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Sample records for human lung microsomes

  1. Spectroscopic quantitation of cytochrome P-450 in human lung microsomes.

    PubMed

    Wheeler, C W; Guenthner, T M

    1990-01-01

    The cytochrome P-450 content of human lung microsomes was measured by difference spectroscopy of the carbon monoxide-complexed hemoprotein. These measurements were only possible after the microsome preparation had been subjected to centrifugation over a discontinuous sucrose gradient, to remove an opaque black contaminant. The specific concentration of total cytochrome P-450 in human lung microsomes is essentially identical to that of microsomes prepared under identical conditions from untreated baboon lungs, but is only 0.7% of the specific content found in lung microsomes from untreated rabbits. These measurements correspond well to the observed metabolic capacities of the various microsome samples.

  2. 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.

  3. Stereoselective biotransformation of ketamine in equine liver and lung microsomes

    PubMed Central

    Schmitz, A.; Portier, C. J.; Thormann, W.; Theurillat, R.; Mevissen, M.

    2010-01-01

    Stereoselectivity has to be considered for pharmacodynamic and pharmacokinetic features of ketamine. Stereoselective biotransformation of ketamine was investigated in equine microsomes in vitro. Concentration curves were constructed over time, and enzyme activity was determined for different substrate concentrations using equine liver and lung microsomes. The concentrations of R/S-ketamine and R/S-norketamine were determined by enantioselective capillary electrophoresis. A two-phase model based on Hill kinetics was used to analyze the biotransformation of R/S-ketamine into R/S-norketamine and, in a second step, into R/S-downstream metabolites. In liver and lung microsomes, levels of R-ketamine exceeded those of S-ketamine at all time points and S-norketamine exceeded R-norketamine at time points below the maximum concentration. In liver and lung microsomes, significant differences in the enzyme velocity (Vmax) were observed between Sand R-norketamine formation and between Vmax of S-norketamine formation when S-ketamine was compared to S-ketamine of the racemate. Our investigations in microsomal reactions in vitro suggest that stereoselective ketamine biotransformation in horses occurs in the liver and the lung with a slower elimination of S-ketamine in the presence of R-ketamine. Scaling of the in vitro parameters to liver and lung organ clearances provided an excellent fit with previously published in vivo data and confirmed a lung first-pass effect. PMID:19000264

  4. Influence of induction and flavones on lung microsomal oxygen metabolism

    SciTech Connect

    Gregory, E.M.; Barlow, R.; Ebel, R.E.

    1986-05-01

    NADPH oxidase activity, H/sub 2/O/sub 2/ production and O/sub 2/ production were studied using microsomes isolated from lungs of control rats and rats treated with phenobarbital (PB), 3-methylcholanthrene (3-MC), 5,6-benzoflavone (..beta..-NF), or chlordecone (CD). 3-MC, ..beta..-NF or Cd treatment resulted in induction of lung P-450 and P-450 reductase. NADPH oxidase activity was slightly higher in 3-MC or ..beta..-NF lung microsomes than in control, PB or CD microsomes. 5 mM metyrapone inhibited oxidase activity of these microsomes by 20-50% depending upon their source. The rates of H/sub 2/O/sub 2/ and O/sub 2/ production did not appear to vary significantly as a function of induction and metyrapone had only a marginal effect on these rates. The rates of H/sub 2/O/sub 2/ and O/sub 2/ production at 37/sup 0/ were 1.8 +/- 0.2 and 1.8 +/- 0.3 nmol/min/mg, respectively. 50 ..mu..M ..beta..-NF stimulated H/sub 2/O/sub 2/ production by about 2-fold regardless of microsomal source while the stimulation of O/sub 2/ production was marginal. In each case, the effect of ..beta..-NF was greater than a comparable amount of 7,8-benzoflavone (..cap alpha..-NF). NADPH oxidase activity was dramatically stimulated by ..beta..-NF. For example, at 37/sup 0/ 100 ..mu..M ..beta..-NF increased the oxidase activity of control lung microsomes by about 9-fold from 3.4 to 31.6 nmol/min/mg. Lung microsomes from induced rats were comparably stimulated by ..beta..-NF. This stimulation was concentration dependent although not hyperbolic. Metyrapone did not inhibit the ..beta..-NF stimulated NADPH oxidase activity.

  5. 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

  6. Relationship between phenytoin and tolbutamide hydroxylations in human liver microsomes.

    PubMed Central

    Doecke, C J; Veronese, M E; Pond, S M; Miners, J O; Birkett, D J; Sansom, L N; McManus, M E

    1991-01-01

    1. The metabolic interaction of phenytoin and tolbutamide in human liver microsomes was investigated. 2. Phenytoin 4-hydroxylation (mean Km 29.6 microM, n = 3) was competitively inhibited by tolbutamide (mean Ki 106.2 microM, n = 3) and tolbutamide methylhydroxylation (mean Km 85.6 microM, n = 3) was competitively inhibited by phenytoin (mean Ki 22.6 microM, n = 3). 3. A significant correlation was obtained between phenytoin and tolbutamide hydroxylations in microsomes from 18 human livers (rs = 0.82, P less than 0.001). 4. Sulphaphenazole was a potent inhibitor of both phenytoin and tolbutamide hydroxylations with IC50 values of 0.4 microM and 0.6 microM, respectively. 5. Mephenytoin was a poor inhibitor of both phenytoin and tolbutamide hydroxylations with IC50 values greater than 400 microM for both reactions. 6. Anti-rabbit P450IIC3 IgG inhibited both phenytoin and tolbutamide hydroxylations in human liver microsomes by 62 and 68%, respectively. 7. These in vitro studies are consistent with phenytoin 4-hydroxylation and tolbutamide methylhydroxylation being catalysed by the same cytochrome P450 isozyme(s) in human liver microsomes. PMID:2049228

  7. Morphine metabolism in human skin microsomes.

    PubMed

    Heilmann, S; Küchler, S; Schäfer-Korting, M

    2012-01-01

    For patients with severe skin wounds, topically applied morphine is an option to induce efficient analgesia due to the presence of opioid receptors in the skin. However, for topical administration it is important to know whether the substance is biotransformed in the skin as this can eventually reduce the concentration of the active agent considerably. We use skin microsomes to elucidate the impact of skin metabolism on the activity of topically applied morphine. We are able to demonstrate that morphine is only glucuronidated in traces, indicating that the biotransformation in the skin can be neglected when morphine is applied topically. Hence, there is no need to take biotransformation into account when setting up the treatment regimen.

  8. [Metabolism of mitomycin C by human liver microsomes in vitro].

    PubMed

    Hao, Fu-rong; Yan, Min-fen; Hu, Zhuo-han; Jin, Yi-zun

    2007-02-01

    To provide the profiles of metabolism of mitomycin C (MMC) by human liver microsomes in vitro, MMC was incubated with human liver microsomes, then the supernatant component was isolated and detected by HPLC. Types of metabolic enzymes were estimated by the effect of NADPH or dicumarol (DIC) on metabolism of MMC. Standard, reaction, background control (microsomes was inactivated), negative control (no NADPH), and inhibitor group (adding DIC) were assigned, the results were analyzed by Graphpad Prism 4. 0 software. Reaction group compared with background control and negative control groups, 3 NADPH-dependent absorption peaks were additionally isolated by HPLC after MMC were incubated with human liver microsomes. Their retention times were 10. 0, 14. 0, 14. 8 min ( named as Ml, M2, M3) , respectively. Their formation was kept as Sigmoidal dose-response and their Km were 0. 52 (95% CI, 0. 40 - 0.67) mmol x L(-1), 0. 81 (95% CI, 0. 59 - 1. 10) mmol x L(-1), 0. 54 (95% CI, 0. 41 -0. 71) mmol x L(-1) , respectively. The data indicated that the three absorption peaks isolated by HPLC were metabolites of MMC. DIC can inhibit formation of M2, it' s dose-effect fitted to Sigmoidal curve and it' s IC50 was 59. 68 (95% CI, 40. 66 - 87. 61) micromol x L(-1) , which indicated DT-diaphorase could take part in the formation of M2. MMC can be metabolized by human liver microsomes in vitro, and at least three metabolites of MMC could be isolated by HPLC in the experiment, further study showed DT-diaphorase participated in the formation of M2.

  9. Inhibitory capacity of human serum on induced microsomal lipoperoxidation.

    PubMed

    Hicks, J J; Medina-Navarro, R

    1995-01-01

    The capacity of human serum for inhibiting in vitro the membrane lipoperoxidation induced by a controlled system (ADP/NADPH + H+/Fe3+) was demonstrated. A concentration of 8 nmol of malondialdehyde was produced in 20 min in rat liver microsomes (1.5 mg of protein) after exposure to an induced lipoperoxidation mixture. Addition of 100 microliters (13.89 mg of protein) of human serum decreased malondialdehyde production nearly 50%. An increase of 25.97% of the inhibitory capacity of serum was obtained by the in vitro addition of 10 microliters/ml of vitamin E. Ten volunteers were supplemented with 400 mg of vitamin E and 1 g of vitamin C/daily for 2 weeks. Their serum inhibitory capacity increased in 12% (p < 0.05). The serum inhibitory capacity for microsomal lipoperoxidation is described herein, and we propose its utilization as an index to determine the individual nonspecific antioxidative defenses against free radical injury and lipoperoxidation in relation to exposure to air pollutants, tobacco smoke, and several acute and chronic diseases, including the hypoxia-reperfusion phenomena.

  10. 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

  11. Comparative metabolism of mycophenolic acid by glucuronic acid and glucose conjugation in human, dog, and cat liver microsomes.

    PubMed

    Slovak, J E; Mealey, K; Court, M H

    2017-04-01

    Use of the immunosuppressant mycophenolic acid (MPA) in cats is limited because MPA elimination depends on glucuronidation, which is deficient in cats. We evaluated formation of major (phenol glucuronide) and minor (acyl glucuronide, phenol glucoside, and acyl glucoside) MPA metabolites using liver microsomes from 16 cats, 26 dogs, and 48 humans. All MPA metabolites were formed by human liver microsomes, while dog and cat liver microsomes formed both MPA glucuronides, but only one MPA glucoside (phenol glucoside). Intrinsic clearance (CLint) of MPA for phenol glucuronidation by cat liver microsomes was only 15-17% that of dog and human liver microsomes. However, CLint for acyl glucuronide and phenol glucoside formation in cat liver microsomes was similar to or greater than that for dog and human liver microsomes. While total MPA conjugation CLint was generally similar for cat liver microsomes compared with dog and human liver microsomes, relative contributions of each pathway varied between species with phenol glucuronidation predominating in dog and human liver microsomes and phenol glucosidation predominating in cat liver microsomes. MPA conjugation variation between cat liver microsomes was threefold for total conjugation and for phenol glucosidation, sixfold for phenol glucuronidation, and 11-fold for acyl glucuronidation. Our results indicate that total MPA conjugation is quantitatively similar between liver microsomes from cats, dogs, and humans despite large differences in the conjugation pathways that are utilized by these species.

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

    NASA Astrophysics Data System (ADS)

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

    2007-12-01

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

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

    PubMed

    Pudney, Christopher R; Khara, Basile; Johannissen, Linus O; Scrutton, Nigel S

    2011-12-01

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

  14. HMG-CoA reductase activity in human liver microsomes: comparative inhibition by statins.

    PubMed

    Dansette, P M; Jaoen, M; Pons, C

    2000-05-01

    The aim of this study was to compare a number of vastatins, HMG-CoA reductase inhibitors, in human liver microsomes. HMG-CoA reductase activity was four times lower than the activity in untreated rat liver microsomes. Vastatins could be classified in this in vitro assay in three classes both in human and rat microsomes: the first one including cerivastatin with an IC50 of 6 nM, the second one with atorvastatin and fluvastatin (IC50) between 40 and 100 nM) and the third one containing pravastatin, simvastatin and lovastatin (IC50 between 100 and 300 nM).

  15. 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.

  16. Effect of benidipine on simvastatin metabolism in human liver microsomes.

    PubMed

    Sugiyama, Yuka; Mimura, Nobuhito; Kuwabara, Takashi; Kobayashi, Hiroyuki; Ushiki, Junko; Fuse, Eiichi

    2007-06-01

    Benidipine, which is a calcium channel blocker that has clinical advantages in the treatment of hypertension, is metabolized by CYP3A4 in humans. The effect of benidipine on the metabolism of simvastatin by human liver microsomes was investigated in order to predict the potential of in vivo drug-drug interactions between benidipine and other substrates of CYP3A4. The results were compared with data generated with azelnidipine, which is also metabolized by CYP3A4. Both benidipine and azelnidipine inhibited simvastatin metabolism in vitro in a concentration-dependent manner. Assuming competitive inhibition, the K(i) values based on the unbound concentrations, were calculated to be 0.846 and 0.0181 microM for benidipine and azelnidipine, respectively. If simvastatin (10 mg) and benidipine (8 mg, the clinically recommended highest dose) were to be administered concomitantly, the ratio of the areas under the concentration-time curves of simvastatin with and without benidipine (AUC((+I))/AUC) was predicted to be 1.01. On the other hand, if simvastatin (10 mg) and azelnidipine (8 mg) were co-administered, the AUC((+I))/AUC for simvastatin was predicted to be 1.72, which is close to the observed value (1.9) in healthy volunteers. These data suggest that benidipine is unlikely to cause a drug interaction by inhibiting CYP3A4 activity in the liver.

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

    PubMed

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

    2014-02-01

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

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

    PubMed Central

    Carlile, David J; Hakooz, Nancy; Bayliss, Martin K; Houston, J Brian

    1999-01-01

    Aims To assess the utility of human hepatic microsomes for predicting in vivo intrinsic clearance (CLint) via the use of four cytochrome P450 2C9 substrates: phenytoin, tolbutamide (S)-ibuprofen (two pathways) and diclofenac, and to examine the role of exogenous albumin within the microsomal incubation. Methods V max, Km and CLint (defined as V max/Km ratio) were estimated under initial rate conditions for five pathways of metabolism in a bank of 15 human hepatic microsomal samples and were scaled to in vivo units using the microsomal protein index. Non-metabolic related binding in microsomes was measured for phenytoin and tolbutamide in the presence and absence of albumin. Results Microsomal CLint values differed by over two orders of magnitude, with the means ranging from 0.18 (phenytoin) to 40.70 (diclofenac) μl min−1 mg−1 microsomal protein. When these data were scaled and compared with published in vivo studies a similar rank order was obtained, however, the actual CLint tended to be underpredicted. While the in vivo unbound Km for phenytoin, 1–5 μm is substantially lower than the value determined in microsomes based on total concentrations (56 μm), correction for the in vitro binding reduces this value to 20 μm and 6 μm in the absence and presence of albumin, respectively. Similar trends were seen with tolbutamide Km. Conclusions An appreciation of the utility of in vitro prediction can be best achieved when the range of CLint values predicted from the individual hepatic microsomal samples are compared with the range of individual in vivo CLint values reported in the literature. The degree of underprediction is less evident using the range than the mean data and no consistent advantage in adding albumin to the incubation media is apparent. PMID:10383540

  19. Phosphatidylglycerol of rat lung. Intracellular sites of formation de novo and acyl species pattern in mitochondria, microsomes and surfactant.

    PubMed Central

    Schlame, M; Rüstow, B; Kunze, D; Rabe, H; Reichmann, G

    1986-01-01

    The subcellular site of phosphatidylglycerol (PG) formation for lung surfactant has not been convincingly clarified. To approach this problem we analysed the acyl species pattern of lung PG in mitochondria, microsomes and surfactant by h.p.l.c. separation of its 1,2-diacyl-3-naphthylurethane derivatives. Both mitochondrial and microsomal PG proved identical with surfactant PG, containing the major species 1-palmitoyl-2-oleoyl-PG and 1,2-dipalmitoyl-PG. The fatty acid composition of mitochondrial PG differs markedly from that of diphosphatidylglycerol. This may be taken as an indication that mitochondrial PG is synthesized on purpose to form surfactant, rather than being only the precursor of diphosphatidylglycerol. In vitro, sn-[U-14C]glycerol 3-phosphate incorporation into PG of mitochondria or microsomes occurs in the presence of CTP, ATP and CoA but independently of the supply of exogenous lipoidic precursors. Although the rate in vitro of autonomous PG synthesis, and the endogenous PG content, are higher in mitochondria than in microsomes, it is assumed that both subcellular fractions are involved in PG formation for surfactant. PMID:3827844

  20. Metabolism and Metabolic Inhibition of Xanthotoxol in Human Liver Microsomes

    PubMed Central

    Shi, Xianbao; Zhang, Gang; Guo, Feng

    2016-01-01

    Cytochrome p450 (CYP450) enzymes are predominantly involved in Phase I metabolism of xenobiotics. In this study, the CYP450 isoforms involved in xanthotoxol metabolism were identified using recombinant CYP450s. In addition, the inhibitory effects of xanthotoxol on eight CYP450 isoforms and its pharmacokinetic parameters were determined using human liver microsomes. CYP1A2, one of CYP450s, played a key role in the metabolism of xanthotoxol compared to other CYP450s. Xanthotoxol showed stronger inhibition on CYP3A4 and CYP1A2 compared to other isoenzymes with the IC50 of 7.43 μM for CYP3A4 and 27.82 μM for CYP1A2. The values of inhibition kinetic parameters (Ki) were 21.15 μM and 2.22 μM for CYP1A2 and CYP3A4, respectively. The metabolism of xanthotoxol obeyed the typical monophasic Michaelis-Menten kinetics and Vmax, Km, and CLint values were calculated as 0.55 nmol·min−1·mg−1, 8.46 μM, and 0.06 mL·min−1·mg−1. In addition, the results of molecular docking showed that xanthotoxol was bound to CYP1A2 with hydrophobic and π-π bond and CYP3A4 with hydrogen and hydrophobic bond. We predicted the hepatic clearance (CLH) and the CLH value was 15.91 mL·min−1·kg−1 body weight. These data were significant for the application of xanthotoxol and xanthotoxol-containing herbs. PMID:27034690

  1. In vitro covalent binding of new brain tracer, para-125I-amphetamine, to rat liver and lung microsomes

    SciTech Connect

    Joulin, Y.; Delaforge, M.; Hoellinger, H.; Moretti, J.L.; Sonnier, M.; Cesaro, P. )

    1990-01-01

    p-125I-amphetamine (I-Amp) is retained significantly in liver and lung during brain tomoscintigraphy. To attempt to explain this clinical observation, we have investigated the interaction of I-Amp with rat liver and lung microsomal proteins. Studies using spectral shift technique indicate that low concentration of I-Amp gives a type I complex and high concentration appears very stable type II complex with cytochrome P-450 Fe III. In the presence of NADPH, I-Amp gives rise to a 455 nm absorbing complex with similar properties to the Fe-RNO complexes. This complex formation was greatly enhanced with phenobarbital treated liver microsomes. The in vitro binding study shows that I-Amp and/or its metabolites was covalently bound to macromolecules in the presence of the molecular oxygen and NADPH-generating system. Incubation in the presence of glutathione, cystein and radical scavengers decreases binding. Mixed function oxydase (MFO) inhibitors diminish the amount of covalent binding and alter the extent of metabolite formation. The total covalent binding level increased with liver microsomes from PB pretreated rats as it was observed with the 455nm complex formation. The radioactivity distribution on microsomal proteins was examinated with SDS polyacrylamide gel electrophoresis and autoradiography. This experiment proves that the radiolabelled compounds are bound on the cytochrome P-450. The radioactivity bound increased when the PB induced rat liver microsomes were used. All these results indicate that I-Amp was activated by an oxydative process dependent on the MFO system which suggests a N-oxydation of I-Amp and the formation of reactive entities which covalently bind to proteins.

  2. Species pattern of phosphatidylinositol from lung surfactant and a comparison of the species pattern of phosphatidylinositol and phosphatidylglycerol synthesized de novo in lung microsomal fractions.

    PubMed Central

    Rüstow, B; Nakagawa, Y; Rabe, H; Waku, K; Kunze, D

    1988-01-01

    1. Phosphatidylinositol (PI) is a minor component of lung surfactant which may be able to replace the functionally important phosphatidylglycerol (PG) [Beppu, Clements & Goerke (1983) J. Appl. Physiol. 55, 496-502] without disturbing lung function. The dipalmitoyl species is one of the main species for both PI (14.4%) and PG (16.9%). Besides the C16:0--C16:0 species, the C16:0--C18:0, C16:0--C18:1, C16:0--C18:2 and C18:0--C18:1 species showed comparable proportions in the PG and PI fractions. These similarities of the species patterns and the acidic character of both phospholipids could explain why surfactant PG may be replaced by PI. 2. PI and PG were radiolabelled by incubation of microsomal fractions with [14C]glycerol 3-phosphate (Gro3P). For 11 out of 14 molecular species of PI and PG we measured comparable proportions of radioactivity. The radioactivity of these 11 species accounted together for more than 80% of the total. The addition of inositol to the incubation system decreased the incorporation in vitro of Gro3P into PG and CDP-DG (diacylglycerol) of lung microsomes (microsomal fractions), but did not change the distribution of radioactivity among the molecular species of PG. These results supported the idea that both acidic surfactant phospholipids may be synthesized de novo from a common CDP-DG pool in lung microsomes. PMID:3178759

  3. Time-dependent activation of the semicarbazide-sensitive amine oxidase (SSAO) from ox lung microsomes.

    PubMed Central

    Lizcano, J M; Tipton, K F; Unzeta, M

    2000-01-01

    The activity of ox lung microsomal semicarbazide-sensitive amine oxidase (EC 1.4.3.6; SSAO) towards benzylamine increased 20-fold during incubation at 37 degrees C. After an initial lag-period, activation was first-order with time and complete after approx. 20 h. No significant changes in activity towards methylamine, histamine or 2-phenylethylamine were observed, although mixed-substrate experiments were consistent with the same enzyme being involved in the oxidation of all these substrates, both before and after time-dependent activation. The enzyme-tryptophan fluorescence increased on incubation at 37 degrees C in parallel with the increase in activity towards benzylamine. Treatment of the activated-enzyme preparation with 6 M guanidinium chloride followed by dialysis, caused both the activity towards benzylamine and the fluorescence to fall to that occurring before activation. However, incubation of this preparation at 37 degrees C resulted in increases in fluorescence and activity similar to those seen with the unactivated enzyme. Benzylamine oxidation was inhibited, uncompetitively with respect to oxygen, by high substrate concentrations but no such inhibition was observed with the other amines. Activation resulted in an increase in V(max) for benzylamine oxidation, with no significant alterations in the K(m) or the K(si) for high-substrate inhibition. Kinetic studies were consistent with sequential mechanisms being followed for the oxidation of both benzylamine and methylamine but the dependence on oxygen concentration was complex. These results might indicate that benzylamine follows a different reaction pathway from the other substrates, with substrate-specific activation involving a reaction step that is rate-limiting for benzylamine oxidation but not for the others. PMID:11042135

  4. 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 ...

  5. Identification of human cytochrome P450 isoforms involved in the 7-hydroxylation of chlorpromazine by human liver microsomes.

    PubMed

    Yoshii, K; Kobayashi, K; Tsumuji, M; Tani, M; Shimada, N; Chiba, K

    2000-01-01

    Studies to identify the cytochrome P450 (CYP) isoform(s) involved in chlorpromazine 7-hydroxylation were performed using human liver microsomes and cDNA-expressed human CYPs. The kinetics of chlorpromazine 7-hydroxylation in human liver microsomes showed a simple Michaelis-Menten behavior. The apparent Km and Vmax values were 3.4+/-1.0 microM and 200.5+/-83.7 pmol/min/mg, respectively. The chlorpromazine 7-hydroxylase activity in human liver microsomes showed good correlations with desipramine 2-hydroxylase activity (r = 0.763, p < 0.05), a marker activity for CYP2D6, and phenacetin O-deethylase activity (r = 0.638, p < 0.05), a marker activity for CYP1A2. Quinidine (an inhibitor of CYP2D6) completely inhibited while alpha-naphthoflavone (an inhibitor of CYP1A2) marginally inhibited the chlorpromazine 7-hydroxylase activity in a human liver microsomal sample showing high CYP2D6 activity. On the other hand, alpha-naphthoflavone inhibited the chlorpromazine 7-hydroxylase activity to 55-65% of control in a human liver microsomal sample showing low CYP2D6 activity. Among eleven cDNA-expressed CYPs studied, CYP2D6 and CYP1A2 exhibited significant activity for the chlorpromazine 7-hydroxylation. The Km values for the chlorpromazine 7-hydroxylation of both cDNA-expressed CYP2D6 and CYP1A2 were in agreement with the Km values of human liver microsomes. These results suggest that chlorpromazine 7-hydroxylation is catalyzed mainly by CYP2D6 and partially by CYP1A2.

  6. Metabolism of (+)-terpinen-4-ol by cytochrome P450 enzymes in human liver microsomes.

    PubMed

    Haigou, Risa; Miyazawa, Mitsuo

    2012-01-01

    We examined the in vitro metabolism of (+)-terpinen-4-ol by human liver microsomes and recombinant enzymes. The biotransformation of (+)-terpinen-4-ol was investigated by gas chromatography-mass spectrometry (GC-MS). (+)-Terpinen-4-ol was found to be oxidized to (+)-(1R,2S,4S)-1,2-epoxy-p-menthan-4-ol, (+)-(1S,2R,4S)-1,2-epoxy-p-menthan-4-ol, and (4S)-p-menth-1-en-4,8-diol by human liver microsomal P450 enzymes. The identities of (+)-terpinen-4-ol metabolites were determined through the relative abundance of mass fragments and retention times on GC-MS. Of 11 recombinant human P450 enzymes tested, CYP1A2, CYP2A6, and CYP3A4 were found to catalyze the oxidation of (+)-terpinen-4-ol. Based on several lines of evidence, CYP2A6 and CYP3A4 were determined to be major enzymes involved in the oxidation of (+)-terpinen-4-ol by human liver microsomes. First, of the 11 recombinant human P450 enzymes tested, CYP1A2, CYP2A6 and CYP3A4 catalyzed oxidation of (+)-terpinen-4-ol. Second, oxidation of (+)-terpinen-4-ol was inhibited by (+)-menthofuran and ketoconazole, inhibitors known to be specific for these enzymes. Finally, there was a good correlation between CYP2A6 and CYP3A4 activities and (+)-terpinen-4-ol oxidation activities in the 10 human liver microsomes.

  7. Detection of human lung cytochromes P450 that are immunochemically related to cytochrome P450IIE1 and cytochrome P450IIIA.

    PubMed

    Wheeler, C W; Wrighton, S A; Guenthner, T M

    1992-07-07

    We have used monoclonal antibodies that were prepared against and specifically recognize human hepatic cytochromes P450 as probes for solid phase radioimmunoassay and Western immunoblotting to directly demonstrate the presence in human lung microsomes of cytochromes P450 immunochemically related to human liver cytochromes P450IIE1 (CYP2E1) and P450IIIA (CYP3A). The detected levels of these cytochromes are much lower than levels in human liver microsomes, but similar to the levels seen in microsomes from untreated baboon lung. Proteins immunochemically related to two other constitutive hepatic cytochromes P450, cytochrome P450IIC8 (CYP2C8) and cytochrome P450IIC9 (CYP2C9), were not detectable in lung microsomes.

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

    PubMed

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

    2013-09-05

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

  9. Lack of in vitro interactions using human liver microsomes between rabeprazole and anticancer drugs.

    PubMed

    Tamaro, Ilaria; Genazzani, Armando; Canonico, Pierluigi; Grosa, Giorgio

    2009-01-01

    The potential interactions between rabeprazole, a widely used proton pump inhibitor, and anticancer drugs (5-fluorouracil, docetaxel, cyclophosphamide, gemcitabine, methotrexate, doxorubicin, etoposide) or drugs commonly present in the therapy of oncological patients (fluoxetine and ondansetron), were studied using in vitro human liver microsomes. The interactions between rabeprazole and the anticancer drugs were evaluated by measuring their concentrations in test and control incubations with HPLC-DAD-UV methods. To achieve this aim, nine HPLC-DAD-UV methods were developed using different stationary and mobile phases. The methods were then validated for the following parameters: selectivity, linearity, precision, and accuracy. As expected rabeprazole did not significantly inhibit the metabolism of the evaluated drugs in human liver microsomal preparations at the selected concentrations. These results shows that rabeprazole probably could be devoid of pharmacokinetic interactions with common drugs used during chemotherapy.

  10. 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.

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

    PubMed

    Zielinski, Jana; Mevissen, Meike

    2015-04-01

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

  12. Interspecies differences in metabolism of deoxypodophyllotoxin in hepatic microsomes from human, monkey, rat, mouse and dog.

    PubMed

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

    2016-08-01

    Deoxypodophyllotoxin (DPT) is a natural lignan product which has drawn much attention due to its pharmacological properties including antitumor effect. The purpose of this study was to investigate interspecies differences in metabolism of DPT in hepatic microsomes from human (HLM), cynomolgus monkey (CyLM), rat (RLM), mouse (MLM) and dog (DLM). Incubation of DPT with hepatic microsomes from five species in the presence of NADPH resulted in formation of seven metabolites, five of which were compared with the synthetic standards. M2 was the most abundant metabolite in microsomes from all species. Rank order of intrinsic clearance for M2 formation was RLM > CyLM > MLM > HLM > DLM. In HLM, sulfaphenazole showed the strongest inhibition effect on M2 formation, but neither ticlopidine nor ketoconazole inhibited M2 formation in HLM. Results from cDNA-expressed human CYP450s experiments showed that clearance of M2 formation was much higher in CYP2C9 and CYP2C19 than that in CYP3A4. Contributions of the three CYP450 isoforms to M2 formation in HLM were estimated using relative activity factor (RAF) method or correction by amount of CYP450 isoforms in HLM. M2 formation in HLM was mainly attributed to CYP2C9, followed by CYP2C19. Involvement of CYP3A4 was minor.

  13. Development of QSAR models for microsomal stability: identification of good and bad structural features for rat, human and mouse microsomal stability

    NASA Astrophysics Data System (ADS)

    Hu, Yongbo; Unwalla, Ray; Denny, R. Aldrin; Bikker, Jack; Di, Li; Humblet, Christine

    2010-01-01

    High throughput microsomal stability assays have been widely implemented in drug discovery and many companies have accumulated experimental measurements for thousands of compounds. Such datasets have been used to develop in silico models to predict metabolic stability and guide the selection of promising candidates for synthesis. This approach has proven most effective when selecting compounds from proposed virtual libraries prior to synthesis. However, these models are not easily interpretable at the structural level, and thus provide little insight to guide traditional synthetic efforts. We have developed global classification models of rat, mouse and human liver microsomal stability using in-house data. These models were built with FCFP_6 fingerprints using a Naïve Bayesian classifier within Pipeline Pilot. The test sets were correctly classified as stable or unstable with satisfying accuracies of 78, 77 and 75% for rat, human and mouse models, respectively. The prediction confidence was assigned using the Bayesian score to assess the applicability of the models. Using the resulting models, we developed a novel data mining strategy to identify structural features associated with good and bad microsomal stability. We also used this approach to identify structural features which are good for one species but bad for another. With these findings, the structure-metabolism relationships are likely to be understood faster and earlier in drug discovery.

  14. 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.

  15. Hydration of arene and alkene oxides by epoxide hydrase in human liver microsomes.

    PubMed

    Kapitulnik, J; Levin, W; Morecki, R; Dansette, P M; Jerina, D M; Conney, A H

    1977-02-01

    The comparative hydration of styrene 7,8-oxide, octene 1,2-oxide, naphthalene 1,2-oxide, phenanthrene 9,10-oxide, benzo[a]anthracene 5,6-oxide, 3-methylcholanthrene 11,12-oxide, dibenzo[a,h]anthracene 5,6-oxide, and benzo[a, 7,8-, 9,10-, and 11,12-oxides to their respective dihydrodiols was investigated in microsomes from nine human autopsy livers. The substrate specificity of the epoxide hydrase in human liver microsomes was very similar to that of the epoxide hydrase in rat liver microsomes. Phenanthrene 9,10-oxide was the best substrate for the human and rat epoxide hydrases and dibenzo[a,h]anthracene 5,6-oxide and benzo[a-a)pyrene 11, 12-oxide were the poorest substrates. Plotting epoxide hydrase activity obtained with one substrate against epoxide hydrase activity for another substrate for each of the nine human livers revealed excellent correlations for all combinations of the 11 substrates studied (r = 0.87 to 0.99). The data suggest the presence in human liver of a single epoxide hydrase with broad substrate specificity. However, the results do not exclude the possible presence in human liver of several epoxide hydrases that are under similar regulatory control. These results suggest the need for further investigation to determine whether there is a safe epoxide of a drug whose in vivo metabolism is predictive of the capacity of different individuals to metabolize a wide variety of epoxides of drugs and environmental chemicals.

  16. Metabolism of sesamin by cytochrome P450 in human liver microsomes.

    PubMed

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

    2010-12-01

    Metabolism of sesamin by cytochrome P450 (P450) was examined using yeast expression system and human liver microsomes. Saccharomyces cerevisiae cells expressing each of human P450 isoforms (CYP1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, and 3A4) were cultivated with sesamin, and monocatechol metabolite was observed in most of P450s. Kinetic analysis using the microsomal fractions of the recombinant S. cerevisiae cells revealed that CYP2C19 had the largest k(cat)/K(m) value. Based on the kinetic data and average contents of the P450 isoforms in the human liver, the putative contribution of P450s for sesamin metabolism was large in the order of CYP2C9, 1A2, 2C19, and 2D6. A good correlation was observed between sesamin catecholization activity and CYP2C9-specific activity in in vitro studies using 10 individual human liver microsomes, strongly suggesting that CYP2C9 is the most important P450 isoform for sesamin catecholization in human liver. Inhibition studies using each anti-P450 isoform-specific antibody confirmed that CYP2C9 was the most important, and the secondary most important P450 was CYP1A2. We also examined the inhibitory effect of sesamin for P450 isoform-specific activities and found a mechanism-based inhibition of CYP2C9 by sesamin. In contrast, no mechanism-based inhibition by sesamin was observed in CYP1A2-specific activity. Our findings strongly suggest that further studies are needed to reveal the interaction between sesamin and therapeutic drugs mainly metabolized by CYP2C9.

  17. 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

  18. Determination of cytochrome P450 enzymes involved in the metabolism of (-)-terpinen-4-ol by human liver microsomes.

    PubMed

    Miyazawa, M; Haigou, R

    2011-12-01

    The in vitro metabolism of (-)-terpinen-4-ol was examined in human liver microsomes and recombinant enzymes. The biotransformation of (-)-terpinen-4-ol was investigated by gas chromatography-mass spectrometry. (-)-Terpinen-4-ol was found to be oxidized to (-)-(1S,2R,4R)-1,2-epoxy-p-menthan-4-ol, major metabolic product by human liver microsomal P450 enzymes. The formation of metabolites of (-)-terpinen-4-ol was determined by relative abundance of mass fragments and retention times on GC. CYP2A6 in human liver microsomes was a major enzyme involved in the oxidation of (-)-terpinen-4-ol by human liver microsomes, based on the following lines of evidence. First, of 11 recombinant human P450 enzymes tested, CYP2A6 had the highest activity for oxidation of (-)-terpinen-4-ol. Second, oxidation of (-)-terpinen-4-ol was inhibited by (+)-menthofuran. Finally, there was a good correlation between CYP2A6 maker activity and (-)-terpinen-4-ol oxidation activities in liver microsomes of 10 human samples. Kinetic analysis showed that the V(max)/K(m) values for (-)-(1S,2R,4R)-1,2-epoxy-p-menthan-4-ol catalysed by liver microsomes of human sample HH-18 was 2.49 μL/min/nmol. Human recombinant CYP2A6 catalysed (-)-(1S,2R,4R)-1,2-epoxy-p-menthan-4-ol with V(max) values of 13.9 nmol/min/nmol P450 and apparent K(m) values of 91 μM.

  19. In Vitro Metabolism of Artepillin C by Rat and Human Liver Microsomes.

    PubMed

    Carrão, Daniel Blascke; de Albuquerque, Nayara Cristina Perez; Marques, Lucas Maciel Mauriz; Crotti, Antônio Eduardo Miller; Pilon, Alan Cesar; Bolzani, Vanderlan Da Silva; Berretta, Andresa Aparecida; de Oliveira, Anderson Rodrigo Moraes

    2017-01-10

    Artepillin C, a natural product present in the Brazilian green propolis, has several biological properties. Among these properties, the antitumor action of this product is noteworthy and makes it a promising drug candidate for the treatment of several types of cancer. This paper describes the in vitro metabolism of Artepillin C in rat and human liver microsomes. The rat model suggested a sigmoidal profile for the metabolism, adapted to the Hill's kinetic model. The enzymatic kinetic parameters were as follows: maximal velocity = 0.757 ± 0.021 µmol/mg protein/min, Hill coefficient = 10.90 ± 2.80, and substrate concentration at which half-maximal velocity of a Hill enzyme is achieved = 33.35 ± 0.55 µM. Based on these results, the calculated in vitro intrinsic clearance for Artepillin C was 16.63 ± 1.52 µL/min/mg protein. The in vitro metabolism assay conducted on the human model did not fit any enzymatic kinetic model. Two novel metabolites were formed in both mammal microsomal models and their chemical structures were elucidated for the first time. The main human cytochrome P450 isoforms involved in Artepillin C metabolism had been identified, and the results suggest a majority contribution of CYP2E1 and CYP2C9 in the formation of the two metabolites.

  20. Metabolism of (-)-cis- and (-)-trans-rose oxide by cytochrome P450 enzymes in human liver microsomes.

    PubMed

    Nakahashi, Hiroshi; Yamamura, Yuuki; Usami, Atsushi; Rangsunvigit, Pramoch; Malakul, Pomthong; Miyazawa, Mitsuo

    2015-12-01

    The in vitro metabolism of (-)-cis- and (-)-trans-rose oxide was investigated using human liver microsomes and recombinant cytochrome P450 (P450 or CYP) enzymes for the first time. Both isomers of rose oxide were incubated with human liver microsomes, and the formation of the respective 9-oxidized metabolite were determined using gas chromatography-mass spectrometry (GC-MS). Of 11 different recombinant human P450 enzymes used, CYP2B6 and CYP2C19 were the primary enzymes catalysing the metabolism of (-)-cis- and (-)-trans-rose oxide. CYP1A2 also efficiently oxidized (-)-cis-rose oxide at the 9-position but not (-)-trans-rose oxide. α-Naphthoflavone (a selective CYP1A2 inhibitor), thioTEPA (a CYP2B6 inhibitor) and anti-CYP2B6 antibody inhibited (-)-cis-rose oxide 9-hydroxylation catalysed by human liver microsomes. On the other hand, the metabolism of (-)-trans-rose oxide was suppressed by thioTEPA and anti-CYP2B6 at a significant level in human liver microsomes. However, omeprazole (a CYP2C19 inhibitor) had no significant effects on the metabolism of both isomers of rose oxide. Using microsomal preparations from nine different human liver samples, (-)-9-hydroxy-cis- and (-)-9-hydroxy-trans-rose oxide formations correlated with (S)-mephenytoin N-demethylase activity (CYP2B6 marker activity). These results suggest that CYP2B6 plays important roles in the metabolism of (-)-cis- and (-)-trans-rose oxide in human liver microsomes.

  1. 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

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

    PubMed

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

    2000-10-01

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

  3. Involvement of CYP2D6 in oxidative metabolism of cinnarizine and flunarizine in human liver microsomes.

    PubMed

    Narimatsu, S; Kariya, S; Isozaki, S; Ohmori, S; Kitada, M; Hosokawa, S; Masubuchi, Y; Suzuki, T

    1993-06-30

    Oxidative metabolism of cinnarizine (CZ) and its fluorine derivative flunarizine (FZ), both of which are selective calcium entry blockers, was examined in human liver microsomes. The ring-hydroxylations and the N-desalkylations constituted primary metabolic pathways in microsomal metabolism of CZ and FZ. Among these pathways, the ring-hydroxylase (p-hydroxylation) activities at the cinnamyl moiety of both drugs were highly correlated with debrisoquine 4-hydroxylase activity and CYP2D6 content. Quinidine, a selective inhibitor of CYP2D6, suppressed the ring-hydroxylase activities of CZ and FZ. These results suggest that CYP2D6 is involved in the ring-hydroxylation of the cinnamyl moiety of both CZ and FZ in human liver microsomes.

  4. Aromatization of 7 alpha-methyl-19-nortestosterone by human placental microsomes in vitro.

    PubMed

    LaMorte, A; Kumar, N; Bardin, C W; Sundaram, K

    1994-02-01

    Part of the biological effects of testosterone (T) are mediated by its enzymatic reduction to 5 alpha-dihydrotestosterone (DHT) or aromatization to estradiol (E2). 7 alpha-Methyl-19-nortestosterone (MENT) is a synthetic androgen that is considerably more potent than T. Previous studies have shown that MENT is not 5 alpha-reduced. The studies reported here were undertaken to determine whether MENT undergoes enzymatic aromatization in vitro. Human placental microsomes were used as the source of the aromatase. Radioactive or nonradioactive T or MENT was incubated with the microsomes in the presence of NADPH and the metabolites extracted out with ethyl ether. Following evaporation of ether, the residue was dissolved in benzene-petroleum ether and extracted with 0.4 N NaOH which selectively removes phenolic metabolites of the androgens. When either radioactive T or MENT was incubated with the aromatase in the presence of NADPH, there was a 20-fold increase in the amount of radioactivity extracted with NaOH. In contrast, if the incubation was carried out in the absence of NADPH or in the presence of R76713, an aromatase inhibitor, most of the radioactivity remained in the benzene-petroleum ether phase. To further identify the enzymatic reaction products, thin layer chromatography (TLC) was performed. The Rf value for MENT was 0.22 while that of the major reaction product was 0.34, which corresponded with the RF value of the estrogen, 7 alpha-methyl-estradiol (MeE2). This was further verified by using a second solvent system for the chromatographic separation. In an effort to ascertain whether the metabolites bind to estrogen receptors (ER), rat uterine cytosol was used. NaOH extracts of medium following incubation of nonradioactive MENT with microsomes showed competitive inhibition of [3H]E2 binding to rat uterine ER. Furthermore, after [3H]MENT was incubated with microsomes, the radioactive metabolite extracted in NaOH showed specific binding to the ER which could

  5. Identification of metabolic pathways involved in the biotransformation of tolperisone by human microsomal enzymes.

    PubMed

    Dalmadi, Balázs; Leibinger, János; Szeberényi, Szabolcs; Borbás, Tímea; Farkas, Sándor; Szombathelyi, Zsolt; Tihanyi, Károly

    2003-05-01

    The in vitro metabolism of tolperisone, 1-(4-methyl-phenyl)-2-methyl-3-(1-piperidino)-1-propanone-hydrochloride, a centrally acting muscle relaxant, was examined in human liver microsomes (HLM) and recombinant enzymes. Liquid chromatography-mass spectrometry measurements revealed methyl-hydroxylation (metabolite at m/z 261; M1) as the main metabolic route in HLM, however, metabolites of two mass units greater than the parent compound and the hydroxy-metabolite were also detected (m/z 247 and m/z 263, respectively). The latter was identified as carbonyl-reduced M1, the former was assumed to be the carbonyl-reduced parent compound. Isoform-specific cytochrome P450 (P450) inhibitors, inhibitory antibodies, and experiments with recombinant P450s pointed to CYP2D6 as the prominent enzyme in tolperisone metabolism. CYP2C19, CYP2B6, and CYP1A2 are also involved to a smaller extent. Hydroxymethyl-tolperisone formation was mediated by CYP2D6, CYP2C19, CYP1A2, but not by CYP2B6. Tolperisone competitively inhibited dextromethorphan O-demethylation and bufuralol hydroxylation (K(i) = 17 and 30 microM, respectively). Tolperisone inhibited methyl p-tolyl sulfide oxidation (K(i) = 1200 microM) in recombinant flavin-containing monooxygenase 3 (FMO3) and resulted in a 3-fold (p < 0.01) higher turnover number using rFMO3 than that of control microsomes. Experiments using nonspecific P450 inhibitors-SKF-525A, 1-aminobenzotriazole, 1-benzylimidazole, and anti-NADPH-P450-reductase antibodies-resulted in 61, 47, 49, and 43% inhibition of intrinsic clearance in HLM, respectively, whereas hydroxymethyl-metabolite formation was inhibited completely by nonspecific chemical inhibitors and by 80% with antibodies. Therefore, it was concluded that tolperisone undergoes P450-dependent and P450-independent microsomal biotransformations to the same extent. On the basis of metabolites formed and indirect evidences of inhibition studies, a considerable involvement of a microsomal reductase is

  6. The CYP2A3 gene product catalyzes coumarin 7-hydroxylation in human liver microsomes

    SciTech Connect

    Yamano, Shigeru; Tatsuno, Jun; Gonzalez, F.J. )

    1990-02-06

    Three cDNAs, designated IIA3, IIA3v, and IIA4, coding for P450s in the CYP2A gene subfamily were isolated from a {lambda}gt11 library prepared from human hepatic mRNA. Only three nucleotide differences and a single amino acid difference, Leu{sup 160}{yields}His, were found between IIA3 and IIA3v, indicating that they are probably allelic variants. IIA4 displayed 94% amino acid similarity with IIA3 and IIA3v. The three cDNAs were inserted into vaccinia virus, and recombinant viruses were used to infect human hepatoma Hep G2 cells. Only IIA3 was able to produce an enzyme that had a reduced CO-bound spectrum with a {lambda}{sub max} at 450 nm. This expressed enzyme was able to carry out coumarin 7-hydroxylation and ethoxycoumarin O-deethylation. cDNA-expressed IIA3v and IIA4 failed to incorporate heme and were enzymatically inactive. Analysis of IIA proteins in human liver microsomes, using antibody against rat IIA2, revealed two proteins of 49 and 50 kDa, the former of which appeared to correlate with human microsomal coumarin 7-hydroxylase activity. A more striking correlation was found between IIa mRNA and enzyme activity. The rat antibody was able to completely abolish coumarin 7-hydroxylase activity in 12 liver samples. These data establish that the CYP2A3 gene product is primarily responsible for coumarin 7-hydroxylase activity in human liver. The level of expression of this activity varied up to 40-fold between livers. Levels of IIA mRNA also varied significantly between liver specimens, and three specimens had no detectable mRNA.

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

    PubMed

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

    1997-11-01

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

  8. 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.

  9. Pramipexole Derivatives as Potent and Selective Dopamine D3 Receptor Agonists with Improved Human Microsomal Stability

    PubMed Central

    Jiang, Cheng; Levant, Beth; Li, Xiaoqin; Zhao, Ting; Wen, Bo; Luo, Ruijuan; Sun, Duxin

    2014-01-01

    We report herein the synthesis and evaluation of a series of new pramipexole derivatives as highly potent and selective dopamine-3 (D3) receptor agonists. A number of these new compounds bind to the D3 receptor with subnanomolar affinities and show excellent selectivity (>10,000) for the D3 receptor over the D1 and D2 receptors. Compound 23 for example, binds to the D3 receptor with a Ki value of 0.53 nM and shows a selectivity of >20,000 over the D2 receptor and the D1 receptor in the binding assays using a rat brain preparation. It has excellent stability in human liver microsomes and in vitro functional assays showed it to be a full agonist for the human D3 receptor. PMID:25338762

  10. Mechanistic insights from comparing intrinsic clearance values between human liver microsomes and hepatocytes to guide drug design.

    PubMed

    Di, Li; Keefer, Christopher; Scott, Dennis O; Strelevitz, Timothy J; Chang, George; Bi, Yi-An; Lai, Yurong; Duckworth, Jonathon; Fenner, Katherine; Troutman, Matthew D; Obach, R Scott

    2012-11-01

    Metabolic stability of drug candidates are often determined in both liver microsome and hepatocyte assays. Comparison of intrinsic clearance values between the two assays provides additional information to guide drug design. Intrinsic clearance values from human liver microsomes and hepatocytes were compared for a set of commercial drugs with known metabolic pathways and transporter characteristics. The results showed that for compounds that were predominately metabolized by CYP mediated mechanisms, the intrinsic clearance values from the two assays were comparable. For compounds with non-CYP pathways, such as UGT and AO, intrinsic clearance was faster in hepatocytes than in microsomes. Substrates of uptake or efflux transporters in this study did not have significant differences of intrinsic clearance between microsomes and hepatocytes, when uptake into the hepatocytes was not the rate-limiting step. When hepatic uptake was rate limiting, intrinsic clearance in microsomes was faster than that in hepatocytes, which was more prevalent for compounds with rapid metabolism. Low passive permeability can limit the exposure to drug molecules to the metabolizing enzymes in the hepatocytes in relationship to the rate of metabolism. The faster the rate of metabolism, the higher permeability is needed for molecule to enter the cells and not becoming rate-limiting. The findings are very useful for drug discovery programs to gain additional insights on mechanistic information to help drug design without added experiments. Follow-up studies can then be designed to address specific questions.

  11. 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.

  12. 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.

  13. 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.

  14. 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...

  15. Glucuronidation versus oxidation of the flavonoid galangin by human liver microsomes and hepatocytes.

    PubMed

    Otake, Yoko; Hsieh, Faye; Walle, Thomas

    2002-05-01

    In a previous study, we used human liver microsomes for the first time to study cytochrome P450 (P450)-mediated oxidation of the flavonoid galangin. The combination of CYP1A2 and CYP2C9 produced a V(max)/K(m) value of 13.6 +/- 1.1 microl/min/mg of protein. In the present extended study, we determined glucuronidation rates for galangin with the same microsomes. Two major and one minor glucuronide were identified by liquid chromatography/mass spectrometry. The V(max)/K(m) values for the two major glucuronides conjugated in the 7- and 3-positions were 155 +/- 30 and 427 +/- 26 microl/min/mg of protein, thus, exceeding that of oxidation by 11 and 31 times, respectively. This highly efficient glucuronidation appeared to be catalyzed mainly by the UDP-glucuronosyltransferase (UGT)1A9 isoform but also by UGT1A1 and UGT2B15. Sulfation of galangin by the human liver cytosol, mediated mainly but not exclusively by sulfotransferase (SULT) 1A1, also appeared to be efficient. These conclusions were strongly supported by experiments using the S9 fraction of the human liver, in which all three metabolic pathways could be directly compared. When galangin metabolism was examined in fresh plated hepatocytes from six donors, glucuronidation clearly predominated followed by sulfation. Oxidation occurred only to a minor extent in two of the donors. This study for the first time establishes that glucuronidation and sulfation of galangin, and maybe other flavonoids, are more efficient than P450-mediated oxidation, clearly being the metabolic pathways of choice in intact cells and therefore likely also in vivo.

  16. 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

  17. 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.

  18. Quaternary ammonium-linked glucuronidation of tamoxifen by human liver microsomes and UDP-glucuronosyltransferase 1A4.

    PubMed

    Kaku, Teppei; Ogura, Kenichiro; Nishiyama, Takahito; Ohnuma, Tomokazu; Muro, Kei; Hiratsuka, Akira

    2004-06-01

    Tamoxifen (TAM), a nonsteroidal antiestrogen, is the most widely used drug for chemotherapy of hormone-dependent breast cancer in women. In the present study, we found a new potential metabolic pathway of TAM via N-linked glucuronic acid conjugation for excretion in humans. TAM N(+)-glucuronide was isolated from a reaction mixture consisting of TAM and human liver microsomes fortified with UDP-glucuronic acid (UDPGA) and identified with a synthetic specimen by high-performance liquid chromatography-electrospray ionization-mass spectrometry. However, no TAM-glucuronidating activity was detected in microsomes from rat, mouse, monkey, dog, and guinea pig livers. A strong correlation (r(2) =0.92 ) was observed between N-glucuronidating activities toward TAM and trifluoperazine, a probe substrate for human UDP-glucuronosyltransferase (UGT) 1A4, in human liver microsomes from eight donors (five females, three males). However, no correlation ( (r(2) =0.02 )) was observed in the activities between 7-hydroxy-4-(trifluoromethyl)coumarin and TAM. Only UGT1A4 catalyzed the N-linked glucuronidation of TAM among recombinant UGTs (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9, UGT2B4, UGT2B7, UGT2B15, and UGT2B17) expressed in insect cells. Apparent K(m) values for TAM N-glucuronidation by human liver microsomes and recombinant UGT1A4 were 35.8 and 32.4 microM, respectively. These results strongly suggested that UGT1A4 could play a role in metabolism and excretion of TAM without Phase I metabolism in human liver. TAM N(+)-glucuronide still had binding affinity similar to TAM itself for human estrogen receptors, ERalpha and ERbeta, suggesting that TAM N(+)-glucuronide might contribute to the biological activity of TAM in vivo.

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

    PubMed

    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 K i 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.

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

    PubMed

    Suzuki, Takashi; Swift, Larry L

    2016-06-03

    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.

  1. 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

  2. Evidence for Human Lung Stem Cells

    PubMed Central

    Kajstura, Jan; Rota, Marcello; Hall, Sean R.; Hosoda, Toru; D’Amario, Domenico; Sanada, Fumihiro; Zheng, Hanqiao; Ogórek, Barbara; Rondon-Clavo, Carlos; Ferreira-Martins, João; Matsuda, Alex; Arranto, Christian; Goichberg, Polina; Giordano, Giovanna; Haley, Kathleen J.; Bardelli, Silvana; Rayatzadeh, Hussein; Liu, Xiaoli; Quaini, Federico; Liao, Ronglih; Leri, Annarosa; Perrella, Mark A.; Loscalzo, Joseph; Anversa, Piero

    2011-01-01

    BACKGROUND Although progenitor cells have been described in distinct anatomical regions of the lung, description of resident stem cells has remained elusive. METHODS Surgical lung-tissue specimens were studied in situ to identify and characterize human lung stem cells. We defined their phenotype and functional properties in vitro and in vivo. RESULTS Human lungs contain undifferentiated human lung stem cells nested in niches in the distal airways. These cells are self-renewing, clonogenic, and multipotent in vitro. After injection into damaged mouse lung in vivo, human lung stem cells form human bronchioles, alveoli, and pulmonary vessels integrated structurally and functionally with the damaged organ. The formation of a chimeric lung was confirmed by detection of human transcripts for epithelial and vascular genes. In addition, the self-renewal and long-term proliferation of human lung stem cells was shown in serial-transplantation assays. CONCLUSIONS Human lungs contain identifiable stem cells. In animal models, these cells participate in tissue homeostasis and regeneration. They have the undemonstrated potential to promote tissue restoration in patients with lung disease. (Funded by the National Institutes of Health.) PMID:21561345

  3. 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.

  4. Metabolic interaction between ethanol, high-dose alprazolam and its two main metabolites using human liver microsomes in vitro.

    PubMed

    Tanaka, Einosuke; Nakamura, Takako; Terada, Masaru; Shinozuka, Tatsuo; Honda, Katsuya

    2007-08-01

    Alprazolam is widely used as a short-acting antidepressant and anxiolytic agent and its effect appears at very low doses while ethanol is used as a social drug worldwide. Sometimes, toxic interactions occur following combined administration of these two drugs. In this study we have investigated the interaction between ethanol and high-dose alprazolam using human liver microsomes in vitro. The interaction effects between ethanol and alprazolam were examined by a mixed-function oxidation reaction using a human liver microsomal preparation. Alprazolam and its two main metabolites (alpha-hydroxyalprazolam: alpha-OH alprazolam, 4-hydroxyalprazolam: 4-OH alprazolam) were measured by HPLC/UV. The production of 4-OH alprazolam, one main metabolite of alprazolam, was weakly inhibited by higher dose of ethanol, but not alpha-OH alprazolam. These results using a human liver microsomal preparation show that the production of 4-OH alprazolam is weakly inhibited by ethanol but not alpha-OH alprazolam. Toxic levels may be reached by simultaneous administration of ethanol and high-dose alprazolam.

  5. No association between microsomal triglyceride transfer protein (MTP) haplotype and longevity in humans.

    PubMed

    Nebel, Almut; Croucher, Peter J P; Stiegeler, Rieke; Nikolaus, Susanna; Krawczak, Michael; Schreiber, Stefan

    2005-05-31

    Human longevity is a multifactorial condition with a significant genetic contribution. A recent association study in two independent samples of long-lived U.S. Caucasians [long-lived individuals (LLI)] identified a SNP haplotype of the microsomal triglyceride transfer protein (MTP, 4q25) that was underrepresented among LLI when compared with younger controls. This suggested that variation in the MTP gene might modify human longevity. Because of its function in lipid metabolism, the MTP gene product could plausibly play a pivotal role in the physiology of aging. However, the association observed in the U.S. samples could not be replicated by the same authors in a larger French LLI sample. We have therefore investigated the MTP "risk" haplotype in our own collection of 1,589 German nonagenarians, centenarians, and appropriately matched controls. No statistically significant differences were observed between LLI and controls at the allele, genotype, or haplotype level. This indicates that a noteworthy influence of the respective MTP haplotype on human longevity in the German population is unlikely. Furthermore, in comparison with all other U.S. and European samples analyzed, the MTP "risk" haplotype was found to be overrepresented only in the U.S. controls. This implies that the putative association is more likely to reflect recent changes in the genetic structure of the U.S. Caucasian population as a whole, rather than genetic effects upon survival to old age. In our view, the original study therefore highlights potential problems that arise when the case-control design is used as a means to map longevity genes in humans.

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

    PubMed

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

    2016-01-01

    1. Raloxifene is an antiestrogen that has been marketed for the treatment of osteoporosis, and is metabolized into 6- and 4'-glucuronides by UDP-glucuronosyltransferase (UGT) enzymes. In this study, the in vitro glucuronidation of raloxifene in humans and monkeys was examined using liver and intestinal microsomes and recombinant UGT enzymes (UGT1A1, UGT1A8 and UGT1A9). 2. Although the K(m) and CL(int) values for the 6-glucuronidation of liver and intestinal microsomes were similar between humans and monkeys, and species differences in Vmax values (liver microsomes, humans > monkeys; intestinal microsomes, humans < monkeys) were observed, no significant differences were noted in the K(m) or S50, Vmax and CL(int) or CLmax values for the 4'-glucuronidation of liver and intestinal microsomes between humans and monkeys. 3. The activities of 6-glucuronidation in recombinant UGT enzymes were UGT1A1 > UGT1A8 >UGT1A9 for humans, and UGT1A8 > UGT1A1 > UGT1A9 for monkeys. The activities of 4'-glucuronidation were UGT1A8 > UGT1A1 > UGT1A9 in humans and monkeys. 4. These results demonstrated that the profiles for the hepatic and intestinal glucuronidation of raloxifene by microsomes were moderately different between humans and monkeys.

  7. Metabolism of xanthohumol and isoxanthohumol, prenylated flavonoids from hops (Humulus lupulus L.), by human liver microsomes.

    PubMed

    Nikolic, Dejan; Li, Yongmei; Chadwick, Lucas R; Pauli, Guido F; van Breemen, Richard B

    2005-03-01

    The female flowers of hops (Humulus lupulus L.) used to flavor beer contain the prenylated flavonoids xanthohumol (XN) and isoxanthohumol (IX). IX is moderately estrogenic in vitro and XN has pharmacological properties that might make it useful as a cancer chemopreventive agent. The metabolism of these dietary flavonoids was investigated in vitro using human liver microsomes. Hydroxylation of a prenyl methyl group was the primary route of oxidative metabolism forming either cis or trans hydroxylated metabolites of IX but only the trans isomer of XN. The double bond on the prenyl group of both compounds formed an epoxide which was opened by an intramolecular reaction with the neighboring hydroxyl group. The potent phytoestrogen 8-prenylnaringenin (8-PN) was detected as a demethylation product of IX. However, the analogous demethylation reaction was not observed for XN. Since XN can be converted to IX through acid-catalyzed cyclization in the stomach, XN might contribute to the in vivo levels of estrogenic 8-PN following consumption of hops extracts.

  8. Metabolism of 8-prenylnaringenin, a potent phytoestrogen from hops (Humulus lupulus), by human liver microsomes.

    PubMed

    Nikolic, Dejan; Li, Yongmei; Chadwick, Lucas R; Grubjesic, Simonida; Schwab, Pia; Metz, Peter; van Breemen, Richard B

    2004-02-01

    The female flowers of hops are used throughout the world as a flavoring agent for beer. Recently, there has been increasing interest in the potential estrogenic properties of hop extracts. Among the possible estrogenic compounds in hops, 8-prenylnaringenin is perhaps most significant due to its high in vitro potency exceeding that of other known phytoestrogens. Since data regarding the pharmacokinetic properties of this compound are lacking, we investigated the in vitro metabolism of 8-prenylnaringenin by human liver microsomes. A total of 12 metabolites were identified, and biotransformation occurred on the prenyl group and the flavanone skeleton. The major site of oxidation was on the terminal methyl groups, and of the two possible isomers, the transisomer was more abundant. The double bond on the prenyl group was also oxidized to an epoxide that was opened by intramolecular reaction with the neighboring hydroxyl group. On the flavanone skeleton, the major site of oxidation was at 3'position on the B ring. Other metabolites included oxidation at carbon-3 as well as desaturation of the C ring to produce 8-prenylapigenin. An unusual hydroxy quinone product formed by ipso hydroxylation of the B ring of 8-prenylnaringenin was also detected. This product was probably an intermediate for the B ring cleavage product, 8-prenylchromone.

  9. In vitro metabolism of phenoxypropoxybiguanide analogues in human liver microsomes to potent antimalarial dihydrotriazines.

    PubMed

    Shearer, Todd W; Kozar, Michael P; O'Neil, Michael T; Smith, Philip L; Schiehser, Guy A; Jacobus, David P; Diaz, Damaris S; Yang, Young-Sun; Milhous, Wilbur K; Skillman, Donald R

    2005-04-21

    Phenoxypropoxybiguanides, such as 1 (PS-15), are prodrugs analogous to the relationship of proguanil and its active metabolite cycloguanil. Unlike cycloguanil, however, 1a (WR99210), the active metabolite of 1, has retained in vitro potency against newly emerging antifolate-resistant malaria parasites. Unfortunately, manufacturing processes and gastrointestinal intolerance have prevented the clinical development of 1. In vitro antimalarial activity and in vitro metabolism studies have been performed on newly synthesized phenoxypropoxybiguanide analogues. All of the active dihydrotriazine metabolites exhibited potent antimalarial activity with in vitro IC(50) values less than 0.04 ng/mL. In vitro metabolism studies in human liver microsomes identified the production of not only the active dihydrotriazine metabolite, but also a desalkylation on the carbonyl chain, and multiple hydroxylated metabolites. The V(max) for production of the active metabolites ranged from 10.8 to 27.7 pmol/min/mg protein with the K(m) ranging from 44.8 to 221 microM. The results of these studies will be used to guide the selection of a lead candidate.

  10. Cryptotanshinone and dihydrotanshinone I exhibit strong inhibition towards human liver microsome (HLM)-catalyzed propofol glucuronidation.

    PubMed

    Cong, Ming; Hu, Cui-Min; Cao, Yun-Feng; Fang, Zhong-Ze; Tang, Shu-Hong; Wang, Jia-Rui; Luo, Jun-Sheng

    2013-03-01

    Danshen is one of the most famous herbs in the world, and more and more danshen-prescribed drugs interactions have been reported in recent years. Evaluation of inhibition potential of danshen's major ingredients towards UDP-glucuronosyltransferases (UGTs) will be helpful for understanding detailed mechanisms for danshen-drugs interaction. Therefore, the aim of the present study is to investigate the inhibitory situation of cryptotanshinone and dihydrotanshinone I towards UGT enzyme-catalyzed propofol glucuronidation. In vitro the human liver microsome (HLM) incubation system was used, and the results showed that cryptotanshinone and dihydrotanshinone I exhibited dose-dependent inhibition towards HLM-catalyzed propofol glucuronidation. Dixon plot and Lineweaver-Burk plot showed that the inhibition type was best fit to competitive inhibition type for both cryptotanshinone and dihydrotanshinone I. The second plot using the slopes from the Lineweaver-Burk plot versus the concentrations of cryptotanshinone or dihydrotanshinone I was employed to calculate the inhibition parameters (Ki) to be 0.4 and 1.7μM, respectively. Using the reported maximum plasma concentration (Cmax), the altered in vivo exposure of propofol increased by 10% and 8.2% for the co-administration of dihydrotanshinone I and cryptotanshinone, respectively. All these results indicated the possible danshen-propofol interaction due to the inhibition of dihydrotanshinone I and cryptotanshinone towards the glucuronidation reaction of propofol.

  11. Glucuronidation of 4-tert-octylphenol in humans, monkeys, rats, and mice: an in vitro analysis using liver and intestine microsomes.

    PubMed

    Hanioka, Nobumitsu; Isobe, Takashi; Ohkawara, Susumu; Tanaka-Kagawa, Toshiko; Jinno, Hideto

    2017-03-01

    4-tert-Octylphenol (4-tOP) is an endocrine-disrupting chemical. It is mainly metabolized into glucuronide by UDP-glucuronosyltransferase (UGT) enzymes in mammals. In the present study, the glucuronidation of 4-tOP in humans, monkeys, rats, and mice was examined in an in vitro system using microsomal fractions. The kinetics of 4-tOP glucuronidation by liver microsomes followed the Michaelis-Menten model for humans and monkeys, and the biphasic model for rats and mice. The K m, V max, and CL int values of human liver microsomes were 0.343 µM, 11.6 nmol/min/mg protein, and 33.8 mL/min/mg protein, respectively. The kinetics of intestine microsomes followed the Michaelis-Menten model for humans, monkeys, and rats, and the biphasic model for mice. The K m, V max, and CL int values of human intestine microsomes were 0.743 µM, 0.571 nmol/min/mg protein, and 0.770 mL/min/mg protein, respectively. The CL int values estimated by Eadie-Hofstee plots were in the order of mice (high-affinity phase) (3.0) > humans (1.0) ≥ monkeys (0.9) > rats (high-affinity phase) (0.4) for liver microsomes, and monkeys (10) > mice (high-affinity phase) (5.6) > rats (1.4) > humans (1.0) for intestine microsomes. The percentages of the CL int values of intestine microsomes to liver microsomes were in the order of monkeys (27 %) > rats (high-affinity phase in liver microsomes) (7.9 %) > mice (high-affinity phase in liver and intestine microsomes) (4.2 %) > humans (2.3 %). These results suggest that the metabolic abilities of UGT enzymes expressed in the liver and intestine toward 4-tOP markedly differ among species and imply that species differences are strongly associated with the toxicities of alkylphenols.

  12. Cytokines in human lung fibrosis.

    PubMed

    Martinet, Y; Menard, O; Vaillant, P; Vignaud, J M; Martinet, N

    1996-01-01

    Fibrosis is a pathological process characterized by the replacement of normal tissue by mesenchymal cells and the extracellular matrix produced by these cells. The sequence of events leading to fibrosis of an organ involves the subsequent processes of injury with inflammation and disruption of the normal tissue architecture, followed by tissue repair with accumulation of mesenchymal cells in the area of derangement. The same sequence of events occurs in wound healing with normal granulation tissue and scar formation, but, while normal scar formation is very localized and transient, in contrast, in fibrosis, the repair process is exaggerated and usually widespread and can be chronic. Inflammatory cells (mainly mononuclear phagocytes), platelets, endothelial cells, and type II pneumocytes play a direct and indirect role in tissue injury and repair. The evaluation of three human fibrotic lung diseases, two diffuse [idiopathic pulmonary fibrosis (IPF), and the adult respiratory distress syndrome (ARDS)], and one focal (tumor stroma in lung cancer), has shown that several cytokines participate to the local injury and inflammatory reaction [interleukin-1 (IL-1), interleukin-8 (IL-8), monocyte chemotactic protein-1 (MCP-1), tumor necrosis factor-alpha (TNF-alpha)], while other cytokines are involved in tissue repair and fibrosis [platelet-derived growth factor (PDGF), insulin-like growth factor-1 (IGF-1), transforming growth factor-beta (TGF-beta), and basic-fibroblast growth factor (b-FGF)]. A better understanding of the cytokines and cytokine networks involved in lung fibrosis leads to the possibility of new therapeutic approaches.

  13. A probabilistic method to report predictions from a human liver microsomes stability QSAR model: a practical tool for drug discovery.

    PubMed

    Aliagas, Ignacio; Gobbi, Alberto; Heffron, Timothy; Lee, Man-Ling; Ortwine, Daniel F; Zak, Mark; Khojasteh, S Cyrus

    2015-04-01

    Using data from the in vitro liver microsomes metabolic stability assay, we have developed QSAR models to predict in vitro human clearance. Models were trained using in house high-throughput assay data reported as the predicted human hepatic clearance by liver microsomes or pCLh. Machine learning regression methods were used to generate the models. Model output for a given molecule was reported as its probability of being metabolically stable, thus allowing for synthesis prioritization based on this prediction. Use of probability, instead of the regression value or categories, has been found to be an efficient way for both reporting and assessing predictions. Model performance is evaluated using prospective validation. These models have been integrated into a number of desktop tools, and the models are routinely used to prioritize the synthesis of compounds. We discuss two therapeutic projects at Genentech that exemplify the benefits of a probabilistic approach in applying the models. A three-year retrospective analysis of measured liver microsomes stability data on all registered compounds at Genentech reveals that the use of these models has resulted in an improved metabolic stability profile of synthesized compounds.

  14. A probabilistic method to report predictions from a human liver microsomes stability QSAR model: a practical tool for drug discovery

    NASA Astrophysics Data System (ADS)

    Aliagas, Ignacio; Gobbi, Alberto; Heffron, Timothy; Lee, Man-Ling; Ortwine, Daniel F.; Zak, Mark; Khojasteh, S. Cyrus

    2015-04-01

    Using data from the in vitro liver microsomes metabolic stability assay, we have developed QSAR models to predict in vitro human clearance. Models were trained using in house high-throughput assay data reported as the predicted human hepatic clearance by liver microsomes or pCLh. Machine learning regression methods were used to generate the models. Model output for a given molecule was reported as its probability of being metabolically stable, thus allowing for synthesis prioritization based on this prediction. Use of probability, instead of the regression value or categories, has been found to be an efficient way for both reporting and assessing predictions. Model performance is evaluated using prospective validation. These models have been integrated into a number of desktop tools, and the models are routinely used to prioritize the synthesis of compounds. We discuss two therapeutic projects at Genentech that exemplify the benefits of a probabilistic approach in applying the models. A three-year retrospective analysis of measured liver microsomes stability data on all registered compounds at Genentech reveals that the use of these models has resulted in an improved metabolic stability profile of synthesized compounds.

  15. Human lung lysozyme: sources and properties.

    PubMed

    Konstan, M W; Chen, P W; Sherman, J M; Thomassen, M J; Wood, R E; Boat, T F

    1981-01-01

    Lysozyme in human airway secretions is thought to defend the lung against airborne bacteria. Although lysozyme has been purified and characterized from human tears, milk, saliva, and other sources (1-5), human lung lysozyme has received little attention except for measurements of concentrations in sputum (6, 7), immunocytochemical and histochemical localization (8-12),and studies of secretion by alveolar macrophages (13). This study was designed to identify the sources of secreted lung lysozyme, to quantitate the secretory activities of the various sources,and to compare the properties of lysozyme from lung cells with those from other tissues.

  16. 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.

  17. Bioactivation of Trimethoprim to Protein-Reactive Metabolites in Human Liver Microsomes.

    PubMed

    Goldman, Jennifer L; Koen, Yakov M; Rogers, Steven A; Li, Kelin; Leeder, James S; Hanzlik, Robert P

    2016-10-01

    The formation of drug-protein adducts via metabolic activation and covalent binding may stimulate an immune response or may result in direct cell toxicity. Protein covalent binding is a potentially pivotal step in the development of idiosyncratic adverse drug reactions (IADRs). Trimethoprim (TMP)-sulfamethoxazole (SMX) is a combination antibiotic that commonly causes IADRs. Recent data suggest that the contribution of the TMP component of TMP-SMX to IADRs may be underappreciated. We previously demonstrated that TMP is bioactivated to chemically reactive intermediates that can be trapped in vitro by N-acetyl cysteine (NAC), and we have detected TMP-NAC adducts (i.e., mercapturic acids) in the urine of patients taking TMP-SMX. However, the occurrence and extent of TMP covalent binding to proteins was unknown. To determine the ability of TMP to form protein adducts, we incubated [(14)C]TMP with human liver microsomes in the presence and absence of NADPH. We observed protein covalent binding that was NADPH dependent and increased with incubation time and concentration of both protein and TMP. The estimated covalent binding was 0.8 nmol Eq TMP/mg protein, which is comparable to the level of covalent binding for several other drugs that have been associated with covalent binding-induced toxicity and/or IADRs. NAC and selective inhibitors of CYP2B6 and CYP3A4 significantly reduced TMP covalent binding. These results demonstrate for the first time that TMP bioactivation can lead directly to protein adduct formation, suggesting that TMP has been overlooked as a potential contributor of TMP-SMX IADRs.

  18. 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.

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

    PubMed

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

    2014-07-03

    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.

  20. Propranolol oxidation by human liver microsomes--the use of cumene hydroperoxide to probe isoenzyme specificity and regio- and stereoselectivity.

    PubMed

    Otton, S V; Gillam, E M; Lennard, M S; Tucker, G T; Woods, H F

    1990-11-01

    1. Three oxidations of the enantiomers of propranolol were studied in human liver microsomes under two reaction conditions. Previous in vitro studies had established that two of the livers were from poor metaboliser (PM) phenotypes for the debrisoquine 4-hydroxylase (cytochrome P-450IID6) and the remaining seven were from extensive metaboliser (EM) phenotypes. 2. In the presence of NADPH and oxygen 4- and 5-hydroxylation of propranolol occurred in microsomes from all nine livers, as did propranolol N-desisopropylation. R(+)-propranolol was oxidized preferentially along the three pathways, although enantioselectivity observed for N-desisopropylation may have arisen not only from stereoselectivity in formation rates, but also from stereoselectivity in subsequent microsomal metabolism, possibly by monoamine oxidase. The involvement of monoamine oxidase in the further microsomal metabolism of N-desisopropylpropranolol was indicated by inhibition of the metabolism of this compound when incubated with phenelzine. 3. Cumene hydroperoxide has been proposed to support only the activity of cytochrome P450IID6. This is consistent with the observations that a) propranolol 4- and 5-hydroxylation occurred in microsomes from the EM livers only and b) side-chain oxidation was not observed under these conditions in either PM or EM livers. 4. Using cumene hydroperoxide to support the reactions, the 4-hydroxylation of propranolol showed little enantioselectivity, whereas S(-)-propranolol was 5-hydroxylated about twice as fast as the R(+)-enantiomer. There were highly significant correlations between the rates of 4- and 5-hydroxylation of R(+)-propranolol (r = 0.96, P less than 0.001, n = 7 livers) and of S(-)-propranolol (r = 0.98, P less than 0.001). Both oxidations were described by single-site Michaelis-Menten kinetics. 5. The findings suggest that P450IID6 is involved in both the 4- and 5-hydroxylations of propranolol, but that these metabolites can also be formed by other P450

  1. High-performance liquid chromatographic assay for N-glucuronidation of nicotine and cotinine in human liver microsomes.

    PubMed

    Nakajima, Miki; Kwon, Jun-Tack; Tanaka, Eriko; Yokoi, Tsuyoshi

    2002-03-01

    A method for the determination of N-glucuronidation of nicotine and cotinine in human liver microsomes by high-performance liquid chromatography was developed. Nicotine or cotinine was incubated with human liver microsomes and UDP-glucuronic acid in a 200-microl incubation mixture. The nicotine N-glucuronide (Nic-glu) and cotinine N-glucuronide (Cot-glu) formed were analyzed by ion-pair chromatography with a C-18 column. The sensitivity of quantification at 260 nm absorption was improved by using a noise-base clean Uni-3, and the limit of quantification was 10 pmol/200 microl mixture for both Nic-glu and Cot-glu. Linear standard curves were obtained within the concentration ranges 25-1000 pmol/200 microl mixture for Nic-glu and 100-5000 pmol/200 microl mixture for Cot-glu. The intraassay precision and accuracy were < or =11.1% coefficient of variation (CV) and 97.5-106.6% for Nic-glu and < or =4.6% CV and 96.7-100.4% for Cot-glu. The interassay precision and accuracy were < or =7.2% CV and 98.2-106.1% for Nic-glu and < or =4.6% CV and 96.8-99.3% for Cot-glu. This is the first report of the in vitro determination of Nic-glu and Cot-glu in human liver microsomes. Furthermore, this highly sensitive HPLC method can be used for the determination of Nic-glu and Cot-glu in biological specimens in vivo.

  2. Oxidation of the flavonoids galangin and kaempferide by human liver microsomes and CYP1A1, CYP1A2, and CYP2C9.

    PubMed

    Otake, Yoko; Walle, Thomas

    2002-02-01

    There is very limited information on cytochrome P450 (P450)-mediated oxidative metabolism of dietary flavonoids in humans. In this study, we used human liver microsomes and recombinant P450 isoforms to examine the metabolism of two flavonols, galangin and kaempferide, and one flavone, chrysin. Both galangin and kaempferide, but not chrysin, were oxidized by human liver microsomes to kaempferol, with K(m) values of 9.5 and 17.8 microM, respectively. These oxidations were catalyzed mainly by CYP1A2 but also by CYP2C9. Consistent with these observations, the human liver microsomal metabolism of galangin and kaempferide were inhibited by the P450 inhibitors furafylline and sulfaphenazole. In addition, CYP1A1, although less efficient, was also able to oxidize the two flavonols. Thus, dietary flavonols are likely to undergo oxidative metabolism mainly in the liver but also extrahepatically.

  3. 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

  4. Tiny Device Mimics Human Lung Function

    SciTech Connect

    McDonald, Rebecca; Harris, Jennifer; Nath, Pulak

    2016-04-25

    Scientists at Los Alamos National Laboratory are developing a miniature, tissue-engineered artificial lung that mimics the response of the human lung to drugs, toxins and other agents. “We breathe in and out thousands of times every day. And while we have control over what we eat or drink, we don’t always have control over what we breathe in,” said Jennifer Harris of Biosecurity and Public Health at Los Alamos, "and so we’re making this miniature lung to be able to test on actual human cells whether something in the environment, or a drug, is toxic or harmful to us." Nicknamed “PuLMo” for Pulmonary Lung Model (Pulmo is also the Latin word for "lung")the device consists of two major parts, the bronchiolar unit and the alveolar unit—just like the human lung. The units are primarily made from various polymers and are connected by a microfluidic “circuit board” that manages fluid and air flow. “When we build our lung, we not only take into account the aspects of different cell types, the tissues that are involved, we also take into account that a lung is supposed to breathe, so PuLMo actually breathes,” said Pulak Nath of Applied Modern Physics, who leads engineering efforts for the project. The most exciting application of PuLMo is a potentially revolutionary improvement in the reliability of drug-toxicity assessments and the prediction of new pharmaceutical success in humans, according to Harris. The PuLMo may also be designed to mimic lung disease conditions, such as Chronic Obstructive Pulmonary Disease (COPD) and asthma, and may be used to study lung air-flow dynamics to better understand the mechanisms of toxins and drug delivery and the effects of smoking, particularly the less-understood effects of e-cigarettes.

  5. Human lung ex vivo infection models.

    PubMed

    Hocke, Andreas C; Suttorp, Norbert; Hippenstiel, Stefan

    2017-03-01

    Pneumonia is counted among the leading causes of death worldwide. Viruses, bacteria and pathogen-related molecules interact with cells present in the human alveolus by numerous, yet poorly understood ways. Traditional cell culture models little reflect the cellular composition, matrix complexity and three-dimensional architecture of the human lung. Integrative animal models suffer from species differences, which are of particular importance for the investigation of zoonotic lung diseases. The use of cultured ex vivo infected human lung tissue may overcome some of these limitations and complement traditional models. The present review gives an overview of common bacterial lung infections, such as pneumococcal infection and of widely neglected pathogens modeled in ex vivo infected lung tissue. The role of ex vivo infected lung tissue for the investigation of emerging viral zoonosis including influenza A virus and Middle East respiratory syndrome coronavirus is discussed. Finally, further directions for the elaboration of such models are revealed. Overall, the introduced models represent meaningful and robust methods to investigate principles of pathogen-host interaction in original human lung tissue.

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

    PubMed

    Kim, Ju-Hyun; Kwon, Soon-Sang; Kong, Tae Yeon; Cheong, Jae Chul; Kim, Hee Seung; In, Moon Kyo; Lee, Hye Suk

    2017-03-10

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

  7. Polysaccharide peptides from Coriolus versicolor competitively inhibit tolbutamide 4-hydroxylation in specific human CYP2C9 isoform and pooled human liver microsomes.

    PubMed

    Yeung, John H K; Or, Penelope M Y

    2011-10-15

    Polysaccharide peptide (PSP), isolated from COV-1 strain of Coriolus versicolor, is commonly used as an adjunct in cancer chemotherapy in China. Previous studies have shown that PSP decreased antipyrine clearance and inhibited CYP2C11-mediated tolbutamide 4-hydroxylation in the rat both in vitro and in vivo. In this study, the effects of water extractable fraction of PSP on tolbutamide 4-hydroxylation was investigated in pooled human liver microsomes and in specific human CYP2C9 isoform. PSP (2.5-20μM) dose-dependently decreased the biotransformation of tolbutamide to 4-hydroxy-tolbutamide. Enzyme kinetics studies showed inhibition of tolbutamide 4-hydroxylase activity was competitive and concentration-dependent. In pooled human liver microsomes, PSP had a K(i) value of 14.2μM compared to sulfaphenazole, a human CYP2C9 inhibitor, showed a K(i) value of 0.32μM. In human CYP2C9 isoform, the K(i) value of PSP was 29.5μM and the K(i) value of sulfaphenazole was 0.04μM. This study demonstrated that PSP can competitively inhibit tolbutamide 4-hydroxylation in both pooled human liver microsomes and specific human CYP2C9 in vitro. This study compliments previous findings in the rat that PSP can inhibit human tolbutamide 4-hydroxylase, but the relatively high K(i) values in human CYP2C9 would suggest a low potential for PSP to cause herb-drug interaction.

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

    PubMed

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

    2006-06-01

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

  9. Metabolism of 2,6-dinitro[3-3H]toluene by human and rat liver microsomal and cytosolic fractions.

    PubMed

    Chapman, D E; Michener, S R; Powis, G

    1992-08-01

    1. 2,6-Dinitrotoluene (2,6-DNT) metabolism by human liver and male Fischer F344 rat liver subcellular fractions under aerobic (100% oxygen) and anaerobic (100% nitrogen) incubation conditions was examined. Under aerobic conditions the major 2,6-DNT metabolite formed by hepatic microsomes was 2,6-dinitrobenzyl alcohol (2,6-DNBalc); under anaerobic conditions 2-amino-6-nitrotoluene (2Am6NT) was the major metabolite. 2. Rates of 2,6-DNBalc formation by human and rat liver microsomes under aerobic conditions were 247 and 132 pmol/min per mg protein, respectively. Rates of 2Am6NT formation by human and rat liver microsomes under anaerobic conditions were 292 and 285 pmol/min per mg protein, respectively. Anaerobic reduction of 2,6-DNT to 2Am6NT by rat and human liver microsomes was inhibited by carbon monoxide and metyrapone, which indicates that microsomal metabolism of 2,6-DNT to 2Am6NT is mediated by cytochrome P-450. 3. Liver cytosolic fractions also metabolized 2,6-DNT to 2Am6NT under anaerobic conditions. Formation of 2Am6NT by human and rat liver cytosols was supported by hypoxanthine, NADPH and NADH. Allopurinol inhibited the hypoxanthine-supported anaerobic metabolism of 2,6-DNT by rat, but not human, liver cytosol. Dicumarol inhibited the NADPH-supported anaerobic metabolism of 2,6-DNT by human, but not rat, liver cytosol. These results indicate that xanthine oxidase contributes to the hypoxanthine-supported anaerobic metabolism of 2,6-DNT by human liver cytosol.

  10. Albendazole treatment of echinococcosis in humans: effects on microsomal metabolism and drug tolerance.

    PubMed

    Steiger, U; Cotting, J; Reichen, J

    1990-03-01

    We prospectively studied the effect of albendazole on microsomal reserve and on first-pass activation to albendazole sulfoxide in patients with hydatid disease. An aminopyrine breath test was performed in 12 patients while they were receiving albendazole treatment and while they were not. Excretion of 14CO2 in breath averaged 0.70%.kg.mmol-1 +/- 0.20%.kg.mmol-1 without treatment and 0.54%.kg.mmol-1 +/- 0.14%.kg.mmol-1 with treatment (p less than 0.005). Plasma levels of albendazole sulfoxide were measured 4 hours after the morning dose during the first and second half of the 4-week treatment cycles. In nine of the 12 patients albendazole sulfoxide levels decreased during the second half of the cycle by an average of 0.84 +/- 0.76 mumol/L (p less than 0.02). Transaminase levels increased in 10 of the 12 patients during long-term albendazole treatment, and major side effects, including hepatotoxicity, neutropenia, and alopecia, were observed in three patients. We conclude that albendazole partially inhibits microsomal enzyme function but induces its own metabolism. Hepatotoxicity and other possible severe side effects necessitate close therapeutic monitoring of patients who are given albendazole.

  11. TFPIβ is the GPI-anchored TFPI isoform on human endothelial cells and placental microsomes

    PubMed Central

    Girard, Thomas J.; Tuley, Elodee

    2012-01-01

    Tissue factor pathway inhibitor (TFPI) produces factor Xa-dependent feedback inhibition of factor VIIa/tissue factor-induced coagulation. Messages for 2 isoforms of TFPI have been identified. TFPIα mRNA encodes a protein with an acidic N-terminus, 3 Kunitz-type protease inhibitor domains and a basic C-terminus that has been purified from plasma and culture media. TFPIβ mRNA encodes a form in which the Kunitz-3 and C-terminal domains of TFPIα are replaced with an alternative C-terminus that directs the attachment of a glycosylphosphatidylinositol (GPI) anchor, but whether TFPIβ protein is actually expressed is not clear. Moreover, previous studies have suggested that the predominant form of TFPI released from cells by phosphatidylinositol-specific phospholipase C (PIPLC) treatment is TFPIα, implying it is bound at cell surfaces to a separate GPI-anchored coreceptor. Our studies show that the form of TFPI released by PIPLC treatment of cultured endothelial cells and placental microsomes is actually TFPIβ based on (1) migration on SDS-PAGE before and after deglycosylation, (2) the lack of a Kunitz-3 domain, and (3) it contains a GPI anchor. Immunoassays demonstrate that, although endothelial cells secrete TFPIα, greater than 95% of the TFPI released by PIPLC treatment from the surface of endothelial cells and from placental microsomes is TFPIβ. PMID:22144186

  12. Comparative metabolism study of β-lapachone in mouse, rat, dog, monkey, and human liver microsomes using liquid chromatography-tandem mass spectrometry.

    PubMed

    Lee, Sangkyu; Kim, In Sook; Kwak, Tae Hwan; Yoo, Hye Hyun

    2013-09-01

    β-Lapachone (3,4-dihydro-2,2-dimethyl-2H-naphthol[1,2-b]pyran-5,6-dione) is a natural compound extracted from the bark of the lapacho tree (Tabebuia avellanedae) and is undergoing phase II clinical trials as an antitumor drug candidate. The present study characterized in vitro metabolites of β-lapachone in mouse, rat, dog, monkey and human liver microsomes. β-Lapachone (10 μM) was incubated with mouse, rat, dog, monkey, and human liver microsomes in the presence of NADPH. The reaction mixtures were analyzed by LC/MS and the metabolites were identified based on their elemental composition and product ion spectra. A total of 6 metabolites (M1-M6) were detected in liver microsomes with a slight difference between species. M1 and M6 were identified as a decarbonated metabolite and a carboxylated metabolite, respectively; M2, M3, and M4 were identified as monohydroxylated metabolites; and M5 was identified as an O-methylated metabolite. M5, an O-methylated metabolite was found in rat and human liver microsomes, which is thought to be formed from a catechol intermediate by MB-COMT-mediated methylation and reported here for the first time.

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

    PubMed Central

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

    2016-01-01

    Objective: Tinospora cordifolia is used for treatment of several diseases in Indian system of medicine. In the present study, the inhibition potential of T. cordifolia extracts and its constituent tinosporaside to cause herb-drug interactions through rat and human liver cytochrome enzymes was evaluated. Materials and Methods: Bioactive compound was quantified through reverse phase high-performance liquid chromatography, to standardize the plant extracts and interaction potential of standardized extract. Interaction potential of the test sample was evaluated through cytochrome P450-carbon monoxide complex (CYP450-CO) assay with pooled rat liver microsome. Influence on individual recombinant human liver microsomes such as CYP3A4, CYP2D6, CYP2C9, and CYP1A2 isozymes was analyzed through fluorescence microplate assay, and respective IC50 values were determined. Results: The content of tinosporaside was found to be 1.64% (w/w) in T. cordifolia extract. Concentration-dependent inhibition was observed through T. cordifolia extract. Observed IC50 (μg/ml) value was 136.45 (CYP3A4), 144.37 (CYP2D6), 127.55 (CYP2C9), and 141.82 (CYP1A2). Tinosporaside and extract showed higher IC50 (μg/ml) value than the known inhibitors. T. cordifolia extract showed significantly less interaction potential and indicates that the selected plant has not significant herb-drug interactions relating to the inhibition of major CYP450 isozymes. Conclusions: Plant extract showed significantly higher IC50 value than respective positive inhibitors against CYP3A4, 2D6, 2C9, and 1A2 isozymes. Consumption of T. cordifolia may not cause any adverse effects when consumed along with other xenobiotics. PMID:27721546

  14. Adrenochrome reaction implicates oxygen radicals in metabolism of cyclosporine A and FK-506 in rat and human liver microsomes.

    PubMed

    Ahmed, S S; Strobel, H W; Napoli, K L; Grevel, J

    1993-06-01

    The role of oxygen radicals in the metabolism of cyclosporine A (CyA), FR900506 (FK-506) and carbon tetrachloride (CCl4) catalyzed by the cytochrome P450 system was investigated in vitro in rat and human microsomal preparations. Varying concentrations of CyA, FK-506 and CCl4 (100 microM-1.0 mM) were added to microsomal preparations, and lipid peroxidation was measured by malondialdehyde (MDA) formation as detected by the thiobarbituric acid assay. The effects of oxygen radical scavengers [superoxide dismutase (SOD) and catalase (CAT)] and an antioxidant [glutathione (GLUT)] were tested on various incubations of CyA, FK-506 and CCl4 to assess the role of oxygen radicals in lipid peroxidation. CyA-dependent MDA formation was moderately inhibited by SOD in the rat model and increased by SOD in the human model. In both models, CAT slightly inhibited CyA-dependent MDA formation and GLUT significantly inhibited MDA formation. FK-506-dependent MDA formation, studied only in the rat model, paralleled CyA-induced MDA formation but showed greater inhibition with CAT and less inhibition with SOD or GLUT. In both models, CCl4-dependent MDA formation was significantly inhibited by GLUT and showed no sensitivity to SOD or CAT. In addition, the adrenochrome reaction, which measures the oxidation of epinephrine to adrenochrome, was used to measure the increased oxygen radical-flux resulting from the metabolism of CyA, FK-506 and CCl4. CyA with epinephrine showed the highest oxidative activity, followed by FK-506 and then CCl4, which showed the least formation of adrenochrome. These results indicated a role for oxygen radicals in CyA and FK-506 metabolism.

  15. Lung retention of cerium in humans.

    PubMed Central

    Pairon, J C; Roos, F; Iwatsubo, Y; Janson, X; Billon-Galland, M A; Bignon, J; Brochard, P

    1994-01-01

    A retrospective study was conducted to evaluate lung retention of particles containing cerium in subjects with and without previous occupational exposure to mineral dusts. Analytical transmission electron microscopy was performed on 459 samples of bronchoalveolar lavage (BAL) fluid and 75 samples of lung tissue. Study of the distribution of mineralogical species in human samples showed that particles containing cerium were encountered in less than 10% of subjects. The proportion of subjects with particles containing cerium in their biological samples was not different between controls and subjects with previous occupational exposure to fibrous or nonfibrous mineral dusts. This was considered as the background level of lung retention of cerium in the general population. By contrast, determination of the absolute concentration of particles containing cerium in BAL fluid and lung tissue samples showed that 1.2% (from BAL fluid) and 1.5% (from lung tissue) of subjects with previous exposure to mineral particles had high lung retention of particles containing cerium. This study is believed to be the first one in which lung retention of cerium was estimated in the general population. PMID:8130849

  16. Tiny Device Mimics Human Lung Function

    ScienceCinema

    McDonald, Rebecca; Harris, Jennifer; Nath, Pulak

    2016-07-12

    Scientists at Los Alamos National Laboratory are developing a miniature, tissue-engineered artificial lung that mimics the response of the human lung to drugs, toxins and other agents. “We breathe in and out thousands of times every day. And while we have control over what we eat or drink, we don’t always have control over what we breathe in,” said Jennifer Harris of Biosecurity and Public Health at Los Alamos, "and so we’re making this miniature lung to be able to test on actual human cells whether something in the environment, or a drug, is toxic or harmful to us." Nicknamed “PuLMo” for Pulmonary Lung Model (Pulmo is also the Latin word for "lung")the device consists of two major parts, the bronchiolar unit and the alveolar unit—just like the human lung. The units are primarily made from various polymers and are connected by a microfluidic “circuit board” that manages fluid and air flow. “When we build our lung, we not only take into account the aspects of different cell types, the tissues that are involved, we also take into account that a lung is supposed to breathe, so PuLMo actually breathes,” said Pulak Nath of Applied Modern Physics, who leads engineering efforts for the project. The most exciting application of PuLMo is a potentially revolutionary improvement in the reliability of drug-toxicity assessments and the prediction of new pharmaceutical success in humans, according to Harris. The PuLMo may also be designed to mimic lung disease conditions, such as Chronic Obstructive Pulmonary Disease (COPD) and asthma, and may be used to study lung air-flow dynamics to better understand the mechanisms of toxins and drug delivery and the effects of smoking, particularly the less-understood effects of e-cigarettes.

  17. 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

  18. MALDI Profiling of Human Lung Cancer Subtypes

    PubMed Central

    Nistal, Manuel; Calvo, Enrique; Madero, Rosario; Díaz, Esther; Camafeita, Emilio; de Castro, Javier; López, Juan Antonio; González-Barón, Manuel; Espinosa, Enrique; Fresno Vara, Juan Ángel

    2009-01-01

    Background Proteomics is expected to play a key role in cancer biomarker discovery. Although it has become feasible to rapidly analyze proteins from crude cell extracts using mass spectrometry, complex sample composition hampers this type of measurement. Therefore, for effective proteome analysis, it becomes critical to enrich samples for the analytes of interest. Despite that one-third of the proteins in eukaryotic cells are thought to be phosphorylated at some point in their life cycle, only a low percentage of intracellular proteins is phosphorylated at a given time. Methodology/Principal Findings In this work, we have applied chromatographic phosphopeptide enrichment techniques to reduce the complexity of human clinical samples. A novel method for high-throughput peptide profiling of human tumor samples, using Parallel IMAC and MALDI-TOF MS, is described. We have applied this methodology to analyze human normal and cancer lung samples in the search for new biomarkers. Using a highly reproducible spectral processing algorithm to produce peptide mass profiles with minimal variability across the samples, lineal discriminant-based and decision tree–based classification models were generated. These models can distinguish normal from tumor samples, as well as differentiate the various non–small cell lung cancer histological subtypes. Conclusions/Significance A novel, optimized sample preparation method and a careful data acquisition strategy is described for high-throughput peptide profiling of small amounts of human normal lung and lung cancer samples. We show that the appropriate combination of peptide expression values is able to discriminate normal lung from non-small cell lung cancer samples and among different histological subtypes. Our study does emphasize the great potential of proteomics in the molecular characterization of cancer. PMID:19890392

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

    PubMed

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

    2015-03-01

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

  20. In vitro inhibitory effect of 1-aminobenzotriazole on drug oxidations in human liver microsomes: a comparison with SKF-525A.

    PubMed

    Emoto, Chie; Murase, Shigeo; Sawada, Yasufusa; Iwasaki, Kazuhide

    2005-10-01

    1-Aminobenzotriazole (ABT) is extensively used as a non-specific cytochrome P450 (CYP) inhibitor. In this study, the inhibitory effect of ABT on CYP-dependent drug oxidations was investigated in human liver microsomes (HLM) and compared with that of SKF-525A, another non-specific inhibitor. The following probe activities for human CYP isoforms were determined using pooled HLM: phenacetin O-deethylation (CYP1A2); diclofenac 4'-hydroxylation (CYP2C9); S-mephenytoin 4'-hydroxylation, (CYP2C19); bufuralol 1'-hydroxylation (CYP2D6); chlorzoxazone 6-hydroxylation (CYP2E1); midazolam 1'-hydroxylation, nifedipine oxidation, and testosterone 6beta-hydroxylation (CYP3A). ABT had the strongest inhibitory effect on the CYP3A-dependent drug oxidations and the weakest effect on the diclofenac 4'-hydroxylation. SKF-525A potently inhibited the bufuralol 1'-hydroxylation, but weakly inhibited chlorzoxazone 6-hydroxylation. The inhibitory effects of ABT and SKF-525A were increased by preincubation in some probe reactions, and this preincubation effect was greater in ABT than in SKF-525A. The remarkable IC50 shift (> 10 times) by preincubation with ABT was observed on the phenacetin O-deethylation, chlorzoxazone 6-hydroxylation, and midazolam 1'-hydroxylation. In conclusion, ABT and SKF-525A had a wide range of IC50 values in inhibiting the drug oxidations by HLM with and without preincubation.

  1. Microsomal and cytosolic scaling factors in dog and human kidney cortex and application for in vitro-in vivo extrapolation of renal metabolic clearance.

    PubMed

    Scotcher, Daniel; Billington, Sarah; Brown, Jay; Jones, Christopher; Brown, Colin D A; Rostami-Hodjegan, Amin; Galetin, Aleksandra

    2017-03-07

    In vitro-in vivo extrapolation of drug metabolism data obtained in enriched preparations of sub-cellular fractions rely upon robust estimates of physiologically relevant scaling factors for prediction of clearance in vivo. The purpose of the current study was to measure the microsomal and cytosolic protein per gram of kidney (MPPGK and CPPGK) in dog and human kidney cortex using appropriate protein recovery markers, and evaluate functional activity of human cortex microsomes. Cytochrome P450 (CYP) content and glucose-6-phosphatase activity were used as microsomal protein markers, whereas glutathione-S-transferase activity was a cytosolic marker. Functional activity of human microsomal samples was assessed by measuring mycophenolic acid glucuronidation. MPPGK was 33.9 and 44.0 mg/g dog kidney cortex, and 41.1 and 63.6 mg/g dog liver (n=17), using CYP content and glucose-6-phosphatase activity, respectively. There were no trends between kidney, liver and intestinal scalars from the same animals. Species differences were evident, as human MPPGK and CPPGK were 26.2 and 53.3 mg/g kidney cortex (n=38), respectively. MPPGK was 2-fold higher than the commonly used in vitro-in vivo extrapolation scalar; difference was mainly attributed to tissue source (mixed kidney regions vs cortex). Robust human MPPGK and CPPGK scalars were measured for the first time. The work emphasized the importance of regional differences (cortex vs. whole kidney specific MPPGK, tissue weight and blood flow) and a need to account for these to improve assessment of renal metabolic clearance and its extrapolation to in vivo.

  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. 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.

  4. Primary deficiency of microsomal triglyceride transfer protein in human abetalipoproteinemia is associated with loss of CD1 function.

    PubMed

    Zeissig, Sebastian; Dougan, Stephanie K; Barral, Duarte C; Junker, Yvonne; Chen, Zhangguo; Kaser, Arthur; Ho, Madelyn; Mandel, Hannah; McIntyre, Adam; Kennedy, Susan M; Painter, Gavin F; Veerapen, Natacha; Besra, Gurdyal S; Cerundolo, Vincenzo; Yue, Simon; Beladi, Sarah; Behar, Samuel M; Chen, Xiuxu; Gumperz, Jenny E; Breckpot, Karine; Raper, Anna; Baer, Amanda; Exley, Mark A; Hegele, Robert A; Cuchel, Marina; Rader, Daniel J; Davidson, Nicholas O; Blumberg, Richard S

    2010-08-01

    Abetalipoproteinemia (ABL) is a rare Mendelian disorder of lipid metabolism due to genetic deficiency in microsomal triglyceride transfer protein (MTP). It is associated with defects in MTP-mediated lipid transfer onto apolipoprotein B (APOB) and impaired secretion of APOB-containing lipoproteins. Recently, MTP was shown to regulate the CD1 family of lipid antigen-presenting molecules, but little is known about immune function in ABL patients. Here, we have shown that ABL is characterized by immune defects affecting presentation of self and microbial lipid antigens by group 1 (CD1a, CD1b, CD1c) and group 2 (CD1d) CD1 molecules. In dendritic cells isolated from ABL patients, MTP deficiency was associated with increased proteasomal degradation of group 1 CD1 molecules. Although CD1d escaped degradation, it was unable to load antigens and exhibited functional defects similar to those affecting the group 1 CD1 molecules. The reduction in CD1 function resulted in impaired activation of CD1-restricted T and invariant natural killer T (iNKT) cells and reduced numbers and phenotypic alterations of iNKT cells consistent with central and peripheral CD1 defects in vivo. These data highlight MTP as a unique regulator of human metabolic and immune pathways and reveal that ABL is not only a disorder of lipid metabolism but also an immune disease involving CD1.

  5. 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.

  6. 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

  7. Stable isotope labeling strategy for curcumin metabolite study in human liver microsomes by liquid chromatography-tandem mass spectrometry.

    PubMed

    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 (18)O 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 (18)O labeled curcumin was successfully synthesized. The non-labeled and (18)O 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.

  8. Search for α3β₂/₃γ2 subtype selective ligands that are stable on human liver microsomes.

    PubMed

    Namjoshi, Ojas A; Wang, Zhi-jian; Rallapalli, Sundari K; Johnson, Edward Merle; Johnson, Yun-Teng; Ng, Hanna; Ramerstorfer, Joachim; Varagic, Zdravko; Sieghart, Werner; Majumder, Samarpan; Roth, Bryan L; Rowlett, James K; Cook, James M

    2013-01-01

    Selective modulation of specific benzodiazepine receptor (BzR) gamma amino butyric acid-A (GABA(A)) receptor ion channels has been identified as an important method for separating out the variety of pharmacological effects elicited by BzR-related drugs. Importantly, it has been demonstrated that both α2β(2/3)γ2 (α2BzR) and α3BzR (and/or α2/α3) BzR subtype selective ligands exhibit anxiolytic effects with little or no sedation. Previously we have identified several such ligands; however, three of our parent ligands exhibited significant metabolic liability in rodents in the form of a labile ester group. Here eight analogs are reported which were designed to circumvent this liability by utilizing a rational replacement of the ester moiety based on medicinal chemistry precedents. In a metabolic stability study using human liver microsomes, four compounds were found to undergo slower metabolic transformation, as compared to their corresponding ester analogs. These compounds were also evaluated in in vitro efficacy assays. Additionally, bioisostere 11 was evaluated in a rodent model of anxiety. It exhibited anxiolytic activity at doses of 10 and 100mg/kg and was devoid of sedative properties.

  9. 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

    2016-09-05

    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.

  10. Analysis of R- and S-Hydroxywarfarin Glucuronidation Catalyzed by Human Liver Microsomes and Recombinant UDP-Glucuronosyltransferases

    PubMed Central

    Bratton, Stacie M.; Mosher, Carrie M.; Khallouki, Farid; Finel, Moshe; Court, Michael H.; Moran, Jeffery H.

    2012-01-01

    Coumadin (R-, S-warfarin) is a challenging drug to accurately dose, both initially and for maintenance, because of its narrow therapeutic range and wide interpatient variability and is typically administered as a racemic (Rac) mixture, which complicates the biotransformation pathways. The goal of the current work was to identify the human UDP-glucuronosyltransferases (UGTs) involved in the glucuronidation of the separated R- and S-enantiomers of 6-, 7-, and 8-hydroxywarfarin and the possible interactions between these enantiomers. The kinetic and inhibition constants for human recombinant 1A family UGTs toward these separated enantiomers have been assessed using high-performance liquid chromatography (HPLC)-UV-visible analysis, and product confirmations have been made using HPLC-mass spectrometry/mass spectrometry. We found that separated R- and S-enantiomers of 6-, 7-, and 8-hydroxywarfarin demonstrate significantly different glucuronidation kinetics and can be mutually inhibitory. In some cases significant substrate inhibition was observed, as shown by Km, Vmax, and Ki, comparisons. In particular, UGT1A1 and extrahepatic UGT1A10 have significantly higher capacities than other isoforms for S-7-hydroxywarfarin and R-7-hydroxywarfarin glucuronidation, respectively. Activity data generated using a set of well characterized human liver microsomes supported the recombinant enzyme data, suggesting an important (although not exclusive) role for UGT1A1 in glucuronidation of the main warfarin metabolites, including Rac-6- and 7-hydroxywarfarin and their R- and S-enantiomers in the liver. This is the first demonstration that the R- and S-enantiomers of hydroxywarfarins are glucuronidated, with significantly different enzymatic affinity and capacity, and supports the importance of UGT1A1 as the major hepatic isoform involved. PMID:21972237

  11. 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

  12. Cell-free synthesis of functional human epidermal growth factor receptor: Investigation of ligand-independent dimerization in Sf21 microsomal membranes using non-canonical amino acids

    PubMed Central

    Quast, Robert B.; Ballion, Biljana; Stech, Marlitt; Sonnabend, Andrei; Varga, Balázs R.; Wüstenhagen, Doreen A.; Kele, Péter; Schiller, Stefan M.; Kubick, Stefan

    2016-01-01

    Cell-free protein synthesis systems represent versatile tools for the synthesis and modification of human membrane proteins. In particular, eukaryotic cell-free systems provide a promising platform for their structural and functional characterization. Here, we present the cell-free synthesis of functional human epidermal growth factor receptor and its vIII deletion mutant in a microsome-containing system derived from cultured Sf21 cells. We provide evidence for embedment of cell-free synthesized receptors into microsomal membranes and asparagine-linked glycosylation. Using the cricket paralysis virus internal ribosome entry site and a repetitive synthesis approach enrichment of receptors inside the microsomal fractions was facilitated thereby providing analytical amounts of functional protein. Receptor tyrosine kinase activation was demonstrated by monitoring receptor phosphorylation. Furthermore, an orthogonal cell-free translation system that provides the site-directed incorporation of p-azido-L-phenylalanine is characterized and applied to investigate receptor dimerization in the absence of a ligand by photo-affinity cross-linking. Finally, incorporated azides are used to generate stable covalently linked receptor dimers by strain-promoted cycloaddition using a novel linker system. PMID:27670253

  13. Pharmacokinetic study in pigs and in vitro metabolic characterization in pig- and human-liver microsomes reveal marked differences in disposition and metabolism of tiletamine and zolazepam (Telazol).

    PubMed

    Kumar, Atul; Mann, Henry J; Remmel, Rory P; Beilman, Greg J; Kaila, Nitin

    2014-04-01

    1. An equal-dose combination of tiletamine and zolazepam (Telazol®) is used as a veterinary anesthetic. There also have been reports of human abuse of Telazol®. The pharmacokinetics and metabolic fate of tiletamine and zolazepam and the rationale for their administration as an equal-dose combination are unclear. 2. The single-dose pharmacokinetics of intramuscular tiletamine and zolazepam (3 mg/kg each) in 16 Yorkshire-crossbred pigs were determined. The metabolites of tiletamine and zolazepam in pig plasma and urine were identified by mass spectrometry. The metabolic stability of tiletamine and zolazepam and the kinetics of formation of their metabolites by pig- and human-liver microsomes were determined. 3. Higher concentrations of zolazepam were observed in pig plasma and it was cleared more slowly compared to tiletamine (apparent clearance: 11 versus 134 l/h; half-life: 2.76 versus 1.97 h). Three metabolites of zolazepam and one metabolite of tiletamine were identified in pig urine, plasma and in microsomal incubations. In vitro formation of each of these metabolites in microsomes was biphasic involving a high-affinity/low-capacity and a low-affinity/high-capacity enzyme. The in vitro metabolic stability of tiletamine was considerably lower compared to zolazepam. 4. These results collectively point to major pharmacokinetic and metabolic differences between the two components of this fixed-dose anesthetic combination.

  14. 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.

  15. Metabolic heterogeneity in human lung tumors

    PubMed Central

    Hensley, Christopher T.; Faubert, Brandon; Yuan, Qing; Lev-Cohain, Naama; Jin, Eunsook; Kim, Jiyeon; Jiang, Lei; Ko, Bookyung; Skelton, Rachael; Loudat, Laurin; Wodzak, Michelle; Klimko, Claire; McMillan, Elizabeth; Butt, Yasmeen; Ni, Min; Oliver, Dwight; Torrealba, Jose; Malloy, Craig R.; Kernstine, Kemp; Lenkinski, Robert E.; DeBerardinis, Ralph J.

    2015-01-01

    SUMMARY Non-small cell lung cancer (NSCLC) is heterogeneous in the genetic and environmental parameters that influence cell metabolism in culture. Here, we assessed the impact of these factors on human NSCLC metabolism in vivo using intra-operative 13C-glucose infusions in nine NSCLC patients to compare metabolism between tumors and benign lung. While enhanced glycolysis and glucose oxidation were common among these tumors, we observed evidence for oxidation of multiple nutrients in each of them, including lactate as a potential carbon source. Moreover, metabolically heterogeneous regions were identified within and between tumors, and surprisingly, our data suggested potential contributions of non-glucose nutrients in well-perfused tumor areas. Our findings not only demonstrate the heterogeneity in tumor metabolism in vivo but also highlight the strong influence of the microenvironment on this feature. PMID:26853473

  16. A novel SCID mouse model for studying spontaneous metastasis of human lung cancer to human tissue.

    PubMed

    Teraoka, S; Kyoizumi, S; Seyama, T; Yamakido, M; Akiyama, M

    1995-05-01

    We established a novel severe combined immunodeficient (SCID) mouse model for the study of human lung cancer metastasis to human lung. Implantation of both human fetal and adult lung tissue into mammary fat pads of SCID mice showed a 100% rate of engraftment, but only fetal lung implants revealed normal morphology of human lung tissue. Using these chimeric mice, we analyzed human lung cancer metastasis to both mouse and human lungs by subcutaneous inoculation of human squamous cell carcinoma and adenocarcinoma cell lines into the mice. In 60 to 70% of SCID mice injected with human-lung squamous-cell carcinoma, RERF-LC-AI, cancer cells were found to have metastasized to both mouse lungs and human fetal lung implants but not to human adult lung implants 80 days after cancer inoculation. Furthermore, human-lung adenocarcinoma cells, RERF-LC-KJ, metastasized to the human lung implants within 90 days in about 40% of SCID mice, whereas there were no metastases to the lungs of the mice. These results demonstrate the potential of this model for the in vivo study of human lung cancer metastasis.

  17. Morphometric examination of native lungs in human lung allograft recipients.

    PubMed

    Wiebe, B M; Burton, C M; Milman, N; Iversen, M; Andersen, C B

    2006-11-01

    The aim of the study was to estimate the degree of lung damage in patients with alpha(1)-antitrypsin (alpha1AT) deficiency, chronic obstructive pulmonary disease (COPD), and cystic fibrosis (CF) at the time of lung transplantation. Using unbiased stereological methods, lung-, bronchial- and vessel-volume, capillary length, and alveolar surface area and densities were estimated in recipient lungs from 21 consecutive patients with pre-transplant diagnoses including COPD (n=7), alpha1AT deficiency (n=6) and CF (n=8). Six unused adult donor lungs served as controls. Information relating to patient demography and pre-transplant lung function was obtained by retrospective chart review. Disease groups differed significantly with respect to demographics and pre-transplant lung function. Total lung volume was similar in all groups. Bronchial volume was significantly larger in CF patients compared to the control group (p<0.0001) and to the other two diagnostic groups: alpha1AT deficiency (p=0.0001) and COPD (p<0.0001). Alveolar surface density and capillary length density were significantly lower in patients with alpha1AT deficiency and COPD compared to controls (p<0.0001, respectively) and to patients with CF (p<0.0002, respectively). There were no correlations between clinical lung function and morphometric measurements. We conclude that unbiased microscopic stereological morphometry is an evolving science with the potential to elucidate pulmonary disease pathogenesis.

  18. Identification of a novel glutathione adduct of diclofenac, 4'-hydroxy-2'-glutathion-deschloro-diclofenac, upon incubation with human liver microsomes.

    PubMed

    Yu, Li J; Chen, Yue; Deninno, Michael P; O'Connell, Thomas N; Hop, Cornelis E C A

    2005-04-01

    Clinical use of the nonsteroidal anti-inflammatory drug diclofenac (DF) is associated with an incidence of idiosyncratic hepatoxicity. The formation of reactive metabolites of DF in vivo has been proposed to be responsible for such toxicity. One type of reactive metabolite, a benzoquinone imine of DF formed through oxidation by cytochromes P450, can be trapped by glutathione in vitro in liver microsomes to form glutathione (GS) adducts. Three GS adducts from DF were reported in the literature, namely, 5-hydroxy (OH)-4-glutathione-DF, 4'-OH-3'-glutathione-DF and 5-OH-6-glutathione-DF, and they all have the same molecular weight of 616. Recently, we developed a sensitive and high throughput method for the detection of GS adducts from liver microsome incubation. This method uses a constant neutral loss scan of m/z 129, a "structure-characteristic" fragment for GS adduct, on an automated chip-based nanoelectrospray (Advion NanoMate 100) attached to a tandem mass spectrometer (Sciex API 3000). The analysis of GS adducts from human liver microsome incubation with DF by the NanoMate 100-API 3000 method unambiguously revealed a new adduct ion with m/z 583 (MH+), in addition to the known adduct peak with m/z 617 (MH+). This new adduct was further confirmed to be 4'-OH-2'-glutathion-deschloro-diclofenac by liquid chromatography (LC) tandem mass spectrometry (MS), LC/MS-NMR, and comparison to a synthetic standard.

  19. Photodynamic effects of new silicon phthalocyanines: in vitro studies utilizing rat hepatic microsomes and human erythrocyte ghosts as model membrane sources.

    PubMed

    Zaidi, S I; Agarwal, R; Eichler, G; Rihter, B D; Kenney, M E; Mukhtar, H

    1993-08-01

    Photodynamic therapy (PDT) of cancer is a modality that relies upon the irradiation of tumors with visible light following selective uptake of a photosensitizer by the tumor tissue. There is considerable emphasis to define new photosensitizers suitable for PDT of cancer. In this study we evaluated six phthalocyanines (Pc) for their photodynamic effects utilizing rat hepatic microsomes and human erythrocyte ghosts as model membrane sources. Of the newly synthesized Pc, two showed significant destruction of cytochrome P-450 and monooxygenase activities, and enhancement of lipid peroxidation, when added to microsomal suspension followed by irradiation with approximately 675 nm light. These two Pc named SiPc IV (HOSiPcOSi[CH3]2[CH2]3N[CH3]2) and SiPc V (HOSiPc-OSi[CH3]2[CH2]3N[CH3]3+I-) showed dose-dependent photodestruction of cytochrome P-450 and monooxygenase activities in liver microsomes, and photoenhancement of lipid peroxidation, lipid hydroperoxide formation and lipid fluorescence in microsomes and erythrocyte ghosts. Compared to chloroaluminum phthalocyanine tetrasulfonate, SiPc IV and SiPc V produced far more pronounced photodynamic effects. Sodium azide, histidine, and 2,5-dimethylfuran, the quenchers of singlet oxygen, afforded highly significant protection against SiPc IV- and SiPc V-mediated photodynamic effects. However, to a lesser extent, the quenchers of superoxide anion, hydrogen peroxide and hydroxyl radical also showed some protective effects. These results suggest that SiPc IV and SiPc V may be promising photosensitizers for the PDT of cancer.

  20. 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...

  1. Development of Monoclonal Antibodies to Human Microsomal Epoxide Hydrolase and Analysis of “Preneoplastic Antigen”-Like Molecules

    PubMed Central

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

    2012-01-01

    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 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; 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

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

    PubMed

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

    2011-05-01

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

  3. Oxidative metabolism of flunarizine and cinnarizine by microsomes from B-lymphoblastoid cell lines expressing human cytochrome P450 enzymes.

    PubMed

    Kariya, S; Isozaki, S; Uchino, K; Suzuki, T; Narimatsu, S

    1996-11-01

    The oxidative metabolism of cinnarizine [(E)-1-(diphenylmethyl)-4-(3-phenyl-2-propyl)piperazine, CZ] and flunarizine [(E)-1-[bis(4-fluorophenyl)methyl]-4-(3-phenyl-2-propyl)piperazine, FZ] was examined in microsomes from lymphoblastoid cells that expressed human cytochrome P450 (CYP) enzymes. Among 10 kinds of CYP enzymes examined, only CYP2D6 catalyzed p-hydroxylation of the cinnamyl phenyl ring of CZ (C-2 formation) and FZ (F-2 formation), and only CYP2B6 exhibited activity for p-hydroxylation (C-4 formation) of the diphenylmethyl group of CZ at a substrate concentration of 50 microM. On the other hand, CYP2C9 together with CYP1A1, -1A2 and/or -2A6 mediated N-desalkylation at the 1- and 4-positions of the piperazine ring of the two drugs that formed C-1 and C-3 from CZ and F-1 and F-3 from FZ, respectively, whereas CYP2C8, -2C19, -2E1 or -3A4 did not show detectable activity for these reactions under the conditions used. We then examined kinetics for the oxidative metabolism of CZ and FZ using CYP2B6 and -2D6 that have considerable activities. CYP2D6 with Km values of 2 to 4 microM had intrinsic clearance values (Vmax/Km) of 0.31 and 0.14 ml/min/nmol CYP for C-2 and F-2 formation, respectively, while CYP2B6 with a Km value of 17 microM exhibited the clearance value of 0.10 ml/min/nmol CYP for C-4 formation. These results suggest that CYP2D6 mainly mediates p-hydroxylation of the cinnamyl phenyl rings of CZ and FZ, and CYP2B6 mediates that of the diphenylmethyl group of CZ.

  4. Inhibitory effects of seven components of danshen extract on catalytic activity of cytochrome P450 enzyme in human liver microsomes.

    PubMed

    Qiu, Furong; Zhang, Rong; Sun, Jianguo; Jiye, A; Hao, Haiping; Peng, Ying; Ai, Hua; Wang, Guangji

    2008-07-01

    The potential for herb-drug interactions has recently received greater attention worldwide, considering the fact that the use of herbal products becomes more and more widespread. The goal of this work was to examine the potential for the metabolism-based drug interaction arising from seven active components (danshensu, protocatechuic aldehyde, protocatechuic acid, salvianolic acid B, tanshinone I, tanshinone IIA, and cryptotanshinone) of danshen extract. Probe substrates of cytochrome P450 enzymes were incubated in human liver microsomes (HLMs) with or without each component of danshen extract. IC(50) and K(i) values were estimated, and the types of inhibition were determined. Among the seven components of danshen extract, tanshinone I, tanshinone IIA, and cryptotanshinone were potent competitive inhibitors of CYP1A2 (K(i) = 0.48, 1.0, and 0.45 microM, respectively); danshensu was a competitive inhibitor of CYP2C9 (K(i) = 35 microM), and cryptotanshinone was a moderate mixed-type inhibitor of CYP2C9 (K(i) = 8 microM); cryptotanshinone inhibited weakly and in mixed mode against CYP2D6 activity (K(i) = 68 microM), and tanshinone I was a weak inhibitor of CYP2D6 (IC(50) = 120 microM); and protocatechuic aldehyde was a weak inhibitor of CYP3A4 (IC(50) = 130 and 160 microM for midazolam and testosterone, respectively). These findings provided some useful information for safe and effective use of danshen preparations in clinical practice. Our data indicated that it was necessary to study the in vivo interactions between drugs and pharmaceuticals with danshen extract.

  5. Drug-drug Interaction between Losartan and Paclitaxel in Human Liver Microsomes with Different CYP2C8 Genotypes.

    PubMed

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

    2015-06-01

    The cytochrome P450 (CYP) 2C8*3 allele is associated with reduced metabolic activity of paclitaxel. This study was aimed to investigate the inhibitory effect of losartan on paclitaxel metabolism in human liver microsomes (HLMs) and to determine the impact of the CYP2C8*3 polymorphism. HLMs that contained the CYP2C8*1 homozygote (HL60) or CYP2C8*3 heterozygote (HL54) genotype were used for the inhibition study. Losartan, at a concentration of 50 μmol/L, significantly inhibited paclitaxel metabolism by 29% and 57% in the HL60 (p < 0.001) and HL54 (p < 0.01), respectively. When using HL60, losartan and the CYP3A4-selective inhibitors, erythromycin and ketoconazole, caused a greater inhibition of the paclitaxel metabolism than quercetin, a CYP2C8-selective inhibitor. This demonstrated that the paclitaxel metabolism was mainly catalysed by CYP3A4 in HL60. There were no significant differences found for the inhibitory effects caused by the four inhibitors of the paclitaxel metabolism in HL54, indicating that both CYP2C8 and CYP3A4 play important roles in paclitaxel metabolism in HL54. These findings suggest that 50 μmol/L of losartan inhibits both CYP2C8 and CYP3A4 in HLMs. In summary, losartan inhibited paclitaxel metabolism, with concentrations over 50 μmol/L in HLMs. The CYP2C8*3 allele carriers are likely susceptible to the interactions of losartan and CYP3A4 inhibitors to paclitaxel metabolism.

  6. Metabolism of methyl tert-butyl ether and other gasoline ethers by human liver microsomes and heterologously expressed human cytochromes P450: identification of CYP2A6 as a major catalyst.

    PubMed

    Hong, J Y; Wang, Y Y; Bondoc, F Y; Lee, M; Yang, C S; Hu, W Y; Pan, J

    1999-10-01

    To reduce the production of carbon monoxide and other pollutants in motor vehicle exhaust, methyl tert-butyl ether (MTBE), ethyl tert-butyl ether (ETBE), and tert-amyl methyl ether (TAME) are added to gasoline as oxygenates for more complete combustion. Previously, we demonstrated that human liver is active in metabolizing MTBE to tert-butyl alcohol (TBA) and that cytochrome P450 (CYP) enzymes play a critical role in the metabolism of MTBE. The present study demonstrates that human liver is also active in the oxidative metabolism of ETBE and TAME. A large interindividual variation in metabolizing these gasoline ethers was observed in 15 human liver microsomal samples. The microsomal activities in metabolizing MTBE, ETBE, and TAME were highly correlated among each other (r, 0.91-0. 96), suggesting that these ethers are metabolized by the same enzyme(s). Correlation analysis of the ether-metabolizing activities with individual CYP enzyme activities in the liver microsomes showed that the highest degree of correlation was with human CYP2A6 (r, 0. 90-0.95), which is constitutively expressed in human livers and known to be polymorphic. CYP2A6 displayed the highest turnover number in metabolizing gasoline ethers among a battery of human CYP enzymes expressed in human B-lymphoblastoid cells. Kinetic studies on MTBE metabolism with three human liver microsomes exhibited apparent Km values that ranged from 28 to 89 microM and the V(max) values from 215 to 783 pmol/min/mg, with similar catalytic efficiency values (7.7 to 8.8 microl/min/mg protein). Metabolism of MTBE, ETBE, and TAME by human liver microsomes was inhibited by coumarin, a known substrate of human CYP2A6, in a concentration-dependent manner. Monoclonal antibody against human CYP2A6 caused a significant inhibition (75% to 95%) of the metabolism of MTBE, ETBE, and TAME in human liver microsomes. Taken together, these results clearly indicate that in human liver, CYP2A6 is the major enzyme responsible for the

  7. 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

  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. Amiloride-inhibited Na -H exchange in human kidney medulla microsomes

    SciTech Connect

    LaBelle, E.F.

    1986-08-01

    Membrane vesicles from the medulla of the human kidney are capable of amiloride-inhibited sodium-proton exchange. Sodium uptake into the vesicles in stimulated by low intravesicular pH (6.0) and inhibited by both low extravesicular pH (6.0) and amiloride (2 mM). Sodium uptake is a linear function of the vesicular protein concentration and can be reversed by raising the osmotic strength of the solution to shrink the vesicle or by adding the inophore nigericin. Acridine orange was accumulated by the vesicles in response to a pH gradient (pH 6.0, inside, 8.0 outside), and the accumulation of acridine orange could be reduced by increase extravesicular sodium. Thus sodium could stimulate proton movement just as protons could stimulate sodium movement across the vesicular membrane.

  10. In Vitro Glucuronidation of Fenofibric Acid by Human UDP-Glucuronosyltransferases and Liver Microsomes

    PubMed Central

    Tojcic, Jelena; Benoit-Biancamano, Marie-Odile; Court, Michael H.; Straka, Robert J.; Caron, Patrick

    2009-01-01

    Fenofibric acid (FA), the active moiety of fenofibrate, is an agonist of the peroxisome proliferator-activated nuclear receptor α that modulates triglyceride and cholesterol profiles. Lipid response to fenofibrate and FA serum concentrations is highly variable. Although FA is reported to be almost exclusively inactivated by UDP-glucuronosyltransferases (UGTs) into FA-glucuronide (FA-G), the contribution of UGT isoenzymes has never been systematically assessed. Heterologously expressed human UGT1A and UGT2B and their coding variants were tested for FA glucuronidation using liquid chromatography/mass spectrometry. Recombinant UGT2B7 presented the highest Vmax/Km value (2.10 μl/min/mg), 16-fold higher than the activity of other reactive UGTs, namely, UGT1A3, UGT1A6, and UGT1A9 (0.13, 0.09, and 0.02 μl/min/mg, respectively). UGT2B7.1 (His268) and UGT2B7.2 (Tyr268) enzyme activity was similar, whereas UGT1A3.2 (R11A47), UGT1A3.3 (Trp11), and UGT1A9.3 (Thr33) showed 61 to 96% reduced Vmax/Km values compared with the respective (1) reference proteins. FA-G formation by a human liver bank (n = 48) varied by 10-fold, but the rate of formation was not associated with common genetic variations in UGT1A3, UGT1A6, UGT1A9, and UGT2B7. Correlation with activities for the probe substrates zidovudine (UGT2B7; r2 = 0.75), mycophenolic acid (UGT1A9; r2 = 0.42), fulvestrant (UGT1A3; r2 = 0.36), but not serotonin (UGT1A6; r2 = 0.06) indicated a primary role for UGT2B7 and lesser roles of UGT1A9 and UGT1A3 in hepatic FA glucuronidation. This was confirmed by a strong correlation of FA-G formation with UGT2B7 protein content and inhibition by fluconazole, a known UGT2B7 selective inhibitor. Additional studies are required to identify genetic factors contributing to the observed FA glucuronidation variability. PMID:19661212

  11. Prediction of the in vivo interaction between midazolam and macrolides based on in vitro studies using human liver microsomes.

    PubMed

    Ito, Kiyomi; Ogihara, Kanako; Kanamitsu, Shin-Ichi; Itoh, Tomoo

    2003-07-01

    Clinical studies have revealed that plasma concentrations of midazolam after oral administration are greatly increased by coadministration of erythromycin and clarithromycin, whereas azithromycin has little effect on midazolam concentrations. Several macrolide antibiotics are known to be mechanism-based inhibitors of CYP3A, a cytochrome P450 isoform responsible for midazolam hydroxylation. The aim of the present study was to quantitatively predict in vivo drug interactions in humans involving macrolide antibiotics with different inhibitory potencies based on in vitro studies. alpha- and 4-Hydroxylation of midazolam by human liver microsomes were evaluated as CYP3A-mediated metabolic reactions, and the effect of preincubation with macrolides was examined. The hydroxylation of midazolam was inhibited in a time- and concentration-dependent manner following preincubation with macrolides in the presence of NADPH, whereas almost no inhibition was observed without preincubation. The kinetic parameters for enzyme inactivation (K'app and kinact) involved in midazolam alpha-hydroxylation were 12.6 microM and 0.0240 min-1, respectively, for erythromycin, 41.4 microM and 0.0423 min-1, respectively, for clarithromycin, and 623 microM and 0.0158 min-1, respectively, for azithromycin. Similar results were obtained for the 4-hydroxylation pathway. These parameters and the reported pharmacokinetic parameters of midazolam and macrolides were then used to simulate in vivo interactions based on a physiological flow model. The area under the concentration-time curve (AUC) of midazolam after oral administration was predicted to increase 2.9- or 3.0-fold following pretreatment with erythromycin (500 mg t.i.d. for 5 or 6 days, respectively) and 2.1- or 2.5-fold by clarithromycin (250 mg b.i.d. for 5 days or 500 mg b.i.d. for 7 days, respectively), whereas azithromycin (500 mg o.d. for 3 days) was predicted to have little effect on midazolam AUC. These results agreed well with the reported

  12. Identification of cytochrome p450 enzymes involved in the metabolism of 4'-methyl-alpha-pyrrolidinopropiophenone, a novel scheduled designer drug, in human liver microsomes.

    PubMed

    Springer, Dietmar; Paul, Liane D; Staack, Roland F; Kraemer, Thomas; Maurer, Hans H

    2003-08-01

    4'-Methyl-alpha-pyrrolidinopropiophenone (MPPP) is a new drug of abuse. It is believed to have an abuse potential similar to that of amphetamines. Previous studies with Wistar rats had shown that MPPP was metabolized mainly by hydroxylation in position 4' followed by dehydrogenation to the corresponding carboxylic acid. The aim of the study presented here was to identify the human hepatic cytochrome p450 (p450) enzymes involved in the biotransformation of MPPP to 4'-hydroxymethyl-pyrrolidinopropiophenone. Baculovirus-infected insect cell microsomes and human liver microsomes were used for this purpose. Only CYP2C19 and CYP2D6 catalyzed this hydroxylation. The apparent Km and Vmax values for the latter were 9.8 +/- 2.5 microM and 13.6 +/- 0.7 pmol/min/pmol p450, respectively. CYP2C19 was not saturable over the tested substrate range (2-1000 microM) and interestingly showed a biphasic kinetic profile with apparent Km,1 and Vmax,1 values of 47.2 +/- 12.5 microM and 8.1 +/- 1.4 pmol/min/pmol p450, respectively. Experiments with pooled human liver microsomes also revealed biphasic nonsaturable kinetics with apparent Km,1 and Vmax,1 values of 57.0 +/- 20.9 microM and 199.7 +/- 59.7 pmol/min/mg of protein for the high affinity enzyme, respectively. Incubation of 2 microM MPPP with 3 microM of the CYP2D6-specific inhibitor quinidine resulted in significant (p < 0.01) turnover inhibition (11.8 +/- 1.6% of control). Based on kinetic data corrected for the relative activity factors, CYP2D6 is the enzyme mainly responsible for MPPP hydroxylation, confirmed by CYP2D6 inhibition studies.

  13. 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.

  14. Antithyroid microsomal antibody

    MedlinePlus

    Thyroid antimicrosomal antibody; Antimicrosomal antibody; Microsomal antibody; Thyroid peroxidase antibody; TPOAb ... Granulomatous thyroiditis Hashimoto thyroiditis High levels of these antibodies have also been linked with an increased risk ...

  15. In vitro inhibition of human CYP2E1 and CYP3A by quercetin and myricetin in hepatic microsomes is not gender dependent.

    PubMed

    Östlund, Johanna; Zlabek, Vladimir; Zamaratskaia, Galia

    2017-04-15

    This is the first in vitro study to investigate gender-related differences in the regulation of human cytochrome P450 by the flavonoids. Activities of CYP2E1 and CYP3A were measured in the presence of quercetin, myricetin, or isorhamnetin in hepatic microsomal pools from male and female donors. Hydroxylation of p-nitrophenol (PNPH) was measured to determine CYP2E1 activity, and O-dealkylation of 7-benzyloxy-4-trifluoromethylcoumarin (BFC) was measured to determine CYP3A activity. Quercetin, but not myricetin or isorhamnetin, competitively inhibited PNPH activity in human recombinant cDNA-expressed CYP2E1 with the Ki=52.1±6.31μM. In the human microsomes, slight inhibition of PNPH activity by quercetin was not considered as physiologically relevant. Quercetin inhibited BFC activity in human recombinant cDNA-expressed CYP3A4 competitively with the Ki=15.4±1.52μM, and myricetin - noncompetitively with the Ki=74.6±7.99μM. The degree of inhibition by quercetin was similar between genders. Myricetin showed somewhat stronger inhibition in female pools, but the Ki values were higher than physiologically relevant concentrations. Isorhamnetin did not affect either PNPH or BFC activity. We concluded that observed inhibition of CYP2E1 and CYP3A by some flavonols were not gender-dependent.

  16. 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.

  17. Characterization of niflumic acid as a selective inhibitor of human liver microsomal UDP-glucuronosyltransferase 1A9: application to the reaction phenotyping of acetaminophen glucuronidation.

    PubMed

    Miners, John O; Bowalgaha, Kushari; Elliot, David J; Baranczewski, Pawel; Knights, Kathleen M

    2011-04-01

    Enzyme selective inhibitors represent the most valuable experimental tool for reaction phenotyping. However, only a limited number of UDP-glucuronosyltransferase (UGT) enzyme-selective inhibitors have been identified to date. This study characterized the UGT enzyme selectivity of niflumic acid (NFA). It was demonstrated that 2.5 μM NFA is a highly selective inhibitor of recombinant and human liver microsomal UGT1A9 activity. Higher NFA concentrations (50-100 μM) inhibited UGT1A1 and UGT2B15 but had little effect on the activities of UGT1A3, UGT1A4, UGT1A6, UGT2B4, UGT2B7, and UGT2B17. NFA inhibited 4-methylumbelliferone and propofol (PRO) glucuronidation by recombinant UGT1A9 and PRO glucuronidation by human liver microsomes (HLM) according to a mixed (competitive-noncompetitive) mechanism, with K(i) values ranging from 0.10 to 0.40 μM. Likewise, NFA was a mixed or noncompetitive inhibitor of recombinant and human liver microsomal UGT1A1 (K(i) range 14-18 μM), whereas competitive inhibition (K(i) 62 μM) was observed with UGT2B15. NFA was subsequently applied to the reaction phenotyping of human liver microsomal acetaminophen (APAP) glucuronidation. Consistent with previous reports, APAP was glucuronidated by recombinant UGT1A1, UGT1A6, UGT1A9, and UGT2B15. NFA concentrations in the range of 2.5 to 100 μM inhibited APAP glucuronidation by UGT1A1, UGT1A9, and UGT2B15 but not by UGT1A6. The mean V(max) for APAP glucuronidation by HLM was reduced by 20, 35, and 40%, respectively, in the presence of 2.5, 50, and 100 μM NFA. Mean K(m) values decreased in parallel with V(max), although the magnitude of the decrease was smaller. Taken together, the NFA inhibition data suggest that UGT1A6 is the major enzyme involved in APAP glucuronidation.

  18. 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

  19. Inhaled cellulosic and plastic fibers found in human lung tissue.

    PubMed

    Pauly, J L; Stegmeier, S J; Allaart, H A; Cheney, R T; Zhang, P J; Mayer, A G; Streck, R J

    1998-05-01

    We report the results of studies undertaken to determine whether inhaled plant (i.e., cellulosic; e.g., cotton) and plastic (e.g., polyester) fibers are present in human lungs and, if so, whether inhaled fibers are also present in human lung cancers. Specimens of lung cancer of different histological types and adjacent nonneoplastic lung tissue were obtained from patients undergoing a lung resection for removal of a tumor. With the protection of a laminar flow hood and safeguards to prevent contamination by extraneous fibers, fresh, nonfixed, and nonstained samples of lung tissue were compressed between two glass microscope slides. Specimens in these dual slide chambers were examined with a microscope configured to permit viewing with white light, fluorescent light, polarizing light, and phase-contrast illumination. Near-term fetal bovine lungs and nonlung human tumors were used as controls. In contrast to the observations of these control tissues, morphologically heterogeneous fibers were seen repetitively in freshly excised human lung tissue using polarized light. Inhaled fibers were present in 83% of nonneoplastic lung specimens (n = 67/81) and in 97% of malignant lung specimens (n = 32/33). Thus, of the 114 human lung specimens examined, fibers were observed in 99 (87%). Examination of histopathology slides of lung tissue with polarized light confirmed the presence of inhaled cellulosic and plastic fibers. Of 160 surgical histopathology lung tissue slides, 17 were selected for critical examination; of these, fibers were identified in 13 slides. The inhalation of mineral (e.g., asbestos) fibers has been described by many investigators; we believe, however, that this is the first report of inhaled nonmineral (e.g., plant and plastic) fibers. These bioresistant and biopersistent cellulosic and plastic fibers are candidate agents contributing to the risk of lung cancer.

  20. 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.

  1. 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

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

    PubMed

    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.

  3. 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.

  4. 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

  5. Human sweat metabolomics for lung cancer screening.

    PubMed

    Calderón-Santiago, Mónica; Priego-Capote, Feliciano; Turck, Natacha; Robin, Xavier; Jurado-Gámez, Bernabé; Sanchez, Jean C; Luque de Castro, María D

    2015-07-01

    Sweat is one of the less employed biofluids for discovery of markers in spite of its increased application in medicine for detection of drugs or for diagnostic of cystic fibrosis. In this research, human sweat was used as clinical sample to develop a screening tool for lung cancer, which is the carcinogenic disease with the highest mortality rate owing to the advanced stage at which it is usually detected. In this context, a method based on the metabolite analysis of sweat to discriminate between patients with lung cancer versus smokers as control individuals is proposed. The capability of the metabolites identified in sweat to discriminate between both groups of individuals was studied and, among them, a trisaccharide phosphate presented the best independent performance in terms of the specificity/sensitivity pair (80 and 72.7%, respectively). Additionally, two panels of metabolites were configured using the PanelomiX tool as an attempt to reduce false negatives (at least 80% specificity) and false positives (at least 80% sensitivity). The first panel (80% specificity and 69% sensitivity) was composed by suberic acid, a tetrahexose, and a trihexose, while the second panel (69% specificity and 80% sensitivity) included nonanedioic acid, a trihexose, and the monoglyceride MG(22:2). Thus, the combination of the five metabolites led to a single panel providing 80% specificity and 79% sensitivity, reducing the false positive and negative rates to almost 20%. The method was validated by estimation of within-day and between-days variability of the quantitative analysis of the five metabolites.

  6. Human embryonic stem cells and lung regeneration.

    PubMed

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

    2008-10-01

    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.

  7. Metabolism of the major Echinacea alkylamide N-isobutyldodeca-2E,4E,8Z,10Z-tetraenamide by human recombinant cytochrome P450 enzymes and human liver microsomes.

    PubMed

    Toselli, F; Matthias, A; Bone, K M; Gillam, E M J; Lehmann, R P

    2010-08-01

    Echinacea preparations are used for the treatment and prevention of upper respiratory tract infections. The phytochemicals believed responsible for the immunomodulatory properties are the alkylamides found in ethanolic extracts, with one of the most abundant being the N-isobutyldodeca-2E,4E,8Z,10Z-tetraenamide (1). In this study, we evaluated the human cytochrome P450 enzymes involved in the metabolism of this alkylamide using recombinant P450s, human liver microsomes and pure synthetic compound. Epoxidation, N-dealkylation and hydroxylation products were detected, with different relative amounts produced by recombinant P450s and microsomes. The major forms showing activity toward the metabolism of 1 were CYP1A1, CYP1A2 (both producing the same epoxide and N-dealkylation product), CYP2A13 (producing two epoxides), and CYP2D6 (producing two epoxides and an hydroxylated metabolite). Several other forms showed less activity. In incubations with human liver microsomes and selective inhibitors, CYP2E1 was found to be principally responsible for producing the dominant, hydroxylation product, whereas CYP2C9 was the principal source of the epoxides and CYP1A2 was responsible for the dealkylation product. In summary, in this study the relative impacts of the main human xenobiotic-metabolizing cytochrome P450s on the metabolism of a major Echinacea alkylamide have been established and the metabolites formed have been identified.

  8. DETECTION AND QUANTITATION OF FALLOUT PARTICLES IN A HUMAN LUNG.

    PubMed

    WEGST, A V; PELLETIER, C A; WHIPPLE, G H

    1964-02-28

    Portions of an adult human lung were studied by autoradiography in order to detect the presence of fallout particles. The radioactivity in the remainder of the tissue was determined with a gamma-ray spectrometer. Four particles were found and their activities were determined. From the measurement for total-fission-product activity in the lung tissue it was calculated that there were approximately 264 particles in the right lung at the time of death.

  9. 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.

  10. Inhaled histamine increases human lung mucociliary transport

    SciTech Connect

    Mussatto, D.J.; Garrard, C.S.; Trumbull, J.J.; Bowers, M.W.; Sanders, C.J.; Yeates, D.B.; Lourenco, R.V.

    1986-03-01

    Histamine, a mediator of airways constriction, alters ciliary beat frequency, bronchial mucus production, and epithelial ion transport; and in dogs, increases mucociliary transport. To evaluate the effect of inhaled histamine on human tracheobronchial mucociliary clearance, the authors measured lung mucociliary clearance (LMC) and tracheal mucociliary transport rate (TMTR) in 5 healthy, nonsmoking subjects in a randomized, double-blind, cross-over study. The concentration of inhaled histamine which produced a 20% fall in FEV/sub 1/ was established for each subject. On a separate day the subjects inhaled a 9 ..mu..m MMAD /sup 99m/Tc-Fe/sub 2/O/sub 3/ aerosol. LMC and TMTR were then measured for 2.5h using a gamma camera and a tracheal multidetector probe. Simultaneously, the subjects were challenged every 26 +/- 4 min with either PBS or histamine in PBS. The Fe/sub 2/O/sub 3/ retained after 24h for histamine (14.4 +/- 7.6%) and PBS studies (13.1 +/- 8.6%) indicated no difference in deposition of Fe/sub 2/O/sub 3/ (ANOVA). Fe/sub 2/O/sub 3/ clearance at 30 min was increased in the histamine studies (61 +/- 21% compared to the PBS studies (44 +/- 29%; p < 0.02, ANOVA)). TMTR was also increased with histamine (7.6 +/- 3.4 mm/min) compared to PBS (4.6 +/- 1.7 mm/min; p < 0.001, ANOVA). Results indicate an acute stimulatory effect of inhaled histamine on mucous transport in humans.

  11. Identification of Metabolite Biomarkers of the Designer Hallucinogen 25I-NBOMe in Mouse Hepatic Microsomal Preparations and Human Urine Samples Associated with Clinical Intoxication.

    PubMed

    Poklis, Justin L; Dempsey, Sara K; Liu, Kai; Ritter, Joseph K; Wolf, Carl; Zhang, Shijun; Poklis, Alphonse

    2015-10-01

    'NBOMe' (dimethoxyphenyl-N-[(2-methoxyphenyl)methyl]ethanamine) derivatives are a new class of designer hallucinogenic drugs widely available on the Internet. Currently, 2-(4-iodo-2,5-dimethoxyphenyl)-N-[(2-methoxyphenyl)methyl]ethanamine (25I-NBOMe) is the most popular abused derivative in the USA. There are little published data on the absorption, metabolism and elimination of 25I-NBOMe, or any of the other NBOMe derivatives. Therefore, there are no definitive metabolite biomarkers. We present the identification of fifteen 25I-NBOMe metabolites in phase I and II mouse hepatic microsomal preparations, and analysis of two human urine samples from 25I-NBOMe-intoxicated patients to test the utility of these metabolites as biomarkers of 25I-NBOMe use. The synthesis of two major urinary metabolites, 2-iodo-4-methoxy-5-[2-[(2-methoxyphenyl) methylamino]ethyl]phenol (2-O-desmethyl-5-I-NBOMe, M5) and 5-iodo-4-methoxy-2-[2-[(2-methoxyphenyl)methylamino]ethyl]phenol (5-O-desmethyl-2-I-NBOMe), is also presented. Seven phase II glucuronidated metabolites of the O-desmethyl or the hydroxylated phase I metabolites were identified. One human urine sample contained 25I-NBOMe as well as all 15 metabolites identified in mouse hepatic microsomal preparations. Another human urine sample contained no parent 25I-NBOMe, but was found to contain three O-desmethyl metabolites. We recommend β-glucuronidase enzymatic hydrolysis of urine prior to 25I-NBOMe screening and the use of M5 as the primary biomarker in drug testing.

  12. The inhibitory effect of tannic acid on cytochrome P450 enzymes and NADPH-CYP reductase in rat and human liver microsomes.

    PubMed

    Yao, Hsien-Tsung; Chang, Yi-Wei; Lan, Shih-Jung; Yeh, Teng-Kuang

    2008-02-01

    Tannic acid has been shown to decrease mutagenicity and/or carcinogenicity of several amine derivatives and polycyclic aromatic hydrocarbons in rodents. The purpose of this study was to evaluate the effect of tannic acid on cytochrome P450 (CYP)-catalyzed oxidations using rat liver microsomes (RLM) and human liver microsomes (HLM) as the enzyme sources. In RLM, tannic acid showed a non-selective inhibitory effect on 7-methoxyresorufin O-demethylation (MROD), 7-ethoxyresorufin O-deethylation (EROD), tolbutamide hydroxylation, p-nitrophenol hydroxylation and testosterone 6beta-hydroxylation activities with IC(50) values ranged from 14.9 to 27.4 microM. In HLM, tannic acid inhibited EROD, MROD and phenacetin O-deethylation activities with IC(50) values ranged from 5.1 to 7.5 microM, and diclofenac 4-hydroxylation, dextromethorphan O-demethylation, chlorzoxazone 6-hydroxylation and testosterone 6beta-hydroxylation with IC(50) values ranged from 20 to 77 microM. In baculovirus-insect cell-expressed human CYP 1A1 and 1A2, the IC(50) values of tannic acid for CYP 1A1- and 1A2-catalyzed EROD activities were 23.1 and 2.3 microM, respectively, indicating that tannic acid preferably inhibited the activity of CYP1A2. Tannic acid inhibited human CYP1A2 non-competitively with a Ki value of 4.8 microM. Tannic acid was also found to inhibit NADPH-CYP reductase in RLM and HLM with IC(50) values of 11.8 and 17.4 microM, respectively. These results suggested that the inhibition of CYP enzyme activities by tannic acid may be partially attributed to its inhibition of NADPH-CYP reductase activity.

  13. Identification of Metabolite Biomarkers of the Designer Hallucinogen 25I-NBOMe in Mouse Hepatic Microsomal Preparations and Human Urine Samples Associated with Clinical Intoxication

    PubMed Central

    Poklis, Justin L.; Dempsey, Sara K.; Liu, Kai; Ritter, Joseph K.; Wolf, Carl; Zhang, Shijun; Poklis, Alphonse

    2015-01-01

    ‘NBOMe’ (dimethoxyphenyl-N-[(2-methoxyphenyl)methyl]ethanamine) derivatives are a new class of designer hallucinogenic drugs widely available on the Internet. Currently, 2-(4-iodo-2,5-dimethoxyphenyl)-N-[(2-methoxyphenyl)methyl]ethanamine (25I-NBOMe) is the most popular abused derivative in the USA. There are little published data on the absorption, metabolism and elimination of 25I-NBOMe, or any of the other NBOMe derivatives. Therefore, there are no definitive metabolite biomarkers. We present the identification of fifteen 25I-NBOMe metabolites in phase I and II mouse hepatic microsomal preparations, and analysis of two human urine samples from 25I-NBOMe-intoxicated patients to test the utility of these metabolites as biomarkers of 25I-NBOMe use. The synthesis of two major urinary metabolites, 2-iodo-4-methoxy-5-[2-[(2-methoxyphenyl) methylamino]ethyl]phenol (2-O-desmethyl-5-I-NBOMe, M5) and 5-iodo-4-methoxy-2-[2-[(2-methoxyphenyl)methylamino]ethyl]phenol (5-O-desmethyl-2-I-NBOMe), is also presented. Seven phase II glucuronidated metabolites of the O-desmethyl or the hydroxylated phase I metabolites were identified. One human urine sample contained 25I-NBOMe as well as all 15 metabolites identified in mouse hepatic microsomal preparations. Another human urine sample contained no parent 25I-NBOMe, but was found to contain three O-desmethyl metabolites. We recommend β-glucuronidase enzymatic hydrolysis of urine prior to 25I-NBOMe screening and the use of M5 as the primary biomarker in drug testing. PMID:26378134

  14. Mechanism-based inhibition of cytochrome P450 (CYP)2A6 by chalepensin in recombinant systems, in human liver microsomes and in mice in vivo

    PubMed Central

    Ueng, Yune-Fang; Chen, Chien-Chih; Chung, Yu-Ting; Liu, Tsung-Yun; Chang, Yu-Ping; Lo, Wei-Sheng; Murayama, Norie; Yamazaki, Hiroshi; Souček, Pavel; Chau, Gar-Yang; Chi, Chin-Wen; Chen, Ruei-Ming; Li, Ding-Tzai

    2011-01-01

    BACKGROUND AND PURPOSE Chalepensin is a pharmacologically active furanocoumarin compound found in rue, a medicinal herb. Here we have investigated the inhibitory effects of chalepensin on cytochrome P450 (CYP) 2A6 in vitro and in vivo. EXPERIMENTAL APPROACH Mechanism-based inhibition was studied in vitro using human liver microsomes and bacterial membranes expressing genetic variants of human CYP2A6. Effects in vivo were studied in C57BL/6J mice. CYP2A6 activity was assayed as coumarin 7-hydroxylation (CH) using HPLC and fluorescence measurements. Metabolism of chalepensin was assessed with liquid chromatography/mass spectrometry (LC/MS). KEY RESULTS CYP2A6.1, without pre-incubation with NADPH, was competitively inhibited by chalepensin. After pre-incubation with NADPH, inhibition by chalepensin was increased (IC50 value decreased by 98%). This time-dependent inactivation (kinact 0.044 min−1; KI 2.64 µM) caused the loss of spectrally detectable P450 content and was diminished by known inhibitors of CYP2A6, pilocarpine or tranylcypromine, and by glutathione conjugation. LC/MS analysis of chalepensin metabolites suggested an unstable epoxide intermediate was formed, identified as the corresponding dihydrodiol, which was then conjugated with glutathione. Compared with the wild-type CYP2A6.1, the isoforms CYP2A6.7 and CYP2A6.10 were less inhibited. In mouse liver microsomes, pre-incubation enhanced inhibition of CH activity. Oral administration of chalepensin to mice reduced hepatic CH activity ex vivo. CONCLUSIONS AND IMPLICATIONS Chalepensin was a substrate and a mechanism-based inhibitor of human CYP2A6. Formation of an epoxide could be a key step in this inactivation. ‘Poor metabolizers’ carrying CYP2A6*7 or *10 may be less susceptible to inhibition by chalepensin. Given in vivo, chalepensin decreased CYP2A activity in mice. PMID:21418183

  15. Influence of human serum albumin on the bile acid-mediated inhibition of liver microsomal type 1 11β-hydroxysteroid dehydrogenase.

    PubMed

    Maeda, Yorio; Funagayama, Mayumi; Shinohara, Akio; Koshimoto, Chihiro; Furusawa, Hidemi; Nakahara, Hiroshi; Yamaguchi, Yukiko; Saitoh, Tomokazu; Yamamoto, Takashi; Komaki, Kansei

    2014-09-01

    The influence of human serum albumin (HSA) on the bile acid-mediated inhibition of liver microsomal type 1 11β-hydroxysteroid dehydrogenase (11β-HSD1) was studied in vitro. A rat liver microsomal fraction was prepared, and the 11β-HSD1 enzyme activity in the presence of various concentrations of bile acids and HSA was determined using hydrocortisone as the substrate. The products of the reaction were extracted and analyzed using high-performance liquid chromatography. The magnitude of the inhibition decreased with the addition of HSA in a dose-dependent manner. Four percent human albumin decreased the inhibitory effects of 100 μM chenodeoxycholic acid and lithocholic acid from 89.9 ± 5.6 to 54.5 ± 6.1% and from 83.8 ± 4.8 to 20.8 ± 4.2%, respectively. In contrast, ursodeoxycholic acid and deoxycholic acid showed no inhibitory effect on the enzyme activity in the presence of 4% human serum albumin, and the addition of 1% γ-globulin to the assay mixture in the presence of bile acids did not affect the enzyme activity. Our in vitro study showed that the addition of HSA ameliorated the inhibition of 11β-HSD1 and that the magnitude of the change is dependent on the species of bile acid, presumably based on the numbers of hydroxyl groups. These results suggest that HSA seems to protect the bile acid-mediated inhibition of 11β-HSD1 in the healthy subject. On the other hand, in the patients with obstructive biliary diseases, not only elevated serum bile acid but also the accompanying hypoalbuminemia is important to evaluate the pathophysiology of the bile acid-mediated inhibition of 11β-HSD1 of the disease.

  16. Structure-activity relationship study of human liver microsomes-catalyzed hydrolysis rate of ester prodrugs of MENT by comparative molecular field analysis (CoMFA).

    PubMed

    Bursi, Roberta; Grootenhuis, Arijan; van der Louw, Jaap; Verhagen, Jos; de Gooyer, Marcel; Jacobs, Peter; Leysen, Dirk

    2003-03-01

    A series of MENT esters (3-71) was designed, prepared and tested to study the structure-activity relationship (SAR) of the hydrolysis rate with human liver microsomes of these prodrugs. Compounds were obtained covering a wide range of metabolic stability. The results are useful for the proper selection of prodrugs for different pharmaceutical formulations to deliver the potent and prostate-sparing androgen MENT. The MENT esters can especially be administered for male hormone replacement therapy and male contraception. Comparative molecular field analysis (CoMFA) was applied to a dataset of 28 esters, for which ED50 values could be obtained. The CoMFA model where the electrostatic and H-bond molecular fields were combined turned out to be most predictive. Despite the limited size of the dataset, CoMFA can help to rationalize the SAR of the ester hydrolysis rate of ester prodrugs of MENT.

  17. 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.

  18. 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.

  19. The accumulation of nickel in human lungs.

    PubMed Central

    Edelman, D A; Roggli, V L

    1989-01-01

    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. PMID:2759060

  20. Participation of CYP2C8 and CYP3A4 in the N-demethylation of imatinib in human hepatic microsomes

    PubMed Central

    Nebot, Noelia; Crettol, Severine; d'Esposito, Fabrizio; Tattam, Bruce; Hibbs, David E; Murray, Michael

    2010-01-01

    BACKGROUND AND PURPOSE Imatinib is a clinically important inhibitor of tyrosine kinases that are dysregulated in chronic myelogenous leukaemia and gastrointestinal stromal tumours. Inter-individual variation in imatinib pharmacokinetics is extensive, and influences drug safety and efficacy. Hepatic cytochrome P450 (CYP) 3A4 has been implicated in imatinib N-demethylation, but the clearance of imatinib decreases during prolonged therapy. CYP3A phenotype correlates with imatinib clearance at the commencement of therapy, but not at steady state. The present study evaluated the possibility that multiple CYPs may contribute to imatinib oxidation in liver. EXPERIMENTAL APPROACH Imatinib biotransformation in human liver microsomes (n = 20) and by cDNA-expressed CYPs was determined by LC–MS. Relationships between imatinib N-demethylation and other drug metabolizing CYPs were assessed. KEY RESULTS N-desmethylimatinib formation was correlated with microsomal oxidation of the CYP3A4 substrates testosterone (ρ= 0.60; P < 0.01) and midazolam (ρ= 0.46; P < 0.05), and the CYP2C8 substrate paclitaxel (ρ= 0.58; P < 0.01). cDNA-derived CYPs 2C8, 3A4, 3A5 and 3A7 supported imatinib N-demethylation, but 10 other CYPs were inactive; in kinetic studies, CYP2C8 was a high-affinity enzyme with a catalytic efficiency ∼15-fold greater than those of CYPs 3A4 and 3A5. The CYP3A inhibitors ketoconazole and troleandomycin, and the CYP2C8 inhibitors quercetin and paclitaxel decreased imatinib oxidation. From molecular modelling, the imatinib structure could be superimposed on a pharmacophore for CYP2C8 substrates. CONCLUSIONS AND IMPLICATIONS CYP2C8 and CYPs 3A contribute to imatinib N-demethylation in human liver. The involvement of CYP2C8 may account in part for the wide inter-patient variation in imatinib pharmacokinetics observed in clinical practice. PMID:20977456

  1. Flow Cytometric Analysis of Mononuclear Phagocytes in Nondiseased Human Lung and Lung-Draining Lymph Nodes

    PubMed Central

    Desch, A. Nicole; Gibbings, Sophie L.; Goyal, Rajni; Kolde, Raivo; Bednarek, Joe; Bruno, Tullia; Slansky, Jill E.; Jacobelli, Jordan; Mason, Robert; Ito, Yoko; Messier, Elise; Randolph, Gwendalyn J.; Prabagar, Miglena; Atif, Shaikh M.; Segura, Elodie; Xavier, Ramnik J.; Bratton, Donna L.; Janssen, William J.; Henson, Peter M.

    2016-01-01

    Rationale: The pulmonary mononuclear phagocyte system is a critical host defense mechanism composed of macrophages, monocytes, monocyte-derived cells, and dendritic cells. However, our current characterization of these cells is limited because it is derived largely from animal studies and analysis of human mononuclear phagocytes from blood and small tissue resections around tumors. Objectives: Phenotypic and morphologic characterization of mononuclear phagocytes that potentially access inhaled antigens in human lungs. Methods: We acquired and analyzed pulmonary mononuclear phagocytes from fully intact nondiseased human lungs (including the major blood vessels and draining lymph nodes) obtained en bloc from 72 individual donors. Differential labeling of hematopoietic cells via intrabronchial and intravenous administration of antibodies within the same lobe was used to identify extravascular tissue-resident mononuclear phagocytes and exclude cells within the vascular lumen. Multiparameter flow cytometry was used to identify mononuclear phagocyte populations among cells labeled by each route of antibody delivery. Measurements and Main Results: We performed a phenotypic analysis of pulmonary mononuclear phagocytes isolated from whole nondiseased human lungs and lung-draining lymph nodes. Five pulmonary mononuclear phagocytes were observed, including macrophages, monocyte-derived cells, and dendritic cells that were phenotypically distinct from cell populations found in blood. Conclusions: Different mononuclear phagocytes, particularly dendritic cells, were labeled by intravascular and intrabronchial antibody delivery, countering the notion that tissue and blood mononuclear phagocytes are equivalent systems. Phenotypic descriptions of the mononuclear phagocytes in nondiseased lungs provide a precedent for comparative studies in diseased lungs and potential targets for therapeutics. PMID:26551758

  2. 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.

  3. Linear dimensions and volumes of human lungs

    DOE PAGES

    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

  4. 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

  5. Elucidation of a novel bioactivation pathway of a 3,4-unsubstituted isoxazole in human liver microsomes: formation of a glutathione adduct of a cyanoacrolein derivative after isoxazole ring opening.

    PubMed

    Yu, Jian; Folmer, James J; Hoesch, Valerie; Doherty, James; Campbell, James B; Burdette, Doug

    2011-02-01

    Studies on the biotransformation of isoxazole rings have shown that molecules containing a C3-substituted isoxazole or a 1,2-benzisoxazole can undergo a two-electron reductive ring cleavage to form an imine. In the absence of a C3 substituent, the isoxazole ring opens via deprotonation of the C3 proton followed by N-O bond cleavage to yield an α-cyanoenol analog. We report the identification of a novel bioactivation pathway of a 3,4-unsubstituted isoxazole in human liver microsomes. After the enzyme-catalyzed cleavage of the 3,4-unsubstituted isoxazole ring of N-((2-isopropyl-7-methyl-1-oxoisoindolin-5-yl)methyl)isoxazole-5-carboxamide (P) in human liver microsomes, the formed α-cyanoenol (M1) condenses with formaldehyde to generate an α,β-unsaturated Michael acceptor intermediate (a cyanoacrolein derivative, VII), which further reacts with the cysteinyl thiol of glutathione to yield a GSH adduct of a cyanoacrolein derivative (M3). The same adduct also is formed when M1, generated in 0.1 N NaOH aqueous solution, reacts with formaldehyde and GSH. (13)C-labeled methanol was used to confirm that methanol from the drug stock solution was oxidized by liver microsomal enzymes to formaldehyde and the carbon atom from methanol was finally incorporated in the corresponding GSH adduct. The formation of isoxazole ring-opened products (M1 and M2) in human liver microsomes is NADPH-dependent. M1 and M2 were found in human liver microsomes preincubated with 1-aminobenzotriazole (1 mM) and NADPH (5 mM) at ∼ 10% of the levels found in the samples in the absence of 1-aminobenzotriazole, suggesting that this biotransformation pathway is primarily catalyzed by cytochrome P450. The formation of M3 also was inhibited by 1-aminobenzotriazole at a similar level.

  6. Large-scale multiplex absolute protein quantification of drug-metabolizing enzymes and transporters in human intestine, liver, and kidney microsomes by SWATH-MS: Comparison with MRM/SRM and HR-MRM/PRM.

    PubMed

    Nakamura, Kenji; Hirayama-Kurogi, Mio; Ito, Shingo; Kuno, Takuya; Yoneyama, Toshihiro; Obuchi, Wataru; Terasaki, Tetsuya; Ohtsuki, Sumio

    2016-08-01

    The purpose of the present study was to examine simultaneously the absolute protein amounts of 152 membrane and membrane-associated proteins, including 30 metabolizing enzymes and 107 transporters, in pooled microsomal fractions of human liver, kidney, and intestine by means of SWATH-MS with stable isotope-labeled internal standard peptides, and to compare the results with those obtained by MRM/SRM and high resolution (HR)-MRM/PRM. The protein expression levels of 27 metabolizing enzymes, 54 transporters, and six other membrane proteins were quantitated by SWATH-MS; other targets were below the lower limits of quantitation. Most of the values determined by SWATH-MS differed by less than 50% from those obtained by MRM/SRM or HR-MRM/PRM. Various metabolizing enzymes were expressed in liver microsomes more abundantly than in other microsomes. Ten, 13, and eight transporters listed as important for drugs by International Transporter Consortium were quantified in liver, kidney, and intestinal microsomes, respectively. Our results indicate that SWATH-MS enables large-scale multiplex absolute protein quantification while retaining similar quantitative capability to MRM/SRM or HR-MRM/PRM. SWATH-MS is expected to be useful methodology in the context of drug development for elucidating the molecular mechanisms of drug absorption, metabolism, and excretion in the human body based on protein profile information.

  7. Quaternary ammonium-linked glucuronidation of trans-4-hydroxytamoxifen, an active metabolite of tamoxifen, by human liver microsomes and UDP-glucuronosyltransferase 1A4.

    PubMed

    Ogura, Kenichiro; Ishikawa, Yuko; Kaku, Teppei; Nishiyama, Takahito; Ohnuma, Tomokazu; Muro, Kei; Hiratsuka, Akira

    2006-04-28

    Tamoxifen (TAM), a nonsteroidal antiestrogen, is the most widely used drug for chemotherapy of hormone-dependent breast cancer in women. Trans-4-hydroxy-TAM (trans-4-HO-TAM), one of the TAM metabolites in humans, has been considered to be an active metabolite of TAM because of its higher affinity toward estrogen receptors (ERs) than the parent drug and other side-chain metabolites. In the present study, we found a new potential metabolic pathway of trans-4-HO-TAM and its geometrical isomer, cis-4-HO-TAM, via N-linked glucuronic acid conjugation for excretion in humans. N+-Glucuronides of 4-HO-TAM isomers were isolated along with O-glucuronides from a reaction mixture consisting of trans- or cis-4-HO-TAM and human liver microsomes fortified with UDP-glucuronic acid and identified with their respective synthetic specimens by high performance liquid chromatography-electrospray ionization time-of-flight mass spectrometry. Although N- and O-glucuronidating activities of human liver microsomes toward trans-4-HO-TAM were nearly comparable, O-glucuronidation was predominant for cis-4-HO-TAM conjugation. Only UGT1A4 catalyzed the N-linked glucuronidation of 4-HO-TAM among recombinant human UGT isoforms (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B4, UGT2B7, UGT2B15, and UGT2B17) expressed in insect cells. In contrast, all UGT isoforms, except for UGT1A3 and UGT1A4, catalyzed O-glucuronidation of 4-HO-TAM. Although O-glucuronidation of 4-HO-TAM greatly decreased binding affinity for human ERs, 4-HO-TAM N+-glucuronide still had binding affinity similar to 4-HO-TAM itself, suggesting that N+-glucuronide might contribute to the biological activity of TAM in vivo.

  8. Evaluation of pharmaceutical excipients as cosolvents in 4-methyl umbelliferone glucuronidation in human liver microsomes: applications for compounds with low solubility.

    PubMed

    Argikar, Upendra A; Liang, Guiqing; Bushee, Jennifer L; Hosagrahara, Vinayak P; Lee, Wendy

    2011-01-01

    Standard incubation procedures for carrying out microsomal assays involve the use of less than 1% w/v organic solvents to minimize the potential inhibitory effects of organic solvents on metabolic activity. This presents a practical limitation for poorly soluble xenobiotics, which cannot be incubated at concentrations high enough to obtain a V(max), and therefore subsequent values for K(m) and Cl(int) cannot be calculated. Our goal was to study the application of a variety of pharmaceutical excipients to aid the solubilization of compounds in vitro in glucuronidation incubations, without affecting the reaction kinetics. In vitro glucuronidation incubations were carried out in human liver microsomes with 4-methylumbelliferone (4-MU) and the kinetics of 4-MU glucuronidation in the presence of excipients were compared to that in control incubations without any excipients. In addition, IC(75) values were calculated for each excipient. We observed that HPBCD (Hydroxypropyl-β-cyclodextrin) may be employed in in vitro glucuronidation incubations up to 0.5% w/v without affecting the Cl(int) of 4-MU. Although NMP (N-methyl-2-pyrrolidone) and DMA (N,N-dimethylacetamide); showed low IC(75) values approximately 0.1% w/v each, neither excipients altered the Cl(int) of 4-MUG (4-methylumbelliferyl-β-D-glucuronide) formation. Our studies point toward possible applications of pharmaceutical excipients to carry out in vitro glucuronidation of substrates with poor aqueous solubility, in order to estimate Cl(int) and subsequently scaled organ clearance values.

  9. Human lung expresses unique gamma-glutamyl transpeptidase transcripts.

    PubMed Central

    Wetmore, L A; Gerard, C; Drazen, J M

    1993-01-01

    gamma-Glutamyl transpeptidase (EC 2.3.2.2, gamma GT) is a membrane-bound ectoenzyme that plays an important role in the metabolism of glutathione. It is composed of two subunits, both of which are encoded by a common mRNA. We examined the expression of gamma GT in human lung tissue by Northern blot analysis and screening a cDNA library made from human lung poly(A)+ RNA. Our results show that there are two gamma GT mRNA populations in human lung tissue. We define these as group I (2.4 kb) and group II (approximately 1.2 kb) transcripts. In the present communication, we characterize the unique lung transcript. Sequence analysis of representative clones shows that group I transcripts are virtually identical to those previously isolated from liver and placenta but possess a unique 5' untranslated region. In marked contrast, group II transcripts appear to be human-lung-specific. Group II transcripts appear on Northern blots probed with full-length or 3'-biased gamma GT cDNA. Sequence analysis of group II clones shows them to be homologous with group I clones in the region that encodes the reading frame for the light chain; however, they possess a series of unique 5' untranslated regions, which suggests that they arise from lung-specific message processing. Additionally, approximately 50% of the isolated group II clones contain 34 nt substitutions compared with the "wild-type" gamma GT transcripts. These data indicate that human lung expresses unique gamma GT transcripts of unknown function as well as the classical form. The abundant group II transcripts may encode part of a heterodimer related to gamma GT or represent processed lung-specific pseudogenes. Images Fig. 1 PMID:7689219

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

    PubMed

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

    2016-04-30

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

  11. Cyclosporin A drug interactions. Screening for inducers and inhibitors of cytochrome P-450 (cyclosporin A oxidase) in primary cultures of human hepatocytes and in liver microsomes.

    PubMed

    Pichard, L; Fabre, I; Fabre, G; Domergue, J; Saint Aubert, B; Mourad, G; Maurel, P

    1990-01-01

    In previous papers we demonstrated that cyclosporin A (CsA) was specifically oxidized in rabbit and human liver by cytochrome P-450IIIA. We therefore anticipated that any drug that is an inducer or an inhibitor of this cytochrome should lead to interaction with CsA when given in association with it. In order to confirm this hypothesis, primary cultures of human hepatocytes and human liver microsomes were used to "reproduce" in vitro clinically significant interactions observed between CsA and drugs known either as specific inducers (i.e., rifampicin) or as specific inhibitors (i.e., erythromycin) of P-450IIIA. Our results were in close agreement with the clinical reports. Human hepatocytes maintained in primary cultures for 72 hr in the presence of 50 microM rifampicin exhibited increased levels of P-450IIIA, determined by Western blot using specific antibodies, and concomitant increase in CsA oxidase activity, determined by HPLC analysis of extra and intracellular media. Conversely, these cultures exhibited erythromycin concentration-dependent decreases in CsA oxidase activity when incubated in the presence of 5, 20, and 100 microM erythromycin. In addition, a Lineweaver-Burk analysis of the erythromycin-mediated inhibition of CsA oxidase activity in human liver microsomes revealed competitive inhibition (with Ki of 75 microM) as expected, this macrolide being a specific substrate of P-450IIIA. Using this experimental approach, 59 molecules representative of 17 different therapeutic classes were screened for inducers and inhibitors of CsA oxidase activity. Our results allowed us to elucidate the molecular mechanism of previously observed, but unexplained, drug interactions involving CsA, and to detect drugs that should interfere with CsA metabolism as inducers or inhibitors. Drugs detected as potential inducers of CsA oxidase included: rifampicin, sulfadimidine, phenobarbital, phenytoin, phenylbutazone, dexamethasone, sulfinpyrazone, and carbamazepine. Drugs

  12. Traditional Herbal Formulas to as Treatments for Musculoskeletal Disorders: Their Inhibitory Effects on the Activities of Human Microsomal Cytochrome P450s and UDP-glucuronosyltransferases

    PubMed Central

    Jin, Seong Eun; Seo, Chang-Seob; Shin, Hyeun-Kyoo; Ha, Hyekyung

    2016-01-01

    Objective: The aim of this study was to assess the influence of traditional herbal formulas, including Bangpungtongseong-san (BPTSS; Fangfengtongsheng-san, Bofu-tsusho-san), Ojeok-san (OJS; Wuji-san, Goshaku-san), and Oyaksungi-san (OYSGS; Wuyaoshungi-san, Uyakujyunki-san), on the activities of the human cytochrome P450s (CYP450s) and UDP-glucuronosyltransferases (UGTs), which are drug-metabolizing enzymes. Materials and Methods: The activities of the major human CYP450 isozymes (CYP1A2, CYP3A4, CYP2B6, CYP2C9, CYP2C19, CYP2D6, and CYP2E1) and UGTs (UGT1A1, UGT1A4, and UGT2B7) were investigated using in vitro fluorescence-based and luminescence-based enzyme assays, respectively. The inhibitory effects of the herbal formulas were characterized, and their IC50 values were determined. Results: BPTSS inhibited the activities of CYP1A2, CYP2C19, CYP2E1, and UGT1A1 while it exerted relatively weak inhibition on CYP2B6, CYP2C9, CYP2D6, and CYP3A4. BPTSS also negligibly inhibited the activities of UGT1A4 and UGT2B7, with IC50 values in the excess of 1000 μg/mL. OJS and OYSGS inhibited the activity of CYP2D6, whereas they exhibited no inhibition of the UGT1A4 activity at doses <1000 μg/mL. In addition, OJS inhibited the CYP1A2 activity but exerted a relatively weak inhibition on the activities of CYP2C9, CYP2C19, CYP2E1, and CYP3A4. Conversely, OJS negligibly inhibited the activities of CYP2B6, UGT1A1, and UGT2B7 with IC50 values in excess of 1000 μg/mL. OYSGS weakly inhibited the activities of CYP1A2, CYP2C19, CYP2E1, CYP3A4, and UGT1A1, with a negligible inhibition on the activities of CYP2B6, CYP2C9, and UGT2B7, with IC50 values in excess of 1000 μg/mL. Conclusions: These results provide information regarding the safety and effectiveness of BPTSS, OJS, and OYSGS when combined with conventional drugs. SUMMARY Bangpungtongseong-san inhibited the activities of human microsomal CYP1A2, CYP2C19, CYP2E1, and UGT1A1, with a negligibly inhibition on the activities of CYP2B6

  13. A liquid chromatographic-electrospray-tandem mass spectrometric method for quantitation of quetiapine in human plasma and liver microsomes: application to study in vitro metabolism.

    PubMed

    Lin, Shen-Nan; Chang, Yan; Moody, David E; Foltz, Rodger L

    2004-09-01

    Quetiapine is an atypical antipsychotic agent for the treatment of schizophrenia. After an oral dose it is absorbed rapidly and extensively metabolized in the liver, resulting in low plasma concentrations of the parent drug. A sensitive analytical method is needed. A liquid chromatographic-electrospray-tandem mass spectrometric (LC-ESI-MS-MS) method combined with a simple liquid-liquid extraction has been developed for the measurement of quetiapine in human plasma and in human liver microsomes (HLM). Clozapine is used as internal standard. Plasma samples or microsomes quenched with methanol (100 microL) were made basic and extracted with 3 mL n-butyl chloride. The reconstituted extracts were analyzed by LC-ESI-MS-MS. Selective reaction monitoring of MH(+) at m/z 384 and 327 resulted in strong fragment ions at m/z 253 and 192 for quetiapine and clozapine, respectively. Recovery of quetiapine and clozapine ranged from 62 to 73%. Intrarun accuracy and precision determined at 1.0 (lower limit of quantitation), 2.5, 200, and 400 ng/mL did not exceed 7% deviation from target and the %CV did not exceed 5.5%. The % target +/- %CV for interrun accuracy and precision were at least 95% +/- 7.4% at concentrations of 2.5, 200, and 400 ng/mL. Plasma samples (2.5 and 400 ng/mL) stored at room temperature for 24 h or after 3 cycles of freeze/thaw were all stable (maximum % deviation < or = 11.0%). Processed extracts (2.5 and 400 ng/mL) stored for 7 days at -20 degrees C or 6 days on the autosampler were all stable (maximum % deviation < or = 11.5%). The method has been used to study quetiapine utilization during incubation with HLM or with cDNA-expressed human cytochrom P450s (CYP). Quetiapine is extensively metabolized by CYP 3A4 and CYP 2D6 and to a lesser extent by CYP 3A7, CYP 3A5, and CYP 2C19.

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

    PubMed

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

    2008-02-01

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

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

    PubMed

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

    2017-03-05

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

  16. 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

  17. 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.

  18. 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)

  19. 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...

  20. Metabolic activation of the phenothiazine antipsychotics chlorpromazine and thioridazine to electrophilic iminoquinone species in human liver microsomes and recombinant P450s.

    PubMed

    Wen, Bo; Zhou, Mingyan

    2009-10-07

    The phenothiazine-derived antipsychotics, namely chlorpromazine and thioridazine, have been associated with very rare but severe incidences of hepatotoxicity in patients. While the mechanism of idiosyncratic hepatotoxicity remains unknown, it is possible that metabolic activation and subsequent covalently binding of reactive metabolites to cellular proteins play a causative role. Studies were initiated to determine whether chlorpromazine and thioridazine undergo cytochrome P450 (P450)-mediated bioactivation in human liver microsomes to electrophilic intermediates. LC/MS/MS analysis of incubations containing chlorpromazine or thioridazine in the presence of NADPH and glutathione (GSH) revealed the formation of GSH conjugates derived from the addition of the sulfydryl nucleophile to monohydroxy metabolites of chlorpromazine and thioridazine, respectively. Formation of reactive intermediates of chlorpromazine and thioridazine was primarily mediated by heterologously expressed recombinant CYP2D6, and to a less extent, CYP1A2. The 7-hydroxyl metabolites of chlorpromazine and thioridazine were also detected by tandem mass spectrometry. A tentative pathway states that after initial 7-hydroxylation, a bioactivation sequence involves P450-catalyzed oxidation of the phenothiazine core to an electrophilic quinone imine intermediate, which is subsequently attacked by glutathione yielding the sulfydryl conjugates. The results from the current investigation constitute the first report on the cytochrome P450-catalyzed bioactivation of the phenothiazine antipsychotics chlorpromazine and thioridazine.

  1. Asbestos fibers in human lung: forensic significance

    SciTech Connect

    Ehrenreich, T.; Selikoff, I.J.

    1981-03-01

    Asbestos is a fibrous mineral which, because of its unique properties, has innumerable applications in many industries and is used in a large variety of consumer products. It has become ubiquitous and is woven, literally and figuratively, into the fabric of our present-day civilization. However, its presence is sometimes unknown and unsuspected by those who are exposed to asbestos by virtue of occupation or environment and inhale its fibers. Exposed workers and even urban dwellers may have a variable lung burden of asbestos fibers. There is indisputable clinical, pathological, experimental and epidemiological proof that, after varying periods of latency, asbestos may cause benign and malignant disease often leading to disability or death. Forensic investigation of suspected asbestos-related deaths includes a life-time occupational history, a complete autopsy, and identification of the asbestos fiber tissue burden. The latter usually requires special procedures.

  2. Cytochrome P450 dependent metabolism of the new designer drug 1-(3-trifluoromethylphenyl)piperazine (TFMPP). In vivo studies in Wistar and Dark Agouti rats as well as in vitro studies in human liver microsomes.

    PubMed

    Staack, Roland F; Paul, Liane D; Springer, Dietmar; Kraemer, Thomas; Maurer, Hans H

    2004-01-15

    1-(3-Trifluoromethylphenyl)piperazine (TFMPP) is a designer drug with serotonergic properties. Previous studies with male Wistar rats (WI) had shown, that TFMPP was metabolized mainly by aromatic hydroxylation. In the current study, it was examined whether this reaction may be catalyzed by cytochrome P450 (CYP)2D6 by comparing TFMPP vs. hydroxy TFMPP ratios in urine from female Dark Agouti rats, a model of the human CYP2D6 poor metabolizer phenotype (PM), male Dark Agouti rats, an intermediate model, and WI, a model of the human CYP2D6 extensive metabolizer phenotype. Furthermore, the human hepatic CYPs involved in TFMPP hydroxylation were identified using cDNA-expressed CYPs and human liver microsomes. Finally, TFMPP plasma levels in the above mentioned rats were compared. The urine studies suggested that TFMPP hydroxylation might be catalyzed by CYP2D6 in humans. Studies using human CYPs showed that CYP1A2, CYP2D6 and CYP3A4 catalyzed TFMPP hydroxylation, with CYP2D6 being the most important enzyme accounting for about 81% of the net intrinsic clearance, calculated using the relative activity factor approach. The hydroxylation was significantly inhibited by quinidine (77%) and metabolite formation in poor metabolizer genotype human liver microsomes was significantly lower (63%) compared to pooled human liver microsomes. Analysis of the plasma samples showed that female Dark Agouti rats exhibited significantly higher TFMPP plasma levels compared to those of male Dark Agouti rats and WI. Furthermore, pretreatment of WI with the CYP2D inhibitor quinine resulted in significantly higher TFMPP plasma levels. In conclusion, the presented data give hints for possible differences in pharmacokinetics in human PM and human CYP2D6 extensive metabolizer phenotype subjects relevant for risk assessment.

  3. 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.

  4. In vitro profiling of the metabolism and drug-drug interaction of tofogliflozin, a potent and highly specific sodium-glucose co-transporter 2 inhibitor, using human liver microsomes, human hepatocytes, and recombinant human CYP.

    PubMed

    Yamane, Mizuki; Kawashima, Kosuke; Yamaguchi, Koji; Nagao, Shunsuke; Sato, Mika; Suzuki, Masayuki; Honda, Kiyofumi; Hagita, Hitoshi; Kuhlmann, Olaf; Poirier, Agnes; Fowler, Stephen; Funk, Christoph; Simon, Sandrine; Aso, Yoshinori; Ikeda, Sachiya; Ishigai, Masaki

    2015-03-01

    Abstract 1. The metabolism and drug-drug interaction (DDI) risk of tofogliflozin, a potent and highly specific sodium-glucose co-transporter 2 inhibitor, were evaluated by in vitro studies using human liver microsomes, human hepatocytes, and recombinant human CYPs. 2. The main metabolite of tofogliflozin was the carboxylated derivative (M1) in human hepatocytes, which was the same as in vivo. The metabolic pathway of tofogliflozin to M1 was considered to be as follows: first, tofogliflozin was catalyzed to the primary hydroxylated derivative (M4) by CYP2C18, CYP4A11 and CYP4F3B, then M4 was oxidized to M1. 3. Tofogliflozin had no induction potential on CYP1A2 and CYP3A4. Neither tofogliflozin nor M1 had inhibition potential on CYPs, with the exception of a weak CYP2C19 inhibition by M1. 4. Not only are multiple metabolic enzymes involved in the tofogliflozin metabolism, but the drug is also excreted into urine after oral administration, indicating that tofogliflozin is eliminated through multiple pathways. Thus, the exposure of tofogliflozin would not be significantly altered by DDI caused by any co-administered drugs. Also, tofogliflozin seems not to cause significant DDI of co-administered drugs because tofogliflozin has no CYP induction or inhibition potency, and the main metabolite M1 has no clinically relevant CYP inhibition potency.

  5. Microsomal metabolism of picene.

    PubMed

    Platt, K L; Petrovic, P; Seidel, A; Beermann, D; Oesch, F

    1988-01-01

    Picene, a polycyclic aromatic hydrocarbon (PAH) of environmental relevance has recently been predicted to be carcinogenic, based on quantum mechanical calculation, although in several animal studies no carcinogenicity could be detected. In order to find out if the metabolism of this PAH can provide an explanation for its lack of carcinogenicity, picene was incubated with the hepatic microsomal fraction of Sprague-Dawley rats, which had been pretreated with Aroclor 1254. Sixteen ethyl acetate-extractable metabolites could be separated by reversed-phase high-performance liquid chromatography. Comparison of the chromatographic behavior and the UV and mass spectral properties of the metabolites with those of synthetic derivatives of picene allowed the identification of trans-1,2-, -3,4-, -5,6-dihydrodiol as well as 2- and 4-phenol as microsomal metabolites of picene. At a substrate concentration of 2.7 microM and an amount of 68 micrograms microsomal protein per ml incubation volume, 4-picenol was the main microsomal metabolite with 32.2% of total metabolic conversion, followed by the 1,2-(bay-region)dihydrodiol with 16.7%, the 3,4-(M-region)dihydrodiol with 15.9%, 2-picenol with 9.1% and the 5,6-(K-region)dihydrodiol with 1.6%. In this respect the metabolism of picene is not significantly different from that of the carcinogenic PAH benzo[a]pyrene and dibenz[a,h]anthracene. The M-region dihydrodiols, potential precursors of electrophilically reactive dihydrodiol bay-region epoxides, are formed from all three PAHs at 11-16% of total metabolic conversion. From the 2.8- to 4.4-fold lower amounts of polar and water-soluble metabolites of picene as compared to dibenz[a,h]anthracene and benzo[a]pyrene it is deduced that dihydrodiol epoxides are generated from picene to a much smaller extent than from the two carcinogenic PAHs. The lacking carcinogenicity of picene could therefore result from the inability of microsomal enzymes to transform its M-region dihydrodiol to

  6. 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

  7. In vitro metabolism of canagliflozin in human liver, kidney, intestine microsomes, and recombinant uridine diphosphate glucuronosyltransferases (UGT) and the effect of genetic variability of UGT enzymes on the pharmacokinetics of canagliflozin in humans.

    PubMed

    Francke, Stephan; Mamidi, Rao N V S; Solanki, Bhavna; Scheers, Ellen; Jadwin, Andrew; Favis, Reyna; Devineni, Damayanthi

    2015-09-01

    O-glucuronidation is the major metabolic elimination pathway for canagliflozin. The objective was to identify enzymes and tissues involved in the formation of 2 major glucuronidated metabolites (M7 and M5) of canagliflozin and subsequently to assess the impact of genetic variations in these uridine diphosphate glucuronosyltransferases (UGTs) on in vivo pharmacokinetics in humans. In vitro incubations with recombinant UGTs revealed involvement of UGT1A9 and UGT2B4 in the formation of M7 and M5, respectively. Although M7 and M5 were formed in liver microsomes, only M7 was formed in kidney microsomes. Participants from 7 phase 1 studies were pooled for pharmacogenomic analyses. A total of 134 participants (mean age, 41 years; men, 63%; white, 84%) were included in the analysis. In UGT1A9*3 carriers, exposure of plasma canagliflozin (Cmax,ss , 11%; AUCτ,ss , 45%) increased relative to the wild type. An increase in exposure of plasma canagliflozin (Cmax,ss , 21%; AUCt,ss , 18%) was observed in participants with UGT2B4*2 genotype compared with UGT2B4*2 noncarriers. Metabolites further delineate the role of both enzymes. The pharmacokinetic findings in participants carrying the UGT1A9*3 and UGT2B4*2 allele implicate that UGT1A9 and UGT2B4 are involved in the metabolism of canagliflozin to M7 and M5, respectively.

  8. Activated protooncogenes in human lung tumors from smokers.

    PubMed

    Reynolds, S H; Anna, C K; Brown, K C; Wiest, J S; Beattie, E J; Pero, R W; Iglehart, J D; Anderson, M W

    1991-02-15

    Fourteen primary human lung tumor DNAs from smokers were analyzed for transforming activity by two DNA transfection assays. Activated protooncogenes were detected in 3 of 11 tumor DNAs by the NIH 3T3 focus assay, whereas activated protooncogenes were detected in 11 of 13 tumor DNAs by the NIH 3T3 cotransfection-nude mouse tumorigenicity assay. K- or NRAS genes activated by point mutation at codons 12 or 61 were detected in a large cell carcinoma, a squamous cell carcinoma, and 5 adenocarcinomas. An HRAS oncogene activated by a different mechanism was detected in an epidermoid carcinoma. One adenocarcinoma was found to contain an activated RAF gene. Two unidentified transforming genes were detected in a squamous cell carcinoma DNA and two adenocarcinoma DNAs. Eight of 10 lung adenocarcinomas that had formed metastases at the time of surgery were found to contain RAS oncogenes. No significant increase in metastasis was observed in the lung adenocarcinomas that contained one or more 6-kilobase EcoRI alleles of the LMYC gene. Overall, 12 of 14 (86%) of the lung tumor DNAs from smokers were found to contain activated protooncogenes. RAS oncogenes appear to play a role in the development of metastases in lung adenocarcinomas.

  9. Human lung morphology models for particle deposition studies.

    PubMed

    Martonen, T B; Schroeter, J D; Hwang, D; Fleming, J S; Conway, J H

    2000-01-01

    Knowledge of human lung morphology is of paramount importance in calculating deposition patterns of inhaled particulate matter (PM) to be used in the definition of ambient air quality standards. Due to the inherently complex nature of the branching structure of the airway network, practical assumptions must be made for modeling purposes. The most commonly used mathematical models reported in the literature that describe PM deposition use Weibel's model A morphology. This assumes the airways of the lung to be a symmetric, dichotomously branching system. However, computer simulations of this model, when compared to scintigraphy images, have shown it to lack physiological realism (Martonen et al., 1994a). Therefore, a more physiologically realistic model of the lung is needed to improve the current PM dosimetry models. Herein, a morphological model is presented that is based on laboratory data from planar gamma camera and single-photon emission computed tomography (SPECT) images. Key elements of this model include: The parenchymal wall of the lung is defined in mathematical terms, the whole lung is divided into distinct left and right components, a set of branching angles is derived from experimental measurements, and the branching network is confined within the discrete left and right components (i.e., there is no overlapping of airways). In future work, this new, more physiologically realistic morphological model can be used to calculate PM deposition patterns for risk assessment protocols.

  10. Utility of intersystem extrapolation factors in early reaction phenotyping and the quantitative extrapolation of human liver microsomal intrinsic clearance using recombinant cytochromes P450.

    PubMed

    Chen, Yuan; Liu, Liling; Nguyen, Khanh; Fretland, Adrian J

    2011-03-01

    Reaction phenotyping using recombinant human cytochromes P450 (P450) has great utility in early discovery. However, to fully realize the advantages of using recombinant expressed P450s, the extrapolation of data from recombinant systems to human liver microsomes (HLM) is required. In this study, intersystem extrapolation factors (ISEFs) were established for CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4 using 11 probe substrates, based on substrate depletion and/or metabolite formation kinetics. The ISEF values for CYP2C9, CYP2D6, and CYP3A4 determined using multiple substrates were similar across substrates. When enzyme kinetics of metabolite formation for CYP1A2, 2C9, 2D6, and 3A4 were used, the ISEFs determined were generally within 2-fold of that determined on the basis of substrate depletion. Validation of ISEFs was conducted using 10 marketed drugs by comparing the extrapolated data with published data. The major isoforms responsible for the metabolism were identified, and the contribution of the predominant P450s was similar to that of previously reported data. In addition, phenotyping data from internal compounds, extrapolated using the rhP450-ISEF method, were comparable to those obtained using an HLM-based inhibition assay approach. Moreover, the intrinsic clearance (CL(int)) calculated from extrapolated rhP450 data correlated well with measured HLM CL(int). The ISEF method established in our laboratory provides a convenient tool in early reaction phenotyping for situations in which the HLM-based inhibition approach is limited by low turnover and/or unavailable metabolite formation. Furthermore, this method allows for quantitative extrapolation of HLM intrinsic clearance from rhP450 phenotyping data simultaneously to obtaining the participating metabolizing enzymes.

  11. 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.

  12. Identification of cytochrome P450 enzymes involved in the metabolism of 3',4'-methylenedioxy-alpha-pyrrolidinopropiophenone (MDPPP), a designer drug, in human liver microsomes.

    PubMed

    Springer, D; Staack, R F; Paul, L D; Kraemer, T; Maurer, H H

    2005-03-01

    The metabolism of 3',4'-methylenedioxy-a-pyrrolidinopropiophenone (MDPPP), a novel designer drug, to its demethylenated major metabolite 3',4'-dihydroxy-pyrrolidinopropiophenone (di-HO-PPP) was studied in pooled human liver microsomes (HLM) and in cDNA-expressed human hepatic cytochrome P450 (CYP) enzymes. CYP2C19 catalysed the demethylenation with apparent Km and Vmax values of 120.0+/-13.4 microM and 3.2+/-0.1 pmol/min/pmol CYP, respectively (mean+/-standard deviation). CYP2D6 catalysed the demethylenation with apparent Km and Vmax values of 13.5+/-1.5 microM and 1.3+/-0.1 pmol/min/pmol CYP, respectively. HLM exhibited a clear biphasic profile with an apparent Km,1 value of 7.6+/-9.0 and a Vmax,1 value of 11.1+/-3.6 pmol/min/mg protein, respectively. Percentages of intrinsic clearances of MDPPP by specific CYPs were calculated using the relative activity factor (RAF) approach with (S)-mephenytoin-4'-hydroxylation or bufuralol-1'-hydroxylation as index reactions for CYP2C19 or CYP2D6, respectively. MDPPP, di-HO-PPP and the standard 4'-methyl-pyrrolidinohexanophenone (MPHP) were separated and analysed by liquid chromatography-mass spectrometry in the selected-ion monitoring (SIM) mode. The CYP2D6-specific chemical inhibitor quinidine (3 microM) significantly (p<0.001) inhibited di-HO-PPP formation by 75.8%+/-1.7% (mean+/-standard error of the mean) in incubation mixtures with HLM and 2 microM MDPPP. It can be concluded from the data obtained from kinetic and inhibition studies that polymorphically expressed CYP2D6 and CYP2C19 are almost equally responsible for MDPPP demethylenation.

  13. Discrimination and quantification of autofluorescence spectra of human lung cells

    NASA Astrophysics Data System (ADS)

    Rahmani, Mahya; Khani, Mohammad Mehdi; Khazaei Koohpar, Zeinab; Molik, Paria

    2016-10-01

    To study laser-induced autofluorescence spectroscopy of the human lung cell line, we evaluated the native fluorescence properties of cancer QU-DB and normal MRC-5 human lung cells during continuous exposure to 405 nm laser light. Two emission bands centered at ~470 nm and ~560 nm were observed. These peaks are most likely attributable to mitochondrial fluorescent reduced nicotinamide adenine dinucleotide and riboflavin fluorophores, respectively. This article highlights lung cell autofluorescence characterization and signal discrimination by collective investigation of different spectral features. The absolute intensity, the spectral shape factor or redox ratio, the full width of half-maximum and the full width of quarter maximum was evaluated. Moreover, the intensity ratio, the area under the peak and the area ratio as a contrast factor for normal and cancerous cells were also calculated. Among all these features it seems that the contrast factor precisely and significantly discriminates the spectral differences of normal and cancerous lung cells. On the other hand, the relative quantum yield for both cell types were found by comparing the quantum yield of an unknown compound with known fluorescein sodium as a reference solution.

  14. Noninvasive determination of ozone distribution in the human lung airways

    SciTech Connect

    Hu Shuchieh.

    1991-01-01

    The response of lung epithelium to ozone exposure depends on cell sensitivity as well as on delivered dose. Because lung damage by ozone is site-specific, it was the main objective of this study to develop and utilize a bolus-response method for the noninvasive determination of longitudinal ozone distribution in the human lung. Ozone transport in a rigid single-pathway anatomic model of the lung was simulated numerically by the method of orthogonal collocation on finite elements to predict the data that would be obtained in human subjects. The simulation results provided evidence supporting the safety of the bolus-response technique. A bolus-response inhalation system including a fast-responding chemiluminescent O[sub 3] analyzer and a small-scale O[sub 3] bolus generator was specifically developed for this project. Measurements of ozone absorption were carried out on nine healthy male subjects at a constant inspiratory and expiratory flow of 250 m[ell]/sec as well as elevated respiratory flows ranging from 150 to 1,000 m[ell]/sec. By evaluating the mathematical moments of the inspired bolus and expired response data, the fraction of inhaled ozone that was absorbed was computed in the first 200 m[ell] of the human lung. The resulting data indicated that under quiet breathing conditions, the fraction of inhaled O[sub 3] absorbed by the upper airways is roughly 50%. By increasing the respired flow from 150 to 1,000 m[ell]/sec, the fraction of ozone absorbed into upper airways decreased from 65 to 15%. Virtually all of the remaining O[sub 3] was absorbed in the lower airways.

  15. 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

  16. 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

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

    DOE PAGES

    Smith, Jordan N.; Mehinagic, Denis; Nag, Subhasree; ...

    2017-01-21

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

  18. 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

  19. Studies to further investigate the inhibition of human liver microsomal CYP2C8 by the acyl-β-glucuronide of gemfibrozil.

    PubMed

    Jenkins, S M; Zvyaga, T; Johnson, S R; Hurley, J; Wagner, A; Burrell, R; Turley, W; Leet, J E; Philip, T; Rodrigues, A D

    2011-12-01

    In previous studies, gemfibrozil acyl-β-glucuronide, but not gemfibrozil, was found to be a mechanism-based inhibitor of cytochrome P450 2C8. To better understand whether this inhibition is specific for gemfibrozil acyl-β-glucuronide or whether other glucuronide conjugates are potential substrates for inhibition of this enzyme, we evaluated several pharmaceutical compounds (as their acyl glucuronides) as direct-acting and metabolism-dependent inhibitors of CYP2C8 in human liver microsomes. Of 11 compounds that were evaluated as their acyl glucuronide conjugates, only gemfibrozil acyl-β-glucuronide exhibited mechanism-based inhibition, indicating that CYP2C8 mechanism-based inhibition is very specific to certain glucuronide conjugates. Structural analogs of gemfibrozil were synthesized, and their glucuronide conjugates were prepared to further examine the mechanism of inhibition. When the aromatic methyl groups on the gemfibrozil moiety were substituted with trifluoromethyls, the resulting glucuronide conjugate was a weaker inhibitor of CYP2C8 and mechanism-based inhibition was abolished. However, the glucuronide conjugates of monomethyl gemfibrozil analogs were mechanism-based inhibitors of CYP2C8, although not as potent as gemfibrozil acyl-β-glucuronide itself. The ortho-monomethyl analog was a more potent inhibitor than the meta-monomethyl analog, indicating that CYP2C8 favors the ortho position for oxidation and potential inhibition. Molecular modeling of gemfibrozil acyl-β-glucuronide in the CYP2C8 active site is consistent with the ortho-methyl position being the favored site of covalent attachment to the heme. Moreover, hydrogen bonding to four residues (Ser100, Ser103, Gln214, and Asn217) is implicated.

  20. Quantitative analysis of cytochrome P450 isoforms in human liver microsomes by the combination of proteomics and chemical probe-based assay.

    PubMed

    Liu, Xidong; Hu, Lianghai; Ge, Guangbo; Yang, Bo; Ning, Jing; Sun, Shixin; Yang, Ling; Pors, Klaus; Gu, Jingkai

    2014-08-01

    Cytochrome P450 (CYP) is one of the most important drug-metabolizing enzyme families, which participates in the biotransformation of many endogenous and exogenous compounds. Quantitative analysis of CYP expression levels is important when studying the efficacy of new drug molecules and assessing drug-drug interactions in drug development. At present, chemical probe-based assay is the most widely used approach for the evaluation of CYP activity although there are cross-reactions between the isoforms with high sequence homologies. Therefore, quantification of each isozyme is highly desired in regard to meeting the ever-increasing requirements for carrying out pharmacokinetics and personalized medicine in the academic, pharmaceutical, and clinical setting. Herein, an absolute quantification method was employed for the analysis of the seven isoforms CYP1A2, 2B6, 3A4, 3A5, 2C9, 2C19, and 2E1 using a proteome-derived approach in combination with stable isotope dilution assay. The average absolute amount measured from twelve human liver microsomes samples were 39.3, 4.3, 54.0, 4.6, 10.3, 3.0, and 9.3 (pmol/mg protein) for 1A2, 2B6, 3A4, 3A5, 2C9, 2C19, and 2E1, respectively. Importantly, the expression level of CYP3A4 showed high correlation (r = 0.943, p < 0.0001) with the functional activity, which was measured using bufalin-a highly selective chemical probe we have developed. The combination of MRM identification and analysis of the functional activity, as in the case of CYP3A4, provides a protocol which can be extended to other functional enzyme studies with wide application in pharmaceutical research.

  1. C-reactive protein modulates human lung fibroblast migration.

    PubMed

    Kikuchi, Kazuhiko; Kohyama, Tadashi; Yamauchi, Yasuhiro; Kato, Jun; Takami, Kazutaka; Okazaki, Hitoshi; Desaki, Masashi; Nagase, Takahide; Rennard, Stephen I; Takizawa, Hajime

    2009-02-01

    C-reactive protein (CRP) has been classically used as a marker of inflammation. The aim of this study was to investigate the effect of CRP on migration of human fetal lung fibroblasts (HFL-1) to human plasma fibronectin (HFn). Using the blindwell chamber technique, CRP inhibited HFL-1 migration in a dose-dependent fashion (at 1 microg/mL, inhibition: 32.5% +/- 7.1%; P < .05). Western blot analysis showed that CRP inhibited the p38 mitogen-activated protein kinase (MAPK) activity in the presence of HFn. Moreover, the MAPK inhibitors SB202190 (25 microM) and SB203580 (25 microM) inhibited HFn-induced cell migration, suggesting an important role of p38 MAPK in HFn-induced migration. Taken together, these results suggest that the inhibitory effect of CRP is mediated by blocking MAPK. In summary, this study demonstrates that CRP directly modulates human lung fibroblasts migration. Thus, CRP may contribute to regulation of wound healing and may be endogenous antifibrotic factor acting on lung fibrosis.

  2. Human lung small-cell carcinoma contains bombesin.

    PubMed Central

    Erisman, M D; Linnoila, R I; Hernandez, O; DiAugustine, R P; Lazarus, L H

    1982-01-01

    The presence of immunoreactive bombesin in a human lung small-cell carcinoma grown in nude mice was established by several criteria: (i) Radioimmunoassay of tissue extracts for bombesin revealed approximately 6.5 pmol/g of tissue; (ii) bombesin was found in 12-14% of the tumor cells by immunohistochemical localization; (iii) gel filtration of small-cell carcinoma extract on Sephadex G-75 and Bio-Gel P-4 gave only a single peak of immunoreactivity, which occurred at the elution volume of bombesin; and (iv) reverse-phase HPLC of acid-solubilized extracts separated the immunoreactive material into three discrete peaks, one of which eluted with a retention time identical to that of synthetic bombesin. The presence of bombesin may represent the ectopic expression of this peptide in small-cell carcinoma, because immunoreactive bombesin was found in human fetal and neonatal lung but apparently not in adult lung tissue [Wharton, J., Polak, J. M., Bloom, S. R., Ghatei, M. A., Solcia, E., Brown, M. R. & Pearse, A. G. E. (1978) Nature (London) 273, 769-770]. The immunoreactive bombesin previously found in mammalian tissues is considerably larger than amphibian bombesin; these data substantiate the presence of a mammalian form of bombesin in a human tumor that may have a structure similar to that of the amphibian peptide. Images PMID:6285381

  3. 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.

  4. 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.

  5. Proteogenomic Analysis of Human Chromosome 9-Encoded Genes from Human Samples and Lung Cancer Tissues

    PubMed Central

    Ahn, Jung-Mo; Kim, Min-Sik; Kim, Yong-In; Jeong, Seul-Ki; Lee, Hyoung-Joo; Lee, Sun Hee; Paik, Young-Ki; Pandey, Akhilesh; Cho, Je-Yoel

    2014-01-01

    The Chromosome-centric Human Proteome Project (C-HPP) was recently initiated as an international collaborative effort. Our team adopted chromosome 9 (Chr 9) and performed a bioinformatics and proteogenomic analysis to catalog Chr 9-encoded proteins from normal tissues, lung cancer cell lines and lung cancer tissues. Approximately 74.7% of the Chr 9 genes of the human genome were identified, which included approximately 28% of missing proteins (46 of 162) on Chr 9 compared with the list of missing proteins from the neXtProt master table (2013-09). In addition, we performed a comparative proteomics analysis between normal lung and lung cancer tissues. Based on the data analysis, 15 proteins from Chr 9 were detected only in lung cancer tissues. Finally, we conducted a proteogenomic analysis to discover Chr 9-residing single nucleotide polymorphisms (SNP) and mutations described in the COSMIC cancer mutation database. We identified 21 SNPs and 4 mutations containing peptides on Chr 9 from normal human cells/tissues and lung cancer cell lines, respectively. In summary, this study provides valuable information of the human proteome for the scientific community as part of C-HPP. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the data set identifier PXD. PMID:24274035

  6. Characterization of muscarinic cholinergic receptor subtypes in human peripheral lung

    SciTech Connect

    Bloom, J.W.; Halonen, M.; Yamamura, H.I.

    1988-02-01

    The authors have characterized the muscarinic cholinergic receptor subtypes in human peripheral lung membranes using the selective muscarinic antagonist (/sup 3/H)pirenzepine ((/sup 3/H)PZ) and the classical muscarinic antagonist (/sup 3/H)(-)-quinuclidinyl benzilate. High-affinity binding with pharmacologic specificity was demonstrated for both radioligands. The high affinity Kd for (/sup 3/H)PZ binding determined from saturation isotherms was 5.6 nM, and the Kd for (/sup 3/H)(-)-quinuclidinyl benzilate binding was 14.3 pM. Approximately 62% of the total muscarinic binding sites in human peripheral lung bind (/sup 3/H)PZ with high affinity. There was no significant effect of the guanine nucleotide, guanyl-5'-yl imidodiphosphate, on the inhibition of (/sup 3/H)(-)-quinyclidinyl benzilate binding by the muscarinic agonist carbachol in peripheral lung membranes. If the muscarinic receptor with high affinity for PZ has an important role in bronchoconstriction, its characterization could result in the development of more selective bronchodilators.

  7. A numerical study of gas transport in human lung models

    NASA Astrophysics Data System (ADS)

    Lin, Ching-Long; Hoffman, Eric A.

    2005-04-01

    Stable Xenon (Xe) gas has been used as an imaging agent for decades in its radioactive form, is chemically inert, and has been used as a ventilation tracer in its non radioactive form during computerized tomography (CT) imaging. Magnetic resonance imaging (MRI) using hyperpolarized Helium (He) gas and Xe has also emerged as a powerful tool to study regional lung structure and function. However, the present state of knowledge regarding intra-bronchial Xe and He transport properties is incomplete. As the use of these gases rapidly advances, it has become critically important to understand the nature of their transport properties and to, in the process, better understand the role of gas density in general in determining regional distribution of respiratory gases. In this paper, we applied the custom developed characteristic-Galerkin finite element method, which solves the three-dimensional (3D) incompressible variable-density Navier-Stokes equations, to study the transport of Xe and He in the CT-based human lung geometries, especially emulating the washin and washout processes. The realistic lung geometries are segmented and reconstructed from CT images as part of an effort to build a normative atlas (NIH HL-064368) documenting airway geometry over 4 decades of age in healthy and disease-state adult humans. The simulation results show that the gas transport process depends on the gas density and the body posture. The implications of these results on the difference between washin and washout time constants are discussed.

  8. Rewiring of human lung cell lineage and mitotic networks in lung adenocarcinomas

    PubMed Central

    Kim, Il-Jin; Quigley, David; To, Minh D.; Pham, Patrick; Lin, Kevin; Jo, Brian; Jen, Kuang-Yu; Raz, Dan; Kim, Jae; Mao, Jian-Hua; Jablons, David; Balmain, Allan

    2015-01-01

    Analysis of gene expression patterns in normal tissues and their perturbations in tumors can help to identify the functional roles of oncogenes or tumor suppressors and identify potential new therapeutic targets. Here, gene expression correlation networks were derived from 92 normal human lung samples and patient-matched adenocarcinomas. The networks from normal lung show that NKX2-1 is linked to the alveolar type 2 lineage, and identify PEBP4 as a novel marker expressed in alveolar type 2 cells. Differential correlation analysis shows that the NKX2-1 network in tumors includes pathways associated with glutamate metabolism, and identifies Vaccinia-related kinase (VRK1) as a potential drug target in a tumor-specific mitotic network. We show that VRK1 inhibition cooperates with inhibition of PARP signaling to inhibit growth of lung tumor cells. Targeting of genes that are recruited into tumor mitotic networks may provide a wider therapeutic window than that seen by inhibition of known mitotic genes. PMID:23591868

  9. 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

  10. Glucuronidation of fenamates: kinetic studies using human kidney cortical microsomes and recombinant UDP-glucuronosyltransferase (UGT) 1A9 and 2B7.

    PubMed

    Gaganis, Paraskevi; Miners, John O; Knights, Kathleen M

    2007-05-15

    Mefenamic acid, a non-steroidal anti-inflammatory drug (NSAID), is used commonly to treat menorrhagia. This study investigated the glucuronidation kinetics of flufenamic, mefenamic and niflumic acid using human kidney cortical microsomes (HKCM) and recombinant UGT1A9 and UGT2B7. Using HKCM Michaelis-Menten (MM) kinetics were observed for mefenamic (K(m)(app) 23 microM) and niflumic acid (K(m)(app) 123 microM) glucuronidation, while flufenamic acid exhibited non-hyperbolic (atypical) glucuronidation kinetics. Notably, the intrinsic renal clearance of mefenamic acid (CL(int) 17+/-5.5 microL/minmg protein) was fifteen fold higher than that of niflumic acid (CL(int) 1.1+/-0.8 microL/minmg protein). These data suggest that renal glucuronidation of mefenamic acid may result in high intrarenal exposure to mefenamic acyl-glucuronide and subsequent binding to renal proteins. Diverse kinetics were observed for fenamate glucuronidation by UGT2B7 and UGT1A9. Using UGT2B7 MM kinetics were observed for flufenamic (K(m)(app) 48 microM) and niflumic acid (K(m)(app) 135 microM) glucuronidation and atypical kinetics with mefenamic acid. Similarity in K(m)(app) between HKCM and UGT2B7 suggests that UGT2B7 may be the predominant renal UGT isoform catalysing niflumic acid glucuronidation. In contrast, UGT1A9 glucuronidation kinetics were characterised by negative cooperativity with mefenamic (S(50) 449 microM, h 0.4) and niflumic acid (S(50) 7344 microM, h 0.4) while atypical kinetics were observed with flufenamic acid. Additionally, potent inhibition of the renal glucuronidation of the UGT substrate 'probe' 4-methylumbelliferone by flufenamic, mefenamic and niflumic acid was observed. These data suggest that inhibitory metabolic interactions may occur between fenamates and other substrates metabolised by UGT2B7 and UGT1A9 in human kidney.

  11. Effects of green tea catechins on cytochrome P450 2B6, 2C8, 2C19, 2D6 and 3A activities in human liver and intestinal microsomes.

    PubMed

    Misaka, Shingen; Kawabe, Keisuke; Onoue, Satomi; Werba, José Pablo; Giroli, Monica; Tamaki, Sekihiro; Kan, Toshiyuki; Kimura, Junko; Watanabe, Hiroshi; Yamada, Shizuo

    2013-01-01

    The effects of green tea catechins on the main drug-metabolizing enzymatic system, cytochrome P450 (CYP), have not been fully elucidated. The objective of the present study was to evaluate the effects of green tea extract (GTE, total catechins 86.5%, w/w) and (-)-epigallocatechin-3-gallate (EGCG) on the activities of CYP2B6, CYP2C8, CYP2C19, CYP2D6 and CYP3A in vitro, using pooled human liver and intestinal microsomes. Bupropion hydroxylation, amodiaquine N-deethylation, (S)-mephenytoin 4'-hydroxylation, dextromethorphan O-demethylation and midazolam 1'-hydroxylation were assessed in the presence or absence of various concentrations of GTE and EGCG to test their effects on CYP2B6, CYP2C8, CYP2C19, CYP2D6 and CYP3A activities, respectively. Each metabolite was quantified using UPLC/ESI-MS, and the inhibition kinetics of GTE and EGCG on CYP enzymes was analyzed. In human liver microsomes, IC50 values of GTE were 5.9, 4.5, 48.7, 25.1 and 13.8 µg/mL, for CYP2B6, CYP2C8, CYP2C19, CYP2D6 and CYP3A, respectively. ECGC also inhibited these CYP isoforms with properties similar to those of GTE, and produced competitive inhibitions against CYP2B6 and CYP2C8, and noncompetitive inhibition against CYP3A. In human intestinal microsomes, IC50 values of GTE and EGCG for CYP3A were 18.4 µg/mL and 31.1 µM, respectively. EGCG moderately inhibited CYP3A activity in a noncompetitive manner. These results suggest that green tea catechins cause clinically relevant interactions with substrates for CYP2B6 and CYP2C8 in addition to CYP3A.

  12. 2-Diethylaminoethyl-2,2-diphenylvalerate-HCl (SKF525A) revisited: comparative cytochrome P450 inhibition in human liver microsomes by SKF525A, its metabolites, and SKF-acid and SKF-alcohol.

    PubMed

    Franklin, Michael R; Hathaway, Laura B

    2008-12-01

    When incubated with human liver microsomes, 2-diethylaminoethyl-2,2-diphenylvalerate-HCl (SKF525A) undergoes cytochrome P450 (P450)-dependent oxidative N-deethylation to the secondary amine metabolite 2-ethylaminoethyl-2,2-diphenylvalerate (SKF8742). P450-selective inhibitors indicated CYP3As catalyzed this reaction, and the deethylation rate correlated best with the CYP3A activity across a range of human liver microsomes. SKF525A and its metabolite and primary amine analog all inhibited CYP2B6-, CYP2C9-, CYP2C19-, CYP2D6-, and CYP3A-selective reactions to varying degrees but had little effect on CYP1A2, CYP2A6, and CYP2E1 reactions. Only the inhibition of CYP3A showed major enhancement when the inhibitors were preincubated with NADPH-fortified microsomes, and the extent of metabolic intermediate (MI) complex formation approximated typical CYP3A content. Two "lost with time" SKF525A derivatives devoid of the ethylamine moiety, 2,2-diphenylpropylethanol (SKF-Alcohol) and 2,2-diphenylpropylacetic acid (SKF-Acid) did not form an MI complex and were identified as selective inhibitors of CYP2C9. Although without detectable metabolism, their CYP2C9 inhibition fitted best with a competitive mechanism. Thus, not all the human P450s are inhibited by SKF525A and related compounds, and the mechanisms contributing to those that are inhibited vary with the isoform. P450 MI-complex formation only seems to play a role with CYP3As.

  13. 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.

  14. 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.

  15. 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.

  16. Stereo-selective metabolism of methadone by human liver microsomes and cDNA-expressed cytochrome P450s: a reconciliation.

    PubMed

    Chang, Yan; Fang, Wenfang B; Lin, Shen-Nan; Moody, David E

    2011-01-01

    In vitro metabolism of methadone was investigated in cytochrome P450 (CYP) supersomes and phenotyped human liver microsomes (HLMs) to reconcile past findings on CYP involvement in stereo-selective metabolism of methadone. Racaemic methadone was used for incubations; (R)- and (S)-methadone turnover and (R)- and (S)-EDDP formation were determined using chiral liquid chromatography-tandem mass spectrometry. CYP supersome activity for methadone use and EDDP formation ranked CYP2B6 > 3A4 > 2C19 > 2D6 > 2C18, 3A7 > 2C8, 2C9, 3A5. After abundance scaling, CYP3A4, 2B6 and 2C19 accounted for 63-74, 12-32 and 1. 4-14% of respective activity. CYP2B6, 2D6 and 2C18 demonstrated a preference for (S)-EDDP formation; CYP2C19, 3A7 and 2C8 for (R)-EDDP; 3A4 none. Correlation analysis with 15 HLMs supported the involvement of CYP2B6 and 3A. The significant correlation of S/R ratio with CYP2B6 activity confirmed its stereo-selectivity. CYP2C19 and 2D6 inhibitors and monoclonal antibody (mAb) did not inhibit EDDP formation in HLM. Chemical and mAb inhibition of CYP3A in high 3A activity HLM reduced EDDP formation by 60-85%; inhibition of CYP2B6 in 2B6 high-activity HLM reduced (S)-EDDP formation by 80% and (R)-EDDP formation by 55%. Inhibition changed methadone metabolism in a stereo-selective manner. When CYP3A was inhibited, 2B6 mediated (S)-EDDP formation predominated; S/R stereo-selectivity increased. When 2B6 was inhibited (S)-EDDP formation fell and stereo-selectivity decreased. The results confirmed the primary roles of CYPs 3A4 and 2B6 in methadone metabolism; CYP2C8 and 2C9 did not appear involved; 2C19 and 2D6 have minimal roles. CYP2B6 is the primary determinant of stereo-selective metabolism; stereo-selective inhibition might play a role in varied plasma concentrations of the two enantiomers.

  17. [The lungs in human immunodeficiency virus type 1 infection].

    PubMed

    Barić, D; Vrkić, L

    1997-01-01

    This report describes a case of two patients who were admitted to the Zadar hospital and according to clinical symptoms directed to the Department of Lung Diseases. Both patients were temporarily employed abroad. It has been established that they were infected with human immunodeficiency virus type 1 (HIV-1). One of the patients has been moved to the Department of Infectious Diseases and later to Zagreb, while the other has returned abroad. On admission to the hospital of the Zadar Medical Center none of them answered the question about being engaged in risky behavior. In 1990 there were 699 registered patients hospitalized and 745 registered in the protocol of the Outpatient Clinic of the Department of Lung Diseases. 0.069% of patients were HIV-1-infected. In 1991, there were 520 hospitalized and 453 outpatients, whereas 0.102% were HIV-1-infected and registered subjects. It must be pointed out that these are only numbers of registration and not subjects, because there were patients who were examined or hospitalized twice or more times during the corresponding calendar year. The aim of this study was to point to a new differentially-diagnostic problem present especially at the Department of Lung Diseases after AIDS has become part of our reality. There still remains a problem in regard to detection of HIV-1 seropositivity in patients at departments with opportunistic infections such as tuberculosis.

  18. 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

  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. 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.

  1. 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).

  2. The role of cytokines in human lung fibrosis.

    PubMed

    Vaillant, P; Menard, O; Vignaud, J M; Martinet, N; Martinet, Y

    1996-04-01

    Fibrosis is a disorder characterized by a qualitative and quantitative alteration of the deposition of extracellular matrix with accumulation of mesenchymal cells in replacement of normal tissue. The sequence of events leading to fibrosis of an organ involves the subsequent processes of injury with inflammation and disruption of the normal tissue architecture, followed by tissue repair with accumulation of mesenchymal cells in this area. A similar sequence of events occurs in wound healing with formation of normal, limited and transient granulation tissue, while in fibrosis, a maladaptive repair leads to an extensive, exaggerated process with functional impairment. Inflammatory cells (mainly mononuclear phagocytes), platelets, endothelial cells, and type II pneumocytes play a direct and indirect role in tissue injury and repair. The evaluation of several human fibrotic lung diseases, five diffuse (idiopathic pulmonary fibrosis (IPF); adult respiratory distress syndrome (ARDS); coal workers' pneumoconiosis (CWP); Hermansky-Pudlak syndrome (HPS); systemic sclerosis (SS)) and two focal (tumour stroma in lung cancer; and obliterative bronchiolitis (OB) after lung transplantation), has shown that several cytokines participate in the local injury and inflammatory reaction (interleukin-1 (IL-1), interleukin-8 (IL-8), monocyte chemotactic protein-1 (MCP-1), and tumour necrosis factor-alpha (TNF-alpha)), while other cytokines are involved in tissue repair and fibrosis (platelet-derived growth factor (PDGF), insulin-like growth factor-1 (IGF-1), transforming growth factor-beta (TGF-beta), and basic-fibroblast growth factor (b-FGF)). A better understanding of the cytokines and cytokine networks involved in lung fibrosis leads to the possibility of new therapeutic approaches.

  3. 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

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

    PubMed

    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.

  5. The Role of Serotonin Transporter in Human Lung Development and in Neonatal Lung Disorders

    PubMed Central

    Sen, P.; Parks, W. T.; Langston, C.

    2017-01-01

    Introduction. Failure of the vascular pulmonary remodeling at birth often manifests as pulmonary hypertension (PHT) and is associated with a variety of neonatal lung disorders including a uniformly fatal developmental disorder known as alveolar capillary dysplasia with misalignment of pulmonary veins (ACD/MPV). Serum serotonin regulation has been linked to pulmonary vascular function and disease, and serotonin transporter (SERT) is thought to be one of the key regulators in these processes. We sought to find evidence of a role that SERT plays in the neonatal respiratory adaptation process and in the pathomechanism of ACD/MPV. Methods. We used histology and immunohistochemistry to determine the timetable of SERT protein expression in normal human fetal and postnatal lungs and in cases of newborn and childhood PHT of varied etiology. In addition, we tested for a SERT gene promoter defect in ACD/MPV patients. Results. We found that SERT protein expression begins at 30 weeks of gestation, increases to term, and stays high postnatally. ACD/MPV patients had diminished SERT expression without SERT promoter alteration. Conclusion. We concluded that SERT/serotonin pathway is crucial in the process of pulmonary vascular remodeling/adaptation at birth and plays a key role in the pathobiology of ACD/MPV. PMID:28316463

  6. 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.

  7. Growth suppressive efficacy of human lak cells against human lung-cancer implanted into scid mice.

    PubMed

    Teraoka, S; Kyoizumi, S; Suzuki, T; Yamakido, M; Akiyama, M

    1995-06-01

    The purpose of our study was to determine the efficacy of immunotherapy using human lymphokine activated killer (LAK) cells against a human-lung squamous-cell carcinoma cell line (RERF-LC-AI) implanted into severe combined immunodeficient (SCID) mice. A statistically significant growth suppressive effect on RERF-LC-AI implanted into SCID mice was observed when human LAK cells were administered into the caudal vein of the mice treated with a continuous supply (initiated prior to LAK cells injection) of rIL-2. The human LAK cells stained with PKH 2, a fluorescent dye, for later detection using flow cytometry were administered into the caudal vein of RERF-LC-AI bearing SCID mice; the cells persisted for 7 days in the implanted lung cancer tissue and in the mouse peripheral blood, but for 5 days in the mouse spleen. The number of infiltrated human LAK cells in each tissue increased dose-dependently with the number of injected cells. The results indicate that the antitumor effect most likely occurred during the early implantation period of the human LAK cells. These results demonstrate the applicability of this model to the in vivo study of human lung cancer therapy.

  8. The cytotoxicity and genotoxicity of soluble and particulate cobalt in human lung epithelial cells.

    PubMed

    Xie, Hong; Smith, Leah J; Holmes, Amie L; Zheng, Tongzhang; Pierce Wise, John

    2016-05-01

    Cobalt is a toxic metal used in various industrial applications leading to adverse lung effects by inhalation. Cobalt is considered a possible human carcinogen with the lung being a primary target. However, few studies have considered cobalt-induced toxicity in human lung cells, especially normal lung epithelial cells. Therefore, in this study, we sought to determine the cytotoxicity and genotoxicity of particulate and soluble cobalt in normal primary human lung epithelial cells. Cobalt oxide and cobalt chloride were used as representative particulate and soluble cobalt compounds, respectively. Exposure to both particulate and soluble cobalt induced a concentration-dependent increase in cytotoxicity, genotoxicity, and intracellular cobalt ion levels. Based on intracellular cobalt ion levels, we found that soluble and particulate cobalt induced similar cytotoxicity while soluble cobalt was more genotoxic than particulate cobalt. These data indicate that cobalt compounds are cytotoxic and genotoxic to human lung epithelial cells.

  9. 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

  10. Establishment of a human lung cancer cell line with high metastatic potential to multiple organs: gene expression associated with metastatic potential in human lung cancer.

    PubMed

    Nakano, Tetsuhiro; Shimizu, Kimihiro; Kawashima, Osamu; Kamiyoshihara, Mitsuhiro; Kakegawa, Seiichi; Sugano, Masayuki; Ibe, Takashi; Nagashima, Toshiteru; Kaira, Kyoichi; Sunaga, Noriaki; Ohtaki, Youichi; Atsumi, Jun; Takeyoshi, Izumi

    2012-11-01

    Convenient and reliable multiple organ metastasis model systems might contribute to understanding the mechanism(s) of metastasis of lung cancer, which may lead to overcoming metastasis and improvement in the treatment outcome of lung cancer. We isolated a highly metastatic subline, PC14HM, from the human pulmonary adenocarcinoma cell line, PC14, using an in vivo selection method. The expression of 34,580 genes was compared between PC14HM and parental PC14 by cDNA microarray analysis. Among the differentially expressed genes, expression of four genes in human lung cancer tissues and adjacent normal lung tissues were compared using real-time reverse transcription polymerase chain reaction. Although BALB/c nude mice inoculated with parental PC14 cells had few metastases, almost all mice inoculated with PC14HM cells developed metastases in multiple organs, including the lung, bone and adrenal gland, the same progression seen in human lung cancer. cDNA microarray analysis revealed that 981 genes were differentially (more than 3-fold) expressed between the two cell lines. Functional classification revealed that many of those genes were associated with cell growth, cell communication, development and transcription. Expression of three upregulated genes (HRB-2, HS3ST3A1 and RAB7) was higher in human cancer tissue compared to normal lung tissue, while expression of EDG1, which was downregulated, was lower in the cancer tissue compared to the normal lung. These results suggest that the newly established PC14HM cell line may provide a mouse model of widespread metastasis of lung cancer. This model system may provide insights into the key genetic determinants of widespread metastasis of lung cancer.

  11. Determination of 4-hydroxy-3-methoxymethamphetamine as a metabolite of methamphetamine in rats and human liver microsomes using gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry.

    PubMed

    Kuwayama, Kenji; Tsujikawa, Kenji; Miyaguchi, Hajime; Kanamori, Tatsuyuki; Iwata, Yuko T; Inoue, Hiroyuki

    2009-06-01

    The aim of this study was to determine whether methamphetamine (MA) is metabolized to 4-hydroxy-3-methoxymethamphetamine (HMMA), which is known as the main metabolite of 3,4-methylenedioxymethamphetamine (MDMA). After MA was intravenously administered to rats, the plasma, urine, and bile were collected periodically. HMMA together with MA and its main metabolites, amphetamine and 4-hydroxymethamphetamine, were detected in the rat plasma, urine, and bile by gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry. In addition, HMMA was produced when MA was incubated with human liver microsomes. HMMA may be produced as a metabolite of MA when humans have consumed MA, although the amount of HMMA would be small compared with that of MA, amphetamine, or 4-hydroxymethamphetamine. The results of the present study will be helpful in determining the type of drug used.

  12. Preferential elevation of Prx I and Trx expression in lung cancer cells following hypoxia and in human lung cancer tissues.

    PubMed

    Kim, H J; Chae, H Z; Kim, Y J; Kim, Y H; Hwangs, T S; Park, E M; Park, Y M

    2003-10-01

    Transient/chronic microenvironmental hypoxia that exists within a majority of solid tumors has been suggested to have a profound influence on tumor growth and therapeutic outcome. Since the functions of novel antioxidant proteins, peroxiredoxin I (Prx I) and II, have been implicated in regulating cell proliferation, differentiation, and apoptosis, it was of our special interest to probe a possible role of Prx I and II in the context of hypoxic tumor microenvironment. Since both Prx I and II use thioredoxin (Trx) as an electron donor and Trx is a substrate for thioredoxin reductase (TrxR), we investigated the regulation of Trx and TrxR as well as Prx expression following hypoxia. Here we show a dynamic change of glutathione homeostasis in lung cancer A549 cells and an up-regulation of Prx I and Trx following hypoxia. Western blot analysis of 10 human lung cancer and paired normal lung tissues also revealed an elevated expression of Prx I and Trx proteins in lung cancer tissues. Immunohistochemical analysis of the lung cancer tissues confirmed an augmented Prx I and Trx expression in cancer cells with respect to the parenchymal cells in adjacent normal lung tissue. Based on these results, we suggest that the redox changes in lung tumor microenvironment could have acted as a trigger for the up-regulation of Prx I and Trx in lung cancer cells. Although the clinical significance of our finding awaits more rigorous future study, preferential augmentation of the Prx I and Trx in lung cancer cells may well represent an attempt of cancer cells to manipulate a dynamic redox change in tumor microenvironment in a manner that is beneficial for their proliferation and malignant progression.

  13. 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.

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

    PubMed

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

    2013-11-01

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

  15. Effects of anthocyanidins and anthocyanins on the expression and catalytic activities of CYP2A6, CYP2B6, CYP2C9, and CYP3A4 in primary human hepatocytes and human liver microsomes.

    PubMed

    Srovnalova, Alzbeta; Svecarova, Michaela; Zapletalova, Michaela Kopecna; Anzenbacher, Pavel; Bachleda, Petr; Anzenbacherova, Eva; Dvorak, Zdenek

    2014-01-22

    Anthocyanidins and anthocyanins are pharmacologically active constituents of various berry fruits, such as blueberry and cranberry. These compounds are also contained in massively used nutritional supplements based on extracts or dry matter from berry fruits. The current study evaluated the effects of anthocyanidins and anthocyanins on the expression and catalytic activity of major drug-metabolizing enzymes CYP2C9, CYP2A6, CYP2B6, and CYP3A4 in primary cultures of human hepatocytes and human liver microsomes. Expression of mRNA was quantified by qRT-PCR. Expression of proteins was evaluated by Western blotting and immunochemiluminescence. The catalytic activity of CYP enzymes was measured by HPLC using specific enzyme substrates. Tested anthocyanidins (6) and anthocyanins (21) did not induce the expression of mRNA and protein of CYP2C9, CYP2A6, CYP2B6, and CYP3A4 genes in human hepatocytes. Catalytic activities of CYP2C9, CYP2A6, CYP2B6, and CYP3A4 enzymes were inhibited by all anthocyanidins to different extents (e.g., delphinidin inhibits CYP3A4 by >90% at 100 μM with IC50 = 32 μM). Of 21 anthocyanins tested, only cyanidin-3-O-rhamnoside (CYP3A4 by >75% at 100 μM with IC50 = 44 μM) and two glycosides of delphinidin significantly inhibited examined cytochromes P450. It may be concluded that in the ranges of common ingestion of either food or dietary supplement an induction or significant inhibition of CYP2C9, CYP2A6, CYP2B6, and CYP3A4 activity is most probably not expected.

  16. In vitro metabolism of two heterocyclic amines, 2-amino-9H-pyrido[2,3-b]indole (A(alpha)C) and 2-amino-3-methyl-9H-pyridol2,3-b]indole (MeA(alpha)C) in human and rat hepatic microsomes.

    PubMed

    Frederiksen, Hanne; Frandsen, Henrik

    2002-03-01

    2-Amino-9H-pyrido[2,3-b]indole (A(alpha)C) and 2-amino-3-methyl-9H-pyrido[2,3-b]indole (MeA(alpha)C) are two mutagenic and carcinogenic heterocyclic amines formed during ordinary cooking. In this study, we have investigated the in vitro metabolism of tritium-labelled A(alpha)C and MeA(alpha)C in hepatic microsomes from human pools, rats induced with polychlorinated biphenyl (PCB) (Aroclor 1254) and control rats. The microsomes were incubated with A(alpha)C and MeAaC and the detoxified and activated metabolites of A(alpha)C and MeA(alpha)C were separated and characterised by HPLC-MS. A(alpha)C is metabolised to two major and three minor detoxified metabolites, while MeA(alpha)C is metabolised to three major and one minor detoxified metabolites. Some A(alpha)C and MeA(alpha)C are activated by oxidation to the reactive metabolites N2-OH-A(alpha)C and N2-OH-MeA(alpha)C, respectively. These reactive N2-OH-metabolites react partially in the incubation system with formation of protein adducts, dimers and the parent compound by reduction of the N2-OH-metabolites. The distribution between the detoxified and activated metabolites in the different types of hepatic microsomes showed same pattern for both A(alpha)C and MeA(alpha)C. In PCB-induced rat microsomes, the major part of the metabolites are detoxified, only a little amount is activated. In control rat microsomes there is a fifty-fifty distribution between detoxification and activation, while the major part of the metabolites from the human microsomes are activated and reacts to form dimers and protein adducts. These data show that, in human hepatic microsomes compared to rat hepatic microsomes, a major part of A(alpha)C and MeA(alpha)C are metabolically activated to the reactive N2-OH-A(alpha)C and N2-OH-MeA(alpha)C.

  17. 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.

  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. Application of a Neutral Community Model To Assess Structuring of the Human Lung Microbiome

    PubMed Central

    Venkataraman, Arvind; Bassis, Christine M.; Beck, James M.; Young, Vincent B.; Curtis, Jeffrey L.; Huffnagle, Gary B.

    2015-01-01

    ABSTRACT  DNA from phylogenetically diverse microbes is routinely recovered from healthy human lungs and used to define the lung microbiome. The proportion of this DNA originating from microbes adapted to the lungs, as opposed to microbes dispersing to the lungs from other body sites and the atmosphere, is not known. We use a neutral model of community ecology to distinguish members of the lung microbiome whose presence is consistent with dispersal from other body sites and those that deviate from the model, suggesting a competitive advantage to these microbes in the lungs. We find that the composition of the healthy lung microbiome is consistent with predictions of the neutral model, reflecting the overriding role of dispersal of microbes from the oral cavity in shaping the microbial community in healthy lungs. In contrast, the microbiome of diseased lungs was readily distinguished as being under active selection. We also assessed the viability of microbes from lung samples by cultivation with a variety of media and incubation conditions. Bacteria recovered by cultivation from healthy lungs represented species that comprised 61% of the 16S rRNA-encoding gene sequences derived from bronchoalveolar lavage samples. Importance  Neutral distribution of microbes is a distinguishing feature of the microbiome in healthy lungs, wherein constant dispersal of bacteria from the oral cavity overrides differential growth of bacteria. No bacterial species consistently deviated from the model predictions in healthy lungs, although representatives of many of the dispersed species were readily cultivated. In contrast, bacterial populations in diseased lungs were identified as being under active selection. Quantification of the relative importance of selection and neutral processes such as dispersal in shaping the healthy lung microbiome is a first step toward understanding its impacts on host health. PMID:25604788

  1. In vitro immunological degranulation of human basophils is modulated by lung histamine and Apis mellifica.

    PubMed Central

    Poitevin, B; Davenas, E; Benveniste, J

    1988-01-01

    1. The effect of high dilutions of two homeopathic drugs Lung histamine (Lung his) and Apis mellifica (Apis mel) used for the treatment of allergic diseases has been assessed on in vitro human basophil degranulation. Experiments were conducted blind. 2. Basophil degranulation induced by 1.66 X 10(-9) M anti-IgE antibody was significantly inhibited in the presence of 5 Lung his (5th centesimal dilution of Lung his) and 15 Lung his (15th centesimal dilution of Lung his) by 28.8% and 28.6% respectively and by 65.8% in the presence of 9 Apis mel (9th centesimal dilution of Apis mel). Basophil degranulation induced by 1.66 X 10(-16) to 1.66 X 10(-18) M anti-IgE antibody was also inhibited by high dilutions of Lung his and Apis mel with an inhibition of nearly 100% with 18 Lung his (18th centesimal dilution of Lung his) and 10 Apis mel (10th centesimal dilution of Apis mel). An alternance of inhibition, inactivity and stimulation was observed when basophils were incubated in the presence of serial dilutions of Lung his and Apis mel. 3. The investigation of the clinical efficacy of high dilutions of Lung his and Apis mel should be envisaged in allergic diseases in parallel with in vitro and ex vivo biological assays. PMID:3382588

  2. Differential response of the epithelium and interstitium in developing human fetal lung explants to hyperoxia.

    PubMed

    Bustani, Porus; Hodge, Rachel; Tellabati, Ananth; Li, Juan; Pandya, Hitesh; Kotecha, Sailesh

    2006-03-01

    Hyperoxia is closely linked with the development of chronic lung disease of prematurity (CLD), but the exact mechanisms whereby hyperoxia alters the lung architecture in the developing lung remain largely unknown. We developed a fetal human lung organ culture model to investigate (a) the morphologic changes induced by hyperoxia and (b) whether hyperoxia resulted in differential cellular responses in the epithelium and interstitium. The effects of hyperoxia on lung morphometry were analyzed using computer-assisted image analysis. The lung architecture remained largely unchanged in normoxia lasting as long as 4 d. In contrast, hyperoxic culture of pseudoglandular fetal lungs resulted in significant dilatation of airways, thinning of the epithelium, and regression of the interstitium including the pulmonary vasculature. Although there were no significant differences in Ki67 between normoxic and hyperoxic lungs, activated caspase-3 was significantly increased in interstitial cells, but not epithelial cells, under hyperoxic conditions. These changes show that exposure of pseudoglandular lungs to hyperoxia modulates the lung architecture to resemble saccular lungs.

  3. 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

  4. Expression of inducible nitric oxide in human lung epithelial cells.

    PubMed

    Robbins, R A; Barnes, P J; Springall, D R; Warren, J B; Kwon, O J; Buttery, L D; Wilson, A J; Geller, D A; Polak, J M

    1994-08-30

    Nitric oxide (NO) is increased in the exhaled air of subjects with several airway disorders. To determine if cytokines could stimulate epithelial cells accounting for the increased NO, the capacity of the proinflammatory cytokines (cytomix: tumor necrosis factor-alpha, interleukin-1 beta, and interferon-gamma) to increase inducible nitric oxide synthase (iNOS) was investigated in A549 and primary cultures of human bronchial epithelial cells. Cytomix induced a time-dependent increase in nitrite levels in culture supernatant fluids (p < 0.05). Increased numbers of cells stained for iNOS and increased iNOS mRNA was detected in the cytokine-stimulated cells compared to control (p < 0.05). Dexamethasone diminished the cytokine-induced increase in nitrite, iNOS by immunocytochemistry, and iNOS mRNA. These data demonstrate that cytokines, such as those released by mononuclear cells, can induce lung epithelial iNOS expression and NO release, and that this is attenuated by dexamethasone.

  5. Toxicity of Cerium Oxide Nanoparticles in Human Lung Cancer Cells

    SciTech Connect

    Weisheng, Lin; Huang, Yue-wern; Zhou, Xiao Dong; Ma, Yinfa

    2006-12-31

    With the fast development of nanotechnology, the nanomaterials start to cause people's attention for potential toxic effect. In this paper, the cytotoxicity and oxidative stress caused by 20-nm cerium oxide (CeO2) nanoparticles in cultured human lung cancer cells was investigated. The sulforhodamine B method was employed to assess cell viability after exposure to 3.5, 10.5, and 23.3 μg/ml of CeO2 nanoparticles for 24, 48, and 72 h. Cell viability decreased significantly as a function of nanoparticle dose and exposure time. Indicators of oxidative stress and cytotoxicity, including total reactive oxygen species, glutathione, malondialdehyde, α-tocopherol, and lactate dehydrogenase, were quantitatively assessed. It is concluded from the results that free radicals generated by exposure to 3.5 to 23.3 μg/ml CeO2 nanoparticles produce significant oxidative stress in the cells, as reflected by reduced glutathione and α-tocopherol levels; the toxic effects of CeO2 nanoparticles are dose dependent and time dependent; elevated oxidative stress increases the production of malondialdehyde and lactate dehydrogenase, which are indicators of lipid peroxidation and cell membrane damage, respectively.

  6. Human metapneumovirus in lung transplant recipients: characteristics and outcomes.

    PubMed

    Niggli, Fabian; Huber, Lars C; Benden, Christian; Schuurmans, Macé M

    2016-01-01

    Human metapneumovirus (hMPV) causes serious respiratory tract infections in lung transplant recipients (LTRs). We evaluated the characteristics and adverse drug reactions (ADR) of oral ribavirin therapy for hMPV infections in LTRs. LTRs with respiratory symptoms or suspected infection of unknown origin were routinely sampled with nasopharyngeal swabs (NPS) for virological and bacteriological analysis as part of a diagnostic workup. Medical records of hMPV polymerase chain reaction (PCR)-positive LTRs at the University Hospital of Zurich were reviewed retrospectively. Between January 2012 and June 2014, 12 (80%) of 15 consecutive patients with documented hMPV infection received oral ribavirin therapy (800 mg/d, after 48 h: 400 mg/d). Mean duration of therapy was 28.6 days (range: 11-54). Mean duration of viral shedding was 16.3 days (range: 5-48). In general, oral ribavirin was well tolerated in LTRs. The most common ADR was moderate anaemia. All patients recovered from infection without immediate serious sequelae within 3 months of infection.

  7. Identification of CCDC6-RET fusion in the human lung adenocarcinoma cell line, LC-2/ad.

    PubMed

    Matsubara, Daisuke; Kanai, Yoshihiko; Ishikawa, Shumpei; Ohara, Shiori; Yoshimoto, Taichiro; Sakatani, Takashi; Oguni, Sachiko; Tamura, Tomoko; Kataoka, Hiroaki; Endo, Shunsuke; Murakami, Yoshinori; Aburatani, Hiroyuki; Fukayama, Masashi; Niki, Toshiro

    2012-12-01

    Rearranged during transfection (RET) fusions have been newly identified in approximately 1% of patients with primary lung tumors. However, patient-derived lung cancer cell lines harboring RET fusions have not yet been established or identified, and therefore, the effectiveness of an RET inhibitor on lung tumors with endogenous RET fusion has not yet been studied. In this study, we report identification of CCDC6-RET fusion in the human lung adenocarcinoma cell line LC-2/ad. LC-2/ad showed distinctive sensitivity to the RET inhibitor, vandetanib, among 39 non-small lung cancer cell lines. The xenograft tumor of LC-2/ad showed cribriform acinar structures, a morphologic feature of primary RET fusion-positive lung adenocarcinomas. LC-2/ad cells could provide useful resources to analyze molecular functions of RET-fusion protein and its response to RET inhibitors.

  8. Flow Cytometric Analysis of Myeloid Cells in Human Blood, Bronchoalveolar Lavage, and Lung Tissues

    PubMed Central

    Yu, Yen-Rei A.; Hotten, Danielle F.; Malakhau, Yuryi; Volker, Ellen; Ghio, Andrew J.; Noble, Paul W.; Kraft, Monica; Hollingsworth, John W.; Gunn, Michael D.

    2016-01-01

    Clear identification of specific cell populations by flow cytometry is important to understand functional roles. A well-defined flow cytometry panel for myeloid cells in human bronchoalveolar lavage (BAL) and lung tissue is currently lacking. The objective of this study was to develop a flow cytometry–based panel for human BAL and lung tissue. We obtained and performed flow cytometry/sorting on human BAL cells and lung tissue. Confocal images were obtained from lung tissue using antibodies for cluster of differentiation (CD)206, CD169, and E cadherin. We defined a multicolor flow panel for human BAL and lung tissue that identifies major leukocyte populations. These include macrophage (CD206+) subsets and other CD206− leukocytes. The CD206− cells include: (1) three monocyte (CD14+) subsets, (2) CD11c+ dendritic cells (CD14−, CD11c+, HLA-DR+), (3) plasmacytoid dendritic cells (CD14−, CD11c−, HLA-DR+, CD123+), and (4) other granulocytes (neutrophils, mast cells, eosinophils, and basophils). Using this panel on human lung tissue, we defined two populations of pulmonary macrophages: CD169+ and CD169− macrophages. In lung tissue, CD169− macrophages were a prominent cell type. Using confocal microscopy, CD169+ macrophages were located in the alveolar space/airway, defining them as alveolar macrophages. In contrast, CD169− macrophages were associated with airway/alveolar epithelium, consistent with interstitial-associated macrophages. We defined a flow cytometry panel in human BAL and lung tissue that allows identification of multiple immune cell types and delineates alveolar from interstitial-associated macrophages. This study has important implications for defining myeloid cells in human lung samples. PMID:26267148

  9. Direct demonstration of 25- and 50-microm arteriovenous pathways in healthy human and baboon lungs.

    PubMed

    Lovering, Andrew T; Stickland, Michael K; Kelso, Amy J; Eldridge, Marlowe W

    2007-04-01

    Postmortem microsphere studies in adult human lungs have demonstrated the existence of intrapulmonary arteriovenous pathways using nonphysiological conditions. The aim of the current study was to determine whether large diameter (>25 and 50 microm) intrapulmonary arteriovenous pathways are functional in human and baboon lungs under physiological perfusion and ventilation pressures. We used fresh healthy human donor lungs obtained for transplantation and fresh lungs from baboons (Papio c. anubis). Lungs were ventilated with room air by using a peak inflation pressure of 15 cm H(2)O and a positive end-expiratory pressure of 5 cm H(2)O. Lungs were perfused between 10 and 20 cm H(2)O by using a phosphate-buffered saline solution with 5% albumin. We infused a mixture of 25- and 50-microm microspheres (0.5 and 1 million total for baboons and human studies, respectively) into the pulmonary artery and collected the entire pulmonary venous outflow. Under these conditions, evidence of intrapulmonary arteriovenous anastomoses was found in baboon (n = 3/4) and human (n = 4/6) lungs. In those lungs showing evidence of arteriovenous pathways, 50-microm microspheres were always able to traverse the pulmonary circulation, and the fraction of transpulmonary passage ranged from 0.0003 to 0.42%. These data show that intrapulmonary arteriovenous pathways >50 microm in diameter are functional under physiological ventilation and perfusion pressures in the isolated lung. These pathways provide an alternative conduit for pulmonary blood flow that likely bypasses the areas of gas exchange at the capillary-alveolar interface that could compromise both gas exchange and the ability of the lung to filter out microemboli.

  10. Zinc transporters are differentially expressed in human non-small cell lung cancer

    PubMed Central

    Yang, Jingxuan; Li, Min

    2016-01-01

    Lung cancer is one of the most common human malignancies worldwide, but its oncogenesis process remains unclear. Recent studies demonstrated that zinc (Zn) and Zn transporters were associated with the development and progression of human cancers. The role of Zn transporters including ZIPs and ZnTs in lung cancer, however, has never been evaluated. Thus, we aimed to investigate the expression levels of all human Zn transporters, including 14 ZIPs and 10 ZnTs, in eight different lung cancer cell lines and paired human tumor tissues. We observed great variations in ZIPs and ZnTs mRNA levels across cell lines and human lung cancer specimens. ZIPs showed a tendency to be upregulated, while ZnTs exhibited a downward expression trend. ZIP4 was overexpressed in six lung cancer cell lines and 59% (26/44) of tumor tissues, which was consistent with results from lung cancer datasets including TCGA database. Our results indicated that the dysregulation of Zn transporters may contribute to lung tumorigenesis. PMID:27611948

  11. Endogenous Semaphorin-7A Impedes Human Lung Fibroblast Differentiation

    PubMed Central

    Esnault, Stephane; Torr, Elizabeth E.; Bernau, Ksenija; Johansson, Mats W.; Kelly, Elizabeth A.; Sandbo, Nathan; Jarjour, Nizar N.

    2017-01-01

    Semaphorin-7A is a glycosylphosphatidylinositol-anchored protein, initially characterized as an axon guidance protein. Semaphorin-7A also contributes to immune cell regulation and may be an essential pro-fibrotic factor when expressed by non-fibroblast cell types (exogenous). In mouse models, semaphorin-7A was shown to be important for TGF-ß1-induced pulmonary fibrosis characterized by myofibroblast accumulation and extracellular matrix deposition, but the cell-specific role of semaphorin-7A was not examined in fibroblasts. The purpose of this study is to determine semaphorin-7A expression by fibroblasts and to investigate the function of endogenously expressed semaphorin-7A in primary human lung fibroblasts (HLF). Herein, we show that non-fibrotic HLF expressed high levels of cell surface semaphorin-7A with little dependence on the percentage of serum or recombinant TGF-ß1. Semaphorin-7A siRNA strongly decreased semaphorin-7A mRNA expression and reduced cell surface semaphorin-7A. Reduction of semaphorin-7A induced increased proliferation and migration of non-fibrotic HLF. Also, independent of the presence of TGF-ß1, the decline of semaphorin-7A by siRNA was associated with increased α-smooth muscle actin production and gene expression of periostin, fibronectin, laminin, and serum response factor (SRF), indicating differentiation into a myofibroblast. Conversely, overexpression of semaphorin-7A in the NIH3T3 fibroblast cell line reduced the production of pro-fibrotic markers. The inverse association between semaphorin-7A and pro-fibrotic fibroblast markers was further analyzed using HLF from idiopathic pulmonary fibrosis (IPF) (n = 6) and non-fibrotic (n = 7) lungs. Using these 13 fibroblast lines, we observed that semaphorin-7A and periostin expression were inversely correlated. In conclusion, our study indicates that endogenous semaphorin-7A in HLF plays a role in maintaining fibroblast homeostasis by preventing up-regulation of pro-fibrotic genes. Therefore

  12. DEPOSITION DISTRICUTION AMONG THE PARALLEL PATHWAYS IN THE HUMAN LUNG CONDUCTING AIRWAY STRUCTURE.

    EPA Science Inventory

    DEPOSITION DISTRIBUTION AMONG THE PARALLEL PATHWAYS IN THE HUMAN LUNG CONDUCTING AIRWAY STRUCTURE. Chong S. Kim*, USEPA National Health and Environmental Effects Research Lab. RTP, NC 27711; Z. Zhang and C. Kleinstreuer, Department of Mechanical and Aerospace Engineering, North C...

  13. The roles of diol epoxide and o-quinone pathways in mouse lung tumorigenesis induced by benzo(a)pyrene: relevance to human lung carcinogenesis

    EPA Science Inventory

    There is sufficient epidemiological evidence supported by experimental data that some PAH-containing complex environmental mixtures pose risks to human health by increasing lung cancer incidence. The International Agency for Research on Cancer has determined that human respirator...

  14. Receptor tyrosine kinase EphA5 is a functional molecular target in human lung cancer

    SciTech Connect

    Staquicini, Fernanda I.; Qian, Ming D.; Salameh, Ahmad; Dobroff, Andrey S.; Edwards, Julianna K.; Cimino, Daniel F.; Moeller, Benjamin J.; Kelly, Patrick; Nunez, Maria I.; Tang, Ximing; Liu, Diane D.; Lee, J. Jack; Hong, Waun Ki; Ferrara, Fortunato; Bradbury, Andrew R. M.; Lobb, Roy R.; Edelman, Martin J.; Sidman, Richard L.; Wistuba, Ignacio I.; Arap, Wadih; Pasqualini, Renata

    2015-03-20

    Lung cancer is often refractory to radiotherapy, but molecular mechanisms of tumor resistance remain poorly defined. Here we show that the receptor tyrosine kinase EphA5 is specifically overexpressed in lung cancer and is involved in regulating cellular responses to genotoxic insult. In the absence of EphA5, lung cancer cells displayed a defective G1/S cell cycle checkpoint, were unable to resolve DNA damage, and became radiosensitive. Upon irradiation, EphA5 was transported into the nucleus where it interacted with activated ATM (ataxia-telangiectasia mutated) at sites of DNA repair. In conclusion, we demonstrate that a new monoclonal antibody against human EphA5 sensitized lung cancer cells and human lung cancer xenografts to radiotherapy and significantly prolonged survival, thus suggesting the likelihood of translational applications.

  15. Regulation of cytochrome P4501A1 expression by hyperoxia in human lung cell lines: Implications for hyperoxic lung injury

    SciTech Connect

    Bhakta, Kushal Y. Jiang, Weiwu; Couroucli, Xanthi I.; Fazili, Inayat S.; Muthiah, Kathirvel; Moorthy, Bhagavatula

    2008-12-01

    Supplemental oxygen, used to treat pulmonary insufficiency in newborns, contributes to the development of bronchopulmonary dysplasia (BPD). Cytochrome P4501A enzymes are induced by hyperoxia in animal models, but their role in human systems is unknown. Here we investigated the molecular mechanisms of induction of CYP1A1 by hyperoxia in human lung cell lines. Three human lung cell lines were exposed to hyperoxia (95% O2) for 0-72 h, and CYP1A1 activities, apoprotein contents, and mRNA levels were determined. Hyperoxia significantly induced CYP1A1 activity and protein contents (2-4 fold), and mRNA levels (30-40 fold) over control in each cell line. Transfection of a CYP1A1 promoter/luciferase reporter construct, followed by hyperoxia (4-72 h), showed marked (2-6 fold) induction of luciferase expression. EMSA and siRNA experiments strongly suggest that the Ah receptor (AHR) is involved in the hyperoxic induction of CYP1A1. MTT reduction assays showed attenuation of cell injury with the CYP1A1 inducer beta-naphthoflavone (BNF). Our results strongly suggest that hyperoxia transcriptionally activates CYP1A1 expression in human lung cell lines by AHR-dependent mechanisms, and that CYP1A1 induction is associated with decreased toxicity. This novel finding of induction of CYP1A1 in the absence of exogenous AHR ligands could lead to novel interventions in the treatment of BPD.

  16. Read-through transcripts in normal human lung parenchyma are down-regulated in lung adenocarcinoma

    PubMed Central

    Cotroneo, Chiara E.; Galvan, Antonella; Noci, Sara; Piazza, Rocco; Pirola, Alessandra; Spinelli, Roberta; Incarbone, Matteo; Palleschi, Alessandro; Rosso, Lorenzo; Santambrogio, Luigi; Dragani, Tommaso A.; Colombo, Francesca

    2016-01-01

    Read-through transcripts result from the continuous transcription of adjacent, similarly oriented genes, with the splicing out of the intergenic region. They have been found in several neoplastic and normal tissues, but their pathophysiological significance is unclear. We used high-throughput sequencing of cDNA fragments (RNA-Seq) to identify read-through transcripts in the non-involved lung tissue of 64 surgically treated lung adenocarcinoma patients. A total of 52 distinct read-through species was identified, with 24 patients having at least one read-through event, up to a maximum of 17 such transcripts in one patient. Sanger sequencing validated 28 of these transcripts and identified an additional 15, for a total of 43 distinct read-through events involving 35 gene pairs. Expression levels of 10 validated read-through transcripts were measured by quantitative PCR in pairs of matched non-involved lung tissue and lung adenocarcinoma tissue from 45 patients. Higher expression levels were observed in normal lung tissue than in the tumor counterpart, with median relative quantification ratios between normal and tumor varying from 1.90 to 7.78; the difference was statistically significant (P < 0.001, Wilcoxon's signed-rank test for paired samples) for eight transcripts: ELAVL1–TIMM44, FAM162B–ZUFSP, IFNAR2–IL10RB, INMT–FAM188B, KIAA1841–C2orf74, NFATC3–PLA2G15, SIRPB1–SIRPD, and SHANK3–ACR. This report documents the presence of read-through transcripts in apparently normal lung tissue, with inter-individual differences in patterns and abundance. It also shows their down-regulation in tumors, suggesting that these chimeric transcripts may function as tumor suppressors in lung tissue. PMID:27058892

  17. Autoradiographic visualization of muscarinic receptor subtypes in human and guinea pig lung

    SciTech Connect

    Mak, J.C.; Barnes, P.J. )

    1990-06-01

    Muscarinic receptor subtypes have been localized in human and guinea pig lung sections by an autoradiographic technique, using (3H)(-)quinuclidinyl benzilate (( 3H)QNB) and selective muscarinic antagonists. (3H)QNB was incubated with tissue sections for 90 min at 25 degrees C, and nonspecific binding was determined by incubating adjacent serial sections in the presence of 1 microM atropine. Binding to lung sections had the characterization expected for muscarinic receptors. Autoradiography revealed that muscarinic receptors were widely distributed in human lung, with dense labeling over submucosal glands and airway ganglia, and moderate labeling over nerves in intrapulmonary bronchi and of airway smooth muscle of large and small airways. In addition, alveolar walls were uniformly labeled. In guinea pig lung, labeling of airway smooth muscle was similar, but in contrast to human airways, epithelium was labeled but alveolar walls were not. The muscarinic receptors of human airway smooth muscle from large to small airways were entirely of the M3-subtype, whereas in guinea pig airway smooth muscle, the majority were the M3-subtype with a very small population of the M2-subtype present. In human bronchial submucosal glands, M1- and M3-subtypes appeared to coexist in the proportions of 36 and 64%, respectively. In human alveolar walls the muscarinic receptors were entirely of the M1-subtype, which is absent from the guinea pig lung. No M2-receptors were demonstrated in human lung. The localization of M1-receptors was confirmed by direct labeling with (3H)pirenzepine. With the exception of the alveolar walls in human lung, the localization of muscarinic receptor subtypes on structures in the lung is consistent with known functional studies.

  18. LGL1 modulates proliferation, apoptosis, and migration of human fetal lung fibroblasts.

    PubMed

    Zhang, Hui; Sweezey, Neil B; Kaplan, Feige

    2015-02-15

    Rapid growth and formation of new gas exchange units (alveogenesis) are hallmarks of the perinatal lung. Bronchopulmonary dysplasia (BPD), common in very premature infants, is characterized by premature arrest of alveogenesis. Mesenchymal cells (fibroblasts) regulate both lung branching and alveogenesis through mesenchymal-epithelial interactions. Temporal or spatial deficiency of late-gestation lung 1/cysteine-rich secretory protein LD2 (LGL1/CRISPLD2), expressed in and secreted by lung fibroblasts, can impair both lung branching and alveogenesis (LGL1 denotes late gestation lung 1 protein; LGL1 denotes the human gene; Lgl1 denotes the mouse/rat gene). Absence of Lgl1 is embryonic lethal. Lgl1 levels are dramatically reduced in oxygen toxicity rat models of BPD, and heterozygous Lgl1(+/-) mice exhibit features resembling human BPD. To explore the role of LGL1 in mesenchymal-epithelial interactions in developing lung, we developed a doxycycline (DOX)-inducible RNA-mediated LGL1 knockdown cellular model in human fetal lung fibroblasts (MRC5(LGL1KD)). We assessed the impact of LGL1 on cell proliferation, cell migration, apoptosis, and wound healing. DOX-induced MRC5(LGL1KD) suppressed cell growth and increased apoptosis of annexin V(+) staining cells and caspase 3/7 activity. LGL1-conditioned medium increased migration of fetal rat primary lung epithelial cells and human airway epithelial cells. Impaired healing by MRC5(LGL1KD) cells of a wound model was attenuated by addition of LGL1-conditioned medium. Suppression of LGL1 was associated with dysregulation of extracellular matrix genes (downregulated MMP1, ColXVα1, and ELASTIN) and proapoptosis genes (upregulated BAD, BAK, CASP2, and TNFRSF1B) and inhibition of 44/42MAPK phosphorylation. Our findings define a role for LGL1 in fibroblast expansion and migration, epithelial cell migration, and mesenchymal-epithelial signaling, key processes in fetal lung development.

  19. Human Lung Carcinoma Reaction against Metabolic Serum Deficiency Stress

    PubMed Central

    Nakhjavani, Maryam; Nikounezhad, Nastaran; Ashtarinezhad, Azadeh; Shirazi, Farshad H.

    2016-01-01

    Cancer treatment is still of the greatest challenges that health care providers and patients are facing. One of the unsolved problems in cancer treatment is cells’ reaction to metabolic stress caused by harsh nutritional conditions around tumor. In order to be able to treat this disease properly, it is important to understand the true nature of the disease. In fact, the cells inside the central part of the tumor lack sufficient access to blood vessels, nutrients, and growth signals. After tumor shrinkage, the cells are exposed to favorable environmental conditions and might regrow and cause tumor recurrence. The main purpose of this study was to investigate the effect of serum starvation, as a type of metabolic stress, on human lung cancer cell line, A549. These cells were treated with 10% (control), 0.5% and 0.25% serum for 1 to 5 days. At 24 h intervals, the cells were released with 10% serum supplemented media. Starved or released cells were studied for their cycle and morphology. The results showed that the cells were actually arrested at G1 phase and following exposure to optimal conditions, the cells could be back to their cycle again. Furthermore, sub-G1 apoptotic cells population was not increased within the starvation period, while control cells had significant increase in sub-G1 cells. Morphological studies also showed that starved cells could make denser colonies while control cells were entering death phase. These observations provide some evidence for the generation of some effective resistance phenomena in cancer cells against harsh metabolic conditions. PMID:28243278

  20. Homo-timeric structural model of human microsomal prostaglandin E synthase-1 and characterization of its substrate/inhibitor binding interactions

    NASA Astrophysics Data System (ADS)

    Xing, Li; Kurumbail, Ravi G.; Frazier, Ronald B.; Davies, Michael S.; Fujiwara, Hideji; Weinberg, Robin A.; Gierse, James K.; Caspers, Nicole; Carter, Jeffrey S.; McDonald, Joseph J.; Moore, William M.; Vazquez, Michael L.

    2009-01-01

    Inducible, microsomal prostaglandin E synthase 1 (mPGES-1), the terminal enzyme in the prostaglandin (PG) biosynthetic pathway, constitutes a promising therapeutic target for the development of new anti-inflammatory drugs. To elucidate structure-function relationships and to enable structure-based design, an mPGES-1 homology model was developed using the three-dimensional structure of the closest homologue of the MAPEG family (Membrane Associated Proteins in Eicosanoid and Glutathione metabolism), mGST-1. The ensuing model of mPGES-1 is a homo-trimer, with each monomer consisting of four membrane-spanning segments. Extensive structure refinement revealed an inter-monomer salt bridge (K26-E77) as well as inter-helical interactions within each monomer, including polar hydrogen bonds (e.g. T78-R110-T129) and hydrophobic π-stacking (F82-F103-F106), all contributing to the overall stability of the homo-trimer of mPGES-1. Catalytic co-factor glutathione (GSH) was docked into the mPGES-1 model by flexible optimization of both the ligand and the protein conformations, starting from the initial location ascertained from the mGST-1 structure. Possible binding site for the substrate, prostaglandin H2 (PGH2), was identified by systematically probing the refined molecular structure of mPGES-1. A binding model was generated by induced fit docking of PGH2 in the presence of GSH. The homology model prescribes three potential inhibitor binding sites per mPGES-1 trimer. This was further confirmed experimentally by equilibrium dialysis study which generated a binding stoichiometric ratio of approximately three inhibitor molecules to three mPGES-1 monomers. The structural model that we have derived could serve as a useful tool for structure-guided design of inhibitors for this emergently important therapeutic target.

  1. In vivo electrical bioimpedance characterization of human lung tissue during the bronchoscopy procedure. A feasibility study.

    PubMed

    Sanchez, Benjamin; Vandersteen, Gerd; Martin, Irene; Castillo, Diego; Torrego, Alfons; Riu, Pere J; Schoukens, Johan; Bragos, Ramon

    2013-07-01

    Lung biopsies form the basis for the diagnosis of lung cancer. However, in a significant number of cases bronchoscopic lung biopsies fail to provide useful information, especially in diffuse lung disease, so more aggressive procedures are required. Success could be improved using a guided electronic biopsy based on multisine electrical impedance spectroscopy (EIS), a technique which is evaluated in this paper. The theoretical basis of the measurement method and the instrument developed are described, characterized and calibrated while the performance of the instrument is assessed by experiments to evaluate the noise and nonlinear source of errors from measurements on phantoms. Additional preliminary results are included to demonstrate that it is both feasible and safe to monitor in vivo human lung tissue electrical bioimpedance (EBI) during the bronchoscopy procedure. The time required for performing bronchoscopy is not extended because the bioimpedance measurements, present no complications, tolerance problems or side effects among any of the patients measured.

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

    PubMed Central

    Ji, Hye Young; Liu, Kwang Hyeon; Jeong, Ji Hyeon; Lee, Dae-Young; Shim, Hyun Joo; Son, Miwon; Lee, Hye Suk

    2012-01-01

    DA-9701 is a new botanical drug composed of the extracts of Corydalis tuber and Pharbitidis semen, and it is used as an oral therapy for the treatment of functional dyspepsia in Korea. The inhibitory potentials of DA-9701 and its component herbs, Corydalis tuber and Pharbitidis semen, on the activities of seven major human cytochrome P450 (CYP) enzymes and four UDP-glucuronosyltransferase (UGT) enzymes in human liver microsomes were investigated using liquid chromatography-tandem mass spectrometry. DA-9701 and Corydalis tuber extract slightly inhibited UGT1A1-mediated etoposide glucuronidation, with 50% inhibitory concentration (IC50) values of 188 and 290 μg/mL, respectively. DA-9701 inhibited CYP2D6-catalyzed bufuralol 1′-hydroxylation with an inhibition constant (Ki) value of 6.3 μg/mL in a noncompetitive manner. Corydalis tuber extract competitively inhibited CYP2D6-mediated bufuralol 1′-hydroxylation, with a Ki value of 3.7 μg/mL, whereas Pharbitidis semen extract showed no inhibition. The volume in which the dose could be diluted to generate an IC50 equivalent concentration (volume per dose index) value of DA-9701 for inhibition of CYP2D6 activity was 1.16 L/dose, indicating that DA-9701 may not be a potent CYP2D6 inhibitor. Further clinical studies are warranted to evaluate the in vivo extent of the observed in vitro interactions. PMID:22548118

  3. Electron microscopy analysis of mineral fibers in human lung tissue

    SciTech Connect

    Friedrichs, K.H.; Brockmann, M.; Fischer, M.; Wick, G. )

    1992-01-01

    In the present study, lung samples from 126 autopsied cases were examined to determine the content of mineral fibers using analytical transmission electron microscopy (ATEM). The cases were divided into four groups (22 lungs of persons exposed to ambient environmental pollution, 32 cases of mesothelioma, 38 cases of primary lung cancer, and 34 asbestosis cases, 13 of these with additional pleural plaques). Fibers were counted, measured, and mineralogically identified using a combination of X-ray microanalysis and electron diffraction of the non-oriented fiber. Concentration of fibrous particles (defined as particles above 1 micron in length with roughly parallel long sides and an aspect ratio of 5:1 and greater) was calculated as fibers 10(6)/g dry lung weight. The concentration of chrysotile was found to be similar throughout the groups except for two cases in the asbestosis group with comparably high numbers of chrysotile. However, a remarkable difference for amphiboles could be observed between the groups. Asbestos bodies were mostly found in the asbestosis group. There was a rather good correlation between numbers of amphibole fibers and asbestos bodies, with an average ratio of 10:1. For comparison purposes between occupationally exposed/non-exposed individuals, a transition was found in the concentration range of 3-10(7) asbestos fibers/g dried lung weight.

  4. Isolation, cultivation and identification of human lung adenocarcinoma stem cells

    PubMed Central

    ZHANG, DE-GENG; JIANG, AI-GUI; LU, HUI-YU; ZHANG, LI-XIN; GAO, XIAO-YAN

    2015-01-01

    Recently, an increasing number of studies have demonstrated that lung cancer is a stem cell disease. However, ideal cell surface markers for isolating stem cells in lung cancer are yet to be identified. In the present study, a cell population with a cluster of differentiation (CD)133+ phenotype was successfully isolated from a single cell suspension of lung adenocarcinoma tissue using magnetic-activated cell sorting (MACS) and enriched in a serum-free culture. In comparison to CD133− cells, the CD133+ cells exhibited an enhanced capacity for self-renewal and differentiation, and a greater potential for in vivo tumor formation, in non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice. Tumors could be induced in NOD/SCID mice by the transplantation of 102 stem-like cells per mouse. The results of the present study demonstrated that CD133 may serve as a specific cell surface marker for lung adenocarcinoma stem cells, and that MACS combined with serum-free culture is an effective method for isolating and enriching lung cancer stem cells. PMID:25435932

  5. Pulsatile uptake of CO in the human lung

    PubMed Central

    Menkes, Harold A.; Sera, Kazuaki; Rogers, Robert M.; Hyde, Richard W.; Forster, Robert E.; DuBois, Arthur B.

    1970-01-01

    The instantaneous uptake of CO in the lungs was measured with a water-filled body plethysmograph in normal man. First, control measurements of plethysmograph pressure were made while the subject held his breath for 7 sec after breathing gas mixtures prepared to bring his alveolar PO2 and PCO2 close to mixed venous levels. Then, CO uptake measurements were made while he held his breath after inhaling the same gas mixtures with added CO (2.0%). The change in lung volume on CO minus the change in lung volume during the control measurement was a measure of the CO uptake in the lungs. Cardiopneumatic changes in lung gas volume were subtracted electrically. All of five subjects showed pulsatile CO uptake. The mean CO uptake was 103 ml/min. A peak uptake of 2.0 (range 1.6-2.3) times the mean uptake occurred 0.3-0.4 sec after the R wave of the EKG and a minimum uptake of 0.4 (range 0.2-0.5) times the mean uptake occurred during the tenth of a second before the R wave of the EKG. These results suggest that pulmonary capillary blood volume is pulsatile during the cardiac cycle. PMID:5411784

  6. Gli promotes epithelial-mesenchymal transition in human lung adenocarcinomas

    PubMed Central

    Jin, Joy Q.; Woodard, Gavitt A.; Tolani, Bhairavi; Luh, Thomas M.; Giroux-Leprieur, Etienne; Mo, Minli; Chen, Zhao; Che, Juanjuan; Zhang, Zhenfa; Zhou, Yong; Wang, Lei; Hao, Xishan; Jablons, David; Wang, Changli; He, Biao

    2016-01-01

    Adenocarcinoma is the most common type of lung cancer. Epithelial-mesenchymal transition (EMT) is required for tumor invasion/metastasis and the components that control this process are potential therapeutic targets. This study we examined the role of Gli in lung adenocarcinoma and whether its activation regulates metastasis through EMT in lung adenocarcinoma. We found that tumors with high Gli expression had significantly lower E-Cadherin expression in two independent cohorts of patients with lung adenocarcinoma that we studied. In vitro up-regulation of SHh resulted in increased cell migration while small molecule inhibitors of Smo or Gli significantly reduced cell mobility both in a wound healing assay and in a 3D cell invasion assay. Inhibition of Gli in vivo decreased tumor growth and induced an increase in E-Cadherin expression. Our results indicate that Gli may be critical for lung adenocarcinoma metastasis and that a novel Gli inhibitor shows promise as a therapeutic agent by preventing cell migration and invasion in vitro and significantly reducing tumor growth and increasing E-Cadherin expression in vivo. PMID:27533453

  7. Linear dimensions and volumes of human lungs obtained from CT images.

    PubMed

    Kramer, Gary H; Capello, Kevin; Bearrs, Brock; Lauzon, Aimée; Normandeau, Lysanne

    2012-04-01

    This work provides the results of a collaboration between the Human Monitoring Laboratory (HML) and the Centre Hospitalier de l'Université de Montréal (CHUM) in which CHUM provided CT lung image sets from 166 patients for the analysis of linear dimensions and lung volume. This work has shown that a large amount of data exists in the medical community that can be of value to the health physics community. The intent of this study was to determine the range of linear dimensional parameters that could be used for torso phantom development for males and females; understand and characterize the variability of linear lung dimensions for males and females; replace the brief table in ICRP 23 with more modern data for males and females; identify an empirical formula that would predict linear dimensions of human lungs from age, height and/or weight for males and females; characterize the left, right, and total lung volumes of males and females in this data set; and compare the lung volumes of males and females to published equations for determining lung volumes. It was found that linear dimensions of lungs are essentially independent of age, height, and weight, so predictive equations cannot be formulated; however, the ranges of those parameters have now been established for the population studied herein. The data presented here are more modern than the brief table that appeared in ICRP 23, and the average values could be used as future guidelines. Whole lung volumes have been determined from the voxel lung phantoms, and empirical formulae have been developed for males and females in this data set; these compare favorably with the published values in ICRP 66.

  8. Regenerative potential of human airway stem cells in lung epithelial engineering.

    PubMed

    Gilpin, Sarah E; Charest, Jonathan M; Ren, Xi; Tapias, Luis F; Wu, Tong; Evangelista-Leite, Daniele; Mathisen, Douglas J; Ott, Harald C

    2016-11-01

    Bio-engineered organs for transplantation may ultimately provide a personalized solution for end-stage organ failure, without the risk of rejection. Building upon the process of whole organ perfusion decellularization, we aimed to develop novel, translational methods for the recellularization and regeneration of transplantable lung constructs. We first isolated a proliferative KRT5(+)TP63(+) basal epithelial stem cell population from human lung tissue and demonstrated expansion capacity in conventional 2D culture. We then repopulated acellular rat scaffolds in ex vivo whole organ culture and observed continued cell proliferation, in combination with primary pulmonary endothelial cells. To show clinical scalability, and to test the regenerative capacity of the basal cell population in a human context, we then recellularized and cultured isolated human lung scaffolds under biomimetic conditions. Analysis of the regenerated tissue constructs confirmed cell viability and sustained metabolic activity over 7 days of culture. Tissue analysis revealed extensive recellularization with organized tissue architecture and morphology, and preserved basal epithelial cell phenotype. The recellularized lung constructs displayed dynamic compliance and rudimentary gas exchange capacity. Our results underline the regenerative potential of patient-derived human airway stem cells in lung tissue engineering. We anticipate these advances to have clinically relevant implications for whole lung bioengineering and ex vivo organ repair.

  9. First experience with a paracorporeal artificial lung in humans.

    PubMed

    Camboni, Daniele; Philipp, Alois; Arlt, Matthias; Pfeiffer, Michael; Hilker, Michael; Schmid, Christof

    2009-01-01

    Lung transplantation is the only treatment option for patients suffering form end-stage respiratory failure. To date, no mechanical device is available to support patients on the waiting list up to months. Here, we summarize our experience with our first two patients, who were supported with a paracorporeal artificial lung (PAL) placed in parallel to the pulmonary circulation with connection to the pulmonary artery and to the left atrium. A low resistance membrane oxygenator (iLA, Novalung, Hirrlingen, Germany) was attached in both patients. Our first patient suffering from a pulmonary veno-occlusive disease was supported for 18 days until he died due to severe sepsis. Our second patient with a primary pulmonary hypertension of unknown origin was supported 62 days followed by successful lung transplantation. In conclusion, the experience obtained with these first two patients under PAL encourages further studies and introduction of this promising concept into clinical practice.

  10. Alterations in Gene Expression and DNA Methylation during Murine and Human Lung Alveolar Septation

    PubMed Central

    Cuna, Alain; Halloran, Brian; Faye-Petersen, Ona; Kelly, David; Crossman, David K.; Cui, Xiangqin; Pandit, Kusum; Kaminski, Naftali; Bhattacharya, Soumyaroop; Ahmad, Ausaf; Mariani, Thomas J.

    2015-01-01

    DNA methylation, a major epigenetic mechanism, may regulate coordinated expression of multiple genes at specific time points during alveolar septation in lung development. The objective of this study was to identify genes regulated by methylation during normal septation in mice and during disordered septation in bronchopulmonary dysplasia. In mice, newborn lungs (preseptation) and adult lungs (postseptation) were evaluated by microarray analysis of gene expression and immunoprecipitation of methylated DNA followed by sequencing (MeDIP-Seq). In humans, microarray gene expression data were integrated with genome-wide DNA methylation data from bronchopulmonary dysplasia versus preterm and term lung. Genes with reciprocal changes in expression and methylation, suggesting regulation by DNA methylation, were identified. In mice, 95 genes with inverse correlation between expression and methylation during normal septation were identified. In addition to genes known to be important in lung development (Wnt signaling, Angpt2, Sox9, etc.) and its extracellular matrix (Tnc, Eln, etc.), genes involved with immune and antioxidant defense (Stat4, Sod3, Prdx6, etc.) were also observed. In humans, 23 genes were differentially methylated with reciprocal changes in expression in bronchopulmonary dysplasia compared with preterm or term lung. Genes of interest included those involved with detoxifying enzymes (Gstm3) and transforming growth factor-β signaling (bone morphogenetic protein 7 [Bmp7]). In terms of overlap, 20 genes and three pathways methylated during mouse lung development also demonstrated changes in methylation between preterm and term human lung. Changes in methylation correspond to altered expression of a number of genes associated with lung development, suggesting that DNA methylation of these genes may regulate normal and abnormal alveolar septation. PMID:25387348

  11. Human Organotypic Lung Tumor Models: Suitable For Preclinical 18F-FDG PET-Imaging

    PubMed Central

    Fecher, David; Hofmann, Elisabeth; Buck, Andreas; Bundschuh, Ralph; Nietzer, Sarah; Dandekar, Gudrun; Walles, Thorsten; Walles, Heike; Lückerath, Katharina; Steinke, Maria

    2016-01-01

    Development of predictable in vitro tumor models is a challenging task due to the enormous complexity of tumors in vivo. The closer the resemblance of these models to human tumor characteristics, the more suitable they are for drug-development and –testing. In the present study, we generated a complex 3D lung tumor test system based on acellular rat lungs. A decellularization protocol was established preserving the architecture, important ECM components and the basement membrane of the lung. Human lung tumor cells cultured on the scaffold formed cluster and exhibited an up-regulation of the carcinoma-associated marker mucin1 as well as a reduced proliferation rate compared to respective 2D culture. Additionally, employing functional imaging with 2-deoxy-2-[18F]fluoro-D-glucose positron emission tomography (FDG-PET) these tumor cell cluster could be detected and tracked over time. This approach allowed monitoring of a targeted tyrosine kinase inhibitor treatment in the in vitro lung tumor model non-destructively. Surprisingly, FDG-PET assessment of single tumor cell cluster on the same scaffold exhibited differences in their response to therapy, indicating heterogeneity in the lung tumor model. In conclusion, our complex lung tumor test system features important characteristics of tumors and its microenvironment and allows monitoring of tumor growth and -metabolism in combination with functional imaging. In longitudinal studies, new therapeutic approaches and their long-term effects can be evaluated to adapt treatment regimes in future. PMID:27501455

  12. MAGNETIC RESONANCE ELASTOGRAPHY OF HUMAN LUNG PARENCHYMA: TECHNICAL DEVELOPMENT, THEORETICAL MODELING AND IN VIVO VALIDATION

    PubMed Central

    Mariappan, Yogesh K; Glaser, Kevin J; Hubmayr, Rolf D; Manduca, Armando; Ehman, Richard L; McGee, Kiaran P

    2011-01-01

    Purpose To develop a novel MR-based method for visualizing the elastic properties of human lung parenchyma in vivo and to evaluate the ability of this method to resolve differences in parenchymal stiffness at different respiration states in healthy volunteers. Materials and Methods A spin-echo MR Elastography (MRE) pulse sequence was developed to provide both high shear wave motion sensitivity and short TE for improved visualization of lung parenchyma. The improved motion sensitivity of this approach was modeled and tested with phantom experiments. In vivo testing was then performed on ten healthy volunteers at the respiratory states of residual volume (RV) and total lung capacity (TLC). Results Shear wave propagation was visualized within the lungs of all volunteers and was processed to provide parenchymal shear stiffness maps of all ten subjects. Density corrected stiffness values at TLC (1.83 ± 0.22 kPa) were higher than those at the RV (1.14 ± 0.14 kPa) with the difference being statistically significant (p<0.0001). Conclusion 1H-based MR Elastography can noninvasively measure the shear stiffness of human lung parenchyma in vivo and can quantitate the change in shear stiffness due to respiration. The values obtained were consistent with previously reported in vitro assessments of cadaver lungs. Further work is required to increase the flexibility of the current acquisition and to investigate the clinical potential of lung MRE. PMID:21591003

  13. Receptor tyrosine kinase EphA5 is a functional molecular target in human lung cancer

    DOE PAGES

    Staquicini, Fernanda I.; Qian, Ming D.; Salameh, Ahmad; ...

    2015-03-20

    Lung cancer is often refractory to radiotherapy, but molecular mechanisms of tumor resistance remain poorly defined. Here we show that the receptor tyrosine kinase EphA5 is specifically overexpressed in lung cancer and is involved in regulating cellular responses to genotoxic insult. In the absence of EphA5, lung cancer cells displayed a defective G1/S cell cycle checkpoint, were unable to resolve DNA damage, and became radiosensitive. Upon irradiation, EphA5 was transported into the nucleus where it interacted with activated ATM (ataxia-telangiectasia mutated) at sites of DNA repair. In conclusion, we demonstrate that a new monoclonal antibody against human EphA5 sensitized lungmore » cancer cells and human lung cancer xenografts to radiotherapy and significantly prolonged survival, thus suggesting the likelihood of translational applications.« less

  14. Correlating 3D morphology with molecular pathology: fibrotic remodelling in human lung biopsies.

    PubMed

    Kellner, Manuela; Wehling, Judith; Warnecke, Gregor; Heidrich, Marko; Izykowski, Nicole; Vogel-Claussen, Jens; Lorbeer, Raoul-Amadeus; Antonopoulos, Georgios; Janciauskiene, Sabina; Grothausmann, Roman; Knudsen, Lars; Ripken, Tammo; Meyer, Heiko; Kreipe, Hans; Ochs, Matthias; Jonigk, Danny; Kühnel, Mark Philipp

    2015-12-01

    Assessing alterations of the parenchymal architecture is essential in understanding fibrosing interstitial lung diseases. Here, we present a novel method to visualise fibrotic remodelling in human lungs and correlate morphological three-dimensional (3D) data with gene and protein expression in the very same sample. The key to our approach is a novel embedding resin that clears samples to full optical transparency and simultaneously allows 3D laser tomography and preparation of sections for histology, immunohistochemistry and RNA isolation. Correlating 3D laser tomography with molecular diagnostic techniques enables new insights into lung diseases. This approach has great potential to become an essential tool in pulmonary research.

  15. Biosynthesized Platinum Nanoparticles Inhibit the Proliferation of Human Lung-Cancer Cells in vitro and Delay the Growth of a Human Lung-Tumor Xenograft in vivo

    PubMed Central

    Yogesh, Bendale; Vineeta, Bendale; Rammesh, Natu; Saili, Paul

    2016-01-01

    Objectives: Lung cancer remains a deadly disease with unsatisfactory overall survival. Cisplatin, a standard platinum (Pt)-based chemotherapeutic agent, has the potential to inhibit the growth of lung cancer. Its use, however, is occasionally limited by severe organ toxicity. However, until now, no systematic study has been conducted to verify its efficacy with proper experimental support in vivo. Therefore, we examined whether biosynthesized Pt nanoparticles (NPs) inhibited human lung cancer in vitro and in vivo to validate their use in alternative and complementary medicine. Methods: We evaluated the in vitro and the in vivo anticancer efficiencies of biosynthesized Pt NPs in a subcutaneous xenograft model with A549 cells. Severe combined immune deficient mice (SCID) were divided into four groups: group 1 being the vehicle control group and groups 2, 3 and 4 being the experimental groups. Once the tumor volume had reached 70 ─ 75 mm3, the progression profile of the tumor growth kinetics and the body weights of the mice were measured every week for 6 weeks after oral administration of Pt NPs. Doses of Pt NPs of 500, 1,000 and 2,000 mg/kg of body weight were administered to the experimental groups and a dose of honey was administered to the vehicle control group. The efficacy was quantified by using the delay in tumor growth following the administration of Pt NPs of A549 human-lung-cancer xenografts growing in SCID mice. Results: The in vitro cytotoxicity evaluation indicated that Pt NPs, in a dose-dependent manner, inhibited the growth of A549 cells, and the in vivo evaluation showed that Pt NPs at the mid and high doses effectively inhibited and delayed the growth of lung cancer in SCID mice. Conclusion: These findings confirm the antitumor properties of biosynthesized Pt NPs and suggest that they may be a cost-effective alternative for the treatment of patients with lung cancer. PMID:27386144

  16. Immunohistological study of human lungs by immunoperoxidase technique.

    PubMed Central

    Fox, B; Shousha, S; James, K R; Miller, G C

    1982-01-01

    An unlabelled antibody peroxidase-antiperoxidase method for the detection of IgG, IgM, complement (C3 and Clq), fibrinogen and albumin was applied to routinely processed paraffin sections of lung from 27 cases. The results in 11 cases were compared with those obtained by immunofluorescence using frozen sections. Tissue was obtained from surgical specimens of cases with interstitial pneumonia comprising 10 of the usual type (UIP) and three of the desquamative type (DIP). Tissue was also obtained from the specimens of cases with sarcoidosis (two cases) and granulomatous inflammation of unknown cause (one case). There were 11 control cases, nine with primary carcinoma of the lung and two with metastatic tumours of the lung. Immunoglobulins of various types and complement were seen in diseased lung tissue. Although most of these deposits were probably due to a non-immunological mechanism there was evidence of the possible implication of immune complexes in three cases of UIP and in the interstitial pneumonia present in the two cases of sarcoidosis. The immunoperoxidase technique is a more sensitive method than immunofluorescence and has the additional advantage of the easy identification of the precise sites of the various deposits. Images PMID:7040481

  17. ALKBH3, a human AlkB homologue, contributes to cell survival in human non-small-cell lung cancer

    PubMed Central

    Tasaki, M; Shimada, K; Kimura, H; Tsujikawa, K; Konishi, N

    2011-01-01

    Background: We have demonstrated for the first time that a novel human AlkB homologue, ALKBH3, contributes to prostate cancer development, but its clinical and biological roles in lung cancer remain unclear. Methods: Expression of both mRNA and protein of PCA-1 was examined by RT–PCR and western blotting. We also assessed association with senescence and in vivo ALKBH3 treatment on orthotopic tumour cell inoculation, and analysed it clinicopathologically. Results: We have since found novel biological roles for ALKBH3 in human lung cancers, particularly in adenocarcinoma. Our immunohistochemical analysis of human adenocarcinomas and squamous cell carcinomas of the lung not only showed overexpression of ALKBH3 in these tumours but the percentage of cells positive for ALKBH3 also correlated statistically to recurrence-free survival in adenocarcinoma. Knockdown of ALKBH3 by siRNA transfection induced expression of p21WAF1/Cip1 and p27Kip1 in the human lung adenocarcinoma cell line A549, resulting in cell cycle arrest, senescence and strong suppression of cell growth in vitro. In vivo, peritoneal tumour growth and dissemination was inhibited in nude mice, previously inoculated with the A549 cell line, by intraperitoneal injection of ALKBH3 siRNA + atelocollagen, as demonstrated by the reduction in both number and diameter of tumours developing in the peritoneum. Conclusion: We suggest that ALKBH3 contributes significantly to cancer cell survival and may be a therapeutic target for human adenocarcinoma of the lung. PMID:21285982

  18. KL-6, a human MUC1 mucin, promotes proliferation and survival of lung fibroblasts

    SciTech Connect

    Ohshimo, Shinichiro; Yokoyama, Akihito . E-mail: yokoyan@hiroshima-u.ac.jp; Hattori, Noboru; Ishikawa, Nobuhisa; Hirasawa, Yutaka; Kohno, Nobuoki

    2005-12-30

    The serum level of KL-6, a MUC1 mucin, is a clinically useful marker for various interstitial lung diseases. Previous studies demonstrated that KL-6 promotes chemotaxis of human fibroblasts. However, the pathophysiological role of KL-6 remains poorly understood. Here, we further investigate the functional aspects of KL-6 in proliferation and apoptosis of lung fibroblasts. KL-6 accelerated the proliferation and inhibited the apoptosis of all human lung fibroblasts examined. An anti-KL-6 monoclonal antibody counteracted both of these effects induced by KL-6 on human lung fibroblasts. The pro-fibroproliferative and anti-apoptotic effects of KL-6 are greater than and additive to those of the maximum effective concentrations of platelet-derived growth factor, basic fibroblast growth factor, and transforming growth factor-{beta}. These findings indicate that increased levels of KL-6 in the epithelial lining fluid may stimulate fibrotic processes in interstitial lung diseases and raise the possibility of applying an anti-KL-6 antibody to treat interstitial lung diseases.

  19. EFFECT OF ANTIOXIDANT SUPPLEMENTATION ON OZONE-INDUCED LUNG INJURY IN HUMAN SUBJECTS

    EPA Science Inventory

    Epidemiological, in vitro and animal studies suggest that dietary antioxidants can modulate the cellular and physiologic effects of ozone (O3) inhalation in humans. To determine whether antioxidants can influence human susceptibility to O3-induced changes in lung function and a...

  20. Modeling Mycobacterium tuberculosis early granuloma formation in experimental human lung tissue.

    PubMed

    Parasa, Venkata Ramanarao; Rahman, Muhammad Jubayer; Ngyuen Hoang, Anh Thu; Svensson, Mattias; Brighenti, Susanna; Lerm, Maria

    2014-02-01

    The widely used animal models for tuberculosis (TB) display fundamental differences from human TB. Therefore, a validated model that recapitulates human lung TB is attractive for TB research. Here, we describe a unique method for establishment of TB infection in an experimental human lung tissue model. The model is based on cell lines derived from human lungs and primary macrophages from peripheral blood, and displays characteristics of human lung tissue, including evenly integrated macrophages throughout the epithelium, production of extracellular matrix, stratified epithelia and mucus secretion. Establishment of experimental infection in the model tissue with Mycobacterium tuberculosis, the bacterium that causes TB, resulted in clustering of macrophages at the site of infection, reminiscent of early TB granuloma formation. We quantitated the extent of granuloma formation induced by different strains of mycobacteria and validated our model against findings in other TB models. We found that early granuloma formation is dependent on ESAT-6, which is secreted via the type VII secretion machinery of virulent mycobacteria. Our model, which can facilitate the discovery of the interactions between mycobacteria and host cells in a physiological environment, is the first lung tissue model described for TB.

  1. 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

    2016-08-29

    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.

  2. Chronic cadmium exposure in vitro induces cancer cell characteristics in human lung cells

    SciTech Connect

    Person, Rachel J.; Tokar, Erik J.; Xu, Yuanyuan; Orihuela, Ruben; Ngalame, Ntube N. Olive; Waalkes, Michael P.

    2013-12-01

    Cadmium is a known human lung carcinogen. Here, we attempt to develop an in vitro model of cadmium-induced human lung carcinogenesis by chronically exposing the peripheral lung epithelia cell line, HPL-1D, to a low level of cadmium. Cells were chronically exposed to 5 μM cadmium, a noncytotoxic level, and monitored for acquired cancer characteristics. By 20 weeks of continuous cadmium exposure, these chronic cadmium treated lung (CCT-LC) cells showed marked increases in secreted MMP-2 activity (3.5-fold), invasion (3.4-fold), and colony formation in soft agar (2-fold). CCT-LC cells were hyperproliferative, grew well in serum-free media, and overexpressed cyclin D1. The CCT-LC cells also showed decreased expression of the tumor suppressor genes p16 and SLC38A3 at the protein levels. Also consistent with an acquired cancer cell phenotype, CCT-LC cells showed increased expression of the oncoproteins K-RAS and N-RAS as well as the epithelial-to-mesenchymal transition marker protein Vimentin. Metallothionein (MT) expression is increased by cadmium, and is typically overexpressed in human lung cancers. The major MT isoforms, MT-1A and MT-2A were elevated in CCT-LC cells. Oxidant adaptive response genes HO-1 and HIF-1A were also activated in CCT-LC cells. Expression of the metal transport genes ZNT-1, ZNT-5, and ZIP-8 increased in CCT-LC cells culminating in reduced cadmium accumulation, suggesting adaptation to the metal. Overall, these data suggest that exposure of human lung epithelial cells to cadmium causes acquisition of cancer cell characteristics. Furthermore, transformation occurs despite the cell's ability to adapt to chronic cadmium exposure. - Highlights: • Chronic cadmium exposure induces cancer cell characteristics in human lung cells. • This provides an in vitro model of cadmium-induced human lung cell transformation. • This occurred with general and lung specific changes typical for cancer cells. • These findings add insight to the relationship

  3. GSTCD and INTS12 regulation and expression in the human lung.

    PubMed

    Obeidat, Ma'en; Miller, Suzanne; Probert, Kelly; Billington, Charlotte K; Henry, Amanda P; Hodge, Emily; Nelson, Carl P; Stewart, Ceri E; Swan, Caroline; Wain, Louise V; Soler Artigas, María; Melén, Erik; Ushey, Kevin; Hao, Ke; Lamontagne, Maxime; Bossé, Yohan; Postma, Dirkje S; Tobin, Martin D; Sayers, Ian; Hall, Ian P

    2013-01-01

    Genome-Wide Association Study (GWAS) meta-analyses have identified a strong association signal for lung function, which maps to a region on 4q24 containing two oppositely transcribed genes: glutathione S-transferase, C-terminal domain containing (GSTCD) and integrator complex subunit 12 (INTS12). Both genes were found to be expressed in a range of human airway cell types. The promoter regions and transcription start sites were determined in mRNA from human lung and a novel splice variant was identified for each gene. We obtained the following evidence for GSTCD and INTS12 co-regulation and expression: (i) correlated mRNA expression was observed both via Q-PCR and in a lung expression quantitative trait loci (eQTL) study, (ii) induction of both GSTCD and INTS12 mRNA expression in human airway smooth muscle cells was seen in response to TGFβ1, (iii) a lung eQTL study revealed that both GSTCD and INTS12 mRNA levels positively correlate with percent predicted FEV1, and (iv) FEV1 GWAS associated SNPs in 4q24 were found to act as an eQTL for INTS12 in a number of tissues. In fixed sections of human lung tissue, GSTCD protein expression was ubiquitous, whereas INTS12 expression was predominantly in epithelial cells and pneumocytes. During human fetal lung development, GSTCD protein expression was observed to be highest at the earlier pseudoglandular stage (10-12 weeks) compared with the later canalicular stage (17-19 weeks), whereas INTS12 expression levels did not alter throughout these stages. Knowledge of the transcriptional and translational regulation and expression of GSTCD and INTS12 provides important insights into the potential role of these genes in determining lung function. Future work is warranted to fully define the functions of INTS12 and GSTCD.

  4. GATA2 is epigenetically repressed in human and mouse lung tumors and is not requisite for survival of KRAS mutant lung cancer

    PubMed Central

    Tessema, Mathewos; Yingling, Christin M.; Snider, Amanda M.; Do, Kieu; Juri, Daniel E.; Picchi, Maria A.; Zhang, Xiequn; Liu, Yushi; Leng, Shuguang; Tellez, Carmen S.; Belinsky, Steven A.

    2014-01-01

    Introduction GATA2 was recently described as a critical survival factor and therapeutic target for KRAS mutant non-small cell lung cancer (NSCLC). However, whether this role is affected by epigenetic repression of GATA2 in lung cancer is unclear. Methods GATA2 expression and promoter CpG island methylation were evaluated using human and mouse NSCLC cell lines and tumor-normal pairs. In vitro assays were used to study GATA2 repression on cell survival and during tobacco carcinogen-induced transformation. Results GATA2 expression in KRAS wild-type (n=15) and mutant (n=10) NSCLC cell lines and primary lung tumors (n=24) was significantly lower, 1.3–33.6-fold (p=2.2×10−9), compared to corresponding normal lung. GATA2 promoter was unmethylated in normal lung (0/10) but frequently methylated in lung tumors (96%, 159/165) and NSCLC cell lines (97%, 30/31). This highly prevalent aberrant methylation was independently validated using TCGA data for 369 NSCLC tumor-normal pairs. In vitro studies using an established carcinogen-induced pre-malignancy model revealed that GATA2 expression was initially repressed by chromatin remodeling followed by cytosine methylation during transformation. Similarly, expression of Gata2 in NNK-induced mouse lung tumors (n=6) and cell lines (n=5) was 5-fold and 100-fold lower, respectively, than normal mouse lung. Finally, siRNA-mediated knockdown of GATA2 in KRAS mutant [human (n=4) and murine (n=5)] and wild-type [human (n=4)] NSCLC cell lines showed that further reduction of expression (up to 95%) does not induce cell death. Conclusion GATA2 is epigenetically repressed in human and mouse lung tumors and its further inhibition is not a valid therapeutic strategy for KRAS mutant lung cancer. PMID:24807155

  5. NRF2/miR-140 signaling confers radioprotection to human lung fibroblasts

    PubMed Central

    Duru, Nadire; Gernapudi, Ramkishore; Zhang, Yongshu; Yao, Yuan; Lo, Pang-Kuo; Wolfson, Benjamin; Zhou, Qun

    2016-01-01

    Breast and lung cancer patients who are treated with radiotherapy often have severe side effects, including radiation-induced lung damage and secondary cancers. Activation of the NRF2 pathway is a well-known mechanism that protects cells against radiation induced oxidative stress, but its role in radiation-induced lung damage is not well understood. Using human lung fibroblasts (HLFs) we found that ionizing radiation (IR) leads to BRCA1-dependent activation of NRF2 through the inhibition of KEAP1 function, promoting the nuclear accumulation of NRF2, and activating critical radioprotective mechanisms. We discovered that NRF2 directly binds to the miR-140 promoter and increases its expression in response to IR treatment. Gain and loss of function studies further showed the ability of miR-140 to regulate lung fibroblast self-renewal upon irradiation, a potential mechanism to contribute to the regulation of DNA repair. We verified our in vitro findings using primary lung fibroblast cultures from wild type and Nrf2 (KO) mice. Using these models we showed that IR induces overexpression of Brca1, Nrf2 and miR-140 in lung tissue after irradiation. These data reveal a novel radioprotective mechanism in which IR promotes NRF2 nuclear translocation and subsequent activation of miR-140 transcription in HLFs. PMID:26300493

  6. The cytotoxicity and genotoxicity of soluble and particulate cobalt in human lung fibroblast cells.

    PubMed

    Smith, Leah J; Holmes, Amie L; Kandpal, Sanjeev Kumar; Mason, Michael D; Zheng, Tongzhang; Wise, John Pierce

    2014-08-01

    Cobalt exposure is increasing as cobalt demand rises worldwide due to its use in enhancing rechargeable battery efficiency, super-alloys, and magnetic products. Cobalt is considered a possible human carcinogen with the lung being a primary target. However, few studies have considered cobalt-induced toxicity in human lung cells. Therefore, in this study, we sought to determine the cytotoxicity and genotoxicity of particulate and soluble cobalt in human lung cells. Cobalt oxide and cobalt chloride were used as representative particulate and soluble cobalt compounds, respectively. Exposure to both particulate and soluble cobalt induced a concentration-dependent increase in cytotoxicity, genotoxicity, and intracellular cobalt ion levels. Based on intracellular cobalt ion levels, we found that soluble cobalt was more cytotoxic than particulate cobalt while particulate and soluble cobalt induced similar levels of genotoxicity. However, soluble cobalt induced cell cycle arrest indicated by the lack of metaphases at much lower intracellular cobalt concentrations compared to cobalt oxide. Accordingly, we investigated the role of particle internalization in cobalt oxide-induced toxicity and found that particle-cell contact was necessary to induce cytotoxicity and genotoxicity after cobalt exposure. These data indicate that cobalt compounds are cytotoxic and genotoxic to human lung fibroblasts, and solubility plays a key role in cobalt-induced lung toxicity.

  7. RBFOX3 regulates Claudin-1 expression in human lung tissue via attenuation of proteasomal degradation

    PubMed Central

    Kim, Yong-Eun; Choi, Sunkyung

    2017-01-01

    RBFOX3, a nuclear RNA-binding protein, is well known as a regulator of alternative pre-mRNA splicing during neuronal development. However, other functions of RBFOX3 are poorly understood. Here, we investigated the function of RBFOX3 in the cytoplasm with respect to regulation of Claudin-1 expression. In human lung tissue, Claudin-1 is higher in RBFOX3-positive cells than in RBFOX3-negative cells. Immunostaining and mRNA quantification revealed that protein levels, but not mRNA levels, of Claudin-1 are increased by RBFOX3. In addition, cycloheximide treatment of human lung cancer cells revealed that RBFOX3 increases the stability of Claudin-1 through attenuation of its ubiquitination. Our study provides insights into the molecular mechanisms by which RBFOX3 regulates Claudin-1 expression in human lung tissue. PMID:28126724

  8. Glucocorticoid Clearance and Metabolite Profiling in an In Vitro Human Airway Epithelium Lung Model

    PubMed Central

    Rivera-Burgos, Dinelia; Sarkar, Ujjal; Lever, Amanda R.; Avram, Michael J.; Coppeta, Jonathan R.; Wishnok, John S.; Borenstein, Jeffrey T.

    2016-01-01

    The emergence of microphysiologic epithelial lung models using human cells in a physiologically relevant microenvironment has the potential to be a powerful tool for preclinical drug development and to improve predictive power regarding in vivo drug clearance. In this study, an in vitro model of the airway comprising human primary lung epithelial cells cultured in a microfluidic platform was used to establish a physiologic state and to observe metabolic changes as a function of glucocorticoid exposure. Evaluation of mucus production rate and barrier function, along with lung-specific markers, demonstrated that the lungs maintained a differentiated phenotype. Initial concentrations of 100 nM hydrocortisone (HC) and 30 nM cortisone (C) were used to evaluate drug clearance and metabolite production. Measurements made using ultra-high-performance liquid chromatography and high-mass-accuracy mass spectrometry indicated that HC metabolism resulted in the production of C and dihydrocortisone (diHC). When the airway model was exposed to C, diHC was identified; however, no conversion to HC was observed. Multicompartmental modeling was used to characterize the lung bioreactor data, and pharmacokinetic parameters, including elimination clearance and elimination half-life, were estimated. Polymerse chain reaction data confirmed overexpression of 11-β hydroxysteroid dehydrogenase 2 (11βHSD2) over 11βHSD1, which is biologically relevant to human lung. Faster metabolism was observed relative to a static model on elevated rates of C and diHC formation. Overall, our results demonstrate that this lung airway model has been successfully developed and could interact with other human tissues in vitro to better predict in vivo drug behavior. PMID:26586376

  9. Glucocorticoid Clearance and Metabolite Profiling in an In Vitro Human Airway Epithelium Lung Model.

    PubMed

    Rivera-Burgos, Dinelia; Sarkar, Ujjal; Lever, Amanda R; Avram, Michael J; Coppeta, Jonathan R; Wishnok, John S; Borenstein, Jeffrey T; Tannenbaum, Steven R

    2016-02-01

    The emergence of microphysiologic epithelial lung models using human cells in a physiologically relevant microenvironment has the potential to be a powerful tool for preclinical drug development and to improve predictive power regarding in vivo drug clearance. In this study, an in vitro model of the airway comprising human primary lung epithelial cells cultured in a microfluidic platform was used to establish a physiologic state and to observe metabolic changes as a function of glucocorticoid exposure. Evaluation of mucus production rate and barrier function, along with lung-specific markers, demonstrated that the lungs maintained a differentiated phenotype. Initial concentrations of 100 nM hydrocortisone (HC) and 30 nM cortisone (C) were used to evaluate drug clearance and metabolite production. Measurements made using ultra-high-performance liquid chromatography and high-mass-accuracy mass spectrometry indicated that HC metabolism resulted in the production of C and dihydrocortisone (diHC). When the airway model was exposed to C, diHC was identified; however, no conversion to HC was observed. Multicompartmental modeling was used to characterize the lung bioreactor data, and pharmacokinetic parameters, including elimination clearance and elimination half-life, were estimated. Polymerse chain reaction data confirmed overexpression of 11-β hydroxysteroid dehydrogenase 2 (11βHSD2) over 11βHSD1, which is biologically relevant to human lung. Faster metabolism was observed relative to a static model on elevated rates of C and diHC formation. Overall, our results demonstrate that this lung airway model has been successfully developed and could interact with other human tissues in vitro to better predict in vivo drug behavior.

  10. Comparative effects of cadmium, zinc, and lead in vitro on pulmonary, adrenal, and hepatic microsomal metabolism in the guinea pig

    SciTech Connect

    Colby, H.D.; Johnson, P.B.; Zulkoski, J.S.; Pope, M.R.; Miles, P.R.

    1981-11-01

    The in vitro effects of Cd, Zn, and Pb on pulmonary, adrenal, and hepatic microsomal enzyme activities in guinea pigs were compared. Cd and Zn produced concentration-dependent (20-200 ..mu..M) decreases in benzphetamine demethylase and biphenyl hydrozylase activities in adrenal, liver, and lung. Pb had no significant effect on either enzyme in any of the tissues studied. Adrenal and pulmonary enzymes were more sensitive to the effects of Cd and Zn than were hepatic enzymes. Benzo(a)pyrene hydroxylase and ethoxycoumarin demethylase activities were decreased by Zn, Cd and Pb in adrenal, liver, and lung microsomes. The inhibitory effects on benzo(a)pyrene and ethoxycoumarin methabolism were far greater than those on benzphetamine or biphenyl metabolism. The relative potencies of the metals as inhibitors of xenobiotic metabolism were Zn > Cd > Pb. Cd and Zn also inhibited steroid 21-hydroxylase activity in adrenal microsomes, but Pb had no effect on steroid metabolism. In addition, microsomal epoxide hydratase activity in adrenal, liver, and lung was inhibited by Cd but not by Zn or Pb. The results demonstrate that adrenal and pulmonary microsomal enzyme, like those in liver, are inhibited by various metals. Inhibition of mixed-function oxidases by metals in vitro is apparently not related to changes in cytochrome P-450 levels or substrate binding to cytochrome P-450. In addition, the actions of Cd, Zn, and Pb in each tissue are highly dependent on the substrates employed.

  11. Preclinical evaluation of human secretoglobin 3A2 in mouse models of lung development and fibrosis

    PubMed Central

    Cai, Yan; Winn, Melissa E.; Zehmer, John K.; Gillette, William K.; Lubkowski, Jacek T.; Pilon, Aprile L.

    2013-01-01

    Secretoglobin (SCGB) 3A2 is a member of the SCGB gene superfamily of small secreted proteins, predominantly expressed in lung airways. We hypothesize that human SCGB3A2 may exhibit anti-inflammatory, growth factor, and antifibrotic activities and be of clinical utility. Recombinant human SCGB3A2 was expressed, purified, and biochemically characterized as a first step to its development as a therapeutic agent in clinical settings. Human SCGB3A2, as well as mouse SCGB3A2, readily formed a dimer in solution and exhibited novel phospholipase A2 inhibitory activity. This is the first demonstration of any quantitative biochemical measurement for the evaluation of SCGB3A2 protein. In the mouse as an experimental animal, human SCGB3A2 exhibited growth factor activity by promoting embryonic lung development in both ex vivo and in vivo systems and antifibrotic activity in the bleomycin-induced lung fibrosis model. The results suggested that human SCGB3A2 can function as a growth factor and an antifibrotic agent in humans. When SCGB3A2 was administered to pregnant female mice through the tail vein, the protein was detected in the dam's serum and lung, as well as the placenta, amniotic fluids, and embryonic lungs at 10 min postadministration, suggesting that SCGB3A2 readily crosses the placenta. The results warrant further development of recombinant SCGB3A2 as a therapeutic agent in treating patients suffering from lung diseases or preterm infants with respiratory distress. PMID:24213919

  12. Effect of Human and Sheep Lung Orientation on Primary Blast Injury Induced by Single Blast

    DTIC Science & Technology

    2010-09-01

    kPa 200 500 700 Positive phase duration, ms 2 2 2 From the wo rk do ne by O ’ Brien et al. an d Coop er et al. [ Refs. 5, 6], pressure w...Effect of human and sheep lung orientation on primary blast injury induced by single blast A. Bouamoul, K. Williams DRDC Valcartier, 2459 Pie...main goal o f this stud y is to verify if the injuries observed in the animal are trul y representative of human lung injuries for simple blast

  13. The lung mycobiome: an emerging field of the human respiratory microbiome

    PubMed Central

    Nguyen, Linh D. N.; Viscogliosi, Eric; Delhaes, Laurence

    2015-01-01

    The lung microbiome, which is believed to be stable or at least transient in healthy people, is now considered as a poly-microorganism component contributing to disease pathogenesis. Most research studies on the respiratory microbiome have focused on bacteria and their impact on lung health, but there is evidence that other non-bacterial organisms, comprising the viruses (virome) and fungi (mycobiome), are also likely to play an important role in healthy people as well as in patients. In the last few years, the lung mycobiome (previously named the fungal microbiota or microbiome) has drawn closer attention. There is growing evidence that the lung mycobiome has a significant impact on clinical outcome of chronic respiratory diseases (CRD) such as asthma, chronic obstructive pulmonary disease, cystic fibrosis, and bronchiectasis. Thanks to advances in culture independent methods, especially next generation sequencing, a number of fungi not detected by culture methods have been molecularly identified in human lungs. It has been shown that the structure and diversity of the lung mycobiome vary in different populations (healthy and different diseased individuals) which could play a role in CRD. Moreover, the link between lung mycobiome and different biomes of other body sites, especially the gut, has also been unraveled. By interacting with the bacteriome and/or virome, the respiratory mycobiome appears to be a cofactor in inflammation and in the host immune response, and therefore may contribute to the decline of the lung function and the disease progression. In this review, we report the recent limited explorations of the human respiratory mycobiome, and discuss the mycobiome’s connections with other local microbial communities, as well as the relationships with the different biomes of other body sites. These studies suggest several outlooks for this understudied emerging field, which will certainly call for a renewal of our understanding of pulmonary diseases. PMID

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. Chronic Exposure to Particulate Chromate Induces Premature Centrosome Separation and Centriole Disengagement in Human Lung Cells

    PubMed Central

    Martino, Julieta; Holmes, Amie L.; Xie, Hong; Wise, Sandra S.; Wise, John Pierce

    2015-01-01

    Particulate hexavalent chromium (Cr(VI)) is a well-established human lung carcinogen. Lung tumors are characterized by structural and numerical chromosome instability. Centrosome amplification is a phenotype commonly found in solid tumors, including lung tumors, which strongly correlates with chromosome instability. Human lung cells exposed to Cr(VI) exhibit centrosome amplification but the underlying phenotypes and mechanisms remain unknown. In this study, we further characterize the phenotypes of Cr(VI)-induced centrosome abnormalities. We show that Cr(VI)-induced centrosome amplification correlates with numerical chromosome instability. We also show chronic exposure to particulate Cr(VI) induces centrosomes with supernumerary centrioles and acentriolar centrosomes in human lung cells. Moreover, chronic exposure to particulate Cr(VI) affects the timing of important centriolar events. Specifically, chronic exposure to particulate Cr(VI) causes premature centriole disengagement in S and G2 phase cells. It also induces premature centrosome separation in interphase. Altogether, our data suggest that chronic exposure to particulate Cr(VI) targets the protein linkers that hold centrioles together. These centriolar linkers are important for key events of the centrosome cycle and their premature disruption might underlie Cr(VI)-induced centrosome amplification. PMID:26293554

  19. The cytotoxicity and genotoxicity of soluble and particulate cobalt in human lung fibroblast cells

    SciTech Connect

    Smith, Leah J.; Holmes, Amie L.; Kandpal, Sanjeev Kumar; Mason, Michael D.; Zheng, Tongzhang; Wise, John Pierce

    2014-08-01

    Cobalt exposure is increasing as cobalt demand rises worldwide due to its use in enhancing rechargeable battery efficiency, super-alloys, and magnetic products. Cobalt is considered a possible human carcinogen with the lung being a primary target. However, few studies have considered cobalt-induced toxicity in human lung cells. Therefore, in this study, we sought to determine the cytotoxicity and genotoxicity of particulate and soluble cobalt in human lung cells. Cobalt oxide and cobalt chloride were used as representative particulate and soluble cobalt compounds, respectively. Exposure to both particulate and soluble cobalt induced a concentration-dependent increase in cytotoxicity, genotoxicity, and intracellular cobalt ion levels. Based on intracellular cobalt ion levels, we found that soluble cobalt was more cytotoxic than particulate cobalt while particulate and soluble cobalt induced similar levels of genotoxicity. However, soluble cobalt induced cell cycle arrest indicated by the lack of metaphases at much lower intracellular cobalt concentrations compared to cobalt oxide. Accordingly, we investigated the role of particle internalization in cobalt oxide-induced toxicity and found that particle-cell contact was necessary to induce cytotoxicity and genotoxicity after cobalt exposure. These data indicate that cobalt compounds are cytotoxic and genotoxic to human lung fibroblasts, and solubility plays a key role in cobalt-induced lung toxicity. - Highlights: • Particulate and soluble cobalt are cytotoxic and genotoxic to human lung cells. • Soluble cobalt induces more cytotoxicity compared to particulate cobalt. • Soluble and particulate cobalt induce similar levels of genotoxicity. • Particle-cell contact is required for particulate cobalt-induced toxicity.

  20. Viral infection of human lung macrophages increases PDL1 expression via IFNβ.

    PubMed

    Staples, Karl J; Nicholas, Ben; McKendry, Richard T; Spalluto, C Mirella; Wallington, Joshua C; Bragg, Craig W; Robinson, Emily C; Martin, Kirstin; Djukanović, Ratko; Wilkinson, Tom M A

    2015-01-01

    Lung macrophages are an important defence against respiratory viral infection and recent work has demonstrated that influenza-induced macrophage PDL1 expression in the murine lung leads to rapid modulation of CD8+ T cell responses via the PD1 receptor. This PD1/PDL1 pathway may downregulate acute inflammatory responses to prevent tissue damage. The aim of this study was to investigate the mechanisms of PDL1 regulation by human macrophages in response to viral infection. Ex-vivo viral infection models using influenza and RSV were established in human lung explants, isolated lung macrophages and monocyte-derived macrophages (MDM) and analysed by flow cytometry and RT-PCR. Incubation of lung explants, lung macrophages and MDM with X31 resulted in mean cellular infection rates of 18%, 18% and 29% respectively. Viral infection significantly increased cell surface expression of PDL1 on explant macrophages, lung macrophages and MDM but not explant epithelial cells. Infected MDM induced IFNγ release from autologous CD8+ T cells, an effect enhanced by PDL1 blockade. We observed increases in PDL1 mRNA and IFNβ mRNA and protein release by MDM in response to influenza infection. Knockdown of IFNβ by siRNA, resulted in a 37.5% reduction in IFNβ gene expression in response to infection, and a significant decrease in PDL1 mRNA. Furthermore, when MDM were incubated with IFNβ, this cytokine caused increased expression of PDL1 mRNA. These data indicate that human macrophage PDL1 expression modulates CD8+ cell IFNγ release in response to virus and that this expression is regulated by autologous IFNβ production.

  1. Possible risks to human lungs from magnetometric dust clearance experiments

    NASA Astrophysics Data System (ADS)

    Sterling, T. D.

    1981-03-01

    Cohen, Arai and Brain did a magnetization study on smokers and nonsmokers from which they conclude that the dust clearance ability of the cigarette smoker's lungs is markedly impaired. Their conclusion may be incorrect because they overlooked that during the magnetization phase of their experiment, iron oxide clusters were preferentially formed in smoker's bronchi because of their high mucus content and consequent low resistance to redistribution of particles. Prudence dictates avoidance of the Cohen, Arai, Brain type study until health hazards related to this work are investigated.

  2. Comparative Microscopic Study of Human and Rat Lungs After Overexposure to Welding Fume

    PubMed Central

    ANTONINI, JAMES M.; ROBERTS, JENNY R.; SCHWEGLER-BERRY, DIANE; MERCER, ROBERT R.

    2015-01-01

    particles were metal complexes with iron, chromium, and nickel being the most common metals present. In conclusion, long-term exposure to specific welding fume can lead to serious chronic lung disease characterized by significant particle deposition and persistence as demonstrated in both a human case study and rat model. Not only were the lung responses similar in the human and rat lungs, as evidenced by inflammatory cell influx and pulmonary disease, but the composition of individual welding particles and agglomerations in situ was comparable. PMID:23798603

  3. Comparative microscopic study of human and rat lungs after overexposure to welding fume.

    PubMed

    Antonini, James M; Roberts, Jenny R; Schwegler-Berry, Diane; Mercer, Robert R

    2013-11-01

    particles were metal complexes with iron, chromium, and nickel being the most common metals present. In conclusion, long-term exposure to specific welding fume can lead to serious chronic lung disease characterized by significant particle deposition and persistence as demonstrated in both a human case study and rat model. Not only were the lung responses similar in the human and rat lungs, as evidenced by inflammatory cell influx and pulmonary disease, but the composition of individual welding particles and agglomerations in situ was comparable.

  4. Inhibition of fibroblast growth factor receptor 3-dependent lung adenocarcinoma with a human monoclonal antibody

    PubMed Central

    Yin, Yongjun; Ren, Xiaodi; Smith, Craig; Guo, Qianxu; Malabunga, Maria; Guernah, Ilhem; Zhang, Yiwei; Shen, Juqun; Sun, Haijun; Chehab, Nabil; Loizos, Nick; Ludwig, Dale L.; Ornitz, David M.

    2016-01-01

    ABSTRACT Activating mutations in fibroblast growth factor receptor 3 (FGFR3) have been identified in multiple types of human cancer and in congenital birth defects. In human lung cancer, fibroblast growth factor 9 (FGF9), a high-affinity ligand for FGFR3, is overexpressed in 10% of primary resected non-small cell lung cancer (NSCLC) specimens. Furthermore, in a mouse model where FGF9 can be induced in lung epithelial cells, epithelial proliferation and ensuing tumorigenesis is dependent on FGFR3. To develop new customized therapies for cancers that are dependent on FGFR3 activation, we have used this mouse model to evaluate a human monoclonal antibody (D11) with specificity for the extracellular ligand-binding domain of FGFR3, that recognizes both human and mouse forms of the receptor. Here, we show that D11 effectively inhibits signaling through FGFR3 in vitro, inhibits the growth of FGFR3-dependent FGF9-induced lung adenocarcinoma in mice, and reduces tumor-associated morbidity. Given the potency of FGF9 in this mouse model and the absolute requirement for signaling through FGFR3, this study validates the D11 antibody as a potentially useful and effective reagent for treating human cancers or other pathologies that are dependent on activation of FGFR3. PMID:27056048

  5. hPSC-derived lung and intestinal organoids as models of human fetal tissue.

    PubMed

    Aurora, Megan; Spence, Jason R

    2016-12-15

    In vitro human pluripotent stem cell (hPSC) derived tissues are excellent models to study certain aspects of normal human development. Current research in the field of hPSC derived tissues reveals these models to be inherently fetal-like on both a morphological and gene expression level. In this review we briefly discuss current methods for differentiating lung and intestinal tissue from hPSCs into individual 3-dimensional units called organoids. We discuss how these methods mirror what is known about in vivo signaling pathways of the developing embryo. Additionally, we will review how the inherent immaturity of these models lends them to be particularly valuable in the study of immature human tissues in the clinical setting of premature birth. Human lung organoids (HLOs) and human intestinal organoids (HIOs) not only model normal development, but can also be utilized to study several important diseases of prematurity such as respiratory distress syndrome (RDS), bronchopulmonary dysplasia (BPD), and necrotizing enterocolitis (NEC).

  6. Investigation of antiaromatase activity using hepatic microsomes of Nile tilapia (Oreochromis niloticus).

    PubMed

    Sassa-Deepaeng, Tanongsak; Chaisri, Wasana; Pikulkaew, Surachai; Okonogi, Siriporn

    2017-03-19

    Microsomal aromatase enzymes of humans and rats have been used in antiaromatase assays, but enzyme activity is species-specific. The current study extracted hepatic microsomes of Nile tilapia (Oreochromis niloticus) to investigate and compare the antiaromatase activity of chrysin, quercetin, and quercitrin. This activity was evaluated using a dibenzylfluorescein (DBF) assay. Results revealed that the age and body weight of Nile tilapia affected the yield of extracted microsomes. Extraction of hepatic microsomes of Nile tilapia was most effective when using a reaction medium with a pH of 8.0. A DBF assay using Nile tilapia microsomes revealed significant differences in levels of antiaromatase activity for chrysin, quercetin, and quercitrin. Chrysin was the most potent aromatase inhibitor, with an IC50 of 0.25 mg/mL. In addition, chrysin is an aromatase inhibitor that also inhibits the proliferation of cancer cells. Hepatic microsomes of Nile tilapia can be used to investigate and compare the antiaromatase activity of different compounds.

  7. Human lung hydrolases delineate Mycobacterium tuberculosis-macrophage interactions and the capacity to control infection.

    PubMed

    Arcos, Jesús; Sasindran, Smitha J; Fujiwara, Nagatoshi; Turner, Joanne; Schlesinger, Larry S; Torrelles, Jordi B

    2011-07-01

    Pulmonary surfactant contains homeostatic and antimicrobial hydrolases. When Mycobacterium tuberculosis is initially deposited in the terminal bronchioles and alveoli, as well as following release from lysed macrophages, bacilli are in intimate contact with these lung surfactant hydrolases. We identified and measured several hydrolases in human alveolar lining fluid and lung tissue that, at their physiological concentrations, dramatically modified the M. tuberculosis cell envelope. Independent of their action time (15 min to 12 h), the effects of the hydrolases on the M. tuberculosis cell envelope resulted in a significant decrease (60-80%) in M. tuberculosis association with, and intracellular growth of the bacteria within, human macrophages. The cell envelope-modifying effects of the hydrolases also led to altered M. tuberculosis intracellular trafficking and induced a protective proinflammatory response to infection. These findings add a new concept to our understanding of M. tuberculosis-macrophage interactions (i.e., the impact of lung surfactant hydrolases on M. tuberculosis infection).

  8. Mechanism of action of ozone on the human lung

    SciTech Connect

    Hazucha, M.J.; Bates, D.V.; Bromberg, P.A. )

    1989-10-01

    Fourteen healthy normal volunteers were randomly exposed to air and 0.5 ppm of ozone (O3) in a controlled exposure chamber for a 2-h period during which 15 min of treadmill exercise sufficient to produce a ventilation of approximately 40 l/min was alternated with 15-min rest periods. Before testing an esophageal balloon was inserted, and lung volumes, flow rates, maximal inspiratory (at residual volume and functional residual capacity) and expiratory (at total lung capacity and functional residual capacity) mouth pressures, and pulmonary mechanics (static and dynamic compliance and airway resistance) were measured before and immediately after the exposure period. After the postexposure measurements had been completed, the subjects inhaled an aerosol of 20% lidocaine until response to citric acid aerosol inhalation was abolished. All of the measurements were immediately repeated. We found that the O3 exposure (1) induced a significant mean decrement of 17.8% in vital capacity (this change was the result of a marked fall in inspiratory capacity without significant increase in residual volume), (2) significantly increased mean airway resistance and specific airway resistance but did not change dynamic or static pulmonary compliance or viscous or elastic work, (3) significantly reduced maximal transpulmonary pressure (by 19%) but produced no changes in inspiratory or expiratory maximal mouth pressures, and (4) significantly increased respiratory rate (in 5 subjects by more than 6 breaths/min) and decreased tidal volume.

  9. Bronchoalveolar lavage cell pattern from healthy human lung

    PubMed Central

    Heron, M; Grutters, J C; ten Dam-Molenkamp, K M; Hijdra, D; van Heugten-Roeling, A; Claessen, A M E; Ruven, H J T; van den Bosch, J M M; van Velzen-Blad, H

    2012-01-01

    Bronchoalveolar lavage (BAL) is widely accepted as a key diagnostic procedure in interstitial lung diseases (ILD). We performed a study to obtain reference intervals of differential cell patterns in BAL fluid with special attention to the origin of lavage fluid, e.g. bronchial/alveolar, to atopy and smoking status and to age of the healthy people. We performed bronchoalveolar lavage in 55 healthy subjects with known atopy status (age: 18–64 years, non-smokers/smokers: 34/21) and determined differential cell counts and lymphocyte subsets in BAL fluid and blood. Moreover, in a subgroup of non-smoking healthy individuals we measured the expression of the regulatory T cell marker forkhead box protein 3 (FoxP3) on blood and BAL fluid lymphocytes in addition to a comprehensive set of activation markers. Differential cell counts from the alveolar lavage fraction differed significantly from calculated pooled fractions (n = 11). In contrast, marginal differences were found between atopic and non-atopic subjects. Interestingly, the BAL fluid CD4+/CD8+ ratio correlated strongly with age (r2 = 0·50, P < 0·0001). We consider the bronchial and alveolar fraction to be lavage fluid from fundamentally different compartments and recommend analysis of the alveolar fraction in diagnostic work-up of ILD. In addition, our data suggest that age corrected BAL fluid CD4+/CD8+ ratios should be used in the clinical evaluation of patients with interstitial lung diseases. PMID:22288596

  10. Studies on the mechanism of activation of microsomal benzo(a)pyrene hydroxylation by flavonoids

    SciTech Connect

    Huang, M.T.; Chang, R.L.; Fortner, J.G.; Conney, A.H.

    1981-07-10

    7,8-benzoflavone or flavone stimulates the hydroxylation of benzo(a)pyrene by liver microsomes from rabbit, hamster, and man severalfold. Little or no activation by the flavonoid occurs in liver microsomes from rat or guinea pig. Intact liver microsomal membranes are not required for the activation. Although 7,8-benzoflavone does not stimulate the NADPH-dependent reduction of cytochrome c by rabbit liver microsomes, the NADPH-dependent reduction of cytochrome P-450 is stimulated by 7,8-benzoflavone either in the presence or absence of benzo(a)pyrene. Purified cytochrome P-450 reductase causes an increase in the rate of benzo(a)pyrene hydroxylation in cholate-solubilized liver microsomes from all of the species studied. In cholate-solubilized microsomes from all of the species susceptible for flavonoid activation, 7,8-benzoflavone decreases the K/sub m/ for cytochrome P-450 reductase and increases the V/sub max/ for benzo(a)pyrene hydroxylation. With cholate-solubilized human liver microsomes, the K/sub m/ for cytochrome P-450 reductase in the absence of flavonoids was about 3-fold higher than in the presence of 100 ..mu..M 7,8-benzoflavone or 500 ..mu..M flavone. 7,8-benzoflavone and flavone stimulate the hydroxylation of benzo(a)pyrene in liver microsomes at least in part by enhancing the interaction between cytochrome P-450 and cytochrome P-450 reductase. 7,8-benzoflavone does not influence the K/sub m/ for benzo(a)pyrene or NADPH, but the V/sub max/ values for benzo(a)pyrene are increased from 2.5- to 4-fold in rabbit liver microsomes. 7,8-benzoflavone does not stimulate the cumene hydroperoxide-dependent hydroxylation of benzo(a)pyrene by rabbit liver microsomes. In two partially purified cytochrome P-450 fractions from rabbit liver microsomes, flavone has a specific stimulatory effect on one of the reconstituted partially purified cytochrome P-450 systems, but an inhibitory effect on the other.

  11. Solubility of indium-tin oxide in simulated lung and gastric fluids: Pathways for human intake.

    PubMed

    Andersen, Jens Christian Østergård; Cropp, Alastair; Paradise, Diane Caroline

    2017-02-01

    From being a metal with very limited natural distribution, indium (In) has recently become disseminated throughout the human society. Little is known of how In compounds behave in the natural environment, but recent medical studies link exposure to In compounds to elevated risk of respiratory disorders. Animal tests suggest that exposure may lead to more widespread damage in the body, notably the liver, kidneys and spleen. In this paper, we investigate the solubility of the most widely used In compound, indium-tin oxide (ITO) in simulated lung and gastric fluids in order to better understand the potential pathways for metals to be introduced into the bloodstream. Our results show significant potential for release of In and tin (Sn) in the deep parts of the lungs (artificial lysosomal fluid) and digestive tract, while the solubility in the upper parts of the lungs (the respiratory tract or tracheobronchial tree) is very low. Our study confirms that ITO is likely to remain as solid particles in the upper parts of the lungs, but that particles are likely to slowly dissolve in the deep lungs. Considering the prolonged residence time of inhaled particles in the deep lung, this environment is likely to provide the major route for uptake of In and Sn from inhaled ITO nano- and microparticles. Although dissolution through digestion may also lead to some uptake, the much shorter residence time is likely to lead to much lower risk of uptake.

  12. Nicotine prevents the apoptosis induced by menadione in human lung cancer cells

    SciTech Connect

    Zhang Tao; Lu Heng; Shang Xuan; Tian Yihao; Zheng Congyi; Wang Shiwen; Cheng Hanhua . E-mail: hhcheng@whu.edu.cn; Zhou Rongjia . E-mail: rjzhou@whu.edu.cn

    2006-04-14

    Approximately 50% of long-term cigarette smokers die prematurely from the adverse effects of smoking, including on lung cancer and other illnesses. Nicotine is a main component in tobacco and has been implicated as a potential factor in the pathogenesis of human lung cancer. However, the mechanism of nicotine action in the development of lung cancer remains largely unknown. In the present study, we designed a nicotine-apoptosis system, by pre-treatment of nicotine making lung cancer cell A549 to be in a physiological nicotine environment, and observed that nicotine promoted cell proliferation and prevented the menadione-induced apoptosis, and exerts its role of anti-apoptosis by shift of apoptotic stage induced by menadione from late apoptotic stage to early apoptotic stage, in which NF-{kappa}B was up-regulated. Interference analysis of NF-{kappa}B in A549 cells showed that knock down of NF-{kappa}B resulted in apoptosis promotion and counteracted the protective effect of nicotine. The findings suggest that nicotine has potential effect in lung cancer genesis, especially in patients with undetectable early tumor development and development of specific NF-{kappa}B inhibitors would represent a potentially exciting new pharmacotherapy for tobacco-related lung cancer.

  13. Relaxin induces an extracellular matrix-degrading phenotype in human lung fibroblasts in vitro and inhibits lung fibrosis in a murine model in vivo.

    PubMed Central

    Unemori, E N; Pickford, L B; Salles, A L; Piercy, C E; Grove, B H; Erikson, M E; Amento, E P

    1996-01-01

    Pulmonary fibrosis is the common end stage of a number of pneumopathies. In this study, we examined the ability of the human cytokine, relaxin, to block extracellular matrix deposition by human lung fibroblasts in vitro, and to inhibit lung fibrosis in a bleomycin-induced murine model. In vitro, relaxin (1-100 ng/ml) inhibited the transforming growth factor-beta-mediated over-expression of interstitial collagen types I and III by human lung fibroblasts by up to 45% in a dose-dependent manner. Relaxin did not affect basal levels of collagen expression in the absence of TGF-beta-induced stimulation. Relaxin also blocked transforming growth factor-beta-induced upregulation of fibronectin by 80% at the highest relaxin dose tested (100 ng/ml). The expression of matrix metalloproteinase-1, or procollagenase, was stimulated in a biphasic, dose-dependent manner by relaxin. In vivo, relaxin, at a steady state circulating concentration of approximately 50 ng/ml, inhibited bleomycin-mediated alveolar thickening compared with the vehicle only control group (P < 0.05). Relaxin also restored bleomycin-induced collagen accumulation, as measured by lung hydroxyproline content, to normal levels (P < 0.05). In summary, relaxin induced a matrix degradative phenotype in human lung fibroblasts in vitro and inhibited bleomycin-induced fibrosis in a murine model in vivo. These data indicate that relaxin may be efficacious in the treatment of pathologies characterized by lung fibrosis. PMID:8981919

  14. Sulfation of chlorotyrosine and nitrotyrosine by human lung endothelial and epithelial cells: Role of the human SULT1A3

    SciTech Connect

    Yasuda, Shin; Yasuda, Tomoko; Liu, Ming-Yih; Shetty, Sreerama; Idell, Steven; Boggaram, Vijayakumar; Suiko, Masahito; Sakakibara, Yoichi; Fu Jian; Liu, Ming-Cheh

    2011-03-01

    During inflammation, potent reactive oxidants formed may cause chlorination and nitration of both free and protein-bound tyrosine. In addition to serving as biomarkers of inflammation-mediated oxidative stress, elevated levels of chlorotyrosine and nitrotyrosine have been linked to the pathogenesis of lung and vascular disorders. The current study was designed to investigate whether the lung cells are equipped with mechanisms for counteracting these tyrosine derivatives. By metabolic labeling, chlorotyrosine O-[{sup 35}S]sulfate and nitrotyrosine O-[{sup 35}S]sulfate were found to be generated and released into the labeling media of human lung endothelial and epithelial cells labeled with [{sup 35}S]sulfate in the presence of added chlorotyrosine and nitrotyrosine. Enzymatic assays using the eleven known human cytosolic sulfotransferases (SULTs) revealed SULT1A3 as the enzyme responsible for catalyzing the sulfation of chlorotyrosine and nitrotyrosine. Reverse transcription-polymerase chain reaction (RT-PCR) analysis demonstrated the expression of SULT1A3 in the lung endothelial and epithelial cells used in this study. Kinetic constants of the sulfation of chlorotyrosine and nitrotyrosine by SULT1A3 were determined. Collectively, these results suggest that sulfation by SULT1A3 in lung endothelial and epithelial cells may play a role in the inactivation and/or disposal of excess chlorotyrosine and nitrotyrosine generated during inflammation.

  15. Association between human papillomavirus and EGFR mutations in advanced lung adenocarcinoma

    PubMed Central

    Li, Ming; Deng, Fang; Qian, Li-Ting; Meng, Shui-Ping; Zhang, Yang; Shan, Wu-Lin; Zhang, Xiao-Lei; Wang, Bao-Long

    2016-01-01

    Previous studies have demonstrated an association between human papillomavirus (HPV) and mutations in the epidermal growth factor receptor (EGFR) gene in lung cancer patients; however, few studies have investigated this association in advanced lung adenocarcinoma patients undergoing gefitinib treatment. The present study investigated the association between HPV and EGFR mutations in advanced lung adenocarcinoma patients. A total of 95 advanced lung adenocarcinoma patients were enrolled in the study. The HPV infection status and presence of EGFR mutations in tumor tissue was evaluated. Patient clinical characteristics were also determined and compared with HPV infection and EGFR mutation status to analyze their impact on progression-free survival. HPV DNA was identified in 27/95 (28.4%) lung adenocarcinoma tumors and was most common in patients with lymph node metastasis (P=0.016). A total of 44/95 (46.3%) cases exhibited EGFR mutations, which were predominantly observed in female patients and non-smokers. The presence of HPV DNA was significantly associated with EGFR mutations (P=0.012) and multivariate analysis also revealed that HPV DNA was significantly associated with EGFR mutations (odds ratio=3.971) in advanced lung adenocarcinoma. Patients with both HPV infections and EGFR mutations exhibit a marked decrease in the risk of lung cancer progression when compared with those without HPV infection or EGFR mutations (adjusted HR=0.640; 95% confidence interval: 0.488–0.840; P=0.001). HPV infection was significantly associated with EGFR mutations in advanced lung adenocarcinoma patients. Furthermore, patients with HPV infections exhibited the longest progression-free survival times, which may be due to good response to tyrosine kinase inhibitor- or platinum-based-adjuvant therapy in these patients. Patients with EGFR mutations exhibited a better prognosis when compared with those exhibiting wild-type EGFR, regardless of HPV status. PMID:27602120

  16. Human receptor kinetics and lung tissue retention of the enhanced-affinity glucocorticoid fluticasone furoate

    PubMed Central

    Valotis, Anagnostis; Högger, Petra

    2007-01-01

    Fluticasone furoate (FF) – USAN approved name, a new topically active glucocorticoid has been recently identified. The aim of this study was to characterise the binding affinity of this compound to the human lung glucocorticoid receptor in relation to other glucocorticoids. Additionally, we sought to determine the binding behaviour of fluticasone furoate to human lung tissue. The glucocorticoid receptor binding kinetics of fluticasone furoate revealed a remarkably fast association and a slow dissociation resulting in a relative receptor affinity (RRA) of 2989 ± 135 with reference to dexamethasone (RRA: 100 ± 5). Thus, the RRA of FF exceeds the RRAs of all currently clinically used corticosteroids such as mometasone furoate (MF; RRA 2244), fluticasone propionate (FP; RRA 1775), ciclesonide's active metabolite (RRA 1212 – rat receptor data) or budesonide (RRA 855). FP and FF displayed pronounced retention in human lung tissue in vitro. Lowest tissue binding was found for MF. There was no indication of instability or chemical modification of FF in human lung tissue. These advantageous binding attributes may contribute to a highly efficacious profile for FF as a topical treatment for inflammatory disorders of the respiratory tract. PMID:17650349

  17. Effects of combinations of diesel exhaust and ozone exposure on lung function in human volunteers.

    EPA Science Inventory

    Ozone (03) exposure induces changes in human lung function, typically seen as a decrease in forced expiratory volume in one sec (FEV1) and forced vital capacity (FVC). Because people are usually exposed to other ambient air pollutants simultaneously with 03, there may be interact...

  18. TISSUE REMODELING IN THE HUMAN LUNG IN RELATION TO PARTICLE CONCENTRATION AND METAL CONTENT

    EPA Science Inventory

    TISSUE REMODELING IN THE HUMAN LUNG IN RELATION TO PARTICLE CONCENTRATION AND METAL CONTENT. J Gallagher1, J Inmon1, S Schlaegle2, A Levine2, T Rogers3, J Scott1, F Green4, M Schenker5, K Pinkerton5 1NHEERL, US-EPA, RTP, NC, USA; 2RJ Lee Group Inc, Monroeville, Pa, USA; ...

  19. OZONE-INDUCED RESPIRATORY SYMPTOMS AND LUNG FUNCTION DECREMENTS IN HUMANS: EXPOSURE-RESPONSE MODELS

    EPA Science Inventory

    Short duration exposure to ozone (<8 hr) is known to result in lung function decrements and respiratory symptoms in humans. The magnitudes of these responses are functions of ozone concentration (C), activity level measured by minute ventilation (Ve), duration of exposure (T), a...

  20. Diesel Exhaust Modulates Ozone-induced Lung Function Decrements in Healthy Human Volunteers

    EPA Science Inventory

    The potential effects of combinations of dilute whole diesel exhaust (DE) and ozone (03), each a common component of ambient airborne pollutant mixtures, on lung function were examined. Healthy young human volunteers were exposed for 2 hr to pollutants while exercising (~50 L/min...

  1. Effect of clarythromycin on the distant metastases of human lung cancer cells in SCID mice.

    PubMed

    Parajuli, P; Yano, S; Hanibuchi, M; Nokihara, H; Shinohara, T; Sone, S

    1998-02-01

    Recently, the use of macrolides is suggested to be therapeutically effective in prolonging the survival of patients with inoperable non-small cell lung cancer. The purpose of this study was to examine therapeutic effects of a macrolide, clarythromycin (CAM) on the metastastic developments of two different human non-small cell lung cancers (squamous cell lung carcinoma RERF-LC-AI, and adenocarcinoma PC-14) in severe combined immunodeficient (SCID) mice depleted or undepleted of natural killer (NK) cells, respectively. CAM, injected subcutaneously at doses of 5 and 10 mg/kg body weight/day from day 7 to 41 after i.v. inoculation of human lung cancer cells, was not effective in inhibiting their distant organ metastases in SCID mice. CAM at concentrations of less than 10 micrograms/ml did not have a direct influence on the proliferation of these tumor cells in vitro. Although CAM alone was not effective in augmenting NK activity, it augmented the IL-2-induced killer (LAK) activity against Daudi cells in vitro. These results suggest that CAM alone may not be enough to control the spread of non-small cell lung cancer in the patient with T cell dysfunction.

  2. Differences in Redox Regulatory Systems in Human Lung and Liver Tumors Suggest Different Avenues for Therapy

    PubMed Central

    Tobe, Ryuta; Carlson, Bradley A.; Tsuji, Petra A.; Lee, Byeong Jae; Gladyshev, Vadim N.; Hatfield, Dolph L.

    2015-01-01

    A common characteristic of many cancer cells is that they suffer from oxidative stress. They, therefore, require effective redox regulatory systems to combat the higher levels of reactive oxygen species that accompany accelerated growth compared to the normal cells of origin. An elevated dependence on these systems in cancers suggests that targeting these systems may provide an avenue for retarding the malignancy process. Herein, we examined the redox regulatory systems in human liver and lung cancers by comparing human lung adenocarcinoma and liver carcinoma to their respective surrounding normal tissues. Significant differences were found in the two major redox systems, the thioredoxin and glutathione systems. Thioredoxin reductase 1 levels were elevated in both malignancies, but thioredoxin was highly upregulated in lung tumor and only slightly upregulated in liver tumor, while peroxiredoxin 1 was highly elevated in lung tumor, but downregulated in liver tumor. There were also major differences within the glutathione system between the malignancies and their normal tissues. The data suggest a greater dependence of liver on either the thioredoxin or glutathione system to drive the malignancy, while lung cancer appeared to depend primarily on the thioredoxin system. PMID:26569310

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

    PubMed

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

    2017-01-12

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

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

    PubMed

    Peng, Hui; Zhu, Qin-shi; Zhong, Shuping; Levy, Daniel

    2015-01-01

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

  5. High-throughput screening to estimate single or multiple enzymes involved in drug metabolism: microtitre plate assay using a combination of recombinant CYP2D6 and human liver microsomes.

    PubMed

    Yamamoto, T; Suzuki, A; Kohno, Y

    2003-08-01

    1. The purpose of this study was to estimate readily involvement of single or multiple enzymes in the metabolism of a drug through inhibitory assessment. Inhibitory effects of various compounds on CYP2D6 activity assayed by formation of fluorescent metabolite from 3-[2-(N,N-diethyl-N-methyl-ammonium)ethyl]-7-methoxy-4-methyl-coumarin (AMMC) were assessed using microtitre plate (MTP) assays with a combination of recombinant CYP2D6 and human liver microsomes (HLM). 2. Among various compounds studied, antipsychotic drugs extensively inhibited recombinant CYP2D6 activity and the IC50 values were generally lower than those of antidepressants and antiarrhythmic drugs. 3. After pre-incubation, the IC50 values of mianserin, chlorpromadine, risperidone, thioridazine, alprenolol, propafenone and dextromethorphan increased but the values of timolol, S-metoprolol and propranolol substantially decreased compared with those in case of co-incubation. 4. The IC50 values of typical substrates of CYP2D6 (bufuralol and dextromethorphan at lower substrate concentration) in inhibition studies using HLM, were similar to those in the case of recombinant CYP2D6, but the values of the compounds that are metabolized by multiple CYP forms (perphenazine and chlorpromazine) in HLM were much larger. 5. If the ratio (HLM/rCYP ratio) of IC50 values between HLM and recombinant CYP2D6 exceeds approximately 2, it suggests that other CYP forms in addition to CYP2D6 might be involved in the metabolism of the test compounds. From the advantage such as speed, high throughput and ease of the technique, the MTP assay using a combination of the recombinant CYP2D6 and HLM is useful to estimate the involvement of single or multiple enzymes in the metabolism of drugs at the stage of drug discovery.

  6. Oral recombinant human or mouse lactoferrin reduces Mycobacterium tuberculosis TDM induced granulomatous lung pathology.

    PubMed

    Hwang, Shen-An; Kruzel, Marian L; Actor, Jeffrey K

    2017-02-01

    Trehalose 6'6-dimycolate (TDM) is the most abundant glycolipid on the cell wall of Mycobacterium tuberculosis (MTB). TDM is capable of inducing granulomatous pathology in mouse models that resembles those induced by MTB infection. Using the acute TDM model, this work investigates the effect of recombinant human and mouse lactoferrin to reduce granulomatous pathology. C57BL/6 mice were injected intravenously with TDM at a dose of 25 μg·mouse(-1). At day 4 and 6, recombinant human or mouse lactoferrin (1 mg·(100 μL)(-1)·mouse(-1)) were delivered by gavage. At day 7 after TDM injection, mice were evaluated for lung pathology, cytokine production, and leukocyte populations. Mice given human or mouse lactoferrin had reduced production of IL-12p40 in their lungs. Mouse lactoferrin increased IL-6 and KC (CXCL1) in lung tissue. Increased numbers of macrophages were observed in TDM-injected mice given human or mouse lactoferrin. Granulomatous pathology, composed of mainly migrated leukocytes, was visually reduced in mice that received human or mouse lactoferrin. Quantitation of granulomatous pathology demonstrated a significant decrease in mice given human or mouse lactoferrin compared with TDM control mice. This report is the first to directly compare the immune modulatory effects of both heterologous recombinant human and homologous mouse lactoferrin on the development of TDM-induced granulomas.

  7. Deformable image registration of heterogeneous human lung incorporating the bronchial tree

    SciTech Connect

    Al-Mayah, Adil; Moseley, Joanne; Velec, Mike; Hunter, Shannon; Brock, Kristy

    2010-09-15

    Purpose: To investigate the effect of the bronchial tree on the accuracy of biomechanical-based deformable image registration of human lungs. Methods: Three dimensional finite element models have been developed using four dimensional computed tomography image data of ten lung cancer patients. Each model is built of a body, left and right lungs, tumor, and bronchial trees. Triangular shell elements are used for the bronchial trees while tetrahedral elements are used for other components. Hyperelastic material properties based on experimental investigation on human lungs are used for the lung parenchyma. Different material properties are assigned for the bronchial tree using five values for the modulus of elasticity of 0.01, 0.12, 0.5, 10, and 18 MPa. Lungs are modeled to slide inside chest cavities by applying frictionless contact surfaces between each lung and corresponding chest cavity. The accuracy of the models is examined using an average of 40 bronchial bifurcation points identified on inhale and exhale images. Relative accuracy is evaluated by comparing the displacement of all nodes within the lungs as well as the dosimetric difference at the exhale position predicted by the model. Results: There is no significant effect of bronchial tree on the model accuracy based on the bifurcation points analysis. However, on the local level, using an average of 38 000 nodes, there is a maximum difference of 8.5 mm in the deformation of the bronchial trees, as the modulus of elasticity of the bronchial trees increases from 0.01 to 18 MPa; however, more than 96% of nodes are within a 2.5 mm difference in each direction. The average dose difference at the predicted exhale position is less than 35 cGy between the models. Conclusions: The bronchial tree has little effect on the global deformation and the accuracy of deformable image registration of lungs. Hence, the homogenous model is a reasonable assumption. Since there are some local deformation differences between nodes

  8. Regional pulmonary perfusion following human heart-lung transplantation

    SciTech Connect

    Lisbona, R.; Hakim, T.S.; Dean, G.W.; Langleben, D.; Guerraty, A.; Levy, R.D. )

    1989-08-01

    Ventilation and perfusion scans were obtained in six subjects who had undergone heart-lung transplantation with consequent denervation of the cardiopulmonary axis. Two of the subjects had developed obliterative bronchiolitis, which is believed to be a form of chronic rejection. Their pulmonary function tests demonstrated airflow obstruction and their scintigraphic studies were abnormal. In the remaining four subjects without obstructive airways disease, ventilation and planar perfusion scans were normal. Single photon emission computed tomography imaging of pulmonary perfusion in these patients revealed a layered distribution of blood flow indistinguishable from that of normal individuals. It is concluded that neurogenic mechanisms have little influence on the pattern of local pulmonary blood flow at rest.

  9. 2058 Expressed sequence tags (ESTs) from a human fetal lung cDNA library

    SciTech Connect

    Kazunori, Sudo |; Katsuya Chinen; Yusuke Nakamura

    1994-11-15

    ESTs (expressed sequence tags) provide complementary resources for structural and functional analyses of the human genome. The authors have performed single-pass sequencing of 2058 randomly selected, directionally cloned cDNAs isolated from a fetal-lung cDNA library constructed with oligo (dT) primers. Computer analyses of the 5{prime}-end sequences revealed that 60.4% of the clones were considered to be identical to previously reported human genes or ESTs; 9.0% of them showed significant homology to known genes in human, other mammals, or lower organisms; 30.6% showed no homology to any genes or DNA sequences in the public database. These data and reagents will be useful for future investigations of gene expression during prenatal development of human lung. 11 refs., 1 fig., 2 tabs.

  10. CYLD Promotes TNF-α-Induced Cell Necrosis Mediated by RIP-1 in Human Lung Cancer Cells

    PubMed Central

    Lin, Xing; Chen, Qianshun; Huang, Chen

    2016-01-01

    Lung cancer is one of the most common cancers in the world. Cylindromatosis (CYLD) is a deubiquitination enzyme and contributes to the degradation of ubiquitin chains on RIP1. The aim of the present study is to investigate the levels of CYLD in lung cancer patients and explore the molecular mechanism of CYLD in the lung cancer pathogenesis. The levels of CYLD were detected in human lung cancer tissues and the paired paracarcinoma tissues by real-time PCR and western blotting analysis. The proliferation of human lung cancer cells was determined by MTT assay. Cell apoptosis and necrosis were determined by FACS assay. The results demonstrated that low levels of CYLD were detected in clinical lung carcinoma specimens. Three pairs of siRNA were used to knock down the endogenous CYLD in lung cancer cells. Knockdown of CYLD promoted cell proliferation of lung cancer cells. Otherwise overexpression of CYLD induced TNF-α-induced cell death in A549 cells and H460 cells. Moreover, CYLD-overexpressed lung cancer cells were treated with 10 μM of z-VAD-fmk for 12 hours and the result revealed that TNF-α-induced cell necrosis was significantly enhanced. Additionally, TNF-α-induced cell necrosis in CYLD-overexpressed H460 cells was mediated by receptor-interacting protein 1 (RIP-1) kinase. Our findings suggested that CYLD was a potential target for the therapy of human lung cancers. PMID:27738385

  11. SEGEL: A Web Server for Visualization of Smoking Effects on Human Lung Gene Expression.

    PubMed

    Xu, Yan; Hu, Brian; Alnajm, Sammy S; Lu, Yin; Huang, Yangxin; Allen-Gipson, Diane; Cheng, Feng

    2015-01-01

    Cigarette smoking is a major cause of death worldwide resulting in over six million deaths per year. Cigarette smoke contains complex mixtures of chemicals that are harmful to nearly all organs of the human body, especially the lungs. Cigarette smoking is considered the major risk factor for many lung diseases, particularly chronic obstructive pulmonary diseases (COPD) and lung cancer. However, the underlying molecular mechanisms of smoking-induced lung injury associated with these lung diseases still remain largely unknown. Expression microarray techniques have been widely applied to detect the effects of smoking on gene expression in different human cells in the lungs. These projects have provided a lot of useful information for researchers to understand the potential molecular mechanism(s) of smoke-induced pathogenesis. However, a user-friendly web server that would allow scientists to fast query these data sets and compare the smoking effects on gene expression across different cells had not yet been established. For that reason, we have integrated eight public expression microarray data sets from trachea epithelial cells, large airway epithelial cells, small airway epithelial cells, and alveolar macrophage into an online web server called SEGEL (Smoking Effects on Gene Expression of Lung). Users can query gene expression patterns across these cells from smokers and nonsmokers by gene symbols, and find the effects of smoking on the gene expression of lungs from this web server. Sex difference in response to smoking is also shown. The relationship between the gene expression and cigarette smoking consumption were calculated and are shown in the server. The current version of SEGEL web server contains 42,400 annotated gene probe sets represented on the Affymetrix Human Genome U133 Plus 2.0 platform. SEGEL will be an invaluable resource for researchers interested in the effects of smoking on gene expression in the lungs. The server also provides useful information

  12. Lung dosimetry and risk assessment of nanoparticles: Evaluating and extending current models in rats and humans

    SciTech Connect

    Kuempel, E.D.; Tran, C.L.; Castranova, V.; Bailer, A.J.

    2006-09-15

    Risk assessment of occupational exposure to nanomaterials is needed. Human data are limited, but quantitative data are available from rodent studies. To use these data in risk assessment, a scientifically reasonable approach for extrapolating the rodent data to humans is required. One approach is allometric adjustment for species differences in the relationship between airborne exposure and internal dose. Another approach is lung dosimetry modeling, which provides a biologically-based, mechanistic method to extrapolate doses from animals to humans. However, current mass-based lung dosimetry models may not fully account for differences in the clearance and translocation of nanoparticles. In this article, key steps in quantitative risk assessment are illustrated, using dose-response data in rats chronically exposed to either fine or ultrafine titanium dioxide (TiO{sub 2}), carbon black (CB), or diesel exhaust particulate (DEP). The rat-based estimates of the working lifetime airborne concentrations associated with 0.1% excess risk of lung cancer are approximately 0.07 to 0.3 mg/m{sup 3} for ultrafine TiO{sub 2}, CB, or DEP, and 0.7 to 1.3 mg/m{sup 3} for fine TiO{sub 2}. Comparison of observed versus model-predicted lung burdens in rats shows that the dosimetry models predict reasonably well the retained mass lung burdens of fine or ultrafine poorly soluble particles in rats exposed by chronic inhalation. Additional model validation is needed for nanoparticles of varying characteristics, as well as extension of these models to include particle translocation to organs beyond the lungs. Such analyses would provide improved prediction of nanoparticle dose for risk assessment.

  13. Effects of sodium cromoglycate and nedocromil sodium on histamine secretion from human lung mast cells.

    PubMed Central

    Leung, K B; Flint, K C; Brostoff, J; Hudspith, B N; Johnson, N M; Lau, H Y; Liu, W L; Pearce, F L

    1988-01-01

    Sodium cromoglycate and nedocromil sodium produced a dose dependent inhibition of histamine secretion from human pulmonary mast cells obtained by bronchoalveolar lavage and by enzymatic dissociation of lung parenchyma. Both compounds were significantly more active against the lavage cells than against the dispersed lung cells, and nedocromil sodium was an order of magnitude more effective than sodium cromoglycate against both cell types. Tachyphylaxis was observed with the parenchymal cells but not with the lavage cells. Nedocromil sodium and sodium cromoglycate also inhibited histamine release from the lavage cells of patients with sarcoidosis and extrinsic asthma. PMID:2462755

  14. Expression of Rab1A is upregulated in human lung cancer and associated with tumor size and T stage

    PubMed Central

    Qin, Xiaoyu; Huang, Tinglei; Huang, Bo; Zhang, Yanjie; Jiang, Bin

    2016-01-01

    Rab1A expression is associated with malignant phenotypes in several human tumors; however, the role of Rab1A in lung cancer is still unclear. In this study, we attempted to establish the role of Rab1A in major human lung cancer subtypes. Rab1A expression in different histological types of human lung cancer was analyzed in lung cancer tissues with paired adjacent noncancerous tissues and a large panel of lung cancer cell lines. The effect of Rab1A expression on multiple cancer-associated signaling pathways was also examined. The results demonstrated that Rab1A was significantly overexpressed in the different histological types of lung cancer as compared to non-cancerous tissues, and Rab1A expression was correlated with tumor volume and stage. In a large panel of lung cancer cell lines, high Rab1A expression was observed as compared to a normal lung/bronchus epithelial cell line. However, Rab1A protein levels were not correlated with mTORC1 (P-S6K1), mTORC2 (P-AKT), MEK (P-ERK), JNK (P-c-Jun) or p38MAPK (P-MK2) signaling. Rab1A knockdown had no effect on mTOR signaling or cell growth. These data suggested that Rab1A may be involved in the pathogenesis of human lung cancer in an mTOR- and MAPK-independent manner. PMID:27902464

  15. Characterization of cDNAs, mRNAs, and proteins related to human liver microsomal cytochrome P-450 (S)-mephenytoin 4'-hydroxylase.

    PubMed

    Ged, C; Umbenhauer, D R; Bellew, T M; Bork, R W; Srivastava, P K; Shinriki, N; Lloyd, R S; Guengerich, F P

    1988-09-06

    A cytochrome P-450 (P-450) multigene family codes for several related human liver enzymes, including the P-450 responsible for (S)-mephenytoin 4'-hydroxylation. This enzyme activity has previously been shown to be associated with a genetic polymorphism. Genomic (Southern) blot analysis using non-overlapping 5' and 3' portions of a cDNA clone suggests that approximately seven related sequences are present in this gene family. In this study four cDNA clones, all nearly full-length, were isolated from a bacteriophage lambda gt11 library prepared from a single human liver. These clones can be grouped into two categories that are approximately 85% identical at the level of DNA sequence. The cDNA clones in one category (MP-4, MP-8) both match the N-terminal sequences of the P-450MP-1 and P-450MP-2 proteins, which had previously been shown to be catalytically active in (S)-mephenytoin 4'-hydroxylation. These two cDNAs, MP-4 and MP-8, differ in only two bases in the coding region but are quite distinct in their 3' noncoding regions. Another protein (P-450MP-3) was isolated on the basis of its immunochemical similarity to P-450MP-1 but was found to be catalytically inactive; amino acid sequencing of tryptic peptides of P-450MP-3 showed a correspondence to the second category of cDNA clones (MP-12, MP-20), which differ from each other in only four (nonsilent) base changes. Oligonucleotides specific for the two groups of cDNA clones were used as probes of human liver mRNAs--individual liver samples examined expressed both types of mRNAs but no correlation was observed between the abundance levels of any mRNA and catalytic activity. Further, oligonucleotide probes indicated that mRNAs corresponding to both the MP-4 and MP-8 clones were apparently present in individual liver samples. A monoclonal antibody was isolated that recognized P-450MP-1 but not P-450MP-2 or P-450MP-3; the amount of protein detected by the antibody in different liver samples was not correlated with the

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

    PubMed

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

    2013-08-19

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

  17. Genotoxicity-Related Chemistry of Human Metabolites of Benzo[ghi]perylene (B[ghi]P) Investigated using Electro-optical Arrays and DNA/Microsome Biocolloid Reactors with LC-MS/MS

    PubMed Central

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

    2013-01-01

    There is limited and sometimes contradictory information about the genotoxicity of polycyclic aromatic hydrocarbon benzo[ghi]perylene (B[ghi]P). Using recently developed metabolic toxicity screening arrays and a biocolloid reactor-LC-MS/MS approach, both featuring films of DNA and human metabolic enzymes, we demonstrated relatively low reactivity of metabolically activated B[ghi]P towards DNA. Electro-optical toxicity screening arrays showed that B[ghi]P metabolites damage DNA at a 3-fold lower rate than benzo[a]pyrene (B[a]P), whose metabolites have a strong and well-understood propensity for DNA damage. Metabolic studies using magnetic bead biocolloid reactors coated with microsomal enzymes in 96-well plates showed that cyt P450s 1A1 and 1B1 provide high activity for B[ghi]P and B[a]P conversion. Consistent with published results, the major metabolism of B[ghi]P involved oxidations at 3,4 and 11,12 positions, leading to formation of B[ghi]P 3,4-oxide and B[ghi]P 3,4,11,12-bisoxide. B[ghi]P 3,4-oxide was synthesized and reacted with deoxyadenosine at N6 and N7 positions and with deoxyguanosine at the N2 position. B[ghi]P 3,4-oxide is hydrolytically unstable and transforms into the 3,4-diol or converts to 3- or 4-hydroxy B[ghi]P. LC-MS/MS of reaction products from the magnetic biocolloid reactor particles coated with DNA and human enzymes revealed for the first time that a major DNA adduct results from reaction between B[ghi]P 3,4,11,12-bisoxide and deoxyguanosine. Results also demonstrated 5-fold lower formation rates of the major DNA adduct for B[ghi]P metabolites compared to B[a]P. Overall, results from both ECL array and biocolloid reactor-LC-MS/MS consistently suggest a lower human genotoxicity profile of B[ghi]P than B[a]P. PMID:23879290

  18. Laminin 5 expression protects against anoikis at aerogenous spread and lepidic growth of human lung adenocarcinoma.

    PubMed

    Kodama, Keiji; Ishii, Gen'ichiro; Miyamoto, Shin'ichi; Goya, Masato; Zhang, Shi-Chuan; Sangai, Takafumi; Yoshikawa, Takeshi; Hasebe, Takahiro; Hitomi, Yoshiaki; Izumi, Keisuke; Ochiai, Atsushi

    2005-10-10

    Adenocarcinoma of the lung is characterized by frequent aerogenous spread (AE) and advancement along the alveolar wall (BAC growth). To elucidate the mechanism of AE metastasis and BAC growth in human lung adenocarcinoma, we established an in vivo orthotopic animal model and an in vitro culture. Investigation of expression levels of integrins, laminins and Type IV collagens, which are the major regulating molecules for cell attachment and anoikis was carried out and a clear correlation between the expression level of laminin 5 (LN5) and the BAC growth was observed using an orthotopic animal model. Introduction of LN5 cDNA to A549 cells increased anoikis resistance in an expression dependent manner. Cells with LN5 overexpression resisted with anoikis after treatment with PI3K-Akt and ERK inhibitors. The amount of phosphorylated focal adhesion kinase (FAK) was also higher in LN5 overexpressing cells. Major tyrosine residues of the EGF receptor at 1068, 1086 and 1173, except at 1148, remained phosphorylated only in the LN5 overexpressing cells even without EGF stimulation, that indicates the ligand independent activation of EGF receptor. BAC growth ratio and AE was confirmed to be significantly correlated with LN5 expression in surgically resected human lung adenocarcinomas by immunohistochemistry. Our results indicate that the activation of the EGF receptor by overexpressing LN5-integrin-FAK signaling pathway may play a crucial role in BAC growth and AE metastasis in human lung adenocarcinoma.

  19. Antimicrobial proteins and peptides in human lung diseases: A friend and foe partnership with host proteases.

    PubMed

    Lecaille, Fabien; Lalmanach, Gilles; Andrault, Pierre-Marie

    2016-03-01

    Lung antimicrobial proteins and peptides (AMPs) are major sentinels of innate immunity by preventing microbial colonization and infection. Nevertheless bactericidal activity of AMPs against Gram-positive and Gram-negative bacteria is compromised in patients with chronic obstructive pulmonary disease (COPD), cystic fibrosis (CF) and asthma. Evidence is accumulating that expression of harmful human serine proteases, matrix metalloproteases and cysteine cathepsins is markedely increased in these chronic lung diseases. The local imbalance between proteases and protease inhibitors compromises lung tissue integrity and function, by not only degrading extracellular matrix components, but also non-matrix proteins. Despite the fact that AMPs are somewhat resistant to proteolytic degradation, some human proteases cleave them efficiently and impair their antimicrobial potency. By contrast, certain AMPs may be effective as antiproteases. Host proteases participate in concert with bacterial proteases in the degradation of key innate immunity peptides/proteins and thus may play immunomodulatory activities during chronic lung diseases. In this context, the present review highlights the current knowledge and recent discoveries on the ability of host enzymes to interact with AMPs, providing a better understanding of the role of human proteases in innate host defense.

  20. Teroxirone inhibited growth of human non-small cell lung cancer cells by activating p53

    SciTech Connect

    Wang, Jing-Ping; Lin, Kai-Han; Liu, Chun-Yen; Yu, Ya-Chu; Wu, Pei-Tsun; Chiu, Chien-Chih; Su, Chun-Li; Chen, Kwun-Min; Fang, Kang

    2013-11-15

    In this work, we demonstrated that the growth of human non-small-cell-lung-cancer cells H460 and A549 cells can be inhibited by low concentrations of an epoxide derivative, teroxirone, in both in vitro and in vivo models. The cytotoxicity was mediated by apoptotic cell death through DNA damage. The onset of ultimate apoptosis is dependent on the status of p53. Teroxirone caused transient elevation of p53 that activates downstream p21 and procaspase-3 cleavage. The presence of caspase-3 inhibitor reverted apoptotic phenotype. Furthermore, we showed the cytotoxicity of teroxirone in H1299 cells with stable ectopic expression of p53, but not those of mutant p53. A siRNA-mediated knockdown of p53 expression attenuated drug sensitivity. The in vivo experiments demonstrated that teroxirone suppressed growth of xenograft tumors in nude mice. Being a potential therapeutic agent by restraining cell growth through apoptotic death at low concentrations, teroxirone provides a feasible perspective in reversing tumorigenic phenotype of human lung cancer cells. - Highlights: • Teroxirone repressed tumor cell growth in nude mice of human lung cancer cells. • The apoptotic cell death reverted by caspase-3 inhibitor is related to p53 status. • Teroxirone provides a good candidate for lung cancer treatment.

  1. Polymorphism of selected enzymes involved in detoxification and biotransformation in relation to lung cancer.

    PubMed

    Gresner, Peter; Gromadzinska, Jolanta; Wasowicz, Wojciech

    2007-07-01

    Available data indicate that there are significant differences in individual susceptibility to lung cancer within the human population. It is believed to be underlie by inherited genetic predispositions related to the genetic polymorphism of several enzymes involved in the detoxification and xenobiotic metabolism. In this review, we collect and discuss the evidence reported up to date on the association between lung cancer and genetic polymorphism of cytochromes P450, N-acetyltransferase, glutathione S-transferases, microsomal epoxide hydrolase, NAD(P)H:quinone oxidoreductase, myeloperoxidase and glutathione peroxidase. All these genes might appear to be candidates for lung cancer susceptibility genes, nevertheless, the present state of the art still offers only a limited explanation of the link between such polymorphisms and increased risk of lung cancer.

  2. YBX1 regulates tumor growth via CDC25a pathway in human lung adenocarcinoma

    PubMed Central

    Yu, Wendan; Li, Jinxiu; Tang, Zhipeng; Yu, Zhenlong; Zhao, Lei; Zhang, Yixiang; Wang, Ziyi; Wang, Peng; Li, Yechi; Li, Fengzhou; Sun, Zhe; Xuan, Yang; Tang, Ranran; Deng, Wu-guo; Guo, Wei; Gu, Chundong

    2016-01-01

    Y-box binding protein 1 (YBX1) is involved in the multi-tumor occurrence and development. However, the regulation of YBX1 in lung tumorigenesis and the underlying mechanisms, especially its relationship with CDC25a, was remains unclear. In this study, we analyzed the expression and clinical significance of YBX1 and CDC25a in lung adenocarcinoma and identified their roles in the regulation of lung cancer growth. The retrospective analysis of 116 patients with lung adenocarcinoma indicated that YBX1 was positively correlated with CDC25a expression. The Cox-regression analysis showed only high-ranking TNM stage and low CDC25a expression were an independent risk factor of prognosis in enrolled patients. High expression of YBX1 or CDC25a protein was also observed in lung adenocarcinoma cells compared with HLF cells. ChIP assay demonstrated the binding of endogenous YBX1 to the CDC25a promoter region. Overexpression of exogenous YBX1 up-regulated the expression of the CDC25a promoter-driven luciferase. By contrast, inhibition of YBX1 by siRNA markedly decreased the capability of YBX1 binding to CDC25a promoter in A549 and H322 cells. Inhibition of YBX1 expression also blocked cell cycle progression, suppressed cell proliferation and induced apoptosis via the CDC25a pathway in vitro. Moreover, inhibition of YBX1 by siRNA suppressed tumorigenesis in a xenograft mouse model and down-regulated the expression of YBX1, CDC25a, Ki67 and cleaved caspase 3 in the tumor tissues of mice. Collectively, these results demonstrate inhibition of YBX1 suppressed lung cancer growth partly via the CDC25a pathway and high expression of YBX1/CDC25a predicts poor prognosis in human lung adenocarcinoma. PMID:27384875

  3. PKCδ/midkine pathway drives hypoxia-induced proliferation and differentiation of human lung epithelial cells.

    PubMed

    Zhang, Hanying; Okamoto, Miyako; Panzhinskiy, Evgeniy; Zawada, W Michael; Das, Mita

    2014-04-01

    Epithelial cells are key players in the pathobiology of numerous hypoxia-induced lung diseases. The mechanisms mediating such hypoxic responses of epithelial cells are not well characterized. Earlier studies reported that hypoxia stimulates protein kinase C (PKC)δ activation in renal cancer cells and an increase in expression of a heparin-binding growth factor, midkine (MK), in lung alveolar epithelial cells. We reasoned that hypoxia might regulate MK levels via a PKCδ-dependent pathway and hypothesized that PKCδ-driven MK expression is required for hypoxia-induced lung epithelial cell proliferation and differentiation. Replication of human lung epithelial cells (A549) was significantly increased by chronic hypoxia (1% O2) and was dependent on expression of PKCδ. Hypoxia-induced proliferation of epithelial cells was accompanied by translocation of PKCδ from Golgi into the nuclei. Marked attenuation in MK protein levels by rottlerin, a pharmacological antagonist of PKC, and by small interfering RNA-targeting PKCδ, revealed that PKCδ is required for MK expression in both normoxic and hypoxic lung epithelial cells. Sequestering MK secreted into the culture media with a neutralizing antibody reduced hypoxia-induced proliferation demonstrating that an increase in MK release from cells is linked with epithelial cell division under hypoxia. In addition, recombinant MK accelerated transition of hypoxic epithelial cells to cells of mesenchymal phenotype characterized by elongated morphology and increased expression of mesenchymal markers, α-smooth muscle actin, and vimentin. We conclude that PKCδ/MK axis mediates hypoxic proliferation and differentiation of lung epithelial cells. Manipulation of PKCδ and MK activity in epithelial cells might be beneficial for the treatment of hypoxia-mediated lung diseases.

  4. PKCδ/midkine pathway drives hypoxia-induced proliferation and differentiation of human lung epithelial cells

    PubMed Central

    Zhang, Hanying; Okamoto, Miyako; Panzhinskiy, Evgeniy; Zawada, W. Michael

    2014-01-01

    Epithelial cells are key players in the pathobiology of numerous hypoxia-induced lung diseases. The mechanisms mediating such hypoxic responses of epithelial cells are not well characterized. Earlier studies reported that hypoxia stimulates protein kinase C (PKC)δ activation in renal cancer cells and an increase in expression of a heparin-binding growth factor, midkine (MK), in lung alveolar epithelial cells. We reasoned that hypoxia might regulate MK levels via a PKCδ-dependent pathway and hypothesized that PKCδ-driven MK expression is required for hypoxia-induced lung epithelial cell proliferation and differentiation. Replication of human lung epithelial cells (A549) was significantly increased by chronic hypoxia (1% O2) and was dependent on expression of PKCδ. Hypoxia-induced proliferation of epithelial cells was accompanied by translocation of PKCδ from Golgi into the nuclei. Marked attenuation in MK protein levels by rottlerin, a pharmacological antagonist of PKC, and by small interfering RNA-targeting PKCδ, revealed that PKCδ is required for MK expression in both normoxic and hypoxic lung epithelial cells. Sequestering MK secreted into the culture media with a neutralizing antibody reduced hypoxia-induced proliferation demonstrating that an increase in MK release from cells is linked with epithelial cell division under hypoxia. In addition, recombinant MK accelerated transition of hypoxic epithelial cells to cells of mesenchymal phenotype characterized by elongated morphology and increased expression of mesenchymal markers, α-smooth muscle actin, and vimentin. We conclude that PKCδ/MK axis mediates hypoxic proliferation and differentiation of lung epithelial cells. Manipulation of PKCδ and MK activity in epithelial cells might be beneficial for the treatment of hypoxia-mediated lung diseases. PMID:24500281

  5. Biopersistence of man-made vitreous silicate fibers in the human lung.

    PubMed Central

    Sébastien, P

    1994-01-01

    There is now a substantial body of experimental data on the pulmonary biopersistence of man-made vitreous silicate fibers (MMVSF), but human data are seriously lacking. Our knowledge in this field is essentially limited to a few reports of measurements of fibers retained in lung tissue samples taken at autopsy from workers manufacturing these products. Three types of exposure were studied: fibrous glass, mineral wool, and refractory ceramic fibers. Overall, the available data do not provide evidence for substantial long-term retention of fibers in the human lung after occupational exposure to MMVSF dusts. A word of caution, however; the amount of data supporting the previous statement is much greater for fibrous glass than for either mineral wool or refractory ceramic fibers. There is no human data on the key question of the kinetics of pulmonary clearance of inhaled MMVSF. PMID:7882938

  6. Effect of transforming growth factor beta on synthesis of glycosaminoglycans by human lung fibroblasts

    SciTech Connect

    Dubaybo, B.A.; Thet, L.A. )

    1990-09-01

    The processes of lung growth, injury, and repair are characterized by alterations in fibroblast synthesis and interstitial distribution of extracellular matrix components. Transforming growth factor beta (TGF-beta), which is postulated to play a role in modulating lung repair, alters the distribution of several matrix components such as collagen and fibronectin. We studied the effect of TGF-beta on the synthesis and distribution of the various glycosaminoglycans (GAGs) and whether these effects may explain its role in lung repair. Human diploid lung fibroblasts (IMR-90) were exposed to various concentrations of TGF-beta (0-5 nM) for variable periods of time (0-18 h). Newly synthesized GAGs were labeled with either (3H)glucosamine or (35S)sulfate. Individual GAGs were separated by size exclusion chromatography after serial enzymatic and chemical digestions and quantitated using scintillation counting. There was a dose-dependent increase in total GAG synthesis with maximal levels detected after 6 h of exposure. This increase was noted in all individual GAG types measured and was observed in both the cell associated GAGs (cell-matrix fraction) as well as the GAGs released into the medium (medium fraction). In the cell-matrix fraction, TGF-beta increased the proportion of heparan sulfate that was membrane bound as well as the proportion of dermatan sulfate in the intracellular compartment. In the medium fraction, TGF-beta increased the proportion of hyaluronic acid, chondroitin sulfate and dermatan sulfate released. We conclude that the role of TGF-beta in lung growth and repair may be related to increased synthesis of GAGs by human lung fibroblasts as well as alterations in the distribution of individual GAGs.

  7. 4-Methoxyestradiol-induced oxidative injuries in human lung epithelial cells

    SciTech Connect

    Cheng Yahsin; Chang, Louis W.; Cheng Lichuan; Tsai, M.-H.; Lin Pinpin . E-mail: pplin@nhri.org.tw

    2007-05-01

    Epidemiological studies indicated that people exposed to dioxins were prone to the development of lung diseases including lung cancer. Animal studies demonstrated that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) increased liver tumors and promoted lung metaplasia in females. Metabolic changes in 17{beta}-estradiol (E{sub 2}) resulted from an interaction between TCDD and E{sub 2} could be associated with gender difference. Previously, we reported that methoxylestradiols (MeOE{sub 2}), especially 4-MeOE{sub 2}, accumulated in human lung cells (BEAS-2B) co-treated with TCDD and E{sub 2}. In the present study, we demonstrate unique accumulation of 4-MeOE{sub 2}, as a result of TCDD/E{sub 2} interaction and revealed its bioactivity in human lung epithelial cell line (H1355). 4-Methoxyestradiol treatment significantly decreased cell growth and increased mitotic index. Elevation of ROS and SOD activity, with a concomitant decrease in the intracellular GSH/GSSG ratio, was also detected in 4-MeOE{sub 2}-treated cells. Quantitative comet assay showed increased oxidative DNA damage in the 4-MeOE{sub 2}-treated H1355 cells, which could be significantly reduced by the anti-oxidant N-acetylcysteine (NAC). However, inhibition of cell growth and increase in mitotic arrest induced by 4-MeOE{sub 2} were unaffected by NAC. We concluded that 4-MeOE{sub 2} accumulation resulting from TCDD and E{sub 2} interaction would contribute to the higher vulnerability on lung pathogenesis in females when exposed to TCDD.

  8. 4DCT-based assessment of regional airflow distribution in healthy human lungs during tidal breathing

    NASA Astrophysics Data System (ADS)

    Choi, Jiwoong; Jahani, Nariman; Choi, Sanghun; Hoffman, Eric; Lin, Ching-Long

    2014-11-01

    Nonlinear dynamics of regional airflow distribution in healthy human lungs are studied with four-dimensional computed tomography (4DCT) quantitative imaging of four subjects. During the scanning session, subjects continuously breathed with tidal volumes controlled by the dual piston system. For each subject, 10 instantaneous volumetric image data sets (5 inspiratory and 5 expiratory phases) were reconstructed. A mass-preserving image registration was then applied to pairs of these image data to construct a breathing lung model. Regional distributions of local flow rate fractions are computed from time-varying local air volumes. The 4DCT registration-based method provides the link between local and global air volumes of the lung, allowing derivation of time-varying regional flow rates during the tidal breathing for computational fluid dynamics analysis. The local flow rate fraction remains greater in the lower lobes than in the upper lobes, being qualitatively consistent with those derived from three static CT (3SCT) images (Yin et al. JCP 2013). However, unlike 3SCT, the 4DCT data exhibit lung hysteresis between inspiration and expiration, providing more sensitive measures of regional ventilation and lung mechanics. NIH Grants U01-HL114494, R01-HL094315 and S10-RR022421.

  9. Subamolide a induces mitotic catastrophe accompanied by apoptosis in human lung cancer cells.

    PubMed

    Hung, Jen-Yu; Wen, Ching-Wen; Hsu, Ya-Ling; Lin, En-Shyh; Huang, Ming-Shyan; Chen, Chung-Yi; Kuo, Po-Lin

    2013-01-01

    This study investigated the anticancer effects of subamolide A (Sub-A), isolated from Cinnamomum subavenium, on human nonsmall cell lung cancer cell lines A549 and NCI-H460. Treatment of cancer cells with Sub-A resulted in decreased cell viability of both lung cancer cell lines. Sub-A induced lung cancer cell death by triggering mitotic catastrophe with apoptosis. It triggered oxidant stress, indicated by increased cellular reactive oxygen species (ROS) production and decreased glutathione level. The elevated ROS triggered the activation of ataxia-telangiectasia mutation (ATM), which further enhanced the ATF3 upregulation and subsequently enhanced p53 function by phosphorylation at Serine 15 and Serine 392. The antioxidant, EUK8, significantly decreased mitotic catastrophe by inhibiting ATM activation, ATF3 expression, and p53 phosphorylation. The reduction of ATM and ATF3 expression by shRNA decreased Sub-A-mediated p53 phosphorylation and mitotic catastrophe. Sub-A also caused a dramatic 70% reduction in tumor size in an animal model. Taken together, cell death of lung cancer cells in response to Sub-A is dependent on ROS generation, which triggers mitotic catastrophe followed by apoptosis. Therefore, Sub-A may be a novel anticancer agent for the treatment of nonsmall cell lung cancer.

  10. Beryllium detection in human lung tissue using electron probe X-ray microanalysis.

    PubMed

    Butnor, Kelly J; Sporn, Thomas A; Ingram, Peter; Gunasegaram, Sue; Pinto, John F; Roggli, Victor L

    2003-11-01

    Chronic berylliosis is an uncommon disease that is caused by the inhalation of beryllium particles, dust, or fumes. The distinction between chronic berylliosis and sarcoidosis can be difficult both clinically and histologically, as both entities can have similar presentations and exhibit nonnecrotizing granulomatous inflammation of the lungs. The diagnosis of chronic berylliosis relies on a history of exposure to beryllium, roentgenographic evidence of diffuse nodular disease, and demonstration of beryllium hypersensitivity by ancillary studies, such as lymphocyte proliferation testing. Additional support may be gained by the demonstration of beryllium in lung tissue. Unlike other exogenous particulates, such as asbestos, detection of beryllium in human lung tissue is problematic. The low atomic number of beryllium usually makes it unsuitable for conventional microprobe analysis. We describe a case of chronic berylliosis in which beryllium was detected in lung tissue using atmospheric thin-window energy-dispersive X-ray analysis (ATW EDXA). A woman with a history of occupational exposure to beryllium at a nuclear weapons testing facility presented with progressive cough and dyspnea and a nodular pattern on chest roentgenograph. Open lung biopsy showed nonnecrotizing granulomatous inflammation that was histologically indistinguishable from sarcoidosis. Scanning electron microscopy and ATW EDXA demonstrated particulates containing beryllium within the granulomas. This application of EDXA offers significant advantages over existing methods of beryllium detection in that it is nondestructive, more widely available, and can be performed using routine paraffin sections.

  11. Novel intrapulmonary model for orthotopic propagation of human lung cancers in athymic nude mice.

    PubMed

    McLemore, T L; Liu, M C; Blacker, P C; Gregg, M; Alley, M C; Abbott, B J; Shoemaker, R H; Bohlman, M E; Litterst, C C; Hubbard, W C

    1987-10-01

    A major impediment to the study of human lung cancer pathophysiology, as well as to the discovery and development of new specific antitumor agents for the treatment of lung cancer, has been the lack of appropriate experimental animal models. This paper describes a new model for the propagation of human lung tumor cells in the bronchioalveolar regions of the right lungs of athymic NCr-nu/nu mice via an intrabronchial (i.b.) implantation procedure. Over 1000 i.b. implantations have been performed to date, each requiring 3 to 5 min for completion and having a surgery-related mortality of approximately 5%. The model was used successfully for the orthotopic propagation of four established human lung cancer cell lines including: an adenosquamous cell carcinoma (NCI-H125); an adenocarcinoma (A549); a large cell undifferentiated carcinoma (NCI-H460), and a bronchioloalveolar cell carcinoma (NCI-H358). When each of the four cell lines was implanted i.b. using a 1.0 X 10(6) tumor cell inoculum, 100 +/- 0% (SD) tumor-related mortality was observed within 9 to 61 days. In contrast, when the conventional s.c. method for implantation was used at the same tumor cell inoculum, only minimal (2.5 +/- 5%) tumor-related mortality was observed within 140 days (P less than 0.001). Similarly, when a 1.0 X 10(5) or 1.0 X 10(4) cell inoculum was used, a dose-dependent, tumor-related mortality was observed when cells were implanted i.b. (56 +/- 24% or 25 +/- 17%) as compared with the s.c. method (5 +/- 5.7% or 0.0 +/- 0%) (P less than 0.02 and P less than 0.05, respectively). Most (greater than 90%) of the lung tumors propagated by i.b. implantation were localized to the right lung fields as documented by necropsy and/or high-resolution chest roentgenography techniques which were developed for these studies. The intrapulmonary model was also used for establishment and propagation of xenografts derived directly from enzymatically digested, fresh human lung tumor specimens obtained at the

  12. Development of ferret as a human lung cancer model by injecting4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (NNK)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Development of new animal lung cancer models that are relevant to human lung carcinogenesis is important for lung cancer research. Previously we have shown the induction of lung tumor in ferrets (Mustela putorius furo) exposed to both tobacco smoke and a tobacco carcinogen (4-(N-methyl-N-nitrosamino...

  13. Differential Transcriptomic Analysis of Spontaneous Lung Tumors in B6C3F1 Mice: Comparison to Human Non–Small Cell Lung Cancer

    PubMed Central

    Pandiri, Arun R.; Sills, Robert C.; Ziglioli, Vincent; Ton, Thai-Vu T.; Hong, Hue–Hua L.; Lahousse, Stephanie A.; Gerrish, Kevin E.; Auerbach, Scott S.; Shockley, Keith R.; Bushel, Pierre R.; Peddada, Shyamal D.; Hoenerhoff, Mark J.

    2016-01-01

    Lung cancer is the leading cause of cancer-related death in people and is mainly due to environmental factors such as smoking and radon. The National Toxicology Program (NTP) tests various chemicals and mixtures for their carcinogenic hazard potential. In the NTP chronic bioassay using B6C3F1 mice, the incidence of lung tumors in treated and control animals is second only to the liver tumors. In order to study the molecular mechanisms of chemically induced lung tumors, an understanding of the genetic changes that occur in spontaneous lung (SL) tumors from untreated control animals is needed. The authors have evaluated the differential transcriptomic changes within SL tumors compared to normal lungs from untreated age-matched animals. Within SL tumors, several canonical pathways associated with cancer (eukaryotic initiation factor 2 signaling, RhoA signaling, PTEN signaling, and mammalian target of rapamycin signaling), metabolism (Inositol phosphate metabolism, mitochondrial dysfunction, and purine and pyramidine metabolism), and immune responses (FcγR-mediated phagocytosis, clathrin-mediated endocytosis, interleukin 8 signaling, and CXCR4 signaling) were altered. Meta-analysis of murine SL tumors and human non–small cell lung cancer transcriptomic data sets revealed a high concordance. These data provide important information on the differential transcriptomic changes in murine SL tumors that will be critical to our understanding of chemically induced lung tumors and will aid in hazard analysis in the NTP 2-year carcinogenicity bioassays. PMID:22688403

  14. The histone demethylase PHF8 is an oncogenic protein in human non-small cell lung cancer

    SciTech Connect

    Shen, Yuzhou; Pan, Xufeng; Zhao, Heng

    2014-08-15

    Highlights: • PHF8 overexpresses in human NSCLC and predicts poor survival. • PHF8 regulates lung cancer cell growth and transformation. • PHF8 regulates apoptosis in human lung cancer cells. • PHF8 promotes miR-21 expression in human lung cancer. • MiR-21 is critically essential for PHF8 function in human lung cancer cells. - Abstract: PHF8 is a JmjC domain-containing protein and erases repressive histone marks including H4K20me1 and H3K9me1/2. It binds to H3K4me3, an active histone mark usually located at transcription start sites (TSSs), through its plant homeo-domain, and is thus recruited and enriched in gene promoters. PHF8 is involved in the development of several types of cancer, including leukemia, prostate cancer, and esophageal squamous cell carcinoma. Herein we report that PHF8 is an oncogenic protein in human non-small cell lung cancer (NSCLC). PHF8 is up-regulated in human NSCLC tissues, and high PHF8 expression predicts poor survival. Our in vitro and in vivo evidence demonstrate that PHF8 regulates lung cancer cell proliferation and cellular transformation. We found that PHF8 knockdown induces DNA damage and apoptosis in lung cancer cells. PHF8 promotes miR-21 expression in human lung cancer, and miR-21 knockdown blocks the effects of PHF8 on proliferation and apoptosis of lung cancer cells. In summary, PHF8 promotes lung cancer cell growth and survival by regulating miR-21.

  15. Cyclooxygenase-2 is associated with malignant phenotypes in human lung cancer

    PubMed Central

    Li, Weiying; Yue, Wentao; Wang, Hui; Lai, Baitang; Yang, Xuehui; Zhang, Chunyan; Wang, Yue; Gu, Meng

    2016-01-01

    The objective of the present study was to investigate whether cyclooxygenase-2 (COX-2) is associated with malignancy, and to investigate its molecular mechanisms in human lung cancer tumor malignancy. The present study used RNA interference (RNAi) methodology and celecoxib, a COX-2 inhibitor, to investigate the effect of COX-2 knockdown on the proliferation and invasion abilities of lung cancer cells and the molecular mechanisms involved. Human lung adenocarcinoma A549-si10 and LTEP-A2 cells transfected with a specific small interfering RNA (A549-si10 and LTEP-A2-si10, respectively) grew more slowly compared with parental cell lines and cells transfected with pU6. The colony formation of A549-si10 and LTEP-A2-si10 cells was also reduced. In addition, A549-si10 and LTEP-A2-si10 cells were characterized by decreased metastatic and invasive abilities. The proliferation and invasive potential of parental A549 and LTEP-A2 cells was inhibited following treatment with celecoxib. In vivo, a COX-2 knockdown resulted in a decrease of proliferation and reduction of vascular endothelial growth factor (VEGF), matrix metalloproteinase-2 (MMP-2) and endothelial growth factor receptor (EGFR) expression in A549 xenografts. In conclusion, the present study revealed that COX-2 plays a extremely important role in tumor growth, infiltration and metastasis via the regulation of VEGF, MMP-2 and EGRF expression. Therefore, COX-2 is a potential therapeutic target for lung cancer. PMID:27895738

  16. Sinomenine inhibits A549 human lung cancer cell invasion by mediating the STAT3 signaling pathway

    PubMed Central

    Jiang, Shulong; Gao, Yebo; Hou, Wei; Liu, Rui; Qi, Xin; Xu, Xia; Li, Jie; Bao, Yanju; Zheng, Honggang; Hua, Baojin

    2016-01-01

    Increasing evidence suggests that the failure of lung cancer treatment may occur as a result of tumor invasion and metastasis. Signal transducer and activator of transcription 3 (STAT3), an epithelial-mesenchymal transition-inducing transcription factor, is a key signaling molecule involved in the proliferation, apoptosis, invasion and metastasis of tumor cells. Sinomenine is an alkaloid compound with an antineoplastic potential against a variety of cancer cells. The aim of the present study was to assess the antitumor mechanisms of sinomenine in the A549 human lung cancer cell line. The results demonstrated that sinomenine manifested dose-dependent cytotoxicity and induced apoptosis in A549 cells. The protein expression of Janus kinase 2, STAT3, phosphorylated-STAT3, Snail, N-cadherin and vimentin decreased in sinomenine-treated cells, while E-cadherin protein expression increased. The regulation of STAT3, N-cadherin and E-cadherin by sinomenine was further confirmed by reverse transcription-quantitative polymerase chain reaction and immunofluorescent staining. It was demonstrated that sinomenine exerts inhibitory effects on A549 human lung cancer cell invasion, possibly through the inhibition of STAT3 signaling. These results provide a novel insight into the role of sinomenine in the treatment of non-small cell lung cancer. PMID:27446441

  17. Human airway organoid engineering as a step toward lung regeneration and disease modeling.

    PubMed

    Tan, Qi; Choi, Kyoung Moo; Sicard, Delphine; Tschumperlin, Daniel J

    2017-01-01

    Organoids represent both a potentially powerful tool for the study cell-cell interactions within tissue-like environments, and a platform for tissue regenerative approaches. The development of lung tissue-like organoids from human adult-derived cells has not previously been reported. Here we combined human adult primary bronchial epithelial cells, lung fibroblasts, and lung microvascular endothelial cells in supportive 3D culture conditions to generate airway organoids. We demonstrate that randomly-seeded mixed cell populations undergo rapid condensation and self-organization into discrete epithelial and endothelial structures that are mechanically robust and stable during long term culture. After condensation airway organoids generate invasive multicellular tubular structures that recapitulate limited aspects of branching morphogenesis, and require actomyosin-mediated force generation and YAP/TAZ activation. Despite the proximal source of primary epithelium used in the airway organoids, discrete areas of both proximal and distal epithelial markers were observed over time in culture, demonstrating remarkable epithelial plasticity within the context of organoid cultures. Airway organoids also exhibited complex multicellular responses to a prototypical fibrogenic stimulus (TGF-β1) in culture, and limited capacity to undergo continued maturation and engraftment after ectopic implantation under the murine kidney capsule. These results demonstrate that the airway organoid system developed here represents a novel tool for the study of disease-relevant cell-cell interactions, and establishes this platform as a first step toward cell-based therapy for chronic lung diseases based on de novo engineering of implantable airway tissues.

  18. Measurement of the depth-dependent resonance of water-loaded human lungs

    NASA Astrophysics Data System (ADS)

    Martin, J. S.; Rogers, P. H.; Cudahy, E. A.

    2005-04-01

    An experiment was conducted to determine the response of the human lung to water-borne sound in the range of 20 to 500 Hz. A small pool inside a hyperbaric chamber was used to simulate four ambient pressure conditions spanning the range of recreational diving depths. Ten subjects were tested on two occasions each using three separate measures to evaluate the response of the subjects' lungs. With some notable exceptions, results were consistent between subjects and between measures. These indicate that human lungs can be reasonably modeled as a lumped single-degree-of-freedom system over the lower portion of the band of interest. Here, the surrounding fluid provides the dominant mass and the dominant stiffness is provided by the entrapped air with a small additional contribution from tissue elasticity. Measured resonances increase with the square root of ambient pressure from an average of 40 Hz with a quality factor of 1.8 at near-surface pressure to 73 Hz with a quality factor of 2.6 at an equivalent depth of 36.4 m. There is evidence of other resonances within or near the band of interest that may be attributable to nonvolumetric chest/lung modes, Helmholtz resonance, and/or resonance of gastrointestinal bubbles. .

  19. Human glutathione S-transferases. Characterization of the anionic forms from lung and placenta.

    PubMed Central

    Dao, D D; Partridge, C A; Kurosky, A; Awasthi, Y C

    1984-01-01

    Anionic glutathione S-transferases were purified from human lung and placenta. Chemical and immunochemical characterization, including polyacrylamide-gel electrophoresis, gave strong evidence that the anionic lung and placental enzymes are chemically similar, if not identical, proteins. The electrophoretic mobilities of both proteins were identical in conventional alkaline gels as well as in gels containing sodium dodecyl sulphate. Gel filtration of the intact active enzyme established an Mr value of 45000; however, with sodium dodecyl sulphate/polyacrylamide-gel electrophoresis under dissociating conditions a subunit Mr of 22500 was obtained. Amino acid sequence analysis of the N-terminal region of the placental enzyme revealed a single polypeptide sequence identical with that of lung. Results obtained from immunoelectrophoresis, immunotitration, double immunodiffusion and rocket immunoelectrophoresis also indicated the anionic lung and placental enzymes to be closely similar. The chemical similarity of these two proteins was further supported by protein compositional analysis and fragment analysis after chemical hydrolysis. Immunochemical comparison of the anionic lung and placental enzymes with human liver glutathione S-transferases revealed cross-reactivity with the anionic omega enzyme, but no cross-reactivity was detectable with the cationic enzymes. Comparison of the N-terminal region of the human anionic enzyme with reported sequences of rat liver glutathione S-transferases gave strong evidence of chemical similarity, indicating that these enzymes are evolutionarily related. However, computer analysis of the 30-residue N-terminal sequence did not show any significant chemical similarity to any other reported protein sequence, pointing to the fact that the glutathione S-transferases represent a unique class of proteins. Images Fig. 2. Fig. 5. Fig. 6. Fig. 7. PMID:6466318

  20. Thioredoxin reductase 1 knockdown enhances selenazolidine cytotoxicity in human lung cancer cells via mitochondrial dysfunction

    PubMed Central

    Poerschke, Robyn L.; Moos, Philip J.

    2010-01-01

    Thioredoxin reductase (TR1) is a selenoprotein that is involved in cellular redox status control and deoxyribonucleotide biosynthesis. Many cancers, including lung, overexpress TR1, making it a potential cancer therapy target. Previous work has shown that TR1 knockdown enhances the sensitivity of cancer cells to anticancer treatments, as well as certain selenocompounds. However, it is unknown if TR1 knockdown produces similar effect on the sensitivity of human lung cancer cells. To further elucidate the role of TR1 in the mechanism of selenocompounds in lung cancer, a lentiviral microRNA delivery system to knockdown TR1 expression in A549 human lung adenocarcinoma cells was utilized. Cell viability was assessed after 48 hr treatment with the selenocysteine prodrug selenazolidines 2-butylselenazolidine-4(R)-carboxylic acid (BSCA) and 2-cyclohexylselenazolidine-4-(R)-carboxylic acid (ChSCA), selenocystine (SECY), methylseleninic acid (MSA), 1,4-phenylenebis(methylene)selenocyanate (p-XSC), and selenomethionine (SEM). TR1 knockdown increased the cytotoxicity of BSCA, ChSCA, and SECY but did not sensitize cells to MSA, SEM, or p-XSC. GSH and TR1 depletion together decreased cell viability, while no change was observed with GSH depletion alone. Reactive oxygen species generation was induced only in TR1 knockdown cells treated with the selenazolidines or SECY. These three compounds also decreased total intracellular glutathione levels and oxidized thioredoxin, but in a TR1 independent manner. TR1 knockdown increased selenazolidine and SECY-induced mitochondrial membrane depolarization, as well as DNA strand breaks and AIF translocation from the mitochondria. These results indicate the ability of TR1 to modulate the cytotoxic effects of BSCA, ChSCA and SECY in human lung cancer cells through mitochondrial dysfunction. PMID:20920480

  1. The isolation and culture of endothelial colony-forming cells from human and rat lungs.

    PubMed

    Alphonse, Rajesh S; Vadivel, Arul; Zhong, Shumei; Zong, Shumei; McConaghy, Suzanne; Ohls, Robin; Yoder, Mervin C; Thébaud, Bernard

    2015-11-01

    Blood vessels are crucial for the normal development, lifelong repair and homeostasis of tissues. Recently, vascular progenitor cell-driven 'postnatal vasculogenesis' has been suggested as an important mechanism that contributes to new blood vessel formation and organ repair. Among several described progenitor cell types that contribute to blood vessel formation, endothelial colony-forming cells (ECFCs) have received widespread attention as lineage-specific 'true' vascular progenitors. Here we describe a protocol for the isolation of pulmonary microvascular ECFCs from human and rat lung tissue. Our technique takes advantage of an earlier protocol for the isolation of circulating ECFCs from the mononuclear cellular fraction of peripheral blood. We adapted the earlier protocol to isolate resident ECFCs from the distal lung tissue. After enzymatic dispersion of rat or human lung samples into a cellular suspension, CD31-expressing cells are positively selected using magnetic-activated cell sorting and plated in endothelial-specific growth conditions. The colonies arising after 1-2 weeks in culture are carefully separated and expanded to yield pure ECFC cultures after a further 2-3 weeks. The resulting cells demonstrate the defining characteristics of ECFCs such as (i) 'cobblestone' morphology of cultured cell monolayers; (ii) acetylated low-density lipoprotein uptake and Ulex europaeus lectin binding; (iii) tube-like network formation in Matrigel; (iv) expression of endothelial cell-specific surface markers and the absence of hematopoietic or myeloid surface antigens; (v) self-renewal potential displayed by the most proliferative cells; and (vi) contribution to de novo vessel formation in an in vivo mouse implant model. Assuming typical initial cell adhesion and proliferation rates, the entire procedure can be completed within 4 weeks. Isolation and culture of lung vascular ECFCs will allow assessment of the functional state of these cells in experimental and human

  2. Transfusion of Human Platelets Treated with Mirasol Pathogen Reduction Technology Does Not Induce Acute Lung Injury in Mice.

    PubMed

    Caudrillier, Axelle; Mallavia, Beñat; Rouse, Lindsay; Marschner, Susanne; Looney, Mark R

    2015-01-01

    Pathogen reduction technology (PRT) has been developed in an effort to make the blood supply safer, but there is controversy as to whether it may induce structural or functional changes to platelets that could lead to acute lung injury after transfusion. In this study, we used a commercial PRT system to treat human platelets that were then transfused into immunodeficient mice, and the development of acute lung injury was determined. P-selectin expression was higher in the Mirasol PRT-treated platelets compared to control platelets on storage day 5, but not storage day 1. Transfusion of control vs. Mirasol PRT-treated platelets (day 5 of storage, 109 platelets per mouse) into NOD/SCID mice did not result in lung injury, however transfusion of storage day 5 platelets treated with thrombin receptor-activating peptide increased both extravascular lung water and lung vascular permeability. Transfusion of day 1 platelets did not produce lung injury in any group, and LPS priming 24 hours before transfusion had no effect on lung injury. In a model of transfusion-related acute lung injury, NOD/SCID mice were susceptible to acute lung injury when challenged with H-2Kd monoclonal antibody vs. isotype control antibody. Using lung intravital microscopy, we did not detect a difference in the dynamic retention of platelets in the lung circulation in control vs. Mirasol PRT-treated groups. In conclusion, Mirasol PRT produced an increase in P-selectin expression that is storage-dependent, but transfusion of human platelets treated with Mirasol PRT into immunodeficient mice did not result in greater platelet retention in the lungs or the development of acute lung injury.

  3. Developing Novel Therapeutic Approaches in Small Cell Lung Carcinoma Using Genetically Engineered Mouse Models and Human Circulating Tumor Cells

    DTIC Science & Technology

    2015-10-01

    Using Genetically Engineered Mouse Models and Human Circulating Tumor Cells PRINCIPAL INVESTIGATOR: Jeffrey Engelman MD PhD CONTRACTING...SUBTITLE Developiing Novel Therapeutic Approaches in Small Cell Lung 5a. CONTRACT NUMBER Carcinoma Using Genetically Engineered Mouse Models and 5b...biomarkers. 15. SUBJECT TERMS Small cell lung cancer (SCLC), Genetically engineered mouse model (GEMM), BH3 mimetic, TORC inhibitor, Apoptosis

  4. Lung-targeting drug delivery system of baicalin-loaded nanoliposomes: development, biodistribution in rabbits, and pharmacodynamics in nude mice bearing orthotopic human lung cancer

    PubMed Central

    Wei, Yumeng; Liang, Jing; Zheng, Xiaoli; Pi, Chao; Liu, Hao; Yang, Hongru; Zou, Yonggen; Ye, Yun; Zhao, Ling

    2017-01-01

    The present study aims to develop a kind of novel nanoliposomes for the lung-targeting delivery system of baicalin as a Chinese medicine monomer. Baicalin-loaded nanoliposomes were prepared by the effervescent dispersion and lyophilized techniques. Baicalin-loaded nanoliposomes had an average particle size of 131.7±11.7 nm with 0.19±0.02 polydispersity index, 82.8%±1.24% entrapment efficiency and 90.47%±0.93% of yield and sustaining drug release effect over 24 h and were stable for 12 months at least. In vitro no hemolytic activity was observed for the experimental drug concentration. After intravenous administration of baicalin-loaded nanoliposomes to rabbits, drug concentration in the lungs was the highest among the tested organs at all time points and was significantly higher than that of its solution. For the targeting parameters, the relative intake rate and the ratio of peak concentration of lung were 4.837 and 2.789, respectively. Compared with plasma, liver, spleen, and kidney, the ratios of targeting efficacy (Te)liposomes to (Te)injection of lung were increased by a factor of 14.131, 1.893, 3.357, and 3.470, respectively. Furthermore, the results showed that the baicalin-loaded nanoliposomes did not induce lung injury. Importantly, baicalin-loaded nanoliposomes showed better antitumor therapeutic efficacy in the nude mice bearing orthotopic human lung cancer with the median survival time of blank liposomes (11.40±0.16 days), baicalin solution (17.30±0.47 days), and baicalin-loaded nanoliposomes (25.90±0.53 days). Therefore, the liposome is a promising drug carrier with an excellent lung-targeting property and therapeutic effect for the treatment of lung disease, such as lung cancer. PMID:28096670

  5. The Audible Human Project: Modeling Sound Transmission in the Lungs and Torso

    NASA Astrophysics Data System (ADS)

    Dai, Zoujun

    Auscultation has been used qualitatively by physicians for hundreds of years to aid in the monitoring and diagnosis of pulmonary diseases. Alterations in the structure and function of the pulmonary system that occur in disease or injury often give rise to measurable changes in lung sound production and transmission. Numerous acoustic measurements have revealed the differences of breath sounds and transmitted sounds in the lung under normal and pathological conditions. Compared to the extensive cataloging of lung sound measurements, the mechanism of sound transmission in the pulmonary system and how it changes with alterations of lung structural and material properties has received less attention. A better understanding of sound transmission and how it is altered by injury and disease might improve interpretation of lung sound measurements, including new lung imaging modalities that are based on an array measurement of the acoustic field on the torso surface via contact sensors or are based on a 3-dimensional measurement of the acoustic field throughout the lungs and torso using magnetic resonance elastography. A long-term goal of the Audible Human Project (AHP ) is to develop a computational acoustic model that would accurately simulate generation, transmission and noninvasive measurement of sound and vibration within the pulmonary system and torso caused by both internal (e.g. respiratory function) and external (e.g. palpation) sources. The goals of this dissertation research, fitting within the scope of the AHP, are to develop specific improved theoretical understandings, computational algorithms and experimental methods aimed at transmission and measurement. The research objectives undertaken in this dissertation are as follows. (1) Improve theoretical modeling and experimental identification of viscoelasticity in soft biological tissues. (2) Develop a poroviscoelastic model for lung tissue vibroacoustics. (3) Improve lung airway acoustics modeling and its

  6. Three-Dimensionally Engineered Normal Human Lung Tissue-Like Assemblies: Target Tissues for Human Respiratory Viral Infections

    NASA Technical Reports Server (NTRS)

    Goodwin, Thomas J.; McCarthy, M.; Lin, Y-H.; Deatly, A. M.

    2008-01-01

    In vitro three-dimensional (3D) human lung epithelio-mesenchymal tissue-like assemblies (3D hLEM TLAs) from this point forward referred to as TLAs were engineered in Rotating Wall Vessel (RWV) technology to mimic the characteristics of in vivo tissues thus providing a tool to study human respiratory viruses and host cell interactions. The TLAs were bioengineered onto collagen-coated cyclodextran microcarriers using primary human mesenchymal bronchial-tracheal cells (HBTC) as the foundation matrix and an adult human bronchial epithelial immortalized cell line (BEAS-2B) as the overlying component. The resulting TLAs share significant characteristics with in vivo human respiratory epithelium including polarization, tight junctions, desmosomes, and microvilli. The presence of tissue-like differentiation markers including villin, keratins, and specific lung epithelium markers, as well as the production of tissue mucin, further confirm these TLAs differentiated into tissues functionally similar to in vivo tissues. Increasing virus titers for human respiratory syncytial virus (wtRSVA2) and the detection of membrane bound glycoproteins over time confirm productive infection with the virus. Therefore, we assert TLAs mimic aspects of the human respiratory epithelium and provide a unique capability to study the interactions of respiratory viruses and their primary target tissue independent of the host s immune system.

  7. Multi-walled carbon nanotube-induced gene signatures in the mouse lung: potential predictive value for human lung cancer risk and prognosis

    PubMed Central

    Guo, Nancy L; Wan, Ying-Wooi; Denvir, James; Porter, Dale W; Pacurari, Maricica; Wolfarth, Michael G; Castranova, Vincent; Qian, Yong

    2012-01-01

    Concerns over the potential for multi-walled carbon nanotubes (MWCNT) to induce lung carcinogenesis have emerged. This study sought to (1) identify gene expression signatures in the mouse lungs following pharyngeal aspiration of well-dispersed MWCNT and (2) determine if these genes were associated with human lung cancer risk and progression. Genome-wide mRNA expression profiles were analyzed in mouse lungs (n=160) exposed to 0, 10, 20, 40, or 80 µg of MWCNT by pharyngeal aspiration at 1, 7, 28, and 56 days post-exposure. By using pairwise-Statistical Analysis of Microarray (SAM) and linear modeling, 24 genes were selected, which have significant changes in at least two time points, have a more than 1.5 fold change at all doses, and are significant in the linear model for the dose or the interaction of time and dose. Additionally, a 38-gene set was identified as related to cancer from 330 genes differentially expressed at day 56 post-exposure in functional pathway analysis. Using the expression profiles of the cancer-related gene set in 8 mice at day 56 post-exposure to 10 µg of MWCNT, a nearest centroid classification accurately predicts human lung cancer survival with a significant hazard ratio in training set (n=256) and test set (n=186). Furthermore, both gene signatures were associated with human lung cancer risk (n=164) with significant odds ratios. These results may lead to development of a surveillance approach for early detection of lung cancer and prognosis associated with MWCNT in the workplace. PMID:22891886

  8. Interactive lung segmentation in abnormal human and animal chest CT scans

    SciTech Connect

    Kockelkorn, Thessa T. J. P. Viergever, Max A.; Schaefer-Prokop, Cornelia M.; Bozovic, Gracijela; Muñoz-Barrutia, Arrate; Rikxoort, Eva M. van; Brown, Matthew S.; Jong, Pim A. de; Ginneken, Bram van

    2014-08-15

    Purpose: Many medical image analysis systems require segmentation of the structures of interest as a first step. For scans with gross pathology, automatic segmentation methods may fail. The authors’ aim is to develop a versatile, fast, and reliable interactive system to segment anatomical structures. In this study, this system was used for segmenting lungs in challenging thoracic computed tomography (CT) scans. Methods: In volumetric thoracic CT scans, the chest is segmented and divided into 3D volumes of interest (VOIs), containing voxels with similar densities. These VOIs are automatically labeled as either lung tissue or nonlung tissue. The automatic labeling results can be corrected using an interactive or a supervised interactive approach. When using the supervised interactive system, the user is shown the classification results per slice, whereupon he/she can adjust incorrect labels. The system is retrained continuously, taking the corrections and approvals of the user into account. In this way, the system learns to make a better distinction between lung tissue and nonlung tissue. When using the interactive framework without supervised learning, the user corrects all incorrectly labeled VOIs manually. Both interactive segmentation tools were tested on 32 volumetric CT scans of pigs, mice and humans, containing pulmonary abnormalities. Results: On average, supervised interactive lung segmentation took under 9 min of user interaction. Algorithm computing time was 2 min on average, but can easily be reduced. On average, 2.0% of all VOIs in a scan had to be relabeled. Lung segmentation using the interactive segmentation method took on average 13 min and involved relabeling 3.0% of all VOIs on average. The resulting segmentations correspond well to manual delineations of eight axial slices per scan, with an average Dice similarity coefficient of 0.933. Conclusions: The authors have developed two fast and reliable methods for interactive lung segmentation in

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

    PubMed

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

    2011-09-01

    The 'relative activity factor' (RAF) compares the activity per unit of microsomal protein in recombinantly expressed cytochrome P450 enzymes (rhCYP) and human liver without separating the potential sources of variation (i.e. abundance of enzyme per mg of protein or variation of activity per unit enzyme). The dimensionless 'inter-system extrapolation factor' (ISEF) dissects differences in activity from those in CYP abundance. Detailed protocols for the determination of this scalar, which is used in population in vitro-in vivo extrapolation (IVIVE), are currently lacking. The present study determined an ISEF for CYP2C9 and, for the first time, systematically evaluated the effects of probe substrate, cytochrome b5 and methods for assessing the intrinsic clearance (CL(int) ). Values of ISEF for S-warfarin, tolbutamide and diclofenac were 0.75 ± 0.18, 0.57 ± 0.07 and 0.37 ± 0.07, respectively, using CL(int) values derived from the kinetic values V(max) and K(m) of metabolite formation in rhCYP2C9 + reductase + b5 BD Supersomes™. The ISEF values obtained using rhCYP2C9 + reductase BD Supersomes™ were more variable, with values of 7.16 ± 1.25, 0.89 ± 0.52 and 0.50 ± 0.05 for S-warfarin, tolbutamide and diclofenac, respectively. Although the ISEF values obtained from rhCYP2C9 + reductase + b5 for the three probe substrates were statistically different (p < 0.001), the use of the mean value of 0.54 resulted in predicted oral clearance values for all three substrates within 1.4 fold of the observed literature values. For consistency in the relative activity across substrates, use of a b5 expressing recombinant system, with the intrinsic clearance calculated from full kinetic data is recommended for generation of the CYP2C9 ISEF. Furthermore, as ISEFs have been found to be sensitive to differences in accessory proteins, rhCYP system specific ISEFs are recommended.

  10. A System for Open-Access 3He Human Lung Imaging at Very Low Field

    PubMed Central

    RUSET, I.C.; TSAI, L.L.; MAIR, R.W.; PATZ, S.; HROVAT, M.I.; ROSEN, M.S.; MURADIAN, I.; NG, J.; TOPULOS, G.P.; BUTLER, J.P.; WALSWORTH, R.L.; HERSMAN, F.W.

    2010-01-01

    We describe a prototype system built to allow open-access very-low-field MRI of human lungs using laser-polarized 3He gas. The system employs an open four-coil electromagnet with an operational B0 field of 4 mT, and planar gradient coils that generate gradient fields up to 0.18 G/cm in the x and y direction and 0.41 G/cm in the z direction. This system was used to obtain 1H and 3He phantom images and supine and upright 3He images of human lungs. We include discussion on challenges unique to imaging at 50 –200 kHz, including noise filtering and compensation for narrow-bandwidth coils. PMID:20354575

  11. Natural innate cytokine response to immunomodulators and adjuvants in human precision-cut lung slices

    SciTech Connect

    Switalla, S.; Lauenstein, L.; Prenzler, F.; Knothe, S.; Foerster, C.; Fieguth, H.-G.; Pfennig, O.; Schaumann, F.; Martin, C.; Guzman, C.A.; Ebensen, T.; Mueller, M.; Hohlfeld, J.M.; Krug, N.; Braun, A.; Sewald, K.

    2010-08-01

    Prediction of lung innate immune responses is critical for developing new drugs. Well-established immune modulators like lipopolysaccharides (LPS) can elicit a wide range of immunological effects. They are involved in acute lung diseases such as infections or chronic airway diseases such as COPD. LPS has a strong adjuvant activity, but its pyrogenicity has precluded therapeutic use. The bacterial lipopeptide MALP-2 and its synthetic derivative BPPcysMPEG are better tolerated. We have compared the effects of LPS and BPPcysMPEG on the innate immune response in human precision-cut lung slices. Cytokine responses were quantified by ELISA, Luminex, and Meso Scale Discovery technology. The initial response to LPS and BPPcysMPEG was marked by coordinated and significant release of the mediators IL-1{beta}, MIP-1{beta}, and IL-10 in viable PCLS. Stimulation of lung tissue with BPPcysMPEG, however, induced a differential response. While LPS upregulated IFN-{gamma}, BPPcysMPEG did not. This traces back to their signaling pathways via TLR4 and TLR2/6. The calculated exposure doses selected for LPS covered ranges occurring in clinical studies with human beings. Correlation of obtained data with data from human BAL fluid after segmental provocation with endotoxin showed highly comparable effects, resulting in a coefficient of correlation > 0.9. Furthermore, we were interested in modulating the response to LPS. Using dexamethasone as an immunosuppressive drug for anti-inflammatory therapy, we found a significant reduction of GM-CSF, IL-1{beta}, and IFN-{gamma}. The PCLS-model offers the unique opportunity to test the efficacy and toxicity of biological agents intended for use by inhalation in a complex setting in humans.

  12. Effect of lung flooding and high-intensity focused ultrasound on lung tumours: an experimental study in an ex vivo human cancer model and simulated in vivo tumours in pigs

    PubMed Central

    2014-01-01

    Background High-intensity focused ultrasound is a valuable tool for minimally invasive tumour ablation. However, due to the air content in ventilated lungs, lung tumours have never been treated with high-intensity focused ultrasound. Lung flooding enables efficient lung sonography and tumour imaging in ex vivo human and in vivo porcine lung cancer models. The current study evaluates the effectiveness of lung flooding and sonography-guided high-intensity focused ultrasound for lung tumour ablation in ex vivo human and in vivo animal models. Methods Lung flooding was performed in four human lung lobes which were resected from non-small cell lung cancers. B-mode imaging and temperature measurements were simultaneously obtained during high-intensity focused ultrasonography of centrally located lung cancers. The tumour was removed immediately following insonation and processed for nicotinamide adenine dinucleotide phosphate-diaphorase and H&E staining. In addition, the left lungs of three pigs were flooded. Purified BSA in glutaraldehyde was injected centrally into the left lower lung lobe to simulate a lung tumour. The ultrasound was focused transthoracically through the flooded lung into the simulated tumour with the guidance of sonography. The temperature of the tumour was simultaneously measured. The vital signs of the animal were monitored during the procedure. Results A well-demarcated lesion of coagulation necrosis was produced in four of four human lung tumours. There did not appear to be any damage to the surrounding lung parenchyma. After high-intensity focused ultrasound insonation, the mean temperature increase was 7.5-fold higher in the ex vivo human tumour than in the flooded lung tissue (52.1 K ± 8.77 K versus 7.1 K ± 2.5 K). The transthoracic high-intensity focused ultrasound of simulated tumours in the in vivo model resulted in a mean peak temperature increase up to 53.7°C (±4.5). All of the animals survived the procedure without

  13. Microenvironmental modulation of asymmetric cell division in human lung cancer cells.

    PubMed

    Pine, Sharon R; Ryan, Bríd M; Varticovski, Lyuba; Robles, Ana I; Harris, Curtis C

    2010-02-02

    Normal tissue homeostasis is maintained through asymmetric cell divisions that produce daughter cells with differing self-renewal and differentiation potentials. Certain tumor cell subfractions can self-renew and repopulate the heterogeneous tumor bulk, suggestive of asymmetric cell division, but an equally plausible explanation is that daughter cells of a symmetric division subsequently adopt differing cell fates. Cosegregation of template DNA during mitosis is one mechanism by which cellular components are segregated asymmetrically during cell division in fibroblast, muscle, mammary, intestinal, and neural cells. Asymmetric cell division of template DNA in tumor cells has remained elusive, however. Through pulse-chase experiments with halogenated thymidine analogs, we determined that a small population of cells within human lung cancer cell lines and primary tumor cell cultures asymmetrically divided their template DNA, which could be visualized in single cells and in real time. Template DNA cosegregation was enhanced by cell-cell contact. Its frequency was density-dependent and modulated by environmental changes, including serum deprivation and hypoxia. In addition, we found that isolated CD133(+) lung cancer cells were capable of tumor cell repopulation. Strikingly, during cell division, CD133 cosegregated with the template DNA, whereas the differentiation markers prosurfactant protein-C and pan-cytokeratins were passed to the opposing daughter cell, demonstrating that segregation of template DNA correlates with lung cancer cell fate. Our results demonstrate that human lung tumor cell fate decisions may be regulated during the cell division process. The characterization and modulation of asymmetric cell division in lung cancer can provide insight into tumor initiation, growth, and maintenance.

  14. Nuclear distribution of claudin-2 increases cell proliferation in human lung adenocarcinoma cells.

    PubMed

    Ikari, Akira; Watanabe, Ryo; Sato, Tomonari; Taga, Saeko; Shimobaba, Shun; Yamaguchi, Masahiko; Yamazaki, Yasuhiro; Endo, Satoshi; Matsunaga, Toshiyuki; Sugatani, Junko

    2014-09-01

    Claudin-2 is expressed in human lung adenocarcinoma tissue and cell lines, although it is absent in normal lung tissue. However, the role of claudin-2 in cell proliferation and the regulatory mechanism of intracellular distribution remain undefined. Proliferation of human adenocarcinoma A549 cells was decreased by claudin-2 knockdown together with a decrease in the percentage of S phase cells. This knockdown decreased the expression levels of ZONAB and cell cycle regulators. Claudin-2 was distributed in the nucleus in human adenocarcinoma tissues and proliferating A549 cells. The nuclear distribution of ZONAB and percentage of S phase cells were higher in cells exogenously expressing claudin-2 with a nuclear localization signal than in cells expressing claudin-2 with a nuclear export signal. Nuclear claudin-2 formed a complex with ZO-1, ZONAB, and cyclin D1. Nuclear distribution of S208A mutant, a dephosphorylated form of claudin-2, was higher than that of wild type. We suggest that nuclear distribution of claudin-2 is up-regulated by dephosphorylation and claudin-2 serves to retain ZONAB and cyclin D1 in the nucleus, resulting in the enhancement of cell proliferation in lung adenocarcinoma cells.

  15. Tsr Chemoreceptor Interacts With IL-8 Provoking E. coli Transmigration Across Human Lung Epithelial Cells

    PubMed Central

    Han, Bing; Li, Manshu; Xu, Yonghao; Islam, Diana; Khang, Julie; Del Sorbo, Lorenzo; Lee, Warren; Szaszi, Katalin; Zhong, Nanshan; Slutsky, Arthur S.; Li, Yimin; Zhang, Haibo

    2016-01-01

    Bacterial colonization of epithelial surfaces and subsequent transmigration across the mucosal barrier are essential for the development of infection. We hypothesized that the methyl-accepting proteins (MCPs), known as chemoreceptors expressed on Escherichia coli (E. coli) bacterial surface, play an important role in mediating bacterial transmigration. We demonstrated a direct interaction between human interleukin-8 (IL-8) and Tsr receptor, a major MCP chemoreceptor. Stimulation of human lung epithelial cell monolayer with IL-8 resulted in increased E. coli adhesion and transmigration of the native strain (RP437) and a strain expressing only Tsr (UU2373), as compared to a strain (UU2599) with Tsr truncation. The augmented E. coli adhesion and migration was associated with a higher expression of carcinoembryonic antigen-related cell adhesion molecule 6 and production of inflammatory cytokines/chemokines, and a lower expression of the tight junction protein claudin-1 and the plasma membrane protein caveolin-1 in lung epithelial cells. An increased E. coli colonization and pulmonary cytokine production induced by the RP437 and UU2373 strains was attenuated in mice challenged with the UU2599 strain. Our results suggest a critical role of the E. coli Tsr chemoreceptor in mediating bacterial colonization and transmigration across human lung epithelium during development of pulmonary infections. PMID:27506372

  16. CFD modeling of turbulent flow and particle deposition in human lungs.

    PubMed

    Radhakrishnan, H; Kassinos, S

    2009-01-01

    Understanding transport and deposition of inhaled particles in the human airways plays a crucial role in the targeted therapy of pulmonary diseases, and the administration of inhaled medicines. Numerous researchers have studied the inhalation of particles using experiments or computer models. Even though experiments have shown that the airflow in the trachea and the upper branches of the lung is turbulent, the flow is taken to be laminar in most computer models. Only few recently published papers have looked at the turbulent transport of air in the human airways. Even fewer results have been published on the effect of the upper airway structures on the turbulent airflow in the lungs or on the effect of the turbulence on particle deposition. The previously published turbulent models have also mainly used RANS methods to predict the flow. To study the unsteady flow and particle deposition in a human lung, an LES model with a dynamic Smagorinsky sub-grid scale model was used. The model equations were solved to study steady inspirational flow at different flow rates for different particle sizes. Results indicate that the upper airway geometry produces turbulence in the flow and the deposition of particles is mainly affected by the particle size and Stokes number.

  17. Evaluation of the anti-inflammatory effects of β-adrenoceptor agonists on human lung macrophages.

    PubMed

    Gill, Sharonjit K; Marriott, Helen M; Suvarna, S Kim; Peachell, Peter T

    2016-12-15

    The principal mechanism by which bronchodilator β-adrenoceptor agonists act is to relax airways smooth muscle although they may also be anti-inflammatory. However, the extent of anti-inflammatory activity and the cell types affected by these agonists are uncertain. The purpose of this study was to evaluate whether β-adrenoceptor agonists prevent pro-inflammatory cytokine generation from activated human lung macrophages. Macrophages were isolated and purified from human lung. The cells were pre-treated with both short-acting (isoprenaline, salbutamol, terbutaline) and long-acting (formoterol, salmeterol, indacaterol) β-agonists before activation with lipopolysaccharide (LPS) to induce cytokine (TNFα, IL-6, IL-8 and IL-10) generation. The experiments showed that short-acting β-agonists were poor inhibitors of cytokine generation. Of the long-acting β-agonists studied, formoterol was also a weak inhibitor of cytokine generation whereas only indacaterol and salmeterol showed moderate inhibitory activity. Further experiments using the β2-adrenoceptor antagonist ICI-118,551 suggested that the effects of indacaterol were likely to be mediated by β2-adrenoceptors whereas those of salmeterol were not. These findings were corroborated by functional desensitization studies in which the inhibitory effects of indacaterol appeared to be receptor-mediated whereas those of salmeterol were not. Taken together, the data indicate that the anti-inflammatory effects of β-adrenoceptor agonists on human lung macrophages are modest.

  18. MicroRNA-221 promotes human non-small cell lung cancer cell H460 growth.

    PubMed

    Xu, Yiming; Zhong, Chongjun; Ding, Shengguang; Huang, Haitao; Shen, Zhenya

    2015-01-01

    MicroRNA (miRNA-221) has been reported to be a regulator of cell proliferation. Here we intended to investigate the role of miRNA-221 in regulating the growth of human non-small cell lung cancer cell line H460. H460 cells were transfected with miRNA-221 mimics/inhibitors or their respective negative controls. Real-time quantitative PCRs (qRT-PCRs) were used to confirm the effects of miRNA-221 mimics and inhibitors in H460 cells while Cell Counting Kit 8 (CCK-8) and 5-Ethynyl-2'-deoxyuridine (EdU) assay were used to access the cell viability and proliferation. P27 and P57, as putative targets of miRNA-221, were determined by qRT-PCRs in H460 cells. We found that overexpression of miRNA-221 led to increased proliferative rate and cell viability in H460 cells while down-regulation of miRNA-221 decreased those effects. P27 but not P57 was identified as a potential target gene of miRNA-221 in H460 as P27 was negatively regulated by miRNA-221 in the protein level. In conclusion, this study suggests that miRNA-221 controls human non-small cell lung cancer cell H460 growth potentially by targeting P57. Inhibition of miRNA-221 represents a novel potential treatment for human non-small cell lung cancer.

  19. Comparative effects of medetomidine enantiomers on in vitro and in vivo microsomal drug metabolism.

    PubMed

    Pelkonen, O; Puurunen, J; Arvela, P; Lammintausta, R

    1991-09-01

    The effects of dexmedetomidine, a selective alpha 2-adrenoceptor agonist, and its levo enantiomer (MPV-1441), on in vitro microsomal P450-dependent drug-metabolizing activities as well as on in vivo aminopyrine elimination and hexobarbital sleeping time were studied. Both enantiomers inhibited the oxidative metabolism of several model substrates and testosterone in rat liver microsomal incubations. Microsomal activities derived from control animals or rats pretreated with phenobarbital were more sensitive to inhibitory effects of dexmedetomidine than those from rats treated with 3-methylcholanthrene. Enzyme activities in human liver microsomes were also inhibited by dexmedetomidine. Retardation of the elimination of aminopyrine was dose-dependent; elimination was marginally retarded with doses up to 100 micrograms/kg (from 17 to 23 min.; both enantiomers). Higher doses of the levo enantiomer prolonged aminopyrine half-life to 78 (1 mg/kg) and 162 min. (10 mg/kg). The hexobarbital sleeping time was prolonged by the dose of 1 mg/kg of the levo enantiomer (128 min. versus 20 min. in controls), while the dose of 0.1 mg/kg had no effect (23 versus 20 min.). These studies indicate that both enantiomers of medetomidine are inhibitors of microsomal drug metabolism in vitro, but significant effects on aminopyrine elimination or hexobarbital sleeping time are apparent only at doses, which do not allow the use of dexmedetomidine because of excessive sedative effect.

  20. Molecular and Cellular Effects of Hydrogen Peroxide on Human Lung Cancer Cells: Potential Therapeutic Implications

    PubMed Central

    2016-01-01

    Lung cancer has a very high mortality-to-incidence ratio, representing one of the main causes of cancer mortality worldwide. Therefore, new treatment strategies are urgently needed. Several diseases including lung cancer have been associated with the action of reactive oxygen species (ROS) from which hydrogen peroxide (H2O2) is one of the most studied. Despite the fact that H2O2 may have opposite effects on cell proliferation depending on the concentration and cell type, it triggers several antiproliferative responses. H2O2 produces both nuclear and mitochondrial DNA lesions, increases the expression of cell adhesion molecules, and increases p53 activity and other transcription factors orchestrating cancer cell death. In addition, H2O2 facilitates the endocytosis of oligonucleotides, affects membrane proteins, induces calcium release, and decreases cancer cell migration and invasion. Furthermore, the MAPK pathway and the expression of genes related to inflammation including interleukins, TNF-α, and NF-κB are also affected by H2O2. Herein, we will summarize the main effects of hydrogen peroxide on human lung cancer leading to suggesting it as a potential therapeutic tool to fight this disease. Because of the multimechanistic nature of this molecule, novel therapeutic approaches for lung cancer based on the use of H2O2 may help to decrease the mortality from this malignancy. PMID:27375834

  1. An alternatively spliced surfactant protein B mRNA in normal human lung: disease implication.

    PubMed Central

    Lin, Z; Wang, G; Demello, D E; Floros, J

    1999-01-01

    We identified an alternatively-spliced surfactant protein B (SP-B) mRNA from normal human lung with a 12 nt deletion at the beginning of exon 8. This deletion causes a loss of four amino acids in the SP-B precursor protein. Sequence comparison of the 3' splice sites reveals only one difference in the frequency of U/C in the 11 predominantly-pyrimidine nucleotide tract, 73% for the normal and 45% for the alternatively-spliced SP-B mRNA (77-99% for the consensus sequence). Analysis of SP-B mRNA in lung indicates that the abundance of the alternatively-spliced form is very low and varies among individuals. Although the relative abundance of the deletion form of SP-B mRNA remains constant among normal lungs, it is found with relatively higher abundance in the lungs of some individuals with diseases such as congenital alveolar proteinosis, respiratory distress syndrome, bronchopulmonary dysplasia, alveolar capillary dysplasia and hypophosphatasia. This observation points to the possibility that the alternative splicing is a potential regulatory mechanism of SP-B and may play a role in the pathogenesis of disease under certain circumstances. PMID:10493923

  2. Nanosomes carrying doxorubicin exhibit potent anticancer activity against human lung cancer cells

    PubMed Central

    Srivastava, Akhil; Amreddy, Narsireddy; Babu, Anish; Panneerselvam, Janani; Mehta, Meghna; Muralidharan, Ranganayaki; Chen, Allshine; Zhao, Yan Daniel; Razaq, Mohammad; Riedinger, Natascha; Kim, Hogyoung; Liu, Shaorong; Wu, Si; Abdel-Mageed, Asim B.; Munshi, Anupama; Ramesh, Rajagopal

    2016-01-01

    Successful chemotherapeutic intervention for management of lung cancer requires an efficient drug delivery system. Gold nanoparticles (GNPs) can incorporate various therapeutics; however, GNPs have limitations as drug carriers. Nano-sized cellular vesicles like exosomes (Exo) can ferry GNP-therapeutic complexes without causing any particle aggregation or immune response. In the present study, we describe the development and testing of a novel Exo-GNP-based therapeutic delivery system -‘nanosomes’- for lung cancer therapy. This system consists of GNPs conjugated to anticancer drug doxorubicin (Dox) by a pH-cleavable bond that is physically loaded onto the exosomes (Exo-GNP-Dox). The therapeutic efficacy of Dox in nanosomes was assessed in H1299 and A549 non-small cell lung cancer cells, normal MRC9 lung fibroblasts, and Dox-sensitive human coronary artery smooth muscle cells (HCASM). The enhanced rate of drug release under acidic conditions, successful uptake of the nanosomes by the recipient cells and the cell viability assays demonstrated that nanosomes exhibit preferential cytotoxicity towards cancer cells and have minimal activity on non-cancerous cells. Finally, the underlying mechanism of cytotoxicity involved ROS-mediated DNA damage. Results from this study mark the establishment of an amenable drug delivery vehicle and highlight the advantages of a natural drug carrier that demonstrates reduced cellular toxicity and efficient delivery of therapeutics to cancer cells. PMID:27941871

  3. Inflammatory and immune processes in the human lung in health and disease: evaluation by bronchoalveolar lavage.

    PubMed Central

    Hunninghake, G. W.; Gadek, J. E.; Kawanami, O.; Ferrans, V. J.; Crystal, R. G.

    1979-01-01

    Bronchoalveolar lavage is an invaluable means of accurately evaluating the inflammatory and immune processes of the human lung. Although lavage recovers only those cells and proteins present on the epithelial surface of the lower respiratory tract, comparison with open lung biopsies shows that these constituents are representative of the inflammatory and immune systems of the alveolar structures. With the use of these techniques, sufficient materials are obtained from normal individuals to allow characterization of not only the types of cells and proteins present but their functions as well. Such observations have been useful in defining the inflammatory and immune capabilities of the normal lung and provide a basis for the study of lung disease. Lavage methods have been used to characterize inflammatory and immune processes of the lower respiratory tract in destructive, infectious, neoplastic, and interstitial disorders. From the data already acquired, it is apparent that bronchoalveolar lavage will yield major insights into the pathogenesis, staging, and therapy decisions involved in these disorders. (Am J Pathol 97:149--206, 1979). Images Figure 9 Figure 1 Figure 2 Figure 10 Figure 7 Figure 8 Figure 4 Figure 5 Figure 6 Figure 3 PMID:495693

  4. Tumor-associated neutrophils stimulate T cell responses in early-stage human lung cancer

    PubMed Central

    Eruslanov, Evgeniy B.; Bhojnagarwala, Pratik S.; Quatromoni, Jon G.; Stephen, Tom Li; Ranganathan, Anjana; Deshpande, Charuhas; Akimova, Tatiana; Vachani, Anil; Litzky, Leslie; Hancock, Wayne W.; Conejo-Garcia, José R.; Feldman, Michael; Albelda, Steven M.; Singhal, Sunil

    2014-01-01

    Infiltrating inflammatory cells are highly prevalent within the tumor microenvironment and mediate many processes associated with tumor progression; however, the contribution of specific populations remains unclear. For example, the nature and function of tumor-associated neutrophils (TANs) in the cancer microenvironment is largely unknown. The goal of this study was to provide a phenotypic and functional characterization of TANs in surgically resected lung cancer patients. We found that TANs constituted 5%–25% of cells isolated from the digested human lung tumors. Compared with blood neutrophils, TANs displayed an activated phenotype (CD62LloCD54hi) with a distinct repertoire of chemokine receptors that included CCR5, CCR7, CXCR3, and CXCR4. TANs produced substantial quantities of the proinflammatory factors MCP-1, IL-8, MIP-1α, and IL-6, as well as the antiinflammatory IL-1R antagonist. Functionally, both TANs and neutrophils isolated from distant nonmalignant lung tissue were able to stimulate T cell proliferation and IFN-γ release. Cross-talk between TANs and activated T cells led to substantial upregulation of CD54, CD86, OX40L, and 4-1BBL costimulatory molecules on the neutrophil surface, which bolstered T cell proliferation in a positive-feedback loop. Together our results demonstrate that in the earliest stages of lung cancer, TANs are not immunosuppressive, but rather stimulate T cell responses. PMID:25384214

  5. Aluminum is More Cytotoxic than Lunar Dust in Human Skin and Lung Fibroblasts

    NASA Technical Reports Server (NTRS)

    Hammond, D.; Shehata, T.; Hammond, D.; Shehata, T.; Wise, J.P.; Martino, J; Wise, J.P.; Wise, J.P.

    2009-01-01

    NASA plans to build a permanent space station on the moon to explore its surface. The surface of the moon is covered in lunar dust, which consists of fine particles that contain silicon, aluminum and titanium, among others. Because this will be a manned base, the potential toxicity of this dust has to be studied. Also, toxicity standards for potential exposure have to be set. To properly address the potential toxicity of lunar dust we need to understand the toxicity of its individual components, as well as their combined effects. In order to study this we compared NASA simulant JSC-1AVF (volcanic ash particles), that simulates the dust found on the moon, to aluminum, the 3rd most abundant component in lunar dust. We tested the cytotoxicity of both compounds on human lung and skin fibroblasts (WTHBF-6 and BJhTERT cell lines, respectively). Aluminum oxide was more cytotoxic than lunar dust to both cell lines. In human lung fibroblasts 5, 10 and 50 g/sq cm of aluminum oxide induced 85%, 61% and 30% relative survival, respectively. For human skin fibroblasts the same concentrations induced 58%, 41% and 58% relative survival. Lunar dust was also cytotoxic to both cell lines, but its effects were seen at higher concentrations: 50, 100, 200 and 400 g/sq cm of lunar dust induced a 69%, 46%, 35% and 30% relative survival in the skin cells and 53%, 16%, 8% and 2% on the lung cells. Overall, for both compounds, lung cells were more sensitive than skin cells. This work was supported by a NASA EPSCoR grant through the Maine Space Grant Consortium (JPW), the Maine Center for Toxicology and Environmental Health., a Fulbright Grant (JM) and a Delta Kappa Gamma Society International World Fellowship (JM).

  6. Vessel co-option is common in human lung metastases and mediates resistance to anti-angiogenic therapy in preclinical lung metastasis models.

    PubMed

    Bridgeman, Victoria L; Vermeulen, Peter B; Foo, Shane; Bilecz, Agnes; Daley, Frances; Kostaras, Eleftherios; Nathan, Mark R; Wan, Elaine; Frentzas, Sophia; Schweiger, Thomas; Hegedus, Balazs; Hoetzenecker, Konrad; Renyi-Vamos, Ferenc; Kuczynski, Elizabeth A; Vasudev, Naveen S; Larkin, James; Gore, Martin; Dvorak, Harold F; Paku, Sandor; Kerbel, Robert S; Dome, Balazs; Reynolds, Andrew R

    2017-02-01

    Anti-angiogenic therapies have shown limited efficacy in the clinical management of metastatic disease, including lung metastases. Moreover, the mechanisms via which tumours resist anti-angiogenic therapies are poorly understood. Importantly, rather than utilizing angiogenesis, some metastases may instead incorporate pre-existing vessels from surrounding tissue (vessel co-option). As anti-angiogenic therapies were designed to target only new blood vessel growth, vessel co-option has been proposed as a mechanism that could drive resistance to anti-angiogenic therapy. However, vessel co-option has not been extensively studied in lung metastases, and its potential to mediate resistance to anti-angiogenic therapy in lung metastases is not established. Here, we examined the mechanism of tumour vascularization in 164 human lung metastasis specimens (composed of breast, colorectal and renal cancer lung metastasis cases). We identified four distinct histopathological growth patterns (HGPs) of lung metastasis (alveolar, interstitial, perivascular cuffing, and pushing), each of which vascularized via a different mechanism. In the alveolar HGP, cancer cells invaded the alveolar air spaces, facilitating the co-option of alveolar capillaries. In the interstitial HGP, cancer cells invaded the alveolar walls to co-opt alveolar capillaries. In the perivascular cuffing HGP, cancer cells grew by co-opting larger vessels of the lung. Only in the pushing HGP did the tumours vascularize by angiogenesis. Importantly, vessel co-option occurred with high frequency, being present in >80% of the cases examined. Moreover, we provide evidence that vessel co-option mediates resistance to the anti-angiogenic drug sunitinib in preclinical lung metastasis models. Assuming that our interpretation of the data is correct, we conclude that vessel co-option in lung metastases occurs through at least three distinct mechanisms, that vessel co-option occurs frequently in lung metastases, and that vessel

  7. EGF receptor mutations in lung cancer: from humans to mice and maybe back to humans.

    PubMed

    Arteaga, Carlos L

    2006-06-01

    Deletions in exon 19 and nucleotide substitutions in exon 21 are the most common mutations of the EGFR (ErbB1) in NSCLC. These mutations endow the receptor with constitutive kinase activity. Most tumors expressing these mutants respond well to EGFR tyrosine kinase inhibitors, suggesting that they are dependent on mutant EGFR signaling. Two groups developed transgenic mice in which expression of these mutants is temporally induced in mouse lung. Mice expressing EGFR mutants develop bronchioloalveolar cancer and lung adenocarcinoma, which are highly sensitive to EGFR inhibitors. These mouse models provide important opportunities for studying the biology of NSCLC and the refinement of anti-EGFR therapies.

  8. hPSC-derived lung and intestinal organoids as models of human fetal tissue

    PubMed Central

    Aurora, Megan; Spence, Jason R.

    2016-01-01

    In vitro human pluripotent stem cell (hPSC) derived tissues are excellent models to study certain aspects of normal human development. Current research in the field of hPSC derived tissues reveals these models to be inherently fetal-like on both a morphological and gene expression level. In this review we briefly discuss current methods for differentiating lung and intestinal tissue from hPSCs into individual 3-dimensional units called organoids. We discuss how these methods mirror what is known about in vivo signaling pathways of the developing embryo. Additionally, we will review how the inherent immaturity of these models lends them to be particularly valuable in the study of immature human tissues in the clinical setting of premature birth. Human lung organoids (HLOs) and human intestinal organoids (HIOs) not only model normal development, but can also be utilized to study several important diseases of prematurity such as respiratory distress syndrome (RDS), bronchopulmonary dysplasia (BPD), and necrotizing enterocolitis (NEC). PMID:27287882

  9. Smoking-Related Gene Expression in Laser Capture Microdissected Human Lung

    PubMed Central

    Tan, Xiang-Lin; Wang, Tao; Xiong, Shengli; Kumar, Shalini V.; Han, Weiguo; Spivack, Simon D.

    2014-01-01

    Purpose Inter-individual differences in quantitative expression could underlie a propensity for lung cancer. To determine precise individual gene expression signatures on a lung compartment-specific basis, we investigated the expression of carcinogen metabolism genes encoding cytochromes P450 (CYP) 1B1, 2A13, glutathione S-transferase (GST) P1, and a tumor suppressor gene p16 in laser capture microdissected samples of human alveolar compartment (AC) and bronchial epithelial compartment (BEC) lung tissue from 62 smokers and non-smokers. Experimental Design Tobacco exposure was determined by plasma nicotine, cotinine, and smoking history. Precise mRNA expression was determined using our RNA-specific qRT-PCR strategy, and correlated with detailed demographic and clinical characteristics. Results Several correlations of mRNA expression included: (a) CYP1B1 in AC (positively with plasma nicotine level, P = 0.008; plasma cotinine level, P = 0.001); (b) GSTP1 in AC (positively with plasma cotinine level, P = 0.003); (c) GSTP1 in BEC (negatively with smoke dose, P = 0.043; occupational risk, P = 0.019). CYP2A13 was rarely expressed in AC, and not expressed in BEC. p16 expression was not correlated with any measured factor. For each gene, subjects showed expression that was individually concordant between these compartments. No clear association of mRNA expression with lung cancer risk was observed in this pilot analysis. Conclusions The association between lung mRNA expression and tobacco exposure implies that gene-tobacco interaction is a measurable quantitative trait, albeit with wide inter-individual variation. Gene expression tends to be concordant for alveolar and bronchial compartments for these genes in an individual, controlling for proximate tobacco exposure. PMID:19996203

  10. Smoking-Related Gene Expression in Laser Capture-Microdissected Human Lung.

    PubMed

    Tan, Xiang-Lin; Wang, Tao; Xiong, Shengli; Kumar, Shalini V; Han, Weiguo; Spivack, Simon D

    2009-12-15

    PURPOSE: Interindividual differences in quantitative expression could underlie a propensity for lung cancer. To determine precise individual gene expression signatures on a lung compartment-specific basis, we investigated the expression of carcinogen metabolism genes encoding cytochromes P450 (CYP) 1B1, 2A13, GSTP1, and a tumor suppressor gene p16 in laser capture-microdissected samples of human alveolar compartment (AC) and bronchial epithelial compartment (BEC) lung tissue from 62 smokers and nonsmokers. EXPERIMENTAL DESIGN: Tobacco exposure was determined by plasma nicotine, cotinine, and smoking history. Precise mRNA expression was determined using our RNA-specific qRT-PCR strategy, and correlated with detailed demographic and clinical characteristics. RESULTS: Several correlations of mRNA expression included (a) CYP1B1 in AC (positively with plasma nicotine level, P = 0.008; plasma cotinine level, P = 0.001), (b) GSTP1 in AC (positively with plasma cotinine level, P = 0.003), and (c) GSTP1 in BEC (negatively with smoke dose, P = 0.043; occupational risk, P = 0.019). CYP2A13 was rarely expressed in AC and not expressed in BEC. p16 expression was not correlated with any measured factor. For each gene, subjects showed expression that was individually concordant between these compartments. No clear association of mRNA expression with lung cancer risk was observed in this pilot analysis. CONCLUSIONS: The association between lung mRNA expression and tobacco exposure implies that gene-tobacco interaction is a measurable quantitative trait, albeit with wide interindividual variation. Gene expression tends to be concordant for alveolar and bronchial compartments for these genes in an individual, controlling for proximate tobacco exposure. (Clin Cancer Res 2009;15(24):7562-70).

  11. The human lung during the embryonic period: vasculogenesis and primitive erythroblasts circulation.

    PubMed

    Pereda, J; Sulz, L; San Martin, S; Godoy-Guzmán, C

    2013-05-01

    Vascularization and blood cell circulation are crucial steps during lung development. However, how blood vessels are generated and when lung circulation is initiated is still a matter of debate. A morpho-functional analysis of pulmonary vasculature was done using human lung samples between 31 and 56 days post-fertilization (pf). The immunolocalization and expression of CD31, CD34, FLT-1, KDR and the vascular growth factor (VEGF) were investigated. The results showed that at day 31 pf, a capillary plexus is already installed, and a few primitive erythroblasts were seen for the first time within the lumen of some blood vessels. Around day 45 pf, an increase in the amount of primitive erythroblasts was detected in the parenchyma surrounding the distal segment of the bronchial tree. The expression of FLT-1, KDR, CD31 and CD34 was observed in endothelial cells of the capillary plexus and the VEGF was detected in the endodermal epithelium. Our results support the hypothesis that the initial formation of the capillary plexus around the tip of the growing airway bud occurs by vasculogenesis, probably regulated by VEGF and KDR. We also showed a very early onset of blood circulation, starting from day 34 pf, concomitant with the generation of new lung buds. In addition, the increasing number of primitive erythroblasts from week 6 onward, associated with a change in the shape of the blood vessels, suggests a remodeling process and that the generation of new distal vessels at the tip of the lung bud occurs mainly by a process of angiogenesis.

  12. The human lung during the embryonic period: vasculogenesis and primitive erythroblasts circulation

    PubMed Central

    Pereda, J; Sulz, L; San Martin, S; Godoy-Guzmán, C

    2013-01-01

    Vascularization and blood cell circulation are crucial steps during lung development. However, how blood vessels are generated and when lung circulation is initiated is still a matter of debate. A morpho-functional analysis of pulmonary vasculature was done using human lung samples between 31 and 56 days post-fertilization (pf). The immunolocalization and expression of CD31, CD34, FLT-1, KDR and the vascular growth factor (VEGF) were investigated. The results showed that at day 31 pf, a capillary plexus is already installed, and a few primitive erythroblasts were seen for the first time within the lumen of some blood vessels. Around day 45 pf, an increase in the amount of primitive erythroblasts was detected in the parenchyma surrounding the distal segment of the bronchial tree. The expression of FLT-1, KDR, CD31 and CD34 was observed in endothelial cells of the capillary plexus and the VEGF was detected in the endodermal epithelium. Our results support the hypothesis that the initial formation of the capillary plexus around the tip of the growing airway bud occurs by vasculogenesis, probably regulated by VEGF and KDR. We also showed a very early onset of blood circulation, starting from day 34 pf, concomitant with the generation of new lung buds. In addition, the increasing number of primitive erythroblasts from week 6 onward, associated with a change in the shape of the blood vessels, suggests a remodeling process and that the generation of new distal vessels at the tip of the lung bud occurs mainly by a process of angiogenesis. PMID:23520979

  13. Arsenic promotes centrosome abnormalities and cell colony formation in p53 compromised human lung cells

    SciTech Connect

    Liao Weiting; Lin Pinpin; Cheng, T.-S.; Yu, H.-S.; Chang, Louis W.

    2007-12-01

    Epidemiological evidence indicated that residents, especially cigarette smokers, in arseniasis areas had significantly higher lung cancer risk than those living in non-arseniasis areas. Thus, an interaction between arsenic and cigarette smoking in lung carcinogenesis was suspected. p53 dysfunction or mutation in lung epithelial cells was frequently observed in cigarette smokers. Our present study was to explore the differential effects by arsenic on H1355 cells (human lung adenocarcinoma cell line with mutation in p53), BEAS-2B (immortalized lung epithelial cell with functional p53) and pifithrin-{alpha}-treated BEAS-2B cells (p53-inhibited cells). These cells were treated with different doses of sodium arsenite (0, 0.1, 1, 5 and 10 {mu}M) for 48 h. A greater reduction in cell viability was observed in the BEAS-2B cells vs. p53 compromised cells (H1355 or p53-inhibited BEAS-2B). Similar observation was also made on 7-day cell survival (growth) study. TUNEL analysis confirmed that there was indeed a significantly reduced arsenite-induced apoptosis found in p53-compromised cells. Centrosomal abnormality has been attributed to eventual chromosomal missegregation, aneuploidy and tumorigenesis. In our present study, reduced p21 and Gadd45a expressions and increased centrosomal abnormality (atopic and multiple centrosomes) were observed in both arsenite-treated H1355 and p53-inhibited BEAS-2B cells as compared with similarly treated BEAS-2B cells. Increased anchorage-independent growth (colony formation) of BEAS-2B cells co-treated with pifithrin-{alpha} and 5 {mu}M sodium arsenite was also observed in soft agar. Our present investigation demonstrated that arsenic would act specifically on p53 compromised cells (either with p53 dysfunction or inhibited) to induce centrosomal abnormality and colony formation. These findings provided strong evidence on the carcinogenic promotional role of arsenic, especially under the condition of p53 dysfunction.

  14. Spectroscopic issues in optical polarization of 3He gas for Magnetic Resonance Imaging of human lungs

    NASA Astrophysics Data System (ADS)

    Dohnalik, T.; Głowacz, B.; Olejniczak, Z.; Pałasz, T.; Suchanek, M.; Wojna, A.

    2013-10-01

    The Magnetic Resonance Imaging (MRI) of human lungs for diagnostic purposes became possible by using nuclear spin hyperpolarized noble gases, such as 3He. One of the methods to polarize 3He is the Metastability Exchange Optical Pumping (MEOP), which up to now has been performed at low pressure of about 1 mbar and in low magnetic field below 0.1 T (standard conditions). The equilibrium nuclear polarization can reach up to 80%, but it is dramatically reduced during the subsequent gas compression to the atmospheric pressure that is necessary for the lungs examination. Further polarization losses occur during the transportation of the gas to the hospital scanner. It was shown recently that up to 50% polarization can be obtained at elevated pressure exceeding 20 mbar, by using magnetic field higher than 0.1 T (nonstandard conditions). Therefore, following the construction of the low-field MEOP polarizer located in the lab, a dedicated portable unit was developed, which uses the magnetic field of the 1.5 T MR medical scanner and works in the continuous-flow regime. The first in Poland MRI images of human lungs in vivo were obtained on the upgraded to 3He resonance frequency Siemens Sonata medical scanner. An evident improvement in the image quality was achieved when using the new technique. The paper shows how spectroscopic measurements of 3He carried out in various experimental conditions led both to useful practical results and to significant progress in understanding fundamental processes taking place during MEOP.

  15. Human Lung Hydrolases Delineate Mycobacterium tuberculosis–Macrophage Interactions and the Capacity To Control Infection

    PubMed Central

    Arcos, Jesus; Sasindran, Smitha J.; Fujiwara, Nagatoshi; Turner, Joanne; Schlesinger, Larry S.; Torrelles, Jordi B.

    2014-01-01

    Pulmonary surfactant contains homeostatic and antimicrobial hydrolases. When Mycobacterium tuberculosis is initially deposited in the terminal bronchioles and alveoli, as well as following release from lysed macrophages, bacilli are in intimate contact with these lung surfactant hydrolases. We identified and measured several hydrolases in human alveolar lining fluid and lung tissue that, at their physiological concentrations, dramatically modified the M. tuberculosis cell envelope. Independent of their action time (15 min to 12 h), the effects of the hydrolases on the M. tuberculosis cell envelope resulted in a significant decrease (60–80%) in M. tuberculosis association with, and intracellular growth of the bacteria within, human macrophages. The cell envelope-modifying effects of the hydrolases also led to altered M. tuberculosis intracellular trafficking and induced a protective proin-flammatory response to infection. These findings add a new concept to our understanding of M. tuberculosis–macrophage inter-actions (i.e., the impact of lung surfactant hydrolases on M. tuberculosis infection). PMID:21602490

  16. Development of a nonlinear fiber-optic spectrometer for human lung tissue exploration.

    PubMed

    Peyrot, Donald A; Lefort, Claire; Steffenhagen, Marie; Mansuryan, Tigran; Ducourthial, Guillaume; Abi-Haidar, Darine; Sandeau, Nicolas; Vever-Bizet, Christine; Kruglik, Sergei G; Thiberville, Luc; Louradour, Frédéric; Bourg-Heckly, Geneviève

    2012-05-01

    Several major lung pathologies are characterized by early modifications of the extracellular matrix (ECM) fibrillar collagen and elastin network. We report here the development of a nonlinear fiber-optic spectrometer, compatible with an endoscopic use, primarily intended for the recording of second-harmonic generation (SHG) signal of collagen and two-photon excited fluorescence (2PEF) of both collagen and elastin. Fiber dispersion is accurately compensated by the use of a specific grism-pair stretcher, allowing laser pulse temporal width around 70 fs and excitation wavelength tunability from 790 to 900 nm. This spectrometer was used to investigate the excitation wavelength dependence (from 800 to 870 nm) of SHG and 2PEF spectra originating from ex vivo human lung tissue samples. The results were compared with spectral responses of collagen gel and elastin powder reference samples and also with data obtained using standard nonlinear microspectroscopy. The excitation-wavelength-tunable nonlinear fiber-optic spectrometer presented in this study allows performing nonlinear spectroscopy of human lung tissue ECM through the elastin 2PEF and the collagen SHG signals. This work opens the way to tunable excitation nonlinear endomicroscopy based on both distal scanning of a single optical fiber and proximal scanning of a fiber-optic bundle.

  17. Carbon nanotubes induce apoptosis resistance of human lung epithelial cells through FLICE-inhibitory protein.

    PubMed

    Pongrakhananon, Varisa; Luanpitpong, Sudjit; Stueckle, Todd A; Wang, Liying; Nimmannit, Ubonthip; Rojanasakul, Yon

    2015-02-01

    Chronic exposure to single-walled carbon nanotubes (SWCNT) has been reported to induce apoptosis resistance of human lung epithelial cells. As resistance to apoptosis is a foundation of neoplastic transformation and cancer development, we evaluated the apoptosis resistance characteristic of the exposed lung cells to understand the pathogenesis mechanism. Passage control and SWCNT-transformed human lung epithelial cells were treated with known inducers of apoptosis via the intrinsic (antimycin A and CDDP) or extrinsic (FasL and TNF-α) pathway and analyzed for apoptosis by DNA fragmentation, annexin-V expression, and caspase activation assays. Whole-genome microarray was performed to aid the analysis of apoptotic gene signaling network. The SWCNT-transformed cells exhibited defective death receptor pathway in association with cellular FLICE-inhibitory protein (c-FLIP) overexpression. Knockdown or chemical inhibition of c-FLIP abrogated the apoptosis resistance of SWCNT-transformed cells. Whole-genome expression signature analysis confirmed these findings. This study is the first to demonstrate carbon nanotube-induced defective death receptor pathway and the role of c-FLIP in the process.

  18. Cyclic mechanical deformation stimulates human lung fibroblast proliferation and autocrine growth factor activity.

    PubMed

    Bishop, J E; Mitchell, J J; Absher, P M; Baldor, L; Geller, H A; Woodcock-Mitchell, J; Hamblin, M J; Vacek, P; Low, R B

    1993-08-01

    Cellular hypertrophy and hyperplasia and increased extracellular matrix deposition are features of tissue hypertrophy resulting from increased work load. It is known, for example, that mechanical forces play a critical role in lung development, cardiovascular remodeling following pressure overload, and skeletal muscle growth. The mechanisms involved in these processes, however, remain unclear. Here we examined the effect of mechanical deformation on fibroblast function in vitro. IMR-90 human fetal lung fibroblasts grown on collagen-coated silastic membranes were subjected to cyclical mechanical deformation (10% increase in culture surface area; 1 Hz) for up to 5 days. Cell number was increased by 39% after 2 days of deformation (1.43 +/- .01 x 10(5) cells/membrane compared with control, 1.03 +/- 0.02 x 10(5) cells; mean +/- SEM; P < 0.02) increasing to 163% above control by 4 days (2.16 +/- 0.16 x 10(5) cells compared with 0.82 +/- 0.03 x 10(5) cells; P < 0.001). The medium from mechanically deformed cells was mitogenic for IMR-90 cells, with maximal activity in the medium from cells mechanically deformed for 2 days (stimulating cell replication by 35% compared with media control; P < 0.002). These data suggest that mechanical deformation stimulates human lung fibroblast replication and that this effect is mediated by the release of autocrine growth factors.

  19. Synthesis of magnetic and fluorescent bifunctional nanocomposites and their applications in detection of lung cancer cells in humans.

    PubMed

    Ma, Jingwei; Fan, Qishi; Wang, Lianhui; Jia, Nengqin; Gu, Zhidong; Shen, Hebai

    2010-06-15

    We developed a novel strategy to detect lung cancer cells by utilizing magnetic and fluorescent bifunctional nanocomposites (BNPs) in combination with monoclonal anti-carcinoembryonic antigen (CEA) antibodies. The BNPs, consisting of silica-coated superparamagnetic nanoparticles and quantum dots (QDs), exhibited high luminescence and were easily separated in an external magnetic field. The binding specificity of the antibody-conjugated BNPs (immunonanoparticles) were confirmed via incubating with human lung adenocarcinoma SPCA-1 cells, human leukemic K562 cells and human embryonic lung fibroblasts MRC-5 cells. Further experiments demonstrated that the as-prepared immunonanoparticles can efficiently capture and detect cancer cells in pleural effusion from lung cancer patients. These results suggest that this method, of which the detection procedures are completed within 1h, could be applied to the rapid and cost-effective monitoring of cancer cells in clinical samples.

  20. Synchrotron-based Micro-CT Imaging of the Human Lung Acinus

    PubMed Central

    Litzlbauer, Horst Detlef; Korbel, Kathrin; Kline, Timothy L.; Jorgensen, Steven M.; Eaker, Diane R.; Bohle, Rainer M.; Ritman, Erik L.; Langheinrich, Alexander C.

    2012-01-01

    Structural data about the human lung fine structure are mainly based on stereological methods applied to serial sections. As these methods utilize 2D images, which are often not contiguous, they suffer from inaccuracies which are overcome by analysis of 3D micro-CT images of the never-sectioned specimen. The purpose of our study was to generate a complete data set of the intact 3-dimensional architecture of the human acinus using high-resolution synchrotron-based micro-CT (synMCT). A human lung was inflation-fixed by formaldehyde ventilation and then scanned in a 64-slice CT over its apex to base extent. Lung samples (8-mm diameter, 10-mm height, n = 12) were punched out, stained with osmium tetroxide, and scanned using synMCT at (4μm)3 voxel size. The lung functional unit (acinus, n = 8) was segmented from the 3D tomographic image using an automated tree-analysis software program. Morphometric data of the lung were analyzed by ANOVA. Intraacinar airways branching occurred over 11 generations. The mean acinar volume was 131.3 ± 29.2 mm3 (range 92.5 – 171.3 mm3) and the mean acinar surface was calculated with 1012 ± 26 cm2. The airway internal diameter (starting from the bronchiolus terminalis) decreases distally from 0.66 ± 0.04 mm to 0.34 ± 0.06 mm (p < 0.001) and remains constant after the 7th generation (p < 0.5). The length of each generation ranges between 0.52 – 0.93 mm and did not show significant differences between the second and 11th generation. The branching angle between daughter branches varies between 113–134° without significant differences between the generations (p < 0.3). This study demonstrates the feasibility of quantitating the 3D structure of the human acinus at the spatial resolution readily achievable using synMCT. PMID:20687188

  1. Synchrotron-Based Micro-CT Imaging of the Human Lung Acinus

    SciTech Connect

    Litzlbauer, H.; Korbel, K; Kline, T; Jorgensen, S; Eaker, D; Bohle, R; Ritman, E; Langheinrich, A

    2010-01-01

    Structural data about the human lung fine structure are mainly based on stereological methods applied to serial sections. As these methods utilize 2D images, which are often not contiguous, they suffer from inaccuracies which are overcome by analysis of 3D micro-CT images of the never-sectioned specimen. The purpose of our study was to generate a complete data set of the intact three-dimensional architecture of the human acinus using high-resolution synchrotron-based micro-CT (synMCT). A human lung was inflation-fixed by formaldehyde ventilation and then scanned in a 64-slice CT over its apex to base extent. Lung samples (8-mm diameter, 10-mm height, N = 12) were punched out, stained with osmium tetroxide, and scanned using synMCT at (4 {micro}m){sup 3} voxel size. The lung functional unit (acinus, N = 8) was segmented from the 3D tomographic image using an automated tree-analysis software program. Morphometric data of the lung were analyzed by ANOVA. Intra-acinar airways branching occurred over 11 generations. The mean acinar volume was 131.3 {+-} 29.2 mm{sup 3} (range, 92.5-171.3 mm{sup 3}) and the mean acinar surface was calculated with 1012 {+-} 26 cm{sup 2}. The airway internal diameter (starting from the bronchiolus terminalis) decreases distally from 0.66 {+-} 0.04 mm to 0.34 {+-} 0.06 mm (P < 0.001) and remains constant after the seventh generation (P < 0.5). The length of each generation ranges between 0.52 and 0.93 mm and did not show significant differences between the second and eleventh generation. The branching angle between daughter branches varies between 113-degree and 134-degree without significant differences between the generations (P < 0.3). This study demonstrates the feasibility of quantitating the 3D structure of the human acinus at the spatial resolution readily achievable using synMCT.

  2. Analysis of the activation routes induced by different metal oxide nanoparticles on human lung epithelial cells

    PubMed Central

    Simón-Vázquez, Rosana; Lozano-Fernández, Tamara; Dávila-Grana, Angela; González-Fernández, África

    2016-01-01

    Nanoparticles (Nps) can induce toxicity in the lung by accidental or intentional exposure. The main objective of the study reported here was to characterize the effect that four metal oxide Nps (CeO2, TiO2, Al2O3 and ZnO) had at the cellular level on a human lung epithelial cell line. This goal was achieved by studying the capacity of the Nps to activate the main mitogen-activated protein kinases (MAPKs) and the nuclear factor NFκB. Only ZnO Nps were able to activate all of the MAPKs and the release of Zn2+ ions was the main cause of activation. ZnO and Al2O3 Nps activated the NFκB pathway and induced the release of inflammatory cytokines. CeO2 and TiO2 Nps were found to have safer profiles. The graphical abstract was obtained using Servier Medical Art. PMID:28031965

  3. Lung Mucosa Lining Fluid Modification of Mycobacterium tuberculosis to Reprogram Human Neutrophil Killing Mechanisms.

    PubMed

    Arcos, Jesús; Diangelo, Lauren E; Scordo, Julia M; Sasindran, Smitha J; Moliva, Juan I; Turner, Joanne; Torrelles, Jordi B

    2015-09-15

    We have shown that human alveolar lining fluid (ALF) contains homeostatic hydrolases capable of altering the Mycobacterium tuberculosis cell wall and subsequently its interaction with human macrophages. Neutrophils are also an integral part of the host immune response to M. tuberculosis infection. Here we show that the human lung mucosa influences M. tuberculosis interaction with neutrophils, enhancing the intracellular killing of ALF-exposed M. tuberculosis and up-regulating the expression of tumor necrosis factor and interleukin 8. In contrast, ALF-exposed M. tuberculosis does not induce neutrophil apoptosis or necrosis, degranulation, or release of extracellular traps, and it decreases the oxidative response. These results suggest an important role for the human alveolar mucosa: increasing the innate capacity of the neutrophil to recognize and kill M. tuberculosis by favoring the use of intracellular mechanisms, while at the same time limiting neutrophil extracellular inflammatory responses to minimize their associated tissue damage.

  4. Lung Mucosa Lining Fluid Modification of Mycobacterium tuberculosis to Reprogram Human Neutrophil Killing Mechanisms

    PubMed Central

    Arcos, Jesús; Diangelo, Lauren E.; Scordo, Julia M.; Sasindran, Smitha J.; Moliva, Juan I.; Turner, Joanne; Torrelles, Jordi B.

    2015-01-01

    We have shown that human alveolar lining fluid (ALF) contains homeostatic hydrolases capable of altering the Mycobacterium tuberculosis cell wall and subsequently its interaction with human macrophages. Neutrophils are also an integral part of the host immune response to M. tuberculosis infection. Here we show that the human lung mucosa influences M. tuberculosis interaction with neutrophils, enhancing the intracellular killing of ALF-exposed M. tuberculosis and up-regulating the expression of tumor necrosis factor and interleukin 8. In contrast, ALF-exposed M. tuberculosis does not induce neutrophil apoptosis or necrosis, degranulation, or release of extracellular traps, and it decreases the oxidative response. These results suggest an important role for the human alveolar mucosa: increasing the innate capacity of the neutrophil to recognize and kill M. tuberculosis by favoring the use of intracellular mechanisms, while at the same time limiting neutrophil extracellular inflammatory responses to minimize their associated tissue damage. PMID:25748325

  5. ER stress and autophagy are involved in the apoptosis induced by cisplatin in human lung cancer cells

    PubMed Central

    SHI, SHAOMIN; TAN, PING; YAN, BINGDI; GAO, RONG; ZHAO, JIANJUN; WANG, JING; GUO, JIA; LI, NING; MA, ZHONGSEN

    2016-01-01

    Cisplatin [cis-diamminedichloroplatinum II (CDDP)] is one of the most classical and effective chemotherapeutic drugs for the treatment of cancers including lung cancer. However, the presence of cisplatin resistance in cancer lowers its curative effect and limits its usage in the clinic. The aim of the present study was to investigate the underlying mechanisms of cisplatin resistance in lung cancer involving endoplasmic reticulum (ER) stress and autophagy. In the present study, we detected the effect of cisplatin on cell viability, ER stress and autophagy in lung cancer cell lines A549 and H460. We also tested the effects of ER stress and autophagy on apoptosis induced by cisplatin. The results showed that cisplatin induced apoptosis, ER stress and autophagy in lung cancer cell lines. In addition, the inhibition of ER stress by 4-phenylbutyric acid (4-PBA) or tauroursodeoxycholic acid sodium (TUDC) enhanced cisplatin-induced apoptosis in the human lung cancer cells. Meanwhile, combination treatment with the autophagic inhibitor 3-methyladenine (3-MA) or chloroquine (CQ) further increased the apoptosis induced by cisplatin in the human lung cancer cells. The present study provides a novel treatment strategy - cisplatin in combination with an autophagic inhibitor or an ER stress inhibitor leads to increased apoptosis in human lung cancer cells. PMID:26985651

  6. Global DNA methylation and PTEN hypermethylation alterations in lung tissues from human silicosis

    PubMed Central

    Zhang, Xianan; Jia, Xiaowei; Mei, Liangying; Zheng, Min; Yu, Chen

    2016-01-01

    Background Silicosis is a respiratory disease caused by long-term silica dust exposure. Our previous study has demonstrated that silica mediates the activation of phosphatidylinositol 3-kinase (PI3K)/phosphatase and tensin homolog deleted on chromosome 10 (PTEN)/serine or threonine kinase (AKT)/mitogen-activated protein kinases (MAPK)/AP-1 pathway in human embryo lung fibroblasts (HELFs). The purpose of this study is to identify genome-wide aberrant DNA methylation profiling in lung tissues from silicosis patients. Methods We performed Illumina Human Methylation 450K Beadchip arrays to investigate the methylation alteration in formalin-fixed, paraffin-embedded (FFPE) lung specimens, immunohistochemistry to detect the level of c-Jun and PTEN proteins; methylation specific PCR (MS-PCR) to identify PTEN and c-Jun promoter methylation in HELFs. Results We found 86,770 CpG sites and 79,660 CpG sites significantly differed in methylation status in early-stage and advanced-stage compared with GEO normal lung methylation data. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed the methylated status of MAPK signaling pathway was considered changed. The number of PTEN and c-Jun CpG promoter methylated-sites were increased in advanced-stage. Early-stage showed the positive expression of c-Jun and PTEN protein and negative or mild expression in advanced-stage. PTEN promoter was no differentially methylated and c-Jun promoter differed at 12 and 24 h in HELFs. Conclusions Abnormal DNA methylation on genome-scale was implicated in silicosis, and PTEN promoter hypermethylation might be associated with decrease of PTEN protein. PMID:27621875

  7. Exposure of Human Lung Cells to Tobacco Smoke Condensate Inhibits the Nucleotide Excision Repair Pathway

    PubMed Central

    Holcomb, Nathaniel; Goswami, Mamta; Han, Sung Gu; Clark, Samuel; Orren, David K.; Gairola, C. Gary; Mellon, Isabel

    2016-01-01

    Exposure to tobacco smoke is the number one risk factor for lung cancer. Although the DNA damaging properties of tobacco smoke have been well documented, relatively few studies have examined its effect on DNA repair pathways. This is especially true for the nucleotide excision repair (NER) pathway which recognizes and removes many structurally diverse DNA lesions, including those introduced by chemical carcinogens present in tobacco smoke. The aim of the present study was to investigate the effect of tobacco smoke on NER in human lung cells. We studied the effect of cigarette smoke condensate (CSC), a surrogate for tobacco smoke, on the NER pathway in two different human lung cell lines; IMR-90 lung fibroblasts and BEAS-2B bronchial epithelial cells. To measure NER, we employed a slot-blot assay to quantify the introduction and removal of UV light-induced 6–4 photoproducts and cyclobutane pyrimidine dimers. We find a dose-dependent inhibition of 6–4 photoproduct repair in both cell lines treated with CSC. Additionally, the impact of CSC on the abundance of various NER proteins and their respective RNAs was investigated. The abundance of XPC protein, which is required for functional NER, is significantly reduced by treatment with CSC while the abundance of XPA protein, also required for NER, is unaffected. Both XPC and XPA RNA levels are modestly reduced by CSC treatment. Finally, treatment of cells with MG-132 abrogates the reduction in the abundance of XPC protein produced by treatment with CSC, suggesting that CSC enhances proteasome-dependent turnover of the protein that is mediated by ubiquitination. Together, these findings indicate that tobacco smoke can inhibit the same DNA repair pathway that is also essential for the removal of some of the carcinogenic DNA damage introduced by smoke itself, increasing the DNA damage burden of cells exposed to tobacco smoke. PMID:27391141

  8. Ca{sup 2+} influx and ATP release mediated by mechanical stretch in human lung fibroblasts

    SciTech Connect

    Murata, Naohiko; Ito, Satoru; Furuya, Kishio; Takahara, Norihiro; Naruse, Keiji; Aso, Hiromichi; Kondo, Masashi; Sokabe, Masahiro; Hasegawa, Yoshinori

    2014-10-10

    Highlights: • Uniaxial stretching activates Ca{sup 2+} signaling in human lung fibroblasts. • Stretch-induced intracellular Ca{sup 2+} elevation is mainly via Ca{sup 2+} influx. • Mechanical strain enhances ATP release from fibroblasts. • Stretch-induced Ca{sup 2+} influx is not mediated by released ATP or actin cytoskeleton. - Abstract: One cause of progressive pulmonary fibrosis is dysregulated wound healing after lung inflammation or damage in patients with idiopathic pulmonary fibrosis and severe acute respiratory distress syndrome. The mechanical forces are considered to regulate pulmonary fibrosis via activation of lung fibroblasts. In this study, the effects of mechanical stretch on the intracellular Ca{sup 2+} concentration ([Ca{sup 2+}]{sub i}) and ATP release were investigated in primary human lung fibroblasts. Uniaxial stretch (10–30% in strain) was applied to fibroblasts cultured in a silicone chamber coated with type I collagen using a stretching apparatus. Following stretching and subsequent unloading, [Ca{sup 2+}]{sub i} transiently increased in a strain-dependent manner. Hypotonic stress, which causes plasma membrane stretching, also transiently increased the [Ca{sup 2+}]{sub i}. The stretch-induced [Ca{sup 2+}]{sub i} elevation was attenuated in Ca{sup 2+}-free solution. In contrast, the increase of [Ca{sup 2+}]{sub i} by a 20% stretch was not inhibited by the inhibitor of stretch-activated channels GsMTx-4, Gd{sup 3+}, ruthenium red, or cytochalasin D. Cyclic stretching induced significant ATP releases from fibroblasts. However, the stretch-induced [Ca{sup 2+}]{sub i} elevation was not inhibited by ATP diphosphohydrolase apyrase or a purinergic receptor antagonist suramin. Taken together, mechanical stretch induces Ca{sup 2+} influx independently of conventional stretch-sensitive ion channels, the actin cytoskeleton, and released ATP.

  9. Unsteady-state airflow and particle deposition in a three-generation human lung geometry.

    PubMed

    Nazridoust, Kambiz; Asgharian, Bahman

    2008-04-01

    The study of particle transport and deposition in the human lung is critical in health risk assessment of air pollutants and in pharmaceutical drug delivery. Several computational fluid dynamics (CFD) studies have investigated particle deposition in the lung for simplified airflow scenarios. A shortcoming with most CFD studies is uncertainty regarding flow boundary conditions, which directly impacts airflow and particle deposition. The influence of inlet and outlet conditions on airflow and particle deposition in lung common airways was assessed here. Common airways consisted of nine airways of the human lung ahead of lobes: the trachea, main, and lobar bronchi connected as a network of cylindrical tubes with dimensions based on morphometric measurements. Three different boundary conditions were used: (1) prescribed constant flow rate at the trachea entrance and atmospheric pressure at terminal branch exits, (2) atmospheric pressure at the trachea inlet and prescribed outlet flow rates corresponding to uniform lobar expansion, and (3) the same as case (2) with exit flow rates according to nonuniform lobar expansion. Unsteady airflow fields were numerically solved for a 2-s inhalation. Spherical particles of 1 nm to 10 microm diameter were injected at the trachea inlet, and particle deposition patterns during inhalation were evaluated. A Lagrangian particle tracking method was used that included particle inertia, gravity, and Brownian motion. Predicted flows showed similar trends but with a notable difference in magnitude. Lower particle deposition was found in case (1) for all particle sizes. The differences among these cases indicated the significance of realistic boundary conditions for accurate assessment of the flow field and particle deposition.

  10. Inhibition of human lung cancer cell proliferation and survival by wine

    PubMed Central

    2014-01-01

    Background Compounds of plant origin and food components have attracted scientific attention for use as agents for cancer prevention and treatment. Wine contains polyphenols that were shown to have anti-cancer and other health benefits. The survival pathways of Akt and extracellular signal-regulated kinase (Erk), and the tumor suppressor p53 are key modulators of cancer cell growth and survival. In this study, we examined the effects of wine on proliferation and survival of human Non-small cell lung cancer (NSCLC) cells and its effects on signaling events. Methods Human NSCLC adenocarcinoma A549 and H1299 cells were used. Cell proliferation was assessed by thymidine incorporation. Clonogenic assays were used to assess cell survival. Immunoblotting was used to examine total and phosphorylated levels of Akt, Erk and p53. Results In A549 cells red wine inhibited cell proliferation and reduced clonogenic survival at doses as low as 0.02%. Red wine significantly reduced basal and EGF-stimulated Akt and Erk phosphorylation while it increased the levels of total and phosphorylated p53 (Ser15). Control experiments indicated that the anti-proliferative effects of wine were not mediated by the associated contents of ethanol or the polyphenol resveratrol and were independent of glucose transport into cancer cells. White wine also inhibited clonogenic survival, albeit at a higher doses (0.5-2%), and reduced Akt phosphorylation. The effects of both red and white wine on Akt phosphorylation were also verified in H1299 cells. Conclusions Red wine inhibits proliferation of lung cancer cells and blocks clonogenic survival at low concentrations. This is associated with inhibition of basal and EGF-stimulated Akt and Erk signals and enhancement of total and phosphorylated levels of p53. White wine mediates similar effects albeit at higher concentrations. Our data suggest that wine may have considerable anti-tumour and chemoprevention properties in lung cancer and deserves further

  11. Cantharidin induces apoptosis of H460 human lung cancer cells through mitochondria-dependent pathways.

    PubMed

    Hsia, Te-Chun; Yu, Chien-Chih; Hsu, Shu-Chun; Tang, Nou-Ying; Lu, Hsu-Feng; Huang, Yi-Ping; Wu, Shin-Hwar; Lin, Jaung-Geng; Chung, Jing-Gung

    2014-07-01

    Lung cancer is one of the leading causes of death in cancer-related diseases. Cantharidin (CTD) is one of the components of natural mylabris (Mylabris phalerata Pallas). Numerous studies have shown that CTD induced cytotoxic effects on cancer cells. However, there is no report to demonstrate that CTD induced apoptosis in human lung cancer cells. Herein, we investigated the effect of CTD on the cell death via the induction of apoptosis in H460 human lung cancer cells. Flow cytometry assay was used for examining the percentage of cell viability, sub-G1 phase of the cell cycle, reactive oxygen species (ROS) and Ca²⁺ productions and the levels of mitochondrial membrane potential (∆Ψm). Annexin V/PI staining and DNA gel electrophoresis were also used for examining cell apoptosis. Western blot analysis was used to examine the changes of apoptosis associated protein expression and confocal microscopy for examining the translocation apoptosis associated protein. Results indicated that CTD significantly induced cell morphological changes and decreased the percentage of viable H460 cells. CTD induced apoptosis based on the occurrence of sub-G1 phase and DNA fragmentation. We found that CTD increased gene expression (mRNA) of caspase-3 and -8. Moreover, CTD increased ROS and Ca2+ production and decreased the levels of ∆Ψm. Western blot analysis results showed that CTD increased the expression of cleavage caspase-3 and -8, cytochrome c, Bax and AIF but inhibited the levels of Bcl-xL. CTD promoted ER stress associated protein expression such as GRP78, IRE1α, IRE1β, ATF6α and caspase-4 and it also promoted the expression of calpain 2 and XBP-1, but inhibited calpain 1 that is associated with apoptosis pathways. Based on those observations, we suggest that CTD may be used as a novel anticancer agent for the treatment of lung cancer in the future.

  12. Multiphoton microscopy based cryo-imaging of inflated frozen human lung sections at -60°C in healthy and COPD lungs

    NASA Astrophysics Data System (ADS)

    Abraham, Thomas; Kayra, Damian; Zhang, Angela; Suzuki, Masaru; McDonough, John; Elliott, W. M.; Cooper, Joel D.; Hogg, James C.

    2013-02-01

    Lung is a complex gas exchanger with interfacial area (where the gas exchange takes place) is about the size of a tennis court. Respiratory function is linked to the biomechanical stability of the gas exchange or alveolar regions which directly depends on the spatial distributions of the extracellular matrix fibers such fibrillar collagens and elastin fibers. It is very important to visualize and quantify these fibers at their native and inflated conditions to have correct morphometric information on differences between control and diseased states. This can be only achieved in the ex vivo states by imaging directly frozen lung specimens inflated to total lung capacity. Multiphoton microscopy, which uses ultra-short infrared laser pulses as the excitation source, produces multiphoton excitation fluorescence (MPEF) signals from endogenously fluorescent proteins (e.g. elastin) and induces specific second harmonic generation (SHG) signals from non-centrosymmetric proteins such as fibrillar collagens in fresh human lung tissues [J. Struct. Biol. (2010)171,189-196]. Here we report for the first time 3D image data obtained directly from thick frozen inflated lung specimens (~0.7- 1.0 millimeter thick) visualized at -60°C without prior fixation or staining in healthy and diseased states. Lung specimens donated for transplantation and released for research when no appropriate recipient was identified served as controls, and diseased lung specimens donated for research by patients receiving lung transplantation for very severe COPD (n=4) were prepared as previously described [N. Engl. J. Med. (2011) 201, 1567]. Lung slices evenly spaced between apex and base were examined using multiphoton microscopy while maintained at -60°C using a temperature controlled cold stage with a temperature resolution of 0.1°C. Infrared femto-second laser pulses tuned to 880nm, dry microscopic objectives, and non-de-scanned detectors/spectrophotometer located in the reflection geometry were

  13. Effect of cadmium on the expression levels of interleukin-1α and interleukin-10 cytokines in human lung cells

    PubMed Central

    ODEWUMI, CAROLINE; LATINWO, LEKAN M.; SINCLAIR, ANDRE; BADISA, VEERA L.D.; ABDULLAH, AHKINYALA; BADISA, RAMESH B.

    2015-01-01

    Cadmium is an environmentally hazardous metal, which causes toxicity in humans. Inhalation of cigarette smoke and industrial fumes containing cadmium are sources of cadmium exposure. It is responsible for the malfunction of various organs, leading to disease particularly in the lungs, liver and kidneys. In the present study, the effect of cadmium chloride (CdCl2) on cell viability, and the expression levels of interleukin (IL)-1α and IL-10 cytokines at various concentrations and incubation durations were assessed in MRC-9 human normal lung and A549 human lung cancer cells to elucidate the mechanism of cadmium toxicity. Cell viability was measured using a crystal violet dye binding assay. The expression levels of the cytokines were measured by cytokine specific enzyme-linked immunosorbent assay kits. The viability assay results revealed higher sensitivity of the A549 lung cancer cells to CdCl2 compared with the normal MRC-9 lung cells. In the normal MRC-9 lung cells, higher expression levels of the cytokines were observed at the lowest CdCl2 concentration at a shorter exposure time compared with the lung cancer cells. Higher levels of the cytokines were observed in the A549 lung cancer cells at all other times and concentrations compared with the MRC-9 cells, indicating higher levels of inflammation. The cytokine levels were reduced at higher CdCl2 concentrations and longer exposure durations, demonstrating the toxic effect of cadmium. The results indicated that CdCl2 affected the expression levels of the cytokines and led to cytotoxicity in human lung cells, and suggested that compounds which reduce inflammation may prevent cadmium toxicity. PMID:26397147

  14. Targeting Interleukin-13 with Tralokinumab Attenuates Lung Fibrosis and Epithelial Damage in a Humanized SCID Idiopathic Pulmonary Fibrosis Model

    PubMed Central

    Zhang, Huilan; Oak, Sameer R.; Coelho, Ana Lucia; Herath, Athula; Flaherty, Kevin R.; Lee, Joyce; Bell, Matt; Knight, Darryl A.; Martinez, Fernando J.; Sleeman, Matthew A.; Herzog, Erica L.; Hogaboam, Cory M.

    2014-01-01

    The aberrant fibrotic and repair responses in the lung are major hallmarks of idiopathic pulmonary fibrosis (IPF). Numerous antifibrotic strategies have been used in the clinic with limited success, raising the possibility that an effective therapeutic strategy in this disease must inhibit fibrosis and promote appropriate lung repair mechanisms. IL-13 represents an attractive target in IPF, but its disease association and mechanism of action remains unknown. In the present study, an overexpression of IL-13 and IL-13 pathway markers was associated with IPF, particularly a rapidly progressive form of this disease. Targeting IL-13 in a humanized experimental model of pulmonary fibrosis using tralokinumab (CAT354) was found to therapeutically block aberrant lung remodeling in this model. However, targeting IL-13 was also found to promote lung repair and to restore epithelial integrity. Thus, targeting IL-13 inhibits fibrotic processes and enhances repair processes in the lung. PMID:24325475

  15. Chromosomal and Genetic Analysis of a Human Lung Adenocarcinoma Cell Line OM

    PubMed Central

    Li, Yong-Wu; Bai, Lin; Dai, Lyu-Xia; He, Xu; Zhou, Xian-Ping

    2016-01-01

    Background: Lung cancer has become the leading cause of death in many regions. Carcinogenesis is caused by the stepwise accumulation of genetic and chromosomal changes. The aim of this study was to investigate the chromosome and gene alterations in the human lung adenocarcinoma cell line OM. Methods: We used Giemsa banding and multiplex fluorescence in situ hybridization focusing on the human lung adenocarcinoma cell line OM to analyze its chromosome alterations. In addition, the gains and losses in the specific chromosome regions were identified by comparative genomic hybridization (CGH) and the amplifications of cancer-related genes were also detected by polymerase chain reaction (PCR). Results: We identified a large number of chromosomal numerical alterations on all chromosomes except chromosome X and 19. Chromosome 10 is the most frequently involved in translocations with six different interchromosomal translocations. CGH revealed the gains on chromosome regions of 3q25.3-28, 5p13, 12q22-23.24, and the losses on 3p25-26, 6p25, 6q26-27, 7q34-36, 8p22-23, 9p21-24, 10q25-26.3, 12p13.31-13.33 and 17p13.1-13.3. And PCR showed the amplification of genes: Membrane metalloendopeptidase (MME), sucrase-isomaltase (SI), butyrylcholinesterase (BCHE), and kininogen (KNG). Conclusions: The lung adenocarcinoma cell line OM exhibited multiple complex karyotypes, and chromosome 10 was frequently involved in chromosomal translocation, which may play key roles in tumorigenesis. We speculated that the oncogenes may be located at 3q25.3-28, 5p13, 12q22-23.24, while tumor suppressor genes may exist in 3p25-26, 6p25, 6q26-27, 7q34-36, 8p22-23, 9p21-24, 10q25-26.3, 12p13.31-13.33, and 17p13.1-13.3. Moreover, at least four genes (MME, SI, BCHE, and KNG) may be involved in the human lung adenocarcinoma cell line OM. PMID:26879013

  16. Chronic inorganic arsenic exposure in vitro induces a cancer cell phenotype in human peripheral lung epithelial cells

    SciTech Connect

    Person, Rachel J.; Olive Ngalame, Ntube N.; Makia, Ngome L.; Bell, Matthew W.; Waalkes, Michael P.; Tokar, Erik J.

    2015-07-01

    Inorganic arsenic is a human lung carcinogen. We studied the ability of chronic inorganic arsenic (2 μM; as sodium arsenite) exposure to induce a cancer phenotype in the immortalized, non-tumorigenic human lung peripheral epithelial cell line, HPL-1D. After 38 weeks of continuous arsenic exposure, secreted matrix metalloproteinase-2 (MMP2) activity increased to over 200% of control, levels linked to arsenic-induced cancer phenotypes in other cell lines. The invasive capacity of these chronic arsenic-treated lung epithelial (CATLE) cells increased to 320% of control and colony formation increased to 280% of control. CATLE cells showed enhanced proliferation in serum-free media indicative of autonomous growth. Compared to control cells, CATLE cells showed reduced protein expression of the tumor suppressor gene PTEN (decreased to 26% of control) and the putative tumor suppressor gene SLC38A3 (14% of control). Morphological evidence of epithelial-to-mesenchymal transition (EMT) occurred in CATLE cells together with appropriate changes in expression of the EMT markers vimentin (VIM; increased to 300% of control) and e-cadherin (CDH1; decreased to 16% of control). EMT is common in carcinogenic transformation of epithelial cells. CATLE cells showed increased KRAS (291%), ERK1/2 (274%), phosphorylated ERK (p-ERK; 152%), and phosphorylated AKT1 (p-AKT1; 170%) protein expression. Increased transcript expression of metallothioneins, MT1A and MT2A and the stress response genes HMOX1 (690%) and HIF1A (247%) occurred in CATLE cells possibly in adaptation to chronic arsenic exposure. Thus, arsenic induced multiple cancer cell characteristics in human peripheral lung epithelial cells. This model may be useful to assess mechanisms of arsenic-induced lung cancer. - Highlights: • Chronic arsenic exposure transforms a human peripheral lung epithelia cell line. • Cells acquire characteristics in common with human lung adenocarcinoma cells. • These transformed cells provide a

  17. Novel metastasis model of human lung cancer in SCID mice depleted of NK cells.

    PubMed

    Yano, S; Nishioka, Y; Izumi, K; Tsuruo, T; Tanaka, T; Miyasaka, M; Sone, S

    1996-07-17

    Metastasis is a critical problem in the treatment of human lung cancer. Thus, a suitable animal model of metastasis of human lung cancer is required for in vivo biological and preclinical studies. In this study, we tried to establish a suitable model for this, using SCID mice. Neither human SCLC H69/VP cells (5 x 10(6)) nor squamous-cell carcinoma RERF-LC-AI cells (1 x 10(6)), injected through a tail vein, formed metastases in untreated SCID mice. Pre-treatment of SCID mice with anti-asialo GM1 serum resulted in only a few metastases of H69/VP cells, but pre-treatment with anti-mouse IL-2 receptor beta chain Ab (TM-beta 1) resulted in numerous lymph-node metastases 56 days after tumor inoculation. H69/VP-M cells, an in vivo-selected variant line, formed significant numbers of lymph-node metastases even in SCID mice pre-treated with anti-asialo GM1 serum. SCID mice depleted of NK cells by treatment with TM-beta 1 showed different patterns of metastasis when inoculated intravenously with the 2 different human lung cancer cell lines (H69/VP and RERF-LC-AI cells): H69/VP cells formed metastases mainly in systemic lymph nodes and the liver, whereas RERF-LC-AI cells formed metastases mainly in the liver and kidneys, with only a few in lymph nodes. A histopathological study showed that the metastatic colonies consisted of cancer cells. The numbers of metastatic colonies formed by the 2 cell lines increased with the number of cells inoculated. TM-beta 1 treatment of SCID mice efficiently removed NK cells from peripheral blood for at least 6 weeks, whereas, after treatment of the mice with anti-asialo GM1 serum, NK cells were recovered within 9 days. These findings suggest that NK-cell-depleted SCID mice may be useful as a model in biological and pre-clinical studies on metastasis of human lung cancer.

  18. Development of an Ex Vivo Tissue Platform To Study the Human Lung Response to Coxiella burnetii

    PubMed Central

    Graham, Joseph G.; Winchell, Caylin G.; Kurten, Richard C.

    2016-01-01

    Coxiella burnetii is an intracellular bacterial pathogen that causes human Q fever, an acute debilitating flu-like illness that can also present as chronic endocarditis. Disease typically occurs following inhalation of contaminated aerosols, resulting in an initial pulmonary infection. In human cells, C. burnetii generates a replication niche termed the parasitophorous vacuole (PV) by directing fusion with autophagosomes and lysosomes. C. burnetii requires this lysosomal environment for replication and uses a Dot/Icm type IV secretion system to generate the large PV. However, we do not understand how C. burnetii evades the intracellular immune surveillance that triggers an inflammatory response. We recently characterized human alveolar macrophage (hAM) infection in vitro and found that avirulent C. burnetii triggers sustained interleukin-1β (IL-1β) production. Here, we evaluated infection of ex vivo human lung tissue, defining a valuable approach for characterizing C. burnetii interactions with a human host. Within whole lung tissue, C. burnetii preferentially replicated in hAMs. Additionally, IL-1β production correlated with formation of an apoptosis-associated speck-like protein containing a caspase activation and recruitment domain (ASC)-dependent inflammasome in response to infection. We also assessed potential activation of a human-specific noncanonical inflammasome and found that caspase-4 and caspase-5 are processed during infection. Interestingly, although inflammasome activation is closely linked to pyroptosis, lytic cell death did not occur following C. burnetii-triggered inflammasome activation, indicating an atypical response after intracellular detection. Together, these studies provide a novel platform for studying the human innate immune response to C. burnetii. PMID:26902725

  19. A comparison of the antigen-presenting capabilities of class II MHC-expressing human lung epithelial and endothelial cells.

    PubMed Central

    Cunningham, A C; Zhang, J G; Moy, J V; Ali, S; Kirby, J A

    1997-01-01

    Human lung alveolar epithelial cells constitutively express class II major histocompatibility complex (MHC). Human lung microvascular endothelial and small airway epithelial cells can be induced to express class II MHC by stimulation with the pro-inflammatory cytokine interferon-gamma. The levels of class II MHC on lung epithelial and endothelial cells were comparable to those seen on an Epstein-Barr virus (EBV)-transformed B-cell line. However, the costimulatory molecules B7-1 and B7-2 were not expressed. The ability of the class II MHC expressing human lung parenchymal cells to present alloantigen to CD4+ T lymphocytes was investigated. Freshly isolated human alveolar epithelial cells (type II pneumocytes) and monolayers of interferon-gamma-stimulated small airway epithelial and lung microvascular endothelial cells were co-cultured with allogeneic CD4+ T lymphocytes and proliferation determined by [3H]thymidine incorporation. A clear difference was observed between effects of the epithelial and endothelial cells on CD4+ T-lymphocyte activation. Alveolar and small airway epithelial cells failed to stimulate the proliferation of allogeneic CD4+ T lymphocytes whereas lung microvascular endothelial cells did stimulate proliferation. This difference could not be explained by the levels of class II MHC or the lack of B7-1 and B7-2 solely. Microvascular endothelial cells, and not alveolar or small airway epithelial cells, possess B7-independent costimulatory pathways. PMID:9301537

  20. Paracrine control of differentiation in the alveolar carcinoma, A549, by human foetal lung fibroblasts.

    PubMed

    Speirs, V; Ray, K P; Freshney, R I

    1991-10-01

    Synthesis of pulmonary surfactant (PS) is necessary for normal functioning of the lungs and its production is indicative of normal differentiated lung. The human alveolar carcinoma, A549, has been found to synthesis and secrete PS in vitro. The purpose of this study was to optimise the culture conditions for PS synthesis by A549 as well as to determine the potential role of foetal lung fibroblasts in the induction of PS by glucocorticoids. A549 cells growing in filter wells produced higher levels of PS in response to steroid, a 5-fold increase on the filter well compared to only a 1.5-fold increase when the cells were cultured on a conventional plastic substrate. A549 cells grown in filter wells responded to coculture with fibroblasts whether in direct contact or separated co-culture. A 20-fold increase in PS over control values was observed in separated steroid-treated co-cultures, suggesting the presence of a diffusible factor. A partially purified factor was isolated from fibroblast conditioned medium which was capable of inducing differentiation and other phenotypic changes in A549, namely induction of PS, reduction of plasminogen activator activity and reduction in the in vivo growth of A549 xenografts in nude mice. These results suggest that, under the correct conditions, A549 cells, although transformed, still retain the capacity to respond to differentiation-inducing signals from normal fibroblasts.

  1. Paracrine control of differentiation in the alveolar carcinoma, A549, by human foetal lung fibroblasts.

    PubMed Central

    Speirs, V.; Ray, K. P.; Freshney, R. I.

    1991-01-01

    Synthesis of pulmonary surfactant (PS) is necessary for normal functioning of the lungs and its production is indicative of normal differentiated lung. The human alveolar carcinoma, A549, has been found to synthesis and secrete PS in vitro. The purpose of this study was to optimise the culture conditions for PS synthesis by A549 as well as to determine the potential role of foetal lung fibroblasts in the induction of PS by glucocorticoids. A549 cells growing in filter wells produced higher levels of PS in response to steroid, a 5-fold increase on the filter well compared to only a 1.5-fold increase when the cells were cultured on a conventional plastic substrate. A549 cells grown in filter wells responded to coculture with fibroblasts whether in direct contact or separated co-culture. A 20-fold increase in PS over control values was observed in separated steroid-treated co-cultures, suggesting the presence of a diffusible factor. A partially purified factor was isolated from fibroblast conditioned medium which was capable of inducing differentiation and other phenotypic changes in A549, namely induction of PS, reduction of plasminogen activator activity and reduction in the in vivo growth of A549 xenografts in nude mice. These results suggest that, under the correct conditions, A549 cells, although transformed, still retain the capacity to respond to differentiation-inducing signals from normal fibroblasts. Images Figure 5 PMID:1654985

  2. Functional and cytometric examination of different human lung epithelial cell types as drug transport barriers

    PubMed Central

    Min, Kyoung Ah; Rosania, Gus R.; Kim, Chong-Kook; Shin, Meong Cheol

    2016-01-01

    To develop inhaled medications, various cell culture models have been used to examine the transcellular transport or cellular uptake properties of small molecules. For the reproducible high throughput screening of the inhaled drug candidates, a further verification of cell architectures as drug transport barriers can contribute to establishing appropriate in vitro cell models. In the present study, side-by-side experiments were performed to compare the structure and transport function of three lung epithelial cells (Calu-3, normal human bronchial primary cells (NHBE), and NL-20). The cells were cultured on the nucleopore membranes in the air-liquid interface (ALI) culture conditions, with cell culture medium in the basolateral side only, starting from day 1. In transport assays, paracellular transport across all three types of cells appeared to be markedly