 MHz, and 0.81 (0.76-0.88 for 3.5 MHz. The Nakagami parameter decreased with the increasing central frequency (r = -0.67, p < 0.0001. However, the effect of ultrasound frequency on the statistical distribution of the backscattered envelopes was not found in the phantom results (r = -0.147, p = 0.0727. The current results demonstrated that the backscattered statistics of normal livers is frequency-dependent. Moreover, the coherent scatterers may be the primary factor to dominate the frequency dependence of the backscattered statistics in a liver.

  19. Effect of ultrasound frequency on the Nakagami statistics of human liver tissues.

    Science.gov (United States)

    Tsui, Po-Hsiang; Zhou, Zhuhuang; Lin, Ying-Hsiu; Hung, Chieh-Ming; Chung, Shih-Jou; Wan, Yung-Liang

    2017-01-01

    The analysis of the backscattered statistics using the Nakagami parameter is an emerging ultrasound technique for assessing hepatic steatosis and fibrosis. Previous studies indicated that the echo amplitude distribution of a normal liver follows the Rayleigh distribution (the Nakagami parameter m is close to 1). However, using different frequencies may change the backscattered statistics of normal livers. This study explored the frequency dependence of the backscattered statistics in human livers and then discussed the sources of ultrasound scattering in the liver. A total of 30 healthy participants were enrolled to undergo a standard care ultrasound examination on the liver, which is a natural model containing diffuse and coherent scatterers. The liver of each volunteer was scanned from the right intercostal view to obtain image raw data at different central frequencies ranging from 2 to 3.5 MHz. Phantoms with diffuse scatterers only were also made to perform ultrasound scanning using the same protocol for comparisons with clinical data. The Nakagami parameter-frequency correlation was evaluated using Pearson correlation analysis. The median and interquartile range of the Nakagami parameter obtained from livers was 1.00 (0.98-1.05) for 2 MHz, 0.93 (0.89-0.98) for 2.3 MHz, 0.87 (0.84-0.92) for 2.5 MHz, 0.82 (0.77-0.88) for 3.3 MHz, and 0.81 (0.76-0.88) for 3.5 MHz. The Nakagami parameter decreased with the increasing central frequency (r = -0.67, p statistical distribution of the backscattered envelopes was not found in the phantom results (r = -0.147, p = 0.0727). The current results demonstrated that the backscattered statistics of normal livers is frequency-dependent. Moreover, the coherent scatterers may be the primary factor to dominate the frequency dependence of the backscattered statistics in a liver.

  20. Alloxan-Induced Diabetes Causes Morphological and Ultrastructural Changes in Rat Liver that Resemble the Natural History of Chronic Fatty Liver Disease in Humans

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    Amanda Natália Lucchesi

    2015-01-01

    Full Text Available Purpose. This study evaluated the long-term effects of alloxan-induced diabetes in rat liver. Methods. Thirty nondiabetic control rats (NC and 30 untreated diabetic (UD rats were divided into three subgroups sacrificed after 6, 14, or 26 weeks. Clinical and laboratory parameters were assessed. Fresh liver weight and its relationship with body weight were obtained, and liver tissue was analyzed. Results. UD rats showed sustained hyperglycemia, high glycosylated hemoglobin, and low plasma insulin. High serum levels of AST and ALT were observed in UD rats after 2 weeks, but only ALT remained elevated throughout the experiment. Fresh liver weight was equal between NC and UD rats, but the fresh liver weight/body weight ratio was significantly higher in UD rats after 14 and 26 weeks. UD rats showed liver morphological changes characterized by hepatic sinusoidal enlargement and micro- and macrovesicular hepatocyte fatty degeneration with progressive liver structure loss, steatohepatitis, and periportal fibrosis. Ultrastructural changes of hepatocytes, such as a decrease in the number of intracytoplasmic organelles and degeneration of mitochondria, rough endoplasmic reticulum, and nuclei, were also observed. Conclusion. Alloxan-induced diabetes triggered liver morphological and ultrastructural changes that closely resembled human disease, ranging from steatosis to steatohepatitis and liver fibrosis.

  1. All-Trans-Retinoic Acid Enhances Mitochondrial Function in Models of Human Liver

    Science.gov (United States)

    Tripathy, Sasmita; Chapman, John D; Han, Chang Y; Hogarth, Cathryn A; Arnold, Samuel L.M.; Onken, Jennifer; Kent, Travis; Goodlett, David R

    2016-01-01

    All-trans-retinoic acid (atRA) is the active metabolite of vitamin A. The liver is the main storage organ of vitamin A, but activation of the retinoic acid receptors (RARs) in mouse liver and in human liver cell lines has also been shown. Although atRA treatment improves mitochondrial function in skeletal muscle in rodents, its role in modulating mitochondrial function in the liver is controversial, and little data are available regarding the human liver. The aim of this study was to determine whether atRA regulates hepatic mitochondrial activity. atRA treatment increased the mRNA and protein expression of multiple components of mitochondrial β-oxidation, tricarboxylic acid (TCA) cycle, and respiratory chain. Additionally, atRA increased mitochondrial biogenesis in human hepatocytes and in HepG2 cells with and without lipid loading based on peroxisome proliferator activated receptor gamma coactivator 1α and 1β and nuclear respiratory factor 1 mRNA and mitochondrial DNA quantification. atRA also increased β-oxidation and ATP production in HepG2 cells and in human hepatocytes. Knockdown studies of RARα, RARβ, and PPARδ revealed that the enhancement of mitochondrial biogenesis and β-oxidation by atRA requires peroxisome proliferator activated receptor delta. In vivo in mice, atRA treatment increased mitochondrial biogenesis markers after an overnight fast. Inhibition of atRA metabolism by talarozole, a cytochrome P450 (CYP) 26 specific inhibitor, increased the effects of atRA on mitochondrial biogenesis markers in HepG2 cells and in vivo in mice. These studies show that atRA regulates mitochondrial function and lipid metabolism and that increasing atRA concentrations in human liver via CYP26 inhibition may increase mitochondrial biogenesis and fatty acid β-oxidation and provide therapeutic benefit in diseases associated with mitochondrial dysfunction. PMID:26921399

  2. The Use of an Acellular Oxygen Carrier in a Human Liver Model of Normothermic Machine Perfusion.

    Science.gov (United States)

    Laing, Richard W; Bhogal, Ricky H; Wallace, Lorraine; Boteon, Yuri; Neil, Desley A H; Smith, Amanda; Stephenson, Barney T F; Schlegel, Andrea; Hübscher, Stefan G; Mirza, Darius F; Afford, Simon C; Mergental, Hynek

    2017-11-01

    Normothermic machine perfusion of the liver (NMP-L) is a novel technique that preserves liver grafts under near-physiological conditions while maintaining their normal metabolic activity. This process requires an adequate oxygen supply, typically delivered by packed red blood cells (RBC). We present the first experience using an acellular hemoglobin-based oxygen carrier (HBOC) Hemopure in a human model of NMP-L. Five discarded high-risk human livers were perfused with HBOC-based perfusion fluid and matched to 5 RBC-perfused livers. Perfusion parameters, oxygen extraction, metabolic activity, and histological features were compared during 6 hours of NMP-L. The cytotoxicity of Hemopure was also tested on human hepatic primary cell line cultures using an in vitro model of ischemia reperfusion injury. The vascular flow parameters and the perfusate lactate clearance were similar in both groups. The HBOC-perfused livers extracted more oxygen than those perfused with RBCs (O2 extraction ratio 13.75 vs 9.43 % ×10 per gram of tissue, P = 0.001). In vitro exposure to Hemopure did not alter intracellular levels of reactive oxygen species, and there was no increase in apoptosis or necrosis observed in any of the tested cell lines. Histological findings were comparable between groups. There was no evidence of histological damage caused by Hemopure. Hemopure can be used as an alternative oxygen carrier to packed red cells in NMP-L perfusion fluid.

  3. Human Liver Cells Expressing Albumin and Mesenchymal Characteristics Give Rise to Insulin-Producing Cells

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    Irit Meivar-Levy

    2011-01-01

    Full Text Available Activation of the pancreatic lineage in the liver has been suggested as a potential autologous cell replacement therapy for diabetic patients. Transcription factors-induced liver-to-pancreas reprogramming has been demonstrated in numerous species both in vivo and in vitro. However, human-derived liver cells capable of acquiring the alternate pancreatic repertoire have never been characterized. It is yet unknown whether hepatic-like stem cells or rather adult liver cells give rise to insulin-producing cells. Using an in vitro experimental system, we demonstrate that proliferating adherent human liver cells acquire mesenchymal-like characteristics and a considerable level of cellular plasticity. However, using a lineage-tracing approach, we demonstrate that insulin-producing cells are primarily generated in cells enriched for adult hepatic markers that coexpress both albumin and mesenchymal markers. Taken together, our data suggest that adult human hepatic tissue retains a substantial level of developmental plasticity, which could be exploited in regenerative medicine approaches.

  4. Criteria for viability assessment of discarded human donor livers during ex vivo normothermic machine perfusion.

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    Michael E Sutton

    Full Text Available Although normothermic machine perfusion of donor livers may allow assessment of graft viability prior to transplantation, there are currently no data on what would be a good parameter of graft viability. To determine whether bile production is a suitable biomarker that can be used to discriminate viable from non-viable livers we have studied functional performance as well as biochemical and histological evidence of hepatobiliary injury during ex vivo normothermic machine perfusion of human donor livers. After a median duration of cold storage of 6.5 h, twelve extended criteria human donor livers that were declined for transplantation were ex vivo perfused for 6 h at 37 °C with an oxygenated solution based on red blood cells and plasma, using pressure controlled pulsatile perfusion of the hepatic artery and continuous portal perfusion. During perfusion, two patterns of bile flow were identified: (1 steadily increasing bile production, resulting in a cumulative output of ≥ 30 g after 6 h (high bile output group, and (2 a cumulative bile production <20 g in 6 h (low bile output group. Concentrations of transaminases and potassium in the perfusion fluid were significantly higher in the low bile output group, compared to the high bile output group. Biliary concentrations of bilirubin and bicarbonate were respectively 4 times and 2 times higher in the high bile output group. Livers in the low bile output group displayed more signs of hepatic necrosis and venous congestion, compared to the high bile output group. In conclusion, bile production could be an easily assessable biomarker of hepatic viability during ex vivo machine perfusion of human donor livers. It could potentially be used to identify extended criteria livers that are suitable for transplantation. These ex vivo findings need to be confirmed in a transplant experiment or a clinical trial.

  5. Gene discovery for the carcinogenic human liver fluke, Opisthorchis viverrini

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    Gasser Robin B

    2007-06-01

    Full Text Available Abstract Background Cholangiocarcinoma (CCA – cancer of the bile ducts – is associated with chronic infection with the liver fluke, Opisthorchis viverrini. Despite being the only eukaryote that is designated as a 'class I carcinogen' by the International Agency for Research on Cancer, little is known about its genome. Results Approximately 5,000 randomly selected cDNAs from the adult stage of O. viverrini were characterized and accounted for 1,932 contigs, representing ~14% of the entire transcriptome, and, presently, the largest sequence dataset for any species of liver fluke. Twenty percent of contigs were assigned GO classifications. Abundantly represented protein families included those involved in physiological functions that are essential to parasitism, such as anaerobic respiration, reproduction, detoxification, surface maintenance and feeding. GO assignments were well conserved in relation to other parasitic flukes, however, some categories were over-represented in O. viverrini, such as structural and motor proteins. An assessment of evolutionary relationships showed that O. viverrini was more similar to other parasitic (Clonorchis sinensis and Schistosoma japonicum than to free-living (Schmidtea mediterranea flatworms, and 105 sequences had close homologues in both parasitic species but not in S. mediterranea. A total of 164 O. viverrini contigs contained ORFs with signal sequences, many of which were platyhelminth-specific. Examples of convergent evolution between host and parasite secreted/membrane proteins were identified as were homologues of vaccine antigens from other helminths. Finally, ORFs representing secreted proteins with known roles in tumorigenesis were identified, and these might play roles in the pathogenesis of O. viverrini-induced CCA. Conclusion This gene discovery effort for O. viverrini should expedite molecular studies of cholangiocarcinogenesis and accelerate research focused on developing new interventions

  6. Control of oxygen tension recapitulates zone-specific functions in human liver microphysiology systems.

    Science.gov (United States)

    Lee-Montiel, Felipe T; George, Subin M; Gough, Albert H; Sharma, Anup D; Wu, Juanfang; DeBiasio, Richard; Vernetti, Lawrence A; Taylor, D Lansing

    2017-10-01

    This article describes our next generation human Liver Acinus MicroPhysiology System (LAMPS). The key demonstration of this study was that Zone 1 and Zone 3 microenvironments can be established by controlling the oxygen tension in individual devices over the range of ca. 3 to 13%. The oxygen tension was computationally modeled using input on the microfluidic device dimensions, numbers of cells, oxygen consumption rates of hepatocytes, the diffusion coefficients of oxygen in different materials and the flow rate of media in the MicroPhysiology System (MPS). In addition, the oxygen tension was measured using a ratiometric imaging method with the oxygen sensitive dye, Tris(2,2'-bipyridyl) dichlororuthenium(II) hexahydrate (RTDP) and the oxygen insensitive dye, Alexa 488. The Zone 1 biased functions of oxidative phosphorylation, albumin and urea secretion and Zone 3 biased functions of glycolysis, α1AT secretion, Cyp2E1 expression and acetaminophen toxicity were demonstrated in the respective Zone 1 and Zone 3 MicroPhysiology System. Further improvements in the Liver Acinus MicroPhysiology System included improved performance of selected nonparenchymal cells, the inclusion of a porcine liver extracellular matrix to model the Space of Disse, as well as an improved media to support both hepatocytes and non-parenchymal cells. In its current form, the Liver Acinus MicroPhysiology System is most amenable to low to medium throughput, acute through chronic studies, including liver disease models, prioritizing compounds for preclinical studies, optimizing chemistry in structure activity relationship (SAR) projects, as well as in rising dose studies for initial dose ranging. Impact statement Oxygen zonation is a critical aspect of liver functions. A human microphysiology system is needed to investigate the impact of zonation on a wide range of liver functions that can be experimentally manipulated. Because oxygen zonation has such diverse physiological effects in the liver, we

  7. The adult livers of immunodeficient mice support human hematopoiesis: evidence for a hepatic mast cell population that develops early in human ontogeny.

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    Marcus O Muench

    Full Text Available The liver plays a vital role in hematopoiesis during mammalian prenatal development but its hematopoietic output declines during the perinatal period. Nonetheless, hepatic hematopoiesis is believed to persist into adulthood. We sought to model human adult-liver hematopoiesis by transplantation of fetal and neonatal hematopoietic stem cells (HSCs into adult immunodeficient mice. Livers were found to be engrafted with human cells consisting primarily of monocytes and B-cells with lesser contributions by erythrocytes, T-cells, NK-cells and mast-cells. A resident population of CD117(++CD203c(+ mast cells was also documented in human midgestation liver, indicating that these cells comprise part of the liver's resident immune cell repertoire throughout human ontogeny. The murine liver was shown to support human multilineage hematopoiesis up to 321 days after transplant. Evidence of murine hepatic hematopoiesis was also found in common mouse strains as old as 2 years. Human HSC engraftment of the murine liver was demonstrated by detection of high proliferative-potential colony-forming cells in clonal cultures, observation of CD38-CD34(++ and CD133(+CD34(++ cells by flow cytometry, and hematopoietic reconstitution of secondary transplant recipients of chimeric liver cells. Additionally, chimeric mice with both hematopoietic and endothelial reconstitution were generated by intrasplenic injection of immunodeficient mice with liver specific expression of the urokinase-type plasminogen activator (uPA transgene. In conclusion, the murine liver is shown to be a hematopoietic organ throughout adult life that can also support human hematopoiesis in severely immunodeficient strains. Further humanization of the murine liver can be achieved in mice harboring an uPA transgene, which support engraftment of non-hematopoietic cells types. Thus, offering a model system to study the interaction of diverse human liver cell types that regulate hematopoiesis and immune

  8. A shift in paradigm towards human biology-based systems for cholestatic-liver diseases.

    Science.gov (United States)

    Noor, Fozia

    2015-12-01

    Cholestatic-liver diseases (CLDs) arise from diverse causes ranging from genetic factors to drug-induced cholestasis. The so-called diseases of civilization (obesity, diabetes, metabolic disorders, non-alcoholic liver disease, cardiovascular diseases, etc.) are intricately implicated in liver and gall bladder diseases. Although CLDs have been extensively studied, there seem to be important gaps in the understanding of human disease. Despite the fact that many animal models exist and substantial clinical data are available, translation of this knowledge towards therapy has been disappointingly limited. Recent advances in liver cell culture such as in vivo-like 3D cultivation of human primary hepatic cells, human induced pluripotent stem cell-derived hepatocytes; and cutting-edge analytical techniques such as 'omics' technologies and high-content screenings could play a decisive role in deeper mechanistic understanding of CLDs. This Topical Review proposes a roadmap to human biology-based research using omics technologies providing quantitative information on mechanisms in an adverse outcome/disease pathway framework. With modern sensitive tools, a shift in paradigm in human disease research seems timely and even inevitable to overcome species barriers in translation. © 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.

  9. Role of Tumor Associated Fibroblasts in Human Liver Regeneration, Cirrhosis, and Cancer

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    Daniela Cesselli

    2011-01-01

    Full Text Available Tumor associated fibroblasts (TAFs are considered a microenvironmental element critical for tumor growth and progression. Experimental studies suggest that their origin could be from mesenchymal stem cells (MSCs derived from the bone marrow. However, the role played by TAFs in cirrhosis, hepatocellular carcinoma development, and progression is largely unknown, and in vitro human models are missing. This paper for the first time demonstrates that (1 human neoplastic livers possess a population of multipotent adult stem cells (MASCs with properties of TAFs; (2 a population of MASC-derived TAFs is already present in cirrhotic, not yet neoplastic, livers; (3 MASCs isolated from nonneoplastic and noncirrhotic liver scan acquire a TAF phenotype when grown in a medium conditioned by tumor cell lines, supporting the notion that TAF could originate from resident primitive cells (MASCs, possibly through a paracrine mechanism.

  10. Scaffold-free 3D bio-printed human liver tissue stably maintains metabolic functions useful for drug discovery

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    Hideki Kizawa

    2017-07-01

    Full Text Available The liver plays a central role in metabolism. Although many studies have described in vitro liver models for drug discovery, to date, no model has been described that can stably maintain liver function. Here, we used a unique, scaffold-free 3D bio-printing technology to construct a small portion of liver tissue that could stably maintain drug, glucose, and lipid metabolism, in addition to bile acid secretion. This bio-printed normal human liver tissue maintained expression of several kinds of hepatic drug transporters and metabolic enzymes that functioned for several weeks. The bio-printed liver tissue displayed glucose production via cAMP/protein kinase A signaling, which could be suppressed with insulin. Bile acid secretion was also observed from the printed liver tissue, and it accumulated in the culture medium over time. We observed both bile duct and sinusoid-like structures in the bio-printed liver tissue, which suggested that bile acid secretion occurred via a sinusoid-hepatocyte-bile duct route. These results demonstrated that our bio-printed liver tissue was unique, because it exerted diverse liver metabolic functions for several weeks. In future, we expect our bio-printed liver tissue to be applied to developing new models that can be used to improve preclinical predictions of long-term toxicity in humans, generate novel targets for metabolic liver disease, and evaluate biliary excretion in drug development.

  11. Scaffold-free 3D bio-printed human liver tissue stably maintains metabolic functions useful for drug discovery.

    Science.gov (United States)

    Kizawa, Hideki; Nagao, Eri; Shimamura, Mitsuru; Zhang, Guangyuan; Torii, Hitoshi

    2017-07-01

    The liver plays a central role in metabolism. Although many studies have described in vitro liver models for drug discovery, to date, no model has been described that can stably maintain liver function. Here, we used a unique, scaffold-free 3D bio-printing technology to construct a small portion of liver tissue that could stably maintain drug, glucose, and lipid metabolism, in addition to bile acid secretion. This bio-printed normal human liver tissue maintained expression of several kinds of hepatic drug transporters and metabolic enzymes that functioned for several weeks. The bio-printed liver tissue displayed glucose production via cAMP/protein kinase A signaling, which could be suppressed with insulin. Bile acid secretion was also observed from the printed liver tissue, and it accumulated in the culture medium over time. We observed both bile duct and sinusoid-like structures in the bio-printed liver tissue, which suggested that bile acid secretion occurred via a sinusoid-hepatocyte-bile duct route. These results demonstrated that our bio-printed liver tissue was unique, because it exerted diverse liver metabolic functions for several weeks. In future, we expect our bio-printed liver tissue to be applied to developing new models that can be used to improve preclinical predictions of long-term toxicity in humans, generate novel targets for metabolic liver disease, and evaluate biliary excretion in drug development.

  12. The Human Amnion Epithelial Cell Secretome Decreases Hepatic Fibrosis in Mice with Chronic Liver Fibrosis

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    Majid Alhomrani

    2017-10-01

    Full Text Available Background: Hepatic stellate cells (HSCs are the primary collagen-secreting cells in the liver. While HSCs are the major cell type involved in the pathogenesis of liver fibrosis, hepatic macrophages also play an important role in mediating fibrogenesis and fibrosis resolution. Previously, we observed a reduction in HSC activation, proliferation, and collagen synthesis following exposure to human amnion epithelial cells (hAEC and hAEC-conditioned media (hAEC-CM. This suggested that specific factors secreted by hAEC might be effective in ameliorating liver fibrosis. hAEC-derived extracellular vesicles (hAEC-EVs, which are nanosized (40–100 nm membrane bound vesicles, may act as novel cell–cell communicators. Accordingly, we evaluated the efficacy of hAEC-EV in modulating liver fibrosis in a mouse model of chronic liver fibrosis and in human HSC.Methods: The hAEC-EVs were isolated and characterized. C57BL/6 mice with CCl4-induced liver fibrosis were administered hAEC-EV, hAEC-CM, or hAEC-EV depleted medium (hAEC-EVDM. LX2 cells, a human HSC line, and bone marrow-derived mouse macrophages were exposed to hAEC-EV, hAEC-CM, and hAEC-EVDM. Mass spectrometry was used to examine the proteome profile of each preparation.Results: The extent of liver fibrosis and number of activated HSCs were reduced significantly in CCl4-treated mice given hAEC-EVs, hAEC-CM, and hAEC EVDM compared to untreated controls. Hepatic macrophages were significantly decreased in all treatment groups, where a predominant M2 phenotype was observed. Human HSCs cultured with hAEC-EV and hAEC-CM displayed a significant reduction in collagen synthesis and hAEC-EV, hAEC-CM, and hAEC-EVDM altered macrophage polarization in bone marrow-derived mouse macrophages. Proteome analysis showed that 164 proteins were unique to hAEC-EV in comparison to hAEC-CM and hAEC-EVDM, and 51 proteins were co-identified components with the hAEC-EV fraction.Conclusion: This study provides novel data

  13. Open-Porous Hydroxyapatite Scaffolds for Three-Dimensional Culture of Human Adult Liver Cells

    Science.gov (United States)

    Schmelzer, Eva; Over, Patrick; Nettleship, Ian; Gerlach, Joerg C.

    2016-01-01

    Liver cell culture within three-dimensional structures provides an improved culture system for various applications in basic research, pharmacological screening, and implantable or extracorporeal liver support. Biodegradable calcium-based scaffolds in such systems could enhance liver cell functionality by providing endothelial and hepatic cell support through locally elevated calcium levels, increased surface area for cell attachment, and allowing three-dimensional tissue restructuring. Open-porous hydroxyapatite scaffolds were fabricated and seeded with primary adult human liver cells, which were embedded within or without gels of extracellular matrix protein collagen-1 or hyaluronan. Metabolic functions were assessed after 5, 15, and 28 days. Longer-term cultures exhibited highest cell numbers and liver specific gene expression when cultured on hydroxyapatite scaffolds in collagen-1. Endothelial gene expression was induced in cells cultured on scaffolds without extracellular matrix proteins. Hydroxyapatite induced gene expression for cytokeratin-19 when cells were cultured in collagen-1 gel while culture in hyaluronan increased cytokeratin-19 gene expression independent of the use of scaffold in long-term culture. The implementation of hydroxyapatite composites with extracellular matrices affected liver cell cultures and cell differentiation depending on the type of matrix protein and the presence of a scaffold. The hydroxyapatite scaffolds enable scale-up of hepatic three-dimensional culture models for regenerative medicine applications. PMID:27403430

  14. Human Parechovirus as a Cause of Isolated Pediatric Acute Liver Failure.

    Science.gov (United States)

    Bigelow, Amee M; Scott, John P; Hong, Johnny C; Cronin, David C; Vitola, Bernadette E; Fons, Roger A; Petersen, Tara L

    2016-11-01

    Among infants, almost half of acute liver failure cases are classified as indeterminate, whereas only a small number of cases show a documented viral infection. We present the first reported case of isolated acute hepatic failure in an infant in the setting of a human parechovirus (HPeV) infection. HPeV also may have been contributory to the posttransplant complication of 2 intussusceptions. This is a 10-month-old girl who presented with only symptoms of fussiness and was noted to have progressive decline in synthetic liver function as well as worsening coagulopathy requiring a liver transplant. The acute liver failure was in the setting of a positive serum RNA HPeV, subtype 3 (HPeV-3), after extensive diagnostic testing with genetic, autoimmune, and infectious causes otherwise negative. After liver transplantation, the postoperative course was complicated by both an ileal-ileal intussusception as well as a jejunal intussusception. Viral testing in pediatric acute liver failure is often performed, but the workup is frequently incomplete. This case report would support more extensive viral testing in this population of patients. In the setting of HPeV, clinicians could be alerted to the possibility of delayed gastrointestinal pathology in the posttransplant phase. Wider use of routine HPeV testing may more clearly define the variable clinical presentations and outcomes. Copyright © 2016 by the American Academy of Pediatrics.

  15. Differential genomic effects of six different TiO2 nanomaterials on human liver HepG2 cells

    Science.gov (United States)

    Engineered nanoparticles are reported to cause liver toxicity in vivo. To better assess the mechanism of the in vivo liver toxicity, we used the human hepatocarcinoma cells (HepG2) as a model system. Human HepG2 cells were exposed to 6 TiO2 nanomaterials (with dry primary partic...

  16. Side Population Cells Derived from Adult Human Liver Generate Hepatocyte-like Cells In Vitro

    Science.gov (United States)

    HUSSAIN, SUNNY ZAHEED; STROM, STEPHEN C.; KIRBY, MARTHA R.; BURNS, SEAN; LANGEMEIJER, SASKIA; UEDA, TAKAHIRO; HSIEH, MATTHEW; TISDALE, JOHN F.

    2009-01-01

    We sought to determine whether hepatic side population (SP) cells derived from adult human liver possess the potential of a novel candidate hepatic stem cell. Human cadaveric donor liver was subjected to collagenase perfusion and hepatocytes were separated from nonparenchymal cells by differential centrifugation. SP cells were isolated from the nonparenchymal portion after Hoechst 33342 staining. Since CD45 is a panleukocyte antigen, CD45-negative SP cells were separated from the vast majority of CD45-positive SP cells (90%), and hepatic growth medium was used to culture both groups. Both CD45-negative and CD45-positive hepatic SP cells generated colonies in the hepatic growth medium in 2–3 weeks. The colonies yielded large cells morphologically consistent with human hepatocytes, demonstrating granule-rich cytoplasm, dense, often double nuclei, and intracellular lipofuscin pigment. The cultured cells from both sources were positive for markers of human hepatocytes: HepPar, cytokeratin 8 (CK8), and human albumin. Reverse transcriptase–polymerase chain reaction (RT-PCR) performed on both groups demonstrated positivity for additional liver markers including human albumin, CK18, α-1 anti-trypsin, and the human cytochrome P450 enzyme CYP2B6. Double immunostaining (CD45 and HepPar) and RT-PCR confirmed that the hepatocyte-like cells derived from the CD45-negative SP cells acquired HepPar positivity but had no detectable CD45 antigen expression. In contrast, the cultured cells derived from the CD45-positive SP cells also acquired HepPar positivity, but only a minimal fraction expressed the CD45 antigen. We conclude that hepatic SP cells derived from the nonparenchymal portion of human liver are a potential source of human hepatocytes irrespective of their CD45 status, and further animal studies will be required to assess their regenerative potential. PMID:16187169

  17. Inhibition of microsomal oxidation of alcohols and of hydroxyl-radical-scavenging agents by the iron-chelating agent desferrioxamine.

    Science.gov (United States)

    Cederbaum, A I; Dicker, E

    1983-01-15

    Rat liver microsomes (microsomal fractions) catalyse the oxidation of straight-chain aliphatic alcohols and of hydroxyl-radical-scavenging agents during NADPH-dependent electron transfer. The iron-chelating agent desferrioxamine, which blocks the generation of hydroxyl radicals in other systems, was found to inhibit the following microsomal reactions: production of formaldehyde from either dimethyl sulphoxide or 2-methylpropan-2-ol (t-butylalcohol); generation of ethylene from 4-oxothiomethylbutyric acid; release of 14CO2 from [I-14C]benzoate; production of acetaldehyde from ethanol or butanal (butyraldehyde) from butan-1-ol. Desferrioxamine also blocked the increase in the oxidation of all these substrates produced by the addition of iron-EDTA to the microsomes. Desferrioxamine had no effect on a typical mixed-function-oxidase activity, the N-demethylation of aminopyrine, nor on the peroxidatic activity of catalase/H2O2 with ethanol. H2O2 appears to be the precursor of the oxidizing radical responsible for the oxidation of the alcohols and the other hydroxyl-radical scavengers. Chelation of microsomal iron by desferrioxamine most likely decreases the generation of hydroxyl radicals, which results in an inhibition of the oxidation of the alcohols and the hydroxyl-radical scavengers. Whereas desferrioxamine inhibited the oxidation of 2-methylpropan-2-ol, dimethyl sulphoxide, 4-oxothiomethylbutyrate and benzoate by more than 90%, the oxidation of ethanol and butanol could not be decreased by more than 45-60%. Higher concentrations of desferrioxamine were required to block the metabolism of the primary alcohols than to inhibit the metabolism of the other substrates. The desferrioxamine-insensitive rate of oxidation of ethanol was not inhibited by competitive hydroxyl-radical scavengers. These results suggest that primary alcohols may be oxidized by two pathways in microsomes, one dependent on the interaction of the alcohols with hydroxyl radicals (desferrioxamine

  18. Risk assessment of paracetamol-induced liver toxicity based on human in vitro data.

    NARCIS (Netherlands)

    Groothuis, Geny; Mafirakureva, Nyashadzaishe; Proost, Johannes; Jetten, M; Kleinjans, Jos; Lommen, A; Peijnenburg, A; Vredenburg, G; Vermeulen, N; Russel, Frans G. M.

    Currently risk assessment is based on animal experiments with limited success. The aim of this study was to explore the feasibility to replace the use of animals in risk assessment for drug-induced liver injury, by hazard identification and kinetic modeling based on human in vitro data for

  19. The fate of the vitelline and umbilical veins during the development of the human liver

    NARCIS (Netherlands)

    Hikspoors, Jill P. J. M.; Peeters, Mathijs M. J. P.; Mekonen, Hayelom K.; Kruepunga, Nutmethee; Mommen, Greet M. C.; Cornillie, Pieter; Köhler, S. Eleonore; Lamers, Wouter H.

    2017-01-01

    Differentiation of endodermal cells into hepatoblasts is well studied, but the remodeling of the vitelline and umbilical veins during liver development is less well understood. We compared human embryos between 3 and 10 weeks of development with pig and mouse embryos at comparable stages, and used

  20. Rate of hepatitis C viral clearance by human livers in human patients: Liver transplantation modeling primary infection and implications for studying entry inhibition.

    Directory of Open Access Journals (Sweden)

    Michael G Hughes

    Full Text Available To better understand the dynamics of early hepatitis C virus (HCV infection, we determined how rapidly non-cirrhotic HCV-uninfected liver allografts clear HCV from the circulation of cirrhotic HCV-infected patients at the time of transplantation but before administration of immunosuppression. Specifically, we characterized serum HCV kinetics during the first 90 min of reperfusion for 19 chronically HCV-infected patients transplanted with an HCV-uninfected liver by measuring serum viral load immediately prior to reperfusion (t = 0 and then every 15 min for a total of 90 min (t = 90. Immunosuppression was withheld until all samples were taken to better model primary infection. During this period, rates of viral clearance varied more than 20-fold with a median rate constant of 0.0357 1/min, range 0.0089-0.2169; half-life (minutes median 19.4, range 3.2-77.8. The majority of viral clearance occurred within the first 60 min. The amount of blood transfused during this 90-min period (a potential confounding variable of this human liver transplant model of primary infection accounted for 53% and 59% of k (r = 0.53, p = 0.05 and half-life (r = 0.59, p = 0.03 variability, respectively. No other clinical variables tested (age, allograft weight, and degree of reperfusion injury as assessed by peak postoperative ALT or AST accounted for the remaining variability (p>0.05.In a human liver transplant model of primary infection, HCV rapidly clears the bloodstream. With approximately 90% of clearance occurring in the first 90 minutes of reperfusion, studies of HCV entry inhibition could utilize rate of clearance during this early period as an outcome measure.

  1. Differences in Redox Regulatory Systems in Human Lung and Liver Tumors Suggest Different Avenues for Therapy

    Directory of Open Access Journals (Sweden)

    Ryuta Tobe

    2015-11-01

    Full Text Available A common characteristic of many cancer cells is that they suffer from oxidative stress. They, therefore, require effective redox regulatory systems to combat the higher levels of reactive oxygen species that accompany accelerated growth compared to the normal cells of origin. An elevated dependence on these systems in cancers suggests that targeting these systems may provide an avenue for retarding the malignancy process. Herein, we examined the redox regulatory systems in human liver and lung cancers by comparing human lung adenocarcinoma and liver carcinoma to their respective surrounding normal tissues. Significant differences were found in the two major redox systems, the thioredoxin and glutathione systems. Thioredoxin reductase 1 levels were elevated in both malignancies, but thioredoxin was highly upregulated in lung tumor and only slightly upregulated in liver tumor, while peroxiredoxin 1 was highly elevated in lung tumor, but downregulated in liver tumor. There were also major differences within the glutathione system between the malignancies and their normal tissues. The data suggest a greater dependence of liver on either the thioredoxin or glutathione system to drive the malignancy, while lung cancer appeared to depend primarily on the thioredoxin system.

  2. Subterminal hydroxylation of lauric acid by microsomes from a marine fish.

    Science.gov (United States)

    Lemaire, P; Lafaurie, M; Weissbart, D; Durst, F; Pflieger, P; Mioskowski, C; Salaün, J P

    1992-03-01

    Microsomes from the liver of sea bass (Dicentrarchus labrax) were shown to hydroxylate lauric acid at subterminal positions. The cytochrome P-450 system converted lauric acid to several mono-hydroxylated metabolites including omega-1 hydroxylaurate, which was the major metabolite (44% of total products). In addition, omega-2, omega-3, omega-4 and a small amount (2.3%) of omega hydroxylaurates were found. Reaction products were identified using thin-layer chromatography (TLC) and gas chromatography/mass spectrometry (GC/MS). Oxidation reactions were dependent upon O2 and NADPH, and did not occur with boiled microsomes or in the presence of a mixture of CO/O2. Hydroxylation proceeded linearly up to 20 min at 28 degrees C for protein concentrations below 380 micrograms. Treatment of fish with benzo(a)pyrene (BP) (20 mg/kg) drastically increased xenobiotic metabolism (ECOD, EROD and BPMO activities), but no difference in laurate hydroxylase activity was observed between untreated and treated fish. Starvation strongly enhanced laurate hydroxylase activity, and resumption of feeding reduced by half this increase of activity. In all of the experiments we did not observe any modification of the regioselectivity of lauric acid hydroxylation by this microsomal in-chain hydroxylating system. We suggest that cytochrome P-450 enzymes involved in lauric acid and xenobiotics metabolism are regulated independently.

  3. Mouse microsomal triglyceride transfer protein large subunit: cDNA cloning, tissue-specific expression, and chromosomal localization

    Energy Technology Data Exchange (ETDEWEB)

    Nakamuta, Makoto; Chang, Benny Hung-Junn; Hoogeveen, R. [Baylor College of Medicine, Houston, TX (United States)] [and others

    1996-04-15

    Microsomal triglyceride transfer protein (MTP) catalyzes the transfer of triglyceride, cholesteryl ester, and phospholipid between membranes. It is essential for the secretion of apolipoprotein B from the cell. Mutations in MTP are a major cause of abetalipoproteinemia. The mouse is a popular animal model for lipoprotein metabolism. We have cloned and sequenced mouse MTP cDNA. The DNA-deduced amino acid sequence indicates that mouse protein shows 93, 86, and 83% sequence indicates that mouse MTP contains 894 amino acids; the mouse protein shows 93, 86, and 83% sequence identity to the hamster, human, and bovine sequences, respectively. Northern blot analysis indicates that mouse MTP mRNA is expressed at high levels in the small intestine and at substantially lower levels in the liver and that it is not detectable in six other tissues examined. The mouse MTP gene has been localized to the distal region of chromosome 3 by Southern blots of interspecific backcross panels using progeny derived from matings of (C57BL/6J x SPRET/Ei)F1 x SPRET/Ei. Comparison of MTP sequences from human, bovine, hamster, and mouse indicates that the C-terminal region of MTP is better conserved than its N-terminal region. 21 refs., 2 figs.

  4. Human liver cell trafficking mutants: characterization and whole exome sequencing.

    Directory of Open Access Journals (Sweden)

    Fei Yuan

    Full Text Available The HuH7 liver cell mutant Trf1 is defective in membrane trafficking and is complemented by the casein kinase 2α subunit CK2α''. Here we identify characteristic morphologies, trafficking and mutational changes in six additional HuH7 mutants Trf2-Trf7. Trf1 cells were previously shown to be severely defective in gap junction functions. Using a Lucifer yellow transfer assay, remarkable attenuation of gap junction communication was revealed in each of the mutants Trf2-Trf7. Electron microscopy and light microscopy of thiamine pyrophosphatase showed that several mutants exhibited fragmented Golgi apparatus cisternae compared to parental HuH7 cells. Intracellular trafficking was investigated using assays of transferrin endocytosis and recycling and VSV G secretion. Surface binding of transferrin was reduced in all six Trf2-Trf7 mutants, which generally correlated with the degree of reduced expression of the transferrin receptor at the cell surface. The mutants displayed the same transferrin influx rates as HuH7, and for efflux rate, only Trf6 differed, having a slower transferrin efflux rate than HuH7. The kinetics of VSV G transport along the exocytic pathway were altered in Trf2 and Trf5 mutants. Genetic changes unique to particular Trf mutants were identified by exome sequencing, and one was investigated in depth. The novel mutation Ile34Phe in the GTPase RAB22A was identified in Trf4. RNA interference knockdown of RAB22A or overexpression of RAB22AI34F in HuH7 cells caused phenotypic changes characteristic of the Trf4 mutant. In addition, the Ile34Phe mutation reduced both guanine nucleotide binding and hydrolysis activities of RAB22A. Thus, the RAB22A Ile34Phe mutation appears to contribute to the Trf4 mutant phenotype.

  5. [Establishment of nude mice liver metastatic model of human primary malignant melanoma of the small intestine].

    Science.gov (United States)

    Tuo, Shuai; Zhang, Ning; Liu, Qiu-Zhen

    2008-07-01

    To provide ideal animal models for exploring pathogenesis and experimental therapy of primary malignant melanoma of the small intestine. The histologically intact primary and liver metastatic fragments derived from surgical specimens of one patient with metastatic malignant melanoma of the small intestine were orthotopically implanted in the small intestinal mucous layer of nude mice. The take rate, invasion and liver metastasis were observed. Morphology (light microscopy, electron microscopy), immunophenotype analysis, flow cytometry and karyotype analysis were applied for the original human tumors and the transplanted tumors. The primary and liver metastatic fragments of malignant melanoma of the small intestine were successfully implanted in nude mice. After continuous passages in nude mice,an orthotopic model of human primary malignant melanoma of the small intestine(from the primary focus)in nude mice (termed HSIM-0501) and a liver metastatic model of human primary malignant melanoma of the small intestine (from the liver metastatic focus) in nude mice (termed HSIM-0502) were established. Histological examination of transplanted tumors revealed high-grade melanoma. S-100 protein and HMB45 were positive. Massive melanin granules and melanin complex were seen in cytoplasm of tumor cells.Chromosomal modal number was between 55 and 59. DNA index (DI) was 1.49-1.61, representing heteroploid. HSIM-0501 and HSIM-0502 were maintained for 25 and 27 passages in nude mice respectively. Three hundred and seventeen nude mice were used for transplantation. Both the take rate after transplantation and resuscitation rate of liquid nitrogen cryopreservation were 100%. HSIM-0501 exhibited 46.2% liver metastasis and 36.7% lymph node metastases. In HSIM-0502, both liver and lymph node metastases were 100%.The transplanted tumors autonomically and invasively grew in the small intestines of nude mice and hematogenous metastasis, lymph node metastasis and celiac planting metastasis

  6. Disruption of clock gene expression in human colorectal liver metastases

    NARCIS (Netherlands)

    S.A. Huisman (Sander); K.R. Ahmadi (Kourosh); J.N.M. IJzermans (Jan); C. Verhoef (Kees); G.T.J. van der Horst (Gijsbertus); R.W.F. de Bruin (Ron)

    2016-01-01

    textabstractThe circadian timing system controls about 40 % of the transcriptome and is important in the regulation of a wide variety of biological processes including metabolic and proliferative functions. Disruption of the circadian clock could have significant effect on human health and has an

  7. IL-7 licenses activation of human liver intrasinusoidal mucosal-associated invariant T cells.

    Science.gov (United States)

    Tang, Xin-Zi; Jo, Juandy; Tan, Anthony T; Sandalova, Elena; Chia, Adeline; Tan, Kai Chah; Lee, Kang Hoe; Gehring, Adam J; De Libero, Gennaro; Bertoletti, Antonio

    2013-04-01

    Human mucosal-associated invariant T (MAIT) cells are a T cell population characterized by the expression of a semi-invariant TCR capable of recognizing bacterial products in the context of MR1. MAIT cells are enriched in the human liver, which is constantly exposed to bacterial products from the intestine. Whether this specific parenchymal localization influences their function remains unknown. We analyzed MAIT cells resident in the vascular bed of livers and showed that they represented the majority of T cells expressing NK markers and the dominant IL-17A(+) T cell subset in the human liver sinusoids. In comparison with MAIT cells purified from peripheral blood, intrasinusoidal MAIT cells expressed markers of T cell activation; however, TCR-mediated cytokine production was equally suppressed in both circulating and intrasinusoidal MAIT cells. MAIT cells also expressed high levels of IL-7R, and we showed that IL-7, a cytokine produced by hepatocytes during inflammation, regulated TCR-mediated activation of MAIT cells, licensing them to dramatically increase Th1 cytokines and IL-17A production. Our quantitative and functional data indicate that MAIT cells are a specialized cell population highly adapted to exert their immune functions in the vascular network of the liver.

  8. [Protective effect of intraperitoneal transplantation of human liver-derived stem cells at different times against concanavalin A-induced acute liver injury in mice].

    Science.gov (United States)

    Bi, Y Z; Fan, Z; Chen, D F; Li, S S; Wang, Q Y; Gao, P F; Wang, Q Q; Duan, Z P; Chen, Y; Kong, L B; Wang, Y B; Hong, F

    2017-03-20

    Objective: To investigate the protective effect of intraperitoneal transplantation of human liver-derived stem cells at different times against concanavalin A (ConA)-induced acute liver injury in mice. Methods: A total of 88 male C57BL/6 mice were randomly divided into normal control group (group C), ConA model group (group M), and human liver-derived stem cells (HYX1)+ConA group (group E); according to the interval between phosphate buffer/HYX1 injection and ConA injection, Groups M and E were further divided into 3-hour groups (M1 and E1 groups), 6-hour groups (M2 and E2 groups), 12-hour groups (M3 and E3 groups), 24-hour groups (M4 and E4 groups), and 48-hour groups (M5 and E5 groups). The levels of alanine aminotransferase (ALT), aspartate transaminase (AST), and total bilirubin (TBil) in peripheral blood were measured, liver tissue sections were used to observe pathological changes, and the Ishak score for liver inflammation was determined. The independent samples t-test was used for comparison between groups, and P 0.05). The pathological sections of liver tissue showed that compared with group M, group E had significant reductions in the degree of necrosis and Ishak score (both P transplantation of human liver-derived stem cells has a protective effect against ConA-induced acute liver injury in mice, and the injection at 6 and 12 hours in advance has the best protective effect.

  9. Nicotine induces fibrogenic changes in human liver via nicotinic acetylcholine receptors expressed on hepatic stellate cells

    Energy Technology Data Exchange (ETDEWEB)

    Soeda, Junpei; Morgan, Maelle; McKee, Chad; Mouralidarane, Angelina; Lin, ChingI [University College London, Centre for Hepatology, Royal Free Hospital, London NW3 2PF (United Kingdom); Roskams, Tania [Department of Morphology and Molecular Pathology, University of Leuven (Belgium); Oben, Jude A., E-mail: j.oben@ucl.ac.uk [University College London, Centre for Hepatology, Royal Free Hospital, London NW3 2PF (United Kingdom); Department of Gastroenterology and Hepatology, Guy' s and St Thomas' Hospital, London SE1 7EH (United Kingdom)

    2012-01-06

    Highlights: Black-Right-Pointing-Pointer Cigarette smoke may induce liver fibrosis via nicotine receptors. Black-Right-Pointing-Pointer Nicotine induces proliferation of hepatic stellate cells (HSCs). Black-Right-Pointing-Pointer Nicotine activates hepatic fibrogenic pathways. Black-Right-Pointing-Pointer Nicotine receptor antagonists attenuate HSC proliferation. Black-Right-Pointing-Pointer Nicotinic receptor antagonists may have utility as novel anti-fibrotic agents. -- Abstract: Background and aims: Cigarette smoke (CS) may cause liver fibrosis but possible involved mechanisms are unclear. Among the many chemicals in CS is nicotine - which affects cells through nicotinic acetylcholine receptors (nAChR). We studied the effects of nicotine, and involved pathways, on human primary hepatic stellate cells (hHSCs), the principal fibrogenic cells in the liver. We then determined possible disease relevance by assaying nAChR in liver samples from human non-alcoholic steatohepatitis (NASH). Methods: hHSC were isolated from healthy human livers and nAChR expression analyzed - RT-PCR and Western blotting. Nicotine induction of hHSC proliferation, upregulation of collagen1-{alpha}2 and the pro-fibrogenic cytokine transforming growth factor beta 1 (TGF-{beta}1) was determined along with involved intracellular signaling pathways. nAChR mRNA expression was finally analyzed in whole liver biopsies obtained from patients diagnosed with non-alcoholic steatohepatitis (NASH). Results: hHSCs express muscle type ({alpha}1, {beta}1, delta and epsilon) and neuronal type ({alpha}3, {alpha}6, {alpha}7, {beta}2 and {beta}4) nAChR subunits at the mRNA level. Among these subunits, {alpha}3, {alpha}7, {beta}1 and {epsilon} were predominantly expressed as confirmed by Western blotting. Nicotine induced hHSC proliferation was attenuated by mecamylamine (p < 0.05). Additionally, collagen1-{alpha}2 and TGF-{beta}1 mRNA expression were significantly upregulated by nicotine and inhibited by

  10. Enantioselective metabolism of hydroxychloroquine employing rats and mice hepatic microsomes

    Directory of Open Access Journals (Sweden)

    Carmem Dickow Cardoso

    2009-12-01

    Full Text Available Hydroxychloroquine (HCQ is an important chiral drug used, mainly, in the treatment of rheumatoid arthritis, systemic lupus erythematosus and malaria, and whose pharmacokinetic and pharmacodynamic properties look to be stereoselective. Respecting the pharmacokinetic properties, some previous studies indicate that the stereoselectivity could express itself in the processes of metabolism, distribution and excretion and that the stereoselective metabolism looks to be a function of the studied species. So, the in vitro metabolism of HCQ was investigated using hepatic microsomes of rats and mice. The microsomal fraction of livers of Wistar rats and Balb-C mice was separated by ultracentrifugation and 500 μL were incubated for 180 minutes with 10 μL of racemic HCQ 1000 μg mL-1. Two stereospecific analytical methods, high performance liquid chromatography (HPLC and capillary electrophoresis (CE, were used to separate and quantify the formed metabolites. It was verified that the main formed metabolite is the (--(R-desethyl hydroxychloroquine for both animal species.A hidroxicloroquina (HCQ é um importante fármaco quiral usado, principalmente, no tratamento de artrite reumatóide, lupus eritematoso sistêmico e malária e cujas propriedades farmacocinéticas e farmacodinâmicas parecem ser estereosseletivas. Em relação às propriedades farmacocinéticas, alguns estudos prévios indicam que a estereosseletividade pode se expressar nos processos de metabolismo, distribuição e excreção e que o metabolismo estereosseletivo parece ser função da espécie estudada. Sendo assim, o metabolismo in vitro da HCQ foi investigado usando microssomas de fígado de ratos e de camundongos. A fração microssômica de fígados de ratos Wistar e de camundongos Balb-C foi isolada por ultracentrifugação e 500 μL foram incubados por 180 minutos com 10 μL de HCQ racêmica 1000 μg mL-1. Dois métodos analíticos estereoespecíficos, por cromatografia líquida de

  11. Proteomic Profiling of Human Liver Biopsies: Hepatitis C Virus-Induced Fibrosis and Mitochondrial Dysfunction

    Energy Technology Data Exchange (ETDEWEB)

    Diamond, Deborah L.; Jacobs, Jon M.; Paeper, Bryan; Proll, Sean; Gritsenko, Marina A.; Carithers, Jr., Robert L.; Larson , Anne M.; Yeh, Matthew M.; Camp, David G.; Smith, Richard D.; Katze, Michael G.

    2007-09-01

    Liver biopsies from HCV-infected patients offer the unique opportunity to study human liver biology and disease in vivo. However, the low protein yields associated with these small samples present a significant challenge for proteomic analysis. In this study we describe the application of an ultra-sensitive proteomics platform for performing robust quantitative proteomic studies on microgram amounts of HCV-infected human liver tissue from 15 patients at different stages of fibrosis. A high quality liver protein data base containing 5,920 unique protein identifications supported high throughput quantitative studies using 16O:18O stable isotope labeling in combination with the accurate mass and time (AMT) tag approach. A total of 1,641 liver biopsy proteins were quantified and ANOVA identified 210 proteins exhibiting statistically significant differences associated with fibrosis stage. Hierarchical clustering revealed that biopsies representative of later fibrosis stages (e.g. Batts-Ludwig stages 3-4) exhibited a distinct protein expression profile indicating an apparent down-regulation of many proteins when compared to samples from earlier fibrosis stages (e.g. Batts-Ludwig stages 0-2). Functional analysis of these signature proteins suggests that impairment of key mitochondrial processes including fatty acid oxidation and oxidative phosphorylation, and response to oxidative stress and reactive oxygen species occurs during advanced stage 3-4 fibrosis. In conclusion, the results reported here represent a significant advancement in clinical proteomics providing to our knowledge, the first demonstration of global proteomic alterations accompanying liver disease progression in patients chronically infected with HCV. Our findings contribute to a generally emerging theme associating oxidative stress and hepatic mitochondrial dysfunction with HCV pathogenesis.

  12. Systemic human T cell developmental processes in humanized mice cotransplanted with human fetal thymus/liver tissue and hematopoietic stem cells.

    Science.gov (United States)

    Joo, Sung-Yeon; Chung, Yun Shin; Choi, Bongkum; Kim, Miyoung; Kim, Jong-Hwa; Jun, Tae-Gook; Chang, Jun; Sprent, Jonathan; Surh, Charles D; Joh, Jae-won; Kim, Sung Joo

    2012-12-15

    In many humanized mouse models, there are few T cells in the engrafted human cell, whereas the number of B cells is high. We attempted to overcome this limitation and investigate whether the entire process of human T cell development arose similarly to the process in humans, as previously reported. To produce an advanced humanized mice model, we transplanted human fetal liver/thymus tissue subrenally and injected human CD34(+) stem cells intravenously into NOD/SCID/IL2Rgamma null (NSG) mice. Humanized mice transplanted with fetal thymus/liver tissues and fetal liver-derived CD34(+) stem cells (FLT+FLCD34) showed higher levels of human cells and T cells than mice transplanted with fetal liver-derived CD34(+) stem cells only (FLCD34). In the transplanted thymus tissue of FLT+FLCD34 mice, thymus seeding progenitors (TSPs), early thymic progenitors (ETPs), pre-T cells, and all the other human T cell populations were identified. In the periphery, FLT+FLCD34 mice have high levels of CD45RA(+) T cells; conversely, FLCD34 mice have higher levels of CD45RO(+) T cells. The CD45RO(+) T cells of FLCD34 mice proliferated rapidly after stimulation and exhibited innate T cells properties, expressing PLZF (promyelocytic leukemia zinc finger protein). Human T cells educated by mouse MHC II in mice without a human thymus differ from normal human T cells. On the basis of these findings, numerous T cell-tropic human diseases could be explored in our humanized mice and molecular aspects of human T cell development could be also studied extensively.

  13. MicroRNA-146b-5p Identified in Porcine Liver Donation Model is Associated with Early Allograft Dysfunction in Human Liver Transplantation

    Science.gov (United States)

    Li, Cheukfai; Zhao, Qiang; Zhang, Wei; Chen, Maogen; Ju, Weiqiang; Wu, Linwei; Han, Ming; Ma, Yi; Zhu, Xiaofeng; Wang, Dongping; Guo, Zhiyong; He, Xiaoshun

    2017-01-01

    Background Poor transplant outcome was observed in donation after brain death followed by circulatory death (DBCD), since the donor organs suffered both cytokine storm of brain death and warm ischemia injury. MicroRNAs (miRNAs) have emerged as promising disease biomarkers, so we sought to establish a miRNA signature of porcine DBCD and verify the findings in human liver transplantation. Material/Methods MiRNA expression was determined with miRNA sequencing in 3 types of the porcine model of organ donation, including donation after brain death (DBD) group, donation after circulatory death (DCD) group, and DBCD group. Bioinformatics analysis was performed to reveal the potential regulatory behavior of target miRNA. Human liver graft biopsy samples after reperfusion detected by fluorescence in situ hybridization were used to verify the expression of target miRNA. Results We compared miRNA expression profiles of the 3 donation types. The porcine liver graft miR-146b was significantly increased and selected in the DBCD group versus in the DBD and DCD groups. The donor liver expression of human miR-146b-5p, which is homologous to porcine miR-146b, was further examined in 42 cases of human liver transplantations. High expression of miR-146b-5p successfully predicted the post-transplant early allograft dysfunction (EAD) with the area under the ROC curve (AUC) 0.759 (P=0.004). Conclusions Our results revealed the miRNA signature of DBCD liver grafts for the first time. The miR-146b-5p may have important clinical implications for monitoring liver graft function and predicating transplant outcomes. PMID:29227984

  14. Improving animal and human health through understanding liver fluke immunology.

    Science.gov (United States)

    Piedrafita, D; Spithill, T W; Smith, R E; Raadsma, H W

    2010-08-01

    Sheep, goats and cattle represent the most numerous and economically important agricultural species worldwide used as sources for milk, fibre and red meat. In addition, in the developing world, these species often represent the sole asset base for small-holder livestock farmers and cattle/buffaloes often provide the majority of draught power for crop production. Production losses caused by helminth diseases of these animals are a major factor in extending the cycle of poverty in developing countries and a major food security issue for developed economies. Fasciola spp. are one of the most important zoonotic diseases with a global economic impact in livestock production systems and a poorly defined but direct effect on human health. Improvements in human and animal health will require a concerted research effort into the development of new accurate and simple diagnostic tests and increased vaccine and drug development against Fasciola infections. Here, the use of definitive natural host breeds with contrasting resistance to Fasciola infections is discussed as a resource to contrast parasite-host interactions and identify parasite immune evasion strategies. Such studies are likely to boost the discovery of new vaccine, drug and diagnostic candidates and provide the foundation for future genetic selection of resistant animals.

  15. Long-Term Culture of Genome-Stable Bipotent Stem Cells from Adult Human Liver

    Science.gov (United States)

    Huch, Meritxell; Gehart, Helmuth; van Boxtel, Ruben; Hamer, Karien; Blokzijl, Francis; Verstegen, Monique M.A.; Ellis, Ewa; van Wenum, Martien; Fuchs, Sabine A.; de Ligt, Joep; van de Wetering, Marc; Sasaki, Nobuo; Boers, Susanne J.; Kemperman, Hans; de Jonge, Jeroen; Ijzermans, Jan N.M.; Nieuwenhuis, Edward E.S.; Hoekstra, Ruurdtje; Strom, Stephen; Vries, Robert R.G.; van der Laan, Luc J.W.; Cuppen, Edwin; Clevers, Hans

    2015-01-01

    Summary Despite the enormous replication potential of the human liver, there are currently no culture systems available that sustain hepatocyte replication and/or function in vitro. We have shown previously that single mouse Lgr5+ liver stem cells can be expanded as epithelial organoids in vitro and can be differentiated into functional hepatocytes in vitro and in vivo. We now describe conditions allowing long-term expansion of adult bile duct-derived bipotent progenitor cells from human liver. The expanded cells are highly stable at the chromosome and structural level, while single base changes occur at very low rates. The cells can readily be converted into functional hepatocytes in vitro and upon transplantation in vivo. Organoids from α1-antitrypsin deficiency and Alagille syndrome patients mirror the in vivo pathology. Clonal long-term expansion of primary adult liver stem cells opens up experimental avenues for disease modeling, toxicology studies, regenerative medicine, and gene therapy. PMID:25533785

  16. Detection of Intrahepatic Human Islets Following Combined Liver-Islet Allotransplantation

    Science.gov (United States)

    Ricordi, Camillo; Tzakis, Andreas; Alejandro, Rodolfo; Zeng, Yijun; Demetris, Anthony J.; Carroll, Patricia; Fung, John J.; Mintz, Daniel H.; Starzl, Thomas E.

    2010-01-01

    Summary This article describes the localization of intact insulin-containing intrahepatic islets after combined liver-islet allotransplantation. The patient was a 36-year-old woman who underwent upper abdominal exenteration for neuroendocrine carcinoma; 289,000 islets were transplanted via portal vein infusion immediately after complete revascularization of the liver. Immunosuppression was with low-dose FK-506. OKT3 and steroids were used to treat one rejection episode 2 weeks after transplantation, but the patient subsequently developed multiple infections and died 109 days after transplantation. At autopsy, the transplanted liver did not show any sign of rejection and well-preserved islets were present in portal triads sampled from the anterior inferior edge of the right lobe. Immunohistochemical labeling confirmed the presence of insulin-containing cells. This finding indicated that human islets can survive after intrahepatic allotransplantation, despite positive cross-match with no HLA antigen match, suggesting that upper abdominal exenteration and liver transplantation may constitute a protective factor for the survival of allogeneic human islets. PMID:1641394

  17. Immunological quantitation and localization of ACAT-1 and ACAT-2 in human liver and small intestine.

    Science.gov (United States)

    Chang, C C; Sakashita, N; Ornvold, K; Lee, O; Chang, E T; Dong, R; Lin, S; Lee, C Y; Strom, S C; Kashyap, R; Fung, J J; Farese, R V; Patoiseau, J F; Delhon, A; Chang, T Y

    2000-09-08

    By using specific anti-ACAT-1 antibodies in immunodepletion studies, we previously found that ACAT-1, a 50-kDa protein, plays a major catalytic role in the adult human liver, adrenal glands, macrophages, and kidneys but not in the intestine. Acyl-coenzyme A:cholesterol acyltransferase (ACAT) activity in the intestine may be largely derived from a different ACAT protein. To test this hypothesis, we produced specific polyclonal anti-ACAT-2 antibodies that quantitatively immunodepleted human ACAT-2, a 46-kDa protein expressed in Chinese hamster ovary cells. In hepatocyte-like HepG2 cells, ACAT-1 comprises 85-90% of the total ACAT activity, with the remainder attributed to ACAT-2. In adult intestines, most of the ACAT activity can be immunodepleted by anti-ACAT-2. ACAT-1 and ACAT-2 do not form hetero-oligomeric complexes. In differentiating intestinal enterocyte-like Caco-2 cells, ACAT-2 protein content increases by 5-10-fold in 6 days, whereas ACAT-1 protein content remains relatively constant. In the small intestine, ACAT-2 is concentrated at the apices of the villi, whereas ACAT-1 is uniformly distributed along the villus-crypt axis. In the human liver, ACAT-1 is present in both fetal and adult hepatocytes. In contrast, ACAT-2 is evident in fetal but not adult hepatocytes. Our results collectively suggest that in humans, ACAT-2 performs significant catalytic roles in the fetal liver and in intestinal enterocytes.

  18. New psychoactive substances: Studies on the metabolism of XLR-11, AB-PINACA, FUB-PB-22, 4-methoxy-α-PVP, 25-I-NBOMe, and meclonazepam using human liver preparations in comparison to primary human hepatocytes, and human urine.

    Science.gov (United States)

    Richter, Lilian H J; Maurer, Hans H; Meyer, Markus R

    2017-10-05

    New psychoactive substances (NPS) are an increasing problem in clinical and forensic toxicology. The knowledge of their metabolism is important for toxicological risk assessment and for developing toxicological urine screenings. Considering the huge numbers of NPS annually appearing on the market, metabolism studies should be realized in a fast, simple, cost efficient, and reliable way. Primary human hepatocytes (PHH) were recommended to be the gold standard for in vitro metabolism studies as they are expected to contain natural enzyme clusters, co-substrates, and drug transporters. In addition, they were already successfully used for metabolism studies of NPS. However, they also have disadvantages such as high costs and limited applicability without special equipment. The aims of the present study were therefore first to investigate exemplarily the phase I and phase II metabolism of six NPS (XLR-11, AB-PINACA, FUB-PB-22, 4-methoxy-α-PVP, 25-I-NBOMe, and meclonazepam) from different drug classes using pooled human S9 fraction (pS9) or pooled human liver microsomes combined with cytosol (pHLM/pHLC) after addition of the co-substrates for the main metabolic phase I and II reactions. Second to compare results to published data generated using primary human hepatocytes and human urine samples. Results of the incubations with pS9 or pHLM/pHLC were comparable in number and abundance of metabolites. Formation of metabolites, particularly after multi-step reactions needed a longer incubation time. However, incubations using human liver preparations resulted in a lower number of total detected metabolites compared to PHH, but they were still able to allow the identification of the main human urinary excretion products. Human liver preparations and particularly the pooled S9 fraction could be shown to be a sufficient and more cost-efficient alternative in context of metabolism studies also for developing toxicological urine screenings. It might be recommended to use the

  19. Rapid production of human liver scaffolds for functional tissue engineering by high shear stress oscillation-decellularization

    NARCIS (Netherlands)

    Mazza, G. (Giuseppe); Al-Akkad, W. (Walid); Telese, A. (Andrea); Longato, L. (Lisa); Urbani, L. (Luca); Robinson, B. (Benjamin); Hall, A. (Andrew); Kong, K. (Kenny); Frenguelli, L. (Luca); Marrone, G. (Giusi); Willacy, O. (Oliver); Shaeri, M. (Mohsen); A.J. Burns (Alan); Malago, M. (Massimo); Gilbertson, J. (Janet); Rendell, N. (Nigel); Moore, K. (Kevin); Hughes, D. (David); Notingher, I. (Ioan); Jell, G. (Gavin); Del Rio Hernandez, A. (Armando); P. de Coppi (Paolo); Rombouts, K. (Krista); Pinzani, M. (Massimo)

    2017-01-01

    textabstractThe development of human liver scaffolds retaining their 3-dimensional structure and extra-cellular matrix (ECM) composition is essential for the advancement of liver tissue engineering. We report the design and validation of a new methodology for the rapid and accurate production of

  20. Completion of hepatitis C virus replication cycle in heterokaryons excludes dominant restrictions in human non-liver and mouse liver cell lines.

    Directory of Open Access Journals (Sweden)

    Anne Frentzen

    2011-04-01

    Full Text Available Hepatitis C virus (HCV is hepatotropic and only infects humans and chimpanzees. Consequently, an immunocompetent small animal model is lacking. The restricted tropism of HCV likely reflects specific host factor requirements. We investigated if dominant restriction factors expressed in non-liver or non-human cell lines inhibit HCV propagation thus rendering these cells non-permissive. To this end we explored if HCV completes its replication cycle in heterokaryons between human liver cell lines and non-permissive cell lines from human non-liver or mouse liver origin. Despite functional viral pattern recognition pathways and responsiveness to interferon, virus production was observed in all fused cells and was only ablated when cells were treated with exogenous interferon. These results exclude that constitutive or virus-induced expression of dominant restriction factors prevents propagation of HCV in these cell types, which has important implications for HCV tissue and species tropism. In turn, these data strongly advocate transgenic approaches of crucial human HCV cofactors to establish an immunocompetent small animal model.

  1. Effect of New Water-Soluble Dendritic Phthalocyanines on Human Colorectal and Liver Cancer Cell Lines

    Directory of Open Access Journals (Sweden)

    Ebru YABAŞ

    2017-08-01

    Full Text Available Human hepatocellular carcinoma (HepG2 cells and colorectal adenocarcinoma (DLD-1 cells were treated with the synthesized water soluble phthalocyanine derivatives to understand the effect of the compounds both on colorectal and liver cancer cells. The compounds inhibited cell proliferation and displayed cytotoxic effect on these cancer cell lines however; the effect of the compounds on healthy control fibroblast cell line was comparatively lower. The compounds can be employed for cancer treatment as anticancer agents.

  2. An orphan esterase ABHD10 modulates probenecid acyl glucuronidation in human liver.

    Science.gov (United States)

    Ito, Yusuke; Fukami, Tatsuki; Yokoi, Tsuyoshi; Nakajima, Miki

    2014-12-01

    Probenecid, a widely used uricosuric agent, is mainly metabolized to probenecid acyl glucuronide (PRAG), which is considered a causal substance of severe allergic or anaphylactoid reactions. PRAG can be hydrolyzed (deglucuronidated) to probenecid. The purpose of this study was to identify enzymes responsible for probenecid acyl glucuronidation and PRAG deglucuronidation in human livers and to examine the effect of deglucuronidation in PRAG formation. In human liver homogenates (HLHs), the intrinsic clearance (CLint) of PRAG deglucuronidation was much greater (497-fold) than that of probenecid acyl glucuronidation. Evaluation of PRAG formation by recombinant UDP-glucuronosyltransferase (UGT) isoforms and an inhibition study using HLHs as an enzyme source demonstrated that multiple UGT isoforms, including UGT1A1, UGT1A9, and UGT2B7, catalyzed probenecid acyl glucuronidation. We found that recombinant α/β hydrolase domain containing 10 (ABHD10) substantially catalyzed PRAG deglucuronidation activity, whereas carboxylesterases did not. Similar inhibitory patterns by chemicals between HLHs and recombinant ABHD10 supported the major contribution of ABHD10 to PRAG deglucuronidation in human liver. Interestingly, it was demonstrated that the CLint value of probenecid acyl glucuronidation in HLHs was increased by 1.7-fold in the presence of phenylmethylsulfonyl fluoride, which potently inhibited ABHD10 activity. In conclusion, we found that PRAG deglucuronidation catalyzed by ABHD10 suppressively regulates PRAG formation via multiple UGT enzymes in human liver. The balance of activities by these enzymes is important for the formation of PRAG, which may be associated with the adverse reactions observed after probenecid administration. Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.

  3. Role of inflammation and infection in the pathogenesis of human acute liver failure: Clinical implications for monitoring and therapy.

    Science.gov (United States)

    Donnelly, Mhairi C; Hayes, Peter C; Simpson, Kenneth J

    2016-07-14

    Acute liver failure is a rare and devastating clinical condition. At present, emergency liver transplantation is the only life-saving therapy in advanced cases, yet the feasibility of transplantation is affected by the presence of systemic inflammation, infection and resultant multi-organ failure. The importance of immune dysregulation and acquisition of infection in the pathogenesis of acute liver failure and its associated complications is now recognised. In this review we discuss current thinking regarding the role of infection and inflammation in the pathogenesis of and outcome in human acute liver failure, the implications for the management of such patients and suggest directions for future research.

  4. Partial isolation and identification of hepatic stimulator substance mRNA extracted from human fetal liver

    Science.gov (United States)

    Yang, Xiao-Ming; Xie, Ling; Xing, Gui-Chun; Wu, Zu-Ze; He, Fu-Chu

    1998-01-01

    AIM: To partially isolate and identify hepatic stimulator substance mRNA from human fetal liver tissues. METHODS: The poly (A) mRNA was extracted from human fetal liver tissues of 4-5 month gestation, fractionated by size on sucrose gradient centrifugation, translated into protein from each fraction in vitro and then its products were tested for HSS activity. RESULTS: Twenty-two 500 μg total RNA was obtained from human fetal liver tissues and pooled. mRNA of 420 μg was yielded, processed by oligo (dT)-cellulose column chromatography, then was size-fractionated by ultracentrifution on a continuous sucrose density gradient (5%-25%), and separated into 18 fractions. Translated products of mRNA in fraction 8 and 9 could produce a two-fold increase in the incorporation of 3H-TdR into DNA of SMMC-7721 hepatoma cells and in a heat resistant and organ-specific way. CONCLUSION: The partially purified HSS mRNA was obtained and this would facilitate the cloning of HSS using expression vectors. PMID:11819247

  5. Phenotype Determines Nanoparticle Uptake by Human Macrophages from Liver and Blood.

    Science.gov (United States)

    MacParland, Sonya A; Tsoi, Kim M; Ouyang, Ben; Ma, Xue-Zhong; Manuel, Justin; Fawaz, Ali; Ostrowski, Mario A; Alman, Benjamin A; Zilman, Anton; Chan, Warren C W; McGilvray, Ian D

    2017-03-28

    A significant challenge to delivering therapeutic doses of nanoparticles to targeted disease sites is the fact that most nanoparticles become trapped in the liver. Liver-resident macrophages, or Kupffer cells, are key cells in the hepatic sequestration of nanoparticles. However, the precise role that the macrophage phenotype plays in nanoparticle uptake is unknown. Here, we show that the human macrophage phenotype modulates hard nanoparticle uptake. Using gold nanoparticles, we examined uptake by human monocyte-derived macrophages that had been driven to a "regulatory" M2 phenotype or an "inflammatory" M1 phenotype and found that M2-type macrophages preferentially take up nanoparticles, with a clear hierarchy among the subtypes (M2c > M2 > M2a > M2b > M1). We also found that stimuli such as LPS/IFN-γ rather than with more "regulatory" stimuli such as TGF-β/IL-10 reduce per cell macrophage nanoparticle uptake by an average of 40%. Primary human Kupffer cells were found to display heterogeneous expression of M1 and M2 markers, and Kupffer cells expressing higher levels of M2 markers (CD163) take up significantly more nanoparticles than Kupffer cells expressing lower levels of surface CD163. Our results demonstrate that hepatic inflammatory microenvironments should be considered when studying liver sequestration of nanoparticles, and that modifying the hepatic microenvironment might offer a tool for enhancing or decreasing this sequestration. Our findings also suggest that models examining the nanoparticle/macrophage interaction should include studies with primary tissue macrophages.

  6. Allicin induces p53-mediated autophagy in Hep G2 human liver cancer cells.

    Science.gov (United States)

    Chu, Yung-Lin; Ho, Chi-Tang; Chung, Jing-Gung; Rajasekaran, Raghu; Sheen, Lee-Yan

    2012-08-29

    Garlic has been used throughout history for both culinary and medicinal purpose. Allicin is a major component of crushed garlic. Although it is sensitive to heat and light and easily metabolized into various compounds such as diallyl disulfide, diallyl trisulfide, and diallyl sulfide, allicin is still a major bioactive compound of crushed garlic. The mortality of hepatocellular carcinoma is quite high and ranks among the top 10 cancer-related deaths in Taiwan. Although numerous studies have shown the cancer-preventive properties of garlic and its components, there is no study on the effect of allicin on the growth of human liver cancer cells. In this study, we focused on allicin-induced autophagic cell death in human liver cancer Hep G2 cells. Our results indicated that allicin induced p53-mediated autophagy and inhibited the viability of human hepatocellular carcinoma cell lines. Using Western blotting, we observed that allicin decreased the level of cytoplasmic p53, the PI3K/mTOR signaling pathway, and the level of Bcl-2 and increased the expression of AMPK/TSC2 and Beclin-1 signaling pathways in Hep G2 cells. In addition, the colocalization of LC3-II with MitoTracker-Red (labeling mitochondria), resulting in allicin-induced degradation of mitochondria, could be observed by confocal laser microscopy. In conclusion, allicin of garlic shows great potential as a novel chemopreventive agent for the prevention of liver cancer.

  7. The mechanism of microsomal azoreduction: predictions based on electronic aspects of structure-activity relationships.

    Science.gov (United States)

    Zbaida, S

    1995-01-01

    ferrous cytochrome P450 substrate complex is rate limiting for the overall reaction. Structure-activity relationships of azo compounds depend on (1) the electron transport component and (2) the oxidation-reduction potential of the dye, which determines its ability to accept electrons from cytochrome P-450. Nesnow et al. examined a group of 36 aryl azo dyes for their ability to be reduced by rat liver microsomal azoreductase.(ABSTRACT TRUNCATED AT 400 WORDS)

  8. Blood concentrations of acrylonitrile in humans after oral administration extrapolated from in vivo rat pharmacokinetics, in vitro human metabolism, and physiologically based pharmacokinetic modeling.

    Science.gov (United States)

    Takano, Ryohji; Murayama, Norie; Horiuchi, Kana; Kitajima, Masato; Kumamoto, Masatoshi; Shono, Fumiaki; Yamazaki, Hiroshi

    2010-11-01

    The present study defined a simplified physiologically based pharmacokinetic (PBPK) model for acrylonitrile in humans based on in vitro metabolic parameters determined using relevant liver microsomes, coefficients derived in silico, physiological parameters derived from the literature, and a prior previously developed PBPK model in rats. The model basically consists of a chemical absorption compartment, a metabolizing compartment, and a central compartment for acrylonitrile. Evaluation of a previous rat model was performed by comparisons with experimental pharmacokinetic values from blood and urine obtained from rats in vivo after oral treatment with acrylonitrile (30 mg/kg, a no-observed-adverse-effect level) for 14 days. Elimination rates of acrylonitrile in vitro were established using data from rat liver microsomes and from pooled human liver microsomes. Acrylonitrile was expected to be absorbed and cleared rapidly from the body in silico, as was the case for rats confirmed experimentally in vivo with repeated low-dose treatments. These results indicate that the simplified PBPK model for acrylonitrile is useful for a forward dosimetry approach in humans. This model may also be useful for simulating blood concentrations of other related compounds resulting from exposure to low chemical doses. Copyright © 2010 Elsevier Inc. All rights reserved.

  9. Hepatic Differentiation of Human Induced Pluripotent Stem Cells in a Perfused 3D Porous Polymer Scaffold for Liver Tissue Engineering

    DEFF Research Database (Denmark)

    Hemmingsen, Mette; Muhammad, Haseena Bashir; Mohanty, Soumyaranjan

    to limitations of primary hepatocytes regarding availability and maintenance of functionality, stem cells and especially human induced pluripotent stem cells (hIPS cells) are an attractive cell source for liver tissue engineering. The aim of this part of NanoBio4Trans is to optimize culture and hepatic......A huge shortage of liver organs for transplantation has motivated the research field of tissue engineering to develop bioartificial liver tissue and even a whole liver. The goal of NanoBio4Trans is to create a vascularized bioartificial liver tissue, initially as a liver-support system. Due...... differentiation of hIPS-derived definitive endoderm (DE) cells in a 3D porous polymer scaffold built-in a perfusable bioreactor. The use of a microfluidic bioreactor array enables the culture of 16 independent tissues in one experimental run and thereby an optimization study to be performed....

  10. Protocol for Isolation of Primary Human Hepatocytes and Corresponding Major Populations of Non-parenchymal Liver Cells.

    Science.gov (United States)

    Kegel, Victoria; Deharde, Daniela; Pfeiffer, Elisa; Zeilinger, Katrin; Seehofer, Daniel; Damm, Georg

    2016-03-30

    Beside parenchymal hepatocytes, the liver consists of non-parenchymal cells (NPC) namely Kupffer cells (KC), liver endothelial cells (LEC) and hepatic Stellate cells (HSC). Two-dimensional (2D) culture of primary human hepatocyte (PHH) is still considered as the "gold standard" for in vitro testing of drug metabolism and hepatotoxicity. It is well-known that the 2D monoculture of PHH suffers from dedifferentiation and loss of function. Recently it was shown that hepatic NPC play a central role in liver (patho-) physiology and the maintenance of PHH functions. Current research focuses on the reconstruction of in vivo tissue architecture by 3D- and co-culture models to overcome the limitations of 2D monocultures. Previously we published a method to isolate human liver cells and investigated the suitability of these cells for their use in cell cultures in Experimental Biology and Medicine(1). Based on the broad interest in this technique the aim of this article was to provide a more detailed protocol for the liver cell isolation process including a video, which will allow an easy reproduction of this technique. Human liver cells were isolated from human liver tissue samples of surgical interventions by a two-step EGTA/collagenase P perfusion technique. PHH were separated from the NPC by an initial centrifugation at 50 x g. Density gradient centrifugation steps were used for removal of dead cells. Individual liver cell populations were isolated from the enriched NPC fraction using specific cell properties and cell sorting procedures. Beside the PHH isolation we were able to separate KC, LEC and HSC for further cultivation. Taken together, the presented protocol allows the isolation of PHH and NPC in high quality and quantity from one donor tissue sample. The access to purified liver cell populations could allow the creation of in vivo like human liver models.

  11. "Non alcoholic fatty liver disease and eNOS dysfunction in humans".

    Science.gov (United States)

    Persico, Marcello; Masarone, Mario; Damato, Antonio; Ambrosio, Mariateresa; Federico, Alessandro; Rosato, Valerio; Bucci, Tommaso; Carrizzo, Albino; Vecchione, Carmine

    2017-03-07

    NAFLD is associated to Insulin Resistance (IR). IR is responsible for Endothelial Dysfunction (ED) through the impairment of eNOS function. Although eNOS derangement has been demonstrated in experimental models, no studies have directly shown that eNOS dysfunction is associated with NAFLD in humans. The aim of this study is to investigate eNOS function in NAFLD patients. Fifty-four NAFLD patients were consecutively enrolled. All patients underwent clinical and laboratory evaluation and liver biopsy. Patients were divided into two groups by the presence of NAFL or NASH. We measured vascular reactivity induced by patients' platelets on isolated mice aorta rings. Immunoblot assays for platelet-derived phosphorylated-eNOS (p-eNOS) and immunohistochemistry for hepatic p-eNOS have been performed to evaluate eNOS function in platelets and liver specimens. Flow-mediated-dilation (FMD) was also performed. Data were compared with healthy controls. Twenty-one (38, 8%) patients had NAFL and 33 (61, 7%) NASH. No differences were found between groups and controls except for HOMA and insulin (p liver (p liver tissue didn't match with FMD.

  12. [Acute liver failure due to human herpesvirus 6 in an infant].

    Science.gov (United States)

    Tronconi, G M; Mariani, B; Pajno, R; Fomasi, M; Cococcioni, L; Biffi, V; Bove, M; Corsin, P; Garbetta, G; Barera, G

    2012-01-01

    We report a case of a 4-months infant with fever in the absence of other specific symptoms that has rapidly and unexpectedly developed acute liver failure (ALF) with coagulopathy and complicated with bone marrow failure without encephalopathy. The main viral infection agents (hepatitis virus A, B, C, Citomegalovirus, Ebstain Barr virus, Parvovirus B19, Adenovirus), drug-induced hepatotoxicity and metabolic disorders associated to ALF were excluded. Quantitative determination of Human Herpesvirus 6 (HHV6) genome was positive with a significant number of copies for mL. A favorable evolution of the clinical symptoms and a progressive hematochemical resolution were obtained. Plasma and Vitamin K were administrated as a support therapy for treating coagulopathy. The present case report and the cases' review from the literature, evidence the importance of always including screening for HHV6 infection in the diagnostic approach to acute onset of liver failure. HHV6 is a common virus in the pediatric population with a greater number of cases of fulminant viral non-A, non-B, non-C hepatitis in immunocompetent patients due to this virus: these forms have often a high mortality rate and maybe necessitate liver transplantation; for this reason correct etiological agent identification is mandatory for the prognosis and it has to be based on the quantitative search of the virus's genome. Pathogenesis of liver-induced damage associated to HHV6 remains unclear; however in vitro studies demonstrate the potential hepatotoxicity effects of this virus.

  13. Molecular Recognition of Human Liver Cancer Cells Using DNA Aptamers Generated via Cell-SELEX.

    Directory of Open Access Journals (Sweden)

    Jiehua Xu

    Full Text Available Most clinical cases of liver cancer cannot be diagnosed until they have evolved to an advanced stage, thus resulting in high mortality. It is well recognized that the implementation of early detection methods and the development of targeted therapies for liver cancer are essential to reducing the high mortality rates associated with this disease. To achieve these goals, molecular probes capable of recognizing liver cancer cell-specific targets are needed. Here we describe a panel of aptamers able to distinguish hepatocarcinoma from normal liver cells. The aptamers, which were selected by cell-based SELEX (Systematic Evolution of Ligands by Exponential Enrichment, have Kd values in the range of 64-349 nM toward the target human hepatoma cell HepG2, and also recognize ovarian cancer cells and lung adenocarcinoma. The proteinase treatment experiment indicated that all aptamers could recognize target HepG2 cells through surface proteins. This outcome suggested that these aptamers could be used as potential probes for further research in cancer studies, such as developing early detection assays, targeted therapies, and imaging agents, as well as for the investigation of common membrane proteins in these distinguishable cancers.

  14. Anticarcinogenic effects of glycoalkaloids from potatoes against human cervical, liver, lymphoma, and stomach cancer cells.

    Science.gov (United States)

    Friedman, Mendel; Lee, Kap-Rang; Kim, Hyun-Jeong; Lee, In-Seon; Kozukue, Nobuyuke

    2005-07-27

    Methods were devised for the isolation of large amounts of pure alpha-chaconine and alpha-solanine from Dejima potatoes and for the extraction and analysis of total glycoalkaloids from five fresh potato varieties (Dejima, Jowon, Sumi, Toya, and Vora Valley). These compounds were then evaluated in experiments using a tetrazolium microculture (MTT) assay to assess the anticarcinogenic effects of (a) the isolated pure glycoalkaloids separately, (b) artificial mixtures of the two glycoalkaloids, and (c) the total glycoalkaloids isolated from each of the five potato varieties. All samples tested reduced the numbers of the following human cell lines: cervical (HeLa), liver (HepG2), lymphoma (U937), stomach (AGS and KATO III) cancer cells and normal liver (Chang) cells. The results show that (a) the effects of the glycoalkaloids were concentration dependent in the range of 0.1-10 mug/mL (0.117-11.7 nmol/mL); (b) alpha-chaconine was more active than was alpha-solanine; (c) some mixtures exhibited synergistic effects, whereas other produced additive ones; (d) the different cancer cells varied in their susceptibilities to destruction; and (e) the destruction of normal liver cells was generally lower than that of cancer liver cells. The decreases in cell populations were also observed visually by reversed-phase microscopy. The results complement related observations on the anticarcinogenic potential of food ingredients.

  15. Acute liver failure due to Human Herpesvirus 6 in an infant

    Directory of Open Access Journals (Sweden)

    G.M. Tronconi

    2012-10-01

    Full Text Available We report a case of a 4-months infant with fever in the absence of other specific symptoms that has rapidly and unexpectedly developed acute liver failure (ALF with coagulopathy and complicated with bone marrow failure without encephalopathy. The main viral infection agents (hepatitis virus A, B, C, Citomegalovirus, Ebstain Barr virus, Parvovirus B19, Adenovirus, drug-induced hepatotoxicity and metabolic disorders associated to ALF were excluded. Quantitative determination of Human Herpesvirus 6 (HHV6 genome was positive with a significant number of copies for mL. A favorable evolution of the clinical symptoms and a progressive hematochemical resolution were obtained. Plasma and Vitamin K were administrated as a support therapy for treating coagulopathy. The present case report and the cases’ review from the literature, evidence the importance of always including screening for HHV6 infection in the diagnostic approach to acute onset of liver failure. HHV6 is a common virus in the pediatric population with a greater number of cases of fulminant viral non-A, non-B, non-C hepatitis in immunocompetent patients due to this virus: these forms have often a high mortality rate and maybe necessitate liver transplantation; for this reason correct etiological agent identification is mandatory for the prognosis and it has to be based on the quantitative search of the virus’s genome. Pathogenesis of liver-induced damage associated to HHV6 remains unclear; however in vitro studies demonstrate the potential hepatotoxicity effects of this virus.

  16. High frequency of Human Cytomegalovirus DNA in the Liver of Infants with Extrahepatic Neonatal Cholestasis

    Directory of Open Access Journals (Sweden)

    Escanhoela Cecília AF

    2005-12-01

    Full Text Available Abstract Background Biliary atresia (BA is the most severe hepatic disorder in newborns and its etiopathogenesis remains unknown. Viral involvement has been proposed, including the human cytomegalovirus (HCMV. The aims of the study were to use the polymerase chain reaction (PCR to screen the liver tissue of infants with extrahepatic cholestasis for HCMV and to correlate the results with serological antibodies against HCMV and histological findings. Methods A retrospective study in a tertiary care setting included 35 patients (31 BA, 1 BA associated with a choledochal cyst, 2 congenital stenosis of the distal common bile duct and 1 hepatic cyst. HCMV serology was determined by ELISA. Liver and porta hepatis were examined histologically. Liver samples from infants and a control group were screened for HCMV DNA. Results Twelve patients had HCMV negative serology, 9 were positive for IgG antibodies and 14 were positive for IgG and IgM. Nine liver and seven porta hepatis samples were positive for HCMV DNA but none of the control group were positive (general frequency of positivity was 34.3% – 12/35. There was no correlation between HCMV positivity by PCR and the histological findings. The accuracy of serology for detecting HCMV antibodies was low. Conclusion These results indicate an elevated frequency of HCMV in pediatric patients with extrahepatic neonatal cholestasis. They also show the low accuracy of serological tests for detecting active HCMV infection and the lack of correlation between HCMV positivity by PCR and the histopathological changes.

  17. Murine Cyp3a knockout chimeric mice with humanized liver: prediction of the metabolic profile of nefazodone in humans.

    Science.gov (United States)

    Nakada, Naoyuki; Kawamura, Akio; Kamimura, Hidetaka; Sato, Koya; Kazuki, Yasuhiro; Kakuni, Masakazu; Ohbuchi, Masato; Kato, Kota; Tateno, Chise; Oshimura, Mitsuo; Usui, Takashi

    2016-01-01

    Chimeric mice with humanized livers (PXB mice) are used to investigate the metabolism and pharmacokinetics of drugs in humans. However, residual murine enzymatic activities derived from the liver and the presence of mouse small intestinal metabolism can hamper the prediction of human drug metabolism. Recently murine Cytochrome P450 3a gene knockout chimeric mice with humanized livers (Cyp3a KO CM) were developed. To evaluate the prediction of drug metabolism, nefazodone (NEF) was administered orally at 10 mg/kg to the following mouse strains: Cyp3a KO CM, murine Cyp3a gene knockout (Cyp3a KO), PXB and severe combined immunodeficiency (SCID) mice. Liquid chromatography-mass spectrometry was used for metabolic profiling of plasma, urine and bile. The prediction of human metabolite levels such as hydroxy nefazodone (OH-NEF), triazoledione form (TD), m-chlorophenylpiperazine and dealkyl metabolites in Cyp3a KO CM was superior to that in Cyp3a KO, PXB or SCID mice. Further, clinical exposure levels of NEF, OH-NEF and TD were reproduced in Cyp3a KO CM. In contrast, NEF was rapidly metabolized to TD in both PXB and SCID mice but not in Cyp3a KO mice, suggesting that murine CYP3A is involved in the elimination of NEF in these mice. These findings demonstrate that the metabolic profile of NEF in Cyp3a KO CM differs qualitatively and quantitatively from that in PXB mice due to the higher metabolic rate of NEF and its metabolites via murine CYP3A. Therefore Cyp3a KO CM might be useful in predicting the metabolic profiles of drug candidates in humans. Copyright © 2016 John Wiley & Sons, Ltd.

  18. Mechanism of secretion of biliary lipids. I. Role of bile canalicular and microsomal membranes in the synthesis and transport of biliary lecithin and cholesterol.

    Science.gov (United States)

    Gregory, D H; Vlahcevic, Z R; Schatzki, P; Swell, L

    1975-01-01

    The role of bile canalicular and microsomal membranes in the synthesis and transport of biliary lipids was investigated by using the isolated perfused rat liver model. Labeled lecithin precursors ((3H)-palmitic acid, (14C)linoleic acid, (3H)choline, and 32PO4) and a cholesterol precursor ((3H)mevalonic acid) were administered with and without sodium taurocholate. The incorporation pattern of these labeled precursors into linoleyl and arachidonyl lecithins and cholesterol fractions of microsomes, bile canaliculi, and bile were examined at 30-min intervals up to 90 min. Marker enzymes and electron microscopy indicated that isolated subfractions of plasma membranes were enriched with bile canaliculi (less than 10 percent microsomal contamination). Taurocholate significantly stimulated the incorporation of 32PO4, (3H)choline, (3H)palmitic acid, and (14C)linoleic acid into linoleyl and arachidonyl lecithin with parallel incorporation curves for microsomal and bile canalicular membranes throughout the 90-min study period. During the 30-60-min period, however, these same lecithin fractions in bile significantly exceeded the specific activity of the membrane lecithins. The enzyme CDP-choline diglyceride transferase was virtually absent from canaliculi relative to microsomes, indicating that canaliculi lack the capacity for de novo lecithin synthesis. Incorporation of (3H)mevalonic acid into membranous and biliary cholesterol followed a pattern similar to that for lecithin. These data provide evidence that (a) biliary lecithin and cholesterol are derived from a microsomal subpool regulated by the flux of enterohepatic bile acids, (b) the role of the bile canalicular membranes with respect to biliary lipids is primarily transport rather than synthesis, and (c) lecithin and cholesterol are transported together from microsomes to bile. The findings are consistent with the existence of a cytoplasmic lipid complex within the hepatocyte which is actively involved in the

  19. Purification of nonspecific lipid transfer protein (sterol carrier protein 2) from human liver and its deficiency in livers from patients with cerebro-hepato-renal (Zellweger) syndrome

    NARCIS (Netherlands)

    Amerongen, A. van; Helms, J.B.; Krift, T.P. van der; Schutgens, R.B.H.; Wirtz, K.W.A.

    1987-01-01

    The nonspecific lipid transfer protein (i.e., sterol carrier protein 2) from human liver was purified to homogeneity using ammonium sulfate precipitation, CM-cellulose chromatography, molecular sieve chromatography and fast protein liquid chromatography. Its amino acid composition was determined and

  20. Liver enzyme-mediated oxidation of Echinacea purpurea alkylamides: production of novel metabolites and changes in immunomodulatory activity.

    Science.gov (United States)

    Cech, Nadja B; Tutor, Katrina; Doty, Bethany A; Spelman, Kevin; Sasagawa, Masa; Raner, Gregory M; Wenner, Cynthia A

    2006-12-01

    The medicinal plant Echinacea is widely used to treat upper respiratory infections and is reported to stimulate the human immune system. A major constituent class of Echinacea, the alkylamides, has immunomodulatory effects. Recent studies show that alkylamides are oxidized by cytochrome P450 enzymes, but the immunomodulatory activity of these products is unknown. The objectives of this study were to characterize the products formed by incubation of an Echinacea extract and an isolated alkylamide with human liver microsomes, and to evaluate the influence of Echinacea alkylamides and metabolites on cytokine production by Jurkat human T cells. A novel class of carboxylic acid alkylamide metabolites was identified and shown to be the major constituents present after 2-h incubation of alkylamides with human liver microsomes. Echinacea alkylamides suppressed IL-2 secretion by stimulated T cells, and this effect was significantly lessened upon oxidation of the alkylamides to carboxylic acids and hydroxylated metabolites. These findings highlight the importance of considering the influence of liver enzyme metabolism when evaluating the immunomodulatory effects of alkylamides.

  1. Mechanisms and regulation of vitamin C uptake: studies of the hSVCT systems in human liver epithelial cells

    National Research Council Canada - National Science Library

    Jack C. Reidling; Veedamali S. Subramanian; Tamara Dahhan; Mohammed Sadat; Hamid M. Said

    2008-01-01

    .... Although the human liver plays a pivotal role in regulating and maintaining vitamin C homeostasis, vitamin C transport physiology and regulation of the hSVCT systems in this organ have not been well defined...

  2. Establishment of a general NAFLD scoring system for rodent models and comparison to human liver pathology.

    Science.gov (United States)

    Liang, Wen; Menke, Aswin L; Driessen, Ann; Koek, Ger H; Lindeman, Jan H; Stoop, Reinout; Havekes, Louis M; Kleemann, Robert; van den Hoek, Anita M

    2014-01-01

    The recently developed histological scoring system for non-alcoholic fatty liver disease (NAFLD) by the NASH Clinical Research Network (NASH-CRN) has been widely used in clinical settings, but is increasingly employed in preclinical research as well. However, it has not been systematically analyzed whether the human scoring system can directly be converted to preclinical rodent models. To analyze this, we systematically compared human NAFLD liver pathology, using human liver biopsies, with liver pathology of several NAFLD mouse models. Based upon the features pertaining to mouse NAFLD, we aimed at establishing a modified generic scoring system that is applicable to broad spectrum of rodent models. The histopathology of NAFLD was analyzed in several different mouse models of NAFLD to define generic criteria for histological assessment (preclinical scoring system). For validation of this scoring system, 36 slides of mouse livers, covering the whole spectrum of NAFLD, were blindly analyzed by ten observers. Additionally, the livers were blindly scored by one observer during two separate assessments longer than 3 months apart. The criteria macrovesicular steatosis, microvesicular steatosis, hepatocellular hypertrophy, inflammation and fibrosis were generally applicable to rodent NAFLD. The inter-observer reproducibility (evaluated using the Intraclass Correlation Coefficient) between the ten observers was high for the analysis of macrovesicular steatosis and microvesicular steatosis (ICC = 0.784 and 0.776, all p<0.001, respectively) and moderate for the analysis of hypertrophy and inflammation (ICC = 0.685 and 0.650, all p<0.001, respectively). The intra-observer reproducibility between the different observations of one observer was high for the analysis of macrovesicular steatosis, microvesicular steatosis and hypertrophy (ICC = 0.871, 0.871 and 0.896, all p<0.001, respectively) and very high for the analysis of inflammation (ICC = 0.931, p<0.001). We

  3. Low dose perfluorooctanoate exposure promotes cell proliferation in a human non-tumor liver cell line

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Hongxia; Cui, Ruina [Key Laboratory of Animal Ecology and Conservation Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101 (China); Guo, Xuejiang [State Key Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing 210029 (China); Hu, Jiayue [Key Laboratory of Animal Ecology and Conservation Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101 (China); Dai, Jiayin, E-mail: daijy@ioz.ac.cn [Key Laboratory of Animal Ecology and Conservation Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101 (China)

    2016-08-05

    Highlights: • Differential expression of proteins induced by PFOA in HL-7702 was identified. • Most of the differentially expressed proteins are related to cell proliferation. • A low dose of PFOA stimulates HL-7702 cell proliferation. • A high dose of PFOA inhibits HL-7702 cell proliferation. - Abstract: Perfluorooctanoate (PFOA) is a well-known persistent organic pollutant widely found in the environment, wildlife and humans. Medical surveillance and experimental studies have investigated the potential effects of PFOA on human livers, but the hepatotoxicity of PFOA on humans and its underlying mechanism remain to be clarified. We exposed a human liver cell line (HL-7702) to 50 μM PFOA for 48 h and 96 h, and identified 111 significantly differentially expressed proteins by iTRAQ analysis. A total of 46 proteins were related to cell proliferation and apoptosis. Through further analysis of the cell cycle, apoptosis and their related proteins, we found that low doses of PFOA (50–100 μM) promoted cell proliferation and numbers by promoting cells from the G1 to S phases, whereas high doses of PFOA (200–400 μM) led to reduced HL-7702 cell numbers compared with that of the control mainly due to cell cycle arrest in the G0/G1 phase. To our knowledge, this is the first report on the promotion of cell cycle progression in human cells following PFOA exposure.

  4. Oxidation of an organosulfur xenobiotic by microsomes from soybean cotyledons.

    Science.gov (United States)

    Blee, E; Durst, F

    1986-03-28

    Methiocarb, an aromatic-alkyl sulfide insecticide was enzymatically oxidized into its sulfoxide by microsomes from soybean cotyledons. No further oxidation into sulfone was detected. Distribution of the sulfoxidase activity was studied in soybean seedlings and found maximal in cotyledons. Subcellular fractionation of cotyledons homogenates indicated that the activity was almost entirely associated with the microsomal fraction. Sulfoxidation of methiocarb did not require cofactors such as NAD(P)H. Nevertheless, the sulfoxidase did not act as a peroxidase.

  5. Liver Immunology

    Science.gov (United States)

    Bogdanos, Dimitrios P.; Gao, Bin; Gershwin, M. Eric

    2014-01-01

    The liver is the largest organ in the body and is generally regarded by non-immunologists as not having lymphoid function. However, such is far from accurate. This review highlights the importance of the liver as a lymphoid organ. Firstly, we discuss experimental data surrounding the role of liver as a lymphoid organ. The liver facilitates a tolerance rather than immunoreactivity, which protects the host from antigenic overload of dietary components and drugs derived from the gut and is also instrumental to fetal immune tolerance. Loss of liver tolerance leads to autoaggressive phenomena which if are not controlled by regulatory lymphoid populations may lead to the induction of autoimmune liver diseases. Liver-related lymphoid subpopulations also act as critical antigen-presenting cells. The study of the immunological properties of liver and delineation of the microenvironment of the intrahepatic milieu in normal and diseased livers provides a platform to understand the hierarchy of a series of detrimental events which lead to immune-mediated destruction of the liver and the rejection of liver allografts. The majority of emphasis within this review will be on the normal mononuclear cell composition of the liver. However, within this context, we will discus select, but not all, immune mediated liver disease and attempt to place these data in the context of human autoimmunity. PMID:23720323

  6. Effects of hypophysectomy and GH administration on bovine and human GH binding to rat liver membranes.

    Science.gov (United States)

    Messina, J L; Edén, S; Kostyo, J L

    1985-07-01

    Experiments were conducted to investigate the specific binding of highly purified bovine and human growth hormones (bGH and hGH) to purified liver plasma membranes of male rats at various times after hypophysectomy and after the acute intravenous administration of bGH. Liver membranes prepared from hypophysectomized male rats showed a two- to threefold increase in the specific binding of either [125I]iodo-bGH or [125I]iodo-hGH, when compared with membranes prepared from the livers of age-matched normal male rats. The increase in GH binding was apparent within 3 days after hypophysectomy and persisted for a number of weeks after the operation. The increase in GH binding produced by hypophysectomy appeared to be due to an increase in the number of binding sites present on the membranes. The intravenous injection of 200 micrograms of bGH into hypophysectomized male rats 5-60 min before they were killed markedly reduced the ability of liver membranes prepared from these animals to bind [125I]iodo-bGH specifically. This decrease in GH binding seen after the injection of bGH may have been due to the development of a slowly dissociating hormone-binding site complex, which thereby reduced the number of available binding sites. This conclusion is supported by the finding that bGH, which is bound in vitro to isolated liver membranes, dissociates slowly and incompletely in the presence of an excess of unlabeled hormone. Moreover, the degree to which the bound hormone can dissociate appears to depend on the length of time that association is allowed to occur.(ABSTRACT TRUNCATED AT 250 WORDS)

  7. Unique cell type-specific junctional complexes in vascular endothelium of human and rat liver sinusoids.

    Directory of Open Access Journals (Sweden)

    Cyrill Géraud

    Full Text Available Liver sinusoidal endothelium is strategically positioned to control access of fluids, macromolecules and cells to the liver parenchyma and to serve clearance functions upstream of the hepatocytes. While clearance of macromolecular debris from the peripheral blood is performed by liver sinusoidal endothelial cells (LSECs using a delicate endocytic receptor system featuring stabilin-1 and -2, the mannose receptor and CD32b, vascular permeability and cell trafficking are controlled by transcellular pores, i.e. the fenestrae, and by intercellular junctional complexes. In contrast to blood vascular and lymphatic endothelial cells in other organs, the junctional complexes of LSECs have not yet been consistently characterized in molecular terms. In a comprehensive analysis, we here show that LSECs express the typical proteins found in endothelial adherens junctions (AJ, i.e. VE-cadherin as well as α-, β-, p120-catenin and plakoglobin. Tight junction (TJ transmembrane proteins typical of endothelial cells, i.e. claudin-5 and occludin, were not expressed by rat LSECs while heterogenous immunreactivity for claudin-5 was detected in human LSECs. In contrast, junctional molecules preferentially associating with TJ such as JAM-A, B and C and zonula occludens proteins ZO-1 and ZO-2 were readily detected in LSECs. Remarkably, among the JAMs JAM-C was considerably over-expressed in LSECs as compared to lung microvascular endothelial cells. In conclusion, we show here that LSECs form a special kind of mixed-type intercellular junctions characterized by co-occurrence of endothelial AJ proteins, and of ZO-1 and -2, and JAMs. The distinct molecular architecture of the intercellular junctional complexes of LSECs corroborates previous ultrastructural findings and provides the molecular basis for further analyses of the endothelial barrier function of liver sinusoids under pathologic conditions ranging from hepatic inflammation to formation of liver metastasis.

  8. Ovarian senescence increases liver fibrosis in humans and zebrafish with steatosis

    Directory of Open Access Journals (Sweden)

    Elena Turola

    2015-09-01

    Full Text Available Contrasting data exist on the effect of gender and menopause on the susceptibility, development and liver damage progression in non-alcoholic fatty liver disease (NAFLD. Our aim was to assess whether menopause is associated with the severity of liver fibrosis in individuals with NAFLD and to explore the issue of ovarian senescence in experimental liver steatosis in zebrafish. In 244 females and age-matched males with biopsy-proven NAFLD, we assessed anthropometric, biochemical and metabolic features, including menopausal status (self-reported; liver biopsy was scored according to ‘The Pathology Committee of the NASH Clinical Research Network’. Young and old male and female zebrafish were fed for 24 weeks with a high-calorie diet. Weekly body mass index (BMI, histopathological examination and quantitative real-time PCR analysis on genes involved in lipid metabolism, inflammation and fibrosis were performed. In the entire cohort, at multivariate logistic regression, male gender [odds ratio (OR: 1.408, 95% confidence interval (95% CI: 0.779-2.542, P=0.25] vs women at reproductive age was not associated with F2-F4 fibrosis, whereas a trend was observed for menopause (OR: 1.752, 95% CI: 0.956-3.208, P=0.06. In women, menopause (OR: 2.717, 95% CI: 1.020-7.237, P=0.04 was independently associated with F2-F4 fibrosis. Similarly, in overfed zebrafish, old female fish with failing ovarian function [as demonstrated by extremely low circulating estradiol levels (1.4±0.1 pg/µl and prevailing presence of atretic follicles in the ovaries] developed massive steatosis and substantial fibrosis (comparable with that occurring in males, whereas young female fish developed less steatosis and were totally protected from the development of fibrosis. Ovarian senescence significantly increases the risk of fibrosis severity both in humans with NAFLD and in zebrafish with experimental steatosis.

  9. Three-Dimensional Culture of Human Embryonic Stem Cell Derived Hepatic Endoderm and Its Role in Bioartificial Liver Construction

    Directory of Open Access Journals (Sweden)

    Ruchi Sharma

    2010-01-01

    Full Text Available The liver carries out a range of functions essential for bodily homeostasis. The impairment of liver functions has serious implications and is responsible for high rates of patient morbidity and mortality. Presently, liver transplantation remains the only effective treatment, but donor availability is a major limitation. Therefore, artificial and bioartificial liver devices have been developed to bridge patients to liver transplantation. Existing support devices improve hepatic encephalopathy to a certain extent; however their usage is associated with side effects. The major hindrance in the development of bioartificial liver devices and cellular therapies is the limited availability of human hepatocytes. Moreover, primary hepatocytes are difficult to maintain and lose hepatic identity and function over time even with sophisticated tissue culture media. To overcome this limitation, renewable cell sources are being explored. Human embryonic stem cells are one such cellular resource and have been shown to generate a reliable and reproducible supply of human hepatic endoderm. Therefore, the use of human embryonic stem cell-derived hepatic endoderm in combination with tissue engineering has the potential to pave the way for the development of novel bioartificial liver devices and predictive drug toxicity assays.

  10. Liver Afferents Contribute to Water Drinking-Induced Sympathetic Activation in Human Subjects: A Clinical Trial

    Science.gov (United States)

    May, Marcus; Gueler, Faikah; Barg-Hock, Hannelore; Heiringhoff, Karl-Heinz; Engeli, Stefan; Heusser, Karsten; Diedrich, André; Brandt, André; Strassburg, Christian P.; Tank, Jens; Sweep, Fred C. G. J.; Jordan, Jens

    2011-01-01

    Water drinking acutely increases sympathetic activity in human subjects. In animals, the response appears to be mediated through transient receptor potential channel TRPV4 activation on osmosensitive hepatic spinal afferents, described as osmopressor response. We hypothesized that hepatic denervation attenuates water drinking-induced sympathetic activation. We studied 20 liver transplant recipients (44±2.6 years, 1.2±0.1 years post transplant) as model of hepatic denervation and 20 kidney transplant recipients (43±2.6 years, 0.8±0.1 years post transplant) as immunosuppressive drug matched control group. Before and after 500 ml water ingestion, we obtained venous blood samples for catecholamine analysis. We also monitored brachial and finger blood pressure, ECG, and thoracic bioimpedance. Plasma norepinephrine concentration had changed by 0.01±0.07 nmol/l in liver and by 0.21±0.07 nmol/l in kidney transplant recipients (pwater drinking. While blood pressure and systemic vascular resistance increased in both groups, the responses tended to be attenuated in liver transplant recipients. Our findings support the idea that osmosensitive hepatic afferents are involved in water drinking-induced sympathetic activation in human subjects. Trial Registration ClinicalTrials.gov NCT01237431 PMID:22016786

  11. HCV-induced immune responses influence the development of operational tolerance after liver transplantation in humans.

    Science.gov (United States)

    Bohne, Felix; Londoño, María-Carlota; Benítez, Carlos; Miquel, Rosa; Martínez-Llordella, Marc; Russo, Carolina; Ortiz, Cecilia; Bonaccorsi-Riani, Eliano; Brander, Christian; Bauer, Tanja; Protzer, Ulrike; Jaeckel, Elmar; Taubert, Richard; Forns, Xavier; Navasa, Miquel; Berenguer, Marina; Rimola, Antoni; Lozano, Juan-José; Sánchez-Fueyo, Alberto

    2014-06-25

    Pathogen-induced immune responses prevent the establishment of transplantation tolerance in experimental animal models. Whether this occurs in humans as well remains unclear. The development of operational tolerance in liver transplant recipients with chronic hepatitis C virus (HCV) infection allows us to address this question. We conducted a clinical trial of immunosuppression withdrawal in HCV-infected adult liver recipients to elucidate (i) the mechanisms through which allograft tolerance can be established in the presence of an ongoing inflammatory response and (ii) whether anti-HCV heterologous immune responses influence this phenomenon. Of 34 enrolled liver recipients, drug withdrawal was successful in 17 patients (50%). Tolerance was associated with intrahepatic overexpression of type I interferon and immunoregulatory genes and with an expansion of exhausted PD1/CTLA4/2B4-positive HCV-specific circulating CD8(+) T cells. These findings were already present before immunosuppression was discontinued and were specific for HCV infection. In contrast, the magnitude of HCV-induced proinflammatory gene expression and the breadth of anti-HCV effector T cell responses did not influence drug withdrawal outcome. Our data suggest that in humans, persistent viral infections exert immunoregulatory effects that could contribute to the restraining of alloimmune responses, and do not necessarily preclude the development of allograft tolerance. Copyright © 2014, American Association for the Advancement of Science.

  12. Liver afferents contribute to water drinking-induced sympathetic activation in human subjects: a clinical trial.

    Directory of Open Access Journals (Sweden)

    Marcus May

    Full Text Available Water drinking acutely increases sympathetic activity in human subjects. In animals, the response appears to be mediated through transient receptor potential channel TRPV4 activation on osmosensitive hepatic spinal afferents, described as osmopressor response. We hypothesized that hepatic denervation attenuates water drinking-induced sympathetic activation. We studied 20 liver transplant recipients (44±2.6 years, 1.2±0.1 years post transplant as model of hepatic denervation and 20 kidney transplant recipients (43±2.6 years, 0.8±0.1 years post transplant as immunosuppressive drug matched control group. Before and after 500 ml water ingestion, we obtained venous blood samples for catecholamine analysis. We also monitored brachial and finger blood pressure, ECG, and thoracic bioimpedance. Plasma norepinephrine concentration had changed by 0.01±0.07 nmol/l in liver and by 0.21±0.07 nmol/l in kidney transplant recipients (p<0.05 between groups after 30-40 minutes of water drinking. While blood pressure and systemic vascular resistance increased in both groups, the responses tended to be attenuated in liver transplant recipients. Our findings support the idea that osmosensitive hepatic afferents are involved in water drinking-induced sympathetic activation in human subjects.ClinicalTrials.gov NCT01237431.

  13. [Vitamin D metabolism and signaling in human hepatocellular carcinoma and surrounding non-tumorous liver].

    Science.gov (United States)

    Horváth, Evelin; Balla, Bernadett; Kósa, János; Lakatos, Péter András; Lazáry, Áron; Németh, Dániel; Jozilan, Hasan; Somorácz, Áron; Korompay, Anna; Gyöngyösi, Benedek; Borka, Katalin; Kiss, András; Kupcsulik, Péter; Schaff, Zsuzsa; Szalay, Ferenc

    2016-11-01

    1,25-Dihydroxy vitamin D 3 mediates antitumor effects in hepatocellular carcinoma. We examined mRNA and protein expression differences in 1,25-Dihydroxy vitamin D 3 -inactivating CYP24A1, mRNA of activating CYP27B1 enzymes, and that of VDR between human hepatocellular carcinoma and surrounding non-tumorous liver. Snap-frozen tissues from 13 patients were studied for mRNA and protein expression of CYP24A1. Paraffin-embedded tissues from 36 patients were used to study mRNA of VDR and CYP27B1. mRNA expression was measured by RT-PCR, CYP24A1 protein was detected by immunohistochemistry. Expression of VDR and CYP27B1 was significantly lower in hepatocellular carcinoma compared with non-tumorous liver (pexpressed CYP24A1 mRNA, but neither of the non-tumorous liver. The gene activation was followed by CYP24A1 protein synthesis. The presence of CYP24A1 mRNA and the reduced expression of VDR and CYP27B1 mRNA in human hepatocellular carcinoma samples indicate decreased bioavailability of 1,25-Dihydroxy vitamin D 3 , providing an escape mechanism from the anti-tumor effect. Orv. Hetil., 2016, 157(48), 1910-1918.

  14. Investigation of the metabolites of the HIF stabilizer FG-4592 (roxadustat) in five different in vitro models and in a human doping control sample using high resolution mass spectrometry.

    Science.gov (United States)

    Hansson, Annelie; Thevis, Mario; Cox, Holly; Miller, Geoff; Eichner, Daniel; Bondesson, Ulf; Hedeland, Mikael

    2017-02-05

    FG-4592 is a hypoxia-inducible factor (HIF) stabilizer, which can increase the number of red blood cells in the body. It has not been approved by regulatory authorities, but is available for purchase on the Internet. Due to its ability to improve the oxygen transportation mechanism in the body, FG-4592 is of interest for doping control laboratories, but prior to this study, little information about its metabolism was available. In this study, the metabolism of FG-4592 was investigated in a human doping control sample and in five in vitro models: human hepatocytes and liver microsomes, equine liver microsomes and S9 fraction and the fungus Cunninghamella elegans. By using liquid chromatography coupled to a Q-TOF mass spectrometer operated in MS(E) and MSMS modes, twelve different metabolites were observed for FG-4592. One monohydroxylated metabolite was detected in both the human and equine liver microsome incubations. For the fungus Cunninghamella elegans eleven different metabolites were observed of which the identical monohydroxylated metabolite had the highest response. This rich metabolic profile and the higher levels of metabolites produced by Cunninghamella elegans demonstrates its usefulness as a metabolite producing medium. In the doping control urine sample, one metabolite, which was the result of a direct glucuronidation, was observed. No metabolites were detected in neither the human hepatocyte nor in the equine liver S9 fraction incubates. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. Liver and Muscle Contribute Differently to the Plasma Acylcarnitine Pool During Fasting and Exercise in Humans

    DEFF Research Database (Denmark)

    Xu, G.; Hansen, J S; Zhao, Jian-xin

    2016-01-01

    BACKGROUND: Plasma acylcarnitine levels are elevated by physiological conditions such as fasting and exercise but also in states of insulin resistance and obesity. AIM: To elucidate the contribution of liver and skeletal muscle to plasma acylcarnitines in the fasting state and during exercise...... in humans. METHODS: In 2 independent studies, young healthy males were fasted overnight and performed an acute bout of exercise to investigate either acylcarnitines in skeletal muscle biopsies and arterial-to-venous plasma differences over the exercising and resting leg (n = 9) or the flux over the hepato......:1-carnitines were released from the exercising leg and simultaneously; C6, C8, C10, C10:1, C14, and C16:1 were taken up by the hepato-splanchnic. CONCLUSION: These data provide novel insight to the organo-specific release/uptake of acylcarnitines. The liver is a major contributor to systemic short chain...

  16. GMP-grade human fetal liver-derived mesenchymal stem cells for clinical transplantation.

    Science.gov (United States)

    Larijani, Bagher; Aghayan, Hamid-Reza; Goodarzi, Parisa; Arjmand, Babak

    2015-01-01

    Stem cell therapy seems a promising avenue in regenerative medicine. Within various stem cells, mesenchymal stem cells have progressively used for cellular therapy. Because of the age-related decreasing in the frequency and differentiating capacity of adult MSCs, fetal tissues such as fetal liver, lung, pancreas, spleen, etc. have been introduced as an alternative source of MSCs for cellular therapy. On the other hand, using stem cells as advanced therapy medicinal products, must be performed in compliance with cGMP as a quality assurance system to ensure the safety, quality, and identity of cell products during translation from the basic stem cell sciences into clinical cell transplantation. In this chapter the authors have demonstrated the manufacturing of GMP-grade human fetal liver-derived mesenchymal stem cells.

  17. Viral load affects the immune response to HBV in mice with humanized immune system and liver.

    Science.gov (United States)

    Dusséaux, Mathilde; Masse-Ranson, Guillemette; Darche, Sylvie; Ahodantin, James; Li, Yan; Fiquet, Oriane; Beaumont, Elodie; Moreau, Pierrick; Rivière, Lise; Neuveut, Christine; Soussan, Patrick; Roingeard, Philippe; Kremsdorf, Dina; Di Santo, James P; Strick-Marchand, Helene

    2017-08-26

    Hepatitis B virus (HBV) infects hepatocytes, but the mechanisms of the immune response against the virus, and how it affects disease progression, are unclear. We performed studies with BALB/c Rag2(-/-)Il2rg(-/-)Sirpa(NOD)Alb-uPA(tg/tg) mice, stably engrafted with human hepatocytes (HUHEP) with or without a human immune system (HIS). HUHEP and HIS-HUHEP mice were given an intraperitoneal injection of HBV. Mononuclear cells were isolated from spleen and liver for analysis by flow cytometry. Liver was analyzed by immunohistochemistry and mRNA levels were measured by quantitative reverse transcription PCR. Plasma levels of HBV DNA was quantified by quantitative PCR, and antigen-specific antibodies were detected by immunocytochemistry of HBV transfected BHK-21 cells. Following HBV infection, a complete viral life cycle, with production of HBV DNA, hepatitis B e, core (HBc) and surface (HBs) antigens, and covalently closed circular DNA, was observed in HUHEP and HIS-HUHEP mice. HBV replicated unrestricted in HUHEP mice resulting in high viral titers without pathologic effects. In contrast, HBV-infected HIS-HUHEP mice developed chronic hepatitis with 10-fold lower titers and antigen-specific IgGs, (anti-HBs, anti-HBc), consistent with partial immune control. HBV-infected HIS-HUHEP livers contained infiltrating Kupffer cells, mature activated natural killer cells (CD69+), and PD-1+ effector memory T cells (CD45RO+). Reducing the viral inoculum resulted in more efficient immune control. Plasma from HBV-infected HIS-HUHEP mice had increased levels of inflammatory and immune-suppressive cytokines (C-X-C motif chemokine ligand 10 and interleukin 10), which correlated with populations of intrahepatic CD4+ T cells (CD45RO+PD-1+). Mice with high levels of viremia had HBV-infected liver progenitor cells. Giving the mice the nucleoside analogue entecavir reduced viral loads and decreased liver inflammation. In HIS-HUHEP mice, HBV infection completes a full life cycle and

  18. Liver Transplant

    Science.gov (United States)

    ... The Progression of Liver Disease Diagnosing Liver Disease – Liver Biopsy and Liver Function Tests Clinical Trials Liver Transplant ... The Progression of Liver Disease Diagnosing Liver Disease: Liver Biopsy and Liver Function Tests Clinical Trials Liver Transplant ...

  19. The liver isoform of carnitine palmitoyltransferase 1 is not targeted to the endoplasmic reticulum.

    OpenAIRE

    Broadway, Neil M; Pease, Richard J.; Birdsey, Graeme; Shayeghi, Majid; Turner, Nigel A; David Saggerson, E

    2003-01-01

    Liver microsomal fractions contain a malonyl-CoA-inhibitable carnitine acyltransferase (CAT) activity. It has been proposed [Fraser, Corstorphine, Price and Zammit (1999) FEBS Lett. 446, 69-74] that this microsomal CAT activity is due to the liver form of carnitine palmitoyltransferase 1 (L-CPT1) being targeted to the endoplasmic reticulum (ER) membrane as well as to mitochondria, possibly by an N-terminal signal sequence [Cohen, Guillerault, Girard and Prip-Buus (2001) J. Biol. Chem. 276, 54...

  20. Warm ischemic injury is reflected in the release of injury markers during cold preservation of the human liver.

    Directory of Open Access Journals (Sweden)

    Bote G Bruinsma

    Full Text Available Liver transplantation plays a pivotal role in the treatment of patients with end-stage liver disease. Despite excellent outcomes, the field is strained by a severe shortage of viable liver grafts. To meet high demands, attempts are made to increase the use of suboptimal livers by both pretransplant recovery and assessment of donor livers. Here we aim to assess hepatic injury in the measurement of routine markers in the post-ischemic flush effluent of discarded human liver with a wide warm ischemic range.Six human livers discarded for transplantation with variable warm and cold ischemia times were flushed at the end of preservation. The liver grafts were flushed with NaCl or Lactated Ringer's, 2 L through the portal vein and 1 L through the hepatic artery. The vena caval effluent was sampled and analyzed for biochemical markers of injury; lactate dehydrogenase (LDH, alanine transaminase (ALT, and alkaline phosphatase (ALP. Liver tissue biopsies were analyzed for ATP content and histologically (H&E examined.The duration of warm ischemia in the six livers correlated significantly to the concentration of LDH, ALT, and ALP in the effluent from the portal vein flush. No correlation was found with cold ischemia time. Tissue ATP content at the end of preservation correlated very strongly with the concentration of ALP in the arterial effluent (P<0.0007, R2 = 0.96.Biochemical injury markers released during the cold preservation period were reflective of the duration of warm ischemic injury sustained prior to release of the markers, as well as the hepatic energy status. As such, assessment of the flush effluent at the end of cold preservation may be a useful tool in evaluating suboptimal livers prior to transplantation, particularly in situations with undeterminable ischemic durations.

  1. Effect of extracellular vesicles of human adipose tissue on insulin signaling in liver and muscle cells.

    Science.gov (United States)

    Kranendonk, Mariëtte E G; Visseren, Frank L J; van Herwaarden, Joost A; Nolte-'t Hoen, Esther N M; de Jager, Wilco; Wauben, Marca H M; Kalkhoven, Eric

    2014-10-01

    Insulin resistance (IR) is a key mechanism in obesity-induced cardiovascular disease. To unravel mechanisms whereby human adipose tissue (AT) contributes to systemic IR, the effect of human AT-extracellular vesicles (EVs) on insulin signaling in liver and muscle cells was determined. EVs released from human subcutaneous (SAT) and omental AT (OAT)-explants ex vivo were used for stimulation of hepatocytes and myotubes in vitro. Subsequently, insulin-induced Akt phosphorylation and expression of gluconeogenic genes (G6P, PEPCK) was determined. AT-EV adipokine levels were measured by multiplex immunoassay, and AT-EVs were quantified by high-resolution flow cytometry. In hepatocytes, AT-EVs from the majority of patients inhibited insulin-induced Akt phosphorylation, while EVs from some patients stimulated insulin-induced Akt phosphorylation. In myotubes AT-EVs exerted an ambiguous effect on insulin signaling. Hepatic Akt phosphorylation related negatively to G6P-expression by both SAT-EVs (r = -0.60, P = 0.01) and OAT-EVs (r = -0.74, P = 0.001). MCP-1, IL-6, and MIF concentrations were higher in OAT-EVs compared to SAT-EVs and differently related to lower Akt phosphorylation in hepatocytes. Finally, the number of OAT-EVs correlated positively with liver enzymes indicative for liver dysfunction. Human AT-EVs can stimulate or inhibit insulin signaling in hepatocytes- possibly depending on their adipokine content- and may thereby contribute to systemic IR. Copyright © 2014 The Obesity Society.

  2. Protective Role of Commiphora molmol Extract against Liver and ...

    African Journals Online (AJOL)

    The toxicity of CCl4 resulted from the bio-activation of. CCl4 into trichloromethyl free radical by cytochrome P450 system in the liver microsomes and consequently this causes lipid peroxidation of membranes that leads to liver injury [4]. Exposure to CCl4 also causes kidney damage that may finally lead to cancer [5]. Several.

  3. An Experimental Study to Measure the Mechanical Properties of the Human Liver.

    Science.gov (United States)

    Karimi, Alireza; Shojaei, Ahmad

    2017-10-31

    Since the liver is one of the most important organs of the body that can be injured during trauma, that is, during accidents like car crashes, understanding its mechanical properties is of great interest. Experimental data is needed to address the mechanical properties of the liver to be used for a variety of applications, such as the numerical simulations for medical purposes, including the virtual reality simulators, trauma research, diagnosis objectives, as well as injury biomechanics. However, the data on the mechanical properties of the liver capsule is limited to the animal models or confined to the tensile/compressive loading under single direction. Therefore, this study was aimed at experimentally measuring the axial and transversal mechanical properties of the human liver capsule under both the tensile and compressive loadings. To do that, 20 human cadavers were autopsied and their liver capsules were excised and histologically analyzed to extract the mean angle of a large fibers population (bundle of the fine collagen fibers). Thereafter, the samples were cut and subjected to a series of axial and transversal tensile/compressive loadings. The results revealed the tensile elastic modulus of 12.16 ± 1.20 (mean ± SD) and 7.17 ± 0.85 kPa under the axial and transversal loadings respectively. Correspondingly, the compressive elastic modulus of 196.54 ± 13.15 and 112.41 ± 8.98 kPa were observed under the axial and transversal loadings respectively. The compressive axial and transversal maximum/failure stress of the capsule were 32.54 and 37.30 times higher than that of the tensile ones respectively. The capsule showed a stiffer behavior under the compressive load compared to the tensile one. In addition, the axial elastic modulus of the capsule was found to be higher than that of the transversal one. The findings of the current study have implications not only for understanding the mechanical properties of the human capsule tissue under tensile

  4. Gene expression of 17beta-estradiol-metabolizing isozymes: comparison of normal human mammary gland to normal human liver and to cultured human breast adenocarcinoma cells.

    Science.gov (United States)

    Lehmann, Leane; Wagner, Jörg

    2008-01-01

    Metabolic activation of 17beta-estradiol (E2) to catechols and quinones together with lack of deactivation constitute risk factors in human breast carcinogenesis. E2-catchols are generated by cytochrome P450-dependent monooxygenases (CYPs). Deactivation of E2, E2-catechols, and E2-quinones is mediated by UDP-glucuronosyltransferase (UGT), sulfotransferase (SULT), catechol-O-methyltransferase (COMT), glutathione-S-transferase (GST), and NADPH-quinone-oxidoreductase (QR) isozymes, respectively. The aim of the present study was to quantify mRNA levels of E2-metabolizing isozymes expressed in MCF-7 cells cultured in the presence/absence of steroids by reverse transcription/competitive PCR in relation to the housekeeping gene hypoxanthine-guanine phosphoribosyltransferase and compare them with expression levels in normal human mammary gland (MG) and liver tissue. CYP1A1, 1B1, SULT1A1, 1A2, membrane-bound and soluble COMT, GSTT1, QR1, and UGT2B7 were detected in both tissues and MCF-7 cells; however, most enzymes were expressed at least tenfold higher in liver. Yet, CYP1B1 was expressed as high in breast as in liver and UGTs were not detected in MCF-7 cells cultured with steroids. MCF-7 cells cultured steroid-free additionally expressed CYP1A2 as well as UGT1A4, 1A8, and 1A9. Normal human liver but not MG expressed CYP1A2, 3A4, UGT1A1, 1A3, 1A4, 1A9, and SULT2A1. UGT1A8 was only detected in MCF7 cells but was not found in human liver. Thus, our study provides a comprehensive overview of expression levels of E2-metabolizing enzymes in a popular in vitro model and in human tissues, which will contribute to the interpretation of in vitro studies concerning the activation/deactivation of E2.

  5. Nutritional modulation of mouse and human liver bud growth through a branched-chain amino acid metabolism.

    Science.gov (United States)

    Koike, Hiroyuki; Zhang, Ran-Ran; Ueno, Yasuharu; Sekine, Keisuke; Zheng, Yun-Wen; Takebe, Takanori; Taniguchi, Hideki

    2017-03-15

    Liver bud progenitors experience a transient amplification during the early organ growth phase, yet the mechanism responsible is not fully understood. Collective evidence highlights the specific requirements in stem cell metabolism for expanding organ progenitors during organogenesis and regeneration. Here, transcriptome analyses show that progenitors of the mouse and human liver bud growth stage specifically express the gene branched chain aminotransferase 1, encoding a known breakdown enzyme of branched-chain amino acids (BCAAs) for energy generation. Global metabolome analysis confirmed the active consumption of BCAAs in the growing liver bud, but not in the later fetal or adult liver. Consistently, maternal dietary restriction of BCAAs during pregnancy significantly abrogated the conceptus liver bud growth capability through a striking defect in hepatic progenitor expansion. Under defined conditions, the supplementation of L-valine specifically among the BCAAs promoted rigorous growth of the human liver bud organoid in culture by selectively amplifying self-renewing bi-potent hepatic progenitor cells. These results highlight a previously underappreciated role of branched-chain amino acid metabolism in regulating mouse and human liver bud growth that can be modulated by maternal nutrition in vivo or cultural supplement in vitro. © 2017. Published by The Company of Biologists Ltd.

  6. A CD13-targeting peptide integrated protein inhibits human liver cancer growth by killing cancer stem cells and suppressing angiogenesis.

    Science.gov (United States)

    Zheng, Yan-Bo; Gong, Jian-Hua; Liu, Xiu-Jun; Li, Yi; Zhen, Yong-Su

    2017-05-01

    CD13 is a marker of angiogenic endothelial cells, and recently it is proved to be a biomarker of human liver cancer stem cells (CSCs). Herein, the therapeutic effects of NGR-LDP-AE, a fusion protein composed of CD13-targeting peptide NGR and antitumor antibiotic lidamycin, on human liver cancer and its mechanism were studied. Western blot and immunofluorescence assay demonstrated that CD13 (WM15 epitope) was expressed in both human liver cancer cell lines and vascular endothelial cells, while absent in normal liver cells. MTT assay showed that NGR-LDP-AE displayed potent cytotoxicity to cultured tumor cell lines with IC50 values at low nanomolar level. NGR-LDP-AE inhibited tumorsphere formation of liver cancer cells, and the IC50 values were much lower than that in MTT assay, indicating selectively killing of CSCs. In endothelial tube formation assay, NGR-LDP-AE at low cytotoxic dose significantly inhibited the formation of intact tube networks. Animal experiment demonstrated that NGR-LDP-AE inhibited the growth of human liver cancer xenograft. Immunohistochemical analysis showed that NGR-LDP-AE induced the down-regulation of CD13. In vitro experiment using cultured tumor cells also confirmed this result. NGR-LDP-AE activated both apoptotic and autophagic pathways in cultured tumor cells, while the induced autophagy protected cells from death. Conclusively, NGR-LDP-AE exerts its antitumor activity via killing liver CSCs and inhibiting angiogenesis. With one targeting motif, NGR-LDP-AE acts on both liver CSCs and angiogenic endothelial cells. It is a promising dual targeting fusion protein for liver cancer therapy, especially for advanced or relapsed cancers. © 2017 Wiley Periodicals, Inc.

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

    Science.gov (United States)

    Waskell, L; Gonzales, J

    1982-07-01

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

  8. Microsomal activation of thioacetamide-S-oxide to a metabolite(s) that covalently binds to calf thymus DNA and other polynucleotides.

    Science.gov (United States)

    Vadi, H V; Neal, R A

    1981-04-01

    In the presence of NADPH liver microsomes isolated from phenobarbital-pretreated rats catalyze the conversion of [3H]thioacetamide-S-oxide to a reactive intermediate(s) which covalently binds to calf thymus DNA, calf liver RNA, polyguanylic acid (poly(G)) and polyadenylic acid (poly(A)). The highest level of binding of radioactivity was obtained with poly(G), followed by poly(A), RNA and DNA. The incorporation of radioactivity into DNA was linear for 30 min and there was a requirement for NADPH for time-dependent covalent binding to occur. Performing the microsomal incubations in an atmosphere of 80% CO/20% O2 or adding partially purified anti cytochrome P-450 immune serum to the microsomal incubations inhibited the total metabolism of thioacetamide-S-oxide and had a small, but insignificant, inhibitory effect on binding of radioactivity to calf thymus DNA. Using a reconstituted monooxygenase system containing cytochrome P-450 purified from phenobarbital-treated rats we were unable to detect any metabolism of thioacetamide-S-oxide. Only background levels of radio-activity were incorporated into calf thymus DNA when microsomes isolated from phenobarbital-treated rats were incubated with [3H]thioacetamide in the presence of NADPH. These results suggest that thioacetamide-S-oxide is an obligatory intermediate in the metabolic activation of thioacetamide to a reactive metabolite(s) which binds to calf thumus DNA.

  9. Liver-related deaths among persons infected with the human immunodeficiency virus: The D:A:D Study

    DEFF Research Database (Denmark)

    Weber, R; Sabin, CA; Friis-Møller, Nina

    2006-01-01

    BACKGROUND: An increasing proportion of deaths among human immunodeficiency virus (HIV)-infected persons with access to combination antiretroviral therapy (cART) are due to complications of liver diseases. METHODS: We investigated the frequency of and risk factors associated with liver......-related deaths in the Data Collection on Adverse Events of Anti-HIV Drugs study, which prospectively evaluated 76 893 person-years of follow-up in 23 441 HIV-infected persons. Multivariable Poisson regression analyses identified factors associated with liver-related, AIDS-related, and other causes of death....... RESULTS: There were 1246 deaths (5.3%; 1.6 per 100 person-years); 14.5% were from liver-related causes. Of these, 16.9% had active hepatitis B virus (HBV), 66.1% had hepatitis C virus (HCV), and 7.1% had dual viral hepatitis co-infections. Predictors of liver-related deaths were latest CD4 cell count...

  10. Liver-related deaths among persons infected with the human immunodeficiency virus: The D:A:D Study

    DEFF Research Database (Denmark)

    Weber, R; Sabin, CA; Friis-Møller, Nina

    2006-01-01

    -related deaths in the Data Collection on Adverse Events of Anti-HIV Drugs study, which prospectively evaluated 76 893 person-years of follow-up in 23 441 HIV-infected persons. Multivariable Poisson regression analyses identified factors associated with liver-related, AIDS-related, and other causes of death......BACKGROUND: An increasing proportion of deaths among human immunodeficiency virus (HIV)-infected persons with access to combination antiretroviral therapy (cART) are due to complications of liver diseases. METHODS: We investigated the frequency of and risk factors associated with liver....... RESULTS: There were 1246 deaths (5.3%; 1.6 per 100 person-years); 14.5% were from liver-related causes. Of these, 16.9% had active hepatitis B virus (HBV), 66.1% had hepatitis C virus (HCV), and 7.1% had dual viral hepatitis co-infections. Predictors of liver-related deaths were latest CD4 cell count...

  11. ADAM12 in human liver cancers: TGF-beta-regulated expression in stellate cells is associated with matrix remodeling

    DEFF Research Database (Denmark)

    Le Pabic, Hélène; Bonnier, Dominique; Wewer, Ulla M

    2003-01-01

    -activated protein kinase kinase (MEK) inhibitor UO126 prevented ADAM12 induction by TGF-beta, suggesting the involvement of PI3K and MEK activities. In vivo, the steady-state of both ADAM9 and ADAM12 mRNA levels was nearly undetectable in both normal livers and benign tumors and increased in hepatocellular...... carcinomas (up to 3- and 6-fold, respectively) and liver metastases from colonic carcinomas (up to 40- and 60-fold, respectively). The up-regulation of both ADAM9 and ADAM12 was correlated with an increase in matrix metalloproteinase 2 expression and activity. In conclusion, in liver cancers ADAM9 and ADAM12......"A disintegrin and metalloproteinases" (ADAMs) form a family of cell-surface glycoproteins with potential protease and cell-adhesion activities. We have investigated ADAM expression in human liver cancers and their regulation by several cytokines involved in liver injury. Using degenerative RT...

  12. Hepatic energy metabolism in human diabetes mellitus, obesity and non-alcoholic fatty liver disease.

    Science.gov (United States)

    Koliaki, Chrysi; Roden, Michael

    2013-10-15

    Alterations of hepatic mitochondrial function have been observed in states of insulin resistance and non-alcoholic fatty liver disease (NAFLD). Patients with overt type 2 diabetes mellitus (T2DM) can exhibit reduction in hepatic adenosine triphosphate (ATP) synthesis and impaired repletion of their hepatic ATP stores upon ATP depletion by fructose. Obesity and NAFLD may also associate with impaired ATP recovery after ATP-depleting challenges and augmented oxidative stress in the liver. On the other hand, patients with obesity or NAFLD can present with upregulated hepatic anaplerotic and oxidative fluxes, including β-oxidation and tricarboxylic cycle activity. The present review focuses on the methods and data on hepatic energy metabolism in various states of human insulin resistance. We propose that the liver can adapt to increased lipid exposition by greater lipid storing and oxidative capacity, resulting in increased oxidative stress, which in turn could deteriorate hepatic mitochondrial function in chronic insulin resistance and NAFLD. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  13. Featured Article: Isolation, characterization, and cultivation of human hepatocytes and non-parenchymal liver cells

    Science.gov (United States)

    Pfeiffer, Elisa; Kegel, Victoria; Zeilinger, Katrin; Hengstler, Jan G; Nüssler, Andreas K; Seehofer, Daniel

    2015-01-01

    Primary human hepatocytes (PHH) are considered to be the gold standard for in vitro testing of xenobiotic metabolism and hepatotoxicity. However, PHH cultivation in 2D mono-cultures leads to dedifferentiation and a loss of function. It is well known that hepatic non-parenchymal cells (NPC), such as Kupffer cells (KC), liver endothelial cells (LEC), and hepatic stellate cells (HSC), play a central role in the maintenance of PHH functions. The aims of the present study were to establish a protocol for the simultaneous isolation of human PHH and NPC from the same tissue specimen and to test their suitability for in vitro co-culture. Human PHH and NPC were isolated from tissue obtained by partial liver resection by a two-step EDTA/collagenase perfusion technique. The obtained cell fractions were purified by Percoll density gradient centrifugation. KC, LEC, and HSC contained in the NPC fraction were separated using specific adherence properties and magnetic activated cell sorting (MACS®). Identified NPC revealed a yield of 1.9 × 106 KC, 2.7 × 105 LEC and 4.7 × 105 HSC per gram liver tissue, showing viabilities >90%. Characterization of these NPC showed that all populations went through an activation process, which influenced the cell fate. The activation of KC strongly depended on the tissue quality and donor anamnesis. KC became activated in culture in association with a loss of viability within 4–5 days. LEC lost specific features during culture, while HSC went through a transformation process into myofibroblasts. The testing of different culture conditions for HSC demonstrated that they can attenuate, but not prevent dedifferentiation in vitro. In conclusion, the method described allows the isolation and separation of PHH and NPC in high quality and quantity from the same donor. PMID:25394621

  14. Applying of hierarchical clustering to analysis of protein patterns in the human cancer-associated liver.

    Science.gov (United States)

    Petushkova, Natalia A; Pyatnitskiy, Mikhail A; Rudenko, Vladislav A; Larina, Olesya V; Trifonova, Oxana P; Kisrieva, Julya S; Samenkova, Natalia F; Kuznetsova, Galina P; Karuzina, Irina I; Lisitsa, Andrey V

    2014-01-01

    There are two ways that statistical methods can learn from biomedical data. One way is to learn classifiers to identify diseases and to predict outcomes using the training dataset with established diagnosis for each sample. When the training dataset is not available the task can be to mine for presence of meaningful groups (clusters) of samples and to explore underlying data structure (unsupervised learning). We investigated the proteomic profiles of the cytosolic fraction of human liver samples using two-dimensional electrophoresis (2DE). Samples were resected upon surgical treatment of hepatic metastases in colorectal cancer. Unsupervised hierarchical clustering of 2DE gel images (n = 18) revealed a pair of clusters, containing 11 and 7 samples. Previously we used the same specimens to measure biochemical profiles based on cytochrome P450-dependent enzymatic activities and also found that samples were clearly divided into two well-separated groups by cluster analysis. It turned out that groups by enzyme activity almost perfectly match to the groups identified from proteomic data. Of the 271 reproducible spots on our 2DE gels, we selected 15 to distinguish the human liver cytosolic clusters. Using MALDI-TOF peptide mass fingerprinting, we identified 12 proteins for the selected spots, including known cancer-associated species. Our results highlight the importance of hierarchical cluster analysis of proteomic data, and showed concordance between results of biochemical and proteomic approaches. Grouping of the human liver samples and/or patients into differing clusters may provide insights into possible molecular mechanism of drug metabolism and creates a rationale for personalized treatment.

  15. Regulation of coagulation factor XI expression by microRNAs in the human liver.

    Directory of Open Access Journals (Sweden)

    Salam Salloum-Asfar

    Full Text Available High levels of factor XI (FXI increase the risk of thromboembolic disease. However, the genetic and environmental factors regulating FXI expression are still largely unknown. The aim of our study was to evaluate the regulation of FXI by microRNAs (miRNAs in the human liver. In silico prediction yielded four miRNA candidates that might regulate FXI expression. HepG2 cells were transfected with miR-181a-5p, miR-23a-3p, miR-16-5p and miR-195-5p. We used mir-494, which was not predicted to bind to F11, as a negative control. Only miR-181a-5p caused a significant decrease both in FXI protein and F11 mRNA levels. In addition, transfection with a miR-181a-5p inhibitor in PLC/PRF/5 hepatic cells increased both the levels of F11 mRNA and extracellular FXI. Luciferase assays in human colon cancer cells deficient for Dicer (HCT-DK demonstrated a direct interaction between miR-181a-5p and 3'untranslated region of F11. Additionally, F11 mRNA levels were inversely and significantly correlated with miR-181a-5p levels in 114 healthy livers, but not with miR-494. This study demonstrates that FXI expression is directly regulated by a specific miRNA, miR-181a-5p, in the human liver. Future studies are necessary to further investigate the potential consequences of miRNA dysregulation in pathologies involving FXI.

  16. Applying of hierarchical clustering to analysis of protein patterns in the human cancer-associated liver.

    Directory of Open Access Journals (Sweden)

    Natalia A Petushkova

    Full Text Available There are two ways that statistical methods can learn from biomedical data. One way is to learn classifiers to identify diseases and to predict outcomes using the training dataset with established diagnosis for each sample. When the training dataset is not available the task can be to mine for presence of meaningful groups (clusters of samples and to explore underlying data structure (unsupervised learning.We investigated the proteomic profiles of the cytosolic fraction of human liver samples using two-dimensional electrophoresis (2DE. Samples were resected upon surgical treatment of hepatic metastases in colorectal cancer. Unsupervised hierarchical clustering of 2DE gel images (n = 18 revealed a pair of clusters, containing 11 and 7 samples. Previously we used the same specimens to measure biochemical profiles based on cytochrome P450-dependent enzymatic activities and also found that samples were clearly divided into two well-separated groups by cluster analysis. It turned out that groups by enzyme activity almost perfectly match to the groups identified from proteomic data. Of the 271 reproducible spots on our 2DE gels, we selected 15 to distinguish the human liver cytosolic clusters. Using MALDI-TOF peptide mass fingerprinting, we identified 12 proteins for the selected spots, including known cancer-associated species.Our results highlight the importance of hierarchical cluster analysis of proteomic data, and showed concordance between results of biochemical and proteomic approaches. Grouping of the human liver samples and/or patients into differing clusters may provide insights into possible molecular mechanism of drug metabolism and creates a rationale for personalized treatment.

  17. Keratin Hypersumoylation Alters Filament Dynamics and Is a Marker for Human Liver Disease and Keratin Mutation*

    Science.gov (United States)

    Snider, Natasha T.; Weerasinghe, Sujith V. W.; Iñiguez-Lluhí, Jorge A.; Herrmann, Harald; Omary, M. Bishr

    2011-01-01

    Keratin polypeptide 8 (K8) associates noncovalently with its partners K18 and/or K19 to form the intermediate filament cytoskeleton of hepatocytes and other simple-type epithelial cells. Human K8, K18, and K19 variants predispose to liver disease, whereas site-specific keratin phosphorylation confers hepatoprotection. Because stress-induced protein phosphorylation regulates sumoylation, we hypothesized that keratins are sumoylated in an injury-dependent manner and that keratin sumoylation is an important regulatory modification. We demonstrate that K8/K18/K19, epidermal keratins, and vimentin are sumoylated in vitro. Upon transfection, K8, K18, and K19 are modified by poly-SUMO-2/3 chains on Lys-285/Lys-364 (K8), Lys-207/Lys-372 (K18), and Lys-208 (K19). Sumoylation affects filament organization and stimulus-induced keratin solubility and is partially inhibited upon mutation of one of three known K8 phosphorylation sites. Extensive sumoylation occurs in cells transfected with individual K8, K18, or K19 but is limited upon heterodimerization (K8/K18 or K8/K19) in the absence of stress. In contrast, keratin sumoylation is significantly augmented in cells and tissues during apoptosis, oxidative stress, and phosphatase inhibition. Poly-SUMO-2/3 conjugates are present in chronically injured but not normal, human, and mouse livers along with polyubiquitinated and large insoluble keratin-containing complexes. Notably, common human K8 liver disease-associated variants trigger keratin hypersumoylation with consequent diminished solubility. In contrast, modest sumoylation of wild type K8 promotes solubility. Hence, conformational changes induced by keratin natural mutations and extensive tissue injury result in K8/K18/K19 hypersumoylation, which retains keratins in an insoluble compartment, thereby limiting their cytoprotective function. PMID:21062750

  18. Keratin hypersumoylation alters filament dynamics and is a marker for human liver disease and keratin mutation.

    Science.gov (United States)

    Snider, Natasha T; Weerasinghe, Sujith V W; Iñiguez-Lluhí, Jorge A; Herrmann, Harald; Omary, M Bishr

    2011-01-21

    Keratin polypeptide 8 (K8) associates noncovalently with its partners K18 and/or K19 to form the intermediate filament cytoskeleton of hepatocytes and other simple-type epithelial cells. Human K8, K18, and K19 variants predispose to liver disease, whereas site-specific keratin phosphorylation confers hepatoprotection. Because stress-induced protein phosphorylation regulates sumoylation, we hypothesized that keratins are sumoylated in an injury-dependent manner and that keratin sumoylation is an important regulatory modification. We demonstrate that K8/K18/K19, epidermal keratins, and vimentin are sumoylated in vitro. Upon transfection, K8, K18, and K19 are modified by poly-SUMO-2/3 chains on Lys-285/Lys-364 (K8), Lys-207/Lys-372 (K18), and Lys-208 (K19). Sumoylation affects filament organization and stimulus-induced keratin solubility and is partially inhibited upon mutation of one of three known K8 phosphorylation sites. Extensive sumoylation occurs in cells transfected with individual K8, K18, or K19 but is limited upon heterodimerization (K8/K18 or K8/K19) in the absence of stress. In contrast, keratin sumoylation is significantly augmented in cells and tissues during apoptosis, oxidative stress, and phosphatase inhibition. Poly-SUMO-2/3 conjugates are present in chronically injured but not normal, human, and mouse livers along with polyubiquitinated and large insoluble keratin-containing complexes. Notably, common human K8 liver disease-associated variants trigger keratin hypersumoylation with consequent diminished solubility. In contrast, modest sumoylation of wild type K8 promotes solubility. Hence, conformational changes induced by keratin natural mutations and extensive tissue injury result in K8/K18/K19 hypersumoylation, which retains keratins in an insoluble compartment, thereby limiting their cytoprotective function.

  19. Isolation of viable human hepatic progenitors from adult livers is possible even after 48 hours of cold ischemia.

    Science.gov (United States)

    Aupet, Sophie; Simoné, Gael; Heyd, Bruno; Bachellier, Philippe; Vidal, Isabelle; Richert, Lysiane; Martin, Hélène

    2013-07-01

    Liver transplantation, utilized routinely for end-stage liver disease, has been constrained by the paucity of organ donors, and is being complemented by alternative strategies such as liver cell transplantation. One of the most promising forms of liver cell transplantation is hepatic stem cell therapies, as the number of human hepatic stem cells (hHpSCs) and other early hepatic progenitor cells (HPCs) are sufficient to provide treatment for multiple patients from a single liver source. In the present study, human adult livers were exposed to cold ischemia and then processed after numbers, albeit somewhat lower, were obtained from those exposed to 48 h of cold ischemia. The yields are similar to those reported from livers with minimal exposure to ischemia. When cultured on plastic dishes and in Kubota's Medium, a serum-free medium designed for early lineage stage HPCs, colonies of rapidly expanding cells formed. They were confirmed to be probable hHpSCs by their ability to survive and expand on plastic and in Kubota's Medium for months, by co-expression of EpCAM and neural cell adhesion molecule, minimal if any albumin expression, with EpCAM found throughout the cells, and no expression of alpha-fetoprotein. The yields of viable EpCAM(+) cells were surprisingly large, and the numbers from a single donor liver are sufficient to treat approximately 50-100 patients given the numbers of EpCAM(+) cells currently used in hepatic stem cell therapies. Thus, cold ischemic livers for up to 48 h are a new source of cells that might be used for liver cell therapies.

  20. Production of factor VIII by human liver sinusoidal endothelial cells transplanted in immunodeficient uPA mice.

    Directory of Open Access Journals (Sweden)

    Marina E Fomin

    Full Text Available Liver sinusoidal endothelial cells (LSECs form a semi-permeable barrier between parenchymal hepatocytes and the blood. LSECs participate in liver metabolism, clearance of pathological agents, immunological responses, architectural maintenance of the liver and synthesis of growth factors and cytokines. LSECs also play an important role in coagulation through the synthesis of Factor VIII (FVIII. Herein, we phenotypically define human LSECs isolated from fetal liver using flow cytometry and immunofluorescence microscopy. Isolated LSECs were cultured and shown to express endothelial markers and markers specific for the LSEC lineage. LSECs were also shown to engraft the liver when human fetal liver cells were transplanted into immunodeficient mice with liver specific expression of the urokinase-type plasminogen activator (uPA transgene (uPA-NOG mice. Engrafted cells expressed human Factor VIII at levels approaching those found in human plasma. We also demonstrate engraftment of adult LSECs, as well as hepatocytes, transplanted into uPA-NOG mice. We propose that overexpression of uPA provides beneficial conditions for LSEC engraftment due to elevated expression of the angiogenic cytokine, vascular endothelial growth factor. This work provides a detailed characterization of human midgestation LSECs, thereby providing the means for their purification and culture based on their expression of CD14 and CD32 as well as a lack of CD45 expression. The uPA-NOG mouse is shown to be a permissive host for human LSECs and adult hepatocytes, but not fetal hepatoblasts. Thus, these mice provide a useful model system to study these cell types in vivo. Demonstration of human FVIII production by transplanted LSECs encourages further pursuit of LSEC transplantation as a cellular therapy for the treatment of hemophilia A.

  1. [Damage effect of Polygonum multiflorum fractions on human normal liver cells L02 and liver cancer cells HepG2].

    Science.gov (United States)

    Zhang, Ruichen; Zhang, Chao; Sun, Zhenxiao; Deng, Qiaohong

    2012-06-01

    To investigate the damage effect of different fractions from Polygonum multiflorum on normal human liver and liver cancer cells, in order to seek for fractions that can obviously kill cancer cells but have less impact on normal liver cells, and make a preliminary study on different mechanism of the two kinds of cells. P. multiflorum water-eluted fraction (RW), 50% ethanol-eluted fraction (R50) and 95% ethanol-eluted fraction (R95) were successively obtained from 70% ethanol extracts of P. multiflorum, after being eluted by water, 50% ethanol and 95% ethanol and then absorbed by AB-8 macroporous resin. Normal human liver L02 cells and liver cancer HepG2 cells were incubated with cell supernatants from different fractions and cells. MTT method and inverted microscope were adopted to observe the impact of L02 on growth of HepG2 cells, screening fractions with damage effect and detect their doses and time effect. Giemsa stain showed changes in cell nucleus after administration and flow cytometry analysis was used to detect cycle and apoptosis of L02 cells. MTT method and inverted microscope showed that R50 had significant growth inhibition effects on L02 and HepG2 cells. According to giemsa stain and flow cytometry analysis, R50 showed different effect on inducing the two cells: there are much more apoptotic HepG2 cells than apoptotic L02 cells in each time phase (the proportion of the apoptosis cells in HepG2 group were 83.62%, 60.52% and 74.49%, and ID2 31.02%, 20.57% and 25.32% after treated with R50 for 24, 48, 72 h. Both cells showed less than 5% of apoptotic cells in the negative control group in each time phase). However, there is no significant impact on cycle of both cells. R50 from P. multiflorum extracts had different damage effects on human liver L02 cells and liver cancer HepG2 cells, which was caused by different degree of induction on apoptosis of the two cells in nature.

  2. Human fetal liver stromal cells that overexpress bFGF support growth and maintenance of human embryonic stem cells.

    Directory of Open Access Journals (Sweden)

    Jiafei Xi

    Full Text Available In guiding hES cell technology toward the clinic, one key issue to be addressed is to culture and maintain hES cells much more safely and economically in large scale. In order to avoid using mouse embryonic fibroblasts (MEFs we isolated human fetal liver stromal cells (hFLSCs from 14 weeks human fetal liver as new human feeder cells. hFLSCs feeders could maintain hES cells for 15 passages (about 100 days. Basic fibroblast growth factor (bFGF is known to play an important role in promoting self-renewal of human embryonic stem (hES cells. So, we established transgenic hFLSCs that stably express bFGF by lentiviral vectors. These transgenic human feeder cells--bFGF-hFLSCs maintained the properties of H9 hES cells without supplementing with any exogenous growth factors. H9 hES cells culturing under these conditions maintained all hES cell features after prolonged culture, including the developmental potential to differentiate into representative tissues of all three embryonic germ layers, unlimited and undifferentiated proliferative ability, and maintenance of normal karyotype. Our results demonstrated that bFGF-hFLSCs feeder cells were central to establishing the signaling network among bFGF, insulin-like growth factor 2 (IGF-2, and transforming growth factor β (TGF-β, thereby providing the framework in which hES cells were instructed to self-renew or to differentiate. We also found that the conditioned medium of bFGF-hFLSCs could maintain the H9 hES cells under feeder-free conditions without supplementing with bFGF. Taken together, bFGF-hFLSCs had great potential as feeders for maintaining pluripotent hES cell lines more safely and economically.

  3. Processing of acetylated human low-density lipoprotein by parenchymal and non-parenchymal liver cells. Involvement of calmodulin?

    OpenAIRE

    Van Berkel, Theo J.C.; Nagelkerke, Jan F.; Harkes, Leen; Kruijt, Johan K.

    1982-01-01

    1. Modified lipoproteins have been implicated to play a significant role in the pathogenesis of atherosclerosis. In view of this we studied the fate and mechanism of uptake in vivo of acetylated human low-density lipoprotein (acetyl-LDL). Injected intravenously into rats, acetyl-LDL is rapidly cleared from the blood. At 10min after intravenous injection, 83% of the injected dose is recovered in liver. Separation of the liver into a parenchymal and non-parenchymal cell fraction indicates that ...

  4. The Role of Human Cytochrome P450 Enzymes in the Formation of 2-Hydroxymetronidazole: CYP2A6 is the High Affinity (Low Km) Catalyst

    OpenAIRE

    Pearce, Robin E.; Cohen-Wolkowiez, Michael; Sampson, Mario R.; Kearns, Gregory L.

    2013-01-01

    Despite metronidazole’s widespread clinical use since the 1960s, the specific enzymes involved in its biotransformation have not been previously identified. Hence, in vitro studies were conducted to identify and characterize the cytochrome P450 enzymes involved in the formation of the major metabolite, 2-hydroxymetronidazole. Formation of 2-hydroxymetronidazole in human liver microsomes was consistent with biphasic, Michaelis-Menten kinetics. Although several cDNA-expressed P450 enzymes catal...

  5. A human liver microphysiology platform for investigating physiology, drug safety, and disease models.

    Science.gov (United States)

    Vernetti, Lawrence A; Senutovitch, Nina; Boltz, Robert; DeBiasio, Richard; Shun, Tong Ying; Gough, Albert; Taylor, D Lansing

    2016-01-01

    This paper describes the development and characterization of a microphysiology platform for drug safety and efficacy in liver models of disease that includes a human, 3D, microfluidic, four-cell, sequentially layered, self-assembly liver model (SQL-SAL); fluorescent protein biosensors for mechanistic readouts; as well as a microphysiology system database (MPS-Db) to manage, analyze, and model data. The goal of our approach is to create the simplest design in terms of cells, matrix materials, and microfluidic device parameters that will support a physiologically relevant liver model that is robust and reproducible for at least 28 days for stand-alone liver studies and microfluidic integration with other organs-on-chips. The current SQL-SAL uses primary human hepatocytes along with human endothelial (EA.hy926), immune (U937) and stellate (LX-2) cells in physiological ratios and is viable for at least 28 days under continuous flow. Approximately, 20% of primary hepatocytes and/or stellate cells contain fluorescent protein biosensors (called sentinel cells) to measure apoptosis, reactive oxygen species (ROS) and/or cell location by high content analysis (HCA). In addition, drugs, drug metabolites, albumin, urea and lactate dehydrogenase (LDH) are monitored in the efflux media. Exposure to 180 μM troglitazone or 210 μM nimesulide produced acute toxicity within 2-4 days, whereas 28 μM troglitazone produced a gradual and much delayed toxic response over 21 days, concordant with known mechanisms of toxicity, while 600 µM caffeine had no effect. Immune-mediated toxicity was demonstrated with trovafloxacin with lipopolysaccharide (LPS), but not levofloxacin with LPS. The SQL-SAL exhibited early fibrotic activation in response to 30 nM methotrexate, indicated by increased stellate cell migration, expression of alpha-smooth muscle actin and collagen, type 1, alpha 2. Data collected from the in vitro model can be integrated into a database with access to related

  6. Toxico-kinetics, recovery efficiency and microsomal changes following administration of deltamethrin to black Bengal goats.

    Science.gov (United States)

    Juliet, S; Chakraborty, A K; Koley, K M; Mandal, T K; Bhattacharyya, A

    2001-03-01

    A study of the toxico-kinetics, recovery percentage from different substrates, cytotoxicity and role of cytochrome P450 and b5 of liver microsome in the metabolism of deltamethrin were carried out in female black Bengal goat. The ALD50 value of deltamethrin in goat by intravenous route lies between 0.2 and 0.6 mg kg-1. Intravenous disposition kinetics using a dose of 0.2 mg kg-1 showed that the maximum blood concentration of deltamethrin was recorded at 0.5 min, followed by rapid decline, and a minimum concentration was detected at 6 min after administration. The following values were obtained: Vdarea 0.148 (+/- 0.02) litre kg-1; t1/2 (alpha) 0.22 (+/- 0.02) min; t1/2 (beta) 2.17 (+/- 0.37) min; Kel 1.05 (+/- 0.24) min-1; AUC 4.30 (+/- 0.45) micrograms min ml-1; ClB 0.05 (+/- 0.006) litre kg-1 min-1; T-B 1.93 (+/- 0.58); fc 0.40 (+/- 0.05). After 10 min, liver retained the maximum residue, and heart, adrenal gland, kidney, spleen, fat and brain also held the insecticide; liver, fat, heart and spleen retained residue after 30 min, and bone, liver and fat retained residue after 60 min of intravenous administration. Oral absorption of deltamethrin was poor and inconsistent, and approximately 65% of administered dose was recovered from faeces and gastrointestinal contents. The excretion of deltamethrin through urine was meagre, and only 0.01 and 0.013% of the administered dose was recovered after 3 and 5 days of oral administration respectively. All the tissues retained the residue after 3 days; while fat, rumen, reticulum, omasum, abomasum, large and small intestine and bone retained the residue after 5 days of oral administration; and the percentage recoveries were 1.73 and 0.027 respectively. Deltamethrin reduced the level of cytochrome P450 content of liver microsomal pellet of goat after 5 days of oral administration. Histopathological examination of liver, kidney, heart, spleen brain and lung sections of treated goats did not reveal any pathological changes.

  7. Lucigenin as a substrate of microsomal NAD(P)H-oxidoreductases.

    Science.gov (United States)

    Schepetkin, I A

    1999-01-01

    NADPH oxidation and cytochrome c reduction with and without lucigenin as well as NAD(P)H/lucigenin-dependent chemiluminescence of rat liver microsomes were studied. An increased rate of NADPH oxidation and cytochrome c reduction in the presence of lucigenin was related to one-electron lucigenin reduction by microsomal NADPH reductases. The apparent Michaelis constant values for lucigenin (Km appLuc) were 3.6 and 5.0 microM in normoxygenic (pO2 = 150 +/- 5 mm Hg) and 8.7 and 8.3 microM in hypoxygenic (pO2 = 45 +/- 4 mm Hg) media in the reactions of lucigenin-dependent NADPH oxidation and cytochrome c reduction, respectively. The maximal level of NADPH/lucigenin-dependent chemiluminescence was registered at lucigenin concentration close to the mean K Luc/m app in the lucigenin-reductase reaction. Increasing the lucigenin concentration from 5 to 100 microM was associated with a decrease in the chemiluminescence intensity; this could be due to the inactivation of cytochrome P450. In the presence of superoxide dismutase (SOD), the rate of lucigenin-dependent cytochrome c reduction and NADPH/lucigenin-dependent chemiluminescence were decreased by 10 and 30%, respectively. The addition of lucigenin to microsomes which contain the reduced hemoprotein--CO complex was followed by the disappearance of the differential absorption spectrum specific for the carboxy complex and by increase in chemiluminescence intensity versus the control (without carboxy complex). Thus, lucigenin-dependent chemiluminescence of microsomes may be due to some enzymes including lucigenin reductase (NADPH-cytochrome P450 reductase, NADH-cytochrome b5 reductase), generation of O2-. in the redox cycle of lucigenin radicals, dioxetane formation by (di)oxygenases, and catalytic action of the cytochrome P450 heme on dioxetane decomposition followed by light quantum emission. Thus, lucigenin cannot be used to measure the basal O2-. formation in tissue homogenates with high levels of NAD(P)H-oxidoreductases.

  8. Functional Human Liver Preservation and Recovery by Means of Subnormothermic Machine Perfusion

    NARCIS (Netherlands)

    Bruinsma, Bote G.; Avruch, James H.; Weeder, Pepijn D.; Sridharan, Gautham V.; Uygun, Basak E.; Karimian, Negin G.; Porte, Robert J.; Markmann, James F.; Yeh, Heidi; Uygun, Korkut

    There is currently a severe shortage of liver grafts available for transplantation. Novel organ preservation techniques are needed to expand the pool of donor livers. Machine perfusion of donor liver grafts is an alternative to traditional cold storage of livers and holds much promise as a modality

  9. Human vs. robot operator error in a needle-based navigation system for percutaneous liver interventions

    Science.gov (United States)

    Maier-Hein, Lena; Walsh, Conor J.; Seitel, Alexander; Hanumara, Nevan C.; Shepard, Jo-Anne; Franz, A. M.; Pianka, F.; Müller, Sascha A.; Schmied, Bruno; Slocum, Alexander H.; Gupta, Rajiv; Meinzer, Hans-Peter

    2009-02-01

    Computed tomography (CT) guided percutaneous punctures of the liver for cancer diagnosis and therapy (e.g. tumor biopsy, radiofrequency ablation) are well-established procedures in clinical routine. One of the main challenges related to these interventions is the accurate placement of the needle within the lesion. Several navigation concepts have been introduced to compensate for organ shift and deformation in real-time, yet, the operator error remains an important factor influencing the overall accuracy of the developed systems. The aim of this study was to investigate whether the operator error and, thus, the overall insertion error of an existing navigation system could be further reduced by replacing the user with the medical robot Robopsy. For this purpose, we performed navigated needle insertions in a static abdominal phantom as well as in a respiratory liver motion simulator and compared the human operator error with the targeting error performed by the robot. According to the results, the Robopsy driven needle insertion system is able to more accurately align the needle and insert it along its axis compared to a human operator. Integration of the robot into the current navigation system could thus improve targeting accuracy in clinical use.

  10. Human endometrial regenerative cells alleviate carbon tetrachloride-induced acute liver injury in mice

    Directory of Open Access Journals (Sweden)

    Shanzheng Lu

    2016-10-01

    Full Text Available Abstract Background The endometrial regenerative cell (ERC is a novel type of adult mesenchymal stem cell isolated from menstrual blood. Previous studies demonstrated that ERCs possess unique immunoregulatory properties in vitro and in vivo, as well as the ability to differentiate into functional hepatocyte-like cells. For these reasons, the present study was undertaken to explore the effects of ERCs on carbon tetrachloride (CCl4–induced acute liver injury (ALI. Methods An ALI model in C57BL/6 mice was induced by administration of intraperitoneal injection of CCl4. Transplanted ERCs were intravenously injected (1 million/mouse into mice 30 min after ALI induction. Liver function, pathological and immunohistological changes, cell tracking, immune cell populations and cytokine profiles were assessed 24 h after the CCl4 induction. Results ERC treatment effectively decreased the CCl4-induced elevation of serum alanine aminotransferase (ALT and aspartate aminotransferase (AST activities and improved hepatic histopathological abnormalities compared to the untreated ALI group. Immunohistochemical staining showed that over-expression of lymphocyte antigen 6 complex, locus G (Ly6G was markedly inhibited, whereas expression of proliferating cell nuclear antigen (PCNA was increased after ERC treatment. Furthermore, the frequency of CD4+ and CD8+ T cell populations in the spleen was significantly down-regulated, while the percentage of splenic CD4+CD25+FOXP3+ regulatory T cells (Tregs was obviously up-regulated after ERC treatment. Moreover, splenic dendritic cells in ERC-treated mice exhibited dramatically decreased MHC-II expression. Cell tracking studies showed that transplanted PKH26-labeled ERCs engrafted to lung, spleen and injured liver. Compared to untreated controls, mice treated with ERCs had lower levels of IL-1β, IL-6, and TNF-α but higher level of IL-10 in both serum and liver. Conclusions Human ERCs protect the liver from acute injury

  11. Protective effects of Spirulina on the liver function and hyperlipidemia of rats and human

    Directory of Open Access Journals (Sweden)

    Mostafa Mohamed El-Sheekh

    2014-02-01

    Full Text Available In the present study, the effects of Spirulina on subchronic treatments (two weeks of hyperlipidemia and liver function of the rats and humans were investigated. The hyperlipidemia was induced in the rats using 25% of soya bean oil and 25% butter. The butter induced more hyperlipidemia than soya bean oil. Spirulina was used at the concentrations of 0, 2.5, 5.0 and 10 % of diet weight of the rats. The decrease in hyperlipidemia by Spirulina was dependent on its concentration in the diet. In case of human studies, about four g/day of Spirulina was taken via oral administration by Egyptian volunteers patients with hyperlipidemia. Spirulina decreased the levels of hyperlipidemia in these patients. The effects were dependent on the amount and number of administered dose of Sprirulina. The results suggested that the Spirulina treatment could induce marked reduction of aminotransferase through correcting lipid profile and increasing high density lipoprotein.

  12. High expression of MDR1, MRP1, and MRP3 in the hepatic progenitor cell compartment and hepatocytes in severe human liver disease

    NARCIS (Netherlands)

    Ros, Jenny E.; Libbrecht, Louis; Geuken, Mariska; Jansen, Peter L. M.; Roskams, Tania A. D.

    2003-01-01

    An increase in bile ductular structures is observed in diverse human liver diseases. These structures harbour the progenitor cell compartment of the liver. Since ATP-binding cassette (ABC) transporters may have a cytoprotective role in liver disease, an immunohistochemical study was performed on

  13. Long-term outcomes of liver transplant patients with human immunodeficiency virus infection and end-stage-liver-disease: single center experience

    Directory of Open Access Journals (Sweden)

    Vernadakis S

    2011-08-01

    Full Text Available Abstract Objective Orthotopic-liver-transplantation (OLT in patients with Human-Immunodeficiency-Virus infection (HIV and end-stage-liver-disease (ESDL is rarely reported. The purpose of this study is to describe our institutional experience on OLT for HIV positive patients. Material and methods This is a retrospective study of all HIV-infected patients who underwent OLT at the University Hospital of Essen, from January 1996 to December 2009. Age, sex, HIV transmission-way, CDC-stage, etiology of ESDL, concomitant liver disease, last CD4cell count and HIV-viral load prior to OLT were collected and analysed. Standard calcineurin-inhibitors-based immunosuppression was applied. All patients received anti-fungal and anti-pneumocystis carinii pneumonia prophylaxis post-OLT. Results Eight transplanted HIV-infected patients with a median age of 46 years (range 35-61 years were included. OLT indications were HCV (n = 5, HBV (n = 2, HCV/HBV/HDV-related cirrhosis (n = 1 and acute liver-failure (n = 1. At OLT, CD4 cell-counts ranged from 113-621 cells/μl, and HIV viral-loads from Conclusions OLT in HIV-infected patients and ESLD is an acceptable therapeutic option in selected patients. Long-term survival can be achieved without HIV disease-progression under antiretroviral therapy and management of the viral hepatitis co-infection.

  14. Studying Closed Hydrodynamic Models of "In Vivo" DNA Perfusion in Pig Liver for Gene Therapy Translation to Humans.

    Science.gov (United States)

    Sendra, Luis; Miguel, Antonio; Pérez-Enguix, Daniel; Herrero, María José; Montalvá, Eva; García-Gimeno, María Adelaida; Noguera, Inmaculada; Díaz, Ana; Pérez, Judith; Sanz, Pascual; López-Andújar, Rafael; Martí-Bonmatí, Luis; Aliño, Salvador F

    2016-01-01

    Expressing exogenous genes after naked DNA delivery into hepatocytes might achieve sustained and high expression of human proteins. Tail vein DNA injection is an efficient procedure for gene transfer in murine liver. Hydrodynamic procedures in large animals require organ targeting, and improve with liver vascular exclusion. In the present study, two closed liver hydrofection models employing the human alpha-1-antitrypsin (hAAT) gene are compared to reference standards in order to evaluate their potential clinical interest. A solution of naked DNA bearing the hAAT gene was retrogradely injected in 7 pig livers using two different closed perfusion procedures: an endovascular catheterization-mediated procedure (n = 3) with infrahepatic inferior vena cava and portal vein blockage; and a surgery-mediated procedure (n = 4) with completely sealed liver. Gene transfer was performed through the suprahepatic inferior cava vein in the endovascular procedure and through the infrahepatic inferior vena cava in the surgical procedure. The efficiency of the procedures was evaluated 14 days after hydrofection by quantifying the hAAT protein copies per cell in tissue and in plasma. For comparison, samples from mice (n = 7) successfully hydrofected with hAAT and healthy human liver segments (n = 4) were evaluated. Gene decoding occurs efficiently using both procedures, with liver vascular arrest improving its efficiency. The surgically closed procedure (sealed organ) reached higher tissue protein levels (4x10^5- copies/cell) than the endovascular procedure, though the levels were lower than in human liver (5x10^6- copies/cell) and hydrofected mouse liver (10^6- copies/cell). However, protein levels in plasma were lower (pcases. Hydrofection of hAAT DNA to "in vivo" isolated pig liver mediates highly efficient gene delivery and protein expression in tissue. Both endovascular and surgically closed models mediate high tissue protein expression. Impairment of protein secretion to plasma

  15. Studying Closed Hydrodynamic Models of "In Vivo" DNA Perfusion in Pig Liver for Gene Therapy Translation to Humans.

    Directory of Open Access Journals (Sweden)

    Luis Sendra

    Full Text Available Expressing exogenous genes after naked DNA delivery into hepatocytes might achieve sustained and high expression of human proteins. Tail vein DNA injection is an efficient procedure for gene transfer in murine liver. Hydrodynamic procedures in large animals require organ targeting, and improve with liver vascular exclusion. In the present study, two closed liver hydrofection models employing the human alpha-1-antitrypsin (hAAT gene are compared to reference standards in order to evaluate their potential clinical interest.A solution of naked DNA bearing the hAAT gene was retrogradely injected in 7 pig livers using two different closed perfusion procedures: an endovascular catheterization-mediated procedure (n = 3 with infrahepatic inferior vena cava and portal vein blockage; and a surgery-mediated procedure (n = 4 with completely sealed liver. Gene transfer was performed through the suprahepatic inferior cava vein in the endovascular procedure and through the infrahepatic inferior vena cava in the surgical procedure. The efficiency of the procedures was evaluated 14 days after hydrofection by quantifying the hAAT protein copies per cell in tissue and in plasma. For comparison, samples from mice (n = 7 successfully hydrofected with hAAT and healthy human liver segments (n = 4 were evaluated.Gene decoding occurs efficiently using both procedures, with liver vascular arrest improving its efficiency. The surgically closed procedure (sealed organ reached higher tissue protein levels (4x10^5- copies/cell than the endovascular procedure, though the levels were lower than in human liver (5x10^6- copies/cell and hydrofected mouse liver (10^6- copies/cell. However, protein levels in plasma were lower (p<0.001 than the reference standards in all cases.Hydrofection of hAAT DNA to "in vivo" isolated pig liver mediates highly efficient gene delivery and protein expression in tissue. Both endovascular and surgically closed models mediate high tissue protein

  16. Studying Closed Hydrodynamic Models of “In Vivo” DNA Perfusion in Pig Liver for Gene Therapy Translation to Humans

    Science.gov (United States)

    Sendra, Luis; Miguel, Antonio; Pérez-Enguix, Daniel; Montalvá, Eva; García-Gimeno, María Adelaida; Noguera, Inmaculada; Díaz, Ana; Pérez, Judith; Sanz, Pascual; López-Andújar, Rafael; Martí-Bonmatí, Luis; Aliño, Salvador F.

    2016-01-01

    Introduction Expressing exogenous genes after naked DNA delivery into hepatocytes might achieve sustained and high expression of human proteins. Tail vein DNA injection is an efficient procedure for gene transfer in murine liver. Hydrodynamic procedures in large animals require organ targeting, and improve with liver vascular exclusion. In the present study, two closed liver hydrofection models employing the human alpha-1-antitrypsin (hAAT) gene are compared to reference standards in order to evaluate their potential clinical interest. Material and Methods A solution of naked DNA bearing the hAAT gene was retrogradely injected in 7 pig livers using two different closed perfusion procedures: an endovascular catheterization-mediated procedure (n = 3) with infrahepatic inferior vena cava and portal vein blockage; and a surgery-mediated procedure (n = 4) with completely sealed liver. Gene transfer was performed through the suprahepatic inferior cava vein in the endovascular procedure and through the infrahepatic inferior vena cava in the surgical procedure. The efficiency of the procedures was evaluated 14 days after hydrofection by quantifying the hAAT protein copies per cell in tissue and in plasma. For comparison, samples from mice (n = 7) successfully hydrofected with hAAT and healthy human liver segments (n = 4) were evaluated. Results Gene decoding occurs efficiently using both procedures, with liver vascular arrest improving its efficiency. The surgically closed procedure (sealed organ) reached higher tissue protein levels (4x10^5- copies/cell) than the endovascular procedure, though the levels were lower than in human liver (5x10^6- copies/cell) and hydrofected mouse liver (10^6- copies/cell). However, protein levels in plasma were lower (p<0.001) than the reference standards in all cases. Conclusion Hydrofection of hAAT DNA to “in vivo” isolated pig liver mediates highly efficient gene delivery and protein expression in tissue. Both endovascular and

  17. Human fetuin/alpha2HS-glycoprotein level as a novel indicator of liver cell function and short-term mortality in patients with liver cirrhosis and liver cancer.

    Science.gov (United States)

    Kalabay, László; Jakab, Lajos; Prohászka, Zoltán; Füst, George; Benkö, Zsuzsa; Telegdy, László; Lörincz, Zsolt; Závodszky, Péter; Arnaud, Philippe; Fekete, Béla

    2002-04-01

    Human fetuin/alpha2HS-glycoprotein (AHSG) is synthesized by hepatocytes. We intended to determine whether liver dysfunction or acute phase reaction is dominant in the regulation of its serum concentrations and to see if decreased AHGS levels are associated with short-term mortality. We determined the serum AHSG levels in patients with acute alcoholic, acute A, B, and Epstein-Barr virus hepatitis, alcoholic cirrhosis, and hepatocellular cancer and correlated them to conventional laboratory parameters of inflammation and liver function. Patients were followed for 1 month. Serum AHSG was determined by radial immunodiffusion. Compared to controls, significantly lower AHSG levels were found in patients with liver cirrhosis and hepatocellular cancer but not the acute viral hepatitides. Strong positive correlation with serum transferrin, albumin and prothrombin was found. Febrile episodes were not associated with significantly decreased AHSG levels. Concentrations below 300 microg/ml were associated with high mortality rate (52.0%; relative risk, 5.497; 95% confidence interval, 2.472-12.23; P AHSG levels showed the greatest difference between deceased and survived patients with cirrhosis and cancer. Moreover, other acute phase reactants did not differ significantly. The multiple logistic regression analysis indicated that the decrease of serum AHSG is independent of all other variables that were found decreased in deceased patients. Decreased serum AHSG concentration is due rather to hepatocellular dysfunction than the acute phase reaction and is an outstanding predictor of short-term mortality in patients with liver cirrhosis and liver cancer.

  18. Rheumatic Manifestations in Autoimmune Liver Disease.

    Science.gov (United States)

    Selmi, Carlo; Generali, Elena; Gershwin, Merrill Eric

    2018-02-01

    Autoimmune liver diseases coexist with rheumatic disorders in approximately 30% of cases and may also share pathogenic mechanisms. Autoimmune liver diseases result from an immune-mediated injury of different tissues, with autoimmune hepatitis (AIH) targeting hepatocytes, and primary biliary cholangitis (PBC) and primary sclerosing cholangitis targeting cholangiocytes. Sjogren syndrome is diagnosed in 7% of AIH cases and serologic autoimmunity profiles are a common laboratory abnormality, particularly in the case of serum antimitochondrial (PBC) or anti-liver kidney microsomal antibodies (AIH). Therapeutic strategies may overlap between rheumatic and autoimmune liver diseases and practitioners should be vigilant in managing bone loss. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Foxa1 reduces lipid accumulation in human hepatocytes and is down-regulated in nonalcoholic fatty liver.

    Directory of Open Access Journals (Sweden)

    Marta Moya

    Full Text Available Triglyceride accumulation in nonalcoholic fatty liver (NAFL results from unbalanced lipid metabolism which, in the liver, is controlled by several transcription factors. The Foxa subfamily of winged helix/forkhead box (Fox transcription factors comprises three members which play important roles in controlling both metabolism and homeostasis through the regulation of multiple target genes in the liver, pancreas and adipose tissue. In the mouse liver, Foxa2 is repressed by insulin and mediates fasting responses. Unlike Foxa2 however, the role of Foxa1 in the liver has not yet been investigated in detail. In this study, we evaluate the role of Foxa1 in two human liver cell models, primary cultured hepatocytes and HepG2 cells, by adenoviral infection. Moreover, human and rat livers were analyzed to determine Foxa1 regulation in NAFL. Results demonstrate that Foxa1 is a potent inhibitor of hepatic triglyceride synthesis, accumulation and secretion by repressing the expression of multiple target genes of these pathways (e.g., GPAM, DGAT2, MTP, APOB. Moreover, Foxa1 represses the fatty acid transporter protein FATP2 and lowers fatty acid uptake. Foxa1 also increases the breakdown of fatty acids by inducing peroxisomal fatty acid β-oxidation and ketone body synthesis. Finally, Foxa1 is able to largely up-regulate UCP1, thereby dissipating energy and consistently decreasing the mitochondria membrane potential. We also report that human and rat NAFL have a reduced Foxa1 expression, possibly through a protein kinase C-dependent pathway. We conclude that Foxa1 is an antisteatotic factor that coordinately tunes several lipid metabolic pathways to block triglyceride accumulation in hepatocytes. However, Foxa1 is down-regulated in human and rat NAFL and, therefore, increasing Foxa1 levels could protect from steatosis. Altogether, we suggest that Foxa1 could be a novel therapeutic target for NAFL disease and insulin resistance.

  20. Effect of β-naphthoflavone and MCPA on liver and kidney drug-metabolizing enzymes from the carp, Cyprinus carpio

    OpenAIRE

    Riviere, J.L.; Devaux, A.; Gonin, O.; Monod, Gilles

    1990-01-01

    The effects of β-naphthoflavone (β-NF) and a chlorophenoxyacetic acid herbicide (MCPA) on hepatic and renal monooxygenase activities and conjugating enzymes from immature carp (Cyprinus carpio) were studied. β-NF increased hepatic monooxygenase activities but the patterns of differential induction generally obtained in rat liver microsomes with two series of homologous substrates, alkoxycoumarins and alkylresorufins, were not found to be similar in carp liver microsomes. On the other hand, MC...

  1. Triple Staining Including FOXA2 Identifies Stem Cell Lineages Undergoing Hepatic and Biliary Differentiation in Cirrhotic Human Liver.

    Science.gov (United States)

    Rogler, Charles E; Bebawee, Remon; Matarlo, Joe; Locker, Joseph; Pattamanuch, Nicole; Gupta, Sanjeev; Rogler, Leslie E

    2017-01-01

    Recent investigations have reported many markers associated with human liver stem/progenitor cells, "oval cells," and identified "niches" in diseased livers where stem cells occur. However, there has remained a need to identify entire lineages of stem cells as they differentiate into bile ducts or hepatocytes. We have used combined immunohistochemical staining for a marker of hepatic commitment and specification (FOXA2 [Forkhead box A2]), hepatocyte maturation (Albumin and HepPar1), and features of bile ducts (CK19 [cytokeratin 19]) to identify lineages of stem cells differentiating toward the hepatocytic or bile ductular compartments of end-stage cirrhotic human liver. We identified large clusters of disorganized, FOXA2 expressing, oval cells in localized liver regions surrounded by fibrotic matrix, designated as "micro-niches." Specific FOXA2-positive cells within the micro-niches organize into primitive duct structures that support both hepatocytic and bile ductular differentiation enabling identification of entire lineages of cells forming the two types of structures. We also detected expression of hsa-miR-122 in primitive ductular reactions expected for hepatocytic differentiation and hsa-miR-23b cluster expression that drives liver cell fate decisions in cells undergoing lineage commitment. Our data establish the foundation for a mechanistic hypothesis on how stem cell lineages progress in specialized micro-niches in cirrhotic end-stage liver disease.

  2. Cadmium Chloride Induces DNA Damage and Apoptosis of Human Liver Carcinoma Cells via Oxidative Stress

    Directory of Open Access Journals (Sweden)

    Anthony Skipper

    2016-01-01

    Full Text Available Cadmium is a heavy metal that has been shown to cause its toxicity in humans and animals. Many documented studies have shown that cadmium produces various genotoxic effects such as DNA damage and chromosomal aberrations. Ailments such as bone disease, renal damage, and several forms of cancer are attributed to overexposure to cadmium.  Although there have been numerous studies examining the effects of cadmium in animal models and a few case studies involving communities where cadmium contamination has occurred, its molecular mechanisms of action are not fully elucidated. In this research, we hypothesized that oxidative stress plays a key role in cadmium chloride-induced toxicity, DNA damage, and apoptosis of human liver carcinoma (HepG2 cells. To test our hypothesis, cell viability was determined by MTT assay. Lipid hydroperoxide content stress was estimated by lipid peroxidation assay. Genotoxic damage was tested by the means of alkaline single cell gel electrophoresis (Comet assay. Cell apoptosis was measured by flow cytometry assessment (Annexin-V/PI assay. The result of MTT assay indicated that cadmium chloride induces toxicity to HepG2 cells in a concentration-dependent manner, showing a 48 hr-LD50 of 3.6 µg/mL. Data generated from lipid peroxidation assay resulted in a significant (p < 0.05 increase of hydroperoxide production, specifically at the highest concentration tested. Data obtained from the Comet assay indicated that cadmium chloride causes DNA damage in HepG2 cells in a concentration-dependent manner. A strong concentration-response relationship (p < 0.05 was recorded between annexin V positive cells and cadmium chloride exposure. In summary, these in vitro studies provide clear evidence that cadmium chloride induces oxidative stress, DNA damage, and programmed cell death in human liver carcinoma (HepG2 cells.

  3. Customised in vitro model to detect human metabolism-dependent idiosyncratic drug-induced liver injury.

    Science.gov (United States)

    Tolosa, Laia; Jiménez, Nuria; Pérez, Gabriela; Castell, José V; Gómez-Lechón, M José; Donato, M Teresa

    2017-07-31

    Drug-induced liver injury (DILI) has a considerable impact on human health and is a major challenge in drug safety assessments. DILI is a frequent cause of liver injury and a leading reason for post-approval drug regulatory actions. Considerable variations in the expression levels of both cytochrome P450 (CYP) and conjugating enzymes have been described in humans, which could be responsible for increased susceptibility to DILI in some individuals. We herein explored the feasibility of the combined use of HepG2 cells co-transduced with multiple adenoviruses that encode drug-metabolising enzymes, and a high-content screening assay to evaluate metabolism-dependent drug toxicity and to identify metabolic phenotypes with increased susceptibility to DILI. To this end, HepG2 cells with different expression levels of specific drug-metabolism enzymes (CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, GSTM1 and UGT2B7) were exposed to nine drugs with reported hepatotoxicity. A panel of pre-lethal mechanistic parameters (mitochondrial superoxide production, mitochondrial membrane potential, ROS production, intracellular calcium concentration, apoptotic nuclei) was used. Significant differences were observed according to the level of expression and/or the combination of several drug-metabolism enzymes in the cells created ad hoc according to the enzymes implicated in drug toxicity. Additionally, the main mechanisms implicated in the toxicity of the compounds were also determined showing also differences between the different types of cells employed. This screening tool allowed to mimic the variability in drug metabolism in the population and showed a highly efficient system for predicting human DILI, identifying the metabolic phenotypes associated with increased DILI risk, and indicating the mechanisms implicated in their toxicity.

  4. Isovolemic hemodilution with glutaraldehyde-polymerized human placenta hemoglobin (PolyPHb) attenuated rat liver ischemia/reperfusion injury.

    Science.gov (United States)

    You, Zhen; Li, Qian; Li, Bei; Yang, Chengmin; Liu, Jin; Li, Tao

    2014-04-01

    This study was to investigate whether glutaraldehyde-polymerized human placenta hemoglobin (PolyPHb) could attenuate ischemia/reperfusion (I/R)-induced liver injury. Isovolemic hemodilution of SD rats was performed by exchanging 15% total blood volume with PolyPHb. I/R was induced by left liver lobes pedicle cross-clamping for 60 min and reperfusion for 2 h. Blood pressure moderately elevated after PolyPHb infusion and returned to basal level within 10 min. The hepatic histopathological damage and the activities of liver injury markers were reduced by PolyPHb. The TUNEL staining and caspase assay indicated hepatic apoptosis was also inhibited. Therefore, our findings suggest PolyPHb can reduce liver I/R injury.

  5. (99m) Tc-labelled human serum albumin cannot replace (125) I-labelled human serum albumin to determine plasma volume in patients with liver disease

    DEFF Research Database (Denmark)

    Henriksen, Ulrik Lütken; Henriksen, Jens H; Bendtsen, Flemming

    2013-01-01

    -labelled human serum albumin (99mTc-HSA) and iodine-labelled human serum albumin (125I-HSA), as the former may have advantages at repeated measurements and the latter is the classical gold standard. Study population and methods In 88 patients, (64 with liver disease, mainly cirrhosis, and 24 patients without...

  6. Serum glycoproteins in the liver diseases. VII. Further studies on the properties of desialylated glycoprotein binding activity in particulate fraction of human liver homogenate.

    Science.gov (United States)

    Arima, T

    1979-08-01

    Binding of desialylated alpha 1-acid glycoprotein by human liver particulate fraction exhibited a dependence on the presence of calcium chloride whereas Cu+, Mn+, Zn+ Fe+ and Co+ inhibited the binding. The other cations such as K+, Na+, Ba+, Mg+ or Pb+ were determined to be non-effective on the binding activity. The pH of the assay for binding was not critical in the range of 6.5 to 9.5. The binding process required the presence of terminal sialic acid on the particulate protein. Fifty nine per cent of binding activity in the original liver paticulate fraction were recovered in acetone powder. Extraction of the acetone powder with a buffer containing EDTA resulted in an increased total binding activity. After extraction with 1--10% Triton X-100, 60% of the activity were still detected in insoluble fraction.

  7. The Proteome of Human Liver Peroxisomes: Identification of Five New Peroxisomal Constituents by a Label-Free Quantitative Proteomics Survey

    OpenAIRE

    Thomas Gronemeyer; Sebastian Wiese; Rob Ofman; Christian Bunse; Magdalena Pawlas; Heiko Hayen; Martin Eisenacher; Christian Stephan; Meyer, Helmut E.; Waterham, Hans R.; Ralf Erdmann; Wanders, Ronald J; Bettina Warscheid

    2013-01-01

    The peroxisome is a key organelle of low abundance that fulfils various functions essential for human cell metabolism. Severe genetic diseases in humans are caused by defects in peroxisome biogenesis or deficiencies in the function of single peroxisomal proteins. To improve our knowledge of this important cellular structure, we studied for the first time human liver peroxisomes by quantitative proteomics. Peroxisomes were isolated by differential and Nycodenz density gradient centrifugation. ...

  8. Detection of human herpesvirus-7 by qualitative nested-PCR: comparison between healthy individuals and liver transplant recipients

    OpenAIRE

    Thomasini, Ronaldo Luis; Martins, Juliana de Moraes; Parola, Daniela Corte; Bonon, Sandra Helena Alves; Boin, Ilka de Fátima Santana Ferreira; Leonardi, Luis Sérgio; Leonardi, Marília; Costa, Sandra Cecília Botelho

    2008-01-01

    Diagnosis of human herpesvirus-7 active infection in transplant patients has proved difficult, because this virus is ubiquitous and can cause persistent infections in the host. The significance of viral DNA detected in leukocytes by PCR is unclear and cross-reaction in serological tests may occur. This study aimed to evaluate nested-PCR to detect human herpesvirus-7 active infection in liver transplant recipients compared to healthy individuals. human herpesvirus-7 nested-PCR was performed on...

  9. Metabolism of 17-(allylamino)-17-demethoxygeldanamycin (NSC 330507) by murine and human hepatic preparations.

    Science.gov (United States)

    Egorin, M J; Rosen, D M; Wolff, J H; Callery, P S; Musser, S M; Eiseman, J L

    1998-06-01

    17-(Allylamino)-17-demethoxygeldanamycin (17AAG), a compound that is proposed for clinical development, shares the ability of geldanamycin to bind to heat shock protein 90 and GRP94, thereby depleting cells of p185erbB2, mutant p53, and Raf-1. Urine and plasma from mice treated i.v. with 17AAG contained six materials with absorption spectra similar to that of 17AAG. Therefore, in vitro metabolism of 17AAG by mouse and human hepatic preparations was studied to characterize: (a) the enzymes responsible for 17AAG metabolism; and (b) the structures of the metabolites produced. These materials had retention times on high-performance liquid chromatography of approximately 2, 4, 5, 6, 7, and 9 min. When incubated in an aerobic environment with 17AAG, murine hepatic supernatant (9000 x g) produced each of these compounds; the 4-min metabolite was the major product. This metabolism required an electron donor, and NADPH was favored over NADH. Metabolic activity resided predominantly in the microsomal fraction. Metabolism was decreased by approximately 80% in anaerobic conditions and was essentially ablated by CO. Microsomes prepared from human livers produced essentially the same metabolites as produced by murine hepatic microsomes, but the 2-min metabolite was the major product, and the 4-min metabolite was next largest. There was no metabolism of 17AAG by human liver cytosol. Metabolism of 17AAG by human liver microsomes also required an electron donor, with NADPH being preferred over NADH, was inhibited by approximately 80% under anaerobic conditions, and was essentially ablated by CO. Liquid chromatography/mass spectrometry analysis of human and mouse in vitro reaction mixtures indicated the presence of materials with molecular weights of 545, 601, and 619, compatible with 17-(amino)-17-demethoxygeldanamycin (17AG), an epoxide, and a diol, respectively. The metabolite with retention time of 4 min was identified as 17AG by cochromatography and mass spectral concordance

  10. Toll-like receptor 8 agonist and bacteria trigger potent activation of innate immune cells in human liver.

    Directory of Open Access Journals (Sweden)

    Juandy Jo

    2014-06-01

    Full Text Available The ability of innate immune cells to sense and respond to impending danger varies by anatomical location. The liver is considered tolerogenic but is still capable of mounting a successful immune response to clear various infections. To understand whether hepatic immune cells tune their response to different infectious challenges, we probed mononuclear cells purified from human healthy and diseased livers with distinct pathogen-associated molecules. We discovered that only the TLR8 agonist ssRNA40 selectively activated liver-resident innate immune cells to produce substantial quantities of IFN-γ. We identified CD161(Bright mucosal-associated invariant T (MAIT and CD56(Bright NK cells as the responding liver-resident innate immune cells. Their activation was not directly induced by the TLR8 agonist but was dependent on IL-12 and IL-18 production by ssRNA40-activated intrahepatic monocytes. Importantly, the ssRNA40-induced cytokine-dependent activation of MAIT cells mirrored responses induced by bacteria, i.e., generating a selective production of high levels of IFN-γ, without the concomitant production of TNF-α or IL-17A. The intrahepatic IFN-γ production could be detected not only in healthy livers, but also in HBV- or HCV-infected livers. In conclusion, the human liver harbors a network of immune cells able to modulate their immunological responses to different pathogen-associated molecules. Their ability to generate a strong production of IFN-γ upon stimulation with TLR8 agonist opens new therapeutic opportunities for the treatment of diverse liver pathologies.

  11. Toll-like receptor 8 agonist and bacteria trigger potent activation of innate immune cells in human liver.

    Science.gov (United States)

    Jo, Juandy; Tan, Anthony T; Ussher, James E; Sandalova, Elena; Tang, Xin-Zi; Tan-Garcia, Alfonso; To, Natalie; Hong, Michelle; Chia, Adeline; Gill, Upkar S; Kennedy, Patrick T; Tan, Kai Chah; Lee, Kang Hoe; De Libero, Gennaro; Gehring, Adam J; Willberg, Christian B; Klenerman, Paul; Bertoletti, Antonio

    2014-06-01

    The ability of innate immune cells to sense and respond to impending danger varies by anatomical location. The liver is considered tolerogenic but is still capable of mounting a successful immune response to clear various infections. To understand whether hepatic immune cells tune their response to different infectious challenges, we probed mononuclear cells purified from human healthy and diseased livers with distinct pathogen-associated molecules. We discovered that only the TLR8 agonist ssRNA40 selectively activated liver-resident innate immune cells to produce substantial quantities of IFN-γ. We identified CD161(Bright) mucosal-associated invariant T (MAIT) and CD56(Bright) NK cells as the responding liver-resident innate immune cells. Their activation was not directly induced by the TLR8 agonist but was dependent on IL-12 and IL-18 production by ssRNA40-activated intrahepatic monocytes. Importantly, the ssRNA40-induced cytokine-dependent activation of MAIT cells mirrored responses induced by bacteria, i.e., generating a selective production of high levels of IFN-γ, without the concomitant production of TNF-α or IL-17A. The intrahepatic IFN-γ production could be detected not only in healthy livers, but also in HBV- or HCV-infected livers. In conclusion, the human liver harbors a network of immune cells able to modulate their immunological responses to different pathogen-associated molecules. Their ability to generate a strong production of IFN-γ upon stimulation with TLR8 agonist opens new therapeutic opportunities for the treatment of diverse liver pathologies.

  12. The interaction of bacterial magnetosomes and human liver cancer cells in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Pingping, E-mail: wangpp@mail.iee.ac.cn [Beijing Key Laboratory of Bioelectromagnetism, Institute of Electrical Engineering, Chinese Academy of Sciences, Beijing 100190 (China); Chen, Chuanfang [Beijing Key Laboratory of Bioelectromagnetism, Institute of Electrical Engineering, Chinese Academy of Sciences, Beijing 100190 (China); Chen, Changyou [Beijing Key Laboratory of Bioelectromagnetism, Institute of Electrical Engineering, Chinese Academy of Sciences, Beijing 100190 (China); University of Chinese Academy of Sciences, Beijing 100049 (China); Li, Yue; Pan, Weidong; Song, Tao [Beijing Key Laboratory of Bioelectromagnetism, Institute of Electrical Engineering, Chinese Academy of Sciences, Beijing 100190 (China)

    2017-04-01

    As the biogenic magnetic nanomaterial, bacterial magnetic nanoparticles, namely magnetosomes, provide many advantages for potential biomedical applications. As such, interactions among magnetosomes and target cells should be elucidated to develop their bioapplications and evaluate their biocompatibilities. In this study, the interaction of magnetosomes and human liver cancer HepG2 cells was examined. Prussian blue staining revealed numerous stained particles in or on the cells. Intracellular iron concentrations, measured through inductively coupled plasma optical emission spectroscopy, increased with the increasing concentration of the magnetosomes. Transmission electron microscopy images showed that magnetosomes could be internalized in cells, mainly encapsulated in membrane vesicles, such as endosomes and lysosomes, and partly found as free particles in the cytosol. Some of the magnetosomes on cellular surfaces were encapsulated through cell membrane ruffling, which is the initiating process of endocytosis. Applying low temperature treatment and using specific endocytic inhibitors, we validated that macropinocytosis and clathrin-mediated endocytosis were involved in magnetosome uptake by HepG2 cells. Consequently, we revealed the interaction and intrinsic endocytic mechanisms of magnetosomes and HepG2 cells. This study provides a basis for the further research on bacterial magnetosome applications in liver diseases. - Highlights: • Bacterial magnetosomes interact with HepG2 cells in a dose-dependent manner. • Magnetosomes are wrapped by membrane ruffling on cell surface. • Internalized magnetosomes mainly localize in endosomes and lysosomes. • Macropinocytosis and CME are involved in the cellular uptake of magnetosomes.

  13. Targeting liver X receptors in human health: deadlock or promising trail?

    Science.gov (United States)

    Viennois, Emilie; Pommier, Aurélien J C; Mouzat, Kévin; Oumeddour, Abdelkader; El Hajjaji, Fatim-Zohra; Dufour, Julie; Caira, Françoise; Volle, David H; Baron, Silvère; Lobaccaro, Jean-Marc A

    2011-02-01

    Liver X receptors (LXR) are transcription factors that belong to the nuclear receptor superfamily. Natural derivatives of cholesterol, known as oxysterols, have been identified as agonistic ligands of LXR. They are thus mainly considered to be intracellular cholesterol 'sensors' whose activation leads to decreased plasma cholesterol. Their implication in other physiologic processes currently prevents their use as therapeutic targets, because of potentially deleterious side effects. The various LXR agonists and antagonists, along with the physiological functions of LXR. Putative clinical targets including atherosclerosis, diabetes, Alzheimer's disease, skin disorders, reproductive disorders and cancer. LXR are promising pharmacological targets because of the high potential to develop ligands owing to the variety of natural or synthetic agonists. Three aspects should be developed to select a LXR-ligand for treatment of human disease: bio-availability; isoform specificity; tissue specificity. This will allow the development of selective liver X modulators (SLiMs). The challenge is to overcome deleterious side effects to establish LXR as new pharmacological targets.

  14. Mode of action and human relevance of THF-induced mouse liver tumors.

    Science.gov (United States)

    Choi, Christopher J; Rushton, Erik K; Vardy, Audrey; Higgins, Larry; Augello, Andrea; Parod, Ralph J

    2017-07-05

    In a National Toxicology Program (NTP) bioassay, inhalation of tetrahydrofuran (THF) induced liver tumors in female B6C3F1 mice but not in male mice or rats of either sex. Since THF is not genotoxic, the NTP concluded this carcinogenic activity was likely mediated via non-genotoxic modes of action (MOA). Based on evidence that THF and phenobarbital share a similar MOA, female Car/Pxr knock-out mice were orally exposed to THF to evaluate the potential role of CAR activation in the MOA for THF-induced liver tumors. Because data from this oral study with Car/Pxr knock-out mice (C57Bl/6) and the inhalation studies with wild type mice (B6C3F1) reported by NTP and others were derived from different strains, oral studies with wild type B6C3F1 and C57Bl/6 mice were conducted to ensure THF responses in both strains were comparable. As seen in inhalation studies with THF, oral exposure of wild type female mice to a maximum tolerated dose of THF increased total P450 content, CAR-related P450 activities, and hepatocyte proliferation; these effects were not observed in Car/Pxr knock-out female mice. This finding supports the hypothesis THF-induced carcinogenicity is likely mediated via CAR activation that has limited, if any, relevance to humans. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  15. The interaction of bacterial magnetosomes and human liver cancer cells in vitro

    Science.gov (United States)

    Wang, Pingping; Chen, Chuanfang; Chen, Changyou; Li, Yue; Pan, Weidong; Song, Tao

    2017-04-01

    As the biogenic magnetic nanomaterial, bacterial magnetic nanoparticles, namely magnetosomes, provide many advantages for potential biomedical applications. As such, interactions among magnetosomes and target cells should be elucidated to develop their bioapplications and evaluate their biocompatibilities. In this study, the interaction of magnetosomes and human liver cancer HepG2 cells was examined. Prussian blue staining revealed numerous stained particles in or on the cells. Intracellular iron concentrations, measured through inductively coupled plasma optical emission spectroscopy, increased with the increasing concentration of the magnetosomes. Transmission electron microscopy images showed that magnetosomes could be internalized in cells, mainly encapsulated in membrane vesicles, such as endosomes and lysosomes, and partly found as free particles in the cytosol. Some of the magnetosomes on cellular surfaces were encapsulated through cell membrane ruffling, which is the initiating process of endocytosis. Applying low temperature treatment and using specific endocytic inhibitors, we validated that macropinocytosis and clathrin-mediated endocytosis were involved in magnetosome uptake by HepG2 cells. Consequently, we revealed the interaction and intrinsic endocytic mechanisms of magnetosomes and HepG2 cells. This study provides a basis for the further research on bacterial magnetosome applications in liver diseases.

  16. Human Liver Stem Cells Suppress T-Cell Proliferation, NK Activity, and Dendritic Cell Differentiation

    Directory of Open Access Journals (Sweden)

    Stefania Bruno

    2016-01-01

    Full Text Available Human liver stem cells (HLSCs are a mesenchymal stromal cell-like population resident in the adult liver. Preclinical studies indicate that HLSCs could be a good candidate for cell therapy. The aim of the present study was to evaluate the immunogenicity and the immunomodulatory properties of HLSCs on T-lymphocytes, natural killer cells (NKs, and dendritic cells (DCs in allogeneic experimental settings. We found that HLSCs inhibited T-cell proliferation by a mechanism independent of cell contact and dependent on the release of prostaglandin E2 (PGE2 and on indoleamine 2,3-dioxygenase activity. When compared with mesenchymal stromal cells (MSCs, HLSCs were more efficient in inhibiting T-cell proliferation. At variance with MSCs, HLSCs did not elicit NK degranulation. Moreover, HLSCs inhibited NK degranulation against K562, a NK-sensitive target, by a mechanism dependent on HLA-G release. When tested on DC generation from monocytes, HLSCs were found to impair DC differentiation and DCs ability to induce T-cell proliferation through PGE2. This study shows that HLSCs have immunomodulatory properties similar to MSCs, but, at variance with MSCs, they do not elicit a NK response.

  17. Evaluating the MoA/human relevance framework for F-344 rat liver epithelioid granulomas with mineral oil hydrocarbons.

    Science.gov (United States)

    Adenuga, David; Goyak, Katy; Lewis, R Jeffrey

    2017-10-01

    Toxicology feeding studies of mineral oil hydrocarbons (MOHs), within the carbon number range C22-C28, results in species-specific epithelioid granulomas in the liver of F-344 rats but not in other rat strains, or species. While MOH has been detected, and some pathological effects have been shown to occur in other organs/tissues of F-344 rats and other rat strains/species, it is generally accepted that the effect of toxicological concern is species-specific inflammatory liver granuloma. As oil retention and other MOH-related nontoxic pathological changes in the liver are observed in humans, some have hypothesized that the potential for oil accumulation over a lifetime, through dietary sources, may predispose humans to similar liver effects as observed in F-344 rats. To address this concern, a mode of action/human relevance framework (MoA/HRF) analysis for MOH-induced epithelioid granuloma in the F-344 rat model was developed. The key events for the development of liver epithelioid granulomas were identified as increased MOH intestinal absorption, preferential tissue retention and ultimately formation of necrotic granulomas encased by infiltrating inflammatory lymphocytes. The hypothesized MoA was evaluated using the modified Bradford Hill considerations for causality and was considered to be established in the F-344 rodent model. However, key strain/species differences in the rate of intestinal absorption, tissue retention of MOH and inflammatory response to MOH in the liver were identified. Overall, the F-344 rat MoA was not considered to be relevant to humans, consistent with data showing no evidence for the formation of epithelioid granulomas with humans even in cases of massive ingestion of MOHs.

  18. Identification of potential biomarkers of hepatitis B-induced acute liver failure using hepatic cells derived from human skin precursors.

    Science.gov (United States)

    Rodrigues, Robim M; Sachinidis, Agapios; De Boe, Veerle; Rogiers, Vera; Vanhaecke, Tamara; De Kock, Joery

    2015-09-01

    Besides their role in the elucidation of pathogenic processes of medical and pharmacological nature, biomarkers can also be used to document specific toxicological events. Hepatic cells generated from human skin-derived precursors (hSKP-HPC) were previously shown to be a promising in vitro tool for the evaluation of drug-induced hepatotoxicity. In this study, their capacity to identify potential liver-specific biomarkers at the gene expression level was investigated with particular emphasis on acute liver failure (ALF). To this end, a set of potential ALF-specific biomarkers was established using clinically relevant liver samples obtained from patients suffering from hepatitis B-associated ALF. Subsequently, this data was compared to data obtained from primary human hepatocyte cultures and hSKP-HPC, both exposed to the ALF-inducing reference compound acetaminophen. It was found that both in vitro systems revealed a set of molecules that was previously identified in the ALF liver samples. Yet, only a limited number of molecules was common between both in vitro systems and the ALF liver samples. Each of the in vitro systems could be used independently to identify potential toxicity biomarkers related to ALF. It seems therefore more appropriate to combine primary human hepatocyte cultures with complementary in vitro models to efficiently screen out potential hepatotoxic compounds. Copyright © 2014 Elsevier Ltd. All rights reserved.

  19. Purification, properties, and immunocytochemical localization of human liver peroxisomal enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase.

    Science.gov (United States)

    Reddy, M K; Usuda, N; Reddy, M N; Kuczmarski, E R; Rao, M S; Reddy, J K

    1987-01-01

    A molecular understanding of genetic disease in which peroxisomal functions are impaired depends on analysis of the structure of normal and mutant enzymes of peroxisomes. We report experiments describing the isolation, characterization, and immunocytochemical localization of enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme (PBE) of the peroxisomal fatty acid beta-oxidation system from normal human liver and compared it with that of rat liver enzyme. The human enzyme, purified approximately equal to 2300-fold by ion-exchange chromatography, is homogeneous as judged by NaDodSO4/PAGE. This PBE is localized exclusively in the matrix of peroxisomes in liver cells by the protein A/gold immunocytochemical method. The human PBE is similar to rat enzyme in size (Mr, approximately equal to 79,000), isoelectric point (pI, 9.8), pH optima, molecular structure as observed by rotary shadowing, and peptide pattern on NaDodSO4/PAGE after proteolytic digestion with Staphylococcus aureus V8 protease. The human and rat enzymes differed in their immunological properties by having partial identity with each other; this is reflected in their slightly dissimilar composition of the amino acids aspartic acid, threonine, glutamic acid, tyrosine, and glycine. COOH-terminal amino acid were similar for both the enzymes: -Gly-Ser-Leu-Ile-COOH. These results suggest that the human and rat liver PBE may be different in their amino acid sequences at their antigenic sites. Images PMID:3106963

  20. Peritransplant energy changes and their correlation to outcome after human liver transplantation

    NARCIS (Netherlands)

    Bruinsma, Bote G; Avruch, James H; Sridharan, Gautham V; Weeder, Pepijn D; Jacobs, Marie Louise; Crisalli, Kerry; Amundsen, Beth; Porte, Robert J; Markmann, James F; Uygun, Korkut; Yeh, Heidi

    BACKGROUND: The ongoing shortage of donor livers for transplantation and the increased use of marginal livers necessitate the development of accurate pretransplant tests of viability. Considering the importance energy status during transplantation, we aimed to correlate peritransplant energy

  1. Feasibility analysis of a Plasma Focus neutron source for BNCT treatment of transplanted human liver

    Science.gov (United States)

    Benzi, V.; Mezzetti, F.; Rocchi, F.; Sumini, M.

    2004-01-01

    Boron Neutron Capture Therapy preliminary treatments on transplanted human liver have been recently conducted at Pavia University. The need of high fluences of thermal neutrons imposed the use of the available thermal channel of a TRIGA reactor properly modified for this application. We analyse the possibility of using the Plasma Focus (PF) machine as a pulsed neutron source for this medical application instead of a nuclear reactor. Thermalization of the fast (2.45 MeV for D-D reactions) neutrons produced by the PF is gained with a paraffin or polyethylene moderator which contains both the neutron source and the irradiation chamber. The design parameters of a PF optimized for such an application are discussed, as well as other considerations on the advantages that this machine can bring to this kind of cancer therapy.

  2. Inhibition Of Microsomal Lipid Peroxidation And Protein Oxidation ...

    African Journals Online (AJOL)

    The antioxidant activities of 53 medicinal plants used in Bamun Folk Medicine for the management of jaundice and hepatitis were investigated. The studies were done using rat hepatic microsomes for lipid peroxidation and bovine serum albumin (BSA) for carbonyl group formation. Silymarine was used as reference ...