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Sample records for human lung microsomes

  1. Metabolism of the anti-tuberculosis drug ethionamide by mouse and human FMO1, FMO2 and FMO3 and mouse and human lung microsomes

    SciTech Connect

    Henderson, Marilyn C.; Siddens, Lisbeth K.; Morre, Jeffrey T.; Krueger, Sharon K.; Williams, David E.

    2008-12-15

    Tuberculosis (TB) results from infection with Mycobacterium tuberculosis and remains endemic throughout the world with one-third of the world's population infected. The prevalence of multi-drug resistant strains necessitates the use of more toxic second-line drugs such as ethionamide (ETA), a pro-drug requiring bioactivation to exert toxicity. M. tuberculosis possesses a flavin monooxygenase (EtaA) that oxygenates ETA first to the sulfoxide and then to 2-ethyl-4-amidopyridine, presumably through a second oxygenation involving sulfinic acid. ETA is also a substrate for mammalian flavin-containing monooxygenases (FMOs). We examined activity of expressed human and mouse FMOs toward ETA, as well as liver and lung microsomes. All FMOs converted ETA to the S-oxide (ETASO), the first step in bioactivation. Compared to M. tuberculosis, the second S-oxygenation to the sulfinic acid is slow. Mouse liver and lung microsomes, as well as human lung microsomes from an individual expressing active FMO, oxygenated ETA in the same manner as expressed FMOs, confirming this reaction functions in the major target organs for therapeutics (lung) and toxicity (liver). Inhibition by thiourea, and lack of inhibition by SKF-525A, confirm ETASO formation is primarily via FMO, particularly in lung. ETASO production was attenuated in a concentration-dependent manner by glutathione. FMO3 in human liver may contribute to the toxicity and/or affect efficacy of ETA administration. Additionally, there may be therapeutic implications of efficacy and toxicity in human lung based on the FMO2 genetic polymorphism, though further studies are needed to confirm that suggestion.

  2. Metabolism of N-methylcarbazole by rat lung microsomes.

    PubMed

    Ibe, B O; Raj, J U

    1994-01-01

    N-methylcarbazole (NMC) is a procarcinogenic component of tobacco smoke particulate matter. It is metabolized by liver microsomes into some hydroxylated metabolites such as the potent mutagen N-hydroxymethylcarbazole (NHMC). Lung metabolism and toxicity of NMC is not known. Since the lung is the primary organ of inhalation of tobacco smoke, NMC metabolism by lung microsomes was studied in comparison with the metabolism by liver microsomes. Liver or lung microsomes (1 mg/mL) were incubated with 0.5 mM NMC for 30 min at 37 degrees C. NMC metabolites were extracted with ethyl acetate and analyzed by reversed-phase high-performance liquid chromatography. Rat lung microsomes metabolized NMC with a similar profile to liver microsomes, although lung microsomes produced greater number of metabolites. The potent mutagen NHMC was also the major NMC metabolite produced by lung microsomes, as confirmed by particle beam mass spectrometry. However, lung microsomes produced only 10% of NHMC produced by liver microsomes. Metabolism of NMC by lung microsomes also led to depletion of the endogenous antioxidant glutathione by 34% compared to controls, indicating a significant generation of some reactive intermediates during NMC metabolism by lung microsomes. The data show that the lung participates directly in producing the potent mutagen NHMC from NMC present in tobacco smoke. PMID:7925139

  3. Paraquat and NADPH-dependent lipid peroxidation in lung microsomes

    SciTech Connect

    Misra, H.P.; Gorsky, L.D.

    1981-10-10

    Since there exists some controversy in the literature as to whether paraquat augments microsomal lipid peroxidation via superoxide anion (O/sub 2//sup -/), the role of paraquat and active oxygen species in NADPH-dependent lung microsomal lipid peroxidation was investigated. Incubation of buffered aerobic mixture of bovine lung microsome and NADPH, in the presence or absence of exogenously added iron, resulted in a progressive formation of lipid peroxides whose accumulation could be followed at 535 nm as malondialdehyde. Paraquat strongly inhibited this lipid peroxidation, Thus, malondialydehyde formation was 50% inhibited by 4 X 10/sup -5/ M paraquat in the reaction mixture. The malondialdehyde color development by lipid peroxides was not affected by this concentration of paraquat. Lipid peroxidation was also strongly inhibited by singlet oxygen scavengers, e.g. dimethylfuran and diphenylfuran, and by catalase. Hydroxyl radical scavengers, e.g. mannitol, benzoate, and ethanol, had little effect in malondialydehyde production. Superoxide dismutase, which removes O/sub 2//sup -/ efficiently, did not inhibit malondialdehyde production by lung microsomes and rather enhanced its formation. A scheme in which paraquat and active O/sub 2/ species may be involved with microsomal lipid peroxidation is presented.

  4. Cyclosporin metabolism by human gastrointestinal mucosal microsomes.

    PubMed Central

    Webber, I R; Peters, W H; Back, D J

    1992-01-01

    The in vitro metabolism of the immunosuppressant cyclosporin (CsA) by human gastrointestinal mucosal microsomes has been studied. Macroscopically normal intestinal (n = 4) and liver (n = 2) tissue was obtained from kidney transplant donors, and microsomes prepared. Intestinal metabolism was most extensive with duodenal protein (15% conversion to metabolites M1/M17 after 2 h incubation at 37 degrees C; metabolite measurement by h.p.l.c). Western blotting confirmed the presence of P-4503A (enzyme subfamily responsible for CsA metabolism) in duodenum and ileum tissue, but not in colon tissue. The results of this study indicate that the gut wall may play a role in the first-pass metabolism of CsA, and could therefore be a contributory factor to the highly variable oral bioavailability of CsA. PMID:1389941

  5. Prostaglandin synthesis by chicken and rat lung microsomes

    SciTech Connect

    Craig-Schmidt, M.C.; Faircloth, S.A.; Wu-Wang, C.Y.

    1986-03-01

    A comparison between chicken and rat lung was made for microsomal prostaglandin (PG) synthesis from 1-/sup 14/C-arachidonic acid. Microsomal protein (2.0 mg) from chicken or rat lung was incubated in the presence of 20 ..mu..g of 1-/sup 14/C-arachidonic acid (specific activity = 3 x 10/sup 6/ dpm/..mu..mol for chicken; 6 x 10/sup 6/ dpm/..mu..mol for rat), 0.05 M Tris-HCl buffer (pH = 8.0), 0.5 mM epinephrine, and 1 mM reduced glutathione in a total volume of 0.5 ml in a 37/sup 0/C water bath with shaking for 15 min. After acidification with 1 M HCl to pH 3, prostaglandins were extracted with ethyl acetate. The products of the reactions were separated by reversed phase chromatography, and the radioactivity of each prostanoid fraction was determined. The predominant prostanoid synthesized by chicken lung microsomes was PGE/sub 2/, followed by much lower amounts of thromboxane B/sub 2/ (TXB/sub 2/), PGF/sub 2//sub ..cap alpha../ and PGD/sub 2/. In at lung, 6-keto-PGF/sub 1//sub ..cap alpha../ was the predominant product formed, with minor amounts of 6-keto-PGE/sub 1/, TXB/sub 2/, PGF/sub 2//sub ..cap alpha../ and PGD/sub 2/. In rat lung, 6-keto-FGF/sub 1//sub ..cap alpha../ was the predominant product formed, with minor amounts of 6-keto-PGF/sub 1//sub ..cap alpha../ was the predominant product formed, with minor amounts of 6-keto-PGE/sub 1/, TXB/sub 2/, PGF/sub 2//sub ..cap alpha../, PGE/sub 2/ and PGD/sub 2/ being formed. Enzyme specific activity (pmol of PG produced per mg microsomal protein per min) was 11.9 for PGE/sub 2/ produced by chicken lung and 16. 7 for 6-keto-P/sub 1//sub ..cap alpha../ produced by rat lung. Thus, there appears to be a species variation in chicken compared to rat for the lung prostanoids which are known to cause bronchial dilation.

  6. Comparative study of the hydrolytic metabolism of methyl-, ethyl-, propyl-, butyl-, heptyl- and dodecylparaben by microsomes of various rat and human tissues.

    PubMed

    Ozaki, Hitomi; Sugihara, Kazumi; Watanabe, Yoko; Fujino, Chieri; Uramaru, Naoto; Sone, Tomomichi; Ohta, Shigeru; Kitamura, Shigeyuki

    2013-12-01

    Hydrolytic metabolism of methyl-, ethyl-, propyl-, butyl-, heptyl- and dodecylparaben by various tissue microsomes and plasma of rats, as well as human liver and small-intestinal microsomes, was investigated and the structure-metabolic activity relationship was examined. Rat liver microsomes showed the highest activity toward parabens, followed by small-intestinal and lung microsomes. Butylparaben was most effectively hydrolyzed by the liver microsomes, which showed relatively low hydrolytic activity towards parabens with shorter and longer alkyl side chains. In contrast, small-intestinal microsomes exhibited relatively higher activity toward longer-side-chain parabens, and showed the highest activity towards heptylparaben. Rat lung and skin microsomes showed liver-type substrate specificity. Kidney and pancreas microsomes and plasma of rats showed small-intestinal-type substrate specificity. Liver and small-intestinal microsomal hydrolase activity was completely inhibited by bis(4-nitrophenyl)phosphate, and could be extracted with Triton X-100. Ces1e and Ces1d isoforms were identified as carboxylesterase isozymes catalyzing paraben hydrolysis by anion exchange column chromatography of Triton X-100 extract from liver microsomes. Ces1e and Ces1d expressed in COS cells exhibited significant hydrolase activities with the same substrate specificity pattern as that of liver microsomes. Small-intestinal carboxylesterase isozymes Ces2a and Ces2c expressed in COS cells showed the same substrate specificity as small-intestinal microsomes, being more active toward longer-alkyl-side-chain parabens. Human liver microsomes showed the highest hydrolytic activity toward methylparaben, while human small-intestinal microsomes showed a broadly similar substrate specificity to rat small-intestinal microsomes. Human CES1 and CES2 isozymes showed the same substrate specificity patterns as human liver and small-intestinal microsomes, respectively. PMID:23742084

  7. Protein Targets of Reactive Electrophiles in Human Liver Microsomes

    PubMed Central

    Shin, Nah-Young; Liu, Qinfeng; Stamer, Sheryl L.; Liebler, Daniel C.

    2008-01-01

    Liver microsomes are widely used to study xenobiotic metabolism in vitro and covalent binding to microsomal proteins serves as a surrogate marker for toxicity mediated by reactive metabolites. We have applied liquid chromatography-tandem mass spectrometry (LC-MS-MS) to identify protein targets of the biotin-tagged model electrophiles 1-biotinamido-4-(4′-[maleimidoethylcyclohexane]-carboxamido)butane (BMCC) and N-iodoacetyl-N-biotinylhexylenediamine (IAB) in human liver microsomes. The biotin-tagged peptides resulting from in-gel tryptic digestion were enriched by biotin-avidin chromatography and LC-MS-MS was used to identify 376 microsomal cysteine thiol targets of BMCC and IAB in 263 proteins. Protein adduction was selective and reproducible and only 90 specific cysteine sites in 70 proteins (approximately 25% of the total) were adducted by both electrophiles. Differences in adduction selectivity correlated with different biological effects of the compounds, as IAB, but not BMCC induced ER stress in HEK293 cells. Targeted LC-MS-MS analysis of microsomal glutathione-S-transferase cysteine 50, a target of both IAB and BMCC, detected time-dependent adduction by the reactive acetaminophen metabolite N-acetyl-p-benzoquinoneimine during microsomal incubations. The results indicate that electrophiles selectively adduct microsomal proteins, but display differing target selectivities that correlate with differences in toxicity. Analysis of selected microsomal protein adduction reactions thus could provide a more specific indication of potential toxicity than bulk covalent binding of radiolabeled compounds. PMID:17480101

  8. Characterisation of theophylline metabolism in human liver microsomes.

    PubMed Central

    Robson, R A; Matthews, A P; Miners, J O; McManus, M E; Meyer, U A; Hall, P M; Birkett, D J

    1987-01-01

    1. A radiometric high performance liquid chromatographic method is described for the assay of theophylline metabolism in vitro by the microsomal fraction of human liver. 2. Formation of the three metabolites of theophylline (3-methylxanthine, 1-methylxanthine and 1,3-dimethyluric acid) were linear with protein concentrations to 4 mg ml-1 and with incubation times up to 180 min. 3. The coefficients of variation for the formation of 3-methylxanthine, 1-methylxanthine and 1,3-dimethyluric acid were 1.2%, 1% and 1.6%, respectively. 4. Theophylline is metabolised by microsomal enzymes with a requirement for NADPH. 5. The mean (n = 7) Km values for 1-demethylation, 3-demethylation and 8-hydroxylation were 545, 630 and 788 microM, respectively, and the mean Vmax values were 2.65, 2.84 and 11.23 pmol min-1 mg-1, respectively. 6. There was a high correlation between the Km and Vmax values for the two demethylation pathways suggesting that the demethylations are performed by the same enzyme. 7. Overall the in vitro studies are consistent with the in vivo results which suggest the involvement of two cytochrome P-450 isozymes in the metabolism of theophylline. PMID:3663445

  9. Etoxazole is Metabolized Enantioselectively in Liver Microsomes of Rat and Human in Vitro.

    PubMed

    Yao, Zhoulin; Qian, Mingrong; Zhang, Hu; Nie, Jing; Ye, Jingqing; Li, Zuguang

    2016-09-01

    Acaricide etoxazole belongs to the ovicides/miticides diphenyloxazole class, affecting adults to lay sterile eggs by inhibiting chitin biosynthesis possibly. The reverse-phase HPLC-MS/MS method was used to determine the etoxazole enantiomers. The enantioselective degradation behavior of rac-etoxazole in liver microsomes of rat and human in vitro with NADPH was dramatically different. The t1/2 of (R)-etoxazole was 15.23 min in rat liver microsomes and 30.54 min in human liver microsomes, while 21.73 and 23.50 min were obtained for (S)-etoxazole, respectively. The Vmax of (R)-etoxazole was almost 5-fold of (S)-etoxazole in liver microsomes of rat in vitro. However, the Vmax of (S)-etoxazole was almost 2-fold of (R)-etoxazole in liver microsomes of human in vitro. The CLint of etoxazole was also shown the enantioselectivity on the contrary in liver microsomes of rat and human. These results indicated that the metabolism of two etoxazole enantiomers was selective in liver microsomes of rat and human in vitro, and enantioselectivity in the two kinds of liver microsomes was in the difference in degradation performance. The reason might be related to the composition and content involved in the enzyme system. PMID:27479246

  10. Purification and specificity of a human microsomal epoxide hydratase

    PubMed Central

    Oesch, Franz

    1974-01-01

    Epoxide hydratase was solubilized from human liver microsomal fractions and purified to an extent where the specific activity was 40-fold greater than that of the liver homogenate. Combination of homogenate and purified preparation showed that the increase in activity was not due to the removal of an inhibitor. Monosubstituted oxiranes with a lipophilic substituent larger than an ethyl group (isopropyl, t-butyl, n-hexyl, phenyl) readily interacted as substrates or inhibitors with this purified human epoxide hydratase, whereas those with a small substituent (methyl, ethyl, vinyl) were inactive, probably reflecting greater affinity of the former epoxides owing to lipophilic binding sites near the active site of the enzyme. In a series of oxiranes having a lipophilic substituent of sufficient size (styrene oxides), monosubstituted as well as 1,1- and cis-1,2-disubstituted oxiranes readily served as substrates or inhibitors of the enzyme, but not the trans-1,2-disubstituted, tri- or tetra-substituted oxiranes. trans-Substitution at the oxirane ring apparently prevents access of the oxirane ring to the active site by steric hindrance. Epoxide hydratase was also solubilized from microsomal fractions of rat and guinea-pig liver and purified by the same procedure. Structural requirements for effective interaction of substrates, inhibitors and activators were qualitatively identical for epoxide hydratase from the three sources. However, several quantitative differences were observed. Thus human hepatic epoxide hydratase seems to be very similar to, although not identical with, the enzyme from guinea pig or rat. Studies with epoxide hydratase from the latter two species therefore appear to be significant with respect to man. In addition, knowledge of structural requirements for epoxides to serve as substrates for human epoxide hydratase may prove useful for drug design. Compounds which need aromatic or olefinic moieties for their desired effect would not be expected to lead

  11. In vitro metabolism of (-)-camphor using human liver microsomes and CYP2A6.

    PubMed

    Gyoubu, Kunihiko; Miyazawa, Mitsuo

    2007-02-01

    The in vitro metabolism of (-)-camphor was examined in human liver microsomes and recombinant enzymes. Biotransformation of (-)-camphor was investigated by gas chromatography-mass spectrometry (GC-MS). (-)-Camphor was oxidized to 5-exo-hydroxyfenchone by human liver microsomal cytochrome (P450) enzymes. The formation of metabolites of (-)-camphor was determined by the relative abundance of mass fragments and retention time on gas chromatography (GC). CYP2A6 was the major enzyme involved in the hydroxylation of (-)-camphor by human liver microsomes, based on the following lines of evidence. First, of eleven recombinant human P450 enzymes tested, CYP2A6 catalyzed the oxidation of (-)-camphor. Second, oxidation of (-)-camphor was inhibited by (+)-menthofuran and anti-CYP2A6 antibody. Finally, there was a good correlation between CYP2A6 contents and (-)-camphor hydroxylation activities in liver microsomes of 9 human samples. PMID:17268056

  12. In vitro glucuronidation kinetics of deoxynivalenol by human and animal microsomes and recombinant human UGT enzymes.

    PubMed

    Maul, Ronald; Warth, Benedikt; Schebb, Nils Helge; Krska, Rudolf; Koch, Matthias; Sulyok, Michael

    2015-06-01

    The mycotoxin deoxynivalenol (DON), formed by Fusarium species, is one of the most abundant mycotoxins contaminating food and feed worldwide. Upon ingestion, the majority of the toxin is excreted by humans and animal species as glucuronide conjugate. First in vitro data indicated that DON phase II metabolism is strongly species dependent. However, kinetic data on the in vitro metabolism as well as investigations on the specific enzymes responsible for DON glucuronidation in human are lacking. In the present study, the DON metabolism was investigated using human microsomal fractions and uridine-diphosphoglucuronyltransferases (UGTs) as well as liver microsomes from five animal species. Only two of the twelve tested human recombinant UGTs led to the formation of DON glucuronides with a different regiospecificity. UGT2B4 predominantly catalyzed the formation of DON-15-O-glucuronide (DON-15GlcA), while for UGT2B7 the DON-3-O-glucuronide (DON-3GlcA) metabolite prevailed. For human UGTs, liver, and intestinal microsomes, the glucuronidation activities were low. The estimated apparent intrinsic clearance (Clapp,int) for all human UGT as well as tissue homogenates was <1 mL/min mg protein. For the animal liver microsomes, moderate Clapp,int between 1.5 and 10 mL/min mg protein were calculated for carp, trout, and porcine liver. An elevated glucuronidation activity was detected for rat and bovine liver microsomes leading to Clapp,int between 20 and 80 mL/min mg protein. The obtained in vitro data points out that none of the animal models is suitable for estimating the human DON metabolism with respect to the metabolite pattern and formation rate. PMID:24927789

  13. 3-ketocholanoic acid is the major in vitro human hepatic microsomal metabolite of lithocholic acid.

    PubMed

    Deo, Anand K; Bandiera, Stelvio M

    2009-09-01

    3alpha-Hydroxy-5 beta-cholan-24-oic (lithocholic) acid is a relatively minor component of hepatic bile acids in humans but is highly cytotoxic. Hepatic microsomal oxidation offers a potential mechanism for effective detoxification and elimination of bile acids. The aim of the present study was to investigate the biotransformation of lithocholic acid by human hepatic microsomes and to assess the contribution of cytochrome P450 (P450) enzymes in human hepatic microsomes using human recombinant P450 enzymes and chemical inhibitors. Metabolites were identified, and metabolite formation was quantified using a liquid chromatography/mass spectrometry-based assay. Incubation of lithocholic acid with human liver microsomes resulted in the formation of five metabolites, which are listed in order of their rates of formation: 3-oxo-5 beta-cholan-24-oic (3-ketocholanoic) acid, 3 alpha,6 alpha-dihydroxy-5 beta-cholan-24-oic (hyodeoxycholic) acid, 3 alpha,7 beta-dihydroxy-5 beta-cholan-24-oic (ursodeoxycholic) acid, 3 alpha,6 beta-dihydroxy-5 beta-cholan-24-oic (murideoxycholic) acid, and 3 alpha-hydroxy-6-oxo-5 beta-cholan-24-oic (6-ketolithocholic) acid. 3-Ketocholanoic acid was the major metabolite, exhibiting apparent K(m) and V(max) values of 22 muM and 336 pmol/min/mg protein, respectively. Incubation of lithocholic acid with a of human recombinant P450 enzymes revealed that all five metabolites were formed by recombinant CYP3A4. Chemical inhibition studies with human liver microsomes and recombinant P450 enzymes confirmed that CYP3A4 was the predominant enzyme involved in hepatic microsomal biotransformation of lithocholic acid. In summary, the results indicate that oxidation of the third carbon of the cholestane ring is the preferred position of oxidation by P450 enzymes for lithocholic acid biotransformation in humans and suggest that formation of lithocholic acid metabolites leads to enhanced hepatic detoxification and elimination. PMID:19487251

  14. Changes in alveolar lavage materials and lung microsomal xenobiotic metabolism following exposures to HCl-washed or unwashed crystalline silica.

    PubMed

    Miles, P R; Bowman, L; Jones, W G; Berry, D S; Vallyathan, V

    1994-12-01

    Intratracheal exposures of rats to crystalline silica washed with HCl to remove iron contaminants have previously been shown to increase lung surfactant phospholipids (PL) and proteins and to alter the pulmonary microsomal cytochrome P450 system. We compared these effects of HCl-washed silica with those produced by exposures to unwashed silica and alumina. Both silica preparations produce increases in lung weights and alveolar lavage PL and proteins, but to different degrees. The increases produced by HCl-washed vs unwashed silica are lung weights, 2.2- vs 1.3-fold; lavage PL, 25.9- vs 3.7-fold; and lavage proteins, 11.1- vs 3.2-fold, respectively. Although the two silica particles increase lung microsomal protein concentrations (expressed per gram lung) by 50-60%, their effects on cytochrome P-450-mediated xenobiotic metabolism are quite different. Exposure to HCl-washed silica leads to a 2.3-fold increase in 7-ethoxyresorufin O-deethylation, a reaction catalyzed by cytochrome P4501A1, and a 0.5- to 0.6-fold reduction in 7-ethoxycoumarin O-deethylation, a reaction which may be catalyzed by cytochrome P-4502B1. Unwashed silica does not alter the metabolism of either xenobiotic when results are expressed per milligram microsomal protein. Administration of alumina produces only minor increases in lung weight and lavage PL and no effect on microsomal xenobiotic metabolism. These results show that the increases in alveolar lavage PL and proteins induced by administration of unwashed silica are exaggerated by 3- to 7-fold if the silica is treated with HCl. Furthermore, exposure to HCl-washed silica results in significant alterations of the lung microsomal cytochrome P450 system, but the unwashed silica has little effect. Although the reason(s) for these different effects is not known, measurements of iron levels and formation of hydroxyl radicals using ESR demonstrate that there is more iron associated with the unwashed than with the HCl-washed silica. PMID:7992313

  15. Serotonin (5-hydroxytryptamine) glucuronidation in vitro: assay development, human liver microsome activities and species differences.

    PubMed

    Krishnaswamy, S; Duan, S X; Von Moltke, L L; Greenblatt, D J; Sudmeier, J L; Bachovchin, W W; Court, M H

    2003-02-01

    1. The main purpose was to develop a high-performance liquid chromatography (HPLC)-based method to assay serotonin glucuronidation activity using liver microsomal fractions. Application of this method was then demonstrated by determining serotonin UDP-glucuronosyltransferase (UGT) enzyme kinetics using human liver microsomes and recombinant human UGT1A6. Interspecies differences were also evaluated using liver microsomes from 10 different mammalian species. 2. Incubation of liver microsomes with serotonin, UDP-glucuronic acid and magnesium resulted in the formation of a single product peak using HPLC with fluorescence and ultraviolet absorbance detection. This peak was confirmed as serotonin glucuronide based on sensitivity to beta-glucuronidase and by obtaining the expected mass of 352 with positive-ion mass spectrometry. 3. Following a preparative HPLC isolation, the structure of this metabolite was established as serotonin-5-O-glucuronide by (1)H-NMR spectroscopy. 4. Enzyme kinetic studies showed apparent K(m) and V(max) of 8.8 +/- 0.3 mM and 43.4 +/- 0.4 nmoles min(-1) mg(-1) protein, respectively, for human liver microsomes, and 5.9 +/- 0.2 mM and 15.8 +/- 0.2 nmoles min(-1) mg(-1), respectively, for recombinant UGT1A6. 5. The order of serotonin-UGT activities in animal liver microsomes was rat > mouse > human > cow > pig > horse > dog > rabbit > monkey > ferret. Cat livers showed no serotonin-UGT activity. Heterozygous and homozygous mutant Gunn rat livers had 40 and 13%, respectively, of the activity of the normal Wistar rat, indicating a significant contribution by a rat UGT1A isoform to serotonin glucuronidation. 6. This assay provides a novel sensitive and specific technique for the measurement of serotonin-UGT activity in vitro. PMID:12623759

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

    PubMed

    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. PMID:2519819

  17. Metabolism of (+)- and (-)-menthols by CYP2A6 in human liver microsomes.

    PubMed

    Miyazawa, Mitsuo; Marumoto, Shinsuke; Takahashi, Toshiyuki; Nakahashi, Hiroshi; Haigou, Risa; Nakanishi, Kyousuke

    2011-01-01

    The in vitro metabolism of (+)-(1S,3S,4R) and (-)-(1R,3R,4S)-menthol enantiomers was examined by incubation with human liver microsomes, and the oxidative metabolites thus formed were analyzed using gas chromatography-mass spectrometry (GC-MS). The (+)- and (-)-menthols were found to be oxidized to the respective (+)-(1S,3S,4S)- and (-)-(1R,3R,4R)-trans-p-menthane-3,8-diol derivatives by human liver microsomal P450 enzymes. Cytochrome P450 (CYP) 2A6 was determined to be the major enzyme involved in the hydroxylation of (+)- and (-)-menthols by human liver microsomes on the basis of the following lines of evidence. First, of 11 recombinant human P450 enzymes tested, CYP2A6 catalyzed the oxidation of (+)- and (-)-menthols. Second, oxidation of (+)- and (-)-menthols was inhibited by (+)-menthofuran and anti-CYP2A6 antibody. Finally, (+)- and (-)-menthol activities were found to correlate with contents of CYP2A6 in liver microsomes of 9 human samples. PMID:21343660

  18. Characterization of fimasartan metabolites in human liver microsomes and human plasma.

    PubMed

    Lee, Ji-Yoon; Choi, Young Jae; Oh, Soo Jin; Chi, Yong Ha; Paik, Soo Heui; Lee, Ki Ho; Jung, Jae-Kyung; Ryu, Chang Seon; Kim, Kwon-Bok; Kim, Dong-Hyun; Yoon, Young-Ran; Kim, Sang Kyum

    2016-01-01

    1. The metabolites of fimasartan (FMS), a new angiotensin II receptor antagonist, were characterized in human liver microsomes (HLM) and human subjects. 2. We developed a method for a simultaneous quantitative and qualitative analysis using predictive multiple reaction monitoring information-dependent acquisition-enhanced product ion scanning. To characterize metabolic reactions, FMS metabolites were analyzed using quadrupole-time of flight mass spectrometer in full-scan mode. 3. The structures of metabolites were confirmed by comparison of chromatographic retention times and mass spectra with those of authentic metabolite standards. 4. In the cofactor-dependent microsomal metabolism study, the half-lives of FMS were 56.7, 247.9 and 53.3 min in the presence of NADPH, UDPGA and NADPH + UDPGA, respectively. 5. The main metabolic routes in HLM were S-oxidation, oxidative desulfuration, n-butyl hydroxylation and N-glucuronidation. 6. In humans orally administered with 120 mg FMS daily for 7 days, the prominent metabolites were FMS S-oxide and FMS N-glucuronide in the 0-8-h pooled plasma sample of each subject. 7. This study characterizes, for the first time, the metabolites of FMS in humans to provide information for its safe use in clinical medicine. PMID:26068523

  19. KINETICS OF BROMODICHLOROMETHANE METABOLISM BY CYTOCHROME P450 ISOENZYMES IN HUMAN LIVER MICROSOMES

    EPA Science Inventory

    Kinetics of Bromodichloromethane Metabolism by
    Cytochrome P450 Isoenzymes in Human Liver Microsomes

    Guangyu Zhao and John W. Allis

    ABSTRACT
    The kinetic constants for the metabolism of bromodichloromethane (BDCM) by three cytochrome P450 (CYP) isoenzymes have ...

  20. METABOLISM OF MYCLOBUTANIL AND TRIADIMEFON BY HUMAN AND RAT CYTOCHROME P450 ENZYMES AND LIVER MICROSOMES.

    EPA Science Inventory

    Metabolism of two triazole-containing antifungal azoles was studied using expressed human and rat cytochrome P450s (CYP) and liver microsomes. Substrate depletion methods were used due to the complex array of metabolites produced from myclobutanil and triadimefon. Myclobutanil wa...

  1. Content and activity of human liver microsomal protein and prediction of individual hepatic clearance in vivo

    PubMed Central

    Zhang, Haifeng; Gao, Na; Tian, Xin; Liu, Tingting; Fang, Yan; Zhou, Jun; Wen, Qiang; Xu, Binbin; Qi, Bing; Gao, Jie; Li, Hongmeng; Jia, Linjing; Qiao, Hailing

    2015-01-01

    The lack of information concerning individual variation in content and activity of human liver microsomal protein is one of the most important obstacles for designing personalized medicines. We demonstrated that the mean value of microsomal protein per gram of liver (MPPGL) was 39.46 mg/g in 128 human livers and up to 19-fold individual variations existed. Meanwhile, the metabolic activities of 10 cytochrome P450 (CYPs) were detected in microsomes and liver tissues, respectively, which showed huge individual variations (200-fold). Compared with microsomes, the activities of liver tissues were much suitable to express the individual variations of CYP activities. Furthermore, individual variations in the in vivo clearance of tolbutamide were successfully predicted with the individual parameter values. In conclusion, we offer the values for MPPGL contents in normal liver tissues and build a new method to assess the in vitro CYP activities. In addition, large individual variations exist in predicted hepatic clearance of tolbutamide. These findings provide important physiological parameters for physiologically-based pharmacokinetics models and thus, establish a solid foundation for future development of personalized medicines. PMID:26635233

  2. Metabolism of chamaechromone in vitro with human liver microsomes and recombinant human drug-metabolizing enzymes.

    PubMed

    Lou, Yan; Hu, Haihong; Qiu, Yunqing; Zheng, Jinqi; Wang, Linrun; Zhang, Xingguo; Zeng, Su

    2014-04-01

    Chamaechromone is a major component in the dried roots of Stellera chamaejasme with antihepatitis B virus and insecticidal activity. In this study, metabolic profiles of chamaechromone were investigated in human liver microsomes. One monohydroxide and two monoglucuronides of chamaechromone were identified. The enzyme kinetics for both hydroxylation and glucuronidation were fitted to the Michaelis-Menten equation. The hydroxylation of chamaechromone was inhibited by α-naphthoflavone, and predominantly catalyzed by recombinant human cytochrome P450 1A2, whereas the glucuronidation was inhibited by quercetin, 1-naphthol, and fluconazole, and mainly catalyzed by recombinant human UDP-glucuronosyltransferase 1A3, 1A7, 1A9, and 2B7. PMID:24687737

  3. Physiological Content and Intrinsic Activities of 10 Cytochrome P450 Isoforms in Human Normal Liver Microsomes.

    PubMed

    Zhang, Hai-Feng; Wang, Huan-Huan; Gao, Na; Wei, Jun-Ying; Tian, Xin; Zhao, Yan; Fang, Yan; Zhou, Jun; Wen, Qiang; Gao, Jie; Zhang, Yang-Jun; Qian, Xiao-Hong; Qiao, Hai-Ling

    2016-07-01

    Due to a lack of physiologic cytochrome P450 (P450) isoform content, P450 activity is typically only determined at the microsomal level (per milligram of microsomal protein) and not at the isoform level (per picomole of P450 isoform), which could result in the misunderstanding of variations in P450 activity between individuals and further hinder development of personalized medicine. We found that there were large variations in protein content, mRNA levels, and intrinsic activities of the 10 P450s in 100 human liver samples, in which CYP2E1 and CYP2C9 showed the highest expression levels. P450 gene polymorphisms had different effects on activity at two levels: CYP3A5*3 and CYP2A6*9 alleles conferred increased activity at the isoform level but decreased activity at the microsomal level; CYP2C9*3 had no effect at the isoform level but decreased activity at the microsomal level. The different effects at each level stem from the different effects of each polymorphism on the resulting P450 protein. Individuals with CYP2A6*1/*4, CYP2A6*1/*9, CYP2C9*1/*3, CYP2D6 100C>T TT, CYP2E1 7632T>A AA, CYP3A5*1*3, and CYP3A5*3*3 genotypes had significantly lower protein content, whereas CYP2D6 1661G>C mutants had a higher protein content. In conclusion, we first offered the physiologic data of 10 P450 isoform contents and found that some single nucleotide polymorphisms had obvious effects on P450 expression in human normal livers. The effects of gene polymorphisms on intrinsic P450 activity at the isoform level were quite different from those at the microsomal level, which might be due to changes in P450 protein content. PMID:27189963

  4. Oxidative metabolism of BDE-99 by human liver microsomes: predominant role of CYP2B6.

    PubMed

    Erratico, Claudio A; Szeitz, András; Bandiera, Stelvio M

    2012-10-01

    Hydroxylated polybrominated diphenyl ethers (PBDEs) have been found in human serum, suggesting that they are formed by in vivo oxidative metabolism of PBDEs. However, the biotransformation of 2,2',4,4',5-pentabromodiphenyl ether (BDE-99), a major PBDE detected in human tissue and environmental samples, is poorly understood. In the present study, the oxidative metabolism of BDE-99 was assessed using pooled and single-donor human liver microsomes, a panel of human recombinant cytochrome P450 (CYP) enzymes, and CYP-specific antibodies. Hydroxylated metabolites were quantified using a liquid chromatography/tandem mass spectrometry-based method. In total, 10 hydroxylated metabolites of BDE-99 were produced by human liver microsomes. Six metabolites were identified as 2,4,5-tribromophenol (2,4,5-TBP), 4-OH-BDE-90, 5'-OH-BDE-99, 6'-OH-BDE-99, 4'-OH-BDE-101, and 2-OH-BDE-123 using authentic standards. Three monohydroxy- and one dihydroxy-pentabrominated metabolites were unidentified. Rates of formation of the three major metabolites (2,4,5-TBP, 5'-OH-BDE-99, and 4'-OH-BDE-101) by human liver microsomes ranged from 24.4 to 44.8 pmol/min/mg protein. Additional experiments demonstrated that the dihydroxylated metabolite was a primary metabolite of BDE-99 and was not produced by hydroxylation of a monohydroxy metabolite. Among the panel of recombinant CYP enzymes tested, formation of all 10 hydroxylated metabolites was catalyzed solely by CYP2B6. A combined approach using antibodies to CYP2B6 and single-donor liver microsomes expressing a wide range of CYP2B6 levels confirmed that CYP2B6 was responsible for the biotransformation of BDE-99. Collectively, the results show that the oxidative metabolism of BDE-99 by human liver microsomes is catalyzed solely by CYP2B6 and is an important determinant of the toxicity and bioaccumulation of BDE-99 in humans. PMID:22738989

  5. Hydrolytic metabolism of phenyl and benzyl salicylates, fragrances and flavoring agents in foods, by microsomes of rat and human tissues.

    PubMed

    Ozaki, Hitomi; Sugihara, Kazumi; Tamura, Yuki; Fujino, Chieri; Watanabe, Yoko; Uramaru, Naoto; Sone, Tomomichi; Ohta, Shigeru; Kitamura, Shigeyuki

    2015-12-01

    Salicylates are used as fragrance and flavor ingredients for foods, as UV absorbers and as medicines. Here, we examined the hydrolytic metabolism of phenyl and benzyl salicylates by various tissue microsomes and plasma of rats, and by human liver and small-intestinal microsomes. Both salicylates were readily hydrolyzed by tissue microsomes, predominantly in small intestine, followed by liver, although phenyl salicylate was much more rapidly hydrolyzed than benzyl salicylate. The liver and small-intestinal microsomal hydrolase activities were completely inhibited by bis(4-nitrophenyl)phosphate, and could be extracted with Triton X-100. Phenyl salicylate-hydrolyzing activity was co-eluted with carboxylesterase activity by anion exchange column chromatography of the Triton X-100 extracts of liver and small-intestinal microsomes. Expression of rat liver and small-intestinal isoforms of carboxylesterase, Ces1e and Ces2c (AB010632), in COS cells resulted in significant phenyl salicylate-hydrolyzing activities with the same specific activities as those of liver and small-intestinal microsomes, respectively. Human small-intestinal microsomes also exhibited higher hydrolyzing activity than liver microsomes towards these salicylates. Human CES1 and CES2 isozymes expressed in COS cells both readily hydrolyzed phenyl salicylate, but the activity of CES2 was higher than that of CES1. These results indicate that significant amounts of salicylic acid might be formed by microsomal hydrolysis of phenyl and benzyl salicylates in vivo. The possible pharmacological and toxicological effects of salicylic acid released from salicylates present in commercial products should be considered. PMID:26321725

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

    PubMed

    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. PMID:19713138

  7. Comparative metabolism of chloroacetamide herbicides and selected metabolites in human and rat liver microsomes.

    PubMed Central

    Coleman, S; Linderman, R; Hodgson, E; Rose, R L

    2000-01-01

    Acetochlor [2-chloro-N-(ethoxymethyl)-N-(2-ethyl-6-methyl-phenyl)-acetamide], alachlor [N-(methoxymethyl)-2-chloro-N-(2, 6-diethyl-phenyl)acetamide], butachlor [N-(butoxymethyl)-2-chloro-N-(2,6-diethyl-phenyl)acetamide], and metolachlor [2-chloro-N-(2-ethyl-6-methylphenyl)-N-(2-methoxy-1-methylethyl) acetamide] are pre-emergent herbicides used in the production of agricultural crops. These herbicides are carcinogenic in rats: acetochlor and alachlor cause tumors in the nasal turbinates, butachlor causes stomach tumors, and metolachlor causes liver tumors. It has been suggested that the carcinogenicity of these compounds involves a complex metabolic activation pathway leading to a DNA-reactive dialkylbenzoquinone imine. Important intermediates in this pathway are 2-chloro-N-(2,6-diethylphenyl)acetamide (CDEPA) produced from alachlor and butachlor and 2-chloro-N-(2-methyl-6-ethylphenyl)acetamide (CMEPA) produced from acetochlor and metolachlor. Subsequent metabolism of CDEPA and CMEPA produces 2,6-diethylaniline (DEA) and 2-methyl-6-ethylaniline (MEA), which are bioactivated through para-hydroxylation and subsequent oxidation to the proposed carcinogenic product dialkylbenzoquinone imine. The current study extends our earlier studies with alachlor and demonstrates that rat liver microsomes metabolize acetochlor and metolachlor to CMEPA (0.065 nmol/min/mg and 0.0133 nmol/min/mg, respectively), whereas human liver microsomes can metabolize only acetochlor to CMEPA (0.023 nmol/min/mg). Butachlor is metabolized to CDEPA to a much greater extent by rat liver microsomes (0.045 nmol/min/mg) than by human liver microsomes (< 0.001 nmol/min/mg). We have determined that both rat and human livers metabolize both CMEPA to MEA (0.308 nmol/min/mg and 0.541 nmol/min/mg, respectively) and CDEPA to DEA (0.350 nmol/min/mg and 0.841 nmol/min/mg, respectively). We have shown that both rat and human liver microsomes metabolize MEA (0.035 nmol/min/mg and 0.069 nmol/min/mg, respectively

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

    PubMed

    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. PMID:26123050

  9. The NADPH- and iron-dependent lipid peroxidation in human placental microsomes.

    PubMed

    Milczarek, Ryszard; Sokolowska, Ewa; Hallmann, Anna; Klimek, Jerzy

    2007-01-01

    In pregnant females, placenta is the most important source of lipid hydroperoxides and other reactive oxygen species (ROS). The increased production of lipid peroxides is often linked to preeclampsia. In our study, we revealed that NADPH- and iron-dependent lipid peroxidation in human placental microsomes (HPM) occurred. In the presence of Fe2+ ion, HPM produced small amounts of thiobarbituric acid-reactive substances (TBARS) - a final product of lipid peroxidation. NADPH caused a strong increase of iron stimulated TBARS formation. TBARS formation was inhibited by superoxide dismutase, butylated hydroxytoluene and alpha-tocopherol but not by mannitol or catalase. TBARS and superoxide radical production was inhibited in similar manner by cytochrome P450 inhibitors. The results obtained led us to the following conclusions: (1) microsomal lipid peroxidation next to mitochondrial lipid peroxidation may by an important source of lipid hydroperoxides in blood during pregnancy and (2) superoxide radical released by microsomal cytochrome P450 is an important factor in NADPH- and iron-dependent lipid peroxidation in HPM. PMID:16896536

  10. In vitro metabolism of 2-ethylhexyldiphenyl phosphate (EHDPHP) by human liver microsomes.

    PubMed

    Ballesteros-Gómez, Ana; Erratico, Claudio A; Eede, Nele Van den; Ionas, Alin C; Leonards, Pim E G; Covaci, Adrian

    2015-01-01

    2-ethylhexyl diphenyl phosphate (EHDPHP) is used as flame retardant and plasticizer additive in a variety of consumer products. Since EHDPHP is toxic to aquatic organisms and has been detected in environmental samples, concerns about human exposure and toxicity are emerging. With the aim of identifying human-specific metabolites, the biotransformation of EHDPHP was investigated using human liver microsomes. Using an in silico program (Meteor) for the prediction of metabolites, untargeted screening tools (agilent Mass Hunter) and a suitable analysis platform based on ultra-high performance liquid chromatography (UPLC) and quadrupole time-of-flight high resolution mass spectrometer (QTOF-MS), for the first time a wide variety of phases-I and II metabolites of EHDPHP were identified. Mono- and di-hydroxylated metabolites, keto metabolites, mixed keto and hydroxylated metabolites and diphenyl phosphate were the major phase-I metabolites of EHDPHP. Glucuronidated metabolites of phase-I metabolites of EHDPHP were also formed by human liver microsomes. Using these results, we propose a general metabolism pathway for EHDPHP in humans and a number of candidate biomarkers for assessing the human exposure to this ubiquitous phosphate flame retardant and plasticizer in future biomonitoring studies. Furthermore, we provide a template analytical approach based on the combination of untargeted and targeted screening and UPLC-QTOF-MS analysis suitable for use in future metabolism studies. PMID:25448284

  11. Benzene metabolism by human liver microsomes in relation to cytochrome P450 2E1 activity.

    PubMed

    Seaton, M J; Schlosser, P M; Bond, J A; Medinsky, M A

    1994-09-01

    Low levels of benzene from sources including cigarette smoke and automobile emissions are ubiquitous in the environment. Since the toxicity of benzene probably results from oxidative metabolites, an understanding of the profile of biotransformation of low levels of benzene is critical in making a valid risk assessment. To that end, we have investigated metabolism of a low concentration of [14C]benzene (3.4 microM) by microsomes from human, mouse and rat liver. The extent of phase I benzene metabolism by microsomal preparations from 10 human liver samples and single microsomal preparations from both mice and rats was then related to measured activities of cytochrome P450 (CYP) 2E1. Measured CYP 2E1 activities, as determined by hydroxylation of p-nitrophenol, varied 13-fold (0.253-3.266 nmol/min/mg) for human samples. The fraction of benzene metabolized in 16 min ranged from 10% to 59%. Also at 16 min, significant amounts of oxidative metabolites were formed. Phenol was the main metabolite formed by all but two human microsomal preparations. In those samples, both of which had high CYP 2E1 activity, hydroquinone was the major metabolite formed. Both hydroquinone and catechol formation showed a direct correlation with CYP 2E1 activity over the range of activities present. A simulation model was developed based on a mechanism of competitive inhibition between benzene and its oxidized metabolites, and was fit to time-course data for three human liver preparations. Model calculations for initial rates of benzene metabolism ranging from 0.344 to 4.442 nmol/mg/min are directly proportional to measured CYP 2E1 activities. The model predicted the dependence of benzene metabolism on the measured CYP 2E1 activity in human liver samples, as well as in mouse and rat liver samples. These results suggest that differences in measured hepatic CYP 2E1 activity may be a major factor contributing to both interindividual and interspecies variations in hepatic metabolism of benzene

  12. Detection of Phenolic Metabolites of Styrene in Mouse Liver and Lung Microsomal IncubationsS⃞

    PubMed Central

    Shen, Shuijie; Zhang, Fan; Gao, Lingbo; Zeng, Su

    2010-01-01

    Metabolic activation is considered to be a critical step for styrene-induced pulmonary toxicity. Styrene-7,8-oxide is a primary oxidative metabolite generated by vinyl epoxidation of styrene. In addition, urinary 4-vinylphenol (4-VP), a phenolic metabolite formed by aromatic hydroxylation, has been detected in workers and experimental animals after exposure to styrene. In the present study, new oxidative metabolites of styrene, including 2-vinylphenol (2-VP), 3-vinylphenol (3-VP), vinyl-1,4-hydroquinone, and 2-hydroxystyrene glycol were detected in mouse liver microsomal incubations. The production rates of 2-VP, 3-VP, 4-VP, and styrene glycol were 0.0527 ± 0.0045, 0.0019 ± 0.0006, 0.0053 ± 0.0002, and 4.42 ± 0.33 nmol/(min · mg protein) in mouse liver microsomes, respectively. Both disulfiram (100 μM) and 5-phenyl-1-pentyne (5 μM) significantly inhibited the formation of the VPs and styrene glycol. 2-VP, 3-VP, and 4-VP were metabolized in mouse liver microsomes at rates of 2.50 ± 0.30, 2.63 ± 0.13, and 3.45 ± 0.11 nmol/(min · mg protein), respectively. The three VPs were further metabolized to vinylcatechols and/or vinyl-1,4-hydroquinone and the corresponding glycols. Pulmonary toxicity of 2-VP, 3-VP, and 4-VP was evaluated in CD-1 mice, and 4-VP was found to be more toxic than 2-VP and 3-VP. PMID:20724499

  13. NADPH- and iron-dependent lipid peroxidation inhibit aromatase activity in human placental microsomes.

    PubMed

    Milczarek, Ryszard; Sokołowska, Ewa; Hallmann, Anna; Kaletha, Krystian; Klimek, Jerzy

    2008-06-01

    During pregnancy placenta is the most significant source of lipid hydroperoxides and other reactive oxygen species (ROS). The increased production of lipid peroxides and other ROS is often linked to pre-eclampsia. It is already proved that placental endoplasmic reticulum may be an important place of lipid peroxides and superoxide radical production. In the present study we revealed that NADPH- and iron-dependent lipid peroxidation in human placental microsomes (HPM) inhibit placental aromatase--a key enzyme of estrogen biosynthesis in human placenta. We showed that significant inhibition of this enzyme is caused by small lipid peroxidation (TBARS (thiobarbituric acid-reactive substances)<4nmol/mg microsomal protein (m.p.)). More intensive lipid peroxidation (TBARS>9nmol/mg microsomal protein) diminishes aromatase activity to value being less than 5% of initial value. NADPH- and iron-dependent lipid peroxidation also causes disappearance of cytochrome P450 parallel to observed aromatase activity inhibition. EDTA, alpha-tocopherol, MgCl(2) and superoxide dismutase (SOD) prevent aromatase activity inhibition and cytochrome P450(AROM) degradation. Mannitol and catalase have not effect on TBARS synthesis, aromatase activity and cytochrome P450 degradation. In view of the above we postulate that the inhibition of aromatase activity observed is mainly a consequence of cytochrome P450(AROM) degradation induced by lipid radicals. The role of hydroxyl radical in cytochrome P450 degradation is negligible in our experimental conditions. The results presented here also suggest that the inhibition of aromatase activity can also take place in placenta at in vivo conditions. PMID:18499441

  14. In vivo effects of 3-methylcholanthrene, phenobarbital, pyrethrum and 2,4,5-T isooctylester on liver, lung and kidney microsomal mixed-function oxidase system of guinea-pig: a comparative study.

    PubMed

    Işcan, M; Arinç, E; Vural, N; Işcan, M Y

    1984-01-01

    The optimum conditions (pH, microsomal protein amount and substrate concentration) of guinea-pig liver, lung and kidney microsomal aniline 4-hydroxylase, ethylmorphine N-demethylase and benzo[a]pyrene hydroxylase activities were determined. Male guinea-pigs weighing 500-700 g were administered 3-methylcholanthrene (25 mg/kg, i.p. 3 days), phenobarbital (75 mg/kg, i.p. 3 days), pyrethrum (120 mg/kg, i.p. 2 days) and 2,4,5-T isooctylester (200 mg/kg, i.p. 3 days). 3-Methylcholanthrene treatment caused significant increases in liver microsomal benzo[a]pyrene hydroxylase and kidney microsomal aniline 4-hydroxylase activities. However, with phenobarbital treatment the only significant increase was observed in liver microsomal ethylmorphine N-demethylase activity. Pyrethrum treatment decreased kidney microsomal ethylmorphine N-demethylase activity significantly. 2,4,5-T isooctylester treatment increased liver microsomal aniline 4-hydroxylase and lung microsomal ethylmorphine N-demethylase activities significantly. Liver microsomal NADPH-cytochrome c reductase activity was increased significantly by phenobarbital and pyrethrum treatment. The other treatments did not cause any significant changes in microsomal NADPH-cytochrome c reductase activities of liver, lung and kidney. Cytochrome P-450 content of guinea-pig liver microsomes were increased significantly about 2.5-fold and 2-fold by treatment with 3-methylcholanthrene and phenobarbital, respectively. 3-Methylcholanthrene also caused 1 nm spectral shift in the absorption maxima of CO difference spectrum of the dithionite-reduced liver microsomal cytochrome P-450, forming P-449. PMID:6141874

  15. Inhibition of Cytochrome P450 by Propolis in Human Liver Microsomes

    PubMed Central

    Ryu, Chang Seon; Oh, Soo Jin; Oh, Jung Min; Lee, Ji-Yoon; Lee, Sang Yoon; Chae, Jung-woo; Kwon, Kwang-il; Kim, Sang Kyum

    2016-01-01

    Although propolis is one of the most popular functional foods for human health, there have been no comprehensive studies of herb-drug interactions through cytochrome P450 (CYP) inhibition. The purpose of this study was to determine the inhibitory effects of propolis on the activities of CYP1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1 and 3A4 using pooled human liver microsomes (HLMs). Propolis inhibited CYP1A2, CYP2E1 and CYP2C19 with an IC50 value of 6.9, 16.8, and 43.1 μg/mL, respectively, whereas CYP2A6, 2B6, 2C9, 2D6, and 3A4 were unaffected. Based on half-maximal inhibitory concentration shifts between microsomes incubated with and without nicotinamide adenine dinucleotide phosphate, propolis-induced CYP1A2, CYP2C19, and CYP2E1 inhibition was metabolism-independent. To evaluate the interaction potential between propolis and therapeutic drugs, the effects of propolis on metabolism of duloxetine, a serotonin-norepinephrine reuptake inhibitor, were determined in HLMs. CYP1A2 and CYP2D6 are involved in hydroxylation of duloxetine to 4-hydroxy duloxetine, the major metabolite, which was decreased following propolis addition in HLMs. These results raise the possibility of interactions between propolis and therapeutic drugs metabolized by CYP1A2. PMID:27437087

  16. Vanadium-mediated lipid peroxidation in microsomes from human term placenta

    SciTech Connect

    Byczkowski, J.Z.; Wan, B.; Kulkarni, A.P.

    1988-11-01

    Vanadium is considered an essential element present in living organisms in trace amounts but it is toxic when introduced in excessive doses to animals and humans. Vanadium compounds are extensively used in modern industry and occupational exposure to high doses of vanadium is quite common. In pregnant mice, vanadium accumulates preferentially in the placenta and to lower extent in fetal skeleton and mammary gland during exposure to radioactive vanadium. Accumulation of vanadium in fetoplacental unit may present threat to the fetus by interacting with enzymes and ion-transporting systems in membranes. It is also possible that accumulation of vanadium with its concomitant reduction to vanadyl may lead to lipid peroxidation, followed by damage to biological membranes, lysosomal enzymes release and destruction of placental tissue. To explore some of these possibilities the authors decided to examine whether vanadate can undergo redox cycling in microsomes from human term placenta (HTP) that can lead to lipid peroxidation.

  17. Selective serotonin reuptake inhibitors and theophylline metabolism in human liver microsomes: potent inhibition by fluvoxamine.

    PubMed Central

    Rasmussen, B B; Maënpää, J; Pelkonen, O; Loft, S; Poulsen, H E; Lykkesfeldt, J; Brøsen, K

    1995-01-01

    1. Fluvoxamine and seven other selective serotonin reuptake inhibitors (SRRI) were tested for their ability to inhibit a number of human cytochrome P450 isoforms (CYPs). 2. None of the drugs showed potent inhibition of CYP2A6 (coumarin 7-hydroxylase) or CYP2E1 (chlorzoxazone 6-hydroxylase), while norfluoxetine was the only potent inhibitor of CYP3A having IC50 values of 11 microM and 19 microM for testosterone 6 beta-hydroxylase and cortisol 6 beta-hydroxylase, respectively. 3. Norfluoxetine, sertraline and fluvoxamine inhibited CYP1A1 (7-ethoxyresorufin O-deethylase) in microsomes from human placenta (IC50 values 29 microM, 35 microM and 80 microM, respectively). Fluvoxamine was a potent inhibitor of CYP1A2-mediated 7-ethoxyresorufin O-deethylase activity (IC50 = 0.3 microM) in human liver. 4. In microsomes from three human livers fluvoxamine potently inhibited all pathways of theophylline biotransformation, the apparent inhibitor constant, Ki, was 0.07-0.13 microM, 0.05-0.10 microM and 0.16-0.29 microM for inhibition of 1-methylxanthine, 3-methylxanthine and 1,3-dimethyluric acid formation, respectively. Seven other SSRIs showed either weak or no inhibition of theophylline metabolism. 5. Ethanol inhibited the formation of 1,3-dimethyluric acid with K(i) value of 300 microM, a value which is consistent with inhibition of CYP2E1. Ethanol and fluvoxamine both inhibited 8-hydroxylation by about 45% and, in combination, the compounds decreased the formation of 1,3-dimethyluric acid by 90%, indicating that CYP1A2 and CYP2E1 are equally important isoforms for the 8-hydroxylation of theophylline. 6. It is concluded that pharmacokinetic interaction between fluvoxamine and theophylline is due to potent inhibition of CYP1A2. PMID:7742153

  18. Aromatase inhibition by synthetic lactones and flavonoids in human placental microsomes and breast fibroblasts--a comparative study.

    PubMed

    van Meeuwen, J A; Nijmeijer, S; Mutarapat, T; Ruchirawat, S; de Jong, P C; Piersma, A H; van den Berg, M

    2008-05-01

    Interference of exogenous chemicals with the aromatase enzyme can be useful as a tool to identify chemicals that could act either chemopreventive for hormone-dependent cancer or adverse endocrine disruptive. Aromatase is the key enzyme in the biosynthesis of steroids, as it converts androgens to estrogens. Certain flavonoids, plant derived chemicals, are known catalytic aromatase inhibitors. Various systems are in use to test aromatase inhibitory properties of compounds. Commonly used are microsomes derived from ovary or placental tissue characterized by high aromatase activity. To a lesser extent whole cell systems are used and specifically cell systems that are potential target tissue in breast cancer development. In this study aromatase inhibitory properties of fadrozole, 8-prenylnaringenin and a synthetic lactone (TM-7) were determined in human placental microsomes and in human primary breast fibroblasts. In addition, apigenin, chrysin, naringenin and two synthetic lactones (TM-8 and TM-9) were tested in human microsomes only. Comparison of the aromatase inhibitory potencies of these compounds between the two test systems showed that the measurement of aromatase inhibition in human placental microsomes is a good predictor of aromatase inhibition in human breast fibroblasts. PMID:18201740

  19. Aromatase inhibition by synthetic lactones and flavonoids in human placental microsomes and breast fibroblasts - A comparative study

    SciTech Connect

    Meeuwen, J.A. van Nijmeijer, S.; Mutarapat, T.; Ruchirawat, S.; Jong, P.C. de; Piersma, A.H.; Berg, M. van den

    2008-05-01

    Interference of exogenous chemicals with the aromatase enzyme can be useful as a tool to identify chemicals that could act either chemopreventive for hormone-dependent cancer or adverse endocrine disruptive. Aromatase is the key enzyme in the biosynthesis of steroids, as it converts androgens to estrogens. Certain flavonoids, plant derived chemicals, are known catalytic aromatase inhibitors. Various systems are in use to test aromatase inhibitory properties of compounds. Commonly used are microsomes derived from ovary or placental tissue characterized by high aromatase activity. To a lesser extent whole cell systems are used and specifically cell systems that are potential target tissue in breast cancer development. In this study aromatase inhibitory properties of fadrozole, 8-prenylnaringenin and a synthetic lactone (TM-7) were determined in human placental microsomes and in human primary breast fibroblasts. In addition, apigenin, chrysin, naringenin and two synthetic lactones (TM-8 and TM-9) were tested in human microsomes only. Comparison of the aromatase inhibitory potencies of these compounds between the two test systems showed that the measurement of aromatase inhibition in human placental microsomes is a good predictor of aromatase inhibition in human breast fibroblasts.

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

    PubMed

    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. PMID:24311535

  1. In vitro metabolic clearance of pyrethroid pesticides by rat and human hepatic microsomes and cytochrome P450 isoforms

    EPA Science Inventory

    Species differences in the intrinsic clearance (CLint) and the enzymes involved in the metabolism of pyrethroid pesticides were examined in rat and human hepatic microsomes. The pyrethroids bifenthrin, S-bioallethrin, bioresmethrin, β-cyfluthrin, cypermethrin, cis-per...

  2. Calcium channel antagonists and cyclosporine metabolism: in vitro studies with human liver microsomes.

    PubMed Central

    Tjia, J F; Back, D J; Breckenridge, A M

    1989-01-01

    The effects of four Ca2+ channel antagonists on the metabolism of cyclosporine (CsA) by human liver microsomes (n = 4) in vitro have been examined. Nicardipine produced marked inhibition of both M17 and M21 (IC50 = 7.0 microM) formation. In contrast nifedipine produced less than 20% inhibition of M17 and M21 even at the highest concentration examined (50 microM). Diltiazem data were comparable to those for nifedipine. Verapamil (50 microM) produced 30 and 28% inhibition of M17 and M21 formation, respectively. These findings give a basis to the increase in CsA blood concentrations seen in transplant patients who are also given nicardipine. PMID:2789931

  3. The effect of enzyme inhibition on the metabolism and activation of tacrine by human liver microsomes.

    PubMed Central

    Spaldin, V; Madden, S; Pool, W F; Woolf, T F; Park, B K

    1994-01-01

    1. Tacrine (1,2,3,4-tetrahydro-9-aminoacridine-hydrochloride: THA) underwent metabolism in vitro by a panel (n = 12) of human liver microsomes genotyped for CYP2D6, in the presence of NADPH, to both protein-reactive and stable metabolites. 2. There was considerable variation in the extent of THA metabolism amongst human livers. Protein-reactive metabolite formation showed a 10-fold variation (0.6 +/- 0.1%-5.2 +/- 0.8% of incubated radioactivity mg-1 protein) whilst stable metabolites showed a 3-fold variation (24.3 +/- 1.7%-78.6 +/- 2.6% of incubated radioactivity). 3. Using cytochrome P450 isoform specific inhibitors CYP1A2 was identified as the major enzyme involved in all routes of THA metabolism. 4. There was a high correlation between aromatic and alicyclic hydroxylation (r = 0.92, P < 0.0001) consistent with these biotransformations being catalysed by the same enzymes. 5. Enoxacin (ENOX), cimetidine (CIM) and chloroquine (CQ) inhibited THA metabolism by a preferential decrease in the bioactivation to protein-reactive, and hence potentially toxic, species. The inhibitory potency of ENOX and CIM was increased significantly upon pre-incubation with microsomes and NADPH. 6. Covalent binding correlated with 7-OH-THA formation before (r = 0.792, P < 0.0001) and after (r = 0.73, P < 0.0001) inhibition by CIM, consistent with a two-step mechanism in the formation of protein-reactive metabolite(s) via a 7-OH intermediate. 7. The use of enzyme inhibitors may provide a useful tool for examining the relationship between the metabolism and toxicity of THA in vivo. PMID:7946932

  4. Reductive metabolism of nabumetone by human liver microsomal and cytosolic fractions: exploratory prediction using inhibitors and substrates as marker probes.

    PubMed

    Matsumoto, Kaori; Hasegawa, Tetsuya; Koyanagi, Junichi; Takahashi, Tamiko; Akimoto, Masayuki; Sugibayashi, Kenji

    2015-06-01

    The metabolic reduction of nabumetone was examined by inhibition and correlation studies using human liver microsomes and cytosol. This reduction was observed in both fractions, with the V(max) values for reduction activity being approximately fourfold higher, and the V(max)/K(m) values approximately three-fold higher, in the microsomes than in the cytosol. The reduction of nabumetone was inhibited by 18β-glycyrrhetinic acid, an 11β-hydroxysteroid dehydrogenase (11β-HSD) inhibitor, in the microsomal fraction. The reduction activity was also inhibited by quercetin and menadione [carbonyl reductase (CBR) inhibitors], and by phenolphthalein and medroxyprogesterone acetate [potent inhibitors of aldo-keto reductase (AKR) 1C1, 1C2 and 1C4] in the cytosol. A good correlation (r² = 0.93) was observed between the reduction of nabumetone and of cortisone, as a marker of 11β-HSD activity, in the microsomal fractions. There was also an excellent relationship between reduction of nabumetone and of the AKR1C substrates, acetohexamide, and ethacrynic acid (r 2 = 0.92 and 0.93, respectively), in the cytosol fractions. However, a poor correlation was observed between the formation of 4-(6-methoxy-2-naphthyl)-butan-2-ol (MNBO) from nabumetone and CBR activity (with 4-benzoyl pyridine reduction as a CBR substrate) in the cytosol fractions (r² = 0.24). These findings indicate that nabumetone may be metabolized by 11β-HSD in human liver microsomes, and primarily by AKR1C4 in human liver cytosol, although multiple enzymes in the AKR1C subfamily may be involved. It cannot be completely denied that CBR is involved to some extent in the formation of MNBO from nabumetone in the cytosol fraction. PMID:24659525

  5. Human microsomal epoxide hydrolase: genetic polymorphism and functional expression in vitro of amino acid variants

    PubMed Central

    Hassett, Christopher; Aicher, Lauri; Sidhu, Jaspreet S.

    2016-01-01

    Human microsomal epoxide hydrolase (mEH) is a biotransformation enzyme that metabolizes reactive epoxide intermediates to more water-soluble trans-dihydrodiol derivatives. We compared protein-coding sequences from six full-length human mEH DNA clones and assessed potential amino acid variation at seven positions. The prevalence of these variants was assessed in at least 37 unrelated individuals using polymerase chain reaction experiments. Only Tyr/His 113 (exon 3) and His/Arg 139 (exon 4) variants were observed. The genotype frequencies determined for residue 113 alleles indicate that this locus may not be in Hardy – Weinberg equilibrium, whereas frequencies observed for residue 139 alleles were similar to expected values. Nucleotide sequences coding for the variant amino acids were constructed in an mEH cDNA using site-directed mutagenesis, and each was expressed in vitro by transient transfection of COS-1 cells. Epoxide hydrolase mRNA level, catalytic activity, and immunoreactive protein were evaluated for each construct. The results of these analyses demonstrated relatively uniform levels of mEH RNA expression between the constructs. mEH enzymatic activity and immunoreactive protein were strongly correlated, indicating that mEH specific activity was similar for each variant. However, marked differences were noted in the relative amounts of immunoreactive protein and enzymatic activity resulting from the amino acid substitutions. These data suggest that common human mEH amino acid polymorphisms may alter enzymatic function, possibly by modifying protein stability. PMID:7516776

  6. Selective Inhibition of Bakuchicin Isolated from Psoralea corylifolia on CYP1A in Human Liver Microsomes

    PubMed Central

    Kim, Sun Joo; Oh, Heung Chan; Kim, Youn-Chul; Jeong, Gil-Saeng; Lee, Sangkyu

    2016-01-01

    Bakuchicin is a furanocoumarin isolated from Psoralea corylifolia and shows several biological activities. Although there have been studies on the biological effects of bakuchicin, its modulation potency of CYP activities has not been previously investigated. Here, we investigated the inhibitory effects of bakuchicin on the activities of CYP isoforms by using a cocktail of probe substrates in pooled human liver microsomes (HLMs) and human recombinant cDNA-expressed CYP. Bakuchicin strongly inhibited CYP1A-mediated phenacetin O-deethylation with an IC50 value of 0.43 μM in HLMs. It was confirmed by human recombinant cDNA-expressed CYP1A1 and CYP1A2 with a Ki value of 0.11 μM and 0.32 μM, respectively. A Lineweaver-Burk plot indicated that the inhibition mechanism of bakuchicin was competitive inhibition. Overall, this is the first study to investigate the potential CYP1A1 and CYP1A2 inhibition associated with bakuchicin and to report its competitive inhibitory effects on HLMs. PMID:26977174

  7. Identification of di-2-ethylhexyl terephthalate (DEHTP) metabolites using human liver microsomes for biomonitoring applications.

    PubMed

    Silva, Manori J; Samandar, Ella; Calafat, Antonia M; Ye, Xiaoyun

    2015-06-01

    Di-2-ethylhexyl terephthalate (DEHTP), a structural isomer of the plasticizer di-2-ethylhexyl phthalate (DEHP), is used in food packaging and medical devices, among other applications, and is a potential replacement for DEHP and other ortho-phthalate plasticizers. Identifying sensitive and specific biomarkers of DEHTP is necessary to assess humans' background exposure to DEHTP. Using mass spectrometry, we investigated the metabolism of DEHTP by human liver microsomes to identify in vitro DEHTP metabolites. We unequivocally identified terephthalic acid (TPA) and mono-2-ethylhydroxyhexyl terephthalate (MEHHTP), using authentic standards, and tentatively identified mono-2-ethylhexyl terephthalate (MEHTP) and two other oxidative metabolites of DEHTP: mono-2-ethyloxohexyl terephthalate (MEOHTP), and mono-2-ethyl-5-carboxypentyl terephthalate (MECPTP) from their mass spectrometry fragmentation patterns. We also evaluated the formation of in vitro metabolites of DEHP. DEHTP and DEHP produced similar metabolites, but their metabolite profiles differed considerably. DEHTP metabolized to form TPA, a metabolite of several terephthalates, as the major in vitro metabolite, followed by MEHTP, MEHHTP, MEOHTP and MECPTP. MEHTP, MEHHTP, MEOHTP and MECPTP, which are specific metabolites of DEHTP, may be suitable biomarkers for assessing exposure to DEHTP. Nonetheless, data on the urinary excretion fraction and temporal stability of these metabolites, among other considerations, are needed to demonstrate their utility as exposure biomarkers. PMID:25687528

  8. Discovery of Novel Splice Variants and Regulatory Mechanisms for Microsomal Triglyceride Transfer Protein in Human Tissues

    PubMed Central

    Suzuki, Takashi; Swift, Larry L.

    2016-01-01

    Microsomal triglyceride transfer protein (MTP) is a unique lipid transfer protein essential for the assembly of triglyceride-rich lipoproteins by the liver and intestine. Previous studies in mice identified a splice variant of MTP with an alternate first exon. Splice variants of human MTP have not been reported. Using PCR approaches we have identified two splice variants in human tissues, which we have named MTP-B and MTP-C. MTP-B has a unique first exon (Ex1B) located 10.5 kb upstream of the first exon (Ex1A) for canonical MTP (MTP-A); MTP-C contains both first exons for MTP-A and MTP-B. MTP-B was found in a number of tissues, whereas MTP-C was prominent in brain and testis. MTP-B does not encode a protein; MTP-C encodes the same protein encoded by MTP-A, although MTP-C translation is strongly inhibited by regulatory elements within its 5′-UTR. Using luciferase assays, we demonstrate that the promoter region upstream of exon 1B is quite adequate to drive expression of MTP. We conclude that alternate splicing plays a key role in regulating cellular MTP levels by introducing distinct promoter regions and unique 5′-UTRs, which contain elements that alter translation efficiency, enabling the cell to optimize MTP activity. PMID:27256115

  9. Discovery of Novel Splice Variants and Regulatory Mechanisms for Microsomal Triglyceride Transfer Protein in Human Tissues.

    PubMed

    Suzuki, Takashi; Swift, Larry L

    2016-01-01

    Microsomal triglyceride transfer protein (MTP) is a unique lipid transfer protein essential for the assembly of triglyceride-rich lipoproteins by the liver and intestine. Previous studies in mice identified a splice variant of MTP with an alternate first exon. Splice variants of human MTP have not been reported. Using PCR approaches we have identified two splice variants in human tissues, which we have named MTP-B and MTP-C. MTP-B has a unique first exon (Ex1B) located 10.5 kb upstream of the first exon (Ex1A) for canonical MTP (MTP-A); MTP-C contains both first exons for MTP-A and MTP-B. MTP-B was found in a number of tissues, whereas MTP-C was prominent in brain and testis. MTP-B does not encode a protein; MTP-C encodes the same protein encoded by MTP-A, although MTP-C translation is strongly inhibited by regulatory elements within its 5'-UTR. Using luciferase assays, we demonstrate that the promoter region upstream of exon 1B is quite adequate to drive expression of MTP. We conclude that alternate splicing plays a key role in regulating cellular MTP levels by introducing distinct promoter regions and unique 5'-UTRs, which contain elements that alter translation efficiency, enabling the cell to optimize MTP activity. PMID:27256115

  10. Human Enteric Microsomal CYP4F Enzymes O-Demethylate the Antiparasitic Prodrug Pafuramidine

    PubMed Central

    Wang, Michael Zhuo; Wu, Judy Qiju; Bridges, Arlene S.; Zeldin, Darryl C.; Kornbluth, Sally; Tidwell, Richard R.; Hall, James Edwin; Paine, Mary F.

    2008-01-01

    CYP4F enzymes, including CYP4F2 and CYP4F3B, were recently shown to be the major enzymes catalyzing the initial oxidative O-demethylation of the antiparasitic prodrug pafuramidine (DB289) by human liver microsomes. As suggested by a low oral bioavailability, DB289 could undergo first-pass biotransformation in the intestine, as well as in the liver. Using human intestinal microsomes (HIM), we characterized the enteric enzymes that catalyze the initial O-demethylation of DB289 to the intermediate metabolite, M1. M1 formation in HIM was catalyzed by cytochrome P450 (P450) enzymes, as evidenced by potent inhibition by 1-aminoben-zotriazole and the requirement for NADPH. Apparent Km and Vmax values ranged from 0.6 to 2.4 μM and from 0.02 to 0.89 nmol/min/mg protein, respectively (n = 9). Of the P450 chemical inhibitors evaluated, ketoconazole was the most potent, inhibiting M1 formation by 66%. Two inhibitors of P450-mediated arachidonic acid metabolism, HET0016 (N-hydroxy-N′-(4-n-butyl-2-methylphenyl)formamidine) and 17-octadecynoic acid, inhibited M1 formation in a concentration-dependent manner (up to 95%). Immunoinhibition with an antibody raised against CYP4F2 showed concentration-dependent inhibition of M1 formation (up to 92%), whereas antibodies against CYP3A4/5 and CYP2J2 had negligible to modest effects. M1 formation rates correlated strongly with arachidonic acid ω-hydroxylation rates (r2 = 0.94, P < 0.0001, n = 12) in a panel of HIM that lacked detectable CYP4A11 protein expression. Quantitative Western blot analysis revealed appreciable CYP4F expression in these HIM, with a mean (range) of 7 (3–18) pmol/mg protein. We conclude that enteric CYP4F enzymes could play a role in the first-pass biotransformation of DB289 and other xenobiotics. PMID:17709372

  11. Kinetics of tris (1-chloro-2-propyl) phosphate (TCIPP) metabolism in human liver microsomes and serum.

    PubMed

    Van den Eede, Nele; Tomy, Gregg; Tao, Fang; Halldorson, Thor; Harrad, Stuart; Neels, Hugo; Covaci, Adrian

    2016-02-01

    Tris(1-chloro-2-propyl) phosphate (TCIPP) is an emerging contaminant which is ubiquitous in the indoor and outdoor environment. Moreover, its presence in human body fluids and biota has been evidenced. Since no quantitative data exist on the biotransformation or stability of TCIPP in the human body, we performed an in vitro incubation of TCIPP with human liver microsomes (HLM) and human serum (HS). Two metabolites, namely bis(2-chloro-isopropyl) phosphate (BCIPP) and bis(1-chloro-2-propyl) 1-hydroxy-2-propyl phosphate (BCIPHIPP), were quantified in a kinetic study using HLM or HS (only BCIPP, the hydrolysis product) and LC-MS. The Michaelis-Menten model fitted best the NADPH-dependent formation of BCIPHIPP and BCIPP in HLM, with respective V(MAX) of 154 ± 4 and 1470 ± 110 pmol/min/mg protein and respective apparent K(m) of 80.2 ± 4.4 and 96.1 ± 14.5 μM. Hydrolases, which are naturally present in HLM, were also involved in the production of BCIPP. A HS paraoxonase assay could not detect any BCIPP formation above 38.6 ± 10.8 pmol/min/μL serum. Our data indicate that BCIPP is the major metabolite of TCIPP formed in the liver. To our knowledge, this is the first quantitative assessment of the stability of TCIPP in tissues of humans or any other species. Further research is needed to confirm whether these biotransformation reactions are associated with a decrease or increase in toxicity. PMID:26473552

  12. Bioactivation of bisphenol A and its analogs (BPF, BPAF, BPZ and DMBPA) in human liver microsomes.

    PubMed

    Schmidt, Jan; Kotnik, Petra; Trontelj, Jurij; Knez, Željko; Mašič, Lucija Peterlin

    2013-06-01

    Bisphenol A analogs are a class of chemicals known as diphenylmethanes, which contain two benzene rings separated by one central carbon atom, usually with a para-hydroxy group on both benzene rings. Bisphenol A (BPA) can induce an uterotrophic response in immature CD-1 mice and elicits estrogenic responses in many other experimental systems. Besides highlighting endocrine effects, a number of metabolic studies provide strong support for the idea that reactive species of BPA are formed in vitro and in vivo that can form covalent adducts with nucleophilic macromolecules and/or produce oxidative stress. We used a liquid chromatography with a triple quadrupole tandem mass spectrometry (LC-MS/MS) for the detection of metabolites and glutathione conjugates of BPA and its analogs (BPF, BPAF, BPZ and DMBPA) in human liver microsomes (HLM) or with recombinant CYP isozymes in the presence of NADPH and GSH as a trapping agent. We have confirmed that BPA and its structural analogs form hydroxylated metabolites and electrophilic species during bioactivation in HLM and CYP isozymes. These results provided important mechanistic insight into the metabolic fate of BPA structural analogs in vitro. PMID:23470418

  13. Baculovirus expression and biochemical characterization of the human microsomal triglyceride transfer protein.

    PubMed Central

    Ritchie, P J; Decout, A; Amey, J; Mann, C J; Read, J; Rosseneu, M; Scott, J; Shoulders, C C

    1999-01-01

    The microsomal triglyceride transfer protein (MTP) complexed to protein disulphide isomerase (PDI) is obligatory for the assembly of chylomicrons and very-low-density lipoproteins. The determination of the atomic structure of the MTP-PDI heterodimer has important implications for the treatment of those forms of hyperlipidaemia associated with the overproduction of very-low-density lipoproteins, which predispose to premature coronary heart disease. To perform structural studies of the human MTP-PDI complex it was necessary to produce milligram quantities of pure protein. We chose the baculovirus expression system for this purpose. Insects cells were co-infected with recombinant viruses encoding FLAG-tagged MTP and His-tagged PDI; the resulting heterodimer was purified by affinity chromatography. From 5 litres of insect cells, 4-6 mg of more than 95% pure recombinant protein was obtained. CD and attenuated total reflection Fourier-transform infrared spectroscopy indicate that the purified protein has around 34% alpha-helical and 33% beta-structure content. The recombinant protein had a comparable triglyceride transfer activity to that of bovine MTP-PDI. The production of polyclonal antibodies raised against the MTP and PDI subunits of the purified protein is described. The present study demonstrates the feasibility of expressing two proteins at high levels in insect cells and describes a transferable methodology for the purification of the resulting protein complex. PMID:10036224

  14. In Vitro Metabolism of 17-(Dimethylaminoethylamino)-17-demethoxygeldanamycin in Human Liver Microsomes

    PubMed Central

    Zheng, Nan; Zou, Peng; Wang, Shaomeng

    2011-01-01

    The objective of this study was to investigate the oxidative metabolism pathways of 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG), a geldanamycin (GA) derivative and 90-kDa heat shock protein inhibitor. In vitro metabolic profiles of 17-DMAG were examined by using pooled human liver microsomes (HLMs) and recombinant CYP450 isozymes in the presence or absence of reduced GSH. In addition to 17-DMAG hydroquinone and 19-glutathionyl 17-DMAG, several oxidative metabolites of 17-DMAG were detected and characterized by liquid chromatography-tandem mass spectrometry. Different from previously reported primary biotransformations of GA and GA derivatives, 17-DMAG was not metabolized primarily through the reduction of benzoquinone and GSH conjugation in HLMs. In contrast, the primary biotransformations of 17-DMAG in HLMs were hydroxylation and demethylation on its side chains. The most abundant metabolite was produced by demethylation from the methoxyl at position 12. The reaction phenotyping study showed that CYP3A4 and 3A5 were the major cytochrome P450 isozymes involved in the oxidative metabolism of 17-DMAG, whereas CYP2C8, 2D6, 2A6, 2C19, and 1A2 made minor contributions to the formation of metabolites. On the basis of the identified metabolite profiles, the biotransformation pathways for 17-DMAG in HLMs were proposed. PMID:21177985

  15. Metabolic inhibition and kinetics of raloxifene by pharmaceutical excipients in human liver microsomes.

    PubMed

    Kim, Ae Ra; Lim, Soo-Jeong; Lee, Beom-Jin

    2009-02-23

    This study was originally undertaken to establish the in vitro metabolic conditions and then evaluate the effect of pharmaceutical excipients (PEs) on drug metabolism in uridine diphosphoglucuronic acid-supplemented human liver microsomes. Poorly bioavailable raloxifene was chosen as a model drug. Intact drug and its two glucuronide metabolites were successfully isolated using gradient HPLC analysis and LC/MS analysis. Formation of raloxifene metabolites was affected by buffer compositions, incubation time and initial raloxifene concentrations. Under optimized metabolic conditions, 41.0% of raloxifene was converted to its metabolites after 2h incubation. This metabolic inhibition of raloxifene by the PEs occurred in a dose-dependent manner and accordingly formed two glucuronide metabolites. In the metabolic kinetics using Lineweaver-Burk analyses, Cremophor EL competitively inhibited formation of metabolites while sodium lauryl sulfate (SLS), polyvinylpyrrolidone K30 (PVP) and Tween 80 significantly inhibited in a mixed competition. Although some PEs showed inhibition on glucuronidation of raloxifene in vitro, their effects on in vivo bioavailability of raloxifene need to be confirmed directly due to the dilution factors and other complicated situations influencing the bioavailability. PMID:18977285

  16. Comparative metabolic study between two selective estrogen receptor modulators, toremifene and tamoxifen, in human liver microsomes.

    PubMed

    Watanabe, Miyuki; Watanabe, Noriko; Maruyama, Sakiko; Kawashiro, Takashi

    2015-10-01

    Toremifene (TOR) and Tamoxifen (TAM) are widely used as endocrine therapy for estrogen receptor positive breast cancer. Poor metabolizers of TAM are likely to have worse clinical outcomes than patients who exhibit normal TAM metabolism due to lower plasma level of its active metabolite, 4-hydroxy-N-desmethyl (4OH-NDM) tamoxifen (endoxifen). In this study, we examined the role of individual cytochrome P450 (CYP) isoforms in the metabolism of TOR to N-desmethyl (NDM), 4-hydroxy (4OH) and 4OH-NDM metabolites in comparison with TAM using human liver microsomes (HLMs) with selective chemical inhibitors for each CYP isoform and recombinant CYP proteins. Similar levels of NDM metabolites were formed for both TOR and TAM, and N-demethylation of both compounds was primarily carried out by CYP3A4. We found that the formation of 4OH-NDM-TOR was catalyzed both by CYP2C9 and CYP2D6, whereas the formation of 4OH-TAM and endoxifen was specifically catalyzed by CYP2D6 in HLMs. Our results suggest that the potential contribution of CYP2D6 in the bioactivation pathway of TOR may be lower compared to TAM, and may have a different impact on clinical outcome than CYP2D6 polymorphisms. PMID:26423799

  17. Metabolic profile of glyburide in human liver microsomes using LC-DAD-Q-TRAP-MS/MS.

    PubMed

    Ravindran, Selvan; Basu, Sudipta; Gorti, Santosh Kapil Kumar; Surve, Prashant; Sloka, Navya

    2013-05-01

    The sulfonylurea urea drug glyburide (glibenclamide) is widely used for the treatment of diabetes milletus and gestational diabetes. In previous studies monohydroxylated metabolites were identified and characterized for glyburide in different species, but the metabolite owing to the loss of cyclohexyl ring was identified only in mouse. Glyburide upon incubation with hepatic microsomes resulted in 10 metabolites for human. The current study identifies new metabolites of glyburide along with the hydroxylated metabolites that were reported earlier. The newly identified drug metabolites are dihydroxylated metabolites, a metabolite owing to the loss of cyclohexyl ring and one owing to hydroxylation with dehydrogenation. Among the 10 identified metabolites, there were six monohydroxylated metabolites, one dihydroxylated metabolite, two metabolites owing to hydroxylation and dehydrogenation, and one metabolite owing to the loss of cyclohexyl ring. New metabolites of glyburide were identified and characterized using liquid chromatography-diode array detector-quadruple-ion trap-mass spectrometry/mass spectrometry (LC-DAD-Q-TRAP-MS/MS). An enhanced mass scan-enhanced product ion scan with information-dependent acquisition mode in a Q-TRAP-MS/MS system was used to characterize the metabolites. Liquid chromatography with diode array detection was used as a complimentary technique to confirm and identify the metabolites. Metabolites formed in higher amounts were detected in both diode array detection and mass spectrometry detection. PMID:23070832

  18. Interaction between oblongifolin C and UDP-glucuronosyltransferase isoforms in human liver and intestine microsomes.

    PubMed

    Gao, Cui; Shi, Rong; Wang, Tianming; Tan, Hongsheng; Xu, Hongxi; Ma, Yueming

    2015-01-01

    1. Oblongifolin C (OC) is a potential natural anticancer candidate, and its metabolic profile has not yet been established. 2. One major OC glucuronidation metabolite (OCG) has been identified in a pool of human liver microsomes (HLMs). Chemical inhibition experiments suggested that OCG was mainly formed by UGT1A. A screen of recombinant UDP-glucuronosyltransferase isoforms (UGTs) indicated that UGT1A1 primarily mediates OC conjugation, with minor contributions from UGT1A3 and UGT1A8. Enzyme kinetic studies showed that UGT1A1 was the main UGT isoform involved in OCG in HLMs. 3. Further investigation suggested that OC is a broad inhibitor of UGTs. Additionally, OC competitively inhibited UGT1A6 with a Ki value of 3.49 ± 0.57 μM, whereas non-competitively inhibited UGT1A10 with a Ki value of 2.12 ± 0.18 μM. 4. Understanding the interaction between OC and UGTs will greatly contribute to future investigations regarding the inter-individual differences in OC metabolism in clinical trials and potential drug-drug interactions. PMID:25714435

  19. In vitro glucuronidation of 2,2-bis(bromomethyl)-1,3-propanediol by microsomes and hepatocytes from rats and humans.

    PubMed

    Rad, Golriz; Hoehle, Simone I; Kuester, Robert K; Sipes, I Glenn

    2010-06-01

    2,2-Bis(bromomethyl)-1,3-propanediol (BMP) is a brominated flame retardant used in unsaturated polyester resins. In a 2-year bioassay BMP was shown to be a multisite carcinogen in rats and mice. Because glucuronidation is the key metabolic transformation of BMP by rats, in this study the in vitro hepatic glucuronidation of BMP was compared across several species. In addition, the glucuronidation activities of human intestinal microsomes and specific human hepatic UDP-glucuronosyltransferase (UGT) enzymes for BMP were determined. To explore other possible routes of metabolism for BMP, studies were conducted with rat and human hepatocytes. Incubation of hepatic microsomes with BMP in the presence of UDP-glucuronic acid resulted in the formation of a BMP monoglucuronide. The order of hepatic microsomal glucuronidation activity of BMP was rats, mice > hamsters > monkeys > humans. The rate of glucuronidation by rat hepatic microsomes was 90-fold greater than that of human hepatic microsomes. Human intestinal microsomes converted BMP to BMP glucuronide at a rate even lower than that of human hepatic microsomes. Among the human UGT enzymes tested, only UGT2B7 had detectable glucuronidation activity for BMP. BMP monoglucuronide was the only metabolite formed when BMP was incubated with suspensions of freshly isolated hepatocytes from male F-344 rats or with cryopreserved human hepatocytes. Glucuronidation of BMP in human hepatocytes was extremely low. Overall, the results support in vivo studies in rats in which BMP glucuronide was the only metabolite found. The poor glucuronidation capacity of humans for BMP suggests that the pharmacokinetic profile of BMP in humans will be dramatically different from that of rodents. PMID:20200232

  20. In Vitro Glucuronidation of 2,2-Bis(bromomethyl)-1,3-propanediol by Microsomes and Hepatocytes from Rats and Humans

    PubMed Central

    Rad, Golriz; Hoehle, Simone I.; Kuester, Robert K.

    2010-01-01

    2,2-Bis(bromomethyl)-1,3-propanediol (BMP) is a brominated flame retardant used in unsaturated polyester resins. In a 2-year bioassay BMP was shown to be a multisite carcinogen in rats and mice. Because glucuronidation is the key metabolic transformation of BMP by rats, in this study the in vitro hepatic glucuronidation of BMP was compared across several species. In addition, the glucuronidation activities of human intestinal microsomes and specific human hepatic UDP-glucuronosyltransferase (UGT) enzymes for BMP were determined. To explore other possible routes of metabolism for BMP, studies were conducted with rat and human hepatocytes. Incubation of hepatic microsomes with BMP in the presence of UDP-glucuronic acid resulted in the formation of a BMP monoglucuronide. The order of hepatic microsomal glucuronidation activity of BMP was rats, mice ≫ hamsters > monkeys ⋙ humans. The rate of glucuronidation by rat hepatic microsomes was 90-fold greater than that of human hepatic microsomes. Human intestinal microsomes converted BMP to BMP glucuronide at a rate even lower than that of human hepatic microsomes. Among the human UGT enzymes tested, only UGT2B7 had detectable glucuronidation activity for BMP. BMP monoglucuronide was the only metabolite formed when BMP was incubated with suspensions of freshly isolated hepatocytes from male F-344 rats or with cryopreserved human hepatocytes. Glucuronidation of BMP in human hepatocytes was extremely low. Overall, the results support in vivo studies in rats in which BMP glucuronide was the only metabolite found. The poor glucuronidation capacity of humans for BMP suggests that the pharmacokinetic profile of BMP in humans will be dramatically different from that of rodents. PMID:20200232

  1. The metabolism of 17 alpha-ethinyloestradiol by human liver microsomes: formation of catechol and chemically reactive metabolites.

    PubMed

    Purba, H S; Maggs, J L; Orme, M L; Back, D J; Park, B K

    1987-04-01

    The metabolism of 17 alpha-ethinyloestradiol (EE2) to catechol and reactive metabolites by human liver microsomes was investigated. 2-Hydroxyethinyloestradiol (2-OHEE2) was either the sole or principal metabolite. Small amounts of 6-hydroxyethinyloestradiol and 16-hydroxyethinyloestradiol were produced by some of the livers. EE2 (10 microM) underwent substantial (5-20% of incubated drug), though highly variable, NADPH-dependent metabolism to material irreversibly bound to microsomal protein. 2-OHEE2 appeared to be the pro-reactive metabolite. The maximum EE2 2-hydroxylase activity was 0.67 nmol min-1 mg-1 microsomal protein, with a Km value of 8.6 microM. Oestradiol, which is mainly hydroxylated to 2-hydroxyoestradiol, was the most potent inhibitor of hydroxylase activity and exhibited competitive inhibition. Progesterone, which undergoes 2-hydroxylation to a minor extent was also a competitive inhibitor, whereas cholesterol and cortisol did not have any appreciable inhibitory effect. Primaquine was the most potent non-steroidal inhibitor but was non-competitive. Other non-steroidal compounds investigated, e.g. antipyrine, did not show any significant effect on EE2 2-hydroxylation. The results of this study suggest that EE2 2-hydroxylation is metabolised by a form(s) of cytochrome P-450 which has affinity for endogenous steroids. PMID:3555579

  2. Critically Assessing the Predictive Power of QSAR Models for Human Liver Microsomal Stability.

    PubMed

    Liu, Ruifeng; Schyman, Patric; Wallqvist, Anders

    2015-08-24

    To lower the possibility of late-stage failures in the drug development process, an up-front assessment of absorption, distribution, metabolism, elimination, and toxicity is commonly implemented through a battery of in silico and in vitro assays. As in vitro data is accumulated, in silico quantitative structure-activity relationship (QSAR) models can be trained and used to assess compounds even before they are synthesized. Even though it is generally recognized that QSAR model performance deteriorates over time, rigorous independent studies of model performance deterioration is typically hindered by the lack of publicly available large data sets of structurally diverse compounds. Here, we investigated predictive properties of QSAR models derived from an assembly of publicly available human liver microsomal (HLM) stability data using variable nearest neighbor (v-NN) and random forest (RF) methods. In particular, we evaluated the degree of time-dependent model performance deterioration. Our results show that when evaluated by 10-fold cross-validation with all available HLM data randomly distributed among 10 equal-sized validation groups, we achieved high-quality model performance from both machine-learning methods. However, when we developed HLM models based on when the data appeared and tried to predict data published later, we found that neither method produced predictive models and that their applicability was dramatically reduced. On the other hand, when a small percentage of randomly selected compounds from data published later were included in the training set, performance of both machine-learning methods improved significantly. The implication is that 1) QSAR model quality should be analyzed in a time-dependent manner to assess their true predictive power and 2) it is imperative to retrain models with any up-to-date experimental data to ensure maximum applicability. PMID:26170251

  3. Glucuronidation of the aspirin metabolite salicylic acid by expressed UDP-glucuronosyltransferases and human liver microsomes.

    PubMed

    Kuehl, Gwendolyn E; Bigler, Jeannette; Potter, John D; Lampe, Johanna W

    2006-02-01

    Acetylsalicylic acid (aspirin) is a common nonsteroidal anti-inflammatory drug used for treatment of pain and arthritis. In the body, acetylsalicylic acid is rapidly deacetylated to form salicylic acid. Both compounds have been proposed as anti-inflammatory agents. Major metabolites of salicylic acid are its acyl and phenolic glucuronide conjugates. Formation of these conjugates, catalyzed by UDP-glucuronosyltransferases (UGTs), decreases the amount of pharmacologically active salicylic acid present. We aimed to identify the UGTs catalyzing the glucuronidation of salicylic acid using both heterologously expressed enzymes and pooled human liver microsomes (HLMs) and to develop a liquid chromatography-tandem mass spectrometry method to quantify glucuronidation activity of UGTs 1A1, 1A3, 1A4, 1A6, 1A7, 1A8, 1A9, 1A10, 2B4, 2B7, 2B15, and 2B17 Supersomes. All UGTs tested, except 1A4, 2B15, and 2B17, catalyzed salicylic acid phenolic and acyl glucuronidation. Ratios of salicylic acid phenolic to acyl glucuronide formation varied more than 12-fold from 0.5 for UGT1A6 to 6.1 for UGT1A1. These results suggest that all UGTs except 1A4, 2B15, and 2B17 might be involved in the glucuronidation of salicylic acid in vivo. From comparisons of apparent Km values determined in pooled HLMs and in expressed UGTs, UGT2B7 was suggested as a likely catalyst of salicylic acid acyl glucuronidation, whereas multiple UGTs were suggested as catalysts of phenolic glucuronidation. The results of this UGT screening may help target future evaluation of the effects of UGT polymorphisms on response to aspirin in clinical and population-based studies. PMID:16258079

  4. The Nonspecific Binding of Tyrosine Kinase Inhibitors to Human Liver Microsomes.

    PubMed

    Burns, Kushari; Nair, Pramod C; Rowland, Andrew; Mackenzie, Peter I; Knights, Kathleen M; Miners, John O

    2015-12-01

    Drugs and other chemicals frequently bind nonspecifically to the constituents of an in vitro incubation mixture, particularly the enzyme source [e.g., human liver microsomes (HLM)]. Correction for nonspecific binding (NSB) is essential for the accurate calculation of the kinetic parameters Km, Clint, and Ki. Many tyrosine kinase inhibitors (TKIs) are lipophilic organic bases that are nonionized at physiologic pH. Attempts to measure the NSB of several TKIs to HLM by equilibrium dialysis proved unsuccessful, presumably due to the limited aqueous solubility of these compounds. Thus, the addition of detergents to equilibrium dialysis samples was investigated as an approach to measure the NSB of TKIs. The binding of six validation set nonionized lipophilic bases (felodipine, isradipine, loratidine, midazolam, nifedipine, and pazopanib) to HLM (0.25 mg/ml) was shown to be unaffected by the addition of CHAPS (6 mM) to the dialysis medium. This approach was subsequently applied to measurement of the binding of axitinib, dabrafenib, erlotinib, gefitinib, ibrutinib, lapatinib, nilotinib, nintedanib, regorafenib, sorafenib, and trametinib to HLM (0.25 mg/ml). As with the validation set drugs, attainment of equilibrium was demonstrated in HLM-HLM and buffer-buffer control dialysis experiments. Values of the fraction unbound to HLM ranged from 0.14 (regorafenib and sorafenib) to 0.93 (nintedanib), and were generally consistent with the known physicochemical determinants of drug NSB. The extensive NSB of many TKIs to HLM underscores the importance of correction for TKI binding to HLM and, presumably, other enzyme sources present in in vitro incubation mixtures. PMID:26443648

  5. Biotransformation of lovastatin--III. Effect of cimetidine and famotidine on in vitro metabolism of lovastatin by rat and human liver microsomes.

    PubMed

    Vyas, K P; Kari, P H; Wang, R W; Lu, A Y

    1990-01-01

    The effects of the H2-receptor antagonists, cimetidine and famotidine, on the microsomal metabolism of [14C]lovastatin were investigated. Liver microsomes were prepared from control, phenobarbital- and 3-methylcholanthrene-pretreated rats and humans (male and female). Concentration-dependent inhibition of the metabolism of lovastatin (0.1 mM) was observed with cimetidine (0.1 to 1.0 mM). In contrast, famotidine at a similar concentration was a very weak inhibitor. The formation of 6'beta-hydroxy-lovastatin, the major microsomal metabolite of lovastatin, was similarly inhibited. The results suggest that in vivo metabolic interaction with concomitantly administered lovastatin is less likely with famotidine than with cimetidine. Phenobarbital pretreatment produced 58% stimulation in overall metabolism, whereas 3-methylcholanthrene pretreatment had no effect relative to control rats (5.4 nmol/mg protein/min). Liver microsomes from phenobarbital-pretreated rats produced 67% more of the 6'beta-hydroxy-lovastatin but 63-66% less of the 3''-hydroxy and 6'-exomethylene metabolites. Liver microsomes from 3-methylcholanthrene-treated rats also produced less 3"-hydroxy-lovastatin (49%) but similar quantities of the other two metabolites. 6'beta-Hydroxy-lovastatin was a major metabolite with human liver microsomes. Interestingly with these microsomes, hydroxylation at the 3''-position of the molecule was a negligible pathway and hydrolysis to the hydroxy acid form was not observed. The formation of 6'-exomethylene-lovastatin was also catalyzed by human liver microsomes (0.5 to 0.8 nmol/mg protein/min). PMID:2297361

  6. In vitro metabolism of benzo[a]pyrene and dibenzo[def,p]chrysene in rodent and human hepatic microsomes

    PubMed Central

    Crowell, S.R.; Hanson-Drury, S.; Williams, D.E.; Corley, R.A.

    2014-01-01

    Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous and often carcinogenic contaminants released into the environment during natural and anthropogenic combustion processes. Benzo[a]pyrene (B[a]P) is the prototypical carcinogenic PAH, and dibenzo[def,p]chrysene (DBC) is a less prevalent, but highly potent transplacental carcinogenic PAH. Both are metabolically activated by isoforms of the cytochrome P450 enzyme superfamily to form reactive carcinogenic and cytotoxic metabolites. Metabolism of B[a]P and DBC was studied in hepatic microsomes of male Sprague-Dawley rats, naïve and pregnant female B6129SF1/J mice, and female humans, corresponding to available pharmacokinetic data. Michaelis–Menten saturation kinetic parameters including maximum rates of metabolism (VMAX, nmol/min/mg microsomal protein), affinity constants (KM, μM), and rates of intrinsic clearance (CLINT, ml/min/kg body weight) were calculated from substrate depletion data. CLINT was also estimated from substrate depletion data using the alternative in vitro half-life method. VMAX and CLINT were higher for B[a]P than DBC, regardless of species. Clearance for both B[a]P and DBC was highest in naïve female mice and lowest in female humans. Clearance rates of B[a]P and DBC in male rat were more similar to female human than to female mice. Clearance of DBC in liver microsomes from pregnant mice was reduced compared to naïve mice, consistent with reduced active P450 protein levels and elevated tissue concentrations and residence times for DBC observed in previous in vivo pharmacokinetic studies. These findings suggest that rats are a more appropriate model organism for human PAH metabolism, and that pregnancy's effects on metabolism should be further explored. PMID:24769260

  7. Stereochemical aspects of vinylcyclohexene bioactivation in rodent hepatic microsomes and purified human cytochrome P450 enzyme systems.

    PubMed

    Fontaine, S M; Mash, E A; Hoyer, P B; Sipes, I G

    2001-02-01

    The racemic mixture of 4-vinylcyclohexene (VCH) forms ovotoxic epoxides [VCH-1,2-epoxide, VCH-7,8-epoxide, and vinylcyclohexene diepoxide (VCD)] by cytochrome P450 (CYP) in B6C3F(1) female mice. These epoxides deplete primordial and primary follicles. The current studies compared in vitro epoxidation of (R)-VCH with that of (S)-VCH in hepatic microsomes prepared from adult female B6C3F(1) mice and Fischer 344 rats. Bioactivation of VCH in the rat was significantly less compared with that in the mouse. (R)-VCH formed significantly more VCH-1,2-epoxide as compared with (S)-VCH in both species, and less VCH-7,8-epoxide in the mouse. Neither of the enantiomers formed detectable amounts of VCD in the mouse or rat. Hepatic microsomes prepared from mice and rats pretreated with CYP-inducing agents (phenobarbital and acetone) were also incubated with (R)-VCH or (S)-VCH. Although monoepoxide formation was not increased enantioselectively in the mouse, VCD was formed preferentially from (R)-VCH as compared with (S)-VCH. Pretreatment with VCH resulted in nonstereoselective increases in both monoepoxide and diepoxide formation. In the rat, these pretreatments resulted in nonstereoselective increases in monoepoxide formation, but VCD formation was not detectable. Incubations with human CYP2E1 enzyme revealed that (R)-VCH formed significantly more VCH-1,2-epoxide and less VCH-7,8-epoxide than (S)-VCH. Human CYP2A6 was limited in its ability to form epoxides from either enantiomer of VCH. Human CYP2B6 preferentially formed VCH-7,8-epoxide compared with VCH-1,2-epoxide, and to a greater extent from (R)-VCH than from (S)-VCH. These results demonstrate regioselectivity and enantioselectivity in the bioactivation of VCH in rodent hepatic microsomes as well as in expressed human CYP enzymes. PMID:11159809

  8. Inhibitory effects of psychotropic drugs on mexiletine metabolism in human liver microsomes: prediction of in vivo drug interactions.

    PubMed

    Hara, Y; Nakajima, M; Miyamoto, K-I; Yokoi, T

    2005-06-01

    Mexiletine, an anti-arrhythmic agent, is used for the control of ventricular arrhythmias and for neuropathic pain from cancer or diabetes mellitus. It is sometimes used together with psychotropic drugs in patients with depression, schizophrenia or sleep disorder. It is metabolized mainly by cytochrome P450 (CYP) 2 D 6 and, to a minor extent, by CYP1A2. To predict possible drug interactions between mexiletine and psychotropic drugs, the inhibitory effects of 14 psychotropic drugs (phenytoin, carbamazepine, fluvoxamine, paroxetine, fluoxetine, citalopram, sertraline, imipramine, desipramine, haloperidol, thioridazine, olanzapine, etizolam, and quazepam) on mexiletine metabolism in human liver microsomes were determined. Fluoxetine (Ki=0.6+/- 0.1 microM), sertraline (Ki=7.6+/- 0.8 microM) and desipramine (Ki=3.2+/- 0.5 microM) competitively inhibited the mexiletine p-hydroxylation in human liver microsomes. Thioridazine (Kis=0.5+/- 0.2 microM; Kii =3.6+/-1.6 microM) and paroxetine (Kis=1.7+/- 0.7 microM; Kii=3.6+/- 0.9 microM) exhibited a mixed-type inhibition (competitive and non-competitive) toward mexiletine p-hydroxylation in human liver microsomes. The changes of the in vivo clearance of mexiletine by the psychotropic drugs were predicted by 1+(I/Ki) using the in vitro Ki and unbound inhibitor concentrations in liver. The values were calculated as 2.4 for paroxetine, 5.5 for fluoxetine, 1.1 for sertraline, 2.8 for desipramine and 2.2 for thioridazine. In addition, paroxetine exhibited a mechanism-based inactivation with Ki=0.7 microM and Kinact=0.15 min(-1). The present study predicted the possibility of drug interactions between mexiletine and paroxetine, fluoxetine, desipramine, and thioridazine in clinical use. PMID:16192107

  9. Validation of 4-nitrophenol as an in vitro substrate probe for human liver CYP2E1 using cDNA expression and microsomal kinetic techniques.

    PubMed

    Tassaneeyakul, W; Veronese, M E; Birkett, D J; Gonzalez, F J; Miners, J O

    1993-12-01

    The involvement of human cytochrome P450 (CYP) 2E1 in the hydroxylation of 4-nitrophenol (4NP) to 4-nitrocatechol (4NC) has been investigated using cDNA expression and liver microsomal kinetic and inhibitor techniques. 4NP hydroxylation by human liver microsomes and cDNA-expressed human CYP2E1 exhibited Michaelis-Menten kinetics; the respective apparent Km values were 30 +/- 7 and 21 microM. Mutual competitive inhibition was observed for 4NP and chlorzoxazone (CZ) (an alternative human CYP2E1 substrate) in liver microsomes, with close similarities between the calculated apparent Km and Ki values for each individual compound. 4NP and CZ hydroxylase activities in microsomes from 18 liver donors varied to a similar extent (3.3- and 3.0-fold, respectively) and 4NP hydroxylase activity correlated significantly (rs > or = 0.75, P < 0.005) with both CZ hydroxylation and immunoreactive CYP2E1 content. The prototypic CYP2E1 inhibitor, diethyldithiocarbamate, was a potent inhibitor of 4NC formation and decreased 4NP hydroxylation by cDNA-expressed CYP2E1 and human liver microsomes in parallel. Probes for other human CYP isoforms namely (alpha-naphthoflavone, coumarin, sulphaphenazole, quinidine, troleandomycin and mephenytoin) caused < 15% inhibition of liver microsomal 4NP hydroxylation. These data confirm that, as in animal species, 4NP hydroxylation is catalysed largely by CYP2E1 in human liver and 4NP may therefore be used as an in vitro substrate probe for the human enzyme. PMID:8267647

  10. Biotransformation of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) by human liver microsomes: identification of cytochrome P450 2B6 as the major enzyme involved.

    PubMed

    Erratico, Claudio A; Szeitz, András; Bandiera, Stelvio M

    2013-05-20

    Polybrominated diphenyl ethers (PBDEs) were widely used flame retardants that have become persistent environmental pollutants. In the present study, we investigated the in vitro oxidative metabolism of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47), a major PBDE detected in human tissue and environmental samples. Biotransformation of BDE-47 by pooled and individual human liver microsomes and by human recombinant cytochrome P450 (P450) enzymes was assessed using a liquid chromatography/tandem mass spectrometry-based method. Of the nine hydroxylated metabolites of BDE-47 produced by human liver microsomes, seven metabolites were identified using authentic standards. A monohydroxy-tetrabrominated and a dihydroxy-tetrabrominated metabolite remain unidentified. Kinetic analysis of the rates of metabolite formation revealed that the major metabolites were 5-hydroxy-2,2',4,4'-tetrabromodiphenyl ether (5-OH-BDE-47), 6-hydroxy-2,2',4,4'-tetrabromodiphenyl ether (6-OH-BDE-47), and possibly the unidentified monohydroxy-tetrabrominated metabolite. Among the human recombinant P450 enzymes tested, P450 2B6 was the most active enzyme in the formation of the hydroxylated metabolites of BDE-47. Moreover, the formation of all metabolites of BDE-47 by pooled human liver microsomes was inhibited by a P450 2B6-specific antibody and was highly correlated with P450 2B6-mediated activity in single donor liver microsomes indicating that P450 2B6 was the major P450 responsible for the biotransformation of BDE-47. Additional experiments involving the incubation of liver microsomes with individual monohydroxy-tetrabrominated metabolites in place of BDE-47 demonstrated that 2,4-dibromophenol was a product of BDE-47 and several primary metabolites, but the dihydroxy-tetrabrominated metabolite was not formed by sequential hydroxylation of any of the monohydroxy-tetrabrominated metabolites tested. The present study provides a comprehensive characterization of the oxidative metabolism of BDE-47 by

  11. Metabolism of Anandamide by Human Cytochrome P450 2J2 in the Reconstituted System and Human Intestinal Microsomes.

    PubMed

    Walker, Vyvyca J; Griffin, Alisha P; Hammar, Dagan K; Hollenberg, Paul F

    2016-06-01

    According to the Centers for Disease Control and Prevention, the incidence of inflammatory bowel diseases (IBD) is about 1 in 250 people in the United States. The disease is characterized by chronic or recurring inflammation of the gut. Because of the localization of the endocannabinoid system in the gastrointestinal tract, it may be a potential pharmacologic target for the treatment of IBD and other diseases. Fatty acid amide hydrolase (FAAH) is a potential candidate because it is upregulated in IBD. FAAH hydrolyzes and, as a consequence, inactivates anandamide (AEA), a prominent endocannabinoid. Inhibition of FAAH would lead to increases in the amount of AEA oxidized by cytochrome P450s (P450s). CYP2J2, the major P450 epoxygenase expressed in the heart, is also expressed in the intestine and has previously been reported to oxidize AEA. We have investigated the possibility that it may play a role in AEA metabolism in the gut and have demonstrated that purified human CYP2J2 metabolizes AEA to form the 20-hydroxyeicosatetraenoic acid ethanolamide (HETE-EA) and several epoxygenated products, including the 5,6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acid ethanolamides (EET-EAs), in the reconstituted system. Kinetic studies suggest that the KM values for these products range from approximately 10 to 468 μM and the kcat values from 0.2 to 23.3 pmol/min per picomole of P450. Human intestinal microsomes, which express CYP2J2, metabolize AEA to give the 5,6-, 8,9-, and 11,12-EET-EAs, as well as 20-HETE-EA. Studies using specific P450 inhibitors suggest that although CYP2J2 metabolizes AEA, it is not the primary P450 responsible for AEA metabolism in human intestines. PMID:27000802

  12. Identification of Human UDP-Glucuronosyltransferase 1A4 as the Major Isozyme Responsible for the Glucuronidation of 20(S)-Protopanaxadiol in Human Liver Microsomes

    PubMed Central

    Li, Jia; He, Chunyong; Fang, Lianxiang; Yang, Li; Wang, Zhengtao

    2016-01-01

    20(S)-protopanaxadiol (PPD), one of the representative aglycones of ginsenosides, has a broad spectrum of pharmacological activities. Although phase I metabolism has been investigated extensively, information regarding phase II metabolism of this compound remains to be elucidated. Here, a glucuronidated metabolite of PPD in human liver microsomes (HLMs) and rat liver microsomes (RLMs) was unambiguously identified as PPD-3-O-β-d-glucuronide by nuclear magnetic resonance spectroscopy and high resolution mass spectrometry. The chemical inhibition and recombinant human UDP-Glucuronosyltransferase (UGT) isoforms assay showed that the PPD glucuronidation was mainly catalyzed by UGT1A4 in HLM, whereas UGT1A3 showed weak catalytic activity. In conclusion, PPD-3-O-β-d-glucuronide was first identified as the principal glucuronidation metabolite of PPD in HLMs, which was catalyzed by UGT1A4. PMID:27005621

  13. Effects of capsaicin and dihydrocapsaicin on human and rat liver microsomal CYP450 enzyme activities in vitro and in vivo.

    PubMed

    Zhang, Qing-Hao; Hu, Jin-Ping; Wang, Bao-Lian; Li, Yan

    2012-01-01

    Capsaicin and dihydrocapsaicin, the two most abundant members of capsaicinoids in chili peppers, are widely used as food additives and for other purposes. In this study, we examined the inhibitory potentials of capsaicin and dihydrocapsaicin against CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4/5 activities in human liver microsomes. The effects of these two capsaicinoids on CYP450 enzymes were also evaluated in vivo in rats. The results demonstrated that capsaicin and dihydrocapsaicin moderately inhibited five isozymes (IC₅₀) values ranging from 4.4 to 61.8 μM), with the exception of CYP2E1 (IC₅₀ > 200 μM). Both capsaicinoids exhibited competitive, mixed, and noncompetitive inhibition on these isozymes (K (i) = 3.1 ± 0.5 - 78.6 ± 8.4 μM). Time-dependent inhibition of CYP3A4/5 by capsaicin was found. After multiple administrations of capsaicin and dihydrocapsaicin (1, 4, and 10 mg/kg) to rats, chlorzoxazone 6-hydroxylase activity and the expression of CYP2E1 were increased in liver microsomes. Our findings indicated that the possibility of food-drug interactions mediated by capsaicin and dihydrocapsaicin could not be excluded, and provided the useful information for evaluating the anticarcinogenic potentials of these two capsaicinoids. PMID:22375877

  14. Nonenzymatic formation of a novel hydroxylated sulfamethoxazole derivative in human liver microsomes: implications for bioanalysis of sulfamethoxazole metabolites.

    PubMed

    Sanderson, Joseph P; Hollis, Frank J; Maggs, James L; Clarke, Stephen E; Naisbitt, Dean J; Park, B Kevin

    2008-12-01

    Sulfamethoxazole is metabolized by microsomal CYP2C9 to a hydroxylamine that is thought to be responsible for the relatively high incidence of hypersensitivity reactions associated with the drug. Accurate quantification of the hydroxylamine requires the loss of metabolite through autoxidation to be blocked with ascorbate. In this study, a partly nonenzymatically generated arylhydroxylated derivative of sulfamethoxazole was identified by liquid chromatography/mass spectrometry in incubations of human liver microsomes, and it was found to coelute with the isomeric hydroxylamine under the conditions of three published high-performance liquid chromatography (HPLC) assays. Partial inhibition of the aryl hydroxylation by 1-aminobenzotriazole suggested some involvement of cytochrome P450. However, the formation of this compound was ascorbate-dependent, and it was enhanced by the addition of Fe2+/EDTA and inhibited by desferrioxamine but not by mannitol. These findings are consistent with the phenol being generated via an Fe2+/ascorbate/O2-oxygenating system that does not involve hydroxyl radicals. It was also produced by H2O2/ascorbate. Because the compound shares close chromatographic similarities with the hydroxylamine metabolite, it is possible that previous studies may have inaccurately characterized or quantified sulfamethoxazole metabolism. PMID:18765684

  15. Metabolites profiling of 10 bufadienolides in human liver microsomes and their cytotoxicity variation in HepG2 cell.

    PubMed

    Han, Lingyu; Wang, Hongjie; Si, Nan; Ren, Wei; Gao, Bo; Li, Yan; Yang, Jian; Xu, Miao; Zhao, Haiyu; Bian, Baolin

    2016-04-01

    Bufadienolides, a class of polyhydroxy steroids, exhibit significant antitumor activity. In this study, a total of 39 metabolites from 10 bufadienolides were detected and identified by ultrahigh-performance liquid chromatography (UHPLC) coupled with an LTQ Orbitrap mass spectrometer. The results showed that hydroxylation and dehydrogenation were the major metabolic pathways of bufadienolides in human liver microsomes (HLMs). CYP3A4 was found to be the major metabolic enzyme and CYP2D6 only mediated the dehydrogenation reaction. A systematic validated cytotoxicity evaluation method for bufadienolide metabolites at equal equivalents was established. Hellebrigenin (1), hellebrigenol (2), arenobufagin (3), bufotalin (5), and bufalin (6) were selected to determine their cytotoxicity against HepG2 cells before and after incubation in HLMs. All the test samples were enriched by a validated solid-phase extraction (SPE) method. Although the cytotoxicities of metabolites were weaker than those of the parent compounds to different degrees, their effects were still strong. PMID:26869342

  16. Relationship of Metabolism and Cell Proliferation to the Mode of Action of Fluensulfone-Induced Mouse Lung Tumors: Analysis of Their Human Relevance Using the IPCS Framework

    PubMed Central

    Strupp, Christian; Banas, Deborah A.; Cohen, Samuel M.; Gordon, Elliot B.; Jaeger, Martina; Weber, Klaus

    2012-01-01

    Species-specific lung tumors in the mouse are induced by a number of chemicals. The underlying cause appears to be a high metabolic activity of mouse lung, due to relatively high abundance of Clara cells in mice compared with humans and the mouse-specific cytochrome P450 isoform 2f2 in the Clara cells. The chemicals are activated to reactive intermediates, leading to local cytotoxicity or mitogenicity resulting in increased cell proliferation and tumors. Rats have lower metabolic activity than mice (already below the threshold needed to cause lung tumors upon lifetime exposure) and activity in humans is lower than in rats. The carcinogenic risk for human lung is low for this mode of action (MOA). Fluensulfone has shown an increased incidence of lung adenomas in mice, but not in rats, at high doses. Fluensulfone is not genotoxic. MOA studies were conducted investigating key events of the postulated MOA. Fluensulfone is extensively metabolized by mouse lung microsomes, whereas no metabolic activity is seen with human lung microsomes. Cyp 2f2 is a major contributor in fluensulfone’s metabolism and Cyp 2e1 is not involved. Furthermore, administration of fluensulfone to mice led to an early increase in Clara cell proliferation. The International Programme on Chemical Safety (IPCS) MOA and human relevance framework was used to evaluate the collective data on fluensulfone. We concluded that fluensulfone leads to species-specific mouse lung tumors and that these tumors are likely not relevant to human hazard or risk. PMID:22491425

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

    PubMed

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

    2016-06-01

    Isocorynoxeine (ICN) is one of the major bioactive tetracyclic oxindole alkaloids found in Uncaria rhynchophylla (Miq.) Jacks. that is widely used for the treatment of hypertension, vascular dementia, and stroke. The present study was undertaken to assess the plasma pharmacokinetic characteristics of major ICN metabolites, and the role of simulated gastric and intestinal fluid (SGF and SIF), human and rat liver microsomes (HLMs and RLMs), and seven recombinant human CYP enzymes in the major metabolic pathway of ICN. A rapid, sensitive and accurate UHPLC/Q-TOF MS method was validated for the simultaneous determination of ICN and its seven metabolites in rat plasma after oral administration of ICN at 40mg/kg. It was found that 18.19-dehydrocorynoxinic acid (DCA) and 5-oxoisocorynoxeinic acid (5-O-ICA) were both key and predominant metabolites, rather than ICN itself, due to the rapid and extensive metabolism of ICN in vivo. The further study indicated that ICN was mainly metabolized in human or rat liver, and CYPs 2C19, 3A4 and 2D6 were the major enzymes responsible for the biotransformation of ICN to DCA and 5-O-ICA in human. These findings are of significance in understanding of the pharmacokinetic nature of tetracyclic oxindole alkaloids, and provide helpful information for the clinical co-administration of the herbal preparations containing U. rhynchophylla with antihypertensive drugs that are mainly metabolized by CYP3A4 and CYP2C19. PMID:27094112

  18. Formation of GSH-trapped reactive metabolites in human liver microsomes, S9 fraction, HepaRG-cells, and human hepatocytes.

    PubMed

    Lassila, Toni; Rousu, Timo; Mattila, Sampo; Chesné, Christophe; Pelkonen, Olavi; Turpeinen, Miia; Tolonen, Ari

    2015-11-10

    The objective was to compare several in vitro human liver-derived subcellular and cellular incubation systems for the formation of GSH-trapped reactive metabolites. Incubations of pooled human liver microsomes, human liver S9 fractions, HepaRG-cells, and human hepatocytes were performed with glutathione as a trapping agent. Experiments with liver S9 were performed under two conditions, using only NADPH and using a full set of cofactors enabling also conjugative metabolism. Ten structurally different compounds were used as a test set, chosen as either "positive" (ciprofloxacin, clozapine, diclofenac, ethinyl estradiol, pulegone, and ticlopidine) or "negative" (caffeine, citalopram, losartan, montelukast) compounds, based on their known adverse reactions on liver or bone marrow. GSH conjugates were observed for seven of the ten compounds; while no conjugates were observed for caffeine, citalopram, or ciprofloxacin. Hepatocyte and HepaRG assays produced a clearly lower number and lower relative abundance of GSH conjugates compared to assays with microsomes and S9 fractions. The major GSH conjugates were different for many compounds in cellular subfractions and cell-based systems. Hepatocytes generally produced a higher number of GSH conjugates than HepaRG cells, although the differences were minor. The results show that the hepatic enzyme system used for screening of GSH-trapped reactive metabolites do have a high impact on the results, and results between different systems are comparable only qualitatively. PMID:26263063

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

    SciTech Connect

    Watanabe, Yoko; Kojima, Hiroyuki; Takeuchi, Shinji; Uramaru, Naoto; Sanoh, Seigo; Sugihara, Kazumi; Kitamura, Shigeyuki; Ohta, Shigeru

    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

  20. Amlodipine metabolism in human liver microsomes and roles of CYP3A4/5 in the dihydropyridine dehydrogenation.

    PubMed

    Zhu, Yanlin; Wang, Fen; Li, Quan; Zhu, Mingshe; Du, Alicia; Tang, Wei; Chen, Weiqing

    2014-02-01

    Amlodipine is a commonly prescribed calcium channel blocker for the treatment of hypertension and ischemic heart disease. The drug is slowly cleared in humans primarily via dehydrogenation of its dihydropyridine moiety to a pyridine derivative (M9). Results from clinical drug-drug interaction studies suggest that CYP3A4/5 mediate metabolism of amlodipine. However, attempts to identify a role of CYP3A5 in amlodipine metabolism in humans based on its pharmacokinetic differences between CYP3A5 expressers and nonexpressers failed. Objectives of this study were to determine the metabolite profile of amlodipine (a racemic mixture and S-isomer) in human liver microsomes (HLM), and to identify the cytochrome P450 (P450) enzyme(s) involved in the M9 formation. Liquid chromatography/mass spectrometry analysis showed that amlodipine was mainly converted to M9 in HLM incubation. M9 underwent further O-demethylation, O-dealkylation, and oxidative deamination to various pyridine derivatives. This observation is consistent with amlodipine metabolism in humans. Incubations of amlodipine with HLM in the presence of selective P450 inhibitors showed that both ketoconazole (an inhibitor of CYP3A4/5) and CYP3cide (an inhibitor of CYP3A4) completely blocked the M9 formation, whereas chemical inhibitors of other P450 enzymes had little effect. Furthermore, metabolism of amlodipine in expressed human P450 enzymes showed that only CYP3A4 had significant activity in amlodipine dehydrogenation. Metabolite profiles and P450 reaction phenotyping data of a racemic mixture and S-isomer of amlodipine were very similar. The results from this study suggest that CYP3A4, rather than CYP3A5, plays a key role in metabolic clearance of amlodipine in humans. PMID:24301608

  1. In vitro characterization of 4'-(p-toluenesulfonylamide)-4-hydroxychalcone using human liver microsomes and recombinant cytochrome P450s.

    PubMed

    Lee, Boram; Wu, Zhexue; Lee, Taeho; Tan, Xue Fei; Park, Ki Hun; Liu, Kwang-Hyeon

    2016-01-01

    1. 4'-(p-Toluenesulfonylamide)-4-hydroxychalcone (TSAHC) is a synthetic sulfonylamino chalcone compound possessing anti-cancer properties. The aim of this study was to elucidate the metabolism of TSAHC in human liver microsomes (HLMs) and to characterize the cytochrome P450 (P450) enzymes that are involved in the metabolism of TSAHC. 2. TSAHC was incubated with HLMs or recombinant P450 isoforms (rP450) in the presence of an nicotinamide adenine dinucleotide phosphate, reduced form (NADPH)-regenerating system. The metabolites were identified and analyzed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). P450 isoforms, responsible for TSAHC metabolite formation, were characterized by chemical inhibition and correlation studies in HLMs and enzyme kinetic studies with a panel of rP450 isoforms. 3. Two hydroxyl metabolites, that is M1 and M2, were produced from the human liver microsomal incubations (K(m) and V(max) values were 2.46 µM and 85.1 pmol/min/mg protein for M1 and 9.98 µM and 32.1 pmol/min/mg protein for M2, respectively). The specific P450 isoforms responsible for two hydroxy-TSAHC formations were identified using a combination of chemical inhibition, correlation analysis and metabolism by expressed recombinant P450 isoforms. The known P450 enzyme activities and the rate of TSAHC metabolite formation in the 15 HLMs showed that TSAHC metabolism is correlated with CYP2C and CYP3A activity. The P450 isoform-selective inhibition study in HLMs and the incubation study of cDNA-expressed enzymes also showed that two hydroxyl metabolites M1 and M2 biotransformed from TSAHC are mainly mediated by CYP2C and CYP3A, respectively. These findings suggest that CYP2C8, CYP2C9, CYP2C19, CYP3A4 and CYP3A5 isoforms are major enzymes contributing to TSAHC metabolism. PMID:26330107

  2. Catalytic characterization of human microsomal glutathione S-transferase 2: identification of rate-limiting steps.

    PubMed

    Ahmad, Shabbir; Niegowski, Damian; Wetterholm, Anders; Haeggström, Jesper Z; Morgenstern, Ralf; Rinaldo-Matthis, Agnes

    2013-03-12

    Microsomal glutathione S-transferase 2 (MGST2) is a 17 kDa trimeric integral membrane protein homologous to leukotriene C4 synthase (LTC4S). MGST2 has been suggested to catalyze the biosynthesis of the pro-inflammatory mediator leukotriene C4 (LTC4) in cells devoid of LTC4S. A detailed biochemical study of MGST2 is critical for the understanding of its cellular function and potential role as an LTC4-producing enzyme. Here we have characterized the substrate specificity and catalytic properties of purified MGST2 by steady-state and pre-steady-state kinetic experiments. In comparison with LTC4S, which has a catalytic efficiency of 8.7 × 10(5) M(-1) s(-1), MGST2, with a catalytic efficiency of 1.8 × 10(4) M(-1) s(-1), is considerably less efficient in producing LTC4. However, the two enzymes display a similar KM(LTA4) of 30-40 μM. While LTC4S has one activated glutathione (GSH) (forming a thiolate) per enzyme monomer, the MGST2 trimer seems to display only third-of-the-sites reactivity for thiolate activation, which in part would explain its lower catalytic efficiency. Furthermore, MGST2 displays GSH-dependent peroxidase activity of ∼0.2 μmol min(-1) mg(-1) toward several lipid hydroperoxides. MGST2, but not LTC4S, is efficient in catalyzing conjugation of the electrophilic substrate 1-chloro-2,4-dinitrobenzene (CDNB) and the lipid peroxidation product 4-hydroxy-2-nonenal with GSH. Using stopped-flow pre-steady-state kinetics, we have characterized the full catalytic reaction of MGST2 with CDNB and GSH as substrates, showing an initial rapid equilibrium binding of GSH followed by thiolate formation. Burst kinetics for the CDNB-GSH conjugation step was observed only at low GSH concentrations (thiolate anion formation becoming rate-limiting under these conditions). Product release is rapid and does not limit the overall reaction. Therefore, in general, the chemical conjugation step is rate-limiting for MGST2 at physiological GSH concentrations. MGST2 and LTC4S

  3. Postmortem inflation and fixation of human lungs

    PubMed Central

    Wright, B. M.; Slavin, G.; Kreel, L.; Callan, K.; Sandin, Brenda

    1974-01-01

    Wright, B. M., Slavin, G., Kreel, L., Callan, K., and Sandin, Brenda (1974).Thorax, 29, 189-194. Postmortem inflation and fixation of human lungs. A method of fixing lungs by inflating them with heated formalin vapour is described. This method facilitates postmortem correlations between radiographic and histological appearances. Images PMID:4598582

  4. Characterization of in vitro metabolic profiles of cinitapride obtained with liver microsomes of humans and various mammal species using UHPLC and chemometric methods for data analysis.

    PubMed

    Marquez, Helena; Albertí, Joan; Salvà, Miquel; Saurina, Javier; Sentellas, Sonia

    2012-05-01

    An ultra-high performance liquid chromatographic method has been utilized to obtain metabolic profiles of cinitapride with liver microsomes of humans and various mammal species such as rats, mice, mini pigs, dogs, and monkeys. Metabolites have been generated by incubation of cinitapride in the presence of microsomes using nicotinamide adenine dinucleotide phosphate as a cofactor. Incubation times from 15 to 60 min have been assayed. Cinitapride and its metabolites have been separated by reversed-phase C(18) mode using ammonium formate aqueous solution (pH 6.5) and acetonitrile as the components of the mobile phase. Concentrations of metabolites in the incubated samples have resulted in an excellent source of multivariate data to be used to extract metabolic information. Statistic parameters and principal component analysis have been used to compare the in vitro metabolism of humans with the other species. PMID:22362276

  5. High-performance liquid chromatographic assay for 4-nitrophenol hydroxylation, a putative cytochrome P-4502E1 activity, in human liver microsomes.

    PubMed

    Tassaneeyakul, W; Veronese, M E; Birkett, D J; Miners, J O

    1993-06-23

    A high-performance liquid chromatographic method which measures formation of product 4-nitrocatechol (4NC) has been developed and applied to the study of human liver microsomal 4-nitrophenol (4NP) hydroxylation. Following diethyl ether extraction, 4NC and the assay internal standard (salicylamide) were separated by reversed-phase (C18) liquid chromatography. Extraction efficiencies of 4NC and internal standard were both > 90%. The assay, which has a limit of detection of 15 pmol injected (or an incubation 4NC concentration of 0.25 microM), is accurate, reproducible and straightforward. With a chromatographic time of 12 min, 40-50 samples may be analyzed per day. Rates of 4NC formation were linear with time and protein concentration to 50 min and 0.5 mg/ml, respectively. Preliminary studies of 4NP hydroxylation showed that this reaction followed single enzyme Michaelis-Menten kinetics in human liver microsomes. PMID:8376495

  6. Metabolic studies of prostanozol with the uPA-SCID chimeric mouse model and human liver microsomes.

    PubMed

    Geldof, Lore; Lootens, Leen; Decroix, Lieselot; Botrè, Francesco; Meuleman, Philip; Leroux-Roels, Geert; Deventer, Koen; Van Eenoo, Peter

    2016-03-01

    Anabolic androgenic steroids are prohibited by the World Anti-Doping Agency because of their adverse health and performance enhancing effects. Effective control of their misuse by detection in urine requires knowledge about their metabolism. In case of designer steroids, ethical objections limit the use of human volunteers to perform excretion studies. Therefore the suitability of alternative models needs to be investigated. In this study pooled human liver microsomes (HLM) and an uPA(+/+)-SCID chimeric mouse model were used to examine the metabolism of the designer steroid prostanozol as a reference standard. Metabolites were detected by GC-MS (full scan) and LC-MS/MS (precursor ion scan). In total twenty-four prostanozol metabolites were detected with the in vitro and in vivo metabolism studies, which could be grouped into two broad classes, those with a 17-hydroxy- and those with a 17-keto-substituent. Major first phase metabolic sites were tentatively identified as C-3'; C-4 and C-16. Moreover, 3'- and 16β-hydroxy-17-ketoprostanozol could be unequivocally identified, since authentic reference material was available, in both models. Comparison with published data from humans showed a good correlation, except for phase II metabolism. As metabolites were in contrast to the human studies predominantly present in the free fraction. Two types of metabolites ((di)hydroxylated prostanozol metabolites) that have not been described before could be confirmed in a real positive doping control sample. Hence, the results provide further evidence for the applicability of chimeric mice and HLM to perform metabolism studies of designer steroids. PMID:26774429

  7. Garlic inhibits microsomal triglyceride transfer protein gene expression in human liver and intestinal cell lines and in rat intestine.

    PubMed

    Lin, Marie C; Wang, Er-Jia; Lee, Catherine; Chin, K T; Liu, Depei; Chiu, Jen-Fu; Kung, Hsiang-Fu

    2002-06-01

    Epidemiologic studies have suggested that fresh garlic has lipid-lowering activity. Because the microsomal triglyceride transfer protein (MTP) plays a pivotal role in the assembly and secretion of apolipoprotein B (apoB)-containing lipoproteins, we evaluated the effect of garlic on the expression of the MTP gene in vitro in cell lines and in vivo in rats. Fresh garlic extract (FGE) reduced MTP mRNA levels in both the human hepatoma HepG2 and intestinal carcinoma Caco-2 cells in dose-dependent fashion; significant reductions were detected with 3 g/L FGE. Maximal 72 and 59% reductions, respectively, were observed with 6 g/L FGE. To evaluate the in vivo effect of garlic on MTP gene expression, rats were given a single oral dose of fresh garlic homogenate (FGH), with hepatic and intestinal MTP mRNA measured 3 h after dosing. Rats fed FGH had significantly (46% of the control) lower intestinal MTP mRNA levels compared with the control rats, whereas hepatic MTP mRNA levels were not affected. These results suggest a new mechanism for the hypolipidemic effect of fresh garlic. Long-term dietary supplementation of fresh garlic may exert a lipid-lowering effect partly through reducing intestinal MTP gene expression, thus suppressing the assembly and secretion of chylomicrons from intestine to the blood circulation. PMID:12042427

  8. Comparative effects of the antimycotic drugs ketoconazole, fluconazole, itraconazole and terbinafine on the metabolism of cyclosporin by human liver microsomes.

    PubMed Central

    Back, D J; Tjia, J F

    1991-01-01

    Four antimycotic drugs, the azoles ketoconazole, itraconazole and fluconazole, and the allylamine terbinafine have been studied for their effect on the metabolism of cyclosporin by human liver microsomes (n = 3) in vitro. Ketoconazole caused marked inhibition of cyclosporin hydroxylase (to metabolites M17 and M1) with IC50 and Ki values of 0.24 +/- 0.01 and 0.022 +/- 0.004 microM, respectively. Based on IC50 values, itraconazole was ten times less potent (IC50 value of 2.2 +/- 0.2 microM) and both fluconazole and terbinafine had values above 100 microM. Ki values for itraconazole and fluconazole were 0.7 +/- 0.2 and 40 +/- 5.6 microM, respectively. No kinetic parameters were calculated for terbinafine because of the lack of inhibitory effects. Based on these data, ketoconazole is confirmed as being a potent inhibitor of cyclosporin metabolism and this has clinical relevance. Although inhibition by fluconazole was much less than that by itraconazole at equimolar concentrations, it should be noted that in patients plasma concentrations of fluconazole are much greater than those of itraconazole. Clinical interactions of cyclosporin with both fluconazole and itraconazole have been reported. In contrast to the azoles, terbinafine does not have the same potential for interaction. PMID:1659439

  9. Inhibitory effect of six herbal extracts on CYP2C8 enzyme activity in human liver microsomes.

    PubMed

    Albassam, Ahmed A; Mohamed, Mohamed-Eslam F; Frye, Reginald F

    2015-05-01

    1. Herbal supplements widely used in the US were screened for the potential to inhibit CYP2C8 activity in human liver microsomes. The herbal extracts screened were garlic, echinacea, saw palmetto, valerian, black cohosh and cranberry. N-desethylamodiaquine (DEAQ) and hydroxypioglitazone metabolite formation were used as indices of CYP2C8 activity. 2. All herbal extracts showed inhibition of CYP2C8 activity for at least one of three concentrations tested. A volume per dose index (VDI) was calculated to determine the volume in which a dose should be diluted to obtain IC50 equivalent concentration. Cranberry and saw palmetto had a VDI value > 5.0 l per dose unit, suggesting a potential for interaction. 3. Inhibition curves were constructed and the IC50 (mean ± SE) values were 24.7 ± 2.7 μg/ml for cranberry and 15.4 ± 1.7 μg/ml for saw palmetto. 4. The results suggest a potential for cranberry or saw palmetto extracts to inhibit CYP2C8 activity. Clinical studies are needed to evaluate the significance of this interaction. PMID:25430798

  10. Diazepam metabolism by human liver microsomes is mediated by both S-mephenytoin hydroxylase and CYP3A isoforms.

    PubMed Central

    Andersson, T; Miners, J O; Veronese, M E; Birkett, D J

    1994-01-01

    1. The primary metabolism of diazepam was studied in human liver microsomes in order to investigate the kinetics and to identify the cytochrome P450 (CYP) isoforms responsible for the formation of the main diazepam metabolites, temazepam and N-desmethyldiazepam. 2. The formation kinetics of both metabolites were atypical and consistent with the occurrence of substrate activation. A sigmoid Vmax model equivalent to the Hill equation was used to fit the data. The degree of sigmoidicity was greater for temazepam formation than for N-desmethyldiazepam formation, so that the ratio of desmethyldiazepam:temazepam formation increased as the substrate (diazepam) concentration decreased. 3. alpha-Naphthoflavone activated both reactions but with a greater effect on temazepam formation than on N-desmethyldiazepam formation. In the presence of 25 microM alpha-naphthoflavone the kinetics for both pathways were approximated by Michaelis-Menten kinetics. 4. Studies with a series of CYP isoform selective inhibitors and with an inhibitory anti-CYP2C antibody indicated that temazepam formation was carried out mainly by CYP3A isoforms, whereas the formation of N-desmethyldiazepam was mediated by both CYP3A isoforms and S-mephenytoin hydroxylase. PMID:7981013

  11. In vitro enantioselective human liver microsomal metabolism and prediction of in vivo pharmacokinetic parameters of tetrabenazine by DLLME-CE.

    PubMed

    Bocato, Mariana Zuccherato; de Lima Moreira, Fernanda; de Albuquerque, Nayara Cristina Perez; de Gaitani, Cristiane Masetto; de Oliveira, Anderson Rodrigo Moraes

    2016-09-01

    A new capillary electrophoresis method for the enantioselective analysis of cis- and trans- dihydrotetrabenazine (diHTBZ) after in vitro metabolism by human liver microsomes (HLMs) was developed. The chiral electrophoretic separations were performed by using tris-phosphate buffer (pH 2.5) containing 1% (w/v) carboxymethyl-β-CD as background electrolyte with an applied voltage of +15kV and capillary temperature kept at 15°C. Dispersive liquid-liquid microextraction was employed to extract the analytes from HLMs. Dichloromethane was used as extraction solvent (75μL) and acetone as disperser solvent (150μL). The method was validated according to official guidelines and showed to be linear over the concentration range of 0.29-19.57μmolL(-1) (r=0.9955) for each metabolite enantiomer. Within- and between-day precision and accuracy evaluated by relative standard deviation and relative error were lower than 15% for all enantiomers. The stability assay showed that the analytes kept stable under handling, storage and in metabolism conditions. After method validation, an enantioselective in vitro metabolism and in vivo pharmacokinetic prediction was carried out. This study showed a stereoselective metabolism and the observed kinetic profile indicated a substrate inhibition behavior. DiHTBZ enantiomers were catalyzed mainly by CYP2C19 and the predicted clearance suggests that liver metabolism is the main route for TBZ elimination which supports the literature data. PMID:27381871

  12. Stable Isotope Labeling Strategy for Curcumin Metabolite Study in Human Liver Microsomes by Liquid Chromatography-Tandem Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Gao, Dan; Chen, Xiaowu; Yang, Xiaomei; Wu, Qin; Jin, Feng; Wen, Hongliang; Jiang, Yuyang; Liu, Hongxia

    2015-04-01

    The identification of drug metabolites is very important in drug development. Nowadays, the most widely used methods are isotopes and mass spectrometry. However, the commercial isotopic labeled reagents are usually very expensive, and the rapid and convenient identification of metabolites is still difficult. In this paper, an 18O isotope labeling strategy was developed and the isotopes were used as a tool to identify drug metabolites using mass spectrometry. Curcumin was selected as a model drug to evaluate the established method, and the 18O labeled curcumin was successfully synthesized. The non-labeled and 18O labeled curcumin were simultaneously metabolized in human liver microsomes (HLMs) and analyzed by liquid chromatography/mass spectrometry (LC-MS). The two groups of chromatograms obtained from metabolic reaction mixture with and without cofactors were compared and analyzed using Metabolynx software (Waters Corp., Milford, MA, USA). The mass spectra of the newly appearing chromatographic peaks in the experimental sample were further analyzed to find the metabolite candidates. Their chemical structures were confirmed by tandem mass spectrometry. Three metabolites, including two reduction products and a glucuronide conjugate, were successfully detected under their specific HLMs metabolic conditions, which were in accordance with the literature reported results. The results demonstrated that the developed isotope labeling method, together with post-acquisition data processing using Metabolynx software, could be used for fast identification of new drug metabolites.

  13. Cysteine amide adduct formation from carboxylic acid drugs via UGT-mediated bioactivation in human liver microsomes.

    PubMed

    Harada, H; Toyoda, Y; Endo, T; Kobayashi, M

    2015-10-01

    Although chemical trapping has been widely used to evaluate cytochrome P450-mediated drug bioactivation, thus far, only a few in vitro-trapping studies have been performed on UDP-glucuronosyltransferase (UGT)-mediated drug bioactivation. In this study, we used cysteine (Cys) as trapping agent to gain new insights into the UGT-mediated bioactivation involving acyl glucuronides of carboxylic acid drugs. Diclofenac, ketoprofen and ibuprofen were incubated in human liver microsomes with UDPGA and Cys, followed by analysis using ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF/MS). The N-acyl-Cys amide adduct of diclofenac was characterized by mass analysis and was detectable even in photodiode array analysis. Our data indicated that the formation of such adducts reflects the reactivity of the corresponding acyl glucuronides. In addition, it was suggested that the adduct formation requires an N-terminal Cys moiety with both a free amine and a free thiol group, from the results using various cysteine derivatives. We propose that the S-acyl-Cys thioester adduct can form via transacylation of an acyl glucuronide and can then form to an N-acyl-Cys amide adduct through intramolecular S- to N-acyl rearrangement. This series of the reactions has important implications as a possible bioactivation mechanism for covalent binding of carboxylic acid drugs to macromolecules. PMID:26601426

  14. Surfactant protein D in human lung diseases.

    PubMed

    Hartl, D; Griese, M

    2006-06-01

    The lung is continuously exposed to inhaled pollutants, microbes and allergens. Therefore, the pulmonary immune system has to defend against harmful pathogens, while an inappropriate inflammatory response to harmless particles must be avoided. In the bronchoalveolar space this critical balance is maintained by innate immune proteins, termed surfactant proteins. Among these, surfactant protein D (SP-D) plays a central role in the pulmonary host defence and the modulation of allergic responses. Several human lung diseases are characterized by decreased levels of bronchoalveolar SP-D. Thus, recombinant SP-D has been proposed as a therapeutical option for cystic fibrosis, neonatal lung disease and smoking-induced emphysema. Furthermore, SP-D serum levels can be used as disease activity markers for interstitial lung diseases. This review illustrates the emerging role of SP-D translated from in vitro studies to human lung diseases. PMID:16684127

  15. In Vitro Metabolism of 20(R)-25-Methoxyl-Dammarane-3, 12, 20-Triol from Panax notoginseng in Human, Monkey, Dog, Rat, and Mouse Liver Microsomes

    PubMed Central

    Li, Wei; Liu, Li; Sun, Baoshan; Guo, Zhenghong; Shi, Caihong; Zhao, Yuqing

    2014-01-01

    The present study characterized in vitro metabolites of 20(R)-25-methoxyl-dammarane-3β, 12β, 20-triol (20(R)-25-OCH3-PPD) in mouse, rat, dog, monkey and human liver microsomes. 20(R)-25-OCH3-PPD was incubated with liver microsomes in the presence of NADPH. The reaction mixtures and the metabolites were identified on the basis of their mass profiles using LC-Q/TOF and were quantified using triple quadrupole instrument by multiple reaction monitoring. A total of 7 metabolites (M1–M7) of the phase I metabolites were detected in all species. 25(R)-OCH3-PPD was metabolized by hydroxylation, dehydrogenation, and O-demethylation. Enzyme kinetic of 20(R)-25-OCH3-PPD metabolism was evaluated in rat and human hepatic microsomes. Incubations studies with selective chemical inhibitors demonstrated that the metabolism of 20(R)-25-OCH3-PPD was primarily mediated by CYP3A4. We conclude that 20(R)-25-OCH3-PPD was metabolized extensively in mammalian species of mouse, rat, dog, monkey, and human. CYP3A4-catalyzed oxygenation metabolism played an important role in the disposition of 25(R)-OCH3-PPD, especially at the C-20 hydroxyl group. PMID:24736630

  16. Metabolic profiling of the Uncaria hook alkaloid geissoschizine methyl ether in rat and human liver microsomes using high-performance liquid chromatography with tandem mass spectrometry.

    PubMed

    Kushida, Hirotaka; Matsumoto, Takashi; Igarashi, Yasushi; Nishimura, Hiroaki; Watanabe, Junko; Maemura, Kazuya; Kase, Yoshio

    2015-01-01

    Geissoschizine methyl ether (GM) is an indole alkaloid found in Uncaria hook, which is a galenical constituent of yokukansan, a traditional Japanese medicine. GM has been identified as the active component responsible for anti-aggressive effects. In this study, the metabolic profiling of GM in rat and human liver microsomes was investigated. Thirteen metabolites of GM were elucidated and identified using a high-performance liquid chromatography with tandem mass spectrometry method, and their molecular structures were proposed on the basis of the characteristics of their precursor ions, product ions, and chromatographic retention times. There were no differences in the metabolites between the rat and human liver microsomes. Among the 13 identified metabolites, there were two demethylation metabolites, one dehydrogenation metabolite, three methylation metabolites, three oxidation metabolites, two water-adduct metabolites, one di-demethylation metabolite, and one water-adduct metabolite followed by oxidation. The metabolic pathways of GM were proposed on the basis of this study. This study will be helpful in understanding the metabolic routes of GM and related Uncaria hook alkaloids, and provide useful information on the pharmacokinetics and pharmacodynamics. This is the first report that describes the separation and identification of GM metabolites in rat and human liver microsomes. PMID:25633336

  17. High-performance liquid chromatography determination of N- and O-demethylase activities of chemicals in human liver microsomes: application of postcolumn fluorescence derivatization using Nash reagent.

    PubMed

    Kobayashi, K; Yamamoto, T; Taguchi, M; Chiba, K

    2000-09-10

    Formaldehyde is liberated in the process of cytochrome P450 (CYP) mediated demethylation of a wide variety of compounds containing the CH(3)N or CH(3)O functionality. A highly sensitive method using a high-performance liquid chromatography (HPLC) system with postcolumn derivatization was developed to measure the liberated formaldehyde as N- and O-demethylase activity of drugs in human liver microsomes. Following the chromatographic separation of formaldehyde on a C18 column, the formaldehyde was reacted with the Nash reagent in the postcolumn reactor at 100 degrees C and detected by the fluorescence method. The results showed that the present method has excellent precision and accuracy. The intra- and interassay variances of this method were less than 10%. The newly developed HPLC method was found to be about 80-fold more sensitive than the colorimetric method in detection of formaldehyde. The N-demethylase activity of sertraline in rat liver microsomes determined by the present method did not differ from those detected by previous methods quantifying produced desmethyl metabolite. The present method has been successfully applied to determine the N-demethylase activities of several drugs, including aminopyrine, erythromycin, fluoxetine, S-mephenytoin, and sertraline, in human liver microsomes. This assay should be useful for generic analysis of N- and O-demethylase activities of xenobiotic and endobiotic chemicals by CYP enzymes. PMID:10964418

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

    PubMed

    Kwon, Soon-Sang; Kim, Ju-Hyun; Jeong, Hyeon-Uk; Cho, Yong Yeon; Oh, Sei-Ryang; Lee, Hye Suk

    2016-01-01

    Aschantin is a bioactive neolignan found in Magnolia flos with antiplasmodial, Ca(2+)-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. PMID:27128896

  19. UDP-glucuronosyltransferase 1A1 is the principal enzyme responsible for puerarin metabolism in human liver microsomes.

    PubMed

    Luo, Cheng-Feng; Cai, Bin; Hou, Ning; Yuan, Mu; Liu, Shi-Ming; Ji, Hong; Xiong, Long-Gen; Xiong, Wei; Luo, Jian-Dong; Chen, Min-Sheng

    2012-11-01

    Puerarin has multiple pharmacological effects and is widely prescribed for patients with cardiovascular diseases, including hypertension, cerebral ischemia, myocardial ischemia, diabetes mellitus, and arteriosclerosis. While puerarin is a useful therapeutic agent, its mechanisms of action have not been well defined. Understanding puerarin metabolism, in particular its interactions with metabolizing enzymes, will contribute to our understanding of its toxic and therapeutic effects and may help to elucidate potential negative drug-drug interactions. In this study, the major metabolite of puerarin was obtained from the urine of rats administered puerarin, by a semi-preparative high-performance liquid chromatography method. The major metabolite was identified as puerarin-7-O-glucuronide. In vitro, we used a UDP-glucuronosyltransferase (UGT) reaction screening method with 12 recombinant human UGTs to demonstrate that formation of puerarin-7-O-glucuronide was catalyzed by UGT1A1, 1A9, 1A10, 1A3, 1A6, 1A7, and 1A8. UGT1A1, 1A9, and 1A10 significantly catalyzed puerarin-7-O-glucuronide formation, and the activity of UGT1A1 was significantly higher than those of 1A9 and 1A10. The V (max) of UGT1A1 was two- to threefold higher than the levels of UGT1A9 or 1A10, with a lower K ( m ) value and a higher V (max)/K ( m ) value. The kinetics of puerarin-7-O-glucuronide formation catalyzed by UGT1A1 were similar to those of the pooled human liver microsomes (HLMs), with V (max) values of 186.3 and 149.2 pmol/min/mg protein, and K ( m ) values of 811.3 and 838.9 μM, respectively. Furthermore, bilirubin and β-estradiol, probe substrates for UGT1A1, significantly inhibited the formation of puerarin-7-O-glucuronide in HLMs. PMID:22648071

  20. Gene polymorphisms and contents of cytochrome P450s have only limited effects on metabolic activities in human liver microsomes.

    PubMed

    Gao, Na; Tian, Xin; Fang, Yan; Zhou, Jun; Zhang, Haifeng; Wen, Qiang; Jia, Linjing; Gao, Jie; Sun, Bao; Wei, Jingyao; Zhang, Yunfei; Cui, Mingzhu; Qiao, Hailing

    2016-09-20

    Extensive inter-individual variations in pharmacokinetics are considered as a major reason for unpredictable drug responses. As the most important drug metabolic enzymes, inter-individual variations of cytochrome P450 (CYP) activities are not clear in human liver. In this paper, metabolic activities, gene polymorphisms and protein contents of 10 CYPs were determined in 105 human normal liver microsomes. The results indicated substantial inter-individual variations in CYP activities, with the greatest being CYP2C19 activity (>600-fold). Only half of 10 CYP isoforms and 26 gene polymorphism sites had limited effects on metabolic activities, such as CYP2A6, CYP2B6, CYP2C9, CYP2D6 and CYP3A4/5, others had almost no effects. Compared with their respective wild type, Km, Vmax, and CLint decreased by 51.6%, 88.7% and 70.7% in CYP2A6*1/*4 genotype, Vmax and CLint decreased by 32.8% and 60.2% in CYP2C9*1/*3 genotype, Km increased by 118.4% and CLint decreased by 65.2% in CYP2D6 100TT genotype, respectively. Moreover, there were only 4 CYP isoforms, CYP1A2, CYP2A6, CYP2E1 and CYP3A5, which had moderate or weak correlations between Vmax values and corresponding contents. In conclusions, the genotypes and contents of some CYPs have only limited effects on metabolic activities, which imply that there are other more important factors to influence inter-individual variations. PMID:27339126

  1. OXIDATIVE AND HYDROLYTIC METABOLISM OF TYPE I PYRETHROIDS IN RAT AND HUMAN HEPATIC MICROSOMES

    EPA Science Inventory

    Pyrethroids are a class of neurotoxic insecticides used in a variety of agricultural and household activities. Increased potential for human exposure to pyrethroids has prompted pharmacokinetic research. To that end, our laboratory has determined the in vitro clearance of the T...

  2. Isolation and identification of intestinal CYP3A inhibitors from cranberry (Vaccinium macrocarpon) using human intestinal microsomes.

    PubMed

    Kim, Eunkyung; Sy-Cordero, Arlene; Graf, Tyler N; Brantley, Scott J; Paine, Mary F; Oberlies, Nicholas H

    2011-02-01

    Cranberry juice is used routinely, especially among women and the elderly, to prevent and treat urinary tract infections. These individuals are likely to be taking medications concomitantly with cranberry juice, leading to concern about potential drug-dietary substance interactions, particularly in the intestine, which, along with the liver, is rich in expression of the prominent drug metabolizing enzyme, cytochrome P450 3A (CYP3A). Using a systematic in vitro-in vivo approach, a cranberry juice product was identified recently that elicited a pharmacokinetic interaction with the CYP3A probe substrate midazolam in 16 healthy volunteers. Relative to water, cranberry juice inhibited intestinal first-pass midazolam metabolism. In vitro studies were initiated to identify potential enteric CYP3A inhibitors from cranberry via a bioactivity-directed fractionation approach involving dried whole cranberry [Vaccinium macrocarpon Ait. (Ericaceae)], midazolam, and human intestinal microsomes (HIM). Three triterpenes (maslinic acid, corosolic acid, and ursolic acid) were isolated. The inhibitory potency (IC(50)) of maslinic acid, corosolic acid, and ursolic acid was 7.4, 8.8, and < 10 µM, respectively, using HIM as the enzyme source and 2.8, 4.3, and < 10 µM, respectively, using recombinant CYP3A4 as the enzyme source. These in vitro inhibitory potencies, which are within the range of those reported for two CYP3A inhibitory components in grapefruit juice, suggest that these triterpenes may have contributed to the midazolam-cranberry juice interaction observed in the clinical study. PMID:20717876

  3. Isolation and Identification of Intestinal CYP3A Inhibitors from Cranberry (Vaccinium macrocarpon) using Human Intestinal Microsomes

    PubMed Central

    Kim, Eunkyung; Sy-Cordero, Arlene; Graf, Tyler N.; Brantley, Scott J.; Paine, Mary F.; Oberlies, Nicholas H.

    2010-01-01

    Cranberry juice is used routinely, especially among women and the elderly, to prevent and treat urinary tract infections. These individuals are likely to be taking medications concomitantly with cranberry juice, leading to concern about potential drug-dietary substance interactions, particularly in the intestine, which, along with the liver, is rich in expression of the prominent drug metabolizing enzyme, cytochrome P450 3A (CYP3A). Using a systematic in vitro-in vivo approach, a cranberry juice product was identified recently that elicited a pharmacokinetic interaction with the CYP3A probe substrate midazolam in 16 healthy volunteers. Relative to water, a cranberry juice inhibited intestinal first-pass midazolam metabolism. In vitro studies were initiated to identify potential enteric CYP3A inhibitors from cranberry via a bioactivity-directed fractionation approach involving dried whole cranberry [Vaccinium macrocarpon Ait. (Ericaceae)], midazolam, and human intestinal microsomes (HIM). Three triterpenes (maslinic acid, corosolic acid, and ursolic acid) were isolated. The inhibitory potency (IC50) of maslinic acid, corosolic acid, and ursolic acid was 7.4, 8.8, and <10 μM, respectively, using HIM as the enzyme source and was 2.8, 4.3, and <10 μM, respectively, using recombinant CYP3A4 as the enzyme source. These in vitro inhibitory potencies, which are within the range of those reported for two CYP3A inhibitory components in grapefruit juice, suggest that these triterpenes may have contributed to the midazolam-cranberry juice interaction observed in the clinical study. PMID:20717876

  4. Development of monoclonal antibodies to human microsomal epoxide hydrolase and analysis of "preneoplastic antigen"-like molecules.

    PubMed

    Duan, Hongying; Yoshimura, Kazunori; Kobayashi, Nobuharu; Sugiyama, Kazuo; Sawada, Jun-Ichi; Saito, Yoshiro; Morisseau, Christophe; Hammock, Bruce D; Akatsuka, Toshitaka

    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. PMID:22310175

  5. Metabolism-mediated drug interaction potential of HS-23, a new herbal drug for the treatment of sepsis in human hepatocytes and liver microsomes.

    PubMed

    Jeong, Hyeon-Uk; Lee, Ji Young; Kwon, Soon-Sang; Kim, Ju Hyun; Kim, Young-Mok; Hong, Sung-Woon; Yeon, Sung Hum; Lee, Sun-Mee; Cho, Yong-Yeon; Lee, Hye Suk

    2015-02-01

    HS-23, an extract of the dried flower buds of Lonicera japonica, is a new botanical drug currently being evaluated in a phase I clinical study in Korea for the treatment of sepsis. The in vitro induction and inhibition potentials of HS-23 on the drug-metabolizing enzymes using human hepatocytes and liver microsomes were assessed to evaluate herb-drug interaction according to botanical drug guideline and drug interaction guidance of FDA. HS-23 slightly inhibited CYP2A6, CYP2B6, CYP2C9, CYP2C19, and CYP3A4 enzyme activities in human liver microsomes with IC50 values of 80.6, 160.7, 169.5, 85.4, and 76.6 μg/mL, respectively. HS-23 showed negligible inhibition of CYP1A2, CYP2C8, CYP2D6, UGT1A1, UGT1A4, UGT1A9, and UGT2B7 activities in human liver microsomes. Based on these results, HS-23 may not inhibit the metabolism of CYP2A6, CYP2B6, CYP2C9, CYP2C19, and CYP3A4-catalyzed drugs in humans. HS-23 did not affect the mRNA expression of CYP1A2, CYP2B6, and CYP3A4 after 48 h treatment at three concentrations (0.5, 5, and 50 μg/mL) in three independent human hepatocytes, indicating that HS-23 has no effect on herb-drug interactions that up- or down-regulate CYP1A2, CYP2B6, and CYP3A4. These results indicate that the administration of HS-23 in human may not cause clinically relevant inhibition and induction of these cytochrome P450 (CYP) and UDP-glucuronosyltransferase (UGT) enzymes and HS-23 may be promising therapeutic agent for treatment of sepsis. PMID:25052959

  6. Metabolism of a 14C/3H-labeled GABAA receptor partial agonist in rat, dog and human liver microsomes: evaluation of a dual-radiolabel strategy.

    PubMed

    Shaffer, Christopher L; Langer, Connie S

    2007-03-12

    The metabolism of 2-{[2-(3-fluoropyrid-2-yl)-1H-imidazol-1-yl]methyl}-1-propyl-5-cyano-1H-benzimidazole (1), a potent subtype-selective GABA(A) receptor partial agonist, was investigated in rat, dog and human liver microsomes. Due to its significant metabolic cleavage at C(8) observed in preliminary biotransformation studies with non-radiolabeled 1, both [(14)C]1 and [(3)H]1 were synthesized with respective radioisotopes placed on either side of C(8) to determine if all microsomal metabolites formed after C(8)N-dealkylation of 1 (or its core-intact metabolites) could be detected and quantified adequately. Both radiolabeled forms of 1, used separately in mono-radiolabel studies in cross-species microsomes and concomitantly in dual-radiolabel studies in rat microsomes, permitted the detection and quantification of all metabolites of 1, and a combination of radioactive and mass spectral data allowed structural elucidation of its Phase I metabolites. As expected, the sum of (14)C-only metabolites equaled that of (3)H-only metabolites in all incubations. In-line radiometric analysis worked extremely well (and was very reproducible) for quantifying either (14)C- or (3)H-compounds within separate incubations when using mono-radiolabeled 1. However, although the in-line radiodetector provided a comprehensive qualitative metabolic profile using dual-radiolabled 1, its inability to exclude completely (14)C- from (3)H-generated counts caused a degree of ambiguity pertaining to metabolite quantification. Thus, off-line liquid scintillation counting of collected dual-radiolabeled incubation LC-fractions was employed to quantify both (14)C- and (3)H-metabolites simultaneously, while in-line radiodetection was only used for qualitative analyses accompanying MS and MS/MS experiments. These studies demonstrated the analytical feasibility of using a dual-radiolabel approach for subsequent in vivo ADME studies with 1. PMID:17150324

  7. Immunolocalization of elastase in human emphysematous lungs.

    PubMed Central

    Damiano, V V; Tsang, A; Kucich, U; Abrams, W R; Rosenbloom, J; Kimbel, P; Fallahnejad, M; Weinbaum, G

    1986-01-01

    The current working hypothesis concerning the pathogenesis of human pulmonary emphysema proposes that neutrophils migrate through the alveolar interstitium and degranulate, releasing proteolytic enzymes into the interstitium. These enzymes, in particular elastase, can bind to and degrade interstitial elastin. This report describes an immunohistochemical, ultrastructural technique that utilizes polyclonal antibodies to localize neutrophil elastase in human lungs. Using both the immunoperoxidase and the immunogold methods on thin, embedded sections of surgically resected human emphysematous lung tissue, elastase was localized in neutrophils in the lung interstitium and extracellularly in association with interstitial elastic fibers in human lungs that showed local emphysema of varying severity. Quantitative morphometric data were obtained from the lungs of eight patients undergoing lobectomy for removal of pulmonary carcinomas. Patients had preoperative forced expiratory volume (FEV1)% levels ranging from 55 to 77. There was a correlation between a quantitative measure of the local distribution of neutrophil elastase in contact with alveolar interstitial elastin and the local presence of emphysematous change as determined by mean linear intercept of the various histologic sections. These data support the validity of the "protease-protease inhibitor balance hypothesis" as an explanation of the pathogenesis of human pulmonary emphysema. Images PMID:3525610

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

    SciTech Connect

    Duan, Hongying; Takagi, Akira; Kayano, Hidekazu; Koyama, Isamu; Morisseau, Christophe; Hammock, Bruce D.; Akatsuka, Toshitaka

    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

  9. Differentiation of xenografted human fetal lung parenchyma

    PubMed Central

    Pavlovic, Jelena; Floros, Joanna; Phelps, David S.; Wigdahl, Brian; Welsh, Patricia; Weisz, Judith; Shearer, Debra A.; Pree, Alphonse Leure du; Myers, Roland; Howett, Mary K.

    2009-01-01

    The goal of this study was to characterize xenografted human fetal lung tissue with respect to developmental stage-specific cytodifferentiation. Human fetal lung tissue (pseudoglandular stage) was grafted either beneath the renal capsule or the skin of athymic mice (NCr-nu). Tissues were analyzed from 3 to 42 days post-engraftment for morphological alterations by light and electron microscopy (EM), and for surfactant protein mRNA and protein by reverse transcription-polymerase chain reaction (RT-PCR) and immunocytochemistry (ICC), respectively. The changes observed resemble those seen in human lung development in utero in many respects, including the differentiation of epithelium to the saccular stage. Each stage occurred over approximately one week in the graft in contrast to the eight weeks of normal in utero development. At all time points examined, all four surfactant proteins (SP-A, SP-B, SP-C, and SP-D) were detected in the epithelium by ICC. Lamellar bodies were first identified by EM in 14 day xenografts. By day 21, a significant increase in lamellar body expression was observed. Cellular proliferation, as marked by PCNA ICC and elastic fiber deposition resembled those of canalicular and saccular in utero development. This model in which xenografted lung tissue in different stages of development is available may facilitate the study of human fetal lung development and the impact of various pharmacological agents on this process. PMID:17555893

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

    PubMed

    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. PMID:22181347

  11. Metabolism of the chlorofluorocarbon substitute 1,1-dichloro-2,2,2-trifluoroethane by rat and human liver microsomes: the role of cytochrome P450 2E1.

    PubMed

    Urban, G; Speerschneider, P; Dekant, W

    1994-01-01

    1,1-Dichloro-2,2,2-trifluoroethane (HCFC-123) has been developed as a substitute for ozone-depleting chlorofluorocarbons. The atmospheric lifetime of HCFC-123 is expected to be much shorter than those of chlorofluorocarbons; however, due to its lower stability and the presence of carbon-hydrogen bonds, metabolism of HCFC-123 in mammals and metabolism-dependent toxicity is likely. We compared the metabolism of HCFC-123 and its analog halothane in rat and human liver microsomes. 19F-NMR studies showed that trifluoroacetic acid is a major metabolite of HCFC-123. Besides trifluoroacetic acid, chlorodifluoroacetic acid and inorganic fluoride were identified as products of the enzymatic oxidation of HCFC-123 in rat and human liver microsomes by 19F-NMR and mass spectrometry. The metabolites were not detected in incubations with halothane. HCFC-123 and halothane were transformed by liver microsomes from untreated rats at low rates. Microsomes from ethanol-and pyridine-treated rats metabolized both HCFC-123 and halothane at much higher rates. These microsomes also exhibited high rates of p-nitrophenol oxidation. p-Nitrophenol is a model substrate mainly oxidized by P450 2E1 to p-nitrocatechol. Samples of human liver microsomes showed considerable differences in the extent of HCFC-123, p-nitrophenol oxidation, and chlorzoxazone hydroxylation. In human liver microsomes, rabbit anti-rat P450 2E1 IgG recognized a single protein band corresponding in apparent molecular weight to human P450 2E1. Immunoblot analysis revealed considerable heterogenity in the P450 2E1 protein content of the human liver samples. Trifluoroacetic acid formation from HCFC-123 and halothane and p-nitrocatechol formation from p-nitrophenol were significantly reduced by the P450 2E1 inhibitor diethyldithiocarbamate.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8199305

  12. Antithyroid microsomal antibody

    MedlinePlus

    Thyroid antimicrosomal antibody; Antimicrosomal antibody; Microsomal antibody; Thyroid peroxidase antibody; TPOAb ... test is done to confirm the cause of thyroid problems, including Hashimoto thyroiditis . The test is also ...

  13. In vitro metabolism of l-corydalmine, a potent analgesic drug, in human, cynomolgus monkey, beagle dog, rat and mouse liver microsomes.

    PubMed

    Tang, Xiange; Di, Xinyu; Zhong, Zeyu; Xie, Qiushi; Chen, Yang; Wang, Fan; Ling, Zhaoli; Xu, Ping; Zhao, Kaijing; Wang, Zhongjian; Liu, Li; Liu, Xiaodong

    2016-09-01

    l-Corydalmine (l-CDL) was under development as an oral analgesic agent, exhibiting potent analgesic activity in preclinical models. The objective of this study was to compare metabolic profiles of l-CDL in liver microsomes from mouse, rat, monkey, dog and human. Six metabolites (M1-M6) were identified using LC-Q/TOF in liver microsomes from the five species. The metabolism of l-CDL included O-demethylation (M1-3) and hydroxylation (M4-6). The desmethyl metabolites were the major ones among the five species, which accounted for more than 84%. Data from chemical inhibition in human liver microsomes (HLM) and human recombinant CYP450s demonstrated that CYP2D6 exhibited strong catalytic activity towards M1 and M2 formations, while CYP2C9 and CYP2C19 also catalyzed M2 formation. Formations of M3 and hydroxyl metabolites (M4 and M5) were mainly catalyzed by CYP3A4. Further studies showed that M1 and M2 were main metabolites in HLM. The kinetics of M1 and M2 formations in HLM and recombinant CYP450s were also investigated. The results showed that M1 and M2 formations in HLM and recombinant CYP2D6 characterized biphasic kinetics, whereas sigmoid Vmax model was better used to fit M2 formation by recombinant CYP2C9 and CYP2C19. The contributions of CYP2D6 to M1 and M2 formations in HLM were estimated to be 75.3% and 50.7%, respectively. However, the contributions of CYP2C9 and CYP2C19 to M2 formation were only 5.0% and 4.1%, respectively. All these data indicated that M1 and M2 were main metabolites in HLM, and CYP2D6 was the primary enzyme responsible for their formations. PMID:27239758

  14. Inhibitory effects of curcumin on activity of cytochrome P450 2C9 enzyme in human and 2C11 in rat liver microsomes.

    PubMed

    Wang, Zhe; Sun, Wei; Huang, Cheng-Ke; Wang, Li; Xia, Meng-Ming; Cui, Xiao; Hu, Guo-Xin; Wang, Zeng-Shou

    2015-04-01

    Cytochrome P450 2C9 (CYP2C9), one of the most important phase I drug metabolizing enzymes, could catalyze the reactions that convert diclofenanc into diclofenac 4'-hydroxylation. Evaluation of the inhibitory effects of compounds on CYP2C9 is clinically important because inhibition of CYP2C9 could result in serious drug-drug interactions. The objective of this work was to investigate the effects of curcumin on CYP2C9 in human and cytochrome P450 2C11 (CYP2C11) in rat liver microsomes. The results showed that curcumin inhibited CYP2C9 activity (10 µmol L(-1) diclofenac) with half-maximal inhibition or a half-maximal inhibitory concentration (IC50) of 15.25 µmol L(-1) and Ki = 4.473 µmol L(-1) in human liver microsomes. Curcumin's mode of action on CYP2C9 activity was noncompetitive for the substrate diclofenanc and uncompetitive for the cofactor NADPH. In contrast to its potent inhibition of CYP2C9 in human, diclofenanc had lesser effects on CYP2C11 in rat, with an IC50 ≥100 µmol L(-1). The observations imply that curcumin has the inhibitory effects on CYP2C9 activity in human. These in vitro findings suggest that more attention should be paid to special clinical caution when intake of curcumin combined with other drugs in treatment. PMID:24517573

  15. Predicting the oral bioavailability of 19-nortestosterone progestins in vivo from their metabolic stability in human liver microsomal preparations in vitro.

    PubMed

    Kuhnz, W; Gieschen, H

    1998-11-01

    It was the aim of this study to investigate whether assessment of the metabolic stability of selected progestins of the 19-nortestosterone type in human microsomal liver preparations was a suitable approach to predict the oral bioavailability of these drugs in humans. The Michaelis-Menten parameters Vmax and KM,app for norethisterone, levonorgestrel, gestodene, desogestrel, 3-keto-desogestrel, norgestimate, and dienogest were determined in in vitro incubations with human liver microsomes. Using these data, both the in vitro intrinsic clearance (CLint) and, after application of a suitable scaling factor, the scaled in vivo CLint were calculated. For progestins for which human in vivo data were available, the in vitro results were correlated with in vivo CLint values and oral bioavailability. A comparison of the scaled in vivo CLint values with the corresponding in vivo CLint values showed a reasonable correlation, although the latter values were generally approximately 2-fold higher than the former. Excluding desogestrel, which is subject to substantial intestinal metabolism in vivo, there was a linear relationship (r = -0.986) between increasing in vitro CLint values for the progestins and decreasing bioavailability in vivo. Other methods of assessing the metabolic stability of the progestins in vitro, such as evaluation of metabolic half-lives at single initial concentrations, showed either no correlation or a less satisfactory correlation with bioavailability data. PMID:9806955

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

    SciTech Connect

    Duan, Hongying; Yoshimura, Kazunori; Kobayashi, Nobuharu; Sugiyama, Kazuo; Sawada, Jun-ichi; Saito, Yoshiro; Morisseau, Christophe; Hammock, Bruce D.; Akatsuka, Toshitaka

    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

  17. Hepatic and intestinal glucuronidation of mono(2-ethylhexyl) phthalate, an active metabolite of di(2-ethylhexyl) phthalate, in humans, dogs, rats, and mice: an in vitro analysis using microsomal fractions.

    PubMed

    Hanioka, Nobumitsu; Isobe, Takashi; Kinashi, Yu; Tanaka-Kagawa, Toshiko; Jinno, Hideto

    2016-07-01

    Mono(2-ethylhexyl) phthalate (MEHP) is an active metabolite of di(2-ethylhexyl) phthalate (DEHP) and has endocrine-disrupting effects. MEHP is metabolized into glucuronide by UDP-glucuronosyltransferase (UGT) enzymes in mammals. In the present study, the hepatic and intestinal glucuronidation of MEHP in humans, dogs, rats, and mice was examined in an in vitro system using microsomal fractions. The kinetics of MEHP glucuronidation by liver microsomes followed the Michaelis-Menten model for humans and dogs, and the biphasic model for rats and mice. The K m and V max values of human liver microsomes were 110 µM and 5.8 nmol/min/mg protein, respectively. The kinetics of intestinal microsomes followed the biphasic model for humans, dogs, and mice, and the Michaelis-Menten model for rats. The K m and V max values of human intestinal microsomes were 5.6 µM and 0.40 nmol/min/mg protein, respectively, for the high-affinity phase, and 430 µM and 0.70 nmol/min/mg protein, respectively, for the low-affinity phase. The relative levels of V max estimated by Eadie-Hofstee plots were dogs (2.0) > mice (1.4) > rats (1.0) ≈ humans (1.0) for liver microsomes, and mice (8.5) > dogs (4.1) > rats (3.1) > humans (1.0) for intestinal microsomes. The percentages of the V max values of intestinal microsomes to liver microsomes were mice (120 %) > rats (57 %) > dogs (39 %) > humans (19 %). These results suggest that the metabolic abilities of UGT enzymes expressed in the liver and intestine toward MEHP markedly differed among species, and imply that these species differences are strongly associated with the toxicity of DEHP. PMID:26514348

  18. 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.

    PubMed

    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. PMID:26014283

  19. Kinetics of Ethylene and Ethylene Oxide in Subcellular Fractions of Lungs and Livers of Male B6C3F1 Mice and Male Fischer 344 Rats and of Human Livers

    PubMed Central

    Csanády, György András; Kessler, Winfried; Klein, Dominik; Pankratz, Helmut; Pütz, Christian; Richter, Nadine; Filser, Johannes Georg

    2011-01-01

    Ethylene (ET) is metabolized in mammals to the carcinogenic ethylene oxide (EO). Although both gases are of high industrial relevance, only limited data exist on the toxicokinetics of ET in mice and of EO in humans. Metabolism of ET is related to cytochrome P450-dependent mono-oxygenase (CYP) and of EO to epoxide hydrolase (EH) and glutathione S-transferase (GST). Kinetics of ET metabolism to EO and of elimination of EO were investigated in headspace vessels containing incubations of subcellular fractions of mouse, rat, or human liver or of mouse or rat lung. CYP-associated metabolism of ET and GST-related metabolism of EO were found in microsomes and cytosol, respectively, of each species. EH-related metabolism of EO was not detectable in hepatic microsomes of rats and mice but obeyed saturation kinetics in hepatic microsomes of humans. In ET-exposed liver microsomes, metabolism of ET to EO followed Michaelis-Menten-like kinetics. Mean values of Vmax [nmol/(min·mg protein)] and of the apparent Michaelis constant (Km [mmol/l ET in microsomal suspension]) were 0.567 and 0.0093 (mouse), 0.401 and 0.031 (rat), and 0.219 and 0.013 (human). In lung microsomes, Vmax values were 0.073 (mouse) and 0.055 (rat). During ET exposure, the rate of EO production decreased rapidly. By modeling a suicide inhibition mechanism, rate constants for CYP-mediated catalysis and CYP inactivation were estimated. In liver cytosol, mean GST activities to EO expressed as Vmax/Km [μl/(min·mg protein)] were 27.90 (mouse), 5.30 (rat), and 1.14 (human). The parameters are most relevant for reducing uncertainties in the risk assessment of ET and EO. PMID:21785163

  20. Effects of mitragynine and 7-hydroxymitragynine (the alkaloids of Mitragyna speciosa Korth) on 4-methylumbelliferone glucuronidation in rat and human liver microsomes and recombinant human uridine 5’-diphospho-glucuronosyltransferase isoforms

    PubMed Central

    Haron, Munirah; Ismail, Sabariah

    2015-01-01

    Background: Glucuronidation catalyzed by uridine 5’- diphospho-glucuronosyltransferase (UGT) is a major phase II drug metabolism reaction which facilitates drug elimination. Inhibition of UGT activity can cause drug-drug interaction. Therefore, it is important to determine the inhibitory potentials of drugs on glucuronidation. Objective: The objective was to evaluate the inhibitory potentials of mitragynine, 7-hydroxymitragynine, ketamine and buprenorphine, respectively on 4-methylumbelliferone (4-MU) glucuronidation in rat liver microsomes, human liver microsomes and recombinant human UGT1A1 and UGT2B7 isoforms. Materials and Methods: The effects of the above four compounds on the formation of 4-MU glucuronide from 4-MU by rat liver microsomes, human liver microsomes, recombinant human UGT1A1 and UGT2B7 isoforms were determined using high-performance liquid chromatography with ultraviolet detection. Results: For rat liver microsomes, ketamine strongly inhibited 4-MU glucuronidation with an IC50 value of 6.21 ± 1.51 μM followed by buprenorphine with an IC50 value of 73.22 ± 1.63 μM. For human liver microsomes, buprenorphine strongly inhibited 4-MU glucuronidation with an IC50 value of 6.32 ± 1.39 μM. For human UGT1A1 isoform, 7-hydroxymitragynine strongly inhibited 4-MU glucuronidation with an IC50 value of 7.13 ± 1.16 μM. For human UGT2B7 isoform, buprenorphine strongly inhibited 4-MU glucuronidation followed by 7-hydroxymitragynine and ketamine with respective IC50 values of 5.14 ± 1.30, 26.44 ± 1.31, and 27.28 ± 1.18 μM. Conclusions: These data indicate the possibility of drug-drug interaction if 7-hydroxymitragynine, ketamine, and buprenorphine are co-administered with drugs that are UGT2B7 substrates since these three compounds showed significant inhibition on UGT2B7 activity. In addition, if 7-hydroxymitragynine is to be taken with other drugs that are highly metabolized by UGT1A1, there is a possibility of drug-drug interaction to occur. PMID

  1. Metabolism studies on prim-O-glucosylcimifugin and cimifugin in human liver microsomes by ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry.

    PubMed

    Jia, Peipei; Zhang, Yuqian; Zhang, Qiaoyue; Sun, Yupeng; Yang, Haotian; Shi, He; Zhang, Xiaoxu; Zhang, Lantong

    2016-09-01

    Prim-O-glucosylcimifugin (PGCN) and cimifugin (CN) are major constituents of Radix Saposhnikoviae that have antipyretic, analgesic and anti-inflammatory pharmacological activities. However, there were few reports with respect to the metabolism of PGCN and CN in vitro. In this paper, we describe a strategy using ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) for fast analysis of the metabolic profile of PGCN and CN in human liver microsomes. In total, five phase I metabolites of PGCN, seven phase I metabolites and two phase II metabolites of CN were identified in the incubation of human liver microsomes. The results revealed that the main phase I metabolic pathways of PGCN were hydroxylation and hydrolysis reactions. The phase I metabolic pathways of CN were found to be hydroxylation, demethylation and dehydrogenation. Meanwhile, the results indicated that O-glucuronidation was the major metabolic pathway of CN in phase II metabolism. The specific UDP-glucuronosyltransferase (UGT) enzymes responsible for CN glucuronidation metabolites were identified using recombinant UGT enzymes. The results indicated that UGT1A1, UGT1A9, UGT2B4 and UGT2B7 might play major roles in the glucuronidation of CN. Overall, this study may be useful for the investigation of metabolic mechanism of PGCN and CN, and it can provide reference and evidence for further pharmacodynamic experiments. Copyright © 2016 John Wiley & Sons, Ltd. PMID:26910368

  2. Simultaneous absolute quantification of 11 cytochrome P450 isoforms in human liver microsomes by liquid chromatography tandem mass spectrometry with in silico target peptide selection.

    PubMed

    Kawakami, Hirotaka; Ohtsuki, Sumio; Kamiie, Junichi; Suzuki, Takashi; Abe, Takaaki; Terasaki, Tetsuya

    2011-01-01

    Cytochrome P450 (CYP) proteins are involved in the biological oxidation and reduction of xenobiotics, affecting the pharmacological efficiency of drugs. This study aimed to establish a method to simultaneously quantify 11 CYP isoforms by multiplexed-multiple reaction monitoring analysis with liquid chromatography tandem mass spectrometry and in silico peptide selection to clarify CYP isoform expression profiles in human liver tissue. CYP1A2, 2A6, and 2D6 target peptides were identified by shot-gun proteomic analysis, and those of other isoforms were selected by in silico peptide selection criteria. The established quantification method detected target peptides at 10  fmol, and the dynamic range of calibration curves was at least 500-fold. The quantification value of CYP1A2 in Supersomes was not significantly different between the established method and quantitative immunoblot analysis. The absolute protein expression levels of 11 CYP isoforms were determined from one pooled and 10 individual human liver microsomes. In the individual microsomes, CYP2C9 showed the highest protein expression level, and CYP1A2, 2A6, 2C19, and 3A4 protein expression exhibited more than a 20-fold difference among individuals. This highly sensitive and selective quantification method is a useful tool for the analysis of highly homologous CYP isoforms and the contribution made by each CYP isoform to drug metabolism. PMID:20564338

  3. Lung adenocarcinoma and human papillomavirus infection.

    PubMed

    Chen, Yen-Ching; Chen, Jen-Hau; Richard, Kradin; Chen, Pao-Yang; Christiani, David C

    2004-09-15

    Over the past three decades, the incidence of lung adenocarcinoma has increased worldwide. Most individuals with lung adenocarcinoma (especially women) are nonsmokers. Reported risk factors for the development of lung adenocarcinoma include cigarette smoking; exposure to cooking fumes, air pollution, second-hand smoke, asbestos, and radon; nutritional status; genetic susceptibility; immunologic dysfunction; tuberculosis infection; and asthma. Human papillomavirus (HPV) infection is a known risk factor for the development of squamous cell carcinoma (SCC), but it has not been thoroughly assessed as a potential risk factor for the development of pulmonary adenocarcinoma. More than 50% of people are infected with HPV during their lifetimes, either via intrauterine or postnatal infection. Recent studies involving Taiwanese patients have demonstrated a possible association between HPV infection and the risk of developing pulmonary adenocarcinoma. HPV transmission pathways have not yet been conclusively identified. The observation of certain types of HPV in association with cervical and oral SCC raises the possibility of sexual transmission of HPV from the cervix to the oral cavity, with subsequent transmission to the larynx and then to the lung. HPV infection and metaplasia in lung tissue may increase an individual's susceptibility to the tumorigenesis of pulmonary adenocarcinoma. Further epidemiologic and pathologic investigations will be necessary to establish a causal relation. PMID:15368331

  4. Human models of acute lung injury

    PubMed Central

    Proudfoot, Alastair G.; McAuley, Danny F.; Griffiths, Mark J. D.; Hind, Matthew

    2011-01-01

    Acute lung injury (ALI) is a syndrome that is characterised by acute inflammation and tissue injury that affects normal gas exchange in the lungs. Hallmarks of ALI include dysfunction of the alveolar-capillary membrane resulting in increased vascular permeability, an influx of inflammatory cells into the lung and a local pro-coagulant state. Patients with ALI present with severe hypoxaemia and radiological evidence of bilateral pulmonary oedema. The syndrome has a mortality rate of approximately 35% and usually requires invasive mechanical ventilation. ALI can follow direct pulmonary insults, such as pneumonia, or occur indirectly as a result of blood-borne insults, commonly severe bacterial sepsis. Although animal models of ALI have been developed, none of them fully recapitulate the human disease. The differences between the human syndrome and the phenotype observed in animal models might, in part, explain why interventions that are successful in models have failed to translate into novel therapies. Improved animal models and the development of human in vivo and ex vivo models are therefore required. In this article, we consider the clinical features of ALI, discuss the limitations of current animal models and highlight how emerging human models of ALI might help to answer outstanding questions about this syndrome. PMID:21357760

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

    PubMed

    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. PMID:24548679

  6. Antithyroid microsomal antibody

    MedlinePlus

    ... Thyroid antimicrosomal antibody; Antimicrosomal antibody; Microsomal antibody; Thyroid peroxidase antibody; TPOAb Images Blood test References Guber HA, Faraq AF. Evaluation of endocrine function. In: McPherson RA, Pincus MR, eds. Henry's Clinical ...

  7. Identification of metabolites of 4-nonylphenol isomer 4-(3',6'-dimethyl-3'-heptyl) phenol by rat and human liver microsomes.

    PubMed

    Ye, Xiaoyun; Bishop, Amber M; Needham, Larry L; Calafat, Antonia M

    2007-08-01

    Nonylphenol (NP) has been widely used for more than 50 years in the synthesis of NP ethoxylates, which are important nonionic surfactants. NP is considered an endocrine disruptor based on in vitro and in vivo animal studies. However, the toxic effects of NP in humans are unknown. Information regarding the metabolic fate of 4-t-nonylphenol (4-tNP), a mixture of commercial NP branched isomers, in mammalian species is limited. This information is critical for the identification of adequate biomarkers of exposure to NP that could be used for exposure and risk assessment. We identified metabolites of one 4-tNP isomer, namely, 4-(3',6'-dimethyl-3'-heptyl) phenol (P363-NP), using rat and human liver microsomes. The P363-NP metabolites were extracted by on-line solid-phase extraction and then separated and detected using high-performance liquid chromatography/tandem mass spectrometry. Using the genuine standard, we unambiguously identified 4-(3',6'-dimethyl-3'-heptyl) catechol (P363-NC) as the main P363-NP metabolite when using human liver microsomes. Based on their chromatographic behavior and mass spectral fragmentation patterns, several other metabolites were tentatively identified, including a hydroxylated P363-NP with the alcohol functional group on the branched alkyl chain and its oxidative metabolite, a catechol with a hydroxylated alkyl side chain. Furthermore, the metabolite profile of P363-NP using rat and human enzymes was compared. Our findings suggest that P363-NC could be used as a biomarker to assess exposure to 4-tNP, although additional research to evaluate its suitability as a biomarker is warranted. PMID:17460028

  8. Comparison of lung preservation solutions in human lungs using an ex vivo lung perfusion experimental model

    PubMed Central

    Medeiros, Israel L.; Pêgo-Fernandes, Paulo M.; Mariani, Alessandro W.; Fernandes, Flávio G.; Unterpertinger, Fernando V.; Canzian, Mauro; Jatene, Fabio B.

    2012-01-01

    OBJECTIVE: Experimental studies on lung preservation have always been performed using animal models. We present ex vivo lung perfusion as a new model for the study of lung preservation. Using human lungs instead of animal models may bring the results of experimental studies closer to what could be expected in clinical practice. METHOD: Brain-dead donors whose lungs had been declined by transplantation teams were used. The cases were randomized into two groups. In Group 1, Perfadex® was used for pulmonary preservation, and in Group 2, LPDnac, a solution manufactured in Brazil, was used. An ex vivo lung perfusion system was used, and the lungs were ventilated and perfused after 10 hours of cold ischemia. The extent of ischemic-reperfusion injury was measured using functional and histological parameters. RESULTS: After reperfusion, the mean oxygenation capacity was 405.3 mmHg in Group 1 and 406.0 mmHg in Group 2 (p = 0.98). The mean pulmonary vascular resistance values were 697.6 and 378.3 dyn·s·cm-5, respectively (p = 0.035). The mean pulmonary compliance was 46.8 cm H2O in Group 1 and 49.3 ml/cm H2O in Group 2 (p = 0.816). The mean wet/dry weight ratios were 2.06 and 2.02, respectively (p = 0.87). The mean Lung Injury Scores for the biopsy performed after reperfusion were 4.37 and 4.37 in Groups 1 and 2, respectively (p = 1.0), and the apoptotic cell counts were 118.75/mm2 and 137.50/mm2, respectively (p = 0.71). CONCLUSION: The locally produced preservation solution proved to be as good as Perfadex®. The clinical use of LPDnac may reduce costs in our centers. Therefore, it is important to develop new models to study lung preservation. PMID:23018310

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

    SciTech Connect

    Choi, Hye Jin; Lee, Dong-Hyung; Park, Seong-Hwan; Kim, Juil; Do, Kee Hun; An, Tae Jin; Ahn, Young Sup; Park, Chung Berm; Moon, Yuseok

    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.

  10. Effect of voriconazole and other azole antifungal agents on CYP3A activity and metabolism of tacrolimus in human liver microsomes.

    PubMed

    Zhang, Shimin; Pillai, Venkateswaran C; Mada, Sripal Reddy; Strom, Steve; Venkataramanan, Raman

    2012-05-01

    Azole antifungal agents are known to inhibit cytochrome P450 3A (CYP3A) enzymes. Limited information is available regarding the effect of voriconazole on CYP3A activity. We examined the effect of voriconazole on CYP3A activity in human liver microsomes as measured by the formation of 6β-hydroxytestosterone from testosterone. We also evaluated the interaction between voriconazole and tacrolimus, an immunosuppressive drug, using human liver microsomes. The effect of voriconazole on CYP3A activity and tacrolimus metabolism was compared to that of other azole antifungal agents. CYP3A4 activity and the metabolism of tacrolimus were measured in the absence and in the presence of various concentrations of voriconazole (0-1.43 mM), fluconazole (0-1.63 mM), itraconazole (0-14 µM) and ketoconazole (0-0.19 µM). At a concentration of 21.2 ± 15.4 µM and 29.8 ± 12.3 µM, voriconazole inhibited the formation of 6β-hydroxytestosterone from testosterone and the metabolism of tacrolimus by 50%, respectively. The rank order of inhibition of 6β-hydroxytestosterone formation from testosterone and the metabolism of tacrolimus, is ketoconazole > itraconazole > voriconazole > fluconazole. Our observations suggest that voriconazole at clinically relevant concentrations will inhibit the hepatic metabolism of tacrolimus and increase the concentration of tacrolimus more than two-fold. Close monitoring of the blood concentrations and adjustment in the dose of tacrolimus are warranted when transplant patients receiving tacrolimus are treated with voriconazole. PMID:22106961

  11. Size- and time-dependent alteration in metabolic activities of human hepatic cytochrome P450 isozymes by gold nanoparticles via microsomal coincubations

    PubMed Central

    2014-01-01

    Nano-sized particles are known to interfere with drug-metabolizing cytochrome P450 (CYP) enzymes, which can be anticipated to be a potential source of unintended adverse reactions, but the mechanisms underlying the inhibition are still not well understood. Herein we report a systematic investigation of the impacts of gold nanoparticles (AuNPs) on five major CYP isozymes under in vitro incubations of human liver microsomes (HLMs) with tannic acid (TA)-stabilized AuNPs in the size range of 5 to 100 nm. It is found that smaller AuNPs show more pronounced inhibitory effects on CYP2C9, CYP2C19, CYP2D6, and CYP3A4 in a dose-dependent manner, while 1A2 is the least susceptible to the AuNP inhibition. The size- and dose-dependent CYP-specific inhibition and the nonspecific drug-nanogold binding in the coincubation media can be significantly reduced by increasing the concentration ratio of microsomal proteins to AuNPs, probably via a noncompetitive mode. Remarkably, AuNPs are also found to exhibit a slow time-dependent inactivation of 2D6 and 3A4 in a β-nicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium salt hydrate (NADPH)-independent manner. During microsomal incubations, UV–vis spectroscopy, dynamic light scattering, and zeta-potential measurements were used to monitor the changes in particle properties under the miscellaneous AuNP/HLM/CYP dispersion system. An improved stability of AuNPs by mixing HLM with the gold nanocolloid reveals that the stabilization via AuNP-HLM interactions may occur on a faster time scale than the salt-induced nanoaggregation by incubation in phosphate buffer. The results suggest that the AuNP induced CYP inhibition can be partially attributed to its adhesion onto the enzymes to alter their structural conformations or onto the HLM membrane therefore impairing the integral membrane proteins. Additionally, AuNPs likely block the substrate pocket on the CYP surface, depending on both the particle characteristics and the

  12. Size- and time-dependent alteration in metabolic activities of human hepatic cytochrome P450 isozymes by gold nanoparticles via microsomal coincubations

    NASA Astrophysics Data System (ADS)

    Ye, Meiling; Tang, Ling; Luo, Mengjun; Zhou, Jing; Guo, Bin; Liu, Yangyuan; Chen, Bo

    2014-11-01

    Nano-sized particles are known to interfere with drug-metabolizing cytochrome P450 (CYP) enzymes, which can be anticipated to be a potential source of unintended adverse reactions, but the mechanisms underlying the inhibition are still not well understood. Herein we report a systematic investigation of the impacts of gold nanoparticles (AuNPs) on five major CYP isozymes under in vitro incubations of human liver microsomes (HLMs) with tannic acid (TA)-stabilized AuNPs in the size range of 5 to 100 nm. It is found that smaller AuNPs show more pronounced inhibitory effects on CYP2C9, CYP2C19, CYP2D6, and CYP3A4 in a dose-dependent manner, while 1A2 is the least susceptible to the AuNP inhibition. The size- and dose-dependent CYP-specific inhibition and the nonspecific drug-nanogold binding in the coincubation media can be significantly reduced by increasing the concentration ratio of microsomal proteins to AuNPs, probably via a noncompetitive mode. Remarkably, AuNPs are also found to exhibit a slow time-dependent inactivation of 2D6 and 3A4 in a β-nicotinamide adenine dinucleotide 2'-phosphate reduced tetrasodium salt hydrate (NADPH)-independent manner. During microsomal incubations, UV-vis spectroscopy, dynamic light scattering, and zeta-potential measurements were used to monitor the changes in particle properties under the miscellaneous AuNP/HLM/CYP dispersion system. An improved stability of AuNPs by mixing HLM with the gold nanocolloid reveals that the stabilization via AuNP-HLM interactions may occur on a faster time scale than the salt-induced nanoaggregation by incubation in phosphate buffer. The results suggest that the AuNP induced CYP inhibition can be partially attributed to its adhesion onto the enzymes to alter their structural conformations or onto the HLM membrane therefore impairing the integral membrane proteins. Additionally, AuNPs likely block the substrate pocket on the CYP surface, depending on both the particle characteristics and the

  13. Metabolism of Nω -methylserotonin, a serotonergic constituent of black cohosh (Cimicifuga racemosa, L. (Nutt.)), by human liver microsomes.

    PubMed

    Nikolić, Dejan; Li, Jinghu; van Breemen, Richard B

    2014-12-01

    The roots/rhizomes of black cohosh (Cimicifuga racemosa L. (Nutt.) (syn. Actaea racemosa L.) are a popular dietary supplements among women for management of menopausal symptoms. Although not estrogenic, Nω -methylserotonin has been identified in black cohosh as a potent agonist of serotonin 5-HT1A and 5-HT7 receptors. In the present study, in vitro metabolism of Nω -methylserotonin was investigated to gain insights into aspects of the bioavailability of this compound. The major metabolic pathway was determined to be conversion into 5-hydroxyindole acetaldehyde catalyzed by the monoamine oxidase A (MAO-A). 5-Hydroxyindole acetaldehyde could be further oxidized to form 5-hydroxyindole acetic acid by the action of microsomal aldehyde dehydrogenase or reduced to 5-hydroxy tryptophol by the action of aldehyde reductase. The cytochrome P450 enzymes had only a minor role in the metabolism of Nω -methylserotonin and then only when MAO-A was inhibited. In many aspects, the metabolism of Nω -methylserotonin was similar to the metabolism of serotonin, suggesting that this compound is unlikely to elicit CNS effects due to rapid metabolism by the widely distributed MAO-A. PMID:24817649

  14. Lung flooding enables efficient lung sonography and tumour imaging in human ex vivo and porcine in vivo lung cancer model

    PubMed Central

    2013-01-01

    Background Sonography has become the imaging technique of choice for guiding intraoperative interventions in abdominal surgery. Due to artefacts from residual air content, however, videothoracoscopic and open intraoperative ultrasound-guided thermoablation of lung malignancies are impossible. Lung flooding is a new method that allows complete ultrasound imaging of lungs and their tumours. Methods Fourteen resected tumourous human lung lobes were examined transpleurally with B-mode ultrasound before (in atelectasis) and after lung flooding with isotonic saline solution. In two swine, the left lung was filled with 15 ml/kg isotonic saline solution through the left side of a double-lumen tube. Lung tumours were simulated by transthoracic ultrasound-guided injection of 5 ml of purified bovine serum albumin in glutaraldehyde, centrally into the left lower lung lobe. The rate of tumour detection, the severity of disability caused by residual gas, and sonomorphology of the lungs and tumours were assessed. Results The ex vivo tumour detection rate was 100% in flooded human lung lobes and 43% (6/14) in atelectatic lungs. In all cases of atelectasis, sonographic tumour imaging was impaired by residual gas. Tumours and atelectatic tissue were isoechoic. In 28% of flooded lungs, a little residual gas was observed that did not impair sonographic tumour imaging. In contrast to tumours, flooded lung tissue was hyperechoic, homogeneous, and of fine-grained structure. Because of the bronchial wall three-laminar structure, sonographic differentiation of vessels and bronchi was possible. In all cases, malignant tumours in the flooded lung appeared well-demarcated from the lung parenchyma. Adenocarcinoma, squamous, and large cell carcinomas were hypoechoic. Bronchioloalveolar cell carcinoma was slightly hyperechoic. Transpleural sonography identifies endobronchial tumour growth and bronchial wall destruction. With transthoracic sonography, the flooded animal lung can be completely

  15. Preconditioning allows engraftment of mouse and human embryonic lung cells, enabling lung repair in mice.

    PubMed

    Rosen, Chava; Shezen, Elias; Aronovich, Anna; Klionsky, Yael Zlotnikov; Yaakov, Yasmin; Assayag, Miri; Biton, Inbal Eti; Tal, Orna; Shakhar, Guy; Ben-Hur, Herzel; Shneider, David; Vaknin, Zvi; Sadan, Oscar; Evron, Shmuel; Freud, Enrique; Shoseyov, David; Wilschanski, Michael; Berkman, Neville; Fibbe, Willem E; Hagin, David; Hillel-Karniel, Carmit; Krentsis, Irit Milman; Bachar-Lustig, Esther; Reisner, Yair

    2015-08-01

    Repair of injured lungs represents a longstanding therapeutic challenge. We show that human and mouse embryonic lung tissue from the canalicular stage of development (20-22 weeks of gestation for humans, and embryonic day 15-16 (E15-E16) for mouse) are enriched with progenitors residing in distinct niches. On the basis of the marked analogy to progenitor niches in bone marrow (BM), we attempted strategies similar to BM transplantation, employing sublethal radiation to vacate lung progenitor niches and to reduce stem cell competition. Intravenous infusion of a single cell suspension of canalicular lung tissue from GFP-marked mice or human fetal donors into naphthalene-injured and irradiated syngeneic or SCID mice, respectively, induced marked long-term lung chimerism. Donor type structures or 'patches' contained epithelial, mesenchymal and endothelial cells. Transplantation of differentially labeled E16 mouse lung cells indicated that these patches were probably of clonal origin from the donor. Recipients of the single cell suspension transplant exhibited marked improvement in lung compliance and tissue damping reflecting the energy dissipation in the lung tissues. Our study provides proof of concept for lung reconstitution by canalicular-stage human lung cells after preconditioning of the pulmonary niche. PMID:26168294

  16. Enhanced Lung Epithelial Specification of Human iPSCs on Decellularized Lung Matrix

    PubMed Central

    Gilpin, Sarah E.; Ren, Xi; Okamoto, Tatsuya; Guyette, Jacques P.; Mou, Hongmei; Rajagopal, Jayaraj; Mathisen, Douglas J.; Vacanti, Joseph P.; Ott, Harald C.

    2014-01-01

    Background Whole lung scaffolds can be created by perfusion decellularization of cadaveric donor lungs. The resulting matrices can then be recellularized to regenerate functional organs. This study evaluates the capacity of acellular lung scaffolds to support recellularization with human induced pluripotent stem cell (iPSC)-derived lung progenitors. Methods Whole rat and human lungs were decellularized by constant-pressure perfusion with 0.1% SDS solution. Resulting lung scaffolds were either cryosectioned into slices or left intact. Human iPSCs were differentiated to definitive endoderm, anteriorized to a foregut fate, and then ventralized to an Nkx2.1-expressing population. Cells were seeded onto slices and whole lungs, which were maintained under constant-perfusion biomimetic culture. Lineage specification was assessed by quantitative PCR and immunofluorescent staining. Regenerated left lungs were transplanted in orthotopic position. Results Activin-A treatment followed by TGF-β inhibition induced differentiation of human iPSCs to anterior foregut endoderm as confirmed by FOXA2, SOX17, and SOX2 expression. Cells cultured on decellularized lung slices demonstrated proliferation and lineage commitment after 5 days. Nkx2.1-expressing cells were identified at 40–60% efficiency. Within whole lung scaffolds and under perfusion culture, cells further up-regulated Nkx2.1 expression. After orthotopic transplantation, grafts were perfused and ventilated via host vasculature and airways. Conclusions Decellularized lung matrix supports the culture and lineage commitment of human iPSC-derived lung progenitor cells. Whole organ scaffolds and biomimetic culture enable co-seeding of iPSC-derived endothelial and epithelial progenitors and enhance early lung fate. Orthotopic transplantation may enable further in vivo graft maturation. PMID:25149047

  17. In vitro studies on the oxidative metabolism of 20(s)-ginsenoside Rh2 in human, monkey, dog, rat, and mouse liver microsomes, and human liver s9.

    PubMed

    Li, Liang; Chen, Xiaoyan; Zhou, Jialan; Zhong, Dafang

    2012-10-01

    20(S)-Ginsenoside Rh2 (Rh2)-containing products are widely used in Asia, Europe, and North America. However, extremely limited metabolism information greatly impedes the complete understanding of its clinical safety and effectiveness. The present study aims to systematically investigate the oxidative metabolism of Rh2 using a complementary set of in vitro models. Twenty-five oxidative metabolites were found using liquid chromatography-electrospray ionization ion-trap mass spectrometry. Six metabolites and a metabolic intermediate were synthesized. The metabolites were structurally identified as 26-hydroxy Rh2 (M1-1), (20S,24S)-epoxydammarane-12,25-diol-3-β-d-glucopyranoside (M1-3), (20S,24R)-epoxydammarane-12,25-diol-3-β-d-glucopyranoside (M1-5), 26,27-dihydroxy Rh2 (M3-6), (20S,24S)-epoxydammarane-12,25,26-triol-3-β-d-glucopyranoside (M3-10), (20S,24R)-epoxydammarane-12,25,26-triol-3-β-d-glucopyranoside (M3-11), and 26-aldehyde Rh2 on the basis of detailed mass spectrometry and nuclear magnetic resonance data analysis. Double-bond epoxidation followed by rearrangement and vinyl-methyl group hydroxylation represent the initial metabolic pathways generating monooxygenated metabolites M1-1 to M1-5. Further sequential metabolites (M2-M5) from the dehydrogenation and/or oxygenation of M1 were also detected. CYP3A4 was the predominant enzyme involved in the oxidative metabolism of Rh2, whereas alcohol dehydrogenase and aldehyde dehydrogenase mainly catalyzed the metabolic conversion of alcohol to the corresponding carboxylic acid. No significant differences were observed in the phase I metabolite profiles of Rh2 among the five species tested. Reactive epoxide metabolite formation in both humans and animals was evident. However, GSH conjugate M6 was detected only in cynomolgus monkey liver microsomal incubations. In conclusion, Rh2 is a good substrate for CYP3A4 and could undergo extensive oxidative metabolism under the catalysis of CYP3A4. PMID:22829543

  18. Cyp2D6 catalyzes 5-hydroxylation of 1-(2-pyrimidinyl)-piperazine, an active metabolite of several psychoactive drugs, in human liver microsomes.

    PubMed

    Raghavan, Nirmala; Zhang, Donglu; Zhu, Mingshe; Zeng, Jianing; Christopher, Lisa

    2005-02-01

    1-(2-Pyrimidinyl)-piperazine (1-PP) is an active metabolite of several psychoactive drugs including buspirone. 1-PP is also the major metabolite in the human circulation and in rat brains following oral administration of buspirone. This study was conducted to identify the enzyme responsible for the metabolic conversion of 1-PP to 5-hydroxy-1-(2-pyrimidinyl)-piperazine (HO-1-PP) in human liver microsomes (HLMs). The product HO-1-PP was quantified by a validated liquid chromatography-tandem mass spectrometry method. In the presence of NADPH, 1-PP (100 microM) was incubated separately with human cDNA-expressed cytochrome P450 isozymes (including CYP2D6, 3A4, 1A2, 2A6, 2C9, 2C19, 2E1, and 2B6) at 37 degrees C. CYP2D6 catalyzed the formation of HO-1-PP from 1-PP. This catalytic activity was >95% inhibited by quinidine, a CYP2D6 inhibitor. HO-1-PP formation rates correlated well with the bufuralol 1-hydroxylase (CYP2D6) activities of individual HLMs. The formation of HO-1-PP followed a Michaelis-Menten kinetics with a K(m) of 171 microM and V(max) of 313 pmol/min x mg protein in HLMs. Collectively, these results indicate that polymorphic CYP2D6 is responsible for the conversion of 1-PP to HO-1-PP. PMID:15507542

  19. Relevance of particle-induced rat lung tumors for assessing lung carcinogenic hazard and human lung cancer risk.

    PubMed Central

    Mauderly, J L

    1997-01-01

    Rats and other rodents are exposed by inhalation to identify agents that might present hazards for lung cancer in humans exposed by inhalation. In some cases, the results are used in attempts to develop quantitative estimates of human lung cancer risk. This report reviews evidence for the usefulness of the rat for evaluation of lung cancer hazards from inhaled particles. With the exception of nickel sulfate, particulate agents thought to be human lung carcinogens cause lung tumors in rats exposed by inhalation. The rat is more sensitive to carcinogenesis from nonfibrous particles than mice or Syrian hamsters, which have both produced false negatives. However, rats differ from mice and nonhuman primates in both the pattern of particle retention in the lung and alveolar epithelial hyperplastic responses to chronic particle exposure. Present evidence warrants caution in extrapolation from the lung tumor response of rats to inhaled particles to human lung cancer hazard, and there is considerable uncertainty in estimating unit risks for humans from rat data. It seems appropriate to continue using rats in inhalation carcinogenesis assays of inhaled particles, but the upper limit of exposure concentrations must be set carefully to avoid false-positive results. A positive finding in both rats and mice would give greater confidence that an agent presents a carcinogenic hazard to man, and both rats and mice should be used if the agent is a gas or vapor. There is little justification for including Syrian hamsters in assays of the intrapulmonary carcinogenicity of inhaled agents. PMID:9400748

  20. Quantitative Anatomy of the Growing Lungs in the Human Fetus

    PubMed Central

    Szpinda, Michał; Siedlaczek, Waldemar; Szpinda, Anna; Woźniak, Alina; Mila-Kierzenkowska, Celestyna; Badura, Mateusz

    2015-01-01

    Using anatomical, digital, and statistical methods we examined the three-dimensional growth of the lungs in 67 human fetuses aged 16–25 weeks. The lung dimensions revealed no sex differences. The transverse and sagittal diameters and the base circumference were greater in the right lungs while the lengths of anterior and posterior margins and the lung height were greater in the left lungs. The best-fit curves for all the lung parameters were natural logarithmic models. The transverse-to-sagittal diameter ratio remained stable and averaged 0.56 ± 0.08 and 0.52 ± 0.08 for the right and left lungs, respectively. For the right and left lungs, the transverse diameter-to-height ratio significantly increased from 0.74 ± 0.09 to 0.92 ± 0.08 and from 0.56 ± 0.07 to 0.79 ± 0.09, respectively. The sagittal diameter-to-height ratio significantly increased from 1.41 ± 0.23 to 1.66 ± 0.18 in the right lung, and from 1.27 ± 0.17 to 1.48 ± 0.22 in the left lung. In the fetal lungs, their proportionate increase in transverse and sagittal diameters considerably accelerates with relation to the lung height. The lung dimensions in the fetus are relevant in the evaluation of the normative pulmonary growth and the diagnosis of pulmonary hypoplasia. PMID:26413517

  1. Oxidative metabolism of BDE-47, BDE-99, and HBCDs by cat liver microsomes: Implications of cats as sentinel species to monitor human exposure to environmental pollutants.

    PubMed

    Zheng, Xiaobo; Erratico, Claudio; Luo, Xiaojun; Mai, Bixian; Covaci, Adrian

    2016-05-01

    BDE-99 is different by cat and human liver microsomes. This suggests that cats are not a suitable sentinel to represent internal exposure of PBDEs for humans, but is likely a promising sentinel for internal HBCDs exposure for humans. PMID:26923239

  2. Cloning of human lung cancer cells.

    PubMed Central

    Walls, G. A.; Twentyman, P. R.

    1985-01-01

    We have carried out a comparison of two different methods for cloning human lung cancer cells. The method of Courtenay & Mills (1978) generally gave higher plating efficiencies (PE) than the method of Carney et al. (1980). The number of colonies increased with incubation time in both methods and the weekly medium replenishment in the Courtenay method was advantageous for longer incubation times of several weeks. In the Courtenay method, the use of August rat red blood cells (RBC) and low oxygen tension were both found to be necessary factors for maximum plating efficiency. The usefulness of heavily irradiated feeder cells in improving PE is less certain; each cell type may have its own requirement. PMID:3904799

  3. Basal expression of the human MAPEG members microsomal glutathione transferase 1 and prostaglandin E synthase genes is mediated by Sp1 and Sp3.

    PubMed

    Ekström, Lena; Lyrenäs, Louise; Jakobsson, Per-Johan; Morgenstern, Ralf; Kelner, Michael J

    2003-06-19

    Microsomal glutathione transferase (MGST1) and prostaglandin E synthase (PGES) are both members of the MAPEG (Membrane Associated Proteins involved in Eicosanoid and Glutathione metabolism) superfamily. In humans, their organ distribution is quite distinct with the former being widely and constitutively expressed whereas PGES is largely inducible. In order to study the basal expression of these genes, we characterized the promoter regions and identified the elements and the transcription factors required using in vitro assays, including reporter analysis of deletion and mutant clones and EMSA. The results indicate that Sp1 is the protein mediating the basal transcription of MGST1. It appears that both the Sp1 and Sp3 proteins are important for the basal expression of PGES. In addition, mutational analysis of two Barbie-box elements in the PGES promoter showed that these were not involved in the down-regulation of PGES by phenobarbital (PB). These results provide the first description of the basal regulation of these genes in humans. PMID:12818425

  4. Computational tools and resources for metabolism-related property predictions. 2. Application to prediction of half-life time in human liver microsomes

    PubMed Central

    Zakharov, Alexey V; Peach, Megan L; Sitzmann, Markus; Filippov, Igor V; McCartney, Heather J; Smith, Layton H; Pugliese, Angelo; Nicklaus, Marc C

    2014-01-01

    Background The most important factor affecting metabolic excretion of compounds from the body is their half-life time. This provides an indication of compound stability of, for example, drug molecules. We report on our efforts to develop QSAR models for metabolic stability of compounds, based on in vitro half-life assay data measured in human liver microsomes. Method A variety of QSAR models generated using different statistical methods and descriptor sets implemented in both open-source and commercial programs (KNIME, GUSAR and StarDrop) were analyzed. The models obtained were compared using four different external validation sets from public and commercial data sources, including two smaller sets of in vivo half-life data in humans. Conclusion In many cases, the accuracy of prediction achieved on one external test set did not correspond to the results achieved with another test set. The most predictive models were used for predicting the metabolic stability of compounds from the open NCI database, the results of which are publicly available on the NCI/CADD Group web server (http://cactus.nci.nih.gov). PMID:23088274

  5. Metabolism of bupropion by baboon hepatic and placental microsomes

    PubMed Central

    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

  6. Production and Assessment of Decellularized Pig and Human Lung Scaffolds

    PubMed Central

    Niles, Jean; Riddle, Michael; Vargas, Gracie; Schilagard, Tuya; Ma, Liang; Edward, Kert; La Francesca, Saverio; Sakamoto, Jason; Vega, Stephanie; Ogadegbe, Marie; Mlcak, Ronald; Deyo, Donald; Woodson, Lee; McQuitty, Christopher; Lick, Scott; Beckles, Daniel; Melo, Esther; Cortiella, Joaquin

    2013-01-01

    The authors have previously shown that acellular (AC) trachea-lung scaffolds can (1) be produced from natural rat lungs, (2) retain critical components of the extracellular matrix (ECM) such as collagen-1 and elastin, and (3) be used to produce lung tissue after recellularization with murine embryonic stem cells. The aim of this study was to produce large (porcine or human) AC lung scaffolds to determine the feasibility of producing scaffolds with potential clinical applicability. We report here the first attempt to produce AC pig or human trachea-lung scaffold. Using a combination of freezing and sodium dodecyl sulfate washes, pig trachea-lungs and human trachea-lungs were decellularized. Once decellularization was complete we evaluated the structural integrity of the AC lung scaffolds using bronchoscopy, multiphoton microscopy (MPM), assessment of the ECM utilizing immunocytochemistry and evaluation of mechanics through the use of pulmonary function tests (PFTs). Immunocytochemistry indicated that there was loss of collagen type IV and laminin in the AC lung scaffold, but retention of collagen-1, elastin, and fibronectin in some regions. MPM scoring was also used to examine the AC lung scaffold ECM structure and to evaluate the amount of collagen I in normal and AC lung. MPM was used to examine the physical arrangement of collagen-1 and elastin in the pleura, distal lung, lung borders, and trachea or bronchi. MPM and bronchoscopy of trachea and lung tissues showed that no cells or cell debris remained in the AC scaffolds. PFT measurements of the trachea-lungs showed no relevant differences in peak pressure, dynamic or static compliance, and a nonrestricted flow pattern in AC compared to normal lungs. Although there were changes in content of collagen I and elastin this did not affect the mechanics of lung function as evidenced by normal PFT values. When repopulated with a variety of stem or adult cells including human adult primary alveolar epithelial type II

  7. DETECTION OF HUMAN LUNG EPITHELIA CELL GROWTH FACTORS PRODUCED BY A LUNG CARCINOMA CELL LINE: USE IN CULTURE OF PRIMARY SOLID LUNG TUMORS

    EPA Science Inventory

    Serum-free medium conditioned for 72 h by a human undifferentiated adenocarcinoma of lung, Cal u 6, stimulated the colony formation of normal human bronchial epithelial cells, newly cultured cells from human solid lung tumors, and established human lung tumor cell lines, includin...

  8. Human embryonic stem cells and lung regeneration

    PubMed Central

    Varanou, A; Page, C P; Minger, S L

    2008-01-01

    Human embryonic stem cells are pluripotent cells derived from the inner cell mass of preimplantation stage embryos. Their unique potential to give rise to all differentiated cell types has generated great interest in stem cell research and the potential that it may have in developmental biology, medicine and pharmacology. The main focus of stem cell research has been on cell therapy for pathological conditions with no current methods of treatment, such as neurodegenerative diseases, cardiac pathology, retinal dysfunction and lung and liver disease. The overall aim is to develop methods of application either of pure cell populations or of whole tissue parts to the diseased organ under investigation. In the field of pulmonary research, studies using human embryonic stem cells have succeeded in generating enriched cultures of type II pneumocytes in vitro. On account of their potential of indefinite proliferation in vitro, embryonic stem cells could be a source of an unlimited supply of cells available for transplantation and for use in gene therapy. Uncovering the ability to generate such cell types will expand our understanding of biological processes to such a degree that disease understanding and management could change dramatically. PMID:18724383

  9. Contribution of carboxylesterase and cytochrome P450 to the bioactivation and detoxification of isocarbophos and its enantiomers in human liver microsomes.

    PubMed

    Zhuang, Xiao-Mei; Wei, Xia; Tan, Yan; Xiao, Wei-Bin; Yang, Hai-Ying; Xie, Jian-Wei; Lu, Chuang; Li, Hua

    2014-07-01

    Organophosphorus pesticides are the most widely used pesticides in modern agricultural systems to ensure good harvests. Isocarbophos (ICP), with a potent acetylcholinesterase inhibitory effect is widely utilized to control a variety of leaf-eating and soil insects. However, the characteristics of the bioactivation and detoxification of ICP in humans remain unclear. In this study, the oxidative metabolism, esterase hydrolysis, and chiral inversion of ICP in human liver microsomes (HLMs) were investigated with the aid of a stereoselective LC/MS/MS method. The depletion of ICP in HLMs was faster in the absence of carboxylesterase inhibitor (BNPP) than in the presence of NADPH and BNPP, with t1/2 of 5.2 and 90 min, respectively. Carboxylesterase was found to be responsible for the hydrolysis of ICP, the major metabolic pathway. CYP3A4, CYP1A2, CYP2D6, CYP2C9, and CYP2C19 were all involved in the secondary metabolism pathway of desulfuration of ICP. Flavin-containing monooxygenase (FMO) did not contribute to the clearance of ICP. The hydrolysis and desulfuration of (±)ICP, (+)ICP, and (-)ICP in HLMs follow Michaelis-Menten kinetics. Individual enantiomers of ICP and its oxidative desulfuration metabolite isocarbophos oxon (ICPO) were found to be inhibitors of acetylcholinesterases at different extents. For example, (±)ICPO is more potent than ICP (IC50 0.031μM vs. 192μM), whereas (+)ICPO is more potent than (-)ICPO (IC50 0.017μM vs. 1.55μM). Given the finding of rapid hydrolysis of ICP and low abundance of oxidative metabolites presence in human liver, the current study highlights that human liver has a greater capacity for detoxification of ICP. PMID:24752505

  10. In vitro assay of six UDP-glucuronosyltransferase isoforms in human liver microsomes, using cocktails of probe substrates and liquid chromatography-tandem mass spectrometry.

    PubMed

    Seo, Kyung-Ah; Kim, Hyo-Ji; Jeong, Eun Sook; Abdalla, Nagi; Choi, Chang-Soo; Kim, Dong-Hyun; Shin, Jae-Gook

    2014-11-01

    UDP-glucuronosyltransferase (UGT)-mediated drug-drug interactions are commonly evaluated during drug development. We present a validated method for the simultaneous evaluation of drug-mediated inhibition of six major UGT isoforms, developed in human liver microsomes through the use of pooled specific UGT probe substrates (cocktail assay) and rapid liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The six probe substrates used in this assay were estradiol (UGT1A1), chenodeoxycholic acid (UGT1A3), trifluoperazine (UGT1A4), 4-hydroxyindole (UGT1A6), propofol (UGT1A9), and naloxone (UGT2B7). In a cocktail incubation, UGT1A1, UGT1A9, and UGT2B7 activities were substantially inhibited by other substrates. This interference could be eliminated by dividing substrates into two incubations: one containing estradiol, trifluoperazine, and 4-hydroxyindole, and the other containing chenodeoxycholic acid, propofol, and naloxone. Incubation mixtures were pooled for the simultaneous analysis of glucuronyl conjugates in a single LC-MS/MS run. The optimized cocktail method was further validated against single-probe substrate assays using compounds known to inhibit UGTs. The degree of inhibition of UGT isoform activities by such known inhibitors in this cocktail assay was not substantially different from that in single-probe assays. This six-isoform cocktail assay may be very useful in assessing the UGT-based drug-interaction potential of candidates in a drug-discovery setting. PMID:25122565

  11. In vitro enantioselective metabolism of TJ0711 hydrochloride by human liver microsomes using a novel chiral liquid chromatography-tandem mass spectrometry method.

    PubMed

    Huang, Jiangeng; Hu, Lei; Xu, Li; Sun, Minghui; Fan, Zhaoze; Qiu, Jun; Li, Gao; Si, Luqin

    2012-04-01

    A novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) method employing chiral analytical techniques was developed and validated for in vitro enantioselective metabolic stability study of racemic 1-[4-(2-methoxyethyl) phenoxy]-3-[[2-(2-methoxyphenoxy) ethyl]amino]-2-propanol hydrochloride (TJ0711 HCl), a newly developed vasodilatory β-blocker. Robust enantiomeric separations were achieved on a chiral SUMICHIRAL OA-2500 column using ethanol and hexane (40:60, v/v) as a mobile phase. Metabolic stability results demonstrated that both TJ0711 enantiomers underwent a rapid phase I metabolism, but preferential metabolism of R-TJ0711 was observed. Our previously reported ultra-performance liquid chromatography-multiple reaction monitoring-information dependent acquisition-enhanced product ion (UPLC-MRM-IDA-EPI) method was finally chosen for metabolite profiling study of TJ0711 enantiomers, because the newly developed HPLC-based method resulted in compromised chromatographic separation, particularly for TJ0711 metabolites. A number of metabolic products were detected and the structures of formed metabolites were predicted. Similar to racemic TJ0711 HCl, demethylation and hydroxylation were proposed to be the principle metabolism pathways during in vitro incubations of each enantiomer with human liver microsomes. PMID:22406105

  12. Regional differences in alveolar density in the human lung are related to lung height.

    PubMed

    McDonough, John E; Knudsen, Lars; Wright, Alexander C; Elliott, W Mark; Ochs, Matthias; Hogg, James C

    2015-06-01

    The gravity-dependent pleural pressure gradient within the thorax produces regional differences in lung inflation that have a profound effect on the distribution of ventilation within the lung. This study examines the hypothesis that gravitationally induced differences in stress within the thorax also influence alveolar density in terms of the number of alveoli contained per unit volume of lung. To test this hypothesis, we measured the number of alveoli within known volumes of lung located at regular intervals between the apex and base of four normal adult human lungs that were rapidly frozen at a constant transpulmonary pressure, and used microcomputed tomographic imaging to measure alveolar density (number alveoli/mm3) at regular intervals between the lung apex and base. These results show that at total lung capacity, alveolar density in the lung apex is 31.6 ± 3.4 alveoli/mm3, with 15 ± 6% of parenchymal tissue consisting of alveolar duct. The base of the lung had an alveolar density of 21.2 ± 1.6 alveoli/mm3 and alveolar duct volume fraction of 29 ± 6%. The difference in alveolar density can be negated by factoring in the effects of alveolar compression due to the pleural pressure gradient at the base of the lung in vivo and at functional residual capacity. PMID:25882386

  13. Human microsomal cyttrochrome P450-mediated reduction of oxysophocarpine, an active and highly toxic constituent derived from Sophora flavescens species, and its intestinal absorption and metabolism in rat.

    PubMed

    Wu, Lili; Zhong, Wanping; Liu, Junjin; Han, Weichao; Zhong, Shilong; Wei, Qiang; Liu, Shuwen; Tang, Lan

    2015-09-01

    Oxysophocarpine (OSC), an active and toxic quinolizidine alkaloid, is highly valued in Sophora flavescens Ait. and Subprostrate sophora Root. OSC is used to treat inflammation and hepatitis for thousands of years in China. This study aims to investigate the CYP450-mediated reduction responsible for metabolizing OSC and to evaluate the absorption and metabolism of OSC in rat in situ. Four metabolites were identified, with sophocarpine (SC) as the major metabolite. SC formation was rapid in human and rat liver microsomes (HLMs and RLMs, respectively). The reduction rates in the liver are two fold higher than in the intestine, both in humans and rats. In HLMs, inhibitors of CYP2C9, 3A4/5, 2D6, and 2B6 had strong inhibitory effects on SC formation. Meanwhile, inhibitors of CYP3A and CYP2D6 had significant inhibition on SC formation in RLMs. Human recombinant CYP3A4/5, 2B6, 2D6, and 2C9 contributed significantly to SC production. The permeability in rat intestine and the excretion rates of metabolites were highest in the duodenum (p<0.05), and the absorbed amount of OSC in duodenum and jejunum was concentration-dependent. The metabolism could be significantly decreased by CYP3A inhibitor ketoconazole. In conclusion, the liver was the main organ responsible for OSC metabolism. First-pass metabolism via CYP3A4/5, 2B6, 2D6, and 2C9 may be the main reason for the poor OSC bioavailability. PMID:26045316

  14. COMPARISON OF LUNG ANTIOXIDANT LEVELS IN HUMANS AND LABORATORY ANIMALS

    EPA Science Inventory

    Basal lung concentrations of ascorbic acid (AA), nonprotein sulfhydryls (NPSH), and a-tocopherol (a-T) were determined in rabbits, guinea pigs, rats, hamsters, mice, domestic pigs and sheep, and in human lung samples obtained from cancer surgery patients. Significant differences ...

  15. DEPOSITION PATTERNS OF POLYDISPERSE AEROSOLS WITHIN HUMAN LUNGS

    EPA Science Inventory

    The efficacy of airborne pharmaceuticals in the treatment of lung diseases can may be improved with the selective deposition of inhaled drugs. erein, a validated mathematical model is used to examine the effects of aerosol polydispersity upon deposition in the human lung. ocalize...

  16. Analytic Intermodel Consistent Modeling of Volumetric Human Lung Dynamics.

    PubMed

    Ilegbusi, Olusegun; Seyfi, Behnaz; Neylon, John; Santhanam, Anand P

    2015-10-01

    Human lung undergoes breathing-induced deformation in the form of inhalation and exhalation. Modeling the dynamics is numerically complicated by the lack of information on lung elastic behavior and fluid-structure interactions between air and the tissue. A mathematical method is developed to integrate deformation results from a deformable image registration (DIR) and physics-based modeling approaches in order to represent consistent volumetric lung dynamics. The computational fluid dynamics (CFD) simulation assumes the lung is a poro-elastic medium with spatially distributed elastic property. Simulation is performed on a 3D lung geometry reconstructed from four-dimensional computed tomography (4DCT) dataset of a human subject. The heterogeneous Young's modulus (YM) is estimated from a linear elastic deformation model with the same lung geometry and 4D lung DIR. The deformation obtained from the CFD is then coupled with the displacement obtained from the 4D lung DIR by means of the Tikhonov regularization (TR) algorithm. The numerical results include 4DCT registration, CFD, and optimal displacement data which collectively provide consistent estimate of the volumetric lung dynamics. The fusion method is validated by comparing the optimal displacement with the results obtained from the 4DCT registration. PMID:26292034

  17. Metabolic activation of 2-methylfuran by rat microsomal systems

    SciTech Connect

    Ravindranath, V.; Boyd, M.R.

    1985-05-01

    2-Methylfuran (2-MF), a constituent of cigarette smoke and coffee, causes necrosis of liver, lungs, and kidneys in rodents. 2-MF is metabolically activated by mixed-function oxidases to acetylacrolein, a reactive metabolite that binds covalently to microsomal protein. The hepatic microsomal metabolism of 2-MF to reactive metabolite required the presence of NADPH and oxygen and was dependent on incubation time and substrate concentration. The microsomal metabolism of 2-MF was inducible by pretreatment of rats with phenobarbital and was inhibited by piperonyl butoxide and N-octyl imidazole, which indicates that the metabolism of 2-MF may be mediated by cytochrome P-450. Acetylacrolein was a potent inhibitor of mixed-function oxidase and completely inhibited the microsomal metabolism of 2-MF, indicating that 2-MF is a suicide substrate for the enzyme. The sulfhydryl nucleophile cysteine was a better trapping agent of the reactive metabolite of 2-MF than N-acetylcysteine or glutathione. Lysine decreased the covalent binding of 2-MF metabolites, presumably by reacting with the aldehyde group of acetylacrolein. In addition, in the presence of NADPH, 2-MF was bioactivated by both pulmonary and renal cortical microsomes to reactive metabolites that were covalently bound to microsomal proteins.

  18. COMPUTER MODEL OF HUMAN LUNG MORPHOLOGY TO COMPLEMENT SPECT ANALYSES

    EPA Science Inventory

    Aerosol therapy protocols could be improved if inhaled pharmacologic drugs were selectively deposited within the human lung. he targeted delivery to specific sites, such as receptors and sensitive airway cells, would enhance the efficacies of airborne pharmaceuticals. he high res...

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

    PubMed

    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. PMID:25684704

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

    PubMed

    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. PMID:25272989

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

    PubMed

    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. PMID:27476943

  2. Towards a porous media model of the human lung

    NASA Astrophysics Data System (ADS)

    DeGroot, Christopher T.; Straatman, Anthony G.

    2012-05-01

    In this article, progress towards building a complete porous media model of the human lung is discussed. While the recent trend in computational fluid dynamics studies of airflow in the human lung has been to continually increase the size and detail of the airway tree under consideration, it is proposed in this work that simulating flow in the human lung as a coupled fluid-porous system is an effective method to simulate the flow in the whole lung. Under the proposed modeling paradigm, a truncated airway tree constitutes a fluid region which is coupled to a porous region that represents the remainder of the lung volume, containing small airways and alveoli. The first part of this work describes pore-level simulations conducted in an alveolated duct geometry, which are present in large quantities in the human lung, to determine its permeability. Next, volume-averaged simulations incorporating the results of the pore-level simulations and using a realistic lung geometry based on computed tomography images are discussed along with future directions for this work.

  3. Persistent Human Cosavirus Infection in Lung Transplant Recipient, Italy

    PubMed Central

    Campanini, Giulia; Rovida, Francesca; Meloni, Federica; Cascina, Alessandro; Ciccocioppo, Rachele; Piralla, Antonio

    2013-01-01

    Human cosavirus is a novel picornavirus recently identified in feces from children in southern Asia. We report infection with human cosavirus in a patient in the Mediterranean area. The patient was an adult double lung transplant recipient who had chronic diarrhea associated with persistent infection with human cosavirus. PMID:24047954

  4. The accumulation of nickel in human lungs

    SciTech Connect

    Edelman, D.A.; Roggli, V.L. )

    1989-05-01

    Using data from published studies, lung concentrations of nickel were compare for persons with and without occupational exposure to nickel. As expected, the concentrations were much higher for persons with occupational exposure. To estimate the effects of nickel-containing tobacco smoke and nickel in the ambient air on the amount of nickel accumulated in lungs over time, a model was derived that took into account various variables related to the deposition of nickel in lungs. The model predicted nickel concentrations that were in the range of those of persons without known nickel exposure. Nickel is a suspected carcinogen and has been associated with an increased risk of respiratory tract cancer among nickel workers. However, before the nickel content of cigarettes can be implicated in the etiology of lung cancer, further studies are needed to evaluate the independent effects of smoking and exposure to nickel.

  5. Solubility of Freon 22 in human blood and lung tissue

    SciTech Connect

    Varene, N.; Choukroun, M.L.; Marthan, R.; Varene, P.

    1989-05-01

    The solubility of Freon 22 in human blood and lung tissue was determined using the chromatographic method of Wagner et al. In normal human blood, the mean Bunsen coefficient of solubility (alpha B) was 0.804 cm3 STPD.cm-3.ATA-1 at 37 degrees C. It increased with hematocrit (Hct) according to the equation alpha B = 0.274 Hct + 0.691. Tissue homogenates were prepared from macroscopically normal lung pieces obtained at thoracotomy from eight patients undergoing resection for lung carcinoma. The Bunsen solubility coefficients were 0.537 +/- 0.068 and 0.635 +/- 0.091 in washed and unwashed lung, respectively. These values can be used in the determination of both cardiac output and pulmonary tissue volume in humans by use of the rebreathing technique.

  6. Linear dimensions and volumes of human lungs

    DOE PAGESBeta

    Hickman, David P.

    2012-03-30

    TOTAL LUNG Capacity is defined as “the inspiratory capacity plus the functional residual capacity; the volume of air contained in the lungs at the end of a maximal inspiration; also equals vital capacity plus residual volume” (from MediLexicon.com). Within the Results and Discussion section of their April 2012 Health Physics paper, Kramer et al. briefly noted that the lungs of their experimental subjects were “not fully inflated.” By definition and failure to obtain maximal inspiration, Kramer et. al. did not measure Total Lung Capacity (TLC). The TLC equation generated from this work will tend to underestimate TLC and does notmore » improve or update total lung capacity data provided by ICRP and others. Likewise, the five linear measurements performed by Kramer et. al. are only representative of the conditions of the measurement (i.e., not at-rest volume, but not fully inflated either). While there was significant work performed and the data are interesting, the data does not represent a maximal situation, a minimal situation, or an at-rest situation. Moreover, while interesting, the linear data generated by this study is limited by the conditions of the experiment and may not be fully comparative with other lung or inspiratory parameters, measures, or physical dimensions.« less

  7. Linear dimensions and volumes of human lungs

    SciTech Connect

    Hickman, David P.

    2012-03-30

    TOTAL LUNG Capacity is defined as “the inspiratory capacity plus the functional residual capacity; the volume of air contained in the lungs at the end of a maximal inspiration; also equals vital capacity plus residual volume” (from MediLexicon.com). Within the Results and Discussion section of their April 2012 Health Physics paper, Kramer et al. briefly noted that the lungs of their experimental subjects were “not fully inflated.” By definition and failure to obtain maximal inspiration, Kramer et. al. did not measure Total Lung Capacity (TLC). The TLC equation generated from this work will tend to underestimate TLC and does not improve or update total lung capacity data provided by ICRP and others. Likewise, the five linear measurements performed by Kramer et. al. are only representative of the conditions of the measurement (i.e., not at-rest volume, but not fully inflated either). While there was significant work performed and the data are interesting, the data does not represent a maximal situation, a minimal situation, or an at-rest situation. Moreover, while interesting, the linear data generated by this study is limited by the conditions of the experiment and may not be fully comparative with other lung or inspiratory parameters, measures, or physical dimensions.

  8. Correlation of Apical Fluid-Regulating Channel Proteins with Lung Function in Human COPD Lungs

    PubMed Central

    Zhao, Meimi; Liu, Shan-Lu; Huang, Yao; Idell, Steven; Li, Xiumin; Ji, Hong-Long

    2014-01-01

    Links between epithelial ion channels and chronic obstructive pulmonary diseases (COPD) are emerging through animal model and in vitro studies. However, clinical correlations between fluid-regulating channel proteins and lung function in COPD remain to be elucidated. To quantitatively measure epithelial sodium channels (ENaC), cystic fibrosis transmembrane conductance regulator (CFTR), and aquaporin 5 (AQP5) proteins in human COPD lungs and to analyze the correlation with declining lung function, quantitative western blots were used. Spearman tests were performed to identify correlations between channel proteins and lung function. The expression of α and β ENaC subunits was augmented and inversely associated with lung function. In contrast, both total and alveolar type I (ATI) and II (ATII)-specific CFTR proteins were reduced. The expression level of CFTR proteins was associated with FEV1 positively. Abundance of AQP5 proteins and extracellular superoxide dismutase (SOD3) was decreased and correlated with spirometry test results and gas exchange positively. Furthermore, these channel proteins were significantly associated with severity of disease. Our study demonstrates that expression of ENaC, AQP5, and CFTR proteins in human COPD lungs is quantitatively associated with lung function and severity of COPD. These apically located fluid-regulating channels may thereby serve as biomarkers and potent druggable targets of COPD. PMID:25329998

  9. An ultra-high performance liquid chromatography-tandem mass spectrometric assay for quantifying 3-ketocholanoic acid: Application to the human liver microsomal CYP3A-dependent lithocholic acid 3-oxidation assay.

    PubMed

    Bansal, Sumit; Chai, Swee Fen; Lau, Aik Jiang

    2016-06-15

    Lithocholic acid (LCA), a hepatotoxic and carcinogenic bile acid, is metabolized to 3-ketocholanoic acid (3-KCA) by cytochrome P450 3A (CYP3A). In the present study, the objectives were to develop and validate an ultra-high performance liquid chromatography-tandem mass spectrometric (UPLC-MS/MS) method to quantify 3-KCA and apply it to the human liver microsomal CYP3A-dependent LCA 3-oxidation assay. Chromatographic separation was achieved on a Waters ACQUITY™ UPLC C18 column (50×2.1mm, 1.7μm) with a gradient system consisting of 0.1% v/v formic acid in water (solvent A) and 0.1% v/v formic acid in acetonitrile (solvent B). The retention time was 3.73min for 3-KCA and 2.73min for cortisol (internal standard). Positive electrospray ionization with multiple reaction monitoring (MRM) mode was used to quantify 3-KCA (m/z 375.4→135.2) and cortisol (m/z 363.5→121.0). The limit of detection of 3-KCA was 10μM, the lower limit of quantification was 33.3μM, and the calibration curve was linear from 0.05-10μM with r(2)>0.99. Intra-day and inter-day accuracy and precision were <13.7%. The quality control samples were stable when assessed after 4h at room temperature, 24h at 4°C, 14days at -20°C, and three freeze-thaw cycles. The liver microsomal matrix did not affect 3-KCA quantification. The amount of KCA formed in the human liver microsomal LCA 3-oxidation assay was linear with respect to the amount of microsomal protein (up to 40μg) and incubation time (5-30min). Enzyme kinetics experiment indicated that LCA 3-oxidation followed the Michaelis-Menten model with an apparent Km of 26±7μM and Vmax of 303±50pmol/min/mg protein. This novel UPLC-MS/MS method for quantifying 3-KCA offers a specific, sensitive, and fast approach to determine liver microsomal LCA 3-oxidation. PMID:27153105

  10. Effect of Cytochrome b5 Content on the Activity of Polymorphic CYP1A2, 2B6, and 2E1 in Human Liver Microsomes

    PubMed Central

    Zhang, Haifeng; Gao, Na; Liu, Tingting; Fang, Yan; Qi, Bing; Wen, Qiang; Zhou, Jun; Jia, Linjing; Qiao, Hailing

    2015-01-01

    Human cytochrome b5 (Cyt b5) plays important roles in cytochrome P450 (CYP)-mediated drug metabolism. However, the expression level of Cyt b5 in normal human liver remains largely unknown. The effect of Cyt b5 on overall CYP activity in human liver microsomes (HLM) has rarely been reported and the relationship between Cyt b5 and the activity of polymorphic CYP has not been systematically investigated. In this study, we found that the median value of Cyt b5 protein was 270.01 pmol/mg from 123 HLM samples, and 12- and 19-fold individual variation was observed in Cyt b5 mRNA and protein levels, respectively. Gender and smoking clearly influenced Cyt b5 content. In addition, we found that Cyt b5 protein levels significantly correlated with the overall activity of CYP1A2, 2B6, and 2E1 in HLM. However, when the CYP activities were sorted by single nucleotide polymorphisms (SNP), the effect of Cyt b5 protein on the kinetic parameters varied greatly. There were significant correlations between Cyt b5 content and Vmax and CLint of CYP1A2 wild-types (3860GG, 2159GG, and 5347CC) as well as homozygous mutants (163AA and 3113GG). In contrast to Vmax and CLint, the Km of CYP2B6 516GG and 785AA genotypes was inversely associated with Cyt b5 content. Correlations between Cyt b5 content and Vmax and CLint of CYP2E1 -1293GG, -1293GC, 7632TT, 7632TA, -333TT, and -352AA genotypes were also observed. In conclusion, Cyt b5 expression levels varied considerably in the Chinese cohort from this study. Cyt b5 had significant impact on the overall activity of CYP1A2, 2B6, and 2E1 in HLM and the effects of Cyt b5 protein on polymorphic CYP1A2, 2B6, and 2E1 activity were SNP-dependent. These findings suggest that Cyt b5 plays an important role in CYP-mediated activities in HLM and may possibly be a contributing factor for the individual variation observed in CYP enzyme activities. PMID:26046844

  11. Immune and Inflammatory Cell Composition of Human Lung Cancer Stroma

    PubMed Central

    Banat, G-Andre; Tretyn, Aleksandra; Pullamsetti, Soni Savai; Wilhelm, Jochen; Weigert, Andreas; Olesch, Catherine; Ebel, Katharina; Stiewe, Thorsten; Grimminger, Friedrich; Seeger, Werner; Fink, Ludger; Savai, Rajkumar

    2015-01-01

    Recent studies indicate that the abnormal microenvironment of tumors may play a critical role in carcinogenesis, including lung cancer. We comprehensively assessed the number of stromal cells, especially immune/inflammatory cells, in lung cancer and evaluated their infiltration in cancers of different stages, types and metastatic characteristics potential. Immunohistochemical analysis of lung cancer tissue arrays containing normal and lung cancer sections was performed. This analysis was combined with cyto-/histomorphological assessment and quantification of cells to classify/subclassify tumors accurately and to perform a high throughput analysis of stromal cell composition in different types of lung cancer. In human lung cancer sections we observed a significant elevation/infiltration of total-T lymphocytes (CD3+), cytotoxic-T cells (CD8+), T-helper cells (CD4+), B cells (CD20+), macrophages (CD68+), mast cells (CD117+), mononuclear cells (CD11c+), plasma cells, activated-T cells (MUM1+), B cells, myeloid cells (PD1+) and neutrophilic granulocytes (myeloperoxidase+) compared with healthy donor specimens. We observed all of these immune cell markers in different types of lung cancers including squamous cell carcinoma, adenocarcinoma, adenosquamous cell carcinoma, small cell carcinoma, papillary adenocarcinoma, metastatic adenocarcinoma, and bronchioloalveolar carcinoma. The numbers of all tumor-associated immune cells (except MUM1+ cells) in stage III cancer specimens was significantly greater than those in stage I samples. We observed substantial stage-dependent immune cell infiltration in human lung tumors suggesting that the tumor microenvironment plays a critical role during lung carcinogenesis. Strategies for therapeutic interference with lung cancer microenvironment should consider the complexity of its immune cell composition. PMID:26413839

  12. Ex Vivo Perfusion Treatment of Infection in Human Donor Lungs.

    PubMed

    Nakajima, D; Cypel, M; Bonato, R; Machuca, T N; Iskender, I; Hashimoto, K; Linacre, V; Chen, M; Coutinho, R; Azad, S; Martinu, T; Waddell, T K; Hwang, D M; Husain, S; Liu, M; Keshavjee, S

    2016-04-01

    Ex vivo lung perfusion (EVLP) is a platform to treat infected donor lungs with antibiotic therapy before lung transplantation. Human donor lungs that were rejected for transplantation because of clinical concern regarding infection were randomly assigned to two groups. In the antibiotic group (n = 8), lungs underwent EVLP for 12 h with high-dose antibiotics (ciprofloxacin 400 mg or azithromycin 500 mg, vancomycin 15 mg/kg, and meropenem 2 g). In the control group (n = 7), lungs underwent EVLP for 12 h without antibiotics. A quantitative decrease in bacterial counts in bronchoalveolar lavage (BAL) was found in all antibiotic-treated cases but in only two control cases. Perfusate endotoxin levels at 12 h were significantly lower in the antibiotic group compared with the control group. EVLP with broad-spectrum antibiotic therapy significantly improved pulmonary oxygenation and compliance and reduced pulmonary vascular resistance. Perfusate endotoxin levels at 12 h were strongly correlated with levels of perfusates tumor necrosis factor α, IL-1β and macrophage inflammatory proteins 1α and 1β at 12 h. In conclusion, EVLP treatment of infected donor lungs with broad-spectrum antibiotics significantly reduced BAL bacterial counts and endotoxin levels and improved donor lung function. PMID:26730551

  13. ACID AIR AND AEROBIOLOGY RELATED TO THE MATURING HUMAN LUNG

    EPA Science Inventory

    The effect of 'acid air' on human health was studied by considering the effects of hygroscopicity upon aerosol deposition in the lung as a function of human subject age. Children are a critical sub-population to be incorporated into health effects analyses following ambient expos...

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

    PubMed

    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

  15. Role of the ABCE1 gene in human lung adenocarcinoma

    PubMed Central

    REN, YI; LI, YINGHUI; TIAN, DALI

    2012-01-01

    ATP-binding cassette transporter E1 (ABCE1), also known as RLI (RNase L inhibitor), is a new type of endoribonuclease inhibitor, which can specifically bind to RNase L and abolish its effect. ABCE1 binds to eIF2α and eIF5 to form a pre-translation initiation complex, suggesting its crucial role in cell growth, development and certain pathological processes. To probe the role of ABCE1 in the development and progress of human lung adenocarcinoma, we first detected the changes of its mRNA and protein expression in tissues, and found a high expression level of ABCE1 in human lung adenocarcinoma tissues and metastatic lymph nodes, which was also correlated with clinical stages. Moreover, human lung adenocarcinoma A549 cells were infected with lentiviral vectors containing ABCE1-specific shRNA, and resulted in significant inhibition of cell growth. Using microarray assay, a number of differentially expressed genes were found after ABCE1 suppression. Our results demonstrated the potential role of ABCE1 in human lung adenocarcinoma, which may provide some molecular basis for the mechanisms of development and progress of human lung adenocarcinoma, and help to find new pharmacological targets. PMID:22267055

  16. Liquid chromatography/nuclear magnetic resonance spectroscopy and liquid chromatography/mass spectrometry identification of novel metabolites of the multidrug resistance modulator LY335979 in rat bile and human liver microsomal incubations.

    PubMed

    Ehlhardt, W J; Woodland, J M; Baughman, T M; Vandenbranden, M; Wrighton, S A; Kroin, J S; Norman, B H; Maple, S R

    1998-01-01

    Compound LY335979 is a P-glycoprotein inhibitor currently entering phase I clinical trials for potential reversal of multidrug resistance to cancer chemotherapy. In early exploratory studies, LY335979 was found to be rapidly transformed in incubations with liver microsomes from rats, dogs, monkeys, and humans. Although the parent compound was completely metabolized, no prominent metabolite peaks were observed. One peak did appear early in the time course, but it did not increase over time. In another preliminary experiment, rats were treated iv with [3H]LY335979 (prepared for pharmacology studies), and urine and bile fractions were collected. Analysis of the urine by reverse-phase HPLC with UV and radioactivity detection revealed that almost all of the material eluted with the solvent front. More than half the radioactivity in bile was accounted for by two peaks eluting earlier than the parent compound (the rest eluted at the solvent front). With both bile and the incubations with microsomes, initial attempts to isolate metabolites were not successful. There was also evidence in both systems of products derived from cleavage of LY335979 (by both further metabolism and degradation). LC/NMR was thus used to analyze materials directly in their respective matrices. An N-oxide metabolite (LY389551) formed by oxidation of the quinoline nitrogen was identified in the microsomal incubations; in bile, three glucuronide metabolites were identified, all of which were conjugates of products formed by oxidation of the quinoline ring of LY335979. There have been few reports in the literature of LC/NMR analysis of bile, which is a more complex matrix than either urine or microsomal suspensions. However, the HPLC techniques developed in this work for the HPLC/UV and LC/MS analyses of LY335979 metabolites in the microsomal matrix and in bile proved readily adaptable for LC/NMR. Using a 500-MHz instrument, basic 1H NMR spectra could be obtained in 2-3 hr with approximately 100 ng of

  17. UPLC/ESI-MS/MS-based determination of metabolism of several new illicit drugs, ADB-FUBINACA, AB-FUBINACA, AB-PINACA, QUPIC, 5F-QUPIC and α-PVT, by human liver microsome.

    PubMed

    Takayama, Takahiro; Suzuki, Mayu; Todoroki, Kenichiro; Inoue, Koichi; Min, Jun Zhe; Kikura-Hanajiri, Ruri; Goda, Yukihiro; Toyo'oka, Toshimasa

    2014-06-01

    The metabolism by human liver microsomes of several new illicit drugs, that is, N-(1-amino-3,3-dimethyl-1-oxobutan-2-yl)-1-(4-fluorobenzyl)-1H-indazole-3- carboxamide (ADB-FUBINACA), N-(1-amino-3-methyl-1-oxobutan-2-yl)-1- (4-fluorobenzyl)-1H-indazole-3-carboxamide (AB-FUBINACA), N-(1-amino-3-methyl-1-oxobutan-2-yl)-1-pentyl-1H-indazole-3-carboxamide (AB-PINACA), quinolin-8-yl 1-pentyl-(1H-indole)-3-carboxylate (QUPIC), quinolin-8-yl 1-(5-fluoropentyl)-(1H-indole)-3-carboxylate (5 F-QUPIC) and α-pyrrolidinovalerothiophenone (α-PVT), which have indole, indazole, quinolinol ester and thiophene structures, was investigated using reversed-phase chromatography and mass spectrometry. The present method is based upon the oxidation by cytochrome p450 superfamily enzymes in the microsomes. The oxidation of ADB-FUBINACA and AB-FUBINACA mainly occurred on the N-(1-amino-alkyl-1-oxobutan) moiety. However, the oxidation of AB-PINACA seemed to occur on the 1-pentyl moiety. On the other hand, QUPIC and 5 F-QUPIC, which have a quinolinol ester structure, predominantly underwent a cleavage reaction to produce indoleacetic acid type metabolites. In contrast, the metabolism reaction of α-PVT was different from that of the other tested drugs, and various oxidation products were observed on the chromatograms. The obtained metabolites are not in conflict with the results predicted by MetaboLynx software. However, the exact structures of the metabolites, except for 1-pentyl-1H-indole-3-carboxylic acid (QUPIC metabolite) and 1-(5-fluoropentyl)-1H-indole-3-carboxylic acid (5 F-QUPIC metabolite), are currently not proven, because we have no authentic compounds for comparison. The proposed approach using human liver microsome seems to provide a new technology for the prediction of possible metabolites occuring in humans. PMID:24861751

  18. A reevaluation of CD22 expression in human lung cancer.

    PubMed

    Pop, Laurentiu M; Barman, Stephen; Shao, Chunli; Poe, Jonathan C; Venturi, Guglielmo M; Shelton, John M; Pop, Iliodora V; Gerber, David E; Girard, Luc; Liu, Xiao-yun; Behrens, Carmen; Rodriguez-Canales, Jaime; Liu, Hui; Wistuba, Ignacio I; Richardson, James A; Minna, John D; Tedder, Thomas F; Vitetta, Ellen S

    2014-01-01

    CD22 is a transmembrane glycoprotein expressed by mature B cells. It inhibits signal transduction by the B-cell receptor and its coreceptor CD19. Recent reports indicate that most human lung cancer cells and cell lines express CD22, making it an important new therapeutic target for lung cancer. The objective of our studies was to independently validate these results with the goal of testing the efficacy of our CD22 immunotoxins on lung cancer cell lines. As determined by quantitative real-time PCR analysis, we found that levels of CD22 mRNA in a panel of human lung cancer cell lines were 200 to 60,000-fold lower than those observed in the human CD22(+) Burkitt lymphoma cells, Daudi. Using flow cytometry with a panel of CD22 monoclonal antibodies and Western blot analyses, we could not detect surface or intracellular expression of CD22 protein in a panel of lung cancer cell lines. In addition, the in vitro proliferation of the lung tumor cell lines was not affected by either CD22 antibodies or our highly potent anti-CD22 immunotoxin. In contrast, CD22(+) Daudi cells expressed high levels of CD22 mRNA and protein, and were sensitive to our CD22 immunotoxin. Importantly, primary non-small cell lung cancers from more than 250 patient specimens did not express detectable levels of CD22 protein as assessed by immunohistochemistry. We conclude that CD22 is not expressed at measurable levels on the surface of lung cancer cells, and that these cells cannot be killed by anti-CD22 immunotoxins. PMID:24395821

  19. Parallel Computation of Airflow in the Human Lung Model

    NASA Astrophysics Data System (ADS)

    Lee, Taehun; Tawhai, Merryn; Hoffman, Eric. A.

    2005-11-01

    Parallel computations of airflow in the human lung based on domain decomposition are performed. The realistic lung model is segmented and reconstructed from CT images as part of an effort to build a normative atlas (NIH HL-04368) documenting airway geometry over 4 decades of age in healthy and disease-state adult humans. Because of the large number of the airway generation and the sheer complexity of the geometry, massively parallel computation of pulmonary airflow is carried out. We present the parallel algorithm implemented in the custom-developed characteristic-Galerkin finite element method, evaluate the speed-up and scalability of the scheme, and estimate the computing resources needed to simulate the airflow in the conducting airways of the human lungs. It is found that the special tree-like geometry enables the inter-processor communications to occur among only three or four processors for optimal parallelization irrespective of the number of processors involved in the computation.

  20. Development and implementation of a stereoselective normal-phase liquid chromatography-tandem mass spectrometry method for the determination of intrinsic metabolic clearance in human liver microsomes.

    PubMed

    Zhang, Yingru; Caporuscio, Christian; Dai, Jun; Witkusa, Michael; Rose, Anne; Santella, Joseph; D'Arienzo, Celia; Wang-Iverson, David B; Tymiak, Adrienne A

    2008-11-01

    The stereoselective determination of stereoisomers in biological samples provides vital information on stereospecific metabolism and pharmacokinetic profiles of the drugs. Despite the unique advantage and the great success of normal-phase (NP) HPLC for the separations of drug stereoisomers using polysaccharide-type chiral stationary phases (CSPs), the technique is rarely applied to quantitative HPLC-MS-MS bioanalysis. This is, at least in part, due to the incompatibility between the usual mobile phase (n-hexane or n-heptane) in normal-phase HPLC and the MS ionization sources which poses a potential detonation hazard. An environmentally friendly and nonflammable alternative solvent, ethoxynonafluorobutane (ENFB), was reported previously to potentially provide an ideal solution for combining the powers of stereoselective NP chromatographic separation and MS-MS detection. In this study, a stereoselective NP-HPLC-MS-MS method was developed using ENFB to quantify a pair of Bristol Myers Squibb (BMS) proprietary drug stereoisomers and their ketone metabolite for an in vitro study, which demonstrated, for the first time, the practical applicability and utility of ENFB for bioanalysis in pharmaceutical industry. The effects of different organic modifiers and temperature, as well as the comparison between ENFB and the usual solvent, heptane, for the separation, are discussed. The resolution of the stereoisomers was achieved using 63% of 3:1 mixture of ethanol and methanol with 37% ENFB on a Chiralpak AD-H column at 50 degrees C. High sensitivity was obtained using the MS-MS detection in the positive ion atmospheric pressure chemical ionization (APCI) mode. The lower limit of quantitation (LLOQ) for the first stereoisomer and the ketone metabolite was 5 ng/mL, and was 10 ng/mL for the second isomer in the human liver microsome-potassium phosphate buffer matrix. The linear dynamic range of 5-1000 ng/mL for both isomers and 10-1000 ng/mL for the metabolite were demonstrated

  1. A predominate role of CYP1A2 for the metabolism of nabumetone to the active metabolite, 6-methoxy-2-naphthylacetic acid, in human liver microsomes.

    PubMed

    Turpeinen, Miia; Hofmann, Ute; Klein, Kathrin; Mürdter, Thomas; Schwab, Matthias; Zanger, Ulrich M

    2009-05-01

    Nabumetone, a widely used nonsteroidal anti-inflammatory drug, requires biotransformation into 6-methoxy-2-naphthylacetic acid (6-MNA), a close structural analog to naproxen, to achieve its analgesic and anti-inflammatory effects. Despite its wide use, the enzymes involved in metabolism have not been identified. In the present study, several in vitro approaches were used to identify the cytochrome P450 (P450) enzyme(s) responsible for 6-MNA formation. In human liver microsomes (HLMs) 6-MNA formation displayed monophasic Michaelis-Menten kinetics with apparent K(m) and V(max) values (mean +/- S.D.) of 75.1 +/- 15.3 microM and 1304 +/- 226 pmol/min/mg protein, respectively, and formation rate of 6-MNA varied approximately 5.5-fold (179-983 pmol/min/mg protein). 6-MNA activity correlated strongly with both CYP1A2-mediated phenacetin O-deethylation activity and CYP1A2 protein content (r = 0.85 and 0.74, respectively; p < 0.0001 for both). Additional correlations were found with model activities of CYP2C19 and CYP3A4. Of 11 cDNA-expressed recombinant P450s used, recombinant CYP1A2 was the major form catalyzing the 6-MNA formation with an apparent K(m) of 45 microM and V(max) of 8.7 pmol/min/pmol P450. Minor fractions were catalyzed by recombinant P450s CYP1A1, CYP2B6, CYP2C19, CYP2D6, and CYP2E1. Experiments with P450-selective chemical inhibitors and monoclonal anti-P450 antibodies showed that furafylline, a mechanism-based inhibitor CYP1A2, and anti-CYP1A2 antibody markedly inhibited 6-MNA formation, whereas inhibitors for other P450s did not show significant inhibitory effects. Taken together, these studies indicate that the formation of the active metabolite of nabumetone, 6-MNA, is predominantly catalyzed by CYP1A2 in HLMs with only minor contribution of other P450s. PMID:19204080

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

    PubMed

    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. PMID:26551762

  3. Deposition of large particles in human lung.

    PubMed

    Svartengren, M; Falk, R; Linnman, L; Philipson, K; Camner, P

    1987-01-01

    Twenty-four nonsmoking males, all without history of pulmonary disease, were randomly divided into four groups of six subjects each. The subjects in each group inhaled monodisperse Teflon particles labelled with 111In (half-life 2.83 days); 8.2, 11.5, 13.7 and 16.4 micron aerodynamic diameter, respectively. Radioactivity in head and throat, lung and stomach was determined after 0, 3 and 24 hrs using a profile scanner. For some subjects radioactivity was also determined using a whole-body scanner at 3.5 and 24 hrs. After the 24-hr determination the subjects inhaled labelled Teflon particles again, this time with a filter in front of the mouth. Average values for total deposition in the body, obtained using a profile scanner, whole-body scanner and filter measurements, agreed fairly well. Lung retention values obtained by whole-body and profile scanning also agreed well. The average deposition in the lung, expressed as a percentage of total deposition, was 49, 31, 21 and 13% for the four particle sizes (8.2-16.4 micron). Alveolar deposition, determined as retention at 24 hrs and expressed in percent of total deposition, was 15, 4, 4 and 1%. For the smallest particle sizes the deposition values agreed with earlier investigations. However, for the larger particles the two deposition values were higher than expected when compared to earlier studies. PMID:3102217

  4. Paracetamol hepatotoxicity and microsomal function.

    PubMed

    Kaushal, R; Dave, K R; Katyare, S S

    1999-03-01

    The effect of paracetamol-induced hepatotoxicity in rats (650 mg/kg) on microsomal function was examined. Paracetamol treatment resulted in lowered Na(+),K(+)-ATPase activity in the microsomes with decrease in V(max) of the low affinity high V(max) component II. However, the temperature kinetics was not influenced significantly. The total phospholipid and cholesterol contents as well as lipid peroxidation in the microsomes were unchanged. However, content of acidic phospholipids: phosphatidylserine and phosphatidylinositol decreased by 50% with a reciprocal increase in the sphingomyelin content; the lysophosphoglyceride content increased by 12-fold. The microsomal membrane appeared to be more fluidized following paracetamol treatment. Paracetamol treatment also resulted in a significant reduction in the sulfhydryl groups content. PMID:21781911

  5. Second-hand smoke and human lung cancer

    PubMed Central

    Besaratinia, Ahmad; Pfeifer, Gerd P.

    2009-01-01

    Since the early 1980s, there has been growing concern about potential health consequences of exposure to second-hand smoke (SHS). Despite SHS being established as a risk factor for lung cancer development, the estimated risk has remained small yet somehow debatable. Human exposure to SHS is complicated because of temporal variabilities in source, composition, and concentration of SHS. The temporality of exposure to SHS is important for human lung carcinogenesis with a latency of many years. To explore the causal effect of SHS in lung carcinogenesis, exposure assessments should estimate chronic exposure to SHS on an individual basis. However, conventional exposure assessment for SHS relies on one-off or short-term measurements of SHS indices. A more reliable approach would be to use biological markers that are specific for SHS exposure and pertinent to lung cancer. This approach requires an understanding of the underlying mechanisms through which SHS could contribute to lung carcinogenesis. This Review is a synopsis of research on SHS and lung cancer, with special focus on hypothetical modes of action of SHS for carcinogenesis, including genotoxic and epigenetic effects. PMID:18598930

  6. In vitro inhibition and enhancement of liver microsomal S-777469 metabolism by long-chain fatty acids and serum albumin: insight into in vitro and in vivo discrepancy of metabolite formation in humans.

    PubMed

    Sekiguchi, Kazutaka; Kanazu, Takushi; Murayama, Norie; Yamazaki, Hiroshi; Yamaguchi, Yoshitaka

    2016-06-01

    1. It was previously demonstrated that 10% of S-777469, a cannabinoid receptor 2 selective agonist, is metabolized to its carboxylic acid metabolite (S-777469 5-carboxylic acid, 5-CA) in humans in vivo, while the formation of 5-CA is extremely low in human cryopreserved hepatocytes and liver microsomes (HLMs). In this study, factors causing the different metabolite formation rates of S-777469 in vitro and in vivo were investigated. 2. Formation of 5-CA and S-777469 5-hydroxymethyl (5-HM), a precursor metabolite of 5-CA, was catalyzed by CYP2C9. Arachidonic acid, α-linolenic acid, oleic acid and myristic acid, which have been reported to exist in liver microsomes, inhibited S-777469 oxidation by CYP2C9, but serum albumin enhanced this reactions. 3. The IC50 values of these fatty acids for 5-CA formation from 5-HM were lower than those of 5-HM formation from S-777469. Serum albumin extensively enhanced 5-CA formation from 5-HM in comparison to 5-HM formation from S-777469. 4. CYP2C9 was the enzyme responsible for S-777469 oxidation in human livers. The suppressive effects of several fatty acids and enhancing action of serum albumin in vitro are likely to be the causal factors for the apparently different rates of in vitro and in vivo metabolite formation of S-777469. PMID:26677906

  7. Histologic, immunohistochemical, and ultrastructural findings in human blast lung injury.

    PubMed

    Tsokos, Michael; Paulsen, Friedrich; Petri, Susan; Madea, Burkhard; Puschel, Klaus; Turk, Elisabeth E

    2003-09-01

    The objective of this autopsy-based study was to investigate the pathology of human blast lung injury using histology, Fat Red 7B staining, immunohistochemistry, and scanning electron microscopy on lung specimens from eight medicolegal autopsy cases of fatal close-range detonations of chemical explosives. The micromorphologic equivalents of human blast lung injury can be summarized as follows: diffuse alveolar overdistension, circumscribed interstitial hemorrhages showing a cufflike pattern around pulmonary vessels, venous air embolism, bone marrow embolism, and pulmonary fat embolism. Hemorrhages within the lung parenchyma that were present in this study in blast victims without coexisting blunt or penetrating chest trauma must be regarded as potentially life-threatening intrapulmonary bleeding sites in survivors. In addition, the potential clinical importance of the presence of massive pulmonary fat embolism, which has, to the best of our knowledge, not been described previously in human blast lung injury, must be emphasized because pulmonary fat embolism may be a leading cause of the rapid respiratory deterioration with progressive hypoxia and development of acute respiratory distress syndrome in blast victims who survive. Furthermore, this study provides evidence that air embolism presenting in blast victims is not a mere ventilation-induced artifact. PMID:12842857

  8. Hyperpolarized 129Xe MRI of the Human Lung

    PubMed Central

    Mugler, John P.; Altes, Talissa A.

    2012-01-01

    By permitting direct visualization of the airspaces of the lung, MR imaging using hyperpolarized gases provides unique strategies for evaluating pulmonary structure and function. Although the vast majority of research in humans has been performed using hyperpolarized 3He, recent contraction in the supply of 3He and consequent increases in price have turned attention to the alternative agent, hyperpolarized 129Xe. Compared to 3He, 129Xe yields reduced signal due to its smaller magnetic moment. Nonetheless, taking advantage of advances in gas-polarization technology, recent studies in humans using techniques for measuring ventilation, diffusion, and partial pressure of oxygen have demonstrated results for hyperpolarized 129Xe comparable to those previously demonstrated using hyperpolarized 3He. In addition, xenon has the advantage of readily dissolving in lung tissue and blood following inhalation, which makes hyperpolarized 129Xe particularly attractive for exploring certain characteristics of lung function, such as gas exchange and uptake, which cannot be accessed using 3He. Preliminary results from methods for imaging 129Xe dissolved in the human lung suggest that these approaches will provide new opportunities for quantifying relationships among gas delivery, exchange, and transport, and thus show substantial potential to broaden our understanding of lung disease. Finally, recent changes in the commercial landscape of the hyperpolarized-gas field now make it possible for this innovative technology to move beyond the research lab. PMID:23355432

  9. DISPERSION OF AEROSOL BOLUSES IN THE HUMAN LUNG: DEPENDENCE ON LUNG VOLUME, BOLUS VOLUME, AND GENDER

    EPA Science Inventory

    The dispersion of aerosol boluses in the human lungs has been studied in health and disease by other investigators as a means of investigating convective mixing. owever, there are only limited data on the roles played in dispersion by critical factors such as the volume of inhale...

  10. Radioactivity and lung cancer-mathematical models of radionuclide deposition in the human lungs

    PubMed Central

    Sturm, Robert

    2011-01-01

    The human respiratory tract is regarded as pathway for radionuclides and other hazardous airborne materials to enter the body. Radioactive particles inhaled and deposited in the lungs cause an irradiation of bronchial/alveolar tissues. At the worst, this results in a malignant cellular transformation and, as a consequence of that, the development of lung cancer. In general, naturally occurring radionuclides (e.g., 222Rn, 40K) are attached to so-called carrier aerosols. The aerodynamic diameters of such radioactively labeled particles generally vary between several nanometers (ultrafine particles) and few micrometers, whereby highest particle fractions adopt sizes around 100 nm. Theoretical simulations of radioactive particle deposition in the human lungs were based on a stochastic lung geometry and a particle transport/deposition model using the random-walk algorithm. Further a polydisperse carrier aerosol (diameter: 1 nm–10 µm, ρ ≈ 1 g cm−3) with irregularly shaped particles and the effect of breathing characteristics and certain respiratory parameters on the transport of radioactive particles to bronchial/alveolar tissues were considered. As clearly shown by the results of deposition modeling, distribution patterns of radiation doses mainly depend on the size of the carrier aerosol. Ultrafine (< 10 nm) and large (> 2 µm) aerosol particles are preferentially deposited in the extrathoracic and upper bronchial region, whereas aerosol particles with intermediate size (10 nm–2 µm) may penetrate to deeper lung regions, causing an enhanced damage of the alveolar tissue by the attached radionuclides. PMID:22263097

  11. Impact of Statins on Gene Expression in Human Lung Tissues

    PubMed Central

    Lane, Jérôme; van Eeden, Stephan F.; Obeidat, Ma’en; Sin, Don D.; Tebbutt, Scott J.; Timens, Wim; Postma, Dirkje S.; Laviolette, Michel; Paré, Peter D.; Bossé, Yohan

    2015-01-01

    Statins are 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors that alter the synthesis of cholesterol. Some studies have shown a significant association of statins with improved respiratory health outcomes of patients with asthma, chronic obstructive pulmonary disease and lung cancer. Here we hypothesize that statins impact gene expression in human lungs and may reveal the pleiotropic effects of statins that are taking place directly in lung tissues. Human lung tissues were obtained from patients who underwent lung resection or transplantation. Gene expression was measured on a custom Affymetrix array in a discovery cohort (n = 408) and two replication sets (n = 341 and 282). Gene expression was evaluated by linear regression between statin users and non-users, adjusting for age, gender, smoking status, and other covariables. The results of each cohort were combined in a meta-analysis and biological pathways were studied using Gene Set Enrichment Analysis. The discovery set included 141 statin users. The lung mRNA expression levels of eighteen and three genes were up-regulated and down-regulated in statin users (FDR < 0.05), respectively. Twelve of the up-regulated genes were replicated in the first replication set, but none in the second (p-value < 0.05). Combining the discovery and replication sets into a meta-analysis improved the significance of the 12 up-regulated genes, which includes genes encoding enzymes and membrane proteins involved in cholesterol biosynthesis. Canonical biological pathways altered by statins in the lung include cholesterol, steroid, and terpenoid backbone biosynthesis. No genes encoding inflammatory, proteases, pro-fibrotic or growth factors were altered by statins, suggesting that the direct effect of statin in the lung do not go beyond its antilipidemic action. Although more studies are needed with specific lung cell types and different classes and doses of statins, the improved health outcomes and survival observed in statin

  12. The association between human papillomavirus infection and female lung cancer

    PubMed Central

    Lin, Frank Cheau-Feng; Huang, Jing-Yang; Tsai, Stella Ching-Shao; Nfor, Oswald Ndi; Chou, Ming-Chih; Wu, Ming-Fang; Lee, Chun-Te; Jan, Cheng-Feng; Liaw, Yung-Po

    2016-01-01

    Abstract Lung cancer is the leading cause of cancer deaths among Taiwanese women. Human papillomavirus (HPV) has been detected in lung cancer tissues. The aim of this study was to investigate the association between HPV infection and lung cancer among the Taiwanese women. The analytical data were collected from the longitudinal health insurance databases (LHID 2005 and 2010) of the National Health Insurance Research Database (NHIRD). The study participants were 30 years and older and included 24,162 individuals who were identified with HPV infection from 2001 to 2004 and 1,026,986 uninfected individuals. Lung cancer incidence among infected and uninfected individuals was compared using the univariate and multivariate regression models. Among the total participants, 24,162 individuals were diagnosed with HPV. After adjusting for age, gender, low income, residential area, and comorbidity, the risk of lung cancer was higher in women (hazard ratio [HR] 1.263, 95% CI 1.015–1.571), while all cancer risks were high in both men and women with corresponding hazard ratios (HR) of 1.161 (95% CI 1.083–1.245) and HR 1.240 (95% CI 1.154–1.331), respectively. This study showed a significant increase in lung cancer risk among Taiwanese women who were exposed to HPV infection. PMID:27281096

  13. BIOMARKERS OF HEALTH EFFECTS IN THE HUMAN LUNG

    EPA Science Inventory

    Little information exists about retained particle/metal burden in human lung and associated biomarkers of internal dose/indicators of health effects. We have shown that anatomical remodeling of the terminal and respiratory bronchioles occur at sites of particle deposition. We ext...

  14. MATHEMATICAL ANALYSIS OF PARTICLE TRANSPORT AND DEPOSITION IN HUMAN LUNGS

    EPA Science Inventory

    MATHEMATICAL ANALYSIS OF PARTICLE TRANSPORT AND DEPOSITION IN HUMAN LUNGS. Jung-il Choi*, Center for Environmental Medicine, University of North Carolina, Chapel Hill, NC 27599; C. S. Kim, USEPA National Health and Environmental Effects Research Lab. RTP, NC 27711

    Partic...

  15. RECONSTRUCTION OF HUMAN LUNG MORPHOLOGY MODELS FROM MAGNETIC RESONANCE IMAGES

    EPA Science Inventory


    Reconstruction of Human Lung Morphology Models from Magnetic Resonance Images
    T. B. Martonen (Experimental Toxicology Division, U.S. EPA, Research Triangle Park, NC 27709) and K. K. Isaacs (School of Public Health, University of North Carolina, Chapel Hill, NC 27514)

  16. Mathematical model of the human lungs during phonation

    NASA Astrophysics Data System (ADS)

    Meshcheryakov, R. V.

    2012-08-01

    Modeling of the human lungs during phonation is considered. The main relationships during physiological phonation process and air passage through vocal folds are established. Results of investigation are presented for statements of various types corresponding to different intonation patterns of the statement.

  17. COMPUTER SIMULATIONS OF HUMAN LUNG STRUCTURES FOR MEDICAL APPLICATIONS

    EPA Science Inventory

    Knowledge of the structure of the human lung has salient health effects applications. he clinical issues encompass (1) aerosol therapy, delivery of inhaled particles to enhance the efficacies of pharmacologic drugs, and (2) nuclear medicine, where planar gamma camera imaging, SPE...

  18. A novel P450-catalyzed transformation of the 2,2,6,6-tetramethyl piperidine moiety to a 2,2-dimethyl pyrrolidine in human liver microsomes: characterization by high resolution quadrupole-time-of-flight mass spectrometry and 1H-NMR.

    PubMed

    Yin, Wenji; Doss, George A; Stearns, Ralph A; Chaudhary, Ashok G; Hop, Cornelis E; Franklin, Ronald B; Kumar, Sanjeev

    2003-02-01

    We describe herein a novel metabolic fate of the 2,2,6,6-tetramethyl-piperidine (2,2,6,6-TMPi) moiety to a ring-contracted 2,2-dimethyl pyrrolidine (2,2-DMPy) in human liver microsomal incubations. The existence of this pathway was demonstrated for three compounds (I-III) of varied structures suggesting that this may be a general biotransformation reaction for the 2,2,6,6-TMPi moiety. The 2,2-DMPy metabolites formed in incubations of the three compounds with human liver microsomes were characterized by online high performance liquid chromatography coupled to a high resolution hybrid quadrupole-time-of-flight mass spectrometer. Suggested elemental composition obtained from accurate mass measurements of the molecular ions and fragment ions of the metabolites clearly indicated the loss of a mass equivalent to C(3)H(6) from the parent 2,2,6,6-TMPi functionality. Additional accurate tandem mass spectrometry data indicated that one of the original two gem-dimethyl groups was intact in the metabolite structure. Proof of a ring-contracted 2,2-DMPy structure was obtained using (1)H-NMR experiments on a metabolite purified from liver microsomal incubations, which showed only two geminal methyl groups, instead of four in the parent compound. Two-dimensional correlation spectroscopy and decoupling experiments established aliphatic protons arranged in a pyrrolidine ring pattern. The fact that the formation of 2,2-DMPy metabolites in human liver microsomes was NADPH-dependent suggested that this novel metabolic reaction was catalyzed by the cytochrome P450 (P450) enzyme(s). Immunoinhibition studies in human liver microsomal incubations using anti-P450 monoclonal antibodies and experiments with insect cell microsomes containing individually expressed recombinant human P450 isozymes indicated that multiple P450 isozymes were capable of catalyzing this novel metabolic transformation. PMID:12527703

  19. Modeling of the Nitric Oxide Transport in the Human Lungs

    PubMed Central

    Karamaoun, Cyril; Van Muylem, Alain; Haut, Benoît

    2016-01-01

    In the human lungs, nitric oxide (NO) acts as a bronchodilatator, by relaxing the bronchial smooth muscles and is closely linked to the inflammatory status of the lungs, owing to its antimicrobial activity. Furthermore, the molar fraction of NO in the exhaled air has been shown to be higher for asthmatic patients than for healthy patients. Multiple models have been developed in order to characterize the NO dynamics in the lungs, owing to their complex structure. Indeed, direct measurements in the lungs are difficult and, therefore, these models are valuable tools to interpret experimental data. In this work, a new model of the NO transport in the human lungs is proposed. It belongs to the family of the morphological models and is based on the morphometric model of Weibel (1963). When compared to models published previously, its main new features are the layered representation of the wall of the airways and the possibility to simulate the influence of bronchoconstriction (BC) and of the presence of mucus on the NO transport in lungs. The model is based on a geometrical description of the lungs, at rest and during a respiratory cycle, coupled with transport equations, written in the layers composing an airway wall and in the lumen of the airways. First, it is checked that the model is able to reproduce experimental information available in the literature. Second, the model is used to discuss some features of the NO transport in healthy and unhealthy lungs. The simulation results are analyzed, especially when BC has occurred in the lungs. For instance, it is shown that BC can have a significant influence on the NO transport in the tissues composing an airway wall. It is also shown that the relation between BC and the molar fraction of NO in the exhaled air is complex. Indeed, BC might lead to an increase or to a decrease of this molar fraction, depending on the extent of the BC and on the possible presence of mucus. This should be confirmed experimentally and might

  20. HT-2 toxin 4-glucuronide as new T-2 toxin metabolite: enzymatic synthesis, analysis, and species specific formation of T-2 and HT-2 toxin glucuronides by rat, mouse, pig, and human liver microsomes.

    PubMed

    Welsch, Tanja; Humpf, Hans-Ulrich

    2012-10-10

    Glucuronides of the mycotoxin T-2 toxin and its phase I metabolite HT-2 toxin are important phase II metabolites under in vivo and in vitro conditions. Since standard substances are essential for the direct quantitation of these glucuronides, a method for the enzymatic synthesis of T-2 and HT-2 toxin glucuronides employing liver microsomes was optimized. Structure elucidation by nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry revealed that besides T-2 toxin glucuronide and HT-2 toxin 3-glucuronide also the newly identified isomer HT-2 toxin 4-glucuronide was formed. Glucuronidation of T-2 and HT-2 toxin in liver microsomes of rat, mouse, pig, and human was compared and metabolites were analyzed directly by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). A distinct, species specific pattern of glucuronidation of T-2 and HT-2 toxin was observed with interesting interindividual differences. Until recently, glucuronides have frequently been analyzed indirectly by quantitation of the aglycone after enzymatic cleavage of the glucuronides by β-glucuronidase. Therefore, the hydrolysis efficiencies of T-2 and HT-2 toxin glucuronides using β-glucuronidases from Helix pomatia, bovine liver, and Escherichia coli were compared. PMID:22967261

  1. Nucleotide Excision Repair Is Not Induced in Human Embryonic Lung Fibroblasts Treated with Environmental Pollutants

    PubMed Central

    Rossner, Pavel; Spatova, Milada; Rossnerova, Andrea; Libalova, Helena; Schmuczerova, Jana; Milcova, Alena; Topinka, Jan; Sram, Radim J.

    2013-01-01

    The cellular response to genotoxic treatment depends on the cell line used. Although tumor cell lines are widely used for genotoxicity tests, the interpretation of the results may be potentially hampered by changes in cellular processes caused by malignant transformation. In our study we used normal human embryonic lung fibroblasts (HEL12469 cells) and tested their response to treatment with benzo[a]pyrene (B[a]P) and extractable organic matter (EOM) from ambient air particles <2.5 µm (PM2.5) collected in two Czech cities differing in levels and sources of air pollution. We analyzed multiple endpoints associated with exposure to polycyclic aromatic hydrocarbons (PAHs) including the levels of bulky DNA adducts and the nucleotide excision repair (NER) response [expression of XPE, XPC and XPA genes on the level of mRNA and proteins, unscheduled DNA synthesis (UDS)]. EOMs were collected in the winter and summer of 2011 in two Czech cities with different levels and sources of air pollution. The effects of the studied compounds were analyzed in the presence (+S9) and absence (–S9) of the rat liver microsomal S9 fraction. The levels of bulky DNA adducts were highest after treatment with B[a]P, followed by winter EOMs; their induction by summer EOMs was weak. The induction of both mRNA and protein expression was observed, with the most pronounced effects after treatment with B[a]P (–S9); the response induced by EOMs from both cities and seasons was substantially weaker. The expression of DNA repair genes was not accompanied by the induction of UDS activity. In summary, our results indicate that the tested compounds induced low levels of DNA damage and affected the expression of NER genes; however, nucleotide excision repair was not induced. PMID:23894430

  2. Aerosol Deposition in the Human Lung in Reduced Gravity

    PubMed Central

    2014-01-01

    Abstract The deposition of aerosol in the human lung occurs mainly through a combination of inertial impaction, gravitational sedimentation, and diffusion. For 0.5- to 5-μm-diameter particles and resting breathing conditions, the primary mechanism of deposition in the intrathoracic airways is sedimentation, and therefore the fate of these particles is markedly affected by gravity. Studies of aerosol deposition in altered gravity have mostly been performed in humans during parabolic flights in both microgravity (μG) and hypergravity (∼1.6G), where both total deposition during continuous aerosol mouth breathing and regional deposition using aerosol bolus inhalations were performed with 0.5- to 3-μm particles. Although total deposition increased with increasing gravity level, only peripheral deposition as measured by aerosol bolus inhalations was strongly dependent on gravity, with central deposition (lung depth<200 mL) being similar between gravity levels. More recently, the spatial distribution of coarse particles (mass median aerodynamic diameter≈5 μm) deposited in the human lung was assessed using planar gamma scintigraphy. The absence of gravity caused a smaller portion of 5-μm particles to deposit in the lung periphery than in the central region, where deposition occurred mainly in the airways. Indeed, 5-μm-diameter particles deposit either by inertial impaction, a mechanism most efficient in the large and medium-sized airways, or by gravitational sedimentation, which is most efficient in the distal lung. On the contrary, for fine particles (∼1 μm), both aerosol bolus inhalations and studies in small animals suggest that particles deposit more peripherally in μG than in 1G, beyond the reach of the mucociliary clearance system. PMID:24870702

  3. Sulf-2, a heparan sulfate endosulfatase, promotes human lung carcinogenesis

    PubMed Central

    Lemjabbar-Alaoui, Hassan; van Zante, Annemieke; Singer, Mark S.; Xue, Qing; Wang, Yang-Qing; Tsay, Durwin; He, Biao; Jablons, David M.; Rosen, Steven D.

    2009-01-01

    Heparan sulfate proteoglycans (HSPGs) bind to multiple growth factors/morphogens and regulate their signaling. 6-O-sulfation (6S) of glucosamine within HS-chains is critical for many of these ligand interactions. Sulf-1 and Sulf-2, which are extracellular neutral-pH sulfatases, provide a novel post-synthetic mechanism for regulation of HSPG function by removing 6S from intact HS-chains. The Sulfs can thereby modulate several signaling pathways, including the promotion of Wnt signaling. We found induction of SULF2 transcripts and Sulf-2 protein in human lung adenocarcinoma and squamous cell carcinoma, the two major classes of non-small cell lung cancers (NSCLC). We confirmed widespread Sulf-2 protein expression in tumor cells of 10/10 surgical specimens of human lung squamous carcinomas. We studied five Sulf-2+ NSCLC cell lines, including two which were derived by cigarette-smoke transformation of bronchial epithelial cells. shRNA-mediated Sulf-2 knockdown in these lines caused an increase in 6S on their cell surface and in parallel reversed their transformed phenotype in vitro, eliminated autocrine Wnt signaling, and strongly blunted xenograft tumor formation in nude mice. Conversely, forced Sulf-2 expression in non-malignant bronchial epithelial cells produced a partially transformed phenotype. Our findings support an essential role for Sulf-2 in lung cancer, the leading cancer killer. PMID:19855436

  4. Selective Toll-Like Receptor Expression in Human Fetal Lung

    PubMed Central

    Petrikin, Joshua E; Gaedigk, Roger; Leeder, J Steven; Truog, William E

    2010-01-01

    Toll-like receptors (TLRs) are critical components of the innate immune system, acting as pattern recognition molecules and triggering an inflammatory response. TLR associated gene products are of interest in modulating inflammatory related pulmonary diseases of the neonate. The ontogeny of TLR related genes in human fetal lung has not been previously described and could elucidate additional functions and identify strategies for attenuating the effects of fetal inflammation. We examined the expression of 84 TLR related genes on 23 human fetal lung samples from three groups with estimated ages of 60 (57-59d), 90 (89-91d), and 130 (117-154d) days. Using a false detection rate algorithm, we identified 32 genes displaying developmental regulation with TLR2 having the greatest up-regulation of TLR genes (9.2 fold increase) and TLR4 unchanged. We confirmed the TLR2 up-regulation by examining an additional 133 fetal lung tissue samples with a fluorogenic polymerase chain reaction assay (TaqMan®) and found an exponential best-fit curve over the time studied. The best-fit curve predicts a 6.1 fold increase from 60d to 130d. We conclude that TLR2 is developmentally expressed from the early pseudoglandular stage of lung development to the canalicular stage. PMID:20581745

  5. In vitro glucuronidation of the primary metabolite of 10-chloromethyl-11-demethyl-12-oxo-calanolide A by human liver microsomes and its interactions with UDP-glucuronosyltransferase substrates.

    PubMed

    Liu, Xin; Sheng, Li; Zhao, Manman; Mi, Jiaqi; Liu, Zhihao; Li, Yan

    2015-02-01

    F18 (10-chloromethyl-11-demethyl-12-oxo-calanolide), an analog of (+)-Calanolide A, is a novel small-molecule nonnucleoside reverse transcriptase inhibitor for the therapy of human immunodeficiency virus (HIV) infection. M3, the most abundant primary metabolite of F18 in human liver microsomes (HLMs) and rat liver microsomes (RLMs), is mainly excreted in bile as a glucuronide conjugate in rats after oral administration. The aim of this study was to identify the UDP glucuronosyltransferase (UGT) isoforms involved in the glucuronidation of M3 by HLMs and recombinant human UGTs and investigate the metabolic interactions of M3 with the substrates of UGTs in HLMs. As a result, UGT1A1 was the major isozyme responsible for the glucuronidation of M3, followed by UGT1A4, UGT1A9 and UGT2B7. M3 exhibited significant inhibition against UGT1A9 and UGT2B7 in both HLMs and recombinant human UGTs. In addition, M3 inhibited UGT1A9 catalyzed mycophenolic acid (MPA) glucuronidation with Ki of 0.39 μM, and M3 also inhibited the glucuronidation of 3'-azido-3'-deoxythymidine (AZT) by a "mixed-type" mechanism with Ki of 16.8 μM. The results suggest that UGT1A1 provides the major contribution to M3 glucuronidation in vitro and M3 has the potential to interact with xenobiotics and endogenous chemicals that are UGT1A9 and UGT 2B7 substrates. PMID:25760535

  6. Comparative Pathobiology of Environmentally Induced Lung Cancers in Humans and Rodents

    PubMed Central

    Pandiri, Arun

    2014-01-01

    Lung cancer is the number one cause of cancer-related deaths in humans worldwide. Environmental factors play an important role in the epidemiology of these cancers. Rodents are the most common experimental model to study human lung cancers and are frequently used in bioassays to identify environmental exposure hazards associated with lung cancer. Lung tumors in rodents are common, particularly in certain strains of mice. Rodent lung tumors are predominantly bronchioloalveolar carcinomas and usually follow a progressive continuum of hyperplasia to adenoma to carcinoma. Human lung cancers are phenotypically more diverse and broadly constitute 2 types: small cell lung cancers or non-small cell lung cancers. Rodent lung tumors resulting from exposure to environmental agents are comparable to certain adenocarcinomas that are a subset of human non-small cell lung cancers. Human pulmonary carcinomas differ from rodent lung tumors by exhibiting greater morphologic heterogeneity (encompassing squamous cell, neuroendocrine, mucinous, sarcomatoid, and multiple cell combinations), higher metastatic rate, higher stromal response, aggressive clinical behavior, and lack of a clear continuum of proliferative lesions. In spite of these differences, rodent lung tumors recapitulate several fundamental aspects of human lung tumor biology at the morphologic and molecular level especially in lung cancers resulting from exposure to environmental carcinogens. PMID:25351923

  7. Inhibition of hepatic microsomal carboxylesterase activity by paraoxon.

    PubMed

    Castle, M C

    1988-01-01

    A large number of therapeutic agents are esters of carboxylic acids and are thus substrates for microsomal carboxylesterase enzymes. These studies characterized the effects of the organophosphate compound, paraoxon, on the hydrolysis of several drug esters (procaine, chloramphenicol succinate, prednisolone succinate, lidocaine, procainamide and methylparaben) by microsomal preparations from guinea-pigs. These investigations demonstrate that carboxylesterase activity toward several drug esters is present in liver, lung and kidney. The liver is by far the major site of hydrolysis of these ester compounds. Since no hydrolysis was observed with the two amide esters, the hydrolysis of carboxylesters and amide esters appears to be mediated by different enzymes in the guinea-pig. At the substrate concentrations studied, the hydrolysis of methylparaben followed zero-order kinetics. When added to isolated microsomal preparations, paraoxon produced a dose-dependent inhibition of hydrolysis of all substrates. Administration of paraoxon to guinea-pigs prior to isolation of microsomes did not produce consistent effects with any substrate. Inhibition of ester hydrolysis was observed with some pretreatments, while either no change or increased hydrolysis was observed with other pretreatment regimens. PMID:3245748

  8. 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.

    PubMed

    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. PMID:25656918

  9. Generation of leukotrienes by purified human lung mast cells.

    PubMed Central

    MacGlashan, D W; Schleimer, R P; Peters, S P; Schulman, E S; Adams, G K; Newball, H H; Lichtenstein, L M

    1982-01-01

    Although mediator release from mast cells and basophils plays a central role in the pathogenesis of human allergic disease, biochemical studies have been restricted to rat peritoneal mast cells and basophilic leukemia cells because they could be easily purified. We have used two new techniques of cell separation to purify human lung mast cells to 98% homogeneity. Lung cell suspensions were obtained by dispersion of chopped lung tissue with proteolytic enzymes. Mast cells were then purified from the suspensions by countercurrent centrifugal elutriation and affinity chromatography. The purified mast cells released both histamine and slow-reacting substance of anaphylaxis (SRS-A) (leukotriene C and D) during stimulation with goat anti-human IgE antibody. Moreover, these preparations were able to generate significant quantities of SRS-A (32 +/- 7 x 10(-17) LTD mole-equivalents/mast cell) at all stages of purification, indicating that a secondary cell is not necessary for the antigen-induced release of SRS. Images PMID:7119113

  10. Activation of proto-oncogenes in human and mouse lung tumors

    SciTech Connect

    Reynolds, S.H.; Anderson, M.W. )

    1991-06-01

    Lung cancer is a leading cause of cancer-related deaths in several nations. Epidemiological studies have indicated that 85% of all lung cancer deaths and 30% of all cancer deaths in the US are associated with tobacco smoking. Various chemicals in tobacco smoke are thought to react with DNA and to ultimately yield heritable mutations. In an effort to understand the molecular mechanisms involved in lung tumorigenesis, the authors have analyzed proto-oncogene activation in a series of human lung tumors from smokers and spontaneously occurring and chemically induced lung tumors in mice. Approximately 86% of the human lung tumors and > 90% of the mouse lung tumors were found to contain activated oncogenes. ras Oncogenes activated by point mutations were detected in many of the human lung adenocarcinomas and virtually all of the mouse lung adenomas and adenocarcinomas. The mutation profiles of the activated K-ras genes detected in the chemically induced mouse lung tumors suggest that the observed mutations result from genotoxic effects of the chemicals. Comparison of the K-ras mutations observed in the human lung adenocarcinomas with mutation profiles observed in the mouse lung tumors suggest that bulky hydrophobic DNA adducts may be responsible for the majority of the mutations observed in the activated human K-ras genes. Other data indicate that approximately 20% of human lung tumors contain potentially novel transforming genes that may also be targets for mutagens in cigarette smoke.

  11. Sex-specific differences in hyperoxic lung injury in mice: Implications for acute and chronic lung disease in humans

    SciTech Connect

    Lingappan, Krithika; Jiang, Weiwu; Wang, Lihua; Couroucli, Xanthi I.; Barrios, Roberto; Moorthy, Bhagavatula

    2013-10-15

    Sex-specific differences in pulmonary morbidity in humans are well documented. Hyperoxia contributes to lung injury in experimental animals and humans. The mechanisms responsible for sex differences in the susceptibility towards hyperoxic lung injury remain largely unknown. In this investigation, we tested the hypothesis that mice will display sex-specific differences in hyperoxic lung injury. Eight week-old male and female mice (C57BL/6J) were exposed to 72 h of hyperoxia (FiO{sub 2} > 0.95). After exposure to hyperoxia, lung injury, levels of 8-iso-prostaglandin F{sub 2} alpha (8-iso-PGF 2α) (LC–MS/MS), apoptosis (TUNEL) and inflammatory markers (suspension bead array) were determined. Cytochrome P450 (CYP)1A expression in the lung was assessed using immunohistochemistry and western blotting. After exposure to hyperoxia, males showed greater lung injury, neutrophil infiltration and apoptosis, compared to air-breathing controls than females. Pulmonary 8-iso-PGF 2α levels were higher in males than females after hyperoxia exposure. Sexually dimorphic increases in levels of IL-6 (F > M) and VEGF (M > F) in the lungs were also observed. CYP1A1 expression in the lung was higher in female mice compared to males under hyperoxic conditions. Overall, our results support the hypothesis that male mice are more susceptible than females to hyperoxic lung injury and that differences in inflammatory and oxidative stress markers contribute to these sex-specific dimorphic effects. In conclusion, this paper describes the establishment of an animal model that shows sex differences in hyperoxic lung injury in a temporal manner and thus has important implications for lung diseases mediated by hyperoxia in humans. - Highlights: • Male mice were more susceptible to hyperoxic lung injury than females. • Sex differences in inflammatory markers were observed. • CYP1A expression was higher in females after hyperoxia exposure.

  12. Autoradiographic localization of beta-adrenoceptors in asthmatic human lung

    SciTech Connect

    Spina, D.; Rigby, P.J.; Paterson, J.W.; Goldie, R.G. )

    1989-11-01

    The autoradiographic distribution and density of beta-adrenoceptors in human non-diseased and asthmatic bronchi were investigated using (125I)iodocyanopindolol (I-CYP). Analysis of the effects of the beta-adrenoceptor antagonists on I-CYP binding demonstrated that betaxolol (20 nM, beta 1-selective) had no significant effect on specific grain density in either nonasthmatic or asthmatic human bronchus, whereas ICI-118551 (20 nM, beta 2-selective) inhibited I-CYP binding by 85 +/- 9% and 89 +/- 3%, respectively. Thus, homogeneous populations of beta 2-adrenoceptors existed in bronchi from both sources. Large populations of beta-adrenoceptors were localized to the bronchial epithelium, submucosal glands, and airway smooth muscle. Asthmatic bronchial tissue featured epithelial damage with exfoliated cells associated with luminal mucus plugs. A thickened basement membrane and airway smooth muscle hyperplasia were also evident. High levels of specific I-CYP binding were also detected over asthmatic bronchial smooth muscle, as assessed by autoradiography and quantitation of specific grain densities. Isoproterenol and fenoterol were 10- and 13-fold less potent, respectively, in bronchi from asthmatic lung than in those from nonasthmatic lung. However, this attenuated responsiveness to beta-adrenoceptor agonists was not caused by reduced beta-adrenoceptor density in asthmatic airways. A defect may exist in the coupling between beta-adrenoceptors and postreceptor mechanisms in severely asthmatic lung.

  13. 3-D segmentation of human sternum in lung MDCT images.

    PubMed

    Pazokifard, Banafsheh; Sowmya, Arcot

    2013-01-01

    A fully automatic novel algorithm is presented for accurate 3-D segmentation of the human sternum in lung multi detector computed tomography (MDCT) images. The segmentation result is refined by employing active contours to remove calcified costal cartilage that is attached to the sternum. For each dataset, costal notches (sternocostal joints) are localized in 3-D by using a sternum mask and positions of the costal notches on it as reference. The proposed algorithm for sternum segmentation was tested on 16 complete lung MDCT datasets and comparison of the segmentation results to the reference delineation provided by a radiologist, shows high sensitivity (92.49%) and specificity (99.51%) and small mean distance (dmean=1.07 mm). Total average of the Euclidean distance error for costal notches positioning in 3-D is 4.2 mm. PMID:24110446

  14. An ex vivo human lung model for ultrasound-guided high-intensity focused ultrasound therapy using lung flooding.

    PubMed

    Wolfram, Frank; Reichenbach, Jürgen R; Lesser, Thomas G

    2014-03-01

    The usability of an ex vivo human lung model for ablation of lung cancer tissue with high-intensity focused ultrasound (HIFU) is described. Lung lobes were flooded with saline, with no gas remaining after complete atelectasis. The tumor was delineated sono-morphologically. Speed of sound, tissue density and ultrasound attenuation were measured for flooded lung and different pulmonary cancer tissues. The acoustic impedance of lung cancer tissue (1.6-1.9 mega-Rayleighs) was higher than that of water, as was its attenuation coefficient (0.31-0.44 dB/cm/MHz) compared with that of the flooded lung (0.12 dB/cm/MHz). After application of HIFU, the temperature in centrally located lung cancer surrounded by the flooded lung increased as high as 80°C, which is sufficient for treatment. On the basis of these preliminary results, ultrasound-guided HIFU ablation of lung cancer, by lung flooding with saline, appears feasible and should be explored in future clinical studies. PMID:24412177

  15. Three-dimensional scaffolds of acellular human and porcine lungs for high throughput studies of lung disease and regeneration

    PubMed Central

    Wagner, Darcy E.; Bonenfant, Nicholas R.; Sokocevic, Dino; DeSarno, Michael; Borg, Zachary; Parsons, Charles; Brooks, Elice M.; Platz, Joseph; Khalpey, Zain; Hoganson, David M.; Deng, Bin; Lam, Ying Wai; Oldinski, Rachael A.; Ashikaga, Takamaru; Weiss, Daniel J.

    2014-01-01

    Acellular scaffolds from complex whole organs such as lung are being increasingly studied for ex vivo organ generation and for in vitro studies of cell-extracellular matrix interactions. We have established effective methods for efficient de- and recellularization of large animal and human lungs including techniques which allow multiple small segments (∼1–3cm3) to be excised that retain 3-dimensional lung structure. Coupled with the use of a synthetic pleural coating, cells can be selectively physiologically inoculated via preserved vascular and airway conduits. Inoculated segments can be further sliced for high throughput studies. Further, we demonstrate thermography as a powerful noninvasive technique for monitoring perfusion decellularization and for evaluating preservation of vascular and airway networks following human and porcine lung decellularization. Collectively, these techniques are a significant step forward as they allow high throughput in vitro studies from a single lung or lobe in a more biologically relevant, three-dimensional acellular scaffold. PMID:24411675

  16. Sex-specific Differences in Hyperoxic Lung Injury in Mice: Implications for Acute and Chronic Lung Disease in Humans

    PubMed Central

    Lingappan, Krithika; Jiang, Weiwu; Wang, Lihua; Couroucli, Xanthi I.; Barrios, Roberto; Moorthy, Bhagavatula

    2014-01-01

    Sex-specific differences in pulmonary morbidity in humans are well documented. Hyperoxia contributes to lung injury in experimental animals and humans. The mechanisms responsible for sex differences in the susceptibility towards hyperoxic lung injury remain largely unknown. In this investigation, we tested the hypothesis that mice will display sex-specific differences in hyperoxic lung injury. Eight week-old male and female mice (C57BL/6J) were exposed to 72 h of hyperoxia (FiO2>0.95). After exposure to hyperoxia, lung injury, levels of 8-iso-prostaglandin F2 alpha (8-iso-PGF 2α) (LC-MS/MS), apoptosis (TUNEL) and inflammatory markers (suspension bead array) were determined. CytochromeP450 (CYP)1A expression in the lung was assessed using immunohistochemistry and western blotting. After exposure to hyperoxia, males showed greater lung injury, neutrophil infiltration and apoptosis, compared to air-breathing controls than females. Pulmonary 8-iso-PGF 2α levels were higher in males than females after hyperoxia exposure. Sexually dimorphic increases in levels of IL-6 (F>M) and VEGF (M>F) in the lungs were also observed. CYP1A1 expression in the lung was higher in female mice compared to males under hyperoxic conditions. Overall, our results support the hypothesis that male mice are more susceptible than females to hyperoxic lung injury and that differences in inflammatory and oxidative stress markers contribute to these sex-specific dimorphic effects. In conclusion, this paper describes the establishment of an animal model that shows sex differences in hyperoxic lung injury in a temporal manner and thus has important implications for lung diseases mediated by hyperoxia in humans. PMID:23792423

  17. Cellular morphometry of the bronchi of human and dog lungs

    SciTech Connect

    Robbins, E.S.

    1991-09-01

    One hundred and forty-seven bronchial samples (generations 3--6) from 66 patients (62 usable; 36 female, 26 male; median age 61) have been dissected by generation from fixed surgical lung specimens obtained after the removal of pathological lesions. In addition, one hundred and fifty-six mongol dog bronchi (generations 2--6) dissected from different lobes of 26 dog lungs have also been similarly prepared. One hundred and twenty-seven human samples have been completely processed for electron microscopy and have yielded 994 electron micrographs of which 655 have been entered into the Computerized Stereological Analysis System (COSAS) and been used for the measurement of the distances of basal and mucous cell nuclei to the epithelial free surface. Similarly 328 micrographs of dog epithelium from 33 bronchial samples have been used to measure the distances of basal and mucous cell nuclei to the epithelial free surface and have been entered into COSAS. Using the COSAS planimetry program, we continue to expand our established data bases which describe the volume density and nuclear numbers per electron micrograph for 5 cell types of the human bronchial epithelial lining of men and women, as well as smokers, non-smokers and ex-smokers and similar parameters for the same 5 epithelial cell types of dog bronchi. Our micrographs of human bronchial epithelium have allowed us to analyze the recent suggestion that the DNA of lymphocytes may be subject to significant damage from Rn progeny while within the lung. Since the last progress report three papers have been submitted for publication. 17 refs., 4 tabs.

  18. The HSP90 Inhibitor Ganetespib Radiosensitizes Human Lung Adenocarcinoma Cells.

    PubMed

    Gomez-Casal, Roberto; Bhattacharya, Chitralekha; Epperly, Michael W; Basse, Per H; Wang, Hong; Wang, Xinhui; Proia, David A; Greenberger, Joel S; Socinski, Mark A; Levina, Vera

    2015-01-01

    The molecular chaperone HSP90 is involved in stabilization and function of multiple client proteins, many of which represent important oncogenic drivers in NSCLC. Utilization of HSP90 inhibitors as radiosensitizing agents is a promising approach. The antitumor activity of ganetespib, HSP90 inhibitor, was evaluated in human lung adenocarcinoma (AC) cells for its ability to potentiate the effects of IR treatment in both in vitro and in vivo. The cytotoxic effects of ganetespib included; G2/M cell cycle arrest, inhibition of DNA repair, apoptosis induction, and promotion of senescence. All of these antitumor effects were both concentration- and time-dependent. Both pretreatment and post-radiation treatment with ganetespib at low nanomolar concentrations induced radiosensitization in lung AC cells in vitro. Ganetespib may impart radiosensitization through multiple mechanisms: such as down regulation of the PI3K/Akt pathway; diminished DNA repair capacity and promotion of cellular senescence. In vivo, ganetespib reduced growth of T2821 tumor xenografts in mice and sensitized tumors to IR. Tumor irradiation led to dramatic upregulation of β-catenin expression in tumor tissues, an effect that was mitigated in T2821 xenografts when ganetespib was combined with IR treatments. These data highlight the promise of combining ganetespib with IR therapies in the treatment of AC lung tumors. PMID:26010604

  19. The HSP90 Inhibitor Ganetespib Radiosensitizes Human Lung Adenocarcinoma Cells

    PubMed Central

    Gomez-Casal, Roberto; Bhattacharya, Chitralekha; Epperly, Michael W.; Basse, Per H.; Wang, Hong; Wang, Xinhui; Proia, David A.; Greenberger, Joel S.; Socinski, Mark A.; Levina, Vera

    2015-01-01

    The molecular chaperone HSP90 is involved in stabilization and function of multiple client proteins, many of which represent important oncogenic drivers in NSCLC. Utilization of HSP90 inhibitors as radiosensitizing agents is a promising approach. The antitumor activity of ganetespib, HSP90 inhibitor, was evaluated in human lung adenocarcinoma (AC) cells for its ability to potentiate the effects of IR treatment in both in vitro and in vivo. The cytotoxic effects of ganetespib included; G2/M cell cycle arrest, inhibition of DNA repair, apoptosis induction, and promotion of senescence. All of these antitumor effects were both concentration- and time-dependent. Both pretreatment and post-radiation treatment with ganetespib at low nanomolar concentrations induced radiosensitization in lung AC cells in vitro. Ganetespib may impart radiosensitization through multiple mechanisms: such as down regulation of the PI3K/Akt pathway; diminished DNA repair capacity and promotion of cellular senescence. In vivo, ganetespib reduced growth of T2821 tumor xenografts in mice and sensitized tumors to IR. Tumor irradiation led to dramatic upregulation of β-catenin expression in tumor tissues, an effect that was mitigated in T2821 xenografts when ganetespib was combined with IR treatments. These data highlight the promise of combining ganetespib with IR therapies in the treatment of AC lung tumors. PMID:26010604

  20. Human Immunodeficiency Virus Infection and Host Defense in the Lungs.

    PubMed

    Charles, Tysheena P; Shellito, Judd E

    2016-04-01

    Immunosuppression associated with human immunodeficiency virus (HIV) infection impacts all components of host defense against pulmonary infection. Cells within the lung have altered immune function and are important reservoirs for HIV infection. The host immune response to infected lung cells further compromises responses to a secondary pathogenic insult. In the upper respiratory tract, mucociliary function is impaired and there are decreased levels of salivary immunoglobulin A. Host defenses in the lower respiratory tract are controlled by alveolar macrophages, lymphocytes, and polymorphonuclear leukocytes. As HIV infection progresses, lung CD4 T cells are reduced in number causing a lack of activation signals from CD4 T cells and impaired defense by macrophages. CD8 T cells, on the other hand, are increased in number and cause lymphocytic alveolitis. Specific antibody responses by B-lymphocytes are decreased and opsonization of microorganisms is impaired. These observed defects in host defense of the respiratory tract explain the susceptibility of HIV-infected persons for oropharyngeal candidiasis, bacterial pneumonia, Pneumocystis pneumonia, and other opportunistic infections. PMID:26974294

  1. GENETIC ASSOCIATION BETWEEN HUMAN CHITINASES AND LUNG FUNCTION IN COPD

    PubMed Central

    Aminuddin, F.; Akhabir, L.; Stefanowicz, D.; Paré, P.D.; Connett, J.E.; Anthonisen, N.R.; Fahy, J.V.; Seibold, M.A.; Burchard, E.G.; Eng, C.; Gulsvik, A.; Bakke, P.; Cho, M. H.; Litonjua, A.; Lomas, D.A.; Anderson, W. H.; Beaty, T.H.; Crapo, J.D.; Silverman, E.K.; Sandford, A.J.

    2013-01-01

    Two primary chitinases have been identified in humans – acid mammalian chitinase (AMCase) and chitotriosidase (CHIT1). Mammalian chitinases have been observed to affect the host’s immune response. The aim of this study was to test for association between genetic variation in the chitinases and phenotypes related to Chronic Obstructive Pulmonary Disease (COPD). Polymorphisms in the chitinase genes were selected based on previous associations with respiratory diseases. Polymorphisms that were associated with lung function level or rate of decline in the Lung Health Study (LHS) cohort were analyzed for association with COPD affection status in four other COPD case-control populations. Chitinase activity and protein levels were also related to genotypes. In the Caucasian LHS population, the baseline forced expiratory volume in one second (FEV1) was significantly different between the AA and GG genotypic groups of the AMCase rs3818822 polymorphism. Subjects with the GG genotype had higher AMCase protein and chitinase activity compared with AA homozygotes. For CHIT1 rs2494303, a significant association was observed between rate of decline in FEV1 and the different genotypes. In the African American LHS population, CHIT1 rs2494303 and AMCase G339T genotypes were associated with rate of decline in FEV1. Although a significant effect of chitinase gene alleles was found on lung function level and decline in the LHS, we were unable to replicate the associations with COPD affection status in the other COPD study groups. PMID:22200767

  2. Organoids as a model system for studying human lung development and disease.

    PubMed

    Nadkarni, Rohan R; Abed, Soumeya; Draper, Jonathan S

    2016-05-01

    The lung is a complex organ comprising multiple cell types that perform a variety of vital processes, including immune defense and gas exchange. Diseases of the lung, such as chronic obstructive pulmonary disease, asthma and lung cancer, together represent one of the largest causes of patient suffering and mortality. Logistical barriers that hamper access to embryonic, normal adult or diseased lung tissue currently hinder the study of lung disease. In vitro lung modeling represents an attractive and accessible avenue for investigating lung development, function and disease pathology, but accurately modeling the lung in vitro requires a system that recapitulates the structural features of the native lung. Organoids are stem cell-derived three-dimensional structures that are supported by an extracellular matrix and contain multiple cell types whose spatial arrangement and interactions mimic those of the native organ. Recently, organoids representative of the respiratory system have been generated from adult lung stem cells and human pluripotent stem cells. Ongoing studies are showing that organoids may be used to model human lung development, and can serve as a platform for interrogating the function of lung-related genes and signalling pathways. In a therapeutic context, organoids may be used for modeling lung diseases, and as a platform for screening for drugs that alleviate respiratory disease. Here, we summarize the organoid-forming capacity of respiratory cells, current lung organoid technologies and their potential use in future therapeutic applications. PMID:26721435

  3. Radiographic Comparison of Human Lung Shape During Normal Gravity and Weightlessness

    NASA Technical Reports Server (NTRS)

    Michels, D. B.; Friedman, P. J.; West, J. B.

    1979-01-01

    Chest radiographs in five seated normal volunteers at 1 G and 0 G were made with a view toward comparing human lung shape during normal gravity and weightlessness. Lung shape was assessed by measuring lung heights and widths in upper, middle and lower lung regions. No significant differences were found between any of the 1-G and 0-G measurements, although there was a slight tendency for the lung to become shorter and wider at 0 G. The evidence that gravity causes regional differences in ventilation by direct action on the lung is consistent with the theoretical analysis of West and Matthews (1972).

  4. Benzylmorpholine Analogs as Selective Inhibitors of Lung Cytochrome P450 2A13 for the Chemoprevention of Lung Cancer in Tobacco Users

    PubMed Central

    Blake, Linda C.; Roy, Anuradha; Neul, David; Schoenen, Frank J.; Aubé, Jeffrey; Scott, Emily E.

    2013-01-01

    Purpose 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), one of the most prevalent and procarcinogenic compounds in tobacco, is bioactivated by respiratory cytochrome P450 (CYP) 2A13, forming DNA adducts and initiating lung cancer. CYP2A13 inhibition offers a novel strategy for chemoprevention of tobacco-associated lung cancer. Methods Twenty-four analogs of a 4-benzylmorpholine scaffold identified by high throughput screening were evaluated for binding and inhibition of both functional human CYP2A enzymes, CYP2A13 and the 94%-identical hepatic CYP2A6, whose inhibition is undesirable. Thus, selectivity is the major challenge in compound design. Results A key feature resulting in CYP2A13-selective binding and inhibition was substitution at the benzyl ortho position, with three analogs being >25-fold selective for CYP2A13 over CYP2A6. Conclusions Two such analogs were negative for genetic and hERG toxicities and metabolically stable in human lung microsomes, but displayed rapid metabolism in human liver and in mouse and rat lung and liver microsomes, likely due to CYP2B-mediated degradation. A specialized knockout mouse mimicking the human lung demonstrates compound persistence in lung and provides an appropriate test model. Compound delivered by inhalation may be effective in the lung but rapidly cleared otherwise, limiting systemic exposure. PMID:23756756

  5. Cellular morphometry of the bronchi of human and dog lungs

    SciTech Connect

    Robbins, E.S.

    1991-03-01

    One hundred and thirty-one bronchial samples from 62 patients have been dissected by generation from fixed surgical lung specimens obtained after the removal of pathological lesions. Complete patient records including occupational and smoking histories, as well as possible exposure to radon, are obtained. In addition, one hundred and sixty-two mongol dog bronchi dissected from different lobes of 23 dog lungs have also been similarly prepared. Ninety-four human samples have been completely processed for electron microscopy and have yielded 994 electron micrographs of which 532 have been entered into the Computerized Stereological Analysis System (COSAS) and been used for the measurement of the distances of basal and mucous cell nuclei to the epithelial free surface. Similarly 240 micrographs of dog epithelium from 31 bronchial samples have been entered into COSAS. We have, using the COSAS planimetry program, established data bases which describe the volume density and nuclear numbers per electron micrograph for 5 cell types of the human bronchial epithelial lining of men and women, as well as smokers, non-smokers and ex-smokers and similar parameters for the epithelial cell types of dog bronchi. The data are being used to develop weighting factors for dosimetry and radon risk analysis. 26 refs., 7 figs., 4 tabs.

  6. Cellular morphometry of the bronchi of human and dog lungs

    SciTech Connect

    Robbins, E.S.

    1990-09-01

    One hundred and twenty one bronchial samples from 58 patients (54 useable; 32 female, 22 male; median age 61) have been dissected by generation from fixed surgical lung specimens obtained after the removal of pathological lesions. Complete patient records including occupational and smoking histories, as well as possible exposure to radon, are being kept. In addition, mongol dog bronchi dissected from different lobes of 23 dog lungs have been used to establish protocols. Ninety human samples have been completely processed for electron microscopy and have yielded 913 electron micrographs of which 471 have been entered into the Computerized Stereological Analysis System (COSAS) and used for the measurement of the distances of basal and mucous cell nuclei to the epithelial free surface. We have, using the COSAS planimetry program, established a small data base which describes the volume density and nuclear numbers per electron micrograph for 5 cell types of the human bronchial epithelial lining of men and women, as well as smokers, nonsmokers and ex-smokers. The data is being used to develop weighting factors for dosimetry and radon risk analysis. The electron micrographs of dog bronchial epithelium are unanalyzed as yet. 4 figs., 2 tabs.

  7. Cellular morphometry of the bronchi of human and dog lungs

    SciTech Connect

    Robbins, E.S.

    1992-09-01

    Quantitative data of the human bronchial epithelial cells at possible risk for malignant transformation in lung cancer is crucial for accurate radon dosimetry and risk analysis. The locations and other parameters of the nuclei which may be damaged by [alpha] particles must be determined and compared in different airway generations, among smokers, non-smokers and ex-smokers, between men and women and in people of different ages. This proposal includes extended morphometric studies on electron micrographs of human epithelium of defined airway generations and in parallel on electron micrographs of the dog bronchial lining. The second part of this proposal describes studies to quantitate the cycling bronchial epithelial population(s) using proliferation markers and immunocytochemistry on frozen and paraffin sections and similar labeling of isolated bronchial epithelial cells sorted flow cytometry.

  8. Physiological and biochemical markers of alveolar epithelial barrier dysfunction in perfused human lungs

    PubMed Central

    Frank, James A.; Briot, Raphael; Lee, Jae Woo; Ishizaka, Akitoshi; Uchida, Tokujiro; Matthay, Michael A.

    2009-01-01

    To study air space fluid clearance (AFC) under conditions that resemble the clinical setting of pulmonary edema in patients, we developed a new perfused human lung preparation. We measured AFC in 20 human lungs rejected for transplantation and determined the contribution of AFC to lung fluid balance. AFC was then compared with air space and perfusate levels of a biological marker of epithelial injury. The majority of human lungs rejected for transplant had intact basal (75%) and β2-adrenergic agonist-stimulated (70%) AFC. For lungs with both basal and stimulated AFC, the basal AFC rate was 19 ± 10%/h, and the β2-adrenergic-stimulated AFC rate was 43 ± 13%/h. Higher rates of AFC were associated with less lung weight gain (Pearson coefficient −0.90, P < 0.0001). Air space and perfusate levels of the type I pneumocyte marker receptor for advanced glycation end products (RAGE) were threefold and sixfold higher, respectively, in lungs without basal AFC compared with lungs with AFC (P < 0.05). These data show that preserved AFC is a critical determinant of favorable lung fluid balance in the perfused human lung, raising the possibility that β2-agonist therapy to increase edema fluid clearance may be of value for patients with acute lung injury and pulmonary edema. Also, although additional studies are needed, a biological marker of alveolar epithelial injury may be useful clinically in predicting preserved AFC. PMID:17351061

  9. Decreased Laminin Expression by Human Lung Epithelial Cells and Fibroblasts Cultured in Acellular Lung Scaffolds from Aged Mice

    PubMed Central

    Godin, Lindsay M.; Sandri, Brian J.; Wagner, Darcy E.; Meyer, Carolyn M.; Price, Andrew P.; Akinnola, Ifeolu; Weiss, Daniel J.; Panoskaltsis-Mortari, Angela

    2016-01-01

    The lung changes functionally and structurally with aging. However, age-related effects on the extracellular matrix (ECM) and corresponding effects on lung cell behavior are not well understood. We hypothesized that ECM from aged animals would induce aging-related phenotypic changes in healthy inoculated cells. Decellularized whole organ scaffolds provide a powerful model for examining how ECM cues affect cell phenotype. The effects of age on ECM composition in both native and decellularized mouse lungs were assessed as was the effect of young vs old acellular ECM on human bronchial epithelial cells (hBECs) and lung fibroblasts (hLFs). Native aged (1 year) lungs demonstrated decreased expression of laminins α3 and α4, elastin and fibronectin, and elevated collagen, compared to young (3 week) lungs. Proteomic analyses of decellularized ECM demonstrated similar findings, and decellularized aged lung ECM contained less diversity in structural proteins compared to young ECM. When seeded in old ECM, hBECs and hLFs demonstrated lower gene expression of laminins α3 and α4, respectively, as compared to young ECM, paralleling the laminin deficiency of aged ECM. ECM changes appear to be important factors in potentiating aging-related phenotypes and may provide clues to mechanisms that allow for aging-related lung diseases. PMID:26954258

  10. Stem cells--potential for repairing damaged lungs and growing human lungs for transplant.

    PubMed

    Bishop, Anne E; Rippon, Helen J

    2006-08-01

    Repair or regeneration of defective lung epithelium would be of great therapeutic potential. It is estimated by the British Lung Foundation that 1 in 7 people in the UK is affected by a lung disease and that 1 in 4 admissions to children's wards are as a result of respiratory problems. Potential cellular sources for the regeneration of lung tissue in vivo or lung tissue engineering in vitro include endogenous pulmonary epithelial stem cells, extrapulmonary circulating stem cells and embryonic stem cells. This article discusses the potential role of each of these stem cell types in future approaches to the treatment of lung injury and disease. PMID:16856797

  11. Clinical grade allogeneic human mesenchymal stem cells restore alveolar fluid clearance in human lungs rejected for transplantation

    PubMed Central

    Curley, G. F.; Hamid, U. I.; Laffey, J. G.; Abbott, J.; McKenna, D. H.; Fang, X.; Matthay, M. A.; Lee, J. W.

    2014-01-01

    The lack of suitable donors for all solid-organ transplant programs is exacerbated in lung transplantation by the low utilization of potential donor lungs, due primarily to donor lung injury and dysfunction, including pulmonary edema. The current studies were designed to determine if intravenous clinical-grade human mesenchymal stem (stromal) cells (hMSCs) would be effective in restoring alveolar fluid clearance (AFC) in the human ex vivo lung perfusion model, using lungs that had been deemed unsuitable for transplantation and had been subjected to prolonged ischemic time. The human lungs were perfused with 5% albumin in a balanced electrolyte solution and oxygenated with continuous positive airway pressure. Baseline AFC was measured in the control lobe and if AFC was impaired (defined as <10%/h), the lungs received either hMSC (5 × 106 cells) added to the perfusate or perfusion only as a control. AFC was measured in a different lung lobe at 4 h. Intravenous hMSC restored AFC in the injured lungs to a normal level. In contrast, perfusion only did not increase AFC. This positive effect on AFC was reduced by intrabronchial administration of a neutralizing antibody to keratinocyte growth factor (KGF). Thus, intravenous allogeneic hMSCs are effective in restoring the capacity of the alveolar epithelium to remove alveolar fluid at a normal rate, suggesting that this therapy may be effective in enhancing the resolution of pulmonary edema in human lungs deemed clinically unsuitable for transplantation. PMID:24532289

  12. Extensive exchange of rat liver microsomal phospholipids.

    PubMed

    Zilversmit, D B; Hughes, M E

    1977-08-15

    Liver microsomal fractions were prepared from rats injected with a single dose of choline [14C]methylchloride or with single or multiple doses of 32Pi. Exchangeability of microsomal phospholipids was determined by incubation with an excess of mitochondria and phospholipid exchange proteins derived from beef heart, beef liver or rat liver. Labeled phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol were found to act as a single pool and were 85--95% exchangeable in 1--2h. High latencies of mannose-6-phosphate phosphohydrolase activities and impermeability of microsomes to EDTA proved that phospholipid exchange proteins did not have access to the intracisternal space. If microsomal membranes are largely composed of phospholipid bilayers, the experiments suggest that one or more of the phospholipid classes in microsomal membranes undergo rapid translocation between the inner and outer portions of the bilayer. PMID:889827

  13. Potential effect of Olea europea leaves, Sonchus oleraceus leaves and Mangifera indica peel extracts on aromatase activity in human placental microsomes and CYP19A1 expression in MCF-7 cell line: Comparative study.

    PubMed

    Shaban, N Z; Hegazy, W A; Abdel-Rahman, S M; Awed, O M; Khalil, S A

    2016-01-01

    Aromatase inhibitors (AIs) provide novel approaches to the adjuvant therapy for postmenopausal women with estrogen-receptor-positive (ER+) breast cancers. In this study, different plant extracts from Olea europaea leaves (OLE), Sonchus oleraceus L. (SOE) and Mangifera indica peels (MPE) were prepared to identify phytoconstituents and measure antioxidant capacities. The effects of these three extracts on aromatase activity in human placental microsomes were evaluated. Additionally, the effects of these extracts on tissue-specific promoter expression of CYP19A1 gene in cell culture model (MCF-7) were assessed using qRT-PCR. Results showed a concentration-dependent decrease in aromatase activity after treatment with OLE and MPE, whereas, SOE showed a biphasic effect. The differential effects of OLE, SOE and MPE on aromatase expression showed that OLE seems to be the most potent suppressor followed by SOE and then MPE. These findings indicate that OLE has effective inhibitory action on aromatase at both the enzymatic and expression levels, in addition to its cytotoxic effect against MCF-7 cells. Also, MPE may be has the potential to be used as a tissue-specific aromatase inhibitor (selective aromatase inhibitor) and it may be promising to develop a new therapeutic agent against ER+ breast cancer. PMID:27585256

  14. Effect of buffer conditions on CYP2C8-mediated paclitaxel 6α-hydroxylation and CYP3A4-mediated triazolam α- and 4-hydroxylation by human liver microsomes.

    PubMed

    Kudo, Toshiyuki; Ozaki, Yuya; Kusano, Tomomi; Hotta, Eri; Oya, Yuka; Komatsu, Seina; Goda, Hitomi; Ito, Kiyomi

    2016-01-01

    1. Buffer conditions in in vitro metabolism studies using human liver microsomes (HLM) have been reported to affect the metabolic activities of several cytochrome P450 (CYP) isozymes in different ways, although there are no reports about the dependence of CYP2C8 activity on buffer conditions. 2. The present study investigated the effect of buffer components (phosphate or Tris-HCl) and their concentration (10-200 mM) on the CYP2C8 and CYP3A4 activities of HLM, using paclitaxel and triazolam, respectively, as marker substrates. 3. The Km (or S50) and Vmax values for both paclitaxel 6α-hydroxylation and triazolam α- and 4-hydroxylation, estimated by fitting analyses based on the Michaelis-Menten or Hill equation, greatly depended on the buffer components and their concentration. 4. The CLint values in phosphate buffer were 1.2-3.0-fold (paclitaxel) or 3.1-6.4-fold (triazolam) higher than in Tris-HCl buffer at 50-100 mM. These values also depended on the buffer concentration, with a maximum 2.3-fold difference observed between 50 and 100 mM which are both commonly used in drug metabolism studies. 5. These findings suggest the necessity for optimization of the buffer conditions in the quantitative evaluation of metabolic clearances, such as in vitro-in vivo extrapolation and also estimating the contribution of a particular enzyme in drug metabolism. PMID:26290405

  15. A model of ventilation of the healthy human lung.

    PubMed

    Steimle, K L; Mogensen, M L; Karbing, D S; Bernardino de la Serna, J; Andreassen, S

    2011-02-01

    This paper presents a model of the lung mechanics which simulates the pulmonary alveolar ventilation. The model includes aspects of: the alveolar geometry; pressure due to the chest wall; pressure due to surface tension determined by surfactant activity; pressure due to lung tissue elasticity; and pressure due to the hydrostatic effects of the lung tissue and blood. The cross-sectional area of the lungs in the supine position derived from computed tomography is used to construct a horizontally layered model, which simulates heterogeneous ventilation distribution from the non-dependent to the dependent layers of the lungs. The model is in agreement with experimentally measured hysteresis of the pressure-volume curve of the lungs, static lung compliance, changes in lung depth during breathing and density distributions at total lung capacity (TLC) and residual volume (RV). In the dependent layers of the lungs, alveolar collapse may occur at RV, depending on the assumptions concerning lung tissue elasticity at very low alveolar volumes. The model simulations showed that ventilation increased with depth in the lungs, although not as pronounced as observed experimentally. The model simulates alveolar ventilation including all of the mentioned components of the respiratory system and to be validated against all the above mentioned experimental data. PMID:20655612

  16. Human lung cancer-derived microparticles enhanced angiogenesis and growth of hepatoma cells in rodent lung parenchyma

    PubMed Central

    Ko, Sheung-Fat; Hsu, Shu-Yuan; Chen, Chih-Hung; Sung, Pei-Hsun; Zhen, Meng-Shen TongYen-Yi; Chen, Yi-Ling; Huang, Tien-Hung; Chen, Sheng-Yi; Kao, Gour-Shenq; Chen, Hong-Hwa; Chang, Chia-Lo; Sun, Cheuk-Kwan; Chang, Hsueh-Wen; Yip, Hon-Kan

    2016-01-01

    This study tested the hypothesis that human lung cancer-derived microparticles (LcD-MPs) played an important role in tumor angiogenesis and growth. Fischer 344 rats (F344, n=18) were equally categorized into group 1 [Sham Control (3.0 mL normal saline intravenous injection (IV))], group 2 [hepatoma cell line (2.0 x 106 cells, IV)], and group 3 [hepatoma cell line + LcD-MPs (3.0 x 106, IV)]. Animals were euthanized by day 28 after hepatoma cells transfusion. Our result showed that the gross pathology confirmed growth of hepatoma cell line in lung parenchyma. The size and weight of the lungs were significantly increased in group 2 and further elevated in group 3 than in group 1 (all p<0.001). Histopathological analysis demonstrated that the lung crowded score and number of small vessel exhibited an identical pattern, whereas the number of alveolar sacs showed an opposite pattern compared to that of total lung weight among the three groups (all p<0.0001). The cellular expressions of CD34+, CXCR4+, c-Kit+, CK19+, VEGF+ and vimentin+ cells in lung parenchyma exhibited an identical pattern compared to those of total lung weight among all groups (all p<0.001). The protein expressions of apoptotic (Bax, cleaved caspase-3 and c-PARP), fibrotic (Smad3, TGF-β), and tumor suppression (PTEN) biomarkers showed an identical pattern, whereas that of anti-apoptotic (Bcl-2) and anti-fibrotic (Smad1/5, BMP-2) biomarkers were displayed an opposite pattern compared to that of total lung weight among all groups (all p<0.001). The MPs could enhance angiogenesis and accelerated hepatoma cell growth in rodent lung parenchyma. PMID:27186261

  17. Human lung cancer-derived microparticles enhanced angiogenesis and growth of hepatoma cells in rodent lung parenchyma.

    PubMed

    Ko, Sheung-Fat; Hsu, Shu-Yuan; Chen, Chih-Hung; Sung, Pei-Hsun; Zhen, Meng-Shen TongYen-Yi; Chen, Yi-Ling; Huang, Tien-Hung; Chen, Sheng-Yi; Kao, Gour-Shenq; Chen, Hong-Hwa; Chang, Chia-Lo; Sun, Cheuk-Kwan; Chang, Hsueh-Wen; Yip, Hon-Kan

    2016-01-01

    This study tested the hypothesis that human lung cancer-derived microparticles (LcD-MPs) played an important role in tumor angiogenesis and growth. Fischer 344 rats (F344, n=18) were equally categorized into group 1 [Sham Control (3.0 mL normal saline intravenous injection (IV))], group 2 [hepatoma cell line (2.0 x 10(6) cells, IV)], and group 3 [hepatoma cell line + LcD-MPs (3.0 x 10(6), IV)]. Animals were euthanized by day 28 after hepatoma cells transfusion. Our result showed that the gross pathology confirmed growth of hepatoma cell line in lung parenchyma. The size and weight of the lungs were significantly increased in group 2 and further elevated in group 3 than in group 1 (all p<0.001). Histopathological analysis demonstrated that the lung crowded score and number of small vessel exhibited an identical pattern, whereas the number of alveolar sacs showed an opposite pattern compared to that of total lung weight among the three groups (all p<0.0001). The cellular expressions of CD34(+), CXCR4(+), c-Kit(+), CK19(+), VEGF(+) and vimentin+ cells in lung parenchyma exhibited an identical pattern compared to those of total lung weight among all groups (all p<0.001). The protein expressions of apoptotic (Bax, cleaved caspase-3 and c-PARP), fibrotic (Smad3, TGF-β), and tumor suppression (PTEN) biomarkers showed an identical pattern, whereas that of anti-apoptotic (Bcl-2) and anti-fibrotic (Smad1/5, BMP-2) biomarkers were displayed an opposite pattern compared to that of total lung weight among all groups (all p<0.001). The MPs could enhance angiogenesis and accelerated hepatoma cell growth in rodent lung parenchyma. PMID:27186261

  18. Micro FT-IR Characterization Of Human Lung Tumor Cells

    NASA Astrophysics Data System (ADS)

    Benedetti, Enzo; Teodori, L.; Vergamini, Piergiorgio; Trinca, M. L.; Mauro, F.; Salvati, F.; Spremolla, Giuliano

    1989-12-01

    FT-IR spectroscopy has opened up a new approach to the analytical study of cell transformation. Investigations carried out in normal and leukemic lymphocytes have evidenced an increase in DNA with respect to proteic components in neoplastic cells.(1) The evaluation of the ratio of the integrated areas(A) of the bands at 1080 cm-1 (mainly DNA) and at 1540 cm-1 (proteic components) has allowed us to establish a parameter which indicates, for values above 1.5, the neoplastic nature of cells. Recently, this approach has been applied to the study of human lung tumor cells. Several monocellular suspension procedures of the tissue fragment (mechanical and/or chemical) were tested to obtain reproducible and reliable spectra able to differentiate clearly between normal and patological cells. Chemical treatment (EDTA, Pepsin, Collagenase, etc.) produced additional bands in the spectra of the cells causing distortion of the profiles of some absorptions, and as a result, mechanical treatment was preferred. The normal and neoplastic cells homogeneously distributed by cytospin preparation on BaF2 windows were examined by means of FT-IR microscopy. An examination of several microareas of each sample yielded reproducible spectra, with values of the A 1080 cm-1 / A 1540 cm-1 parameter within a very narrow range for each sample, even if certain differences still remained among the different cases, in good agreement with the results obtained for leukemic cells.(1) The value of this parameter was found to be lower for cells isolated from the normal area of lung, than in the case of those corresponding to the tumoral area, meaning that an increase occurs in DNA with respect to the proteic components. These insights, which provide a basis to obtain indications at the molecular level, can open up new possibilities in clinical practice, in order to obtain diagnosis confirmation, to detect early stages of disease and to offer additional indications in cases of dubious interpretation.

  19. Nanoparticle diffusion in respiratory mucus from humans without lung disease

    PubMed Central

    Schuster, Benjamin S.; Suk, Jung Soo; Woodworth, Graeme F.; Hanes, Justin

    2013-01-01

    A major role of respiratory mucus is to trap inhaled particles, including pathogens and environmental particulates, to limit body exposure. Despite the tremendous health implications, how particle size and surface chemistry affect mobility in respiratory mucus from humans without lung disease is not known. We prepared polymeric nanoparticles densely coated with low molecular weight polyethylene glycol (PEG) to minimize muco-adhesion, and compared their transport to that of uncoated particles in human respiratory mucus, which we collected from the endotracheal tubes of surgical patients with no respiratory comorbidities. We found that 100 and 200 nm diameter PEG-coated particles rapidly penetrated respiratory mucus, at rates exceeding their uncoated counterparts by approximately 15- and 35-fold, respectively. In contrast, PEG-coated particles ≥ 500 nm in diameter were sterically immobilized by the mucus mesh. Thus, even though respiratory mucus is a viscoelastic solid at the macroscopic level (as measured using a bulk rheometer), nanoparticles that are sufficiently small and muco-inert can penetrate the mucus as if it were primarily a viscous liquid. These findings help elucidate the barrier properties of respiratory mucus and provide design criteria for therapeutic nanoparticles capable of penetrating mucus to approach the underlying airway epithelium. PMID:23384790

  20. Hydrolysis of a series of parabens by skin microsomes and cytosol from human and minipigs and in whole skin in short-term culture.

    PubMed

    Jewell, Christopher; Prusakiewicz, Jeffery J; Ackermann, Chrisita; Payne, N Ann; Fate, Gwendolyn; Voorman, Richard; Williams, Faith M

    2007-12-01

    Parabens are esters of 4-hydroxybenzoic acid and used as anti-microbial agents in a wide variety of toiletries, cosmetics and pharmaceuticals. It is of interest to understand the dermal absorption and hydrolysis of parabens, and to evaluate their disposition after dermal exposure and their potential to illicit localised toxicity. The use of minipig as a surrogate model for human dermal metabolism and toxicity studies, justifies the comparison of paraben metabolism in human and minipig skin. Parabens are hydrolysed by carboxylesterases to 4-hydroxybenzoic acid. The effects of the carboxylesterase inhibitors paraoxon and bis-nitrophenylphosphate provided evidence of the involvement of dermal carboxylesterases in paraben hydrolysis. Loperamide, a specific inhibitor of human carboxylesterase-2 inhibited butyl- and benzylparaben hydrolysis in human skin but not methylparaben or ethylparaben. These results show that butyl- and benzylparaben are more selective substrates for human carboxylesterase-2 in skin than the other parabens examined. Parabens applied to the surface of human or minipig skin were absorbed to a similar amount and metabolised to 4-hydroxybenzoic acid during dermal absorption. These results demonstrate that the minipig is a suitable model for man for assessing dermal absorption and hydrolysis of parabens, although the carboxylesterase profile in skin differs between human and minipig. PMID:17889094

  1. Hydrolysis of a series of parabens by skin microsomes and cytosol from human and minipigs and in whole skin in short-term culture

    SciTech Connect

    Jewell, Christopher; Prusakiewicz, Jeffery J.; Ackermann, Chrisita; Payne, N. Ann; Fate, Gwendolyn; Voorman, Richard; Williams, Faith M.

    2007-12-01

    Parabens are esters of 4-hydroxybenzoic acid and used as anti-microbial agents in a wide variety of toiletries, cosmetics and pharmaceuticals. It is of interest to understand the dermal absorption and hydrolysis of parabens, and to evaluate their disposition after dermal exposure and their potential to illicit localised toxicity. The use of minipig as a surrogate model for human dermal metabolism and toxicity studies, justifies the comparison of paraben metabolism in human and minipig skin. Parabens are hydrolysed by carboxylesterases to 4-hydroxybenzoic acid. The effects of the carboxylesterase inhibitors paraoxon and bis-nitrophenylphosphate provided evidence of the involvement of dermal carboxylesterases in paraben hydrolysis. Loperamide, a specific inhibitor of human carboxylesterase-2 inhibited butyl- and benzylparaben hydrolysis in human skin but not methylparaben or ethylparaben. These results show that butyl- and benzylparaben are more selective substrates for human carboxylesterase-2 in skin than the other parabens examined. Parabens applied to the surface of human or minipig skin were absorbed to a similar amount and metabolised to 4-hydroxybenzoic acid during dermal absorption. These results demonstrate that the minipig is a suitable model for man for assessing dermal absorption and hydrolysis of parabens, although the carboxylesterase profile in skin differs between human and minipig.

  2. Microsomal metabolism of NDMA and analogs

    SciTech Connect

    Wade, D.; Yang, C.S.

    1987-05-01

    The metabolism of N-nitrosodimethylamine (NDMA), dimethylamine (DMA), N-nitro-DMA (N x NO/sub 2/ x DMA), N-nitrosodiethylamine (NDEA), and diethylamine (DEA) was studied using control, acetone (Ac)-, butylated hydroxytoluene (BHT)-, pregnenolone 16- ..cap alpha..-carbonitrile (PCN)-, and phenobarbital (PB)-induced rat liver microsomes. At low substrate concentrations, the NDMA demethylase activity of Ac-induced microsomes was 5-fold greater than that of control, BHT-, and PCN-induced microsomes. The rate of NDMA denitrosation was ca. 10% that of demethylation. N x NO/sub 2/ x DMA was metabolized to HCHO, but not to NO/sub 2//sup -/, and the rate of metabolism was greatest with Ac-induced microsomes; the K/sub m/ and V/sub max/ of Ac-induced microsomes were similar to those of NDMA. For the dealkylation of NDEA, Ac- and BHT-induced microsomes were twice as active as the control. Ratios of dealkylation/denitrosation for NDEA remained constant over a broad range of low substrate concentrations. BHT- or Ac-treatment appeared to cause a selective increase in the ability of microsomes to denitrosate NDEA. The activity of all microsome preparations with the amines, DMA and DEA was less than that with the nitrosamine or nitramine substrates. The results suggest that both the N-nitroso and N-nitro compounds are good substrates for microsomal P-450; the amines, which bear positive charges, are not. Denitrosation appeared to be a more important pathway with NDEA than with NDMA.

  3. Frizzled-8 as a putative therapeutic target in human lung cancer

    SciTech Connect

    Wang, Hua-qing; Xu, Mei-lin; Ma, Jie; Zhang, Yi; Xie, Cong-hua

    2012-01-06

    Highlights: Black-Right-Pointing-Pointer Fzd-8 is over-expressed in human lung cancer. Black-Right-Pointing-Pointer shRNA knock-down of Fzd-8 inhibits proliferation and Wnt pathway in lung cancer cells. Black-Right-Pointing-Pointer shRNA knock-down of Fzd-8 suppresses tumor growth in vivo. Black-Right-Pointing-Pointer shRNA knock-down Fzd-8 sensitizes lung cancer cells to chemotherapy Taxotere. -- Abstract: Lung cancer is the leading cause of cancer related deaths worldwide. It is necessary to better understand the molecular mechanisms involved in lung cancer in order to develop more effective therapeutics for the treatment of this disease. Recent reports have shown that Wnt signaling pathway is important in a number of cancer types including lung cancer. However, the role of Frizzled-8 (Fzd-8), one of the Frizzled family of receptors for the Wnt ligands, in lung cancer still remains to be elucidated. Here in this study we showed that Fzd-8 was over-expressed in human lung cancer tissue samples and cell lines. To investigate the functional importance of the Fzd-8 over-expression in lung cancer, we used shRNA to knock down Fzd-8 mRNA in lung cancer cells expressing the gene. We observed that Fzd-8 shRNA inhibited cell proliferation along with decreased activity of Wnt pathway in vitro, and also significantly suppressed A549 xenograft model in vivo (p < 0.05). Furthermore, we found that knocking down Fzd-8 by shRNA sensitized the lung cancer cells to chemotherapy Taxotere. These data suggest that Fzd-8 is a putative therapeutic target for human lung cancer and over-expression of Fzd-8 may be important for aberrant Wnt activation in lung cancer.

  4. The extracellular calcium-sensing receptor regulates human fetal lung development via CFTR

    PubMed Central

    Brennan, Sarah C.; Wilkinson, William J.; Tseng, Hsiu-Er; Finney, Brenda; Monk, Bethan; Dibble, Holly; Quilliam, Samantha; Warburton, David; Galietta, Luis J.; Kemp, Paul J.; Riccardi, Daniela

    2016-01-01

    Optimal fetal lung growth requires anion-driven fluid secretion into the lumen of the developing organ. The fetus is hypercalcemic compared to the mother and here we show that in the developing human lung this hypercalcaemia acts on the extracellular calcium-sensing receptor, CaSR, to promote fluid-driven lung expansion through activation of the cystic fibrosis transmembrane conductance regulator, CFTR. Several chloride channels including TMEM16, bestrophin, CFTR, CLCN2 and CLCA1, are also expressed in the developing human fetal lung at gestational stages when CaSR expression is maximal. Measurements of Cl−-driven fluid secretion in organ explant cultures show that pharmacological CaSR activation by calcimimetics stimulates lung fluid secretion through CFTR, an effect which in humans, but not mice, was also mimicked by fetal hypercalcemic conditions, demonstrating that the physiological relevance of such a mechanism appears to be species-specific. Calcimimetics promote CFTR opening by activating adenylate cyclase and we show that Ca2+-stimulated type I adenylate cyclase is expressed in the developing human lung. Together, these observations suggest that physiological fetal hypercalcemia, acting on the CaSR, promotes human fetal lung development via cAMP-dependent opening of CFTR. Disturbances in this process would be expected to permanently impact lung structure and might predispose to certain postnatal respiratory diseases. PMID:26911344

  5. Lung Epithelial Cell-Specific Expression of Human Lysosomal Acid Lipase Ameliorates Lung Inflammation and Tumor Metastasis in Lipa(-/-) Mice.

    PubMed

    Zhao, Ting; Ding, Xinchun; Du, Hong; Yan, Cong

    2016-08-01

    Lysosomal acid lipase (LAL), a key enzyme in the metabolic pathway of neutral lipids, has a close connection with inflammation and tumor progression. One major manifestation in LAL-deficient (Lipa(-/-)) mice is an increase of tumor growth and metastasis associated with expansion of myeloid-derived suppressor cells. In the lung, LAL is highly expressed in alveolar type II epithelial cells. To assess how LAL in lung epithelial cells plays a role in this inflammation-related pathogenic process, lung alveolar type II epithelial cell-specific expression of human LAL (hLAL) in Lipa(-/-) mice was established by crossbreeding of CCSP-driven rtTA transgene and (TetO)7-CMV-hLAL transgene into Lipa(-/-) mice (CCSP-Tg/KO). hLAL expression in lung epithelial cells not only reduced tumor-promoting myeloid-derived suppressor cells in the lung, but also down-regulated the synthesis and secretion of tumor-promoting cytokines and chemokines into the bronchoalveolar lavage fluid of Lipa(-/-) mice. hLAL expression reduced the immunosuppressive functions of bronchoalveolar lavage fluid cells, inhibited bone marrow cell transendothelial migration, and inhibited endothelial cell proliferation and migration in Lipa(-/-) mice. As a result, hLAL expression in CCSP-Tg/KO mice corrected pulmonary damage, and inhibited tumor cell proliferation and migration in vitro, and tumor metastasis to the lung in vivo. These results support a concept that LAL is a critical metabolic enzyme in lung epithelial cells that regulates lung homeostasis, immune response, and tumor metastasis. PMID:27461363

  6. 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

    SciTech Connect

    Nagahama, Yu; Obama, Takashi; Usui, Michihiko; Kanazawa, Yukari; Iwamoto, Sanju; Suzuki, Kazushige; Miyazaki, Akira; Yamaguchi, Tomohiro; Yamamoto, Matsuo; Itabe, Hiroyuki

    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.

  7. Quantification of Flavin-containing Monooxygenases 1, 3, and 5 in Human Liver Microsomes by UPLC-MRM-Based Targeted Quantitative Proteomics and Its Application to the Study of Ontogeny.

    PubMed

    Chen, Yao; Zane, Nicole R; Thakker, Dhiren R; Wang, Michael Zhuo

    2016-07-01

    Flavin-containing monooxygenases (FMOs) have a significant role in the metabolism of small molecule pharmaceuticals. Among the five human FMOs, FMO1, FMO3, and FMO5 are the most relevant to hepatic drug metabolism. Although age-dependent hepatic protein expression, based on immunoquantification, has been reported previously for FMO1 and FMO3, there is very little information on hepatic FMO5 protein expression. To overcome the limitations of immunoquantification, an ultra-performance liquid chromatography (UPLC)-multiple reaction monitoring (MRM)-based targeted quantitative proteomic method was developed and optimized for the quantification of FMO1, FMO3, and FMO5 in human liver microsomes (HLM). A post-in silico product ion screening process was incorporated to verify LC-MRM detection of potential signature peptides before their synthesis. The developed method was validated by correlating marker substrate activity and protein expression in a panel of adult individual donor HLM (age 39-67 years). The mean (range) protein expression of FMO3 and FMO5 was 46 (26-65) pmol/mg HLM protein and 27 (11.5-49) pmol/mg HLM protein, respectively. To demonstrate quantification of FMO1, a panel of fetal individual donor HLM (gestational age 14-20 weeks) was analyzed. The mean (range) FMO1 protein expression was 7.0 (4.9-9.7) pmol/mg HLM protein. Furthermore, the ontogenetic protein expression of FMO5 was evaluated in fetal, pediatric, and adult HLM. The quantification of FMO proteins also was compared using two different calibration standards, recombinant proteins versus synthetic signature peptides, to assess the ratio between holoprotein versus total protein. In conclusion, a UPLC-MRM-based targeted quantitative proteomic method has been developed for the quantification of FMO enzymes in HLM. PMID:26839369

  8. The in vitro generation of lung and airway progenitor cells from human pluripotent stem cells

    PubMed Central

    Huang, Sarah X L; Green, Michael D; de Carvalho, Ana Toste; Mumau, Melanie; Chen, Ya-Wen; D’Souza, Sunita L.; Snoeck, Hans-Willem

    2015-01-01

    Lung and airway epithelial cells generated in vitro from human pluripotent stem cells have applications in regenerative medicine, modeling of lung disease, drug screening and studies of human lung development. Here we describe a strategy for directed differentiation of human pluripotent stem cells into developmental lung progenitors, and their subsequent differentiation into predominantly distal lung epithelial cells. The protocol entails four stages that recapitulate lung development and takes approximately 50 days. First, definitive endoderm is induced in the presence of high concentrations of Activin A. Subsequently, lung-biased anterior foregut endoderm is specified by sequential inhibition of BMP, TGF-β and Wnt signaling. Anterior foregut endoderm is then ventralized by applying Wnt, BMP, FGF and RA signaling to obtain lung and airway progenitors. Finally, these are further differentiated into more mature epithelial cells types using Wnt, FGF, c-AMP and glucocorticoid agonism. This protocol is conducted in defined conditions, does not involve genetic manipulation of the cells, and results in cultures where the majority of the cells express markers of various lung and airway epithelial cells, with a predominance of cells identifiable as functional type II alveolar epithelial cells. PMID:25654758

  9. CD11b immunophenotyping identifies inflammatory profiles in the mouse and human lungs.

    PubMed

    Duan, M; Steinfort, D P; Smallwood, D; Hew, M; Chen, W; Ernst, M; Irving, L B; Anderson, G P; Hibbs, M L

    2016-03-01

    The development of easily accessible tools for human immunophenotyping to classify patients into discrete disease endotypes is advancing personalized therapy. However, no systematic approach has been developed for the study of inflammatory lung diseases with often complex and highly heterogeneous disease etiologies. We have devised an internally standardized flow cytometry approach that can identify parallel inflammatory alveolar macrophage phenotypes in both the mouse and human lungs. In mice, lung innate immune cell alterations during endotoxin challenge, influenza virus infection, and in two genetic models of chronic obstructive lung disease could be segregated based on the presence or absence of CD11b alveolar macrophage upregulation and lung eosinophilia. Additionally, heightened alveolar macrophage CD11b expression was a novel feature of acute lung exacerbations in the SHIP-1(-/-) model of chronic obstructive lung disease, and anti-CD11b antibody administration selectively blocked inflammatory CD11b(pos) but not homeostatic CD11b(neg) alveolar macrophages in vivo. The identification of analogous profiles in respiratory disease patients highlights this approach as a translational avenue for lung disease endotyping and suggests that heterogeneous innate immune cell phenotypes are an underappreciated component of the human lung disease microenvironment. PMID:26422753

  10. Lung Beractant Increases Free Cytosolic Levels of Ca2+ in Human Lung Fibroblasts

    PubMed Central

    Guzmán-Silva, Alejandro; Vázquez de Lara, Luis G.; Torres-Jácome, Julián; Vargaz-Guadarrama, Ajelet; Flores-Flores, Marycruz; Pezzat Said, Elias; Lagunas-Martínez, Alfredo; Mendoza-Milla, Criselda; Tanzi, Franco; Moccia, Francesco; Berra-Romani, Roberto

    2015-01-01

    Beractant, a natural surfactant, induces an antifibrogenic phenotype and apoptosis in normal human lung fibroblasts (NHLF). As intracellular Ca2+ signalling has been related to programmed cell death, we aimed to assess the effect of beractant on intracellular Ca2+ concentration ([Ca2+]i) in NHLF in vitro. Cultured NHLF were loaded with Fura-2 AM (3 μM) and Ca2+ signals were recorded by microfluorimetric techniques. Beractant causes a concentration-dependent increase in [Ca2+]i with a EC50 of 0.82 μg/ml. The application of beractant, at a concentration of 500 μg/ml, which has been shown to exert an apoptotic effect in human fibroblasts, elicited different patterns of Ca2+ signals in NHLF: a) a single Ca2+ spike which could be followed by b) Ca2+ oscillations, c) a sustained Ca2+ plateau or d) a sustained plateau overlapped by Ca2+ oscillations. The amplitude and pattern of Ca2+ transients evoked by beractant were dependent on the resting [Ca2+]i. Pharmacological manipulation revealed that beractant activates a Ca2+ signal through Ca2+ release from intracellular stores mediated by phospholipase Cβ (PLCβ), Ca2+ release from inositol 1,4,5-trisphosphate receptors (IP3Rs) and Ca2+ influx via a store-operated pathway. Moreover, beractant-induced Ca2+ release was abolished by preventing membrane depolarization upon removal of extracellular Na+ and Ca2+. Finally, the inhibition of store-operated channels prevented beractant-induced NHLF apoptosis and downregulation of α1(I) procollagen expression. Therefore, beractant utilizes SOCE to exert its pro-apoptotic and antifibrinogenic effect on NHLF. PMID:26230503