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Sample records for brain microsomal nasup

  1. Influence of cadmium on ketamine-induced anesthesia and brain microsomal Na[sup +], K[sup +]-ATPase in mice

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    Shen, Y.; Sangiah, S. (Oklahoma State Univ., Stillwater, OK (United States))

    1994-10-01

    Cadmium is a rare metallic element, present in almost all types of food. Shellfish, wheat and rice accumulate very high amounts. Occupational and environmental pollutants are the main sources of cadmium exposure. Cadmium has a very long biologic half-life. Exposure to Cadmium causes anemia, hypertension, hepatic, renal, pulmonary and cardiovascular disorders as well as being a possible mutagen, teratogen and carcinogen. Acute cadmium treatment increased the hexobarbital sleeping time and inhibited hepatic microsomal drug metabolism due to a decrease in cytochrome P[sub 450] content. Cadmium potentiated ethanol-induced sleep in a dose-dependent manner. Cadmium has been shown to inhibit brain microsomal Na[sup +], K[sup +]-ATPase activity in vitro and in vivo. Cadmium and ethanol additively inhibited brain Na[sup +], K[sup +]-ATPase. This might be a direct interaction between cadmium and ethanol in the central nervous system. Ketamine is an intravenous anesthetic agent. It acts on central nervous system and produces [open quotes]dissociative anaesthesia.[close quotes] Ketamine provides adequate surgical anesthesia and is used alone in humans and/or combination with xylazine, an [alpha][sub 2]-adrenergic agonist in animals. It produces CNS depression, analgesia, amnesia, immobility and a feeling of dissociation from the environment. Ketamine is a non-competitive antagonist of the NMDA subset of the glutamate receptor. This perhaps results in an increase in neuronal activity leading to disorganization of normal neurotransmission and produces dissociative anesthetic state. Because it is different from most other anesthetics, ketamine may be expected to have a unique effect on brain biochemical parameters and enzymes. The purpose of this study was to examine the interactions between cadmium and ketamine on the central nervous system and ATPase, in an attempt to further understand the mechanism of action. 12 refs., 3 figs.

  2. Inhibition of rat brain microsomal cytochrome P450-dependent dealkylation activities by an oxidative stress.

    Science.gov (United States)

    Lagrange, P; El-Bachá, R D; Netter, P; Minn, A

    2001-08-01

    There is increasing evidence that an oxidative stress not only alters cellular lipids and nucleic acids, but also numerous proteins. This oxidation results in alterations of some cellular functions, either by reversible modifications allowing a post-transcriptional regulation of enzyme activities or receptor affinities, or by irreversible modifications of the protein, triggering its inactivation and destruction. In the present work, we examined the effects of an experimental oxidative stress on rat brain microsomal cytochrome P450-dependent dealkylation activities. For that purpose, superoxide anions were produced either by the NADPH-dependent redox cycling of a quinine, menadione, or by the addition of apomorphine, which produces by autoxidation both superoxide anions and apomorphine-derived quinones. The inhibition of brain cytochrome P450-dependent alkoxyresorufin O-dealkylase activities was dependent on both menadione or apomorphine concentrations. Simultaneously, an increase of microsomal carbonyl groups was recorded. Immunoblotting characterization of brain microsomal oxidized protein was carried out, using antibodies raised against 2,4-dinitrophenylhydrazine as a reagent of protein carbonyl groups, and a revelation by a chemiluminescence method. We observed an increase in cerebral CYP1A protein oxidation, related to menadione concentration, suggesting that oxidation of cytochrome P450 protein may result in its catalytic inactivation.

  3. Cytochrome P450 expression in mouse brain: specific isoenzymes involved in Phase I metabolizing system of porphyrinogenic agents in both microsomes and mitochondria.

    Science.gov (United States)

    Lavandera, Jimena; Ruspini, Silvina; Batlle, Alcira; Buzaleh, Ana María

    2015-02-01

    Brain cytochrome P450 (CYP) metabolizes a variety of drugs to produce their pharmacological effects within the brain. We have previously observed that porphyrinogenic agents altered CYP levels in brain. The aim of this work was to further study the involvement of mice brain mitochondrial and microsomal Phase I drug metabolizing system when porphyrinogenic agents, such as Enflurane, Isoflurane, allylisopropylacetamide, veronal, ethanol, and Griseofulvin were administered. To this end, CYP2E1, CYP2B1, and CYP3A4 expression were measured. NADPH cytochrome P450 reductase (CPR) expression was also determined. Western Blots were performed in microsomes and mitochondria of whole brain. Some of the drugs studied altered expression mainly in microsomes. Chronic Isoflurane augmented mitochondrial isoform, although this anaesthetic diminished microsomal expression. Ethanol and topical Griseofulvin affected expression in microsomes but not in mitochondria. CYP2E1 mitochondrial activity was induced by acute Enflurane; while the activity of the microsomal protein was enhanced in alcoholised animals. Ethanol also induced CYP2E1 expression in microsomes, although Isoflurane provoked opposite effects in mitochondria and microsomes. Expression of CPR was also induced. Several reports support an emergent role of CYP enzymes in the pathogenesis of neurological disorders, so CYP response in brain could be one of the multiples factors influencing porphyria acute attacks.

  4. An allometric study of fatty acids and sensitivity to lipid peroxidation of brain microsomes and mitochondria isolated from different bird species.

    Science.gov (United States)

    Gutiérrez, A M; Reboredo, G R; Mosca, S M; Catalá, A

    2008-07-01

    The objective of this investigation was to examine the relationship between body size, fatty acid composition and sensitivity to lipid peroxidation of mitochondria and microsomes isolated from the brain of different size bird species: manon, quail, pigeon, duck and goose, representing a 372-fold range of body mass. Fatty acids of total lipids were determined using gas chromatography and lipid peroxidation was evaluated using a chemiluminescence assay. The allometric study of the fatty acids present in brain mitochondria and microsomes of the different bird species showed a small number of significant allometric trends. In mitochondria the percentage of monounsaturated fatty acids, was significantly lower in the larger birds (r=-0.965; Pmicrosomes but not in mitochondria may indicate a preferential incorporation of this fatty acid in the brain endoplasmic reticulum of the larger bird species. The brain of all birds studied had a high content of docosahexaenoic acid. However brain mitochondria but not microsomes isolated from all the birds analyzed showed a significant decrease of arachidonic and docosahexaenoic acids during lipid peroxidation. The allometric analyses of chemiluminescence were not statistically significant. In conclusion our results show absence of correlation between the sensitivity to lipid peroxidation of brain mitochondria and microsomes with body size and maximum life span.

  5. Protective effects of riboflavin and selenium on brain microsomal Ca2+-ATPase and oxidative damage caused by glyceryl trinitrate in a rat headache model.

    Science.gov (United States)

    Nazıroğlu, Mustafa; Çelik, Ömer; Uğuz, Abdulhadi Cihangir; Bütün, Ayşe

    2015-03-01

    Migraine headaches are considered to be associated with increased mitochondrial energy metabolism. Mitochondrial oxidative stress is also important in migraine headache pathophysiology although riboflavin and selenium (Se) induced a modulator role on mitochondrial oxidative stress in the brain. The current study aimed to determine the effects of Se with/without riboflavin on the microsomal membrane Ca(2+)-ATPase (MMCA), lipid peroxidation, antioxidant, and electroencephalography (EEG) values in glyceryl trinitrate (GTN)-induced brain injury rats. Thirty-two rats were randomly divided into four groups. The first group was used as the control, and the second group was the GTN group. Se and Se plus oral riboflavin were administered to rats constituting the third and fourth groups for 10 days prior to GTN administration. The second, third, and fourth groups received GTN to induce headache. Ten hours after the administration of GTN, the EEG records and brain cortex samples were obtained for all groups. Brain cortex microsomes were obtained from the brain samples. The brain and microsomal lipid peroxidation levels were higher in the GTN group compared to the control group, whereas they were decreased by selenium and selenium + riboflavin treatments. Vitamin A, vitamin C, vitamin E, and reduced glutathione (GSH) concentrations of the brain and MMCA, GSH and glutathione peroxidase values of microsomes were decreased by the GTN administration, although the values and β-carotene concentrations were increased by Se and Se + riboflavin treatments. There was no significant change in EEG records of the four groups. In conclusion, Se with/without riboflavin administration protected against GTN-induced brain oxidative toxicity by inhibiting free radicals and the modulation of MMCA activity and supporting the antioxidant redox system.

  6. Riboflavin and vitamin E increase brain calcium and antioxidants, and microsomal calcium-ATP-ase values in rat headache models induced by glyceryl trinitrate.

    Science.gov (United States)

    Bütün, Ayşe; Nazıroğlu, Mustafa; Demirci, Serpil; Çelik, Ömer; Uğuz, Abdulhadi Cihangir

    2015-04-01

    The essential use of riboflavin is the prevention of migraine headaches, although its effect on migraines is considered to be associated with the increased mitochondrial energy metabolism. Oxidative stress is also important in migraine pathophysiology. Vitamin E is a strong antioxidant in nature and its analgesic effect is not completely clear in migraines. The current study aimed to investigate the effects of glyceryl trinitrate (GTN)-sourced exogen nitric oxide (NO), in particular, and also riboflavin and/or vitamin E on involved in the headache model induced via GTN-sourced exogen NO on oxidative stress, total brain calcium levels, and microsomal membrane Ca(2+)-ATPase levels. GTN infusion is a reliable method to provoke migraine-like headaches in experimental animals and humans. GTN resulted in a significant increase in brain cortex and microsomal lipid peroxidation levels although brain calcium, vitamin A, vitamin C, and vitamin E, and brain microsomal-reduced glutathione (GSH), glutathione peroxidase (GSH-Px), and plasma-membrane Ca(2+)-ATPase values decreased through GTN. The lipid peroxidation, GSH, vitamin A, β-carotene, vitamin C, and vitamin E, and calcium concentrations, GSH-Px, and the Ca(2+)-ATPase activities were increased both by riboflavin and vitamin E treatments. Brain calcium and vitamin A concentrations increased through riboflavin only. In conclusion, riboflavin and vitamin E had a protective effect on the GTN-induced brain injury by inhibiting free radical production, regulation of calcium-dependent processes, and supporting the antioxidant redox system. However, the effects of vitamin E on the values seem more important than in riboflavin.

  7. Electron spin resonance study of free radicals produced from ethanol and acetaldehyde after exposure to a Fenton system or to brain and liver microsomes

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    Gonthier, B.; Jeunet, A.; Barret, L. (Departement de Toxicologie, C.H.R.U. de Grenoble, (France))

    1991-09-01

    Free radical formation from ethanol and acetaldehyde was studied in the presence of a spin-trap and a NADPH generating system with a chemical model, Fenton's reagent, or by enzymatic oxidation of these solvents by rat liver and brain microsomes. The free radicals were detected by electron spin resonance spectroscopy (E.S.R.), using the spin-trapping agent, alpha-(4-pyridyl l-oxide)-N-tertbutyl-nitrone (POBN). Under such conditions, the hydroxyethyl radical derived from ethanol was obtained after both incubation in liver and brain microsomes as well as after exposure to the Fenton system. Enzymatic inhibition and activation showed that the mixed function oxidase system plays an important role in the generation of such a radical, even in the brain. Under all the experimental conditions acetaldehyde could also generate a free radical deriving directly from the parent molecule and modified by enzymatic activation or inhibition. A second, longer lasting radical was also observed in the presence of acetaldehyde. On the basis of a comparative study to a known process causing lipoperoxidation, its lipidic origin was suggested.

  8. Alpha adrenergic modulation of the Na/sup +/ pump of canine vascular smooth muscle

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    Navran, S.S.; Adair, S.E.; Allen, J.C.; Seidel, C.L.

    1986-03-01

    Some vasoactive agents, eg. beta adrenergic agonists and forskolin, stimulate the Na/sup 7/ pump by a cAMP- dependent mechanism. The authors have now demonstrated that phenylephrine (PE) stimulates the Na/sup 7/ pump in intact blood vessels as quantitated by an increased ouabain-sensitive /sup 86/Rb uptake. The stimulation is dose-dependent (ED/sub 50/, 3 x 10/sup -6/M) and blocked by phentolamine (I/sub 50/, 10/sup -7/M), prazosin (I/sub 50/, 10/sup -8/M) yohimbine (I/sub 50/, 10/sup -6/M) or elevated intracellular Na/sup +/. These data suggest that the Na/sup +/ pump stimulation is mediated through alpha/sub 1/ receptors which produce an influx of extracellular Na/sup +/. In vascular smooth muscle cell cultures PE stimulates the Na/sup +/ pump, but only when cells have been deprived of fetal calf serum (FCS). Since FCS is known to stimulate Na/sup +/influx, in the continuous presence of FCS, these cells may already be Na/sup +/-loaded and therefore refractory to further stimulation by alpha-adrenergic agents. Unlike those vasorelaxants whose mechanism involves stimulation of the Na/sup +/ pump, alpha adrenergic agents are vasoconstrictors and therefore the role of Na/sup +/ pump stimulation in this case may be as a mechanism of feedback inhibition of contractility.

  9. Subcellular fractionation of rough microsomes.

    Science.gov (United States)

    Sabatini, David D

    2014-09-02

    When eukaryotic cells are homogenized, the rough endoplasmic reticula are converted into small vesicles, called rough microsomes. Strategies for the isolation of rough microsomes are introduced here, as are methods for evaluating the purity and intactness of an isolated rough microsomal fraction.

  10. Thermodynamics of ion exchange between clinoptilolite and aqueous solutions of Na{sup +}/K{sup +} and Na{sup +}/Ca{sup 2+}

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    Pabalan, R.T. [Southwest Research Institute, San Antonio, TX (United States)

    1994-11-01

    Because of their ion-exchange, adsorption, and molecular sieve properties, zeolite minerals have generated worldwide interest for use in a broad range of applications such as nuclear and municipal waste water treatment, stack-gas cleanup, natural gas purification, petroleum production, and in agriculture and aquaculture. To provide a thermodynamic basis for understanding zeolite-water interactions in geologic systems, ion-exchange experiments were conducted at 25{degrees}C between clinoptilolite, which is the predominant zeolite mineral in altered pyroclastic and volcaniclastic rocks, and aqueous mixtures of Na{sup +}/K{sup +} and Na{sup +}/Ca{sup 2+}. Isotherm points were obtained by equilibrating Na-clinoptilolite, which was prepared from clinoptilolite-rich tuff from Death Valley Junction, California, USA, and Na{sup +}/K{sup +} and Na{sup +}/Ca{sup 2+} chloride solutions having different ionic concentration ratios, but constant total normalities of 0.5, 0.05, or 0.005 N. The experimental data were interpreted using a Margules thermodynamic formulation for zeolite solid solutions, coupled with the Pitzer model for aqueous activity coefficients. The isotherm data for 0.5 N Na{sup +}/K{sup +} and Na{sup +}/Ca{sup 2+} solutions were used to derive equilibrium constants and Gibbs free energies for the ion-exchange reactions, as well as parameters for the Margules model. Using the same parameters derived from the 0.5 N data, isotherms were calculated for the 0.05 and 0.005 N solutions. The predicted values agree very well with experimental data. The results of this study indicate that a Margules solid solution model for zeolites, coupled with an activity coefficient model for aqueous solutions (e.g., Pitzer model), can successfully describe and predict binary ion-exchange equilibria between aqueous solutions and the zeolite mineral clinoptilolite over a wide range of solution composition and concentration.

  11. Effect of an extract of Aloe vera on the biodistribution of sodium pertechnetate (Na{sup 99m}TcO{sub 4}) in rats

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    Holanda, Cecilia Maria de Carvalho Xavier [Universidade Federal do Rio Grande do Norte (UFRN), Natal, RN (Brazil). Dept. of Microbiology and Parasitology. Experimental Radiobiology and Antiparasitic Assays Lab.], e-mail: cechol@ufrnet.br; Costa, Monique Batista da; Silva, Natalia Chilinque Zambao da; Silva Junior, Mauricio Ferreira da; Barbosa, Vanessa Santos de Arruda; Silva, Roseane Pereira da [Universidade Federal do Rio Grande do Norte (UFRN), Natal, RN (Brazil); Medeiros, Aldo da Cunha [Universidade Federal do Rio Grande do Norte (UFRN), Natal, RN (Brazil). Nucleus of Experimental Surgery

    2009-07-01

    Purpose: Aloe vera is a tropical plant popularly known in Brazil as babosa. We have investigated the effect of aqueous extract of Aloe vera on the biodistribution of Na{sup 99m}TcO{sub 4} and laboratorial parameters in Wistar rats. Methods: twelve animals were divided into treated and control groups. In the treated group, Aloe vera was given by gavage (5mg/mL/day) during 10 days. The control group received sorbitol by the same way and period. One hour after the last dose, we injected 0.1mL of Na{sup 99m}TcO{sub 4} by orbital plexus. After 60 min, all the animals were killed. Samples were harvested from the brain, liver, heart, muscle, pancreas, stomach, femur, kidneys, blood, testis and thyroid and the percentage of radioactivity (% ATI/g) was determined. Biochemical dosages were performed. Results: there was a significant increase of %ATI/g in blood, femur, kidneys, liver, stomach, testis and thyroid and also in blood levels of AST and ALT. A significant decrease in levels of glucose, cholesterol, triglycerides, creatinine and urea occurred. The statistical analyses were performed by Mann-Whitney test and T-Student test (p<0.05). Conclusion: The aqueous extract of Aloe vera facilitated the uptake of Na{sup 99m}TcO{sub 4} in organs of rats and it was responsible to a high increase of levels of AST and ALT. (author)

  12. Effects of Na/sup +/ on ultraviolet light-induced photorelaxation and c-GMP levels in rabbit aorta

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    Aceto, J.F.; Raffa, R.B.; Tallarida, R.J.

    1986-03-05

    Isolated strips of rabbit aorta in a state of drug-induced contraction relax reversibly when irradiated with ultraviolet light. The authors previously found that the magnitude of the photorelaxation progressively diminished as the extracellular Na/sup +/ ion concentration was reduced from 145 mM to 85 mM. At 85 mM Na/sup +/, there was minimal photorelaxation, even though the preparation continued to respond to vasoconstricting agents. The reduction in photosensitivity is not an osmotic effect because restoration of osmolarity did not restore photosensitivity. Neither the mechanism underlaying photorelaxation nor its modification by Na/sup +/ is precisely known. In order to examine these further the authors measured cyclic GMP levels in the absence and presence of UV light at both normal and reduced Na/sup +/ levels. At 145 mM Na/sup +/, irradiation resulted in an increase of cGMP from 0.299 to 0.717 fmole/..mu..g protein. At 85 mM Na/sup +/, the corresponding levels were 0.541 and 1.24 fmole/..mu..g protein. Thus, cGMP levels increase (approximately double) with UV irradiation at both reduced and normal Na/sup +/ concentrations even though there is little or no photorelaxation in the reduced Na/sup +/ environment. The reduction in Na/sup +/ may uncouple a link between cGMP elevation and cytoplasmic calcium in the aortic cell.

  13. Effect of TGFβ on Na{sup +}/K{sup +} ATPase activity in megakaryocytes

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    Hosseinzadeh, Zohreh; Schmid, Evi; Shumilina, Ekaterina [Department of Physiology, University of Tübingen (Germany); Laufer, Stefan [Pharmaceutical Chemistry, University of Tübingen (Germany); Borst, Oliver; Gawaz, Meinrad [Cardiology and Cardiovascular Medicine, University of Tübingen (Germany); Lang, Florian, E-mail: florian.lang@uni-tuebingen.de [Department of Physiology, University of Tübingen (Germany)

    2014-09-26

    Highlights: • TGFß1 markedly up-regulates Na{sup +}/K{sup +} ATPase in megakaryocytes. • The effect is abrogated by p38-MAP kinase inhibitor skepinone. • The effect is abrogated by SGK inhibitor EMD638683. • The effect is abrogated by NF-κB inhibitor wogonin. - Abstract: The Na{sup +}/K{sup +} ATPase generates the Na{sup +} and K{sup +} concentration gradients across the plasma membrane and is thus essential for cellular electrolyte homeostasis, cell membrane potential and cell volume maintenance. A powerful regulator of Na{sup +}/K{sup +} ATPase is the serum- and glucocorticoid-inducible kinase 1 (SGK1). The most powerful known regulator of SGK1 expression is TGFß1, which is pivotal in the regulation of megakaryocyte maturation and platelet formation. Signaling involved in the upregulation of SGK1 by TGFß1 includes p38 mitogen activated protein (MAP) kinase. SGK1 in turn phosphorylates the IκB kinase (IKKα/β), which phosphorylates the inhibitor protein IκBα thus triggering nuclear translocation of nuclear factor kappa B (NF-κB). The present study explored whether TGFβ influences Na{sup +}/K{sup +} ATPase activity in megakaryocytes, and if so, whether the effect of TGß1 requires p38 MAP kinase, SGK1 and/or NF-κB. To this end, murine megakaryocytes were treated with TGFß1 and Na{sup +}/K{sup +} ATPase activity determined from K{sup +} induced current utilizing whole cell patch clamp. The pump current (I{sub pump}) was determined in the absence and presence of Na{sup +}/K{sup +} ATPase inhibitor ouabain (100 μM). TGFß1 (60 ng/ml) was added in the absence or presence of p38 MAP kinase inhibitor skepinone-L (1 μM), SGK1 inhibitor EMD638683 (50 μM) or NF-κB inhibitor wogonin (50 nM). As a result, the I{sub pump} was significantly increased by pretreatment of the megakaryocytes with TGFß1, an effect reaching statistical significance within 16 and 24 h and virtually abrogated in the presence of skepinone-L, EMD638683 or wogonin. In conclusion

  14. Molecular dynamics simulations of Na{sup +} and leucine transport by LeuT

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    Chen, Rong, E-mail: rong.chen@anu.edu.au; Chung, Shin-Ho

    2015-08-14

    Molecular dynamics simulations are used to gain insight into the binding of Na{sup +} and leucine substrate to the bacterial amino acid transporter LeuT, focusing on the crystal structures of LeuT in the outward-open and inward-open states. For both conformations of LeuT, a third Na{sup +} binding site involving Glu290 in addition to the two sites identified from the crystal structures is observed. Once the negative charge from Glu290 in the inward-open LeuT is removed, the ion bound to the third site is ejected from LeuT rapidly, suggesting that the protonation state of Glu290 regulates Na{sup +} binding and release. In Cl{sup −}-dependent transporters where Glu290 is replaced by a neutral serine, a Cl{sup −} ion would be required to replace the role of Glu290. Thus, the simulations provide insights into understanding Na{sup +} and substrate transport as well as Cl{sup −}-independence of LeuT. - Highlights: • Ion binding site involving Glu290 is identified in the outward- and inward-open LeuT. • Sodium is released from inward-open LeuT once the side chain of Glu290 is protonated. • Protonation state of Glu290 regulates sodium binding and transport in LeuT.

  15. Effect of tripanossomicide benznidazole (Rochagan) on the biodistribution of sodium pertechnetate (Na{sup 99m}TcO4) in Wistar rats

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    Barbosa, Vanessa Santos de Arruda; Holanda, Cecilia Maria de Carvalho Xavier; Silva, Roseane Pereira da; Medeiros, Aldo Cunha [Universidade Federal do Rio Grande do Norte (UFRN), Natal, RN (Brazil). Centro de Ciencias da Saude]. E-mail: vambio@oi.com.br; Oliveira, Daniel Pereira de; Silva Junior, Mauricio Ferreira da; Oliveira, Elias Herculano de [Universidade Federal do Rio Grande do Norte (UFRN), Natal, RN (Brazil). Centro de Biociencias. Dept. de Microbiologia e Parasitologia; Spyrides, Maria Helena Constantino [Universidade Federal do Rio Grande do Norte (UFRN), Natal, RN (Brazil). Dept. de Estatistica

    2008-12-15

    Benznidazole, a drug with specific anti-Trypanosoma cruzi activity, is used in the treatment of Chagas' disease. The radiopharmaceutical sodium pertechnetate (Na{sup 99m}TcO{sub 4}) is used to obtain diagnostic images of the stomach, thyroid, parathyroids, salivary glands, brain and in the study of esophageal reflux and blood flow. This study aimed at evaluating in vivo the influence of benznidazole treatment on the sodium pertechnetate biodistribution in Wistar rats. The percentage of radioactivity per gram (%ATI/g) of various organs (brain, heart, esophagus, stomach, small intestine, large intestine, spleen, liver, muscle and blood) was determined. Comparing the treated rats with the controls, we observed that sodium pertechnetate biodistribution did not change when administered to rats treated for thirty days with benznidazole. (author)

  16. The physiological significance of HKT1, a Na{sup +} - coupled high affinity K{sup +} transporter in `Triticum aestivum`

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    Box, S.; Schachtman, D.P. [University of Adelaide, SA (Australia). Department of Botany

    1997-12-31

    Full text: Several mechanisms for high affinity K{sup +} uptake by higher plants have been proposed:-an ATP-energised K:+ pump, a K{sup +}/H{sup +} antiport and a H{sup +}coupled carrier. Recently, a Na{sup +}--coupled high affinity K{sup +} transporter, HKT1, was isolated from wheat roots. Whilst Na{sup +}K{sup +} symports have been described in charophyte algae, the cloning of HKT1 from wheat is the first, evidence that this type d transport mechanism may function in higher plants. Is the activity of HKT1 an important mechanism involved in K{sup +} acquisition by wheat? The aim of this study was to assess the physiological significance of Na{sup +}- coupled high affinity K{sup +} uptake in T. aestivum. To determine whether HKT1 plays a significant role in wheat growth, we measured the dry weights and ion content of plants grown in a range of [K{sup +}], with and without Na{sup +}. To directly assess the activity of Na{sup +}- coupled K{sup +} transport, {sup 86}Rb{sup +} and {sup 22}Na{sup +} flux analyses were performed on the elongation zones and whole roots of intact seedlings, expressing a high affinity K{sup +} uptake system. The results of these growth and tracer flux studies will be discussed in relation to the expression of the gene encoding HKT1 in T. aestivum

  17. Biodistribution of the radiopharmaceutical sodium pertechnetate (Na{sup 99m}TcO{sub 4}) after massive small bowel resection in rats; Biodistribuicao do radiofarmaco pertecnetato de sodio (Na{sup 99m}TcO{sub 4}) em ratos submetidos a resseccao extensa de intestino delgado

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    Chacon, Damaso de Araujo; Araujo-Filho, Irami; Villarim-Neto, Arthur; Brandao-Neto, Jose; Medeiros, Aldo Cunha [Universidade Federal do Rio Grande do Norte (UFRN), Natal, RN (Brazil). Programa de Pos-graduacao em Ciencias da Saude]. E-mail: damasochacon@uol.com.br; Rego, Amalia Cinthia Meneses [Universidade Federal do Rio Grande do Norte (UFRN), Natal, RN (Brazil). Programa de Iniciacao Cientifica; Azevedo, Italo Medeiros [Universidade Federal do Rio Grande do Norte (UFRN), Natal, RN (Brazil). Dept. de Cirurgia; Bernardo-Filho, Mario [Universidade do Estado, Rio de Janeiro, RJ (Brazil). Dept. de Biofisica e Biometria

    2007-11-15

    Purpose: To evaluate the biodistribution of sodium pertechnetate (Na{sup 99m}TcO{sub 4}) in organs and tissues, the morphometry of remnant intestinal mucosa and ponderal evolution in rats subjected to massive resection of the small intestine. Methods:Twenty-one Wistar rats were randomly divided into three groups of 7 animals each. The short bowel (SB) group was subjected to massive resection of the small intestine; the control group (C) rats were not operated on, and soft intestinal handling was performed in sham rats. The animals were weighed weekly. On the 30th postoperative day, 0.1 mL of Na{sup 99m}TcO{sub 4}, with mean activity of 0.66 MBq was injected intravenously into the orbital plexus. After 30 minutes, the rats were killed with an overdose of anesthetic, and fragments of the liver, spleen, pancreas, stomach, duodenum, small intestine, thyroid, lung, heart, kidney, bladder, muscle, femur and brain were harvested. The biopsies were washed with 0.9% NaCl.,The radioactivity was counted using Gamma Counter Wizard{sup TM} 1470, Perkin-Elmer. The percentage of radioactivity per gram of tissue (%ATI/g) was calculated. Biopsies of the remaining jejunum were analysed by HE staining to obtain mucosal thickness. Analysis of variance (ANOVA) and the Tukey test for multiple comparisons were used, considering p<0.05 as significant. Results: There were no significant differences in %ATI/g of the Na{sup 99m}TcO{sub 4} in the organs of the groups studied (p>0.05). An increase in the weight of the SB rats was observed after the second postoperative week. The jejunal mucosal thickness of the SB rats was significantly greater than that of C and sham rats (p<0.05). Conclusion: In rats with experimentally-produced short bowel syndrome, an adaptive response by the intestinal mucosa reduced weight loss. The biodistribution of Na{sup 99m}TcO{sub 4} was not affected by massive intestinal resection, suggesting that short bowel syndrome is not the cause of misleading interpretation

  18. Preparation of rough microsomes from rat liver.

    Science.gov (United States)

    Sabatini, David D

    2014-08-01

    This protocol describes how to prepare rat liver rough microsomes that contain undegraded membrane-bound polysomes and can function very well in an in vitro translation system. It uses endogenous ribonuclease inhibitor in all steps, avoiding pelleting rough microsomes in all steps and sacrificing good recovery.

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

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    Menéndez R.

    2000-01-01

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

  20. Microsomal protein synthesis inhibition: an early manifestation of gentamicin nephrotoxicity

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    Bennett, W.M.; Mela-Riker, L.M.; Houghton, D.C.; Gilbert, D.N.; Buss, W.C.

    1988-08-01

    Aminoglycoside antibiotics achieve bacterial killing by binding to bacterial ribosomes and inhibiting protein synthesis. To examine whether similar mechanisms could be present in renal tubular cells prior to the onset of overt proximal tubular necrosis due to these drugs, we isolated microsomes from Fischer rats given 20 mg/kg gentamicin every 12 h subcutaneously for 2 days and from vehicle-injected controls. Concomitant studies of renal structure, function, and mitochondrial respiration were carried out. (3H)leucine incorporation into renal microsomes of treated animals was reduced by 21.9% (P less than 0.01), whereas brain and liver microsomes from the same animals were unaffected. Gentamicin concentration in the renal microsomal preparation was 56 micrograms/ml, a value 7- to 10-fold above concentrations necessary to inhibit bacterial growth. Conventional renal function studies were normal (blood urea, serum creatinine, creatinine clearance). Treated animals showed only a mild reduction of inulin clearance, 0.71 compared with 0.93 ml.min-1.100 g-1 in controls (P less than 0.05), and an increase in urinary excretion of N-acetylglucosaminidase of 20 compared with 14.8 units/l (P less than 0.05). Renal slice transport of p-aminohippuric acid, tetraethylammonium, and the fractional excretion of sodium were well preserved. There was no evidence, as seen by light microscopy, of proximal tubular necrosis. Mitochondrial cytochrome concentrations were normal and respiratory activities only slightly reduced. Processes similar to those responsible for bacterial killing could be involved in experimental gentamicin nephrotoxicity before overt cellular necrosis.

  1. Lipid peroxidation in adrenal and testicular microsomes.

    OpenAIRE

    1981-01-01

    Studies were carried out to determine the actions of and interactions between ascorbate, NADPH, Fe2+, and Fe3+ on lipid peroxidation in adrenal and testicular microsomes. Ascorbate-induced malonaldehyde production was maximal in adrenal and testicular microsomes at an ascorbate concentration of 1 X 10(-4)M. Fe2+, at levels between 10(-6) and 10(-3)M, produced concentration-dependent increases in lipid peroxidation in adrenal and testicular microsomes; Fe2+ had a far greater effect than Fe3+ i...

  2. Crystallization of the NADH-oxidizing domain of the Na{sup +}-translocating NADH:ubiquinone oxidoreductase from Vibrio cholerae

    Energy Technology Data Exchange (ETDEWEB)

    Tao, Minli [Department of Biochemistry, University of Zürich, Winterthurerstrasse 190, 8057 Zürich (Switzerland); Türk, Karin [School of Engineering and Science, International University Bremen, 28759 Bremen (Germany); Diez, Joachim [Swiss Light Source at Paul Scherrer Institut, 5232 Villigen PSI (Switzerland); Grütter, Markus G. [Department of Biochemistry, University of Zürich, Winterthurerstrasse 190, 8057 Zürich (Switzerland); Fritz, Günter, E-mail: guenter.fritz@uni-konstanz.de [Fachbereich Biologie, Universität Konstanz, Postfach M665, Universitätsstrasse 10, 78457 Konstanz (Germany); Steuber, Julia, E-mail: guenter.fritz@uni-konstanz.de [Department of Biochemistry, University of Zürich, Winterthurerstrasse 190, 8057 Zürich (Switzerland)

    2006-02-01

    The FAD domain of the NqrF subunit from the Na{sup +}-translocating NADH dehydrogenase from V. cholerae has been purified and crystallized. A complete data set was recorded at 3.1 Å. The Na{sup +}-translocating NADH:quinone oxidoreductase (Na{sup +}-NQR) from pathogenic and marine bacteria is a respiratory complex that couples the exergonic oxidation of NADH by quinone to the transport of Na{sup +} across the membrane. The NqrF subunit oxidizes NADH and transfers the electrons to other redox cofactors in the enzyme. The FAD-containing domain of NqrF has been expressed, purified and crystallized. The purified NqrF FAD domain exhibited high rates of NADH oxidation and contained stoichiometric amounts of the FAD cofactor. Initial crystallization of the flavin domain was achieved by the sitting-drop technique using a Cartesian MicroSys4000 robot. Optimization of the crystallization conditions yielded yellow hexagonal crystals with dimensions of 30 × 30 × 70 µm. The protein mainly crystallizes in long hexagonal needles with a diameter of up to 30 µm. Crystals diffract to 2.8 Å and belong to space group P622, with unit-cell parameters a = b = 145.3, c = 90.2 Å, α = β = 90, γ = 120°.

  3. The composition of rat liver microsomes. The structural proteins of rat liver microsomes

    Science.gov (United States)

    Ward, K. A.; Pollak, J. K.

    1969-01-01

    1. The structural-protein component of microsomal membranes was isolated by three separate methods. Analysis by polyacrylamide-gel electrophoresis indicated that the microsomal structural component is made up of a heterogeneous group of proteins. These proteins were further characterized by their phospholipid-binding capacity. The electrophoretic patterns of microsomal structural proteins were found to differ significantly from those of mitochondrial structural proteins. 2. The reticulosomal fraction was also characterized by electrophoresis with reference to total microsomal proteins, microsomal structural proteins and ribosomal proteins. The reticulosomes gave an electrophoretic pattern significantly different from those of the other three preparations examined. It is suggested that reticulosomes consist largely of enzymic proteins of the endoplasmic reticulum. ImagesPLATE 1PLATE 2PLATE 3 PMID:5810063

  4. Phosphatidylcholine mobility in bile salt depleted rat liver microsomes

    NARCIS (Netherlands)

    Oliveira Filgueiras, O.M. de; Defize, B.; Echteld, C.J.A. van; Bosch, H. van den

    1980-01-01

    Rat liver microsomes prepared by differential centrifugation are known to contain measurable levels of bile salts. More than 90% of these can be removed by passing the microsomal preparation through a Bio-Gel A-150m column. Bile salt depleted microsomes show a high level (> 95%) of mannose-6-phospha

  5. Role of the Na{sup +}/H{sup +} exchanger on the development of diabetes mellitus and its chronic complications

    Energy Technology Data Exchange (ETDEWEB)

    Sun, Yan-Ming [Department of Cardiac Care Unit, The First Affiliated Hospital of Harbin Medical University, Harbin 150001 (China); Su, Ying [Department of Endocrinology, The First Affiliated Hospital of Harbin Medical University, Harbin 150001 (China); Li, Jia; Tian, Ye [Department of Cardiac Care Unit, The First Affiliated Hospital of Harbin Medical University, Harbin 150001 (China); Wang, Lan-Feng, E-mail: wlfccu@126.com [Department of Cardiac Care Unit, The First Affiliated Hospital of Harbin Medical University, Harbin 150001 (China)

    2012-10-19

    Highlights: Black-Right-Pointing-Pointer NHE protect against intracellular hydrogen overload. Black-Right-Pointing-Pointer NHE protect {beta}-cells against strong acidification. Black-Right-Pointing-Pointer NHE inhibitors improve myocardial ischemia and reperfusion. -- Abstract: Micro- and macrovascular complications are the main cause of morbidity and mortality in diabetes mellitus. The Na{sup +}/H{sup +} exchanger (NHE) is a family of proteins which exchange Na{sup +} for H{sup +} according to their concentration gradients in an electroneutral manner. The exchanger also plays a key role in several other cellular functions including proliferation, differentiation, apoptosis, migration, and cytoskeletal organization. Since not much is known on the relationship between NHE and diabetes mellitus, this review outlines the contribution of NHE to chronic complications of diabetes mellitus, such as diabetic nephropathy; diabetic cardiomyopathy.

  6. Identification of new flavone-8-acetic acid metabolites using mouse microsomes and comparison with human microsomes.

    Science.gov (United States)

    Pham, Minh Hien; Auzeil, Nicolas; Regazzetti, Anne; Dauzonne, Daniel; Dugay, Annabelle; Menet, Marie-Claude; Scherman, Daniel; Chabot, Guy G

    2007-11-01

    Flavone-8-acetic acid (FAA) is a potent anticancer agent in mouse but has not shown activity in humans. Because FAA metabolism could play a role in this interspecies difference, our aim was to identify the metabolites formed in vitro using mouse microsomes compared with those in human microsomes. Mouse microsomes produced six metabolites as detected by reversed-phase high-performance liquid chromatography-mass spectrometry (MS). Three metabolites were identified as the 3'-, 4'-, or 6-hydroxy-FAA, by comparison with retention times and UV and MS spectra of standards. Two metabolites presented a molecular weight of 296 (FAA = 280) indicating the presence of one oxygen but did not correspond to any monohydroxylated FAA derivative. These two metabolites were identified as epoxides because they were sensitive to epoxide hydrolase. The position of the oxygen was determined by the formation of the corresponding phenols under soft acidic conditions: one epoxide yielded the 3'- and 4'-hydroxy-FAA, thus corresponding to the 3',4'-epoxy-FAA, whereas the other epoxide yielded 5- and 6-hydroxy-FAA, thus identifying the 5,6-epoxy-FAA. The last metabolite was assigned to the 3',4'-dihydrodiol-FAA because of its molecular weight (314) and sulfuric acid dehydration that indicated that the 3'- and 4'-positions were involved. Compared with mouse microsomes, human microsomes (2 pools and 15 individual microsomes) were unable to metabolize FAA to a significant extent. In conclusion, we have identified six new FAA metabolites formed by mouse microsomes, whereas human microsomes could not metabolize this flavonoid to a significant extent. The biological importance of the new metabolites identified herein remains to be evaluated.

  7. Hydroxylation of pentamidine by rat liver microsomes.

    Science.gov (United States)

    Berger, B J; Reddy, V V; Le, S T; Lombardy, R J; Hall, J E; Tidwell, R R

    1991-03-01

    The antiprotozoal/antifungal drug pentamidine [1,5-bis(4-amidinophenoxy)pentane] has been recently shown to be metabolized by rat liver fractions to at least six putative metabolites as detected by high-performance liquid chromatography. Two minor metabolites have been previously identified as N-hydroxypentamidine and N,N'-dihydroxypentamidine. In this study, the two major microsomal metabolites have been identified as the 2-pentanol and 3-pentanol analogs of pentamidine [1,5-di(4-amidinophenoxy)-2-pentanol; and 1,5-bis(4-amidinophenoxy)-3-pentanol]. As well, a seventh putative metabolite has been discovered and identified as para-hydroxybenzamidine, a fragment of the original drug. Whereas the cytochromes P-450 have been demonstrated as the enzyme system responsible for pentamidine metabolism, hydroxylation of the drug was not inducible by phenobarbital, beta-naphthoflavone, clofibrate, isosafrole, pregnenolone-16 alpha-carbonitrile, ethanol or pentamidine pretreatment of rats. The kinetics of the production of the two major microsomal metabolites has been determined as Km = 56 +/- 19 microM and Vmax = 126 +/- 21 pmol/min/mg microsomal protein for the 2-pentanol analog, and Km = 28 +/- 0.28 microM and Vmax = 195 +/- 2.4 pmol/min/mg microsomal protein for the 3-pentanol analog. Therefore, the mixed-function oxidases readily convert pentamidine to hydroxylated metabolites, but exactly which isozyme(s) of cytochrome P-450 is responsible is not clear.

  8. Effect of chloride substitution on the order–disorder transition in NaBH{sub 4} and Na{sup 11}BD{sub 4}

    Energy Technology Data Exchange (ETDEWEB)

    Olsen, Jørn Eirik [Institute for Energy Technology, Physics Department, P.O. Box 40, NO-2027 Kjeller (Norway); Karen, Pavel [University of Oslo, Department of Chemistry, P.O. Box 1033 Blindern, NO-0315 Oslo (Norway); Sørby, Magnus H. [Institute for Energy Technology, Physics Department, P.O. Box 40, NO-2027 Kjeller (Norway); Hauback, Bjørn C., E-mail: bjorn.hauback@ife.no [Institute for Energy Technology, Physics Department, P.O. Box 40, NO-2027 Kjeller (Norway)

    2014-02-25

    Graphical abstract: Interactions that order the BD{sub 4}{sup -} tetrahedra below the order–disorder transition became increasingly frustrated by the solute in the Na({sup 11}BD{sub 4}){sub 1−x}Cl{sub x} solid solutions, and the order disappears at x = 0.158. Highlights: • The order–disorder transition temperature for Na(BH{sub 4}){sub 1−x}Cl{sub x} and Na({sup 11}BD{sub 4}){sub 1−x}Cl{sub x} is highly dependent on the Cl-content, x. • The transition is characterized by DSC for Na({sup 11}BD{sub 4}){sub 1−x}Cl{sub x} for x = 0, 0.10 and 0.15. • No transition is observed for x ⩾ 0.20 on cooling to 8 K. • The crystal structures are reported for Na{sup 11}BD{sub 4} at room temperature and 8 K and Na({sup 11}BD{sub 4}){sub 1−x}Cl{sub x} (x = 0.10, 0.15, 0.20 and 0.25) at 8 K from powder neutron diffraction. -- Abstract: Phase transition associated with anion disordering over two orientations in Na{sup 11}BD{sub 4} (NaBH{sub 4}) and its solid solutions with NaCl, Na({sup 11}BD{sub 4}){sub 1−x}Cl{sub x}, is investigated with powder diffraction (neutron and synchrotron radiation), differential scanning calorimetry and Raman spectroscopy. Upon heating, the transition temperature extrapolated to zero rate of heating is 192.2 K for Na{sup 11}BD{sub 4}, ΔS = 4.41 J/mol K, hysteresis 1.7 K and the volume increase 0.43%. Thermal parameters of the transition in Na({sup 11}BD{sub 4}){sub 1−x}Cl{sub x} follow a colligative-property model of an ideal solution, with x = 0.158(1) as the critical concentration at which the ordering interactions and the transition itself are eliminated. On approaching this limit, the tetragonal distortion of the ordered structure decreases somewhat towards the cubic average, and this is associated with a partial disorder of the tetrahedral anions seen by diffraction methods. In fact, a 3% disorder is already present in the pure solvent of the solid solution (Na{sup 11}BD{sub 4}) at 8 K.

  9. Effect of the dilution factor on {sup 18}FDG and Na{sup 18}F samples for bacterial endotoxin test using PTS (portable test system)

    Energy Technology Data Exchange (ETDEWEB)

    Silveira, Marina B.; Costa, Flavia M.; Ferreira, Soraya Z., E-mail: mbs@cdtn.b [Centro de Desenvolvimento da Tecnologia Nuclear (CDTN/CNEN-MG), Belo Horizonte, MG (Brazil). Unidade de Pesquisa e Producao de Radiofarmacos

    2011-07-01

    {sup 18}FDG and Na{sup 18}F are radiopharmaceuticals produced as sterile solutions suitable for intravenous administration, which must contain no more than 175 EV/V. The most commonly used approach to detect endotoxins is the gelclot technique that requires 60 minutes for results. For radiopharmaceuticals containing short-life radionuclides, such as {sup 18}F, there is an increasing interest for faster quality control methods. FDA licensed the Endosafe, PTS, a kinetic chromogenic endotoxin detection system that takes about 15 minutes for results. As other techniques, PTS test is susceptible to interferences which can be solved by product dilution. The aim of this study was to establish the best dilution of {sup 18}FDG and Na{sup 18}F for PTS analysis. Two different dilution factors for {sup 18}FDG and 1:10 for Na{sup 18}F were essayed: 1:10 and 1:100. {sup 18}FDG and Na{sup 18} solutions were prepared by the addition of LAL reagent water. Considering the assay acceptance criteria, the best dilution factor was 1:100 for {sup 18}FDG and 1:10 for Na{sup 18}F. The recovery of the product positive control was 98-12% for {sup 18}FDG 1:100 and 104-120% for Na{sup 18}F 1:10, which were, in both cases, within the specification (50-200%) and very close to 100%. Results obtained with these dilution studies were important to establish the most appropriate and non-interfering dilution factor for {sup 18}FDG and Na{sup 18}F routine endotoxin test. (author)

  10. Burn injury enhances brain prostaglandin E2 production through induction of cyclooxygenase2 and microsomal prostaglandin E synthase in cerebral vascular endothelial cells in rats%前列腺素E2(PGE2)在烧伤后大鼠血管内皮细胞分泌的机制研究

    Institute of Scientific and Technical Information of China (English)

    刘永林; 陈能彬

    2012-01-01

    目的 明确外周烧伤是否可引起中枢神经系统血管内皮细胞前列腺素E2增加及其发生机制.方法 试验大鼠(SD大鼠)接受约25%的全层(Ⅲ度)烧伤,并进行相关治疗,在36小时后,对大鼠脑脊液进行采样,测量其中的前列腺素E2(PGE2)含量,同时对大鼠脑和脊椎组织进行免疫组化研究,对其中与前列腺素合成有关的2种重要酶,环氧化酶2(COX-2)和微粒体前列腺E2合成酶(mPGES)进行分析.结果 在烧伤后的大鼠脑脊液中,前列腺素E2的含量明显增加,但可以被环氧化酶2的选择性抑制剂NS398所抑制.在烧伤后,对大鼠中枢神经系统血管内皮细胞的免疫组化研究可以发现COX-2和mPGES.双重免疫荧光法发现这2种酶主要集中在内皮细胞的核周.结论 外周的烧伤主要通过诱导中枢神经内皮细胞产生COX-2和mPGES.这些酶可以提高脑脊液中的前列腺素E2浓度,从而激活中枢神经系统前列腺素E2的受体,产生烧伤后的全身症状.适当的应用COX-2抑制剂,使烧伤患者的PGE2水平处在对机体有利的合适范围内,不仅可以减少患者的临床的不适症状,以减轻烧伤早期损害和第二次打击时机体失控的炎症反应,而且可以减少患者感染的发生率.%Objective Methods To examine whether peripheral burn injury in rats elevates prostaglandin EZ in the central nervous system and to determine where in the central nervous system enzymes responBible for prostaglandin E2 synthesis arc expressed. Subjects: Spraguc-Dawley rats. Interventions; Rats received either approximately 25% fullthickness burn injury or sham treatment. At 36 hrs after the injury, the cerebrospinal fluid was sampled to measure prostaglandin E2,and the brain and the spinal cord were sampled for immunohistochemical detection of cyclooxygcnasc-2 and microsomal-typc prostaglandin E2 synthasc,enzymes that arc responsible for prostaglandin E2 production. Measurements and Main. Results The

  11. Interrogating Circulating Microsomes and Exosomes Using Metal Nanoparticles.

    Science.gov (United States)

    Zhou, Yi-Ge; Mohamadi, Reza M; Poudineh, Mahla; Kermanshah, Leyla; Ahmed, Sharif; Safaei, Tina Saberi; Stojcic, Jessica; Nam, Robert K; Sargent, Edward H; Kelley, Shana O

    2016-02-10

    A chip-based approach for electrochemical characterization and detection of microsomes and exosomes based on direct electro-oxidation of metal nanoparticles (MNPs) that specifically recognize surface markers of these vesicles is reported. It is found that exosomes and microsomes derived from prostate cancer cells can be identified by their surface proteins EpCAM and PSMA, suggesting the potential of exosomes and microsomes for use as diagnostic biomarkers.

  12. Metabolism of bupropion by baboon hepatic and placental microsomes.

    Science.gov (United States)

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

    2011-08-01

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

  13. Carotenoid incorporation into microsomes: yields, stability and membrane dynamics

    Science.gov (United States)

    Socaciu, Carmen; Jessel, Robert; Diehl, Horst A.

    2000-12-01

    The carotenoids β-carotene (BC), lycopene (LYC), lutein (LUT), zeaxanthin (ZEA), canthaxanthin (CTX) and astaxanthin (ASTA) have been incorporated into pig liver microsomes. Effective incorporation concentrations in the range of about 1-6 nmol/mg microsomal protein were obtained. A stability test at room temperature revealed that after 3 h BC and LYC had decayed totally whereas, gradually, CTX (46%), LUT (21%), ASTA (17%) and ZEA (5%) decayed. Biophysical parameters of the microsomal membrane were changed hardly by the incorporation of carotenoids. A small rigidification may occur. Membrane anisotropy seems to offer only a small tolerance for incorporation of carotenoids and seems to limit the achievable incorporation concentrations of the carotenoids into microsomes. Microsomes instead of liposomes should be preferred as a membrane model to study mutual effects of carotenoids and membrane dynamics.

  14. Development of cholinephosphotransferase in guinea pig lung mitochondria and microsomes.

    Science.gov (United States)

    Stith, I E; Das, S K

    1982-02-02

    Development of mitochondrial and microsomal choline phosphotransferase in the fetal guinea pig lung was investigated. The activity in fetal mitochondria was more than twice of that in fetal microsomes. However, in adult lung, the enzyme was distributed mostly in microsomes. In fetal lung, both the mitochondrial and microsomal enzyme activity was greatest at approx. 81% of the total gestation period (55 days). The specific activity in the microsomal fraction than declined until term, but increased again in the 24-h newborn from 1.0 to 2.3 nmol/min per mg protein. The activity in the mitochondrial fraction declined after 61 days (2.8 nmol/min per mg) to a minimal level at term (0.6 nmol/min per mg). Although the enzyme activity decreased from day 55 (1.2 nmol/min per mg), the amount of phosphatidylcholine gradually increased between day 55 and term.

  15. Ginkgo biloba extract alters the binding of the sodium [{sup 123}I] iodide (Na{sup 123}I) on blood constituents

    Energy Technology Data Exchange (ETDEWEB)

    Aleixo, Luiz Claudio Martins [Universidade do Estado do Rio de Janeiro, Instituto de Biologia Roberto Alcantara Gomes, Departamento de Biofisica e Biometria, 28 de Setembro, 87, 20551-030, Rio de Janeiro, RJ (Brazil); Comissao Nacional de Energia Nuclear, Instituto de Engenharia Nuclear, Cidade Universitaria, Ilha do Fundao, Via Cinco s/n, 21945-450 Rio de Janeiro (Brazil); Moreno, Silvana Ramos Farias, E-mail: srfmoreno@hotmail.com [Departamento de Patologia, Universidade Federal Fluminense, 24030-210, Niteroi, RJ (Brazil); Programa de Pos-Graduacao em Ciencias Medicas, Universidade Federal Fluminense, 24030-210, Niteroi, RJ (Brazil); Freitas, Rosimeire de Souza [Universidade do Estado do Rio de Janeiro, Instituto de Biologia Roberto Alcantara Gomes, Departamento de Biofisica e Biometria, 28 de Setembro, 87, 20551-030, Rio de Janeiro, RJ (Brazil); Thomaz, Helio [Comissao Nacional de Energia Nuclear, Instituto de Engenharia Nuclear, Cidade Universitaria, Ilha do Fundao, Via Cinco s/n, 21945-450 Rio de Janeiro (Brazil); Santos-Filho, Sebastiao David [Universidade do Estado do Rio de Janeiro, Instituto de Biologia Roberto Alcantara Gomes, Departamento de Biofisica e Biometria, 28 de Setembro, 87, 20551-030, Rio de Janeiro, RJ (Brazil)

    2012-01-15

    We evaluated the in vitro effect of an aqueous extract of Ginkgo biloba (EGb) on the distribution in blood cells (BC) and plasma (P) and on the binding of Na{sup 123}I to the blood constituents using precipitation with trichloroacetic acid. The radioactivity percentages insoluble (SF) and insoluble fraction (IF) of blood constituents were determined. The EGb interfered (p<0.05) on the distribution of Na{sup 123}I in the P (from 69.64 to 86.13) and BC (from 30.36 to 13.87) and altered the fixation of the Na{sup 123}I in IF-P and in IF-BC. - Highlights: Black-Right-Pointing-Pointer Interaction between the Ginkgo biloba and blood constituents radiolabeled. Black-Right-Pointing-Pointer Modification of the binding of sodium iodide (Na{sup 123}I) to the blood constituents. Black-Right-Pointing-Pointer This alteration should have influence in a diagnosis of nuclear medicine.

  16. A Biochemical and Morphological Study of Rat Liver Microsomes

    Science.gov (United States)

    Moulé, Y.; Rouiller, C.; Chauveau, J.

    1960-01-01

    Microsomes isolated by differential centrifugation from a rat liver homogenate in 0.88 M sucrose solution have been studied from the biochemical and morphological point of view. 1. Under these experimental conditions, the "total microsome" fraction was obtained by centrifuging the cytoplasmic extract free of nuclei and mitochondria, for 3 hours at 145,000 g. Morphologically, the total microsomes consist mainly of "rough-surfaced membranes" and "smooth" ones. 2. The total microsomes have been divided into 2 subfractions so that the 1st microsomal fraction contains the "rough" vesicles (2 hours centrifugation at 40,000 g) while the 2nd microsomal fraction consists essentially of smooth vesicles, free particles, and ferritin (centrifugation of the supernatant at 145,000 g for 3 hours). 3. By the action of 0.4 per cent sodium deoxycholate in 0.88 M sucrose, it was possible to obtain a pellet for each of the 2 fractions which consisted of dense particles, rich in RNA, poor in lipids, and which represented about 50 to 60 percent of the RNA and 10 to 15 per cent of the proteins. The results have been discussed taking into consideration the hypothesis of the presence of RNA in the membranes of microsomal vesicles. PMID:14424705

  17. Enantioselective Metabolism of Flufiprole in Rat and Human Liver Microsomes.

    Science.gov (United States)

    Lin, Chunmian; Miao, Yelong; Qian, Mingrong; Wang, Qiang; Zhang, Hu

    2016-03-23

    The enantioselective metabolism of flufiprole in rat and human liver microsomes in vitro was investigated in this study. The separation and determination were performed using a liquid chromatography system equipped with a triple-quadrupole mass spectrometer and a Lux Cellulose-2 chiral column. The enantioselective metabolism of rac-flufiprole was dramatically different in rat and human liver microsomes in the presence of the β-nicotinamide adenine dinucleotide phosphate regenerating system. The half-lives (t1/2) of flufiprole in rat and human liver microsomes were 7.22 and 21.00 min, respectively, for R-(+)-flufiprole, whereas the values were 11.75 and 17.75 min, respectively, for S-(-)-flufiprole. In addition, the Vmax of R-(+)-flufiprole was about 3-fold that of S-(-)-flufiprole in rat liver microsomes, whereas its value in the case of S-(-)-flufiprole was about 2-fold that of R-(+)-flufiprole in human liver microsomes. The CLint of rac-flufiprole also showed opposite enantioselectivy in rat and human liver microsomes. The different compositions and contents of metabolizing enzyme in the two liver microsomes might be the reasons for the difference in the metabolic behavior of the two enantiomers.

  18. Effects of abrasion and Na/sup +/ on dactyl-mediated chemoreception in mature kelp crabs, Pugettia producta (Randall)

    Energy Technology Data Exchange (ETDEWEB)

    Hamilton, K.A.; Case, J.F.

    1983-01-01

    Extracellular recordings from the mixed sensory nerves innervating the abraded dactylopodites of the kelp crab, Pugettia producta (Randall), indicate that at least some chemoreceptors and mechanoreceptors remain functional. The chemoreceptors of the abraded dactyls are sensitive to both the concentration and chemical nature of the stimulants. The responses of the chemoreceptors, but not of the mechanoreceptors, are reduced when choline is substituted for sodium in the stimulant solutions. Only chemoreception is blocked by the topical application of tetrodotoxin (TTX) to the dactyls; partial reversal of the blockage occurs with time. The differential blockage of receptor activity by low Na/sup +/ and TTX is consistent with the idea that spike initiation occurs more distally in the dendrites of the chemosensory neurons than in the mechanosensory neurons. The relevance of this to the ability of at least some abraded dactyl setae to remain functional in a long-lived, nonmolting crab is considered.

  19. Morphine metabolism in human skin microsomes.

    Science.gov (United States)

    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.

  20. Selective bioreduction of nitroxides by rat liver microsomes

    Energy Technology Data Exchange (ETDEWEB)

    Rosen, G.M.; Rauckman, E.J.; Hanck, K.W.

    1977-09-01

    Presented are possible explanations for the inability of rat liver microsomes to reduce 2-ethyl-2,4,4-trimethyl-3-oxazolidinyloxy (OXAN) even though the reduction potential for OXAN is identical to that for 2,2,6,6-tetramethylpiperidinoxyl (TEMPO) and di-tert-butyl nitroxide (DTBN), both of which are reduced by these microsomes. It is suggested that the conformation of OXAN prevents these enzymes from reducing the compound. Administration of phenobarbital was found to induce the synthesis of a form of cytochrome P-450, which enabled the rat liver microsomes to reduce the OXAN. (2 diagrams, 9 references)

  1. Metabolism of bupropion by baboon hepatic and placental microsomes

    OpenAIRE

    2011-01-01

    The aim of this investigation was to determine the biotransformation of bupropion by baboon hepatic and placental microsomes, identify the enzyme(s) catalyzing the reaction(s) and determine its kinetics. Bupropion was metabolized by baboon hepatic and placental microsomes to hydroxybupropion (OH-BUP), threo- (TB) and erythrohydrobupropion (EB). OH-bupropion was the major metabolite formed by hepatic microsomes (Km 36 ± 6 µM, Vmax 258 ± 32 pmol mg protein−1 min−1), however the formation of OH-...

  2. Thermodynamics of ion-exchange between Na{sup +}/Sr{sup 2+} solutions and the zeolite mineral clinoptilolite

    Energy Technology Data Exchange (ETDEWEB)

    Pabalan, R.T.; Bertetti, F.P. [Southwest Research Institute, San Antonio, TX (United States)

    1994-12-31

    Ion-exchange experiments were conducted at 25{degrees}C between the zeolite mineral clinoptilolite and aqueous solutions of varying equivalent ratios of Na{sup +} and Sr{sup 2+} and total concentrations of 0.005, 0.05, and 0.5 N. The experiments were designed to investigate the effects of changes in total solution concentration and in the relative concentrations of exchangeable cations on the following ion-exchange equilibrium: Sr{sup 2+} + 2NaZ {r_reversible} SrZ{sub 2} + 2Na{sup +}. Using the isotherm data at 0.05 N solution concentration, a thermodynamic model for the ion-exchange reaction was derived using a Margules formulation for the activity coefficients of zeolite components and the Pitzer ion-interaction approach for activity coefficients of aqueous ions. The results of the forward experiments showed that the ion-exchange isotherm strongly depends on the total solution concentration. Additional experiments demonstrated that the above ion-exchange reaction is reversible. The derived equilibrium constant, K, and Gibbs energy of ion-exchange, {Delta}G{sup 0}, are equal to 0.321{+-}0.021 and 2,820 {+-} 170 J/mol, respectively. Using thermodynamic parameters derived from the 0.05 N isotherm experiment, the model was used to predict isotherm values at 0.005 and 0.5 N, which showed excellent agreement with measured data. Because the thermodynamic model used in this study can be easily extended to ternary and more complicated mixtures, it may be useful for modeling ion-exchange equilibria in multicomponent geochemical systems.

  3. Genetic enhancement of microsomal epoxide hydrolase improves metabolic detoxification but impairs cerebral blood flow regulation.

    Science.gov (United States)

    Marowsky, Anne; Haenel, Karen; Bockamp, Ernesto; Heck, Rosario; Rutishauser, Sibylle; Mule, Nandkishor; Kindler, Diana; Rudin, Markus; Arand, Michael

    2016-12-01

    Microsomal epoxide hydrolase (mEH) is a detoxifying enzyme for xenobiotic compounds. Enzymatic activity of mEH can be greatly increased by a point mutation, leading to an E404D amino acid exchange in its catalytic triad. Surprisingly, this variant is not found in any vertebrate species, despite the obvious advantage of accelerated detoxification. We hypothesized that this evolutionary avoidance is due to the fact that the mEH plays a dualistic role in detoxification and control of endogenous vascular signaling molecules. To test this, we generated mEH E404D mice and assessed them for detoxification capacity and vascular dynamics. In liver microsomes from these mice, turnover of the xenobiotic compound phenanthrene-9,10-oxide was four times faster compared to WT liver microsomes, confirming accelerated detoxification. mEH E404D animals also showed faster metabolization of a specific class of endogenous eicosanoids, arachidonic acid-derived epoxyeicosatrienoic acids (EETs) to dihydroxyeicosatrienoic acids (DHETs). Significantly higher DHETs/EETs ratios were found in mEH E404D liver, urine, plasma, brain and cerebral endothelial cells compared to WT controls, suggesting a broad impact of the mEH mutant on endogenous EETs metabolism. Because EETs are strong vasodilators in cerebral vasculature, hemodynamics were assessed in mEH E404D and WT cerebral cortex and hippocampus using cerebral blood volume (CBV)-based functional magnetic resonance imaging (fMRI). Basal CBV0 levels were similar between mEH E404D and control mice in both brain areas. But vascular reactivity and vasodilation in response to the vasodilatory drug acetazolamide were reduced in mEH E404D forebrain compared to WT controls by factor 3 and 2.6, respectively. These results demonstrate a critical role for mEH E404D in vasodynamics and suggest that deregulation of endogenous signaling pathways is the undesirable gain of function associated with the E404D variant.

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

    Energy Technology Data Exchange (ETDEWEB)

    Kornbrust, D.J.

    1979-01-01

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

  5. Glucuronidation of thyroxine in human liver, jejunum, and kidney microsomes.

    Science.gov (United States)

    Yamanaka, Hiroyuki; Nakajima, Miki; Katoh, Miki; Yokoi, Tsuyoshi

    2007-09-01

    Glucuronidation of thyroxine is a major metabolic pathway facilitating its excretion. In this study, we characterized the glucuronidation of thyroxine in human liver, jejunum, and kidney microsomes, and identified human UDP-glucuronosyltransferase (UGT) isoforms involved in the activity. Human jejunum microsomes showed a lower K(m) value (24.2 microM) than human liver (85.9 microM) and kidney (53.3 microM) microsomes did. Human kidney microsomes showed a lower V(max) value (22.6 pmol/min/mg) than human liver (133.4 pmol/min/mg) and jejunum (184.6 pmol/min/mg) microsomes did. By scaling-up, the in vivo clearances in liver, intestine, and kidney were estimated to be 1440, 702, and 79 microl/min/kg body weight, respectively. Recombinant human UGT1A8 (108.7 pmol/min/unit), UGT1A3 (91.6 pmol/min/unit), and UGT1A10 (47.3 pmol/min/unit) showed high, and UGT1A1 (26.0 pmol/min/unit) showed moderate thyroxine glucuronosyltransferase activity. The thyroxine glucuronosyltransferase activity in microsomes from 12 human livers was significantly correlated with bilirubin O-glucuronosyltransferase (r = 0.855, p microsomes was mainly catalyzed by UGT1A8 and UGT1A10 and to a lesser extent by UGT1A1, and the activity in human kidney microsomes was mainly catalyzed by UGT1A7, UGT1A9, and UGT1A10. The changes of activities of these UGT1A isoforms via inhibition and induction by administered drugs as well as genetic polymorphisms may be a causal factor of interindividual differences in the plasma thyroxine concentration.

  6. Inositol trisphosphate and thapsigargin discriminate endoplasmic reticulum stores of calcium in rat brain

    DEFF Research Database (Denmark)

    Verma, A; Hirsch, D J; Hanley, M R

    1990-01-01

    ATP dependent Ca2+ accumulation into oxalate-loaded rat brain microsomes is potently inhibited by thapsigargin with an IC50 of 2 nM and maximal inhibition at 10 nM. Approximately 15% of the total A23187-releasable microsomal calcium store is insensitive to thapsigargin concentrations up to 100 mi...

  7. The microsomal dicarboxylyl-CoA synthetase.

    Science.gov (United States)

    Vamecq, J; de Hoffmann, E; Van Hoof, F

    1985-09-15

    Dicarboxylic acids are products of the omega-oxidation of monocarboxylic acids. We demonstrate that in rat liver dicarboxylic acids (C5-C16) can be converted into their CoA esters by a dicarboxylyl-CoA synthetase. During this activation ATP, which cannot be replaced by GTP, is converted into AMP and PPi, both acting as feedback inhibitors of the reaction. Thermolabile at 37 degrees C, and optimally active at pH 6.5, dicarboxylyl-CoA synthetase displays the highest activity on dodecanedioic acid (2 micromol/min per g of liver). Cell-fractionation studies indicate that this enzyme belongs to the hepatic microsomal fraction. Investigations about the fate of dicarboxylyl-CoA esters disclosed the existence of an oxidase, which could be measured by monitoring the production of H2O2. In our assay conditions this H2O2 production is dependent on and closely follows the CoA consumption. It appears that the chain-length specificity of the handling of dicarboxylic acids by this catabolic pathway (activation to acyl-CoA and oxidation with H2O2 production) parallels the pattern of the degradation of exogenous dicarboxylic acids in vivo.

  8. Human liver microsomal metabolism of (+)-discodermolide.

    Science.gov (United States)

    Fan, Yun; Schreiber, Emanuel M; Day, Billy W

    2009-10-01

    The polyketide natural product (+)-discodermolide is a potent microtubule stabilizer that has generated considerable interest in its synthetic, medicinal, and biological chemistry. It progressed to early clinical oncology trials, where it showed some efficacy in terms of disease stabilization but also some indications of causing pneumotoxicity. Remarkably, there are no reports of its metabolism. Here, we examined its fate in mixed human liver microsomes. Due to limited availability of the agent, we chose a nanoflow liquid chromatography-electrospray ionization-mass spectrometry analytical approach employing quadrupolar ion trap and quadrupole-quadrupole-time-of-flight instruments for these studies. (+)-Discodermolide was rapidly converted to eight metabolites, with the left-side lactone (net oxidation) and the right-side diene (epoxidation followed by hydrolysis, along with an oxygen insertion product) being the most metabolically labile sites. Other sites of metabolism were the allylic and pendant methyl moieties in the C12-C14 region of the molecule. The results provide information on the metabolic soft spots of the molecule and can be used in further medicinal chemistry efforts to optimize discodermolide analogues.

  9. Kavalactone metabolism in rat liver microsomes.

    Science.gov (United States)

    Fu, Shuang; Rowe, Anthony; Ramzan, Iqbal

    2012-07-01

    The specific CYP enzymes involved in kavalactone (KLT) metabolism and their kinetics have not been fully examined. This study used rat liver microsomes (RLM) to determine kavain (KA), methysticin (MTS) and desmethoxyyangonin (DMY) enzyme kinetic parameters, to elucidate the major CYP450 isoforms involved in KLT metabolism and to examine gender differences in KLT metabolism. Formation of the major KLT metabolites was first-order, consistent with classic enzyme kinetics. In both male and female RLM, clotrimazole (CLO) was the most potent inhibitor of KA and MTS metabolism. This suggests CYP3A1/3A23 (females) and CYP3A2 (males) are the main isoenzymes involved in the metabolism of these KLTs in rats, while the roles of CYP1A2, -2 C6, -2 C9, -2E1 and -3A4 are limited. Desmethoxyyangonin metabolism was equally inhibited by cimetidine (CIM) and CLO in females, and CIM and nortriptyline in males. This implies that DMY metabolism involves CYP2C6 and CYP2C11 in males, and CPY2C12 in females. CYP3A1/3A23 may also be involved in females.

  10. Role of H{sub 2}O{sub 2} on the kinetics of low-affinity high-capacity Na{sup +}-dependent alanine transport in SHR proximal tubular epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Pinto, Vanda; Pinho, Maria Joao [Institute of Pharmacology and Therapeutics, Faculty of Medicine, University of Porto, 4200-319 Porto (Portugal); Jose, Pedro A. [Center for Molecular Physiology Research, Children' s National Medical Center, Department of Pediatrics, George Washington School of Medicine and Health Sciences, Washington, DC (United States); Soares-da-Silva, Patricio, E-mail: pss@med.up.pt [Institute of Pharmacology and Therapeutics, Faculty of Medicine, University of Porto, 4200-319 Porto (Portugal)

    2010-07-30

    Research highlights: {yields} H{sub 2}O{sub 2} in excess is required for the presence of a low-affinity high-capacity component for the Na{sup +}-dependent [{sup 14}C]-L-alanine uptake in SHR PTE cells only. {yields} It is suggested that Na{sup +} binding in renal ASCT2 may be regulated by ROS in SHR PTE cells. -- Abstract: The presence of high and low sodium affinity states for the Na{sup +}-dependent [{sup 14}C]-L-alanine uptake in immortalized renal proximal tubular epithelial (PTE) cells was previously reported (Am. J. Physiol. 293 (2007) R538-R547). This study evaluated the role of H{sub 2}O{sub 2} on the Na{sup +}-dependent [{sup 14}C]-L-alanine uptake of ASCT2 in immortalized renal PTE cells from Wistar Kyoto rat (WKY) and spontaneously hypertensive rat (SHR). Na{sup +} dependence of [{sup 14}C]-L-alanine uptake was investigated replacing NaCl with an equimolar concentration of choline chloride in vehicle- and apocynin-treated cells. Na{sup +} removal from the uptake solution abolished transport activity in both WKY and SHR PTE cells. Decreases in H{sub 2}O{sub 2} levels in the extracellular medium significantly reduced Na{sup +}-K{sub m} and V{sub max} values of the low-affinity high-capacity component in SHR PTE cells, with no effect on the high-affinity low-capacity state of the Na{sup +}-dependent [{sup 14}C]-L-alanine uptake. After removal of apocynin from the culture medium, H{sub 2}O{sub 2} levels returned to basal values within 1 to 3 h in both WKY and SHR PTE cells and these were found stable for the next 24 h. Under these experimental conditions, the Na{sup +}-K{sub m} and V{sub max} of the high-affinity low-capacity state were unaffected and the low-affinity high-capacity component remained significantly decreased 1 day but not 4 days after apocynin removal. In conclusion, H{sub 2}O{sub 2} in excess is required for the presence of a low-affinity high-capacity component for the Na{sup +}-dependent [{sup 14}C]-L-alanine uptake in SHR PTE cells only

  11. Different K{sup +}-Na{sup +} inter-diffusion kinetics between the air side and tin side of an ion-exchanged float aluminosilicate glass

    Energy Technology Data Exchange (ETDEWEB)

    Jiang Liangbao; Guo Xintao; Li Xiaoyu; Li Lei; Zhang Guanli [Beijing Institute of Aeronautical Materials, Beijing 100095 (China); Yan Yue, E-mail: Yue.Yan@biam.ac.cn [Beijing Institute of Aeronautical Materials, Beijing 100095 (China)

    2013-01-15

    Highlights: Black-Right-Pointing-Pointer The difference between the diffusion characteristics of K{sup +}-Na{sup +} ions in float glass on the air and tin sides was first demonstrated. Black-Right-Pointing-Pointer The diffusion coefficient is more sensitive to temperature and time on the tin than on the air sides. Black-Right-Pointing-Pointer The activation energy on the tin side is about three times larger than that on the air side. - Abstract: The difference between the inter-diffusion kinetics of K{sup +}-Na{sup +} in the air and tin sides of an ion-exchanged float aluminosilicate glass was investigated as a function of the exchange temperature and time. The potassium concentration profiles of the ion-exchanged glass surface were experimentally measured by electron microprobe analysis, and the diffusion coefficient was calculated by the Boltzmann-Montano approach. On the tin side of the ion-exchanged glass, the diffusion of K{sup +}-Na{sup +} ions is hindered by tin. The diffusion coefficient is also more sensitive to temperature and time on the tin than on the air sides. The results would be useful in guiding the strengthening process of float glass by one step ion-exchange or two step ion-exchange to obtain engineered stress profile (ESP) glasses.

  12. The influence of post-annealing treatment on the wettability of Ag{sup +}/Na{sup +} ion-exchanged soda-lime glasses

    Energy Technology Data Exchange (ETDEWEB)

    Razzaghi, Ahmad [Department of Physics, Institute for Advanced Studies in Basic Sciences (IASBS), P.O. Box 45195-1159, Zanjan 45137-66731 (Iran, Islamic Republic of); Maleki, Maniya, E-mail: m_maleki@iasbs.ac.ir [Department of Physics, Institute for Advanced Studies in Basic Sciences (IASBS), P.O. Box 45195-1159, Zanjan 45137-66731 (Iran, Islamic Republic of); Azizian-Kalandaragh, Yashar [Department of Physics, University of Mohaghegh Ardabili, P.O. Box 179, Ardabil (Iran, Islamic Republic of)

    2013-04-01

    In this paper, the effect of thermal annealing and the duration of ion-exchange on the wetting parameters of the Ag{sup +}/Na{sup +} ion-exchanged glasses have been reported. The analysis of wetting angle in different post-annealing temperatures shows that the wetting angle is increased by increasing the annealing temperature. The wetting parameters of Ag{sup +}/Na{sup +} ion-exchanged glasses at different ion-exchanged periods of time have been also investigated. Scanning electron microscopy (SEM), UV–Visible spectroscopy and Fourier transform infrared (FTIR) spectroscopy have been used for determination of surface morphology and composition analysis of the prepared samples. The results of SEM show changes in the surface of the samples for different post-annealing temperatures. The optical characterization using UV–Vis spectroscopy shows an increase in the intensity of the absorption peak with increasing the ion-exchange duration. The FTIR spectroscopy confirms the formation of silver oxide material on the surface of Ag{sup +}/Na{sup +} ion-exchanged glasses.

  13. ON THE MECHANISM OF DRUG HYDROXYLATION IN RAT LIVER MICROSOMES

    Science.gov (United States)

    Orrenius, Sten

    1965-01-01

    The TPNH- and O2-dependent drug hydroxylation system of liver microsomes has been studied using normal rats and rats in which the drug-hydroxylating activity has been enhanced by repeated injections of phenobarbital. The oxidative demethylation of aminopyrine is employed as an assay. Optimal conditions for the assay with regard to the concentrations of TPNH and aminopyrine are established. TPN inhibits the reaction in a competitive manner, similarly to its effect on the microsomal TPNH-cytochrome c reductase. Drug hydroxylation, but not the "TPNH oxidase," TPNH-cytochrome c, -2,6-dichlorophenolindophenol, or -neotetrazolium reductase reaction, or the TPNH-dependent lipid peroxidation, is blocked by carbon monoxide. Microsomes from phenobarbital-treated rats exhibit increased activities of the various TPNH-linked reductase reactions, parallel to the increased drug hydroxylation activity, whereas the "TPNH oxidase" activity does not change appreciably. Measurements with microsomes from drug-treated animals reveal a 1:1:1 stoichiometry of aminopyrine-dependent oxygen uptake, TPNH oxidation, and formaldehyde formation. Attempts to solubilize the drug-hydroxylating enzyme system are also presented. It is concluded that the drug-hydroxylating enzyme system involves the microsomal TPNH-cytochrome c reductase and CO-binding pigment, and a hypothetic reaction scheme accounting for the data presented is proposed. PMID:19866674

  14. In vitro identification of metabolitesof verapamil in rat liver microsomes

    Institute of Scientific and Technical Information of China (English)

    LuSUN; Shu-qiuZHANG; Da-fangZHONG

    2004-01-01

    AIM: To investigate the metabolism of verapamil at low concentrations in rat liver microsomes. METHODS: Liver microsomes of Wistar rats were prepared using ultracentrifuge method. The in vitro metabolism of verapamil was studied with the rat liver microsomal incubation at concentration of 1.0 μmol/L and 5.0 μmol/L. The metabolites were separated and assayed by liquid chromatography-ion trap mass spectrometry (LC/MSn), and further identified by comparison of their mass spectra and chromatographic behaviors with reference substances. RESULTS: Eightmetabolites, including two novel metabolites (M4 and MS), were found in rat liver microsomal incubates. They were identified as O-demethyl-verapamil isomers (M1 - M4), N-dealkylated derivatives of verapamil (MS-MT), and N, O-didemethyl-verapamil (MS). CONCLUSION: O-Demethylation and N-dealkylation were the main metabolic pathways of verapamil at low concentrations in rat liver microsomes, and the relative proportion of them in verapamil metabolism changed with different substrate concentrations.

  15. Coordinated role of voltage-gated sodium channels and the Na{sup +}/H{sup +} exchanger in sustaining microglial activation during inflammation

    Energy Technology Data Exchange (ETDEWEB)

    Hossain, Muhammad M. [Department of Environmental and Occupational Medicine and Environmental and Occupational Health Sciences Institute, Rutgers Robert Wood Johnson Medical School, Piscataway, NJ (United States); Sonsalla, Patricia K. [Department of Neurology, Rutgers Robert Wood Johnson Medical School, Piscataway, NJ (United States); Richardson, Jason R., E-mail: jricha3@eohsi.rutgers.edu [Department of Environmental and Occupational Medicine and Environmental and Occupational Health Sciences Institute, Rutgers Robert Wood Johnson Medical School, Piscataway, NJ (United States)

    2013-12-01

    Persistent neuroinflammation and microglial activation play an integral role in the pathogenesis of many neurological disorders. We investigated the role of voltage-gated sodium channels (VGSC) and Na{sup +}/H{sup +} exchangers (NHE) in the activation of immortalized microglial cells (BV-2) after lipopolysaccharide (LPS) exposure. LPS (10 and 100 ng/ml) caused a dose- and time-dependent accumulation of intracellular sodium [(Na{sup +}){sub i}] in BV-2 cells. Pre-treatment of cells with the VGSC antagonist tetrodotoxin (TTX, 1 μM) abolished short-term Na{sup +} influx, but was unable to prevent the accumulation of (Na{sup +}){sub i} observed at 6 and 24 h after LPS exposure. The NHE inhibitor cariporide (1 μM) significantly reduced accumulation of (Na{sup +}){sub i} 6 and 24 h after LPS exposure. Furthermore, LPS increased the mRNA expression and protein level of NHE-1 in a dose- and time-dependent manner, which was significantly reduced after co-treatment with TTX and/or cariporide. LPS increased production of TNF-α, ROS, and H{sub 2}O{sub 2} and expression of gp91{sup phox}, an active subunit of NADPH oxidase, in a dose- and time-dependent manner, which was significantly reduced by TTX or TTX + cariporide. Collectively, these data demonstrate a closely-linked temporal relationship between VGSC and NHE-1 in regulating function in activated microglia, which may provide avenues for therapeutic interventions aimed at reducing neuroinflammation. - Highlights: • LPS causes immediate increase in sodium through VGSC and subsequently through the NHE-1. • Inhibition of VGSC reduces increases in NHE-1 and gp91{sup phox}. • Inhibition of VGSC and NHE-1 reduces NADPH oxidase-mediated Tnf-α, ROS, and H{sub 2}O{sub 2} production. • NHE-1 and Na{sub v}1.6 may be viable targets for therapeutic interventions to reduce neuroinflammation in neurodegenerative disease.

  16. Stereoselective propranolol metabolism in two drug induced rat hepatic microsomes

    Institute of Scientific and Technical Information of China (English)

    Xin Li; Su Zeng

    2000-01-01

    AIM To study the influence of inducers BNF and PB on the stereoselective metabolism of propranolol in rat hepatic microsomes.METHODS Phase Ⅰ metabolism of propranolol was studied by using the microsomes induced by BNF and PB and the non-induced microsome as the control. The enzymatic kinetic parameters of propranolol enantiomers were calculated by regression analysis of Lineweaver-Burk plots.Propranolol concentrations were assayed by HPLC.RESULTS A RP-HPLC method was developed to determine propranolol concentration in rat hepatic microsomes. The linearity equations for R( + )-propranolol and S ( - )-propranolol were A=705.7C+ 311.2C (R =0.9987) and A=697.2C +311.4C (R = 0.9970) respectively. Recoveries of each enantiomer were 98.9%, 99.5%, 101.0% at 60 μmol/L, 120 μmol/L, 240 μmol/L respectively. At the concentration level of 120 μmol/L, propranolol enantiomers were metabolized at different rates in different microsomes. The concentration ratio R (+)/S (-) of control and PB induced microsomes increased with time, whereas that of microsome induced by BNF decreased. The assayed enzyme parameters were: 1. Km. Control group: R( + )30±8, S( - )18 ± 5; BNF group: R( + )34 ± 3, S (-)39±7; PB group: R(+)38±17, S(-)36± 10.2. Vmax. Control group: R(+ )1.5 ±0.2, S( - )2.9±0.3; BNF group: R(+)3.8±0.3, S(-)3.3±0.5; PB group: R( + )0.07±0.03, S( - )1.94±0.07.3.Clint. Control group: R( + )60±3, S(- )170±30; BNF group: R( + )111.0 ±1, S(- ) 84±5; PB group: R(+)2.0 ± 2, S(- )56.0 ± 1. The enzyme parameters compared with unpaired t tests showed that no stereoselectivity was observed in enzymatic affinity of three microsomes to enantiomers and their catalytic abilitieswere quite different and had stereoselectivities. Compared with the control,microsome induced by BNF enhanced enzyme activity to propranolol R ( + )-enantiomer, and microsome induced by PB showed less enzyme activity to propranolol S(- )-enantiomer which remains the same stereoselectivities as

  17. Preparation of microsomes to study Ca2+ channels.

    Science.gov (United States)

    Bezprozvanny, Ilya

    2013-11-01

    Native cerebellar microsomes are used to study the properties of native cerebellar inositol(1,4,5)-trisphosphate receptor 1 (InsP3R1) and ryanodine receptor 1 (RyanR1) Ca(2+) release channels. Additionally, microsomes prepared from Sf9 cells infected with InsP3R- or RyanR-expressing recombinant baculoviruses can be used to compare properties of different InsP3R and RyanR isoforms and to perform structure-function studies of both types of receptors. This protocol describes how to prepare ER microsomes from native cerebellar tissue or Sf9 cells infected with InsP3R- or RyanR-expressing recombinant baculoviruses for planar lipid bilayer (also called black lipid membranes or BLM) experiments. Prepared material is aliquoted and can be stored at -80°C freezer for many months before BLM experiments.

  18. Acyl-CoA synthetase activity links wild-type but not mutant a-Synuclein to brain arachidonate metabolism

    DEFF Research Database (Denmark)

    Golovko, Mikhail; Rosenberger, Thad; Færgeman, Nils J.;

    2006-01-01

    an established steady-state kinetic model. Liver was used as a negative control, and no changes were observed between groups. In Snca-/- brains, there was a marked reduction in 20:4n-6-CoA mass and in microsomal acyl-CoA synthetase (Acsl) activity toward 20:4n-6. Microsomal Acsl activity was completely restored...

  19. Biotransformation of myrislignan by rat liver microsomes in vitro.

    Science.gov (United States)

    Li, Fei; Yang, Xiu-Wei

    2008-02-01

    Myrislignan (1), erythro-(1R,2S)-2-(4-allyl-2,6-dimethoxyphenoxyl)-1-(4-hydroxy-3-methoxyphenyl) propan-1-ol, is a major acyclic neolignan in seeds of Myristica fragrans. Studies have suggested that myrislignan may deter feeding activity, but little is known about its metabolism. We investigated the biotransformation of myrislignan by rat liver microsomes in vitro. Seven metabolites were produced by liver microsomes from rats pre-treated with sodium phenobarbital. These were identified, using spectroscopic methods, as myrislignanometins A-G (2-8), respectively.

  20. Two New Lactones Metabolized from Isoline by Rat Liver Microsomes

    Institute of Scientific and Technical Information of China (English)

    Jun TANG; Zheng Tao WANG; Teruaki AKAO; Norio NAKAMURA; Masao HATTORI

    2003-01-01

    Two new metabolites, namely bisline lactone and isolinecic acid lactone, were isolated from the resultant incubates after a scale-up incubation of isoline with rat liver microsomes. Their structures were determined by spectroscopic data, especially those from 1D and 2D NMR experiments.

  1. Preparation of crude rough microsomes from tissue culture cells.

    Science.gov (United States)

    Sabatini, David D

    2014-09-02

    There are various procedures for isolating microsomal fractions from tissue culture cells. The essential conditions for each step of one procedure are described here. Notes for special circumstances are included so that the procedure can be modified according to the experimental purpose.

  2. A Comparative Study of the Metabolism of 6-Methylbenzo [A] Pyrene and Benzo [A] Pyrene by Rat Liver Microsomes

    Science.gov (United States)

    1984-02-24

    Rat Liver Microsomes Name...pyrena by Rat Liver Microsomes Karen Lee Hamernik, Doctor of Philosophy, 1984 Dissertation directed by: Shan K. Yang, Ph.D., Professor, Department of...Microsomal Enzymes 85 Metabolism of 6-OHMBaP by Rat Liver Microsomes 94 Identification of 6-OHMBaP Metabolites 94 UV absorption spectral analysis

  3. Age-dependent changes in diastolic Ca{sup 2+} and Na{sup +} concentrations in dystrophic cardiomyopathy: Role of Ca{sup 2+} entry and IP{sub 3}

    Energy Technology Data Exchange (ETDEWEB)

    Mijares, Alfredo [Instituto Venezolano de Investigaciones Científicas, Centro de Biofísica y Bioquímica, Caracas (Venezuela, Bolivarian Republic of); Altamirano, Francisco [Department of Molecular Biosciences, School of Veterinary Medicine, University of California, Davis, CA 95616 (United States); Kolster, Juan [Centro de Investigaciones Biomédicas, México D.F. (Mexico); Adams, José A. [Division of Neonatology, Mount Sinai Medical Center, Miami, FL 33140 (United States); López, José R., E-mail: jrlopez@ucdavis.edu [Instituto Venezolano de Investigaciones Científicas, Centro de Biofísica y Bioquímica, Caracas (Venezuela, Bolivarian Republic of); Department of Molecular Biosciences, School of Veterinary Medicine, University of California, Davis, CA 95616 (United States)

    2014-10-03

    Highlights: • Age-dependent increase in [Ca{sup 2+}]{sub d} and [Na{sup +}]{sub d} in mdx cardiomyocytes. • Gadolinium significantly reduced both [Ca{sup 2+}]{sub d} and [Na{sup +}]{sub d} at all ages. • IP{sub 3}-pathway inhibition reduced cations concentrations in dystrophic cardiomyocytes. - Abstract: Duchenne muscular dystrophy (DMD) is a lethal X-inherited disease caused by dystrophin deficiency. Besides the relatively well characterized skeletal muscle degenerative processes, DMD is also associated with a dilated cardiomyopathy that leads to progressive heart failure at the end of the second decade. The aim of the present study was to characterize the diastolic Ca{sup 2+} concentration ([Ca{sup 2+}]{sub d}) and diastolic Na{sup +} concentration ([Na{sup +}]{sub d}) abnormalities in cardiomyocytes isolated from 3-, 6-, 9-, and 12-month old mdx mice using ion-selective microelectrodes. In addition, the contributions of gadolinium (Gd{sup 3+})-sensitive Ca{sup 2+} entry and inositol triphosphate (IP{sub 3}) signaling pathways in abnormal [Ca{sup 2+}]{sub d} and [Na{sup +}]{sub d} were investigated. Our results showed an age-dependent increase in both [Ca{sup 2+}]{sub d} and [Na{sup +}]{sub d} in dystrophic cardiomyocytes compared to those isolated from age-matched wt mice. Gd{sup 3+} treatment significantly reduced both [Ca{sup 2+}]{sub d} and [Na{sup +}]{sub d} at all ages. In addition, blockade of the IP{sub 3}-pathway with either U-73122 or xestospongin C significantly reduced ion concentrations in dystrophic cardiomyocytes. Co-treatment with U-73122 and Gd{sup 3+} normalized both [Ca{sup 2+}]{sub d} and [Na{sup +}]{sub d} at all ages in dystrophic cardiomyocytes. These data showed that loss of dystrophin in mdx cardiomyocytes produced an age-dependent intracellular Ca{sup 2+} and Na{sup +} overload mediated at least in part by enhanced Ca{sup 2+} entry through Gd{sup 3+} sensitive transient receptor potential channels (TRPC), and by IP{sub 3} receptors.

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

    Science.gov (United States)

    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.

  5. The Na{sup +}/K{sup +} -pump in rat peritoneal mast cells: Some aspects of regulatio of activity and cellular fusion

    Energy Technology Data Exchange (ETDEWEB)

    Knudsen, T. [Odense Univ., Dept. of Pharmacology, Inst. of Medical Biology, The Faculty of Health Scineces (Denmark)

    1995-12-31

    The mast cell contains potent mediators of inflammation which are released after IgE-directed and non-IgE-directed stimulation of the cell. This highly specialized cell is therefore ascribed a role in the pathogenesis of disease states in which the inflammatory response plays a role for the development of the clinical symptoms. Thus, besides being of interest in basic research, studies of the cellular processes leading to release of inflammatory mediators from the mast cell also also have important clinical implications. The aim of the present work has been to document the existence of the Na{sup +}/K{sup +}-pump in rat peritoneal mast cells, to investigate the regulation of the pump activity and to explore whether modulation of the pump activity interferes with the cellular stimulus/secretion coupling mechanism. The Na{sup +}/K{sup +}-pump activity following stimulation of the mast cell was also investigated. The pump activity was assessed as the ouabain-sensitive cellular potassium uptake with {sup 86}Rb{sup +} as a tracer for potassium. The histamine release from the mast cell following IgE-directed and non-IgE-directed stimulation of the cell was used as a parameter of cellular degranulation. Histamine was measured by spectrofluorometry. Besides describing aspects of the function and regulation of the Na{sup +}/K{sup +}-pump in the rat peritoneal mast cell, this thesis points to the potential role of sodium transport mechanisms in mast cell physiology. Pharmacological manipulations of such transport mechanisms might in the future add to the treatment of allergic diseases. (au) 253 refs.

  6. Parathyroid hormone inhibition of Na{sup +}/H{sup +} exchanger 3 transcription: Intracellular signaling pathways and transcription factor expression

    Energy Technology Data Exchange (ETDEWEB)

    Neri, Elida Adalgisa; Bezerra, Camila Nogueira Alves, E-mail: camilab@icb.usp.br; Queiroz-Leite, Gabriella Duarte; Polidoro, Juliano Zequini; Rebouças, Nancy Amaral

    2015-06-12

    The main transport mechanism of reabsorption of sodium bicarbonate and fluid in the renal proximal tubules involves Na{sup +}/H{sup +} exchanger 3 (NHE3), which is acutely and chronically downregulated by parathyroid hormone (PTH). Although PTH is known to exert an inhibitory effect on NHE3 expression and transcription, the molecular mechanisms involved remain unclear. Here, we demonstrated that, in opossum kidney proximal tubule (OKP) cells, PTH-induced inhibition of Nhe3 gene promoter occurs even in the core promoter that controls expression of the reporter gene. We found that inhibition of the protein kinase A (PKA) and Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathways transformed PTH from an inhibitor of promoter activity into an activator of that same activity, as did point mutations in the EGR1, Sp1, and Sp3 binding consensus elements in the promoter. In nuclear extracts of PTH-treated OKP cells, we also observed increased expression of EGR1 mRNA and of some Sp3 isoforms. Electrophoretic mobility shift assay showed a supershift of the −61 to −42-bp probe with an anti-EGR1 antibody in PTH-treated cells, suggesting that EGR1 binding is relevant for the inhibitory activity of PTH. We conclude that PTH-induced inhibition of NHE3 transcription is related to higher EGR1 expression; to EGR1 binding to the proximal and core promoters; and to PKA and JAK/STAT pathway activation. This mechanism might be responsible, at least in part, for lower NHE3 expression and sodium reabsorption in renal proximal tubules in the presence of high PTH levels. - Highlights: • PTH regulation of Nhe3 promoter depends on EGR1 binding. • EGR1, PKA and JAK/STAT are involved in PTH inhibition of the Nhe3 promoter. • PTH alters expression of EGR1 and Sp3. • PTH inhibits the Nhe3 promoter by regulating PKA and JAK/STAT signaling.

  7. Synthesis and photoluminescence properties of LaAlO{sub 3}:Mn{sup 4+}, Na{sup +} deep red-emitting phosphor

    Energy Technology Data Exchange (ETDEWEB)

    Cao, Renping; Ceng, Dong; Yu, Xiaoguang; Guo, Siling; Zheng, Guotai [Jinggangshan University, College of Mathematics and Physics, Ji' an (China); Liu, Pan [Jinggangshan University, Scientific Research Office, Ji' an (China)

    2016-04-15

    LaAlO{sub 3}:Mn{sup 4+} and LaAlO{sub 3}:Mn{sup 4+}, Na{sup +} deep red-emitting phosphors are synthesized by a solid-state reaction method in air. Their crystal structures, lifetimes, and luminescence properties are investigated, respectively. PLE spectrum monitored at 730 nm contains three PLE bands peaking at ∝276, 325, and 500 nm within the range 200-550 nm, and PL spectrum with excitation 325 nm exhibits two PL band peaks located at ∝703 and 730 nm owing to anti-stokes vibronic sidebands associated with the excited state {sup 2}E of Mn{sup 4+} ion and the {sup 2}E → {sup 4}A{sub 2} transition of Mn{sup 4+} ion, respectively. The optimal Mn{sup 4+} doping concentration is ∝0.8 mol%. Lifetime of LaAl{sub 0.992}O{sub 3}:0.8 %Mn{sup 4+} phosphor is ∝0.92 ms. Na{sup +} ion as charge compensator can improve obviously the luminescence properties of LaAlO{sub 3}:Mn{sup 4+} phosphor due to the charge compensation. The luminous mechanism of Mn{sup 4+} ion is explained by using Tanabe-Sugano diagram of Mn{sup 4+} ion in octahedral crystal field. The contents of this paper will be helpful to develop novel Mn{sup 4+}-doped materials and improve their luminescence properties. (orig.)

  8. In vitro biotransformation of flavonoids by rat liver microsomes

    DEFF Research Database (Denmark)

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

    1998-01-01

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

  9. [Enzyme kinetics of ligustilide metabolism in rat liver microsomes].

    Science.gov (United States)

    Qian, Min; Shi, Li-fu; Hu, Jin-hong

    2009-04-01

    To study the enzyme kinetics of ligustilide metabolism and the effects of selective CYP450 inhibitors on the metabolism of ligustilide in liver microsomes of rat, a LC-MS method was established for quantitative analysis of ligustilide in liver microsomes incubation system with nitrendipine as internal standard. The determination m/z for ligustilide was 173, and for nitrendipine, 315. An optimum incubation system was found and various selective CYP inhibitors were used to investigate their inhibitory effects on the metabolism of ligustilide. The results showed that enzyme kinetics of ligustilide could be significantly inhibited by ketoconazole, trimethoprim and a-naphthoflavon but scarcely inhibited by omeprazole, 4-methylpyrazole and quinidine. Therefore, CYP3A4, CYP2C9 and CYP1A2 are the major isoenzyme participated in in vitro metabolism of ligustilide.

  10. 4-Dimethylaminoazobenzenes: carcinogenicities and reductive cleavage by microsomal azo reductase.

    Science.gov (United States)

    Lambooy, J P; Koffman, B M

    1985-01-01

    Twenty-four 4-dimethylaminoazobenzenes (DABs) in which systematic structural modifications have been made in the prime ring have been studied for substrate specificity for microsomal azo reductase. The DABs were also evaluated for carcinogenicity and it was found that there was no correlation between carcinogenicity and extent of azo bond cleavage by azo reductase. While any substituent in the prime ring reduces the rate of cleavage of the azo bond relative to the unsubstituted dye, there is a correlation between substituent size and susceptibility to the enzyme. Substituent size was also found to be a significant factor in the induction of hepatomas by the dyes. Preliminary studies have shown that there appears to be a positive correlation between microsomal riboflavin content and the activity of the azo reductase.

  11. Oxidation of esterified arachidonate by rat liver microsomes

    Energy Technology Data Exchange (ETDEWEB)

    Davis, H.W.; Suzuki, T.; Schenkman, J.B.

    1986-03-05

    The authors have previously demonstrated a relationship between phospholipid arachidonate in liver microsomes and malondialdehyde (MDA) formation during lipid peroxidation. In this study arachidonic acid (U-/sup 14/C) was incorporated into rat liver microsomes and NADPH-supported peroxidation was carried out at 37/sup 0/C for 15 minutes. The microsomes were pelleted by centrifugation and the labeled products in the supernatant were isolated by a solid phase method. Pellets were hydrolyzed with phospholipase A/sub 2/ and extracted with diethyl ether and the products from both fractions were separated by reverse phase HPLC. The results show that (1) oxidation occurs in all of the major phospholipids but that phosphatidylethanolamine is the most susceptible; (2) a linear correlation exists between MDA formation and supernatant radioactivity; (3) several different polar products are found in both the supernatant and the hydrolyzed pellet but that the ratios of product peaks in HPLC do not change during the peroxidation, indicating no secondary metabolism or propagation; and (4) cytochrome P-450 is not involved in the peroxidative reactions since no oxidation occurs in the absence of Fe/sup 3 +/ and since product formation is unaffected in the presence of carbon monoxide.

  12. Thin film voltammetry of metabolic enzymes in rat liver microsomes

    Science.gov (United States)

    Krishnan, Sadagopan; Rusling, James F.

    2007-01-01

    We report herein thin film voltammetry and kinetics of electron transfer for redox proteins in rat liver microsomes for the first time. Films were made layer-by-layer from liver microsomes and polycations on pyrolytic graphite electrodes. Cyclic voltammograms were chemically reversible with a midpoint potential of −0.48 V vs SCE at 0.1 V s−1 in pH 7.0 phosphate buffer. Reduction peak potentials shifted negative at higher scan rates, and oxidation-reduction peak current ratios were ∼1 consistent with non-ideal quasireversible thin film voltammetry. Analysis of oxidation-reduction peak separations gave an average apparent surface electron transfer rate constant of 30 s−1. Absence of significant electrocatalytic reduction of O2 or H2O2 and lack of shift in midpoint potential when CO is added that indicates lack of an iron heme cofactor suggest that peaks can be attributed to oxidoreductases present in the microsomes rather than cytochrome P450 enzymes. PMID:18037986

  13. Stereoselective metabolism of tetrahydropalmatine enantiomers in rat liver microsomes.

    Science.gov (United States)

    Zhao, Ming; Li, Li-Ping; Sun, Dong-Li; Sun, Si-Yuan; Huang, Shan-Ding; Zeng, Su; Jiang, Hui-Di

    2012-05-01

    Tetrahydropalmatine (THP), with one chiral center, is an active alkaloid ingredient in Rhizoma Corydalis. The aim of the present paper is to study whether THP enantiomers are metabolized stereoselectively in rat, mouse, dog, and monkey liver microsomes, and then, to elucidate which Cytochrome P450 (CYP) isoforms are predominately responsible for the stereoselective metabolism of THP enantiomers in rat liver microsomes (RLM). The results demonstrated that (+)-THP was preferentially metabolized by liver microsomes from rats, mice, dogs, and monkeys, and the intrinsic clearance (Cl(int)) ratios of (+)-THP to (-)-THP were 2.66, 2.85, 4.24, and 1.67, respectively. Compared with the metabolism in untreated RLM, the metabolism of (-)-THP and (+)-THP was significantly increased in dexamethasone (Dex)-induced and β-naphthoflavone (β-NF)-induced RLM; meanwhile, the Cl(int) ratios of (+)-THP to (-)-THP in Dex-induced and β-NF-induced RLM were 5.74 and 0.81, respectively. Ketoconazole had stronger inhibitory effect on (+)-THP than (-)-THP, whereas fluvoxamine had stronger effect on (-)-THP in untreated and Dex-induced or β-NF-induced RLM. The results suggested that THP enantiomers were predominately metabolized by CYP3A1/2 and CYP1A2 in RLM, and CYP3A1/2 preferred to metabolize (+)-THP, whereas CYP1A2 preferred (-)-THP.

  14. Stereoselective degradation of chiral fungicide myclobutanil in rat liver microsomes.

    Science.gov (United States)

    Yan, Jin; Zhang, Ping; Wang, Xinru; Wang, Yao; Zhou, Zhiqiang; Zhu, Wentao

    2014-01-01

    Myclobutanil, (RS)-2-(4-chlorophenyl)-2-(1H-1, 2, 4-triazol-1-ylmethyl)hexanenitrile is a broad-spectrum systemic triazole fungicide which consists of a pair of enantiomers. The stereoselective degradation of myclobutanil was investigated in rat liver microsomes. The concentrations of myclobutanil enantiomers were determined by high-performance liquid chromatography (HPLC) with a cellulose-tris-(3,5-dimethyl-phenylcarbamate)-based chiral stationary phase (CDMPC-CSP) under reversed phase condition. The t(1/2) of (+)-myclobutanil is 8.49 min, while the t(1/2) of (-)-myclobutanil is 96.27 min. Such consequences clearly indicated that the degradation of myclobutanil in rat liver microsomes was stereoselective and the degradation rate of (+)-myclobutanil was much faster than (-)-myclobutanil. In addition, significant differences between two enantiomers were also observed in enzyme kinetic parameters. The V(max) of (+)-myclobutanil was about 4-fold of (-)-myclobutanil and the CL(int) of (+)-myclobutanil was three times as much as (-)-myclobutanil after incubation in rat liver microsomes. Corresponding consequences may shed light on the environmental and ecological risk assessment for myclobutanil and may improve human health.

  15. Stereoselective glucuronidation of carvedilol by Chinese liver microsomes

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    Objective: To study the stereoselective glucuronidation of carvedilol (CARV) by three Chinese liver microsomes.Methods: The metabolites of CARV were identified by a hydrolysis reaction with β-glucuronidase and HPLC-MS/MS. The enzyme kinetics for CARV enantiomers glucuronidation was determined by a reversed phase-high pressure liquid chromatography (RP-HPLC) assay using (S)-propafenone as internal standard after precolumn derivatization with 2,3,4,6-tetra-O-acetyl-β-D-glucopyranosylisothiocyanate. Results: Two CARV glucuronides were found in three Chinese liver microsomes incubated with CARV. The non-linear regression analysis showed that the values of Km and Vmax for (S)-CARV and (R)-CARV enantiomers were (118±44) μmol/L, (2 500±833) pmol/(min.mg protein) and (24±7) μmol/L, (953+399) pmol/(min.mg protein),respectively. Conclusion: These results suggested that there was a significant (P<0.05) stereoselective glucuronidation of CARV enantiomers in three Chinese liver microsomes, which might partly explain the enantioselective pharmacokinetics of CARV.

  16. Antigen-loaded ER microsomes from APC induce potent immune responses against viral infection.

    Science.gov (United States)

    Sofra, Vassiliki; Mansour, Salah; Liu, Mengya; Gao, Bin; Primpidou, Elisavet; Wang, Ping; Li, Suling

    2009-01-01

    Although matured DC are capable of inducing effective primary and secondary immune responses in vivo, it is difficult to control the maturation and antigen loading in vitro. In this study, we show that ER-enriched microsomal membranes (microsomes) isolated from DC contain more peptide-receptive MHC I and II molecules than, and a similar level of costimulatory molecules to, their parental DC. After loading with defined antigenic peptides, the microsomes deliver antigenic peptide-MHC complexes (pMHC) to both CD4 and CD8 T cells effectively in vivo. The peptide-loaded microsomes accumulate in peripheral lymphoid organs and induce stronger immune responses than peptide-pulsed DC. The microsomal vaccines protect against acute viral infection. Our data demonstrate that peptide-MHC complexes armed microsomes from DC can be an important alternative to DC-based vaccines for protection from viral infection.

  17. Accelerated elimination of pimonidazole following microsomal enzyme induction in mice: a possible approach to reduced neurotoxicity of the pimonidazole-etanidazole combination

    Energy Technology Data Exchange (ETDEWEB)

    Workman, P.

    1989-04-01

    The pimonidazole-etanidazole combination shows promise for improved radiosensitization, although pimonidazole may increase the dose-limiting peripheral neurotoxicity of etanidazole in man. Induction of liver microsomal drug metabolizing enzymes by phenobarbitone resulted in reduced exposure to pimonidazole in plasma, brain, and tumor in mice. Peak tumor concentrations were lowered, but to a lesser extent. Phenobarbitone induction caused no change in the urinary ratio of pimonidazole to its N-oxide metabolite, and in fact, markedly reduced the circulating metabolite concentrations in plasma. There was no effect of phenobarbitone on plasma or tissue pharmacokinetics of etanidazole, which is eliminated by renal clearance. The results suggest that hepatic microsomal enzyme induction may be a possible approach to reducing the toxicity of the pimonidazole-etanidazole combination, and may also provide valuable information on the enzymology of pimonidazole metabolism.

  18. Analysis of the Inhibitory Effect of Gypenoside on Na+,K+-ATPase in Rats' Heart and Brain and Its Kinetics

    Institute of Scientific and Technical Information of China (English)

    HAN Xiao-yan; WEI Hong-bo; ZHANG Fu-cheng

    2007-01-01

    ObjectiYe: To study the effects of gypenoside (Gyp) on the activity of microsomal Na+,K+-ATPase in rat's heart and brain in vitro. Methods: The microsomal Na+, K+-ATPase was prepared from rat's heart and brain by differential centrifugation. The activity of microsomal Na+, K+-ATPase was assayed by colorimetric technique. Enzyme kinetic analysis method was used to analyze the effect of Gyp on the microsomal Na+, K+-ATPase of rats. Results: Gyp reversibly inhibited the brain and heart's microsomal Na+, K+-ATPase in a concentration-dependent manner, and showed a more potent effect on enzyme in the brain. The IC50 of Gyp for the heart and brain were 58.79± 8.05 mg/L and 52.07 ±6.25 mg/L, respectively. The inhibition was enhanced by lowering the Na+, or K+ concentrations or increasing the ATP concentration. Enzyme kinetic studies indicated that the inhibitory effect of Gyp on the enzyme is like that of competitive antagonist of Na+, the counter-competitive inhibitor for the substrate ATP, and the mixed-type inhibitor for K+. Conclusion: Gyp displays its cardiotonic and central inhibitory effects by way of inhibiting heart and brain's microsomal Na+, K+-ATPase activities in rats.

  19. Evaluation of Five Jet Fuels in the Salmonella-Escherichia coli / Microsome Plate Incorporation Assay

    Science.gov (United States)

    2010-09-01

    AFRL-RH-WP-TR-2010-0138 Evaluation of Five Jet Fuels in the Salmonella-Escherichia coli / Microsome Plate Incorporation Assay Edward S...31 Jul 2010 4. TITLE AND SUBTITLE Evaluation of Five Jet Fuels in the Salmonella-Escherichia coli / Microsome Plate Incorporation Assay 5a...the Salmonella typhimurium-Escherichia coli/ microsome plate incorporation assay. The assay was performed using the plate incorporation procedure

  20. Increase in phorbol ester binding in liver microsomes after chronic administration of phenobarbital

    Energy Technology Data Exchange (ETDEWEB)

    Menez, J.F.; Deitrich, R.A. (Univ. of Colorado, Denver (United States))

    1991-03-15

    The effect of chronic administration of phenobarbital on the binding of phorbol-12,13-dibutyrate (({sup 3}H)PDBu), an activator of protein kinase C (PKC), was examined in rat liver microsomes. A significant increase in the number of binding sites was observed in microsomes of Fisher 344 rats. However, no change appeared in liver cytosol binding of PDBu. Consequently, a translocation process of PKC is unlikely. The increase in ({sup 3}H)PDBu binding in liver microsomes is significant 24 h. after one injection of phenobarbital and reaches its maximum in 2 days. In other strains of rats (ACI and lean Zucker), significant differences were found in the increase of ({sup 3}H)PDBu binding in microsomes. Fisher 344 were the most sensitive, lean Zucker rats, the least sensitive. Those results parallel the pentoxy-resorufin O demethylase activity in the microsomes of the same animals. EC{sub 50} values for inhibition of ({sup 3}H)PDBu binding by pentobarbital were determined in control microsomes from Fisher and Zucker rats. In Fisher rats, ({sup 3}H)PDBu binding in microsomes was found to be more sensitive to the inhibitory effect of pentobarbital than in lean Zucker rats, which suggests that the more microsomes are inhibited in vitro the greater the increase in PKC in microsomes following chronic barbiturate treatment.

  1. Metabolic Characterization of a Tripeptide Human Immunodeficiency Virus Type 1 Protease Inhibitor, KNI-272, in Rat Liver Microsomes

    Science.gov (United States)

    Kiriyama, Akiko; Nishiura, Tomoyuki; Yamaji, Hirokazu; Takada, Kanji

    1999-01-01

    KNI-272 is a tripeptide protease inhibitor for treating human immunodeficiency virus type 1 (HIV-1). In in vitro stability studies using rat tissue homogenates, KNI-272 concentrations in the liver, kidney, and brain decreased significantly with time. Moreover, in tissue distribution studies, KNI-272 distributed highly to the liver, kidney, and small intestine in vivo. From these results and reported physiological parameters such as the tissue volume and tissue blood flow rate, we considered the liver to be the main organ which takes part in the metabolic elimination of KNI-272. Then the hepatic metabolism of KNI-272 was more thoroughly investigated by using rat liver microsomes. KNI-272 was metabolized in the rat liver microsomes, and five metabolites were found. The initial metabolic rate constant (kmetabolism) tended to decrease when the KNI-272 concentration in microsomal suspensions increased. The calculated Michaelis-Menten constant (Km) and the maximum velocity of KNI-272 metabolism (Vmax), after correction for the unbound drug concentration, were 1.12 ± 0.09 μg/ml (1.68 ± 0.13 μM) and 0.372 ± 0.008 μg/mg of protein/min (0.558 ± 0.012 nmol/mg of protein per min), respectively. The metabolic clearance (CLint,metabo), calculated as Vmax/Km, was 0.332 ml/mg of protein per min. Moreover, by using selective cytochrome P-450 inhibitors and recombinant human CYP3A4 fractions, KNI-272 was determined to be metabolized mainly by the CYP3A isoform. In addition, ketoconazole, a representative CYP3A inhibitor, inhibited KNI-272 metabolism competitively, and the inhibition constant (Ki) was 4.32 μM. PMID:10049266

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

    Science.gov (United States)

    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.

  3. Interaction between Na-type smectite and Fe{sup 2+} ions. Na{sup +}/Fe{sup 2+} ion exchange in the interlayer space of smectite

    Energy Technology Data Exchange (ETDEWEB)

    Shinozaki, Tomoko [ITC, Tokyo (Japan); Oda, Harue; Shibata, Masahiro; Kamei, Gento

    1998-02-01

    Adsorption-desorption tests for Fe{sup 2+} ions versus Na-type smectite were carried out in the solution under the controlled atmosphere (O{sub 2} < 0.1 ppm, CO{sub 2} < 2 ppm) in order to elucidate the interaction between bentonite and iron containers for underground waste disposal. Smectite after the adsorption test was subjected to the X-ray diffraction and X-ray photoelectron spectroscopy to study the Fe{sup 2+} adsorption site. As a result, the interaction between Fe{sup 2+} and smectite was shown to be reversible ion exchange between Na{sup +} and Fe{sup 2+} in the interlayer space of smectite. The ion exchange distribution coefficient was found to be log K = 1.08 {+-} 0.07 and 0.66 {+-} 0.19 for the ionic strength of 0.05 and 0.0016, respectively. (H. Baba)

  4. Investigation of the swelling behavior of cationic exchange resins saturated with Na{sup +} ions in a C{sub 3}S paste

    Energy Technology Data Exchange (ETDEWEB)

    Lafond, E. [CEA, DEN, DTCD, SPDE, F-30207 Bagnols-sur-Cèze cedex (France); Cau Dit Coumes, C., E-mail: celine.cau-dit-coumes@cea.fr [CEA, DEN, DTCD, SPDE, F-30207 Bagnols-sur-Cèze cedex (France); Gauffinet, S. [UMR5209 Institut Carnot de Bourgogne, Université de Bourgogne Dijon, Faculté des Sciences Mirande, 9 Avenue Alain Savary, BP 47870, 21078 Dijon cedex (France); Chartier, D. [CEA, DEN, DTCD, SPDE, F-30207 Bagnols-sur-Cèze cedex (France); Le Bescop, P. [CEA, DEN, DPC, SECR, F-91192 Gif-sur-Yvette (France); Stefan, L. [AREVA, Back End Business Group, Dismantling & Services, 1 place Jean Millier, 92084 Paris La Défense (France); Nonat, A. [UMR5209 Institut Carnot de Bourgogne, Université de Bourgogne Dijon, Faculté des Sciences Mirande, 9 Avenue Alain Savary, BP 47870, 21078 Dijon cedex (France)

    2015-03-15

    Ion exchange resins (IERs) are widely used by the nuclear industry to decontaminate radioactive effluents. Spent products are usually encapsulated in cementitious materials. However, the solidified waste form can exhibit strong expansion, possibly leading to cracking, if the appropriate binder is not used. In this work, the interactions between cationic resins in the Na{sup +} form and tricalcium silicate are investigated during the early stages of hydration in order to gain a better understanding of the expansion process. It is shown that the IERs exhibit a transient swelling of small magnitude due to the decrease in the osmotic pressure of the external solution. This expansion, which occurs just after setting, is sufficient to damage the material which is poorly consolidated for several reasons: low degree of hydration, precipitation of poorly cohesive sodium-bearing C–S–H, and very heterogeneous microstructure with zones of high porosity.

  5. Inhibition by 5-N-(4-chlorobenzyl)-2`,4`-dimethylbenzamil of Na{sup +}/Ca{sup 2+} exchange and L-type Ca{sup 2+} channels in isolated cardiomyocytes

    Energy Technology Data Exchange (ETDEWEB)

    Sharikabad, M.N.; Broers, O. [Ullevaal Univ. Hospital, Clinical Chemistry dept., Div. of Clinical Pharmacology and Toxicology, Olso (Norway); Cragoe, E.J. Jr.

    1997-02-01

    The inhibitory effect of the amiloride derivative 5-N-(4-chlorobenzyl)-2`,4`-dimethylbenzamil (CBDMB) on calcium (Ca{sup 2+}) uptake via sarcolemmal sodium-calcium (Na{sup +}/Ca{sup 2+}) exchange and L-type Ca{sup 2+} channels was investigated in isolated adult rat ventricular cardiomyocytes under depolarizing conditions in cells preincubated with 1 mM ouabain or 137 mM lithium (Li{sup +}), respectively. Fifteen or 120 min. preincubation with CBDMB inhibited Ca{sup 2+} uptake via Na{sup +}/Ca{sup 2+} exchange in Na{sup +}-loaded depolarized cells completely at 100 {mu}M with an IC{sub 50} of 21 {mu}M. After 120 min. preincubation, CBDMB inhibited Ca{sup 2+} uptake via L-type Ca{sup 2+} channels by 75.1{+-}8.1% (mean and S.E.M.) and IC{sub 50} of 4 {mu}M, whereas no significant inhibition was observed after 15 min. preincubation. (+)-Isradipine (10 {mu}3M) inhibited high potassium (K{sup +}) induced Ca{sup 2+} uptake via L-type Ca{sup 2+} channels by 35% after 15 min. and by 70% after 120 min. preincubation. Inhibition by CBDMB of specific (+)-[{sup 3}]isradipine binding to L-type Ca{sup 2+} channels showed similar concentration dependency as inhibition of Ca{sup 2+} uptake via L-type Ca{sup 2+} channels. In conclusio, CBDMB inhibits sarcolemmal Na{sup +}/Ca{sup 2+} exchange in rat ventricular cardiomyocytes rapidly. However, after longer preincubation periods, L-type Ca{sup 2+} channels are inhibited as well and with higher potency than Na{sup +}/Ca{sup 2+} exchange. (au) 17 refs.

  6. Corticosteroids increase superoxide anion production by rat liver microsomes.

    Science.gov (United States)

    Nelson, D H; Ruhmann-Wennhold, A

    1975-01-01

    Superoxide anion production by liver microsomes from intact, adrenalectomized, and cortisoltreated adrenalectomized rats has been determined. The amount formed was roughly proportionate to the amount of cortisol given, and a similar response was seen in the activity of NADPH-cytochrome c reductase. The amount of measurable superoxide anion was markedly reduced by the addition of superoxide dismutase. The increased production of this potent free radical with cortisol therapy suggests that its formation may contribute to some of the harmful effects of corticosteroids given in more than physiologic amounts. PMID:239969

  7. Metabolism of 3-methylcholanthrene by rat liver microsomes: a reinvestigation

    Energy Technology Data Exchange (ETDEWEB)

    Stoming, T.A. (Medical Coll., Augusta, GA); Bornstein, W.; Bresnick, E.

    1977-01-01

    Metabolites of 3-methylcholanthrene (3-MC) formed by rat liver microsomes were analyzed by high pressure liquid chromatography. The metabolic profile is significantly different from previous studies using thin layer chromatography. The major metabolites include 1- and 2-hydroxy-3-MC. Use of the high pressure liquid chromatographic system allows for the separation of at least seven new metabolites. The amounts of three of these new metabolites are substantially decreased when the potent epoxide hydrase inhibitor 3,3,3-trichloropropene oxide is added to the incubation system. These results then suggest the formation of epoxides of 3-methylcholanthrene other than the K-region oxide.

  8. Microsomal prostaglandin E synthase-1 in rheumatic diseases

    Directory of Open Access Journals (Sweden)

    Marina eKorotkova

    2011-01-01

    Full Text Available Microsomal prostaglandin E synthase-1 (mPGES-1 is a well recognized target for the development of novel anti-inflammatory drugs that can reduce symptoms of inflammation in rheumatic diseases and other inflammatory conditions. In this review, we focus on mPGES-1 in rheumatic diseases with the aim to cover the most recent advances in the understanding of mPGES-1 in rheumatoid arthritis, osteoarthritis and inflammatory myopathies. Novel findings regarding regulation of mPGES1 cell expression as well as enzyme inhibitors are also summarized.

  9. Inhibitory effects of beryllium chloride on rat liver microsomal enzymes.

    Science.gov (United States)

    Teixeira, C F; Yasaka, W J; Silva, L F; Oshiro, T T; Oga, S

    1990-04-30

    A single i.v. dose (0.1 mmol Be2+/kg) of beryllium chloride prolonged the duration of pentobarbital-induced sleep and zoxazolamine-induced paralysis, in rats. The effects are correlated with changes of the pharmacokinetic parameters and with the in vitro inhibition of both aliphatic and aromatic hydroxylation of pentobarbital and zoxazolamine. In vitro N-demethylation of meperidine and aminopyrine was partially inhibited while O-demethylation of quinidine was unaffected by liver microsomes of rats pretreated with beryllium salt. The findings give clues that beryllium chloride inhibits some forms of cytochrome P-450, especially those responsible for hydroxylation of substrates, like pentobarbital and zoxazolamine.

  10. Duration of hexobarbital-induced sleep and monoamine oxidase activities in rat brain: Focus on the behavioral activity and on the free-radical oxidation.

    Science.gov (United States)

    Tseilikman, Vadim E; Kozochkin, Denis A; Manukhina, Eugenia B; Downey, H Fred; Tseilikman, Olga B; Misharina, Maria E; Nikitina, Anna A; Komelkova, Maria V; Lapshin, Maxim S; Kondashevskaya, Marina V; Lazuko, Svetlana S; Kusina, Oxana V; Sahabutdinov, Marat V

    2016-04-01

    The present study is focused on the relationship between monoamine oxidase (MAO) activity and hepatic content of cytochrome P450 (CYP), which reflects the status of microsomal oxidation. For vital integrative evaluation of hepatic microsomal oxidation in rats, the hexobarbital sleep test was used, and content of CYP was measured in hepatic microsomes. Rats with short hexobarbital sleep time (SHST) had higher content of microsomal CYP than rats with long hexobarbital sleep time (LHST). Whole brain MAO-A and MAO-B activities, serotonin and carbonylated protein levels were higher in SHST than in LHST rats. MAO-A and MAO-B activities were higher in brain cortex of SHST rats; MAO-A activity was higher only in hypothalamus and medulla of LHST. The same brain regions of LHST rats had higher concentrations of carbonylated proteins and lipid peroxidation products than in SHST rats. MAO activity was correlated with microsomal oxidation phenotype. Rats with higher hepatic content of CYP had higher activities of MAO-A and MAO-B in the brain and higher plasma serotonin levels than rats with lower microsomal oxidation. In conclusion, data obtained in this study showed a correlation between MAO activity and microsomal oxidation phenotype.

  11. Permeability of Rat Liver Microsomes to Sucrose and Carboxypoly-glucose in vitro

    Science.gov (United States)

    Share, Leonard; Hansrote, Ronald W.

    1960-01-01

    A study was made of the permeability of the microsomes to C14-sucrose and to C14-carboxypolyglucose, a branch-chained glucose polymer with a molecular weight of approximately 50,000. It was concluded that the microsomal membranes are permeable to sucrose on the basis of the following evidence: the volume of distribution of C14-sucrose was 84 per cent of the total microsomal pellet water; the sucrose unavailable volume, the per cent dry weights of the microsomal pellets, and the optical density of microsomal suspensions were independent of the concentration of sucrose in the suspending medium. It is suggested that the microsomal water which is unavailable to sucrose may be bound to protein and/or ribonucleic acid of the microsomes. The volume of distribution of C14-carboxypolyglucose was 44 per cent of the total pellet water, and it is considered that the microsomal membranes may be impermeable to this compound. Pretreatment with ribonuclease resulted in small increases in the volumes of distribution of both C14-sucrose and C14-carboxypolyglucose. PMID:14445567

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

    NARCIS (Netherlands)

    Moonen, H.; Bosch, H. van den

    1979-01-01

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

  13. Identification of saturated and unsaturated fatty acids released during microsomal incubations.

    Science.gov (United States)

    Bushee, Jennifer L; Liang, Guiqing; Dunne, Christine E; Harriman, Shawn P; Argikar, Upendra A

    2014-08-01

    1. In vitro clearance in liver microsomes is routinely measured in drug discovery and development for new chemical entities. Literature reports indicate that long chain fatty acids such as arachidonic, linoleic and oleic acids may be released over a period of time during microsomal incubations. Some fatty acids have been shown to interfere with oxidative and conjugative reactions in microsomes, thus potentially inhibiting microsomal clearance of compounds. 2. The present study was aimed at deciphering the fatty acids present or released from microsomes. Analytical methods were developed to characterize and quantitatively assess the fatty acids without chemical derivatization in rat, monkey and human liver microsomes. Additionally, incubations with uridine-5'-diphosphoglucuronic acid (UDPGA) were utilized to trap the released fatty acids as their glucuronate esters, which were characterized and confirmed by high-resolution LC-MS/MS. 3. Our results indicate for the first time that timnodonic, trans-eicosenoic, gondoic, behenic, and nervonic acid were released during microsomal incubations. Additionally, α- and γ-linolenic, timnodonic, palmitoleic, linoleic, arachidonic, palmitic, oleic, and stearic acid were identified as their corresponding acyl-glucuronides in rat, monkey and human liver microsomes, providing the first direct evidence that the released fatty acids are capable of forming glucuronides under incubation conditions.

  14. In vitro covalent binding of /sup 14/C-mibolerone to rat liver microsomes

    Energy Technology Data Exchange (ETDEWEB)

    Jaglan, P.S.

    1986-01-01

    Mibolerone (17-Hydroxy-7,17-dimethylestr-4-en-3-one; 7 alpha-17 alpha dimethyl-19-nortestosterone) is being marketed by The Upjohn Company for the inhibition of estrus in bitches. The aim of this study was to determine the extent of covalent binding of mibolerone to rat liver microsomes. Liver microsomes were obtained from Control and phenobarbitol-treated female Fisher rats, and were incubated with /sup 14/C-mibolerone at 37/sup 0/C for 10 minutes. No covalent binding to macromolecules was observed when /sup 14/C-mibolerone was incubated with rat liver microsomes. Under identical conditions, /sup 14/C-estradiol was covalently bound to macromolecules. Slightly higher covalent binding of estradiol was observed with microsomes from phenobarbitol-treated rats. Ascorbic acid and glutathione inhibited covalent binding of estradiol to macromolecules in the in vitro microsomal system.

  15. Fungal microsomes in a biotransformation perspective: protein nature of membrane-associated reactions.

    Science.gov (United States)

    Svobodová, Kateřina; Mikesková, Hana; Petráčková, Denisa

    2013-12-01

    Microsomal fraction of fungal cells grabs the attention of many researchers for it contains enzymes that play a role in biotechnologically relevant processes. Microsomal enzymes, namely, CYP450s, were shown to metabolize a wide range of xenobiotic compounds, including PAHs, PCBs, dioxins, and endocrine disruptors, and take part in other fungal biotransformation reactions. However, little is known about the nature and regulation of these membrane-associated reactions. Advanced proteomic and post-genomic techniques make it possible to identify larger numbers of microsomal proteins and thus add to a deeper study of fungal intracellular processes. In this work, proteins that were identified through a shotgun proteomic approach in fungal microsomes under various culture conditions are reviewed. However, further research is still needed to fully understand the role of microsomes in fungal biodegradation and biotransformation reactions.

  16. Isolation and catalytic competence of different animal liver microsomal fractions prepared by calcium-aggregation method

    Directory of Open Access Journals (Sweden)

    Walawalkar Pradnya

    2006-01-01

    Full Text Available Rat, mouse, rabbit, and guinea pig liver microsomes were prepared, using the calcium-aggregation method. The mean specific spectral cytochrome P450 content obtained for rat, mouse, rabbit, and guinea pig liver microsomes were 0.547, 0.394, 0.677, and 0.378 nmol cytochrome P450/mg protein, respectively. All the microsomal samples showed the ability to biotransform p-nitrophenol to p-nitrocatechol. The mean rate of formation of p-nitrocatechol by rat, mouse, rabbit, and guinea pig liver microsomes was 0.272, 0.247, 0.497 and 0.424 nmole/min/nmole cytochrome 450, respectively. This method circumvents the need of an ultracentrifuge for isolation of microsomes, by the conventional differential centrifugation method.

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

    Science.gov (United States)

    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 transformation of organophosphorus pesticides by white rot fungi.

    Science.gov (United States)

    Jauregui, Juan; Valderrama, Brenda; Albores, Arnulfo; Vazquez-Duhalt, Rafael

    2003-12-01

    The enzymatic mechanism for the transformation of organophosphorus pesticides (OPPs) by different white-rot fungi strains was studied. With the exception of Ganoderma applanatum 8168, all strains from a collection of 17 different fungi cultures were able to deplete parathion. Three strains showing the highest activities were selected for further studies: Bjerkandera adusta 8258, Pleurotus ostreatus 7989 and Phanerochaete chrysosporium 3641. These strains depleted 50 to 96% of terbufos, azinphos-methyl, phosmet and tribufos after four-days exposure to the pesticides. In order to identify the cellular localization of the transformation activity, the extracellular and microsomal fractions of Pleuronts ostreatus 7989 were evaluated in vitro. While the activities of ligninolytic enzymes (lignin peroxidase, manganese peroxidase and laccase) were detected in the extracellular fraction, no enzymatic modification of any of the five pesticides tested could be found, suggesting the intracellular origin of the transformation activity. In accordance with this observation the microsomal fraction was found able to transform three OPPs with the following rates: 10 micromol mg prot(-1) h(-1) for phosmet, 5.7 micromol mg prot(-1) h(-1) for terbufos, and 2.2 micromol mg prot(-1) h(-1) for azinphos-methyl. The products from these reactions and from the transformation of trichlorfon and malathion, were identified by mass-spectrometry. These results, supported by specific inhibition experiments and the stringent requirement for NADPH during the in vitro assays suggest the involvement of a cytochrome P450.

  19. PHOTOMETRIC EVIDENCE FOR THE OSMOTIC BEHAVIOR OF RAT LIVER MICROSOMES

    Science.gov (United States)

    Tedeschi, Henry; James, Joseph M.; Anthony, William

    1963-01-01

    Electron microscope observations are consistent with the interpretation that the elements of the endoplasmic reticulum are osmotically active in situ as well as after isolation. More recently, it has been reported that microsomal suspensions equilibrate almost completely with added C14-sucrose and that no osmotic behavior is evident from photometric data. These findings were considered at variance with the electron microscope data. However, equilibration with added label simply attests to a relatively high permeability, and, in addition, the photometric data need not be critical. Osmotic volume changes, measured photometrically, may be masked by concomitant events (e.g., changes in the refractive index of the test solutions at varying osmotic pressures, breakdown of the particles, and agglutination). For these reasons the photometric experiments were repeated. In this work, the reciprocal of optical density of microsomal suspensions was found to vary linearly with the reciprocal of concentration of the medium at constant refractive index. These changes probably correspond to osmotic volume changes, since the effect was found to be (a) independent of substance used and (b) osmotically reversible. The transmission of the suspension was found to vary with the refractive index of the medium, the concentration of particles, and the wavelength of incident light, according to relationships that are similar to or identical with those obtained for mitochondrial suspensions. PMID:14064105

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

    Science.gov (United States)

    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

  1. An intrinsic gut leptin-melanocortin pathway modulates intestinal microsomal triglyceride transfer protein and lipid absorption.

    Science.gov (United States)

    Iqbal, Jahangir; Li, Xiaosong; Chang, Benny Hung-Junn; Chan, Lawrence; Schwartz, Gary J; Chua, Streamson C; Hussain, M Mahmood

    2010-07-01

    Fat is delivered to tissues by apoB-containing lipoproteins synthesized in the liver and intestine with the help of an intracellular chaperone, microsomal triglyceride transfer protein (MTP). Leptin, a hormone secreted by adipose tissue, acts in the brain and on peripheral tissues to regulate fat storage and metabolism. Our aim was to identify the role of leptin signaling in MTP regulation and lipid absorption using several mouse models deficient in leptin receptor (LEPR) signaling and downstream effectors. Mice with spontaneous LEPR B mutations or targeted ablation of LEPR B in proopiomelanocortin (POMC) or agouti gene related peptide (AGRP) expressing cells had increased triglyceride in plasma, liver, and intestine. Furthermore, melanocortin 4 receptor (MC4R) knockout mice expressed a similar triglyceride phenotype, suggesting that leptin might regulate intestinal MTP expression through the melanocortin pathway. Mechanistic studies revealed that the accumulation of triglyceride in the intestine might be secondary to decreased expression of MTP and lipid absorption in these mice. Surgical and chemical blockade of vagal efferent outflow to the intestine in wild-type mice failed to alter the triglyceride phenotype, demonstrating that central neural control mechanisms were likely not involved in the observed regulation of intestinal MTP. Instead, we found that enterocytes express LEPR, POMC, AGRP, and MC4R. We propose that a peripheral, local gut signaling mechanism involving LEPR B and MC4R regulates intestinal MTP and controls intestinal lipid absorption.

  2. Fatty acid hydroxylation in rat kidney cortex microsomes.

    Science.gov (United States)

    Ellin, A; Orrenius, S

    1975-08-30

    Rat kidney microsomes have been found to catalyze the hydroxylation of medium-chained fatty acids to the omega- and (omego-1)-hydroxy derivatives. This reaction, which requires NADPH and molecular oxygen, is a function of monooxygenase system present in the kidney microsomes, containing NADPH-cytochrome c reductase and cytochrome P-450K. NADH is about half as effective as an electron donor as NADPH and there is an additive effect in the presence of both nucleotides. Cytochrome P-450K absorbs light maximally at 452-3 nm, when it is reduced and bound to carbon monoxide. The extinction coefficient of this complex is 91 mM(-1) cm(-1). Electrons from NADPH are transferred to cytochrome P-450K via the NADPH-cytochrome c reductase. The reduction rate of cytochrome P-450K is stimulated by added fatty acids and the reduction kinetics reveal the presence of endogenous substrates bound to cytochrome P-450K. Both cytochrome P-450K concentration and fatty acid hydroxylation activity in kidney microsomes are increased by starvation. On the other hand, phenobarbital treatment of the rats has no effect on either the hemoprotein or the overall hydroxylation reaction and 3,4-benzpyrene administration induces a new species of cytochrome P-450K not involved in fatty acid hydroxylation. Cytochrome P-450K shows, in contrast to liver P-450, high substrate specificity. The only substances forming enzyme-substrate complexes with cytochrome P-450K are the medium-chained fatty acids and certain derivatives of these acids. The chemical requirements for substrate binding include a carbon chain of medium length and at the end of the chain a carbonyl group and a free electron pair on a neighbouring atom. The distance between the binding site for the carbonyl group and the active oxygen is suggested to be in the order of 16 A. This distance fixes the ratio of omega- and (omega-1)-hydroxylated products formed from a certain fatty acid by the single species of cytochrome P-450K involved. The

  3. Preparation and characterization of rodent intestinal microsomes: Comparative assessment of two methods

    Directory of Open Access Journals (Sweden)

    Damre Anagha

    2009-01-01

    Full Text Available Small intestine plays an important role in the first-pass metabolism of orally ingested xenobiotics as a result of expression of both Phase I and Phase II metabolic enzymes, together with associated transporters. Intestinal microsomes thus can be used to study susceptibility of compounds to metabolism in vitro. The present study was undertaken to have a comparative assessment between different methods of preparation of rodent intestinal microsomes. Mouse and rat intestinal microsomes were prepared by two methods, in method A intestines were homogenized, while in method B mucosal cells were scrapped followed by homogenization. Further, microsomes were prepared by centrifugation (10000xg followed by ultra centrifugation (100000xg of the homogenates. The prepared microsomes were characterized for protein concentration using Bradford′s method and CYP450 content using carbon monoxide bubbling method. The protein concentration and CYP450 content in microsomes prepared by method B was significantly higher than method A. In conclusion, superior quality intestinal microsomes can be obtained from rodents by using scrapped intestinal mucosal cells as compared to the intestinal homogenates.

  4. Butylbenzyl phthalate hydrolysis in liver microsomes of humans, monkeys, dogs, rats and mice.

    Science.gov (United States)

    Takahara, Yuka; Kinashi, Yu; Takahara, Yuusuke; Hichiya, Hiroyuki; Okada, Kenji; Murata, Mikio; Shigeyama, Masato; Hanioka, Nobumitsu

    2014-01-01

    Butylbenzyl phthalate (BBzP) is used as a plasticizer to import flexibility to polyvinylchloride plastics. In this study, hydrolysis of BBzP to monobutyl phthalate (MBP) and monobenzyl phthalate (MBzP) in liver microsomes of humans, monkeys, dogs, rats and mice was examined. The kinetics for MBP formation by human, dog and mouse liver microsomes followed the Michaelis-Menten model, whereas the kinetics by monkey and rat liver microsomes fitted the Hill model. The kinetics for MBzP formation fitted the Hill model for all liver microsomes. The Vmax and in vitro clearance (CLint or CLmax) ratios of MBP/MBzP formation varied among animal species, although the Km for MBP and MBzP formation in each liver microsomes were generally comparable. The hydrolysis of BBzP to monoester phthalates in mammalian liver microsomes could be classified into two types: MBzP>MBP type for humans and dogs, and MBP>MBzP type for monkeys, rats and mice. These findings suggest that the formation profile of MBzP and MBP from BBzP by liver microsomes differs extensively among animal species.

  5. Glycerophosphate acylation by microsomes and mitochondria of normal and dystrophic human muscle.

    Science.gov (United States)

    Kunze, D; Rüstow, B; Olthoff, D

    1984-07-16

    The incorporation of [14C]glycerophosphate into phosphatidic acid, diacylglycerol, triacylglycerol and phosphatidylcholine by microsomes and mitochondria prepared from normal and dystrophic human muscle incubated in vitro in the presence of 0.3 mmol/l CDP-choline was measured. In mitochondria only phosphatidic acid and diacylglycerol are labelled; the rate of incorporation into these two compounds showed no difference between dystrophic and normal mitochondria. In dystrophic microsomes the incorporation into phosphatidic acid was delayed and decreased. No incorporation of glycerol into diacylglycerol, phosphatidylcholine and triacylglycerol could be measured. Thus in dystrophic muscle microsomes only PA was labelled during an incubation of up to 45 min. In both types of microsomes the concentration of endogenous free fatty acids and diacylglycerol was nearly identical. The level of phosphatidylcholine was 186 and 79 nmol/mg microsomal protein in normal and dystrophic muscle microsomes, respectively. Whether the results could be explained as secondary changes was discussed. Despite some unsolved problems the conclusion was drawn that a better explanation of the results is to assume a primary defect involving microsomal-bound phosphatidic acid phosphohydrolase and possibly glycerol-P-acyltransferases.

  6. A STUDY OF THE NUCLEOSIDE TRI- AND DIPHOSPHATE ACTIVITIES OF RAT LIVER MICROSOMES

    Science.gov (United States)

    Ernster, Lars; Jones, Lois C.

    1962-01-01

    Rat liver microsomes catalyze the hydrolysis of the triphosphates of adenosine, guanosine, uridine, cytidine, and inosine into the corresponding diphosphates and inorganic orthophosphate. The activities are stimulated by Na2S2O4, and inhibited by atebrin, chlorpromazine, sodium azide, and deaminothyroxine. Sodium deoxycholate inhibits the ATPase activity in a progressive manner; the release of orthophosphate from GTP and UTP is stimulated by low, and inhibited by high, concentrations of deoxycholate, and that from CTP and ITP is unaffected by low, and inhibited by high, concentrations of deoxycholate. Subfractionation of microsomes with deoxycholate into ribosomal, membrane, and soluble fractions reveals a concentration of the triphosphatase activity in the membrane fraction. Rat liver microsomes also catalyze the hydrolysis of the diphosphates of the above nucleosides into the corresponding monophosphates and inorganic orthophosphate. Deoxycholate strongly enhances the GDPase, UDPase, and IDPase activities while causing no activation or even inhibition of the ADPase and CDPase activities. The diphosphatase is unaffected by Na2S2O4 and is inhibited by azide and deaminothyroxine but not by atebrin or chlorpromazine. Upon fractionation of the microsomes with deoxycholate, a large part of the GDPase, UDPase, and IDPase activities is recovered in the soluble fraction. Mechanical disruption of the microsomes with an Ultra Turrax Blender both activates and releases the GDPase, UDPase, and IDPase activities, and the former effect occurs more readily than the latter. The GDPase, UDPase, and IDPase activities of the rat liver cell reside almost exclusively in the microsomal fraction, as revealed by comparative assays of the mitochondrial, microsomal, and final supernatant fractions of the homogenate. The microsomes exhibit relatively low nucleoside monophosphatase and inorganic pyrophosphatase activities, and these are unaffected by deoxycholate or mechanical treatment

  7. Encapsulation of liver microsomes into a thermosensitive hydrogel for characterization of drug metabolism and toxicity.

    Science.gov (United States)

    Yang, Huiying; Zheng, Yuanting; Zhao, Bei; Shao, Tengfei; Shi, Qingling; Zhou, Ning; Cai, Weimin

    2013-12-01

    This study reported the encapsulation of liver microsomes into a thermosensitive hydrogel to characterize drug metabolism and predict drug effects. Pluronic(®)F-127 (F127) and acrylamide-bisacrylamide (Acr-Bis) were utilized as the two precursors. After chemical crosslinking catalyzed by ammonium persulfate (APS) and N,N,N',N'-tetramethylethylenediamine (TEMED), the resulting Pluronic F127-acrylamide-bisacrylamide (FAB) hydrogel could encapsulate microsomes at 4 °C and facilitate metabolic reactions at 37 °C. The gel morphology at different Acr-Bis concentrations was characterized using field emission scanning electron microscopy (FE-SEM). Higher concentrations of Acr-Bis could lead to higher degrees of cross-linking of the gel. A fluorescent staining assay was subsequently used to demonstrate successful encapsulation of microsomes into the gel as well as the free diffusion process of micromolecular substrates. The thermosensitivity of the FAB gel was studied using swelling ratio and protein release assay to verify its ability to encapsulate microsomes. The metabolic activity of microsomes encapsulated in gels was investigated by detecting the metabolites of FDA-approved substrates, including dextromethorphan, chlorzoxazone and testosterone. Compared with the traditional method of microsomal incubation, the FAB gel maintained 60%-70% of microsome activity. Lastly, the classic anticancer prodrug cyclophosphamide (CTX) was chosen as a model drug for the study of drug metabolism and the prediction of drug effects. When the microsomes encapsulated in the FAB gel were used in the cell culture system, CTX induced a higher level of apoptosis in MCF-7 cells compared with traditional microsomes.

  8. Activation of K{sup +} channels and Na{sup +}/K{sup +} ATPase prevents aortic endothelial dysfunction in 7-day lead-treated rats

    Energy Technology Data Exchange (ETDEWEB)

    Fiorim, Jonaina, E-mail: nanafiorim@hotmail.com [Department of Physiological Sciences, Federal University of Espirito Santo, Vitoria, ES (Brazil); Ribeiro Júnior, Rogério Faustino, E-mail: faustino43@oi.com.br [Department of Physiological Sciences, Federal University of Espirito Santo, Vitoria, ES (Brazil); Azevedo, Bruna Fernades, E-mail: brunafernandes.azevedo@gmail.com [Department of Physiological Sciences, Federal University of Espirito Santo, Vitoria, ES (Brazil); Simões, Maylla Ronacher, E-mail: yllars@hotmail.com [Department of Physiological Sciences, Federal University of Espirito Santo, Vitoria, ES (Brazil); Padilha, Alessandra Simão, E-mail: ale_spadilha@yahoo.com.br [Department of Physiological Sciences, Federal University of Espirito Santo, Vitoria, ES (Brazil); Stefanon, Ivanita, E-mail: ivanita@pq.cnpq.br [Department of Physiological Sciences, Federal University of Espirito Santo, Vitoria, ES (Brazil); Alonso, Maria Jesus, E-mail: mariajesus.alonso@urjc.es [Departamento de Ciencias de la Salud III, Universidad Rey Juan Carlos, Alcorcón (Spain); Salaices, Mercedes, E-mail: mercedes.salaices@uam.es [Departamento de Farmacología, Universidad Autónoma de Madrid, Instituto de Investigación Hospital Universitario La Paz (IdiPaz) (Spain); Vassallo, Dalton Valentim, E-mail: daltonv2@terra.com.br [Department of Physiological Sciences, Federal University of Espirito Santo, Vitoria, ES (Brazil)

    2012-07-01

    Seven day exposure to a low concentration of lead acetate increases nitric oxide bioavailability suggesting a putative role of K{sup +} channels affecting vascular reactivity. This could be an adaptive mechanism at the initial stages of toxicity from lead exposure due to oxidative stress. We evaluated whether lead alters the participation of K{sup +} channels and Na{sup +}/K{sup +}-ATPase (NKA) on vascular function. Wistar rats were treated with lead (1st dose 4 μg/100 g, subsequent doses 0.05 μg/100 g, im, 7 days) or vehicle. Lead treatment reduced the contractile response of aortic rings to phenylephrine (PHE) without changing the vasodilator response to acetylcholine (ACh) or sodium nitroprusside (SNP). Furthermore, this treatment increased basal O{sub 2}{sup −} production, and apocynin (0.3 μM), superoxide dismutase (150 U/mL) and catalase (1000 U/mL) reduced the response to PHE only in the treated group. Lead also increased aortic functional NKA activity evaluated by K{sup +}-induced relaxation curves. Ouabain (100 μM) plus L-NAME (100 μM), aminoguanidine (50 μM) or tetraethylammonium (TEA, 2 mM) reduced the K{sup +}-induced relaxation only in lead-treated rats. When aortic rings were precontracted with KCl (60 mM/L) or preincubated with TEA (2 mM), 4-aminopyridine (4-AP, 5 mM), iberiotoxin (IbTX, 30 nM), apamin (0.5 μM) or charybdotoxin (0.1 μM), the ACh-induced relaxation was more reduced in the lead-treated rats. Additionally, 4-AP and IbTX reduced the relaxation elicited by SNP more in the lead-treated rats. Results suggest that lead treatment promoted NKA and K{sup +} channels activation and these effects might contribute to the preservation of aortic endothelial function against oxidative stress. -- Highlights: ► Increased free radicals production ► Increased Na{sup +}/K{sup +} ATPase activity ► Promotes activation of the K{sup +} channels and reduced vascular reactivity ► These effects preserve endothelial function against oxidative

  9. Diffusion of Tritiated Water (HTO) and {sup 22}Na{sup +}-Ions through Non-Degraded Hardened Cement Pastes - II. Modelling Results

    Energy Technology Data Exchange (ETDEWEB)

    Jakob, A

    2002-12-01

    In this report, the procedure and the results of an inverse modelling study on the through-diffusion of tritiated water (HTO) and {sup 2}2Na{sup +}-ions are presented using high-porous hardened cement pastes with a water/cement ratio of 1.3 in the first stage of the cement degradation. For the analysis two alternative models were applied: 1) a diffusion model where a possible sorption of the tracer was entirely neglected, and 2) a diffusion model with linear sorption. The analysis of the through-diffusion phase allowed extracting values for the effective diffusion coefficient (D{sub e}) and the rock-capacity factor ({alpha}). Both models could fit the breakthrough curves equally well, and also mass-balance considerations did not allow to clearly preferring one of the two competing models to the other. But blind-predictions for tracer out-diffusion using the best-fit parameter values deduced from analysing the former through-diffusion phase gave a clear indication that linear sorption had to be included in the diffusion model. The extracted K{sub d} values for HTO are in excellent agreement with values from batch sorption experiments and are of the order of 0.8. 10{sup -3} m{sup 3}/kg. Those for {sup 2}2Na{sup +} are of the order of 1.0. 10{sup -3} m{sup 3}/kg and are by a factor of two larger than values from batch sorption experiments. The values for the effective diffusion coefficients for HTO are of the order of (2-3).10{sup -1}0 m{sup 2}/s, and those for sodium are roughly by a factor of two smaller than values for HTO. On the one hand, the observed tracer uptake could only partially be addressed to isotope exchange; the most obvious process which could account for the remaining part of the uptaken tracer mass is diffusion into a second type of porosity, the dead-end pores. On the other hand, the results and conclusions drawn are encouraging for future investigations; therefore no major deficiency concerning the applied equipment and the modelling methodology

  10. Antitumor activity of two gelsemine metabolites in rat liver microsomes.

    Science.gov (United States)

    Zhao, Qing-Chun; Hua, Wei; Zhang, Lin; Guo, Tao; Zhao, Ming-Hong; Yan, Ming; Shi, Guo-Bing; Wu, Li-Jun

    2010-09-01

    Gelsemine is one of the major alkaloids from Gelsemium elegans Benth., which has been used as an antitumor remedy in clinic. In this paper, metabolism of gelsemine has been investigated in vitro in phenobarbital-treated rat liver microsomes. The metabolites of gelsemine were separated and evaluated using the flash silica gel column, preparative HPLC, using NMR and MS methods. According to the spectral data, two metabolites, M1 and M2, were identified as 4-N-demethylgelsemine and 21-oxogelsemine, respectively. By the MTT method in vitro, the antitumor activities between gelsemine and its metabolites were compared in the HepG2 and HeLa cell lines. Moreover, the main metabolic pathway was further proposed.

  11. In vitro glucuronidation of ochratoxin a by rat liver microsomes.

    Science.gov (United States)

    Han, Zheng; Tangni, Emmanuel K; Di Mavungu, José Diana; Vanhaecke, Lynn; De Saeger, Sarah; Wu, Aibo; Callebaut, Alfons

    2013-12-18

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

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

    Directory of Open Access Journals (Sweden)

    Zheng Han

    2013-12-01

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

  13. Modification of radiation-induced oxidative damage in liposomal and microsomal membrane by eugenol

    Energy Technology Data Exchange (ETDEWEB)

    Pandey, B.N. [Radiation Biology and Health Sciences Division, Bhabha Atomic Research Centre, Mumbai 400 085 (India); Lathika, K.M. [Radiation Biology and Health Sciences Division, Bhabha Atomic Research Centre, Mumbai 400 085 (India); Mishra, K.P. [Radiation Biology and Health Sciences Division, Bhabha Atomic Research Centre, Mumbai 400 085 (India)]. E-mail: kpm@magnum.barc.ernet.in

    2006-03-15

    Radiation-induced membrane oxidative damage, and their modification by eugenol, a natural antioxidant, was investigated in liposomes and microsomes. Liposomes prepared with DPH showed decrease in fluorescence after {gamma}-irradiation, which was prevented significantly by eugenol and correlated with magnitude of oxidation of phospholipids. Presence of eugenol resulted in substantial inhibition in MDA formation in irradiated liposomes/microsomes, which was less effective when added after irradiation. Similarly, the increase in phospholipase C activity observed after irradiation in microsomes was inhibited in samples pre-treated with eugenol. Results suggest association of radio- oxidative membrane damage with alterations in signaling molecules, and eugenol significantly prevented these membrane damaging events.

  14. Modification of radiation-induced oxidative damage in liposomal and microsomal membrane by eugenol

    Science.gov (United States)

    Pandey, B. N.; Lathika, K. M.; Mishra, K. P.

    2006-03-01

    Radiation-induced membrane oxidative damage, and their modification by eugenol, a natural antioxidant, was investigated in liposomes and microsomes. Liposomes prepared with DPH showed decrease in fluorescence after γ-irradiation, which was prevented significantly by eugenol and correlated with magnitude of oxidation of phospholipids. Presence of eugenol resulted in substantial inhibition in MDA formation in irradiated liposomes/microsomes, which was less effective when added after irradiation. Similarly, the increase in phospholipase C activity observed after irradiation in microsomes was inhibited in samples pre-treated with eugenol. Results suggest association of radio- oxidative membrane damage with alterations in signaling molecules, and eugenol significantly prevented these membrane damaging events.

  15. A study of liver microsomal enzymes in rats following propoxur (Baygon) administration.

    Science.gov (United States)

    Nelson, D L; Lamb, D W; Mihail, F

    1984-08-01

    Groups of rats were given either propoxur, were left as untreated controls, or were given phenobarbital, DDT, chlordane or toxaphene which are known to induce liver microsomal detoxification enzymes. Microsomal enzyme activity was measured by testing the ability of liver homogenates to degrade EPN (O-ethyl O-(4-nitrophenyl) phenylphosphonothioate) to p-nitrophenol. The activity of aminopyrine-N-demethylase, cytochrome P-450 and p-nitroanisole-O-demethylase in liver homogenates of rats receiving propoxur was measured. Liver microsomal detoxification enzymes were not induced by propoxur exposure.

  16. Toxic alkaloids and their interaction with microsomal cytochrome P-450 in vitro.

    Science.gov (United States)

    Peeples, A; Dalvi, R R

    1982-12-01

    Studies on the binding spectra of certain alkaloids with rat liver microsomes revealed that brucine, scopolamine and strychnine are type I compounds, whereas boldine, emetine, nicotine, reserpine and sanguinarine show type II binding. In contrast, colchicine and solanine failed to produce any measurable binding spectra. In vitro incubation of colchicine, nicotine or scopolamine with microsomal suspensions and NADPH resulted in demethylation of these alkaloids, while the incubation of boldine, brucine, emetine, reserpine, sanguinarine or solanine showed little or no dealkylation reaction. Furthermore, the effect of these alkaloids on the in vitro microsomal metabolism of a drug, benzphetamine, has also been studied.

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

    Directory of Open Access Journals (Sweden)

    Christopher R Pudney

    2011-12-01

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

  18. Synthesis, conductivity behaviour and second-order nonlinear optics of partially substituted double KDP containing As{sup 5+} and Na{sup +}

    Energy Technology Data Exchange (ETDEWEB)

    Ennaceur, Nasreddine, E-mail: nennaceu@ens-cachan.fr [Laboratoire Physico-chimie de l' Etat Solide, Faculte des Sciences BP 1171, 3000 Sfax, Universite de Sfax (Tunisia); Laboratoire de Photonique Quantique Moleculaire, Institut d' Alembert-Ecole Normale Superieure, 94230 Cachan (France); Ledoux-Rak, Isabelle [Laboratoire de Photonique Quantique Moleculaire, Institut d' Alembert-Ecole Normale Superieure, 94230 Cachan (France); Mhiri, Tahar [Laboratoire Physico-chimie de l' Etat Solide, Faculte des Sciences BP 1171, 3000 Sfax, Universite de Sfax (Tunisia)

    2013-02-01

    A new solid solution K{sub 0.95}Na{sub 0.05}H{sub 2}(PO{sub 4}){sub 0.5}(AsO{sub 4}){sub 0.5} abbreviated (KNDAP) has been prepared by slow evaporation of an aqueous solution at room temperature. Its crystal structure which was solved by the direct method from single crystal X-ray diffraction data can give birth to two kinds of disorder. The first one is statistical or dynamical in the shortest O---H{sup Horizontal-Ellipsis }O hydrogen bond that can facilitate the migration of proton. As for the second kind, it is related to the effect of the substitution of Na{sup +} and As{sup 5+} in the same site occupancy as that of the K{sup +} and P{sup 5+}, respectively. This disorder could promote hard defects in the structure, which can enhance the physical properties, especially, the conductivity.

  19. A Toxoplasma gondii protein with homology to intracellular type Na{sup +}/H{sup +} exchangers is important for osmoregulation and invasion

    Energy Technology Data Exchange (ETDEWEB)

    Francia, Maria E.; Wicher, Sarah [Department of Biological Sciences, University of Idaho, Life Sciences South Room 142, Moscow, ID 83844 (United States); Pace, Douglas A. [Center for Tropical and Emerging Global Diseases and Department of Cellular Biology University of Georgia, Athens, GA 30602 (United States); Sullivan, Jack [Department of Biological Sciences, University of Idaho, Life Sciences South Room 142, Moscow, ID 83844 (United States); Moreno, Silvia N.J. [Center for Tropical and Emerging Global Diseases and Department of Cellular Biology University of Georgia, Athens, GA 30602 (United States); Arrizabalaga, Gustavo, E-mail: gustavo@uidaho.edu [Department of Biological Sciences, University of Idaho, Life Sciences South Room 142, Moscow, ID 83844 (United States)

    2011-06-10

    The obligate intracellular parasite Toxoplasma gondii is exposed to a variety of physiological conditions while propagating in an infected organism. The mechanisms by which Toxoplasma overcomes these dramatic changes in its environment are not known. In yeast and plants, ion detoxification and osmotic regulation are controlled by vacuolar compartments. A novel compartment named the plant-like vacuole or vacuolar compartment (PLV/VAC) has recently been described in T.gondii, which could potentially protect extracellular tachyzoites against salt and other ionic stresses. Here, we report the molecular characterization of the vacuolar type Na{sup +}/H{sup +} exchanger in T. gondii, TgNHE3, and its co-localization with the PLV/VAC proton-pyrophosphatase (TgVP1). We have created a TgNHE3 knockout strain, which is more sensitive to hyperosmotic shock and toxic levels of sodium, possesses a higher intracellular Ca{sup 2+} concentration [Ca{sup 2+}]{sub i}, and exhibits a reduced host invasion efficiency. The defect in invasion correlates with a measurable reduction in the secretion of the adhesin TgMIC2. Overall, our results suggest that the PLV/VAC has functions analogous to those of the vacuolar compartments of plants and yeasts, providing the parasite with a mechanism to resist ionic fluctuations and, potentially, regulate protein trafficking.

  20. Distinct interactions of Na{sup +} and Ca{sup 2+} ions with the selectivity filter of the bacterial sodium channel Na{sub V}Ab

    Energy Technology Data Exchange (ETDEWEB)

    Ke, Song, E-mail: song.ke@univie.ac.at [Department of Pharmacology and Toxicology, University of Vienna, Althanstrasse 14, UZA 2, A-1090 Vienna (Austria); Zangerl, Eva-Maria, E-mail: a0509032@unet.univie.ac.at [Department of Pharmacology and Toxicology, University of Vienna, Althanstrasse 14, UZA 2, A-1090 Vienna (Austria); Stary-Weinzinger, Anna, E-mail: anna.stary@univie.ac.at [Department of Pharmacology and Toxicology, University of Vienna, Althanstrasse 14, UZA 2, A-1090 Vienna (Austria)

    2013-01-25

    Highlights: ► Ca{sup 2+} translocates slowly in the filter, due to lack of “loose” knock-on mechanism. ► Identification of a high affinity binding site in Na{sub V}Ab selectivity filter. ► Changes of EEEE locus triggered by electrostatic interactions with Ca{sup 2+} ions. -- Abstract: Rapid and selective ion transport is essential for the generation and regulation of electrical signaling pathways in living organisms. In this study, we use molecular dynamics simulations and free energy calculations to investigate how the bacterial sodium channel Na{sub V}Ab (Arcobacter butzleri) differentiates between Na{sup +} and Ca{sup 2+} ions. Multiple nanosecond molecular dynamics simulations revealed distinct binding patterns for these two cations in the selectivity filter and suggested a high affinity calcium binding site formed by backbone atoms of residues Leu-176 and Thr-175 (S{sub CEN}) in the sodium channel selectivity filter.

  1. Stimulatory effects of chlordiazepoxide, diazepam and oxazepam on the drug-metabolizing enzymes in microsomes.

    Science.gov (United States)

    Jablońska, J K; Knobloch, K; Majka, J; Wiśniewska-Knypl, J M

    1975-09-01

    5 days' exposure of rats to daily doses of 400 mg/kg body wt. of chlordiazepoxide, diazepam and oxazepam stimulated the microsomal metabolism in the liver, as evidenced by acceleration of both p-hydroxylation of aniline and hydroxylation of benzene. The effect was accompanied by an increased concentration of liver microsomal protein and by the development of tolerance to the drugs. Similar effects were found after exposure of rats to lower doses of the drugs. The metabolism of aniline in vivo in rats treated with chlordiazepoxide was accelerated; this was correlated with development of tolerance to these drugs. It is suggested that both the stimulation of microsomal metabolism and the development of tolerance are associated with the induction of microsomal drug-metabolizing enzymes.

  2. CHARACTERIZATION OF HYDROQUINONE AND CATECHOL FORMATION USING HEPATIC MICROSOMES FROM THREE SPECIES OF FISH

    Science.gov (United States)

    The in vitro metabolism of phenol at 11 degrees C has been studied using juvenile and immature adult rainbow (Orcorhynchus mykiss), immature adult brook (Salvelinus fontinalis), and immature adult lake trout (Salvelinus namacush) hepatic microsomal preparations.

  3. COMPARATIVE PHASE I AND II MICROSOMAL METABOLISM OF PHENOL IN THREE FISH SPECIES

    Science.gov (United States)

    In vitro metabolism of phenol at 11 degrees C has been studied using immature adult rainbow (Oncorhynchus mykiss), brook (Salvelinus fontinalis), and lake trout (Salvelinus namaycush) hepatic microsomal preparations. Incubations were optimized for time, cofactor concentration, pH...

  4. Does de novo synthesis of lysophosphatidylcholine occur in rat lung microsomes?

    NARCIS (Netherlands)

    Aarsman, A.J.; Bosch, H. van den

    1980-01-01

    Incubation of rat lung microsomes with CDP[Me-14C]choline resulted in formation of radioactive lysophosphatidylcholine and phosphatidylcholine. Evidence is provided which suggests that lysophosphatidylcholine formation cannot be ascribed completely to phospholipase A degradation of phosphatidylcholi

  5. Different enzyme kinetics of midazolam in recombinant CYP3A4 microsomes from human and insect sources.

    Science.gov (United States)

    Christensen, Hege; Mathiesen, Liv; Postvoll, Lillian W; Winther, Bjørn; Molden, Espen

    2009-01-01

    In vitro drug metabolism techniques with human CYP c-DNA expressed systems are frequently used to predict human drug metabolism in vivo. The aim of this study was to compare midazolam enzyme kinetics in recombinant expressed CYP3A4 microsomes from human and insect cells. The amounts of 1'- hydroxymidazolam and 4-hydroxymidazolam formed in CYP3A4 microsomes from transfected human liver epithelial cells (T5-3A4 microsomes) and baculovirus-infected insect cells (with and without coexpressed cytochrome b(5)) were analysed by LC-MS. Enzyme kinetic parameters were estimated by nonlinear regression. Mean K(m) for the formation of 1'-hydroxymidazolam was 3- and 4-fold higher in T5-3A4 microsomes than in insect microsomes (pmicrosomes was reflected by significantly lower Cl(int) compared to insect microsomes (pmicrosomes displayed Michaelis-Menten kinetics, while insect microsomes showed substrate inhibition kinetics. The different enzyme kinetics of midazolam observed in recombinant CYP3A4 microsomes from human and insect sources, especially the substantially higher K(m) obtained in human microsomes compared to insect microsomes, should be further evaluated since it may have implications for correlations to in vivo situation.

  6. In vitro characterization of borneol metabolites by GC-MS upon incubation with rat liver microsomes.

    Science.gov (United States)

    Zhang, Rong; Liu, Chang-hui; Huang, Tian-lai; Wang, Ning-sheng; Mi, Sui-qing

    2008-01-01

    The metabolism of borneol is studied by the analysis of incubations of in vitro-prepared rat liver microsomes. A sensitive gas chromatography (GC)-mass spectrometry (MS) method is developed for the identification of borneol and its metabolites. Four novel metabolites, which have not previously been reported, are isolated and confirmed by comparison of the GC-MS method. The biotransformation pathway of borneol in rat liver microsomes is proposed based on the in vitro results.

  7. Effect of x radiation on hydroperoxidase activity of rat liver microsomes

    Energy Technology Data Exchange (ETDEWEB)

    Platonov, A.G.; Deev, L.I.

    1979-06-01

    The effect of single exposure to total-body x radiation on hydroperoxidase activity (HPA) of rat liver microsomes and levels of cytochromes P-450 and b/sub 5/ in them were studied. Cytochrome b/sub 5/ content was measured in view of data indicative of the possible involvement of this enzyme in breaking down hydroperoxides of the microsomal fraction of the liver.

  8. Phospholipide turnover in microsomal membranes of the pancreas during enzyme secretion.

    Science.gov (United States)

    REDMAN, C M; HOKIN, L E

    1959-10-01

    After incubation of pigeon pancreas slices with P(32) and isolation of various fractions by differential centrifugation the deoxycholate extract of the microsome fraction was found to account for over half of the phospholipide P and over half of the P(32) incorporated into the phospholipides. The remaining phospholipide P and P(32) were fairly evenly distributed in the nuclei, zymogen granules, mitochondria, microsomal ribonucleoprotein particles, and the soluble fraction. When enzyme secretion was stimulated with acetylcholine about two-thirds of the increment in radioactivity in the total phospholipides was found in deoxycholate soluble components of the microsome fraction. The remainder of the increment was distributed in the other fractions. This indicates that the cellular component in which the increase in phospholipide turnover occurs on stimulation of secretion is a membranous structure. Evidence is presented which indicates that the increment in radioactivity in the non-microsomal fractions on stimulation of secretion is due to contamination of these fractions with fragments of the stimulated membranous structure. The distribution of P(32) radioactivity in each of the chromatographically separated phospholipides in the various fractions from unstimulated tissue paralleled the distribution of radioactivity in the total phospholipide fraction, indicating that individual phospholipides are not concentrated in different fractions but are associated together in the membranous structures of the microsome fraction. The major proportion of the stimulation of the turnover of the individual phospholipides also occurred in the microsome fraction. The distribution of radioactivity from glycerol-1-C(14) in the total phospholipides and in the individual phospholipides in the various fractions was similar to the distribution of P(32). In the microsome fraction acetylcholine stimulated the incorporation of glycerol-1-C(14) in each phospholipide which showed a stimulation

  9. Microsomal drug-metabolizing enzymes in the olive baboon (papio anabis)

    DEFF Research Database (Denmark)

    1975-01-01

    1.1. The activity of microsomal drug-metabolizing enzymes—azo reductase, nitroreductase, p-hydroxylation, N-demethylation, O-demethylation, NADPH cytochrome c reductase and cytochrome P P-450—in the olive baboon are lower than in other animal species, e.g. mouse, rat, guinea-pig. 2. 2. The level...... and beta-glucuronidase is present more in the lysosomal than in the microsomal fraction....

  10. Kinetics of glyburide metabolism by hepatic and placental microsomes of human and baboon.

    Science.gov (United States)

    Zharikova, Olga L; Ravindran, Selvan; Nanovskaya, Tatiana N; Hill, Ronald A; Hankins, Gary D V; Ahmed, Mahmoud S

    2007-06-15

    Glyburide (glibenclamide) is under investigation for treatment of gestational diabetes. Two metabolites of glyburide have been previously identified in patients, namely, 4-trans-(M1) and 3-cis-(M2) hydroxycyclohexyl glyburide. Recently, the metabolism of glyburide by microsomes of liver and placenta from humans and baboons revealed the formation of four additional metabolites: 4-cis-(M2a), 3-trans-(M3), and 2-trans-(M4) hydroxycyclohexyl glyburide, and ethyl-hydroxy glyburide (M5). The aim of this investigation was to determine the kinetics for the metabolism of glyburide by cytochrome P450 (CYP) isozymes of human and baboon placental and hepatic microsomes. The metabolism of glyburide by microsomes from the four organs revealed saturation kinetics and apparent K(m) values between 4 and 12 microM. However, the rates for formation of the metabolites varied between organs and species. M1 was the major metabolite (36% of total), formed by human hepatic microsomes with V(max) of 80+/-13 pmol mg protein(-1)min(-1), and together with M2, accounted for only 51% of the total. M5 was the major metabolite (87%) formed by human placental microsomes with V(max) of 11 pmol mg protein(-1)min(-1). In baboon liver, M5 had the highest rate of formation (V(max) 135+/-32 pmol mg protein(-1)min(-1), 39% of total), and in its placenta, was M4 (V(max) 0.7+/-0.1 pmol mg protein(-1)min(-1), 65%). The activity of human and baboon hepatic microsomes in metabolizing glyburide was similar, but the activity of human and baboon placental microsomes was 7% and 0.3% of their respective hepatic microsomes. The data obtained suggest that more than 1 CYP isozyme is responsible for catalyzing the hydroxylation of glyburide.

  11. Identification of CYP isozymes involved in benzbromarone metabolism in human liver microsomes.

    Science.gov (United States)

    Kobayashi, Kaoru; Kajiwara, Eri; Ishikawa, Masayuki; Oka, Hidenobu; Chiba, Kan

    2012-11-01

    Benzbromarone (BBR) is metabolized to 1'-hydroxy BBR and 6-hydroxy BBR in the liver. 6-Hydroxy BBR is further metabolized to 5,6-dihydroxy BBR. The aim of this study was to identify the CYP isozymes involved in the metabolism of BBR to 1'-hydroxy BBR and 6-hydroxy BBR and in the metabolism of 6-hydroxy BBR to 5,6-dihydroxy BBR in human liver microsomes. Among 11 recombinant P450 isozymes examined, CYP3A4 showed the highest formation rate of 1'-hydroxy BBR. The formation rate of 1'-hydroxy BBR significantly correlated with testosterone 6β-hydroxylation activity in a panel of 12 human liver microsomes. The formation of 1'-hydroxy BBR was completely inhibited by ketoconazole in pooled human liver microsomes. On the other hand, the highest formation rate of 6-hydroxy BBR was found in recombinant CYP2C9. The highest correlation was observed between the formation rate of 6-hydroxy BBR and diclofenac 4'-hydroxylation activity in 12 human liver microsomes. The formation of 6-hydroxy BBR was inhibited by tienilic acid in pooled human liver microsomes. The formation of 5,6-dihydroxy BBR from 6-hydroxy BBR was catalysed by recombinant CYP2C9 and CYP1A2. The formation rate of 5,6-dihydroxy BBR was significantly correlated with diclofenac 4'-hydroxylation activity and phenacetin O-deethylation activity in 12 human liver microsomes. The formation of 5,6-dihydroxy BBR was inhibited with either tienilic acid or α-naphthoflavone in human liver microsomes. These results suggest that (i) the formation of 1'-hydroxy BBR and 6-hydroxy BBR is mainly catalysed by CYP3A4 and CYP2C9, respectively, and (ii) the formation of 5,6-dihydroxy BBR is catalysed by CYP2C9 and CYP1A2 in human liver microsomes.

  12. Involvement of CYP2B6 in the biotransformation of propofol by human liver microsomes

    Institute of Scientific and Technical Information of China (English)

    TANG Bing; WANG Jun-ke; FENG Wan-yu

    2008-01-01

    Objective To determine whether the cytochrome P4502B6 (CYP2B6) is involved in the oxidation of propofol by human liver microsomes. Methods The change of propofol concentration in an incubation mixture with human liver microsomes was monitored by the high performance liquid chromatography (HPLC), in order to calculate the rate constants of metabolism of propofol. The correlation between the rate constants and the rate of metabolism of CYP2B6 selective substrate bupropion, and the effect of two different CYP2B6 specific inhibitors on the propofol metabolism were examined. Results The mean rate constant of propofol metabolism by liver microsomes obtained from twelve individuals was 3.9 (95 % confidence intervals 3.3, 4.5) nmol·min-1·mg-1 protein. The rate constants of propofol metabolism by liver microsomes were significantly correlated with bupropion hydroxylation (r=0.888, P<0.001). Both selective chemical inhibitors of CYP2B6, orphenadrine and N, N′, N″-triethylenethiophosphoramide (thioTEPA), reduced the rate constants of propofol metabolism by 37.596 (P<0.001) and 42.796 (P<0.001)in liver microsomes, respectively. Conclusions CYP2B6 is predominantly involved in the oxidation of propofol by human liver microsomes.

  13. Enantioselective metabolism of hydroxychloroquine employing rats and mice hepatic microsomes

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    Carmem Dickow Cardoso

    2009-12-01

    Full Text Available Hydroxychloroquine (HCQ is an important chiral drug used, mainly, in the treatment of rheumatoid arthritis, systemic lupus erythematosus and malaria, and whose pharmacokinetic and pharmacodynamic properties look to be stereoselective. Respecting the pharmacokinetic properties, some previous studies indicate that the stereoselectivity could express itself in the processes of metabolism, distribution and excretion and that the stereoselective metabolism looks to be a function of the studied species. So, the in vitro metabolism of HCQ was investigated using hepatic microsomes of rats and mice. The microsomal fraction of livers of Wistar rats and Balb-C mice was separated by ultracentrifugation and 500 μL were incubated for 180 minutes with 10 μL of racemic HCQ 1000 μg mL-1. Two stereospecific analytical methods, high performance liquid chromatography (HPLC and capillary electrophoresis (CE, were used to separate and quantify the formed metabolites. It was verified that the main formed metabolite is the (--(R-desethyl hydroxychloroquine for both animal species.A hidroxicloroquina (HCQ é um importante fármaco quiral usado, principalmente, no tratamento de artrite reumatóide, lupus eritematoso sistêmico e malária e cujas propriedades farmacocinéticas e farmacodinâmicas parecem ser estereosseletivas. Em relação às propriedades farmacocinéticas, alguns estudos prévios indicam que a estereosseletividade pode se expressar nos processos de metabolismo, distribuição e excreção e que o metabolismo estereosseletivo parece ser função da espécie estudada. Sendo assim, o metabolismo in vitro da HCQ foi investigado usando microssomas de fígado de ratos e de camundongos. A fração microssômica de fígados de ratos Wistar e de camundongos Balb-C foi isolada por ultracentrifugação e 500 μL foram incubados por 180 minutos com 10 μL de HCQ racêmica 1000 μg mL-1. Dois métodos analíticos estereoespecíficos, por cromatografia líquida de

  14. Oxidative metabolism of cocaine: comparison of brain and liver.

    Science.gov (United States)

    Benuck, M; Reith, M E; Sershen, H; Wiener, H L; Lajtha, A

    1989-01-01

    Norcocaine (NC) and N-hydroxynorcocaine (NHNC), products of the oxidative metabolism of cocaine, were examined in plasma, brain, and liver of mice injected intraperitoneally with cocaine. Plasma levels of NHNC were altered in vivo by inhibiting esterase activity with diazinon and chloral hydrate or activating esterase activity with phenobarbital, and activating the microsomal P-450 system with phenobarbital. Changes in plasma concentrations of NHNC resulted in similar changes in brain, which were often different from those in liver. After intracisternal administration of cocaine to mice, no appreciable amount of NC or NHNC could be detected in brain; the same results were obtained upon intracisternal and intraventricular administration to rats. Microsomal preparations from mouse brain were found to be considerably less active than those from liver in converting NC to NHNC. We conclude that the cerebral oxidative metabolism of cocaine is not appreciable and that most of the NC and NHNC found in the brain after systemic cocaine administration is derived from plasma rather than formed centrally by brain microsomal enzymes.

  15. Involvement of central microsomal prostaglandin E synthase-1 in IL-1beta-induced anorexia.

    Science.gov (United States)

    Pecchi, E; Dallaporta, M; Thirion, S; Salvat, C; Berenbaum, F; Jean, A; Troadec, J-D

    2006-05-16

    In response to infection or inflammation, individuals develop a set of symptoms referred to as sickness behavior, which includes a decrease in food intake. The characterization of the molecular mechanisms underlying this hypophagia remains critical, because chronic anorexia may represent a significant health risk. Prostaglandins (PGs) constitute an important inflammatory mediator family whose levels increase in the brain during inflammatory states, and their involvement in inflammatory-induced anorexia has been proposed. The microsomal PGE synthase (mPGES)-1 enzyme is involved in the last step of PGE2 biosynthesis, and its expression is stimulated by proinflammatory agents. The present study attempted to determine whether an upregulation of mPGES-1 gene expression may account for the immune-induced anorexic behavior. We focused our study on mPGES-1 expression in the hypothalamus and dorsal vagal complex, two structures strongly activated during peripheral inflammation and involved in the regulation of food intake. We showed that mPGES-1 gene expression was robustly upregulated in these structures after intraperitoneal and intracerebroventricular injections of anorexigenic doses of IL-1beta. This increase was correlated with the onset of anorexia. The concomitant reduction in food intake and central mPGES-1 gene upregulation led us to test the feeding behavior of mice lacking mPGES-1 during inflammation. Interestingly, IL-1beta failed to decrease food intake in mPGES-1(-/-) mice, although these animals developed anorexia in response to a PGE2 injection. Taken together, our results demonstrate that mPGES-1, which is strongly upregulated during inflammation in central structures involved in feeding control, is essential for immune anorexic behavior and thus may constitute a potential therapeutic target.

  16. Four new koumine metabolites in rat liver microsomes.

    Science.gov (United States)

    Zhang, Lin; Du, Lan; Lv, Wen-Wen; Zhao, Qing-Chun; Hua, Wei; An, Ye; Guo, Tao; Wu, Li-Jun

    2013-01-01

    Four new metabolites M-1 [1,2,18,19-tetradehydro-4-demethyl-3,17-epoxy-7,20(2H,19H)-cyclovobasan], M-2 [1,2,4,21,18,19-hexadehydro-4-demethyl-3,17-epoxy-7,20(2H,19H)-cyclovobasan], M-3 [1,2,18,19-tetradehydro-4-demethyl-4-formaldehyde-3,17-epoxy-7,20(2H,19H)-cyclovobasan], and M-4 [1,2,4,21,18,19-hexadehydro-4-demethyl-4-oxy-3,17-epoxy-7,20(2H,19H)-cyclovobasan] were isolated from the chloroform extract of koumine incubated with phenobarbital-treated rat liver microsomes. The structures of M-1, M-2, M-3, and M-4 were elucidated by spectroscopic methods including ESI-TOF-MS, 1D, and 2D NMR experiments. The metabolic pathway of koumine was proposed. The cytotoxic activities between koumine and its metabolites were also compared in the A549 cell line.

  17. Stereoselective degradation of tebuconazole in rat liver microsomes.

    Science.gov (United States)

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

    2012-01-01

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

  18. Cytotoxic mechanisms of Zn{sup 2+} and Cd{sup 2+} involve Na{sup +}/H{sup +} exchanger (NHE) activation by ROS

    Energy Technology Data Exchange (ETDEWEB)

    Koutsogiannaki, Sophia [Laboratory of Animal Physiology, Zoology Department, School of Biology, Faculty of Science, Aristotle University of Thessaloniki, 54124 Thessaloniki (Greece); Evangelinos, Nikolaos [Laboratory of Animal Physiology, Zoology Department, School of Biology, Faculty of Science, Aristotle University of Thessaloniki, 54124 Thessaloniki (Greece); Koliakos, George [Department of Biological Chemistry, Medical School, Aristotle University of Thessaloniki, P.O. Box 17034, 54124 Thessaloniki (Greece); Kaloyianni, Martha [Laboratory of Animal Physiology, Zoology Department, School of Biology, Faculty of Science, Aristotle University of Thessaloniki, 54124 Thessaloniki (Greece)]. E-mail: kaloyian@bio.auth.gr

    2006-07-20

    The signaling mechanism induced by cadmium (Cd) and zinc (Zn) in gill cells of Mytilus galloprovincialis was investigated. Both metals cause an increase in {center_dot}O{sub 2} {sup -} production, with Cd to be more potent (216 {+-} 15%) than Zn (150 {+-} 9.5%), in relation to control value (100%). The metals effect was reversed after incubation with the amiloride analogue, EIPA, a selective Na{sup +}/H{sup +} exchanger (NHE) inhibitor as well as in the presence of calphostin C, a protein kinase C (PKC) inhibitor. The heavy metals effect on {center_dot}O{sub 2} {sup -} production was mediated via the interaction of metal ions with {alpha}{sub 1}- and {beta}-adrenergic receptors, as shown after incubation with their respective agonists and antagonists. In addition, both metals caused an increase in intracellular pH (pHi) of gill cells. EIPA together with either metal significantly reduced the effect of each metal treatment on pHi. Incubation of gill cells with the oxidants rotenone, antimycin A and pyruvate caused a significant increase in pHi ({delta}pHi 0.830, 0.272 and 0.610, respectively), while in the presence of the anti-oxidant N-acetyl cysteine (NAC) a decrease in pHi ({delta}pHi -0.090) was measured, indicating that change in reactive oxygen species (ROS) production by heavy metals affects NHE activity. When rosiglitazone was incubated together with either heavy metal a decrease in O{sub 2} {sup -} production was observed. Our results show a key role of NHE in the signal transduction pathway induced by Zn and Cd in gill cells, with the involvement of ROS, PKC, adrenergic and PPAR-{gamma} receptors. In addition, differences between the two metals concerning NHE activation, O{sub 2} {sup -} production and interaction with adrenergic receptors were observed.

  19. Metabolism of ketoconazole and deacetylated ketoconazole by rat hepatic microsomes and flavin-containing monooxygenases.

    Science.gov (United States)

    Rodriguez, R J; Acosta, D

    1997-06-01

    Ketoconazole (KT) has been reported to cause hepatotoxicity, which is probably not mediated through an immunoallergic mechanism. Although KT is extensively metabolized by hepatic microsomal enzymes, the nature, route of formation, and toxicity of suspected metabolites are largely unknown. Recent reports indicate that N-deacetyl ketoconazole (DAK) is a major initial metabolite in mice, which, like lipophilic 4-alkylpiperazines, is susceptible to successive oxidative attacks on the N-1 position producing ring-opened dialdehydes. The rate of formation of DAK from hepatic rat microsomal incubations of KT was determined by HPLC. The rate of disappearance for KT was almost equal to the rate of DAK formation: 5.96 and 5.88 microM/hr, respectively. Also, the potential bioactivation of DAK was evaluated by measuring substrate activity of DAK with purified pig liver flavin-containing monooxygenase (FMO) and rat liver microsomes. Activity was measured by following DAK-dependent oxygen uptake polarographically at 37 degrees C in pyrophosphate buffer (pH 8.8) containing the glucose-6-phosphate NADPH-generating system. The K(M)'s of DAK were 34.6 and 77.4 microM for the purified FMO and rat microsomal FMO, respectively. Lastly, DAK was found to be metabolized by an NADPH-dependent rat liver microsomal monooxygenases at pH 8.8 to two metabolites as determined by HPLC. Heat inactivation of rat liver microsomal FMO abolished the formation of these metabolites from DAK. SKF-525A and anti-rat NADPH cytochrome P450 reductase did not inhibit this reaction. These results suggest that deacetylation of KT yields a major product, DAK, for further metabolism by microsomal monooxygenases that seem to be FMO-related.

  20. Overexpression of Catalase Enhances Benzo(a)pyrene Detoxification in Endothelial Microsomes

    Science.gov (United States)

    Yang, Fang; Yang, Hong; Ramesh, Aramandla; Goodwin, J. Shawn; Okoro, Emmanuel U.; Guo, ZhongMao

    2016-01-01

    We previously reported that overexpression of catalase upregulated xenobiotic- metabolizing enzyme (XME) expression and diminished benzo(a)pyrene (BaP) intermediate accumulation in mouse aortic endothelial cells (MAECs). Endoplasmic reticulum (ER) is the most active organelle involved in BaP metabolism. To examine the involvement of ER in catalase-induced BaP detoxification, we compared the level and distribution of XMEs, and the profile of BaP intermediates in the microsomes of wild-type and catalase transgenic endothelial cells. Our data showed that endothelial microsomes were enriched in cytochrome P450 (CYP) 1A1, CYP1B1 and epoxide hydrolase 1 (EH1), and contained considerable levels of NAD(P)H: quinone oxidoreductase-1 (NQO1) and glutathione S-transferase-pi (GSTP). Treatment of wild-type MAECs with 1μM BaP for 2 h increased the expression of microsomal CYP1A1, 1B1 and NQO1 by ~300, 64 and 116%, respectively. However, the same treatment did not significantly alter the expression of EH1 and GSTP. Overexpression of catalase did not significantly increase EH1, but upregulated BaP-induced expression of microsomal CYP1A1, 1B1, NQO1 and GSTP in the following order: 1A1>NQO1>GSTP>1B1. Overexpression of catalase did not alter the distribution of each of these enzymes in the microsomes. In contrast to our previous report showing lower level of BaP phenols versus BaP diols/diones in the whole-cell, this report demonstrated that the sum of microsomal BaP phenolic metabolites were ~60% greater than that of the BaP diols/diones after exposure of microsomes to BaP. Overexpression of catalase reduced the concentrations of microsomal BaP phenols and diols/diones by ~45 and 95%, respectively. This process enhanced the ratio of BaP phenol versus diol/dione metabolites in a potent manner. Taken together, upregulation of phase II XMEs and CYP1 proteins, but not EH1 in the ER might be the mechanism by which overexpression of catalase reduces the levels of all the BaP metabolites

  1. Targeted label-free approach for quantification of epoxide hydrolase and glutathione transferases in microsomes.

    Science.gov (United States)

    Song, Wei; Yu, Longjiang; Peng, Zhihong

    2015-06-01

    The aim of this study was to investigate the expression and organ distribution of cytochrome P450 (CYP450) enzymes, microsomal epoxide hydrolase (MEH), and microsomal glutathione-S-transferase (MGST 1, 2, 3) in human liver, lung, intestinal, and kidney microsomes by targeted peptide-based quantification using nano liquid chromatography-tandem multiple reaction monitoring (nano LC-MRM). Applying this method, we analyzed 16 human liver microsomes and pooled lung, kidney, and intestine microsomes. Nine of the CYP450s (CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4, 3A5) could be quantified in liver. Except for CYP3A4 and 3A5 existing in intestine, other CYP450s had little content (<0.1 pmol/mg protein) in extrahepatic tissues. MEH and MGSTs could be quantified both in hepatic and in extrahepatic tissues. The highest concentrations of MEH and MGST 1, 2 were found in liver; conversely MGST 3 was abundant in human kidney and intestine compared to liver. The targeted proteomics assay described here can be broadly and efficiently utilized as a tool for investigating the targeted proteins. The method also provides novel CYP450s, MEH, and MGSTs expression data in human hepatic and extrahepatic tissues that will benefit rational approaches to evaluate metabolism in drug development.

  2. Metabolism of 17alpha-hydroxyprogesterone caproate by hepatic and placental microsomes of human and baboons.

    Science.gov (United States)

    Yan, Ru; Nanovskaya, Tatiana N; Zharikova, Olga L; Mattison, Donald R; Hankins, Gary D V; Ahmed, Mahmoud S

    2008-05-01

    Recent data from our laboratory revealed the formation of an unknown metabolite of 17 hydroxyprogesterone caproate (17-HPC), used for treatment of preterm deliveries, during its perfusion across the dually perfused human placental lobule. Previously, we demonstrated that the drug is not hydrolyzed, neither in vivo nor in vitro, to progesterone and caproate. Therefore, the hypothesis for this investigation is that 17-HPC is actively metabolized by human and baboon (Papio cynocephalus) hepatic and placental microsomes. Baboon hepatic and placental microsomes were investigated to validate the nonhuman primate as an animal model for drug use during pregnancy. Data presented here indicate that human and baboon hepatic microsomes formed several mono-, di-, and tri-hydroxylated derivatives of 17-HPC. However, microsomes of human and baboon placentas metabolized 17-HPC to its mono-hydroxylated derivatives only in quantities that were a fraction of those formed by their respective livers, except for two metabolites (M16' and M17') that are unique for placenta and contributed to 25% and 75% of the total metabolites formed by human and baboon, respectively. The amounts of metabolites formed, relative to each other, by human and baboon microsomes were different suggesting that the affinity of 17-HPC to CYP enzymes and their activity could be species-dependent.

  3. Determination of microsomal CYP2A6 activity by high performance liquid chromatography

    Directory of Open Access Journals (Sweden)

    Lavhekar S

    2007-01-01

    Full Text Available Coumarin has been reported to be a reliable probe for the determination of human microsomal CYP2A6 activity. Coumarin is converted to 7-hydroxycoumarin by CYP2A6. A high pressure liquid chromatographic assay for the estimation of CYP2A6 activity in microsomes was evaluated. A RP C-18 Novapak Waters (15 cm x 3.9 mm, 5 µm column was used for the assay. The mobile phase composition was methanol : 1% glacial acetic acid (35:65 v/v (pH~3.1, with a flow rate of 0.6 ml/min, injection volume of 100 µl and detection at 320 nm. The retention times for coumarin and 7-hydroxycoumarin were 8.7 min and 5.3 min, respectively. The limit of detection (LOD was 0.05 µM, and the limit of quantitation (LOQ was 0.1 µM, for 7-hydroxycoumarin. The percent coefficient of variation associated with 7-hydroxycoumarin determination after duplicate estimation was found to be in the range 0.03 to 3.6% for buffer matrix and 0.1 to 6.5% for microsomal matrix. The mean rate of 7-hydroxycoumarin formation in guinea pig liver microsomes was 0.084 nmol/min/nmol P450. Coumarin 7-hydroxylase activity was absent in rat liver microsomes. No interference was observed from incubation mixture components.

  4. A two-dimensional protein map of Pleurotus ostreatus microsomes-proteome dynamics.

    Science.gov (United States)

    Petráčková, Denisa; Halada, Petr; Bezoušková, Silvia; Křesinová, Zdena; Svobodová, Kateřina

    2016-01-01

    Recent studies documented that several processes in filamentous fungi are connected with microsomal enzyme activities. In this work, microsomal subproteomes of Pleurotus ostreatus were analyzed by two-dimensional (2-D) polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry analysis. To assess proteome dynamics, microsomal proteins were isolated from fungal cultures after 7 and 12 days of cultivation. Additionally, 10 mg/L of 17α-ethinylestradiol (EE2) was treated with the cultures during 2 days. Despite the EE2 degradation by the fungus reached 97 and 76.3 % in 7- and 12-day-old cultures, respectively, only a minor effect on the composition of microsomal proteins was observed. The changes in protein maps related to ageing prevailed over those induced by EE2. Epoxide hydrolase, known to metabolize EE2, was detected in 12-day-old cultures only which suggests differences in EE2 degradation pathways utilized by fungal cultures of different age. The majority (32 %) of identified microsomal proteins were parts of mitochondrial energy metabolism.

  5. Maximum yields of microsomal-type membranes from small amounts of plant material without requiring ultracentrifugation.

    Science.gov (United States)

    Abas, Lindy; Luschnig, Christian

    2010-06-15

    Isolation of a microsomal membrane fraction is a common procedure in studies involving membrane proteins. By conventional definition, microsomal membranes are collected by centrifugation of a postmitochondrial fraction at 100,000g in an ultracentrifuge, a method originally developed for large amounts of mammalian tissue. We present a method for isolating microsomal-type membranes from small amounts of Arabidopsis thaliana plant material that does not rely on ultracentrifugation but instead uses the lower relative centrifugal force (21,000g) of a microcentrifuge. We show that the 21,000g pellet is equivalent to that obtained at 100,000g and that it contains all of the membrane fractions expected in a conventional microsomal fraction. Our method incorporates specific manipulation of sample density throughout the procedure, with minimal preclearance, minimal volumes of extraction buffer, and minimal sedimentation pathlength. These features allow maximal membrane yields, enabling membrane isolation from limited amounts of material. We further demonstrate that conventional ultracentrifuge-based protocols give submaximal yields due to losses during early stages of the procedure; that is, extensive amounts of microsomal-type membranes can sediment prematurely during the typical preclearance steps. Our protocol avoids such losses, thereby ensuring maximal yield and a representative total membrane fraction. The principles of our method can be adapted for nonplant material.

  6. Immunolocalization of a microsomal prostaglandin E synthase in rabbit kidney.

    Science.gov (United States)

    Fuson, Amanda L; Komlosi, Peter; Unlap, Tino M; Bell, P Darwin; Peti-Peterdi, János

    2003-09-01

    PGE2, the major cyclooxygenase (COX) metabolite of arachidonic acid, is an important paracrine regulator of numerous tubular and vascular functions in the kidney. To date, COX activity has been considered the key step in prostaglandin synthesis and is well characterized. However, much less is known about the recently cloned microsomal PGE2 synthase (mPGES), the terminal enzyme of PGE2 synthesis, which converts COX-derived PGH2 to the biologically important PGE2. Present studies provide the detailed localization of mPGES protein in the rabbit kidney using immunohistochemistry. In the cortex, strong mPGES labeling was found in the macula densa (MD) and principal cells of the connecting segment and cortical collecting tubule but not in intercalated cells. The medulla was abundant in mPGES-positive structures, with heavy labeling in the collecting duct system. In descending thin limbs and renal medullary interstitial cells, mPGES expression was less intense, and it was below the limits of detection in the vasa recta. Expression of MD mPGES, similarly to COX-2, was greatly increased in response to low-salt diet and angiotensin I-converting enzyme inhibition by captopril. These findings suggest autocrine regulation of renal salt and water transport by PGE2 in descending thin limb and collecting tubule and a paracrine effect of PGE2 on the glomerular and medullary vasculature. Similar to other organs, mPGES in the kidney is an inducible enzyme and may be similarly regulated and acts in concert with COX-2.

  7. Nonadiabatic ionic--covalent transitions. Exponential-linear model for the charge exchange and neutralization reactions Na+H arrow-right-left Na/sup +/+H/sup -/

    Energy Technology Data Exchange (ETDEWEB)

    Errea, L.F.; Mendez, L.; Mo, O.; Riera, A.

    1986-01-01

    A previous study of charge exchange processes taking place through ionic--covalent transitions is extended to the case of Na+H and Na/sup +/+H/sup -/ collisions. A five-state molecular expansion, with the inclusion of two-electron translation factors, is employed to calculate the charge exchange and neutralization cross sections. Transitions at the first two pseudocrossings between the energy curves, practically determine the cross sections in the energy range 0.16--5 keV amu/sup -1/. We also show that the widely used multichannel Landau--Zener theory is totally inadequate, to treat these transitions.

  8. Separation of 1-acylglycerolphosphate acyltransferase and 1-acylglycerolphosphorylcholine acyltransferase of rat liver microsomes.

    Science.gov (United States)

    Yamashita, S; Nakaya, N; Miki, Y; Numa, S

    1975-01-01

    1-Acylglycerolphosphate acyltransferase (Ec 2.3.1-) and 1-acylglycerolphosphorylcholine acyltransferase (EC 2.3.1.23) of rat liver microsomes were separated from each other. The separation was achieved by sucrose density gradient centrifugation of the enzyme preparation that was obtained by solubilizing microsomes with a nonionic detergent, Triton X-100, and subjecting the solubilized microsomes to molecular-sieve chromatography. The two acyltransferases are distinguishable from each other also with respect to their stabilities to heat and to Triton X-100. Hence, it is concluded that these acyltransferases are distinct enzymes. These results, together with our previous finding that glycerolphosphate acyltransferase is also a separate enzyme, demonstrate the presence of distinct acyltransferases responsible for the acylation of the different acyl acceptors. Furthermore, the acyl-donor specificities of these acyltransferases provide the enzymatic basis for the nonrandom distribution of fatty acids in naturally occurring glycerolipids. PMID:1054842

  9. Characterization of triptolide hydroxylation by cytochrome P450 in human and rat liver microsomes.

    Science.gov (United States)

    Li, W; Liu, Y; He, Y-Q; Zhang, J-W; Gao, Y; Ge, G-B; Liu, H-X; Huo, H; Liu, H-T; Wang, L-M; Sun, J; Wang, Q; Yang, L

    2008-12-01

    Triptolide, the primary active component of a traditional Chinese medicine Tripterygium wilfordii Hook F, has a wide range of pharmacological activities. In the present study, the metabolism of triptolide by cytochrome P450s was investigated in human and rat liver microsomes. Triptolide was converted to four metabolites (M-1, M-2, M-3, and M-4) in rat liver microsomes and three (M-2, M-3, and M-4) in human liver microsomes. All the products were identified as mono-hydroxylated triptolides by liquid chromatography-mass spectrometry (LC-MS). The studies with chemical selective inhibitors, complementary DNA-expressed human cytochrome P450s, correlation analysis, and enzyme kinetics were also conducted. The results demonstrate that CYP3A4 and CYP2C19 could be involved in the metabolism of triptolide in human liver, and that CYP3A4 was the primary isoform responsible for its hydroxylation.

  10. The lipidome and proteome of microsomes from the methylotrophic yeast Pichia pastoris.

    Science.gov (United States)

    Klug, Lisa; Tarazona, Pablo; Gruber, Clemens; Grillitsch, Karlheinz; Gasser, Brigitte; Trötzmüller, Martin; Köfeler, Harald; Leitner, Erich; Feussner, Ivo; Mattanovich, Diethard; Altmann, Friedrich; Daum, Günther

    2014-02-01

    The methylotrophic yeast Pichia pastoris is a popular yeast expression system for the production of heterologous proteins in biotechnology. Interestingly, cell organelles which play an important role in this process have so far been insufficiently investigated. For this reason, we started a systematic approach to isolate and characterize organelles from P. pastoris. In this study, we present a procedure to isolate microsomal membranes at high purity. These samples represent endoplasmic reticulum (ER) fractions which were subjected to molecular analysis of lipids and proteins. Organelle lipidomics included a detailed analysis of glycerophospholipids, fatty acids, sterols and sphingolipids. The microsomal proteome analyzed by mass spectrometry identified typical proteins of the ER known from other cell types, especially Saccharomyces cerevisiae, but also a number of unassigned gene products. The lipidome and proteome analysis of P. pastoris microsomes are prerequisite for a better understanding of functions of this organelle and for modifying this compartment for biotechnological applications.

  11. Evaluation of surfactants as solubilizing agents in microsomal metabolism reactions with lipophilic substrates.

    Science.gov (United States)

    Randall, Kathleen; Cheng, Shun Wen; Kotchevar, Anne Therese

    2011-10-01

    Solubilizing agents are routinely added when investigating the biotransformation of lipophilic substrates using hepatic microsomes. For highly lipophilic compounds, the concentration of solvent or surfactant necessary for dissolution can be detrimental to enzyme activity. This study evaluates the effect of 12 surfactants on microsomal metabolism and the ability of the same surfactants to improve the aqueous solubility of the pentabrominated diphenyl ether BDE-100, a lipophilic environmental contaminant previously found to be recalcitrant to in vitro metabolism. Of the surfactants investigated, Cremophor EL and Tween 80 displayed the best combination of increased BDE-100 solubility and minimal inhibition of microsomal metabolism. However, a comparison of the in vitro metabolism products of BDE-100 in the presence of the two surfactants revealed varying amounts of metabolites depending on the surfactant used.

  12. Temporal evolution of {sup 137}Cs{sup +}, K{sup +} and Na{sup +} in fruits of South American tropical species

    Energy Technology Data Exchange (ETDEWEB)

    Cid, A.S. [LARA — Laboratório de Radioecologia, Instituto de Física, Universidade Federal Fluminense, Av. Gal Milton Tavares de Souza, s/no, Gragoatá, 24210-340, Niterói, RJ (Brazil); Anjos, R.M., E-mail: meigikos@if.uff.br [LARA — Laboratório de Radioecologia, Instituto de Física, Universidade Federal Fluminense, Av. Gal Milton Tavares de Souza, s/no, Gragoatá, 24210-340, Niterói, RJ (Brazil); Zamboni, C.B. [Instituto de Pesquisas Energéticas e Nucleares (IPEN/CNEN), Av. Lineu Prestes 2242, Cidade Universitária, 05508-000, Paulo, SP (Brazil); Velasco, H. [GEA, Instituto de Matemática Aplicada San Luis (IMASL), Universidad Nacional de San Luis, Consejo Nacional de Investigaciones Científicas y Técnicas. Ej. de los Andes 950, D5700HHW San Luis (Argentina); Macario, K. [LARA — Laboratório de Radioecologia, Instituto de Física, Universidade Federal Fluminense, Av. Gal Milton Tavares de Souza, s/no, Gragoatá, 24210-340, Niterói, RJ (Brazil); Rizzotto, M. [GEA, Instituto de Matemática Aplicada San Luis (IMASL), Universidad Nacional de San Luis, Consejo Nacional de Investigaciones Científicas y Técnicas. Ej. de los Andes 950, D5700HHW San Luis (Argentina); and others

    2013-02-01

    Concentrations of {sup 137}Cs, K and Na in fruits of lemon (Citrus limon B.) and of K and Na in fruits of coconut (Cocos nucifera L.) trees were measured by both gamma spectrometry and neutron activation analysis, with the aim to understand the behaviour of monovalent inorganic cations in tropical plants as well as the plant ability to store these elements. Similar amounts of K{sup +} were incorporated by lemon and coconut trees during the growth and ripening processes of its fruits. The K concentration decreased exponentially during the growth of lemons and coconuts, ranging from 13 to 25 g kg{sup −1} dry weight. The incorporation of Na{sup +} differed considerably between the plant species studied. The Na concentration increased linearly during the lemon growth period (0.04 to 0.70 g kg{sup −1} d.w.) and decreased exponentially during the coconut growth period (1.4 to 0.5 g kg{sup −1} d.w.). Even though radiocaesium is not an essential element to plants, our results have shown that {sup 137}Cs incorporation to vegetable tissues is positively correlated to K distribution within the studied tropical plant species, suggesting that the two elements might be assimilated in a similar way, going through the biological cycle together. A mathematical model was developed from the experimental data allowing simulating the incorporation process of monovalent inorganic cations by the fruits of such tropical species. The agreement between the theoretical approach and the experimental values is satisfactory along fruit development. - Highlights: ► Concentrations of {sup 137}Cs, K and Na in fruits of lemon (Citrus limon B.) are presented. ► Concentrations of K and Na in fruits of coconut (Cocos nucifera L.) are also showed. ► We investigated the use of {sup 137}Cs as a tracer for the plant absorption of macronutrients. ► A model was developed to simulate the temporal evolution of {sup 137}Cs, K and Na by fruits. ► This model exhibited close agreement with our

  13. Effects of salinity stress on Bufo balearicus and Bufo bufo tadpoles: Tolerance, morphological gill alterations and Na{sup +}/K{sup +}-ATPase localization

    Energy Technology Data Exchange (ETDEWEB)

    Bernabò, Ilaria; Bonacci, Antonella; Coscarelli, Francesca [Department of Ecology, University of Calabria, Via P. Bucci, 87036 Rende (Cosenza) (Italy); Tripepi, Manuela [University of Pennsylvania, Department of Biology, 201 Leidy Laboratories, Philadelphia, PA 19104 (United States); Brunelli, Elvira, E-mail: brunelli@unical.it [Department of Ecology, University of Calabria, Via P. Bucci, 87036 Rende (Cosenza) (Italy)

    2013-05-15

    Freshwater habitats are globally threatened by human-induced secondary salinization. Amphibians are generally poorly adapted to survive in saline environments. We experimentally investigated the effects of chronic exposure to various salinities (5%, 10%, 15%, 20%, 25%, 30% and 35% seawater, SW) on survival, larval growth and metamorphosis of tadpoles from two amphibian populations belonging to two species: the green toad Bufo balearicus and the common toad Bufo bufo. In addition, gill morphology of tadpoles of both species after acute exposure to hypertonic conditions (20%, 25%, and 30% SW) was examined by light and electron microscopy. Tadpoles experienced 100% mortality above 20% SW in B. balearicus while above 15% SW in B. bufo. We detected also sublethal effects of salinity stress on growth and metamorphosis. B. bufo cannot withstand chronic exposure to salinity above 5% SW, tadpoles grew slower and were significantly smaller than those in control at metamorphosis. B. balearicus tolerated salinity up to 20% SW without apparent effects during larval development, but starting from 15% SW tadpoles metamorphosed later and at a smaller size compared with control. We also revealed a negative relation between increasing salt concentration and gill integrity. The main modifications were increased mucous secretion, detachment of external layer, alteration of epithelial surface, degeneration phenomena, appearance of residual bodies, and macrophage immigration. These morphological alterations of gill epithelium can interfere with respiratory function and both osmotic and acid-base regulation. Significant variations in branchial Na{sup +}/K{sup +}-ATPase activity were also observed between two species; moreover an increase in enzyme activity was evident in response to SW exposure. Epithelial responses to increasing salt concentration were different in the populations belonging to two species: the intensity of histological and ultrastructural pathology in B. bufo was

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

    Science.gov (United States)

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

    2013-12-01

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

  15. Fatty acyl-CoA esters inhibit glucose-6-phosphatase in rat liver microsomes.

    Science.gov (United States)

    Fulceri, R; Gamberucci, A; Scott, H M; Giunti, R; Burchell, A; Benedetti, A

    1995-01-01

    In native rat liver microsomes glucose 6-phosphatase activity is dependent not only on the activity of the glucose-6-phosphatase enzyme (which is lumenal) but also on the transport of glucose-6-phosphate, phosphate and glucose through the respective translocases T1, T2 and T3. By using enzymic assay techniques, palmitoyl-CoA or CoA was found to inhibit glucose-6-phosphatase activity in intact microsomes. The effect of CoA required ATP and fatty acids to form fatty acyl esters. Increasing concentrations (2-50 microM) of CoA (plus ATP and 20 microM added palmitic acid) or of palmitoyl-CoA progressively decreased glucose-6-phosphatase activity to 50% of the control value. The inhibition lowered the Vmax without significantly changing the Km. A non-hydrolysable analogue of palmitoyl-CoA also inhibited, demonstrating that binding of palmitoyl-CoA rather than hydrolysis produces the inhibition. Light-scattering measurements of osmotically induced changes in the size of rat liver microsomal vesicles pre-equilibrated in a low-osmolality buffer demonstrated that palmitoyl-CoA alone or CoA plus ATP and palmitic acid altered the microsomal permeability to glucose 6-phosphate, but not to glucose or phosphate, indicating that T1 is the site of palmitoyl-CoA binding and inhibition of glucose-6-phosphatase activity in native microsomes. The type of inhibition found suggests that liver microsomes may comprise vesicles heterogeneous with respect to glucose-6-phosphate translocase(s), i.e. sensitive or insensitive to fatty acid ester inhibition. PMID:7733874

  16. Metabolism of 4'-(9-acridinylamino)methanesulfon-m-anisidide by rat liver microsomes

    Energy Technology Data Exchange (ETDEWEB)

    Shoemaker, D.D.; Cysyk, R.L.; Gormley, P.E.; DeSouza, J.J.; Malspeis, L.

    1984-05-01

    4'-(9-Acridinylamino)methanesulfon-m-anisidide (m-AMSA) is metabolized by a hepatic microsomal enzyme system composed of rat liver microsomes, a reduced nicotinamide adenine dinucleotide phosphate-generating system, cytosolic protein (or glutathione), and oxygen. Omission of any one of the components, or incubation under an atmosphere of CO or N/sub 2/, results in inhibition of the reaction. Also, the addition of inhibitors of microsomal metabolism (alpha-naphthoflavone, metyrapone, or SKF 525-A) decreases m-AMSA metabolism. Metabolism of m-AMSA is more rapid with microsomes prepared from rats pretreated with phenobarbital or 3-methylcholanthrene. Two microsomal oxidation products of m-AMSA were isolated and identified as N1'-methanesulfonyl-N4'-(9-acridinyl)-3'-methoxy-2',5'-cyclohex adiene-1', 4'-dimine (m-AQDI) and 3'-methoxy-4'-(9-acridinylamino-2',5'-cyclohexadien-1'-one (m-AQI). m-AQDI reacts with glutathione to form a product previously identified in in vivo studies as the principal rat biliary metabolite and which is not cytotoxic to cultured L1210 cells. Thus, the end result of the microsomal metabolism of m-AMSA is detoxification. However, the two primary oxidation products (m-AQDI and m-AQI) are considerably more cytotoxic to L1210 cells in vitro than is m-AMSA. The concentration of m-AMSA required to produce a 5-log kill is 1.0 microgram/ml compared to 0.01 microgram/ml for m-AQDI and m-AQI. These results indicate that m-AMSA might undergo bioactivation to form the active cytotoxic species of the drug.

  17. Species and sex differences in propofol glucuronidation in liver microsomes of humans, monkeys, rats and mice.

    Science.gov (United States)

    Mukai, M; Isobe, T; Okada, K; Murata, M; Shigeyama, M; Hanioka, N

    2015-07-01

    Propofol (2,6-diisopropylphenol) is a short-acting anesthetic commonly used in clinical practice, and is rapidly metabolized into glucuronide by UDP-glucuronosyltransferase (UGT). In the present study, propofol glucuronidation was examined in the liver microsomes of male and female humans, monkeys, rats, and mice. The kinetics of propofol glucuronidation by liver microsomes fit the substrate inhibition model for humans and mice, the Hill model for monkeys, and the isoenzyme (biphasic) model for rats. The K(m), V(max), and CL(int) values of human liver microsomes were 50 μM, 5.6 nmol/min/mg protein, and 110 μL/min/mg protein, respectively, for males, and 46 μM, 6.0 nmol/min/mg protein, and 130 μL/min/mg protein, respectively, for females. The rank order of the CL(int) or CL(max) (in vitro clearance) values of liver microsomes was mice humans > monkeys > rats (high-affinity phase) rats (low-affinity phase) in both males and females. Although no significant sex differences were observed in the values of kinetic parameters in any animal species, the in vitro clearance values of liver microsomes were males females in monkeys, rats (high-affinity phase), and mice. These results demonstrated that the kinetic profile of propofol glucuronidation by liver microsomes markedly differed among humans, monkeys, rats, and mice, and suggest that species and sex differences exist in the roles of UGT isoform(s), including UGT1A9, involved in its metabolism.

  18. Triton X-114 phase separation in the isolation and purification of mouse liver microsomal membrane proteins.

    Science.gov (United States)

    Mathias, Rommel A; Chen, Yuan-Shou; Kapp, Eugene A; Greening, David W; Mathivanan, Suresh; Simpson, Richard J

    2011-08-01

    Integral membrane proteins (IMPs) mediate several cellular functions including cell adhesion, ion and nutrient transport, and cell signalling. IMPs are typically hard to isolate and purify due to their hydrophobic nature and low cellular abundance, however, microsomes are small lipid vesicles rich in IMPs, which form spontaneously when cells are mechanically disrupted. In this study, we have employed mouse liver microsomes as a model for optimising a method for IMP isolation and characterisation. Microsomes were collected by differential centrifugation, purified with sodium carbonate, and subjected to GeLC-MS/MS analysis. A total of 1124 proteins were identified in the microsome fraction, with 47% (524/1124) predicted by TMHMM to contain at least one transmembrane domain (TMD). The ability of phase partitioning using the detergent Triton X-114 (TX-114) to further enrich for membrane proteins was evaluated. Microsomes were subjected to successive rounds of solubility-based phase separation, with proteins partitioning into the aqueous phase, detergent phase, or TX-114-insoluble pellet fraction. GeLC-MS/MS analysis of the three TX-114 fractions identified 1212 proteins, of which 146 were not detected in the un-fractionated microsome sample. Conspicuously, IMPs partitioned to the detergent phase, with 56% (435/770) of proteins identified in that fraction containing at least one TMD. GO Slim characterisation of the microsome proteome revealed enrichment of proteins from the endoplasmic reticulum, mitochondria, Golgi apparatus, endosome, and cytoplasm. Further, enzymes including monooxygenases were well represented with 35 cytochrome P450 identifications (CYPs 1A2, 2A5, 2A12, 2B10, 2C29, 2C37, 2C39, 2C44, 2C50, 2C54. 2C67, 2C68, 2C70, 2D10, 2D11, 2D22, 2D26, 2D9, 2E1, 2F2, 2J5, 2U1, 3A11, 3A13, 3A25, 4A10, 4A12A, 4A12B, 4F13, 4F14, 4F15, 4V3, 51,7B1, and 8B1). Evaluation of biological processes showed enrichment of proteins involved in fatty acid biosynthesis and

  19. The effect of alpha-tocopherol on lipid peroxidation of microsomes and mitochondria from rat testis.

    Science.gov (United States)

    Gavazza, M B; Catalá, A

    2006-04-01

    The testis is a remarkably active metabolic organ; hence it is suitable not only for studies of lipid metabolism in the organ itself but also for the study of lipid peroxidation processes in general. The content of fatty acids in testis is high with a prevalence of polyunsaturated fatty acids (PUFA) which renders this tissue very susceptible to lipid peroxidation. Studies were carried out to evaluate the effect of alpha-tocopherol in vitro on ascorbate-Fe(++) lipid peroxidation of rat testis microsomes and mitochondria. Chemiluminescence and fatty acid composition were used as an index of the oxidative destruction of lipids. Special attention was paid to the changes produced on the highly PUFA [C20:4 n6] and [C22:5 n6]. Lipid peroxidation of testis microsomes or mitochondria induced a significant decrease of both fatty acids. Total chemiluminescence was similar in both kinds of organelles when the peroxidized without (control) and with ascorbate-Fe(++) (peroxidized) groups were compared. Arachidonic acid was protected more efficiently than docosapentaenoic acid at all alpha-tocopherol concentrations tested when rat testis microsomes or mitochondria were incubated with ascorbate-Fe(++). The maximal percentage of inhibition in both organelles was approximately 70%; corresponding to an alpha-tocopherol concentration between 1 and 0.25 mM. IC50 values from the inhibition of alpha-tocopherol on the chemiluminescence were higher in microsomes (0.144 mM) than mitochondria (0.078 mM). The protective effect observed by alpha-tocopherol in rat testis mitochondria was higher compared with microsomes, associated with the higher amount of [C20:4 n6]+[C22:5 n6] in microsomes that in mitochondria. It is proposed that the vulnerability to lipid peroxidation of rat testis microsomes and mitochondria is different because of the different proportion of PUFA in these organelles The peroxidizability index (PI) was positively correlated with the level of long chain fatty acids. The

  20. Cytotoxicity of MEIC chemicals Nos. 11-30 in 3T3 mouse fibroblasts with and without microsomal activation

    DEFF Research Database (Denmark)

    Rasmussen, Eva

    1999-01-01

    The cytotoxicity of MEIC chemicals Nos, 11-30 was evaluated by determination of neutral red uptake in Balb/c 3T3 mouse fibroblasts with and without the addition of a microsomal activation mixture. The use of microsomes significantly decreased the cytotoxicity of malathion, 2,4-dichlorophenoxyacetic...... acid, propranolol, thioridazine, lithium sulfate, copper sulfate and thallium sulfate, whereas the cytotoxicity of 1,1,1-trichloroethylene, phenol, nicotine, and paraquat was significantly increased by use of the microsomal activation mixture. These cytotoxicity data are in line with observations...... in other studies on microsomal modulation of the cytotoxicity of the test substances. Moderate to good correlations were found between the cytotoxicity data and rodent lethality data, and the addition of microsomes slightly improved the in vitro/in vivo concordance. The evidence to support the relevance...

  1. Prediction of Drug-Induced Liver Injury in HepG2 Cells Cultured with Human Liver Microsomes.

    Science.gov (United States)

    Choi, Jong Min; Oh, Soo Jin; Lee, Ji-Yoon; Jeon, Jang Su; Ryu, Chang Seon; Kim, Young-Mi; Lee, Kiho; Kim, Sang Kyum

    2015-05-18

    Drug-induced liver injury (DILI) via metabolic activation by drug-metabolizing enzymes, especially cytochrome P450 (CYP), is a major cause of drug failure and drug withdrawal. In this study, an in vitro model using HepG2 cells in combination with human liver microsomes was developed for the prediction of DILI. The cytotoxicity of cyclophosphamide, a model drug for bioactivation, was augmented in HepG2 cells cultured with microsomes in a manner dependent on exposure time, microsomal protein concentration, and NADPH. Experiments using pan- or isoform-selective CYP inhibitors showed that CYP2B6 and CYP3A4 are responsible for the bioactivation of cyclophosphamide. In a metabolite identification study employing LC-ESI-QTrap and LC-ESI-QTOF, cyclophosphamide metabolites including phosphoramide mustard, a toxic metabolite, were detected in HepG2 cells cultured with microsomes, but not without microsomes. The cytotoxic effects of acetaminophen and diclofenac were also potentiated by microsomes. The potentiation of acetaminophen cytotoxicity was dependent on CYP-dependent metabolism, and the augmentation of diclofenac cytotoxicity was not mediated by either CYP- or UDP-glucuronosyltransferase-dependent metabolism. The cytotoxic effects of leflunomide, nefazodone, and bakuchiol were attenuated by microsomes. The detoxication of leflunomide by microsomes was attributed to mainly CYP3A4-dependent metabolism. The protective effect of microsomes against nefazodone cytotoxicity was dependent on both CYP-mediated metabolism and nonspecific protein binding. Nonspecific protein binding but not CYP-dependent metabolism played a critical role in the attenuation of bakuchiol cytotoxicity. The present study suggests that HepG2 cells cultured with human liver microsomes can be a reliable model in which to predict DILI via bioactivation by drug metabolizing enzymes.

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

    Science.gov (United States)

    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.

  3. Changes in brain glucose use and extracellular ions associated with kainic acid-induced seizures: (/sup 14/C)-2-deoxyglucose and intracranial

    Energy Technology Data Exchange (ETDEWEB)

    Chastain, J.E Jr.

    1986-01-01

    The effect of kainic acid (KA) on brain glucose use with coadministration of diazepam, and the effect of KA on brain extracellular (K/sup +/), Ca/sup 2 +/), and (Na/sup +/) was investigated in rats by means of (/sup 14/C)-2-deoxyglucose (2-DG) and intracranial microdialysis, respectively. Also, the impact of intracranial microdialysis on brain regional metabolic function was studied. Co-treatment with KA and diazepam attenuated KA-induced 3 hr increases and prevented 48 hr decreases in glucose use within all structures measured, particularly the piriform cortex and amygdala. Hippocampal CA/sub 3/, CA/sub 4/, and CA/sub 1/-ventral were least affected by diazepam. The results suggest that diazepam suppresses KA seizure spread from its focus, proposed to be CA/sub 3/. KA-induced ions changes were studied by intracranial microdialysis. Dialysis fibers were implanted within the hippocampus or piriform cortex and perfused 24 hr later. Samples, collected before and after KA, were analyzed for (K/sup +/), (Ca/sup 2 +/), and (Na/sup +/). KA caused an early and prolonged increase in extracellular (K/sup +/) and a negligible decrease in (Ca/sup 2 +/) within the hippocampus. In the piriform cortex, both (K/sup +/) and (Na/sup +/) increase during a period of early seizure signs. The results indicate that ion homostatic control of ion levels is better maintained during parenteral KA-induced seizures than when the brain is activated locally or during ischemia/hypoxia. The effect of intracranial microdialysis was studied by means of 2-DG in control state and KA-induced seizure state. The results indicate that intracranial microdialysis alters brain metabolic function during KA-induced seizures, but not in the control state. At 3 hr post KA, seizure metabolic activity was enhanced within the piriform cortex, and attenuated within the hippocampus.

  4. CYP3A4 mediated in vitro metabolism of vinflunine in human liver microsomes

    Institute of Scientific and Technical Information of China (English)

    Xiao-ping ZHAO; Jiao ZHONG; Xiao-quan LIU; Guang-ji WANG

    2007-01-01

    Aim: To study the metabolism of vinflunine and the effects of selective cyto-chrome P-450 (CYP450) inhibitors on the metabolism of vinflunine in human liver microsomes. Methods: Individual selective CYP450 inhibitors were used to inves-tigate their effects on the metabolism of vinflunine and the principal CYP450 isoform involved in the formation of metabolites M1 and M2 in human liver microsomes.Results: Vinflunine was rapidly metabolized to 2 metabolites: M1 and M2 in human liver microsomes. M1 and M2 were tentatively presumed to be the N-oxide metabo-lite or hydroxylated metabolite and epoxide metabolite of vinflunine, respectively. Ketoconazole uncompetitively inhibited the formation of M1, and competitively inhibited the formation of M2, while α-naphthoflavone, sulfaphenazole, diethyl dithiocarbamate, tranylcypromine and quinidine had little or no inhibitory effect on the formation of M1 and M2. Conclusion: Vinflunine is rapidly metabolized in human liver microsomes, and CYP3A4 is the major human CYP450 involved in the metabolism of vinflunine.

  5. The acylation of 1-acylglycero-3-phosphorylcholines by rat-liver microsomes

    NARCIS (Netherlands)

    Bosch, H. van den; Golde, L.M.G. van; Eibl, H.; Deenen, L.L.M. van

    1967-01-01

    1. 1. The transfer of acyl groups from acyl-coenzyme A derivatives to phosphatidylcholine by rat-liver microsomes was found to be significantly stimulated by the addition of synthetic 1-acylglycero-3-phosphorylcholines. Unsaturated acyl chains were transferred in preference to saturated ones, partic

  6. Microsomal quercetin glucuronidation in rat small intestine depends on age and segment

    Science.gov (United States)

    UDP-glucuronosyltransferase (UGT) activity toward the flavonoid quercetin and UGT protein were characterized in 3 equidistant small intestine (SI) segments from 4, 12, 18, and 28 mo male F344 rats, n=8/age using villin to control for enterocyte content. SI microsomal intrinsic clearance of quercetin...

  7. Age-related increases in F344 rat intestine microsomal quercetin glucuronidation

    Science.gov (United States)

    The objective of this study was to establish the extent age modifies intestinal quercetin glucuronidation capacity. Pooled microsomal fractions of three equidistant small intestine (SI) segments from 4, 12, 18, and 28 mo male F344 rats (n=8/group) were employed to model the enzyme kinetics of UDP-gl...

  8. Glutathione delays varies as-tocopherol oxidation and subsequent lipid peroxidation in rat liver microsomes

    Energy Technology Data Exchange (ETDEWEB)

    Robey, S.; Mavis, R.

    1986-05-01

    A method has been developed for in vitro trace radiolabeling of rat liver microsomes with /sup 3/H-..cap alpha..-tocopherol (..cap alpha..T*) which allows virtually complete oxidation of the ..cap alpha..T* under oxidizing conditions. The supernatant of a 16,000 xg centrifugation of homogenized rat liver, containing the cytosolic rat liver vitamin E (VE) transfer protein, was incubated with an ethanolic solution of ..cap alpha..T* for 10 minutes at 37/sup 0/C. Labeled microsomes were collected in the washed 100,000 xg pellet. Microsomes were then incubated with 30 ..mu..M Fe/sup 2 +/ in an NADPH-generating system, and both production of malondialdehyde (MDA) (a product of lipid peroxidation) and oxidation of ..cap alpha..T* were monitored over a time course in the presence and absence of glutathione (GSH). The results indicate virtually complete oxidation of ..cap alpha..T* precedes significant membrane lipid peroxidation, and that addition of 5 mM GSH delays both ..cap alpha..T* oxidation and subsequent MDA production. This suggests that the previously observed VE-dependent heat labile inhibition of microsomal lipid peroxidation by GSH involves maintaining membrane levels of ..cap alpha..-tocopherol.

  9. Characterization and biosynthesis of cytochrome b5 in rat liver microsomes

    Science.gov (United States)

    Sargent, J. R.; Vadlamudi, B. P.

    1968-01-01

    1. Cytochrome b5 is released from rat liver microsomes by both proteolytic enzymes and by treatments that disrupt phospholipids. Cytochrome P-420 is only released to a marked extent by treatments that disrupt phospholipids. 2. Cytochrome b5 was isolated in a pure state from both the rough and smooth fractions of rat liver microsomes after treatment with trypsin, and was shown to contain two cytochrome components with identical spectral properties. 3. Amino acid analyses of the two components are presented, together with peptide `fingerprint' patterns of tryptic digests of the two components. 4. Studies based on the direct isolation of cytochrome b5 after administration of a single dose of radioactive amino acid to rats demonstrate that the cytochrome is synthesized initially in the rough fraction of microsomes and only subsequently appears in the smooth fraction. 5. Isolated rat liver microsomes are capable of incorporating radioactive amino acids into cytochrome b5 under standard conditions. 6. Under these conditions the amino acid is incorporated into peptide linkage in the cytochrome. PMID:16742610

  10. Metabolism of kadsurenone and 9,10-dihydrokadsurenone in rhesus monkeys and rat liver microsomes

    Energy Technology Data Exchange (ETDEWEB)

    Thompson, K.L.; Chang, M.N.; Chabala, J.C.; Chiu, S.H.; Eline, D.; Hucker, H.B.; Sweeney, B.M.; White, S.D.; Arison, B.H.; Smith, J.L.

    1988-09-01

    The metabolism of the PAF antagonists kadsurenone and tritium-labeled 9,10-dihydrokadsurenone was studied in rhesus monkeys and rat liver microsomes. The monkey metabolites of the two drugs were isolated as their glucuronide conjugates from the urine of iv dosed males. The metabolites from both monkey and microsomal metabolism were purified by reverse phase HPLC and identified by spectral (NMR, UV, and mass spectrometric) analysis. The principal pathway of biotransformation of the tritium-labeled 9,10-dihydrokadsurenone in monkeys was hydroxylation of the C-5 propyl side chain to give two metabolites, 10-hydroxy-9,10-dihydrokadsurenone and 9-hydroxy-9,10-dihydrokadsurenone. These compounds were excreted as glucuronides. Microsomal incubation of tritium-labeled 9,10-dihydrokadsurenone yielded the 10-, 9-, and 8-hydroxy-9,10-dihydrokadsurenone as major metabolites. Kadsurenone was also metabolized at the C-5 side chain, an allyl group. The monoglucuronide of 9,10-dihydroxykadsurenone was isolated from monkey urine. Spectral analysis was not definitive as to the site of conjugation, and the structure of the metabolite was assigned as the C-10 conjugate. A major metabolite of rat liver microsomal incubation of kadsurenone was 9,10-dihydroxykadsurenone.

  11. ISOLATION OF SMOOTH VESICLES AND FREE RIBOSOMES FROM RAT LIVER MICROSOMES

    Science.gov (United States)

    Chauveau, J.; Moulé, Y.; Rouiller, C.; Schneebeli, J.

    1962-01-01

    Microsomes, isolated from rat liver homogenate in 0.88 M sucrose, have been fractionated by differential centrifugation. The 2nd microsomal fraction, sedimented between 60 minutes at 105,000 g and 3 hours at 145,000 g, consists mainly of smooth vesicles, free ribosomes, and ferritin. By utilizing the differences in density existing between the membranes and the granular elements it has been possible to separate the smooth membranes from the free ribosomes and ferritin. The procedure is to resuspend the 2nd microsomal fraction in a sucrose solution of 1.21 or 1.25 density and centrifuge it at 145,000 g for 20 or 40 hours. A centripetal migration of membranes and a centrifugal sedimentation of granular elements are obtained. Phospholipids, as well as the enzymatic activities DPNH-cytochrome c reductase, glucose-6-phosphatase and esterase are localized in the membranes. The free ribosomes have been purified by washing. A concentration of 200 µg RNA per mg nitrogen has been reached. RNA is also present in the membranes. These results are discussed in relation to current views on microsomal structure and chemistry. PMID:13878497

  12. Alcohol-induced oxidative stress in rat liver microsomes: Protective effect of Emblica officinalis.

    Science.gov (United States)

    Reddy, Vaddi Damodara; Padmavathi, Pannuru; Hymavathi, Reddyvari; Maturu, Paramahamsa; Varadacharyulu, N Ch

    2014-06-01

    The protective effect of Emblica officinalis fruit extract (EFE) against alcohol-induced oxidative damage in liver microsomes was investigated in rats. EFE (250mg/kg b.wt/day) and alcohol (5g/kg b.wt/day, 20%, w/v) were administered orally to animals for 60 days. Alcohol administration significantly increased lipid peroxidation, protein carbonyls with decreased sulfhydryl groups in microsomes, which were significantly restored to normal levels in EFE and alcohol co-administered rats. Alcohol administration also markedly decreased the levels of reduced glutathione (GSH), superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) in the liver microsomes, which were prevented with EFE administration. Further, alcohol administration significantly increased the activities of cytochrome P-450, Na(+)/K(+) and Mg(2+) ATPases and also membrane fluidity. But, administration of EFE along with alcohol restored the all above enzyme activities and membrane fluidity to normal level. Thus, EFE showed protective effects against alcohol-induced oxidative damage by possibly reducing the rate of lipid peroxidation and restoring the various membrane bound and antioxidant enzyme activities to normal levels, and also by protecting the membrane integrity in rat liver microsomes. In conclusion, the polyphenolic compounds including flavonoid and tannoid compounds present in EFE might be playing a major role against alcohol-induced oxidative stress in rats.

  13. Oxidation of carbon tetrachloride, bromotrichloromethane, and carbon tetrabromide by rat liver microsomes to electrophilic halogens

    Energy Technology Data Exchange (ETDEWEB)

    Mico, B.A. (National Inst. of General Medical Sciences, Bethesda, MD); Branchflower, R.V.; Pohl, L.R.; Pudzianowski, A.T.; Loew, G.H.

    1982-01-11

    In order to determine whether CCl/sub 4/, CBrCl/sub 3/, CBr/sub 4/ or CHCl/sub 3/ undergo oxidative metabolism to electrophilic halogens by liver microsomes, they were incubated with liver microsomes from phenobarbital pretreated rats in the presence of NADPH and 2,6-dimethylphenol. The analysis of the reaction mixtures by capillary gas chromatography mass spectrometry revealed that 4-chloro-2,6-dimethylphenol was a metabolite of CCl/sub 4/ and CBrCl/sub 3/ whereas 4-bromo-2,6-dimethylphenol was a metabolite of CBr/sub 4/. The formation of the metabolites was significantly decreased when the reactions were conducted with heat denatured microsomes, in the absence of NADPH or under an atmosphere of N/sub 2/. These results indicate that the chlorines of CBrCl/sub 3/ and CCl/sub 4/ and the bromines of CBr/sub 4/ are oxidatively metabolized by rat liver microsomes to electrophilic and potentially toxic metabolites.

  14. Development of vitamin D/sub 3/ 25-hydroxylase activity in rat liver microsomes

    Energy Technology Data Exchange (ETDEWEB)

    Thierry-Palmer, M.; Cullins, S.; Rashada, S.; Gray, T.K.; Free, A.

    1986-05-01

    The authors have determined the ontogeny of vitamin D/sub 3/ 25-hydroxylase activity in rat liver microsomes. Microsomes from fetuses, neonates, and their mothers were incubated with 44 nM /sup 3/H-vitamin D/sub 3/ in the presence of an NADPH generating system, oxygen, KCl, and MgCl/sub 2/. Lipid extracts of the incubation samples were partially purified by thin-layer chromatography. Tritiated 25-hydroxy vitamin D/sub 3/ (250HD/sub 3/) was analyzed by high-pressure liquid chromatography using 94/6 hexane/isopropanol. Production rate for 250HD/sub 3/ in the mothers ranged from 0.22 to 0.30 pmol/mg protein/hr. Activities in the fetuses and neonates were 2.1, 12.9, 32.0, 35.8, and 71.0% of that of their mothers at -3, 0, 2, 7, and 15 days of age. The cytosolic fraction protected the substrate from degradation, stimulated the vitamin D/sub 3/ 25-hydroxylase reaction in neonates and mothers (1.4 to 1.7 fold increase), and was absolutely required for 25-hydroxylase activity in fetuses. These data suggest that microsomal vitamin D/sub 3/ 25-hydroxylase activity develops slowly and approaches full activity near the weaning stage. A cytosolic factor present as early as -3 days of age stimulates the activity of the microsomal vitamin D/sub 3/ 25-hydroxylase.

  15. Biphenyl metabolism by rat liver microsomes. Regioselective effects of inducers, inhibitors, and solvents

    Energy Technology Data Exchange (ETDEWEB)

    Haugen, D.A.

    1981-01-01

    The effects of the inducers phenobarbital and 3-methylcholanthrene, the inhibitors 7,8-benzoflavone and 1-benzyl-imidazole, and the solvents methanol, acetone, and dimethyl sulfoxide on the 2-, 3-, and 4-hydroxylation of biphenyl and the O-de-ethylation of 7-ethoxycoumarin by rat liver microsomes were examined. Phenobarbital pretreatment primarily induced 2- and 3-hydroxylation, the latter most dramatically. 3-Methylcholanthrene pretreatment induced 2- and 3-hydroxylation to similar extents. The inhibitors and solvents had regioselective effects on biphenyl metabolism that were characteristic of the uninduced, phenobarbital-induced, and 3-methylcholanthrene-induced microsomes. The presence of multiple forms of cytochrome P-450 in uninduced microsomes is indicated by the regioselective effects of the solvents and the inhibitors. The 3-methylcholanthrene-dependent increases in 2- and 3-hydroxylation appear due to induction of a single form of cytochrome P-450, as indicated by similar dose-response relationships and similar changes in sensitivitty to the inhibitors. The phenobarbital-dependent increases in 2- and 3-hydroxylation appear due to the induction of two forms of cytochrome P-450, as indicated by different changes in sensitivity to the effects of dimethyl sulfoxide and 7,8-benzoflavone. The results indicate that examination of the regioselectivity of biphenyl metabolism is a useful approach for characterizing microsomal mono-oxygenases, and they suggest that the approach may also be useful in the characterization of purified mono-oxygenase systems. (JMT)

  16. Comparative azo reductase activity of red azo dyes through caecal and hepatic microsomal fraction in rats.

    Science.gov (United States)

    Singh, S; Das, M; Khanna, S K

    1997-09-01

    In order to study the rate of formation of toxic aromatic amines, anaerobic reduction of four red azo dyes viz. amaranth, carmoisine, fast Red E and ponceau 4R was investigated by incubating caecal content and hepatic microsomal fraction of rats with 37.5 microM concentration of dyes in sodium phosphate buffer pH 7.4 using NADPH generating system, glucose oxidase system and nitrogen as the gaseous phase. Caecal suspension exhibited higher azo reductase activity than that of hepatic microsomal fraction using any of the 4 azo dyes. Caecal microbes showed maximal azo reductase activity when ponceau 4R was used as a substrate followed by fast Red E and carmoisine, while with amaranth the activity was minimum. Similarly ponceau 4 R exhibited maximum hepatic microsomal azo reductase activity followed by fast Red E and carmoisine whereas, amaranth had minimum activity. Caecal flora possessed almost 17 fold higher degradative capability of ponceau 4 R and fast Red E colourants than the hepatic microsomal fraction. The higher reductive ability through caecal flora for ponceau 4R and fast Red E signifies the formation of more aromatic amines which may be re-absorbed through the intestine to be either eliminated through urine as conjugates or retained in the target tissues to elicit toxic effects.

  17. Genetically lowered microsomal epoxide hydrolase activity and tobacco-related cancer in 47,000 individuals

    DEFF Research Database (Denmark)

    Lee, Julie; Dahl, Morten; Nordestgaard, Børge G

    2011-01-01

    Two functional polymorphisms of the microsomal epoxide hydrolase (mEH) gene (EPHX1), Tyr113His (rs1051740) and His139Arg (rs2234922), have variably been found to influence susceptibility to various cancer forms. We tested whether genetically lowered mEH activity affects risk of developing cancer...

  18. Isolation and structural elucidation of tiamulin metabolites formed in liver microsomes of pigs

    DEFF Research Database (Denmark)

    Lykkeberg, Anne Kruse; Cornett, Claus; Halling-Sørensen, Bent;

    2006-01-01

    Although the antimicrobial tiamulin is extensively metabolized in pigs, the metabolism is not well investigated. In this work the NADPH dependent metabolism of tiamulin in liver microsomes from pigs has been studied. The tiamulin metabolites formed in the incubations were analysed using LC...

  19. Biosynthesis of intestinal microvillar proteins. Processing of aminopeptidase N by microsomal membranes

    DEFF Research Database (Denmark)

    Danielsen, E M; Norén, Ove; Sjöström, H

    1983-01-01

    The biosynthesis of small-intestinal aminopeptidase N (EC 3.4.11.2) was studied in a cell-free translation system derived from rabbit reticulocytes. When dog pancreatic microsomal fractions were present during translation, most of the aminopeptidase N synthesized was found in a membrane-bound rat......The biosynthesis of small-intestinal aminopeptidase N (EC 3.4.11.2) was studied in a cell-free translation system derived from rabbit reticulocytes. When dog pancreatic microsomal fractions were present during translation, most of the aminopeptidase N synthesized was found in a membrane......-bound rather than a soluble form, indicating that synthesis of the enzyme takes place on ribosomes attached to the rough endoplasmic reticulum. The microsomal fractions process the Mr-115 000 polypeptide, which is the primary translation product of aminopeptidase N, to a polypeptide of Mr 140 000...... that microsomal fractions should be added before about 25% of the polypeptide was synthesized to ensure processing to the high-mannose glycosylated form. This suggests that the signal sequence is situated in the N-terminal part of the aminopeptidase N. The size of the cell-free translation product in the absence...

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

    Science.gov (United States)

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

  1. Microsomal epoxide hydrolase genotypes and the risk for head and neck cancer.

    NARCIS (Netherlands)

    Lacko, M.; Roelofs, H.M.J.; Morsche, R.H.M. te; Voogd, A.C.; Ophuis, MB Oude; Peters, W.H.M.; Manni, J.J.

    2008-01-01

    BACKGROUND: Microsomal epoxide hydrolase (mEH) is an enzyme involved in the metabolism of (pre)carcinogens in tobacco smoke. We investigated whether functional genetic polymorphisms in mEH may have a risk-modifying effect on head and neck carcinogenesis. METHODS: Blood from 429 patients with oral, p

  2. Metabolism and metabolic inhibition of gamboglc acid in rat liver microsomes

    Institute of Scientific and Technical Information of China (English)

    Yi-tong LIU; Kun HAO; Xiao-quan LIU; Guang-Ji WANG

    2006-01-01

    Aim: To study the metabolism of gambogic acid (GA) and the effects of selective cytochrome P-450 (CYP450) inhibitors on the metabolism of GA in rat liver microsomes in vitro. Methods: Rat liver micrp,so,rn,e$ were used to perform metabolism studies. Various selective CYP450 inhibitors were used to investigate their effects on the metabolism of GA and the principal CYP450 isoform involved in the formation of major metabolite M1 in rat liver microsomes. Types of inhibition in an enzyme kinetics model were used to model the interaction. Results: GA was rapidly metabolized to two phase Ⅰ metabolites,, M1 and M2, in rat liver microsomes. M1 and M2 were tentatively presumed to be the hydration metabolite and epoxide metabolite of GA, respectively. α-Naphthoflavone uncompetitively inhibited the formation of M1 while ketoconazole, sulfophenazole, diethyl dithiocarbamate and quinidine had little or no inhibitory effects on the formation of M1. Conclusion: GA is rapidly metabolized in rat liver microsomes and M1 is crucial for the elimination of GA. Cytochrome P-450 1A2 is the major rat CYP involved in the metabolism of GA.

  3. Differential selectivity of cytochrome P450 inhibitors against probe substrates in human and rat liver microsomes

    Science.gov (United States)

    Eagling, Victoria A; Tjia, John F; Back, David J

    1998-01-01

    Aims Chemical inhibitors of cytochrome P450 (CYP) are a useful tool in defining the role of individual CYPs involved in drug metabolism. The aim of the present study was to evaluate the selectivity and rank the order of potency of a range of isoform-selective CYP inhibitors and to compare directly the effects of these inhibitors in human and rat hepatic microsomes. Methods Four chemical inhibitors of human cytochrome P450 isoforms, furafylline (CYP1A2), sulphaphenazole (CYP2C9), diethyldithiocarbamate (CYP2E1), and ketoconazole (CYP3A4) were screened for their inhibitory specificity towards CYP-mediated reactions in both human and rat liver microsomal preparations. Phenacetin O-deethylation, tolbutamide 4-hydroxylation, chlorzoxazone 6-hydroxylation and testosterone 6β-hydroxylation were monitored for enzyme activity. Results Furafylline was a potent, selective inhibitor of phenacetin O-deethylation (CYP1A2-mediated) in human liver microsomes (IC50 = 0.48 μm), but inhibited both phenacetin O-deethylation and tolbutamide 4-hydroxylation (CYP2C9-mediated) at equimolar concentrations in rat liver microsomes (IC50 = 20.8 and 24.0 μm respectively). Sulphaphenazole demonstrated selective inhibition of tolbutamide hydroxylation in human liver microsomes but failed to inhibit this reaction in rat liver microsomes. DDC demonstrated a low level of selectivity as an inhibitory probe for chlorzoxazone 6-hydroxylation (CYP2E1-mediated). DDC also inhibited testosterone 6β-hydroxylation (CYP3A-mediated) in man and rat, and tolbutamide 4-hydroxylase activity in rat. Ketoconazole was a very potent, selective inhibitor of CYP3A4 activity in human liver (IC50 = 0.04 μm). Although inhibiting CYP3A in rat liver it also inhibited all other reactions at concentrations ≤5 μm. Conclusions It is evident that CYP inhibitors do not exhibit the same selectivity in human and rat liver microsomes. This is due to differential selectivity of the inhibitors and/or differences in the CYP

  4. Estrogenic activity of styrene oligomers after metabolic activation by rat liver microsomes.

    Science.gov (United States)

    Kitamura, Shigeyuki; Ohmegi, Motoko; Sanoh, Seigo; Sugihara, Kazumi; Yoshihara, Shin'ichi; Fujimoto, Nariaki; Ohta, Shigeru

    2003-01-01

    In this study we examined estrogenic activity of styrene oligomers after metabolic activation by rat liver microsomes. Trans-1,2-diphenylcyclobutane (TCB), cis-1,2-diphenylcyclobutane (CCB), 1,3-diphenylpropane, 2,4-diphenyl-1-butene, 2,4,6-triphenyl-1-hexene, and 1-alpha-phenyl-4ss-(1 -phenylethyl)tetralin were negative in the yeast estrogen screening assay and estrogen reporter assay using estrogen-responsive human breast cancer cell line MCF-7. However, TCB exhibited estrogenic activity after incubation with liver microsomes of phenobarbital-treated rats in the presence of reduced nicotinamide adenine dinucleotide phosphate (NADPH). Minor activity was observed when liver microsomes of untreated or 3-methylcholanthrene-treated rats were used instead of those from phenobarbital-treated rats. CCB, 1,3-diphenylpropane, and 2,4-diphenyl-1-butene also exhibited estrogenic activity after metabolic activation by liver microsomes, but the activity was lower than that of TCB. 2,4,6-Triphenyl-1-hexene and 1-alpha-phenyl-4ss-(1 -phenylethyl)tetralin did not show estrogenic activity after such incubation. When TCB was incubated with liver microsomes of phenobarbital-treated rats in the presence of NADPH, three metabolites were detected by high-performance liquid chromatography (HPLC). One metabolite isolated by HPLC exhibited a significant estrogenic activity. The active metabolite was identified as trans-1-(4-hydroxyphenyl)-2-phenylcyclobutane by mass and nuclear magnetic resonance spectral analysis. These results suggest that the estrogenic activity of TCB was caused by the formation of the 4-hydroxylated metabolite. PMID:12611662

  5. Effect of benzo(a)pyrene exposure on fluoranthene metabolism by mouse adipose tissue microsomes.

    Science.gov (United States)

    Huderson, Ashley C; Harris, Deacqunita L; Niaz, Mohammad S; Ramesh, Aramandla

    2010-02-01

    The present study has been undertaken to examine whether exposure to benzo(a)pyrene (BaP), a polycyclic aromatic hydrocarbon (PAH) compound, influences the metabolism of fluoranthene (FLA), another PAH compound. Microsomes were isolated from the adipose tissue of mice that received 50 microg/kg BaP and incubated with FLA (3 microM) alone; FLA in combination with BaP at equimolar concentrations, and a control group that received nothing. Post-incubation, samples were extracted with ethyl acetate and analyzed for FLA metabolites by reverse-phase HPLC with fluorescence detection. The rate of FLA metabolism (pmol of metabolite/min/mg protein) was increased when microsomes from BaP-treated mice were exposed to FLA alone and FLA in combination with BaP, compared to controls. On the other hand, the difference in FLA metabolic rate between microsomes that were exposed to FLA + BaP was higher than the ones that received FLA. The microsomes from BaP-pre-treated mice produced a considerably higher proportion of FLA 2, 3-diol, and 2, 3 D FLA when microsomes were incubated with FLA. There were no differences in the FLA metabolite types formed when BaP-pre-treated mice were co-incubated with BaP and FLA than with FLA alone. The enhanced biotransformation of FLA as a result of prior and concomitant exposure to BaP may have implications for assessment of risks arising from human exposure to PAH mixtures.

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

    Science.gov (United States)

    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 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.

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

    Science.gov (United States)

    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.

  8. Metabolic activation of the olfactory toxicant, dichlobenil, in rat olfactory microsomes: comparative studies with p-nitrophenol.

    Science.gov (United States)

    Eriksson, C; Brittebo, E B

    1995-03-18

    The tissue-specific toxicity of the herbicide, dichlobenil (2,6-dichlorobenzonitrile), in the olfactory mucosa is related to a cytochrome P450 (P450)-dependent metabolism, depletion of glutathione and covalent binding of metabolites. Pretreatment of mice with diethyldithiocarbamate (DEDTC) protected against the dichlobenil-induced necrosis. Addition of DEDTC abolished the covalent binding of [14C]-dichlobenil to rat olfactory microsomes, whereas P4502E1-substrates such as ethanol, acetone or p-nitrophenol (NP) had no effect. The NP-hydroxylation in olfactory microsomes was > 6 times higher than that in liver microsomes and was markedly decreased following addition of dichlobenil, DEDTC or metyrapone. In liver microsomes of acetone-treated rats the NP-hydroxylation was markedly decreased following addition of DEDTC, whereas metyrapone and dichlobenil had no effect. In acetone-treated rats, the NP-hydroxylation and the metabolic activation of [14C]-dichlobenil in olfactory microsomes were decreased to 50 and 73% of untreated controls, respectively, whereas in liver microsomes these activities increased > 6 and 3.5-fold, respectively. An antibody to P4502E1 had no effect on the NP-hydroxylation or metabolic activation of [14C]-dichlobenil in olfactory microsomes, whereas the NP-hydroxylation in liver microsomes of acetone-treated rats was markedly decreased. In conclusion, the results do not support a major role for P4502E1 in the metabolic activation of dichlobenil or hydroxylation of NP in rat olfactory microsomes and suggest that these catalytic activities in the olfactory mucosa may represent a common form of P450.

  9. Kinetic studies on toluene metabolism in ethanol- and phenobarbital-induced rat liver microsomes in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Wang Ruisheng; Nakajima, Tamie (Shinshu Univ., Matsumoto (Japan). Dept. of Hygiene)

    1991-01-01

    In vitro metabolism of toluene was investigated at substrate concentrations of 0.03-6.25 mM in liver microsomes from control and ethanol- and phenobarbital (PB)-treated rats. Three metabolites, benzylalcohol (BA), o-and p-cresol, were measured by high-performance liquid chromatograph. BA was the main metabolite of toluene, whereas o- and p-creasol contributed only 1.1-1.5% and 1.7-2.8% of total metabolites, respectively, in microsomes from control rats. Ethanol treatment showed little effect on the percentages of three metabolites, but PB increased the percentages of o- and p-cresol to as high as 5.5% and 8.0%, respectively, following the increase in toluene concentration. There were two different isozymes with different K{sub m} involved in the side-chain hydroxylation of toluene in microsomes from control and ethanol-treated rats. One had a low K{sub m} value (0.13-0.17 mM) and could be greatly induced with ethanol treatment. The other was a high K{sub m} isozyme (0.60-0.87 mM). PB-induced isozyme showed a similar K{sub m} value to that of the high K{sub m} isozyme existing in microsomes from control and ethanol-treated rats. Two isozymes were involved in the formation of p-cresol in microsomes of control rats; the low-K{sub m} type had a similar value (0.15 mM) to the low isozyme of BA formation, but the high K{sub m} isozyme had a larger value (2.04 mM) than the high isozyme of BA. Only one enzyme responsible for o-cresol formation was detected in microsomes of control rats, and had a similar K{sub m} (2.11 mM) to that of the high K{sub m} isozyme of p-cresol. The high K{sub m} and low V{sub max} values of isozymes of aromatic hydroxylation account for the minor proportion of cresols. Ethanol and PB treatments enhanced the metabolism of toluene, but they differ in that the ethanol-inducible isozyme metabolized toluene at a faster rate at low substrate concentrations, whereas PB did so at high toluene concentrations. (orig./MG).

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

    Science.gov (United States)

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

    2013-09-05

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

  11. Immunochemical and immunoelectron microscope studies on localization of NADPH-cytochrome c reductase on rat liver microsomes

    Science.gov (United States)

    1976-01-01

    By the use of ferritin-conjugated antibody (conjugate) indirect immunoelectron microscopy, NADPH-cytochrome c reductase was localized on rat liver microsomes. Most microsomes in the sections had from 1 to 12 conjugates on their outer surfaces. Among the conjugates, 83% was estimated to bind to NADPH-cytochrome c reductase at a molecular ratio of 1:1, 12% at the ratio of 2:1, and 5% at the ratio of 3 or 4:1. The correlation between immunochemical and morphological data confirmed that most of the NADPH-cytochrome c reducatase reacted with the conjugates. Subsequent morphological analyses have revealed that the enzyme is distributed homogeneously on the outer surfaces of microsomes but heterogeneously within microsomes in groups of three to five enzyme molecules. PMID:812876

  12. Gender and Species-Mediated Differences in the In Vitro Metabolism of Triadimefon by Rodent Hepatic Microsomes

    Science.gov (United States)

    Understanding how metabolism kinetics differ between genders and species is important in developing informative pharmacokinetic models and accurately assessing risk. Metabolism of the conazole fungicide Triadimefon (TDN) was studied in hepatic microsomes of SD rats and CD-1 mice...

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

    Science.gov (United States)

    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...

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

    Science.gov (United States)

    Nicks, Diane

    2017-01-01

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

  15. Brain Basics

    Medline Plus

    Full Text Available ... News About Us Home > Health & Education > Educational Resources Brain Basics Introduction The Growing Brain The Working Brain ... to mental disorders, such as depression. The Growing Brain Inside the Brain: Neurons & Neural Circuits Neurons are ...

  16. Brain Basics

    Science.gov (United States)

    ... News About Us Home > Health & Education > Educational Resources Brain Basics Introduction The Growing Brain The Working Brain ... to mental disorders, such as depression. The Growing Brain Inside the Brain: Neurons & Neural Circuits Neurons are ...

  17. Diffusion of HTO, {sup 36}Cl{sup -}, {sup 125}I{sup -} and {sup 22}Na{sup +} in Opalinus Clay: Effect of Confining Pressure, Sample Orientation, Sample Depth and Temperature

    Energy Technology Data Exchange (ETDEWEB)

    Van Loon, L.R.; Soler, J.M

    2004-02-01

    Effective diffusion coefficients (D{sub e}), rock capacity factors ({alpha}) and diffusion-accessible porosities ({epsilon}) were measured using the through-diffusion technique. Transport (diffusion) was measured both perpendicular and parallel to the bedding. Special cells that allowed the application of an axial confining pressure were designed. The pressures applied ranged from 1 to 5 MPa for Mont Terri samples and between 4 and 15 MPa for Benken samples, the upper values representing the in-situ confining pressure at both locations. The test solutions used in the experiments were synthetic Opalinus Clay pore water, which has Na and Cl as main components (Mont Terri: I = 0.39 M; Benken: I = 0.20 M). Pressure only had a small effect on the value of the effective diffusion coefficients. In the case of Mont Terri samples, increasing the pressure from 1 to 5 MPa resulted in a decrease of the effective diffusion coefficient of 20% for HTO, 27% for {sup 36}Cl{sup -}, 29% for {sup 125}I{sup -} and 17 % for {sup 22}Na{sup +}. In the case of Benken samples, increasing the pressure from 4 to 15 MPa resulted in a decrease of D{sub e} of 17% for HTO, 22% for {sup 36}Cl{sup -}, 32% for {sup 125}I{sup -} and 17 % for {sup 22}Na{sup +}. Moreover, the effective diffusion coefficients for for {sup 36}Cl{sup -}are smaller than for HTO, which is consistent with an effect arising from anion exclusion. This ion exclusion effect is smaller in samples from Mont Terri than in samples from Benken, which can be explained by the higher ionic strength of the Mont Terri water used in the experiments. The diffusion of {sup 22}Na{sup +} is similar to that of HTO in the case of Mont Terri OPA. For Benken OPA, the D{sub e} value of {sup 22}Na{sup +} is a factor of 2 higher than that of HTO. This last observation cannot be explained so far but is comparable to experimental data from ANDRA (1999) on Callovo-Oxfordian claystones from the Meuse/Haute Same site. {sup 125}I{sup -} is retarded with

  18. Diffusion of HTO, {sup 36}Cl{sup -}, {sup 125}I{sup -} and {sup 22}Na{sup +} in Opalinus Clay: Effect of Confining Pressure, Sample Orientation, Sample Depth and Temperature

    Energy Technology Data Exchange (ETDEWEB)

    Van Loon, L.R.; Soler, J.M

    2004-02-01

    Effective diffusion coefficients (D{sub e}), rock capacity factors ({alpha}) and diffusion-accessible porosities ({epsilon}) were measured using the through-diffusion technique. Transport (diffusion) was measured both perpendicular and parallel to the bedding. Special cells that allowed the application of an axial confining pressure were designed. The pressures applied ranged from 1 to 5 MPa for Mont Terri samples and between 4 and 15 MPa for Benken samples, the upper values representing the in-situ confining pressure at both locations. The test solutions used in the experiments were synthetic Opalinus Clay pore water, which has Na and Cl as main components (Mont Terri: I = 0.39 M; Benken: I = 0.20 M). Pressure only had a small effect on the value of the effective diffusion coefficients. In the case of Mont Terri samples, increasing the pressure from 1 to 5 MPa resulted in a decrease of the effective diffusion coefficient of 20% for HTO, 27% for {sup 36}Cl{sup -}, 29% for {sup 125}I{sup -} and 17 % for {sup 22}Na{sup +}. In the case of Benken samples, increasing the pressure from 4 to 15 MPa resulted in a decrease of D{sub e} of 17% for HTO, 22% for {sup 36}Cl{sup -}, 32% for {sup 125}I{sup -} and 17 % for {sup 22}Na{sup +}. Moreover, the effective diffusion coefficients for for {sup 36}Cl{sup -}are smaller than for HTO, which is consistent with an effect arising from anion exclusion. This ion exclusion effect is smaller in samples from Mont Terri than in samples from Benken, which can be explained by the higher ionic strength of the Mont Terri water used in the experiments. The diffusion of {sup 22}Na{sup +} is similar to that of HTO in the case of Mont Terri OPA. For Benken OPA, the D{sub e} value of {sup 22}Na{sup +} is a factor of 2 higher than that of HTO. This last observation cannot be explained so far but is comparable to experimental data from ANDRA (1999) on Callovo-Oxfordian claystones from the Meuse/Haute Same site. {sup 125}I{sup -} is retarded with

  19. Identification of metabolites of fosinopril produced by human and rat liver microsomes with liquid chromatography-mass spectrometry.

    Science.gov (United States)

    Uutela, Päivi; Monto, Matti; Iso-Mustajärvi, Ilona; Madetoja, Mari; Yliperttula, Marjo; Ketola, Raimo A

    2014-03-12

    Metabolic profiles of prodrug fosinopril and pharmacologically active metabolite fosinoprilat were studied using human or rat liver microsomes and S9 fractions. Metabolites were identified by ultra high-performance liquid chromatography/quadrupole time-of-flight mass spectrometry (UHPLC/Q-TOF-MS) using electrospray ionization in the positive and negative ion mode. They were characterized by accurate MS and MS/MS spectra and based on their different fragmentation pathways. With human liver microsomes fosinopril was metabolized via hydroxylation, glucuronidation, and hydrolysis to fosinoprilat. As expected the main metabolite was fosinoprilat and it was further hydroxylated and glucuronidated. However, these metabolites were not detected after incubation of fosinoprilat with human liver microsomes, indicating that metabolic reactions occur in sequence and fosinopril is hydrolyzed after glucuronidation or hydroxylation. With the developed UHPLC/Q-TOF-MS method once or twice hydroxylated fosinopril metabolites were detected for the first time and different regioisomers were separated. It was observed that the hydrolysis of fosinopril to fosinoprilat was more efficient with rat than with human liver microsomes, and therefore more hydroxylated fosinoprilat metabolites were detected when rat liver microsomes were used. Glucuronidation of fosinopril was not observed with rat liver microsomes.

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

    Science.gov (United States)

    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.

  1. Effects of calmodulin antagonists on radiation-induced lipid peroxidation in microsomes

    Energy Technology Data Exchange (ETDEWEB)

    Varshney, R.; Kale, R.K. (Jawaharlal Nehru Univ., New Delhi (India). School of Life Sciences)

    1990-11-01

    Rat liver microsomes were irradiated with {gamma}-rays at a dose of 1.31 Gy s{sup -1}. The extent of lipid peroxidation, measured in terms of malondialdehyde (MDA) formed, increased with radiation dose. The presence of calmodulin antagonists during irradiation decreased lipid peroxidation. The order of their protective efficiency was: chlorpromazine (CPZ)>promethazine (PMZ)>trimeprazine (TMZ). Their protective effect was diminished in the presence of ferrous (Fe{sup 2+}) ions and was restored on addition of EDTA. However, calmodulin antagonists considerably inhibited radiation-induced lipid peroxidation in the presence of ferric (Fe{sup 3+}) ions. Calmodulin antagonists also decreased the cytochrome P-450 content of microsomes. These results are discussed with respect to their applicability to radiotherapy. A possible mechanism for the inhibition of radiation-induced lipid peroxidation is suggested. (author).

  2. Effect of sedimentation through sucrose solutions on the protein-synthesizing ability of rat liver microsomes

    Science.gov (United States)

    Leslie, Ruth A.; Mansbridge, J. N.

    1970-01-01

    1. Centrifugation of the postmitochondrial supernatant of rat liver through 1.0m-sucrose produces particles that have 85–95% less incorporating ability in a cell-free protein-synthesizing system than either ribosomes or microsomes. 2. The incorporation of [14C]phenylalanine into protein by particles prepared by sedimentation through 1.0m-sucrose is stimulated about 20-fold by addition of poly U. 3. The content of rapidly labelled RNA of microsomes is decreased during centrifugation through 1.0m-sucrose. 4. It is suggested that degradation of mRNA occurs during the formation of the pellet in the centrifuge tube as a result of a membrane-bound alkaline ribonuclease, after removal of the ribonuclease inhibitor of the soluble fraction. PMID:5451910

  3. Cleavage of honeybee prepromelittin by an endoprotease from rat liver microsomes: identification of intact signal peptide.

    Science.gov (United States)

    Mollay, C; Vilas, U; Kreil, G

    1982-01-01

    It has previously been shown that rat liver microsomes contain a proteolytic enzyme that cleaves honeybee prepromelittin to yield promelittin. This enzyme has now been further purified by centrifugation on a sucrose-deoxycholate gradient and then reconstituted into phospholipid vesicles. Incubation of prepromelittin with vesicles in the presence of melittin yields, in addition to promelittin, a hydrophobic peptide. The latter could be isolated by extraction with l-butanol and paper electrophoresis in 30% formic acid and was shown to be intact signal peptide by analysis of peptic fragments and automated Edman degradation. The microsomal enzyme is thus an endoprotease that hydrolyzes prepromelittin exclusively at the pre-pro junction. The precision of this cleavage of an insect preprotein by a rat liver enzyme indicates that we are dealing with the ubiquitous eukaryotic signal peptidase. PMID:7048315

  4. Formation of carcinogenic and inactive chrysene metabolites by rat liver microsomes of various monooxygenase activities

    Energy Technology Data Exchange (ETDEWEB)

    Jacob, J.; Grimmer, G.; Schmoldt, A.

    1982-12-01

    Microsomal oxidation of chrysene in rat liver occurs at various positions (1,2-; 3,4-; 5,6-). This has been verified by means of gas chromatography/mass spectrometry (GC/MS) and comparison with synthetic reference substances. After various rat pretreatments with inducers of the monooxygenase system the oxidation at the 3,4-position predominated in isolated microsomes. The formation of the ultimate carcinogen of chrysene - 1,2-dihydroxy-3,4-epoxy-1,2,3,4-tetrahydrochrysene - was not detectable in untreated rats. However, it was observed as 1,2,3-trihydroxy-1,2,3,4-tetrahydrochrysene-TMS-ether formed under workup and derivatisation conditions after pretreating the rats with phenobarbital, polychlorinated biphenyl, benzoflavone, or various polycyclic aromatic hydrocarbons. Polychlorinated biphenyls and benzoflavone were the most potent inducers for the formation of this metabolite.

  5. High-performance liquid chromatographic assay detects pentamidine metabolism by Fisher rat liver microsomes.

    Science.gov (United States)

    Tuttle, R H; Hall, J E; Tidwell, R R

    1997-01-24

    Fisher rat liver microsomes metabolized the antimicrobial drug pentamidine to four new compounds detected by gradient elution reversed-phase high-performance liquid chromatography with variable wavelength detection. Coelution experiments with pentamidine metabolite standards determined the new peaks to be previously identified hydroxylated metabolites of pentamidine, with 1,5-bis(4'-amidinophenoxy)-3-pentanol and 1,5-di-(4'-amidinophenoxy)-2-pentanol formed in the greatest amount. The data contradict a previous report that Fisher rat liver homogenates do not metabolize pentamidine. Pentamidine and its known primary metabolites have almost identical absorption spectra; thus, pentamidine metabolism must be evaluated using gradient elution HPLC to resolve pentamidine from its metabolites. The current assay has now been used to demonstrate that Fisher and Sprague-Dawley rat, mouse, rabbit and human liver microsomes all metabolize pentamidine in vitro.

  6. Flavin-containing monooxygenase-mediated metabolism of N-deacetyl ketoconazole by rat hepatic microsomes.

    Science.gov (United States)

    Rodriguez, R J; Proteau, P J; Marquez, B L; Hetherington, C L; Buckholz, C J; O'Connell, K L

    1999-08-01

    Although ketoconazole is extensively metabolized by hepatic microsomal enzymes, the route of formation and toxicity of suspected metabolites are largely unknown. Reports indicate that N-deacetyl ketoconazole (DAK) is a major initial metabolite in mice. DAK may be susceptible to successive oxidative attacks on the N-1 position by flavin-containing monooxygenases (FMO) producing potentially toxic metabolites. Previous laboratory findings have demonstrated that postnatal rat hepatic microsomes metabolize DAK by NADPH-dependent monooxygenases to two metabolites as determined by HPLC. Our current investigation evaluated DAK's metabolism in adult male and female rats and identified metabolites that may be responsible for ketoconazole's hepatotoxicity. DAK was extensively metabolized by rat liver microsomal monooxygenases at pH 8.8 in pyrophosphate buffer containing the glucose 6-phosphate NADPH-generating system to three metabolites as determined by HPLC. The initial metabolite of DAK was a secondary hydroxylamine, N-deacetyl-N-hydroxyketoconazole, which was confirmed by liquid chromatography/mass spectrometry and NMR spectroscopy. Extensive metabolism of DAK occurred at pH 8.8 in pyrophosphate buffer (female 29% and male 53% at 0.25 h; female 55% and male 57% at 0.5 h; and female 62% and male 66% at 1.0 h). Significantly less metabolism of DAK occurred at pH 7.4 in phosphate buffer (female 11%, male 17% at 0.25 h; female 20%, male 31% at 0.5 h; and female 27%, male 37% at 1 h). Heat inactivation of microsomal-FMO abolished the formation of these metabolites from DAK. SKF-525A did not inhibit this reaction. These results suggest that DAK appears to be extensively metabolized by adult FMO-mediated monooxygenation.

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

    Science.gov (United States)

    Marker, E K; Kulkarni, A P

    1986-01-01

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

  8. Mechanisms for epigallocatechin gallate induced inhibition of drug metabolizing enzymes in rat liver microsomes.

    Science.gov (United States)

    Weng, Zuquan; Greenhaw, James; Salminen, William F; Shi, Qiang

    2012-11-15

    Epigallocatechin gallate (EGCG) inhibits drug metabolizing enzymes by unknown mechanisms. Here we examined if the inhibition is due to covalent-binding of EGCG to the enzymes or formation of protein aggregates. EGCG was incubated with rat liver microsomes at 1-100μM for 30min. The EGCG-binding proteins were affinity purified using m-aminophenylboronic acid agarose and probed with antibodies against glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin, cytochrome P450 (CYP) 1A1, CYP1A2, CYP2B1/2, CYP2E1, CYP3A, catechol-O-methyltransferase (COMT) and microsomal glutathione transferase 1 (MGST1). All but actin and soluble COMT were positively detected at ≥1μM EGCG, indicating EGCG selectively bound to a subset of proteins including membrane-bound COMT. The binding correlated well with inhibition of CYP activities, except for CYP2E1 whose activity was unaffected despite evident binding. The antioxidant enzyme MGST1, but not cytosolic GSTs, was remarkably inhibited, providing novel evidence supporting the pro-oxidative effects of EGCG. When microsomes incubated with EGCG were probed on Western blots, all but the actin and CYP2E1 antibodies showed a significant reduction in binding at ≥1μM EGCG, suggesting that a fraction of the indicated proteins formed aggregates that likely contributed to the inhibitory effects of EGCG but were not recognizable by antibodies against the intact proteins. This raised the possibility that previous reports on EGCG regulating protein expression using GAPDH as a reference should be revisited for accuracy. Remarkable protein aggregate formation in EGCG-treated microsomes was also observed by analyzing Coomassie Blue-stained SDS-PAGE gels. EGCG effects were partially abolished in the presence of 1mM glutathione, suggesting they are particularly relevant to the in vivo conditions when glutathione is depleted by toxicant insults.

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

    NARCIS (Netherlands)

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

    1996-01-01

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

  10. Effect of pretreatment with hepatic microsomal enzyme inducers on the toxicity of diazinon in calves.

    Science.gov (United States)

    Abdelsalam, E B; Ford, E J

    1986-11-01

    The pretreatment of calves with a single dose of 10 mg kg-1 dieldrin or 21 daily doses of 10 mg kg-1 phenobarbitone increased the toxicity of diazinon as reflected by the development of more severe clinical signs and greater depression in whole blood cholinesterase activity in the pretreated calves. Induction by dieldrin or phenobarbitone of the hepatic microsomal enzyme amidopyrine-N-demethylase was also accompanied by a concurrent rise in the liver carboxylesterase activity.

  11. Cell-free synthesis of membrane proteins: tailored cell models out of microsomes.

    Science.gov (United States)

    Fenz, Susanne F; Sachse, Rita; Schmidt, Thomas; Kubick, Stefan

    2014-05-01

    Incorporation of proteins in biomimetic giant unilamellar vesicles (GUVs) is one of the hallmarks towards cell models in which we strive to obtain a better mechanistic understanding of the manifold cellular processes. The reconstruction of transmembrane proteins, like receptors or channels, into GUVs is a special challenge. This procedure is essential to make these proteins accessible to further functional investigation. Here we describe a strategy combining two approaches: cell-free eukaryotic protein expression for protein integration and GUV formation to prepare biomimetic cell models. The cell-free protein expression system in this study is based on insect lysates, which provide endoplasmic reticulum derived vesicles named microsomes. It enables signal-induced translocation and posttranslational modification of de novo synthesized membrane proteins. Combining these microsomes with synthetic lipids within the electroswelling process allowed for the rapid generation of giant proteo-liposomes of up to 50 μm in diameter. We incorporated various fluorescent protein-labeled membrane proteins into GUVs (the prenylated membrane anchor CAAX, the heparin-binding epithelial growth factor like factor Hb-EGF, the endothelin receptor ETB, the chemokine receptor CXCR4) and thus presented insect microsomes as functional modules for proteo-GUV formation. Single-molecule fluorescence microscopy was applied to detect and further characterize the proteins in the GUV membrane. To extend the options in the tailoring cell models toolbox, we synthesized two different membrane proteins sequentially in the same microsome. Additionally, we introduced biotinylated lipids to specifically immobilize proteo-GUVs on streptavidin-coated surfaces. We envision this achievement as an important first step toward systematic protein studies on technical surfaces.

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

    Science.gov (United States)

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

    2014-09-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1990-09-01

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

  14. Formation of Schiff base adduct between acetaldehyde and rat liver microsomal phosphatidylethanolamine.

    Science.gov (United States)

    Kenney, W C

    1984-01-01

    Recent studies have established the formation of acetaldehyde adducts of proteins even at low concentrations of acetaldehyde expected to occur in vivo under conditions of ethanol metabolism. Although formation of acetaldehyde adducts with phospholipids has been obtained at high pH values and at high concentrations of acetaldehyde, the occurrence of such adducts under more physiological conditions had yet to be demonstrated. Rat liver microsomes were incubated with 0.2 mM [14C]acetaldehyde at pH 7.4 and 37 degrees C. After treatment with sodium borohydride to reduce any Schiff bases formed, the phospholipids were isolated. The major radioactive component within the phospholipid fraction had chromatographic properties identical to N-ethylphosphatidylethanolamine. In addition, the nitrogenous base derived therefrom by acid hydrolysis was identical to N-ethylethanolamine. These results indicate that a Schiff base adduct between acetaldehyde and microsomal phosphatidylethanolamine had been formed during incubation of low concentrations of acetaldehyde with rat liver microsomes.

  15. Formation of 4-hydroxyochratoxin A from ochratoxin A by rat liver microsomes.

    Science.gov (United States)

    Størmer, F C; Pedersen, J I

    1980-01-01

    Hydroxyochratoxin A was isolated and identified from the urine of rats after injection with ochratoxin A. By incubating ochratoxin A with rat liver microsomes and reduced nicotinamide adenine dinucleotide phosphate, one major (90%) and two minor metabolites, more polar than ochratoxin A, were formed. Thin-layer chromatography revealed that the major metabolite had Rf values identical to those of hydroxyochratoxin A in six different solvent systems. Formation of the metabolites in vitro was inhibited by carbon monoxide and by metyrapone, and the rate of formation increased after pretreatment of the rats with phenobarbital. A type I spectrum appeared upon binding of ochratoxin A to microsomes with a spectral dissociation constant (Ks) of 37.6 microM. These findings strongly suggest the involvement of a cytochrome P-450 in the hydroxylation of ochratoxin A by rat liver microsomes. Apparent Km and Vmax values for the formation of hydroxyochratoxin A were determined to 50 microM and 5.5 nmol/mg of protein per h, respectively. PMID:7396488

  16. Enantioselective Degradation of (2RS, 3RS)-Paclobutrazol in Rat Liver Microsomes.

    Science.gov (United States)

    Wu, Shuchun; Yu, Miao; Zhang, Hu; Han, Jianzhong; Qian, Mingrong

    2015-05-01

    Paclobutrazol, with two stereogenic centers, but gives only (2R, 3R) and (2S, 3S)-enantiomers because of steric-hindrance effects, is an important plant growth regulator in agriculture and horticulture. Enantioselective degradation of paclobutrazol was investigated in rat liver microsomes in vitro. The degradation kinetics and the enantiomer fraction were determined using a Lux Cellulose-1 chiral column on a reverse-phase liquid chromatography-tandem mass spectrometry system. The t1/2 of (2R, 3R)-paclobutrazol is 18.60 min, while the t1/2 of (2S, 3S)-paclobutrazol is 10.93 min. Such consequences clearly indicated that the degradation of paclobutrazol in rat liver microsomes was stereoselective and the degradation rate of (2S, 3S)-paclobutrazol was much faster than (2R, 3R)-paclobutrazol. In addition, significant differences between the two enantiomers were also observed in enzyme kinetic parameters. The Vmax of (2S, 3S)-paclobutrazol was more than 2-fold of (2R, 3R)-paclobutrazol and the Clint of (2S, 3S)-paclobutrazol was higher than that of (2R, 3R)-paclobutrazol after incubation in rat liver microsomes. These results may have potential implications for better environmental and ecological risk assessment for paclobutrazol.

  17. Toxic dark effects of protoporphyrin on the cytochrome P-450 system in rat liver microsomes.

    Science.gov (United States)

    Williams, M; Van der Zee, J; Van Steveninck, J

    1992-01-01

    In erythropoietic protoporphyria, accumulation of protoporphyrin has been found in various tissues and liver cirrhosis occurs frequently in this disease, probably due to toxic dark effects of protoporphyrin. We have studied the effect of porphyrins on various enzymic functions in rat liver microsomes. Incubation of microsomes with protoporphyrin resulted in a concentration-dependent inhibition of the oxidation of 7-ethoxycoumarin and aminopyrine by the cytochrome P-450 system. Kinetic analysis showed a decrease in Vmax., whereas the Km was not affected (non-competitive inhibition). Furthermore, reduction of cytochrome c by the NADPH-cytochrome P-450 reductase and by the NADH-cytochrome b5 reductase was inhibited. However, the activity of the reductases was only affected when the microsomes were pre-incubated with protoporphyrin, and it was found that the inhibition was dependent on the duration of the pre-incubation. Kinetic analysis again revealed non-competitive inhibition. When these experiments were repeated with uroporphyrin, no inhibition could be observed. With Stern-Volmer plots it was demonstrated that this was most likely caused by the localization of the porphyrins: protoporphyrin is localized in the membrane, whereas uroporphyrin remains in solution. From these results it is concluded that accumulation of protoporphyrin in the liver may markedly affect the cytochrome P-450 system and thus its detoxification function. PMID:1332695

  18. Fluoroaluminate treatment of rat liver microsomes inhibits GTP-dependent vesicle fusion.

    Science.gov (United States)

    Comerford, J G; Dawson, A P

    1991-01-01

    1. Inhibition of GTP-dependent membrane fusion of rat liver microsomes requires preincubation of the membranes with GDP (17 microM) and relatively high Mg2+ concentration (0.5 mM) as well as AlCl3 (30 microM) and KF (5 mM). Preincubation is required for maximal inhibition (75%). 2. Vesicle fusion in rat liver microsomes has been demonstrated in the absence of polyethylene glycol (PEG). Further, inhibition by AlF4- of GTP-dependent vesicle fusion in the absence of PEG has been demonstrated. 3. Under similar preincubation conditions AlF4- can bring about inhibition (80%) of the high-affinity PEG-stimulated GTPase activity in rat liver microsomes, previously described by Nicchitta, Joseph & Williamson [(1986) FEBS Lett. 209, 243-248]. 4. Preincubation of small-Mr GTP-binding proteins (Gn proteins) on nitrocellulose strips with GDP (20 pM), AlCl3 (30 microM) and KF (5 mM) results in inhibition of binding of guanosine 5'-[gamma-[35S]thio]triphosphate to Gn proteins. The extent of inhibition of this binding differs for different Gn proteins. PMID:1747106

  19. Effect of GTP on the dolichol pathway for protein glycosylation in rat liver microsomes.

    Science.gov (United States)

    Bossuyt, X; Blanckaert, N

    1993-01-01

    Incubation of native rat liver microsomes with GTP resulted in enhanced incorporation of N-acetylglucosamine (GlcNAc) from UDP-GlcNAc into lipid acceptors. The stimulation of GlcNAc transfer by GTP was specific for GTP; ATP exerted no effect. The GTP effect was blocked by a non-hydrolysable GTP analogue guanosine 5'-[beta gamma-imido]triphosphate, indicating that GTP hydrolysis was crucial. Though dolichyl pyrophosphate NN'-diacetylchitobiose [Dol-PP-(GlcNAc)2] was the main radiolabelled product formed upon incubation of GTP-treated microsomes with UDP-GlcNAc, GTP selectively stimulated UDP-GlcNAc:dolichyl phosphate (Dol-P) N-acetylglucosaminyl 1-phosphotransferase (N-acetylglucosaminyl 1-phosphotransferase). This conclusion was reached on the basis of experiments in which tunicamycin was used to selectively inhibit N-acetylglucosaminyl 1-phosphotransferase. The enhanced transformation of Dol-P to dolichyl pyrophosphate N-acetylglucosamine (Dol-PP-GlcNAc) by GTP ultimately led to enhanced protein glycosylation. GTP-induced stimulation of GlcNAc incorporation in lipid and protein by GTP was observed also in microsomes fully permeabilized with Staph. aureus alpha-toxin. These findings refute the previous proposal [Godelaine, Beaufay, Wibo and Ravoet (1983) J. Cell Biol. 97, 340-350] that increased membrane permeability constitutes the mechanism whereby GTP activates the reactions of the dolichol pathway. PMID:8280061

  20. High affinity binding of (/sup 3/H)cocaine to rat liver microsomes

    Energy Technology Data Exchange (ETDEWEB)

    El-Maghrabi, E.A.; Calligaro, D.O.; Eldefrawi, M.E.

    1988-01-01

    )/sup 3/H)cocaine bound reversible, with high affinity and stereospecificity to rat liver microsomes. Little binding was detected in the lysosomal, mitochondrial and nuclear fractions. The binding kinetics were slow and the kinetically calculated K/sub D/ was 2 nM. Induction of mixed function oxidases by phenobarbital did not produce significant change in (/sup 3/H)cocaine binding. On the other hand, chronic administration of cocaine reduced (/sup 3/H)cocaine binding drastically. Neither treatment affected the affinity of the liver binding protein for cocaine. Microsomes from mouse and human livers had less cocaine-binding protein and lower affinity for cocaine than those from rat liver. Binding of (/sup 3/H)cocaine to rat liver microsomes was insensitive to monovalent cations and > 10 fold less sensitive to biogenic amines than the cocaine receptor in rat striatum. However, the liver protein had higher affinity for cocaine and metabolites except for norcocaine. Amine uptake inhibitors displaced (/sup 3/H)cocaine binding to liver with a different rank order of potency than their displacement of (/sup 3/H)cocaine binding to striatum. This high affinity (/sup 3/H)cocaine binding protein in liver is not likely to be monooxygenase, but may have a role in cocaine-induced hepatotoxicity

  1. The stimulatory effects of asbestos on NADPH-dependent lipid peroxidation in rat liver microsomes.

    Science.gov (United States)

    Fontecave, M; Mansuy, D; Jaouen, M; Pezerat, H

    1987-01-01

    Lipid peroxidation in rat liver microsomes induced by asbestos fibres, crocidolite and chrysotile, is greatly increased in the presence of NADPH, leading to malondialdehyde levels comparable with those induced by CCl4, a very strong inducer of lipid peroxidation. This synergic effect only occurs during the first minutes and could be explained by an increase or a regeneration of the ferrous active sites of asbestos by NADPH, which in turn could rapidly be prevented by the adsorption of microsomal proteins on the surface of the fibres. It is not inhibited by superoxide dismutase, catalase and mannitol, indicating that oxygen radicals are not involved in the reaction. It is also not inhibited by desferrioxamine, indicating that it is not due to a release of free iron ions in solution from the fibres. Lipid peroxidation in NADPH-supplemented microsomes is also greatly increased upon addition of magnetite. This could be linked to the presence of ferrous ions in this solid iron oxide, since the ferric oxides haematite and goethite are completely inactive. PMID:3036068

  2. CYP3A catalyses schizandrin biotransformation in human, minipig and rat liver microsomes.

    Science.gov (United States)

    Cao, Y-F; Zhang, Y-Y; Li, J; Ge, G-B; Hu, D; Liu, H-X; Huang, T; Wang, Y-C; Fang, Z-Z; Sun, D-X; Huo, H; Yin, J; Yang, L

    2010-01-01

    Schizandrin is recognized as the major absorbed effective constituent of Fructus schisandrae, which is extensively applied in Chinese medicinal formula. The present study aimed to profile the phase I metabolites of schizandrin and identify the cytochrome P450 (CYP) isoforms involved. After schizandrin was incubated with human liver microsomes, three metabolites were isolated by high-performance liquid chromatography (HPLC) and their structures were identified to be 8(R)-hydroxyl-schizandrin, 2-demethyl-8(R)-hydroxyl-schizandrin, 3-demethyl-8(R)-hydroxyl-schizandrin, by liquid chromatography-mass spectrometry (LC-MS), (1)H-nuclear magnetic resonance (NMR), and (13)C-NMR, respectively. A combination of correlation analysis, chemical inhibition studies, assays with recombinant CYPs, and enzyme kinetics indicated that CYP3A4 was the main hepatic isoform that cleared schizandrin. Rat and minipig liver microsomes were included when evaluating species differences, and the results showed little difference among the species. In conclusion, CYP3A4 plays a major role in the biotransformation of schizandrin in human liver microsomes. Minipig and rat could be surrogate models for man in schizandrin pharmacokinetic studies. Better knowledge of schizandrin's metabolic pathway could provide the vital information for understanding the pharmacokinetic behaviours of schizandrin contained in Chinese medicinal formula.

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

    Science.gov (United States)

    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.

  4. Identification of UDP-glucuronosyltransferase isoforms responsible for leonurine glucuronidation in human liver and intestinal microsomes.

    Science.gov (United States)

    Tan, Bo; Cai, Weimin; Zhang, Jinlian; Zhou, Ning; Ma, Guo; Yang, Ping; Zhu, Qing; Zhu, Yizhun

    2014-09-01

    Leonurine is a potent component of herbal medicine Herba leonuri. The detail information on leonurine metabolism in human has not been revealed so far. Two primary metabolites, leonurine O-glucuronide and demethylated leonurine, were observed and identified in pooled human liver microsomes (HLMs) and O-glucuronide is the predominant one. Among 12 recombinant human UDP-glucuronosyltransferases (UGTs), UGT1A1, UGT1A8, UGT1A9, and UGT1A10 showed catalyzing activity toward leonurine glucuronidation. The intrinsic clearance (CLint) of UGT1A1 was approximately 15-to 20-fold higher than that of UGT1A8, UGT1A9, and UGT1A10, respectively. Both chemical inhibition study and correlation study demonstrated that leonurine glucuronidation activities in HLMs had significant relationship with UGT1A1 activities. Leonurine glucuronide was the major metabolite in human liver microsomes. UGT1A1 was principal enzyme that responsible for leonurine glucuronidation in human liver and intestine microsomes.

  5. In Vitro Metabolism Studies of Polybrominated Diphenyl Ethers Using Rat and Human Liver Microsomes

    Directory of Open Access Journals (Sweden)

    Shun W. Cheng

    2008-01-01

    Full Text Available A number of studies have recently reported the bioaccumulation of the commonly used fire retardants, Polybrominated Diphenyl Ethers (PBDEs, in humans and wildlife. Exposure of animals to PBDEs has been shown to result in developmental neurological, reproductive abnormalities and the disruption of endocrine function. Thyroid hormone equilibria was also shown to be altered by PBDE exposure. There is evidence that hydroxylated metabolites of PBDEs are directly involved in some of these adverse effects. Although metabolites of PBDEs have been isolated and characterized during in vivo studies, the identification of metabolites from an in vitro system has been problematic. We investigated the in vitro metabolism of four PBDEs, with varying numbers of bromine atoms, in rat and human liver microsomes. The addition of small amounts of a nonionic surfactant to the reaction mixture was necessary to obtain measurable amounts of metabolites due to the low aqueous solubility of the PBDEs. Using gas chromatography/mass spectroscopy, mono and/or dihydroxylated metabolites were identified from three of the four PBDEs with phenobarbitol- and β-naphthoflavone-induced rat liver microsomes. When using uninduced rat or human liver microsomes, metabolites were found with only one of the PBDEs. The ease of PBDE metabolism appears to be inversely related to the number of bromine atoms on the parent compound.

  6. Metabolism of dictamnine in liver microsomes from mouse, rat, dog, monkey, and human.

    Science.gov (United States)

    Wang, Pei; Zhao, Yunli; Zhu, Yingdong; Sun, Jianbo; Yerke, Aaron; Sang, Shengmin; Yu, Zhiguo

    2016-02-05

    Dictamnine, a furoquinoline alkaloid isolated from the root bark of Dictamnus dasycarpus Turcz. (Rutaceae), is reported to have a wide range of pharmacological activities. In this study, the in vitro metabolic profiles of dictamnine in mouse, rat, dog, monkey, and human liver microsomes were investigated and compared. Dictamnine was incubated with liver microsomes in the presence of an NADPH-regenerating system, resulting in the formation of eight metabolites (M1-M8). M1 is an O-desmethyl metabolite. M5 and M6 are formed by a mono-hydroxylation of the benzene ring of dictamnine. M8 was tentatively identified as an N-oxide metabolite. The predominant metabolic pathway of dictamnine occurs through the epoxidation of the 2,3-olefinic to yield a 2,3-epoxide metabolite (M7), followed by the ring of the epoxide opening to give M4. Likewise, cleavage of the furan ring forms M2 and M3. Slight differences were observed in the in vitro metabolic profiles of dictamnine among the five species tested. A chemical inhibition study with a broad and five specific CYP450 inhibitors revealed that most of the dictamnine metabolites in liver microsomes are mediated by CYP450, with CYP3A4 as the predominant enzyme involved in the formation of M7, the major metabolite. These findings provide vital information to better understand the metabolic processes of dictamnine among various species.

  7. Pulmonary surfactant protein A inhibits the lipid peroxidation stimulated by linoleic acid hydroperoxide of rat lung mitochondria and microsomes.

    Science.gov (United States)

    Terrasa, Ana M; Guajardo, Margarita H; de Armas Sanabria, Elizabeth; Catalá, Angel

    2005-07-15

    Reactive oxygen species play an important role in several acute lung injuries. The lung tissue contains polyunsaturated fatty acids (PUFAs) that are substrates of lipid peroxidation that may lead to loss of the functional integrity of the cell membranes. In this study, we compare the in vitro protective effect of pulmonary surfactant protein A (SP-A), purified from porcine surfactant, against ascorbate-Fe(2+) lipid peroxidation stimulated by linoleic acid hydroperoxide (LHP) of the mitochondria and microsomes isolated from rat lung; deprived organelles of ascorbate and LHP were utilized as control. The process was measured simultaneously by chemiluminescence as well as by PUFA degradation of the total lipids isolated from these organelles. The addition of LHP to rat lung mitochondria or microsomes produces a marked increase in light emission; the highest value of activation was produced in microsomes (total chemiluminescence: 20.015+/-1.735 x 10(5) cpm). The inhibition of lipid peroxidation (decrease of chemiluminescence) was observed with the addition of increasing amounts (2.5 to 5.0 microg) of SP-A in rat lung mitochondria and 2.5 to 7.5 microg of SP-A in rat lung microsomes. The inhibitory effect reaches the highest values in the mitochondria, thus, 5.0 microg of SP-A produces a 100% inhibition in this membranes whereas 7.5 microg of SP-A produces a 51.25+/-3.48% inhibition in microsomes. The major difference in the fatty acid composition of total lipids isolated from native and peroxidized membranes was found in the arachidonic acid content; this decreased from 9.68+/-1.60% in the native group to 5.72+/-1.64% in peroxidized mitochondria and from 7.39+/-1.14% to 3.21+/-0.77% in microsomes. These changes were less pronounced in SP-A treated membranes; as an example, in the presence of 5.0 microg of SP-A, we observed a total protection of 20:4 n-6 (9.41+/-3.29%) in mitochondria, whereas 7.5 microg of SP-A produced a 65% protection in microsomes (5

  8. Brain herniation

    Science.gov (United States)

    ... herniation; Uncal herniation; Subfalcine herniation; Tonsillar herniation; Herniation - brain ... Brain herniation occurs when something inside the skull produces pressure that moves brain tissues. This is most ...

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

    Science.gov (United States)

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

    2000-01-01

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

  10. Curcumin blocks prostaglandin E2 biosynthesis through direct inhibition of the microsomal prostaglandin E2 synthase-1.

    Science.gov (United States)

    Koeberle, Andreas; Northoff, Hinnak; Werz, Oliver

    2009-08-01

    Prostaglandin E(2) (PGE(2)) plays a crucial role in the apparent link between tumor growth and chronic inflammation. Cyclooxygenase (COX)-2 and microsomal PGE(2) synthase-1, which are overexpressed in many cancers, are functionally coupled and thus produce massive PGE(2) in various tumors. Curcumin, a polyphenolic beta-diketone from tumeric with anti-carcinogenic and anti-inflammatory activities, was shown to suppress PGE(2) formation and to block the expression of COX-2 and of microsomal PGE(2) synthase-1. Here, we identified microsomal PGE(2) synthase-1 as a molecular target of curcumin and we show that inhibition of microsomal PGE(2) synthase-1 activity is the predominant mechanism of curcumin to suppress PGE(2) biosynthesis. Curcumin reversibly inhibited the conversion of PGH(2) to PGE(2) by microsomal PGE(2) synthase-1 in microsomes of interleukin-1beta-stimulated A549 lung carcinoma cells with an IC(50) of 0.2 to 0.3 micromol/L. Closely related polyphenols (e.g., resveratrol, coniferyl alcohol, eugenol, rosmarinic acid) failed in this respect, and isolated ovine COX-1 and human recombinant COX-2 were not inhibited by curcumin up to 30 micromol/L. In lipopolysaccharide-stimulated human whole blood, curcumin inhibited COX-2-derived PGE(2) formation from endogenous or from exogenous arachidonic acid, whereas the concomitant formation of COX-2-mediated 6-keto PGF(1)alpha and COX-1-derived 12(S)-hydroxy-5-cis-8,10-trans-heptadecatrienoic acid was suppressed only at significant higher concentrations. Based on the key function of PGE(2) in inflammation and carcinogenesis, inhibition of microsomal PGE(2) synthase-1 by curcumin provides a molecular basis for its anticarcinogenic and anti-inflammatory activities.

  11. Brain-specific interaction of a 91-kDa membrane-bound protein with the cytoplasmic tail of the 300-kDa mannose 6-phosphate receptor

    DEFF Research Database (Denmark)

    Rosorius, O; Issinger, O G; Braulke, T

    1996-01-01

    in microsomal and synaptosomal fractions. Furthermore, the formation of cross-link complexes with membrane proteins appeared to be developmentally and regionally regulated in the brain and inhibited upon ATP hydrolysis. The data suggest the requirement of specific protein interactions for MPR 300 functions...

  12. Degradation of methyl and ethyl mercury into inorganic mercury by hydroxyl radical produced from rat liver microsomes

    Energy Technology Data Exchange (ETDEWEB)

    Suda, Ikuo (Kumamoto Univ. (Japan). Dept. of Pharmacology); Hirayama, Kimiko (Kumamoto Univ. (Japan). Coll. of Medical Science)

    1992-07-01

    Liver microsomes were prepared from Wistar rat by the Ca{sup 2+} aggregation method. Under various conditions, ethyl mercury chloride (EtHgCl) or methyl mercury chloride (MeHgCl) was incubated with the microsomal preparations. After the incubation, the amounts of inorganic Hg and hydroxyl radical (.OH) in the preparations were determined. Although the preparations alone produced a small amount of inorganic Hg and .OH, the addition of NADPH to the preparations increased both inorganic Hg and .OH production, which were further accelerated by the addition of KCN. The addition of Fe(III)EDTA, a .OH formation promoter, to the microsome-NADPH-KCN system increased inorganic Hg production, whereas the addition of diethylenetriamine pentaacetic acid, a .OH formation inhibitor, decreased inorganic Hg production. When .OH scavengers such as mannitol and dimethyl sulfoxide were added to this system, the inorganic Hg production decreased. These results suggested that the .OH produced from liver microsomes was responsible for the degradation of MeHg and EtHg. Since both .OH and inorganic Hg production decreased with a concomitant decrease in NADPH-cytochrome P-450 reductase activities, it is suggested that this enzyme may be involved in the microsomal degradation of MeHg and EtHg. (orig.).

  13. Fluoranthene metabolism: Human and rat liver microsomes display different stereoselective formation of the trans-2,3-dihydrodiol

    Energy Technology Data Exchange (ETDEWEB)

    Day, B.W.; Sahali, Y.; Hutchins, D.A.; Wildschuette, M.P.; Pastorelli, R.; Nguyen, T.T.; Naylor, S.; Skipper, P.L.; Wishnok, J.S.; Tannenbaum, S.R. (Univ. of Pittsburgh, PA (United States))

    1992-11-01

    The metabolism of the environmental carcinogen fluoroanthene by human liver microsomes was compared to that by liver microsomes from rats treated with Aroclor 1254. Although the human-derived system gave primarily one product, similar metabolites were noted from each system. Enantiomers of the major metabolic product, in both cases the trans-2,3-dihydrodiol, were separated by chiral stationary-phase chromatography. Absolute configurations were assigned by application of the benzoate exciton chirality rules to the CD spectra of the 4-(dimethylamino)benzoyl esters. Liver microsomes from Aroclor 1254-treated rats produced the R,R enantiomer of the diol in 75-78% enantiomeric excess, while human liver microsomes produced this enantiomer in only 6-12% excess. The activities of these enantiomers were compared in Salmonella typhimurium strain TM677 mutagenicity assays employing the 9000g supernatant of Aroclor 1254-induced rat liver homogenates. Both the syn- and anti-2,3-dihydrodiol 1,10b-epoxides, which had only been inferred to be metabolites in previous studies, were isolated from the microsomal incubations by preparative reverse-phase HPLC. The evident exceptional aqueous stabilities of these diol epoxides were further examined by half-life determination experiments. Their tetrahydrotetrol hydrolysis products were also noted in the metabolite HPLC profiles. The structures of the tetrahydrotetrols were confirmed by total synthesis.

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

    Science.gov (United States)

    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.

  15. Biosynthesis and stereoselective analysis of (-)- and (+)-zaltoprofen glucuronide in rat hepatic microsomes and its application to the kinetic analysis.

    Science.gov (United States)

    Wang, Haina; Ji, Jianbo; Zeng, Su

    2011-08-15

    Zaltoprofen, available commercially as a racemic mixture, is a propionic acid derivative of non-steroidal anti-inflammatory drugs (NSAIDs). Firstly, (+)- and (-)-zaltoprofen glucuronide was biosynthesized and purified. Then a simple and rapid RP-HPLC analysis method for direct determination of (+)- and (-)-zaltoprofen glucuronide in rat hepatic microsomes was developed and validated. The calibration curves of (+)- and (-)-zaltoprofen glucuronide both showed good linearity in the concentration range from 0.15 to 31.13 μM. The lower limit of quantification was 0.15 μM. Finally, this method was used to investigate the enantioselectivity of zaltoprofen glucuronidation in rat hepatic microsomes. The kinetics of zaltoprofen glucuronidation in rat hepatic microsomes for 40 min incubation fit the Michaelis-Menten model. Kinetic analysis indicated that (-)-zaltoprofen had a higher glucuronidation rate in rat liver microsome than that of (+)-zaltoprofen. The catalyzing efficiency (V(max)/K(m)) ratio of (+)-zaltoprofen to (-)-enantiomer is 0.8 times in rat liver microsomes.

  16. Comparative study of the oxidation of propranolol enantiomers in hepatic and small intestinal microsomes from cynomolgus and marmoset monkeys.

    Science.gov (United States)

    Shimizudani, Takeshi; Nagaoka, Kenjiro; Hanioka, Nobumitsu; Yamano, Shigeru; Narimatsu, Shizuo

    2010-01-05

    Oxidative metabolism of propranolol (PL) enantiomers (R-PL and S-PL) to 4-hydroxypropranolol (4-OH-PL), 5-OH-PL and N-deisopropylpropranolol (NDP) was examined in hepatic microsomes from cynomolgus and marmoset monkeys and in small intestinal microsomes from monkeys and humans. In hepatic microsomes, levels of oxidation activities were similar between the two monkey species, and substrate enantioselectivity (R-PLmicrosomes. In small intestinal microsomes, activity levels were much higher in cynomolgus monkeys than in marmosets and humans and reversed substrate enantioselectivity (R-PL>S-PL) was seen in the formation of NDP in cynomolgus monkeys and humans and in the formation of 5-OH-PL in marmosets. The formation of the three metabolites in cynomolgus monkeys and the formation of NDP in marmosets were biphasic, while the formation of 4-OH-PL in humans was monophasic. From the inhibition experiments using CYP antibodies, CYP2C9 and 2C19 were thought to be involved as N-deisopropylases and CYP2D6 and 3A4 as 4-hydroxylases in human small intestine. Furthermore, CYP1A, 2C and 3A enzymes could be involved in cynomolgus monkeys and CYP2C and 3A enzymes in marmosets. These results indicate that the oxidative profile of PL in hepatic and small intestinal microsomes differ considerably among cynomolgus monkeys, marmosets and humans.

  17. Ultraviolet-induced photodegradation of cucumber (Cucumis sativus L. ) microsomal and soluble protein tryptophanyl residues in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Caldwell, C.R. (Dept. of Agriculture, Beltsville, MD (United States))

    1993-03-01

    The in vitro effects of ultraviolet B (280--320 nm) radiation on microsomal membrane proteins and partially purified ribulose bisphosphate carboxylase (Rubisco) from cucumber (Cucumis sativus L.) was investigated by measuring the direct photolytic reduction of tryptophan fluorescence and the formation of fluorescent photooxidation products. Exposure of microsomes and Rubisco to monochromatic 300-nm radiation resulted in the loss of intrinsic tryptophan fluorescence and the production of blue-emitting fluorophores. The major product of tryptophan photolysis was tentatively identified as N-formylkynurenine (N-FK). Even though the rates of tryptophan photodegradation and N-FK formation were similar, the amount of blue fluorescence produced was significantly higher in the microsomes relative to Rubisco. Studies with various free radical scavengers and other modifiers indicated that tryptophan photodegradation requires oxygen species. The optimum wavelengths for loss of tryptophan fluorescence were 290 nm for the microsomes and 280 nm for Rubisco. The temperature dependence of tryptophan fluorescence and rate of tryptophan photodegradation indicated an alteration in the cucumber microsomal membranes at about 24[degrees]C, which influenced protein structure and tryptophan photosensitivity. 29 refs., 6 figs., 1 tab.

  18. Isolation and biochemical characterization of the microsomal fraction from the digestive gland of mussel Mytilus galloprovincialis Lam.

    Science.gov (United States)

    Viarengo, A; Pertica, M; Mancinelli, G; Palmero, S; Orunesu, M

    1986-01-01

    A procedure to prepare microsomes from the mussel digestive gland is proposed. The data concerning the biochemical characterization of this subcellular fraction shows a typical RNA:protein ratio, but the presence of hydrolytic enzymes was also found; therefore a mixture of hydrolase inhibitors to study the different biochemical characteristics was used. The biochemical data demonstrate that glucose-6-phosphatase activity (G6Pase), a typical microsomal marker in mammalian cells, is not present in mussel digestive gland microsomes but a high non-specific phosphatase activity was detected. Benzo[a]pyrene hydroxylase activity was found to be present although in a minimal amount. The evaluation of the molecular weight of the rRNA demonstrates that the larger ribosomal subunit contains RNA of Mr 1.40 X 10(-6) (approximately 26S) and the smaller subunit is composed of RNA of Mr 0.65 X 10(-6) (18S). The data from mussel digestive gland microsomes was compared with that experimentally obtained from rat liver microsomes and discussed from a functional or an evolutionary point of view.

  19. Calcium uptake in brain synaptosomes: a pharmacologic study

    Energy Technology Data Exchange (ETDEWEB)

    Rampe, D.E.

    1986-01-01

    Pinched-off nerve endings (synaptosomes) from rat and guinea pig brain were used as a model to study Ca/sup 2 +/ entry mechanisms in neuronal tissue. Synaptosomes contain high affinity binding sites for both, 1,4-dihydropyridine Ca/sup 2 +/ channel antagonists, and activators. The thermodynamic characteristics of (/sup 3/H)nitrendipine building in synaptosomes were similar to those seen in both cardiac and smooth muscle preparations. Synaptosomes display two distinct K/sup +/-induced Ca/sup 2 +/ entry mechanisms. These are kinetically distinct with the faster of the two terminating in approx. 1 second while the slower persists for approx. minute. The slow phase uptake process is abolished in Na/sup +/-free media, is sensitive to antagonism by 3,4-dichlorobenzamil and displays a more rapid ontogenic appearance relative to the fast phase. It is likely that the slow phase represents Ca/sup 2 +/ entry via Na/sup +//Ca/sup 2 +/ exchange. The rapid inactivation of the fast phase coupled with its voltage dependence suggest that it represents Ca/sup 2 +/ entry via one or more types of voltage dependent Ca/sup 2 +/ channels. These channels may not be dihydropyridin sensitive since neither nitrendipine nor Bay K 8644 were shown to modulate synaptosomal Ca/sup 2 +/ uptake. The benzodiazepine receptor ligands Ro 5-4864, PK 11195 and diazepam all selectively inhibited fast phase Ca/sup 2 +/ entry relative to slow phase entry. In addition, these compounds altered (/sup 3/H)nitrendipine binding affinity. It is concluded that certain benzodiazepine receptor ligands can interact specifically with voltage dependent Ca/sup 2 +/ channels.

  20. Inhibition of Re Du Ning Injection on Enzyme Activities of Rat Liver Microsomes Using Cocktail Method

    Institute of Scientific and Technical Information of China (English)

    Xiao-qian Xu; Ting Geng; She-bing Zhang; Dan-yu Kang; Yan-jing Li; Gang Ding; Wen-zhe Huang; Zhen-zhong Wang; Wei Xiao

    2016-01-01

    Objective Re Du Ning Injection(RDN), a Chinese materia medica injection, is made from the extracts of Lonicerae Japonicae Flos, Gardeniae Fructus, and Artemisiae Annuae Herba. Since last decade, RDN has been widely used in China for the treatment of viral infection, fever, and inflammation. To assess the potential interacting of RDN with co-administered drugs, the inhibitory effects of RDN on the enzyme activities(CYP1A1, CYP1A2, CYP2C11, CYP2D1, and CYP3A1/2) of rat liver microsomes were investigated by a cocktail method. Methods A sensitive and specific LC-MS method capable of simultaneous quantification of five metabolites in rat liver microsomes was developed and validated. Then RDN(0.625%–1.0%) was incubated with rat liver microsomes and specific substrates. The enzyme activities were expressed as the formation rate of the specific metabolites of the substrates(pmol·mg·protein-1·min-1). Results RDN competitively inhibited the activities of CYP1A2 and CYP2C11, with inhibition constant(Ki) values determined to be 0.18% and 0.63%, respectively. RDN exhibited the mixed inhibition on the activity of CYP2D1, with a Ki value of 0.15%. The activities of CYP1A1 and CYP3A1/2 were not markedly inhibited even by 1.0% RDN. Conclusion RDN could inhibit the rat enzyme activities of CYP1A2, 2C11, and 2D1 in vitro with different inhibition modes, which is worthy of promoting safety and efficacy of RDN.

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

    Science.gov (United States)

    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 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 assumed.

  2. Formation of 4'-carboxyl acid metabolite of imrecoxib by rat liver microsomes

    Institute of Scientific and Technical Information of China (English)

    Hai-yan XU; Peng ZHANG; Ai-shen GONG; Yu-ming SUN; Feng-ming CHU; Zong-ru GUO; Da-fang ZHONG

    2006-01-01

    Aim:Imrecoxib is a novel and moderately selective COX-2 inhibitor.The aim of the present in vitro investigation was to study the formation of the major metabolite 4'-carboxylic acid imrecoxib (M2) and identify the enzyrne(s) involved in the reaction.Methods:The formation of M2 was studied in rat liver cytosol in the absence or presence of liver microsomes.The formed metabolite was identified and quantified by LC/MSn.In addition,to characterize the cytochrome P450 (CYP) isozymes involved in M2 formation,the effects of typical CYP inhibitors (such as ketoconazle,quinine,α-naphthoflavone, methylpyrazole,and cimetidine) on the formation rate of M2 were investigated.Results:The formation of M2 from 4'hydroxymethyl imrecoxib (M4) was completely dependent on rat liver microsomes and NADPH.Enzyme kinetic studies demonstrated that the formation rate of M2 conformed to monophasic Michaelis-Menten kinetics.Additional experiments showed that the formation of M2 was induced significantly by dexamethasone and lowered by ketoconazole strongly and concentration-dependently.By comparison.other CYP inhibitors.such as α-naphthoflavone,cimetidine,quinine,and methylpyrazole had no inhibitory effects on this metabolic pathway.Conclusion:These biotransformation studies of M4 and imrecoxib in rat liver at the subcellular level showed that the formation of M2 occurs in rat liver microsomes and is NADPH-dependent.The reaction was mainly catalyzed by CYP 3A in untreated rats and in dexamethasone-induced rats.Other CYP,such as CYP 1A,2C,2D,and 2E,seem unlikely to participate in this metabolic pathway.

  3. Metabolism evaluation of the anticancer candidate AC04 by biomimetic oxidative model and rat liver microsomes.

    Science.gov (United States)

    Pigatto, Maiara Cássia; Alves de Lima, Maria do Carmo; Galdino, Suely Lins; Pitta, Ivan da Rocha; Vessecchi, Ricardo; Assis, Marilda das Dores; dos Santos, Joicy Santamalvina; Dalla Costa, Teresa; Lopes, Norberto Peporine

    2011-09-01

    Jacobsen reagents, in the presence of monooxygen donors, appear as an alternative to produce metabolites from biological active compounds. This reaction may mimic the oxidation and oxygenation reactions of cytochrome P450 (CYP450) enzymes upon various drugs and biologically active compounds. Acridines represent a well-known group of polyaromatic compounds capable of acting as DNA intercalating agents. Viewing to search for new anticancer agents, one promising new acridine, the 5-acridin-9-ylmethylene-3-(4-methyl-benzyl)-thiazolidine-2,4-dione (AC04) (2), has been studied by our group and the in vitro metabolism was investigated in this work, aiming to advance in the pre-clinical pharmacokinetic investigation. A systematic investigation of the gas-phase reaction, supported by computational chemistry, of the AC04 (2) was studied to help the structure elucidation of possible in vivo metabolites. To confirm the methodology, the oxidized product was obtained in large scale for NMR analysis and the data confirmed the structure. In addition, AC04 (2) was submitted to an in vitro metabolism assay employing rat liver microsomes and also, a pilot study was conducted in rats after AC04 intravenous (i.v.) dosing of 1.5 mg/kg. A single oxidized product was obtained from microsomal metabolism and detected in rat plasma by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis corresponding to the same product formed by Jacobsen-catalyzed reaction. These results indicate that Jacobsen oxidation reactions, combined with in vitro metabolism assays employing isolated microsomes, might replace some in vivo metabolism studies, thus reducing the use of animals in new chemical entities pre-clinical investigation.

  4. Environmentally Persistent Free Radicals Inhibit Cytochromes P450 Activity in Rat Liver Microsomes

    Science.gov (United States)

    Reed, James R.; Cawley, George F.; Ardoin, Taylor G.; Dellinger, Barry; Lomnicki, Slawomir M.; Hasan, Farhana; Kiruri, Lucy W.; Backes, Wayne L.

    2014-01-01

    Combustion processes generate particulate matter that affects human health. When incineration fuels include components that are highly enriched in aromatic hydrocarbons (especially halogenated varieties) and redox-active metals, ultrafine particulate matter containing air-stable, environmentally persistent free radicals (EPFRs) are generated. The exposure to fine EPFRs (less than 2.5 μm in diameter) has been shown to negatively influence pulmonary and cardiovascular functions in living organisms. The goal of this study was to determine if these EPFRs have a direct affect on cytochrome P450 function. This was accomplished by direct addition of the EPFRs to rat liver microsomal preparations and measurement of several P450 activities using form-selective substrates. The EPFRs used in this study were formed by heating vapors from an organic compound (either monochlorophenol (MCP230) or 1,2- dichlorobenzene (DCB230)) and 5% copper oxide supported on silica (approximately 0.2 μm in diameter) to 230°C under vacuum. Both types of EPFRs (but not silica, physisorbed silica, or silica impregnated with copper oxide) dramatically inhibited the activities of CYP1A, CYP2B, CYP2E1, CYP2D2 and CYP3A when incubated at concentrations less than 0.1 mg/ml with microsomes and NADPH. Interestingly, at the same concentrations, the EPFRs did not inhibit HO-1 activity or the reduction of cytochrome c by NADPH-cytochrome P450 reductase. CYP2D2-selective metabolism by rat liver microsomes was examined in more detail. The inhibition of CYP2D2-selective metabolism by both DCB230- and MCP230-EPFRs appeared to be largely noncompetitive and was attenuated in the presence of catalase suggesting that reactive oxygen species may be involved in the mechanism of inhibition. PMID:24713513

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

    Science.gov (United States)

    Reed, James R; Cawley, George F; Ardoin, Taylor G; Dellinger, Barry; Lomnicki, Slawomir M; Hasan, Farhana; Kiruri, Lucy W; Backes, Wayne L

    2014-06-01

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

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  8. Comparative metabolism of cinobufagin in liver microsomes from mouse, rat, dog, minipig, monkey, and human.

    Science.gov (United States)

    Ma, Xiao-Chi; Ning, Jing; Ge, Guang-Bo; Liang, Si-Cheng; Wang, Xiu-Li; Zhang, Bao-Jing; Huang, Shan-Shan; Li, Jing-Kui; Yang, Ling

    2011-04-01

    Cinobufagin (CB), a major bioactive component of the traditional Chinese medicine Chansu, has been reported to have potent antitumor activity. In this study, in vitro metabolism of CB among species was compared with respect to metabolic profiles, enzymes involved, and catalytic efficiency by using liver microsomes from human (HLM), mouse (MLM), rat (RLM), dog (DLM), minipig (PLM), and monkey (CyLM). Significant species differences in CB metabolism were revealed. In particular, species-specific deacetylation and epimerization combined with hydroxylation existed in RLM, whereas hydroxylation was a major pathway in HLM, MLM, DLM, PLM, and CyLM. Two monohydroxylated metabolites of CB in human and animal species were identified as 1α-hydroxylcinobufagin and 5β-hydroxylcinobufagin by using liquid chromatography-mass spectrometry and two-dimensional NMR techniques. CYP3A4 was identified as the main isoform involved in CB hydroxylation in HLM on the basis of the chemical inhibition studies and screen assays with recombinant human cytochrome P450s. Furthermore, ketoconazole, a specific inhibitor of CYP3A, strongly inhibited CB hydroxylation in MLM, DLM, PLM, and CyLM, indicating that CYP3A was responsible for CB hydroxylation in these animal species. The apparent substrate affinity and catalytic efficiency for 1α- and 5β-hydroxylation of CB in liver microsomes from various species were also determined. PLM appears to have K(m) and total intrinsic clearance value (V(max)/K(m)) similar to those for HLM, and the total microsomal intrinsic clearance values for CB obeyed the following order: mouse > dog > monkey > human > minipig. These findings provide vital information to better understand the metabolic behaviors of CB among various species.

  9. Reductive metabolism of oxymatrine is catalyzed by microsomal CYP3A4

    Directory of Open Access Journals (Sweden)

    Liu W

    2015-10-01

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

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

    Directory of Open Access Journals (Sweden)

    Ji Hyun Jeong

    2010-09-01

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

  11. Comprehensive identification of cytochrome p450 isoforms from solubility-based fractionated rat liver microsomes.

    Science.gov (United States)

    Yamanaka, Hidenori; Takeyoshi, Masahiro; Minobe, Yasushi; Yakabe, Yoshikuni; Takatsuki, Mineo; Sato, Hisaya

    2007-12-01

    Cytochrome P450 isoforms from male rat liver microsomes were comprehensively identified using nano liquid chromatography tandem mass spectrometry (nanoLC-MS/MS). The enrichment of P450, an endomembrane-anchored heme protein, was achieved by solubility-based protein fractionation, and greatly improved the total number of identified P450 isoforms. LC-MS/MS analysis of fractions resulted in the identification of total 36 P450 isoforms. The combination of proteomic analysis and the solubility-based fractionation would provide powerful tool for the expression analysis of the superfamily proteins having great similarities between the amino acids sequences.

  12. Heat stable cell growth inhibiting factor isolated from rat liver microsomes.

    Directory of Open Access Journals (Sweden)

    Inaba,Kozo

    1979-08-01

    Full Text Available A heat stable cell growth inhibiting factor was isolated from rat liver microsomes by hot salt extraction, ethanol fractionation and the hot phenol method. The factor was contained in the RNA fraction (designated as mhRNA. mhRNA inhibited the growth of mouse fibroblast (L-929 cells at a relatively low concentration (55 microgram/ml of culture medium. The molecular weight of mhRNA was about 27,000 and the base composition was guanine and cytosine rich.

  13. Mechanism of activation of mouse liver microsomal glutations S—transferase by paracetamol treatment

    Institute of Scientific and Technical Information of China (English)

    ZhenY; LouYJ

    2002-01-01

    Microsomal glutathion S-transferase(mGST) is one of the important detoxifcation enzymes in vivo,its modifying activation by drugs has been paid more attention to in pertinent field recently.This study was to explore the influence of paracetamol(Par) on mGST and its possible mechanism in vivo,and to further reveal the biological significance.Par is metabolized to N-acetyl-p-benzoquinone imine(NAPQI) by CYP2E1 and mGST is activated by sulfhydryl modification.

  14. Steady state and time-resolved fluorimetry of benzo( a)pyrene in liposomes and microsomes

    Science.gov (United States)

    Li, K. P.; Glick, M. R.; Indralingam, R.; Winefordner, J. D.

    1989-01-01

    Comparisons of the steady-state fluorescence signals of benzo( a)pyrene (BP) in liposomes and in microsomes indicate that BP molecules involved in the formation of excimers emitting around 520 nm may be the primary species being oxidized during the first 30 min of metabolism. Moreover, these excimers seem to be kinetically differentiable from BP molecules in other locations in the membrane. Since excimers can be formed only when BP molecules are close together, this implies that the metabolizing BP molecules are more likely on the membrane surface than in the midplane region.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-06-01

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

  16. Activite succino-deshydrogenasique des microsomes et mode d'incorporation du succinate 2,3-(14)C dans les acides gras des microsomes de foie de rat in vitro.

    Science.gov (United States)

    Rous, S; Aubry, L; Bonini, F

    1970-03-16

    The incorporations of 2,3-(14)C-succinate 2-(14)C-acetate into fatty acids of different cellular fractions of rat liver were studied. Acetate was incorporated mainly into supernatant and succinate into microsomal fatty acids. Mitochondria only could intensively decarboxylate pyruvate. Avidine inhibited fatty acid synthesis from succinate mainly in the supernatant. It is suggested that succinate is an important physiological precursor of fatty acids in the liver and that an active succino-dehydrogenase is present in microsomes.

  17. Characterization of in vitro metabolites of deoxypodophyllotoxin in human and rat liver microsomes using liquid chromatography/tandem mass spectrometry.

    Science.gov (United States)

    Lee, Sang Kyu; Jun, In Hye; Yoo, Hye Hyun; Kim, Ju Hyun; Seo, Young Min; Kang, Mi Jeong; Lee, Seung Ho; Jeong, Tae Cheon; Kim, Dong Hyun

    2008-01-01

    The in vitro metabolism of deoxypodophyllotoxin (DPT), a medicinal herbal product isolated from Anthriscus sylvestris (Apiaceae), was investigated in rats and human microsomes and human recombinant cDNA-expressed CYPs. The incubation of DPT with pooled human microsomes in the presence of NADPH generated five metabolites while its incubation with dexamethasone (Dex)-induced rat liver resulted in seven metabolites (M1-M7) with major metabolic reactions including mono-hydroxylation, O-demethylation and demethylenation. Reasonable structures of the seven metabolites of DPT could be proposed, based on the electrospray tandem mass spectra. Chemical inhibition by ketoconazole and metabolism studies with human recombinant cDNA-expressed CYPs indicated that CYP 3A4 and 2C19 are the major CYP isozymes in the metabolism of DPT in human liver microsomes.

  18. Influence of cystein deficiency on the inhibition of hepatic microsomal detoxication by methyl mercury in two rat strains.

    Science.gov (United States)

    Beije, B; Arrhenius, E

    1978-06-01

    Two rat strains, Wistar, strain R and Sprague--Dawley, were subjected to cystein deficiency and methyl mercury pretreatment, both separately and in combination, after which the hepatic microsomal N- and C-oxygenation of N,N-dimethylaniline (DMA) was studied. Cystein deficiency caused a reduction in C-oxygenation in strain R microsomes, and this reduction was nearly doubled by methyl mercury pretreatment of the depleted rats. Methyl mercury pretreatment per se of strain R rats on the standard diet gave no effect. By contrast microsomes from cystein deficient SpD rats showed no statistically significant decrease in C-oxygenation, and cystein deficiency did not further enhance the inhibitory effect obtained with methyl mercury pretreatment alone. N-oxygenation was not significantly affected by any treatment of the two strains.

  19. Genetic variants in microsomal epoxide hydrolase and N-acetyltransferase 2 in susceptibility of IBD in the Danish population

    DEFF Research Database (Denmark)

    Ernst, Anja; Andersen, Vibeke; Østergaard, Mette;

    induce or sustain an immune response. Changes in detoxification of substances that causes epithelial damage may confer susceptibility to IBD. Hence, polymorphic enzymes involved in the detoxification processes may be risk factors of IBD. Methods. The two biotransformation enzymes microsomal epoxide...... hydrolase and N-acetyltransferase 2 were genotyped using TaqMan based Real-Time PCR in 388 patients with Crohn's disease (CD), 565 patients with ulcerative colitis (UC) and 796 healthy Danish controls. Results. No association was found between low microsomal epoxide hydrolase activity or slow N......-acetyltransferase 2 acetylator status and IBD. An association between high activity of microsomal epoxide hydrolase and disease diagnosis before age 40 in CD with an OR of 2.2(1.1- 4.2) P=0.02) was found. No other phenotypic associations were found for the two enzymes and IBD, regarding age at onset, disease location...

  20. Chromatographic separation of piracetam and its metabolite in a mixture of microsomal preparations, followed by an MS/MS analysis.

    Science.gov (United States)

    Sahu, Kapendra; Siddiqui, Anees A; Shaharyar, Mohammad; Ahmad, Niyaz; Anwar, Mohammad; Ahmad, Farhan J

    2013-07-01

    A rapid bioanalytical method was evaluated for the simultaneous determination of piracetam and its metabolite (M1) in human microsomal preparations by fast ultra-performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS). In addition, a validated method of M1 in rat plasma was developed and successfully applied on pharmacokinetic studies. The present study was carried out to determine the metabolic pathways of piracetam for phase I metabolism and used cytochrome P450 isoforms responsible for the piracetam metabolism in human liver microsomes (HLMs). While additional potential metabolites of piracetam were suggested by computer-modeling. The resulting 2-(2-oxopyrrolidin-1-yl) acetic acid was the sole metabolite detected after the microsomal treatment. The amide hydrolysis mainly underwent to form a metabolite i.e., 2-(2-oxopyrrolidin-1-yl) acetic acid (M1).

  1. Clearance and clearance inhibition of the HIV-1 protease inhibitors ritonavir and saquinavir in sandwich-cultured rat hepatocytes and rat microsomes.

    NARCIS (Netherlands)

    Treijtel, N.; van Eijkeren, J.C.; Nijmeijer, S.; de Greef-van der Sandt, I.C.; Freidig, A.

    2009-01-01

    The metabolism and active transport of ritonavir and saquinavir were studied using sandwich-cultured rat hepatoyctes and rat liver microsomes. For ritonavir four comparable metabolites were observed in the sandwich-culture and in microsomes. For saquinavir eight metabolites were observed in sandwich

  2. IMPACT OF STRUCTURAL DIFFERENCES ON THE INVITRO GLUCURONIDATION KINETICS OF POTENTIALLY DOPAMINERGIC HYDROXY-2-AMINOTETRALINS AND NAPHTHOXAZINES USING RAT AND HUMAN LIVER-MICROSOMES

    NARCIS (Netherlands)

    SWART, PJ; JANSMAN, FGA; DRENTH, BFH; DEZEEUW, RA; DIJKSTRA, D; HORN, AS

    1991-01-01

    The in vitro glucuronidation of seven monohydroxy-2-aminotetralins and two naphthoxazines has been determined using human and rat liver microsomes. All these compounds stimulate the D2 dopamine receptor. The influence of the position of the phenolic hydroxyl group was studied with rat microsomes in

  3. Distinct rat hepatic microsomal epoxide hydrolases catalyze the hydration of cholesterol 5,6 alpha-oxide and certain xenobiotic alkene and arene oxides.

    Science.gov (United States)

    Levin, W; Michaud, D P; Thomas, P E; Jerina, D M

    1983-02-01

    Metabolism of cholesterol 5,6 alpha-oxide to the 5,6-glycol is catalyzed by a rat liver microsomal epoxide hydrolase that is distinct from the microsomal epoxide hydrolase that metabolizes a wide range of xenobiotic alkene and arene oxides. The two enzymes are antigenically distinct, and the purified microsomal epoxide hydrolase that metabolizes xenobiotic oxides does not catalyze the hydration of cholesterol 5,6 alpha-oxide. In vivo treatment of rats with inducers of microsomal epoxide hydrolase does not enhance the activity of cholesterol 5,6 alpha-oxide hydrolase and, in some cases, actually depresses enzyme activity in the resultant microsomal preparations. Octene 1,2-oxide and benz[a]anthracene 5,6-oxide, both good substrates for xenobiotic epoxide hydrolase, are not competitive inhibitors of cholesterol oxide hydration by rat liver microsomes. The above results establish the existence of a liver microsomal epoxide hydrolase that is under different regulatory control and that appears to have a different substrate specificity than the well-characterized microsomal epoxide hydrolase involved in the metabolism of a widely diverse group of alkene and arene oxides.

  4. Clearance and clearance inhibition of the HIV-1 protease inhibitors ritonavir and saquinavir in sandwich-cultured rat hepatocytes and rat microsomes

    NARCIS (Netherlands)

    Treijtel, N.; Eijkeren, J.C.H.v.; Nijmeijer, S.; Greef de - Sandt, I.C.J. van der; Freidig, A.P.

    2009-01-01

    The metabolism and active transport of ritonavir and saquinavir were studied using sandwich-cultured rat hepatoyctes and rat liver microsomes. For ritonavir four comparable metabolites were observed in the sandwich-culture and in microsomes. For saquinavir eight metabolites were observed in sandwich

  5. Glucuronidation of drugs in humanized UDP-glucuronosyltransferase 1 mice: Similarity with glucuronidation in human liver microsomes.

    Science.gov (United States)

    Kutsuno, Yuki; Sumida, Kyohei; Itoh, Tomoo; Tukey, Robert H; Fujiwara, Ryoichi

    2013-10-01

    Uridine 5'-diphosphate-glucuronosyltransferases (UGTs) are phase II drug-metabolizing enzymes that catalyze glucuronidation of various endogenous and exogenous substrates. Among 19 functional human UGTs, UGT1A family enzymes largely contribute to the metabolism of clinically used drugs. While the UGT1A locus is conserved in mammals such as humans, mice, and rats, species differences in drug glucuronidation have been reported. Recently, humanized UGT1 mice in which the original Ugt1 locus was disrupted and replaced with the human UGT1 locus (hUGT1 mice) have been developed. To evaluate the usefulness of hUGT1 mice to predict human glucuronidation of drugs, UGT activities, and inhibitory effects on UGTs were examined in liver microsomes of hUGT1 mice as well as in those of wild-type mice and humans. Furosemide acyl-glucuronidation was sigmoidal and best fitted to the Hill equation in hUGT1 mice and human liver microsomes, while it was fitted to the substrate inhibition equation in mouse liver microsomes. Kinetic parameters of furosemide glucuronidation were very similar between hUGT1 mice and human liver microsomes. The kinetics of S-naproxen acyl-glucuronidation and inhibitory effects of compounds on furosemide glucuronidation in hUGT1 liver microsomes were also slightly, but similar to those in human liver microsomes, rather than in wild-type mice. While wild-type mice lack imipramine and trifluoperazine N-glucuronidation potential, hUGT1 mice showed comparable N-glucuronidation activity to that of humans. Our data indicate that hUGT1 mice are promising tools to predict not only in vivo human drug glucuronidation but also potential drug-drug interactions.

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

    Science.gov (United States)

    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.

  7. Sulfation of p-nitrophenyl-N-acetyl-beta-D-galactosaminide with a microsomal fraction from cultured chondrocytes

    Energy Technology Data Exchange (ETDEWEB)

    Habuchi, O.; Conrad, H.E.

    1985-10-25

    Chick embryo chondrocyte microsomes containing intact Golgi vesicles took up 3'-phosphoadenosine-5'-phospho(TVS)sulfate ((TVS)PAPS) in a time- and temperature-dependent, substrate-saturable manner. When (TVS)PAPS and p-nitrophenyl-N-acetyl-beta-D-galactosaminide (pNP-GalNAc) were added to the incubation in the absence of detergent, the microsomes catalyzed the transfer of sulfate from (TVS)PAPS to pNP-GalNAc to form pNP-GalNAc-6-TVSO4. The apparent Km values for PAPS in the uptake and the pNP-GalNAc sulfation reactions were 2 X 10(-7) and 2 X 10(-6) M, respectively. The sulfation of pNP-GalNAc by the microsomal preparation was inhibited by detergent. The microsomal fraction also catalyzed the transfer of sulfate from (TVS)PAPS to oligosaccharides prepared from chondroitin. However, in contrast to the sulfation of pNP-GalNAc, the rate of sulfation of these oligosaccharides was low in the absence of detergent and was markedly stimulated when detergent was added. Sulfation of pNP-GalNAc by the freeze-thawed microsomes was inhibited when the octasaccharide prepared from chondroitin was present in the reaction mixture. As the PAPS that had been internalized in the microsomal vesicles was consumed in the sulfation of pNP-GalNAc, more (TVS)PAPS was taken up and the sulfated pNP-GalNAc was released from the vesicles. These observations suggest that pNP-GalNAc may serve as a model membrane-permeable substrate for study of the 6-sulfo-transferase reaction involved in sulfation of chondroitin sulfate in intact Golgi vesicles.

  8. Simultaneous determination of cytochrome P450 1A, 2A and 3A activities in porcine liver microsomes.

    Science.gov (United States)

    Johansson, Monika; Tomankova, Jana; Li, Shengjie; Zamaratskaia, Galia

    2012-09-01

    The aim of this study was to develop a robust method for the simultaneous determination of the activities of three porcine CYP450 enzymes in hepatic microsomes. A cocktail consisting of three selective CYP450 probe substrates, 7-ethoxyresorufin (CYP1A), coumarin (CYP2A) and 7-benzyloxy-4-trifluoromethylcoumarin (BFC; CYP3A), was incubated with porcine liver microsomes. The presence of 7-ethoxyresorufin appears to significantly influence the kinetics of coumarin hydroxylation and BFC O-debenzylation. These results indicate that the use of 7-ethoxyresorufin in substrate cocktails together with coumarin and BFC should be avoided.

  9. [Kinetic characteristics of microsomal NAD-glycohydrolase natural and solubilized with a non-ionic surface-active substance].

    Science.gov (United States)

    Sestini, S; Cinci, G; Ricci, C

    1982-04-30

    Microsomal rat spleen NAD-glycohydrolase was solubilized by Nonidet P40. The solubilized enzyme shows Nicotinamide inhibition and pH dependence at the same extent as unsolubilized microsomal one. It differs from the latter in having a higher affinity for NAD and NADP, and in showing two peaks, instead of one, on electrofocusing: the former with a pH 5 pI without any activity, the latter with a pH 4, 1 pI with a high NAD-ase activity.

  10. Brain Basics

    Medline Plus

    Full Text Available ... Basics will introduce you to some of this science, such as: How the brain develops How genes and the environment affect the brain The basic structure of the brain How different parts of the brain communicate and work with each other How changes in the brain ...

  11. Brain Fingerprinting

    Directory of Open Access Journals (Sweden)

    Ravi Kumar

    2012-12-01

    Full Text Available Brain Fingerprinting is a scientific technique to determine whether or not specific information is stored in an individual's brain by measuring a electrical brain wave response to Word, phrases, or picture that are presented on computer screen. Brain Fingerprinting is a controversial forensic science technique that uses electroencephalography (EEG to determine whether specific information is stored in a subject's brain.

  12. Brain Fingerprinting

    Directory of Open Access Journals (Sweden)

    ravi kumar

    2012-12-01

    Full Text Available Brain Fingerprinting is a scientific technique to determine whether or not specific information is stored in an individual's brain by measuring a electrical brain wave response to Word, phrases, or picture that are presented on computer screen. Brain Fingerprinting is a controversial forensic science technique that uses electroencephalograph y (EEG to determine whether specific information is stored in a subject's brain

  13. Brain Tumors

    Science.gov (United States)

    A brain tumor is a growth of abnormal cells in the tissues of the brain. Brain tumors can be benign, with no cancer cells, ... cancer cells that grow quickly. Some are primary brain tumors, which start in the brain. Others are ...

  14. Origanum majorana Essential Oil Lacks Mutagenic Activity in the Salmonella/Microsome and Micronucleus Assays

    Science.gov (United States)

    Klein-Júnior, Luiz Carlos; Guecheva, Temenouga N.; dos Santos, Luciana D.; Zanette, Régis A.; de Mello, Fernanda B.; de Mello, João Roberto Braga

    2016-01-01

    The present study aimed to investigate the in vitro mutagenic activity of Origanum majorana essential oil. The most abundant compounds identified by GC-MS were γ-terpinene (25.73%), α-terpinene (17.35%), terpinen-4-ol (17.24%), and sabinene (10.8%). Mutagenicity was evaluated by the Salmonella/microsome test using the preincubation procedure on TA98, TA97a, TA100, TA102, and TA1535 Salmonella typhimurium strains, in the absence or in the presence of metabolic activation. Cytotoxicity was detected at concentrations higher than 0.04 μL/plate in the absence of S9 mix and higher than 0.08 μL/plate in the presence of S9 mix and no gene mutation increase was observed. For the in vitro mammalian cell micronucleus test, V79 Chinese hamster lung fibroblasts were used. Cytotoxicity was only observed at concentrations higher than or equal to 0.05 μg/mL. Moreover, when tested in noncytotoxic concentrations, O. majorana essential oil was not able to induce chromosome mutation. The results from this study therefore suggest that O. majorana essential oil is not mutagenic at the concentrations tested in the Salmonella/microsome and micronucleus assays. PMID:27891531

  15. Reductive metabolism of oxymatrine is catalyzed by microsomal CYP3A4

    Science.gov (United States)

    Liu, Wenqin; Shi, Jian; Zhu, Lijun; Dong, Lingna; Luo, Feifei; Zhao, Min; Wang, Ying; Hu, Ming; Lu, Linlin; Liu, Zhongqiu

    2015-01-01

    Oxymatrine (OMT) is a pharmacologically active primary quinolizidine alkaloid with various beneficial and toxic effects. It is confirmed that, after oral administration, OMT could be transformed to the more toxic metabolite matrine (MT), and this process may be through the reduction reaction, but the study on the characteristics of this transformation is limited. The aim of this study was to investigate the characteristics of this transformation of OMT in the human liver microsomes (HLMs) and human intestinal microsomes (HIMs) and the cytochrome P450 (CYP) isoforms involved in this transformation. The current studies demonstrated that OMT could be metabolized to MT rapidly in HLMs and HIMs and CYP3A4 greatly contributed to this transformation. All HLMs, HIMs, and CYP3A4 isoform mediated reduction reaction followed typical biphasic kinetic model, and Km, Vmax, and CL were significant higher in HLMs than those in HIMs. Importantly, different oxygen contents could significantly affect the metabolism of OMT, and with the oxygen content decreased, the formation of metabolite was increased, suggesting this transformation was very likely a reduction reaction. Results of this in vitro study elucidated the metabolic pathways and characteristics of metabolism of OMT to MT and would provide a theoretical basis and guidance for the safe application of OMT. PMID:26586934

  16. Application of microdialysis to study the in vitro metabolism of drugs in liver microsomes.

    Science.gov (United States)

    Gunaratna, C; Kissinger, P T

    1997-10-01

    Current methods for studying in vitro drug metabolism involve add-incubate-separate-measure approach. Separation of the desired analytes requires removal of protein which is typically accomplished by precipitation and centrifugation and extraction of the analytes into an organic phase. The analysis scheme then becomes more complex resulting in a decrease in precision and an increase in assay time. Microdialysis sampling circumvents these problems by allowing researchers to sample the reaction mixture periodically and obtain the complete metabolic profile. In the present study, microdialysis sampling was used to investigate Phase I metabolism of salicylic acid, diazepam and ibuprofen in rat liver microsomes. The major metabolites of these drugs were profiled by LC. Michaelis-Menten enzyme kinetic parameters, Km and Vmax were obtained for the formation of diazepam metabolites by both microdialysis and conventional microsomal incubations and were in good agreement with the values reported in the literature. This study shows that microdialysis has considerable promise as a sampling technique for in vitro drug metabolism studies. By making minor modifications to the instruments, microdialysis can be applied to other in vitro systems such as isolated hepatocytes to study the Phase II metabolism or tissue slices to study drug distribution.

  17. Origanum majorana Essential Oil Lacks Mutagenic Activity in the Salmonella/Microsome and Micronucleus Assays

    Directory of Open Access Journals (Sweden)

    Andrea dos Santos Dantas

    2016-01-01

    Full Text Available The present study aimed to investigate the in vitro mutagenic activity of Origanum majorana essential oil. The most abundant compounds identified by GC-MS were γ-terpinene (25.73%, α-terpinene (17.35%, terpinen-4-ol (17.24%, and sabinene (10.8%. Mutagenicity was evaluated by the Salmonella/microsome test using the preincubation procedure on TA98, TA97a, TA100, TA102, and TA1535 Salmonella typhimurium strains, in the absence or in the presence of metabolic activation. Cytotoxicity was detected at concentrations higher than 0.04 μL/plate in the absence of S9 mix and higher than 0.08 μL/plate in the presence of S9 mix and no gene mutation increase was observed. For the in vitro mammalian cell micronucleus test, V79 Chinese hamster lung fibroblasts were used. Cytotoxicity was only observed at concentrations higher than or equal to 0.05 μg/mL. Moreover, when tested in noncytotoxic concentrations, O. majorana essential oil was not able to induce chromosome mutation. The results from this study therefore suggest that O. majorana essential oil is not mutagenic at the concentrations tested in the Salmonella/microsome and micronucleus assays.

  18. /sup 45/Ca/sup 2 +/ uptake and phospholipid methylation in isolated rat liver microsomes

    Energy Technology Data Exchange (ETDEWEB)

    Kraus-Friedmann, N.; Zimniak, P.

    1983-07-01

    The effects of glucagon, epinephrine and insulin on hepatic phospholipid methylation were studied. Glucagon, either injected into rats or added to perfused livers, stimulated methylation in subsequently isolated microsomes. Epinephrine also increased phospholipid methylation. Insulin by itself did not influence the rate of the reaction, but, when administered prior to glucagon, it blocked the effect of the latter. The possibility that the observed stimulation of phospholipid methylation might be causally linked to the reported stimulation by glucagon of 45Ca2+ uptake in subsequently isolated liver microsomes was examined. Both the substrate and the competitive inhibitor of the methylation reaction, S-adenosylmethionine and S-adenosylhomocysteine, had profound effect on the rate of phospholipid methylation, without having comparable effects on Ca2+ uptake. S-adenosylmethionine in increasing concentration stimulated methylation four-fold, while no significant changes in 45Ca2+ uptake were seen. S-adenosylhomocysteine did not inhibit 45Ca2+ uptake even at levels causing more than 95% decrease in methylation. In conclusion, while both phospholipid methylation and 45Ca2+ uptake seem to be hormonally controlled, the correlation between these two processes was not sufficient to support the notion that the changes in 45Ca2+ uptake are caused by the changes in phospholipid methylation.

  19. The metabolism of pentachlorobenzene by rat liver microsomes: The nature of the reactive intermediates formed

    Energy Technology Data Exchange (ETDEWEB)

    den Besten, C.; Peters, M.M.; van Bladeren, P.J. (Agricultural Univ., Wageningen (Netherlands))

    1989-09-29

    Metabolism of (14C)-pentachlorobenzene by liver microsomes from dexamethasone-induced rats results in the formation of pentachlorophenol and 2,3,4,6-tetrachlorophenol as major primary metabolites in a ratio of 4:1, with 2,3,4,5- and 2,3,5,6-tetrachlorophenols as minor metabolites. The unsubstituted carbon atom is thus the favourite site of oxidative attack, but the chlorine substituted positions still play a sizable role. As secondary metabolites both para- and ortho-tetrachlorohydroquinone are formed (1.4 and 0.9% of total metabolites respectively). During this cytochrome P450-dependent conversion of pentachlorobenzene, 5-15% of the total amount of metabolites becomes covalently bound to microsomal protein. Ascorbic acid inhibits this binding to a considerable extent, indicating that quinone metabolites play an important role in the binding. However, complete inhibition was never reached by ascorbic acid, nor by glutathione, suggesting that other reactive intermediates, presumably epoxides, are also responsible for covalent binding.

  20. [Effect of titanium dioxide nanoparticles on protein expression profiles in rat liver microsomes].

    Science.gov (United States)

    Tananova, O N; Arianova, E A; Gmoshinskiĭ, I V; Aksenov, I V; Zgoda, V G; Khotimchenko, S A

    2012-01-01

    There was studied an influence of intragastric administration of titanium dioxide (anatase form) nanoparticles (NP) on protein expression profiles in rat's liver microsomes by methods of proteomics. Animals received water suspension of NP in doses from 0,1 to 10 mg per kg body weight intragastrically daily during 28 days. Microsomes were isolated from liver by means of preparative ultracentrifugation. Proteins composition was studied by 2D-electrophoresis in acrylamide gel. Protein spots were identified by MALDI-TOF analysis. The results demonstrated appearance of 53 new protein spots and disappearance of 19 spots in animals subjected to NP irrespective of their dose. In addition 25 new spots appeared and 3 disappeared in higher doses of NP when compared to low dose group and control animals. Mass-spectrum analysis showed presence of few polypeptides registered in international database among proteins expressed under influence of NP. One of this dominant expressed proteins corresponded to enzyme glutathione transpherase Mu 2 isoform (M=41,55 kD, pI=8,0). The conclusion was made of well advances of proteomic analysis in artificial NP influences on biosynthetic processes estimation.

  1. [Changes in proteome profiles of rat liver microsomes induced by silicon dioxide nanoparticles].

    Science.gov (United States)

    Tananova, O N; Arianova, E A; Gmoshinskii, I V; Toropygin, I Yu; Khryapova, E V; Trusov, N V; Khotimchenko, S A; Tutel'yan, V A

    2015-01-01

    The effect of daily intragastric administration of an aqueous dispersion of silicon nanoparticles (NPs) (the dose range from 1.0 mg/kg to 100 mg/kg body weight for 28 days) to rats on the proteomic profile of liver microsomes has been investigated by 2D-electrophoresis followed by subsequent mass spectrometry identification. The liver microsomal fraction was isolated by differential centrifugation and its protein composition was analyzed by 2D-polyacrylamide gel electrophoresis. Identification of protein spots was carried out using MALDI-TOF mass spectrometric analysis. The mass spectrometry analysis revealed the protein GRP78 (78 kD glucose-regulated protein precursor), belonging to the family of heat shock proteins. This protein present in animals of the control group was not detected in NP-treated rats of group 2 (1 mg/kg body weight/day) and group 3 (10 mg/kg body weight/day). This protein predominantly localized in the liver cell endoplasmic reticulum and plasma membrane has the chaperone biological activity. Possible mechanisms of the effects of engineered nanoparticles on biosynthetic processes in the body are discussed.

  2. Identification of paraoxonase 3 in rat liver microsomes: purification and biochemical properties.

    Science.gov (United States)

    Rodrigo, Lourdes; Gil, Fernando; Hernandez, Antonio F; Lopez, Olga; Pla, Antonio

    2003-01-01

    Three paraoxonase genes (PON1, PON2 and PON3) have been described so far in mammals. Although considerable information is available regarding PON1, little is known about PON2 and PON3. PON3 has been isolated recently from rabbit serum [Draganov, Stetson, Watson, Billecke and La Du (2000) J. Biol. Chem. 275, 33435-33442] and liver [Ozols (1999) Biochem. J. 338, 265-275]. In the present study, we have identified the presence of PON3 in rat liver microsomes and a method for the purification to homogeneity is presented. PON3 has been purified 177-fold to apparent homogeneity with a final specific activity of 461 units/mg using a method consisting of seven steps: solubilization of the microsomal fraction, hydroxyapatite adsorption, chromatography on DEAE-Sepharose CL-6B, non-specific affinity chromatography on Cibacron Blue 3GA, two DEAE-cellulose steps and a final affinity chromatography on concanavalin A-Sepharose. SDS/PAGE of the final preparation indicated a single protein-staining band with an apparent molecular mass of 43 kDa. The isolated protein was identified by nanoelectrospray MS. Internal amino acid sequences of several peptides were determined and compared with those of human, rabbit and mouse PON3, showing a high similarity. Some biochemical properties of PON3 were also studied, including optimum pH, K(m) and heat and pH stability. PMID:12946270

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

    Science.gov (United States)

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

    2016-07-01

    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.

  4. Omega-hydroxylation of phytanic acid in rat liver microsomes: implications for Refsum disease.

    Science.gov (United States)

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

    2004-07-01

    The 3-methyl-branched fatty acid phytanic acid is degraded by the peroxisomal alpha-oxidation route because the 3-methyl group blocks beta-oxidation. In adult Refsum disease (ARD), peroxisomal alpha-oxidation is defective, which is caused by mutations in the gene coding for phytanoyl-CoA hydroxylase in the majority of ARD patients. As a consequence, phytanic acid accumulates in tissues and body fluids. This study focuses on an alternative route of phytanic acid degradation, omega-oxidation. The first step in omega-oxidation is hydroxylation at the omega-end of the fatty acid, catalyzed by a member of the cytochrome P450 multienzyme family. To study this first step, the formation of hydroxylated intermediates was studied in rat liver microsomes incubated with phytanic acid and NADPH. Two hydroxylated metabolites of phytanic acid were formed, omega- and (omega-1)-hydroxyphytanic acid (ratio of formation, 5:1). The formation of omega-hydroxyphytanic acid was NADPH dependent and inhibited by imidazole derivatives. These results indicate that phytanic acid undergoes omega-hydroxylation in rat liver microsomes and that an isoform of cytochrome P450 catalyzes the first step of phytanic acid omega-oxidation.

  5. Critical appraisal of 13C breath tests for microsomal liver function: aminopyrine revisited.

    Science.gov (United States)

    Pijls, Kirsten E; de Vries, Hanne; Nikkessen, Suzan; Bast, Aalt; Wodzig, Will K W H; Koek, Ger H

    2014-04-01

    As liver diseases are a major health problem and especially the incidence of metabolic liver diseases like non-alcoholic fatty liver disease (NAFLD) is rising, the demand for non-invasive tests is growing to replace liver biopsy. Non-invasive tests such as carbon-labelled breath tests can provide a valuable contribution to the evaluation of metabolic liver function. This review aims to critically appraise the value of the (13) C-labelled microsomal breath tests for the evaluation of metabolic liver function, and to discuss the role of cytochrome P450 enzymes in the metabolism of the different probe drugs, especially of aminopyrine. Although a number of different probe drugs have been used in breath tests, the perfect drug to assess the functional metabolic capacity of the liver has not been found. Data suggest that both the (13) C(2) -aminopyrine and the (13) C-methacetin breath test can play a role in assessing the capacity of the microsomal liver function and may be useful in the follow-up of patients with chronic liver diseases. Furthermore, CYP2C19 seems to be an important enzyme in the N-demethylation of aminopyrine, and polymorphisms in this gene may influence breath test values, which should be kept in mind when performing the (13) C(2) -aminopyrine breath test in clinical practice.

  6. Brain components

    Science.gov (United States)

    ... can make complex movements without thinking. The brain stem connects the brain with the spinal cord and is composed of ... structures: the midbrain, pons, and medulla oblongata. The brain stem provides us with automatic functions that are necessary ...

  7. Brain surgery

    Science.gov (United States)

    Craniotomy; Surgery - brain; Neurosurgery; Craniectomy; Stereotactic craniotomy; Stereotactic brain biopsy; Endoscopic craniotomy ... cut depends on where the problem in the brain is located. The surgeon creates a hole in ...

  8. Brain Malformations

    Science.gov (United States)

    Most brain malformations begin long before a baby is born. Something damages the developing nervous system or causes it ... medicines, infections, or radiation during pregnancy interferes with brain development. Parts of the brain may be missing, ...

  9. Brain Basics

    Medline Plus

    Full Text Available ... science, such as: How the brain develops How genes and the environment affect the brain The basic ... that with brain development in people mental disorders. Genes and environmental cues both help to direct this ...

  10. Brain Basics

    Medline Plus

    Full Text Available ... can lead to mental disorders, such as depression. The Growing Brain Inside the Brain: Neurons & Neural Circuits ... tailored treatments, and possibly prevention of such illnesses. The Working Brain Neurotransmitters Everything we do relies on ...

  11. The effects of various incubation temperatures, particulate isolation, and possible role of calmodulin on the activity of the base exchange enzymes of rat brain.

    Science.gov (United States)

    Buchanan, A G; Kanfer, J N

    1980-10-01

    The involvement of calmodulin in the choline, ethanolamine, and serine exchange activities of rat brain microsomes was investigated. Calmodulin stimulated choline exchange activity to a greater extent than ethanolamine and serine exchange activities. The three base exchange activities were inhibited by antipsychotic drugs believed to prevent calmodulin interaction, but not by calmodulin-binding protein. The solutions employed for tissue homogenization and subsequent isolation of microsomes greatly influenced the base exchange activities. The process of resuspending isolated microsomes and recentrifugation, or "washing," produced major losses of detectable activity. The base exchange enzyme activities were maximal at 45 degrees, and Arrhenius plots revealed a common transition temperature of 31 degrees. The activation energies for the base exchange reactions decreased at temperatures above the observed transition temperature. Kinetic data, Km and Vmax, for the base exchange activities at 27, 37, and 45 degrees are presented.

  12. The relationship between microsomal enzyme induction and liver tumour formation : a study on the effects of xenobiotic and naturally occurring microsomal enzyme inducers on livers of male CF-1 mice

    NARCIS (Netherlands)

    Tennekes, H.A.

    1979-01-01

    The effects of naturally occurring microsomal enzyme inducers on important hepatocellular pathways for the metabolism of foreign compounds (xenobiotics) and also upon the incidence of liver tumours in CF-1 mice treated or not with 10 mg dieldrin.kg -1diet were inves

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-04-01

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

  14. RATE AND CAPACITY OF HEPATIC MICROSOMAL RING HYDROXYLATION OF PHENOL TO HYDROQUINONE AND CATECHOL IN RAINBOW TROUT

    Science.gov (United States)

    Rainbow trout (Oncorhynchus mykiss) liver microsomes were used to study the rate of ring-hydroxylation of phenol PH) by directly measuring the production of hydroquinone (HQ), the primary metabolite, and catechol (CAT), a secondary metabolite. An HPLC method with integrated ultra...

  15. Nitroaryl-1,4-dihydropyridines as antioxidants against rat liver microsomes oxidation induced by iron/ascorbate, nitrofurantoin and naphthalene.

    Science.gov (United States)

    Letelier, María Eugenia; Entrala, Paz; López-Alarcón, Camilo; González-Lira, Víctor; Molina-Berríos, Alfredo; Cortés-Troncoso, Juan; Jara-Sandoval, José; Santander, Paola; Núñez-Vergara, Luis

    2007-12-01

    1,4-Dihydropyridines (DHPs) used in the treatment of cardiovascular diseases, are calcium channel antagonists and also antioxidant agents. These drugs are metabolized through cytochrome P(450) oxidative system, majority localized in the hepatic endoplasmic reticulum. Several lipophilic drugs generate oxidative stress to be metabolized by this cellular system. Thus, DHP antioxidant properties may prevent the oxidative stress associated with hepatic biotransformation of drugs. In this work, we tested the antioxidant capacity of several synthetic nitro-phenyl-DHPs. These compounds (I-IV) inhibited the microsomal lipid peroxidation, UDPGT oxidative activation and microsomal thiols oxidation; all phenomena induced by Fe(3+)/ascorbate, a generator system of oxygen free radicals. As the same manner, these compounds inhibited the oxygen consumption induced by Cu(2+)/ascorbate in the absence of microsomes. Furthermore, compound III (2,6-dimethyl-4-(4-nitrophenyl)-1,4-dihydropyridin-3,5-ethyl-dicarboxylate) and compound V (N-ethyl-2,6-dimethyl-4-(4-nitrophenyl)-1,4-dihydropyridin-3,5-methyl-dicarboxylate) inhibited the microsomal lipid peroxidation induced by Nitrofurantoin and naphthalene in the presence of NADPH. Oxidative stress induced on endoplasmic reticulum may alter the biotransformation of drugs, so, modifying their plasmatic concentrations and therapeutic effects. When drugs which are activated by biotransformation are administered together with antioxidant drugs, such as DHPs, oxidative stress induced in situ may be prevented.

  16. A new metabolite of nodakenetin by rat liver microsomes and its quantification by RP-HPLC method.

    Science.gov (United States)

    Zhang, Peng; Yang, Xiu-Wei

    2010-02-01

    The biotransformation of nodakenetin (NANI) by rat liver microsomes in vitro was investigated. Two major polar metabolites were produced by liver microsomes from phenobarbital-pretreated rats and detected by reversed-phase high-performance liquid chromatography (RP-HPLC) analysis. The chemical structures of two metabolites were firmly identified as 3'(R)-hydroxy-nodakenetin-3'-ol and 3'(S)-hydroxy-nodakenetin-3'-ol, respectively, on the basis of their (1)H-NMR, MS and optical rotation analysis. The latter was a new compound. A sensitive, selective and simple RP-HPLC method has been developed for the simultaneous determination of NANI and its two major metabolites in rat liver microsomes. Chromatographic conditions comprise a C(18) column, a mobile phase with MeOH-H(2)O (40 : 60, v/v), a total run time of 40 min, and ultraviolet absorbance detection at 330 nm. In the rat heat-inactivated liver microsomal supernatant, the lower limits of detection and quantification of metabolite I, metabolite II and NANI were 5.0, 2.0, 10.0 ng/mL and 20.0, 5.0, 50.0 ng/mL, respectively, and their calibration curves were linear over the concentration range 50-400, 20-120 and 150-24000 ng/mL, respectively. The results provided a firm basis for further evaluating the pharmacokinetics and clinical efficacy of NANI.

  17. Species-specific testicular and hepatic microsomal metabolism of benzo(a)pyrene, an ubiquitous toxicant and endocrine disruptor

    Science.gov (United States)

    Information on the metabolism of the environmental toxicant, benzo(a)pyrene (BaP) in the male reproductive system is crucial for understanding BaP-induced infertility. Microsomes were isolated from the liver and testes of rat, mouse, hamster, ram, boar, bull, and monkey and incubated with BaP. Post-...

  18. Cardiac expression of microsomal triglyceride transfer protein is increased in obesity and serves to attenuate cardiac triglyceride accumulation

    DEFF Research Database (Denmark)

    Bartels, Emil D; Nielsen, Jan M; Hellgren, Lars I;

    2009-01-01

    secretion of apolipoproteinB-containing (apoB) lipoproteins. Lipoprotein formation depends on expression of microsomal triglyceride transfer protein (MTP); the mouse expresses two isoforms of MTP, A and B. Since many aspects of the link between obesity-induced cardiac disease and cardiac lipid metabolism...

  19. Inhibition of cumene hydroperoxide-induced lipid peroxidation by a novel pyridoindole antioxidant in rat liver microsomes.

    Science.gov (United States)

    Stefek, M; Masarykova, M; Benes, L

    1992-06-01

    The ability of stobadine, a novel pyridoindole antioxidant, to inhibit lipid peroxidation induced by cumene hydroperoxide was investigated in rat liver microsomes. In the micromolar range stobadine effectively inhibited lipid peroxidation as measured by the formation of thiobarbituric acid reactive products. The peroxidation-related degradation of microsomal cytochrome P-450 was prevented by stobadine in the same pattern. Another line of evidence in support of the antioxidant action of stobadine was given by its inhibition of cumene hydroperoxide-induced oxygen consumption in microsomal incubations. Inhibition of lipid peroxidation was not a function of decreased bioactivation of cumene hydroperoxide, as stobadine did not affect the rate of cytochrome P-450 dependent cleavage of cumene hydroperoxide. Neither had stobadine any effect on cytochrome P-450 peroxidase function characterized by the rate of cumene hydroperoxide-dependent oxidation of TMPD, and no direct spectral interaction with microsomal cytochrome P-450 was observed in the micromolar region. We suggest that it is the ability of stobadine to scavenge alkoxyl and peroxyl radicals that is predominantly responsible for the observed antioxidant effect.

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

    Science.gov (United States)

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

    2014-06-01

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

  1. Interspecies variation in phase I metabolism of bufalin in hepatic microsomes from mouse, rat, dog, minipig, monkey, and human.

    Science.gov (United States)

    Ning, Jing; Hou, Jie; Wang, Ping; Wu, Jing-Jing; Dai, Zi-Ru; Zou, Li-Wei; Li, Wei; Ge, Guang-Bo; Ma, Xiao-Chi; Yang, Ling

    2015-01-01

    1. Bufalin (BF), one of the major bioactive compounds in traditional Chinese medicine (TCM) Chansu, has been found with various pharmacological and toxicological effects. This study aims to investigate the species differences in phase I metabolism of BF in hepatic microsomes from human and five common experimental animals. 2. Metabolite profiling demonstrated that two major metabolites were formed in liver microsomes from human and animal species in NADPH-generating system. Two major metabolites were identified as 5β-hydroxyl-bufalin and 3-keto-bufalin, with the help of authentic standards. CYP3A was assigned as the main isoform involved in both 5β-hydroxylation and 3-oxidation in all studied liver microsomes. The apparent kinetic parameters including substrate affinity and catalytic efficiency for 5β-hydroxylation and 3-oxidation of BF were also determined. 3. In summary, CYP3A mediated 5β-hydroxylation and 3-oxidation were two major metabolic pathways of BF in hepatic microsomes from human and five studied animals, but kinetic analysis demonstrated that the intrinsic clearances of these two metabolic pathways were much different among various species. The qualitative and quantitative interspecies study indicated that minipig exhibited the similar metabolic profile, kinetic behaviors and intrinsic metabolic clearances of BF phase I biotransformation in comparison with that of human.

  2. Oxidation process intensity in microsomal fraction of rat liver under conditions of different supplementation with polyunsaturated fatty acids

    Directory of Open Access Journals (Sweden)

    O. V. Ketsa

    2014-02-01

    Full Text Available The effect of fat compositions with the varying ratio of polyunsaturated fatty acids (PUFAs of families ω-3 and ω-6 on oxidation process intensity in microsomal fraction of rat liver has been investigated. The aim of the study was to investigate the level of markers of oxidative modification of lipids and proteins in microsomal fraction of rat liver. Fat components in the experiment diets were presented by sunflower oil, soybean oil and fish oil. Rats were fed using one of the fillowing 5 diets for the period of 4 weeks: 1 AIN-93 diet with 7% sunflower oil and fish oil, with the inclusion of linoleic acid, eicosapentaenoic acid and docosahexaenoic acid in the ratio of ω-6:ω-3 – 7:1 (control diet; 2 AIN-93 diet with 7% soybean oil, with the inclusion of linoleic acid and α-linolenic acid in the ratio of 7:1; 3 the diet containing only ω-6 PUFAs; 4 the diet containing only ω-3 PUFAs; 5 the diet without PUFAs. The fatty acid compositions of the diets were analysed by gas chromatography. We measured the primary and secondary lipoperoxidation products, proteins carbonyl derivatives and SH-groups of proteins. It was shown that inclusion of linoleic acid and α-linolenic acid in the ratio of 7:1 or ω-6 PUFAs into the animal diet increased lipid peroxidation in microsomal fraction of the rat liver as compared with the control group. Only ω-6 PUFAs increased the oxidative modification of proteins in microsomal fraction of the rat liver as compared with the control rat group. High dose of ω-3 PUFAs – eicosapentaenoic acid and docosahexaenoic acid had no influence on free radical oxidation of lipids and proteins. Using the diet without PUFAs increased oxidation process intensity in microsomal fraction of rat liver. According to our study, ω-6 PUFAs increased the oxidative modification of lipids and proteins in microsomal fraction of the rat liver. ω-3 PUFAs, in particular, eicosapentaenoic acid and docosahexaenoic acid, increased lipid and

  3. Structural modifications of the serotonin 5-HT7 receptor agonist N-(4-cyanophenylmethyl)-4-(2-biphenyl)-1-piperazinehexanamide (LP-211) to improve in vitro microsomal stability: A case study.

    Science.gov (United States)

    Lacivita, Enza; Podlewska, Sabina; Speranza, Luisa; Niso, Mauro; Satała, Grzegorz; Perrone, Roberto; Perrone-Capano, Carla; Bojarski, Andrzej J; Leopoldo, Marcello

    2016-09-14

    The 5-HT7 serotonin receptor is revealing a promising target for innovative therapeutic strategies of neurodevelopmental and neuropsychiatric disorders. Here, we report the synthesis of thirty long-chain arylpiperazine analogs of the selective and brain penetrant 5-HT7 receptor agonist LP-211 (1) designed to enhance stability towards microsomal oxidative metabolism. Commonly used medicinal chemistry strategies were used (i.e., reduction of overall lipophilicity, introduction of electron-withdrawing groups, blocking of potential vulnerable sites of metabolism), and in vitro microsomal stability was tested. The data showed that the adopted design strategy does not directly translate into improvements in stability. Instead, the metabolic stability of the compounds was related to the presence of specific substituents in well-defined regions of the molecule. The collected data allowed for the construction of a machine learning model that, in a given chemical space, is able to describe and quantitatively predict the metabolic stability of the compounds. The majority of the synthesized compounds maintained high affinity for 5-HT7 receptors and showed selectivity towards 5-HT6 and dopamine D2 receptors and different selectivity for 5-HT1A and α1 adrenergic receptors. Compound 50 showed 3-fold higher in vitro stability towards oxidative metabolism than 1 and was able to stimulate neurite outgrowth in neuronal primary cultures through the 5-HT7 receptor in a shorter time and at a lower concentration than the agonist 1. A preliminary disposition study in mice revealed that compound 50 was metabolically stable and was able to pass the blood-brain barrier, thus representing a new tool for studying the pharmacotherapeutic potential of 5-HT7 receptor in vivo.

  4. Topology of nucleotide-sugar:dolichyl phosphate glycosyltransferases involved in the dolichol pathway for protein glycosylation in native rat liver microsomes.

    Science.gov (United States)

    Bossuyt, X; Blanckaert, N

    1993-01-01

    Activities of nucleotide-sugar:dolichyl phosphate glycosyltransferases (UDP-N-acetylglucosamine:dolichyl phosphate N-acetylglucosaminyl 1-phosphotransferase, UDP-glucose:dolichyl phosphate glucosyltransferase and GDP-mannose:dolichyl phosphate mannosyltransferase) are not fully expressed in native microsomes and can be enhanced by pretreatment of the microsomes with detergent. To examine whether the latency of dolichyl phosphate glycosyltransferases in native microsomes reflects a lumenal orientation of the catalytic centre, we examined the effect of proteinase treatment of native microsomes on enzymic activity and investigated the relationship between enzymic activity and alteration of the permeability of the microsomal membrane barrier. The enzymic activities catalysing transfer of N-acetylglucosamine and glucose to lipid acceptors were proteinase-sensitive in native sealed microsomes. When various detergents were used to disrupt the membrane barrier, we found no relationship between activity of dolichyl phosphate glycosyltransferases and the latency of mannose-6-phosphatase, which is a marker of the permeability properties of the microsomal membrane. Permeabilization of the endoplasmic reticulum membrane by the pore-forming Staphylococcus aureus alpha-toxin did not affect glycosyltransferase activities. These results do not support the hypothesis that latency of the transferase activities is dependent on the permeability properties of the endoplasmic-reticulum membrane. Collectively our findings can best be explained by postulating that the active centres of the transferases are cytoplasmically oriented, while activation by detergent may be conformation-dependent. PMID:8280060

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

    Science.gov (United States)

    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.

  6. Studies on the metabolism of chlorotrianisene to a reactive intermediate and subsequent covalent binding to microsomal proteins

    Energy Technology Data Exchange (ETDEWEB)

    Juedes, M.J.

    1989-01-01

    The studies on chlorotrianisene were conducted to determine whether metabolism of chlorotrianisene occurs via the cytochrome P450 monooxygenase system and whether a reactive intermediate is being formed that is capable of binding covalently to microsomal proteins. ({sup 3}H)-chlorotrianisene was incubated with liver microsomes supplemented with NADPH. At the termination of the incubation, the protein was trapped on a glass filter and the unbound chlorotrianisene was removed by extensive washing of the protein with organic solvent. A dramatic stimulation of covalent binding was demonstrated in microsomes from rats treated with methylcholanthrene (60 fold increase) versus control or phenobarbital treatment. Verification of covalent binding was achieved by localization of radiolabeled bands following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the macromolecules in the incubation mixture. Further analysis of the radiolabeled macromolecules separated on SDS-PAGE revealed that these macromolecules were degraded by protease degradation indicating that the macromolecules were proteins. Further investigations were done to determine the cause of the dramatic stimulation of covalent binding detected in microsomes from methylcholanthrene treated rats versus control or phenobarbital treated rats. Further evidence for the participation of P-450c was obtained with a reconstituted cytochrome P-450 system. Incubations of chlorotrianisene with reconstituted P-450c and NADPH-cytochrome P-450 reductase exhibited covalent binding characteristics comparable to those seen in microsomal incubations. Investigations into the nature of the binding site and the reactive intermediate are currently being conducted. By analyzing the BSA adduct, the author intends to isolate the specific amino acid binding site(s).

  7. Selective inactivation by 21-chlorinated steroids of rabbit liver and adrenal microsomal cytochromes P-450 involved in progesterone hydroxylation.

    Science.gov (United States)

    Halpert, J; Jaw, J Y; Balfour, C; Mash, E A; Johnson, E F

    1988-08-01

    The inactivation by 21-chlorinated steroids of rabbit liver cytochromes P-450 involved in the hydroxylation of progesterone has been investigated in intact microsomes encompassing two phenotypes of 21-hydroxylase activity, two phenotypes of 16 alpha-hydroxylase activity, and three phenotypes of 6 beta-hydroxylase activity. In liver microsomes from outbred New Zealand White male rabbits exhibiting a high content of cytochrome P-450 1, 21,21-dichloropregnenolone caused a time- and NADPH-dependent loss of 21-hydroxylase activity. This loss of activity exhibited a number of characteristics of mechanism-based inactivation, including irreversibility, saturation with increasing inhibitor concentrations, and protection by substrate, and was also documented with purified P-450 1 in a reconstituted system. 21,21-Dichloropregnenolone caused no time-dependent loss of 6 beta-hydroxylase activity in microsomes from the New Zealand White rabbits or from control or rifampicin-treated rabbits of the inbred B/J strain. In contrast, in the microsomes from the B/J rabbits, some inactivation of the 16 alpha-hydroxylase was observed (k = 0.04 min-1), regardless of the rifampicin treatment. The other two compounds tested, 21-chloropregnenolone and 21,21-dichloroprogesterone, were less effective than the dichloropregnenolone as inactivators of cytochrome P-450 1. On the other hand, 21,21-dichloroprogesterone, but not 21,21-dichloropregneolone, caused a rapid time-dependent loss of 21-hydroxylase activity in rabbit adrenal microsomes. The results indicate that the introduction of a dichloromethyl group into a substrate bearing a methyl group normally hydroxylated by only one or a few forms of cytochrome P-450 may be a rational means of designing selective inhibitors of the enzyme.

  8. Effect of the beta-glucuronidase inhibitor saccharolactone on glucuronidation by human tissue microsomes and recombinant UDP-glucuronosyltransferases.

    Science.gov (United States)

    Oleson, Lauren; Court, Michael H

    2008-09-01

    Glucuronidation studies using microsomes and recombinant uridine diphosphoglucuronosyltransferases (UGTs) can be complicated by the presence of endogenous beta-glucuronidases, leading to underestimation of glucuronide formation rates. Saccharolactone is the most frequently used beta-glucuronidase inhibitor, although it is not clear whether this reagent should be added routinely to glucuronidation incubations. Here we have determined the effect of saccharolactone on eight different UGT probe activities using pooled human liver microsomes (pHLMs) and recombinant UGTs (rUGTs). Despite the use of buffered incubation solutions, it was necessary to adjust the pH of saccharolactone solutions to avoid effects (enhancement or inhibition) of lowered pH on UGT activity. Saccharolactone at concentrations ranging from 1 to 20 mM did not enhance any of the glucuronidation activities evaluated that could be considered consistent with inhibition of beta-glucuronidase. However, for most activities, higher saccharolactone concentrations resulted in a modest degree of inhibition. The greatest inhibitory effect was observed for glucuronidation of 5-hydroxytryptamine and estradiol by pHLMs, with a 35% decrease at 20 mM saccharolactone concentration. Endogenous beta-glucuronidase activities were also measured using various human tissue microsomes and rUGTs with estradiol-3-glucuronide and estradiol-17-glucuronide as substrates. Glucuronide hydrolysis was observed for pHLMs, lung microsomes and insect-cell expressed rUGTs, but not for kidney, intestinal or human embryonic kidney HEK293 microsomes. However, the extent of hydrolysis was relatively small, representing only 9-19% of the glucuronide formation rate measured in the same preparations. Consequently, these data do not support the routine inclusion of saccharolactone in glucuronidation incubations. If saccharolactone is used, concentrations should be titrated to achieve activity enhancement without inhibition.

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

    Science.gov (United States)

    Hu, Xiao; Huang, Wei; Yang, Yuan

    2015-12-01

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

  10. Anatomy of the Brain

    Science.gov (United States)

    ... Menu Brain Tumor Information Brain Anatomy Brain Structure Neuron Anatomy Brain Tumor Symptoms Diagnosis Types of Tumors Risk Factors ... form Brain Tumor Information Brain Anatomy Brain Structure Neuron Anatomy Brain Tumor Symptoms Diagnosis Types of Tumors Risk Factors ...

  11. Relative efficacies of alpha-tocopherol, N-acetyl-serotonin, and melatonin in reducing non-enzymatic lipid peroxidation of rat testicular microsomes and mitochondria.

    Science.gov (United States)

    Gavazza, Mariana; Catalá, Angel

    2009-01-01

    In this study, we examined the relative efficacies of alpha-tocopherol, N-acetyl-serotonin, and melatonin in reducing ascorbate-Fe(2+) lipid peroxidation (LPO) of rat testicular microsomes and mitochondria. Special attention was paid to the changes produced on the highly polyunsaturated fatty acids (PUFAs) C20:4 n6 and C22:5 n6. The LPO of testicular microsomes or mitochondria produced a significant decrease of C20:4 n6 and C22:5 n6. Both long-chain PUFAs were protected when the antioxidants were incorporated either in microsomes or mitochondria. By comparison of the IC50 values obtained between alpha-tocopherol and both indolamines, it was observed that alpha-tocopherol was the most efficient antioxidant against the LPO induced by ascorbate-Fe(2+) under experimental conditions in vitro, IC50 values from the inhibition of alpha-tocopherol on the chemiluminescence were higher in microsomes (0.14 mM) than in mitochondria (0.08 mM). The protective effect observed by alpha-tocopherol in rat testis mitochondria was higher compared with microsomes, associated with the higher amount of [C20:4 n6] + [C22:5 n6] in microsomes than that in mitochondria. Melatonin and N-acetyl-serotonin were more effective in inhibiting the LPO in mitochondria than that in microsomes. Thus, a concentration of 1 mM of both indolamines was sufficient to inhibit in approximately 70% of the light emission in mitochondria, whereas a greater dosage of 10 times (10 mM) was necessary to produce the same effect in microsomes. It is proposed that the vulnerability to LPO of rat testicular microsomes and mitochondria in the presence of both indolamines is different because of the different proportion of PUFAs in these organelles.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1991-03-11

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

  13. Evaluation of beta-naphthoxyacetic acid for mutagenic activity in the Salmonella/mammalian microsome assay.

    Science.gov (United States)

    Rashid, K A; Mumma, R O

    1986-06-01

    Beta-naphthoxyacetic acid (BNOA) is used as a plant growth regulator on tomatoes and strawberries. It is the active ingredient in Blossom-Set and Berry-Set, two plant hormone sprays for fruit-set. The mutagenic activity of BNOA was evaluated in four strains of Salmonella typhimurium (TA97, TA98, TA100 and TA1535) in the presence and absence of liver microsomal and cytosolic enzymes derived from Aroclor induced rats. BNOA did not produce any significant increase (p less than 0.05) in the reversion of any of the four tester strains in the standard plate incorporation assay. Results of the agar overlay toxicity tests indicates that the chemical shows toxic effects at concentrations above 500 micrograms/plate. It was concluded that under the conditions of these tests, BNOA did not exhibit any mutagenic activity.

  14. Chiral metabolism of propafenone in rat hepatic microsomes treated with two inducers

    Institute of Scientific and Technical Information of China (English)

    Quan Zhou; Tong-Wei Yao; Su Zeng

    2001-01-01

    AIM: To study the influence of inducers of drug metabolism enzyme, β-naphthoflavone (BNF) and dexamethasone (DEX), on the stereoselective metabolism of propafenone in the rat hepatic microsomes. METHODS: Phase I metabolism of propafenone was studied using the microsomes induced by BNF and DEX and the non-induced microsome was used as the control. The enzymatic kinetics parameters of propafenone enantiomers were calculated by regress analysis of Eadie-Hofstee Plots.Propafenone enantiomer concentrations were assayed by a chiral HPLC. RESULTS: The metabolite of propafenone, N-desalkylpropafenone, was found after incubstion of propafenone with the rat hepatic microsomes induced by BNF and DEX. In these two groups, the stereoselectivity favoring R ( - ) isomer was observed in metabolism st Iow substrate concentrations of racemic propafenone, but lost the stereoselectivity st high substrate concentrations.However; in control group, no stereeselectivity was observed. The enzyme kinetic parameters were: ① Km.Control group: R( - ) 83 ± 6, S( + ) 94 ± 7; BNF group: R (-)105 ± 6, S( + )128 ± 14; DEX group: R( - ) 86± 11, S( + ) 118 ± 16; ② vmax. Control group: R( - ) 0.75 ± 0.16, S( + ) 0.72±0.07; BNF group: R( - )1.04± 0.15, S( + )1.07±14; DEX group: R( - ) 0.93 ± 0.06, S( + ) 1.04 ± 0.09; (③)Clint. Control group: R( - ) 8.9± 1.1, S( + ) 7.6±0.7; BNFgroup: R( - )9.9±0.9, S( + )8.3±0.7; DEX group: R( - )10.9± 0.8, S( + ) 8.9 ± 0.9. The enantiomeric differences in Km and Clint were both significant, but not in Vmax, in BNF and DEX group. Whereas enantiomeric differences in three parameters were all insignificant in control group.Furthermore, Km and Umax were both significantly less than those in BNF or DEX group. In the rat liver microsorne induced by DEX, nimodipine (NDP) decreased the stereoselectivity in propafenone metabolism at Iow substrate concentration. The inhibition of NDP on the metabolism of propafenone was stereo.selective with R

  15. Metabolism of the hepatotoxic compound sophoraflavanone G in rat liver microsomes.

    Science.gov (United States)

    Chen, Ping; Zhang, Xiuwen; Huang, Taomin; Yu, Qianqian; Cheng, Nengneng

    2014-07-01

    Our study aimed at investigating the metabolic characteristics of sophoraflavanone G (SFG), one of the hepatotoxic constituents of Sophora flavescens, in rat liver microsomes (RLMs). SFG was metabolized to 3 phase I metabolites, di-hydroxylated SFG (M1), mono-hydroxylated SFG (M2), dehydrogenated product of mono-hydroxylated SFG (M3) and 3 SFG glucuronides (M4, M5, and M6) by RLMs. The formation kinetics of M2 conformed to biphasic kinetics in RLMs. The formation kinetics of M4 and M5 best-fitted the Hill equation kinetics. Chemical inhibition studies found that CYP1A2 and CYP2E1 were the major enzymes responsible for the formation of M2, and the formation of M4 and M5 may be catalyzed by multiple UGT1A isoforms.

  16. ER Adaptor SCAP Translocates and Recruits IRF3 to Perinuclear Microsome Induced by Cytosolic Microbial DNAs

    Science.gov (United States)

    Yu, Huansha; Liu, Xing; Huang, Lulu; Wang, Qiang; Liu, Heng; Cui, Ye; Tang, Yijun; Zhang, Peng; Wang, Chen

    2016-01-01

    Stimulator of interferon genes (STING, also known as MITA, ERIS or MPYS) induces the activation of TBK1 kinase and IRF3 transcription factor, upon sensing of microbial DNAs. How IRF3 is recruited onto the STING signalosome remains unknown. We report here that silencing of the ER adaptor SCAP markedly impairs the IRF3-responsive gene expression induced by STING. Scap knockdown mice are more susceptible to HSV-1 infection. Interestingly, SCAP translocates from ER, via Golgi, to perinuclear microsome in a STING-dependent manner. Mechanistically, the N-terminal transmembrane domain of SCAP interacts with STING, and the C-terminal cytosolic domain of SCAP binds to IRF3, thus recruiting IRF3 onto STING signalosome. Mis-localization of SCAP abolishes its antiviral function. Collectively, this study characterizes SCAP as an essential adaptor in the STING signaling pathway, uncovering a critical missing link in DNAs-triggered host antiviral responses. PMID:26900919

  17. Dataset from proteomic analysis of rat, mouse, and human liver microsomes and S9 fractions

    Directory of Open Access Journals (Sweden)

    Makan Golizeh

    2015-06-01

    Full Text Available Rat, mouse and human liver microsomes and S9 fractions were analyzed using an optimized method combining ion exchange fractionation of digested peptides, and ultra-high performance liquid chromatography (UHPLC coupled to high resolution tandem mass spectrometry (HR-MS/MS. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org via the PRIDE partner repository (Vizcaíno et al., 2013 [1] with the dataset identifiers PXD000717, PXD000720, PXD000721, PXD000731, PXD000733 and PXD000734. Data related to the peptides (trypsin digests only were also uploaded to Peptide Atlas (Farrah et al., 2013 [2] and are available with the dataset identifiers PASS00407, PASS00409, PASS00411, PASS00412, PASS00413 and PASS00414. The present dataset is associated with a research article published in EuPA Open Proteomics [3].

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

    Science.gov (United States)

    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.

  19. ER Adaptor SCAP Translocates and Recruits IRF3 to Perinuclear Microsome Induced by Cytosolic Microbial DNAs.

    Directory of Open Access Journals (Sweden)

    Wei Chen

    2016-02-01

    Full Text Available Stimulator of interferon genes (STING, also known as MITA, ERIS or MPYS induces the activation of TBK1 kinase and IRF3 transcription factor, upon sensing of microbial DNAs. How IRF3 is recruited onto the STING signalosome remains unknown. We report here that silencing of the ER adaptor SCAP markedly impairs the IRF3-responsive gene expression induced by STING. Scap knockdown mice are more susceptible to HSV-1 infection. Interestingly, SCAP translocates from ER, via Golgi, to perinuclear microsome in a STING-dependent manner. Mechanistically, the N-terminal transmembrane domain of SCAP interacts with STING, and the C-terminal cytosolic domain of SCAP binds to IRF3, thus recruiting IRF3 onto STING signalosome. Mis-localization of SCAP abolishes its antiviral function. Collectively, this study characterizes SCAP as an essential adaptor in the STING signaling pathway, uncovering a critical missing link in DNAs-triggered host antiviral responses.

  20. Characterization of covalent binding of N'-nitrosonornicotine in rat liver microsomes

    Energy Technology Data Exchange (ETDEWEB)

    Hughes, M.F.; Brock, W.J.; Marion, L.J.; Vore, M.

    1986-01-01

    The metabolism of the carcinogenic nitrosamine, N'-nitrosonornicotine (NNN), to reactive intermediates which bind covalently was assessed using male Sprague-Dawley rat liver microsomes. The NADPH-dependent covalent binding of (/sup 14/C)NNN was linear with time up to 90 min and protein concentration up to 3.0 mg/ml. The apparent Km and Vmax of the binding were determined from the initial velocities and found to be 0.91 mM and 4.7 pmol/min/mg protein, respectively. Although NNN is not a hepatocarcinogen, this amount of NADPH-dependent covalent binding is 7-fold greater than that reported for dimethylnitrosamine, a potent hepatocarcinogen. Extensive covalent binding of (/sup 14/C)NNN to liver and muscle microsomal protein was also present in the absence of an NADPH-generating system and in the presence of 50% methanol, indicating a non-enzymatically mediated reaction. Addition of the nucleophiles glutathione, cysteine and N-acetylcysteine significantly decreased (p less than 0.01) the non-NADPH-dependent binding, but did not affect NADPH-dependent binding. In vitro addition of the cytochrome P-450 inhibitors metyrapone, piperonyl butoxide and SKF-525A significantly decreased (p less than 0.05) NADPH-dependent binding of (14C)NNN by 27-40%. NADH did not replace NADPH in supporting covalent binding. Replacement of an air atmosphere with nitrogen or CO:O2 (8:2) significantly decreased (p less than 0.05) NADPH-dependent binding of (/sup 14/C)NNN by 40 and 27%, respectively. Aroclor 1254 pre-treatment of the rats did not enhance the NADPH-dependent binding of (/sup 14/C)NNN. These data indicate that cytochrome P-450 is at least in part responsible for the metabolic activation of the carcinogen NNN but also suggest additional mechanisms of activation.

  1. Preparation of ribosome-free membranes from rat liver microsomes by means of lithium chloride

    Science.gov (United States)

    Scott-Burden, T.; Hawtrey, A. O.

    1969-01-01

    1. Treatment of washed rat liver microsomes in a medium containing 0·12m-sucrose, 12·5mm-potassium chloride, 2·5mm-magnesium chloride and 25mm-tris–hydrochloric acid buffer, pH7·6, with 2m-lithium chloride at 5° for 16hr. leads to the formation of membranes free of ribosomes and ribosomal subunits. 2. Confirmation of the absence of ribosomes from lithium chloride-prepared membranes was obtained by treatment of the membranes with sodium deoxycholate, followed by sucrose-density-gradient centrifugation, which showed the complete absence of ribosomes. 3. Treatment of membranes with phenol, followed by sucrose-density-gradient analysis of the isolated RNA, showed the presence of a small amount of 4s material. Repetition of the phenol extraction procedure in the presence of liver cell sap as a ribonuclease inhibitor again showed the presence of only 4s material. The 4s RNA was shown to be transfer RNA by the fact that it had the same capacity for accepting 14C-labelled amino acids as isolated transfer RNA from rat liver pH5 enzyme. 4. Analysis showed that microsomes and membranes possessed similar glucose 6-phosphatase, NADH–2,6-dichlorophenol-indophenol reductase, NADH–neo-tetrazolium reductase, NADH–cytochrome c reductase and ribonuclease activities. 5. 3H-labelled ribosomal RNA binds to membranes. However, isolation of the bound RNA by the phenol extraction procedure, followed by sucrose-density-gradient analysis, shows the RNA to be degraded to 7s material. Very little breakdown of 3H-labelled ribosomal RNA bound to membranes occurs if the binding and isolation are carried out in the presence of liver cell sap. ImagesPLATE 1 PMID:4311814

  2. [Metabolite profiling of two anti-HIV lead compounds in rat liver microsomes].

    Science.gov (United States)

    Wang, Rui; Chen, Jia; Qin, Bing-Jie; Xie, Jian-Wei; Li, Hua; Xie, Lan

    2012-12-01

    The metabolite profiling of DAPA-7012 and DAAN-4442, the lead compounds from two new kinds of non-nucleoside reverse transcriptase inhibitors (NNRTIs), was performed using an ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS/MS), with the assistance of a metabolite data processing software. By utilizing the mass defect filter (MDF) technique, the data acquired from the 0 h-incubation and the 2 h-incubation were compared and analyzed with the MetaboLynx software. After incubation, 14 metabolites of DAPA-7012 and 14 metabolites of DAAN-4442 were found in rat liver microsome. The MS2 spectra for some metabolites were obtained using the MS(E) technique to get fragment ions for structural elucidation. The results indicated that both compounds could undergo extensive metabolism in rat liver microsomes. The major phase I reaction was oxidation/hydroxylation. The major phase II reaction was S-glutathione conjugation. The metabolic pathways were similar between the two lead compounds, though they have different backbone structures. Besides, the 4-NO2 of ring B in DAAN-4442 was susceptible to reduction, the benzyl of ring C in DAPA-7012 was tend to be oxidized. The common metabolic soft spots were primary amine of ring B and two methyl groups of ring C. Early SAR results showed that the primary amine and methyl were necessary substituent groups. The stability of these active groups needs to be improved and optimized. The approach of combining metabolites information and structure-activity analysis can provide a reference for further structural optimization.

  3. In vitro metabolism of the anti-androgenic fungicide vinclozolin by rat liver microsomes.

    Science.gov (United States)

    Sierra-Santoyo, Adolfo; Angeles-Soto, Esperanza; de Lourdes López-González, Ma; Harrison, Randy A; Hughes, Michael F

    2012-03-01

    Vinclozolin (V) is a fungicide used in agricultural settings. V administered to rats is hydrolyzed to 2-[[(3,5-dichlorophenyl)-carbamoyl]oxy]-2-methyl-3-butenoic acid (M1) and 3',5'-dichloro-2-hydroxy-2-methylbut-3-enanilide (M2). V, M1 and M2 have antiandrogenic properties by interacting with the androgen receptor. Data on V, M1 and M2 biotransformation are limited. Our objective was to characterize V metabolism by rat liver microsomes. V was incubated with non-treated adult male Long-Evans rat liver microsomes and NADPH. Several metabolites were detected following the extraction of incubate with acetonitrile and analysis by HPLC/DAD/MSD. One metabolite was identified as [3-(3,5-dichlorophenyl)-5-methyl-5-(1,2-dihydroxyethyl)-1,3-oxazolidine-2,4-dione] (M4), which was gradually converted to 3',5'-dichloro-2,3,4-trihydroxy-2-methylbutylanilide (M5). Both co-eluted in the same HPLC peak. Another metabolite ([M7]) was detected by UV but was unstable for mass spectral analysis. The K(M app) for co-eluted M4/M5 and [M7] was 53.7 and 135.4 μM, the V(max app) was 0.812 and 0.669 nmoles/min/mg protein, and CL(int) was 15.1 and 4.9 ml/min/g protein, respectively. Pilocarpine, orphenadrine and proadifen and anti-rat cytochrome P450 (CYP)2A, 2B and 3A antibodies inhibited M4/M5 and [M7] formation. These results indicate that V is efficiently metabolized by CYP. Determination of the metabolites of V will provide further insight into the relationship between toxicity and tissue dose of V and its metabolites.

  4. Trimeric microsomal glutathione transferase 2 displays one third of the sites reactivity.

    Science.gov (United States)

    Ahmad, Shabbir; Thulasingam, Madhuranayaki; Palombo, Isolde; Daley, Daniel O; Johnson, Kenneth A; Morgenstern, Ralf; Haeggström, Jesper Z; Rinaldo-Matthis, Agnes

    2015-10-01

    Human microsomal glutathione transferase 2 (MGST2) is a trimeric integral membrane protein that belongs to the membrane-associated proteins in eicosanoid and glutathione metabolism (MAPEG) family. The mammalian MAPEG family consists of six members where four have been structurally determined. MGST2 activates glutathione to form a thiolate that is crucial for GSH peroxidase activity and GSH conjugation reactions with electrophilic substrates, such as 1-chloro-2,4-dinitrobenzene (CDNB). Several studies have shown that MGST2 is able to catalyze a GSH conjugation reaction with the epoxide LTA4 forming the pro-inflammatory LTC4. Unlike its closest homologue leukotriene C4 synthase (LTC4S), MGST2 appears to activate its substrate GSH using only one of the three potential active sites [Ahmad S, et al. (2013) Biochemistry. 52, 1755-1764]. In order to demonstrate and detail the mechanism of one-third of the sites reactivity of MGST2, we have determined the enzyme oligomeric state, by Blue native PAGE and Differential Scanning Calorimetry, as well as the stoichiometry of substrate and substrate analog inhibitor binding to MGST2, using equilibrium dialysis and Isothermal Titration Calorimetry, respectively. Global simulations were used to fit kinetic data to determine the catalytic mechanism of MGST2 with GSH and CDNB (1-chloro-2,4-dinitrobenzene) as substrates. The best fit was observed with 1/3 of the sites catalysis as compared with a simulation where all three sites were active. In contrast to LTC4S, MGST2 displays a 1/3 the sites reactivity, a mechanism shared with the more distant family member MGST1 and recently suggested also for microsomal prostaglandin E synthase-1.

  5. Resveratrol inhibits 11β-hydroxysteroid dehydrogenase type 1 activity in rat adipose microsomes.

    Science.gov (United States)

    Tagawa, Noriko; Kubota, Sayaka; Kato, Ikuo; Kobayashi, Yoshiharu

    2013-09-01

    It has been suggested that resveratrol, a polyphenol in wine, can regulate adiposity because it decreases adipose deposition in mice and rats; however, the mechanism underlying this effect has not been fully clarified. In humans and rodents, 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1) is expressed in liver and adipose tissue. 11β-HSD1 converts inactive glucocorticoid into active glucocorticoid in adipocytes. Activated glucocorticoid plays an important role in the pathogenesis of central obesity. The objective of this study was to investigate the effects of resveratrol on 11β-HSD1 activity in rodent adipose tissue. 11β-HSD1 activity in microsomes from rat mesenteric adipose depots and 3T3-L1 adipocytes was determined in the presence of 11-dehydrocorticosterone with or without varying concentrations of resveratrol. Significant inhibition of 11β-HSD1 by resveratrol was observed in rat adipose microsomes and 3T3-L1 adipocytes within 10 min. Time- and dose-dependent effects were also observed. The 11β-HSD1 activity by resveratrol was also inhibited in rat epididymal adipose tissue, and this inhibition was not recovered by estrogen receptor blockers. The kinetic study revealed that resveratrol acted as a non-competitive inhibitor of 11β-HSD1. Ki and IC50 values of resveratrol were 39.6 and 35.2 μM respectively. Further, resveratrol did not affect the activities of 11β-HSD2 and hexose-6-phosphate dehydrogenase. These results suggest that the most likely mechanism of 11β-HSD1 inhibition by resveratrol is via interaction between resveratrol and 11β-HSD1 enzyme, rather than via a transcriptional pathway. We demonstrated that the antiobesity effects of resveratrol may partially be attributed to the inhibition of 11β-HSD1 activity in adipocytes.

  6. Mechanism of ethylbenzene-induced mouse-specific lung tumor: metabolism of ethylbenzene by rat, mouse, and human liver and lung microsomes.

    Science.gov (United States)

    Saghir, Shakil A; Rick, David L; McClymont, E L; Zhang, Fagen; Bartels, Michael J; Bus, James S

    2009-02-01

    This study was conducted to determine species differences in the metabolism of ethylbenzene (EB) in liver and lung. EB (0.22-7.0mM) was incubated with mouse, rat and human liver and lung microsomes and the formation of 1-phenylethanol (1PE), acetophenone (AcPh), 2-ethylphenol (2EP), 4-ethylphenol (4EP), 2,5-ethylquinone, and 3,4-ethylquinone were measured. Reactive metabolites (2,5-dihydroxyethylbenzene-GSH [2EP-GSH] and 3,4-dihydroxyethylbenzene-GSH [4EP-GSH]) were monitored via glutathione (GSH) trapping technique. None of the metabolites were formed at detectable levels in incubations with human lung microsomes. Percent conversion of EB to 1PE ranged from 1% (rat lung; 7.0mM EB) to 58% (mouse lung; 0.22 mM EB). More 1PE was formed in mouse lung than in mouse liver microsomes, although formation of 1PE by rat liver and lung microsomes was similar. Metabolism of EB to 1PE was in the order of mouse > rat > human. Formation of AcPh was roughly an order of magnitude lower than 1PE. Conversion of EB to ring-hydroxylated metabolites was much lower (0.0001% [4EP-GSH; rat lung] to 0.6% [2EP-GSH; mouse lung]); 2EP-GSH was typically 10-fold higher than 4EP-GSH. Formation of 2EP-GSH was higher by lung (highest by mouse lung) than liver microsomes and the formation of 2EP-GSH by mouse liver microsomes was higher than rat and human liver microsomes. Increasing concentrations of EB did lead to a decrease in amount of some formed metabolites. This may indicate some level of substrate- or metabolite-mediated inhibition. High concentrations of 2EP and 4EP were incubated with microsomes to further investigate their oxidation to ethylcatechol (ECat) and ethylhydroquinone (EHQ). Conversion of 2EP to EHQ ranged from 6% to 9% by liver (mouse > human > rat) and from 0.1% to 18% by lung microsomes (mouse > rat > human). Conversion of 4EP to ECat ranged from 2% to 4% by liver (mouse > human approximately rat) and from 0.3% to 7% by lung microsomes (mouse > rat > human). Although ring

  7. Brain Basics

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    Full Text Available ... have been linked to many mental disorders, including autism , obsessive compulsive disorder (OCD) , schizophrenia , and depression . Brain ... studies show that brain growth in children with autism appears to peak early. And as they grow ...

  8. Brain Basics

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    Full Text Available ... depression. The Growing Brain Inside the Brain: Neurons & Neural Circuits Neurons are the basic working unit of ... but sometimes give rise to disabilities or diseases. neural circuit —A network of neurons and their interconnections. ...

  9. Brain Basics

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    Full Text Available ... than ever before. Brain Imaging Using brain imaging technologies such as magnetic resonance imaging (MRI), which uses ... depression experience when starting treatment. Gene Studies Advanced technologies are also making it faster, easier, and more ...

  10. Brain Basics

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    Full Text Available ... Real Life Brain Basics in Real Life—How Depression affects the Brain Meet Sarah Sarah is a ... blues" from time to time. In contrast, major depression is a serious disorder that lasts for weeks. ...

  11. Brain Basics

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    Full Text Available ... the brain cannot effectively coordinate the billions of cells in the body, the results can affect many ... unit of the brain and nervous system. These cells are highly specialized for the function of conducting ...

  12. Brain Diseases

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    The brain is the control center of the body. It controls thoughts, memory, speech, and movement. It regulates the function of many organs. When the brain is healthy, it works quickly and automatically. However, ...

  13. Brain Basics

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    Full Text Available ... related to changes in the anatomy, physiology, and chemistry of the nervous system. When the brain cannot ... their final destination. Chemical signals from other cells guide neurons in forming various brain structures. Neighboring neurons ...

  14. Brain Basics

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    Full Text Available ... in the anatomy, physiology, and chemistry of the nervous system. When the brain cannot effectively coordinate the billions ... the basic working unit of the brain and nervous system. These cells are highly specialized for the function ...

  15. Brain Basics

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    Full Text Available ... affect many aspects of life. Scientists are continually learning more about how the brain grows and works ... early brain development. It may also assist in learning and memory. Problems in making or using glutamate ...

  16. Loss of cholesterol 7 alpha-hydroxylase activity in vitro in the presence of bivalent metal ions and by dialysis of rat liver microsomes.

    Science.gov (United States)

    Sanghvi, A; Grassi, E; Diven, W

    1983-01-01

    A loss in cholesterol 7 alpha-hydroxylase activity [cholesterol 7 alpha-monooxygenase; cholesterol,NADPH:oxygen oxidoreductase (7 alpha-hydroxylating), EC 1.14.13.17] was seen when rat liver microsomes were incubated in the presence of Ca2+, Mg2+, or Mn2+. The loss in enzyme activity was complete within only 5 min of incubation with Ca2+ and Mn2+, whereas Mg2+ required 10 to 15 min of incubation with microsomes to produce a similar inhibition. This effect of metal ions could be blocked if the incubations were carried out in phosphate buffer. Similarly, preincubation of microsomes in the presence of NaF completely prevented the loss in enzyme activity due to Ca2+ and Mg2+ ions, but only partially the loss due to Mn2+. These results suggest metal ion activation of an endogenous microsomal phosphatase, which in turn may inactivate cholesterol 7 alpha-hydroxylase through its dephosphorylation. Further, a dialyzable microsomal factor appears to be essential for stabilizing the enzyme, because dialysis of a microsomal suspension results in a considerable loss of enzyme activity. PMID:6300898

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

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

    2016-01-01

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

  19. Brain Basics

    Medline Plus

    Full Text Available ... such as depression. The Growing Brain Inside the Brain: Neurons & Neural Circuits Neurons are the basic working unit ... final destination. Chemical signals from other cells guide neurons in forming various brain structures. Neighboring neurons make connections with each other ...

  20. Brain Aneurysm

    Science.gov (United States)

    A brain aneurysm is an abnormal bulge or "ballooning" in the wall of an artery in the brain. They are sometimes called berry aneurysms because they ... often the size of a small berry. Most brain aneurysms produce no symptoms until they become large, ...

  1. The Brain.

    Science.gov (United States)

    Hubel, David H.

    1979-01-01

    This article on the brain is part of an entire issue about neurobiology and the question of how the human brain works. The brain as an intricate tissue composed of cells is discussed based on the current knowledge and understanding of its composition and structure. (SA)

  2. The H{sub 1}–H{sub 2} domain of the α{sub 1} isoform of Na{sup +}–K{sup +}–ATPase is involved in ouabain toxicity in rat ventricular myocytes

    Energy Technology Data Exchange (ETDEWEB)

    Xiong, Chen; Li, Jun-xia; Guo, Hui-cai; Zhang, Li-nan; Guo, Wei; Meng, Jing; Wang, Yong-li, E-mail: wangyongli@gmail.com

    2012-07-01

    The composition of different isoforms of Na{sup +}-K{sup +}-ATPase (NKA, Na/K pump) in ventricular myocytes is an important factor in determining the therapeutic effect and toxicity of cardiac glycosides (CGs) on heart failure. The mechanism whereby CGs cause these effects is still not completely clear. In the present study, we prepared two site-specific antibodies (SSA78 and WJS) against the H{sub 1}–H{sub 2} domain of α{sub 1} and α{sub 2} isoforms of NKA in rat heart, respectively, and compared their influences on the effect of ouabain (OUA) in isolated rat ventricular myocytes. SSA78 or WJS, which can specifically bind with the α{sub 1} or α{sub 2} isoform, were assessed with enzyme linked immunosorbent assay (ELISA), Western blot and immunofluorescent staining methods. Preincubation of myocytes with SSA78 inhibited low OUA affinity pump current but not high OUA affinity pump current, reduced the rise in cytosolic calcium concentration ([Ca{sup 2+}]{sub i}), attenuated mitochondrial Ca{sup 2+} overload, restored mitochondrial membrane potential reduction, and delayed the decrease of the myocardial contractile force as well as the occurrence of arrhythmic contraction induced by high concentrations (1 mM) but not low concentrations (1 μM) of OUA. Similarly, preincubation of myocytes with WJS inhibited high OUA affinity pump current, reduced the increase of [Ca{sup 2+}]{sub i} and the contractility induced by 1 μM but not that induced by 1 mM OUA. These results indicate that the H{sub 1}–H{sub 2} domain of the NKA α{sub 1} isoform mediates OUA-induced cardiac toxicity in rat ventricular myocytes, and inhibitors for this binding site may be used as an adjunct to CGs treatment for cardiovascular disease. -- Highlights: ► We prepared two antibodies against the H{sub 1}-H{sub 2} domain of α{sub 1} and α{sub 2} isoforms of NKA. ► The H{sub 1}-H{sub 2} domain of the NKA α{sub 1} isoform mediates OUA-induced cardiac toxicity. ► The H{sub 1}-H{sub 2

  3. Effect of methoxychlor on the antioxidant system in mitochondrial and microsome-rich fractions of rat testis.

    Science.gov (United States)

    Latchoumycandane, C; Mathur, P P

    2002-07-01

    Methoxychlor, an environmental contaminant, which is widely used as a pesticide in many countries, has been shown to induce reproductive abnormalities in male rats. The precise nature and mechanism of action of methoxychlor on the male reproductive system is not clear. In the present study, we have sought to investigate the induction of oxidative stress in the testis of rat after exposure to methoxychlor. Methoxychlor (1, 10, and 100 mg kg(-1) body weight per day) was administered orally to the rats for 45 days. After 24 h of the last treatment the animals were killed using anesthetic ether. The body weight of the animals administered with methoxychlor did not show any significant change. The weights of the testis, epididymis, seminal vesicles and ventral prostate decreased significantly in 100 mg dose but remained unchanged in 1 and 10 mg doses. Mitochondrial and microsome-rich fractions of the testis were obtained by the method of differential centrifugation. The activities of antioxidant enzymes such as superoxide dismutase, catalase, glutathione reductase and glutathione peroxidase decreased significantly in the animals treated with methoxychlor in a dose-dependent manner in the mitochondrial and microsome-rich fractions of rat testis. The levels of hydrogen peroxide generation (H(2)O(2)) and lipid peroxidation increased in mitochondrial and microsome-rich fractions of the testis of the rats treated with methoxychlor. The results suggested that the low to medium doses of methoxychlor elicit depletion of antioxidant enzymes and concomitant increase in the levels of H(2)O(2) and lipid peroxidation differentially in mitochondrial and microsome-rich fractions of rat testis. In conclusion, the adverse effect of methoxychlor on male reproduction could be due to the induction of oxidative stress in testis.

  4. [Effects of six kinds of Chinese herb extracts on the activities of rat liver microsomes in vitro].

    Science.gov (United States)

    Bi, Yun-Feng; Zhu, Hong-Bin; Xing, Jun-Peng; Liu, Zhi-Qiang; Song, Feng-Rui

    2013-07-01

    Effects of six kinds of Chinese herb extracts, including Folium Crataegi extract, Herba Epimedii extract, Folium Acanthopanacis Senticosi extract, Trifolium pratense L. extract, Folium Ginkgo extract and Radix Puerariae extract, on the activities of CYP450 isozymes (CYP1A2, CYP2C, CYP2E1, CYP2D, CYP3A) in rat hepatic microsomals were studied by using a UPLC-MS/MS (MRM) and cocktail probe substrates method. The results showed that effects of six kinds of Chinese herb extracts on each CYP450 isozyme activity were inhibitory. The IC50 of Folium Crataegi extract for the inhibition of rat microsomal CYP2D activity was only for 4.04 microg x mL(-1), which showed the highest inhibition; Trifolium pratense L. extract had strong inhibitory action to CYP2D, the IC50 value was 5.73 microg x mL(-1); Folium Crataegi extract also had strong inhibitory action on CYP2E1, the IC50 value was 10.91 microg x mL(-1). Furthermore, the IC50 of Folium Ginkgo extract for the inhibition of rat microsomal CYP3A, 2D, 2E1 activities were 45.12, 35.45 and 22.41 microg x mL(-1), respectively, and the IC50 of Folium Acanthopanacis Senticosi extract on the inhibition of rat microsomal CYP2E1 activity was 32.89 microg x mL(-1). In addition, mechanism of inhibition experimental results showed that the inhibiting abilities of Folium Crataegi extract and Radix Puerariae extract on each CYP450 isozyme increased with the increasing of the preincubation time, therefore, the inhibitory effects were a mechanism-based inhibition.

  5. Oxidation of 5-methoxy-N,N-diisopropyltryptamine in rat liver microsomes and recombinant cytochrome P450 enzymes.

    Science.gov (United States)

    Narimatsu, Shizuo; Yonemoto, Rei; Masuda, Kazufumi; Katsu, Takashi; Asanuma, Masato; Kamata, Tooru; Katagi, Munehiro; Tsuchihashi, Hitoshi; Kumamoto, Takuya; Ishikawa, Tsutomu; Naito, Shinsaku; Yamano, Shigeru; Hanioka, Nobumitsu

    2008-02-01

    The oxidative metabolism of 5-methoxy-N,N-diisopropyltryptamine (5-MeO-DIPT), a tryptamine-type designer drug, was studied using rat liver microsomal fractions and recombinant cytochrome P450 (CYP) enzymes. 5-MeO-DIPT was biotransformed mainly into a side-chain N-deisopropylated metabolite and partially into an aromatic ring O-demethylated metabolite in liver microsomal fractions from untreated rats of both sexes. This metabolic profile is different from our previous findings in human liver microsomal fractions, in which the aromatic ring O-demethylation was the major pathway whereas the side-chain N-deisopropylation was minor [Narimatsu S, Yonemoto R, Saito K, Takaya K, Kumamoto T, Ishikawa T, et al. Oxidative metabolism of 5-methoxy-N,N-diisopropyltryptamine (Foxy) by human liver microsomes and recombinant cytochrome P450 enzymes. Biochem Pharmacol 2006;71:1377-85]. Kinetic and inhibition studies indicated that the side-chain N-dealkylation is mediated by CYP2C11 and CYP3A2, whereas the aromatic ring O-demethylation is mediated by CYP2D2 and CYP2C6 in untreated male rats. Pretreatment of male rats with beta-naphthoflavone (BNF) produced an aromatic ring 6-hydroxylated metabolite. Recombinant rat and human CYP1A1 efficiently catalyzed 5-MeO-DIPT 6-hydroxylation under the conditions used. These results provide valuable information on the metabolic fate of 5-MeO-DIPT in rats that can be used in the toxicological study of this designer drug.

  6. Biotransformation of chlorpyrifos and diazinon by human liver microsomes and recombinant human cytochrome P450s (CYP).

    Science.gov (United States)

    Sams, C; Cocker, J; Lennard, M S

    2004-10-01

    The cytochrome P450 (CYP)-mediated biotransformation of the organophosphorothioate insecticides chlorpyrifos and diazinon was investigated. Rates of desulphuration to the active oxon metabolite (chlorpyrifos-oxon and diazinon-oxon) and dearylation to non-toxic hydrolysis products were determined in human liver microsome preparations from five individual donors and in recombinant CYP enzymes. Chlorpyrifos and diazinon underwent desulphuration in human liver microsome with mean Km = 30 and 45 microM and V(max) = 353 and 766 pmol min(-1) mg(-1), respectively. Dearylation of these compounds by human liver microsome proceeded with Km = 12 and 28 microM and V(max) = 653 and 1186 pmol min(-1) mg(-1), respectively. The apparent intrinsic clearance (V(max)/Km) of dearylation was 4.5- and 2.5-fold greater than desulphuration for chlorpyrifos and diazinon, respectively. Recombinant human CYP2B6 possessed the highest desulphuration activity for chlorpyrifos, whereas CYP2C19 had the highest dearylation activity. In contrast, both desulphuration and dearylation of diazinon were catalysed at similar rates, in the rank order CYP2C19 > CYP1A2 > CYP2B6 > CYP3A4. Both organophosphorothioates were more readily detoxified (dearylation) than bioactivated (desulphuration) in all human liver microsome preparations. However, the role of individual CYP enzymes in these two biotransformation pathways varied according to the structure of the organophosphorothioate, which was reflected in different activation/detoxification ratios for chlorpyrifos and diazinon. Variability in activity of individual CYP enzymes may influence interindividual sensitivity to the toxic effects of chlorpyrifos and diazinon.

  7. Effect of N-acetylcysteine in COPD patients with different microsomal epoxide hydrolase genotypes

    Directory of Open Access Journals (Sweden)

    Zhang JQ

    2015-05-01

    Full Text Available Jian-Qing Zhang,1 Jia-Qiang Zhang,1 Hua Liu,2 Zhi-Huan Zhao,1 Li-Zhou Fang,1 Ling Liu,1 Wei-Ping Fu,1 Jing-Kui Shu,1 Jia-Gang Feng,1 Lu-Ming Dai1 1Department of Respiratory Critical Care Medicine, 2Department of Clinical Laboratory, First Affiliated Hospital of Kunming Medical University, Kunming, People’s Republic of China Background: The role of the antioxidant N-acetylcysteine (NAC in the treatment of chronic obstructive pulmonary disease (COPD has not been clarified as yet. In early studies, we found that the proportion of smokers with COPD having extremely slow/slow microsomal epoxide hydrolase (EPHX1 enzyme activity is significantly higher than that in healthy smokers. The purpose of this study was to evaluate whether different EPHX1 enzyme activity is related to differential therapeutic effects of treatment with NAC in COPD.Methods: A total of 219 patients with COPD were randomly allocated to an extremely slow/slow EPHX1 enzyme activity group (n=157 or a fast/normal EPHX1 enzyme activity group (n=62 according to their EPHX1 enzyme activity. Both groups were treated with NAC 600 mg twice daily for one year. The main study parameters, including forced expiratory volume in one second (FEV1, St George’s Respiratory Questionnaire (SGRQ, and yearly exacerbation rate, were measured at baseline and at 6-month intervals for one year.Results: Both FEV1 and SGRQ symptom scores were improved after treatment with NAC in the slow activity group when compared with the fast activity group. Further, changes in FEV1 and SGRQ symptom score in patients with mild-to-moderate COPD were more significant than those in patients with severe-to-very severe COPD. The yearly exacerbation rates were reduced in both groups, but the reduction in the slow activity group was significantly lower than in the fast activity group.Conclusion: NAC treatment in COPD patients with extremely slow/slow EPHX1 enzyme activity improves FEV1 and the SGRQ symptom score, especially

  8. Inhibition of genistein glucuronidation by bisphenol A in human and rat liver microsomes.

    Science.gov (United States)

    Coughlin, Janis L; Thomas, Paul E; Buckley, Brian

    2012-03-01

    Genistein is a natural phytoestrogen of the soybean, and bisphenol A (BPA) is a synthetic chemical used in the production of polycarbonate plastics. Both genistein and BPA disrupt the endocrine system in vivo and in vitro. Growing concerns of altered xenobiotic metabolism due to concomitant exposures from soy milk in BPA-laden baby bottles has warranted the investigation of the glucuronidation rate of genistein in the absence and presence (25 μM) of BPA by human liver microsomes (HLM) and rat liver microsomes (RLM). HLM yield V(max) values of 0.93 ± 0.10 nmol · min(-1) · mg(-1) and 0.62 ± 0.05 nmol · min(-1) · mg(-1) in the absence and presence of BPA, respectively. K(m) values for genistein glucuronidation by HLM in the absence and presence of BPA are 15.1 ± 7.9 μM and 21.5 ± 7.7 μM, respectively, resulting in a K(i) value of 58.7 μM for BPA. Significantly reduced V(max) and unchanged K(m) in the presence of BPA in HLM are suggestive of noncompetitive inhibition. In RLM, the presence of BPA resulted in a K(i) of 35.7 μM, an insignificant change in V(max) (2.91 ± 0.26 nmol · min(-1) · mg(-1) and 3.05 ± 0.41 nmol · min(-1) · mg(-1) in the absence and presence of BPA, respectively), and an increase in apparent K(m) (49.4 ± 14 μM with no BPA and 84.0 ± 28 μM with BPA), indicative of competitive inhibition. These findings are significant because they suggest that BPA is capable of inhibiting the glucuronidation of genistein in vitro, and that the type of inhibition is different between HLM and RLM.

  9. Stereoselective Degradation of alpha-Cypermethrin and Its Enantiomers in Rat Liver Microsomes.

    Science.gov (United States)

    Yan, Jin; Zhang, Ping; Wang, Xinru; Xu, Meiqi; Wang, Yao; Zhou, Zhiqiang; Zhu, Wentao

    2016-01-01

    Alpha-cypermethrin (α-CP), [(RS)-a-cyano-3-phenoxy benzyl (1RS)-cis-3-(2, 2-dichlorovinyl)-2, 2-dimethylcyclopropanecarboxylate], comprises a diastereoisomer pair of cypermethrin, which are (+)-(1R-cis-αS)-CP (insecticidal) and (-)-(1S-cis-αR)-CP (inactive). In this experiment, the stereoselective degradation of α-CP was investigated in rat liver microsomes by high-performance liquid chromatography (HPLC) with a cellulose-tris- (3, 5-dimethylphenylcarbamate)-based chiral stationary phase. The results revealed that the degradation of (-)-(1S-cis-αR)-CP was much faster than (+)-(1R-cis-αS)-CP both in enantiomer monomers and rac-α-CP. As for the enzyme kinetic parameters, there were some variances between rac-α-CP and the enantiomer monomers. In rac-α-CP, the Vmax and CLint of (+)-(1R-cis-αS)-CP (5105.22 ± 326.26 nM/min/mg protein and 189.64 mL/min/mg protein) were about one-half of those of (-)-(1S-cis-αR)-CP (9308.57 ± 772.24 nM/min/mg protein and 352.19 mL/min/mg protein), while the Km of the two α-CP enantiomers were similar. However, in the enantiomer monomers of α-CP, the Vmax and Km of (+)-(1R-cis-αS) -CP were 2-fold and 5-fold of (-)-(1S-cis-αR)-CP, respectively, which showed a significant difference with rac-α-CP. The CLint of (+)-(1R-cis-αS)-CP (140.97 mL/min/mg protein) was still about one-half of (-)-(1S-cis-αR)-CP (325.72 mL/min/mg protein) in enantiomer monomers. The interaction of enantiomers of α-CP in rat liver microsomes was researched and the results showed that there were different interactions between the IC50 of (-)- to (+)-(1R-cis-αS)-CP and (+)- to (-)-(1S-cis-αR)-CP(IC50(-)/(+) / IC50(+)/(-)  = 0.61).

  10. SUPRESSION OF MICROSOMAL OXIDATION WEAKENS HISTOCHROME’S DIURETIC EFFECT AT RATS

    Directory of Open Access Journals (Sweden)

    O. S. Talalaeva

    2013-01-01

    Full Text Available Histochrome is the medicinal form of echinochrome (2, 3, 5, 6, 8-pentahydroxy-7-ethyl-1,4-naphthoquinone. Arisen during clinical application of the drug questions concerning its biotransformation have predetermined the aim of this research: to study participation liver monooxygenase system in maintenance of histochrome’s pharmacological activity.Simple and informative method of the lifetime control of liver monooxygenase systems influence on a metabolism of a medical product is the estimation of changes of pharmacological effect of a r esearched preparation on a background microsomal oxidations i nhibitor. In experiments on rats chloramphenicol action on diuretic effect of histochrome, as the most convenient for screening, was i nvestigated.To control group of animals during 10 days were hypodermically entered by histochrome in a doze of 10 mg/kg (n = 15. Experimental animals preliminary oral received 50 mg/kg of chloramphenicol before three hours of histochrome introduction (n = 16. In both groups of animals measured volume daily excretion of water, creathinin, sodium and potassium ions excretions in experimental rats each two days. The initial level of parameters of excretory kidneys functions were estimated before introduction of preparations at animals.Long-term histochrome’s injection was followed by a fivefold increasing of water excretion and simultaneously creathinin growth one. Allocation of ions of sodium was statistically significantly increased by 11-th day of experiment, and potassium ions – since the ninth day of histochrome injection. In conditions preliminary chloramphenicol applications volume daily daily urine output and creathinin excretion were essentially less control parameters. Allocation with urine of ions of sodium was decreased almost twice in comparison with the values, fixed at introduction histochrome. Excretion potassium ions ware corresponded to an initial level during all period of supervision.Taking into

  11. The stability studies and in vitro hepatic microsomal metabolism of some alpha-phenyl-N-substituted nitrones in rats.

    Science.gov (United States)

    Bulut, Gülen; Oktav, Mehmet; Ulgen, Mert

    2004-01-01

    Nitrones are a very important class of synthetic chemicals as synthetic intermediates, antioxidant agents, and metabolic oxidation products of secondary amines and imines used drug, food, cosmetic and printing industry. In the present study, the stability experiments and in vitro metabolism studies using rat microsomal preparations fortified with NADPH were carried out using three different alpha-phenyl-N-substituted nitrones ie alpha-phenyl-N-tert-butylnitrone (PTBN), alpha-(2,6-dichlorophenyl)-N-phenylnitrone (DCPPN) and alpha-phenyl-N-adamantanylnitrone (PADN). The separation of these compounds from the potential degradation, isomerization and metabolic products were performed using a reverse phase HPLC system with a diodearray uv detection. Following stability experiments at 37 degrees C using methanolic nitrone solutions, it was observed that PTBN produced trace amounts of benzaldehyde and the corresponding amide. DCPPN also produced trace amounts of amide. After 12 hours, the amount of the amide significantly increased. PADN produced trace amount of benzaldehyde but not any amide. The proposed compounds were incubated with rat microsomal preparations fortified with NADPH, extracted into dichloromethane (DCM) and finally evaporated under nitrogen in the dark conditions. PTBN was metabolized into corresponding amide whereas DCPPN and PADN did not. With all of the substrates, the corresponding aldehydes are observed with both test and control tubes using denaturated microsomes and without co-factors.

  12. Bilirubin diglucuronide synthesis by a UDP-glucuronic acid-dependent enzyme system in rat liver microsomes.

    Science.gov (United States)

    Blanckaert, N; Gollan, J; Schmid, R

    1979-01-01

    Incubation of rat liver homogenate or microsomal preparations with bilirubin or bilirubin monoglucuronide with (BMG) resulted in formation of bilirubin diglucuronide (BDG). Both synthesis of BMG and its conversion to BDG were critically dependent on the presence of UDP-glucuronic acid. Pretreatment of the animals with phenobarbital stimulated both reactions. When 33 microM bilirubin was incubated with microsomal preparations from phenobarbital-treated rats, 80-90% of the substrate was converted to bilirubin glucuronides; the reaction products consisted of almost equal amounts of BMG and BDG. When phenobarbital pretreatment was omitted or when the substrate concentration was increased to 164 microM bilirubin, proportionally more BMG and less BDG were formed. Homogenate and microsomes from homozygous Gunn rats neither synthesized BMG nor converted BMG to BDG. These findings in vitro suggest an explanation for the observations in vivo that, in conditions of excess bilirubin load or of genetically decreased bilirubin UDP glucuronosyltransferase (EC 2.4.1.17) activity, proportionally more BMG and less BDG are excreted in bile. PMID:109837

  13. Identification of protein complexes of microsomes in rat adipocytes by native gel coupled with LC-ESI-QTOF.

    Science.gov (United States)

    Ke, Ming; Zhang, Yongqian; Xiong, Yan; Saeed, Yasmeen; Deng, Yulin

    2016-04-01

    The study of the composition of microsome proteins/complexes/interactions in adipocytes provides useful information for researchers related to energy metabolism disorders. The native gel coupled with LC-ESI-QTOF approach was employed here for separating protein complexes. We found a series of proteins functionally clustered in biological processes of protein metabolism, cellular carbohydrate catabolism, response to stimulus and wounding, macromolecular complex subunit organization, positive regulation of molecular function, regulation of programmed cell death and biomolecule transport. According to clustering of proteins' electrophoresis profiles across native gel fractions and bioinformatics data retrieval, protein complexes/interactions involved in protein metabolism, cellular carbohydrate catabolism, macromolecular complex subunit organization and biomolecule transport were identified. Besides, the results also revealed some functional linkages, which may provide useful information for discovering previously unknown interactions. The interaction between SSAO and ALDH2 was verified by co-immunoprecipitation. The native gel combining mass spectrometry approach appeared to be a useful tool for investigating microsome proteins and complexes to complement the traditional electrophoresis approaches. The native gel strategy together with our findings should facilitate future studies of the composition of rat adipocyte microsome protein complexes under different conditions.

  14. Enzyme kinetic study of a new cardioprotective agent, KR-32570 using human liver microsomes and recombinant CYP isoforms.

    Science.gov (United States)

    Kim, Hyojin; Seo, Kyung-Ah; Kim, Hyunmi; Lee, Hye Suk; Lee, Choong-Hwan; Shin, Jae-Gook; Liu, Kwang-Hyeon

    2007-04-01

    KR-32570 (5-(2-Methoxy-5-chlorophenyl)furan-2-ylcarbonyl)guanidine) is a new cardioprotective agent for preventing ischemia-reperfusion injury. Human liver microsomal incubation of KR-32570 in the presence of NADPH resulted in the formation of two metabolites, hydroxy-KR-32570 and O-desmethyl-KR-32570. In this study, a kinetic analysis of the metabolism of two metabolites from KR-32570 was performed in human liver microsomes, and recombinant CYP1A2, and CYP3A4. The metabolism for hydroxy- and O-desmethyl-KR-32570 formation from KR-32570 by human liver microsomes was best described by a Michaelis-Menten equation and a Hill equation, respectively. The Cl(int) values of hydroxy- and O-desmethyl-KR-32570 formation were similar to each other (0.03 vs 0.04 microL/min/pmol CYP, respectively). CYP3A4 mediated the formation of hydroxy-KR-32570 from KR-32570 with Cl(int) = 0.24 microL/min/pmol CYP3A4. The intrinsic clearance for O-desmethyl-KR-32570 formation by CYP1A2 was 0.83 AL/min/pmol CYP1A2. These findings suggest that CYP3A4 and CYP1A2 enzymes are major enzymes contributing to the metabolism of KR-32570.

  15. Compartmentation of hepatic fatty-acid-binding protein in liver cells and its effect on microsomal phosphatidic acid biosynthesis.

    Science.gov (United States)

    Bordewick, U; Heese, M; Börchers, T; Robenek, H; Spener, F

    1989-03-01

    Fatty-acid-binding proteins are known to occur in the cytosol of mammalian cells and to bind fatty acids and their CoA-esters. Application of the postembedding protein A-gold labeling method with antibody against the hepatic type fatty-acid-binding protein (hFABP) to cross-sections of liver cells and a newly developed gel-chromatographic immunofluorescence assay established qualitatively (1) that hFABP in mitochondria was confined to outer mitochondrial membranes, (2) the presence of this protein in microsomes and (3) that nuclei were also filled with hFABP. Quantitative data elaborated with a non-competitive ELISA confirmed these results. A significant difference to the distribution of cardiac FABP in heart muscle cells, where this type of protein was found in cytosol, matrix and nuclei, was observed (Börchers et al. (1989) Biochim. Biophys. Acta, in the press). hFABP-containing rat liver microsomes were incubated with long-chain acyl-CoAs in the presence of hFABP (isolated from rat liver cytosol) in a study on the acylation of sn-glycerol-3-phosphate and lysophosphatidic acid. Both acyltransferases were stimulated by addition of hFABP to the incubation medium. The morphological, immunochemical as well as kinetic data infer a direct interaction of hFABP with microsomal membranes in liver cells.

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

    Science.gov (United States)

    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

  17. High resistance to lipid peroxidation of bird heart mitochondria and microsomes: Effects of mass and maximum lifespan.

    Science.gov (United States)

    Gutiérrez, A M; Reboredo, G R; Mosca, S M; Catalá, A

    2009-11-01

    The aim of this investigation was to study the connection between body size, fatty acid composition and sensitivity to lipid peroxidation of heart mitochondria and microsomes isolated from different size bird species: manon (Lonchura striata), quail (Coturnix coturnix var japonica), pigeon (Columba livia), duck (Cairina moschata) and goose (Anser anser), representing a 372-fold range of body mass. Fatty acids of total lipids were determined using gas chromatography and lipid peroxidation was evaluated with a chemiluminescence assay. The fatty acids present in heart organelles of the different bird species analyzed showed a small number of significant allometric trends. In mitochondria, from the individual fatty acid data, palmitoleic acid (C16:1 n7) increased allometrically (r=0.878), while stearic acid (C18:0) was negatively related to body mass (r=-0.903). Interestingly, none of the calculated fatty acid variables, the average fatty acid saturated, monounsaturated, polyunsaturated (PUFA) and the unsaturation index (UI) was established to show significant body size-related variations. In heart microsomes, the content of C18:0 was significantly smaller (r=-0.970) in the birds of greater size. A significant allometric increase in linoleic acid (C18:2 n6) (r=0.986), polyunsaturated (r=0.990) and UI (r=0.904) was observed in the larger birds. The total n6 fatty acids of heart mitochondria did not show significant differences when it was correlated to body mass of the birds. Moreover, positive allometric relationships were shown for microsomes. The total n3 fatty acids of heart mitochondria and microsomes indicated no significant correlations to body mass of birds. The C16:1 n7, C18:0 in mitochondria and C18:0, C18:2 n6, PUFA, UI and PUFA n6 in microsomes showed significant differences when they were correlated to maximum life span (MLSP) of birds. As light emission=chemiluminescence originated from heart organelles was not statistically significant, a lack of

  18. Antioxidant Capacity of Flavonoids in Hepatic Microsomes Is not Reflected by Antioxidant Effects In Vivo

    Directory of Open Access Journals (Sweden)

    Garry Duthie

    2012-01-01

    Full Text Available Flavonoids are polyphenolic compounds with potential antioxidant activity via multiple reduction capacities. Oxidation of cellular lipids has been implicated in many diseases. Consequently, this study has assessed the ability of several dietary flavonoid aglycones to suppress lipid peroxidation of hepatic microsomes derived from rats deficient in the major lipid soluble antioxidant, dα-tocopherol. Antioxidant effectiveness was galangin > quercetin > kaempferol > fisetin > myricetin > morin > catechin > apigenin. However, none of the flavonoids were as effective as dα-tocopherol, particularly at the lowest concentrations used. In addition, there appears to be an important distinction between the in vitro antioxidant effectiveness of flavonoids and their ability to suppress indices of oxidation in vivo. Compared with dα-tocopherol, repletion of vitamin E deficient rats with quercetin, kaempferol, or myricetin did not significantly affect indices of lipid peroxidation and tissue damage. Direct antioxidant effect of flavonoids in vivo was not apparent probably due to low bioavailability although indirect redox effects through stimulation of the antioxidant response element cannot be excluded.

  19. Microsomal PGE2 synthase-1 regulates melanoma cell survival and associates with melanoma disease progression.

    Science.gov (United States)

    Kim, Sun-Hee; Hashimoto, Yuuri; Cho, Sung-Nam; Roszik, Jason; Milton, Denái R; Dal, Fulya; Kim, Sangwon F; Menter, David G; Yang, Peiying; Ekmekcioglu, Suhendan; Grimm, Elizabeth A

    2016-05-01

    COX-2 and its product PGE2 enhance carcinogenesis and tumor progression, which has been previously reported in melanoma. As most COX inhibitors cause much toxicity, the downstream microsomal PGE2 synthase-1 (mPGES1) is a consideration for targeting. Human melanoma TMAs were employed for testing mPGES1 protein staining intensity and percentage levels, and both increased with clinical stage; employing a different Stage III TMA, mPGES1 intensity (not percentage) associated with reduced patient survival. Our results further show that iNOS was also highly expressed in melanoma tissues with high mPGES1 levels, and iNOS-mediated NO promoted mPGES1 expression and PGE2 production. An mPGES1-specific inhibitor (CAY10526) as well as siRNA attenuated cell survival and increased apoptosis. CAY10526 significantly suppressed tumor growth and increased apoptosis in melanoma xenografts. Our findings support the value of a prognostic and predictive role for mPGES1, and suggest targeting this molecule in the PGE2 pathway as another avenue toward improving melanoma therapy.

  20. [Liver diseases with microsomal antibodies. Frequency, clinical and immunological findings and course observations (author's transl)].

    Science.gov (United States)

    Schmidt, G; Kienle, J; Schmidt, D S; Wolf, H

    1978-08-01

    Microsomal antibodies (LKM-antibodies) differ in the indirect immunfluorescence by use of rat organ sections (kidney, stomach, and liver) from mitochondrial antibodies by there reaction with proximal renal tubules and hepatocytes while parietal cells usually fail to stain. In contrast to other humoral autoantibodies LKM-antibodies are rare. In a prospective study on 930 adults and 33 children with two third chronic hepatitis resp. cirrhosis frequency in the first group amounts to 0.38% and in the latter to 6.9% of all liver diseases. Among six patients with LKW-antibodies and chronic active hepatitis there were three children aged 4-13 and three adults between 45-55 years. On common results two children showed beside LKM-antibodies by absence of other immunphenomenons a rise of gamma-globulins in the electrophoresis and immunglobulin G as well as a chronic active hepatitis with necrosis leading in spite of initial rapid progression and persistent antibodies into inactive postnecrotiv cirrhosis. The other four patients showed no common course in respect to clinical, histological and immunological findings.

  1. Metabolism and toxicity of styrene in microsomal epoxide hydrolase-deficient mice.

    Science.gov (United States)

    Carlson, Gary P

    2010-01-01

    Styrene, which is widely used in manufacturing, is both acutely and chronically toxic to mice. Styrene is metabolized by cytochromes P-450 to the toxic metabolite styrene oxide, which is detoxified via hydrolysis with microsomal epoxide hydrolase (mEH) playing a major role. The purpose of these studies was to characterize the importance of this pathway by determining the hepatotoxicity and pneumotoxicity of styrene in wild-type and mEH-deficient (mEH(-/-)) mice. While the mEH(-/-) mice metabolized styrene to styrene oxide at the same rate as the wild-type mice, as expected there was minimal metabolism of styrene oxide to glycol. mEH(-/-) mice were more susceptible to the lethal effects of styrene. Twenty-four hours following the administration of 200 mg/kg ip styrene, mice demonstrated a greater hepatotoxic response due to styrene, as measured by increased serum sorbitol dehydrogenase activity and greater pneumotoxicity as shown by increased protein levels, cell numbers, and lactate dehydrogenase activity in bronchioalveolar lavage fluid. mEH(-/-) mice were also more susceptible to styrene-induced oxidative stress, as indicated by greater decreases in hepatic glutathione levels 3 h after styrene. Styrene oxide at a dose of 150 mg/kg did not produce hepatotoxicity in either wild-type or mEH(-/-) mice. However, styrene oxide produced pneumotoxicity that was similar in the two strains. Thus, mEH plays an important role in the detoxification of styrene but not for exogenously administered styrene oxide.

  2. Effect of regular organic solvents on cytochrome P450-mediated metabolic activities in rat liver microsomes.

    Science.gov (United States)

    Li, Dan; Han, Yonglong; Meng, Xiangle; Sun, Xipeng; Yu, Qi; Li, Yan; Wan, Lili; Huo, Yan; Guo, Cheng

    2010-11-01

    The effects of regular organic solvents on the metabolic activities of various human cytochromes P450 (P450s) have been reported. However, very little is known about their influence on metabolic activities mediated by P450s in the rat liver microsomes (RLM). The purpose of this study was to investigate the effects of organic solvents such as methanol, acetonitrile, dimethyl sulfoxide (DMSO), acetone, and ethanol on CYP1A, CYP2C, CYP2D, CYP2E, and CYP3A-mediated metabolism using RLM. The results showed that the activities of most rat P450 enzymes appeared to be organic solvent-dependent, and the metabolism of the tested probes were remarkably reduced when the concentration of organic solvents was up to 5% v/v, whereas most organic solvents demonstrated no significant interference when the concentration was below 1%, with the exception of DMSO. In addition, organic solvents exhibited different inhibitory effects, for example, CYP2D and CYP2E showed a significant reduction of activities at lower concentrations of organic solvents. Hence, this phenomenon should be taken into consideration when designing in vitro metabolism studies of new chemical entities. Therefore, we recommend acetonitrile as the most suitable solvent for RLM incubations, and the content of organic solvent should be kept lower than 1% v/v.

  3. Biotransformation of isoimperatorin by rat liver microsomes and its quantification by LC-MS/MS method.

    Science.gov (United States)

    Chen, Tian-Li; Zhang, You-Bo; Xu, Wei; Kang, Ting-Guo; Yang, Xiu-Wei

    2014-03-01

    The aim of the present research was to establish a comprehensive strategy to identify the metabolites of isoimperatorin after biotransformation with rat liver microsomes in vitro, and further describe metabolic kinetic characteristics of isoimperatorin and its main metabolites. Utilizing liquid chromatography with time of flight mass spectrometry (LC-TOF-MS), 18 metabolites (M 1-18) were characterized according to the typical fragment ions and literature data. Among them, M-2, 3, 5, 9, 10, and 15 were new compounds. To further verify structures of the metabolites, five main metabolites were obtained from the magnifying biotransformation incubation system, and their chemical structures were elucidated as 8-hydroxyoxypeucedanin (M-3), hydroxypeucedanin hydrate (M-4), E-5-(4-hydroxy-3-methyl-2-alkenyloxy)-psoralen (M-11), Z-5-(4-hydroxy-3-methyl-2-alkenyloxy)-psoralen (M-12), and oxypeucedanin (M-16) by various spectroscopy methods including IR, MS and NMR. A simple new liquid chromatography with triple quadrupole tandem mass spectrometry (LC-QqQ-MS) method was developed for the simultaneous determination of isoimperatorin and its main metabolites. The analysis was performed on a Diamonsil™ ODS C18 column with acetonitrile-water containing 0.1% formic acid as mobile phase. Total run time was 20.0 min. The results suggested that the method we exhibited was successfully applied for analysis of isoimperatorin and its metabolites. The study provides essential data for proposing metabolite pathway and further pharmacological study of isoimperatorin.

  4. Inhibition of cytochrome P450 enzymes by rhein in rat liver microsomes.

    Science.gov (United States)

    Tang, Jing-cheng; Yang, Hua; Song, Xue-ying; Song, Xiao-hong; Yan, Shu-lian; Shao, Jian-qun; Zhang, Tian-lan; Zhang, Jin-nan

    2009-02-01

    Rhein, an active ingredient extensively found in plants such as Aloe, Cassitora L., rhubarb and so on, has been used for a long time in China. Pharmacological tests revealed that rhein not only had a strong antibacterial action, but also may be useful in cancer chemotherapy as a biochemical modulator. Its therapeutic action and toxicity is still the subject of considerable research. With microsome incubation assays in vitro and HPLC methods, the inhibition of rat liver CYP1A2, CYP2C9, CYP2D6, CYP2E1 and CYP3A enzymes by rhein were studied kinetically. The results showed the most inhibition of CYP2E1 by rhein (K(i) = 10 microm, mixed); CYP3A and CYP2C9 were also inhibited by rhein, K(i) = 30 microm (mixed) and K(i) = 38 microm (mixed), respectively; rhein revealed some inhibition of CYP1A2 (K(i) = 62 microm, uncompetitive) and CYP2D6 (K(i) = 74 microm, mixed). Drug-drug interactions, especially cytochrome P450 (CYP)-mediated interactions, cause an enhancement or attenuation in the efficacy of co-administered drugs. Inhibition of the five major CYP enzymes observed for rhein suggested that changes in pharmacokinetics of co-administered drugs were likely to occur. Therefore, caution should be paid to the possible drug interaction of medicinal plants containing rhein and CYP substrates.

  5. Expression of rapeseed microsomal lysophosphatidic acid acyltransferase isozymes enhances seed oil content in Arabidopsis.

    Science.gov (United States)

    Maisonneuve, Sylvie; Bessoule, Jean-Jacques; Lessire, René; Delseny, Michel; Roscoe, Thomas J

    2010-02-01

    In higher plants, lysophosphatidic acid acyltransferase (LPAAT), located in the cytoplasmic endomembrane compartment, plays an essential role in the synthesis of phosphatidic acid, a key intermediate in the biosynthesis of membrane phospholipids in all tissues and storage lipids in developing seeds. In order to assess the contribution of LPAATs to the synthesis of storage lipids, we have characterized two microsomal LPAAT isozymes, the products of homoeologous genes that are expressed in rapeseed (Brassica napus). DNA sequence homologies, complementation of a bacterial LPAAT-deficient mutant, and enzymatic properties confirmed that each of two cDNAs isolated from a Brassica napus immature embryo library encoded a functional LPAAT possessing the properties of a eukaryotic pathway enzyme. Analyses in planta revealed differences in the expression of the two genes, one of which was detected in all rapeseed tissues and during silique and seed development, whereas the expression of the second gene was restricted predominantly to siliques and developing seeds. Expression of each rapeseed LPAAT isozyme in Arabidopsis (Arabidopsis thaliana) resulted in the production of seeds characterized by a greater lipid content and seed mass. These results support the hypothesis that increasing the expression of glycerolipid acyltransferases in seeds leads to a greater flux of intermediates through the Kennedy pathway and results in enhanced triacylglycerol accumulation.

  6. Mutagenicity of products from coal gasification and liquefaction in the Salmonella/microsome assay.

    Science.gov (United States)

    Schoeny, R; Warshawsky, D; Hollingsworth, L; Hund, M; Moore, G

    1981-01-01

    As a first step in the assessment of their possible bio-effects, coal-related materials were tested for mutagenicity in the Salmonella/microsome assay. Of three coal gasification by-products tested, only a tar was mutagenic for any of four Salmonella strains. The following liquefaction materials were mutagenic for strains TA1538, TA98, and/or TA100: A liquefaction vehicle oil and coal hydrogenation filtered liquid, separated bottoms, vacuum overhead, and vacuum bottoms. Neither powdered coal nor water produced as a by-product of the hydrogenation process was positive in the Salmonella test. No coal-related material was mutagenic for the missense mutant TA1535 or for any strain in the absence of metabolic activation provided by rat hepatic homogenates (S9). In all but one instance Aroclor 1254-induced S9 provided the maximum activation for mutagenesis. Fractionation of all samples was undertaken by serial extraction with organic solvents of increasing polarity (hexane, toluene, methylene chloride, acetonitrile). Highly mutagenic materials were found in fractions of the hydrogenation filtered liquid, vacuum overhead, and vacuum bottoms. Thus far non-mutagenic samples have not yielded mutagenic components upon fractionation.

  7. Evaluation of extracts from Coccoloba mollis using the Salmonella/microsome system and in vivo tests

    Directory of Open Access Journals (Sweden)

    Marcela Stefanini Tsuboy

    2010-01-01

    Full Text Available The common everyday use of medicinal plants is an ancient, and still very widespread practice, whereby the need for studies on their possible toxicity and mutagenic properties. The species Coccoloba mollis has been much used in phytotherapy, mainly in cases involving loss of memory and stress. In order to investigate its genotoxic and mutagenic potential, ethanolic extracts from the leaves and roots underwent Salmonella/microsome assaying (TA98 and TA100 strains, with and without exogenous metabolism - S9, besides comet and micronucleus tests in vivo.There was no significant increase in the number of revertants/plate of Salmonella strains in any of the analyzed root-extract concentrations, although the extract itself was extremely toxic to the Salmonella TA98 strain in the tests carried out with S9 (doses varying from 0.005 to 0.5 µg/plate. On the other hand, the leaf-extract induced mutations in the TA98 strain in the absence of S9 in the highest concentration evaluated, although at very low mutagenic potency (0.004 rev/µg. Furthermore, there was no statistically significant increase in the number of comets and micronuclei, in treatments involving Swiss mice. It was obvious that extracts of Coccoloba mollis, under the described experimental conditions, are not mutagenic.

  8. Regioselective glucuronidation of oxyresveratrol, a natural hydroxystilbene, by human liver and intestinal microsomes and recombinant UGTs.

    Science.gov (United States)

    Hu, Nan; Mei, Mei; Ruan, Jianqing; Wu, Wenjin; Wang, Yitao; Yan, Ru

    2014-01-01

    Oxyresveratrol (OXY) is a natural hydroxystilbene that shows similar bioactivity but better water solubility than resveratrol. This study aims to characterize its glucuronidation kinetics in human liver (HLMs) and intestinal (HIMs) microsomes and identify the main UDP-glucuronosyltransferase (UGT) isoforms involved. Three and four mono-glucuronides of OXY were generated in HIMs and HLMs, respectively, with oxyresveratrol-2-O-β-D-glucuronosyl (G4) as the major metabolite in both organs. The kinetics of G4 formation fit a sigmoidal model in HLMs and biphasic kinetics in HIMs. Multiple UGT isoforms catalyzed G4 formation with the highest activity observed with UGT1A9 followed by UGT1A1. G4 formation by both isoforms followed substrate inhibition kinetics. Propofol (UGT1A9 inhibitor) effectively blocked G4 generation in HLMs (IC50 63.7 ± 11.6 µM), whereas the UGT1A1 inhibitor bilirubin only produced partial inhibition in HLMs and HIMs. These findings shed light on the metabolic mechanism of OXY and arouse awareness of drug interactions.

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

    Science.gov (United States)

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

    2015-01-01

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

  10. Partial purification of the microsomal rat liver iodothyronine deiodinase. II. Affinity chromatography.

    Science.gov (United States)

    Mol, J A; van den Berg, T P; Visser, T J

    1988-02-01

    Iodothyronine deiodinase has been solubilized and purified approximately 2400 times from liver microsomal fractions of male Wistar rats pretreated with thyroxine. The deiodinase was solubilized with 1% cholate, and stripped of adhering phospholipids by ammonium sulfate precipitation followed by solubilization with the non-ionic detergent Emulgen 911. The enzyme was further purified by successive ion-exchange chromatography on DEAE-Sephacel and Cellex-P and affinity chromatography on 3,3',5-triiodothyronine-Sepharose. Finally, the deiodinase was reacted with 6-propionyl-2-thiouracil-Sepharose, a derivative of the mechanism-based inhibitor 6-propyl-2-thiouracil. Covalent binding was observed only in the presence of substrate in agreement with the proposed mechanism of deiodination. The deiodinase was eluted from the affinity column by reduction of the enzyme-propylthiouracil mixed disulfide with 50 mM dithiothreitol. The enzyme was approximately 50% pure as judged by SDS-PAGE, exhibiting a subunit molecular weight of 25,000. This preparation was equally enriched in outer ring and inner ring deiodinase activities in keeping with the view that both are intrinsic to a single, type I deiodinase.

  11. Hepatic microsomal cytochromes P450 in mink fed Saginaw Bay carp (SBC)

    Science.gov (United States)

    Melancon, M.J.; LeCaptain, L.; Rattner, B.A.; Heaton, S.; Aulerich, R.; Tillitt, D.; Stegeman, John J.; Woodin, B.

    1992-01-01

    Livers from mink fed diets containing 0% (n = 12), 10% (n = 11), 20% (n = 12) and 40% (n = 10) SBC for 6 months contained 0.1, 2.2, 3.6, and 6.3 ug/g total PCBs, respectively. Hepatic microsomes were prepared and assayed for protein, arylhydrocarbon hydroxylase (AHH), benzyloxyresorufin-O-dealkylase (BROD), ethoxy-ROD (ER0D), pentoxy-ROD (PROD), and ethoxycoumarin-OD (ECOD). Mink fed SBC had increased AHH, EROD, and ECOD (group means 2.2-3.4 X control means), decreased BROD and unchanged PROD (the latter 2 assays indicators for phenobarbital-type induction in mammals). Three samples from each group were examined by western blot using a polyclonal anti-P450llB antibody and a monoclonal anti-P450lA antibody (MAb 1-12-3). Mink fed SBC showed induction of a protein recognized by anti-P450lA (8 X control), but had little protein recognized by anti-P450IlB. The monooxygenase activities and western blot data give a consistent picture of MC-type but not PB-type induction in mink fed SBC.

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

    Directory of Open Access Journals (Sweden)

    Sun Joo Kim

    2016-01-01

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

  13. The effects of carbon tetrachloride on rat liver microsomes during the first hour of poisoning in vivo, and the modifying actions of promethazine

    Science.gov (United States)

    Slater, T. F.; Sawyer, B. C.

    1969-01-01

    The effects of an oral administration of carbon tetrachloride on various liver microsomal and supernatant components were studied 1hr. and 2hr. after dosing. The modifications of such early changes resulting from a concomitant administration of promethazine together with the carbon tetrachloride were also investigated. The microsomal components studied were: cytochromes P-450 and b5; inorganic pyrophosphatase; NADH– and NADPH–cytochrome c reductases; NADH– and NADPH–neotetrazolium reductases; a lipid-peroxidation system associated with the oxidation of NADPH and stimulated by ADP and Fe2+. NAD– and NADP– DT-diaphorases were measured in the supernatant solution remaining after isolation of liver microsomes, and the distribution of RNA phosphorus between the microsomes and supernatant solution was also determined. Carbon tetrachloride produced a rapid fall in inorganic pyrophosphatase activity, a rather slower decrease in cytochrome P-450 content of the microsomes and small increases in the activities of NADH–cytochrome c reductase and neotetrazolium reductases. The activities of NADPH–cytochrome c reductase, the NADPH–ADP/Fe2+-linked lipid-peroxidation system, DT-diaphorases and the content of cytochrome b5 in the microsomes were unchanged. There was also a loss of RNA phosphorus from the microsomes into the supernatant solution. The RNA phosphorus redistribution, the decrease in inorganic pyrophosphatase and the increases in neotetrazolium reductase activities were at least partially prevented by a concomitant dosing with promethazine. However, the decrease in cytochrome P-450 was not affected by promethazine treatment. These early changes are discussed in terms of the liver necrosis produced by carbon tetrachloride and which is greatly retarded in its onset by the administration of promethazine. PMID:5767054

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

    Science.gov (United States)

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

    2010-06-01

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

  15. 3,4,5-Trimethoxyphenylacetaldehyde, an intermediate metabolite of mescaline, is a substrate for microsomal aldehyde oxygenase in the mouse liver.

    Science.gov (United States)

    Watanabe, K; Kayano, Y; Matsunaga, T; Yamamoto, I; Yoshimura, H

    1995-05-01

    3,4,5-Trimethoxyphenylacetaldehyde, an intermediate metabolite of mescaline, was oxidized to 3,4,5-trimethoxyphenylacetic acid by mouse hepatic microsomes. The reaction was NADPH-dependent, and inhibited by SKF 525-A, metyrapone and disulfiram. A P450 isozyme in mouse hepatic microsomes, P450 MUT-2 (CYP2C29), catalyzed the reaction (0.96 nmol/min/nmol P450) in which NADPH and NADPH-cytochrome c reductase were essential for the catalytic activity. The reaction was confirmed to be an oxygenation since molecular oxygen was incorporated into the carboxylic acid metabolite formed under oxygen-18 gas by GC-MS analysis. By addition of antibody against CYP2C29 to the microsomes (3.2 mg/mg microsomal protein) the MALDO activity was inhibited by 35% of the control value with preimmune serum, suggesting that CYP2C29 or an immunologically-related isozyme(s) plays a major role in the NADPH-dependent oxidation of 3,4,5-trimethoxyphenylacetaldehyde to 3,4,5-trimethoxyphenylacetic acid by mouse hepatic microsomes. Pharmacological experiments on mescaline and its deaminated metabolites using mice indicated that the metabolites were much less active or were inactive in cataleptogenic effect and pentobarbital-induced sleep prolongation as compared with the parent compound.

  16. Overt and latent activities of diacylglycerol acytransferase in rat liver microsomes: possible roles in very-low-density lipoprotein triacylglycerol secretion.

    Science.gov (United States)

    Owen, M R; Corstorphine, C C; Zammit, V A

    1997-01-01

    The possibility that triacylglycerol (TAG) synthesis occurs on both aspects of the endoplasmic-reticular membrane during the process of incorporation of TAG into secreted very-low-density lipoprotein (VLDL) [Zammit (1996) Biochem. J. 314, 1-14] was investigated by measuring the latency of diacylglycerol acyltransferase (DGAT) in microsomal fractions obtained from rat liver homogenates. Permeabilization of microsomes with taurocholate resulted in the doubling of the activity, indicating that DGAT activities of approximately equal magnitude occur on either aspect of the microsomal membrane. The taurocholate concentrations required for exposure of the latent activity of DGAT were identical with those that resulted in the exposure of marker enzymes for the lumen of the endoplasmic reticulum. Fractionation of the microsomes into smooth and rough populations indicated that the distribution of overt and latent DGAT activities was the same throughout. The possibility that taurocholate effects may result from non-specific activation of the overt enzyme was excluded by employing the channel-forming peptide alamethicin to effect permeabilization, and by varying the mode of delivery of diacylglycerol substrate to the microsomal membranes. Permeabilization using alamethicin gave a slightly higher latent/overt ratio for DGAT. The possible roles of overt and latent DGAT activities in the synthesis and secretion of TAG by the liver are discussed. PMID:9173878

  17. Evidence for the activation of organophosphate pesticides by cytochromes P450 3A4 and 2D6 in human liver microsomes.

    Science.gov (United States)

    Sams, C; Mason, H J; Rawbone, R

    2000-08-16

    The role of specific cytochrome P450 isoforms in catalysing the oxidative biotransformation of the organophosphorothioate pesticides parathion, chlorpyrifos and diazinon into structures that inhibit cholinesterase has been investigated in human liver microsomes using chemical inhibitors. Pesticides were incubated with human liver microsomes and production of the anticholinergic oxon metabolite was investigated by the inhibition of human serum cholinesterase. Quinidine and ketoconazole at 10 micromol/l inhibited oxidative biotransformation. Compared to control incubations (no inhibitor) where cholinesterase activity was inhibited to between 1 and 4% of control levels, incorporation of the CYP2D6 inhibitor quinidine into the microsomal incubation resulted in cholinesterase activity of 50% for parathion, 38% for diazinon and 30% for chlorpyrifos. Addition of the CYP3A4 inhibitor ketoconazole to microsomal incubations resulted in 66% cholinesterase activity with diazinon, 20% with parathion and 5% with chlorpyrifos. The unexpected finding that CYP2D6, as well as CYP3A4, catalysed oxidative biotransformation was confirmed for chlorpyrifos and parathion using microsomes prepared from a human lymphoblastoid cell line expressing CYP2D6. While parathion has been investigated only as a model compound, chlorpyrifos and diazinon are both very important, widely used pesticides and CYP2D6 appears to be an important enzyme in their bioactivation pathway. CYP2D6 is polymorphic and hence may influence individual susceptibility to exposure to chlorpyrifos and diazinon as well as other structurally similar pesticides.

  18. Characterization of metabolites of meisoindigo in male and female rat kidney microsomes by high-performance liquid chromatography coupled with positive electrospray ionization tandem mass spectrometry.

    Science.gov (United States)

    Huang, Meng; Choo, Lip-Wee; Ho, Paul C

    2008-12-01

    Meisoindigo has been effectively applied for the treatment of chronic myelogenous leukemia (CML). Although the metabolic profile of meisoindigo has been studied in liver, information relevant to extrahepatic metabolism of meisoindigo is absent in kidney so far. In this study, the metabolism of meisoindigo in rat kidney microsomes was qualitatively and quantitatively investigated by liquid chromatography/tandem mass spectrometry (LC/MS/MS), in terms of metabolite identification, metabolic stability, metabolite formation and gender effect. The metabolic profiling was accomplished by integration of multiple reaction monitoring (MRM) with conventional full MS scan followed by MS/MS methodology. The major in vitro metabolites of meisoindigo in rat kidney microsomes were identified as stereoselective 3,3' double-bond reduced meisoindigo, whereas the minor metabolites were regioselective phenyl monohydroxylmeisoindigo. An LC/MS/MS method for quantification of meisoindigo in rat kidney microsomes was also developed and validated. The calculated in vitro half-life (t(1/2)) values of meisoindigo in male and female rat kidney microsomes were 107.8 +/- 17.0 min and 130.0 +/- 12.9 min, respectively. There were no statistically significant differences between different genders in the metabolic stability profiles of meisoindigo. The reductive metabolite-formation profiles of meisoindigo in male and female rat kidney microsomes were plotted semi-quantitatively as well. The information regarding in vitro renal metabolism of meisoindigo provided a better understanding of the role of the kidney in the disposition of meisoindigo.

  19. Bioactivation of coumarin in rat olfactory mucosal microsomes: Detection of protein covalent binding and identification of reactive intermediates through analysis of glutathione adducts.

    Science.gov (United States)

    Zhuo, Xiaoliang; Zhao, Weiping; Zheng, Joanna; Humphreys, W Griffith; Shu, Yue-Zhong; Zhu, Mingshe

    2009-10-07

    The presence of high levels, as well as tissue-specific forms, of cytochrome P450 enzymes in mammalian olfactory mucosa (OM) has important implications in the bioactivation and toxicity of xenobiotics entering the tissue. Previous studies have shown that coumarin, a known olfactory toxicant in rats, is bioactivated by OM microsomal P450s to a number of products, presumably via coumarin-3,4-epoxide and other epoxide intermediates. The aim of the current study was to obtain direct evidence for the formation of such reactive intermediates in rat OM through the detection of protein covalent binding and glutathione (GSH) adduct formation. Protein covalent binding experiments with [(14)C]coumarin (10microM) displayed a 7-9-fold higher NADPH-dependent radioactivity binding in rat OM microsomes (2.5nmol/mg/30min) compared to those in rat and human liver microsomes; the binding value in rat OM microsomes was substantially but not completely reduced by the addition of GSH (5mM). LC/MS analyses detected a number of GSH adducts in GSH-supplemented coumarin metabolism reaction in rat OM microsomes; 3-glutathionyl coumarin was found to be the major one, indicating 3,4-epoxidation as the main bioactivation pathway. Additional GSH adducts were identified, presumably forming via the same pathway or epoxidation on the benzene moiety. Our findings provide direct evidence for the formation of multiple coumarin reactive intermediates in rat OM, leading to protein covalent binding and GSH conjugation.

  20. Evaluation of cytochrome P450 activity in vitro, using dermal and hepatic microsomes from four species and two keratinocyte cell lines in culture.

    Science.gov (United States)

    Rolsted, Kamilla; Kissmeyer, Anne-Marie; Rist, Gerda Marie; Hansen, Steen Honoré

    2008-01-01

    The Cytochrome P450 (CYP450) enzymes are expressed in the skin, and despite a low activity, as compared to the hepatic counterpart, a role during transdermal delivery of a drug cannot be excluded. Additionally, the enzymes may play a role in local toxicity, and further knowledge of dermal CYP450 activity can contribute to elucidate this issue. To achieve this, a cocktail of six selective CYP450 probe substrates were incubated with dermal and hepatic microsomes isolated from mouse, rat, minipig and man. Different species were used to evaluate if a reliable substitute for human tissue was possible. Further, the hepatic microsomes were included in this study, to estimate if the hepatic CYP450 activity is predictive of dermal CYP450 activity. The CYP450 activity was determined in two keratinocyte cell lines as well, as this in vitro model is desirable due to the ease in handling, among other factors. Overall, the metabolism found in the dermal microsomes was very low, and major differences were observed between species. When comparing the activities in dermal and hepatic microsomes, the qualitative pattern was to some extent similar within species, but also a number of differences were observed. The CYP450 metabolic activity in the two keratinocyte cell lines was not comparable to metabolism in the human dermal microsomes.

  1. Acylation of lysophosphatidylcholine and glycerolphosphate and fatty acid pattern in phosphatidylcholine and -ethanolamine in microsomes of normal and dystrophic human muscle.

    Science.gov (United States)

    Kunze, D; Rüstow, B; Kuksis, A; Myher, J J

    1986-02-01

    Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were isolated from microsomes obtained from normal and dystrophic human muscle and the fatty acid (FA) pattern estimated by GLC. In PC a decrease of the fatty acids of 16:0 and 18:2 and an increase of 18:0 and 18:1 was observed. In PE the decrease measured 18:2 and the increase 18:0 and 18:1. The acylation of lysophosphatidylcholine (LPC) and glycerol-3-phosphate (G3P) was measured in a microsomal system containing exogenously added LPC or G3P and labelled palmitic and oleic acid CoA esters. The incorporation of both labelled fatty acids in LPC-forming PC is reduced in dystrophic microsomes. On the other hand the acylation of glycerolphosphate and the formation of phosphatidic acid (PA) is greater in dystrophic microsomes when compared with normal controls. Possible correlations between the shifted FA pattern and the acylation rate by dystrophic microsomes measured in vitro in the two systems are discussed.

  2. Effects of trans n-6 fatty acids on the fatty acid profile of tissues and liver microsomal desaturation in the rat

    Directory of Open Access Journals (Sweden)

    Berdeaux, Olivier

    1996-04-01

    Full Text Available 18:2Δ 9c,12t and 18:2 Δ9t,12c are present in our diet, as result of heat treatment of vegetable oils. A nutritional study was carried out in order to obtain more precise information on the conversion of these two isomers into long chain polyunsaturated fatty acids (PUFA by rat tissues. This in vivo study performed using rat fed with small quantities of mono trans linoleic acid isomers (0.6% of total energy showed that 18:2 Δ9c,12t was converted into 20:4 Δ5c,8c,11c,14t while 18:2 Δ9t,12c was only slightly converted into 20:4 Δ5c,8c,11t,14c. Furthermore 18:2 Δ9t,12c was preferentially elongated into 20:2 Δ11t,14c. Each C20 metabolite of these mono trans 18:2 isomers was isolated as methyl ester by semi-preparative high-performance liquid chromatography (HPLC followed by silver nitrate thin layer chromatography (AgNO3-TLC.The structure of the components was identified using partial hydrazine reduction, AgNO3-TLC of the resulting monoenes and gas-liquid chromatography coupled with mass spectrometry (GC-MS of the 4,4-dimethyloxazoline (DMOX derivatives. Fourier-transform-infrared spectroscopy (GC-FTIR confirmed the frans geometry. Gas-liquid chromatography (GC analyses showed that 18:2 Δ9c,12t and 18:2 Δ9t,12c were present in different tissue lipids (liver, heart, testes, brain and adipose tissue, and without any modification in the amount of 20:4n-6. 20:4 Δ5c, 8c,11c,14t was incorporated in different rat tissues except in brain. Furthermore, its incorporation followed that of its structural analogue, 20:3n-9 in liver phospholipid classes (phosphatidylethanolamine, phosphatidylinositol and phosphatidylcholine. Finally, an in vitro study carried out with rat liver microsomes showed that dietary trans 18:2 isomers could inhibit the Δ6- desaturation of 18:2n-6 to 18:3n-6 and the Δ5-desaturation of 20:3n-6 to 20:4n-6.

  3. Inhibitory effects of amiodarone on simvastatin metabolism in human liver microsomes

    Institute of Scientific and Technical Information of China (English)

    Chao Wan; Jiang wei Zhang; Ning Zhu; Ling Yang

    2009-01-01

    Objective To investigate the effects ofamiodarone (AMD) on simvastatin (SV) in human liver microsomes and the possible underlying mechanisms. Methods Time-, NADPH- and concentration-dependent inhibitions were tested in HLM. The logarithm of relative inhibition values was plotted versus preincubation time (0, 5, 10, 15, 20min) for a series concentration of AMD used (0, 2, 5,25, 50 μ mol/L), and the slopes determined by linear regression. These slope values represente the observed inactivation rate constants (kobs). A double-reciprocal plot was then constructed using the reciprocal of the ko~ (y-axis) and the reciprocal of the associated inhibitor concentration (x-axis) to estimate the values ofkinact and K, which were two principal kinetic constants that were specific for mechanism-based inhibition (MBI).drug-drug interactions (DDI) potential was predicted based on in vitro data and by using the in vitro-in vivo extrapolation. Results The time-, concentration- and NADPH-dependent charactga'istics confirmed that when SV was the substrate of CYP3A4, the inhibition of AMD to CYP3A4 is MBI. Kj and kinact value were calculated to be 5.1 μ mol/L and 0.018min-1 The Clint of SV was reduced 2.96-5.63 fold when it was administrated with AMD. Conclusion Based on the results, AMD would inhibit SV metabolism via the mechanism-based manner, which would lead to DDI when they are taken together. Careful clinical observation is recommended when AMD and SV have to be simultaneously prescribed.

  4. Identification of a Novel Transcript and Regulatory Mechanism for Microsomal Triglyceride Transfer Protein

    Science.gov (United States)

    Suzuki, Takashi; Brown, Judy J.; Swift, Larry L.

    2016-01-01

    Microsomal triglyceride transfer protein (MTP) is essential for the assembly of triglyceride-rich apolipoprotein B-containing lipoproteins. Previous studies in our laboratory identified a novel splice variant of MTP in mice that we named MTP-B. MTP-B has a unique first exon (1B) located 2.7 kB upstream of the first exon (1A) for canonical MTP (MTP-A). The two mature isoforms, though nearly identical in sequence and function, have different tissue expression patterns. In this study we report the identification of a second MTP splice variant (MTP-C), which contains both exons 1B and 1A. MTP-C is expressed in all the tissues we tested. In cells transfected with MTP-C, protein expression was less than 15% of that found when the cells were transfected with MTP-A or MTP-B. In silico analysis of the 5’-UTR of MTP-C revealed seven ATGs upstream of the start site for MTP-A, which is the only viable start site in frame with the main coding sequence. One of those ATGs was located in the 5’-UTR for MTP-A. We generated reporter constructs in which the 5’-UTRs of MTP-A or MTP-C were inserted between an SV40 promoter and the coding sequence of the luciferase gene and transfected these constructs into HEK 293 cells. Luciferase activity was significantly reduced by the MTP-C 5’-UTR, but not by the MTP-A 5’-UTR. We conclude that alternative splicing plays a key role in regulating MTP expression by introducing unique 5’-UTRs, which contain elements that alter translation efficiency, enabling the cell to optimize MTP levels and activity. PMID:26771188

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

    Science.gov (United States)

    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.

  6. Intestine-specific deletion of microsomal triglyceride transfer protein increases mortality in aged mice.

    Directory of Open Access Journals (Sweden)

    Zhe Liang

    Full Text Available BACKGROUND: Mice with conditional, intestine-specific deletion of microsomal triglyceride transfer protein (Mttp-IKO exhibit a complete block in chylomicron assembly together with lipid malabsorption. Young (8-10 week Mttp-IKO mice have improved survival when subjected to a murine model of Pseudomonas aeruginosa-induced sepsis. However, 80% of deaths in sepsis occur in patients over age 65. The purpose of this study was to determine whether age impacts outcome in Mttp-IKO mice subjected to sepsis. METHODS: Aged (20-24 months Mttp-IKO mice and WT mice underwent intratracheal injection with P. aeruginosa. Mice were either sacrificed 24 hours post-operatively for mechanistic studies or followed seven days for survival. RESULTS: In contrast to young septic Mttp-IKO mice, aged septic Mttp-IKO mice had a significantly higher mortality than aged septic WT mice (80% vs. 39%, p = 0.005. Aged septic Mttp-IKO mice exhibited increased gut epithelial apoptosis, increased jejunal Bax/Bcl-2 and Bax/Bcl-XL ratios yet simultaneously demonstrated increased crypt proliferation and villus length. Aged septic Mttp-IKO mice also manifested increased pulmonary myeloperoxidase levels, suggesting increased neutrophil infiltration, as well as decreased systemic TNFα compared to aged septic WT mice. CONCLUSIONS: Blocking intestinal chylomicron secretion alters mortality following sepsis in an age-dependent manner. Increases in gut apoptosis and pulmonary neutrophil infiltration, and decreased systemic TNFα represent potential mechanisms for why intestine-specific Mttp deletion is beneficial in young septic mice but harmful in aged mice as each of these parameters are altered differently in young and aged septic WT and Mttp-IKO mice.

  7. Induction profiles of P450 in rat liver microsomes by pyrazole or methylpyrazole

    Energy Technology Data Exchange (ETDEWEB)

    Krikun, G.; Cederbaum, A.I.

    1986-05-01

    Rats were injected for 2-3 days with pyrazole (P) or 4-methylpyrazole (MP) potent inhibitors of alcohol dehydrogenase. While P treatment induced a P450 isozyme with MW 52,000 as seen on SDS gels, MP induced 2 or 3 P450s. One of the P450s induced by MP appeared to be similar to the one increased by P treatment. The increase of 2-3 bands by MP correlated with a two fold increased in total P450 content. Microsomes from the P treated rats displayed increased activity (per mg protein or per nmole P450) with aniline, p-nitroanisole, dimethylnitrosamine (low Km DMN) and ethanol as substrates, but not with aminopyrine, ethoxycoumarin or DMN (high Km). A stereochemical preference for + 2-butanol over the -isomer was also observed. Kinetic experiments indicated that P treatment increased the Vmax for ethanol, aniline and + 2-butanol. These properties are similar to those found after chronic ethanol treatment. MP treatment resulted in an increased in the oxidation of all the drugs and alcohols tested, primarily due to the increase in content of P450. In analogy to results with P, MP treatment also resulted in stereochemical preference for + vs -2-butanol, and increased turnover numbers with aniline and p-nitroanisole. However in contrast to P, no increase in turnover number with ethanol, + 2-butanol or DMN (low Km) was found after MP treatment. It is probable that these divergent effects are due to the induction of several isozymes, one of which has properties similar to that induced by P. Thus, P induces a P450 similar to that induced by ethanol whereas MP induces that isozyme in addition to others.

  8. CYP-specific bioactivation of four organophosphorothioate pesticides by human liver microsomes.

    Science.gov (United States)

    Buratti, Franca M; Volpe, Maria Teresa; Meneguz, Annarita; Vittozzi, Luciano; Testai, Emanuela

    2003-02-01

    The bioactivation of azinphos-methyl (AZIN), chlorpyrifos (CPF), diazinon (DIA), and parathion (PAR), four widely used organophosphorothioate (OPT) pesticides has been investigated in human liver microsomes (HLM). In addition, the role of human cytochrome P450 (CYPs) in OPT desulfuration at pesticide levels representative of human exposure have been defined by means of correlation and immunoinhibition studies. CYP-mediated oxon formation from the four OPTs is efficiently catalyzed by HLM, although showing a high variability (>40-fold) among samples. Two distinct phases were involved in the desulfuration of AZIN, DIA, and PAR, characterized by different affinity constants (K(mapp1) = 0.13-9 microM and K(mapp2) = 5- 269 microM). Within the range of CPF concentrations tested, only the high-affinity component was evidenced (K(mapp1) = 0.27-0.94 microM). Oxon formation in phenotyped individual HLM showed a significant correlation with CYP1A2-, 3A4-, and 2B6-related activities, at different levels depending on the OPT concentration. Anti-human CYP1A2, 2B6, and 3A4 antibodies significantly inhibited oxon formation, showing the same OPT concentration dependence. Our data indicated that CYP1A2 is mainly involved in OPT desulfuration at low pesticide concentrations, while the role of CYP3A4 is more significant to the low-affinity component of OPT bioactivation. The contribution of CYP2B6 to total hepatic oxon formation was relevant in a wide range of pesticide concentrations, being a very efficient catalyst of both the high- and low-affinity phase. These results suggest CYP1A2 and 2B6 as possible metabolic biomarkers of susceptibility to OPT toxic effect at the actual human exposure levels.

  9. Male mice deficient in microsomal epoxide hydrolase are not susceptible to benzene-induced toxicity.

    Science.gov (United States)

    Bauer, Alison K; Faiola, Brenda; Abernethy, Diane J; Marchan, Rosemarie; Pluta, Linda J; Wong, Victoria A; Gonzalez, Frank J; Butterworth, Byron E; Borghoff, Susan J; Everitt, Jeffrey I; Recio, Leslie

    2003-04-01

    Enzymes involved in benzene metabolism are likely genetic determinants of benzene-induced toxicity. Polymorphisms in human microsomal epoxide hydrolase (mEH) are associated with an increased risk of developing leukemia, specifically those associated with benzene. This study was designed to investigate the importance of mEH in benzene-induced toxicity. Male and female mEH-deficient (mEH-/-) mice and background mice (129/Sv) were exposed to inhaled benzene (0, 10, 50, or 100 ppm) 5 days/week, 6 h/day, for a two-week duration. Total white blood cell counts and bone marrow cell counts were used to assess hematotoxicity and myelotoxicity. Micronucleated peripheral blood cells were counted to assess genotoxicity, and the p21 mRNA level in bone marrow cells was used as a determinant of the p53-regulated DNA damage response. Male mEH-/- mice did not have any significant hematotoxicity or myelotoxicity at the highest benzene exposure compared to the male 129/Sv mice. Significant hematotoxicity or myelotoxicity did not occur in the female mEH-/- or 129/Sv mice. Male mEH-/- mice were also unresponsive to benzene-induced genotoxicity compared to a significant induction in the male 129/Sv mice. The female mEH-/- and 129/Sv mice were virtually unresponsive to benzene-induced genotoxicity. While p21 mRNA expression was highly induced in male 129/Sv mice after exposure to 100-ppm benzene, no significant alteration was observed in male mEH-/- mice. Likewise, p21 mRNA expression in female mEH-/- mice was not significantly induced upon benzene exposure whereas a significant induction was observed in female 129/Sv mice. Thus mEH appears to be critical in benzene-induced toxicity in male, but not female, mice.

  10. Carnosol and carnosic acids from Salvia officinalis inhibit microsomal prostaglandin E2 synthase-1.

    Science.gov (United States)

    Bauer, Julia; Kuehnl, Susanne; Rollinger, Judith M; Scherer, Olga; Northoff, Hinnak; Stuppner, Hermann; Werz, Oliver; Koeberle, Andreas

    2012-07-01

    Prostaglandin E(2) (PGE(2)), the most relevant eicosanoid promoting inflammation and tumorigenesis, is formed by cyclooxygenases (COXs) and PGE(2) synthases from free arachidonic acid. Preparations of the leaves of Salvia officinalis are commonly used in folk medicine as an effective antiseptic and anti-inflammatory remedy and possess anticancer activity. Here, we demonstrate that a standard ethyl acetate extract of S. officinalis efficiently suppresses the formation of PGE(2) in a cell-free assay by direct interference with microsomal PGE(2) synthase (mPGES)-1. Bioactivity-guided fractionation of the extract yielded closely related fractions that potently suppressed mPGES-1 with IC(50) values between 1.9 and 3.5 μg/ml. Component analysis of these fractions revealed the diterpenes carnosol and carnosic acid as potential bioactive principles inhibiting mPGES-1 activity with IC(50) values of 5.0 μM. Using a human whole-blood assay as a robust cell-based model, carnosic acid, but not carnosol, blocked PGE(2) generation upon stimulation with lipopolysaccharide (IC(50) = 9.3 μM). Carnosic acid neither inhibited the concomitant biosynthesis of other prostanoids [6-keto PGF(1α), 12(S)-hydroxy-5-cis-8,10-trans-heptadecatrienoic acid, and thromboxane B(2)] in human whole blood nor affected the activities of COX-1/2 in a cell-free assay. Together, S. officinalis extracts and its ingredients carnosol and carnosic acid inhibit PGE(2) formation by selectively targeting mPGES-1. We conclude that the inhibitory effect of carnosic acid on PGE(2) formation, observed in the physiologically relevant whole-blood model, may critically contribute to the anti-inflammatory and anticarcinogenic properties of S. officinalis.

  11. Polymorphisms of microsomal triglycedde transfer protein in different hepatitis B virus-infected patients

    Institute of Scientific and Technical Information of China (English)

    Zhi-Tao Yang; Xin-Xin Zhang; Xiao-Fei Kong; Dong-Hua Zhang; Shen-Ying Zhang; Jie-Hong Jiang; Qi-Ming Gong; Gen-Di Jin; Zhi-Meng Lu

    2008-01-01

    AIM: To identify the two polymorphisms of microsomal triglyceride transfer protein (MTP) gene in the Chinese population and to explore their correlation with both hepatitis B virus (HBV) self-limited infection and persistent infection.METHODS: A total of 316 subjects with self-limited HBV infection and 316 patients with persistent HBV infection (195 subjects without familial history),matched with age and sex,from the Chinese Han population were enrolled in this study.Polymorphisms of MTP at the promoter region -493 and at H297Q were determined by the allele specific polymerase chain reaction (PCR).RESULTS: The ratio of males to females was 2.13:1 for each group and the average age in the self-limited and chronic infection groups was 38.36 and 38.28 years,respectively.None of the allelic distributions deviated significantly from that predicted by the Hardy-Weinberg equilibrium.There was a linkage disequilibrium between H297Q and -493G/T(D'=0.77).As the X2 test was used,the genotype distribution of MTP-493G/T demonstrated a significant difference between the self-limited infection group and the entire chronic group or the chronic patients with no family history (X2=8.543,P=0.015 and X2=7.199,P=0.019).The allele distribution at the MTP-493 position also demonstrated a significant difference between the study groups without family history (X2=6.212,P=0.013).The T allele emerged as a possible protective factor which may influence the outcomes of HBV infection (OR: 0.59; 95% CI: 0.389-0.897).CONCLUSION: The polymorphism of the MTP gene,T allele at -493,may be involved in determining the HBV infection outcomes,of which the mechanism needs to be further investigated.

  12. Functional analysis of promoter variants in the microsomal triglyceride transfer protein (MTTP) gene.

    Science.gov (United States)

    Rubin, Diana; Schneider-Muntau, Alexandra; Klapper, Maja; Nitz, Inke; Helwig, Ulf; Fölsch, Ulrich R; Schrezenmeir, Jürgen; Döring, Frank

    2008-01-01

    The microsomal triglyceride transfer protein (MTTP) is required for the assembly and secretion of apolipoprotein B (apoB)-containing lipoproteins from the intestine and liver. According to this function, polymorphic sites in the MTTP gene showed associations to low-density lipoprotein (LDL) cholesterol and related traits of the metabolic syndrome. Here we studied the functional impact of common MTTP promoter polymorphisms rs1800804:T>C (-164T>C), rs1800803:A>T (-400A>T), and rs1800591:G>T (-493G>T) using gene-reporter assays in intestinal Caco-2 and liver Huh-7 cells. Significant results were obtained in Huh-7 cells. The common MTTP promoter haplotype -164T/-400A/-493G showed about two-fold lower activity than the rare haplotype -164C/-400T/-493T. MTTP promoter mutant constructs -164T/-400A/-493T and -164T/-400T/-493T exhibited similar activity than the common haplotype. Activities of mutants -164C/-400A/-493G and -164C/-400A/-493T resembled the rare MTTP promoter haplotype. Electrophoretic mobility shift assays (EMSAs) revealed higher binding capacity of the transcriptional factor Sterol regulatory element binding protein1a (SREBP1a) to the -164T probe in comparison to the -164C probe. In conclusion, our study indicates that the polymorphism -164T>C mediates different activities of common MTTP promoter haplotypes via SREBP1a. This suggested that the already described SREBP-dependent modulation of MTTP expression by diet is more effective in -164T than in -164C carriers.

  13. Perspective of microsomal prostaglandin E2 synthase-1 as drug target in inflammation-related disorders.

    Science.gov (United States)

    Koeberle, Andreas; Werz, Oliver

    2015-11-01

    Prostaglandin (PG)E2 encompasses crucial roles in pain, fever, inflammation and diseases with inflammatory component, such as cancer, but is also essential for gastric, renal, cardiovascular and immune homeostasis. Cyclooxygenases (COX) convert arachidonic acid to the intermediate PGH2 which is isomerized to PGE2 by at least three different PGE2 synthases. Inhibitors of COX - non-steroidal anti-inflammatory drugs (NSAIDs) - are currently the only available therapeutics that target PGE2 biosynthesis. Due to adverse effects of COX inhibitors on the cardiovascular system (COX-2-selective), stomach and kidney (COX-1/2-unselective), novel pharmacological strategies are in demand. The inducible microsomal PGE2 synthase (mPGES)-1 is considered mainly responsible for the excessive PGE2 synthesis during inflammation and was suggested as promising drug target for suppressing PGE2 biosynthesis. However, 15 years after intensive research on the biology and pharmacology of mPGES-1, the therapeutic value of mPGES-1 as drug target is still vague and mPGES-1 inhibitors did not enter the market so far. This commentary will first shed light on the structure, mechanism and regulation of mPGES-1 and will then discuss its biological function and the consequence of its inhibition for the dynamic network of eicosanoids. Moreover, we (i) present current strategies for interfering with mPGES-1-mediated PGE2 synthesis, (ii) summarize bioanalytical approaches for mPGES-1 drug discovery and (iii) describe preclinical test systems for the characterization of mPGES-1 inhibitors. The pharmacological potential of selective mPGES-1 inhibitor classes as well as dual mPGES-1/5-lipoxygenase inhibitors is reviewed and pitfalls in their development, including species discrepancies and loss of in vivo activity, are discussed.

  14. Alterations of testosterone metabolism in microsomes from rats with experimental colitis induced by dextran sulfate sodium.

    Science.gov (United States)

    Huang, Yanjuan; Hu, Nan; Gao, Xuejiao; Yan, Zhixiang; Li, Sai; Jing, Wanghui; Yan, Ru

    2015-05-05

    Down-regulation of some hepatic cytochrome P450s (CYP450s) was observed in patients and animals with ulcerative colitis (UC). This study examined changes of CYP450s activities in microsomes of liver (RLMs), intestine (RIMs) and kidney (RRMs) from rats with experimental acute colitis induced by 5% dextran sulfate sodium (DSS) for 7days and those receiving DSS treatment followed by 7-d cessation through measuring 6α-(CYP1A1), 7α-(CYP2A1), 16α-(CYP2C11) and 2β-/6β-(CYP3A2) hydroxytestosterone (OHT) formed from testosterone. Both pro-(IL-1β, IL-6, TNF-α) and anti-(IL-4, IL-10) inflammatory cytokines were elevated in acute colitis, while the production of the former was enhanced and that of the latter declined by DSS withdrawal. In RLMs, the CYP2A1 activity was significantly increased at DSS stimulation and partially returned to normal level when DSS treatment was terminated. Activity of other CYP450s were decreased by acute colitis and remained after DSS withdrawal. In RRMs, formations of 6α-, 16α- and 2β-OHT significantly declined in acute colitis and DSS termination further potentiated the down-regulation, while 7α-OHT formation was suppressed at DSS stimulation and remained after DSS withdrawal. The formation of 6β-OHT only showed significant decrease after DSS withdrawal. Two metabolites (6α- and 6β-OHT) formed in RIMs and 6β-OHT formation was significantly decreased by DSS stimulation and continued after DSS treatment halted. These findings indicate that the alterations of CYP450s activities vary with organ, CYP isoforms and colitis status, which arouse cautions on efficacy and toxicity of drug therapy during disease progression.

  15. Influence of sex hormones on relative quantities of multiple species of cytochrome P-450 in rat liver microsomes. A possible answer to the sexual differences in metabolic activities of rat liver microsomes

    Energy Technology Data Exchange (ETDEWEB)

    Fujita, S.; Peisach, J. (Albert Einstein Coll. of Medicine, Bronx, NY (USA)); Chevion, M. (Hebrew Univ., Jerusalem (Israel). Dept. of Cellular Biochemistry; Hebrew Univ., Jerusalem (Israel). Hadassah Medical School)

    1981-01-01

    EPR spectra of rat liver microsomes from male, female and hormonally-treated castrated hepatectomized rats were studied. The spectra, especially in the region of gsub(max) suggested a heterogeneity of local environments of the low spin ferric heme indicative of multiple structures for cytochrome P-450. Certain features in the spectrum correlated with sexual differences. It is suggested that the changes in the relative amplitudes of the EPR features represent differences in the relative abundance of the individual proteins in the mixture that, in turn, are related to the sexual differences of metabolic patterns for reactions catalyzed by cytochrome P-450.

  16. Brain Autopsy

    Science.gov (United States)

    ... why a family should consider arranging for a brain autopsy upon the death of their loved one. To get a definitive ... study of tissue removed from the body after death. Examination of the whole brain is important in understanding FTD because the patterns ...

  17. Brain Basics

    Medline Plus

    Full Text Available ... Brain Imaging Using brain imaging technologies such as magnetic resonance imaging (MRI), which uses magnetic fields to take pictures of ... to slow or stop them from progressing. Functional magnetic resonance imaging (fMRI) is another important research tool in understanding ...

  18. Brain Basics

    Medline Plus

    Full Text Available ... and are working to compare that with brain development in people mental disorders. Genes and environmental cues both help to direct ... comparing such children to those with normal brain development may help scientists to pinpoint when and where mental disorders begin and perhaps how to slow or stop ...

  19. Brain peroxisomes.

    Science.gov (United States)

    Trompier, D; Vejux, A; Zarrouk, A; Gondcaille, C; Geillon, F; Nury, T; Savary, S; Lizard, G

    2014-03-01

    Peroxisomes are essential organelles in higher eukaryotes as they play a major role in numerous metabolic pathways and redox homeostasis. Some peroxisomal abnormalities, which are often not compatible with life or normal development, were identified in severe demyelinating and neurodegenerative brain diseases. The metabolic roles of peroxisomes, especially in the brain, are described and human brain peroxisomal disorders resulting from a peroxisome biogenesis or a single peroxisomal enzyme defect are listed. The brain abnormalities encountered in these disorders (demyelination, oxidative stress, inflammation, cell death, neuronal migration, differentiation) are described and their pathogenesis are discussed. Finally, the contribution of peroxisomal dysfunctions to the alterations of brain functions during aging and to the development of Alzheimer's disease is considered.

  20. The kinetic basis for age-associated changes in quercetin and genistein glucuronidation by rat liver microsomes.*

    Science.gov (United States)

    Bolling, Bradley W.; Court, Michael H.; Blumberg, Jeffrey B.; Chen, C-Y. Oliver

    2009-01-01

    The dietary bioavailability of the isoflavone genistein is decreased in older rats compared to young adults. Since flavonoids are metabolized extensively by the UDP-glucuronosyltransferases (UGTs), we hypothesized that UGT flavonoid conjugating activity changes with age. The effect of age on flavonoid glucuronidation was determined using hepatic microsomes from male F344 rats. Kinetic models of UGT activity toward the flavonol quercetin and the isoflavone genistein were established using pooled hepatic microsomal fractions of rats at different ages, and glucuronidation rates determined using individual samples. Intrinsic clearance (Vmax/Km) values in 4, 18, and 28 mo old rats were 0.100, 0.078, and 0.087 mL/min/mg for quercetin-7-O-glucuronide, 0.138, 0.133, and 0.088 for quercetin-3′-O-glucuronide, and 0.075, 0.077, and 0.057 for quercetin-4′-O-glucuronide, respectively. While there were no differences in formation rates of total quercetin glucuronides in individual samples, the production of the primary metabolite, quercetin-7-O-glucuronide, at 30 μM quercetin concentration was increased from 3.4 and 3.1 nmol/min/mg at 4 and 18 mo to 3.8 nmol/min/mg at 28 mo, while quercetin-3′-O-glucuronide formation at 28 mo declined by a similar degree (P ≤0.05). At 30 and 300 μM quercetin concentration, the rate of quercetin-4′-O-glucuronide formation peaked at 18 mo at 0.9 nmol/min/mg. Intrinsic clearance values of genistein 7-O-glucuronide increased with age, in contrast to quercetin glucuronidation. Thus, the capacity for flavonoid glucuronidation by rat liver microsomes is dependent on age, UGT isoenzymes, and flavonoid structure. PMID:19446449

  1. Determination of metabolic profile of anti-malarial trioxane CDRI 99/411 in rat liver microsomes using HPLC.

    Science.gov (United States)

    Mishra, Smriti; Manickavasagam, Lakshmi; Jain, Girish Kumar

    2012-01-01

    CDRI 99/411 is a potent 1,2,4-trioxane anti-malarial candidate compound of the Central Drug Research Institute, India. This study aimed to conduct comprehensive in vitro metabolic investigations of CDRI 99/411 to corroborate its preclinical investigations. Preliminary in vitro metabolic investigations were performed to assess the metabolic stability [in vitro half-life (t(1/2) ) and in vitro hepatic intrinsic clearance (Cl(int) )] of CDRI 99/411 in male Sprague-Dawley rat and human liver microsomes using validated high-performance liquid chromatography with photodiode array detector. The observed in vitro t(1/2) of the compound in rat and human liver microsomes was 13 min with in vitro Cl(int) 130.7±25.0 μL/min/mg and 19 min with in vitro Cl(int) 89.3 ± 17.40 μL/min/mg. These observations suggested moderate metabolic degradation and in vitro Cl(int) with insignificant difference (p>0.05) in the metabolic stability profile in rat and human. Hence, in vitro metabolic investigations were performed with rat liver microsomes. It was observed that CDRI 99/411 exhibited sigmoidal kinetics. At nonlinear regression (r ≥ 0.99) EC(50) and Hill slope values were 17 µm and 1.50, respectively. The metabolism of CDRI 99/411 was primarily mediated by CYP3A2 and was inferred by CYP reaction phenotyping with known potent inhibitors. Two metabolites of CDRI 99/411 were detected which were undetectable on incubation with 1-aminobenzotriazole and ketoconazole.

  2. Effect of liver fatty acid binding protein on fatty acid movement between liposomes and rat liver microsomes.

    Science.gov (United States)

    McCormack, M; Brecher, P

    1987-01-01

    Although movement of fatty acids between bilayers can occur spontaneously, it has been postulated that intracellular movement is facilitated by a class of proteins named fatty acid binding proteins (FABP). In this study we have incorporated long chain fatty acids into multilamellar liposomes made of phosphatidylcholine, incubated them with rat liver microsomes containing an active acyl-CoA synthetase, and measured formation of acyl-CoA in the absence or presence of FABP purified from rat liver. FABP increased about 2-fold the accumulation of acyl-CoA when liposomes were the fatty acid donor. Using fatty acid incorporated into liposomes made either of egg yolk lecithin or of dipalmitoylphosphatidylcholine, it was found that the temperature dependence of acyl-CoA accumulation in the presence of FABP correlated with both the physical state of phospholipid molecules in the liposomes and the binding of fatty acid to FABP, suggesting that fatty acid must first desorb from the liposomes before FABP can have an effect. An FABP-fatty acid complex incubated with microsomes, in the absence of liposomes, resulted in greater acyl-CoA formation than when liposomes were present, suggesting that desorption of fatty acid from the membrane is rate-limiting in the accumulation of acyl-CoA by this system. Finally, an equilibrium dialysis cell separating liposomes from microsomes on opposite sides of a Nuclepore filter was used to show that liver FABP was required for the movement and activation of fatty acid between the compartments. These studies show that liver FABP interacts with fatty acid that desorbs from phospholipid bilayers, and promotes movement to a membrane-bound enzyme, suggesting that FABP may act intracellularly by increasing net desorption of fatty acid from cell membranes. PMID:3446187

  3. The kinetic basis for age-associated changes in quercetin and genistein glucuronidation by rat liver microsomes.

    Science.gov (United States)

    Bolling, Bradley W; Court, Michael H; Blumberg, Jeffrey B; Chen, C-Y Oliver

    2010-06-01

    The dietary bioavailability of the isoflavone genistein is decreased in older rats compared to young adults. Since flavonoids are metabolized extensively by the UDP-glucuronosyltransferases (UGTs), we hypothesized that UGT flavonoid conjugating activity changes with age. The effect of age on flavonoid glucuronidation was determined using hepatic microsomes from male F344 rats. Kinetic models of UGT activity toward the flavonol quercetin and the isoflavone genistein were established using pooled hepatic microsomal fractions of rats at different ages, and glucuronidation rates were determined using individual samples. Intrinsic clearance (V(max)/K(m)) values in 4-, 18- and 28-month-old rats were 0.100, 0.078 and 0.087 ml/min/mg for quercetin-7-O-glucuronide; 0.138, 0.133 and 0.088 for quercetin-3'-O-glucuronide; and 0.075, 0.077 and 0.057 for quercetin-4'-O-glucuronide, respectively. While there were no differences in formation rates of total quercetin glucuronides in individual samples, the production of the primary metabolite, quercetin-7-O-glucuronide, at 30 microM quercetin concentration was increased from 3.4 and 3.1 nmol/min/mg at 4 and 18 months to 3.8 nmol/min/mg at 28 months, while quercetin-3'-O-glucuronide formation at 28 months declined by a similar degree (Pmicrosomes is dependent on age, UGT isoenzymes and flavonoid structure.

  4. Separation and characterization of the aldehydic products of lipid peroxidation stimulated by ADP-Fe2+ in rat liver microsomes.

    Science.gov (United States)

    Esterbauer, H; Cheeseman, K H; Dianzani, M U; Poli, G; Slater, T F

    1982-01-01

    1. Methods using t.l.c. and high-pressure liquid chromatography (h.p.l.c.) have been used to separate the complex variety of substances possessing a carbonyl function that are produced during lipid peroxidation. 2. The major type of lipid peroxidation studied was the ADP-Fe2+-stimulated peroxidation of rat liver microsomal phospholipids. Preliminary separation of the polar and non-polar products was achieved by t.l.c.: further separation and identification of individual components was performed by h.p.l.c. Estimations were performed on microsomal pellets and the supernatant mixture after incubation of microsomes for 30 min at 37 degrees C. 3. The polar fraction was larger than the non-polar fraction when expressed as nmol of carbonyl groups/g of liver. In the non-polar supernatant fraction the major contributors were n-alkanals (31% of the total), alpha-dicarbonyl compounds (22%) and 4-hydroxyalkenals (37%) with the extraction method used. 4. Major individual contributors to the non-polar fraction were found to be propanal, 4-hydroxynonenal, hexanal and oct-2-enal. Other components identified include butanal, pent-2-enal, hex-2-enal, hept-2-enal, 4-hydroxyoctenal and 4-hydroxyundecenal. The polar carbonyl fraction was less complex than the non-polar fraction, although the identities of the individual components have not yet been established. 5. Since these carbonyl compounds do not react significantly in the thiobarbituric acid reaction, which largely demonstrates the presence of malonaldehyde, it is concluded that considerable amounts of biologically reactive carbonyl derivatives are released in lipid peroxidation and yet may not be picked up by the thiobarbituric acid reaction. PMID:7159389

  5. Dynamic headspace analysis of volatile metabolites from the reductive dehalogenation of trichloro- and tetrachloroethanes by hepatic microsomes.

    Science.gov (United States)

    Thompson, J A; Ho, B; Mastovich, S L

    1985-03-01

    A dynamic headspace technique was developed to facilitate the identification and quantitation of low levels of volatile metabolites produced in vitro by subcellular preparations. The method is complementary to commonly used static headspace and solvent-extraction techniques, and involves purging the compounds from microsomal suspensions with an inert gas, trapping them on a short column of adsorbant resin, and transferring the metabolites to a gas chromatograph. An apparatus was designed to facilitate the incubations and isolations of volatile compounds. Recoveries of several chlorinated hydrocarbons with boiling points in the range 12 to 186 degrees C were 85% or higher, and the recovery of vinyl chloride (boiling point -13 degrees C) was 25%. The quantitative precision of the method was determined and calibration curves were established for each metabolite, demonstrating that no discrimination occurred over a wide range of concentrations. This technique was employed to investigate the reductive metabolism of 1,1,1-trichloroethane, 1,1,2-trichloroethane, and 1,1,2,2-tetrachloroethane by rat liver microsomes. The metabolites from these substrates were 1,1-dichloroethane, vinyl chloride, and 1,2-dichloroethylene, respectively. These conversions were NADPH-dependent, occurred only under anaerobic conditions, and indicate that chloroethanes with relatively low electron affinities can be reduced slowly by microsomal cytochrome P-450. The rates of formation of vinyl chloride, 1,1-dichloroethane, and 1,2-dichloroethylene with 1.0 mM substrate were 12.5 +/- 2.0, 122 +/- 14, and 147 +/- 12 pmol/min/mg of protein, respectively. The results show that there are distinct advantages of the purge/trap method over the static headspace method for studying volatile metabolites when high sensitivity is required.

  6. Sulfation of chondroitin. Specificity, degree of sulfation, and detergent effects with 4-sulfating and 6-sulfating microsomal systems

    Energy Technology Data Exchange (ETDEWEB)

    Sugumaran, G.; Silbert, J.E.

    1988-04-05

    Microsomal preparations from chondroitin 6-sulfate-producing chick embryo epiphyseal cartilage, and from chondroitin 4-sulfate-producing mouse mastocytoma cells, were incubated with UDP-(14C)glucuronic acid and UDP-N-acetylgalactosamine to form non-sulfated proteo(14C)chondroitin. Aliquots of the incubations were then incubated with 3'-phosphoadenylylphosphosulfate (PAPS) in the presence or absence of various detergents. In the absence of detergents, there was good sulfation of this endogenous proteo(14C)chondroitin by the original microsomes from both sources. Detergents, with the exception of Triton X-100, markedly inhibited sulfation in the mast cell system but not in the chick cartilage system. These results indicate that sulfation and polymerization are closely linked on cell membranes and that in some cases this organization can be disrupted by detergents. When aliquots of the original incubation were heat inactivated, and then reincubated with new microsomes from chick cartilage and/or mouse mastocytoma cells plus PAPS, there was no significant sulfation of this exogenous proteo(14C) chondroitin with either system unless Triton X-100 was added. Sulfation of exogenous chondroitin and chondroitin hexasaccharide was compared with sulfation of endogenous and exogenous proteo(14C)chondroitin. Sulfate incorporation into hexasaccharide and chondroitin decreased as their concentrations (based on uronic acid) approached that of the proteo(14C)chondroitin. At the same time, the degree of sulfation in percent of substituted hexosamine increased. However, the degree of sulfation did not reach that of the endogenous proteo(14C)chondroitin. Hexasaccharide and chondroitin sulfation were stimulated by the presence of Triton X-100. However, in contrast to the exogenous proteo(14C)chondroitin, there was some sulfation of hexasaccharide and chondroitin in the absence of this detergent.

  7. Metabolism of 20(S-Ginsenoside Rg2 by Rat Liver Microsomes: Bioactivation to SIRT1-Activating Metabolites

    Directory of Open Access Journals (Sweden)

    Li-Yuan Ma

    2016-06-01

    Full Text Available 20(S-Ginsenoside Rg2 (1 has recently become a hot research topic due to its potent bioactivities and abundance in natural sources such as the roots, rhizomes and stems-leaves of Panax ginseng. However, due to the lack of studies on systematic metabolic profiles, the prospects for new drug development of 1 are still difficult to predict, which has become a huge obstacle for its safe clinical use. To solve this problem, investigation of the metabolic profiles of 1 in rat liver microsomes was first carried out. To identify metabolites, a strategy of combined analyses based on prepared metabolites by column chromatography and ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF/MS was performed. As a result, four metabolites M1–M4, including a rare new compound named ginsenotransmetin A (M1, were isolated and the structures were confirmed by spectroscopic analyses. A series of metabolites of 1, MA–MG, were also tentatively identified by UPLC-Q-TOF/MS in rat liver microsomal incubate of 1. Partial metabolic pathways were proposed. Among them, 1 and its metabolites M1, M3 and M4 were discovered for the first time to be activators of SIRT1. The SIRT1 activating effects of the metabolite M1 was comparable to those of 1, while the most interesting SIRT1 activatory effects of M3 and M4 were higher than that of 1 and comparable with that of resveratrol, a positive SIRT1 activator. These results indicate that microsome-dependent metabolism may represent a bioactivation pathway for 1. This study is the first to report the metabolic profiles of 1 in vitro, and the results provide an experimental foundation to better understand the in vivo metabolic fate of 1.

  8. Metabolism of 20(S)-Ginsenoside Rg₂ by Rat Liver Microsomes: Bioactivation to SIRT1-Activating Metabolites.

    Science.gov (United States)

    Ma, Li-Yuan; Zhou, Qi-Le; Yang, Xin-Bao; Wang, Hong-Ping; Yang, Xiu-Wei

    2016-06-10

    20(S)-Ginsenoside Rg₂ (1) has recently become a hot research topic due to its potent bioactivities and abundance in natural sources such as the roots, rhizomes and stems-leaves of Panax ginseng. However, due to the lack of studies on systematic metabolic profiles, the prospects for new drug development of 1 are still difficult to predict, which has become a huge obstacle for its safe clinical use. To solve this problem, investigation of the metabolic profiles of 1 in rat liver microsomes was first carried out. To identify metabolites, a strategy of combined analyses based on prepared metabolites by column chromatography and ultra-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF/MS) was performed. As a result, four metabolites M1-M4, including a rare new compound named ginsenotransmetin A (M1), were isolated and the structures were confirmed by spectroscopic analyses. A series of metabolites of 1, MA-MG, were also tentatively identified by UPLC-Q-TOF/MS in rat liver microsomal incubate of 1. Partial metabolic pathways were proposed. Among them, 1 and its metabolites M1, M3 and M4 were discovered for the first time to be activators of SIRT1. The SIRT1 activating effects of the metabolite M1 was comparable to those of 1, while the most interesting SIRT1 activatory effects of M3 and M4 were higher than that of 1 and comparable with that of resveratrol, a positive SIRT1 activator. These results indicate that microsome-dependent metabolism may represent a bioactivation pathway for 1. This study is the first to report the metabolic profiles of 1 in vitro, and the results provide an experimental foundation to better understand the in vivo metabolic fate of 1.

  9. Identification of Three New N-Demethylated and O-Demethyled Bisbenzylisoquinoline Alkaloid Metabolites of Isoliensinine from Dog Hepatic Microsomes

    Directory of Open Access Journals (Sweden)

    Su Zeng

    2012-10-01

    Full Text Available Isoliensinine, a natural phenolic bisbenzyltetrahydroisoquinoline alkaloid, has received considerable attention for its potential biological effects such as antioxidant and anti-HIV activities. From the dog hepatic microsomes of isoliensinine, three new N-demethylated and O-demethylated metabolites, 2-N-desmethyl-isoliensinine (M1, 2'-N-desmethylisoliensinine (M2, and 2'-N-6-O-didesmethylisoliensinine (M3, were identified by high-performance liquid chromatography and data-dependent electrospray ionization tandem mass spectrometry. Possible metabolic pathways for isoliensinine have been proposed. The result should prove very helpful for evaluation of the drug-like properties of isoliensinine and other bisbenzylisoquinoline alkaloids.

  10. [Progress in quantitative methods based on liquid chromatography-mass spectrometry for drug metabolizing enzymes in human liver microsomes].

    Science.gov (United States)

    Wang, Huanhuan; Lu, Yayao; Peng, Bo; Qian, Xiaohong; Zhang, Yangjun

    2015-06-01

    Cytochrome P450 (CYP) enzymes and uridine 5-diphospho-glucuronosyltransferase (UGT) enzymes are critical enzymes for drug metabolism. Both chemical drugs and traditional Chinese medicines are converted to more readily excreted compounds by drug metabolizing enzymes in human livers. Because of the disparate expression of CYP and UGT enzymes among different individuals, accurate quantification of these enzymes is essential for drug pharmacology, drug-drug interactions and drug clinical applications. The research progress in quantitative methods based on liquid chromatography-mass spectrometry for drug metabolizing enzymes in human liver microsomes in the recent decade is reviewed.

  11. Multidimensional LC–MS/MS analysis of liver proteins in rat, mouse and human microsomal and S9 fractions

    Directory of Open Access Journals (Sweden)

    Makan Golizeh

    2015-03-01

    Full Text Available Liver plays a key role in metabolism and detoxification, therefore analysis of its proteome is relevant for toxicology and drug discovery studies. To optimize for high proteome coverage, protein and peptide-level ion exchange fractionation were assessed using rat liver microsomes and S9 fractions. 2D-(SCX-RP-LC–MS/MS analysis with peptide fractionation was subsequently employed for rat, mouse and human samples, yielding between 1400 and 1939 identified proteins, 58% of which were shared between species, and with relatively high sequence coverage. This rich dataset is specifically interesting for the toxicology community, and could serve as an excellent source for targeted assay development.

  12. Metabolic stability and determination of cytochrome P450 isoenzymes' contribution to the metabolism of medetomidine in dog liver microsomes.

    Science.gov (United States)

    Duhamel, Marie-Claude; Troncy, Eric; Beaudry, Francis

    2010-08-01

    Medetomidine is a potent and selective alpha2-adrenergic agonist. The activation of alpha2-adrenergic receptor mediates a variety of effects including sedation, analgesia, relief of anxiety, vasoconstriction and bradycardia. However, our main interest is the sedative effects of medetomidine when used as a premedicant prior surgery in companion animals, especially in dogs. Recently, data suggested that following intravenous infusion at six dosing regiments non-linear pharmacokinetics was observed. Major causes of non-linear pharmacokinetics are the elimination of the drug not following a simple first-order kinetics and/or the elimination half-life changing due to saturation of an enzyme system. The goal of this study was to establish the metabolic stability and determine the metabolic pathway of medetomidine in dog liver microsomes. Consequently, Michaelis-Menten parameters (V(max), K(m)), T(1/2) and CL(i) were determined. The incubations were performed in a microcentrifuge tube and containing various concentrations of medetomidine (10-5000 nM), 1 mg/mL of microsomal proteins suspended in 0.1 M phosphate buffer, pH 7.4. Microsomal suspensions were preincubated with NADPH (1 mM) for 5 min at 37 degrees C prior to fortification with medetomidine. Samples were taken at various time points for kinetic information and the initial velocity (v(i)) was determined after 10 min incubation. The reaction was stopped by the addition of an internal standard solution (100 ng/mL of dextrometorphan in acetone). Medetomidine concentrations were determined using a selective and sensitive HPLC-ESI/MS/MS method. Using non-linear regression, we determined a K(m) value of 577 nM, indicating relatively low threshold enzyme saturation consistent with previous in vivo observation. The metabolic stability was determined at a concentration of 100 nm (dog liver microsomes, also consistent with previous in vivo data. Moreover, results suggest that principally medetomidine is metabolized by the

  13. 7-Methylbenz(a)anthracene deoxyribonucleoside products isolated from DNA after metabolism of the carcinogen by rat liver microsomes in the presence of DNA

    Energy Technology Data Exchange (ETDEWEB)

    Thompson, M.H.; Osborne, M.R.; King, H.W.S.; Brookes, P.

    1976-07-01

    Metabolism of 7-methylbenz(a)anthracene (7MeBA) by 3-methylcholanthrene-induced rat liver microsomes in the presence of added native or denatured DNA resulted in covalent binding of the hydrocarbon to the nucleic acid. Enzymatic degradation and column chromatographic fractionation showed that the hydrocarbon-deoxyribonucleoside products were separable from the products similarly obtained from DNA having 7MeBA bound following treatment of mouse embryo cells in culture with this hydrocarbon. Comparison of the microsome catalyzed hydrocarbon-deoxyribonucleoside products with those obtained by reaction with DNA of 7MeBA-5,6-oxide suggested that this K-region epoxide made a significant contribution to the liver microsome-induced DNA binding.

  14. Further evidence on the intramolecular lactonization in rat liver microsomal metabolism of 12-O-acetylated retronecine-type pyrrolizidine alkaloids.

    Science.gov (United States)

    Tang, Jun; Wang, Zhengtao; Akao, Teruaki; Hattori, Masao

    2013-11-01

    We have previously found evidence of intramolecular lactonization in rat liver microsomal metabolism of isoline, a 12-O-acetylated pyrrolizidine alkaloid. In this study, the metabolism of another 12-O-acetylated pyrrolizidine alkaloid, acetylduciformine, by the proposed transformation pathway was investigated under the same incubation conditions. Two deacetylated metabolites from acetylduciformine were isolated and purified by chromatographic methods, and further characterized based on their physical properties and spectral data. One metabolite (lankongensisine A) was the lactone of another one (duciformine). Both compounds were first obtained as hydrolyzed metabolites from acetylduciformine by rat liver microsomes. More importantly, the present study provided further evidence for the intramolecular lactonization in the microsomal metabolism of 12-O-acetylated retronecine-type PAs.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

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

    2013-05-20

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

  17. Effect of X-radiation on the content, composition and para-nitroanisol-o-demethylase activity of cytochrome P-450 in rat liver microsomes

    Energy Technology Data Exchange (ETDEWEB)

    Deev, L.I.; Akhalaya, M.Ya.; Vasilenko, O.V.; Platonov, A.G.; Topchishvili, G.I. (Moskovskij Gosudarstvennyj Univ. (USSR))

    A study was made of the effect of X-radiation of different doses on the content of P-450 cytochrome in a microsomal fraction of rat liver. When the haemoprotein level markedly decreased an increase in Ksub(m) and a decrease in Vsub(max) were noted in the reaction of O-demethylation of para-nitroanisole by microsomes of the irradiated rat liver. It is suggested that one of the cause of the effect observed is the postirradiation change in the composition of cytochrome P-450 pool resulting from a selective decrease in the level of the radiosensitive forms of haemoprotein.

  18. Brain radiation - discharge

    Science.gov (United States)

    Radiation - brain - discharge; Cancer-brain radiation; Lymphoma - brain radiation; Leukemia - brain radiation ... Decadron) while you are getting radiation to the brain. It may make you hungrier, cause leg swelling ...

  19. Brain tumor - primary - adults

    Science.gov (United States)

    ... Vestibular schwannoma (acoustic neuroma) - adults; Meningioma - adults; Cancer - brain tumor (adults) ... Primary brain tumors include any tumor that starts in the brain. Primary brain tumors can start from brain cells, ...

  20. Brain Basics

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    Full Text Available ... highly developed area at the front of the brain that, in humans, plays a role in executive functions such as ... Higher Death Rate Among Youth with Psychosis Delayed Walking Link ...

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  2. Brain Basics

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    Full Text Available ... Some people who develop a mental illness may recover completely; others may have repeated episodes of illness ... in early detection, more tailored treatments, and possibly prevention of such illnesses. The Working Brain Neurotransmitters Everything ...

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    Full Text Available ... that contains codes to make proteins and other important body chemicals. DNA also includes information to control ... cells required for normal function and plays an important role during early brain development. It may also ...

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    Full Text Available ... of the cell from its surrounding environment and controls what enters and leaves the cell, and responds ... via axons) to form brain circuits. These circuits control specific body functions such as sleep and speech. ...

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    Full Text Available ... in early detection, more tailored treatments, and possibly prevention of such illnesses. The Working Brain Neurotransmitters Everything ... can cause tremors or symptoms found in Parkinson's disease. Serotonin —helps control many functions, such as mood, ...

  6. Brain Basics

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    Full Text Available ... Offices and Divisions Careers@NIMH Advisory Boards and Groups Staff Directories Getting to NIMH National Institutes of ... electrical signals. The brain begins as a small group of cells in the outer layer of a ...

  7. Brain Basics

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    Full Text Available ... These circuits control specific body functions such as sleep and speech. The brain continues maturing well into ... factors that can affect our bodies, such as sleep, diet, or stress. These factors may act alone ...

  8. In vitro metabolism of the alkaloid piplartine by rat liver microsomes.

    Science.gov (United States)

    Marques, Lucas Maciel Mauriz; da Silva, Eduardo Afonso; Gouvea, Dayana Rubio; Vessecchi, Ricardo; Pupo, Mônica Tallarico; Lopes, Norberto Peporine; Kato, Massuo Jorge; de Oliveira, Anderson Rodrigo Moraes

    2014-07-01

    Because piplartine (PPT) has demonstrated biological activities, such as cytotoxic, anxiolytic, antidepressant, antifungal and antiplatelet activities, this molecule is a relevant drug candidate. The metabolic fate of drug candidates is an essential requirement in assessing their safety and efficacy. Based on this requirement, the biotransformation of PPT by cytochrome P450 enzymes (CYP) was investigated for the first time. To determine the in vitro enzymatic kinetic parameters, an HPLC method was developed and validated to quantify PPT. All samples were separated on a reversed-phase C18 column using a mobile phase of acetonitrile:water (40:60, v/v). The method exhibited a linear range of 2.4-157.7 μmol/L, with the following calibration curve: y=0.0934 (±0.0010)x+0.0027, r=0.9975. The lower limit of quantitation was verified to be 2.4 μmol/L, with an RSD below 7%. The precision and accuracy were assessed for both within-day and between-day determinations; neither relative standard (RSD%) deviations nor relative errors (RER) exceeded a value of 15%. The mean absolute recovery was 85%, with an RSD value below 6%. The enzymatic kinetic parameters revealed a sigmoidal profile, with V(max)=4.7±0.3 μmol/mg mL⁻¹/min, h=2.5±0.4, S₅₀=44.7±0.3 μmol/L and CL(max)=0.054 μL/min/mg protein, indicating cooperativity behavior. Employing a mammalian model, PPT metabolism yielded two unreported monohydroxylated products (m/z 334). The identification and structural elucidation of the metabolites were performed by comparing their mass spectra with those spectra of the parent drug. For the first time, the in vitro metabolism studies employing microsomes were demonstrated to be a suitable tool for data regarding enzymatic kinetics and for the metabolites formed in the PPT mammalian metabolism.

  9. Binding of mescaline with subcellular fractions upon incubation of brain cortex slices with [14C] mescaline.

    Science.gov (United States)

    Datta, R K; Antopol, W; Ghosh, J J

    1977-01-01

    Incubation of brain cortex slices in the presence of glucose resulted in the permeation of about 65% of [14C] mescaline into slices. Of this, about one-third radioactivity was bound with nuclei, mitochondria, microsomes, and ribosomes. Dialysis of subcellular fractions did not markedly reduce the amounts of radioactivity bound to the fractions. The permeation into slices and the binding of mescaline to subcellular fractions were fairly time-dependent, but were inhibited by the presence of potassium cyanide, or by the absence of glucose and by heating to 80 degrees C for 1 min.

  10. Identification of stereoisomeric metabolites of meisoindigo in rat liver microsomes by achiral and chiral liquid chromatography/tandem mass spectrometry.

    Science.gov (United States)

    Huang, Meng; Goh, Lin Tang; Ho, Paul C

    2008-11-01

    N-methylisoindigotin, abbreviated as meisoindigo, has been a routine therapeutic agent in the clinical treatment of chronic myelogenous leukemia in China since the 1980s. However, information relevant to in vitro metabolism of meisoindigo is limited. In this study, in vitro stereoisomeric metabolites of meisoindigo in rat liver microsomes were identified for the first time by achiral and chiral liquid chromatography/tandem mass spectrometry, together with proton NMR spectroscopy and synchrotron infrared spectroscopy. The major in vitro phase I metabolites of meisoindigo were tentatively identified as stereoselective-reduced meisoindigo, which comprised a pair of (3-R, 3'-R) and (3-S, 3'-S) enantiomers with lower abundance, as well as another pair of (3-R, 3'-S) and (3-S, 3'-R) enantiomers with higher abundance. One type of minor in vitro metabolites was tentatively identified as stereoselective N-demethyl-reduced meisoindigo including a pair of (3-R, 3'-R) and (3-S, 3'-S) enantiomers, as well as one meso compound. Another type of minor in vitro metabolites was tentatively identified as both stereoselective and regioselective monohydroxyl-reduced meisoindigo. Based on the metabolite profiling, three parallel metabolic pathways of meisoindigo in rat liver microsomes were proposed.

  11. Effect of Water Miscible Organic Solvents on p-Nitrophenol Hydroxylase (CYP2E1) Activity in Rat Liver Microsomes.

    Science.gov (United States)

    Patil, Pranali G; Kamble, S H; Shah, T S; Iyer, K R

    2015-01-01

    Organic solvents used for solubilization of the substrates/NCEs are known to affect the activity of cytochrome P450 enzymes. Further, this effect varies with the solvents used, the substrates and CYP450 isoforms in question. In the present study, we have investigated the effect of ten commonly used water miscible organic solvents (methanol, ethanol, 1-propanol, 2-propanol, acetonitrile, acetone, dimethyl sulphoxide, N,N-dimethyl formamide, dioxane and polyethylene glycol 400) on p-nitrophenol hydroxylase activity at 0, 0.1, 0.25, 0.5, 0.75 and 1% v/v concentration in rat liver microsomes. All the solvents studied showed concentration dependent inhibition of the p-nitrophenol hydroxylase activity except acetonitrile which showed activation of the activity at concentration range studied. Out of ten solvents studied, dioxane was found to be the most inhibitory solvent (inhibition >90% at 0.25% v/v concentration). Overall, solvents like dimethyl sulphoxide, dimethyl formamide and dioxane appeared to be unsuitable for characterizing p-nitrophenol hydroxylase (CYP2E1-mediated) reactions due to a high degree of inhibition. On the other hand, methanol and acetonitrile at concentrations <0.5% v/v appeared to be appropriate solvents for substrate solubilization while evaluating CYP2E1-mediated catalysis. The results of this study imply that caution should be exercised while choosing solvents for dissolution of substrate during enzyme studies in liver microsomes.

  12. [Identification of metabolites of epiberberine in rat liver microsomes and its inhibiting effects on CYP2D6].

    Science.gov (United States)

    Yang, Xiao-Yan; Ye, Jing; Sun, Gui-Xia; Xue, Bao-Juan; Zhao, Yuan-Yuan; Miao, Pei-Pei; Su, Jin; Zhang, Yu-Jie

    2014-10-01

    Epiberberine, one of the most important isoquinoline alkaloid in Coptidis Rhizoma, possesses extensive pharmacological activities. In this paper, the liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to study phase I and phase II metabolites. A Thermo HPLC system (including Surveyor AS, Surveyor LC Pump, Surveyor PDA. USA) was used. The cocktail probe drugs method was imposed to determine the content change of metoprolol, dapsone, phenacetin, chlorzoxazone and tolbutamide simultaneously for evaluating the activity of CYP2D6, CYP3A4, CYP1A2, CYP2E1 and CYP2C9 under different concentrations of epiberberine in rat liver microsomes. The result showed that epiberberine may have phase I and phase II metabolism in the rat liver and two metabolites in phase I and three metabolites in phase II are identified in the temperature incubation system of in vitro liver microsomes. Epiberberine showed significant inhibition on CYP2D6 with IC50 value of 35.22 μmol L(-1), but had no obvious inhibiting effect on the activities of CYP3A4, CYP1A2, CYP2E1 and CYP2C9. The results indicated that epiberberine may be caused drug interactions based on CYP2D6 enzyme. This study aims to provide a reliable experimental basis for its further research and development of epiberberine.

  13. Metabolism of dl-praeruptorin A in rat liver microsomes using HPLC-electrospray ionization tandem mass spectrometry.

    Science.gov (United States)

    Ruan, Hang; Zhang, Zhen; Liang, Xin-fang; Fu, Yan; Su, Mei-qin; Liu, Qi-lin; Wang, Xiu-min; Zhu, Xuan

    2011-08-01

    dl-Praeruptorin A (Pd-Ia) is the major active constituent of the traditional Chinese medicine Peucedanum praeruptorum Dunn. Recently it has been identified as a novel agent in the treatment and prevention of cardiovascular diseases. Accordingly, we investigated the metabolism of Pd-Ia in rat liver microsomes. The involvement of cytochrome P450 (CYP) and CYP isoforms were identified using a CYP-specific inhibitor (SKF-525A), CYP-selective inhibitors (α-naphthoflavone, metyrapone, fluvastatin, quinidine, disulfiram, ketoconazole and ticlopidine) and CYP-selective inducers (phenobarbital, dexamethasone and β-naphthoflavone). Residual concentrations of the substrate and metabolites were determined by HPLC, and further identified by their mass spectra and chromatographic behavior. These experiments showed that CYP450 is involved in Pd-Ia metabolism, and that the major CYP isoform responsible is CYP3A1/2, which acts in a concentration-dependent manner. Four Pd-Ia metabolites (M1, M2, M3, and M4) were detected after incubation with rat liver microsomes. Hydroxylation was the primary metabolic pathway of Pd-Ia, and possible chemical structures of the metabolites were identified. Further research is now needed to link the metabolism of Pd-Ia to its drug-drug interactions.

  14. [Influence of jinlingzi powder with different compatibility on activity of cytochrome P1 A2 from rat liver microsomes].

    Science.gov (United States)

    Cheng, Long; Wang, Lan; Wang, Yanli; Liang, Rixin; Yang, Weipeng; Wang, Wei; Hu, Nan; Yin, Xiaojie; Weng, Xiaogang; Wang, Yiwei; Yang, Qing

    2012-03-01

    To illustrate the compability rule of Jinlingizi powder, by investigating the effects of Jinlingzi Powder with different compatibility on the enzymatic activity of cytochrome P1 A2 (CYP1A2) from rat liver microsome. The different compability of Jinlingizi powder is designed, based on the orthogonal array L9 (3(4)). In vitro test, rat liver microsomes incubation system is applied to detect the 50% inhibitory concentraton of Jinlingzi powder with different compatibility to cytochrome P1A2 (CYP1A2) enzyme. In vivo experiments, rats is treated orally with the different compability of Jinlingizi powder for 5 days, then be injected with probe drug phenacetin. The biosample from liver tissue is obtained by microdialysis probe, then analysisd by HPLC. The concentration-time data are modulated by software WinNonlin. IC50 data show no significant inhibitory activty to cytochrome P1 A2. Acetaminophen and phenacetin PK parameters indicate that the different compability of Jinlingizi powder can modulate the CYP 1A2 mediated metabolism, which is associate with the compatibility of Jinlingzi powder.

  15. Trapping and Identification of the Dichloroacetate Radical from the Reductive Dehalogenation of Trichloroacetate by Mouse and Rat Liver Microsomes

    Energy Technology Data Exchange (ETDEWEB)

    Merdink, James L.(WASHINGTON STATE UNIV TC); Bull, Richard J.(SELF-EMPLOYED CONSULTANTS); Schultz, Irvin R.(BATTELLE (PACIFIC NW LAB))

    1999-12-01

    A key question in the risk assessment of trichloroethylene (TRI) is the extent to which its carcinogenic effects might depend on the formation of dichloroacetate (DCA) as a metabolite. One of the metabolic pathways proposed for the formation of DCA from TRI is by the reductive dehalogenation of trichloroacetate (TCA), via a free radical intermediate. Although proof of this radical has been elusive, the detection of fully dechlorinated metabolites in the urine and the formation of lipid peroxidation by-products in microsomal incubations with TCA argue for its existence. We report here the trapping of the dichloroacetate radical with the spin-trapping agent PBN, and its identification by GC/MS. The PBN/dichloroacetate radical adduct was found to undergo an intramolecular rearrangement during its extraction into organic solvent. An internal condensation reaction between the acetate and the nitroxide radical moieties is hypothesized to form a cyclic adduct with the elimination of an OH radical. The PBN/dichloroacetate radical adduct has been identified by GC/MS in both a chemical Fenton system and in rodent microsomal incubations with TCA as substrate.

  16. Metabolism of indole alkaloid tumor promoter, (-)-indolactam V, which has the fundamental structure of teleocidins, by rat liver microsomes

    Energy Technology Data Exchange (ETDEWEB)

    Hagiwara, N.; Irie, K.; Tokuda, H.; Koshimizu, K.

    1987-07-01

    Metabolic activation and/or deactivation of indole alkaloid tumor promoter, (-)-indolactam V (ILV), was examined using rat liver microsomes. Reaction of ILV with the microsomes supplemented with NADPH and MgCl/sub 2/ gave three major metabolites, which were identified as (-)-N13-desmethylindolactam V and two diastereomers of (-)-2-oxyindolactam V at C-3. The tumor-promoting activities of these metabolites were evaluated by induction of Epstein-Barr virus early antigen and inhibition of specific binding of (/sup 3/H)-12-O-tetradecanoylphorbol-13-acetate to a mouse epidermal particulate fraction, and proved to be conspicuously lower than that of ILV. These results demonstrate that the metabolism of ILV results in detoxification, and that it itself is the tumor-promoting entity. Studies on the enzymes concerned with this metabolism suggested the involvement of cytochrome P-450-containing mixed-function oxidases. Similar deactivation seems to be possible by skin, where the mixed-function oxidases are known to exist.

  17. Effect of water miscible organic solvents on p-nitrophenol hydroxylase (CYP2E1 activity in rat liver microsomes

    Directory of Open Access Journals (Sweden)

    Pranali G Patil

    2015-01-01

    Full Text Available Organic solvents used for solubilization of the substrates/NCEs are known to affect the activity of cytochrome P450 enzymes. Further, this effect varies with the solvents used, the substrates and CYP450 isoforms in question. In the present study, we have investigated the effect of ten commonly used water miscible organic solvents (methanol, ethanol, 1-propanol, 2-propanol, acetonitrile, acetone, dimethyl sulphoxide, N,N-dimethyl formamide, dioxane and polyethylene glycol 400 on p-nitrophenol hydroxylase activity at 0, 0.1, 0.25, 0.5, 0.75 and 1% v/v concentration in rat liver microsomes. All the solvents studied showed concentration dependent inhibition of the p-nitrophenol hydroxylase activity except acetonitrile which showed activation of the activity at concentration range studied. Out of ten solvents studied, dioxane was found to be the most inhibitory solvent (inhibition >90% at 0.25% v/v concentration. Overall, solvents like dimethyl sulphoxide, dimethyl formamide and dioxane appeared to be unsuitable for characterizing p-nitrophenol hydroxylase (CYP2E1-mediated reactions due to a high degree of inhibition. On the other hand, methanol and acetonitrile at concentrations <0.5% v/v appeared to be appropriate solvents for substrate solubilization while evaluating CYP2E1-mediated catalysis. The results of this study imply that caution should be exercised while choosing solvents for dissolution of substrate during enzyme studies in liver microsomes.

  18. The Relationship Between Senescence and Ca2+-ATPase Activity of Microsomal Membrane and Lipid Peroxidation in Harvested Peach Fruit

    Institute of Scientific and Technical Information of China (English)

    GUAN Jun-feng; FAN Xiu-cai; DOU Shi-juan; ZHANG Ji-shu; LI Guang-min

    2006-01-01

    Peach fruit easily soften and have a short storage time at normal temperature. In this study, peach fruit (Prunus persica sieb et Zucc cv. Yingqing) were picked and stored at 25 and 4℃ to investigate the senescence in correlation with Ca2+- ATPase activity of microsomal membrane and lipid peroxidation during ripening and senescence. In comparison with that stored at 25℃, the fruit stored at 4℃ exhibited a higher flesh firmness, lower respiration rate, and generated the late bigger peak value of Ca2+-ATPase activity as well as maintained the higher activity of the enzyme. Meanwhile, the lower levels of super oxygen radical (O2-) production and content of malondialdehyde (MDA), a product of membrane lipid peroxidation were observed. Sodium orthovanadate (SO) and erythrosin B (EB), as Ca2+-ATPase inhibitors, could stimulate the respiration rate. The results suggested that the slower senescence rate of peach fruit was closely related to the higher peak value and longer duration of Ca2+-ATPase activity in microsomal membrane, with the slighter membrane lipid peroxidation and lower O2(-) production rate.

  19. Immunochemical detection of cytochrome P450 enzymes in small intestine microsomes of male and female untreated juvenile cynomolgus monkeys.

    Science.gov (United States)

    Uehara, Shotaro; Murayama, Norie; Nakanishi, Yasuharu; Nakamura, Chika; Hashizume, Takanori; Zeldin, Darryl C; Yamazaki, Hiroshi; Uno, Yasuhiro

    2014-09-01

    The expression of small intestinal cytochromes P450 (P450s) has not been systematically measured in cynomolgus monkeys, which are widely used in preclinical drug studies to predict pharmacokinetics and toxicity in humans: therefore, P450 content of small intestine was quantified in 35 cynomolgus monkeys by immunoblotting using 11 selective antibodies. CYP2D, CYP2J2, CYP3A4 and CYP3A5 were detected in all 35 animals, while CYP1A and CYP2C9/19 were detected in 31 and 17 animals, respectively. CYP2C9 and CYP2C19 were detected with the same antibody. CYP1D, CYP2A, CYP2B6, CYP2C76 and CYP2E1 were not detected in any of the 35 animals examined. On analysis of pooled microsomes (35 animals), CYP3A (3A4+3A5) was most abundant (79% of total immunoquantified CYP1-3 proteins), followed by CYP2J2 (13%), CYP2C9/19 (4%), CYP1A (3%) and CYP2D (0.4%). On the analysis of individual microsome samples, each P450 content varied 2-to-6-fold between animals, and no sex differences were observed in any P450 content. These findings should help to increase the understanding of drug metabolism, especially the first-pass effect, in cynomolgus monkey small intestines.

  20. Role of cytochrome P450 and UDP-glucuronosyltransferases in metabolic pathway of homoegonol in human liver microsomes.

    Science.gov (United States)

    Kwon, Soon Sang; Kim, Ju Hyun; Jeong, Hyeon-Uk; Ahn, Kyung-Seop; Oh, Sei-Ryang; Lee, Hye Suk

    2015-08-01

    Homoegonol is being evaluated for the development of a new antiasthmatic drug. Based on a pharmacokinetic study of homoegonol in rats, homoegonol is almost completely eliminated via metabolism, but no study on its metabolism has been reported in animals and humans. Incubation of homoegonol in human liver microsomes in the presence of the reduced form of nicotinamide adenine dinucleotide phosphate and UDP-glucuronic acid resulted in the formation of five metabolites: 4-O-demethylhomoegonol (M1), hydroxyhomoegonol (M2 and M3), 4-O-demethylhomoegonol glucuronide (M4), and homoegonol glucuronide (M5). We characterized the cytochrome P450 (CYP) and UDP-glucuronosyltransferase (UGT) enzymes responsible for homoegonol metabolism using human liver microsomes, and cDNA-expressed CYP and UGT enzymes. CYP1A2 played a more prominent role than CYP3A4 and CYP2D6 in the 4-O-demethylation of homoegonol to M1. CYP3A4 was responsible for the hydroxylation of homoegonol to M2. The hydroxylation of homoegonol to M3 was insufficient to characterize CYP enzymes. Glucuronidation of homoegonol to M5 was mediated by UGT1A1, UGT1A3, UGT1A4, and UGT2B7 enzymes, whereas M4 was formed from 4-O-demethylhomoegonol by UGT1A1, UGT1A8, UGT1A10, and UGT2B15 enzymes.

  1. Proteomic Profiling of the Microsomal Root Fraction: Discrimination of Pisum sativum L. Cultivars and Identification of Putative Root Growth Markers

    Science.gov (United States)

    Meisrimler, Claudia-Nicole; Wienkoop, Stefanie; Lüthje, Sabine

    2017-01-01

    Legumes are a large and economically important family, containing a variety of crop plants. Alongside different cereals, some fruits, and tropical roots, a number of leguminosae evolved for millennia as crops with human society. One of these legumes is Pisum sativum L., the common garden pea. In the past, breeding has been largely selective on improved above-ground organs. However, parameters, such as root-growth, which determines acquisition of nutrients and water, have largely been underestimated. Although the genome of P. sativum is still not fully sequenced, multiple proteomic studies have been published on a variety of physiological aspects in the last years. The presented work focused on the connection between root length and the influence of the microsomal root proteome of four different pea cultivars after five days of germination (cultivar Vroege, Girl from the Rhineland, Kelvedon Wonder, and Blauwschokker). In total, 60 proteins were identified to have significantly differential abundances in the four cultivars. Root growth of five-days old seedlings and their microsomal proteome revealed a similar separation pattern, suggesting that cultivar-specific root growth performance is explained by differential membrane and ribosomal protein levels. Hence, we reveal and discuss several putative root growth protein markers possibly playing a key role for improved primary root growth breeding strategies. PMID:28257117

  2. Microgravity induces changes in microsome-associated proteins of Arabidopsis seedlings grown on board the international space station.

    Directory of Open Access Journals (Sweden)

    Christian Mazars

    Full Text Available The "GENARA A" experiment was designed to monitor global changes in the proteome of membranes of Arabidopsis thaliana seedlings subjected to microgravity on board the International Space Station (ISS. For this purpose, 12-day-old seedlings were grown either in space, in the European Modular Cultivation System (EMCS under microgravity or on a 1 g centrifuge, or on the ground. Proteins associated to membranes were selectively extracted from microsomes and identified and quantified through LC-MS-MS using a label-free method. Among the 1484 proteins identified and quantified in the 3 conditions mentioned above, 80 membrane-associated proteins were significantly more abundant in seedlings grown under microgravity in space than under 1 g (space and ground and 69 were less abundant. Clustering of these proteins according to their predicted function indicates that proteins associated to auxin metabolism and trafficking were depleted in the microsomal fraction in µg space conditions, whereas proteins associated to stress responses, defence and metabolism were more abundant in µg than in 1 g indicating that microgravity is perceived by plants as a stressful environment. These results clearly indicate that a global membrane proteomics approach gives a snapshot of the cell status and its signaling activity in response to microgravity and highlight the major processes affected.

  3. Microgravity Induces Changes in Microsome-Associated Proteins of Arabidopsis Seedlings Grown on Board the International Space Station

    Science.gov (United States)

    Grat, Sabine; Pichereaux, Carole; Rossignol, Michel; Pereda-Loth, Veronica; Eche, Brigitte; Boucheron-Dubuisson, Elodie; Le Disquet, Isabel; Medina, Francisco Javier; Graziana, Annick; Carnero-Diaz, Eugénie

    2014-01-01

    The “GENARA A” experiment was designed to monitor global changes in the proteome of membranes of Arabidopsis thaliana seedlings subjected to microgravity on board the International Space Station (ISS). For this purpose, 12-day-old seedlings were grown either in space, in the European Modular Cultivation System (EMCS) under microgravity or on a 1 g centrifuge, or on the ground. Proteins associated to membranes were selectively extracted from microsomes and identified and quantified through LC-MS-MS using a label-free method. Among the 1484 proteins identified and quantified in the 3 conditions mentioned above, 80 membrane-associated proteins were significantly more abundant in seedlings grown under microgravity in space than under 1 g (space and ground) and 69 were less abundant. Clustering of these proteins according to their predicted function indicates that proteins associated to auxin metabolism and trafficking were depleted in the microsomal fraction in µg space conditions, whereas proteins associated to stress responses, defence and metabolism were more abundant in µg than in 1 g indicating that microgravity is perceived by plants as a stressful environment. These results clearly indicate that a global membrane proteomics approach gives a snapshot of the cell status and its signaling activity in response to microgravity and highlight the major processes affected. PMID:24618597

  4. Microgravity induces changes in microsome-associated proteins of Arabidopsis seedlings grown on board the international space station.

    Science.gov (United States)

    Mazars, Christian; Brière, Christian; Grat, Sabine; Pichereaux, Carole; Rossignol, Michel; Pereda-Loth, Veronica; Eche, Brigitte; Boucheron-Dubuisson, Elodie; Le Disquet, Isabel; Medina, Francisco Javier; Graziana, Annick; Carnero-Diaz, Eugénie

    2014-01-01

    The "GENARA A" experiment was designed to monitor global changes in the proteome of membranes of Arabidopsis thaliana seedlings subjected to microgravity on board the International Space Station (ISS). For this purpose, 12-day-old seedlings were grown either in space, in the European Modular Cultivation System (EMCS) under microgravity or on a 1 g centrifuge, or on the ground. Proteins associated to membranes were selectively extracted from microsomes and identified and quantified through LC-MS-MS using a label-free method. Among the 1484 proteins identified and quantified in the 3 conditions mentioned above, 80 membrane-associated proteins were significantly more abundant in seedlings grown under microgravity in space than under 1 g (space and ground) and 69 were less abundant. Clustering of these proteins according to their predicted function indicates that proteins associated to auxin metabolism and trafficking were depleted in the microsomal fraction in µg space conditions, whereas proteins associated to stress responses, defence and metabolism were more abundant in µg than in 1 g indicating that microgravity is perceived by plants as a stressful environment. These results clearly indicate that a global membrane proteomics approach gives a snapshot of the cell status and its signaling activity in response to microgravity and highlight the major processes affected.

  5. Effect of charge compensator ions (R{sup +} = Li{sup +}, Na{sup +} and K{sup +}) on Sr{sub 2}MgSi{sub 2}O{sub 7}:Dy{sup 3+} phosphors by solid-state reaction method

    Energy Technology Data Exchange (ETDEWEB)

    Sahu, Ishwar Prasad [Pt. Ravishankar Shukla University, School of Studies in Physics and Astrophysics, Raipur, Chhattisgarh (India)

    2016-09-15

    The Sr{sub 2}MgSi{sub 2}O{sub 7}:Dy{sup 3+} and Sr{sub 2}MgSi{sub 2}O{sub 7}:Dy{sup 3+}, R{sup +} (R{sup +} = Li{sup +}, Na{sup +} and K{sup +}) phosphors were prepared by solid-state reaction method. The crystal structures of sintered phosphors were an akermanite-type structure which belongs to the tetragonal crystallography. The prepared phosphors were excited at 350 nm, and their corresponding emission spectrum were recorded at blue (482 nm) and yellow (575 nm) region due to the {sup 4}F{sub 9/2} → {sup 6}H{sub 15/2} and {sup 4}F{sub 9/2} → {sup 6}H{sub 13/2} transitions, respectively, of Dy{sup 3+} ions. Commission Internationale de L'Eclairage coordinates have been calculated for each sample and its value exhibited that overall emission is near white light. The possible mechanisms of discussed white light emitting phosphors were also investigated. In order to investigate the suitability of the samples as white color light sources for industrial uses, color purity, correlated color temperature (CCT) and color rendering index (CRI) were calculated. Values of color purity, CCT and CRI were found well within the defined acceptable range. With incorporating (R{sup +} = Li{sup +}, Na{sup +} and K{sup +}) as charge compensator ions, the emission intensity of Sr{sub 2}MgSi{sub 2}O{sub 7}:Dy{sup 3+} can be obviously enhanced. The results indicate that prepared phosphors may be a potential application in display devices. (orig.)

  6. Differential expression of human cytochrome P450 enzymes from the CYP3A subfamily in the brains of alcoholic subjects and drug-free controls.

    Science.gov (United States)

    Booth Depaz, Iris M; Toselli, Francesca; Wilce, Peter A; Gillam, Elizabeth M J

    2013-06-01

    Cytochrome P450 enzymes are responsible for the metabolism of most commonly used drugs. Among these enzymes, CYP3A forms mediate the clearance of around 40-50% of drugs and may also play roles in the biotransformation of endogenous compounds. CYP3A forms are expressed both in the liver and extrahepatically. However, little is known about the expression of CYP3A proteins in specific regions of the human brain. In this study, form-selective antibodies raised to CYP3A4 and CYP3A5 were used to characterize the expression of these forms in the human brain. Both CYP3A4 and CYP3A5 immunoreactivity were found to varying extents in the microsomal fractions of cortex, hippocampus, basal ganglia, amygdala, and cerebellum. However, only CYP3A4 expression was observed in the mitochondrial fractions of these brain regions. N-terminal sequencing confirmed the principal antigen detected by the anti-CYP3A4 antibody in cortical microsomes to be CYP3A4. Immunohistochemical analysis revealed that CYP3A4 and CYP3A5 expression was primarily localized in the soma and axonal hillock of neurons and varied according to cell type and cell layer within brain regions. Finally, analysis of the frontal cortex of chronic alcohol abusers revealed elevated expression of CYP3A4 in microsomal but not mitochondrial fractions; CYP3A5 expression was unchanged. The site-specific expression of CYP3A4 and CYP3A5 in the human brain may have implications for the role of these enzymes in both normal brain physiology and the response to drugs.

  7. Brain death.

    Science.gov (United States)

    Wijdicks, Eelco F M

    2013-01-01

    The diagnosis of brain death should be based on a simple premise. If every possible confounder has been excluded and all possible treatments have been tried or considered, irreversible loss of brain function is clinically recognized as the absence of brainstem reflexes, verified apnea, loss of vascular tone, invariant heart rate, and, eventually, cardiac standstill. This condition cannot be reversed - not even partly - by medical or surgical intervention, and thus is final. Many countries in the world have introduced laws that acknowledge that a patient can be declared brain-dead by neurologic standards. The U.S. law differs substantially from all other brain death legislation in the world because the U.S. law does not spell out details of the neurologic examination. Evidence-based practice guidelines serve as a standard. In this chapter, I discuss the history of development of the criteria, the current clinical examination, and some of the ethical and legal issues that have emerged. Generally, the concept of brain death has been accepted by all major religions. But patients' families may have different ideas and are mostly influenced by cultural attitudes, traditional customs, and personal beliefs. Suggestions are offered to support these families.

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

    DEFF Research Database (Denmark)

    Jørgensen, L; Johansen, Torben

    1983-01-01

    The rate of conversion of p-nitrophenetole to p-nitrophenol by rat liver microsomes was studied. Inhibition of the reaction by CO and by SKF 525A and the absolute dependence on NADPH and oxygen indicate that cytochrome P-450 catalyzes the reaction. The apparent Km for oxygen was 0.07 micro...

  9. Species- and gender-dependent differences in the glucuronidation of a flavonoid glucoside and its aglycone determined using expressed UGT enzymes and microsomes.

    Science.gov (United States)

    Dai, Peimin; Luo, Feifei; Wang, Ying; Jiang, Huangyu; Wang, Liping; Zhang, Guiyu; Zhu, Lijun; Hu, Ming; Wang, Xinchun; Lu, Linlin; Liu, Zhongqiu

    2015-12-01

    Flavonoids occur naturally as glucosides and aglycones. Their common phenolic hydroxyl groups may trigger extensive UDP-glucuronosyltransferase (UGT)- catalysed metabolism. Unlike aglycones, glucosides contain glucose moieties. However, the influence of these glucose moieties on glucuronidation of glucosides and aglycones remains unclear. In this study, the flavonoid glucoside tilianin and its aglycone acacetin were used as model compounds. The glucuronidation characteristics and enzyme kinetics of tilianin and acacetin were compared using human UGT isoforms, liver microsomes and intestinal microsomes obtained from different animal species. Tilianin and acacetin were metabolized into different glucuronides, with UGT1A8 produced as the main isoform. Assessment of enzyme kinetics in UGT1A8, human liver microsomes and human intestinal microsomes revealed that compared with tilianin, acacetin displayed lower Km (0.6-, 0.7- and 0.6-fold, respectively), higher Vmax (20-, 60- and 230-fold, respectively) and higher clearance (30-, 80- and 300-fold, respectively). Furthermore, glucuronidation of acacetin and tilianin showed significant species- and gender-dependent differences. In conclusion, glucuronidation of flavonoid aglycones is faster than that of glucosides in the intestine and the liver. Understanding the metabolism and species- and gender-dependent differences between glucosides and aglycones is crucial for the development of drugs from flavonoids.

  10. Phase I metabolism of 3-methylindole, an environmental pollutant, by hepatic microsomes from carp (Cyprinus carpio) and rainbow trout (Oncorhynchus mykiss).

    Science.gov (United States)

    Zlabek, Vladimir; Burkina, Viktoriia; Borrisser-Pairó, Francesc; Sakalli, Sidika; Zamaratskaia, Galia

    2016-05-01

    We studied the in vitro metabolism of 3-methylindole (3MI) in hepatic microsomes from fish. Hepatic microsomes from juvenile and adult carp (Cyprinus carpio) and rainbow trout (Oncorhynchus mykiss) were included in the study. Incubation of 3MI with hepatic microsomes revealed the time-dependent formation of two major metabolites, 3-methyloxindole (3MOI) and indole-3-carbinol (I3C). The rate of 3MOI production was similar in both species at both ages. No differences in kinetic parameters were observed (p = 0.799 for Vmax, and p = 0.809 for Km). Production of I3C was detected only in the microsomes from rainbow trout. Km values were similar in juvenile and adult fish (p = 0.957); Vmax was higher in juvenile rainbow trout compared with adults (p = 0.044). In rainbow trout and carp, ellipticine reduced formation of 3MOI up to 53.2% and 81.9% and ketoconazole up to 65.8% and 91.3%, respectively. The formation of I3C was reduced by 53.7% and 51.5% in the presence of the inhibitors ellipticine and ketoconazole, respectively. These findings suggest that the CYP450 isoforms CYP1A and CYP3A are at least partly responsible for 3MI metabolism. In summary, 3MI is metabolised in fish liver to 3MOI and I3C by CYP450, and formation of these metabolites might be species-dependent.

  11. Glucuronidation of zearalenone, zeranol and four metabolites in vitro: formation of glucuronides by various microsomes and human UDP-glucuronosyltransferase isoforms.

    Science.gov (United States)

    Pfeiffer, Erika; Hildebrand, Andreas; Mikula, Hannes; Metzler, Manfred

    2010-10-01

    Glucuronidation constitutes an important pathway in the phase II metabolism of the mycotoxin zearalenone (ZEN) and the growth promotor α-zearalanol (α-ZAL, zeranol), but the enzymology of their formation is yet unknown. In the present study, ZEN, α-ZAL and four of their major phase I metabolites were glucuronidated in vitro using hepatic microsomes from steer, pig, rat and human, intestinal microsomes from humans, and eleven recombinant human UDP-glucuronosyltransferases (UGTs). After assigning chemical structures to the various glucuronides by using previously published information, the enzymatic activities of the various microsomes and UGT isoforms were determined together with the patterns of glucuronides generated. All six compounds were good substrates for all microsomes studied. With very few exceptions, glucuronidation occurred preferentially at the sterically unhindered phenolic 14-hydroxyl group. UGT1A1, 1A3 and 1A8 had the highest activities and gave rise to the phenolic glucuronide, whereas glucuronidation of the aliphatic hydroxyl group was mostly mediated by UGT2B7 with low activity. Based on these in vitro data, ZEN, α-ZAL and their metabolites must be expected to be readily glucuronidated both in the liver and intestine as well as in other extrahepatic organs of humans and various animal species.

  12. Contrasting Influence of NADPH and a NADPH-Regenerating System on the Metabolism of Carbonyl-Containing Compounds in Hepatic Microsomes

    Science.gov (United States)

    Carbonyl containing xenobiotics may be susceptible to NADPH-dependent cytochrome P450 (P450) and carbonyl-reduction reactions. In vitro hepatic microsome assays are routinely supplied NADPH either by direct addition of NADPH or via an NADPH-regenerating system (NRS). In contrast ...

  13. Effects of dietary phenochlor DP5 on microsomal enzymes, liver, and blood lipids in adult male and female rats after subchronic and perinatal exposures

    Energy Technology Data Exchange (ETDEWEB)

    Poul, J.M.

    1987-08-01

    PCBs have numerous toxic effects on laboratory animals, namely hepatotoxicity, immunotoxicity, reproductive and hormonal effects, mutagenic and carcinogenic potency (Safe 1984). They have been recognized as potent inducers of many microsomal drug metabolizing enzymes in several species. Moreover, treatment of rats with PCBs gave rise to altered lipid metabolism with accumulation of lipids in the liver. In most of these studies male rats have been used. However, sex differences in the effects of xenobiotics on microsomal drug metabolizing enzymes have been shown particularly with PCBs and little was known about differences in the effects of PCBs on lipid metabolism. This study was designed to investigate the effects of a subchronic treatment with Phenochlor DP5 on some microsomal drug metabolizing enzyme activities and on liver and blood lipids of male and female rats. The long-term effects of DP5 administration during pre and postnatal period on adult microsomal enzyme activities and liver and blood lipids of both sexes have also been studied. A possible xenobiotic imprinting of the hepatic monooxygenase system during neonatal period has been shown recently, and it has been recognized that some functional defects which often manifest themselves in adult period may be induced prenatally or before weaning.

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

    Science.gov (United States)

    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.

  15. Brain computer

    Directory of Open Access Journals (Sweden)

    Sarah N. Abdulkader

    2015-07-01

    Full Text Available Brain computer interface technology represents a highly growing field of research with application systems. Its contributions in medical fields range from prevention to neuronal rehabilitation for serious injuries. Mind reading and remote communication have their unique fingerprint in numerous fields such as educational, self-regulation, production, marketing, security as well as games and entertainment. It creates a mutual understanding between users and the surrounding systems. This paper shows the application areas that could benefit from brain waves in facilitating or achieving their goals. We also discuss major usability and technical challenges that face brain signals utilization in various components of BCI system. Different solutions that aim to limit and decrease their effects have also been reviewed.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-01-15

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

  17. Pretreatment with turmeric modulates the inhibitory influence of cisplatin and paclitaxel on CYP2E1 and CYP3A1/2 in isolated rat hepatic microsomes.

    Science.gov (United States)

    Ahmed, Enas M; EL-Maraghy, Shohda A; Teleb, Zakaria A; Shaheen, Amira A

    2014-09-05

    Previous animal studies have shown that turmeric can significantly modulate the activity of several drug metabolizing enzymes, this may dramatically affect the bioavailability of several drugs resulting in over dose or less therapeutic effects. This study was directed to evaluate the inhibitory effects of cisplatin and paclitaxel on two CYP450 enzymes namely CYP2E1 and CYP3A1/2 in hepatic microsomes isolated from normal and turmeric pretreated rats. Cisplatin and paclitaxel were added by different concentrations to hepatic microsomes isolated from untreated and turmeric (100 mg/kg/day) pretreated rats for 15 days after receiving pyrazole or dexamethasone for induction of CYP2E1 and CYP3A1/2 respectively. The kinetic potency of these drugs as CYP inhibitors was determined by analysis of Lineweaver-Burk plot. Addition of cisplatin or paclitaxel by (10, 50 and 100 μM) to hepatic microsomes from normal or turmeric pretreated rats caused a concentration dependent inhibition of CYP2E1, with an evidence of less inhibition in turmeric pretreated microsomes particularly at higher concentration. Both drugs at 100 μM displayed a mixed type of inhibition of CYP2E1 in normal or turmeric pretreated microsomes where paclitaxel was the most potent inhibitor. Cisplatin (10, 50 and 100 μM) caused a concentration dependant inhibition of CYP3A1/2 that was enhanced by turmeric pretreatment. The inhibition of CYP3A1/2 by cisplatin (100 μM) was in non-competitive manner with a smaller Ki value in turmeric pretreated microsomes. The inhibitory influence of paclitaxel (10, 50 and 100 μM) on CYP3A1/2 decreased with increasing the drug concentration and this inhibition was augmented by turmeric pretreatment. Interestingly, the inhibition of this enzyme by paclitaxel (10 μM) was switched from mixed type in normal microsomes to competitive manner in turmeric pretreated ones with a marked reduction of Ki values reflecting greater inhibitory influence of paclitaxel on CYP3A1/2 by turmeric

  18. 2,2',3,3',6,6'-Hexachlorobiphenyl (PCB 136) is enantioselectively oxidized to hydroxylated metabolites by rat liver microsomes.

    Science.gov (United States)

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

    2011-12-19

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

  19. [Inhibitory effect of imperatorin and isoimperatorin on activity of cytochrome P450 enzyme in human and rat liver microsomes].

    Science.gov (United States)

    Cao, Yan; Zhong, Yu-Huan; Yuan, Mei; Li, Hua; Zhao, Chun-Jie

    2013-04-01

    Imperatorin (IM) and isoimperatorin (ISOIM) are major active components of common herbal medicines from Umbelliferae plants, and widely used in clinic. This article studies the inhibitory effect of IM and ISOIM on the activity of cytochrome P450 (CYP) enzyme, and assesses their potential drug-drug interaction. IM and ISOIM were incubated separately with human or rat liver microsomes for 30 min, with phenacetin, bupropion, tolbutamide, S-mephenytoin, dextromethorphan and midazolam as probe substrates. Metabolites of the CYP probe substrates were determined by LC-MS/MS, and IC50 values were calculated to assess the inhibitory effect of the two drugs on human CYP1A2, 2B6, 2C9, 2C19, 2D6 and 3A4 enzymes, as well as on rat CYP1A2, 2B6, 2D2 and 3A1/2, and grade their inhibitory intensity. In human liver microsomes, IM and ISOIM showed different inhibitory effects on all of the six CYP isoenzymes. They were strong inhibitors for 1A2 and 2B6. The IC50 values were 0.05 and 0.20 micromol x L(-1) for 1A2, and 0.18 and 1.07 micromol x L(-1) for 2B6, respectively. They also showed moderate inhibitory effect on 2C19, and weak effect on 2C9, 2D6 and 3A4. In rat liver microsomes, IM and ISOIM were identified as moderate inhibitors for 1A2, with IC50 values of 1.95 and 2.98 micromol x L(-1). They were moderate and weak inhibitors for 2B6, with IC50 values of 6.22 and 21.71 micromol x L(-1), respectively. They also had weaker inhibitory effect on 2D2 and 3A1/2. The results indicated that IM and ISOIM had extensive inhibitory effects on human CYP enzymes. They are strong inhibitors of CYP1 A2 and 2B6 enzymes. However, it is worth noting the interaction arising from the inhibitory effect of CYP enzymes in clinic.

  20. A critical role for astrocytes in hypercapnic vasodilation in brain

    DEFF Research Database (Denmark)

    Howarth, C; Sutherland, B A; Choi, H B

    2017-01-01

    Cerebral blood flow (CBF) is controlled by arterial blood pressure, arterial CO2, arterial O2, and brain activity and is largely constant in the awake state. Although small changes in arterial CO2 are particularly potent to change CBF (1 mmHg variation in arterial CO2 changes CBF by 3-4%), the co......Cerebral blood flow (CBF) is controlled by arterial blood pressure, arterial CO2, arterial O2, and brain activity and is largely constant in the awake state. Although small changes in arterial CO2 are particularly potent to change CBF (1 mmHg variation in arterial CO2 changes CBF by 3...... in brain slices with in vivo work in rats and C57Bl/6J mice to examine the hemodynamic responses to CO2 and somatosensory stimulation before and after inhibition of astrocytic glutathione and PgE2 synthesis. We demonstrate that hypercapnia (increased CO2) evokes an increase in astrocyte [Ca(2+)]i...... and stimulates COX-1 activity. The enzyme downstream of COX-1 that synthesizes PgE2 (microsomal prostaglandin E synthase-1) depends critically for its vasodilator activity on the level of glutathione in the brain. We show that when glutathione levels are reduced, astrocyte calcium-evoked release of PgE2...

  1. Determination of metabolic profile of novel triethylamine containing thiophene S006-830 in rat, rabbit, dog and human liver microsomes.

    Science.gov (United States)

    Hidau, Mahendra Kumar; Singh, Yeshwant; Singh, Shio Kumar

    2016-02-01

    CDRI S006-830 is a potent triethylamine containing thiophene antitubercular compound of the Central Drug Research Institute, India. The present study aimed to conduct comprehensive metabolic investigations of CDRI S006-830 to corroborate its preclinical investigations. Preliminary metabolic investigations were performed to assess the metabolic stability, enzyme kinetics, reaction phenotyping, and metabolite identification of CDRI S006-830 in rat, rabbit, dog, and human liver microsomes using liquid chromatography with mass spectrometry. The observed in vitro t1/2 and Clint values were 9.9 ± 1.29, 4.5 ± 0.52, 4.5 ± 0.86, 17 ± 5.21 min and 69.60 ± 8.37, 152.0 ± 17.26, 152.34 ± 27.63, 33.62 ± 21.04 μL/min/mg in rat, rabbit, dog and human liver microsomes respectively. These observations suggested that CDRI S006-830 rapidly metabolized in the presence of NADPH in liver microsomes of rat, rabbit and dog while moderately metabolized in human liver microsomes. It was observed that CDRI S006-830 exhibited monophasic Michaelis-Menten kinetics. The metabolism of CDRI S006-830 was primarily mediated by CYP3A4 and was deduced by CYP reaction phenotyping with known potent inhibitors. CYP3A4 involvement was also confirmed by cDNA-expressed recombinant human isozyme activity with different CYPs. Four major phase-I metabolites of S006-830, (M-1 to M-4) were detected in rat, rabbit, dog (except M4) and human liver microsomes.

  2. Examination of microsomal cytochrome P450-catalyzed in vitro activation of o-phenylphenol to DNA binding metabolite(s) by 32P-postlabeling technique.

    Science.gov (United States)

    Pathak, D N; Roy, D

    1992-09-01

    It has been previously reported that the reactive metabolites phenylsemiquinone and phenylbenzoquinone are generated during microsomal cytochrome P450-catalyzed redox cycling of o-phenylphenol (OPP). However, covalent modification of DNA by OPP-reactive metabolites has yet not been demonstrated. In the present study we have investigated the covalent binding in DNA by OPP-reactive metabolites using 32P-postlabeling. Analysis of adducts by 32P-postlabeling in products of chemical reaction of DNA with phenylbenzoquinone revealed four major and several minor adducts. The chemical reaction of deoxyguanosine 3'-phosphate with phenylbenzoquinone also showed four major adducts. The chromatographic mobility of major adducts of deoxyguanosine 3'-phosphate-phenylbenzoquinone was identical to that of major adducts of DNA-phenylbenzoquinone. The major adducts are demonstrated to be stable. The total covalent binding in deoxyguanosine 3'-phosphate by phenylbenzoquinone (686,000-687,000 amol/nmol nucleotide) was higher than that observed in DNA (26,500-28,000 amol/nmol nucleotides). Reaction of DNA with OPP or a hydroxylated metabolite of OPP, phenylhydroquinone, in the presence of microsomes and NADPH or cumene hydroperoxide showed four major adducts. Adduct formation in DNA by OPP or phenylhydroquinone in the presence of the microsomal activation system was drastically decreased by known inhibitors of cytochrome P450. The chromatographic mobility of major adducts in DNA by OPP or phenylhydroquinone in the presence of microsomal activation system matched with those major adducts observed in deoxyguanosine 3'-phosphate or DNA reacted with pure phenylbenzoquinone. These data demonstrate that OPP or phenylhydroquinone, a hydroxylated metabolite of OPP, is able to bind covalently to DNA in the presence of a microsomal cytochrome P450 activation system. Phenylbenzoquinone is one of the DNA-binding metabolite(s) of OPP. It is concluded that OPP is genotoxic in an in vitro system and

  3. Biotransformation of a novel antimitotic agent, I-387, by mouse, rat, dog, monkey, and human liver microsomes and in vivo pharmacokinetics in mice.

    Science.gov (United States)

    Ahn, Sunjoo; Kearbey, Jeffrey D; Li, Chien-Ming; Duke, Charles B; Miller, Duane D; Dalton, James T

    2011-04-01

    3-(1H-Indol-2-yl)phenyl)(3,4,5-trimethoxyphenyl)methanone (I-387) is a novel indole compound with antitubulin action and potent antitumor activity in various preclinical models. I-387 avoids drug resistance mediated by P-glycoprotein and showed less neurotoxicity than vinca alkaloids during in vivo studies. We examined the pharmacokinetics and metabolism of I-387 in mice as a component of our preclinical development of this compound and continued interest in structure-activity relationships for antitubulin agents. After a 1 mg/kg intravenous dose, noncompartmental pharmacokinetic analysis in plasma showed that clearance (CL), volume of distribution at steady state (Vd(ss)), and terminal half-life (t(1/2)) of I-387 were 27 ml per min/kg, 5.3 l/kg, and 7 h, respectively. In the in vitro metabolic stability study, half-lives of I-387 were between 10 and 54 min by mouse, rat, dog, monkey, and human liver microsomes in the presence of NADPH, demonstrating interspecies variability. I-387 was most stable in rat liver microsomes and degraded quickly in monkey liver microsomes. Liquid chromatography-tandem mass spectrometry was used to identify phase I metabolites. Hydroxylation, reduction of a ketone group, and O-demethylation were the major metabolites formed by the liver microsomes of the five species. The carbonyl group of I-387 was reduced and identified as the most labile site in human liver microsomes. The results of these drug metabolism and pharmacokinetic studies provide the foundation for future structural modification of this pharmacophore to improve stability of drugs with potent anticancer effects in cancer patients.

  4. Silicon Brains

    Science.gov (United States)

    Hoefflinger, Bernd

    Beyond the digital neural networks of Chap. 16, the more radical mapping of brain-like structures and processes into VLSI substrates has been pioneered by Carver Mead more than 30 years ago [1]. The basic idea was to exploit the massive parallelism of such circuits and to create low-power and fault-tolerant information-processing systems. Neuromorphic engineering has recently seen a revival with the availability of deep-submicron CMOS technology, which allows for the construction of very-large-scale mixed-signal systems combining local analog processing in neuronal cells with binary signalling via action potentials. Modern implementations are able to reach the complexity-scale of large functional units of the human brain, and they feature the ability to learn by plasticity mechanisms found in neuroscience. Combined with high-performance programmable logic and elaborate software tools, such systems are currently evolving into user-configurable non-von-Neumann computing systems, which can be used to implement and test novel computational paradigms. The chapter introduces basic properties of biological brains with up to 200 Billion neurons and their 1014 synapses, where action on a synapse takes ˜10 ms and involves an energy of ˜10 fJ. We outline 10x programs on neuromorphic electronic systems in Europe and the USA, which are intended to integrate 108 neurons and 1012 synapses, the level of a cat's brain, in a volume of 1 L and with a power dissipation design an intelligent technical response.

  5. Robot brains

    NARCIS (Netherlands)

    Babuska, R.

    2011-01-01

    The brain hosts complex networks of neurons that are responsible for behavior in humans and animals that we generally call intelligent. I is not easy to give an exact definition of intelligence – for the purpose of this talk it will suffice to say that we refer to intelligence as a collection of cap

  6. Structural elucidation of in vitro metabolites of bavachinin in rat liver microsomes by LC-ESI-MSn and chemical synthesis.

    Science.gov (United States)

    Xie, Fan; Du, Guoxin; Ma, Shunan; Li, Yiming; Wang, Rui; Guo, Fujiang

    2016-01-01

    1. Bavachinin isolated from Psoralea corylifolia has various activities, such as antimicrobial, antiallergic, antitumor and so on. Our previous study showed that natural bavachinin exhibits peroxisome proliferator-activated receptor γ-agonist activity. 2. In vitro studies on bavachinin metabolism were conducted using rat liver microsomes incubated at 37 °C for 60 min. 3. Structures of eight metabolites of the incubation mixtures were cautiously characterized using electrospray tandem mass spectra and three synthetic compounds. The results indicated that eight metabolites of bavachinin were biotransformed mainly through oxidation. 4. The metabolic pathways of bavachinin were elucidated in vitro. These results contribute to the understanding of bavachinin's in vivo metabolism.

  7. Metabolism of chicoric acid by rat liver microsomes and bioactivity comparisons of chicoric acid and its metabolites.

    Science.gov (United States)

    Liu, Qian; Wang, Yutang; Xiao, ChunXia; Wu, Wanqiang; Liu, Xuebo

    2015-06-01

    Chicoric acid has recently become a hot research topic due to its potent bioactivities. However, there are few studies relevant to this acid's pharmacokinetic characteristics and the pharmacological activities of its metabolites. To compare the abilities of chicoric acid and its metabolites in scavenging free radicals and their effects on the viability of 3T3-L1 preadipocytes, an in vitro study of the metabolism of chicoric acid in rat liver microsomes was performed using liquid tandem mass spectrometry (HPLC-MS/MS). The results indicated that caffeic acid and caftaric acid were the hepatic phase I metabolites of chicoric acid. These three compounds had strong capacities for scavenging free radicals and had been demonstrated to increase intracellular ROS levels in 3T3-L1 preadipocytes, thereby reducing cell vitality. Finally, the pharmacological activities of chicoric acid were significantly stronger than those of its metabolites within a certain concentration range.

  8. Effects of two novel sugar drug candidates on CYP450 isoforms in different sexed Chinese human liver microsome in vitro

    Institute of Scientific and Technical Information of China (English)

    SHI Jie; ZHANG Xin-hui; SU Jia-ru

    2008-01-01

    The sex-based differences between the effects of two novel sugar-based drug candidates, a sulfated polymannuroguluronate (SPMG-911) and an acidic oligosaccharide sugar chain compound (AOSC-971), on the enzymes CYP 1A2, CYP2E1 and CYP3A4 of Chinese human liver microsome were investigated. The results showed that neither SPMG-911 nor AOSC-971 have any effect on CYP3A4, AOSC-971 induced the CYP 2E1 in men but have no effect on CYP1A2, SPMG-911 inhibit the CYP1A2 also in men but have no effect on CYP2E1. The results are useful for their safety evaluation, as well as for the prediction of interdrug interactions associated with the two drugs.

  9. Mechanism on activation of mouse liver microsomal glutathione S—transferase—I by cyclophosphamide treatment in vivo

    Institute of Scientific and Technical Information of China (English)

    ZhenY; LouYJ

    2002-01-01

    Membrane-associated microsomal glutathione S-transferase-I (mGST-I) is activated easily by alkyl agent or electrophilic metabolite.It was expected that toxic drugs and their metabolites derived from biotransformation by cytochrome P-450 maybe bind to and activate the mGST-I that can accelerate the metabolism of drugs to form inactive metabolites and simultaneously protect cell from damages.The aim of the present study was to investigate whether mGST-I is activated by cyclophosphamide(CP) treatment and to explore the possible mechanism in vivo.The results suggested that the main mechanism of mGST-I activation caused by overdose CP treatment is the unique sulfhydryl modification on its Cys-49.

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

    Science.gov (United States)

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

    2016-04-01

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

  11. Inhibition of fipronil and nonane metabolism in human liver microsomes and human cytochrome P450 isoforms by chlorpyrifos.

    Science.gov (United States)

    Joo, Hyun; Choi, Kyoungju; Rose, Randy L; Hodgson, Ernest

    2007-01-01

    Previous studies have established that chlorpyrifos (CPS), fipronil, and nonane can all be metabolized by human liver microsomes (HLM) and a number of cytochrome P450 (CYP) isoforms. However, metabolic interactions between these three substrates have not been described. In this study the effect of either coincubation or preincubation of CPS with HLM or CYP isoforms with either fipronil or nonane as substrate was investigated. In both co- and preincubation experiments, CPS significantly inhibited the metabolism of fipronil or nonane by HLM although CPS inhibited the metabolism of fipronil more effectively than that of nonane. CPS significantly inhibited the metabolism of fipronil by CYP3A4 as well as the metabolism of nonane by CYP2B6. In both cases, preincubation with CPS caused greater inhibition than coincubation, suggesting that the inhibition is mechanism based.

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

    Directory of Open Access Journals (Sweden)

    Ju-Hyun Kim

    2017-03-01

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

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

    Directory of Open Access Journals (Sweden)

    Shiv Bahadur

    2016-01-01

    Full Text Available 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 IC 50 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 IC 50 (μg/ml value was 136.45 (CYP3A4, 144.37 (CYP2D6, 127.55 (CYP2C9, and 141.82 (CYP1A2. Tinosporaside and extract showed higher IC 50 (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 IC 50 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.

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

    Science.gov (United States)

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

    2016-01-01

    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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1986-05-01

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

  16. Application of parallel liquid chromatography/mass spectrometry for high throughput microsomal stability screening of compound libraries.

    Science.gov (United States)

    Xu, Rongda; Nemes, Csaba; Jenkins, Kelly M; Rourick, Robyn A; Kassel, Daniel B; Liu, Charles Z C

    2002-02-01

    Solution-phase and solid-phase parallel synthesis and high throughput screening have enabled biologically active and selective compounds to be identified at an unprecedented rate. The challenge has been to convert these hits into viable development candidates. To accelerate the conversion of these hits into lead development candidates, early assessment of the physicochemical and pharmacological properties of these compounds is being made. In particular, in vitro absorption, distribution, metabolism, and elimination (ADME) assays are being conducted at earlier and earlier stages of discovery with the goal of reducing the attrition rate of these potential drug candidates as they progress through development. In this report, we present an eight-channel parallel liquid chromatography/mass spectrometry (LC/MS) system in combination with custom Visual Basic and Applescript automated data processing applications for high throughput early ADME. The parallel LC/MS system was configured with one set of gradient LC pumps and an eight-channel multiple probe autosampler. The flow was split equivalently into eight streams before the multiple probe autosampler and recombined after the eight columns and just prior to the mass spectrometer ion source. The system was tested for column-to-column variation and for reproducibility over a 17 h period (approximately 500 injections per column). The variations in retention time and peak area were determined to be less than 2 and 10%, respectively, in both tests. The parallel LC/MS system described permits time-course microsomal incubations (t(o), t5, t15, t30) to be measured in triplicate and enables estimations of t 1/2 microsomal stability. The parallel LC/MS system is capable of analyzing up to 240 samples per hour and permits the complete profiling up to two microtiter plates of compounds per day (i.e., 176 test substrate compounds + sixteen controls).

  17. Brain tumor - children

    Science.gov (United States)

    ... children; Neuroglioma - children; Oligodendroglioma - children; Meningioma - children; Cancer - brain tumor (children) ... The cause of primary brain tumors is unknown. Primary brain tumors may ... (spread to nearby areas) Cancerous (malignant) Brain tumors ...

  18. Understanding Brain Tumors

    Science.gov (United States)

    ... to Know About Brain Tumors . What is a Brain Tumor? A brain tumor is an abnormal growth
 ... Tumors” from Frankly Speaking Frankly Speaking About Cancer: Brain Tumors Download the full book Questions to ask ...

  19. Brain Tumors (For Parents)

    Science.gov (United States)

    ... Old Feeding Your 1- to 2-Year-Old Brain Tumors KidsHealth > For Parents > Brain Tumors Print A ... radiation therapy or chemotherapy, or both. Types of Brain Tumors There are many different types of brain ...

  20. Brain and Nervous System

    Science.gov (United States)

    ... Your 1- to 2-Year-Old Brain and Nervous System KidsHealth > For Parents > Brain and Nervous System Print ... brain is quite the juggler. Anatomy of the Nervous System If you think of the brain as a ...

  1. The Creative Brain.

    Science.gov (United States)

    Herrmann, Ned

    1982-01-01

    Outlines the differences between left-brain and right-brain functioning and between left-brain and right-brain dominant individuals, and concludes that creativity uses both halves of the brain. Discusses how both students and curriculum can become more "whole-brained." (Author/JM)

  2. Brain and Addiction

    Science.gov (United States)

    ... Search Term(s): Teens / Drug Facts / Brain and Addiction Brain and Addiction Print Your Brain Your brain is who you are. It’s what ... solve problems, and make decisions. How Does Your Brain Communicate? The brain is a complex communications network ...

  3. Quantum Brain?

    CERN Document Server

    Mershin, A; Skoulakis, E M C

    2000-01-01

    In order to create a novel model of memory and brain function, we focus our approach on the sub-molecular (electron), molecular (tubulin) and macromolecular (microtubule) components of the neural cytoskeleton. Due to their size and geometry, these systems may be approached using the principles of quantum physics. We identify quantum-physics derived mechanisms conceivably underlying the integrated yet differentiated aspects of memory encoding/recall as well as the molecular basis of the engram. We treat the tubulin molecule as the fundamental computation unit (qubit) in a quantum-computational network that consists of microtubules (MTs), networks of MTs and ultimately entire neurons and neural networks. We derive experimentally testable predictions of our quantum brain hypothesis and perform experiments on these.

  4. Animating Brains

    Science.gov (United States)

    Borck, Cornelius

    2016-01-01

    A recent paper famously accused the rising field of social neuroscience of using faulty statistics under the catchy title ‘Voodoo Correlations in Social Neuroscience’. This Special Issue invites us to take this claim as the starting point for a cross-cultural analysis: in which meaningful ways can recent research in the burgeoning field of functional imaging be described as, contrasted with, or simply compared to animistic practices? And what light does such a reading shed on the dynamics and effectiveness of a century of brain research into higher mental functions? Reviewing the heated debate from 2009 around recent trends in neuroimaging as a possible candidate for current instances of ‘soul catching’, the paper will then compare these forms of primarily image-based brain research with older regimes, revolving around the deciphering of the brain’s electrical activity. How has the move from a decoding paradigm to a representational regime affected the conceptualisation of self, psyche, mind and soul (if there still is such an entity)? And in what ways does modern technoscience provide new tools for animating brains? PMID:27292322

  5. Getting the MAX out of Computational Models: The Prediction of Unbound-Brain and Unbound-Plasma Maximum Concentrations.

    Science.gov (United States)

    Mente, Scot; Doran, Angela; Wager, Travis T

    2012-06-14

    The objective of this work was to establish that unbound maximum concentrations may be reasonably predicted from a combination of computed molecular properties assuming subcutaneous (SQ) dosing. Additionally, we show that the maximum unbound plasma and brain concentrations may be projected from a mixture of in vitro absorption, distribution, metabolism, excretion experimental parameters in combination with computed properties (volume of distribution, fraction unbound in microsomes). Finally, we demonstrate the utility of the underlying equations by showing that the maximum total plasma concentrations can be projected from the experimental parameters for a set of compounds with data collected from clinical research.

  6. Anticuerpos anti 21 hidroxilasa séricos en pacientes con anticuerpos antifracción microsomal: Síndrome poliendocrino autoinmune Seric 21- hydroxilase antibodies in patients with anti-microsomal fraction antibodies: Autoimmune polyendocrine syndrome

    Directory of Open Access Journals (Sweden)

    Silvia Botta

    2007-04-01

    Full Text Available El síndrome poliendocrino autoinmune (SPA es la asociación de enfermedades endocrinas autoinmunes con otros desórdenes autoinmunes no endocrinos. Los tipos 1, 2 y 4 presentan adrenalitis autoinmune, esto indica la presencia de autoanticuerpos, y su marcador serológico específico es el anti 21 hidroxilasa (a21-OH. El SPA tipo 2 es la asociación de adrenalitis, enfermedad tiroidea y/o diabetes mellitus inducidas por autoanticuerpos. Como componentes menores, pueden estar asociados entre otros, vitiligo, alopecia y miastenia. Nuestros objetivos fueron: establecer la prevalencia de a21-OH séricos en pacientes con anticuerpos anti fracción microsomal (AFM positivos, enfermedad tiroidea autoinmune y/o afecciones endocrinas y no endocrinas autoinmunes; diagnosticar formas incompletas de SPA y estudiar individuos con probable riesgo de progresión a un SPA completo. Estudiamos 72 pacientes AFM positivos y 60 sujetos tomados como grupo control, AFM negativos. Hallamos a21-OH elevados en dos pacientes: A= 47 U/ml, hipotiroidismo autoinmune y miastenia; y B= 8.75 U/ml, hipotiroidismo autoinmune y vitiligo; ambos con ausencia de insuficiencia adrenal. La prevalencia de a21-OH encontrada fue del 2.8%. Las pacientes A y B corresponden a un SPA tipo 2 incompleto y latente en relación al componente adrenal. Considerando a los a21-OH marcadores de enfermedad autoinmune latente, el eventual riesgo de evolución hacia la afección clínica sugiere la necesidad de estrechos controles clínicos y bioquímicos periódicos.Autoimmune polyendocrine syndrome (APS is the association of autoimmune endocrine diseases, with other autoimmune nonendocrine disorders. APS types 1, 2 and 4 include autoimmune adrenalitis; this suggests the presence of autoantibodies. A specific serological marker for these is the anti 21- hydroxilase autoantibody (a21-OH. APS type 2 is the association of autoimmune adrenalitis, to autoimmune thyroid disease and/or diabetes mellitus, all

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

    DEFF Research Database (Denmark)

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

    1996-01-01

    have been investigated. 3. An incubation system where liver slices are incubated in 12-well culture plates was evaluated with respect to metabolism of metronidazole. Optimal viability was observed for a time period of up to 24 h. The Michaelis-Menten parameters for the metabolism of metronidazole......, whereas the intrinsic clearance with respect to formation of the glucuronic acid conjugate was lower in slices compared with hepatocytes. 4. The metabolism of metronidazole in liver slices, in hepatocytes in primary monolayer culture, in hepatocytes incubated in suspension, and in liver microsomes...... higher in microsomes than in the other liver preparations. The metabolic rates in hepatocytes in primary culture and in suspension with respect to the oxidative metabolites were higher than in liver slices. The metabolic turnover observed in liver slices was predicted to correlate with in vivo data...

  8. Use of high pressure liquid chromatography to study chemically induced alterations in the pattern of benzo(a)pyrene metabolism. [Rat liver microsomes

    Energy Technology Data Exchange (ETDEWEB)

    Freudenthal, R.I.; Leber, A.P.; Emmerling, D.; Clarke, P.

    1975-11-01

    The metabolism of radiolabeled benzo(a)pyrene (BP) by control, 3-methylcholanthrene (3-MC) induced and 1,1,1-trichloropropene-2,3-oxide (TCPO)-inhibited rat liver microsomes was measured using fluorescence, radiometric, and high-pressure liquid chromatographic (HPLC) assays. Significant differences in the total measurable metabolism of BP by the three microsomal enzyme incubations resulted from the use of the three assay procedures. Appreciable differences in the concentration of the metabolite fractions after 3-MC induction and TCPO inhibition are clearly demonstrated. NMR analysis revealed that while the 3-hydroxy-BP fraction is greater than 90 percent pure, the 9-hydroxy fraction contains a number of metabolites having essentially identical retention times.

  9. Structural elucidation of metabolites of ginkgolic acid in rat liver microsomes by ultra-performance liquid chromatography/electrospray ionization tandem mass spectrometry and hydrogen/deuterium exchange.

    Science.gov (United States)

    Liu, Z H; Chen, J; Yu, L S; Jiang, H D; Yao, T W; Zeng, S

    2009-07-01

    Ginkgolic acids have been shown to possess allergenic as well as genotoxic and cytotoxic properties. The question arises whether the metabolism of ginkgolic acids in the liver could decrease or increase their toxicity. In this study, the in vitro metabolism of ginkgolic acid (15:1, GA), one component of ginkgo acids, was investigated as a model compound in Sprague-Dawley rat liver microsomes. The metabolites were analyzed by ultra-performance liquid chromatography coupled with photodiode array detector/negative-ion electrospray ionization tandem mass spectrometry (UPLC-PDA/ESI-MS/MS) and hydrogen/deuterium (H/D) exchange. The result showed that the benzene ring remained unchanged and the oxidations occurred at the side alkyl chain in rat liver microsomes. At least eight metabolites were found. Among them, six phase I metabolites were tentatively identified. This study might be useful for the investigation of toxicological mechanism of ginkgolic acids.

  10. Separation, purification, and properties of cytochrome P-450 from uninduced rat liver microsomes for the studies of metabolism of environmental chemicals

    Energy Technology Data Exchange (ETDEWEB)

    Dialameh, G.H. (Tehran Univ. of Medical Sciences (Iran))

    1988-09-01

    This study reports the authors present results on the development of a procedure for purification of multiple forms of cytochrome P-450 from un-induced rat liver microsomes. These cytochromes are catalytically active when reconstituted with NADPH-cytochrome c reductase and lipid and exhibit substrate specificities. The presence of four distinct forms of cytochrome P-450 in uninduced rat liver microsomes which is the result of this research report, compared with the presence of six forms in induced animals represent the importance of genetic control of these enzymes for the metabolism and detoxification of environmental chemicals. These metabolite patterns are not only different for the various species, but also among different individuals. The molecular basis for this are genetic and environmental factors, which exhibit interesting evolutionary aspects.

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

    Science.gov (United States)

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

    2016-03-09

    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.

  12. Brain glycogen

    DEFF Research Database (Denmark)

    Obel, Linea Lykke Frimodt; Müller, Margit S; Walls, Anne B

    2012-01-01

    activity and memory formation. In line with the great spatiotemporal complexity of the brain and thereof derived focus on the basis for ensuring the availability of the right amount of energy at the right time and place, we here encourage a closer look into the molecular and subcellular mechanisms...... underlying glycogen metabolism. Based on (1) the compartmentation of the interconnected second messenger pathways controlling glycogen metabolism (calcium and cAMP), (2) alterations in the subcellular location of glycogen-associated enzymes and proteins induced by the metabolic status and (3) a sequential...

  13. [Multiphasic character of the kinetics of cytochrome P-450 destruction in microsomal LM2- and LM4-forms in the reaction with cumene hydroperoxide].

    Science.gov (United States)

    Akhrem, A A; Eremin, A N; Usanov, S A; Metelitsa, D I

    1980-01-01

    Cytochrome P-450 destruction kinetics by cumene hydroperoxide has been studied in LM2 and LM4 microsomal and purified forms. Three destruction phases of cytochrome P-450 were shown to be observed irrespective of the source and integration degree, cytochrome P-450 pseudomonomolecular consumption rate constants being dependent in a complex way upon the cumene hydroperoxide initial concentration. The radical character of cytochrome P-450 destruction was proved by experiments with 1-naphtol. The mechanism of radicals formation is discussed.

  14. Cytochrome P450 enzymes involved in the metabolic pathway of the histamine 2 (H2)-receptor antagonist roxatidine acetate by human liver microsomes.

    Science.gov (United States)

    Sasaki, M; Nakayama, M; Numazawa, S; Oguro, T; Honma, S; Iwamura, S; Tsukamoto, K; Yoshida, T

    2001-01-01

    Roxatidine acetate hydrochloride (ROX, 2-acetoxy-N-[3-[m-(1-piperidinylmethyl)phenoxy]propyl]acetamide hydrochloride, CAS 78273-80-0), a histamine 2 (H2)-receptor antagonist, has been clinically applied for the treatment of gastritis, gastric and duodenal ulcers. There is no report on the identification of the metabolic enzyme of M-1 (2-hydroxy-N-[3-[m-(1-piperidinylmethyl)phenoxy]propyl]acetamide), the pharmacologically active metabolite, in humans. In this study, the Cytochrome P450 (CYP or P450) enzymes which participate in the metabolism of ROX were identified using human liver microsomes and S9 fractions. M-1 was converted to M-4 (3-[m-(1-piperidinyl-methyl)phenoxy]propylamine) by the enzyme reaction with the S9 but not with microsomes. M-4 was further metabolized to M-5 (3-[m-(1-piperidinylmethyl)phenoxy]propanol) by microsomes. The metabolism was inhibited by coumarin and anti-CYP2A1 serum. (3-[m-(1-piperidinylmethyl)-phenoxy]propionic acid) and M-3 (m-(1-piperidinylmethyl) phenol) formation from M-5 were inhibited by quinidine and anti-CYP2D6 serum. Moreover, M-5 was converted to M-2 and M-3 by cDNA-expressed CYP2D6. In conclusion, this study shows that microsomal enzymes do not participate in the clearance of the active metabolite M-1, CYP2A6 primarily catalyzes M-5 formation from M-4, and CYP2D6 primarily catalyzes M-2 and M-3 formation from M-5 in humans.

  15. Effects of cytochrome P450 inhibitors on the biotransformation of fluorogenic substrates by adult male rat liver microsomes and cDNA-expressed rat cytochrome P450 isoforms.

    Science.gov (United States)

    Makaji, Emilija; Trambitas, Cristina S; Shen, Pamela; Holloway, Alison C; Crankshaw, Denis J

    2010-02-01

    We have evaluated the use of a panel of six fluorogenic cytochrome P450 (CYP) substrates as a potential tool for rapid screening for global changes in CYP activity in rats under different physiological conditions. The biotransformation of 3-[2-(N,N-diethyl-N-methylammonium)ethyl]-7-methoxy-4-methylcoumarin (AMMC), 7-benzyloxy-4-(trifluoromethyl)-coumarin, 7-benzyloxyquinoline, 3-cyano-7-ethoxycoumarin, 7-methoxy-4-(trifluoromethyl)-coumarin, and 7-ethoxy-4-trifluoromethyl-coumarin by microsomes from adult male rat liver were characterized, their sensitivities to 15 putative inhibitors were determined and compared to similar experiments using nine different complementary DNA (cDNA)-expressed rat CYPs. Inhibitory profiles of the substrates in microsomes were different from each other, with some overlap, suggesting that each substrate is to some extent biotransformed by a different CYP isoform. Ketoconazole and clotrimazole were nonselective inhibitors, while ticlopidine selectively inhibited biotransformation of AMMC. CYP2A1 did not biotransform any of the substrates, and CYP2E1 was insensitive to all the inhibitors tested. Some inhibitors did not affect the biotransformation of the fluorogenic substrates by cDNA-expressed isoforms as predicted by their effects on conventional substrates, e.g., chlorzoxazone and diethyldithiocarbamate were inactive against CYP2E1, and CYP2C6 was not inhibited by sulfaphenazole. When results in microsomes and cDNA-expressed CYPs were compared, only the majority of the biotransformation of AMMC by microsomes could be assigned with full confidence to a specific CYP isoform, namely CYP2D2. Nevertheless, different inhibitory profiles of the substrates indicate that the panel will be useful for rapid functional quantification of global CYP activity in rats under different experimental conditions. Our results also demonstrate the inappropriateness of extrapolating inhibitory data between conventional and fluorogenic CYP substrates.

  16. Metabolism of olaquindox in rat liver microsomes: structural elucidation of metabolites by high-performance liquid chromatography combined with ion trap/time-of-flight mass spectrometry.

    Science.gov (United States)

    Liu, Zhaoying; Huang, Lingli; Dai, Menghong; Chen, Dongmei; Wang, Yulian; Tao, Yanfei;